U.S. patent application number 11/192046 was filed with the patent office on 2006-07-27 for immunogenic compositions for gram positive bacteria such as streptococcus agalactiae.
Invention is credited to Michelle Barocchi, Guiliano Bensi, Guido Grandi, Peter Lauer, Domenico Maione, Vega Masignani, Marirosa Mora, Rino Rappuoli, Daniela Rinaudo, John L. Telford, Immaculada Margarit Y Ros.
Application Number | 20060165716 11/192046 |
Document ID | / |
Family ID | 36692677 |
Filed Date | 2006-07-27 |
United States Patent
Application |
20060165716 |
Kind Code |
A1 |
Telford; John L. ; et
al. |
July 27, 2006 |
Immunogenic compositions for gram positive bacteria such as
streptococcus agalactiae
Abstract
The invention relates to the identification of a new adhesin
islands within the genomes of several Group A and Group B
Streptococcus serotypes and isolates. The adhesin islands are
thought to encode surface proteins which are important in the
bacteria's virulence. Thus, the adhesin island proteins of the
invention may be used in immunogenic compositions for prophylactic
or therapeutic immunization against GAS or GBS infection. For
example, the invention may include an immunogenic composition
comprising one or more of the discovered adhesin island
proteins.
Inventors: |
Telford; John L.; (Siena,
IT) ; Grandi; Guido; (Siena, IT) ; Lauer;
Peter; (Barkeley, CA) ; Mora; Marirosa;
(Siena, IT) ; Y Ros; Immaculada Margarit; (Siena,
IT) ; Maione; Domenico; (Siena, IT) ; Bensi;
Guiliano; (Siena, IT) ; Rinaudo; Daniela;
(Siena, IT) ; Masignani; Vega; (Siena, IT)
; Barocchi; Michelle; (Siena, IT) ; Rappuoli;
Rino; (Siena, IT) |
Correspondence
Address: |
BANNER & WITCOFF
1001 G STREET N W
SUITE 1100
WASHINGTON
DC
20001
US
|
Family ID: |
36692677 |
Appl. No.: |
11/192046 |
Filed: |
July 29, 2005 |
Related U.S. Patent Documents
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Application
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Patent Number |
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60697643 |
Jul 11, 2005 |
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60695453 |
Jul 1, 2005 |
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60693001 |
Jun 21, 2005 |
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60673754 |
Apr 22, 2005 |
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60660321 |
Mar 11, 2005 |
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60640069 |
Dec 30, 2004 |
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60633418 |
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60616833 |
Oct 8, 2004 |
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60609833 |
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60592805 |
Jul 29, 2004 |
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Current U.S.
Class: |
424/190.1 |
Current CPC
Class: |
A61K 39/05 20130101;
A61P 31/04 20180101; A61K 39/092 20130101; A61K 39/0208 20130101;
A61P 43/00 20180101; A61K 2039/523 20130101; C07K 16/1267 20130101;
Y02A 50/30 20180101; A61K 39/085 20130101; A61K 39/095 20130101;
A61K 39/09 20130101; A61K 39/08 20130101; Y02A 50/396 20180101;
A61P 37/04 20180101 |
Class at
Publication: |
424/190.1 |
International
Class: |
A61K 39/02 20060101
A61K039/02 |
Claims
1. An immunogenic composition comprising a purified Group B
Streptococcus (GBS) adhesin island (AI) polypeptide in oligomeric
form.
2. The immunogenic composition of claim 1 wherein the GBS AI
polypeptide is selected from a GBS AI-1.
3. The immunogenic composition of claim 1 wherein the GBS AI
polypeptide is selected from a GBS AI-2.
4. The immunogenic composition of claim 2 wherein the GBS AI
polypeptide is selected from the group consisting of GBS 80, GBS
104, GBS 52, and fragments thereof.
5. The immunogenic composition of claim 3 wherein the GBS AI
polypeptide is selected from the group consisting of GBS 59, GBS
67, GBS 150, 01521, 01523, 01524, and fragments thereof.
6. The immunogenic composition of claim 4 wherein the GBS AI
polypeptide is GBS 80.
7. The immunogenic composition of any of claims 1-6 wherein the
oligomeric form is a hyperoligomer.
8 (22). An immunogenic composition comprising a purified Gram
positive bacteria adhesin island (AI) polypeptide in an oligomeric
form.
9 (23). The immunogenic composition of claim 8 wherein the Gram
positive bacteria is of a genus selected from the group consisting
of Streptococcus, Enterococcus, Staphylococcus, Clostridium,
Corynebacterium, or Listeria.
10 (24). The immunogenic composition of claim 9 wherein the Gram
positive bacteria is of the genus Streptococcus.
11 (35). The immunogenic composition of claim 10 wherein the genus
Streptococcus bacteria is Group A Streptococcus (GAS) bacteria and
the Gram positive bacteria AI polypeptide is a GAS AI
polypeptide.
12 (36). The immunogenic composition of claim 11 wherein the GAS AI
polypeptide is selected from a GAS AI-1.
13 (37). The immunogenic composition of claim 11 wherein the GAS AI
polypeptide is selected from a GAS AI-2.
14 (38). The immunogenic composition of claim 11 wherein the GAS AI
polypeptide is selected from a GAS AI-3.
15 (39). The immunogenic composition of claim 11 wherein the GAS AI
polypeptide is selected from a GAS AI-4.
16 (66). The immunogenic composition of any one of claims 8-15
wherein the oligomeric form is a hyperoligomer.
17. An immunogenic composition comprising a first and a second
Group B Streptococcus (GBS) adhesin island (AI) polypeptide.
18. The immunogenic composition of claim 17 wherein the first GBS
AI polypeptide is encoded by a GBS AI-1.
19. The immunogenic composition of claim 18 wherein the second GBS
AI polypeptide is encoded by a GBS AI-2.
20. The immunogenic composition of claim 18 wherein the first GBS
AI polypeptide is selected from the group consisting of GBS 80, GBS
104, GBS 52, and fragments thereof.
21. The immunogenic composition of claim 19 wherein the second GBS
AI polypeptide is selected from the group consisting of GBS 59, GBS
67, GBS 150, 01521, 01523, 01524, and fragments thereof, and
wherein the first and the second GBS AI polypeptide are not the
same polypeptide.
22. The immunogenic composition of claim 19 wherein the first GBS
AI polypeptide is GBS 80 and the second GBS AI polypeptide is GBS
67.
23. An immunogenic composition comprising a first and a second Gram
positive bacteria adhesin island (AI) polypeptide.
24. The immunogenic composition of claim 23 wherein the Gram
positive bacteria is Streptococcus, Enterococcus, Staphylococcus,
Clostridium, Corynebacterium, or Listeria.
25. The immunogenic composition of claim 23 wherein the first Gram
positive bacteria AI polypeptide is a first Group A Streptococcus
(GAS) AI polypeptide.
26. The immunogenic composition of claim 25 wherein the first GAS
AI polypeptide is a first GAS AI-1 polypeptide.
27. The immunogenic composition of claim 25 wherein the first GAS
AI polypeptide is a first GAS AI-2 polypeptide.
28. The immunogenic composition of claim 25 wherein the first GAS
AI polypeptide is a first GAS AI-3 polypeptide.
29. The immunogenic composition of claim 25 wherein the first GAS
AI polypeptide is a first GAS AI-4 polypeptide.
30. The immunogenic composition of any one of claims 25-29 wherein
the second Gram positive bacteria AI polypeptide is a second GAS AI
polypeptide.
31. The immunogenic composition of claim 30 wherein the second GAS
AI polypeptide is a second GAS AI-1 polypeptide.
32. The immunogenic composition of claim 30 wherein the second GAS
AI polypeptide is a second GAS AI-2 polypeptide.
33. The immunogenic composition of claim 30 wherein the second GAS
AI polypeptide is a second GAS AI-3 polypeptide.
34. The immunogenic composition of claim 30 wherein the second GAS
AI polypeptide is a second GAS AI-4 polypeptide.
35. A modified Gram positive bacterium adapted to produce increased
levels of AI surface protein.
36. The modified Gram positive bacterium of claim 35 wherein the AI
surface protein is in oligomeric form.
37. The modified Gram positive bacterium of claim 36 wherein the
oligomeric form is a hyperoligomer.
38. The modified Gram positive bacterium of any one of claims 35-37
which is a non-pathogenic Gram positive bacterium.
39. The modified Gram positive bacterium of claim 38 wherein the
non-pathogenic Gram positive bacterium is Lactococcus lactis.
40. A method for manufacturing an oligomeric adhesin island (AI)
surface antigen comprising: culturing a Gram positive bacterium
that expresses an oligomeric AI surface antigen and isolating the
expressed oligomeric AI surface antigen.
Description
FIELD OF THE INVENTION
[0001] The invention relates to the identification of adhesin
islands within the genome Streptococcus agalactiae ("GBS") and the
use of adhesin island amino acid sequences encoded by these adhesin
islands in compositions for the treatment or prevention of GBS
infection. Similar sequences have been identified in other Gram
positive bacteria. The invention further includes immunogenic
compositions comprising adhesin island amino acid sequences of Gram
positive bacteria for the treatment or prevention of infection of
Gram positive bacteria. Preferred immunogenic compositions of the
invention include an adhesin island surface protein which may be
formulated or purified in an oligomeric or pilus form.
BACKGROUND OF THE INVENTION
[0002] GBS has emerged in the last 20 years as the major cause of
neonatal sepsis and meningitis that affects 0.5-3 per 1000 live
births, and an important cause of morbidity among older age groups
affecting 5-8 per 100,000 of the population. Current disease
management strategies rely on intrapartum antibiotics and neonatal
monitoring which have reduced neonatal case mortality from >50%
in the 1970's to less than 10% in the 1990's. Nevertheless, there
is still considerable morbidity and mortality and the management is
expensive. 15-35% of pregnant women are asymptomatic carriers and
at high risk of transmitting the disease to their babies. Risk of
neonatal infection is associated with low serotype specific
maternal antibodies and high titers are believed to be protective.
In addition, invasive GBS disease is increasingly recognized in
elderly adults with underlying disease such as diabetes and
cancer.
[0003] The "B" in "GBS" refers to the Lancefield classification,
which is based on the antigenicity of a carbohydrate which is
soluble in dilute acid and called the C carbohydrate. Lancefield
identified 13 types of C carbohydrate, designated A to 0, that
could be serologically differentiated. The organisms that most
commonly infect humans are found in groups A, B, D, and G. Within
group B, strains can be divided into at least 9 serotypes (Ia, Ib,
Ia/c, II, III, IV, V, VI, VII and VIII) based on the structure of
their polysaccharide capsule. In the past, serotypes Ia, Ib, II,
and III were equally prevalent in normal vaginal carriage and early
onset sepsis in newborns. Type V GBS has emerged as an important
cause of GBS infection in the USA, however, and strains of types VI
and VIII have become prevalent among Japanese women.
[0004] The genome sequence of a serotype V strain 2603 V/R has been
published (See Tettelin et al. (2002) Proc. Natl. Acad. Sci. USA,
10.1073/pnas.182380799) and various polypeptides for use a vaccine
antigens have been identified (WO 02/34771). The vaccines currently
in clinical trials, however, are based primarily on polysaccharide
antigens. These suffer from serotype-specificity and poor
immunogenicity, and so there is a need for effective vaccines
against S. agalactiae infection.
[0005] S. agalactiae is classified as a gram positive bacterium, a
collection of about 21 genera of bacteria that colonize humans,
have a generally spherical shape, a positive Gram stain reaction
and lack endospores. Gram positive bacteria are frequent human
pathogens and include Staphylococcus (such as S. aureus),
Streptococcus (such as S. pyogenes (GBS), S. pyogenes (GAS), S.
pneumonaie, S. mutans), Enterococcus (such as E. faecalis and E.
faecium), Clostridium (such as C. difficile), Listeria (such as L.
monocytogenes) and Corynebacterium (such as C. diphtheria).
[0006] It is an object of the invention to provide further and
improved compositions for providing immunity against disease and/or
infection of Gram positive bacteria. The compositions are based on
the identification of adhesin islands within Streptococcal genomes
and the use of amino acid sequences encoded by these islands in
therapeutic or prophylactic compositions. The invention further
includes compositions comprising immunogenic adhesin island
proteins within other Gram positive bacteria in therapeutic or
prophylactic compositions.
SUMMARY OF THE INVENTION
[0007] Applicants have identified a new adhesin island, "GBS
Adhesin Island 1", "AI-1" or "GBS AI-1", within the genomes of
several Group B Streptococcus serotypes and isolates. This adhesin
island is thought to encode surface proteins which are important in
the bacteria's virulence. In addition, Applicants have discovered
that surface proteins within GBS Adhesin Islands form a previously
unseen pilus structure on the surface of GBS bacteria. Amino acid
sequences encoded by such GBS Adhesin Islands may be used in
immunogenic compositions for the treatment or prevention of GBS
infection.
[0008] A preferred immunogenic composition of the invention
comprises an AI-1 surface protein, such as GBS 80, which may be
formulated or purified in an oligomeric (pilus) form. In a
preferred embodiment, the oligomeric form is a hyperoligomer.
Electron micrographs depicting some of the first visualizations of
this pilus structure in a wild type GBS strain are shown in FIGS.
16, 17, 49, and 50. In addition, Applicants have transformed a GBS
strain with a plasmid comprising the AI surface protein GBS 80
which resulted in increased production of that AI surface protein.
The electron micrographs of this mutant GBS strain in FIGS. 13-15
reveal long, hyper-oligomeric structures comprising GBS 80 which
appear to cover portions of the surface of the bacteria and stretch
far out into the supernatant. These hyper-oligomeric pilus
structures comprising a GBS AI surface protein may be purified or
otherwise formulated for use in immunogenic compositions.
[0009] GBS AI-1 comprises a series of approximately five open
reading frames encoding for a collection of amino acid sequences
comprising surface proteins and sortases ("AI-1 proteins").
Specifically, AI-1 includes polynucleotide sequences encoding for
two or more of GBS 80, GBS 104, GBS 52, SAG0647 and SAG0648. One or
more of the AI-1 polynucleotide sequences may be replaced by a
polynucleotide sequence coding for a fragment of the replaced ORF.
Alternatively, one or more of the AI-1 open reading frames may be
replaced by a sequence having sequence homology (sequence identity)
to the replaced ORF.
[0010] AI-1 typically resides on an approximately 16.1 kb
transposon-like element frequently inserted into the open reading
frame for trmA. One or more of the AI-1 surface protein sequences
typically include an LPXTG motif (such as LPXTG (SEQ ID NO: 122))
or other sortase substrate motif. The AI surface proteins of the
invention may affect the ability of the GBS bacteria to adhere to
and invade epithelial cells. AI surface proteins may also affect
the ability of GBS to translocate through an epithelial cell layer.
Preferably, one or more AI surface proteins are capable of binding
to or otherwise associating with an epithelial cell surface. AI
surface proteins may also be able to bind to or associate with
fibrinogen, fibronectin, or collagen.
[0011] The sortase proteins are thought to be involved in the
secretion and anchoring of the LPXTG containing surface proteins.
AI-1 may encode at least one surface protein. Alternatively, AI-1
may encode at least two surface proteins and at least one sortase.
Preferably, AI-1 encodes for at least three surface proteins and at
least two sortases. One or more of the surface proteins may include
an LPXTG motif or other sortase substrate motif.
[0012] The GBS AI-1 protein of the composition may be selected from
the group consisting of GBS 80, GBS 104, GBS 52, SAG0647 and
SAG0648. GBS AI-1 surface proteins GBS 80 and GBS 104 are preferred
for use in the immunogenic compositions of the invention.
[0013] In addition to the open reading frames encoding the AI-1
proteins, AI-1 may also include a divergently transcribed
transcriptional regulator such as araC (i.e., the transcriptional
regulator is located near or adjacent to the AI protein open
reading frames, but it transcribed in the opposite direction). It
is believed that araC may regulate the expression of the GBS AI
operon. (See Korbel et al., Nature Biotechnology (2004) 22(7):
911-917 for a discussion of divergently transcribed regulators in
E. coli).
[0014] A second adhesin island, "Adhesin Island-2", "AI-2" or "GBS
AI-2", has also been identified in numerous GBS serotypes. Amino
acid sequences encoded by the open reading frames of AI-2 may also
be used in immunogenic compositions for the treatment or prevention
of GBS infection.
[0015] GBS AI-2 comprises a series of approximately five open
reading frames encoding for a collection of amino acid sequences
comprising surface proteins and sortases. Specifically, AI-2
includes open reading frames encoding for two or more of GBS 67,
GBS 59, GBS 150, SAG1405, SAG1406, 01520, 01521, 01522, 01523,
01523, 01524 and 01525. The GBS AI-2 sequences may be divided into
two subgroups. In one embodiment, AI-2 includes open reading frames
encoding for two or more of GBS 67, GBS 59, GBS 150, SAG1405, and
SAG1406. This collection of open reading frames may be generally
referred to as GBS AI-2 subgroup 1. Alternatively, AI-2 may include
open reading frames encoding for two or more of 01520, 01521,
01522, 01523, 01523, 01524 and 01525. This collection of open
reading frames may be generally referred to as GBS AI-2 subgroup
2.
[0016] One or more of the AI-2 open reading frame polynucleotide
sequences may be replaced by a polynucleotide sequence coding for a
fragment of the replaced ORF. Alternatively, one or more of the
AI-2 open reading frames may be replaced by a sequence having
sequence homology (sequence identity) to the replaced ORF.
[0017] One or more of the AI-2 surface proteins typically include
an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) or other sortase
substrate motif. These sortase proteins are thought to be involved
in the secretion and anchoring of the LPXTG containing surface
proteins. AI-2 may encode for at least one surface protein.
Alternatively, AI-2 may encode for at least two surface proteins
and at least one sortase. Preferably, AI-2 encodes for at least
three surface proteins and at least two sortases. One or more of
the surface proteins may include an LPXTG motif.
[0018] The AI-2 protein of the composition may be selected from the
group consisting of GBS 67, GBS 59, GBS 150, SAG1405, SAG1406,
01520, 01521, 01522, 01523, 01523, 01524 and 01525. AI-2 surface
proteins GBS 67, GBS 59, and 01524 are preferred AI-2 proteins for
use in the immunogenic compositions of the invention. GBS 67 or GBS
59 is particularly preferred.
[0019] GBS AI-2 may also include a divergently transcribed
transcriptional regulator such as a RofA like protein (for example
rogB). As in AI-1, rogB is thought to regulate the expression of
the AI-2 operon.
[0020] The GBS AI proteins of the invention may be used in
immunogenic compositions for prophylactic or therapeutic
immunization against GBS infection. For example, the invention may
include an immunogenic composition comprising one or more GBS AI-1
proteins and one or more GBS AI-2 proteins.
[0021] The immunogenic compositions may also be selected to provide
protection against an increased range of GBS serotypes and strain
isolates. For example, the immunogenic composition may comprise a
first and second GBS AI protein, wherein a full length
polynucleotide sequence encoding for the first GBS AI protein is
not present in a genome comprising a full length polynucleotide
sequence encoding for the second GBS AI protein. In addition, each
antigen selected for use in the immunogenic compositions will
preferably be present in the genomes of multiple GBS serotypes and
strain isolates. Preferably, each antigen is presnt in the genomes
of at least two (i.e., 3, 4, 5, 6, 7, 8, 9, 10, or more) GBS strain
isolates. More preferably, each antigen is present in the genomes
of at least two (i.e., at least 3, 4, 5 or more) GBS serotypes.
[0022] Within GBS AI-1, Applicants have found that Group B
Streptococcus surface exposure of GBS 104 is dependent on the
concurrent expression of GBS 80. It is thought that GBS 80 is
involved in the transport or localization of GBS 104 to the surface
of the bacteria. The two proteins may be oligomerized or otherwise
chemically or physically associated. It is possible that this
association involves a conformational change in GBS 104 that
facilitates its transition to the surface of the GBS bacteria. In
addition, one or more AI sortases may also be involved in this
surface localization and chemical or physical association. Similar
relationships are thought to exist within GBS AI-2. The
compositions of the invention may therefore include at least two AI
proteins, wherein the two AI proteins are physically or chemically
associated. Preferably, the two AI proteins form an oligomer.
Preferably, one or more of the AI proteins are in a
hyper-oligomeric form. In one embodiment, the associated AI
proteins may be purified or isolated from a GBS bacteria or
recombinant host cell.
[0023] It is also an object of the invention to provide further and
improved compositions for providing prophylactic or therapeutic
protection against disease and/or infection of Gram positive
bacteria. The compositions are based on the identification of
adhesin islands within Streptococcal genomes and the use of amino
acid sequences encoded by these islands in therapeutic or
prophylactic compositions. The invention further includes
compositions comprising immunogenic adhesin island proteins within
other Gram positive bacteria in therapeutic or prophylactic
compositions. Preferred Gram positive adhesin island proteins for
use in the invention may be derived from Staphylococcus (such as S.
aureus), Streptococcus (such as S. agalactiae (GBS), S. pyogenes
(GAS), S. pneumonaie, S. mutans), Enterococcus (such as E. faecalis
and E. faecium), Clostridium (such as C. difficile), Listeria (such
as L. monocytogenes) and Corynebacterium (such as C. diphtheria).
Preferably, the Gram positive adhesin island surface proteins are
in oligomeric or hyperologimeric form.
[0024] For example, Applicants have identified adhesin islands
within the genomes of several Group A Streptococcus serotypes and
isolates. These adhesion islands are thought to encode surface
proteins which are important in the bacteria's virulence, and
Applicants have obtained the first electron micrographs revealing
the presence of these adhesin island proteins in hyperoligomeric
pilus structures on the surface of Group A Streptococcus.
[0025] Group A Streptococcus is a human specific pathogen which
causes a wide variety of diseases ranging from pharyngitis and
impetigo through life threatening invasive disease and necrotizing
fasciitis. In addition, post-streptococcal autoimmune responses are
still a major cause of cardiac pathology in children.
[0026] Group A Streptococcal infection of its human host can
generally occur in three phases. The first phase involves
attachment and/or invasion of the bacteria into host tissue and
multiplication of the bacteria within the extracellular spaces.
Generally this attachment phase begins in the throat or the skin.
The deeper the tissue level infected, the more severe the damage
that can be caused. In the second stage of infection, the bacteria
secretes a soluble toxin that diffuses into the surrounding tissue
or even systemically through the vasculature. This toxin binds to
susceptible host cell receptors and triggers innappropropriate
immune responses by these host cells, resulting in pathology.
Because the toxin can diffuse throughout the host, the necrosis
directly caused by the GAS toxins may be physically located in
sites distant from the bacterial infection. The final phase of GAS
infection can occur long after the original bacteria have been
cleared from the host system. At this stage, the host's previous
immune response to the GAS bacteria due to cross reactivity between
epitopes of a GAS surface protein, M, and host tissues, such as the
heart. A general review of GAS infection can be found in Principles
of Bacterial Pathogeneis, Groisman ed., Chapter 15 (2001).
[0027] In order to prevent the pathogenic effects associated with
the later stages of GAS infection, an effective vaccine against GAS
will preferably facilitate host elimination of the bacteria during
the initial attachment and invasion stage.
[0028] Isolates of Group A Streptococcus are historically
classified according to the M surface protein described above. The
M protein is surface exposed trypsin-sensitive protein generally
comprising two polypeptide chains complexed in an alpha helical
formation. The carboxyl terminus is anchored in the cytoplasmic
membrane and is highly conserved among all group A streptococci.
The amino terminus, which extend through the cell wall to the cell
surface, is responsible for the antigenic variability observed
among the 80 or more serotypes of M proteins.
[0029] A second layer of classification is based on a variable,
trypsin-resistant surface antigen, commonly referred to as the
T-antigen. Decades of epidemiology based on M and T serological
typing have been central to studies on the biological diversity and
disease causing potential of Group A Streptococci. While the
M-protein component and its inherent variability have been
extensively characterized, even after five decades of study, there
is still very little known about the structure and variability of
T-antigens. Antisera to define T types is commercially available
from several sources, including Sevapharma
(http://www.sevapharma.cz/en).
[0030] The gene coding for one form of T-antigen, T-type 6, from an
M6 strain of GAS (D741) has been cloned and characterized and maps
to an approximately 11 kb highly variable pathogenicity island.
Schneewind et al., J. Bacteriol. (1990) 172(6):3310-3317. This
island is known as the Fibronectin-binding, Collagen-binding
T-antigen (FCT) region because it contains, in addition to the T6
coding gene (tee6), members of a family of genes coding for Extra
Cellular Matrix (ECM) binding proteins. Bessen et al., Infection
& Immunity (2002) 70(3):1159-1167. Several of the protein
products of this gene family have been shown to directly bind
either fibronectin and/or collagen. See Hanski et al., Infection
& Immunity (1992) 60(12):5119-5125; Talay et al., Infection
& Immunity (1992(60(9):3837-3844; Jaffe et al. (1996)
21(2):373-384; Rocha et al., Adv Exp Med Biol. (1997) 418:737-739;
Kreikemeyer et al., J Biol Chem (2004) 279(16):15850-15859;
Podbielski et al., Mol. Microbiol. (1999) 31(4):1051-64; and
Kreikemeyer et al., Int. J. Med Microbiol (2004) 294(2-3):177-88.
In some cases direct evidence for a role of these proteins in
adhesion and invasion has been obtained.
[0031] Applicants raised antiserum against a recombinant product of
the tee6 gene and used it to explore the expression of T6 in M6
strain 2724. In immunoblot of mutanolysin extracts of this strain,
the antiserum recognized, in addition to a band corresponding to
the predicted molecular mass of the product, very high molecular
weight ladders ranging in mobility from about 100 kDa to beyond the
resolution of the 3-8% gradient gels used.
[0032] This pattern of high molecular weight products is similar to
that observed in immunoblots of the protein components of the pili
identified in Streptococcus agalactiae (described above) and
previously in Corynebacterium diphtheriae. Electron microscropy of
strain M6.sub.--2724 with antisera specific for the product of tee6
revealed abundant surface staining and long pilus like structures
extending up to 700 nanometers from the bacterial surface,
revealing that the T6 protein, one of the antigens recognized in
the original Lancefiled serotyping system, is located within a GAS
Adhesin Island (GAS AI-1) and forms long covalently linked pilus
structures.
[0033] Applicants have identified at least four different Group A
Streptococcus Adhesin Islands. While these GAS AI sequences can be
identified in numerous M types, Applicants have surprisingly
discovered a correlation between the four main pilus subunits from
the four different GAS AI types and specific T classifications.
While other trypsin-resistant surface exposed proteins are likely
also implicated in the T classification designations, the discovery
of the role of the GAS adhesin islands (and the associated
hyper-oligomeric pilus like structures) in T classification and GAS
serotype variance has important implications for prevention and
treatment of GAS infections. Applicants have identified protein
components within each of the GAS adhesin islands which are
associated with the pilus formation. These proteins are believed to
be involved in the bacteria's initial adherence mechanisms.
Immunological recognition of these proteins may allow the host
immune response to slow or prevent the bacteria's transition into
the more pathogenic later stages of infection.
[0034] In addition, Applicants have discovered that the GBS pili
structures appear to be implicated in the formation of biofilms
(populations of bacteria growing on a surface, often enclosed in an
exopolysaccharide matrix). Biofilms are generally associated with
bacterial resistance, as antibiotic treatments and host immune
response are frequently unable to erradicate all of the bacteria
components of the biofilm. Direction of a host immune response
against surface proteins exposed during the first steps of
bacterial attachment (i.e., before complete biofilm formation) is
preferable.
[0035] The invention therefore provides for improved immunogenic
compositions against GAS infection which may target GAS bacteria
during their initial attachment efforts to the host epithelial
cells and may provide protection against a wide range of GAS
serotypes. The immunogenic compositions of the invention include
GAS AI surface proteins which may be formulated in an oligomeric,
or hyperoligomeric (pilus) form. The immunogenic compositions of
the invention may include one or more GAS AI surface proteins. The
invention also includes combinations of GAS AI surface proteins.
Combinations of GAS AI surface proteins may be selected from the
same adhesin island or they may be selected from different GAS
adhesin islands.
[0036] Amino acid sequence encoded by such GAS Adhesin Islands may
be used in immunogenic compositions for the treatment or prevention
of GAS infection. Preferred immunogenic compositions of the
invention comprise a GAS AI surface protein which has been
formulated or purified in an oligomeric (pilus) form. In a
preferred embodiment, the oligomeric form is a hyperoligomer.
[0037] GAS Adhesin Islands generally include a series of open
reading frames within a GAS genome that encode for a collection of
surface proteins and sortases. A GAS Adhesin Island may encode for
an amino acid sequence comprising at least one surface protein. The
Adhesin Island, therefore, may encode at least one surface protein.
Alternatively, a GAS Adhesin Island may encode for at least two
surface proteins and at least one sortase. Preferably, a GAS
Adhesin Island encodes for at least three surface proteins and at
least two sortases. One or more of the surface proteins may include
an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) or other sortase
substrate motif. One or more GAS AI surface proteins may
participate in the formation of a pilus structure on the surface of
the Gram positive bacteria.
[0038] GAS Adhesin Islands of the invention preferably include a
divergently transcribed transcriptional regulator. The
transcriptional regulator may regulate the expression of the GAS AI
operon. Examples of transcriptional regulators found in GAS AI
sequences include RofA and Nra.
[0039] The GAS AI surface proteins may bind or otherwise adhere to
fibrinogen, fibronectin, or collagen. One or more of the GAS AI
surface proteins may comprise a fimbrial structural subunit.
[0040] One or more of the GAS AI surface proteins may include an
LPXTG motif or other sortase substrate motif. The LPXTG motif may
be followed by a hydrophobic region and a charged C terminus, which
are thought to retard the protein in the cell membrane to
facilitate recognition by the membrane-localized sortase. See
Barnett, et al., J. Bacteriology (2004) 186 (17): 5865-5875.
[0041] GAS AI sequences may be generally categorized as Type 1,
Type 2, Type 3, or Type 4, depending on the number and type of
sortase sequences within the island and the percentage identity of
other proteins (with the exception of RofA and cpa) within the
island. Schematics of the GAS adhesin islands are set forth in FIG.
51A and FIG. 162. "GAS Adhesin Island-1 or "GAS AI-1" comprises a
series of approximately five open reading frames encoding for a
collection of amino acid sequences comprising surface proteins and
sortases ("GAS AI-1 proteins"). GAS AI-1 preferably comprises
surface proteins, a srtB sortase and a rofA divergently transcribed
transcriptional regulator. GAS AI-1 surface proteins may include a
fibronectin binding protein, a collagen adhesion protein and a
fimbrial structural subunit. The fimbrial structural subunit (also
known as tee6) is thought to form the shaft portion of the pilus
like structure, while the collagen adhesion protein (Cpa) is
thought to act as an accessory protein facilitating the formation
of the pilus structure, exposed on the surface of the bacterial
capsule.
[0042] Specifically, GAS AI-1 includes polynucleotide sequences
encoding for two or more of M6_Spy0157, M6_Spy0158, M6_Spy0159,
M6_Spy0160, M6_Spy0161. The GAS AI-1 may also include
polynucleotide sequences encoding for any one of CDC SS
410_fimbrial, ISS3650_fimbrial, DSM2071_fimbrial
[0043] A preferred immunogenic composition of the invention
comprises a GAS AI-1 surface protein which may be formulated or
purified in an oligomeric (pilus) form. In a preferred embodiment,
the oligomeric form is a hyperoligomer. The immunogenic composition
of the invention may alternatively comprise an isolated GAS AI-1
surface protein in oligomeric (pilus) form. The oligomer or
hyperoligomeric pilus structures comprising GAS AI-1 surface
proteins may be purified or otherwise formulated for use in
immunogenic compositions.
[0044] One or more of the GAS AI-1 polynucleotide sequences may be
replaced by a polynucleotide sequence coding for a fragment of the
replaced ORF. Alternatively, one or more of the GAS AI-1 open
reading frames may be replaced by a sequence having sequence
homology (sequence identity) to the replaced ORF.
[0045] One or more of the GAS AI-1 surface proteins typically
include an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) or other
sortase substrate motif. These sortase proteins are thought to be
involved in the secretion and anchoring of the LPXTG containing
surface proteins. GAS AI-1 may encode for at least one surface
protein. Alternatively, GAS AI-1 may encode for at least two
surface proteins and at least one sortase. Preferably, GAS AI-1
encodes for at least three surface proteins and at least two
sortases. One or more of the surface proteins may include an LPXTG
motif.
[0046] GAS AI-1 preferably includes a srtB sortase. GAS srtB
sortases may preferably anchor surface proteins with an LPSTG motif
(SEQ ID NO: 166), particularly where the motif is followed by a
serine.
[0047] The GAS AI-1 protein of the composition may be selected from
the group consisting of M6_Spy0157, M6_Spy0158, M6_Spy0159,
M6_Spy0160 M6_Spy0161, CDC SS 410_fimbrial, ISS3650_fimbrial, and
DSM2071_fimbrial. GAS AI-1 surface proteins M6_Spy0157 (a
fibronectin binding protein), M6_Spy0159 (a collagen adhesion
protein, Cpa), M6_Spy0160 (a fimbrial structural subunit, tee6),
CDC SS 410_fimbrial (a fimbrial structural subunit),
ISS3650_fimbrial (a fimbrial structural subunit), and
DSM2071_fimbrial (a fimbrial structural subunit) are preferred GAS
AI-1 proteins for use in the immunogenic compositions of the
invention. The fimbrial structural subunit tee6 and the collagen
adhesion protein Cpa are preferred GAS AI-1 surface proteins.
Preferably, each of these GAS AI-1 surface proteins includes an
LPXTG sortase substrate motif, such as LPXTG (SEQ ID NO: 122) or
LPXSG (SEQ ID NO: 134) (conservative replacement of threonine with
serine).
[0048] In addition to the open reading frames encoding the GAS AI-1
proteins, GAS AI-1 may also include a divergently transcribed
transcriptional regulator such as rofA (i.e., the transcriptional
regulator is located near or adjacent to the GAS AI protein open
reading frames, but it transcribed in the opposite direction).
[0049] The GAS AI-1 surface proteins may be used alone, in
combination with other GAS AI-1 surface proteins or in combination
with other GAS AI surface proteins. Preferably, the immunogenic
compositions of the invention include the GAS AI-1 fimbrial
structural subunit (tee6) and the GAS AI-1 collagen binding
protein. Still more preferably, the immunogenic compositions of the
invention include the GAS AI-1 fimbrial structural subunit
(tee6).
[0050] A second GAS adhesion island, "GAS Adhesin Island-2" or "GAS
AI-2," has also been identified in GAS serotypes. Amino acid
sequences encoded by the open reading frames of GAS AI-2 may also
be used in immunogenic compositions for the treatment or prevention
of GAS infection.
[0051] A preferred immunogenic composition of the invention
comprises a GAS AI-2 surface protein which may be formulated or
purified in an oligomeric (pilus) form. In a preferred embodiment,
the oligomeric form is a hyperoligomer. A preferred immunogenic
composition of the invention alternatively comprises an isolated
GAS AI-2 surface protein in oligomeric (pilus) form. The oligomer
or hyperoligomeric pilus structures comprising GAS AI-2 surface
proteins may be purified or otherwise formulated for use in
immunogenic compositions.
[0052] GAS AI-2 comprises a series of approximately eight open
reading frames encoding for a collection of amino acid sequences
comprising surface proteins and sortases ("GAS AI-2 proteins"). GAS
AI-2 preferably comprises surface proteins, a srtB sortase, a srtC1
sortase and a rofA divergently transcribed transcriptional
regulator.
[0053] Specifically, GAS AI-2 includes polynucleotide sequences
encoding for two or more of GAS15, Spy0127, GAS16, GAS17, GAS18,
Spy0131, Spy0133, and GAS20.
[0054] One or more of the GAS AI-2 polynucleotide sequences may be
replaced by a polynucleotide sequence coding for a fragment of the
replaced ORF. Alternatively, one or more of the GAS AI-2 open
reading frames may be replaced by a sequence having sequence
homology (sequence identity) to the replaced ORF.
[0055] One or more of the GAS AI-2 surface proteins typically
include an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) or other
sortase substrate motif. These sortase proteins are thought to be
involved in the secretion and anchoring of the LPXTG containing
surface proteins. GAS AI-2 may encode for at least one surface
protein. Alternatively, GAS AI-2 may encode for at least two
surface proteins and at least one sortase. Preferably, GAS AI-2
encodes for at least three surface proteins and at least two
sortases. One or more of the surface proteins may include an LPXTG
motif.
[0056] GAS AI-2 preferably includes a srtB sortase and a srtC1
sortase. As discussed above, GAS srtB sortases may preferably
anchor surface proteins with an LPSTG motif (SEQ ID NO: 166),
particularly where the motif is followed by a serine. GAS srtC1
sortase may preferentially anchor surface proteins with a V(P/V)PTG
(SEQ ID NO: 167) motif. GAS srtC1 may be differentially regulated
by rofA.
[0057] The GAS AI-2 protein of the composition may be selected from
the group consisting of GAS15, Spy0127, GAS16, GAS17, GAS18,
Spy0131, Spy0133, and GAS20. GAS AI-2 surface proteins GAS15 (Cpa),
GAS16 (thought to be a fimbrial protein, M1.sub.--128), GAS18
(M1_Spy0130), and GAS20 are preferred for use in the immunogenic
compositions of the invention. GAS 16 is thought to form the shaft
portion of the pilus like structure, while GAS 15 (the collagen
adhesion protein Cpa) and GAS 18 are thought to act as accessory
proteins facilitating the formation of the pilus structure, exposed
on the surface of the bacterial capsule. Preferably, each of these
GAS AI-2 surface proteins includes an LPXTG sortase substrate
motif, such as LPXTG (SEQ ID NO: 122), VVXTG (SEQ ID NO: 135), or
EVXTG (SEQ ID NO: 136).
[0058] In addition to the open reading frames encoding the GAS AI-2
proteins, GAS AI-2 may also include a divergently transcribed
transcriptional regulator such as rofA (i.e., the transcriptional
regulator is located near or adjacent to the GAS AI protein open
reading frames, but it transcribed in the opposite direction). The
GAS AI-2 surface proteins may be used alone, in combination with
other GAS AI-2 surface proteins or in combination with other GAS AI
surface proteins. Preferably, the immunogenic compositions of the
invention include the GAS AI-2 fimbrial protein (GAS 16), the GAS
AI-2 collagen binding protein (GAS 15) and GAS 18 (M1_Spy0130).
More preferably, the immunogenic compositions of the invention
include the GAS AI-2 fimbrial protein (GAS 16).
[0059] A third GAS adhesion island, "GAS Adhesin Island-3" or "GAS
AI-3," has also been identified in numerous GAS serotypes. Amino
acid sequences encoded by the open reading frames of GAS AI-3 may
also be used in immunogenic compositions for the treatment or
prevention of GAS infection.
[0060] A preferred immunogenic composition of the invention
comprises a GAS AI-3 surface protein which may be formulated or
purified in an oligomeric (pilus) form. In a preferred embodiment,
the oligomeric form is a hyperoligomer. A preferred immunogenic
composition of the invention alternatively comprises an isolated
GAS AI-3 surface protein in oligomeric (pilus) form. The oligomer
or hyperoligomeric pilus structures comprising GAS AI-3 surface
proteins may be purified or otherwise formulated for use in
immunogenic compositions. GAS AI-3 comprises a series of
approximately seven open reading frames encoding for a collection
of amino acid sequences comprising surface proteins and sortases
("GAS AI-3 proteins"). GAS AI-3 preferably comprises surface
proteins, a srtC2 sortase, and a Negative transcriptional regulator
(Nra) divergently transcribed transcriptional regulator. GAS AI-3
surface proteins may include a collagen binding protein, a fimbrial
protein, and a F2 like fibronectin-binding protein. GAS AI-3
surface proteins may also include a hypothetical surface protein.
The fimbrial protein is thought to form the shaft portion of the
pilus like structure, while the collagen adhesion protein (Cpa) and
the hypothetical surface protein are thought to act as accessory
proteins facilitating the formation of the pilus structure, exposed
on the surface of the bacterial capsule. Preferred AI-3 surface
proteins include the fimbrial proein, the collagen binding protein
and the hypothetical protein. Preferably, each of these GAS AI-3
surface proteins include an LPXTG sortase substrate motif, such as
LPXTG (SEQ ID NO: 122), VPXTG (SEQ ID NO: 137), QVXTG (SEQ ID NO:
138) or LPXAG (SEQ ID NO: 139).
[0061] Specifically, GAS AI-3 includes polynucleotide sequences
encoding for two or more of SpyM3.sub.--0098, SpyM3.sub.--0099,
SpyM3.sub.--0100, SpyM3.sub.--0101, SpyM3.sub.--0102,
SpyM3.sub.--0103, SpyM3.sub.--0104, Sps0100, Sps0101, Sps0102,
Sps0103, Sps0104, Sps0105, Sps0106, orf78, orf79, orf80, orf81,
orf82, orf83, orf84, spyM18.sub.--0126, spyM18.sub.--0127,
spyM18.sub.--0128, spyM18.sub.--0129, spyM18.sub.--0130,
spyM18.sub.--0131, spyM18.sub.--0132, SpyoM0100156, SpyoM0100155,
SpyoM0100154, SpyoM01000153, SpyoM01000152, SpyoM01000151,
SpyoM01000150, SpyoM01000149, ISS3040_fimbrial, ISS3776_fimbrial,
and ISS4959_fimbrial. In one embodiment, GAS AI-3 may include open
reading frames encoding for two or more of SpyM3.sub.--0098,
SpyM3.sub.--0099, SpyM3.sub.--0100, SpyM3.sub.--0101,
SpyM3.sub.--0102, SpyM3.sub.--0103, and SpyM3.sub.--0104.
Alternatively, GAS AI-3 may include open reading frames encoding
for two or more of Sps0100, Sps0101, Sps0102, Sps0103, Sps0104,
Sps0105, and Sps0106. Alternatively, GAS AI-3 may include open
reading frames encoding for two or more of orf78, orf79, orf80,
orf81, orf82, orf83, and orf84. Alternatively, GAS AI-3 may include
open reading frames encoding for two or more of spyM18.sub.--0126,
spyM18.sub.--0127, spyM18.sub.--0128, spyM18.sub.--0129,
spyM18.sub.--0130, spyM18.sub.--0131, and spyM18.sub.--0132.
Alternatively, GAS AI-3 may include open reading frames encoding
for two or more of SpyoM01000156, SpyoM01000155, SpyoM01000154,
SpyoM01000153, SpyoM01000152, SpyoM01000151, SpyoM01000150, and
SpyoM01000149. Alternatively, GAS AI-1 may also include
polynucleotide sequences encoding for any one of ISS3040_fimbrial,
ISS3776_fimbrial, and ISS4959_fimbrial.
[0062] One or more of the GAS AI-3 polynucleotide sequences may be
replaced by a polynucleotide sequence coding for a fragment of the
replaced ORF. Alternatively, one or more of the GAS AI-3 open
reading frames may be replaced by a sequence having sequence
homology (sequence identity) to the replaced ORF.
[0063] One or more of the GAS AI-3 surface proteins typically
include an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) or other
sortase substrate motif. These sortase proteins are thought to be
involved in the secretion and anchoring of the LPXTG containing
surface proteins. GAS AI-3 may encode for at least one surface
protein. Alternatively, GAS AI-3 may encode for at least two
surface proteins and at least one sortase. Preferably, GAS AI-3
encodes for at least three surface proteins and at least two
sortases. One or more of the surface proteins may include an LPXTG
motif.
[0064] GAS AI-3 preferably includes a srtC2 type sortase. GAS srtC2
type sortases may preferably anchor surface proteins with a QVPTG
(SEQ ID NO: 140) motif, particularly when the motif is followed by
a hydrophobic region and a charged C terminus tail. GAS SrtC2 may
be differentially regulated by Nra.
[0065] The GAS AI-3 protein of the composition may be selected from
the group consisting of SpyM3.sub.--0098, SpyM3.sub.--0099,
SpyM3.sub.--0100, SpyM3.sub.--0101, SpyM3.sub.--0102,
SpyM3.sub.--0103, SpyM3.sub.--0104, Sps0100, Sps0101, Sps0102,
Sps0103, Sps0104, Sps0105, Sps0106, orf78, orf79, orf80, orf81,
orf82, orf83, orf84, spyM18.sub.--0126, spyM18.sub.--0127,
spyM18.sub.--0128, spyM18.sub.--0129, spyM18.sub.--0130,
spyM18.sub.--0131, spyM18.sub.--0132, SpyoM01000156, SpyoM01000155,
SpyoM01000154, SpyoM01000153, SpyoM01000152, SpyoM01000151,
SpyoM01000150, SpyoM01000149, ISS3040_fimbrial, ISS3776_fimbrial,
and ISS4959_fimbrial. GAS AI-3 surface proteins SpyM3.sub.--0098,
SpyM3.sub.--0100, SpyM3.sub.--0102, SpyM3.sub.--0104, SPs0100,
SPs0102, SPs0104, SPs0106, orf78, orf80, orf82, orf84,
spyM18.sub.--0126, spyM18.sub.--0128, spyM18.sub.--0130,
spyM18.sub.--0132, SpyoM01000155, SpyoM01000153, SpyoM01000151,
SpyoM01000149, ISS3040_fimbrial, ISS3776_fimbrial, and
ISS4959_fimbrial are preferred GAS AI-3 proteins for use in the
immunogenic compositions of the invention.
[0066] In addition to the open reading frames encoding the GAS AI-3
proteins, GAS AI-3 may also include a transcriptional regulator
such as Nra.
[0067] GAS AI-3 may also include a LepA putative signal peptidase I
protein.
[0068] The GAS AI-3 surface proteins may be used alone, in
combination with other GAS AI-3 surface proteins or in combination
with other GAS AI surface proteins. Preferably, the immunogenic
compositions of the invention include the GAS AI-3 fimbrial
protein, the GAS AI-3 collagen binding protein, the GAS AI-3
surface protein (such as SpyM3.sub.--0102, M3_Sps0104, M5_orf82, or
spyM18.sub.--0130), and fibronectin binding protein PrtF2. More
preferably, the immunogenic compositions of the invention include
the GAS AI-3 fimbrial protein, the GAS AI-3 collagen binding
protein, and the GAS AI-3 surface protein. Still more preferably,
the immunogenic compositions of the invention include the GAS AI-3
fimbrial protein.
[0069] Representative examples of the GAS AI-3 fimbrial protein
include SpyM3.sub.--0100, M3_Sps0102, M5_orf80, spyM18.sub.--128,
SpyoM01000153, ISS3040_fimbrial, ISS3776_fimbrial,
ISS4959_fimbrial.
[0070] Representative examples of the GAS AI-3 collagen binding
protein include SpyM3.sub.--0098, M3_Sps0100, M5_orf 78,
spyM18.sub.--0126, and SpyoM01000155.
[0071] Representative examples of the GAS AI-3 fibronectin binding
protein PrtF2 include SpyM3.sub.--0104, M3_Sps0106, M5_orf84 and
spyM18.sub.--0132, and SpyoM01000149.
[0072] A fourth GAS adhesion island, "GAS Adhesin Island-4" or "GAS
AI-4," has also been identified in GAS serotypes. Amino acid
sequences encoded by the open reading frames of GAS AI-4 may also
be used in immunogenic compositions for the treatment or prevention
of GAS infection.
[0073] A preferred immunogenic composition of the invention
comprises a GAS AI-4 surface protein which may be formulated or
purified in an oligomeric (pilus) form. In a preferred embodiment,
the oligomeric form is a hyperoligomer. A preferred immunogenic
composition of the invention alternatively comprises an isolated
GAS AI-4 surface protein in oligomeric (pilus) form. The oligomer
or hyperoligomeric pilus structures comprising GAS AI-3 surface
proteins may be purified or otherwise formulated for use in
immunogenic compositions. The oligomeric or hyperoligomeric pilus
structures comprising GAS AI-4 surface proteins may be purified or
otherwise formulated for use in immunogenic compositions.
[0074] GAS AI-4 comprises a series of approximately eight open
reading frames encoding for a collection of amino acid sequences
comprising surface proteins and sortases ("GAS AI-4 proteins").
This GAS adhesin island 4 ("GAS AI-4") comprises surface proteins,
a srtC2 sortase, and a RofA regulatory protein. GAS AI-4 surface
proteins within may include a fimbrial protein, F1 and F2 like
fibronectin-binding proteins, and a capsular polysaccharide
adhesion protein (Cpa). GAS AI-4 surface proteins may also include
a hypothetical surface protein in an open reading frame (orf).
[0075] The fimbral protein (EftLSL) is thought to form the shaft
portion of the pilus like structure, while the collagen adhesion
protein (Cpa) and the hypothetical protein are thought to act as
accessory proteins facilitating the formation of the pilus
structure, exposed on the surface of the bacterial capsule.
Preferably, each of these GAS AI-4 surface proteins include an
LPXTG sortase substrate motif, such as LPXTG (SEQ ID NO: 122),
VPXTG (SEQ ID NO: 137), QVXTG (SEQ ID NO: 138) or LPXAG (SEQ ID NO:
139).
[0076] Specifically, GAS AI-4 includes polynucleotide sequences
encoding for two or more of 19224134, 19224135, 19224136, 19224137,
19224138, 19224139, 19224140, and 19224141. A GAS AI-4
polynucleotide may also include polynucleotide sequences encoding
for any one of 20010296_fimbrial, 20020069_fimbrial, CDC SS
635_fimbrial, ISS4883_fimbrial, ISS4538_fimbrial. One or more of
the GAS AI-4 polynucleotide sequences may be replaced by a
polynucleotide sequence coding for a fragment of the replaced ORF.
Alternatively, one or more of the GAS AI-4 open reading frames may
be replaced by a sequence having sequence homology (sequence
identity) to the replaced ORF.
[0077] One or more of the GAS AI-4 surface proteins typically
include an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) or other
sortase substrate motif. These sortase proteins are thought to be
involved in the secretion and anchoring of the LPXTG containing
surface proteins. GAS AI-4 may encode for at least one surface
protein. Alternatively, GAS AI-4 may encode for at least two
surface proteins and at least one sortase. Preferably, GAS AI-4
encodes for at least three surface proteins and at least two
sortases. One or more of the surface proteins may include an LPXTG
motif.
[0078] GAS AI-4 includes a SrtC2 type sortase. GAS SrtC2 type
sortases may preferably anchor surface proteins with a QVPTG (SEQ
ID NO: 140) motif, particularly when the motif is followed by a
hydrophobic region and a charged C terminus tail.
[0079] The GAS AI-4 protein of the composition may be selected from
the group consisting of 19224134, 19224135, 19224136, 19224137,
19224138, 19224139, 19224140, 19224141, 20010296_fimbrial,
20020069_fimbrial, CDC SS 635_fimbrial, ISS4883_fimbrial, and
ISS4538_fimbrial. GAS AI-4 surface proteins 19224134, 19224135,
19224137, 19224139, 19224141, 20010296_fimbrial, 20020069_fimbrial,
CDC SS 635_fimbrial, ISS4883_fimbrial, ISS4538_fimbrial are
preferred proteins for use in the immunogenic compositions of the
invention.
[0080] In addition to the open reading frames encoding the GAS AI-4
proteins, GAS AI-4 may also include a divergently transcribed
transcriptional regulator such as RofA (i.e., the transcriptional
regulator is located near or adjacent to the AI protein open
reading frames, but it transcribed in the opposite direction.
[0081] GAS AI-4 may also include a LepA putative signal peptidase I
protein and a MsmRL protein. The GAS AI-4 surface proteins may be
used alone, in combination with other GAS AI-4 surface proteins or
in combination with other GAS AI surface proteins. Preferably, the
immunogenic compositions of the invention include the GAS AI-4
fimbrial protein (EftLSL or 20010296_fimbrial, 20020069_fimbrial,
CDC SS 635_fimbrial, ISS4883_fimbrial, or ISS4538_fimbrial), the
GAS AI-4 collagen binding protein, the GAS AI-4 surface protein
(such as M12 isolate A735 orf 2), and fibronectin binding protein
PrtF1 and PrtF2. More preferably, the immunogenic compositions of
the invention include the GAS AI-4 fimbrial protein, the GAS AI-4
collagen binding protein, and the GAS AI-4 surface protein. Still
more preferably, the immunogenic compositions of the invention
include the GAS AI-4 fimbrial protein.
[0082] The GAS AI proteins of the invention may be used in
immunogenic compositions for prophylactic or therapeutic
immunization against GAS infection. For example, the invention may
include an immunogenic composition comprising one or more GAS AI-1
proteins and one or more of any of GAS AI-2, GAS AI-3, or GAS AI-4
proteins. For example, the invention includes an immunogenic
composition comprising at least two GAS AI proteins where each
protein is selected from a different GAS adhesin island. The two
GAS AI proteins may be selected from one of the following GAS AI
combinations: GAS AI-1 and GAS AI-2; GAS AI-1 and GAS AI-3; GAS
AI-1 and GAS AI-4; GAS AI-2 and GAS AI-3; GAS AI-2 and GAS AI-4;
and GAS AI 3 and GAS AI-4. Preferably the combination includes
fimbrial proteins from one or more GAS adhesin islands.
[0083] The immunogenic compositions may also be selected to provide
protection against an increased range of GAS serotypes and strain
isolates. For example, the immunogenic composition may comprise a
first and second GAS AI protein, wherein a full length
polynucleotide sequence encoding for the first GAS AI protein is
not present in a genome comprising a full length polynucleotide
sequence encoding for the second GAS AI protein. In addition, each
antigen selected for use in the immunogenic compositions will
preferably be present in the genomes of multiple GAS serotypes and
strain isolates. Preferably, each antigen is present in the genomes
of at least two (i.e., 3, 4, 5, 6, 7, 8, 9, 10, or more) GAS strain
isolates. More preferably, each antigen is present in the genomes
of at least two (i.e., at least 3, 4, 5, or more) GAS
serotypes.
[0084] Applicants have also identified adhesin islands within the
genome of Streptococcus pneumoniae. These adhesion islands are
thought to encode surface proteins which are important in the
bacteria's virulence. Amino acid sequence encoded by such S.
pneumoniae Adhesin Islands may be used in immunogenic compositions
for the treatment or prevention of S. pneumoniae infection.
Preferred immunogenic compositions of the invention comprise a S.
pneumoniae AI surface protein which has been formulated or purified
in an oligomeric (pilus) form. In a preferred embodiment, the
oligomeric form is a hyperoligomer. A preferred immunogenic
composition of the invention alternatively comprises an isolated S.
pneumoniae surface protein in oligomeric (pilus) form. The oligomer
or hyperoligomeric pilus structures comprising S. pneumoniae
surface proteins may be purified or otherwise formulated for use in
immunogenic compositions.
[0085] The S. pneumoniae Adhesin Islands generally include a series
of open reading frames within a S. pneumoniae genome that encode
for a collection of surface proteins and sortases. A S. pneumoniae
Adhesin Island may encode for an amino acid sequence comprising at
least one surface protein. Alternatively, the S. pneumoniae Adhesin
Island may encode for at least two surface proteins and at least
one sortase. Preferably, a S. pneumoniae Adhesin Island encodes for
at least three surface proteins and at least two sortases. One or
more of the surface proteins may include an LPTXG motif (such as
LPXTG (SEQ ID NO: 122)) or other sortase substrate motif. One or
more S. pneumoniae AI surface proteins may participate in the
formation of a pilus structure on the surface of the S. pneumoniae
bacteria.
[0086] The S. pneumoniae Adhesin Islands of the invention
preferably include a divergently transcribed transcriptional
regulator. The transcriptional regulator may regulate the
expression of the S. pneumonaie AI operon. An example of a
transcriptional regulator found in S. pneumoniae AI sequences is
rlrA.
[0087] A schematic of the organization of a S. pneumoniae AI locus
is provided in FIG. 137. The locus comprises open reading frames
encoding a transcriptional regulator (rlrA), cell wall surface
proteins (rrgA, rrgB, rrgc) and sortases (srt B, srtC, srtD).
[0088] S. pneumoniae AI sequences may be generally divided into two
groups of homology, S. pneuamoniae AI-a and AI-b. S. pneumoniae
strains that comprise AI-a include 14 CSR 10, 19A Hungary 6, 23 F
Poland 15, 670, 6B Finland 12, and 6B Spain 2. S. pneumoniae AI
strains that comprise AI-b include 19F Taiwan 14, 9V Spain 3, 23F
Taiwan 15 and TIGR 4.
[0089] S. pneumoniae AI from TIGR4 comprises a series of
approximately seven open reading frames encoding for a collection
of amino acid sequences comprising surface proteins and sortases
("S. pneumoniae AI proteins"). Specifically, S. pneumoniae AI from
TIGR4 includes polynucleotide sequences encoding for two or more of
SP0462, SP0463, SP0464, SP0465, SP0466, SP0467, and SP0468.
[0090] One or more of the S. pneumoniae AI from TIGR4
polynucleotide sequences may be replaced by a polynucleotide
sequence coding for a fragment of the replaced ORF. Alternatively,
one or more of the S. pneumoniae AI from TIGR4 open reading frames
may be replaced by a sequence having sequence homology to the
replaced ORF.
[0091] S. pneumoniae strain 670 AI comprises a series of
approximately seven open reading frames encoding for a collection
of amino acid sequences comprising surface proteins and sortases
("S. pneumoniae AI proteins"). Specifically, S. pneumoniae strain
670 AI includes polynucleotide sequences encoding for two or more
of orf1.sub.--670, orf3.sub.--670, orf4.sub.--670, orf5.sub.--670,
orf6.sub.--670, orf7.sub.--670, and orf8.sub.--670.
[0092] One or more of the S. pneumoniae strain 670 AI
polynucleotide sequences may be replaced by a polynucleotide
sequence coding for a fragment of the replaced ORF. Alternatively,
one or more of the S. pneumoniae strain 670 AI open reading frames
may be replaced by a sequence having sequence homology to the
replaced ORF.
[0093] S. pneumoniae AI from 14 CSR10 comprises a series of
approximately seven open reading frames encoding for a collection
of amino acid sequences comprising surface proteins and sortases
("S. pneumoniae AI proteins"). Specifically, S. pneumoniae AI from
14 CSR10 includes polynucleotide sequences encoding for two or more
of ORF2.sub.--14CSR, ORF3.sub.--14CSR, ORF4.sub.--14CSR,
ORF5.sub.--14CSR, ORF6.sub.--14CSR, ORF7.sub.--14CSR, and
ORF8.sub.--14CSR.
[0094] One or more of the S. pneumoniae AI from 14 CSR10
polynucleotide sequences may be replaced by a polynucleotide
sequence coding for a fragment of the replaced ORF. Alternatively,
one or more of the S. pneumoniae AI from 14 CSR10 open reading
frames may be replaced by a sequence having sequence homology to
the replaced ORF.
[0095] S. pneumoniae AI from 19A Hungary 6 comprises a series of
approximately seven open reading frames encoding for a collection
of amino acid sequences comprising surface proteins and sortases
("S. pneumoniae AI proteins"). Specifically, S. pneumoniae AI from
19A Hungary 6 includes polynucleotide sequences encoding for two or
more of ORF2.sub.--19AH, ORF3.sub.--1 gAH, ORF4.sub.--19AH,
ORF5.sub.--19AH, ORF6.sub.--19AH, ORF7.sub.--19AH, and
ORF8.sub.--19AH.
[0096] One or more of the S. pneumoniae AI from 19A Hungary 6
polynucleotide sequences may be replaced by a polynucleotide
sequence coding for a fragment of the replaced ORF. Alternatively,
one or more of the S. pneumoniae AI from 19A Hungary 6 open reading
frames may be replaced by a sequence having sequence homology to
the replaced ORF.
[0097] S. pneumoniae AI from 19F Taiwan 14 comprises a series of
approximately seven open reading frames encoding for a collection
of amino acid sequences comprising surface proteins and sortases
("S. pneumoniae AI proteins"). Specifically, S. pneumoniae AI from
19F Taiwan 14 includes polynucleotide sequences encoding for two or
more of ORF2.sub.--19FTW, ORF3.sub.--19FTW, ORF4.sub.--19FTW,
ORF5.sub.--19FTW, ORF6.sub.--19FTW, ORF7.sub.--19FTW, and
ORF8.sub.--19FTW.
[0098] One or more of the S. pneumoniae AI from 19F Taiwan 14
polynucleotide sequences may be replaced by a polynucleotide
sequence coding for a fragment of the replaced ORF. Alternatively,
one or more of the S. pneumoniae AI from 19F Taiwan 14 open reading
frames may be replaced by a sequence having sequence homology to
the replaced ORF.
[0099] S. pneumoniae AI from 23F Poland 16 comprises a series of
approximately seven open reading frames encoding for a collection
of amino acid sequences comprising surface proteins and sortases
("S. pneumoniae AI proteins"). Specifically, S. pneumoniae AI from
23F Poland 16 includes polynucleotide sequences encoding for two or
more of ORF2.sub.--23FP, ORF3.sub.--23FP, ORF4.sub.--23FP,
ORF5.sub.--23FP, ORF6.sub.--23FP, ORF7.sub.--23FP, and
ORF8.sub.--23FP.
[0100] One or more of the S. pneumoniae AI from 23F Poland 16
polynucleotide sequences may be replaced by a polynucleotide
sequence coding for a fragment of the replaced ORF. Alternatively,
one or more of the S. pneumoniae AI from 23F Poland 16 open reading
frames may be replaced by a sequence having sequence homology to
the replaced ORF.
[0101] S. pneumoniae AI from 23F Taiwan 15 comprises a series of
approximately seven open reading frames encoding for a collection
of amino acid sequences comprising surface proteins and sortases
("S. pneumoniae AI proteins"). Specifically, S. pneumoniae AI from
23F Taiwan 15 includes polynucleotide sequences encoding for two or
more of ORF2.sub.--23FTW, ORF3.sub.--23FTW, ORF4.sub.--23FTW,
ORF5.sub.--23FTW, ORF6.sub.--23FTW, ORF7.sub.--23FTW, and
ORF8.sub.--23FTW.
[0102] One or more of the S. pneumoniae AI from 23F Taiwan 15
polynucleotide sequences may be replaced by a polynucleotide
sequence coding for a fragment of the replaced ORF. Alternatively,
one or more of the S. pneumoniae AI from 23F Taiwan 15 open reading
frames may be replaced by a sequence having sequence homology to
the replaced ORF.
[0103] S. pneumoniae AI from 6B Finland 12 comprises a series of
approximately seven open reading frames encoding for a collection
of amino acid sequences comprising surface proteins and sortases
("S. pneumoniae AI proteins"). Specifically, S. pneumoniae AI from
6B Finland 12 includes polynucleotide sequences encoding for two or
more of ORF2.sub.--6BF, ORF3.sub.--6BF, ORF4.sub.--6BF,
ORF5.sub.--6BF, ORF6.sub.--6BF, ORF7.sub.--6BF, and
ORF8.sub.--6BF.
[0104] One or more of the S. pneumoniae AI from 6B Finland 12
polynucleotide sequences may be replaced by a polynucleotide
sequence coding for a fragment of the replaced ORF. Alternatively,
one or more of the S. pneumoniae AI from 6B Finland 12 open reading
frames may be replaced by a sequence having sequence homology to
the replaced ORF.
[0105] S. pneumoniae AI from 6B Spain 2 comprises a series of
approximately seven open reading frames encoding for a collection
of amino acid sequences comprising surface proteins and sortases
("S. pneumoniae AI proteins"). Specifically, S. pneumoniae AI from
6B Spain 2 includes polynucleotide sequences encoding for two or
more of ORF2.sub.--6BSP, ORF3.sub.--6BSP, ORF4.sub.--6BSP,
ORF5.sub.--6BSP, ORF6.sub.--6BSP, ORF7.sub.--6BSP, and
ORF8.sub.--6BSP.
[0106] One or more of the S. pneumoniae AI from 6B Spain 2
polynucleotide sequences may be replaced by a polynucleotide
sequence coding for a fragment of the replaced ORF. Alternatively,
one or more of the S. pneumoniae AI from 6B Spain 2 open reading
frames may be replaced by a sequence having sequence homology to
the replaced ORF.
[0107] S. pneumoniae AI from 9V Spain 3 comprises a series of
approximately seven open reading frames encoding for a collection
of amino acid sequences comprising surface proteins and sortases
("S. pneumoniae AI proteins"). Specifically, S. pneumoniae AI from
9V Spain 3 includes polynucleotide sequences encoding for two or
more of ORF2.sub.--9VSP, ORF3.sub.--9VSP, ORF4.sub.--9VSP,
ORF5.sub.--9VSP, ORF6.sub.--9VSP, ORF7.sub.--9VSP, and
ORF8.sub.--9VSP.
[0108] One or more of the S. pneumoniae AI from 9V Spain 3
polynucleotide sequences may be replaced by a polynucleotide
sequence coding for a fragment of the replaced ORF. Alternatively,
one or more of the S. pneumoniae AI from 9V Spain 3 open reading
frames may be replaced by a sequence having sequence homology to
the replaced ORF.
[0109] One or more of the S. pneumoniae AI surface proteins
typically include an LPXTG motif (such as LPXTG (SEQ ID NO: 122))
or other sortase substrate motif. These sortase proteins are
thought to be involved in the secretion and anchoring of the LPXTG
containing surface proteins. S. pneumoniae AI may encode for at
least one surface protein. The Adhesin Island, may encode at least
one surface protein. Alternatively, S. pneumoniae AI may encode for
at least two surface proteins and at least one sortase. Preferably,
S. pneumoniae AI encodes for at least three surface proteins and at
least two sortases. One or more of the surface proteins may include
an LPXTG motif.
[0110] The S. pneumoniae AI protein of the composition may be
selected from the group consisting of SP0462, SP0463, SP0464,
SP0465, SP0466, SP0467, SP0468, orf1.sub.--670, orf5.sub.--670,
orf4.sub.--670, orf5.sub.--670, orf6.sub.--670, orf7.sub.--670,
orf8.sub.--670, ORF2.sub.--14CSR, ORF3.sub.--14CSR,
ORF4.sub.--14CSR, ORF5.sub.--14CSR, ORF6.sub.--14CSR,
ORF7.sub.--14CSR, ORF8.sub.--14CSR, ORF2.sub.--19AH,
ORF3.sub.--19AH, ORF4.sub.--19AH, ORF5.sub.--9AH, ORF6.sub.--19AH,
ORF7.sub.--19AH, ORF8.sub.--19AH, ORF2.sub.--19FTW,
ORF3.sub.--19FTW, ORF4.sub.--19FTW, ORF5.sub.--19FTW,
ORF6.sub.--19FTW, ORF7.sub.--19FTW, ORF8.sub.--19FTW,
ORF2.sub.--23FP, ORF3.sub.--23FP, ORF4.sub.--23FP, ORF5.sub.--23FP,
ORF6.sub.--23FP, ORF7.sub.--23FP, ORF8.sub.--23FP,
ORF2.sub.--23FTW, ORF3.sub.--23FTW, ORF4.sub.--23FTW,
ORF5.sub.--23FTW, ORF6.sub.--23FTW, ORF7.sub.--23FTW,
ORF8.sub.--23FTW, ORF2.sub.--6BF, ORF3.sub.--6BF, ORF4.sub.--6BF,
ORF5.sub.--6BF, ORF6.sub.--6BF, ORF7.sub.--6BF, ORF8.sub.--6BF,
ORF2.sub.--6BSP, ORF3.sub.--6BSP, ORF4.sub.--6BSP, ORF5.sub.--6BSP,
ORF6.sub.--6BSP, ORF7.sub.--6BSP, ORF8.sub.--6BSP, ORF2.sub.--9VSP,
ORF3.sub.--9VSP, ORF4.sub.--9VSP, ORF5.sub.--9VSP, ORF6.sub.--9VSP,
ORF7.sub.--9VSP and, ORF8.sub.--9VSP.
[0111] S. pneumoniae AI surface proteins are preferred proteins for
use in the immunogenic compositions of the invention. In one
embodiment, the compositions of the invention comprise combinations
of two or more S pneumoniae AI surface proteins. Preferably such
combinations are selected from two or more of the group consisting
of SP0462, SP0463, SP0464, orf3.sub.--670, orf4.sub.--670,
orf5.sub.--670, ORF3.sub.--14CSR, ORF4.sub.--14CSR,
ORF5.sub.--14CSR, ORF3.sub.--19AH, ORF4.sub.--19AH,
ORF5.sub.--19AH, ORF3.sub.--19FTW, ORF4.sub.--19FTW, ORF5.sub.--1
gFTW, ORF3.sub.--23FP, ORF4.sub.--23FP, ORF5.sub.--23FP,
ORF3.sub.--23FTW, ORF4.sub.--23FTW, ORF5.sub.--23FTW,
ORF3.sub.--6BF, ORF4.sub.--6BF, ORF5.sub.--6BF, ORF3.sub.--6BSP,
ORF4.sub.--6BSP, ORF5.sub.--6BSP, ORF3.sub.--9VSP, ORF4.sub.--9VSP,
and ORF5.sub.--9VSP.
[0112] In addition to the open reading frames encoding the S.
pneumoniae AI proteins, S. pneumoniae AI may also include a
transcriptional regulator.
[0113] The S. pneumoniae AI proteins of the invention may be used
in immunogenic compositions for prophylactic or therapeutic
immunization against S. pneumoniae infection. For example, the
invention may include an immunogenic composition comprising one or
more S. pneumoniae from TIGR4 AI proteins and one or more S.
pneumoniae strain 670 proteins. The immunogenic composition may
comprise one or more AI proteins from any one or more of S.
pneumoniae strains TIGR4, 19A Hungary 6, 6B Finland 12, 6B Spain 2,
9V Spain 3, 14 CSR 10, 19F Taiwan 14, 23F Taiwan 15, 23F Poland 16,
and 670.
[0114] The immunogenic compositions may also be selected to provide
protection against an increased range of S. pneumoniae serotypes
and strain isolates. For example, the immunogenic composition may
comprise a first and second S. pneumoniae AI protein, wherein a
full length polynucleotide sequence encoding for the first S.
pneumoniae AI protein is not present in a genome comprising a full
length polynucleotide sequence encoding for the second S.
pneumoniae AI protein. In addition, each antigen selected for use
in the immunogenic compositions will preferably be present in the
genomes of multiple S. pneumoniae serotypes and strain isolates.
Preferably, each antigen is present in the genomes of at least two
(i.e., 3, 4, 5, 6, 7, 8, 9, 10, or more) S. pneumoniae strain
isolates. More preferably, each antigen is present in the genomes
of at least two (i.e., at least 3, 4, 5, or more) S. pneumoniae
serotypes.
[0115] The immunogenic compositions may also be selected to provide
protection against an increased range of serotypes and strain
isolates of a Gram positive bacteria. For example, the immunogenic
composition may comprise a first and second Gram positive bacteria
AI protein, wherein a full length polynucleotide sequence encoding
for the first Gram positive bacteria AI protein is not present in a
genome comprising a full length polynucleotide sequence encoding
for the second Gram positive bacteria AI protein. In addition, each
antigen selected for use in the immunogenic compositions will
preferably be present in the genomes of multiple serotypes and
strain isolates of the Gram positive bacteria. Preferably, each
antigen is present in the genomes of at least two (i.e., 3, 4, 5,
6, 7, 8, 9, 10, or more) Gram positive bacteria strain isolates.
More preferably, each antigen is present in the genomes of at least
two (i.e., at least 3, 4, 5, or more) Gram positive bacteria
serotypes. One or both of the first and second AI proteins may
preferably be in oligomeric or hyperoligomeric form.
[0116] Adhesin island surface proteins from two or more Gram
positive bacterial genus or species may be combined to provide an
immunogenic composition for prophylactic or therapeutic treatment
of disease or infection of two more Gram positive bacterial genus
or species. Optionally, the adhesin island surface proteins may be
associated together in an oligomeric or hyperoligomeric
structure.
[0117] In one embodiment, the invention comprises adhesin island
surface proteins from two or more Streptococcus species. For
example, the invention includes a composition comprising a GBS AI
surface protein and a GAS adhesin island surface protein. As
another example, the invention includes a composition comprising a
GAS adhesin island surface protein and a S. pneumoniae adhesin
island surface protein. One or both of the GAS AI surface protein
and the S. pneumoniae AI surface protein may be in oligomeric or
hyperoligomeric form. As a further example, the invention includes
a composition comprising a GBS adhesin island surface protein and a
S. pneumoniae adhesin island surface protein.
[0118] In one embodiment, the invention comprises an adhesin island
surface protein from two or more Gram positive bacterial genus. For
example, the invention includes a composition comprising a
Streptococcus adhesin island protein and a Corynebacterium adhesin
island protein. One or more of the Gram positive bacteria AI
surface proteins may be in an oligomeric or hyperoligomeric
form.
[0119] In addition, the AI polynucleotides and amino acid sequences
of the invention may also be used in diagnostics to identify the
presence or absence of GBS (or a Gram positive bacteria) in a
biological sample. They may be used to generate antibodies which
can be used to identify the presence of absence of an AI protein in
a biological sample or in a prophylactic or therapeutic treatment
for GBS (or a Gram positive bacterial) infection. Further, the AI
polynucleotides and amino acid sequences of the invention may also
be used to identify small molecule compounds which inhibit or
decrease the virulence associated activity of the AI.
BRIEF DESCRIPTION OF THE FIGURES
[0120] FIG. 1 presents a schematic depiction of Adhesin Island 1
("AI-1") comprising open reading frames for GBS 80, GBS 52,
SAG0647, SAG0648 and GBS 104.
[0121] FIG. 2 illustrates the identification of AI-1 sequences in
several GBS serotypes and strain isolates (GBS serotype V, strain
isolate 2603; GBS serotype III, strain isolate nem316; GBS serotype
II, strain isolate 18RS21; GBS serotype V, strain isolate CJB111;
GBS serotype III, strain isolate COH1 and GBS serotype 1a, strain
isolate A909). (An AI-1 was not identified in GBS serotype 1b,
strain isolate H36B or GBS serotype 1a, strain isolate 515).
[0122] FIG. 3 presents a schematic depiction of the correlation
between AI-1 and the Adhesin Island 2 ("AI-2") within the GBS
serotype V, strain isolate 2603 genome. (This AI-2 comprises open
reading frames for GBS 67, GBS 59, SAG1406, SAG1405 and GBS
150).
[0123] FIG. 4 illustrates the identification of AI-2 comprising
open reading frames encoding for GBS 67, GBS 59, SAG1406, SAG1404
and GBS 150 (or sequences having sequence homology thereto) in
several GBS serotypes and strain isolates (GBS serotype V, strain
isolate 2603; GBS serotype III, strain isolate NEM316; GBS serotype
1b, strain isolate H36B; GBS serotype V, strain isolate CJB111; GBS
serotype II, strain isolate 18RS21; and GBS serotype 1a, strain
isolate 515).
[0124] FIG. 4 also illustrates the identification of AI-2
comprising open reading frames encoding for 01520 (a sortase),
01521, 01522 (a sortase), 01523 (spb1), 01524 and 01525 (or
sequences having sequence homology thereto).
[0125] FIG. 5 presents data showing that GBS 80 binds to
fibronectin and fibrinogen in ELISA.
[0126] FIG. 6 illustrates that all genes in AI-1 are co-transcribed
as an operon.
[0127] FIG. 7 presents schematic depictions of in-frame deletion
mutations within AI-1.
[0128] FIG. 8 presents FACS data showing that GBS 80 is required
for surface localization of GBS 104.
[0129] FIG. 9 presents FACS data showing that sortases SAG0647 and
SAG0648 play a semi-redundant role in surface exposure of GBS 80
and GBS 104.
[0130] FIG. 10 presents Western Blots of the in-frame deletion
mutants probed with anti-GBS80 and anti-GBS 104 antisera.
[0131] FIG. 11: Electron micrograph of surface exposed pili
structures in Streptococcus agalactiae containing GBS 80.
[0132] FIG. 12: PHD predicted secondary structure of GBS 067.
[0133] FIGS. 13, 14 and 15: Electron micrograph of surface exposed
pili structures of strain isolate COH1 of Streptococcus agalactiae
containing a plasmid insert encoding GBS 80.
[0134] FIGS. 16 and 17: Electron micrograph of surface exposed pili
structure of wild type strain isolate COH1 of Streptococcus
agalactiae.
[0135] FIG. 18: Alignment of polynucleotide sequences of AI-1 from
serotype V, strain isolates 2603 and CJB111; serotype II, strain
isolate 18RS21; serotype HI, strain isolates COH1 and NEM316; and
serotype 1a, strain isolate A909.
[0136] FIG. 19: Alignment of polynucleotide sequences of AI-2 from
serotype V, strain isolates 2603 and CJB111; serotype II, strain
isolate 18RS21; serotype 1b, strain isolate H36B; and serotype 1a,
strain isolate 515.
[0137] FIG. 20: Alignment of polynucleotide sequences of AI-2 from
serotype V, strain isolate 2603 and serotype III, strain isolate
NEM316.
[0138] FIG. 21: Alignment of polynucleotide sequences of AI-2 from
serotype III, strain isolate COH1 and serotype Ia, strain isolate
A909.
[0139] FIG. 22: Alignment of amino acid sequences of AI-1 surface
protein GBS 80 from serotype V, strain isolates 2603 and CJB111;
serotype 1a, strain isolate A909; serotype III, strain isolates
COH1 and NEM316.
[0140] FIG. 23: Alignment of amino acid sequences of AI-1 surface
protein GBS 104 from serotype V, strain isolates 2603 and CJB111;
serotype III, strain isolates COH1 and NEM316; and serotype II,
strain isolate 18RS21.
[0141] FIG. 24: Alignment of amino acid sequences of AI-2 surface
protein GBS 067 from serotype V, strain isolates 2603 and CJB111;
serotype 1a, strain isolate 515; serotype II, strain isolate
18RS21; serotype Ib, strain isolate H36B; and serotype III, strain
isolate NEM316.
[0142] FIG. 25: Illustrates that GBS closely associates with tight
junctions and cross the monolayer of ME180 cervical epithelial
cells by a paracellular route.
[0143] FIG. 26: Illustrates GBS infection of ME180 cells.
[0144] FIG. 27: Illustrates that GBS 80 recombinant protein does
not bind to epithelial cells.
[0145] FIG. 28: Illustrates that deletion of GBS 80 does not effect
the capacity of GBS strain 2603 V/R to adhere and invade ME180
cervical epithelial cells.
[0146] FIG. 29: Illustrates binding of recombinant GBS 104 protein
to epithelial cells.
[0147] FIG. 30: Illustrates that deletion of GBS 104 in the GBS
strain COH1, reduces the capacity of GBS to adhere to ME180
cervical epithelial cells.
[0148] FIG. 31: Illustrates that GBS 80 knockout mutant strain
partially loses the ability to translocate through an epithelial
cell monolayer.
[0149] FIG. 32: Illustrates that deletion of GBS 104, but not GBS
80, reduces the capacity of GBS to invade J774 macrophage-like cell
line.
[0150] FIG. 33: Illustrates that GBS 104 knockout mutant strain
translocates through an epithelial monolayer less efficiently than
the isogenic wild type.
[0151] FIG. 34: Negative stained electron micrographs of GBS
serotype III, strain isolate COH1, containing a plasmid insert to
over-express GBS 80.
[0152] FIG. 35: Electron micrographs of surface exposed pili
structures on GBS serotype III, strain isolate COH1, containing a
plasmid insert to over-express GBS 80, stained with anti-GBS 80
antibodies (visualized with 10 nm gold particles).
[0153] FIG. 36: Electron micrographs of surface exposed pili
structures on GBS serotype III, strain isolate COH1, containing a
plasmid insert to over-express GBS 80, stained with anti-GBS 80
antibodies (visualized with 10 nm gold particles).
[0154] FIG. 37: Electron micrographs of surface exposed pili
structures on GBS serotype III, strain isolate COH1, containing a
plasmid insert to over-express GBS 80, stained with anti-GBS 80
antibodies (visualized with 20 nm gold particles).
[0155] FIG. 38: Electron micrographs of surface exposed pili
structures on GBS serotype III, strain isolate COH1, containing a
plasmid insert to over-express GBS 80, stained with anti-GBS 104
antibodies or preimmune sera (visualized with 10 nm gold
particles).
[0156] FIG. 39: Electron micrographs of surface exposed pili
structures on GBS serotype III, strain isolate COH1, containing a
plasmid insert to over-express GBS 80, stained with anti-GBS 80
antibodies (visualized with 20 nm gold particles) and anti-GBS 104
antibodies (visualized with 10 nm gold particles).
[0157] FIG. 40: Electron micrographs of surface exposed pili
structures on GBS serotype III, strain isolate COH1, containing a
plasmid insert to over-express GBS 80, stained with anti-GBS 80
antibodies (visualized with 20 nm gold particles) and anti-GBS 104
antibodies (visualized with 10 nm gold particles).
[0158] FIG. 41: Illustrates that GBS 80 is necessary for polymer
formation and GBS104 and sortase SAG0648 are necessary for
efficient assembly of pili.
[0159] FIG. 42: Illustrates that GBS 67 is part of a second pilus
and that GBS 80 is polymerized in strain 515.
[0160] FIG. 43: Illustrates that two macro-molecules are visible in
Coh1, one of which is the GBS 80 pilin.
[0161] FIG. 44: Illustrates pilin assembly.
[0162] FIG. 45: Illustrates that GBS 52 is a minor component of the
GBS pilus.
[0163] FIG. 46: Illustrates that the pilus is found in the
supernatant of a bacterial culture.
[0164] FIG. 47: Illustrates that the pilus is found in the
supernatant of bacterial cultures in all phases.
[0165] FIG. 48: Illustrates that in Coh1, only the GBS 80 protein
and one sortase (sag0647 or sag0648) is required for
polymerization.
[0166] FIG. 49: IEM image of GBS 80 staining of a GBS serotype VIII
strain JM9030013 that express pili.
[0167] FIG. 50: IEM image of GBS 104 staining of a GBS serotype
VIII strain JM9030013 that express pili.
[0168] FIG. 51A: Schematic depiction of open reading frames
comprising a GAS AI-2 serotype M1 isolate, GAS AI-3 serotype M3,
M5, M18, and M49 isolates, a GAS AI-4 serotype M12 isolate, and an
GAS AI-1 serotype M6 isolate.
[0169] FIG. 51B: Amino acid alignment of SrtC1-type sortase of a
GAS AI-2 serotype M1 isolate, SrtC2-type sortases of serotype M3,
M5, M118, and M49 isolates, and a SrtC2-type sortase of a GAS AI-4
serotype M12 isolate.
[0170] FIG. 52: Amino acid alignment of the capsular polysaccharide
adhesion proteins of GAS AI-4 serotype M12 (A735), GAS AI-3
serotype M5 (Manfredo), S. pyogenes strain MGAS315 serotype M3, S.
pyogenes strain SSI-1 serotype M3, S. pyogenes strain MGAS8232
serotype M3, and GAS AI-2 serotype M1.
[0171] FIG. 53: Amino acid alignment of F-like fibronectin-binding
proteins of GAS AI-4 serotype M12 (A735) and S. pyogenes strain
MGAS10394 serotype M6.
[0172] FIG. 54: Amino acid alignment of F2-like fibronectin-binding
proteins of GAS AI-4 serotype M12 (A735), S. pyogenes strain
MGAS8232 serotype M3, GAS AI-3 strain M5 (Manfredo), S. pyogenes
strain SSI serotype M3, and S. pyogenes strain MGAS315 serotype
M3.
[0173] FIG. 55: Amino acid alignment of fimbrial proteins of GAS
AI-4 serotype M12 (A735), GAS AI-3 serotype M5 (Manfredo), S.
pyogenes strain MGAS315 serotype M3, S. pyogenes strain SSI
serotype M3, S. pyogenes strain MGAS8232 serotype M3, and S.
pyogenes M1 GAS serotype M1.
[0174] FIG. 56: Amino acid alignment of hypothetical proteins of
GAS AI-4 serotype M12 (A735), S. pyogenes strain MGAS315 serotype
M3, S. pyogenes strain SSI-1 serotype M3, GAS AI-3 serotype M5
(Manfredo), and S. pyogenes strain MGAS8232 serotype M3.
[0175] FIG. 57: Results of FASTA homology search for amino acid
sequences that align with the collagen adhesion protein of GAS AI-1
serotype M6 (MGAS10394).
[0176] FIG. 58: Results of FASTA homology search for amino acid
sequences that align with the fimbrial structural subunit of GAS
AI-1 serotype M6 (MGAS10394).
[0177] FIG. 59: Results of FASTA homology search for amino acid
sequences that align with the hypothetical protein of GAS AI-2
serotype M1 (SF370).
[0178] FIG. 60: Specifies pilin and E box motifs present in GAS
type 3 and 4 adhesin islands.
[0179] FIG. 61: Illustrates that surface expression of GBS 80
protein on GBS strains COH and JM9130013 correlates with formation
of pili structures. Surface expression of GBS 80 was determined by
FACS analysis using an antibody that cross-hybridizes with GBS 80.
Formation of pili structures was determined by immunogold electron
microscopy using gold-labelled anti-GBS 80 antibody.
[0180] FIG. 62: Illustrates that surface exposure is
capsule-dependent for GBS 322 but not for GBS 80.
[0181] FIG. 63: Illustrates the amino acid sequence identity of GBS
59 proteins in GBS strains.
[0182] FIG. 64: Western blotting of whole GBS cell extracts with
anti-GBS 59 antibodies.
[0183] FIG. 65: Western blotting of purified GBS 59 and whole GBS
cell extracts with anti-GBS 59 antibodies.
[0184] FIG. 66: FACS analysis of GBS strains CJB111, 7357B, 515
using GBS 59 antiserum.
[0185] FIG. 67: Illustrates that anti-GBS 59 antibodies are opsonic
for CJB111 GBS strain serotype V.
[0186] FIG. 68: Western blotting of GBS strain JM9130013 total
extracts.
[0187] FIG. 69: Western blotting of GBS stain 515 total extracts
shows that GBS 67 and GBS 150 are parts of a pilus.
[0188] FIG. 70: Western blotting of GBS strain 515 knocked out for
GBS 67 expression
[0189] FIG. 71: FACS analysis of GBS strain 515 and GBS strain 515
knocked out for GBS 67 expression using GBS 67 and GBS 59
antiserum.
[0190] FIG. 72: Illustrates complementation of GBS 515 knocked out
for GBS 67 expression with a construct overexpressing GBS 80.
[0191] FIG. 73: FACS analysis of GAS serotype M6 for
spyM6.sub.--0159 surface expression.
[0192] FIG. 74: FACS analysis of GAS serotype M6 for
spyM6.sub.--0160 surface expression.
[0193] FIG. 75: FACS analysis of GAS serotype M1 for GAS 15 surface
expression.
[0194] FIG. 76: FACS analysis of GAS serotype M1 for GAS 16 surface
expression using a first anti-GAS 16 antiserum.
[0195] FIG. 77: FACS analysis of GAS serotype M1 for GAS 18 surface
expression using a first anti-GAS 18 antiserum.
[0196] FIG. 78: FACS analysis of GAS serotype M1 for GAS 18 surface
expression using a second anti-GAS 18 antiserum.
[0197] FIG. 79: FACS analysis of GAS serotype M1 for GAS 16 surface
expression using a second anti-GAS 16 antisera.
[0198] FIG. 80: FACS analysis of GAS serotype M3 for
spyM3.sub.--0098 surface expression.
[0199] FIG. 81: FACS analysis of GAS serotype M3 for
spyM3.sub.--0100 surface expression.
[0200] FIG. 82: FACS analysis of GAS serotype M3 for
spyM3.sub.--0102 surface expression.
[0201] FIG. 83: FACS analysis of GAS serotype M3 for
spyM3.sub.--0104 surface expression.
[0202] FIG. 84: FACS analysis of GAS serotype M3 for
spyM3.sub.--0106 surface expression.
[0203] FIG. 85: FACS analysis of GAS serotype M12 for 19224134
surface expression.
[0204] FIG. 86: FACS analysis of GAS serotype M12 for 19224135
surface expression.
[0205] FIG. 87: FACS analysis of GAS serotype M12 for 19224137
surface expression.
[0206] FIG. 88: FACS analysis of GAS serotype M12 for 19224141
surface expression.
[0207] FIG. 89: Western blot analysis of GAS 15 expression on GAS
M1 bacteria.
[0208] FIG. 90: Western blot analysis of GAS 15 expression using
GAS 15 immune sera.
[0209] FIG. 91: Western blot analysis of GAS 15 expression using
GAS 15 pre-immune sera.
[0210] FIG. 92: Western blot analysis of GAS 16 expression on GAS
M1 bacteria.
[0211] FIG. 93: Western blot analysis of GAS 16 expression using
GAS 16 immune sera.
[0212] FIG. 94: Western blot analysis of GAS 16 expression using
GAS 16 pre-immune sera.
[0213] FIG. 95: Western blot analysis of GAS 18 on GAS M1
bacteria.
[0214] FIG. 96: Western blot analysis of GAS 18 using GAS 18 immune
sera.
[0215] FIG. 97: Western blot analysis of GAS 18 using GAS 18
pre-immune sera.
[0216] FIG. 98: Western blot analysis of M6_Spy0159 expression on
GAS bacteria.
[0217] FIG. 99: Western blot analysis of 19224135 expression on M12
GAS bacteria.
[0218] FIG. 100: Western blot analysis of 19224137 expression on
M12 GAS bacteria.
[0219] FIG. 101: Full length nucleotide sequence of an S.
pneumoniae strain 670 .mu.l.
[0220] FIG. 102: Western blot analysis of GAS 15, GAS 16, and GAS
18 in GAS M1 strain 2580.
[0221] FIG. 103: Western blot analysis of GAS 15, GAS 16, and GAS
18 in GAS M1 strain 2913.
[0222] FIG. 104: Western blot analysis of GAS 15, GAS 16, and GAS
18 in GAS M1 strain 3280.
[0223] FIG. 105: Western blot analysis of GAS 15, GAS 16, and GAS
18 in GAS M1 strain 3348.
[0224] FIG. 106: Western blot analysis of GAS 15, GAS 16, and GAS
18 in GAS M1 strain 2719.
[0225] FIG. 107: Western blot analysis of GAS 15, GAS 16, and GAS
18 in GAS M1 strain SF370.
[0226] FIG. 108: Western blot analysis of 19224135 and 19224137 in
GAS M12 strain 2728.
[0227] FIG. 109: Western blot analysis of 19224139 in GAS M12
strain 2728 using antisera raised against SpyM3.sub.--0102.
[0228] FIG. 110: Western blot analysis of M6_Spy0159 and M6_Spy0160
in GAS M6 strain 2724.
[0229] FIG. 111: Western blot analysis of M6 Spy0159 and M6 Spy0160
in GAS M6 strain SF370.
[0230] FIG. 112: Western blot analysis of M6_Spy160 in GAS M6
strain 2724.
[0231] FIGS. 113-115: Electron micrographs of surface exposed GAS
15 on GAS M1 strain SF370.
[0232] FIGS. 116-121: Electron micrographs of surface exposed GAS
16 on GAS M1 strain SF370.
[0233] FIGS. 122-125: Electron micrographs of surface exposed GAS
18 on GAS M1 strain SF370 detected using anti-GAS 18 antisera.
[0234] FIG. 126: IEM image of a hyperoligomer on GAS M1 strain
SF370 detected using anti-GAS 18 antisera.
[0235] FIGS. 127-132: IEM images of oligomeric and hyperoligomeric
structures containing M6_Spy0160 extending from the surface of GAS
serotype M6 3650.
[0236] FIGS. 133A and B: Western blot analysis of L. lactis
transformed to express GBS 80 with anti-GBS 80 antiserum.
[0237] FIG. 134: Western blot analyses of L. lactis transformed to
express GBS AI-1 with anti-GBS 80 antiserum.
[0238] FIG. 135: Ponceau staining of same acrylamide gel as used in
FIG. 134.
[0239] FIG. 136A: Western blot analysis of sonicated pellets and
supernatants of cultured L. lactis transformed to express GBS AI-1
polypeptides using anti-GBS 80 antiserum.
[0240] FIG. 136B: Polyacrylamide gel electrophoresis of sonicated
pellets and supernatants of cultured L. lactis transformed to
express GBS AI polypeptides.
[0241] FIG. 137: Depiction of an example S. pneumoniae AI
locus.
[0242] FIG. 138: Schematic of primer hybridization sites within the
S. pneumoniae AI locus of FIG. 137.
[0243] FIG. 139A: The set of amplicons produced from the S.
pneumoniae strain TIGR4 AI locus.
[0244] FIG. 139B: Base pair lengths of amplicons produced from FIG.
139A primers in S. pneumoniae strain TIGR4.
[0245] FIG. 140: CGH analysis of S. pneumoniae strains for the AI
locus.
[0246] FIG. 141: Amino acid sequence alignment of polypeptides
encoded by AI orf 2 in S. pneumoniae AI-positive strains.
[0247] FIG. 142: Amino acid sequence alignment of polypeptides
encoded by AI orf 3 in S. pneumoniae AI-positive strains.
[0248] FIG. 143: Amino acid sequence alignment of polypeptides
encoded by AI orf 4 in S. pneumoniae AI-positive strains.
[0249] FIG. 144: Amino acid sequence alignment of polypeptides
encoded by AI orf 5 in S. pneumoniae AI-positive strains.
[0250] FIG. 145: Amino acid sequence alignment of polypeptides
encoded by AI orf 6 in S. pneumoniae AI-positive strains.
[0251] FIG. 146: Amino acid sequence alignment of polypeptides
encoded by AI orf 7 in S. pneumoniae AI-positive strains.
[0252] FIG. 147: Amino acid sequence alignment of polypeptides
encoded by AI orf 8 in S. pneumoniae AI-positive strains.
[0253] FIG. 148: Diagram comparing amino acid sequences of RrgA in
S. pneumoniae strains.
[0254] FIG. 149: Amino acid sequence comparison of RrgB S.
pneumoniae strains.
[0255] FIG. 150A: Sp0462 amino acid sequence.
[0256] FIG. 150B: Primers used to produce a clone encoding the
Sp0462 polypeptide.
[0257] FIG. 151A: Schematic depiction of recombinant Sp0462
polypeptide.
[0258] FIG. 151B: Schematic depiction of full-length Sp0462
polypeptide.
[0259] FIG. 152A: Western blot probed with serum obtained from S.
pneumoniae-infected patients for Sp0462.
[0260] FIG. 152B: Western blot probed with GBS 80 serum for
Sp0462.
[0261] FIG. 153A: Sp0463 amino acid sequence.
[0262] FIG. 153B: Primers used to produce a clone encoding the
Sp0463 polypeptide.
[0263] FIG. 154A: Schematic depiction of recombinant Sp0463
polypeptide.
[0264] FIG. 154B: Schematic depiction of full-length Sp0463
polypeptide.
[0265] FIG. 155: Western blot detection of recombinant Sp0463
polypeptide.
[0266] FIG. 156: Western blot detection of high molecular weight
Sp0463 polymers.
[0267] FIG. 157A: Sp0464 amino acid sequence.
[0268] FIG. 157B: Primers used to produce a clone encoding the
Sp0464 polypeptide.
[0269] FIG. 158A: Schematic depiction of recombinant Sp0464
polypeptide.
[0270] FIG. 158B: Schematic depiction of full-length Sp0464
polypeptide.
[0271] FIG. 159: Western blot detection of recombinant Sp0464
polypeptide.
[0272] FIG. 160: Amplification products prepared for production of
Sp0462, Sp0463, and Sp0464 clones.
[0273] FIG. 161: Opsonic killing by anti-sera raised against L.
lactis expressing GBS AI
[0274] FIG. 162: Schematic depicting GAS adhesin islands GAS AI-1,
GAS AI-2, GAS AI-3 and GAS AI-4.
[0275] FIGS. 163 A-D: Immunoblots of cell-wall fractions of GAS
strains with antisera specific for LPXTG proteins of M6_ISS3650
(A), M1_SF370 (B), M5_ISS4883 (C) and M12.sub.--20010296 (D).
[0276] FIGS. 163 E-H: Immunoblots of cell-wall fractions of
deletion mutants M1_SF370.DELTA.128 (E) M1_SF370.DELTA.130 (F)
M1_SF370.DELTA.SrtC1 (G) and the M1.sub.--128 deletion strain
complemented with plasmid pAM::128 which contains the M1.sub.--128
gene (H) with antisera specific for the pilin components of
M1_SF370.
[0277] FIGS. 163 I-N: Immunogold labelling and transmission
electron microscopy of: T6 (I) and Cpa (J) in M6_ISS3650;
M1.sub.--128 in M1_SF370 (K) and deletion strain M1_SF370.DELTA.128
(N); M5_orf80 in M5_ISS4883 (L); M12_EftLSL.A in M12.sub.--20010296
(M). The strains used are indicated below the panels. Bars=200
nm.
[0278] FIG. 164: Schematic representation of the FCT region from 7
GAS strains
[0279] FIGS. 165 A-H: Flow cytometry of GAS bacteria treated or not
with trypsin and stained with sera specific for the major pilus
component. Preimmune staining; black lines, untreated bacteria;
green lines and trypsin treated bacteria; blue lines. M6_ISS3650
stained with sera which recognize the M6 protein (A) or anti-M6_T6
(B), M1_SF370 stained with anti-M1 (C) or anti-M1.sub.--128 (D),
M5_ISS4883 stained with anti-PrtF (E) or anti-M5_orf80 (F) and
M12.sub.--20010296 with anti-M12 (G) or anti-EftLSL.A (H)
[0280] FIGS. 166 A-C: Immunoblots of recombinant pilin components
with polyvalent Lancefield T-typing sera. The recombinant proteins
are shown above the blot and the sera pool used is shown below the
blot.
[0281] FIGS. 166 D-G: Immunoblots of pilin proteins with monovalent
T-typing sera. The recombinant proteins are shown below the blot
and the sera used above the blot.
[0282] FIG. 166 H and I Flow cytometry analysis of strain M1_SF370
(H) and the deletion strain M1_SF370.DELTA.128 (I) with T-typing
antisera pool T.
[0283] FIG. 167: Chart describing the number and type of sortase
sequences identified within GAS AIs.
[0284] FIG. 168 A: Immunogold-electronmicroscopy of L. lactis
lacking an expression construct for GBS AI-1 using anti-GBS 80
antibodies.
[0285] FIG. 168 B and C: Immunogold-electronmicroscopy detects GBS
80 in oligomeric (pilus) structures on surface of L. lactis
transformed to express GBS AI-1
[0286] FIG. 169: FACS analysis detects expression of GBS 80 and GBS
104 on the surface of L. lactis transformed to express GBS
AI-1.
[0287] FIG. 170: Phase contrast microscopy and
immuno-electronmicroscopy shows that expression of GBS AI-1 in L.
lactis induces L. lactis aggregation.
[0288] FIG. 171: Purification of GBS pili from L. lactis
transformed to express GBS AI-1.
[0289] FIG. 172: Schematic depiction of GAS M6 (AI-1), M1 (AI-2),
and M12 (AI-4) adhesin islands and portions of the adhesin islands
inserted in the pAM401 construct for expression in L. lactis.
[0290] FIG. 173 A-C: Western blot analysis showing assembly of GAS
pili in L. lactis expressing GAS AI-2 (M1) (A), GAS AI-4 (M12) (B),
and GAS AI-1(M6) (C).
[0291] FIG. 174: FACS analysis of GAS serotype M6 for M6_Spy0157
surface expression.
[0292] FIG. 175: FACS analysis of GAS serotype M12 for 19224139
surface expression.
[0293] FIG. 176 A-E: Immunogold electron microscopy using
antibodies against M6_Spy0160 detects pili on the surface of M6
strain 2724.
[0294] FIG. 176 F: Immunogold electron microscopy using antibodies
against M6_Spy0159 detects M6_Spy0159 surface expression on M6
strain 2724.
[0295] FIG. 177 A-C: Western blot analysis of M1 strain SF370 GAS
bacteria individually deleted for M1.sub.--130, SrtC1, or
M1.sub.--128 using anti-M1.sub.--130 serum (A), anti-M1.sub.--128
serum (B), and anti-M1.sub.--126 serum (C).
[0296] FIG. 178 A-C: Immunogold electron microscopy using
antibodies against M1.sub.--128 to detect surface expression on
wildtype strain SF370 bacteria (A), M1.sub.--128 deleted SF370
bacteria (B), and SrtC1 deleted SF370 bacteria (C).
[0297] FIG. 179 A-C: FACS analysis to detect expression of
M1.sub.--1126 (A), M1.sub.--128 (B), and M1.sub.--130 (C) on the
surface of wildtype SF370 GAS bacteria.
[0298] FIG. 179 D-F: FACS analysis to detect expression of
M1.sub.--126 (D), M1.sub.--128 (E), and M1.sub.--130 (F) on the
surface of M1.sub.--128 deleted SF370 GAS bacteria.
[0299] FIG. 179 G-I: FACS analysis to detect expression of
M1.sub.--126 (G), M1.sub.--128 (H), and M1.sub.--130 (I) on the
surface of SrtC1 deleted SF370 GAS bacteria.
[0300] FIG. 180 A and B: FACS analysis of wildtype (A) and LepA
deletion mutant (B) strains of SF370 bacteria for M1 surface
expression.
[0301] FIG. 181: Western blot analysis detects high molecular
weight polymers in S. pneumoniae TIGR4 using anti-RrgB
antisera.
[0302] FIG. 182: Detection of high molecular weight polymers in S.
pnuemoniae rlrA positive strains.
[0303] FIG. 183: Detection of high molecular weight polymers in S.
pneumoniae TIGR4 by silver staining and Western blot analysis using
anti-RrgB antisera.
[0304] FIG. 184: Deletion of S. pneumoniae TIGR4 adhesin island
sequences interferes with the ability of S. pneumoniae to adhere to
A549 alveolar cells.
[0305] FIG. 185: Negative staining of S. pneumoniae strain TIGR4
showing abundant pili on the bacterial surface.
[0306] FIG. 186: Negative staining of strain TIGR4 deleted for
rrgA-srtD adhesin island sequences showing no pili on the bacterial
surface
[0307] FIG. 187: Negative staining of the TIGR4 mgrA mutant showing
abundant pili on the bacterial surface.
[0308] FIG. 188: Negative staining of the negative control TIGR4
mgrA mutant deleted for adhesin island sequences rrgA-srtD showing
no pili on the bacterial surface.
[0309] FIG. 189: Immuno-gold labelling of S. pneumoniae strain
TIGR4 grown on blood agar solid medium using .alpha.-RrgB (5 nm)
and .alpha.-RrgC (10 nm). Bar represents 200 nm.
[0310] FIG. 190 A and B: Detection of expression and purification
of S. pneumoniae RrgA protein by SDS-PAGE (A) and Western blot
analysis (B).
[0311] FIG. 191: Detection of RrgB by antibodies produced in
mice.
[0312] FIG. 192: Detection of RrgC by antibodies produced in
mice.
[0313] FIG. 193: Purification of S. pneumoniae TIGR 4 pili by a
cultivation and digestion method and detection of the purified
TIGR4 pili.
[0314] FIG. 194: Purification of S. pneumoniae TIGR 4 pili by a
sucrose gradient centrifugation method and detection of the
purified TIGR4 pili.
[0315] FIG. 195: Purification of S. pneumoniae TIGR 4 pili by a gel
filtration method and detection of the purified TIGR4 pili.
[0316] FIG. 196: Alignment of full length S. pneumoniae adhesin
island sequences from ten S. pneumoniae strains.
[0317] FIG. 197 A: Schematic of GBS AI-1 coding sequences.
[0318] FIG. 197 B: Nucleotide sequence of intergenic region between
AraC and GBS 80 (SEQ ID NO: 273.
[0319] FIG. 197 C: FACS analysis results for GBS 80 expression in
GBS strains having different length polyA tracts in the intergenic
region between AraC and GBS 80.
[0320] FIG. 198: Table comparing the percent identity of surface
proteins encoded by a serotype M6 (harbouring a GAS AI-1) adhesin
island relative to other GAS serotypes harbouring an adhesin
island.
[0321] FIG. 199: Table comparing the percent identity of surface
proteins encoded by a serotype M1 (harbouring a GAS AI-2) adhesin
island relative to other GAS serotypes harbouring an adhesin
island.
[0322] FIG. 200: Table comparing the percent identity of surface
proteins encoded by serotypes M3, M18, M5, and M49 (harbouring GAS
AI-3) adhesin islands relative to other GAS serotypes harbouring an
adhesin island.
[0323] FIG. 201: Table comparing the percent identity of surface
proteins encoded by a serotype M12 (harbouring a GAS AI-1) adhesin
island-relative to other GAS serotypes harbouring an adhesin
island.
[0324] FIG. 202: GBS 80 recombinant protein does not bind to
epithelial cells.
[0325] FIG. 203: Deletion of GBS 80 protein does not affect the
ability of GBS to adhere and invade ME180 cervical epithelial
cells.
[0326] FIG. 204: GBS 80 binds to extracellular matrix proteins.
[0327] FIG. 205: Deletion of GBS 104 protein, but not GBS 80,
reduces the capacity of GBS to invade J774 macrophage-like
cells
[0328] FIG. 206: GBS 104 knockout mutant strains of bacteria
translocate through an epithelial monolayer less efficiently that
the isogenic wild type strain.
[0329] FIG. 207: GBS 80 knockout mutant strains of bacteria
partially lose the ability to translocate through an epithelial
monolayer.
[0330] FIG. 208: GBS adherence to HUVEC endothelial cells.
[0331] FIG. 209: Strain growth rate of wildtype, GBS 80-deleted, or
GBS 104 deleted COH1 GBS.
[0332] FIG. 210: Binding of recombinant GBS 104 protein to
epithelial cells by FACS analysis.
[0333] FIG. 211: Deletion of GBS 104 protein in the GBS strain COH1
reduces the ability of GBS to adhere to ME180 cervical epithelial
cells.
[0334] FIG. 212: COH1 strain GBS overexpressing GBS 80 protein has
an impaired capacity to translocate through an epithelial
monolayer.
[0335] FIG. 213: Scanning electron microscopy shows that
overexpression of GBS 80 protein on COH1 strain GBS enhances the
capacity of the COH1 bacteria to form microcolonies on epithelial
cells.
[0336] FIG. 214: Confocal imaging shows that overexpression of GBS
80 proteins on COH1 strain GBS enhances the capacity of the COH1
bacteria to form microcolonies on epithelial cells.
[0337] FIG. 215: Detection of GBS 59 on the surface of GBS strain
515 by immuno-electron microscopy.
[0338] FIG. 216: Detection of GBS 67 on the surface of GBS strain
515 by immuno-electron microscopy.
[0339] FIG. 217: GBS 67 binds to fibronectin.
[0340] FIG. 218: Western blot analysis shows that deletion of both
GBS AI-2 sortase genes abolishes assembly of the pilus.
[0341] FIG. 219: FACS analysis shows that deletion of both GBS AI-2
sortase genes abolishes assembly of the pilus.
[0342] FIG. 220 A-C: Western blot analysis shows that GBS 59, GBS
67, and GBS 150 form high molecular weight complexes.
[0343] FIG. 221 A-C: Western blot analysis shows that GBS 59 is
required for polymer formation of GBS 67 and GBS 150.
[0344] FIG. 222: FACS analysis shows that GBS 59 is required for
surface exposure of GBS 67.
[0345] FIG. 223: Summary Western blots for detection of GBS 59, GBS
67, or GBS 150 in GBS 515 and GBS 515 mutant strain.
[0346] FIG. 224: Description of GBS 59 Allelic variants.
[0347] FIG. 225: GBS 59 is opsonic only against a strain of GBS
expressing a homologous GBS 59.
[0348] FIG. 226 A and B: Results of FACS analysis for surface
expression of GBS 59 using antibodies specific for different GBS 59
isoforms.
[0349] FIG. 227 A and B: Results of FACS analysis for surface
expression of GBS 80, GBS 104, GBS 322, GBS 67, and GBS 59 on 41
various strains of GBS bacteria.
[0350] FIG. 228: Results of FACS analysis for surface expression of
GBS 80, GBS 104, GBS 322, GBS 67, and GBS 59 on 41 strains of GBS
bacteria obtained from the CDC.
[0351] FIG. 229: Expected immunogenicity coverage of different
combinations of GBS 80, GBS 104, GBS 322, GBS 67, and GBS 59 across
strains of GBS bacteria.
[0352] FIG. 230: GBS 59 opsonophagocytic activity is comparable to
that of a mixture of GBS 80, GBS 104, GBS 322 and GBS 67.
[0353] FIG. 231 A-C: Schematic presentation of example hybrid GBS
AIs.
[0354] FIG. 232: Schematic presentation of an example hybrid GBS
AI.
[0355] FIG. 233 A and B: Western blot and FACS analysis detect
expression of GBS 80 and GBS 67 on the surface of L. lactis
transformed with a hybrid GBS AI.
[0356] FIG. 234 A-E Hybrid GBS AI cloning strategy.
[0357] FIG. 235: High magnification of S. pneumoniae strain TIGR4
pili double labeled with .alpha.-RrgB (5 nm) and .alpha.-RrgC (10
nm). Bar represents 100 nm.
[0358] FIG. 236: Immuno-gold labeling of the S. pneumoniae TIGR4
rrgA-srtD deletion mutant with no visible pili on the surface
detectable by .alpha.-RrgB- and .alpha.-RrgC. Bar represents 200
nm.
[0359] FIG. 237: Variability in GBS 67 amino acid sequences between
strains 2603 and H36B.
[0360] FIG. 238: Strain variability in GBS 67 amino acid sequences
of allele I (2603).
[0361] FIG. 239: Stran variability in GBS 67 amino acid sequence of
allele II (H36B).
BRIEF DESCRIPTION OF THE TABLES
[0362] TABLE 1: Active Maternal Immunization Assay for fragments of
GBS 80
[0363] TABLE 2: Passive Maternal Immunization Assay for fragments
of GBS 80
[0364] TABLE 3: Lethal dose 50% of AI-1 mutants from GBS strain
isolate 2603.
[0365] TABLE 4: GAS AI-1 sequences from M6 isolate (MGAS10394).
[0366] TABLE 5: GAS AI-2 sequences from M1 isolate (SF370).
[0367] TABLE 6: GAS AI-3 sequences from M3 isolate (MGAS315).
[0368] TABLE 7: GAS AI-3 sequences from M3 isolate (SSI-1).
[0369] TABLE 8: GAS AI-3 sequences from M18 isolate (MGAS8232).
[0370] TABLE 9: S. pneumoniae AI sequences from TIGR4 sequence.
[0371] TABLE 10: GAS AI-3 sequences from M5 isolate (Manfredo).
[0372] TABLE 11: GAS AI-4 sequences from M12 isolate (A735).
[0373] TABLE 12: Conservation of GBS 80 and GBS 104 amino acid
sequences.
[0374] TABLE 13: Conservation of GBS 322 and GBS 276 amino acid
sequences.
[0375] TABLE 14: Active maternal immunization assay for a
combination of fragments from GBS 322, GBS 80, GBS 104, and GBS
67.
[0376] TABLE 15: Antigen surface exposure of GBS 80, GBS 322, GBS
104, and GBS 67.
[0377] TABLE 16: Active maternal immunization assay for each of GBS
80 and GBS 322 antigens.
[0378] TABLE 17: Active maternal immunization assay for GBS 59.
[0379] TABLE 18: Summary of FACS values for surface expression of
spyM6.sub.--0159.
[0380] TABLE 19: Summary of FACS values for surface expression of
spyM6.sub.--0160.
[0381] TABLE 20: Summary of FACS values for surface expression of
GAS 15.
[0382] TABLE 21: Summary of FACS values for surface expression of
GAS 16.
[0383] TABLE 22: Summary of FACS values for surface expression of
GAS 16 using a second antisera.
[0384] TABLE 23: Summary of FACS values for surface expression of
GAS 18.
[0385] TABLE 24: Summary of FACS values for surface expression of
GAS 18 using a second antisera.
[0386] TABLE 25: Summary of FACS values for surface expression of
SpyM3.sub.--0098.
[0387] TABLE 26: Summary of FACS values for surface expression of
SpyM3.sub.--0100.
[0388] TABLE 27: Summary of FACS values for surface expression of
SpyM3.sub.--0102 in M3 serotypes.
[0389] TABLE 28: Summary of FACS values for surface expression of
SpyM3.sub.--0102 in M6 serotypes.
[0390] TABLE 29: Summary of FACS values for surface expression of
SpyM3.sub.--0104 in M3 serotypes.
[0391] TABLE 30: Summary of FACS values for surface expression of
SpyM3.sub.--0104 in an M12 serotype.
[0392] TABLE 31: Summary of FACS values for surface expression of
SPs.sub.--0106 in M3 serotypes.
[0393] TABLE 32: Summary of FACS values for surface expression of
SPs.sub.--0106 in an M12 serotype.
[0394] TABLE 33: Summary of FACS values for surface expression of
19224134 in an M12 serotype.
[0395] TABLE 34: Summary of FACS values for surface expression of
19224134 in M6 serotypes.
[0396] TABLE 35: Summary of FACS values for surface expression of
19224135 in an M12 serotype.
[0397] TABLE 36: Summary of FACS values for surface expression of
19224137 in an M12 serotype.
[0398] TABLE 37: Summary of FACS values for surface expression of
19224141 in an M12 serotype.
[0399] TABLE 38: S. pneumoniae strain 670 .mu.l sequences.
[0400] TABLE 39: Pecent identity comparison of S. pneumoniae
strains AI sequences.
[0401] TABLE 40: FACS analysis of L. lactis and GBS bacteria
strains expressing GBS AI-1.
[0402] TABLE 41: Sequences of primers used to amplify AI locus.
[0403] TABLE 42: Conservation of amino acid sequences encoded by
the S. pneumoniae AI locus.
[0404] TABLE 43: Protection of Mice Immunized with L. lactis
expressing GBS AI-1.
[0405] TABLE 44: GAS AI-3 sequences from M49 isolate (591).
[0406] TABLE 45: Comparison of Sequences Between the Four GAS
AIs.
[0407] TABLE 46: Antibody Responses against GBS 80 in Serum of Mice
Immunized with L. lactis Expressing GBS AI-1
[0408] TABLE 47: Anti-GBS 80 IgA Antibodies Detected in Mouse
Tissues Following Immunization with L. lactis Expressing GBS
AI-1
[0409] TABLE 48: GBS 67 Protects Mice in an Immunization Assay
[0410] TABLE 49: Exposure Levels of GBS 80, GBS 104, GBS 67, GBS
322, and GBS 59 on GBS Strains
[0411] TABLE 50: High Levels of Surface Protein Expression on GBS
Serotypes
[0412] TABLE 51: Further Protection of Mice Immunized with L.
lactis expressing GBS AI-1
DETAILED DESCRIPTION OF THE INVENTION
[0413] The practice of the present invention will employ, unless
otherwise indicated, conventional methods of chemistry,
biochemistry, molecular biology, immunology and pharmacology,
within the skill of the art. Such techniques are explained fully in
the literature. See, e.g., Remington's Pharmaceutical Sciences,
Mack Publishing Company, Easton, Pa., 19th Edition (1995); Methods
In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press,
Inc.); and Handbook of Experimental Immunology, Vols. I-IV (D. M.
Weir and C. C. Blackwell, eds., 1986, Blackwell Scientific
Publications); Sambrook, et al., Molecular Cloning: A Laboratory
Manual (2nd Edition, 1989); Handbook of Surface and Colloidal
Chemistry (Birdi, K. S. ed., CRC Press, 1997); Short Protocols in
Molecular Biology, 4th ed. (Ausubel et al. eds., 1999, John Wiley
& Sons); Molecular Biology Techniques: An Intensive Laboratory
Course, (Ream et al., eds., 1998, Academic Press); PCR
(Introduction to Biotechniques Series), 2nd ed. (Newton &
Graham eds., 1997, Springer Verlag); Peters and Dalrymple, Fields
Virology (2d ed), Fields et al. (eds.), B. N. Raven Press, New
York, N.Y.
[0414] All publications, patents and patent applications cited
herein, are hereby incorporated by reference in their
entireties.
[0415] As used herein, an "Adhesin Island" or "AI" refers to a
series of open reading frames within a bacterial genome, such as
the genome for Group A or Group B Streptococcus or other gram
positive bacteria, that encodes for a collection of surface
proteins and sortases. An Adhesin Island may encode for amino acid
sequences comprising at least one surface protein. The Adhesin
Island may encode at least one surface protein. Alternatively, an
Adhesin Island may encode for at least two surface proteins and at
least one sortase. Preferably, an Adhesin Island encodes for at
least three surface proteins and at least two sortases. One or more
of the surface proteins may include an LPXTG motif (such as LPXTG
(SEQ ID NO: 122)) or other sortase substrate motif. One or more AI
surface proteins may participate in the formation of a pilus
structure on the surface of the gram positive bacteria.
[0416] Adhesin Islands of the invention preferably include a
divergently transcribed transcriptional regulator (i.e., the
transcriptional regulator is located near or adjacent to the AI
protein open reading frames, but it transcribed in the opposite
direction). The transcriptional regulator may regulate the
expression of the AI operon.
GBS Adhesin Island 1
[0417] As discussed above, Applicants have identified a new adhesin
island, "Adhesin Island 1", "AI-1", or "GBS AI-1", within the
genomes of several Group B Streptococcus serotypes and isolates.
AI-1 comprises a series of approximately five open reading frames
encoding for a collection of amino acid sequences comprising
surface proteins and sortases ("AI-1 proteins"). Specifically, AI-1
includes open reading frames encoding for two or more (i.e., 2, 3,
4 or 5) of GBS 80, GBS 104, GBS 52, SAG0647 and SAG0648. One or
more of the AI-1 open reading frame polynucleotide sequences may be
replaced by a polynucleotide sequence coding for a fragment of the
replaced ORF. Alternatively, one or more of the AI-1 open reading
frames may be replaced by a sequence having sequence homology to
the replaced ORF.
[0418] A schematic of AI-1 is presented in FIG. 1. AI-1 typically
resides on an approximately 16.1 kb transposon-like element
frequently inserted into the open reading frame for trmA. One or
more of the AI-1 surface protein sequences typically include an
LPXTG motif (such as LPXTG (SEQ ID NO: 122)) motif or other sortase
substrate motif. The AI surface proteins of the invention may
affect the ability of the GBS bacteria to adhere to and invade
epithelial cells. AI surface proteins may also affect the ability
of GBS to translocate through an epithelial cell layer. Preferably,
one or more AI surface proteins are capable of binding to or
otherwise associating with an epithelial cell surface. AI surface
proteins may also be able to bind to or associate with fibrinogen,
fibronectin, or collagen.
[0419] The AI-1 sortase proteins are predicted to be involved in
the secretion and anchoring of the LPXTG containing surface
proteins. AI-1 may encode for at least one surface protein.
Alternatively, AI-1 may encode for at least two surface exposed
proteins and at least one sortase. Preferably, AI-1 encodes for at
least three surface exposed proteins and at least two sortases. The
AI-1 protein preferably includes GBS 80 or a fragment thereof or a
sequence having sequence identity thereto.
[0420] As used herein, an LPXTG motif represents an amino acid
sequence comprising at least five amino acid residues. Preferably,
the motif includes a leucine (L) in the first amino acid position,
a proline (P) in the second amino acid position, a threonine (T) in
the fourth amino acid position and a glycine (G) in the fifth amino
acid position. The third position, represented by X, may be
occupied by any amino acid residue. Preferably, the X is occupied
by lysine (K), Glutamate (E), Asparagine (N), Glutamine (Q) or
Alanine (A). Preferably, the X position is occupied by lysine (K).
In some embodiments, one of the assigned LPXTG amino acid positions
is replaced with another amino acid. Preferably, such replacements
comprise conservative amino acid replacements, meaning that the
replaced amino acid residue has similar physiological properties to
the removed amino acid residue. Genetically encoded amino acids may
be divided into four families based on physiological properties:
(1) acidic (asparatate and glutamate), (2) basic (lysine, arginine,
histitidine), (3) non-polar (alanine, valine, leucine, isoleucine,
proline, phenylalanine, methionine, tryptophane) and (4) uncharged
polar (glycine, asparagines, glutamine, cysteine, serine,
threonine, and tyrosine). Phenylalanine, tryptophan and tyrosine
are sometimes classified jointly as aromatic amino acids. For
example, it is reasonably predictable that an isolated replacement
of a leucine with an isoleucine or valine, an asparate with a
glutamate, a threonine with a serine, or a similar conservative
replacement of an amino acid with a structurally related amino acid
will not have a major effect on the biological activity.
[0421] The first amino acid position of the LPXTG motif may be
replaced with another amino acid residue. Preferably, the first
amino acid residue (leucine) is replaced with an alanine (A),
valine (V), isoleucine (I), proline (P), phenylalanine (F),
methionine (M), glutamic acid (E), glutamine (Q), or tryptophan (Y)
residue. In one preferred embodiment, the first amino acid residue
is replaced with an isoleucine (I).
[0422] The second amino acid residue of the LPXTG motif may be
replaced with another amino acid residue. Preferably, the second
amino acid residue praline (P) is replaced with a valine (V)
residue.
[0423] The fourth amino acid residue of the LPXTG motif may be
replaced with another amino acid residue. Preferably, the fourth
amino acid residue (threonine) is replaced with a serine (S) or an
alanine (A).
[0424] In general, an LPXTG motif may be represented by the amino
acid sequence XXXXG, in which X at amino acid position 1 is an L, a
V, an E, an I, an F, or a Q; X at amino acid position 2 is a P if X
at amino acid position 1 is an L, an I, or an F; X at amino acid
position 2 is a V if X at amino acid position 1 is a E or a Q; X at
amino acid position 2 is a V or a P if X at amino acid position 1
is a V; X at amino acid position 3 is any amino acid residue; X at
amino acid position 4 is a T if X at amino acid position 1 is a V,
E, I, F, or Q; and X at amino acid position 4 is a T, S, or A if X
at amino acid position 1 is an L.
[0425] Generally, the LPXTG motif of a GBS AI protein may be
represented by the amino acid sequence XPXTG, in which X at amino
acid position 1 is L, I, or F, and X at amino acid position 3 is
any amino acid residue. Specific examples of LPXTG motifs in GBS AI
proteins may include LPXTG (SEQ ID NO: 122) or IPXTG (SEQ ID NO:
133).
[0426] As discussed further below, the threonine in the fourth
amino acid position of the LPXTG motif may be involved in the
formation of a bond between the LPXTG containing protein and a cell
wall precursor. Accordingly, in preferred LPXTG motifs, the
threonine in the fourth amino acid position is not replaced with
another amino acid or, if the threonine is replaced, the
replacement amino acid is preferably a conservative amino acid
replacement, such as serine.
[0427] Instead of an LPXTG motif, the AI surface proteins of the
invention may contain alternative sortase substrate motifs such as
NPQTN (SEQ ID NO: 142), NPKTN (SEQ ID NO: 168), NPQTG (SEQ ID NO:
169), NPKTG (SEQ ID NO: 170), XPXTGG (SEQ ID NO: 143), LPXTAX (SEQ
ID NO: 144), or LAXTGX (SEQ ID NO: 145). (Similar conservative
amino acid substitutions can also be made to these membrane
motifs).
[0428] The AI surface proteins may be covalently attached to the
bacterial cell wall by membrane-associated transpeptidases, such as
an AI sortase. The sortase may function to cleave the surface
protein, preferably between the threonine and glycine residues of
an LPXTG motif. The sortase may then assist in the formation of an
amide link between the threonine carboxyl group and a cell wall
precursor such as lipid II. The precursor can then be incorporated
into the peptidoglycan via the transglycoslylation and
transpeptidation reactions of bacterial wall synthesis. See Comfort
et al., Infection & Immunity (2004) 72(5): 2710-2722.
[0429] The AI surface proteins may be polymerized into pili by
sortase-catalysed transpeptidation. (See FIG. 44.) Cleavage of AI
surface proteins by sortase between the threonine and glycine
residues of an LPXTG motif yields a thioester-linked acyl
intermediate of sortase. Many AI surface proteins include a pilin
motif amino acid sequence which interacts with the sortase and
LPXTG amino acid sequence. The first lysine residue in a pilin
motif can serve as an amino group acceptor of the cleaved LPXTG
motif and thereby provide a covalent linkage between AI subunits to
form pili. For example, the pilin motif can make a nucleophilic
attack on the acyl enzyme providing a covalent linkage between AI
subunits to form pili and regenerate the sortase enzyme. Examples
of pilin motifs may include ((YPKN(X.sub.10)K; SEQ ID NO: 146),
(YPKN(X.sub.9)K; SEQ ID NO: 147), (YPK(X.sub.7)K; SEQ ID NO: 148),
(YPK(X.sub.11)K; SEQ ID NO: 149), or (PKN(X.sub.9)K; SEQ ID NO:
150)). Preferably, the AI surface proteins of the invention include
a pilin motif amino acid sequence.
[0430] Typically, AI surface proteins of the invention will contain
an N-terminal leader or secretion signal to facilitate
translocation of the surface protein across the bacterial
membrane.
[0431] Group B Streptococci are known to colonize the urinary
tract, the lower gastrointestinal tract and the upper respiratory
tract in humans. Electron micrograph images of GBS infection of a
cervical epithelial cell line (ME180) are presented in FIG. 25. As
shown in these images, the bacteria closely associate with tight
junctions between the cells and appear to cross the monolayer by a
paracellular route. Similar paracellular invasion of ME180 cells is
also shown in the contrast images in FIG. 26. The AI surface
proteins of the invention may effect the ability of the GBS
bacteria to adhere to and invade epithelial cells. AI surface
proteins may also affect the ability of GBS to translocate through
an epithelial cell layer. Preferably, one or more AI surface
proteins are capable of binding to or otherwise associating with an
epithelial cell surface.
[0432] Applicants have discovered that AI-1 surface protein GBS 104
can bind epithelial cells such as ME180 human cervical cells, A549
human lung cells and Caco2 human intestinal cells (See FIGS. 29 and
210). Further, deletion of the GBS 104 sequence in a GBS strain
reduces the capacity of GBS to adhere to ME180 cervical epithelial
cells. (See FIGS. 30 and 211). Deletion of GBS 104 also reduces the
capacity of GBS to invade J774 macrophage-like cells. (See FIGS. 32
and 205). Deletion of GBS 104 also causes GBS to translocate
through epithelial monolayers less efficiently. See FIG. 206. GBS
104 protein therefore appears to bind to ME180 epithelial cells and
to have a role in adhesion to epithelial cells and macrophage cell
lines.
[0433] Similar to the GBS bacteria that are deletion mutants for
GBS 104, GBS 80 knockout mutant strains also partially lose the
ability to translocate through an epithelial monolayer. See FIG.
207. Deletion of either GBS 80 or GBS 104 in COH1 cells diminishes
adherence to HUVEC endothelial cells. See FIG. 208. Deletion of GBS
80 or GBS 104 in COH1 does not, however, affect growth of COH1
either with ME180 cells or in incubation medium (IM). See FIG. 209.
Both GBS 80 and GBS 104, therefore, appear to be involved in
translocation of GBS through epithelial cells.
[0434] GBS 80 does not appear to bind to epithelial cells.
Incubation of epithelial cells in the presence of GBS 80 protein
followed by FACS analysis using an anti-GBS 80 polyclonal antibody
did not detect GBS 80 binding to the epithelial cells. See FIG.
202. Furthermore, deletion of GBS 80 protein does not affect the
ability of GBS to adhere and invade ME180 cervical epithelial
cells. See FIG. 203
[0435] Preferably, one or more of the surface proteins may bind to
one or more extracellular matrix (ECM) binding proteins, such as
fibrinogen, fibronectin, or collagen. As shown in FIGS. 5 and 204,
and Example 1, GBS 80, one of the AI-1 surface proteins, can bind
to the extracellular matrix binding proteins fibronectin and
fibrinogen. While GBS 80 protein apparently does not bind to
certain epithelial cells or affect the capacity of a GBS bacteria
to adhere to or invade cervical epithelial cells (See FIGS. 27 and
28), removal of GBS 80 from a wild type strain decreases the
ability of that strain to translocate through an epithelial cell
layer (see FIG. 31).
[0436] GBS 80 may also be involved in formation of biofilms. COH1
bacteria overexpressing GBS 80 protein have an impaired ability to
translocate through an epithelial monolayer. See FIG. 212. These
COH1 bacteria overexpressing GBS 80 form microcolonies on
epithelial cells. See FIGS. 213 and 214. These microcolonies may be
the initiation of biofilm development.
[0437] AI Surface proteins may also demonstrate functional homology
to previously identified adhesion proteins or extracellular matrix
(ECM) binding proteins. For example, GBS 80, a surface protein in
AI-1, exhibits some functional homology to FimA, a major fimbrial
subunit of a Gram positive bacteria A. naeslundii. FimA is thought
to be involved in binding salivary proteins and may be a component
in a fimbrae on the surface of A. naeslundii. See Yeung et al.
(1997) Infection & Immunity 65:2629-2639; Yeunge et al (1998)
J. Bacteriol 66:1482-1491; Yeung et al. (1988) J. Bacteriol
170:3803-3809; and Li et al. (2001) Infection & Immunity
69:7224-7233.
[0438] A similar functional homology has also been identified
between GBS 80 and proteins involved in pili formation in the Gram
positive bacteria Corynebacterium diphtheriae (SpaA, SpaD, and
SpaH). See, Ton-That et al. (2003) Molecular Microbiology
50(4):1429-1438 and Ton-That et al. (2004) Molecular Microbiology
53(1):251-261. The C. diphtheriae proteins all included a pilin
motif of WxxxVxVYPK (SEQ ID NO: 151; where x indicates a varying
amino acid residue). The lysine (K) residue is particularly
conserved in the C. diphtheriae pilus proteins and is thought to be
involved in sortase catalized oligomerization of the subunits
involved in the C. diphtheriae pilus structure. (The C. diphtheriae
pilin subunit SpaA is thought to occur by sortase-catalyzed amide
bond cross-linking of adjacent pilin subunits. As the
thioester-linked acyl intermediate of sortase requires nucleophilic
attack for release, the conserved lysine within the SpaA pilin
motif might function as an amino group acceptor of cleaved sorting
signals, thereby providing for covalent linkages of the C.
diphtheria pilin subunits. See FIG. 6(d) of Ton-That et al.,
Molecular Microbiology (2003) 50(4): 1429-1438.)
[0439] In addition, an "E box" comprising a conserved glutamic acid
residue has also been identified in the C. diphtheria pilin
associated proteins as important in C. diphtheria pilin assembly.
The E box motif generally comprises YxLxETxAPxGY (SEQ ID NO: 152;
where x indicates a varying amino acid residue). In particular, the
conserved glutamic acid residue within the E box is thought
necessary for C. diphtheria pilus formation.
[0440] Preferably, the AI-1 polypeptides of the immunogenic
compositions comprise an E box motif. Some examples of E box motifs
in the AI-1 polypeptides may include the amino acid sequences
YxLxExxxxxGY (SEQ ID NO: 153), YxLxExxxPxGY (SEQ ID NO: 154), or
YxLxETxAPxGY (SEQ ID NO: 152). Specifically, the E box motif of the
polypeptides may comprise the amino acid sequences YKLKETKAPEGY
(SEQ ID NO: 155), YVLKEIETQSGY (SEQ ID NO: 156), or YKLYEISSPDGY
(SEQ ID NO: 157).
[0441] As discussed in more detail below, a pilin motif containing
a conserved lysine residue and an E box motif containing a
conserved glutamic acid residue have both been identified in GBS
80.
[0442] While previous publications have speculated that pilus-like
structures might be formed on the surface of streptococci, (see,
e.g., Ton-That et al., Molecular Microbiology (2003) 50(4):
1429-1438), these structures have not been previously visible in
negative stain (non-specific) electron micrographs, throwing such
speculations into doubt. For example, FIG. 34 presents electron
micrographs of GBS serotype III, strain isolate COH1 with a plasmid
insert to facilitate the overexpression of GBS 80. This EM photo
was produced with a standard negative stain--no pilus structures
are distinguishable. In addition, the use of such AI surface
proteins in immunogenic compositions for the treatment or
prevention of infection against a Gram positive bacteria has not
been previously described.
[0443] Surprisingly, Applicants have now identified the presence of
GBS 80 in surface exposed pilus formations visible in electron
micrographs. These structures are only visible when the electron
micrographs are specifically stained against an AI surface protein
such as GBS 80. Examples of these electron micrographs are shown in
FIGS. 11, 16 and 17, which reveal the presence of pilus structures
in wild type COH1 Streptococcus agalactiae. Other examples of these
electron micrographs are shown in FIG. 49, which reveals that GBS
80 is associated with pili in a wild type clinical isolate of S.
agalactiae, JM9030013. (See FIG. 49.)
[0444] Applicants have also constructed mutant GBS strains
containing a plasmid comprising the GBS 80 sequence resulting in
the overexpression of GBS 80 within this mutant. The electron
micrographs of FIGS. 13-15 are also stained against GBS 80 and
reveal long, oligomeric structures containing GBS 80 which appear
to cover portions of the surface of the bacteria and stretch far
out into the supernatant.
[0445] In some instances, the formation of pili structures on GBS
appears to be correlated to surface expression of GBS 80. FIG. 61
provides FAC analysis of GBS 80 surface levels on bacterial strains
COH1 and JM9130013 using an anti-GBS 80 antisera. Immunogold
electron microscopy of the COH1 and JM9130013 bacteria using
anti-GBS 80 antisera demonstrates that JM9130013 bacteria, which
have higher values for GBS 80 surface expression, also form longer
pili structures.
[0446] The surface exposure of GBS 80 on GBS is generally not
capsule-dependent. FIG. 62 provides FACS analysis of capsulated and
uncapsulated GBS analyzed with anti-GBS 80 and anti-GBS 322
antibodies. Surface exposure of GBS 80, unlike GBS 322, is not
capsule dependent.
[0447] An Adhesin Island surface protein, such as GBS 80 appears to
be required for pili formation, as well as an Adhesin Island
sortase. Pili are formed in Coh1 bacterial clones that overexpress
GBS 80, but lack GBS 104, or one of the AI-1 sortases sag0647 or
sag0648. However, pili are not formed in Coh1 bacterial clones that
overexpress GBS 80 and lack both sag0647 and sag0648. Thus, for
example, it appears that at least GBS 80 and a sortase, sag0647 or
sag0648, may be necessary for pili formation. (See FIG. 48.)
Overexpression of GBS 80 in GBS strain 515, which lacks an AI-1,
also assembles GBS 80 into pili. GBS strain 515 contains an AI-2,
and thus AI-2 sortases. The AI-2 sortases in GBS strain 515
apparently polymerize GBS 80 into pili. (See FIG. 42.)
Overexpression of GBS 80 in GBS strain 515 cell knocked out for GBS
67 expression also apparently polymerizes GBS 80 into pili. (See
FIG. 72.)
[0448] While GBS 80 appears to be required for GBS AI-1 pili
formation, GBS 104 and sortase SAG0648 appears to be important for
efficent AI-1 pili assembly. For example, high-molecular structures
are not assembled in isogenic COH1 strains which lack expression of
GBS 80 due to gene disruption and are less efficiently assembled in
isogenic COH1 strains which lack the expression of GBS 104 (see
FIG. 41). This GBS strain comprises high molecular weight pili
structures composed of covalently linked GBS 80 and GBS 104
subunits. In addition, deleting SAG0648 in COH1 bacteria interferes
with assembly of some of the high molecular weight pili structures.
Thus, indicating that SAG0648 plays a role in assembly of these
pilin species. (See FIG. 41).
[0449] EM photos confirm the involvement of AI surface protein GBS
104 within the hyperoligomeric structures of a GBS strain adapted
for increased GBS 80 expression. (See FIGS. 34-41 and Example 6).
In a wild type serotype VIII GBS strain, strain JM9030013, IEM
identifies GBS 104 as forming clusters on the bacterial surface.
(See FIG. 50.)
[0450] GBS 52 also appears to be a component of the GBS pili.
Immunoblots using an anti-GBS 80 antisera on total cell extracts of
Coh1 and a GBS 52 null mutant Coh1 reveal a shift in detected
proteins in the Coh1 wild type strain relative to the GBS 52 null
mutant Coh1 strain. The shifted proteins were also detected in the
wild type Coh1 bacteria with an anti-GBS 52 antisera, indicating
that the GBS 52 may be present in the pilus. (See FIG. 45.)
[0451] In one embodiment, the invention includes a composition
comprising oligomeric, pilus-like structures comprising an AI
surface protein such as GBS 80. The oligomeric, pilus-like
structure may comprise numerous units of AI surface protein.
Preferably, the oligomeric, pilus-like structures comprise two or
more AI surface proteins. Still more preferably, the oligomeric,
pilus-like structure comprises a hyper-oligomeric pilus-like
structure comprising at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90,
100, 120, 140, 150, 200 or more) oligomeric subunits, wherein each
subunit comprises an AI surface protein or a fragment thereof. The
oligomeric subunits may be covalently associated via a conserved
lysine within a pilin motif. The oligomeric subunits may be
covalently associated via an LPXTG motif, preferably, via the
threonine amino acid residue.
[0452] AI surface proteins or fragments thereof to be incorporated
into the oligomeric, pilus-like structures of the invention will
preferably include one or both of a pilin motif comprising a
conserved lysine residue and an E box motif comprising a conserved
glutamic acid residue.
[0453] More than one AI surface protein may be present in the
oligomeric, pilus-like structures of the invention. For example,
GBS 80 and GBS 104 may be incorporated into an oligomeric
structure. Alternatively, GBS 80 and GBS 52 may be incorporated
into an oligomeric structure, or GBS 80, GBS 104 and GBS 52 may be
incorporated into an oligomeric structure.
[0454] In another embodiment, the invention includes compositions
comprising two or more AI surface proteins. The composition may
include surface proteins from the same adhesin island. For example,
the composition may include two or more GBS AI-1 surface proteins,
such as GBS 80, GBS 104 and GBS 52. The surface proteins may be
isolated from Gram positve bacteria or they may be produced
recombinantly.
[0455] The oligomeric, pilus like structures may be used alone or
in the combinations of the invention. In one embodiment, the
invention comprises a GBS Adhesin Island protein in oligomeric
form, preferably in a hyperoligomeric form. In one embodiment, the
invention comprises a composition comprising one or more GBS
Adhesin Island 1 ("AI-1") proteins and one or more GBS Adhesin
Island 2 ("AI-2") proteins, wherein one or more of the Adhesin
Island proteins is in the form of an oligomer, preferably in a
hyperoligomeric form.
[0456] The oligomeric, pilus-like structures of the invention may
be combined with one or more additional GBS proteins. In one
embodiment, the oligomeric, pilus-like structures comprise one or
more AI surface proteins in combination with a second GBS protein.
The second GBS protein may be a known GBS antigen, such as GBS 322
(commonly referred to as "sip") or GBS 276. Nucleotide and amino
acid sequences of GBS 322 sequenced from serotype V isolated strain
2603 V/R are set forth in WO 02/35771 as SEQ ID 8539 and SEQ ID
8540 and in the present specification as SEQ ID NOs: 38 and 39. A
particularly preferred GBS 322 polypeptide lacks the N-terminal
signal peptide, amino acid residues 1-24. An example of a preferred
GBS 322 polypeptide is a 407 amino acid fragment and is shown in
SEQ ID NO: 40. Examples of preferred GBS 322 polypeptides are
further described in PCTUS04/______, attorney docket number
PP20665.002 filed Sep. 15, 2004, hereby incorporated by reference,
published as WO 2005/002619.
[0457] Additional GBS proteins which may be combined with the GBS
AI surface proteins of the invention are also described in WO
2005/002619. These GBS proteins include GBS 91, GBS 184, GBS 305,
GBS 330, GBS 338, GBS 361, GBS 404, GBS 690, and GBS 691.
[0458] Additional GBS proteins which may be combined with the GBS
AI surface proteins of the invention are described in WO
02/34771.
[0459] GBS polysaccharides which may be combined with the GBS AI
surface proteins of the invention are described in WO 2004/041157.
For example, the GBS AI surface proteins of the invention may be
combined with a GBS polysaccharides selected from the group
consisting of serotype Ia, Ib, Ia/c, II, III, IV, V, VI, VII and
VIII.
[0460] The oligomeric, pilus-like structures may be isolated or
purified from bacterial cultures in which the bacteria express an
AI surface protein. The invention therefore includes a method for
manufacturing an oligomeric AI surface antigen comprising culturing
a GBS bacterium that expresses the oligomeric AI protein and
isolating the expressed oligomeric AI protein from the GBS
bacteria. The AI protein may be collected from secretions into the
supernatant or it may be purified from the bacterial surface. The
method may further comprise purification of the expressed AI
protein. Preferably, the AI protein is in a hyperoligomeric form.
Macromolecular structures associated with oligomeric pili are
observed in the supernatant of cultured GBS strain Coh1. (See FIG.
46.) These pili are found in the supernatant at all growth phases
of the cultured Coh1 bacteria. (See FIG. 47.)
[0461] The oligomeric, pilus-like structures may be isolated or
purified from bacterial cultures overexpressing an AI surface
protein. The invention therefore includes a method for
manufacturing an oligomeric Adhesin Island surface antigen
comprising culturing a GBS bacterium adapted for increased AI
protein expression and isolation of the expressed oligomeric
Adhesin Island protein from the GBS bacteria. The AI protein may be
collected from secretions into the supernatant or it may be
purified from the bacterial surface. The method may further
comprise purification of the expressed Adhesin Island protein.
Preferably, the Adhesin Island protein is in a hyperoligomeric
form.
[0462] The GBS bacteria are preferably adapted to increase AI
protein expression by at least two (e.g., 2, 3, 4, 5, 8, 10, 15,
20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150 or 200)
times wild type expression levels.
[0463] GBS bacteria may be adapted to increase AI protein
expression by any means known in the art, including methods of
increasing gene dosage and methods of gene upregulation. Such means
include, for example, transformation of the GBS bacteria with a
plasmid encoding the AI protein. The plasmid may include a strong
promoter or it may include multiple copies of the sequence encoding
the AI protein. Optionally, the sequence encoding the AI protein
within the GBS bacterial genome may be deleted. Alternatively, or
in addition, the promoter regulating the GBS Adhesin Island may be
modified to increase expression.
[0464] GBS bacteria harbouring a GBS AI-1 may also be adapted to
increase AI protein expression by altering the number adenosine
nucleotides present at two sites in the intergenic region between
AraC and GBS 80. See FIG. 197 A, which is a schematic showing the
organization of GBS AI-1 and FIG. 197 B, which provides the
sequence of the intergenic region between AraC and GBS 80 in the
AI. The adenosine tracts which applicants have identified as
influencing GBS 80 surface expression are at nucleotide positions
187 and 233 of the sequence shown in FIG. 197 B (SEQ ID NO: 273).
Applicants determined the influence of these adenosine tracts on
GBS 80 surface expression in strains of GBS bacteria harboring four
adenosines at position 187 and six adenosines at position 233, five
adenosines at position 187 and six adenosines position 233, and
five adenosines at position 187 and seven adenosines at position
233. FACS analysis of these strains using anti GBS 80 antiserum
determined that an intergenic region with five adenosines at
position 187 and six adenosines at position 233 had higher
expression levels of GBS 80 on their surface than other stains. See
FIG. 197 C for results obtained from the FACS analysis. Therefore,
manipulating the number of adenosines present at positions 187 and
233 of the AraC and GBS 80 intergenic region may further be used to
adapt GBS to increase AI protein expression.
[0465] The invention further includes GBS bacteria which have been
adapted to produce increased levels of AI surface protein. In
particular, the invention includes GBS bacteria which have been
adapted to produce oligomeric or hyperoligomeric AI surface
protein, such as GBS 80. In one embodiment, the Gram positive
bacteria of the invention are inactivated or attenuated to permit
in vivo delivery of the whole bacteria, with the AI surface protein
exposed on its surface.
[0466] The invention further includes GBS bacteria which have been
adapted to have increased levels of expressed AI protein
incorporated in pili on their surface. The GBS bacteria may be
adapted to have increased exposure of oligomeric or hyperoligomeric
AI proteins on its surface by increasing expression levels of a
signal peptidase polypeptide. Increased levels of a local signal
peptidase expression in Gram positive bacteria (such us LepA in
GAS) are expected to result in increased exposure of pili proteins
on the surface of Gram positive bacteria. Increased expression of a
leader peptidase in GBS may be achieved by any means known in the
art, such as increasing gene dosage and methods of gene
upregulation. The GBS bacteria adapted to have increased levels of
leader peptidase may additionally be adapted to express increased
levels of at least one pili protein.
[0467] Alternatively, the AI proteins of the invention may be
expressed on the surface of a non-pathogenic Gram positive
bacteria, such as Streptococus gordonii (See, e.g., Byrd et al.,
"Biological consequences of antigen and cytokine co-expression by
recombinant Streptococcus gordonii vaccine vectors", Vaccine (2002)
20:2197-2205) or Lactococcus lactis (See, e.g., Mannam et al.,
"Mucosal Vaccine Made from Live, Recombinant Lactococcus lactis
Protects Mice against Pharangeal Infection with Streptococcus
pyogenes" Infection and Immunity (2004) 72(6):3444-3450). As used
herein, non-pathogenic Gram positive bacteria refer to Gram
positive bacteria which are compatible with a human host subject
and are not associated with human pathogenisis. Preferably, the
non-pathogenic bacteria are modified to express the AI surface
protein in oligomeric, or hyper-oligomeric form. Sequences encoding
for an AI surface protein and, optionally, an AI sortase, may be
integrated into the non-pathogenic Gram positive bacterial genome
or inserted into a plasmid. The non-pathogenic Gram positive
bacteria may be inactivated or attenuated to facilitate in vivo
delivery of the whole bacteria, with the AI surface protein exposed
on its surface. Alternatively, the AI surface protein may be
isolated or purified from a bacterial culture of the non-pathogenic
Gram positive bacteria. For example, the AI surface protein may be
isolated from cell extracts or culture supernatants. Alternatively,
the AI surface protein may be isolated or purified from the surface
of the non-pathogenic Gram positive bacteria.
[0468] The non-pathogenic Gram positive bacteria may be used to
express any of the Gram positive bacterial Adhesin Island proteins
described herein, including proteins from a GBS Adhesin Island, a
GAS Adhesin Island, or a S pneumo Adhesin Island. The
non-pathogenic Gram positive bacteria are transformed to express an
Adhesin Island surface protein. Preferably, the non-pathogenic Gram
positive bacteria also express at least one Adhesin Island sortase.
The AI transformed non-pathogenic Gram positive bacteria of the
invention may be used to prevent or treat infection with a
pathogenic Gram positive bacteria, such as GBS, GAS or
Streptococcus pneumoniae. The non-pathogenic Gram positive bacteria
may express the Gram positive bacterial Adheshin Island proteins in
oligomeric forms that further comprise adhesin island proteins
encoded within the genome of the non-pathogenic Gram positive
bacteria.
[0469] Applicants modified L. lactis to demonstrate that it can
express GBS AI polypeptides. L. lactis was transformed with a
construct encoding GBS 80 under its own promoter and terminator
sequences. The transformed L. lactis appeared to express GBS 80 as
shown by Western blot analysis using anti-GBS 80 antiserum. See
lanes 6 and 7 of the Western Blots provided in FIGS. 133A and 133B
(133A and 133B are two different exposures of the same Western
blot). See also Example 13.
[0470] Applicants also transformed L. lactis with a construct
encoding GBS AI-1 polypeptides GBS 80, GBS 52, SAG0647, SAG0648,
and GBS 104 under the GBS 80 promoter and terminator sequences.
These L. lactis expressed high molecular weight structures that
were immunoreactive with anti-GBS 80 in immunoblots. See FIG. 134,
lane 2, which shows detection of a GBS 80 monomer and higher
molecular weight polymers in total transformed L. lactis extracts.
Thus, it appeared that L. lactis is capable of expressing GBS 80 in
oligomeric form. The high molecular weight polymers were not only
detected in L. lactis extracts, but also in the culture
supernatants. See FIG. 135 at lane 4. See also Example 14. Thus,
the GBS AI polypeptides in oligomeric form can be isolated and
purified from either L. lactis cell extracts or culture
supernatants. These oligomeric forms can, for instance, be isolated
from cell extracts or culture supernatants by release by
sonication. See FIGS. 136A and B. See also FIG. 171, which shows
purification of GBS pili from whole extracts of L. lactis
expressing the GBS AI-1 following sonication and gel filtration on
a Sephacryl HR 400 column.
[0471] Furthermore, the L. lactis transformed with the construct
encoding GBS AI-1 polypeptides GBS 80, GBS 52, SAG0647, SAG0648,
and GBS 104 under the GBS 80 promoter and terminator sequences
expressed the GBS AI-1 polypeptides on its surface. FACS analysis
of these transformed L. lactis detected cell surface expression of
both GBS 80 and GBS 104. The surface expression levels of GBS 80
and GBS 104 on the transformed L. lactis were similar to the
surface expression levels of GBS 80 and GBS 104 on GBS strains COH1
and JM9130013, which naturally express GBS AI-1.
[0472] See FIG. 169 for FACS analysis data for L. lactis
transformed with GBS AI-1 and wildtype JM9130013 bacteria using
anti-GBS 80 and GBS 104 antisera. Table 40 provides the results of
FACS analysis of transformed L. lactis, COH1, and JM9130013
bacteria using anti-GBS 80 and anti-GBS 104 antisera. The numbers
provided represent the mean fluorescence value difference
calculated for immune versus pre-immune sera obtained for each
bacterial strain. TABLE-US-00001 TABLE 40 FACS analysis of L.
lactis and GBS bacteria strains expressing GBS AI-1 Anti-GBS 80
Anti-GBS 104 antiserum antiserum GBS AI-1 transformed 298 251 L.
lactis GBS COH1 305 305 GBS JM9130013 461 355
Immunogold-electronmicroscopy performed with anti-GBS 80 primary
antibodies detected the presence of pilus structures on the surface
of the L. lactis bacteria expressing GBS AI-1, confirming the
results of the FACS analysis. See FIG. 168 B and C. Interestingly,
this expression of GBS pili on the surface of the L. lactis induced
L. lactis aggregation. See FIG. 170. Thus, GBS AI polypeptides may
also be isolated and purified from the surface of L. lactis. The
ability of L. lactis to express GBS AI polypeptides on its surface
also demonstrates that it may be useful as a host to deliver GBS AI
antigens.
[0473] In fact, immunization of mice with L. lactis transformed
with GBS AI-1 was protective in a subsequent challenge with GBS.
Female mice were immunized with L. lactis transformed with GBS
AI-1. The immunized female mice were bred and their pups were
challenged with a dose of GBS sufficient to kill 90% of
non-immunized pups. Detailed protocols for intranasal and
subcutaneous immunization of mice with transformed L. lactis can be
found in Examples 18 and 19, respectively. Table 43 provides data
showing that immunization of the female mice with L. lactis
expressing GBS AI-1(LL-AI 1) greatly increased survival rate of
challenged pups relative to both a negative PBS control (PBS) and a
negative L. lactis control (LL 10 E9, which is wild type L. lactis
not transformed to express GBS AI-1). TABLE-US-00002 TABLE 43
Protection of Mice Immunized with L. lactis expressing GBS AI-1
Immunization Survival Route Antigen Alive/Treated Survival % %
Range p value Intraperitoneum Recombinant GBS 80 16/18 89 80-100
<0.001 Subcutaneous LL-AI 1 10 E9 40/49 82 70-90 <0.001 LL-AI
1 10 E10 50/60 83 60-100 <0.001 PBS 4/30 13 0-30 LL 10 E9 3/57 5
0-20 Intranasal LL-AI 1 10 E9 22/60 37 0-100 0.02 LL-AI 1 10 E10
31/49 63 30-90 <0.001 LL 10 E9 2/27 7 0-20
[0474] Table 51 provides further evidence that immunization of mice
with L. lactis transformed with GBS AI-1 is protective against GBS.
TABLE-US-00003 TABLE 51 Further Protection of Mice Immunized with
L. lactis expressing GBS AI-1 Immunization Alive/ Survival %
Antigen route Treated (Pval < 0.0000001) Recombinant GBS 80 IP
48/50 92 Recombinant GBS 80 SC 21/30 70 L. lactis + AI1 10.sup.6
cfu SC 6/66 9 L. lactis + AI1 10.sup.7 cfu SC 47/70 73 L. lactis +
AI1 10.sup.8 cfu SC 116/153 76 L. lactis + AI1 10.sup.9 cfu SC
98/118 83 L. lactis + AI1 10.sup.10 cfu SC 107/129 83 L. lactis
10.sup.10 cfu SC 4/83 5 PBS SC 6/110 5 L. lactis + AI1 10.sup.10
cfu IN 51/97 52 L. lactis 10.sup.11 cfu IN 1/40 7 PBS IN 0/37 0
[0475] Protection of immunized mice with L. lactis expressing the
GBS AI-1 is at least partly due to a newly raised antibody
response. Table 46 provides anti-GBS 80 antibody titers detected in
serum of the mice immunized with L. lactis expressing the GBS AI-1
as described above. Mice immunized with L. lactis expressing the
GBS AI-1 have anti-GBS 80 antibody titres, which are not observed
in mice immunized with L. lactis not transformed to express the GBS
AI-1. Further, as expected from the survival data, mice
subcutaneously immunized with L. lactis transformed to express the
GBS AI-1 have significantly higher serum anti-GBS 80 antibody
titers than mice intranasally immunized with L. lactis transformed
to express the GBS AI-1. TABLE-US-00004 TABLE 46 Antibody Responses
against GBS 80 in Serum of Mice Immunized with L. lactis Expressing
GBS AI-1 Ab Titre Obtained Following Subcutaneous Intranasal
Intraperitoneal Antigen Immunization Immunization Immunization LL
10 E9 0 0 LL-AI 1 10 E9 14000 50 LL-AI 1 10 E10 25000 406
Recombinant GBS 80 120000
[0476] Anti-GBS 80 antibodies of the IgA isotype were specifically
detected in various body fluids of the mice subcutaneously or
intranasally immunized with L. lactis expressing the GBS AI-1.
TABLE-US-00005 TABLE 47 Anti-GBS 80 IgA Antibodies Detected in
Mouse Tissues Following Immunization with L. lactis Expressing GBS
AI-1 Anti-GBS 80 IgA Antibodies Detected in Antigen Immunization
route Serum Vaginal Wash Nasal Wash LL 10 E9 0 0 0 LL-AI 1
Subcutaneous 0 25 20 LL-AI 1 Intranasal 140 0 150 GBS 80
Intraperitoneal 60 0
[0477] Furthermore, opsonophagocytosis assays also demonstrated
that at least some of the antiserum produced against the L. lactis
expressing GBS AI 1 is opsonic for GBS. See FIG. 161.
[0478] To obtain protection of against GBS across a greater number
of strains and serotypes, it is possible to transform L. lactis
with a recombinant GBS AI encoding both GBS AI-1 and AI-2, i.e., a
hybrid GBS AI. By way of example, a hybrid GBS AI may be a GBS AI-1
with a replacement of the GBS 104 gene with a GBS 67 gene. A
schematic of such a hybrid GBS AI is depicted in FIG. 231 A. A
hybrid GBS AI may alternatively be a GBS AI-1 with a replacement of
the GBS 52 gene with a GBS 59 gene. See the schematic at FIG. 231
B. Alternatively, a hybrid GBS AI may be a GBS AI-1 with a
substitution of a GBS 59 polypeptide for the GBS 52 gene and a
substitution of the GBS 104 gene for genes encoding GBS 59 and the
two GBS AI-2 sortases. Another example of a hybrid GBS AI is a GBS
AI-1 with the substitution of a GBS 59 gene for the GBS 52 gene and
a GBS 67 for the GBS 104 gene. See the schematic at FIG. 232. A
further example of a hybrid GBS AI is a GBS AI-1 having a GBS 59
gene and genes encoding the GBS AI-2 sortases in place of the GBS
52 gene. Yet another example of a hybrid GBS AI is a GBS AI-1 with
a substitution of either GBS 52 or GBS 104 with a fusion protein
comprising GBS 322 and one of GBS 59, GBS 67, or GBS 150. Some of
these hybrid GBS AIs may be prepared as briefly outlined in FIG.
234 A-F.
[0479] Applicants have prepared a hybrid GBS AI having a GBS AI-1
sequence with a substitution of a GBS 67 coding sequence for the
GBS 104 gene as depicted in FIG. 231 A. Transformation of L. lactis
with the hybrid GBS AI-1 resulted in L. lactis expression of high
molecular weight polymers containing the GBS 80 and GBS 67
proteins. See FIG. 233 A, which provides Western blot analysis of
L. lactis transformed with the hybrid GBS AI depicted in FIG. 231
A. When L. lactis transformed with the hybrid GBS AI were probed
with antibodies to GBS 80 or GBS 67, high molecular weight
structures were detected. See lanes labelled LL+a) in both the
.alpha.-80 and .alpha.-67 immunoblots. The GBS 80 and GBS 67
proteins were confirmed to be present on the surface of L. lactis
by FACS analysis. See FIG. 233 B, which shows a shift in
fluorescence when GBS 80 and GBS 67 antibodies are used to detect
GBS 80 and GBS 67 surface expression. The same shifts in
fluorescence were not observed in L. lactis control cells, cells
not transformed with the hybrid GBS AI.
[0480] Alternatively, the oligomeric, pilus-like structures may be
produced recombinantly. If produced in a recombinant host cell
system, the AI surface protein will preferably be expressed in
coordination with the expression of one or more of the AI sortases
of the invention. Such AI sortases will facilitate oligomeric or
hyperoligomeric formation of the AI surface protein subunits.
[0481] AI Sortases of the invention will typically have a signal
peptide sequence within the first 70 amino acid residues. They may
also include a transmembrane sequence within 50 amino acid residues
of the C terminus. The sortases may also include at least one basic
amino acid residue within the last 8 amino acids. Preferably, the
sortases have one or more active site residues, such as a catalytic
cysteine and histidine.
[0482] As shown in FIG. 1, AI-1 includes the surface exposed
proteins of GBS 80, GBS 52 and GBS 104 and the sortases SAG0647 and
SAG0648. AI-1 typically appears as an insertion into the 3' end of
the trmA gene.
[0483] In addition to the open reading frames encoding the AI-1
proteins, AI-1 may also include a divergently transcribed
transcriptional regulator such as araC (i.e., the transcriptional
regulator is located near or adjacent to the AI protein open
reading frames, but it transcribed in the opposite direction). It
is believed that araC may regulate the expression of the AI operon.
(See Korbel et al., Nature Biotechnology (2004) 22(7): 911-917 for
a discussion of divergently transcribed regulators in E. coli).
[0484] AI-1 may also include a sequence encoding a rho independent
transcriptional terminator (see hairpin structure in FIG. 1). The
presence of this structure within the adhesin island is thought to
interrupt transcription after the GBS 80 open reading frame,
leading to increased expression of this surface protein.
[0485] A schematic identifying AI-1 within several GBS serotypes is
depicted in FIG. 2. AI-1 sequences were identified in GBS serotype
V, strain isolate 2603; GBS serotype III, strain isolate NEM316;
GBS serotype II, strain isolate 18RS21; GBS serotype V, strain
isolate CJB111; GBS serotype III, strain isolate COH1 and GBS
serotype 1a, strain isolate A909. (Percentages shown are amino acid
identity to the 2603 sequence). (An AI-1 was not identified in GBS
serotype 1b, strain isolate H36B or GBS serotype 1a, strain isolate
515).
[0486] An alignment of AI-1 polynucleotide sequences from serotype
V, strain isolates 2603 and CJB111; serotype II, strain isolate
18RS21; serotype III, strain isolates COH1 and NEM316; and serotype
1a, strain isolate A909 is presented in FIG. 18. An alignment of
amino acid sequences of AI-1 surface protein GBS 80 from serotype
V, strain isolates 2603 and CJB111; serotype 1a, strain isolate
A909; serotype III, strain isolates COH1 and NEM316 is presented in
FIG. 22. An alignment of amino acid sequences of AI-1 surface
protein GBS 104 from serotype V, strain isolates 2603 and CJB111;
serotype III, strain isolates COH1 and NEM316; and serotype II,
strain isolate 18RS21 is presented in FIG. 23. Preferred AI-1
polynucleotide and amino acid sequences are conserved among two or
more GBS serotypes or strain isolates.
[0487] As shown in this figure, the full length of surface protein
GBS 80 is particularly conserved among GBS serotypes V (strain
isolates 2603 and CJBIII), III (strain isolates NEM316 and COH1),
and Ia (strain isolate A909). The GBS 80 surface protein is missing
or fragmented in serotypes II (strain isolate 18RS21), Ib (strain
isolate H36B) and Ia (strain isolate 515).
[0488] Polynucleotide and amino acid sequences for AraC are set
forth in FIG. 30.
GBS Adhesin Island 2
[0489] A second adhesin island, "Adhesin Island 2" or "AI-2" or GBS
AI-2" has also been identified in numerous GBS serotypes. A
schematic depicting the correlation between AI-1 and AI-2 within
the GBS serotype V, strain isolate 2603 is shown in FIG. 3.
(Homology percentages in FIG. 3 represent amino acid identity of
the AI-2 proteins to the AI-1 proteins). Alignments of AI-2
polynucleotide sequences are presented in FIGS. 20 and 21 (FIG. 20
includes sequences from serotype V, strain isolate 2603 and
serotype III, strain isolate NEM316. FIG. 21 includes sequences
from serotype III, strain isolate COH1 and serotype Ia, strain
isolate A909). An alignment of amino acid sequences of AI-2 surface
protein GBS 067 from serotype V, strain isolates 2603 and CJB111;
serotype 1a, strain isolate 515; serotype II, strain isolate
18RS21; serotype Ib, strain isolate H36B; and serotype III, strain
isolate NEM316 is presented in FIG. 24. Preferred AI-2
polynucleotide and amino acid sequences are conserved among two or
more GBS serotypes or strain isolates.
[0490] AI-2 comprises a series of approximately five open reading
frames encoding for a collection of amino acid sequences comprising
surface proteins and sortases. Specifically, AI-2 includes open
reading frames encoding for two or more (i.e., 2, 3, 4, 5 or more)
of GBS 67, GBS 59, GBS 150, SAG1405, SAG1406, 01520, 01521, 01522,
01523, 01523, 01524 and 01525. In one embodiment, AI-2 includes
open reading frames encoding for two or more of GBS 67, GBS 59, GBS
150, SAG1405, and SAG1406. Alternatively, AI-2 may include open
reading frames encoding for two or more of 01520, 01521, 01522,
01523, 01523, 01524 and 01525.
[0491] One or more of the surface proteins typically include an
LPXTG motif (such as LPXTG (SEQ ID NO: 122)) or other sortase
substrate motif. The GBS AI-2 sortase proteins are thought to be
involved in the secretion and anchoring of the LPXTG containing
surface proteins. GBS AI-2 may encode for at least one surface
protein. Alternatively, AI-2 may encode for at least two surface
proteins and at least one sortase. Preferably, GBS AI-2 encodes for
at least three surface proteins and at least two sortases. One or
more of the AI-2 surface proteins may include an LPXTG or other
sortase substrate motif.
[0492] One or more of the surface proteins may also typically
include pilin motif. The pilin motif may be involved in pili
formation. Cleavage of AI surface proteins by sortase between the
threonine and glycine residue of an LPXTG motif yields a
thioester-linked acyl intermediate of sortase. The first lysine
residue in a pilin motif can serve as an amino group acceptor of
the cleaved LPXTG motif and thereby provide a covalent linkage
between AI subunits to form pili. For example, the pilin motif can
make a nucleophilic attack on the acyl enzyme providing a covalent
linkage between AI subunits to form pili and regenerate the sortase
enzyme. Some examples of pilin motifs that may be present in the
GBS AI-2 proteins include ((YPKN(X.sub.8)K; SEQ ID NO: 158),
(PK(X.sub.8)K; SEQ ID NO: 159), (YPK(X.sub.9)K; SEQ ID NO: 160),
(PKN(X.sub.8)K; SEQ ID NO: 161), or (PK(X.sub.10)K; SEQ ID NO:
162)).
[0493] One or more of the surface protein may also include an E box
motif. The E box motif contains a conserved glutamic acid residue
that is believed to be necessary for pilus formation. Some examples
of E box motifs may include the amino acid sequences YxLxETxAPxG
(SEQ ID NO: 163), YxxxExxAxxGY (SEQ ID NO: 164), YxLxExxxPxDY (SEQ
ID NO: 165), or YxLxETxAPxGY (SEQ ID NO: 152).
[0494] As shown in FIG. 3, GBS AI-2 may include the surface exposed
proteins of GBS 67, GBS 59 and GBS 150 and the sortases of SAG1406
and SAG1405. Alternatively, GBS AI-2 may include the proteins
01521, 01524 and 01525 and sortases 01520 and 01522. GBS 067 and
01524 are preferred AI-2 surface proteins.
[0495] AI-2 may also include a divergently transcribed
transcriptional regulator such as a RofA like protein (for example
rogB). As in AI-1, rogB is thought to regulate the expression of
the AI-2 operon.
[0496] A schematic depiction of AI-2 within several GBS serotypes
is depicted in FIG. 4. (Percentages shown are amino acid identity
to the 2603 sequence). While the AI-2 surface proteins GBS 59 and
GBS 67 are more variable across GBS serotypes than the
corresponding AI-1 surface proteins, AI-2 surface protein GBS 67
appears to be conserved in GBS serotypes where the AI-1 surface
proteins are disrupted or missing.
[0497] For example, as discussed above and in FIG. 2, the AI-1 GBS
80 surface protein is fragmented in GBS serotype II, strain isolate
18RS21. Within AI-2 for this same sequence, as shown in FIG. 4, the
GBS 67 surface protein has 99% amino acid sequence homology with
the corresponding sequence in strain isolate 2603. Similarly, the
AI-1 GBS 80 surface protein appears to be missing in GBS serotype
Ib, strain isolate H36B and GBS serotype Ia, strain isolate 515.
Within AI-2 for these sequences, however, the GBS 67 surface
protein has 97-99% amino acid sequence homology with the
corresponding sequence in strain isolate 2603. GBS 67 appears to
have two allelic variants, which can be divided according to
percent homology with strains 2603 and H36B. See FIGS. 237-239.
[0498] Unlike for GBS 67, amino acid sequence identity of GBS 59 is
variable across different GBS strains. As shown in FIGS. 63 and
224, GBS 59 of GBS strain isolate 2603 shares 100% amino acid
residue homology with GBS strain 18RS21, 62% amino acid sequence
homology with GBS strain H36B, 48% amino acid residue homology with
GBS strain 515 and GBS strain CJB111, and 47% amino acid residue
homology with GBS strain NEM316. The amino acid sequence homologies
of the different GBS strains suggest that there are two isoforms of
GBS 59. The first isoform appears to include the GBS 59 protein of
GBS strains CJB111, NEM316, and 515. The second isoform appears to
include the GBS 59 protein of GBS strains 18RS21, 2603, and H36B.
(See FIGS. 63 and 224.) As expected from the variability in GBS 59
isoforms, antibodies specific for the first GBS 59 isoform detect
the first but not the second GBS 59 isoform and antibodies specific
for the second GBS 59 isoform detect the second but not the first
GBS 59 isoform. See FIG. 226A, which shows FACS analysis of 28 GBS
strains having a GBS 59 gene detected using PCR for GBS 59 surface
expression. For each of the 28 GBS strains, FACS analysis was
performed using either an antibody for GBS 59 isoform 1
(.alpha.-Cjb111) or GBS 59 isoform 2 (.alpha.-2603). Only one of
the two antibodies detected GBS 59 surface expression on each GBS
strain. As a negative control, GBS strains in which a GBS 59 gene
was not detectable by PCR did not have significant GBS 59 surface
expression levels. FIG. 226B.
[0499] Also, GBS 59 is opsonic only against GBS strains expressing
a homologous GBS 59 protein. See FIG. 225.
[0500] In one embodiment, the immunogenic composition of the
invention comprises a first and a second isoform of the GBS 59
protein to provide protection across a wide range of GBS serotypes
that express polypeptides from a GBS AI-2. The first isoform may be
the GBS 59 protein of GBS strain CJB111, NEM316, or 515. The second
isoform may be the GBS 59 protein of GBS strain 18RS21, 2603, or
H36B.
[0501] The gene encoding GBS 59 has been identified in a high
number of GBS isolates; the GBS 59 gene was detected in 31 of 40
GBS isolates tested (77.5%). The GBS 59 protein also appears to be
present as part of a pilus in whole extracts derived from GBS
strains. FIG. 64 shows detection of high molecular weight GBS 59
polymers in whole extracts of GBS strains CJB111, 7357B, COH31,
D1363C, 5408, 1999, 5364, 5518, and 515 using antiserum raised
against GBS 59 of GBS strain CJB111. FIG. 65 also shows detection
of these high molecular weight GBS 59 polymers in whole extracts of
GBS strains D136C, 515, and CJB111 with anti-GBS 59 antiserum. (See
also FIG. 220 A for detection of GBS 59 high molecular weight
polymers in strain 515.) FIG. 65 confirms the presence of different
isoforms of GBS 59. Antisera raised against two different GBS 59
isoforms results in different patterns of immunoreactivity
depending on the GBS strain origin of the whole extract. FIG. 65
further shows detection of GBS 59 monomers in purified GBS 59
preparations.
[0502] GBS 59 is also highly expressed on the surface of GBS
strains. GBS 59 was detected on the surface of GBS strains CJB111,
DK1, DK8, Davis, 515, 2986, 5551, 1169, and 7357B by FACS analysis
using mouse antiserum raised against GBS 59 of GBS CJB111. FACS
analysis did not detect surface expression of GBS 59 in GBS strains
SMU071, JM9130013, and COH1, which do not contain a GBS 59 gene.
(See FIG. 66.) Further confirmation that GBS 59 is expressed on the
surface of GBS is detection of GBS 59 by immuno-electron microscopy
on the surface of GBS strain 515 bacteria. See FIG. 215.
[0503] GBS 67 and GBS 150 also appear to be included in high
molecular weight structures, or pili. FIG. 69 shows that anti-GBS
67 and anti-GBS 150 immunoreact with high molecular weight
structures in whole GBS strain 515 extracts. (See also FIG. 220 B
and C.) It is also notable in FIG. 69 that the anti-GBS 59
antisera, raised in a mouse following immunization with GBS 59 of
GBS strain 2603, does not cross-hybridize with GBS 59 in GBS strain
515. GBS 59 of GBS stain 515 is of a different isotype than GBS 59
of GBS stain 2603. See FIG. 63, which illustrates that the homology
of these two GBS 59 polypeptides is 48%, and FIG. 65, which
confirms that GBS 59 antisera raised against GBS strain 2603 does
not cross-hybridize with GBS 59 of GBS strain 515.
[0504] Formation of pili containing GBS 150 does not appear to
require GBS 67 expression. FIG. 70 provides Western blots showing
that higher molecular weight structures in GBS strain 515 total
extracts immunoreact with anti-GBS 67 and anti-GBS 150 antiserum.
In a GBS strain 515 lacking GBS 67 expression, anti-GBS 67
antiserum no longer immunoreacts with polypeptides in total
extracts, while anti-GBS 150 antiserum is still able to
cross-hybridze with high molecular weight structures.
[0505] Likewise, formation of pili containing GBS 59 does not
appear to require GBS 67 expression. As expected, FACS detects GBS
67 cell surface expression on wildtype GBS strain 515, but not GBS
strain 515 cells knocked out for GBS 67. FACS analysis using
anti-GBS 59 antisera, however, detects GBS 59 expression on both
the wildtype GBS strain 515 cells and the GBS strain 515 cells
knocked out for GBS 67. Thus, GBS 59 cell surface expression is
detected on GBS stain 515 cells regardless of GBS 67
expression.
[0506] GBS 67, while present in pili, appears to be localized
around the surface of GBS strain 515 cells. See the immuno-electron
micrographs presented in FIG. 216. GBS 67 binds to fibronectin. See
FIG. 217.
[0507] Formation of pili encoded by GBS AI-2 does require
expression of GBS 59. Deletion of GBS 59 from strain 515 bacteria
eliminates detection of high molecular weight structures by
antibodies that bind to GBS 59 (FIG. 221 A, lane 3), GBS 67 (FIG.
221 B, lane 3), and GBS 150 (FIG. 221 C, lane 3). By contrast,
Western blot analysis of 515 bacteria with a deletion of the GBS 67
gene detects high molecular weight structures using GBS 59 (FIG.
221 A, lane 2) and GBS 150 (FIG. 221 C, lane 2) antisera.
Similarly, Western blot analysis of 515 bacteria with a deletion of
the GBS 150 gene detects high molecular weight structures using GBS
59 (FIG. 221 A, lane 4) and GBS 67 (FIG. 221 B, lane 4). See also
FIG. 223, which provides Western blots of each of the 515 strains
interrogated with antibodies for GBS 59, GBS 67, and GBS 150. FACS
analysis of strain 515 bacteria deleted for either GBS 59 or GBS 67
confirms these results. See FIG. 222, which shows that only
deletion of GBS 59 abolishes surface expression of both GBS 59 and
GBS 67.
[0508] Formation of pili encoded by GBS AI-2 also requires
expression of both GBS adhesin island-2 encoded sortases. See FIG.
218, which provides Western blot analysis of strain 515 bacteria
lacking Srt1, Srt2, or both Srt1 and Srt2. Only deletion of both
Srt1 and Srt2 abolishes pilus assembly as detected by antibodies
that cross-hybridize with each of GBS 59, GBS 67 and GBS 150. The
results of the Western blot analysis were verified by FACS, which
provided similar results. See FIG. 219.
[0509] As shown in FIG. 4, two of the GBS strain isolates (COH1 and
A909) do not appear to contain homologues to the surface proteins
GBS 59 and GBS 67. For these two strains, the percentages shown in
FIG. 4 are amino acid identity to the COH1 protein).
Notwithstanding the difference in the surface protein lengths for
these two strains, AI-2 within these sequences still contains two
sortase proteins and three LPXTG containing surface proteins, as
well as a signal peptidase sequence leading into the first surface
protein. One of the surface proteins in this variant of AI-2, spb1,
has previously been identified as a potential adhesion protein.
(See Adderson et al., Infection and Immunity (2003)
71(12):6857-6863). Alternatively, because of the lack of GBS 59 and
GBS 67 sequences, this variant of AI-2 may be a third type of AI
(Adhesin Island-3, AI-3, or GBS AI-3).
[0510] More than one AI surface protein may be present in the
oligomeric, pilus-like structures of the invention. For example,
GBS 59 and GBS 67 may be incorporated into an oligomeric structure.
Alternatively, GBS 59 and GBS 150 may be incorporated into an
oligomeric structure, or GBS 59, GBS 150 and GBS 67 may be
incorporated into an oligomeric structure.
[0511] In another embodiment, the invention includes compositions
comprising two or more AI surface proteins. The composition may
include surface proteins from the same adhesin island. For example,
the composition may include two or more GBS AI-2 surface proteins,
such as GBS 59, GBS 67 and GBS 150. The surface proteins may be
isolated from Gram positve bacteria or they may be produced
recombinantly.
GAS Adhesin Islands
[0512] As discussed above, Applicants have identified at least four
different GAS Adhesin Islands. These adhesion islands are thought
to encode surface proteins which are important in the bacteria's
virulence, and Applicants have obtained the first electron
micrographs revealing the presence of these adhesin island proteins
in hyperoligomeric pilus structures on the surface of Group A
Streptococcus.
[0513] Group A Streptococcus is a human specific pathogen which
causes a wide variety of diseases ranging from pharyngitis and
impetigo through life threatening invasive disease and necrotizing
fascilitis. In addition, post-streptococcal autoimmune responses
are still a major cause of cardiac pathology in children.
[0514] Group A Streptococcal infection of its human host can
generally occur in three phases. The first phase involves
attachment and/or invasion of the bacteria into host tissue and
multiplication of the bacteria within the extracellular spaces.
Generally this attachment phase begins in the throat or the skin.
The deeper the tissue level infected, the more severe the damage
that can be caused. In the second stage of infection, the bacteria
secretes a soluble toxin that diffuses into the surrounding tissue
or even systemically through the vasculature. This toxin binds to
susceptible host cell receptors and triggers innappropropriate
immune responses by these host cells, resulting in pathology.
Because the toxin can diffuse throughout the host, the necrosis
directly caused by the GAS toxins may be physically located in
sites distant from the bacterial infection. The final phase of GAS
infection can occur long after the original bacteria have been
cleared from the host system. At this stage, the host's previous
immune response to the GAS bacteria due to cross reactivity between
epitopes of a GAS surface protein, M, and host tissues, such as the
heart. A general review of GAS infection can be found in Principles
of Bacterial Pathogeneis, Groisman ed., Chapter 15 (2001).
[0515] In order to prevent the pathogenic effects associated with
the later stages of GAS infection, an effective vaccine against GAS
will preferably facilitate host elimination of the bacteria during
the initial attachment and invasion stage.
[0516] Isolates of Group A Streptococcus are historically
classified according to the M surface protein described above. The
M protein is surface exposed trypsin-sensitive protein generally
comprising two polypeptide chains complexed in an alpha helical
formation. The carboxyl terminus is anchored in the cytoplasmic
membrane and is highly conserved among all group A streptococci.
The amino terminus, which extend through the cell wall to the cell
surface, is responsible for the antigenic variability observed
among the 80 or more serotypes of M proteins.
[0517] A second layer of classification is based on a variable,
trypsin-resistant surface antigen, commonly referred to as the
T-antigen. Decades of epidemiology based on M and T serological
typing have been central to studies on the biological diversity and
disease causing potential of Group A Streptococci. While the
M-protein component and its inherent variability have been
extensively characterized, even after five decades of study, there
is still very little known about the structure and variability of
T-antigens. Antisera to define T types is commercially available
from several sources, including Sevapharma
(http://www.sevapharma.cz/en).
[0518] The gene coding for one form of T-antigen, T-type 6, from an
M6 strain of GAS (D741) has been cloned and characterized and maps
to an approximately 11 kb highly variable pathogenicity island.
Schneewind et al., J. Bacteriol. (1990) 172(6):3310-3317. This
island is known as the Fibronectin-binding, Collagen-binding
T-antigen (FCT) region because it contains, in addition to the T6
coding gene (tee6), members of a family of genes coding for Extra
Cellular Matrix (ECM) binding proteins. Bessen et al., Infection
& Immunity (2002) 70(3):1159-1167. Several of the protein
products of this gene family have been shown to directly bind
either fibronectin and/or collagen. See Hanski et al., Infection
& Immunity (1992) 60(12):5119-5125; Talay et al., Infection
& Immunity (1992(60(9):3837-3844; Jaffe et al. (1996)
21(2):373-384; Rocha et al., Adv Exp Med Biol. (1997) 418:737-739;
Kreikemeyer et al., J Biol Chem (2004) 279(16):15850-15859;
Podbielski et al., Mol. Microbiol. (1999) 31(4):1051-64; and
Kreikemeyer et al., Int. J. Med Microbiol (2004) 294(2-3):177-88.
In some cases direct evidence for a role of these proteins in
adhesion and invasion has been obtained.
[0519] Applicants raised antiserum against a recombinant product of
the tee6 gene and used it to explore the expression of T6 in M6
strain 2724. In immunoblot of mutanolysin extracts of this strain,
the antiserum recognized, in addition to a band corresponding to
the predicted molecular mass of the product, very high molecular
weight ladders ranging in mobility from about 100 kDa to beyond the
resolution of the 3-8% gradient gels used.
[0520] This pattern of high molecular weight products is similar to
that observed in immunoblots of the protein components of the pili
identified in Streptococcus agalactiae (described above) and
previously in Corynebacterium diphtheriae. Electron microscropy of
strain M6.sub.--2724 with antisera specific for the product of tee6
revealed abundant surface staining and long pilus like structures
extending up to 700 nanometers from the bacterial surface,
revealing that the T6 protein, one of the antigens recognized in
the original Lancefiled serotyping system, is located within a GAS
Adhesin Island (GAS AI-1) and forms long covalently linked pilus
structures.
[0521] Applicants have identified at least four different Group A
Streptococcus Adhesin Islands. While these GAS AI sequences can be
identified in numerous M types, Applicants have surprisingly
discovered a correlation between the four main pilus subunits from
the four different GAS AI types and specific T classifications.
While other trypsin-resistant surface exposed proteins are likely
also implicated in the T classification designations, the discovery
of the role of the GAS adhesin islands (and the associated
hyper-oligomeric pilus like structures) in T classification and GAS
serotype variance has important implications for prevention and
treatment of GAS infections. Applicants have identified protein
components within each of the GAS adhesin islands which are
associated with the pilus formation. These proteins are believed to
be involved in the bacteria's initial adherence mechanisms.
Immunological recognition of these proteins may allow the host
immune response to slow or prevent the bacteria's transition into
the more pathogenic later stages of infection.
[0522] In addition, Applicants have discovered that the GBS pili
structures appear to be implicated in the formation of biofilms
(populations of bacteria growing on a surface, often enclosed in an
exopolysaccharide matrix). Biofilms are generally associated with
bacterial resistance, as antibiotic treatments and host immune
response are frequently unable to erradicate all of the bacteria
components of the biofilm. Direction of a host immune response
against surface proteins exposed during the first steps of
bacterial attachment (i.e., before complete biofilm formation) is
preferable.
[0523] The invention therefore provides for improved immunogenic
compositions against GAS infection which may target GAS bacteria
during their initial attachment efforts to the host epithelial
cells and may provide protection against a wide range of GAS
serotypes. The immunogenic compositions of the invention include
GAS AI surface proteins which may be formulated in an oligomeric,
or hyperoligomeric (pilus) form. The invention also includes
combinations of GAS AI surface proteins. Combinations of GAS AI
surface proteins may be selected from the same adhesin island or
they may be selected from different GAS adhesin islands.
[0524] While there is surprising variability in the number and
sequence of the GAS AI components across isolates, GAS AI sequences
may be generally characterized as Type 1, Type 2, Type 3, and Type
4, depending on the number and type of sortase sequence within the
island and the percentage identity of other proteins within the
island. Schematics of the GAS adhesin islands are set forth in FIG.
51A and FIG. 162. In all strains identified so far, the adhesin
island region is flanked by highly conserved open reading frames
M1.sub.--123 and M1.sub.--136. Between three and five genes in each
GAS adhesin island code for ECM binding adhesin proteins containing
LPXTG motifs.
GAS Adhesin Island 1
[0525] As discussed above, Applicants have identified adhesin
islands, "GAS Adhesin Island 1" or "GAS AI-1", within the genome
Group A Streptococcus serotypes and isolates. GAS AI-1 comprises a
series of approximately five open reading frames encoding for a
collection of amino acid sequences comprising surface proteins and
sortases ("GAS AI-1 proteins"). GAS AI-1 preferably comprises
surface proteins, a srtB sortase, and a rofA divergently
transcribed transcriptional regulator. GAS AI-1 surface proteins
may include a fibronectin binding protein, a collagen adhesion
protein and a fimbrial structural subunit. Preferably, each of
these GAS AI-1 surface proteins includes an LPXTG sortase substrate
motif, such as LPXTG (SEQ ID NO: 122) or LPXSG (SEQ ID NO: 134)
(conservative replacement of threonine with serine). Specifically,
GAS AI-1 includes open reading frames encoding for two or more
(i.e., 2, 3, 4 or 5) of M6_Spy0157, M6_Spy0158, M6_Spy0159,
M6_Spy0160, M6_Spy0161.
[0526] Applicants have also identified open reading frames encoding
fimbrial structural subunits in other GAS bacteria harbouring an
AI-1. These open reading frames encode fimbrial structural subunits
CDC SS 410_fimbrial, ISS3650_fimbrial, and DSM2071_fimbrial. A GAS
AI-1 may comprise a polynucleotide encoding any one of CDC SS
410_fimbrial, ISS3650_fimbrial, and DSM2071_fimbrial.
[0527] As discussed above, the hyper-oligomeric pilus structure of
GAS AI-1 appears to be responsible for the T-antigen type 6
classification, and GAS AI-1 corresponds to the FCT region
previously identified for tee6. As in GAS AI-1, the tee6 FCT region
includes open reading frames encoding for a collagen adhesion
protein (cpa, capsular polysaccharide adhesion) and a fibronectin
binding protein (prtF1). Immunoblots of tee6, a GAS AI-1 fimbrial
structural subunit corresponding to M6_Spy160, reveal high
molecular weight structures indicative of the hyper-oligomeric
pilus structures. Immunoblots with antiserum specific for Cpa also
recognize a high molecular weight ladder structure, indicating Cpa
involvement in the GAS AI-1 pilus structure or formation. In EM
photos of GAS bacteria, Cpa antiserum reveals abundant staining on
the surface of the bacteria and occasional gold particles extended
from the surface of the bacteria. In contrast, immunoblots with
antiserum specific for PrtF1 recognize only a single molecular
species with electrophoretic mobility corresponding to its
predicted molecular mass, indicating that PrtF1 may not be
associated with the oligomeric pilus structure. A preferred
immunogenic composition of the invention comprises a GAS AI-1
surface protein which may be formulated or purified in an
oligomeric (pilis) form. In a preferred embodiment, the oligomeric
form is a hyperoligomer. Another preferred immunogenic composition
of the invention comprises a GAS AI-1 surface protein which has
been isolated in an oligomeric (pilis) form. The oligomer or
hyperoligomeric pilus structures comprising the GAS AI-1 surface
proteins may be purified or otherwise formulate for use in
immunogenic compositions.
[0528] One or more of the GAS AI-1 open reading frame
polynucleotide sequences may be replaced by a polynucleotide
sequence coding for a fragment of the replaced ORF. Alternatively,
one or more of the GAS AI-1 open reading frames may be replaced by
a sequence having sequence homology to the replaced ORF.
[0529] One or more of the GAS AI-1 surface protein sequences
typically include an LPXTG motif (such as LPXTG (SEQ ID NO: 122))
or other sortase substrate motif.
[0530] The LPXTG sortase substrate motif of a GAS AI surface
protein may be generally represented by the formula XXXXG, wherein
X at amino acid position 1 is an L, a V, an E, or a Q, wherein X at
amino acid position 2 is a P if X at amino acid position 1 is an L,
wherein X at amino acid position 2 is a V if X at amino acid
position 1 is a E or a Q, wherein X at amino acid position 2 is a V
or a P if X at amino acid position 1 is a V, wherein X at amino
acid position 3 is any amino acid residue, wherein X at amino acid
position 4 is a T if X at amino acid position 1 is a V, E, or Q,
and wherein X at amino acid position 4 is a T, S, or A if X at
amino acid position 1 is an L. Some examples of LPXTG motifs
present in GAS AI surface proteins include LPSXG (SEQ ID NO: 134),
VVXTG (SEQ ID NO: 135), EVXTG (SEQ ID NO: 136), VPXTG (SEQ ID NO:
137), QVXTG (SEQ ID NO: 138), LPXAG (SEQ ID NO: 139), QVPTG (SEQ ID
NO: 140), and FPXTG (SEQ ID NO: 141).
[0531] The GAS AI surface proteins of the invention may affect the
ability of the GAS bacteria to adhere to and invade epithelial
cells. AI surface proteins may also affect the ability of GAS to
translocate through an epithelial cell layer. Preferably, one or
more GAS AI surface proteins are capable of binding to or otherwise
associating with an epithelial cell surface. GAS AI surface
proteins may also be able to bind to or associate with fibrinogen,
fibronectin, or collagen.
[0532] The GAS AI-1 sortase proteins are predicted to be involved
in the secretion and anchoring of the LPXTG containing surface
proteins. GAS AI-1 may encode for at least one surface protein.
Alternatively, GAS AI-1 may encode for at least two surface exposed
proteins and at least one sortase. Preferably, GAS AI-1 encodes for
at least three surface exposed proteins and at least two
sortases.
[0533] The AI surface proteins may be covalently attached to the
bacterial cell wall by membrane-associated transpeptidases, such as
an AI sortase. The sortase may function to cleave the surface
protein, preferably between the threonine and glycine residues of
an LPXTG motif. The sortase may then assist in the formation of an
amide link between the threonine carboxyl group and a cell wall
precursor such as lipid II. The precursor can then be incorporated
into the peptidoglycan via the transglycoslylation and
transpeptidation reactions of bacterial wall synthesis. See Comfort
et al., Infection & Immunity (2004) 72(5): 2710-2722.
[0534] GAS AI-1 preferably includes a srtB sortase. GAS srtB
sortases may preferably anchor surface proteins with an LPSTG motif
(SEQ ID NO: 166), particularly where the motif is followed by a
serine.
[0535] In one embodiment, the invention includes a composition
comprising oligomeric, pilus-like structures comprising a GAS AI-1
surface protein such as M6_Spy0157, M6_Spy0159, M6_Spy0160, CDC SS
410_fimbrial, ISS3650_fimbrial, or DSM2071_fimbrial. The
oligomeric, pilus-like structure may comprise numerous units of AI
surface protein. Preferably, the oligomeric, pilus-like structures
comprise two or more AI surface proteins. Still more preferably,
the oligomeric, pilus-like structure comprises a hyper-oligomeric
pilus-like structure comprising at least two (e.g., 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60,
70, 80, 90, 100, 120, 140, 150, 200 or more) oligomeric subunits,
wherein each subunit comprises an AI surface protein or a fragment
thereof. The oligomeric subunits may be covalently associated via a
conserved lysine within a pilin motif. The oligomeric subunits may
be covalently associated via an LPXTG motif, preferably, via the
threonine or serine amino acid residue, respectively:
[0536] AI surface proteins or fragments thereof to be incorporated
into the oligomeric, pilus-like structures of the invention will
preferably include a pilin motif.
[0537] The oligomeric, pilus like structures may be used alone or
in the combinations of the invention. In one embodiment, the
invention comprises a GAS Adhesin Island protein in oligomeric
form, preferably in a hyperoligomeric form. In one embodiment, the
invention comprises a composition comprising one or more GAS
Adhesin Island 1 ("GAS AI-1") proteins and one or more GAS Adhesin
Island 2 ("GAS AI-2"), GAS Adhesin Island 3 ("GAS AI-3"), or GAS
Adhesin Island 4 ("GAS AI-4") proteins, wherein one or more of the
GAS Adhesin Island proteins is in the form of an oligomer,
preferably in a hyperoligomeric form.
[0538] In addition to the open reading frames encoding the GAS AI-1
proteins, GAS AI-1 may also include a divergently transcribed
transcriptional regulator such as RofA (i.e., the transcriptional
regulator is located near or adjacent to the AI protein open
reading frames, but it transcribed in the opposite direction).
GAS Adhesin Island 2
[0539] A second adhesin island, "GAS Adhesin Island 2" or "GAS
AI-2" has also been identified in Group A Streptococcus serotypes
and isolates. GAS AI-2 comprises a series of approximately eight
open reading frames encoding for a collection of amino acid
sequences comprising surface proteins and sortases ("GAS AI-2
proteins"). Specifically, GAS AI-2 includes open reading frames
encoding for two or more (i.e., 2, 3, 4, 5, 6, 7, or 8) of GAS15,
Spy0127, GAS16, GAS17, GAS18, Spy0131, Spy0133, and GAS20.
[0540] A preferred immunogenic composition of the invention
comprises a GAS AI-2 surface protein which may be formulated or
purified in an oligomeric (pilis) form. In a preferred embodiment,
the oligomeric form is a hyperoligomer. Another preferred
immunogenic composition of the invention comprises a GAS AI-2
surface protein which has been isolated in an oligomeric (pilis)
form. The oligomer or hyperoligomeric pilus structures comprising
the GAS AI-2 surface proteins may be purified or otherwise
formulate for use in immunogenic compositions.
[0541] One or more of the GAS AI-2 open reading frame
polynucleotide sequences may be replaced by a polynucleotide
sequence coding for a fragment of the replaced ORF. Alternatively,
one or more of the GAS AI-2 open reading frames may be replaced by
a sequence having sequence homology to the replaced ORF.
[0542] One or more of the GAS AI-2 surface protein sequences
typically include an LPXTG motif (such as LPXTG (SEQ ID NO: 122))
or other sortase substrate motif. The AI surface proteins of the
invention may affect the ability of the GAS bacteria to adhere to
and invade epithelial cells. AI surface proteins may also affect
the ability of GAS to translocate through an epithelial cell layer.
Preferably, one or more AI surface proteins are capable of binding
to or otherwise associating with an epithelial cell surface. AI
surface proteins may also be able to bind to or associate with
fibrinogen, fibronectin, or collagen.
[0543] The GAS AI-2 sortase proteins are predicted to be involved
in the secretion and anchoring of the LPXTG containing surface
proteins. GAS AI-2 may encode for at least one surface protein.
Alternatively, GAS AI-2 may encode for at least two surface exposed
proteins and at least one sortase. Preferably, GAS AI-2 encodes for
at least three surface exposed proteins and at least two
sortases.
[0544] The AI surface proteins may be covalently attached to the
bacterial cell wall by membrane-associated transpeptidases, such as
an AI sortase. The sortase may function to cleave the surface
protein, preferably between the threonine and glycine residues of
an LPXTG motif. The sortase may then assist in the formation of an
amide link between the threonine carboxyl group and a cell wall
precursor such as lipid II. The precursor can then be incorporated
into the peptidoglycan via the transglycoslylation and
transpeptidation reactions of bacterial wall synthesis. See Comfort
et al., Infection & Immunity (2004) 72(5): 2710-2722.
[0545] In one embodiment, the invention includes a composition
comprising oligomeric, pilus-like structures comprising an AI
surface protein such as GAS 15, GAS 16, or GAS 18. The oligomeric,
pilus-like structure may comprise numerous units of AI surface
protein. Preferably, the oligomeric, pilus-like structures comprise
two or more AI surface proteins. Still more preferably, the
oligomeric, pilus-like structure comprises a hyper-oligomeric
pilus-like structure comprising at least two (e.g., 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60,
70, 80, 90, 100, 120, 140, 150, 200 or more) oligomeric subunits,
wherein each subunit comprises an AI surface protein or a fragment
thereof. The oligomeric subunits may be covalently associated via a
conserved lysine within a pilin motif. The oligomeric subunits may
be covalently associated via an LPXTG motif, preferably, via the
threonine amino acid residue.
[0546] AI surface proteins or fragments thereof to be incorporated
into the oligomeric, pilus-like structures of the invention will
preferably include a pilin motif.
[0547] The oligomeric, pilus like structures may be used alone or
in the combinations of the invention. In one embodiment, the
invention comprises a GAS Adhesin Island protein in oligomeric
form, preferably in a hyperoligomeric form. In one embodiment, the
invention comprises a composition comprising one or more GAS
Adhesin Island 2 ("GAS AI-2") proteins and one or more GAS Adhesin
Island 1 ("GAS AI-1"), GAS Adhesin Island 3 ("GAS AI-3"), or GAS
Adhesin Island 4 ("GAS AI-4") proteins, wherein one or more of the
Adhesin Island proteins is in the form of an oligomer, preferably
in a hyperoligomeric form.
[0548] In addition to the open reading frames encoding the GAS AI-2
proteins, GAS AI-2 may also include a divergently transcribed
transcriptional regulator such as rofA (i.e., the transcriptional
regulator is located near or adjacent to the AI protein open
reading frames, but it transcribed in the opposite direction).
GAS Adhesin Island 3
[0549] A third adhesin island, "GAS Adhesin Island 3" or "GAS AI-3"
has also been identified in several Group A Streptococcus serotypes
and isolates. GAS AI-3 comprises a series of approximately seven
open reading frames encoding for a collection of amino acid
sequences comprising surface proteins and sortases ("GAS AI-3
proteins"). Specifically, GAS AI-3 includes open reading frames
encoding for two or more (i.e., 2, 3, 4, 5, 6, or 7) of
SpyM3.sub.--0098, SpyM3.sub.--0099, SpyM3.sub.--0100,
SpyM3.sub.--0101, SpyM3.sub.--0102, SpyM3.sub.--0103,
SpyM3.sub.--0104, SPs0100, SPs0101, SPs0102, SPs0103, SPs0104,
SPs0105, SPs0106, orf78, orf79, orf80, orf81, orf82, orf83, orf84,
spyM18.sub.--0126, spyM18.sub.--0127, spyM18.sub.--0128,
spyM18.sub.--0129, spyM18.sub.--0130, spyM18.sub.--0131,
spyM18.sub.--0132, SpyoM01000156, SpyoM01000155, SpyoM01000154,
SpyoM01000153, SpyoM01000152, SpyoM01000151, SpyoM01000150, and
SpyoM01000149. In one embodiment, GAS AI-3 includes open reading
frames encoding for two or more (i.e., 2, 3, 4, 5, 6, or 7) of
SpyM3.sub.--0098, SpyM3.sub.--0099, SpyM3.sub.--0100,
SpyM3.sub.--0101, SpyM3.sub.--0102, SpyM3.sub.--0103, and
SpyM3.sub.--0104. In another embodiment, GAS AI-3 includes open
reading frames encoding for two or more (i.e., 2, 3, 4, 5, 6, or 7)
of SPs0100, SPs0101, SPs0102, SPs0103, SPs0104, SPs0105, and
SPs0106. In a further embodiment, GAS AI-3 includes open reading
frames encoding for two or more (i.e., 2, 3, 4, 5, 6, or 7) of
orf78, orf79, orf80, orf81, orf82, orf83, and orf84. In yet another
embodiment, GAS AI-3 includes open reading frames encoding for two
or more (i.e., 2, 3, 4, 5, 6, or 7) of spyM18.sub.--0126,
spyM18.sub.--0127, spyM18.sub.--0128, spyM18.sub.--0129,
spyM18.sub.--0130, spyM18.sub.--0131, and spyM18.sub.--0132. In yet
another embodiment, GAS AI-3 includes open reading frames encoding
for two or more (i.e., 2, 3, 4, 5, 6, or 7) of SpyoM01000156,
SpyoM01000155, SpyoM01000154, SpyoM01000153, SpyoM01000152,
SpyoM01000151, SpyoM01000150, and SpyoM01000149.
[0550] Applicants have also identified open reading frames encoding
fimbrial structural subunits in other GAS bacteria harbouring an
AI-3. These open reading frames encode fimbrial structural subunits
ISS3040_fimbrial, ISS3776_fimbrial, and ISS4959_fimbrial. A GAS
AI-3 may comprise a polynucleotide encoding any one of
ISS3040_fimbrial, ISS3776_fimbrial, and ISS4959_fimbrial.
[0551] One or more of the GAS AI-3 open reading frame
polynucleotide sequences may be replaced by a polynucleotide
sequence coding for a fragment of the replaced ORF. Alternatively,
one or more of the GAS AI-3 open reading frames may be replaced by
a sequence having sequence homology to the replaced ORF.
[0552] A preferred immunogenic composition of the invention
comprises a GAS AI-3 surface protein which may be formulated or
purified in an oligomeric (pilis) form. In a preferred embodiment,
the oligomeric form is a hyperoligomer. Another preferred
immunogenic composition of the invention comprises a GAS AI-3
surface protein which has been isolated in an oligomeric (pilis)
form. The oligomer or hyperoligomeric pilus structures comprising
the GAS AI-3 surface proteins may be purified or otherwise
formulate for use in immunogenic compositions.
[0553] One or more of the GAS AI-3 surface protein sequences
typically include an LPXTG motif (such as LPXTG (SEQ ID NO: 122))
or other sortase substrate motif. The AI surface proteins of the
invention may affect the ability of the GAS bacteria to adhere to
and invade epithelial cells. AI surface proteins may also affect
the ability of GAS to translocate through an epithelial cell layer.
Preferably, one or more AI surface proteins are capable of binding
to or otherwise associating with an epithelial cell surface. AI
surface proteins may also be able to bind to or associate with
fibrinogen, fibronectin, or collagen.
[0554] The GAS AI-3 sortase proteins are predicted to be involved
in the secretion and anchoring of the LPXTG containing surface
proteins. GAS AI-3 may encode for at least one surface protein.
Alternatively, GAS AI-3 may encode for at least two surface exposed
proteins and at least one sortase. Preferably, GAS AI-3 encodes for
at least three surface exposed proteins and at least two
sortases.
[0555] The AI surface proteins may be covalently attached to the
bacterial cell wall by membrane-associated transpeptidases, such as
an AI sortase. The sortase may function to cleave the surface
protein, preferably between the threonine and glycine residues of
an LPXTG motif. The sortase may then assist in the formation of an
amide link between the threonine or alanine carboxyl group and a
cell wall precursor such as lipid II. The precursor can then be
incorporated into the peptidoglycan via the transglycoslylation and
transpeptidation reactions of bacterial wall synthesis. See Comfort
et al., Infection & Immunity (2004) 72(5): 2710-2722.
[0556] The invention includes a composition comprising oligomeric,
pilus-like structures comprising an AI surface protein such as
SpyM3.sub.--0098, SpyM3.sub.--0100, SpyM3.sub.--0102,
SpyM3.sub.--0104, SPs0100, SPs0102, SPs0104, SPs0106, orf78, orf80,
orf82, orf84, spyM18.sub.--0126, spyM18.sub.--0128,
spyM18.sub.--0130, spyM18.sub.--0132, SpyoM01000155, SpyoM01000153,
SpyoM01000151, SpyoM01000149, ISS3040_fimbrial, ISS3776_fimbrial,
and ISS4959_fimbrial. In one embodiment, the invention includes a
composition comprising oligomeric, pilus-like structures comprising
an AI surface protein such as SpyM3.sub.--0098, SpyM3.sub.--0100,
SpyM3.sub.--0102, and SpyM3.sub.--0104. In another embodiment, the
invention includes a composition comprising oligomeric, pilus-like
structures comprising an AI surface protein such as SPs0100,
SPs0102, SPs0104, and SPs0106. In another embodiment, the invention
includes a composition comprising oligomeric, pilus-like structures
comprising an AI surface protein such as orf78, orf80, orf82, and
orf84. In yet another embodiment, the invention includes a
composition comprising oligomeric, pilus-like structures comprising
an AI surface protein such as spyM18.sub.--0126, spyM18.sub.--0128,
spyM18.sub.--0130, and spyM18.sub.--0132. In a further embodiment,
the invention includes a composition comprising oligomeric,
pilus-like structures comprising an AI surface protein such as
SpyoM01000155, SpyoM01000153, SpyoM0000151, and SpyoM1000149. In
yet a further embodiment, the invention includes a composition
comprising oligomeric, pilus-like structures comprising an AI
surface protein such as ISS3040_fimbrial, ISS3776_fimbrial, and
ISS4959_fimbrial. The oligomeric, pilus-like structure may comprise
numerous units of AI surface protein. Preferably, the oligomeric,
pilus-like structures comprise two or more AI surface proteins.
Still more preferably, the oligomeric, pilus-like structure
comprises a hyper-oligomeric pilus-like structure comprising at
least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 150, 200
or more) oligomeric subunits, wherein each subunit comprises an AI
surface protein or a fragment thereof. The oligomeric subunits may
be covalently associated via a conserved lysine within a pilin
motif. The oligomeric subunits may be covalently associated via an
LPXTG motif, preferably, via the threonine amino acid residue.
[0557] AI surface proteins or fragments thereof to be incorporated
into the oligomeric, pilus-like structures of the invention will
preferably include a pilin motif.
[0558] The oligomeric, pilus like structures may be used alone or
in the combinations of the invention. In one embodiment, the
invention comprises a GAS Adhesin Island protein in oligomeric
form, preferably in a hyperoligomeric form. In one embodiment, the
invention comprises a composition comprising one or more GAS
Adhesin Island 3 ("GAS AI-3") proteins and one or more GAS Adhesin
Island 1 ("GAS AI-1"), GAS Adhesin Island 2 ("GAS AI-2"), or GAS
Adhesin Island 4 ("GAS AI-4") proteins, wherein one or more of the
Adhesin Island proteins is in the form of an oligomer, preferably
in a hyperoligomeric form.
[0559] In addition to the open reading frames encoding the GAS AI-3
proteins, GAS AI-3 may also include a transcriptional regulator
such as Nra.
GAS Adhesin Island 4
[0560] A fourth adhesin island, "GAS Adhesin Island 4" or "GAS
AI-4" has also been identified in Group A Streptococcus serotypes
and isolates. GAS AI-4 comprises a series of approximately eight
open reading frames encoding for a collection of amino acid
sequences comprising surface proteins and sortases ("GAS AI-4
proteins"). Specifically, GAS AI-4 includes open reading frames
encoding for two or more (i.e., 2, 3, 4, 5, 6, 7, or 8) of
19224134, 19224135, 19223136, 19223137, 19224138, 19224139,
19224140, and 19224141.
[0561] Applicants have also identified open reading frames encoding
fimbrial structural subunits in other GAS bacteria harbouring an
AI-4. These open reading frames encode fimbrial structural subunits
20010296_fimbrial, 20020069_fimbrial, CDC SS 635_fimbrial,
ISS4883_fimbrial, and ISS4538_fimbrial. A GAS AI-4 may comprise a
polynucleotide encoding any one of 20010296_fimbrial,
20020069_fimbrial, CDC SS 635_fimbrial, ISS4883_fimbrial, and
ISS4538_fimbrial.
[0562] One or more of the GAS AI-4 open reading frame
polynucleotide sequences may be replaced by a polynucleotide
sequence coding for a fragment of the replaced ORF. Alternatively,
one or more of the GAS AI-4 open reading frames may be replaced by
a sequence having sequence homology to the replaced ORF.
[0563] A preferred immunogenic composition of the invention
comprises a GAS AI-4 surface protein which may be formulated or
purified in an oligomeric (pilis) form. In a preferred embodiment,
the oligomeric form is a hyperoligomer. Another preferred
immunogenic composition of the invention comprises a GAS AI-4
surface protein which has been isolated in an oligomeric (pilis)
form. The oligomer or hyperoligomeric pilus structures comprising
the GAS AI-4 surface proteins may be purified or otherwise
formulate for use in immunogenic compositions.
[0564] One or more of the GAS AI-4 surface protein sequences
typically include an LPXTG motif (such as LPXTG (SEQ ID NO: 122))
or other sortase substrate motif. The AI surface proteins of the
invention may effect the ability of the GAS bacteria to adhere to
and invade epithelial cells. AI surface proteins may also affect
the ability of GAS to translocate through an epithelial cell layer.
Preferably, one or more AI surface proteins are capable of binding
to or otherwise associating with an epithelial cell surface. AI
surface proteins may also be able to bind to or associate with
fibrinogen, fibronectin, or collagen.
[0565] The GAS AI-4 sortase proteins are predicted to be involved
in the secretion and anchoring of the LPXTG containing surface
proteins. GAS AI-4 may encode for at least one surface protein.
Alternatively, GAS AI-4 may encode for at least two surface exposed
proteins and at least one sortase. Preferably, GAS AI-4 encodes for
at least three surface exposed proteins and at least two
sortases.
[0566] The AI surface proteins may be covalently attached to the
bacterial cell wall by membrane-associated transpeptidases, such as
an AI sortase. The sortase may function to cleave the surface
protein, preferably between the threonine and glycine residues of
an LPXTG motif. The sortase may then assist in the formation of an
amide link between the threonine carboxyl group and a cell wall
precursor such as lipid II. The precursor can then be incorporated
into the peptidoglycan via the transglycoslylation and
transpeptidation reactions of bacterial wall synthesis. See Comfort
et al., Infection & Immunity (2004) 72(5): 2710-2722.
[0567] In one embodiment, the invention includes a composition
comprising oligomeric, pilus-like structures comprising an AI
surface protein such as 19224134, 19224135, 19224137, 19224139,
19224141, 20010296_fimbrial, 20020069_fimbrial, CDC SS
635_fimbrial, ISS4883_fimbrial, and ISS4538_fimbrial. The
oligomeric, pilus-like structure may comprise numerous units of AI
surface protein. Preferably, the oligomeric, pilus-like structures
comprise two or more AI surface proteins. Still more preferably,
the oligomeric, pilus-like structure comprises a hyper-oligomeric
pilus-like structure comprising at least two (e.g., 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60,
70, 80, 90, 100, 120, 140, 150, 200 or more) oligomeric subunits,
wherein each subunit comprises an AI surface protein or a fragment
thereof. The oligomeric subunits may be covalently associated via a
conserved lysine within a pilin motif. The oligomeric subunits may
be covalently associated via an LPXTG motif, preferably, via the
threonine amino acid residue.
[0568] AI surface proteins or fragments thereof to be incorporated
into the oligomeric, pilus-like structures of the invention will
preferably include a pilin motif.
[0569] The oligomeric, pilus like structures may be used alone or
in the combinations of the invention. In one embodiment, the
invention comprises a GAS Adhesin Island protein in oligomeric
form, preferably in a hyperoligomeric form. In one embodiment, the
invention comprises a composition comprising one or more GAS
Adhesin Island 4 ("GAS AI-4") proteins and one or more GAS Adhesin
Island 1 ("GAS AI-1"), GAS Adhesin Island 2 ("GAS AI-2"), or GAS
Adhesin Island 3 ("GAS AI-3") proteins, wherein one or more of the
Adhesin Island proteins is in the form of an oligomer, preferably
in a hyperoligomeric form.
[0570] In addition to the open reading frames encoding the GAS AI-4
proteins, GAS AI-4 may also include a divergently transcribed
transcriptional regulator such as rofA (i.e., the transcriptional
regulator is located near or adjacent to the AI protein open
reading frames, but it transcribed in the opposite direction).
[0571] The oligomeric, pilus-like structures of the invention may
be combined with one or more additional GAS proteins. In one
embodiment, the oligomeric, pilus-like structures comprise one or
more AI surface proteins in combination with a second GAS
protein.
[0572] The oligomeric, pilus-like structures may be isolated or
purified from bacterial cultures in which the bacteria express an
AI surface protein. The invention therefore includes a method for
manufacturing an oligomeric AI surface antigen comprising culturing
a GAS bacterium that expresses the oligomeric AI protein and
isolating the expressed oligomeric AI protein from the GAS
bacteria. The AI protein may be collected from secretions into the
supernatant or it may be purified from the bacterial surface. The
method may further comprise purification of the expressed AI
protein. Preferably, the AI protein is in a hyperoligomeric
form.
[0573] The oligomeric, pilus-like structures may be isolated or
purified from bacterial cultures overexpressing an AI surface
protein. The invention therefore includes a method for
manufacturing an oligomeric Adhesin Island surface antigen
comprising culturing a GAS bacterium adapted for increased AI
protein expression and isolation of the expressed oligomeric
Adhesin Island protein from the GAS bacteria. The AI protein may be
collected from secretions into the supernatant or it may be
purified from the bacterial surface. The method may further
comprise purification of the expressed Adhesin Island protein.
Preferably, the Adhesin Island protein is in a hyperoligomeric
form.
[0574] The GAS bacteria are preferably adapted to increase AI
protein expression by at least two (e.g., 2, 3, 4, 5, 8, 10, 15,
20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150 or 200)
times wild type expression levels.
[0575] GAS bacteria may be adapted to increase AI protein
expression by any means known in the art, including methods of
increasing gene dosage and methods of gene upregulation. Such means
include, for example, transformation of the GAS bacteria with a
plasmid encoding the AI protein. The plasmid may include a strong
promoter or it may include multiple copies of the sequence encoding
the AI protein. Optionally, the sequence encoding the AI protein
within the GAS bacterial genome may be deleted. Alternatively, or
in addition, the promoter regulating the GAS Adhesin Island may be
modified to increase expression.
[0576] The invention further includes GAS bacteria which have been
adapted to produce increased levels of AI surface protein. In
particular, the invention includes GAS bacteria which have been
adapted to produce oligomeric or hyperoligomeric AI surface
protein. In one embodiment, the Gram positive bacteria of the
invention are inactivated or attenuated to permit in vivo delivery
of the whole bacteria, with the AI surface protein exposed on its
surface.
[0577] The invention further includes GAS bacteria which have been
adapted to have increased levels of expressed AI protein
incorporated in pili on their surface. The GAS bacteria may be
adapted to have increased exposure of oligomeric or hyperoligomeric
AI proteins on its surface by increasing expression levels of LepA
polypeptide, or an equivalent signal peptidase, in the GAS
bacteria. Applicants have shown that deletion of LepA in strain
SF370 bacteria, which harbour a GAS AI-2, abolishes surface
exposure of M and pili proteins on the GAS. Increased levels of
LepA expression in GAS are expected to result in increased exposure
of M and pili proteins on the surface of GAS. Increased expression
of LepA in GAS may be achieved by any means known in the art, such
as increasing gene dosage and methods of gene upregulation. The GAS
bacteria adapted to have increased levels of LepA expression may
additionally be adapted to express increased levels of at least one
pili protein.
[0578] Alternatively, the AI proteins of the invention may be
expressed on the surface of a non-pathogenic Gram positive
bacteria, such as Streptococus gordonii (See, e.g., Byrd et al.,
"Biological consequences of antigen and cytokine co-expression by
recombinant Streptococcus gordonii vaccine vectors", Vaccine (2002)
20:2197-2205) or Lactococcus lactis (See, e.g., Mannam et al.,
"Mucosal Vaccine Made from Live, Recombinant Lactococcus lactis
Protects Mice against Pharangeal Infection with Streptococcus
pyogenes" Infection and Immunity (2004) 72(6):3444-3450). As used
herein, non-pathogenic Gram positive bacteria refer to Gram
positive bacteria which are compatible with a human host subject
and are not associated with human pathogenisis. Preferably, the
non-pathogenic bacteria are modified to express the AI surface
protein in oligomeric, or hyper-oligomeric form. Sequences encoding
for an AI surface protein and, optionally, an AI sortase, may be
integrated into the non-pathogenic Gram positive bacterial genome
or inserted into a plasmid. The non-pathogenic Gram positive
bacteria may be inactivated or attenuated to facilitate in vivo
delivery of the whole bacteria, with the AI surface protein exposed
on its surface. Alternatively, the AI surface protein may be
isolated or purified from a bacterial culture of the non-pathogenic
Gram positive bacteria. For example, the AI surface protein may be
isolated from cell extracts or culture supernatants. Alternatively,
the AI surface protein may be isolated or purified from the surface
of the non-pathogenic Gram positive bacteria.
[0579] The non-pathogenic Gram positive bacteria may be used to
express any of the GAS Adhesin Island proteins described herein.
The non-pathogenic Gram positive bacteria are transformed to
express an Adhesin Island surface protein. Preferably, the
non-pathogenic Gram positive bacteria also express at least one
Adhesin Island sortase. The AI transformed non-pathogenic Gram
positive bacteria of the invention may be used to prevent or treat
infection with pathogenic GAS.
[0580] Applicants modified L. lactis to demonstrate that, like GBS
polypeptides, it can express GAS AI polypeptides. L. lactis was
transformed with pAM401 constructs encoding entire pili gene
clusters of AI-1, AI-2, and AI-4 adhesin islands. Briefly, the
pAM401 is a promoterless high-copy plasmid. The entire pili gene
clusters of an M6 (AI-1), M1 (AI-2), and M12 (AI-4) bacteria were
inserted into the pAM401 construct. The gene clusters were
transcribed under the control their own (M6, M1, or M12) promoter
or the GBS promoter that successfully initiated expression of the
GBS AI-1 adhesin islands in L. lactis, described above. FIG. 172
provides a schematic depiction of GAS M6 (AI-1), M1 (AI-2), and M12
(AI-4) adhesin islands and indicates the portions of the adhesin
island sequences inserted in the pAM401 construct.
[0581] Each of the L. lactis transformed with one of the M6, M1, or
M12 adhesin island gene clusters expressed high molecular weight
structures that were immunoreactive with antibodies that bind to
polypeptides present in their respective pili. FIGS. 173 A-C
provide results of Western blot analysis of surface
protein-enriched extracts of L. lactis transformed with M6 (FIG.
173 A), M1 (FIG. 173 B), or M12 (FIG. 173 C) adhesin island gene
clusters using antibodies that bind to the fimbrial structural
subunit encoded by each cluster. FIG. 173A at lanes 3 and 4 shows
detection of high molecular structures in L. lactis transformed
with an adhesin island pilus gene cluster from an M1 AI-2 using an
antibody that binds to fimbrial structural subunit Spy0128. FIG.
173B at lanes 3 and 4 shows detection of high molecular weight
structures in L. lactis transformed with an adhesin island pilus
gene cluster from an M12 AI-4 using an antibody that binds to
fimbrial structural subunit EftLSL.A. FIG. 173C at lane 3 shows
detection of high molecular weight structures in L. lactis
transformed with an adhesin island pilus gene cluster from an M6
AI-1 using an antibody that binds to fimbrial structural subunit
M6_Spy0160. In FIGS. 173 A-C, "p1" immediately following the
notation of AI subtype indicates that the promoter present in the
Adhesin Island is used to drive transcription of the adhesin island
gene cluster and "p2" indicates that the promoter was the GBS
promoter described above. Thus, it appears that L. lactis is
capable of expressing the fimbrial structural subunits encoded by
GAS adhesin islands in an oligomeric form.
[0582] Alternatively, the oligomeric, pilus-like structures may be
produced recombinantly. If produced in a recombinant host cell
system, the AI surface protein will preferably be expressed in
coordination with the expression of one or more of the AI sortases
of the invention. Such AI sortases will facilitate oligomeric or
hyperoligomeric formation of the AI surface protein subunits.
S. pneumoniae from TIGR4 Adhesin Island
[0583] As discussed above, Applicants have identified adhesin
islands within the genome of S. pneumoniae from TIGR4. The S.
pneumoniae from TIGR4 Adhesin Island comprises a series of
approximately seven open reading frames encoding for a collection
of amino acid sequences comprising surface proteins and sortases.
Specifically, the S. pneumoniae from TIGR4 AI proteins includes
open reading frames encoding for two or more (i.e., 2, 3, 4, 5, 6,
or 7) of SP0462, SP0463, SP0464, SP0465, SP0466, SP0467, and
SP0468.
[0584] A preferred immunogenic composition of the invention
comprises a S. pneumoniae from TIGR4 AI surface protein which may
be formulated or purified in an oligomeric (pilis) form. In a
preferred embodiment, the oligomeric form is a hyperoligomer.
Another preferred immunogenic composition of the invention
comprises a S. pneumoniae from TIGR4 AI surface protein which has
been isolated in an oligomeric (pilis) form. The oligomer or
hyperoligomer pilus structures comprising S. pneumoniae surface
proteins may be purified or otherwise formulated for use in
immunogenic compositions.
[0585] One or more of the S. pneumoniae from TIGR4 AI open reading
frame polynucleotide sequences may be replaced by a polynucleotide
sequence coding for a fragment of the replaced ORF. Alternatively,
one or more of the S. pneumoniae from TIGR4 AI open reading frames
may be replaced by a sequence having sequence homology to the
replaced ORF.
[0586] One or more of the S. pneumoniae from TIGR4 AI surface
protein sequences typically include an LPXTG motif (such as LPXTG
(SEQ ID NO: 122)) or other sortase substrate motif.
[0587] The S. pneumoniae from TIGR4 AI surface proteins of the
invention may affect the ability of the S. pneumoniae bacteria to
adhere to and invade epithelial cells. AI surface proteins may also
affect the ability of S. pneumoniae to translocate through an
epithelial cell layer. Preferably, one or more S. pneumoniae from
TIGR4 AI surface proteins are capable of binding to or otherwise
associating with an epithelial cell surface. S. pneumoniae from
TIGR4 AI surface proteins may also be able to bind to or associate
with fibrinogen, fibronectin, or collagen.
[0588] The S. pneumoniae from TIGR4 AI sortase proteins are
predicted to be involved in the secretion and anchoring of the
LPXTG containing surface proteins. S. pneumoniae from TIGR4 AI may
encode for at least one surface protein. Alternatively, S.
pneumoniae from TIGR4 AI may encode for at least two surface
exposed proteins and at least one sortase. Preferably, S.
pneumoniae from TIGR4 AI encodes for at least three surface exposed
proteins and at least two sortases.
[0589] The AI surface proteins may be covalently attached to the
bacterial cell wall by membrane-associated transpeptidases, such as
an AI sortase. The sortase may function to cleave the surface
protein, preferably between the threonine and glycine residues of
an LPXTG motif. The sortase may then assist in the formation of an
amide link between the threonine carboxyl group and a cell wall
precursor such as lipid II. The precursor can then be incorporated
into the peptidoglycan via the transglycoslylation and
transpeptidation reactions of bacterial wall synthesis. See Comfort
et al., Infection & Immunity (2004) 72(5): 2710-2722.
[0590] In one embodiment, the invention includes a composition
comprising oligomeric, pilus-like structures comprising a S.
pneumoniae from TIGR4 AI surface protein such as SP0462, SP0463,
SP0464, or SP0465. The oligomeric, pilus-like structure may
comprise numerous units of AI surface protein. Preferably, the
oligomeric, pilus-like structures comprise two or more AI surface
proteins. Still more preferably, the oligomeric, pilus-like
structure comprises a hyper-oligomeric pilus-like structure
comprising at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120,
140, 150, 200 or more) oligomeric subunits, wherein each subunit
comprises an AI surface protein or a fragment thereof. The
oligomeric subunits may be covalently associated via a conserved
lysine within a pilin motif. The oligomeric subunits may be
covalently associated via an LPXTG motif, preferably, via the
threonine or serine amino acid residue, respectively.
[0591] AI surface proteins or fragments thereof to be incorporated
into the oligomeric, pilus-like structures of the invention will
preferably include a pilin motif.
[0592] The oligomeric, pilus like structures may be used alone or
in the combinations of the invention. In one embodiment, the
invention comprises a S. pneumoniae from TIGR4 AI protein in
oligomeric form, preferably in a hyperoligomeric form. In one
embodiment, the invention comprises a composition comprising one or
more S. pneumoniae from TIGR4 AI proteins and one or more S.
pneumoniae strain 670 AI proteins, wherein one or more of the S.
pneumoniae AI proteins is in the form of an oligomer, preferably in
a hyperoligomeric form.
[0593] In addition to the open reading frames encoding the S.
pneumoniae from TIGR4 AI proteins, S. pneumoniae from TIGR4 AI may
also include a transcriptional regulator.
S. pneumoniae Strain 670 Adhesin Island
[0594] As discussed above, Applicants have identified adhesin
islands within the genome of S. pneumoniae strain 670. The S.
pneumoniae strain 670 Adhesin Island comprises a series of
approximately seven open reading frames encoding for a collection
of amino acid sequences comprising surface proteins and sortases.
Specifically, the S. pneumoniae strain 670 AI proteins includes
open reading frames encoding for two or more (i.e., 2, 3, 4, 5, 6,
or 7) of orf1.sub.--670, orf3.sub.--670, orf4.sub.--670,
orf5.sub.--670, orf6.sub.--670, orf7.sub.--670, orf8.sub.--670.
[0595] A preferred immunogenic composition of the invention
comprises a S. pneumoniae strain 670 AI surface protein which may
be formulated or purified in an oligomeric (pilis) form. Another
preferred immunogenic composition of the invention comprises a S.
pneumoniae strain 670 AI surface protein which has been isolated in
an oligomeric (pilis) form.
[0596] One or more of the S. pneumoniae strain 670 AI open reading
frame polynucleotide sequences may be replaced by a polynucleotide
sequence coding for a fragment of the replaced ORF. Alternatively,
one or more of the S. pneumoniae strain 670 AI open reading frames
may be replaced by a sequence having sequence homology to the
replaced ORF.
[0597] One or more of the S. pneumoniae strain 670 AI surface
protein sequences typically include an LPXTG motif (such as LPXTG
(SEQ ID NO: 122)) or other sortase substrate motif.
[0598] The S. pneumoniae strain 670 AI surface proteins of the
invention may affect the ability of the S. pneumoniae bacteria to
adhere to and invade epithelial cells. AI surface proteins may also
affect the ability of S. pneumoniae to translocate through an
epithelial cell layer. Preferably, one or more S. pneumoniae strain
670 AI surface proteins are capable of binding to or otherwise
associating with an epithelial cell surface. S. pneumoniae strain
670 AI surface proteins may also be able to bind to or associate
with fibrinogen, fibronectin, or collagen.
[0599] The S. pneumoniae strain 670 AI sortase proteins are
predicted to be involved in the secretion and anchoring of the
LPXTG containing surface proteins. S. pneumoniae strain 670 AI may
encode for at least one surface protein. Alternatively, S.
pneumoniae strain 670 AI may encode for at least two surface
exposed proteins and at least one sortase. Preferably, S.
pneumoniae strain 670 AI encodes for at least three surface exposed
proteins and at least two sortases.
[0600] The AI surface proteins may be covalently attached to the
bacterial cell wall by membrane-associated transpeptidases, such as
an AI sortase. The sortase may function to cleave the surface
protein, preferably between the threonine and glycine residues of
an LPXTG motif. The sortase may then assist in the formation of an
amide link between the threonine carboxyl group and a cell wall
precursor such as lipid II. The precursor can then be incorporated
into the peptidoglycan via the transglycoslylation and
transpeptidation reactions of bacterial wall synthesis. See Comfort
et al., Infection & Immunity (2004) 72(5): 2710-2722.
[0601] In one embodiment, the invention includes a composition
comprising oligomeric, pilus-like structures comprising a S.
pneumoniae strain 670 AI surface protein such as orf3.sub.--670,
orf4.sub.--670, or orf5.sub.--670. The oligomeric, pilus-like
structure may comprise numerous units of AI surface protein.
Preferably, the oligomeric, pilus-like structures comprise two or
more AI surface proteins. Still more preferably, the oligomeric,
pilus-like structure comprises a hyper-oligomeric pilus-like
structure comprising at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90,
100, 120, 140, 150, 200 or more) oligomeric subunits, wherein each
subunit comprises an AI surface protein or a fragment thereof. The
oligomeric subunits may be covalently associated via a conserved
lysine within a pilin motif. The oligomeric subunits may be
covalently associated via an LPXTG motif, preferably, via the
threonine or serine amino acid residue, respectively.
[0602] AI surface proteins or fragments thereof to be incorporated
into the oligomeric, pilus-like structures of the invention will
preferably include a pilin motif.
[0603] The oligomeric, pilus like structures may be used alone or
in the combinations of the invention. In one embodiment, the
invention comprises a S. pneumoniae strain 670 AI protein in
oligomeric form, preferably in a hyperoligomeric form. In one
embodiment, the invention comprises a composition comprising one or
more S. pneumoniae strain 670 AI proteins and one or more S.
pneumoniae from TIGR4 AI proteins, wherein one or more of the S.
pneumoniae AI proteins is in the form of an oligomer, preferably in
a hyperoligomeric form.
[0604] In addition to the open reading frames encoding the S.
pneumoniae strain 670 AI proteins, S. pneumoniae strain 670 AI may
also include a transcriptional regulator.
S. pneumoniae strain 14 CSR 10 Adhesin Island
[0605] As discussed above, Applicants have identified adhesin
islands within the genome of S. pneumoniae strain 14 CSR 10. The S.
pneumoniae strain 14 CSR 10 Adhesin Island comprises a series of
approximately seven open reading frames encoding for a collection
of amino acid sequences comprising surface proteins and sortases.
Specifically, the S. pneumoniae strain 14 CSR 10 AI proteins
includes open reading frames encoding for two or more (i.e., 2, 3,
4, 5, 6, or 7) of ORF2.sub.--14CSR, ORF3.sub.--14CSR,
ORF4.sub.--14CSR, ORF5.sub.--14CSR, ORF6.sub.--14CSR,
ORF7.sub.--14CSR, ORF8.sub.--14CSR.
[0606] A preferred immunogenic composition of the invention
comprises a S. pneumoniae strain 14 CSR 10 AI surface protein which
may be formulated or purified in an oligomeric (pilis) form.
Another preferred immunogenic composition of the invention
comprises a S. pneumoniae strain 14 CSR 10 AI surface protein which
has been isolated in an oligomeric (pilis) form.
[0607] One or more of the S. pneumoniae strain 14 CSR 10 AI open
reading frame polynucleotide sequences may be replaced by a
polynucleotide sequence coding for a fragment of the replaced ORF.
Alternatively, one or more of the S. pneumoniae strain 14 CSR 10 AI
open reading frames may be replaced by a sequence having sequence
homology to the replaced ORF.
[0608] One or more of the S. pneumoniae strain 14 CSR 10 AI surface
protein sequences typically include an LPXTG motif (such as LPXTG
(SEQ ID NO: 122)) or other sortase substrate motif.
[0609] The S. pneumoniae strain 14 CSR 10 AI surface proteins of
the invention may affect the ability of the S. pneumoniae bacteria
to adhere to and invade epithelial cells. AI surface proteins may
also affect the ability of S. pneumoniae to translocate through an
epithelial cell layer. Preferably, one or more S. pneumoniae strain
14 CSR 10 AI surface proteins are capable of binding to or
otherwise associating with an epithelial cell surface. S.
pneumoniae strain 14 CSR 10 AI surface proteins may also be able to
bind to or associate with fibrinogen, fibronectin, or collagen.
[0610] The S. pneumoniae strain 14 CSR 10 AI sortase proteins are
predicted to be involved in the secretion and anchoring of the
LPXTG containing surface proteins. S. pneumoniae strain 14 CSR 10
AI may encode for at least one surface protein. Alternatively, S.
pneumoniae strain 14 CSR 10 AI may encode for at least two surface
exposed proteins and at least one sortase. Preferably, S.
pneumoniae strain 14 CSR 10 AI encodes for at least three surface
exposed proteins and at least two sortases.
[0611] The AI surface proteins may be covalently attached to the
bacterial cell wall by membrane-associated transpeptidases, such as
an AI sortase. The sortase may function to cleave the surface
protein, preferably between the threonine and glycine residues of
an LPXTG motif. The sortase may then assist in the formation of an
amide link between the threonine carboxyl group and a cell wall
precursor such as lipid II. The precursor can then be incorporated
into the peptidoglycan via the transglycoslylation and
transpeptidation reactions of bacterial wall synthesis. See Comfort
et al., Infection & Immunity (2004) 72(5): 2710-2722.
[0612] In one embodiment, the invention includes a composition
comprising oligomeric, pilus-like structures comprising a S.
pneumoniae strain 14 CSR 10 AI surface protein such as orf3_CSR,
orf4_CSR, or orf5_CSR. The oligomeric, pilus-like structure may
comprise numerous units of AI surface protein. Preferably, the
oligomeric, pilus-like structures comprise two or more AI surface
proteins. Still more preferably, the oligomeric, pilus-like
structure comprises a hyper-oligomeric pilus-like structure
comprising at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120,
140, 150, 200 or more) oligomeric subunits, wherein each subunit
comprises an AI surface protein or a fragment thereof. The
oligomeric subunits may be covalently associated via a conserved
lysine within a pilin motif. The oligomeric subunits may be
covalently associated via an LPXTG motif, preferably, via the
threonine or serine amino acid residue, respectively.
[0613] AI surface proteins or fragments thereof to be incorporated
into the oligomeric, pilus-like structures of the invention will
preferably include a pilin motif.
[0614] The oligomeric, pilus like structures may be used alone or
in the combinations of the invention. In one embodiment, the
invention comprises a S. pneumoniae strain 14 CSR 10 AI protein in
oligomeric form, preferably in a hyperoligomeric form. In one
embodiment, the invention comprises a composition comprising one or
more S. pneumoniae strain 14 CSR 10 AI proteins, and one or more AI
proteins of any of S. pneumoniae from TIGR4, 670, 19A Hungary 6, 6B
Finland 12, 6B Spain 2, 9V Spain 3, 19F Taiwan 14, 23F Taiwan 15,
or 23F Poland 16, wherein one or more of the S. pneumoniae AI
proteins is in the form of an oligomer, preferably in a
hyperoligomeric form.
[0615] In addition to the open reading frames encoding the S.
pneumoniae strain 14 CSR 10 AI proteins, S. pneumoniae strain 14
CSR 10 AI may also include a transcriptional regulator.
S. pneumoniae Strain 19A Hungary 6 Adhesin Island
[0616] As discussed above, Applicants have identified adhesin
islands within the genome of S. pneumoniae strain 19A Hungary 6.
The S. pneumoniae strain 19A Hungary 6 Adhesin Island comprises a
series of approximately seven open reading frames encoding for a
collection of amino acid sequences comprising surface proteins and
sortases. Specifically, the S. pneumoniae strain 19A Hungary 6 AI
proteins includes open reading frames encoding for two or more
(i.e., 2, 3, 4, 5, 6, or 7) of ORF2.sub.--19AH, ORF3.sub.--19AH,
ORF4.sub.--19AH, ORF5.sub.--1 gAH, ORF6.sub.--1 gAH,
ORF7.sub.--19AH, ORF8.sub.--19AH.
[0617] A preferred immunogenic composition of the invention
comprises a S. pneumoniae strain 19A Hungary 6 AI surface protein
which may be formulated or purified in an oligomeric (pilis) form.
Another preferred immunogenic composition of the invention
comprises a S. pneumoniae strain 19A Hungary 6 AI surface protein
which has been isolated in an oligomeric (pilis) form.
[0618] One or more of the S. pneumoniae strain 19A Hungary 6 AI
open reading frame polynucleotide sequences may be replaced by a
polynucleotide sequence coding for a fragment of the replaced ORF.
Alternatively, one or more of the S. pneumoniae strain 19A Hungary
6 AI open reading frames may be replaced by a sequence having
sequence homology to the replaced ORF.
[0619] One or more of the S. pneumoniae strain 19A Hungary 6 AI
surface protein sequences typically include an LPXTG motif (such as
LPXTG (SEQ ID NO: 122)) or other sortase substrate motif.
[0620] The S. pneumoniae strain 19A Hungary 6 AI surface proteins
of the invention may affect the ability of the S. pneumoniae
bacteria to adhere to and invade epithelial cells. AI surface
proteins may also affect the ability of S. pneumoniae to
translocate through an epithelial cell layer. Preferably, one or
more S. pneumoniae strain 19A Hungary 6 AI surface proteins are
capable of binding to or otherwise associating with an epithelial
cell surface. S. pneumoniae strain 19A Hungary 6 AI surface
proteins may also be able to bind to or associate with fibrinogen,
fibronectin, or collagen.
[0621] The S. pneumoniae strain 19A Hungary 6 AI sortase proteins
are predicted to be involved in the secretion and anchoring of the
LPXTG containing surface proteins. S. pneumoniae strain 19A Hungary
6 AI may encode for at least one surface protein. Alternatively, S.
pneumoniae strain 19A Hungary 6 AI may encode for at least two
surface exposed proteins and at least one sortase. Preferably, S.
pneumoniae strain 19A Hungary 6 AI encodes for at least three
surface exposed proteins and at least two sortases.
[0622] The AI surface proteins may be covalently attached to the
bacterial cell wall by membrane-associated transpeptidases, such as
an AI sortase. The sortase may function to cleave the surface
protein, preferably between the threonine and glycine residues of
an LPXTG motif. The sortase may then assist in the formation of an
amide link between the threonine carboxyl group and a cell wall
precursor such as lipid II. The precursor can then be incorporated
into the peptidoglycan via the transglycoslylation and
transpeptidation reactions of bacterial wall synthesis. See Comfort
et al., Infection & Immunity (2004) 72(5): 2710-2722.
[0623] In one embodiment, the invention includes a composition
comprising oligomeric, pilus-like structures comprising a S.
pneumoniae strain 19A Hungary 6 AI surface protein such as
orf3.sub.--19AH, orf4.sub.--19AH, or orf5.sub.--19AH. The
oligomeric, pilus-like structure may comprise numerous units of AI
surface protein. Preferably, the oligomeric, pilus-like structures
comprise two or more AI surface proteins. Still more preferably,
the oligomeric, pilus-like structure comprises a hyper-oligomeric
pilus-like structure comprising at least two (e.g., 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60,
70, 80, 90, 100, 120, 140, 150, 200 or more) oligomeric subunits,
wherein each subunit comprises an AI surface protein or a fragment
thereof. The oligomeric subunits may be covalently associated via a
conserved lysine within a pilin motif. The oligomeric subunits may
be covalently associated via an LPXTG motif, preferably, via the
threonine or serine amino acid residue, respectively.
[0624] AI surface proteins or fragments thereof to be incorporated
into the oligomeric, pilus-like structures of the invention will
preferably include a pilin motif.
[0625] The oligomeric, pilus like structures may be used alone or
in the combinations of the invention. In one embodiment, the
invention comprises a S. pneumoniae strain 19A Hungary 6 AI protein
in oligomeric form, preferably in a hyperoligomeric form. In one
embodiment, the invention comprises a composition comprising one or
more S. pneumoniae strain 19A Hungary 6 AI proteins and one or more
AI proteins from one of any one of S. pneumoniae from TIGR4, 670,
14 CSR 10, 6B Finland 12, 6B Spain 2, 9V Spain 3, 19F Taiwan 14,
23F Taiwan 15, or 23F Poland 16 AI GR4 AI proteins, wherein one or
more of the S. pneumoniae AI proteins is in the form of an
oligomer, preferably in a hyperoligomeric form.
[0626] In addition to the open reading frames encoding the S.
pneumoniae strain 19A Hungary 6 AI proteins, S. pneumoniae strain
19A Hungary 6 AI may also include a transcriptional regulator.
S. pneumoniae Strain 19F Taiwan 14 Adhesin Island
[0627] As discussed above, Applicants have identified adhesin
islands within the genome of S. pneumoniae strain 19F Taiwan 14.
The S. pneumoniae strain 19F Taiwan 14 Adhesin Island comprises a
series of approximately seven open reading frames encoding for a
collection of amino acid sequences comprising surface proteins and
sortases. Specifically, the S. pneumoniae strain 19F Taiwan 14 AI
proteins includes open reading frames encoding for two or more
(i.e., 2, 3, 4, 5, 6, or 7) of ORF2.sub.--19FTW, ORF3.sub.--19FTW,
ORF4.sub.--19FTW, ORF5.sub.--19FTW, ORF6.sub.--19FTW,
ORF7.sub.--19FTW, ORF8.sub.--19FTW.
[0628] A preferred immunogenic composition of the invention
comprises a S. pneumoniae strain 19F Taiwan 14 AI surface protein
which may be formulated or purified in an oligomeric (pilis) form.
Another preferred immunogenic composition of the invention
comprises a S. pneumoniae strain 19F Taiwan 14 AI surface protein
which has been isolated in an oligomeric (pilis) form.
[0629] One or more of the S. pneumoniae strain 19F Taiwan 14 AI
open reading frame polynucleotide sequences may be replaced by a
polynucleotide sequence coding for a fragment of the replaced ORF.
Alternatively, one or more of the S. pneumoniae strain 19F Taiwan
14 AI open reading frames may be replaced by a sequence having
sequence homology to the replaced ORF.
[0630] One or more of the S. pneumoniae strain 19F Taiwan 14 AI
surface protein sequences typically include an LPXTG motif (such as
LPXTG (SEQ ID NO: 122)) or other sortase substrate motif.
[0631] The S. pneumoniae strain 19F Taiwan 14 AI surface proteins
of the invention may affect the ability of the S. pneumoniae
bacteria to adhere to and invade epithelial cells. AI surface
proteins may also affect the ability of S. pneumoniae to
translocate through an epithelial cell layer. Preferably, one or
more S. pneumoniae strain 19F Taiwan 14 AI surface proteins are
capable of binding to or otherwise associating with an epithelial
cell surface. S. pneumoniae strain 19F Taiwan 14 AI surface
proteins may also be able to bind to or associate with fibrinogen,
fibronectin, or collagen.
[0632] The S. pneumoniae strain 19F Taiwan 14 AI sortase proteins
are predicted to be involved in the secretion and anchoring of the
LPXTG containing surface proteins. S. pneumoniae strain 19F Taiwan
14 .mu.l may encode for at least one surface protein.
Alternatively, S. pneumoniae strain 19F Taiwan 14 AI may encode for
at least two surface exposed proteins and at least one sortase.
Preferably, S. pneumoniae strain 19F Taiwan 14 AI encodes for at
least three surface exposed proteins and at least two sortases.
[0633] The AI surface proteins may be covalently attached to the
bacterial cell wall by membrane-associated transpeptidases, such as
an AI sortase. The sortase may function to cleave the surface
protein, preferably between the threonine and glycine residues of
an LPXTG motif. The sortase may then assist in the formation of an
amide link between the threonine carboxyl group and a cell wall
precursor such as lipid II. The precursor can then be incorporated
into the peptidoglycan via the transglycoslylation and
transpeptidation reactions of bacterial wall synthesis. See Comfort
et al., Infection & Immunity (2004) 72(5): 2710-2722.
[0634] In one embodiment, the invention includes a composition
comprising oligomeric, pilus-like structures comprising a S.
pneumoniae strain 19F Taiwan 14 AI surface protein such as
orf3.sub.--19FTW, orf4.sub.--19FTW, or orf5.sub.--19FTW. The
oligomeric, pilus-like structure may comprise numerous units of AI
surface protein. Preferably, the oligomeric, pilus-like structures
comprise two or more AI surface proteins. Still more preferably,
the oligomeric, pilus-like structure comprises a hyper-oligomeric
pilus-like structure comprising at least two (e.g., 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60,
70, 80, 90, 100, 120, 140, 150, 200 or more) oligomeric subunits,
wherein each subunit comprises an AI surface protein or a fragment
thereof. The oligomeric subunits may be covalently associated via a
conserved lysine within a pilin motif. The oligomeric subunits may
be covalently associated via an LPXTG motif, preferably, via the
threonine or serine amino acid residue, respectively.
[0635] AI surface proteins or fragments thereof to be incorporated
into the oligomeric, pilus-like structures of the invention will
preferably include a pilin motif.
[0636] The oligomeric, pilus like structures may be used alone or
in the combinations of the invention. In one embodiment, the
invention comprises a S. pneumoniae strain 19F Taiwan 14 AI protein
in oligomeric form, preferably in a hyperoligomeric form. In one
embodiment, the invention comprises a composition comprising one or
more S. pneumoniae strain 19F Taiwan 14 AI proteins and one or more
AI proteins of any one or more of S. pneumoniae from TIGR4, 670,
19A Hungary 6, 6B Finland 12, 6B Spain 2, 9V Spain 3, 14 CSR 10,
23F Taiwan 15, or 23F Poland 16, wherein one or more of the S.
pneumoniae AI proteins is in the form of an oligomer, preferably in
a hyperoligomeric form.
[0637] In addition to the open reading frames encoding the S.
pneumoniae strain 19F Taiwan 14 AI proteins, S. pneumoniae strain
19F Taiwan 14 AI may also include a transcriptional regulator.
S. pneumoniae Strain 23F Poland 16 Adhesin Island
[0638] As discussed above, Applicants have identified adhesin
islands within the genome of S. pneumoniae strain 23F Poland 16.
The S. pneumoniae strain 23F Poland 16 Adhesin Island comprises a
series of approximately seven open reading frames encoding for a
collection of amino acid sequences comprising surface proteins and
sortases. Specifically, the S. pneumoniae strain 23F Poland 16 AI
proteins includes open reading frames encoding for two or more
(i.e., 2, 3, 4, 5, 6, or 7) of ORF2.sub.--23FP, ORF3.sub.--23FP,
ORF4.sub.--23FP, ORF5.sub.--23FP, ORF6.sub.--23FP, ORF7.sub.--23FP,
and ORF8.sub.--23FP.
[0639] A preferred immunogenic composition of the invention
comprises a S. pneumoniae strain 23F Poland 16 AI surface protein
which may be formulated or purified in an oligomeric (pilis) form.
Another preferred immunogenic composition of the invention
comprises a S. pneumoniae strain 23F Poland 16 AI surface protein
which has been isolated in an oligomeric (pilis) form.
[0640] One or more of the S. pneumoniae strain 23F Poland 16 AI
open reading frame polynucleotide sequences may be replaced by a
polynucleotide sequence coding for a fragment of the replaced ORF.
Alternatively, one or more of the S. pneumoniae strain 23F Poland
16 AI open reading frames may be replaced by a sequence having
sequence homology to the replaced ORF.
[0641] One or more of the S. pneumoniae strain 23F Poland 16 AI
surface protein sequences typically include an LPXTG motif (such as
LPXTG (SEQ ID NO: 122)) or other sortase substrate motif.
[0642] The S. pneumoniae strain 23F Poland 16 AI surface proteins
of the invention may affect the ability of the S. pneumoniae
bacteria to adhere to and invade epithelial cells. AI surface
proteins may also affect the ability of S. pneumoniae to
translocate through an epithelial cell layer. Preferably, one or
more S. pneumoniae strain 23F Poland 16 AI surface proteins are
capable of binding to or otherwise associating with an epithelial
cell surface. S. pneumoniae strain 23F Poland 16 AI surface
proteins may also be able to bind to or associate with fibrinogen,
fibronectin, or collagen.
[0643] The S. pneumoniae strain 23F Poland 16 AI sortase proteins
are predicted to be involved in the secretion and anchoring of the
LPXTG containing surface proteins. S. pneumoniae strain 23F Poland
16 AI may encode for at least one surface protein. Alternatively,
S. pneumoniae strain 23F Poland 16 AI may encode for at least two
surface exposed proteins and at least one sortase. Preferably, S.
pneumoniae strain 23F Poland 16 AI encodes for at least three
surface exposed proteins and at least two sortases.
[0644] The AI surface proteins may be covalently attached to the
bacterial cell wall by membrane-associated transpeptidases, such as
an AI sortase. The sortase may function to cleave the surface
protein, preferably between the threonine and glycine residues of
an LPXTG motif. The sortase may then assist in the formation of an
amide link between the threonine carboxyl group and a cell wall
precursor such as lipid II. The precursor can then be incorporated
into the peptidoglycan via the transglycoslylation and
transpeptidation reactions of bacterial wall synthesis. See Comfort
et al., Infection & Immunity (2004) 72(5): 2710-2722.
[0645] In one embodiment, the invention includes a composition
comprising oligomeric, pilus-like structures comprising a S.
pneumoniae strain 23F Poland 16 AI surface protein such as
orf3.sub.--23FP, orf4.sub.--23FP, or orf5.sub.--23FP. The
oligomeric, pilus-like structure may comprise numerous units of AI
surface protein. Preferably, the oligomeric, pilus-like structures
comprise two or more AI surface proteins. Still more preferably,
the oligomeric, pilus-like structure comprises a hyper-oligomeric
pilus-like structure comprising at least two (e.g., 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60,
70, 80, 90, 100, 120, 140, 150, 200 or more) oligomeric subunits,
wherein each subunit comprises an AI surface protein or a fragment
thereof. The oligomeric subunits may be covalently associated via a
conserved lysine within a pilin motif. The oligomeric subunits may
be covalently associated via an LPXTG motif, preferably, via the
threonine or serine amino acid residue, respectively.
[0646] AI surface proteins or fragments thereof to be incorporated
into the oligomeric, pilus-like structures of the invention will
preferably include a pilin motif.
[0647] The oligomeric, pilus like structures may be used alone or
in the combinations of the invention. In one embodiment, the
invention comprises a S. pneumoniae strain 23F Poland 16 AI protein
in oligomeric form, preferably in a hyperoligomeric form. In one
embodiment, the invention comprises a composition comprising one or
more S. pneumoniae strain 23F Poland 16 AI proteins and one or more
AI proteins from any one or more S. pneumoniae strains of TIGR4,
670, 19A Hungary 6, 6B Finland 12, 6B Spain 2, 9V Spain 3, 19F
Taiwan 14, 23F Taiwan 15, or 14 CSR 10, wherein one or more of the
S. pneumoniae AI proteins is in the form of an oligomer, preferably
in a hyperoligomeric form.
[0648] In addition to the open reading frames encoding the S.
pneumoniae strain 23F Poland 16 AI proteins, S. pneumoniae strain
23F Poland 16 AI may also include a transcriptional regulator.
S. pneumoniae Strain 23F Taiwan 15 Adhesin Island
[0649] As discussed above, Applicants have identified adhesin
islands within the genome of S. pneumoniae strain 23F Taiwan 15.
The S. pneumoniae strain 23F Taiwan 15 Adhesin Island comprises a
series of approximately seven open reading frames encoding for a
collection of amino acid sequences comprising surface proteins and
sortases. Specifically, the S. pneumoniae strain 23F Taiwan 15 AI
proteins includes open reading frames encoding for two or more
(i.e., 2, 3, 4, 5, 6, or 7) of ORF2.sub.--23FTW, ORF3.sub.--23FTW,
ORF4.sub.--23FTW, ORF5.sub.--23FTW, ORF6.sub.--23FTW,
ORF7.sub.--23F[W, ORF8.sub.--23FTW.
[0650] A preferred immunogenic composition of the invention
comprises a S. pneumoniae strain 23F Taiwan 15 AI surface protein
which may be formulated or purified in an oligomeric (pilis) form.
Another preferred immunogenic composition of the invention
comprises a S. pneumoniae strain 23F Taiwan 15 AI surface protein
which has been isolated in an oligomeric (pilis) form.
[0651] One or more of the S. pneumoniae strain 23F Taiwan 15 AI
open reading frame polynucleotide sequences may be replaced by a
polynucleotide sequence coding for a fragment of the replaced ORF.
Alternatively, one or more of the S. pneumoniae strain 23F Taiwan
15 AI open reading frames may be replaced by a sequence having
sequence homology to the replaced ORF.
[0652] One or more of the S. pneumoniae strain 23F Taiwan 15 AI
surface protein sequences typically include an LPXTG motif (such as
LPXTG (SEQ ID NO: 122)) or other sortase substrate motif.
[0653] The S. pneumoniae strain 23F Taiwan 15 AI surface proteins
of the invention may affect the ability of the S. pneumoniae
bacteria to adhere to and invade epithelial cells. AI surface
proteins may also affect the ability of S. pneumoniae to
translocate through an epithelial cell layer. Preferably, one or
more S. pneumoniae strain 23F Taiwan 15 AI surface proteins are
capable of binding to or otherwise associating with an epithelial
cell surface. S. pneumoniae strain 23F Taiwan 15 AI surface
proteins may also be able to bind to or associate with fibrinogen,
fibronectin, or collagen.
[0654] The S. pneumoniae strain 23F Taiwan 15 AI sortase proteins
are predicted to be involved in the secretion and anchoring of the
LPXTG containing surface proteins. S. pneumoniae strain 23F Taiwan
15 AI may encode for at least one surface protein. Alternatively,
S. pneumoniae strain 23F Taiwan 15 AI may encode for at least two
surface exposed proteins and at least one sortase. Preferably, S.
pneumoniae strain 23F Taiwan 15 AI encodes for at least three
surface exposed proteins and at least two sortases.
[0655] The AI surface proteins may be covalently attached to the
bacterial cell wall by membrane-associated transpeptidases, such as
an AI sortase. The sortase may function to cleave the surface
protein, preferably between the threonine and glycine residues of
an LPXTG motif. The sortase may then assist in the formation of an
amide link between the threonine carboxyl group and a cell wall
precursor such as lipid II. The precursor can then be incorporated
into the peptidoglycan via the transglycoslylation and
transpeptidation reactions of bacterial wall synthesis. See Comfort
et al., Infection & Immunity (2004) 72(5): 2710-2722.
[0656] In one embodiment, the invention includes a composition
comprising oligomeric, pilus-like structures comprising a S.
pneumoniae strain 23F Taiwan 15 AI surface protein such as
orf3.sub.--23FTW, orf4.sub.--23FTW, or orf5.sub.--23FTW. The
oligomeric, pilus-like structure may comprise numerous units of AI
surface protein. Preferably, the oligomeric, pilus-like structures
comprise two or more AI surface proteins. Still more preferably,
the oligomeric, pilus-like structure comprises a hyper-oligomeric
pilus-like structure comprising at least two (e.g., 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60,
70, 80, 90, 100, 120, 140, 150, 200 or more) oligomeric subunits,
wherein each subunit comprises an AI surface protein or a fragment
thereof. The oligomeric subunits may be covalently associated via a
conserved lysine within a pilin motif. The oligomeric subunits may
be covalently associated via an LPXTG motif, preferably, via the
threonine or serine amino acid residue, respectively.
[0657] AI surface proteins or fragments thereof to be incorporated
into the oligomeric, pilus-like structures of the invention will
preferably include a pilin motif.
[0658] The oligomeric, pilus like structures may be used alone or
in the combinations of the invention. In one embodiment, the
invention comprises a S. pneumoniae strain 23F Taiwan 15 AI protein
in oligomeric form, preferably in a hyperoligomeric form. In one
embodiment, the invention comprises a composition comprising one or
more S. pneumoniae strain 23F Taiwan 15 AI proteins and one or more
AI proteins from any one or more of S. pneumoniae from TIGR4, 670,
19A Hungary 6, 6B Finland 12, 6B Spain 2, 9V Spain 3, 19F Taiwan
14, 14 CSR 10, or 23F Poland 16 AI, wherein one or more of the S.
pneumoniae AI proteins is in the form of an oligomer, preferably in
a hyperoligomeric form.
[0659] In addition to the open reading frames encoding the S.
pneumoniae strain 23F Taiwan 15 AI proteins, S. pneumoniae strain
23F Taiwan 15 AI may also include a transcriptional regulator.
S. pneumoniae Strain 6B Finland 12 Adhesin Island
[0660] As discussed above, Applicants have identified adhesin
islands within the genome of S. pneumoniae strain 6B Finland 12.
The S. pneumoniae strain 6B Finland 12 Adhesin Island comprises a
series of approximately seven open reading frames encoding for a
collection of amino acid sequences comprising surface proteins and
sortases. Specifically, the S. pneumoniae strain 6B Finland 12 AI
proteins includes open reading frames encoding for two or more
(i.e., 2, 3, 4, 5, 6, or 7) of ORF2.sub.--6BF, ORF3.sub.--6BF,
ORF4.sub.--6BF, ORF5.sub.--6BF, ORF6.sub.--6BF, ORF7.sub.--6BF,
ORF8.sub.--6BF.
[0661] A preferred immunogenic composition of the invention
comprises a S. pneumoniae strain 6B Finland 12 AI surface protein
which may be formulated or purified in an oligomeric (pilis) form.
Another preferred immunogenic composition of the invention
comprises a S. pneumoniae strain 6B Finland 12 AI surface protein
which has been isolated in an oligomeric (pilis) form.
[0662] One or more of the S. pneumoniae strain 6B Finland 12 AI
open reading frame polynucleotide sequences may be replaced by a
polynucleotide sequence coding for a fragment of the replaced ORF.
Alternatively, one or more of the S. pneumoniae strain 6B Finland
12 AI open reading frames may be replaced by a sequence having
sequence homology to the replaced ORF.
[0663] One or more of the S. pneumoniae strain 6B Finland 12 AI
surface protein sequences typically include an LPXTG motif (such as
LPXTG (SEQ ID NO: 122)) or other sortase substrate motif.
[0664] The S. pneumoniae strain 6B Finland 12 AI surface proteins
of the invention may affect the ability of the S. pneumoniae
bacteria to adhere to and invade epithelial cells. AI surface
proteins may also affect the ability of S. pneumoniae to
translocate through an epithelial cell layer. Preferably, one or
more S. pneumoniae strain 6B Finland 12 AI surface proteins are
capable of binding to or otherwise associating with an epithelial
cell surface. S. pneumoniae strain 6B Finland 12 AI surface
proteins may also be able to bind to or associate with fibrinogen,
fibronectin, or collagen.
[0665] The S. pneumoniae strain 6B Finland 12 AI sortase proteins
are predicted to be involved in the secretion and anchoring of the
LPXTG containing surface proteins. S. pneumoniae strain 6B Finland
12 AI may encode for at least one surface protein. Alternatively,
S. pneumoniae strain 6B Finland 12 AI may encode for at least two
surface exposed proteins and at least one sortase. Preferably, S.
pneumoniae strain 6B Finland 12 AI encodes for at least three
surface exposed proteins and at least two sortases.
[0666] The AI surface proteins may be covalently attached to the
bacterial cell wall by membrane-associated transpeptidases, such as
an AI sortase. The sortase may function to cleave the surface
protein, preferably between the threonine and glycine residues of
an LPXTG motif. The sortase may then assist in the formation of an
amide link between the threonine carboxyl group and a cell wall
precursor such as lipid II. The precursor can then be incorporated
into the peptidoglycan via the transglycoslylation and
transpeptidation reactions of bacterial wall synthesis. See Comfort
et al., Infection & Immunity (2004) 72(5): 2710-2722.
[0667] In one embodiment, the invention includes a composition
comprising oligomeric, pilus-like structures comprising a S.
pneumoniae strain 6B Finland 12 AI surface protein such as
orf3.sub.--6BF, orf4.sub.--6BF, or orf5.sub.--6BF. The oligomeric,
pilus-like structure may comprise numerous units of AI surface
protein. Preferably, the oligomeric, pilus-like structures comprise
two or more AI surface proteins. Still more preferably, the
oligomeric, pilus-like structure comprises a hyper-oligomeric
pilus-like structure comprising at least two (e.g., 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60,
70, 80, 90, 100, 120, 140, 150, 200 or more) oligomeric subunits,
wherein each subunit comprises an AI surface protein or a fragment
thereof. The oligomeric subunits may be covalently associated via a
conserved lysine within a pilin motif. The oligomeric subunits may
be covalently associated via an LPXTG motif, preferably, via the
threonine or serine amino acid residue, respectively.
[0668] AI surface proteins or fragments thereof to be incorporated
into the oligomeric, pilus-like structures of the invention will
preferably include a pilin motif.
[0669] The oligomeric, pilus like structures may be used alone or
in the combinations of the invention. In one embodiment, the
invention comprises a S. pneumoniae strain 6B Finland 12 AI protein
in oligomeric form, preferably in a hyperoligomeric form. In one
embodiment, the invention comprises a composition comprising one or
more S. pneumoniae strain 6B Finland 12 AI proteins and one or more
AI proteins of any one or more of S. pneumoniae from TIGR4, 670,
19A Hungary 6, 6B Finland 12, 6B Spain 2, 9V Spain 3, 19F Taiwan
14, 23F Taiwan 15, or 23F Poland 16 AI, wherein one or more of the
S. pneumoniae AI proteins is in the form of an oligomer, preferably
in a hyperoligomeric form.
[0670] In addition to the open reading frames encoding the S.
pneumoniae strain 6B Finland 12 AI proteins, S. pneumoniae strain
6B Finland 12 AI may also include a transcriptional regulator.
S. pneumoniae Strain 6B Spain 2 Adhesin Island
[0671] As discussed above, Applicants have identified adhesin
islands within the genome of S. pneumoniae strain 6B Spain 2. The
S. pneumoniae strain 6B Spain 2 Adhesin Island comprises a series
of approximately seven open reading frames encoding for a
collection of amino acid sequences comprising surface proteins and
sortases. Specifically, the S. pneumoniae strain 6B Spain 2 AI
proteins includes open reading frames encoding for two or more
(i.e., 2, 3, 4, 5, 6, or 7) of ORF2.sub.--6BSP, ORF3.sub.--6BSP,
ORF4.sub.--6BSP, ORF5.sub.--6BSP, ORF6.sub.--6BSP, ORF7.sub.--6BSP,
and ORF8.sub.--6BSP.
[0672] A preferred immunogenic composition of the invention
comprises a S. pneumoniae strain 6B Spain 2 AI surface protein
which may be formulated or purified in an oligomeric (pilis) form.
Another preferred immunogenic composition of the invention
comprises a S. pneumoniae strain 6B Spain 2 AI surface protein
which has been isolated in an oligomeric (pilis) form.
[0673] One or more of the S. pneumoniae strain 6B Spain 2 AI open
reading frame polynucleotide sequences may be replaced by a
polynucleotide sequence coding for a fragment of the replaced ORF.
Alternatively, one or more of the S. pneumoniae strain 6B Spain 2
AI open reading frames may be replaced by a sequence having
sequence homology to the replaced ORF.
[0674] One or more of the S. pneumoniae strain 6B Spain 2 AI
surface protein sequences typically include an LPXTG motif (such as
LPXTG (SEQ ID NO: 122)) or other sortase substrate motif.
[0675] The S. pneumoniae strain 6B Spain 2 AI surface proteins of
the invention may affect the ability of the S. pneumoniae bacteria
to adhere to and invade epithelial cells. AI surface proteins may
also affect the ability of S. pneumoniae to translocate through an
epithelial cell layer. Preferably, one or more S. pneumoniae strain
6B Spain 2 AI surface proteins are capable of binding to or
otherwise associating with an epithelial cell surface. S.
pneumoniae strain 6B Spain 2 AI surface proteins may also be able
to bind to or associate with fibrinogen, fibronectin, or
collagen.
[0676] The S. pneumoniae strain 6B Spain 2 AI sortase proteins are
predicted to be involved in the secretion and anchoring of the
LPXTG containing surface proteins. S. pneumoniae strain 6B Spain 2
AI may encode for at least one surface protein. Alternatively, S.
pneumoniae strain 6B Spain 2 AI may encode for at least two surface
exposed proteins and at least one sortase. Preferably, S.
pneumoniae strain 6B Spain 2 AI encodes for at least three surface
exposed proteins and at least two sortases.
[0677] The AI surface proteins may be covalently attached to the
bacterial cell wall by membrane-associated transpeptidases, such as
an AI sortase. The sortase may function to cleave the surface
protein, preferably between the threonine and glycine residues of
an LPXTG motif. The sortase may then assist in the formation of an
amide link between the threonine carboxyl group and a cell wall
precursor such as lipid II. The precursor can then be incorporated
into the peptidoglycan via the transglycoslylation and
transpeptidation reactions of bacterial wall synthesis. See Comfort
et al., Infection & Immunity (2004) 72(5): 2710-2722.
[0678] In one embodiment, the invention includes a composition
comprising oligomeric, pilus-like structures comprising a S.
pneumoniae strain 6B Spain 2 AI surface protein such as
orf3.sub.--6BSP, orf4.sub.--6BSP, or orf5.sub.--6BSP. The
oligomeric, pilus-like structure may comprise numerous units of AI
surface protein. Preferably, the oligomeric, pilus-like structures
comprise two or more AI surface proteins. Still more preferably,
the oligomeric, pilus-like structure comprises a hyper-oligomeric
pilus-like structure comprising at least two (e.g., 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60,
70, 80, 90, 100, 120, 140, 150, 200 or more) oligomeric subunits,
wherein each subunit comprises an AI surface protein or a fragment
thereof. The oligomeric subunits may be covalently associated via a
conserved lysine within a pilin motif. The oligomeric subunits may
be covalently associated via an LPXTG motif, preferably, via the
threonine or serine amino acid residue, respectively.
[0679] AI surface proteins or fragments thereof to be incorporated
into the oligomeric, pilus-like structures of the invention will
preferably include a pilin motif.
[0680] The oligomeric, pilus like structures may be used alone or
in the combinations of the invention. In one embodiment, the
invention comprises a S. pneumoniae strain 6B Spain 2 AI protein in
oligomeric form, preferably in a hyperoligomeric form. In one
embodiment, the invention comprises a composition comprising one or
more S. pneumoniae strain 6B Spain 2 AI proteins and one or more AI
proteins of any one or more of S. pneumoniae from TIGR4, 670, 19A
Hungary 6, 6B Finland 12, 14 CSR 10, 9V Spain 3, 19F Taiwan 14, 23F
Taiwan 15, or 23F Poland 16 AI, wherein one or more of the S.
pneumoniae AI proteins is in the form of an oligomer, preferably in
a hyperoligomeric form.
[0681] In addition to the open reading frames encoding the S.
pneumoniae strain 6B Spain 2 AI proteins, S. pneumoniae strain 6B
Spain 2 AI may also include a transcriptional regulator.
S. pneumoniae Strain 9V Spain 3 Adhesin Island
[0682] As discussed above, Applicants have identified adhesin
islands within the genome of S. pneumoniae strain 9V Spain 3. The
S. pneumoniae strain 9V Spain 3 Adhesin Island comprises a series
of approximately seven open reading frames encoding for a
collection of amino acid sequences comprising surface proteins and
sortases. Specifically, the S. pneumoniae strain 9V Spain 3 AI
proteins includes open reading frames encoding for two or more
(i.e., 2, 3, 4, 5, 6, or 7) of ORF2.sub.--9VSP, ORF3.sub.--9VSP,
ORF4.sub.--9VSP, ORF5.sub.--9VSP, ORF6.sub.--9VSP, ORF7.sub.--9VSP,
and ORF8.sub.--9VSP.
[0683] A preferred immunogenic composition of the invention
comprises a S. pneumoniae strain 9V Spain 3 AI surface protein
which may be formulated or purified in an oligomeric (pilis) form.
Another preferred immunogenic composition of the invention
comprises a S. pneumoniae strain 9V Spain 3 AI surface protein
which has been isolated in an oligomeric (pilis) form.
[0684] One or more of the S. pneumoniae strain 9V Spain 3 AI open
reading frame polynucleotide sequences may be replaced by a
polynucleotide sequence coding for a fragment of the replaced ORF.
Alternatively, one or more of the S. pneumoniae strain 9V Spain 3
AI open reading frames may be replaced by a sequence having
sequence homology to the replaced ORF.
[0685] One or more of the S. pneumoniae strain 9V Spain 3 AI
surface protein sequences typically include an LPXTG motif (such as
LPXTG (SEQ ID NO: 122)) or other sortase substrate motif.
[0686] The S. pneumoniae strain 9V Spain 3 AI surface proteins of
the invention may affect the ability of the S. pneumoniae bacteria
to adhere to and invade epithelial cells. AI surface proteins may
also affect the ability of S. pneumoniae to translocate through an
epithelial cell layer. Preferably, one or more S. pneumoniae strain
9V Spain 3 AI surface proteins are capable of binding to or
otherwise associating with an epithelial cell surface. S.
pneumoniae strain 9V Spain 3 AI surface proteins may also be able
to bind to or associate with fibrinogen, fibronectin, or
collagen.
[0687] The S. pneumoniae strain 9V Spain 3 AI sortase proteins are
predicted to be involved in the secretion and anchoring of the
LPXTG containing surface proteins. S. pneumoniae strain 9V Spain 3
AI may encode for at least one surface protein. Alternatively, S.
pneumoniae strain 9V Spain 3 AI may encode for at least two surface
exposed proteins and at least one sortase. Preferably, S.
pneumoniae strain 9V Spain 3 AI encodes for at least three surface
exposed proteins and at least two sortases.
[0688] The AI surface proteins may be covalently attached to the
bacterial cell wall by membrane-associated transpeptidases, such as
an AI sortase. The sortase may function to cleave the surface
protein, preferably between the threonine and glycine residues of
an LPXTG motif. The sortase may then assist in the formation of an
amide link between the threonine carboxyl group and a cell wall
precursor such as lipid II. The precursor can then be incorporated
into the peptidoglycan via the transglycoslylation and
transpeptidation reactions of bacterial wall synthesis. See Comfort
et al., Infection & Immunity (2004) 72(5): 2710-2722.
[0689] In one embodiment, the invention includes a composition
comprising oligomeric, pilus-like structures comprising a S.
pneumoniae strain 9V Spain 3 AI surface protein such as
orf3.sub.--9VSP, orf4.sub.--9VSP, or orf5.sub.--9VSP. The
oligomeric, pilus-like structure may comprise numerous units of AI
surface protein. Preferably, the oligomeric, pilus-like structures
comprise two or more AI surface proteins. Still more preferably,
the oligomeric, pilus-like structure comprises a hyper-oligomeric
pilus-like structure comprising at least two (e.g., 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60,
70, 80, 90, 100, 120, 140, 150, 200 or more) oligomeric subunits,
wherein each subunit comprises an AI surface protein or a fragment
thereof. The oligomeric subunits may be covalently associated via a
conserved lysine within a pilin motif. The oligomeric subunits may
be covalently associated via an LPXTG motif, preferably, via the
threonine or serine amino acid residue, respectively.
[0690] AI surface proteins or fragments thereof to be incorporated
into the oligomeric, pilus-like structures of the invention will
preferably include a pilin motif.
[0691] The oligomeric, pilus like structures may be used alone or
in the combinations of the invention. In one embodiment, the
invention comprises a S. pneumoniae strain 9V Spain 3 AI protein in
oligomeric form, preferably in a hyperoligomeric form. In one
embodiment, the invention comprises a composition comprising one or
more S. pneumoniae strain 9V Spain 3 AI proteins and one or more AI
proteins from any one or more of S. pneumoniae from TIGR4, 670, 19A
Hungary 6, 6B Finland 12, 6B Spain 2, 14 CSR 10, 19F Taiwan 14, 23F
Taiwan 15, or 23F Poland 16 AI, wherein one or more of the S.
pneumoniae AI proteins is in the form of an oligomer, preferably in
a hyperoligomeric form.
[0692] In addition to the open reading frames encoding the S.
pneumoniae strain 9V Spain 3 AI proteins, S. pneumoniae strain 9V
Spain 3 AI may also include a transcriptional regulator.
[0693] The S. pneumoniae oligomeric, pilus-like structures may be
isolated or purified from bacterial cultures in which the bacteria
express an S. pneumoniae AI surface protein. The invention
therefore includes a method for manufacturing an oligomeric AI
surface antigen comprising culturing a S. pneumoniae bacterium that
expresses the oligomeric AI protein and isolating the expressed
oligomeric AI protein from the S. pneumoniae bacteria. The AI
protein may be collected from secretions into the supernatant or it
may be purified from the bacterial surface. The method may further
comprise purification of the expressed AI protein. Preferably, the
AI protein is in a hyperoligomeric form.
[0694] The oligomeric, pilus-like structures may be isolated or
purified from bacterial cultures overexpressing an AI surface
protein. The invention therefore includes a method for
manufacturing an S. pneumoniae oligomeric Adhesin Island surface
antigen comprising culturing a S. pneumoniae bacterium adapted for
increased AI protein expression and isolation of the expressed
oligomeric Adhesin Island protein from the S. pneumoniae bacteria.
The AI protein may be collected from secretions into the
supernatant or it may be purified from the bacterial surface. The
method may further comprise purification of the expressed Adhesin
Island protein. Preferably, the Adhesin Island protein is in a
hyperoligomeric form.
[0695] The S. pneumoniae bacteria are preferably adapted to
increase AI protein expression by at least two (e.g., 2, 3, 4, 5,
8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125,
150 or 200) times wild type expression levels.
[0696] S. pneumoniae bacteria may be adapted to increase AI protein
expression by any means known in the art, including methods of
increasing gene dosage and methods of gene upregulation. Such means
include, for example, transformation of the S. pneumoniae bacteria
with a plasmid encoding the AI protein. The plasmid may include a
strong promoter or it may include multiple copies of the sequence
encoding the AI protein. Optionally, the sequence encoding the AI
protein within the S. pneumoniae bacterial genome may be deleted.
Alternatively, or in addition, the promoter regulating the S.
pneumoniae Adhesin Island may be modified to increase
expression.
[0697] The invention further includes S. pneumoniae bacteria which
have been adapted to produce increased levels of AI surface
protein. In particular, the invention includes S. pneumoniae
bacteria which have been adapted to produce oligomeric or
hyperoligomeric AI surface protein. In one embodiment, the S.
pneumoniae of the invention are inactivated or attenuated to permit
in vivo delivery of the whole bacteria, with the AI surface protein
exposed on its surface.
[0698] The invention further includes S. pneumoniae bacteria which
have been adapted to have increased levels of expressed AI protein
incorporated in pili on their surface. The S. pneumoniae bacteria
may be adapted to have increased exposure of oligomeric or
hyperoligomeric AI proteins on its surface by increasing expression
levels of a signal peptidase polypeptide. Increased levels of a
local signal peptidase expression in Gram positive bacteria (such
us LepA in GAS) are expected to result in increased exposure of
pili proteins on the surface of Gram positive bacteria. Increased
expression of a leader peptidase in S. pneumoniae may be achieved
by any means known in the art, such as increasing gene dosage and
methods of gene upregulation. The S. pneumoniae bacteria adapted to
have increased levels of leader peptidase may additionally be
adapted to express increased levels of at least one pili
protein.
[0699] Alternatively, the AI proteins of the invention may be
expressed on the surface of a non-pathogenic Gram positive
bacteria, such as Streptococus gordonii (See, e.g., Byrd et al.,
"Biological consequences of antigen and cytokine co-expression by
recombinant Streptococcus gordonii vaccine vectors", Vaccine (2002)
20:2197-2205) or Lactococcus lactis (See, e.g., Mannam et al.,
"Mucosal Vaccine Made from Live, Recombinant Lactococcus lactis
Protects Mice against Pharangeal Infection with Streptococcus
pyogenes" Infection and Immunity (2004) 72(6):3444-3450). As used
herein, non-pathogenic Gram positive bacteria refer to Gram
positive bacteria which are compatible with a human host subject
and are not associated with human pathogenisis. Preferably, the
non-pathogenic bacteria are modified to express the AI surface
protein in oligomeric, or hyper-oligomeric form. Sequences encoding
for an AI surface protein and, optionally, an AI sortase, may be
integrated into the non-pathogenic Gram positive bacterial genome
or inserted into a plasmid. The non-pathogenic Gram positive
bacteria may be inactivated or attenuated to facilitate in vivo
delivery of the whole bacteria, with the AI surface protein exposed
on its surface. Alternatively, the AI surface protein may be
isolated or purified from a bacterial culture of the non-pathogenic
Gram positive bacteria. For example, the AI surface protein may be
isolated from cell extracts or culture supernatants. Alternatively,
the AI surface protein may be isolated or purified from the surface
of the non-pathogenic Gram positive bacteria.
[0700] The non-pathogenic Gram positive bacteria may be used to
express any of the S. pneumoniae Adhesin Island proteins described
herein. The non-pathogenic Gram positive bacteria are transformed
to express an Adhesin Island surface protein. Preferably, the
non-pathogenic Gram positive bacteria also express at least one
Adhesin Island sortase. The AI transformed non-pathogenic Gram
positive bacteria of the invention may be used to prevent or treat
infection with pathogenic S. pneumoniae.
[0701] FIGS. 190 A and B, and 193-195 provide examples of three
methods successfully practiced by applicants to purify pili from S.
pneumoniae TIGR4.
Immunogenic Compositions
[0702] The Gram positive bacteria AI proteins described herein are
useful in immunogenic compositions for the prevention or treatment
of Gram positive bacterial infection. For example, the GBS AI
surface proteins described herein are useful in immunogenic
compositions for the prevention or treatment of GBS infection. As
another example, the GAS AI surface proteins described herein may
be useful in immunogenic compositions for the prevention or
treatment of GAS infection. As another example, the S. pneumoniae
AI surface proteins may be useful in immunogenic cojmpositions for
the prevention or treatment of S. pneumoniae infection.
[0703] Gram positive bacteria AI surface proteins that can provide
protection across more than one serotype or strain isolate may be
used to increase immunogenic effectiveness. For example, a
particular GBS AI surface protein having an amino acid sequence
that is at least 50% (i.e., at least 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) homologous to the
particular GBS AI surface protein of at least 2 (i.e., at least 3,
4, 5, 6, 7, 8, 9, 10, or more) other GBS serotypes or strain
isolates may be used to increase the effectiveness of such
compositions.
[0704] As another example, fragments of Gram positive bacteria AI
surface proteins that can provide protection across more than one
serotype or strain isolate may be used to increase immunogenic
effectiveness. Such a fragment may be identified within a consensus
sequence of a full length amino acid sequence of a Gram positive
bacteria AI surface protein. Such a fragment can be identified in
the consensus sequence by its high degree of homology or identity
across multiple (i.e, at least 3, 4, 5, 6, 7, 8, 9, 10, or more)
Gram positive bacteria serotypes or strain isolates. Preferably, a
high degree of homology is a degree of homology of at least 90%
(i.e., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
or 100%) across Gram positive bacteria serotypes or strain
isolates. Preferably, a high degree of identity is a degree of
identity of at least 90% (i.e., at least 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, or 100%) across Gram positive bacteria
serotypes or strain isolates. In one embodiment of the invention,
such a fragment of a Gram positive bacteria AI surface protein may
be used in the immunogenic compositions.
[0705] In addition, the AI surface protein oligomeric pilus
structures may be formulated or purified for use in immunization.
Isolated AI surface protein oligomeric pilus structures may also be
used for immunization.
[0706] The invention includes an immunogenic composition comprising
a first Gram positive bacteria AI protein and a second Gram
positive bacterial AI protein. One or more of the AI proteins may
be a surface protein. Such surface proteins may contain an LPXTG
motif or other sortase substrate motif.
[0707] The first and second AI proteins may be from the same or
different genus or species of Gram positive bacteria. If within the
same species, the first and second AI proteins may be from the same
or different AI subtypes. If two AIs are of the same subtype, the
AIs have the same numerical designation. For example, all AIs
designated as AI-1 are of the same AI subtype. If two AIs are of a
different subtype, the AIs have different numerical designations.
For example, AI-1 is of a different AI subtype from AI-2, AI-3,
AI-4, etc. Likewise, AI-2 is of a different AI subtype from AI-1,
AI-3, and AI-4, etc.
[0708] For example, the invention includes an immunogenic
composition comprising one or more GBS AI-1 proteins and one or
more GBS AI-2 proteins. One or more of the AI proteins may be a
surface protein. Such surface proteins may contain an LPXTG motif
(such as LPXTG (SEQ ID NO: 122)) and may bind fibrinogen,
fibronectin, or collagen. One or more of the AI proteins may be a
sortase. The GBS AI-1 proteins may be selected from the group
consisting of GBS 80, GBS 104, GBS 52, SAG0647 and SAG0648.
Preferably, the GBS AI-1 proteins include GBS 80 or GBS 104.
[0709] The GBS AI-2 proteins may be selected from the group
consisting of GBS 67, GBS 59, GBS 150, SAG1405, SAG1406, 01520,
01521, 01522, 01523, 01523, 01524 and 01525. In one embodiment, the
GBS AI-2 proteins are selected from the group consisting of GBS 67,
GBS 59, GBS 150, SAG1405, and SAG1406. In another embodiment, the
GBS AI-2 proteins may be selected from the group consisting of
01520, 01521, 01522, 01523, 01523, 01524 and 01525. Preferably, the
GBS AI-2 protein includes GBS 59 or GBS 67.
[0710] As another example, the invention includes an immunogenic
composition comprising one or more of any combination of GAS AI-1,
GAS AI-2, GAS AI-3, or GAS AI-4 proteins. One or more of the GAS AI
proteins may be a sortase. The GAS AI-1 proteins may be selected
from the group consisting of M6_Spy0156, M6_Spy0157, M6_Spy0158,
M6_Spy0159, M6_Spy0160, M6_Spy0161, CDC SS 410_fimbrial,
ISS3650_fimbrial, and DSM2071_fimbrial. Preferably, the GAS AI-1
proteins are selected from the group consisting of M6_Spy0157,
M6_Spy0159, M6_Spy0160, CDC SS 410_fimbrial, ISS3650_fimbrial, and
DSM2071_fimbrial.
[0711] The GAS AI-2 proteins may be selected from the group
consisting of Spy0124, GAS15, Spy0127, GAS16, GAS17, GAS18,
Spy0131, Spy0133, and GAS20. Preferably, the GAS AI-2 proteins are
selected from the group consisting of GAS 15, GAS 16, and GAS
18.
[0712] The GAS AI-3 proteins may be selected from the group
consisting of SpyM3.sub.--0097, SpyM3.sub.--0098, SpyM3.sub.--0099,
SpyM3.sub.--0100, SpyM3.sub.--0101, SpyM3.sub.--0102,
SpyM3.sub.--0103, SpyM3.sub.--0104, SPs0099, SPs0100, SPs0101,
SPs0102, SPs0103, SPs0104, SPs0105, SPs0106, orf77, orf78, orf79,
orf80, orf81, orf82, orf83, orf84, spyM18.sub.--0125,
spyM18.sub.--0126, spyM18.sub.--0127, spyM18.sub.--0128,
spyM18.sub.--0129, spyM18.sub.--0130, spyM18.sub.--0131,
spyM18.sub.--0132, SpyoM01000156, SpyoM01000155, SpyoM01000154,
SpyoM01000153, SpyoM01000152, SpyoM01000151, SpyoM01000150,
SpyoM01000149, ISS3040_fimbrial, ISS3776_fimbrial, and
ISS4959_fimbrial. In one embodiment the GAS AI-3 proteins are
selected from the group consisting of SpyM3.sub.--0097,
SpyM3.sub.--0098, SpyM3.sub.--0099, SpyM3.sub.--0100,
SpyM3.sub.--0101, SpyM3.sub.--0102, SpyM3.sub.--0103, and
SpyM3.sub.--0104. In another embodiment, the GAS AI-3 proteins are
selected from the group consisting of SPs0099, SPs0100, SPs0101,
SPs0102, SPs0103, SPs0104, SPs0105, and SPs0106. In yet another
embodiment, the GAS AI-3 proteins are selected from the group
consisting of orf77, orf78, orf79, orf80, orf81, orf82, orf83, and
orf84. In a further embodiment, the GAS AI-3 proteins are selected
from the group consisting of spyM18.sub.--0125, spyM18.sub.--0126,
spyM18.sub.--0127, spyM18.sub.--0128, spyM18.sub.--0129,
spyM18.sub.--0130, spyM18.sub.--0131, and spyM18.sub.--0132. In yet
another embodiment the GAS AI-3 proteins are selected from the
group consisting of SpyoM01000156, SpyoM01000155, SpyoM01000154,
SpyoM01000153, SpyoM01000152, SpyoM01000151, SpyoM01000150, and
SpyoM01000149.
[0713] The GAS AI-4 proteins may be selected from the group
consisting of 19224133, 19224134, 19224135, 19224136, 19224137,
19224138, 19224139, 19224140, 19224141, 20010296_fimbrial,
20020069_fimbrial, CDC SS 635_fimbrial, ISS4883_fimbrial, and
ISS4538_fimbrial. Preferably, the GAS-AI-4 proteins are selected
from the group consisting of 19224134, 19224135, 19224137,
19224139, 19224141, 20010296_fimbrial, 20020069_fimbrial, CDC SS
635_fimbrial, ISS4883_fimbrial, and ISS4538_fimbrial.
[0714] As yet another example, the invention includes an
immunogenic composition comprising one or more of any combination
of S. pneumonaie from TIGR4, S. pneumonaie strain 670, S.
pneumonaie from 19A Hungary 6, S. pneumonaie from 6B Finland 12, S.
pneumonaie from 6B Spain 2, S. pneumonaie from 9V Spain 3, S.
pneumonaie from 14 CSR 10, S. pneumonaie from 19F Taiwan 14, S.
pneumonaie from 23F Taiwan 15, or S. pneumonaie from 23F Poland 16
AI proteins. One or more of the AI proteins may be a surface
protein. Such surface proteins may contain an LPXTG motif (such as
LPXTG (SEQ ID NO: 122)) and may bind fibrinogen, fibronectin, or
collagen. One or more of the AI proteins may be a sortase.
[0715] The S. pneumonaie from TIGR4 AI proteins may be selected
from the group consisting of SP0462, SP0463, SP0464, SP0465,
SP0466, SP0467, SP0468. Preferably, the S. pneumonaie from TIGR4 AI
proteins include SP0462, SP0463, or SP0464.
[0716] The S. pneumonaie strain 670 AI proteins may be selected
from the group consisting of Orf1.sub.--670, Orf3.sub.--670,
Orf4.sub.--670, Orf5.sub.--670, Orf6.sub.--670, Orf7.sub.--670, and
Orf8.sub.--670. Preferably, the S. pneumonaie strain 670 AI
proteins include Orf3.sub.--670, Orf4.sub.--670, or
Orf5.sub.--670.
[0717] The S. pneumonaie from 19A Hungary 6 AI proteins may be
selected from the group consisting of ORF2.sub.--1 gAH,
ORF3.sub.--1 gAH, ORF4.sub.--19AH, ORF5.sub.--19AH, ORF6.sub.--1
gAH, ORF7.sub.--19AH, or ORF8.sub.--19AH.
[0718] The S. pneumonaie from 6B Finland 12 AI proteins may be
selected from the group consisting of ORF2.sub.--6BF,
ORF3.sub.--6BF, ORF4.sub.--6BF, ORF5.sub.--6BF, ORF6.sub.--6BF,
ORF7.sub.--6BF, or ORF8.sub.--6BF.
[0719] The S. pneumonaie from 6B Spain 2 AI proteins may be
selected from the group consisting of ORF2.sub.--6BSP,
ORF3.sub.--6BSP, ORF4.sub.--6BSP, ORF5.sub.--6BSP, ORF6.sub.--6BSP,
ORF7.sub.--6BSP, or ORF8.sub.--6BSP.
[0720] The S. pneumonaie from 9V Spain 3 AI proteins may be
selected from the group consisting of ORF2.sub.--9VSP,
ORF3.sub.--9VSP, ORF4.sub.--9VSP, ORF5.sub.--9VSP, ORF6.sub.--9VSP,
ORF7.sub.--9VSP, or ORF8.sub.--9VSP.
[0721] The S. pneumonaie from 14 CSR 10 AI proteins may be selected
from the group consisting of ORF2.sub.--14CSR, ORF3.sub.--14CSR,
ORF4.sub.--14CSR, ORF5.sub.--14CSR, ORF6.sub.--14CSR,
ORF7.sub.--14CSR, or ORF8.sub.--14CSR.
[0722] The S. pneumonaie from 19F Taiwan 14 AI proteins may be
selected from the group consisting of ORF2.sub.--1 gFTW,
ORF3.sub.--19FTW, ORF4.sub.--19FTW, ORF5.sub.--19FTW,
ORF6.sub.--19FTW, ORF7.sub.--19FTW, or ORF8.sub.--19FTW.
[0723] The S. pneumonaie from 23F Taiwan 15 AI proteins may be
selected from the group consisting of ORF2.sub.--23FTW,
ORF3.sub.--23FTW, ORF4.sub.--23FTW, ORF5.sub.--23FTW,
ORF6.sub.--23FTW, ORF7.sub.--23FTW, or ORF8.sub.--23FTW.
[0724] The S. pneumonaie from 23F Poland 16 AI proteins may be
selected from the group consisting of ORF2.sub.--23FP,
ORF3.sub.--23FP, ORF4.sub.--23FP, ORF5.sub.--23FP, ORF6.sub.--23FP,
ORF7.sub.--23FP or ORF8.sub.--23FP.
[0725] Preferably, the Gram positive bacteria AI proteins included
in the immunogenic compositions of the invention can provide
protection across more than one serotype or strain isolate. For
example, the immunogenic composition may comprise a first AI
protein, wherein the amino acid sequence of said AI protein is at
least 90% (i.e., at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or
100%) homologous to the amino acid sequence of a second AI protein,
and wherein said first AI protein and said second AI protein are
derived from the genomes of different serotypes of a Gram positive
bacteria. The first AI protein may also be homologous to the amino
acid sequence of a third AI protein, such that the first AI
protein, the second AI protein and the third AI protein are derived
from the genomes of different serotypes of a Gram positive
bacteria. The first AI protein may also be homologous to the amino
acid sequence of a fourth AI protein, such that the first AI
protein, the second AI protein and the third AI protein are derived
from the genomes of different serotypes of a Gram positive
bacteria.
[0726] For example, preferably, the GBS AI proteins included in the
immunogenic compositions of the invention can provide protection
across more than one GBS serotype or strain isolate. For example,
the immunogenic composition may comprise a first GBS AI protein,
wherein the amino acid sequence of said AI protein is at least 90%
(i.e., at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%)
homologous to the amino acid sequence of a second GBS AI protein,
and wherein said first AI protein and said second AI protein are
derived from the genomes of different GBS serotypes. The first GBS
AI protein may also be homologous to the amino acid sequence of a
third GBS AI protein, such that the first AI protein, the second AI
protein and the third AI protein are derived from the genomes of
different GBS serotypes. The first AI protein may also be
homologous to the amino acid sequence of a fourth GBS AI protein,
such that the first AI protein, the second AI protein and the third
AI protein are derived from the genomes of different GBS
serotypes.
[0727] The first AI protein may be selected from an AI-1 protein or
an AI-2 protein. For example, the first AI protein may be a GBS
AI-1 surface protein such as GBS 80. The amino acid sequence of GBS
80 from GBS serotype V, strain isolate 2603 is greater than 90%
homologous to the GBS 80 amino acid sequence from GBS serotype III,
strain isolates NEM316 and COH1 and the GBS 80 amino acid sequence
from GBS serotype 1a, strain isolate A909.
[0728] As another example, the first AI protein may be GBS 104. The
amino acid sequence of GBS 104 from GBS serotype V, strain isolate
2603 is greater than 90% homologous to the GBS 104 amino acid
sequence from GBS serotype III, strain isolates NEM316 and COH1,
the GBS 104 amino acid sequence from GBS serotype 1a, strain
isolate A909, and the GBS 104 amino acid sequence serotype II,
strain isolate 18RS21.
[0729] Table 12 provides the amino acid sequence identity of GBS 80
and GBS 104 across GBS serotypes Ia, Ib, II, III, V, and VIII. The
GBS strains in which genes encoding GBS 80 and GBS 104 were
identified share, on average, 99.88 and 99.96 amino acid sequence
identity, respectively. This high degree of amino acid identity
indicates that an immunogenic composition comprising a first
protein of GBS 80 or GBS 104 may provide protection across more
than one GBS serotype or strain isolate. TABLE-US-00006 TABLE 12
Conservation of GBS 80 and GBS 104 amino acid sequences GBS 80 GBS
104 Serotype Strains cGH % AA identity cGH % AA identity Ia 090 +
99.79 + 100.00 A909 + 100.00 + 100.00 515 - - DK1 - - DK8 - - Davis
- - Ib 7357b + 100.00 + H36B - - II 18RS21 - + 100.00 DK21 - - III
NEM316 + 100.00 + 100.00 COH31 + 100.00 + D136 + 100.00 + M732 +
100.00 + 99.88 COH1 + 99.79 + 99.88 M781 + 99.79 + 99.88 No type
CJB110 + 99.37 + 100.00 1169NT - - V CJB111 + 100.00 + 100.00 2603
+ 100.00 + 100.00 VIII JM130013 + 99.79 + 100.00 SMU014 + 100.00 +
total 14/22 99.88 +/- 0.19 15/22 99.96 +/- 0.056
[0730] As another example, the first AI protein may be an AI-2
protein such as GBS 67. The amino acid sequence of GBS 67 from GBS
serotype V, strain isolate 2603 is greater than 90% homologous to
the GBS 67 amino acid sequence from GBS serotype III, strain
isolate NEM316, the GBS 67 amino acid sequence from GBS serotype
1b, strain isolate H36B, and the GBS 67 amino acid sequence from
GBS serotype II, strain isolate 17RS21.
[0731] As another example, the first AI protein may be an AI-2
protein such as spb1. The amino acid sequence of spb1 from GBS
serotype III, strain isolate COH1 is greater than 90% homologous to
the spb1 amino acid sequence from GBS serotype Ia, strain isolate
A909.
[0732] As yet another example, the first AI protein may be an AI-2
protein such as GBS 59. The amino acid sequence of GBS 59 from GBS
serotype II, strain isolate 18RS21 is 100% homologous to the GBS 59
amino acid sequence from GBS serotype V, strain isolate 2603. The
amino acid sequence of GBS 59 from GBS serotype V, strain isolate
CJB111 is 98% homologous to the GBS 59 amino acid sequence from GBS
serotype III, strain isolate NEM316.
[0733] The compositions of the invention may also be designed to
include Gram positive AI proteins from divergent serotypes or
strain isolates, i.e., to include a first AI protein which is
present in one collection of serotypes or strain isolates of a Gram
positive bacteria and a second AI protein which is present in those
serotypes or strain isolates not represented by the first AI
protein.
[0734] For example, the invention may include an immunogenic
composition comprising a first and second Gram positive bacteria AI
protein, wherein a polynucleotide sequence encoding for the full
length sequence of the first AI protein is not present in a similar
Gram positive bacterial genome comprising a polynucleotide sequence
encoding for the second AI protein.
[0735] The compositions of the invention may also be designed to
include AI proteins from divergent GBS serotypes or strain
isolates, i.e., to include a first AI protein which is present in
one collection of GBS serotypes or strain isolates and a second AI
protein which is present in those serotypes or strain isolates not
represented by the first AI protein.
[0736] For example, the invention may include an immunogenic
composition comprising a first and second GBS AI protein, wherein a
polynucleotide sequence encoding for the full length sequence of
the first GBS AI protein is not present in a genome comprising a
polynucleotide sequence encoding for the second GBS AI protein. For
example, the first AI protein could be GBS 80 (such as the GBS 80
sequence from GBS serotype V, strain isolate 2603). As previously
discussed (and depicted in FIG. 2), the sequence for GBS 80 in GBS
sertoype II, strain isolate 18RS21 is disrupted. In this instance,
the second AI protein could be GBS 104 or GBS 67 (sequences
selected from the GBS serotype II, strain isolate 18RS21).
[0737] Further, the the invention may include an immunogenic
composition comprising a first and second GBS AI protein, wherein
the first GBS AI protein has detectable surface exposure on a first
GBS strain or serotype but not a second GBS strain or serotype and
the second GBS AI protein has detectable surface exposure on a
second GBS strain or serotype but not a first GBS strain or
serotype. For example, the first AI protein could be GBS 80 and the
second AI protein could be GBS 67. As seen in Table 15, there are
some GBS serotypes and strains that have surface exposed GBS 80 but
that do not have surface exposed GBS 67 and vice versa. An
immunogenic composition comprising a GBS 80 and a GBS 67 protein
may provide protection across a wider group of GBS strains and
serotypes. TABLE-US-00007 TABLE 15 Antigen surface exposure of GBS
80, GBS 322, GBS 104, and GBS 67 GBS strains Type GBS 80 GBS 322
GBS 104 GBS 67 DK1* Ia 0 nd 237 478 DK8* 0 213 151 475 Davis* 0 86
271 430 515* 0 227 262 409 090 0 0 0 0 A909 0 0 0 0 2986 0 0 157
397 5551 0 36 384 485 2177 Ib 477 323 328 66 H36B* 0 105 518 444
7357b- 91 102 309 316 2129 57 71 132 0 5518 31 nd 60 28 COH1 III
305 130 305 0 D136C 16 460 226 406 COH31 0 479 71 273 M732 105 292
101 0 M781 65 224 136 0 1998 95 288 205 350 5376 165 76 156 0 5435
93 88 100 0 18RS21 II 0 471 50 103 DK21* 0 342 419 331 3050 43 188
289 460 5401 170 135 494 618 2141 0 76 0 69 CJB111 V 365 58 355 481
2603 62 293 100 105 5364 454 463 379 394 2110 0 11 345 589 2274 IV
113 161 465 484 1999 0 55 492 453 2210 0 0 363 574 2928 VII 0 0 0 0
SMU071 VIII 556 170 393 79 JM9130013 587 133 436 83 2189 0 0 0 0
5408 0 0 159 433 CJB110 NT 71 587 169 245 1169* 0 213 371 443
.DELTA. Mean >100 9/40 22/38 32/40 25/40 22% 58% 80% 62%
[0738] Alternatively, the invention may include an immunogenic
composition comprising a first and second Gram positive bacteria AI
protein, wherein the polynucleotide sequence encoding the sequence
of the first AI protein is less than 90% (i.e., less than 90, 88,
86, 84, 82, 80, 78, 76, 74, 72, 70, 65, 60, 55, 50, 45, 40, 35 or
30 percent) homologous than the corresponding sequence in the
genome of the second AI protein.
[0739] The invention may include an immunogenic composition
comprising a first and second GBS AI protein, wherein the
polynucleotide sequence encoding the sequence of the first GBS AI
protein is less than 90% (i.e., less than 90, 88, 86, 84, 82, 80,
78, 76, 74, 72, 70, 65, 60, 55, 50, 45, 40, 35 or 30 percent)
homologous than the corresponding sequence in the genome of the
second GBS AI protein. For example, the first GBS AI protein could
be GBS 67 (such as the GBS 67 sequence from GBS serotype 1b, strain
isolate H36B). As shown in FIGS. 2 and 4, the GBS 67 sequence for
this strain is less than 90% homologous (87%) to the corresponding
GBS 67 sequence in GBS serotype V, strain isolate 2603. In this
instance, the second GBS AI protein could then be the GBS 80
sequence from GBS serotype V, strain isolate 2603.
[0740] An example immunogenic composition of the invention may
comprise adhesin island proteins GBS 80, GBS 104, GBS 67, and GBS
59, and non-AI protein GBS 322. FACS analysis of different GBS
strains demonstrates that at least one of these five proteins is
always found to be expressed on the surface of GBS bacteria. An
initial FACS analysis of 70 strains of GBS bacteria, obtained from
the CDC in the United States (33 strains), ISS in Italy (17
strains), and Houston/Harvard (20 strains), detected surface
exposure of at least one of GBS 80, GBS 104, GBS 322, GBS 67, or
GBS 59 on the surface of the GBS bacteria. FIG. 227 provides the
FACS data obtained for surface exposure of GBS 80, GBS 104, GBS 67,
GBS 322, and GBS 59 on each of 37 GBS strains. FIG. 228 provides
the FACS data obtained for surface exposure of GBS 80, GBS 104, GBS
67, GBS 322, and GBS 59 on each of 41 GBS strains obtained from the
CDC. As can be seen from FIGS. 227 and 228, each GBS strain had
surface expression of at least one of GBS 80, GBS 104, GBS 67, GBS
322, and GBS 59. The surface exposure of at least one of these
proteins on each bacterial strain indicates that an immunogenic
composition comprising these proteins will provide wide protection
across GBS strains and serotypes.
[0741] The surface exposed GBS 80, GBS 104, GBS 67, GBS 322, and
GBS 59 proteins are also present at high levels as determined by
FACS. Table 49 summarizes the FACS results for the initial 70 GBS
strains examined for GBS 80, GBS 104, GBS 67, GBS 322, and GBS 59
surface expression. A protein was designated as having high levels
of surface expression of a protein if a five-fold shift in
fluorescence was observed when using antibodies for the protein
relative to preimmune control serum. TABLE-US-00008 TABLE 49
Exposure Levels of GBS 80, GBS 104, GBS 67, GBS 322, and GBS 59 on
GBS Strains GBS 80 GBS 104 GBS 67 GBS 59 GBS 322 5-fold shift in
17/70 14/70 49/70 46/70 33/70 fluorescence 24% 20% 70% 66% 47% by
FACS
[0742] Table 50 details which of the surface proteins is highly
expressed on the different GBS serotype. TABLE-US-00009 TABLE 50
High Levels of Surface Protein Expression on GBS Serotypes 5-fold
shift in fluorescence by FACS GBS 80 GBS 104 GBS 67 GBS 59 GBS 322
Ia + Ib + III 4/36 2/36 22/36 20/36 18/36 II + V 11/25 9/25 21/25
21/25 13/25 Others 2/9 3/9 6/9 5/9 2/9
[0743] Alternatively, the immunogenic composition of the invention
may include GBS 80, GBS 104, GBS 67, and GBS 322. Assuming that
protein antigens that are highly accessible to antibodies confer
100% protection with suitable adjuvants, an immunogenic composition
containing GBS 80, GBS 104, GBS 67, GBS 59 and GBS 322 will provide
protection for 89% of GBS strains and serotypes, the same
percentage as an immunogenic composition containing GBS 80, GBS
104, GBS 67, and GBS 322 proteins. See FIG. 229. However, it may be
preferable to include GBS 59 in the composition to increase its
immunogenic strength. As seen from Table 50, GBS 59 is highly
expressed on the surface two-thirds of GBS bacteria examined by
FACS analysis, unlike GBS 80, GBS 104, and GBS 322, which are
highly expressed in less than half of GBS bacteria examined. GBS 59
opsonophagocytic activity is also comparable to that of a mix of
GBS 322, GBS 104, GBS 67, and GBS 80 proteins. See FIG. 230.
[0744] By way of another example, preferably, the GAS AI proteins
included in the immunogenic compositions of the invention can
provide protection across more than one GAS serotype or strain
isolate. For example, the immunogenic composition may comprise a
first GAS AI protein, wherein the amino acid sequence of said AI
protein is at least 90% (i.e., at least 90, 91, 92, 93, 94, 95, 96,
97, 98, 99 or 100%) homologous to the amino acid sequence of a
second GAS AI protein, and wherein said first AI protein and said
second AI protein are derived from the genomes of different GAS
serotypes. The first GAS AI protein may also be homologous to the
amino acid sequence of a third GAS AI protein, such that the first
AI protein, the second AI protein and the third AI protein are
derived from the genomes of different GAS serotypes. The first AI
protein may also be homologous to the amino acid sequence of a
fourth GAS AI protein, such that the first AI protein, the second
AI protein and the third AI protein are derived from the genomes of
different GAS serotypes.
[0745] The compositions of the invention may also be designed to
include GAS AI proteins from divergent serotypes or strain
isolates, i.e., to include a first AI protein which is present in
one collection of serotypes or strain isolates of a GAS bacteria
and a second AI protein which is present in those serotypes or
strain isolates not represented by the first AI protein.
[0746] For example, the first AI protein could be a prtF2 protein
(such as the 19224141 protein from GAS serotype M12, strain isolate
A735). As previously discussed (and depicted in FIG. 164), the
sequence for a prtF2 protein is not present in GAS AI types 1 or 2.
In this instance, the second AI protein could be collagen binding
protein M6_Spy0159 (from M6 isolate (MGAS10394), which comprises an
AI-1) or GAS15 (from M1 isolate (SF370), which comprises an
AI-2).
[0747] Further, the invention may include an immunogenic
composition comprising a first and second GAS AI protein, wherein
the first GAS AI protein has detectable surface exposure on a first
GAS strain or serotype but not a second GAS strain or serotype and
the second GAS AI protein has detectable surface exposure on a
second GAS strain or serotype but not a first GAS strain or
serotype.
[0748] The invention may include an immunogenic composition
comprising a first and second GAS AI protein, wherein the
polynucleotide sequence encoding the sequence of the first GAS AI
protein is less than 90% (i.e., less than 90, 88, 86, 84, 82, 80,
78, 76, 74, 72, 70, 65, 60, 55, 50, 45, 40, 35 or 30 percent)
homologous than the corresponding sequence in the genome of the
second GAS AI protein. Preferably the first and second GAS AI
proteins are subunits of the pilus. More preferably the first and
second GAS AI proteins are selected from the major pilus forming
proteins (i.e., M6_Spy0160 from M6 strain 10394, SPy0128 from M1
strain SF370, SpyM3.sub.--0100 from M3 strain 315, SPs0102 from M3
strain SSI, orf80 from M5 isolate Manfredo, spyM18.sub.--0128 from
M18 strain 8232, SpyoM01000153 from M49 strain 591, 19224137 from
M12 strain A735, fimbrial structural subunit from M77 strain
ISS4959, fimbrial structural subunit from M44 strain ISS3776,
fimbrial structural subunit from M50 strain ISS3776 ISS 4538,
fimbrial structural subunit from M12 strain CDC SS635, fimbrial
structural subunit from M23 strain DSM2071, fimbrial structural
subunit from M6 strain CDC SS410). Table 45 provides the percent
identity between the amino acidic sequences of each of the main
pilus forming subunits from GAS AI-1, AI-2, AI-3, and AI-4
representative strains (i.e., M6_Spy0160 from M6 strain 10394,
SPy0128 from M1 strain SF370, SpyM3.sub.--0100 from M3 strain 315,
SPs0102 from M3 strain SSI, orf80 from M5 isolate Manfredo,
spyM18.sub.--0128 from M18 strain 8232, SpyoM01000153 from M49
strain 591, 19224137 from M12 strain A735, Fimbrial structural
subunit from M77 strain ISS4959, fimbrial structural subunit from
M44 strain ISS3776, fimbrial structural subunit from M50 strain
ISS3776 ISS 4538, fimbrial structural subunit from M12 strain CDC
SS635, fimbrial structural subunit from M23 strain DSM2071,
fimbrial structural subunit from M6 strain CDC SS410).
TABLE-US-00010 TABLE 45 Comparison of Amino Acid Sequences of Major
Pilus Proteins in the Four GAS AIs ##STR1##
[0749] For example, the first main pilus subunit may be selected
from bacteria of GAS serotype M6 strain 10394 and the second main
pilus subunit may be selected from bacteria of GAS serotype M1
strain 370. As can be seen from Table 45, the main pilus subunits
encoded by these strains of bacteria share only 23% nucleotide
identity. An immunogenic composition comprising pilus main subunits
from each of these strains of bacteria is expected to provide
protection across a wider group of GAS strains and serotypes. Other
examples of main pilus subunits that can be used in combination to
provide increased protection across a wider range of GAS strains
and serotypes include proteins encoded by GAS serotype M5 Manfredo
isolate and serotype M6 strain 10394, which share 23% sequence
identity, GAS serotype M18 strain 8232 and serotype M1 strain 370,
which share 38% sequence identity, GAS serotype M3 strain 315 and
serotype M12 strain A735, which share 61% sequence identity, and
GAS serotype M3 strain 315 and serotype M6 strain 10394 which share
25% sequence identity.
[0750] As also can be seen from Table 45, the amino acid sequences
of the four types of main pilus subunits present in GAS are
relatively divergent. FIGS. 198-201 provide further tables
comparing the percent identity of adhesin island-encoded surface
exposed proteins for different GAS serotypes relative to other GAS
serotypes harbouring an adhesin island of the same or a different
subtype (GAS AI-1, GAS AI-2, GAS AI-3, and GAS AI-4). See also
further discussion below.
[0751] Immunizations with the Adhesin Island proteins of the
invention are discussed further in the Examples.
Co-Expression of GBS Adhesin Island Proteins and Role of GBS AI
Proteins in Surface Presentation
[0752] In addition to the use of the GBS adhesin island proteins
for cross strain and cross serotype protection, Applicants have
identified interactions between adhesin island proteins which
appear to affect the delivery or presentation of the surface
proteins on the surface of the bacteria.
[0753] In particular, Applicants have discovered that surface
exposure of GBS 104 is dependent on the concurrent expression of
GBS 80. As discussed further in Example 2, reverse transcriptase
PCR analysis of AI-1 shows that all of the AI genes are
co-transcribed as an operon. Applicants constructed a series of
mutant GBS containing in frame deletions of various AI-1 genes. (A
schematic of the GBS mutants is presented in FIG. 7). FACS analysis
of the various mutants comparing mean shift values using anti-GBS
80 versus anti-GBS 104 antibodies is presented in FIG. 8. Removal
of the GBS 80 operon prevented surface exposure of GBS 104; removal
of the GBS 104 operon did not affect surface exposure of GBS 80.
While not being limited to a specific theory, it is thought that
GBS 80 is involved in the transport or localization of GBS 104 to
the surface of the bacteria. The two proteins may be oligomerized
or otherwise associated. It is possible that this association
involves a conformational change in GBS 104 that facilitates its
transition to the surface of the GBS bacteria.
[0754] Pili structures that comprise GBS 104 appear to be of a
lower molecular weight than pili structures lacking GBS 104. FIG.
68 shows that polyclonal anti-GBS 104 antibodies (see lane marked
.alpha.-104 POLIC.) cross-hybridize with smaller structures than do
polyclonal anti-GBS 80 antibodies (see lane marked .alpha.-GBS 80
POLIC.).
[0755] In addition, Applicants have shown that removal of GBS 80
can cause attenuation, further suggesting the protein contributes
to virulence. As described in more detail in Example 3, the
LD.sub.50's for the .DELTA.80 mutant and the .DELTA.80, .DELTA.104
double mutant were reduced by an order of magnitude compared to
wildtype and .DELTA.104 mutant.
[0756] The sortases within the adhesin island also appear to play a
role in localization and presentation of the surface proteins. As
discussed further in Example 4, FACS analysis of various sortase
deletion mutants showed that removal of sortase SAG0648 prevented
GBS 104 from reaching the surface and slightly reduced the surface
exposure of GBS 80. When sortase SAG0647 and sortase SAG0648 were
both knocked out, neither GBS 80 nor GBS 104 were surface exposed.
Expression of either sortase alone was sufficient for GBS 80 to
arrive at the bacterial surface. Expression of SAG0648, however,
was required for GBS 104 surface localization.
[0757] Accordingly, the compositions of the invention may include
two or more AI proteins, wherein the AI proteins are physically or
chemically associated. For example, the two AI proteins may form an
oligomer. In one embodiment, the associated proteins are two AI
surface proteins, such as GBS 80 and GBS 104. The associated
proteins may be AI surface proteins from different adhesin islands,
including host cell adhesin island proteins if the AI surface
proteins are expressed in a recombinant system. For example, the
associated proteins may be GBS 80 and GBS 67.
Adhesin Island Proteins from Other Gram Positive Bacteria
[0758] Applicants' identification and analysis of the GBS adhesin
islands and the immunological and biological functions of these AI
proteins and their pilus structures provides insight into similar
structures in other Gram positive bacteria.
[0759] As discussed above, "Adhesin Island" or "AI" refers to a
series of open reading frames within a bacterial genome that encode
for a collection of surface proteins and sortases. An Adhesin
Island may encode for amino acid sequences comprising at least one
surface protein. The Adhesin Island may encode at least one surface
protein. Alternatively, an Adhesin Island may encode for at least
two surface proteins and at least one sortase. Preferably, an
Adhesin Island encodes for at least three surface proteins and at
least two sortases. One or more of the surface proteins may include
an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) or other sortase
substrate motif. One or more AI surface proteins may participate in
the formation of a pilus structure on the surface of the Gram
positive bacteria.
[0760] Gram positive adhesin islands of the invention preferably
include a divergently transcribed transcriptional regulator. The
transcriptional regulator may regulate the expression of the AI
operon.
[0761] The invention includes a composition comprising one or more
Gram positive bacteria AI surface proteins. Such AI surface
proteins may be associated in an oligomeric or hyperoligomeric
structure.
[0762] Preferred Gram positive adhesin island proteins for use in
the invention may be derived from Staphylococcus (such as S.
aureus), Streptococcus (such as S. agalactiae (GBS), S. pyogenes
(GAS), S. pneumonaie, S. mutans), Enterococcus (such as E. faecalis
and E. faecium), Clostridium (such as C. difficile), Listeria (such
as L. monocytogenes) and Corynebacterium (such as C.
diphtheria).
[0763] One or more of the Gram positive AI surface protein
sequences typically include an LPXTG motif or other sortase
substrate motif. Gram positive AI surface proteins of the invention
may affect the ability of the Gram positive bacteria to adhere to
and invade epithelial cells. AI surface proteins may also affect
the ability of Gram positive bacteria to translocate through an
epithelial cell layer. Preferably, one or more AI surface proteins
are capable of binding to or otherwise associating with an
epithelial cell surface. Gram positive AI surface proteins may also
be able to bind to or associate with fibrinogen, fibronectin, or
collagen.
[0764] Gram positive AI sortase proteins are predicted to be
involved in the secretion and anchoring of the LPXTG containing
surface proteins. A Gram positive bacteria AI may encode for at
least one surface exposed protein. The Adhesin Island may encode at
least one surface protein. Alternatively, a Gram positive bacteria
AI may encode for at least two surface exposed proteins and at
least one sortase. Preferably, a Gram positive AI encodes for at
least three surface exposed proteins and at least two sortases.
[0765] Gram positive AI surface proteins may be covalently attached
to the bacterial cell wall by membrane-associated transpeptidases,
such as an AI sortase. The sortase may function to cleave the
surface protein, preferably between the threonine and glycine
residues of an LPXTG motif. The sortase may then assist in the
formation of an amide link between the threonine carboxyl group and
a cell wall precursor such as lipid II. The precursor can then be
incorporated into the peptidoglycan via the transglycoslylation and
transpeptidation reactions of bacterial wall synthesis. See Comfort
et al., Infection & Immunity (2004) 72(5): 2710-2722.
Typically, Gram positive bacteria AI surface proteins of the
invention will contain an N-terminal leader or secretion signal to
facilitate translocation of the surface protein across the
bacterial membrane.
[0766] Gram positive bacteria AI surface proteins of the invention
may affect the ability of the Gram positive bacteria to adhere to
and invade target host cells, such as epithelial cells. Gram
positive bacteria AI surface proteins may also affect the ability
of the gram positive bacteria to translocate through an epithelial
cell layer. Preferably, one or more of the Gram positive AI surface
proteins are capable of binding to or other associating with an
epithelial cell surface. Further, one or more Gram positive AI
surface proteins may bind to fibrinogen, fibronectin, or collagen
protein.
[0767] In one embodiment, the invention includes a composition
comprising oligomeric, pilus-like structures comprising a Gram
positive bacteria AI surface protein. The oligomeric, pilus-like
structure may comprise numerous units of the AI surface protein.
Preferably, the oligomeric, pilus-like structures comprise two or
more AI surface proteins. Still more preferably, the oligomeric,
pilus-like structure comprises a hyper-oligomeric pilus-like
structure comprising at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90,
100, 120, 140, 150, 200 or more) oligomeric subunits, wherein each
subunit comprises an AI surface protein or a fragment thereof. The
oligomeric subunits may be covalently associated via a conserved
lysine within a pilin motif. The oligomeric subunits may be
covalently associated via an LPXTG motif, preferably, via the
threonine amino acid residue.
[0768] Gram positive bacteria AI surface proteins or fragments
thereof to be incorporated into the oligomeric, pilus-like
structures of the invention will preferably include one or both of
a pilin motif comprising a conserved lysine residue and an E box
motif comprising a conserved glutamic acid residue.
[0769] The oligomeric, pilus like structures may be used alone or
in the combinations of the invention. In one embodiment, the
invention comprises a Gram positive bacteria Adhesin Island in
oligomeric form, preferably in a hyperoligomeric form.
[0770] The oligomeric, pilus-like structures of the invention may
be combined with one or more additional Gram positive AI proteins
(from the same or a different Gram positive species or genus). In
one embodiment, the oligomeric, pilus-like structures comprise one
or more Gram positive bacteria AI surface proteins in combination
with a second Gram positive bacteria protein. The second Gram
positive bacteria protein may be a known antigen, and need not
normally be associated with an AI protein.
[0771] The oligomeric, pilus-like structures may be isolated or
purified from bacterial cultures overexpressing a Gram positive
bacteria AI surface protein. The invention therefore includes a
method for manufacturing an oligomeric Adhesin Island surface
antigen comprising culturing a Gram positive bacteria adapted for
increased AI protein expression and isolation of the expressed
oligomeric Adhesin Island protein from the Gram positive bacteria.
The AI protein may be collected from secretions into the
supernatant or it may be purified from the bacterial surface. The
method may further comprise purification of the expressed Adhesin
Island protein. Preferably, the Adhesin Island protein is in a
hyperoligomeric form.
[0772] Gram positive bacteria are preferably adapted to increase AI
protein expression by at least two (e.g., 2, 3, 4, 5, 8, 10, 15,
20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150 or 200)
times wild type expression levels.
[0773] Gram positive bacteria may be adapted to increase AI protein
expression by means known in the art, including methods of
increasing gene dosage and methods of gene upregulation. Such means
include, for example, transformation of the Gram positive bacteria
with a plasmid encoding the AI protein. The plasmid may include a
strong promoter or it may include multiple copies of the sequence
encoding the AI protein. Optionally, the sequence encoding the AI
protein within the Gram positive bacterial genome may be deleted.
Alternatively, or in addition, the promoter regulating the Gram
positive Adhesin Island may be modified to increase expression.
[0774] The invention further includes Gram positive bacteria which
have been adapted to produce increased levels of AI surface
protein. In particular, the invention includes Gram positive
bacteria which have been adapted to produce oligomeric or
hyperoligomeric AI surface protein. In one embodiment, the Gram
positive bacteria of the invention are inactivated or attenuated to
permit in vivo delivery of the whole bacteria, with the AI surface
protein exposed on its surface.
[0775] The invention further includes Gram positive bacteria which
have been adapted to have increased levels of expressed AI protein
incorporated in pili on their surface. The Gram positive bacteria
may be adapted to have increased exposure of oligomeric or
hyperoligomeric AI proteins on its surface by increasing expression
levels of a signal peptidase polypeptide. Increased levels of a
local signal peptidase expression in Gram positive bacteria (such
us LepA in GAS) are expected to result in increased exposure of
pili proteins on the surface of Gram positive bacteria. Increased
expression of a leader peptidase in Gram positive may be achieved
by any means known in the art, such as increasing gene dosage and
methods of gene upregulation. The Gram positive bacteria adapted to
have increased levels of leader peptidase may additionally be
adapted to express increased levels of at least one pili
protein.
[0776] Alternatively, the AI proteins of the invention may be
expressed on the surface of a non-pathogenic Gram positive
bacteria, such as Streptococus gordonii (See, e.g., Byrd et al.,
"Biological consequences of antigen and cytokine co-expression by
recombinant Streptococcus gordonii vaccine vectors", Vaccine (2002)
20:2197-2205) or Lactococcus lactis (See, e.g., Mannam et al.,
"Mucosal VaccineMade from Live, Recombinant Lactococcus lactis
Protects Mice against Pharangeal Infection with Streptococcus
pyogenes" Infection and Immunity (2004) 72(6):3444-3450). It has
already been demonstrated, above, that L. lactis expresses GBS and
GAS AI polypeptides in oligomeric form and on its surface.
[0777] Alternatively, the oligomeric, pilus-like structures may be
produced recombinantly. If produced in a recombinant host cell
system, the Gram positive bacteria AI surface protein will
preferably be expressed in coordination with the expression of one
or more of the AI sortases of the invention. Such AI sortases will
facilitate oligomeric or hyperoligomeric formation of the AI
surface protein subunits.
[0778] Gram positive AI Sortases of the invention will typically
have a signal peptide sequence within the first 70 amino acid
residues. They may also include a transmembrane sequence within 50
amino acid residues of the C terminus. The sortases may also
include at least one basic amino acid residue within the last 8
amino acids. Preferably, the sortases have one or more active site
residues, such as a catalytic cysteine and histidine.
[0779] Adhesin island surface proteins from two or more Gram
positive bacterial genus or species may be combined to provide an
immunogenic composition for prophylactic or therapeutic treatment
of disease or infection of two more Gram positive bacterial genus
or species. Optionally, the adhesin island surface proteins may be
associated together in an oligomeric or hyperoligomeric
structure.
[0780] In one embodiment, the invention comprises an adhesin island
surface proteins from two or more Streptococcus species. For
example, the invention includes a composition comprising a GBS AI
surface protein and a GAS adhesin island surface protein. As
another example, the invention includes a composition comprising a
GAS adhesin island surface protein and a S. pneumoniae adhesin
island surface protein.
[0781] In one embodiment, the invention comprises an adhesin island
surface protein from two or more Gram positive bacterial genus. For
example, the invention includes a composition comprising a
Streptococcus adhesin island protein and a Corynebacterium adhesin
island protein.
[0782] Examples of AI sequences in several Gram positive bacteria
are discussed further below.
Streptococcus pyogenes (GAS)
[0783] As discussed above, Applicants have identified at least four
different GAS Adhesin Islands. These adhesion islands are thought
to encode surface proteins which are important in the bacteria's
virulence, and Applicants have obtained the first electron
micrographs revealing the presence of these adhesin island proteins
in hyperoligomeric pilus structures on the surface of Group A
Streptococcus.
[0784] Group A Streptococcus is a human specific pathogen which
causes a wide variety of diseases ranging from pharyngitis and
impetigo through life threatening invasive disease and necrotizing
fasciitis. In addition, post-streptococcal autoimmune responses are
still a major cause of cardiac pathology in children.
[0785] Group A Streptococcal infection of its human host can
generally occur in three phases. The first phase involves
attachment and/or invasion of the bacteria into host tissue and
multiplication of the bacteria within the extracellular spaces.
Generally this attachment phase begins in the throat or the skin.
The deeper the tissue level infected, the more severe the damage
that can be caused. In the second stage of infection, the bacteria
secrete a soluble toxin that diffuses into the surrounding tissue
or even systemically through the vasculature. This toxin binds to
susceptible host cell receptors and triggers innappropropriate
immune responses by these host cells, resulting in pathology.
Because the toxin can diffuse throughout the host, the necrosis
directly caused by the GAS toxins may be physically located in
sites distant from the bacterial infection. The final phase of GAS
infection can occur long after the original bacteria have been
cleared from the host system. At this stage, the host's previous
immune response to the GAS bacteria due to cross reactivity between
epitopes of a GAS surface protein, M, and host tissues, such as the
heart. A general review of GAS infection can be found in Principles
of Bacterial Pathogeneis, Groisman ed., Chapter 15 (2001).
[0786] In order to prevent the pathogenic effects associated with
the later stages of GAS infection, an effective vaccine against GAS
will preferably facilitate host elimination of the bacteria during
the initial attachment and invasion stage.
[0787] Isolates of Group A Streptococcus are historically
classified according to the M surface protein described above. The
M protein is surface exposed trypsin-sensitive protein generally
comprising two polypeptide chains complexed in an alpha helical
formation. The carboxyl terminus is anchored in the cytoplasmic
membrane and is highly conserved among all group A streptococci.
The amino terminus, which extends through the cell wall to the cell
surface, is responsible for the antigenic variability observed
among the 80 or more serotypes of M proteins.
[0788] A second layer of classification is based on a variable,
trypsin-resistant surface antigen, commonly referred to as the
T-antigen. Decades of epidemiology based on M and T serological
typing have been central to studies on the biological diversity and
disease causing potential of Group A Streptococci. While the
M-protein component and its inherent variability have been
extensively characterized, even after five decades of study, there
is still very little known about the structure and variability of
T-antigens. Antisera to define T types are commercially available
from several sources, including Sevapharma
(http://www.sevapharma.cz/en).
[0789] The gene coding for one form of T-antigen, T-type 6, from an
M6 strain of GAS (D741) has been cloned and characterized and maps
to an approximately 11 kb highly variable pathogenicity island.
Schneewind et al., J. Bacteriol. (1990) 172(6):3310-3317. This
island is known as the Fibronectin-binding, Collagen-binding
T-antigen (FCT) region because it contains, in addition to the T6
coding gene (tee6), members of a family of genes coding for Extra
Cellular Matrix (ECM) binding proteins. Bessen et al., Infection
& Immunity (2002) 70(3):1159-1167. Several of the protein
products of this gene family have been shown to directly bind
either fibronectin and/or collagen. See Hanski et al., Infection
& Immunity (1992) 60(12):5119-5125; Talay et al., Infection
& Immunity (1992(60(9):3837-3844; Jaffe et al. (1996)
21(2):373-384; Rocha et al., Adv Exp Med Biol. (1997) 418:737-739;
Kreikemeyer et al., J Biol Chem (2004) 279(16):15850-15859;
Podbielski et al., Mol. Microbiol. (1999) 31(4):1051-64; and
Kreikemeyer et al., Int. J. Med Microbiol (2004) 294(2-3):177-88.
In some cases direct evidence for a role of these proteins in
adhesion and invasion has been obtained.
[0790] Applicants raised antiserum against a recombinant product of
the tee6 gene and used it to explore the expression of T6 in M6
strain ISS3650. In immunoblot of mutanolysin extracts of this
strain, the antiserum recognized, in addition to a band
corresponding to the predicted molecular mass of the tee6 gene
product, very high molecular weight ladders ranging in mobility
from about 100 kDa to beyond the resolution of the 3-8% gradient
gels used. See FIG. 163A, last lane labeled "M6 Tee6."
[0791] This pattern of high molecular weight products is similar to
that observed in immunoblots of the protein components of the pili
identified in Streptococcus agalactiae (described above) and
previously in Corynebacterium diphtheriae. Electron microscropy of
strain M6 ISS3650 with antisera specific for the product of tee6
revealed abundant surface staining and long pilus like structures
extending up to 700 nanometers from the bacterial surface,
revealing that the T6 protein, one of the antigens recognized in
the original Lancefield serotyping system, is located within a GAS
Adhesin Island (GAS AI-1) and forms long covalently linked pilus
structures. See FIG. 163I.
[0792] In addition to the tee6 gene, the FCT region in M6_ISS3650
(GAS AI-1) contains two other genes (prtF1 and cpa) predicted to
code for surface exposed proteins; these proteins are characterized
as containing the cell wall attachment motif LPXTG. Western blot
analysis using antiserum specific for PrtF1 detected a single
molecular species with electrophoretic mobility corresponding to
the predicted molecular mass of the protein and one smaller band of
unknown origin. Western blot analysis using antisera specific for
Cpa recognized a high molecular weight covalently linked ladder
(FIG. 163A, second lane). Immunogold labelling of Cpa with specific
antiserum followed by transmission electron microscopy detected an
abundance of Cpa at the cell surface and only occasional structures
extending from the cell surface (FIG. 163J).
[0793] Four classes of FCT region can be discerned by the types and
order of the genes contained within the region. The FCT region of
strains of types M3, M5, M18 and M49 have a similar organization
whereas those of M6, M1 and M12 differ. See FIG. 164. As discussed
below, these four FCT regions correlate to four GAS Adhesin Island
types (AI-1, AI-2, AI-3 and AI-4).
[0794] Applicants discovery of genes coding for pili in the FCT
region of strain M6_ISS3650 prompted them to examine the predicted
surface exposed proteins in the variant FCT regions of three other
GAS strains of having different M-type (M1_SF370, M5_ISS4883 and
M12.sub.--20010296) representing the other three FCT variants. Each
gene present in the FCT region of each bacteria was cloned and
expressed. Antisera specific for each recombinant protein was then
used to probe mutanolysin extracts of the respective strains (6).
In M1 strain SF370, there are three predicted surface proteins (Cpa
(also referred to as M1.sub.--126 and GAS 15), M1.sub.--128 (a
fimbrial protein also referred to as Spy0128 and GAS 16), and
M1.sub.--130 (also referred to as Spy0130 and GAS 18)) (GAS AI-2).
Antisera specific for each surface protein reacted with a ladder of
high molecular weight material (FIG. 163B). Immunogold staining of
M1 strain SF370 with antiserum specific for M1.sub.--128 revealed
pili structures similar to those seen when M6 strain ISS3650 was
immunogold stained with antiserum specific for tee6 (See FIG.
1163K). Antisera specific for surface proteins Cpa and M1.sub.--130
revealed abundant surface staining and occasional structures
extending from the surface of M1 strain SF370 bacteria (FIG.
163S).
[0795] The M1.sub.--128 protein appears to be necessary for
polymerization of Cpa and M1.sub.--130 proteins. If the
M1.sub.--128 gene in M1_SF370 was deleted, Western blot analysis
using antibodies that hybridize to Cpa and M1.sub.--130 no longer
detected high molecular weight ladders comprising the Cpa and
M1.sub.--130 proteins (FIG. 163 E). See also FIGS. 177 A-C which
provide the results of Western blot analysis of the M1.sub.--128
(.DELTA.128) deleted bacteria using anti-M1.sub.--130 antiserum
(FIG. 177 A), anti-M1.sub.--128 antiserum (FIG. 177 B), and
anti-M1.sub.--126 antiserum (FIG. 177 C). High molecular weight
ladders, indicative of pilus formation on the surface of M1 strain
SF370, could not be detected by any of the three antisera in
.DELTA.128 bacteria. If the .DELTA.128 bacteria were transformed
with a plasmid containing the gene for M1.sub.--128, Western blot
analysis using antisera specific for Cpa and M1.sub.--130 again
detected high molecular weight ladders (FIG. 163 H).
[0796] In agreement with the Western blot analysis, immunoelectron
microscopy failed to detect pilus assembly on the .DELTA.128 strain
SF370 bacteria using M1.sub.--128 antisera (FIG. 178 B). Although
.DELTA.128 SF370 bacteria were unable to form pili, M1.sub.--126
(cpa) and M1.sub.--130, which contain sortase substrate motifs,
were present on the bacteria's surface. FACS analysis of the
M1.sub.--128 deleted (.DELTA.128) strain SF370 bacteria also
detected both M1.sub.--126 and M1.sub.--130 on the surface of the
.DELTA.128 strain SF370 bacteria. See FIG. 179 D and F, which show
a shift in fluorescence when antibodies immunoreactive to
M1.sub.--126 and M1.sub.--130 are used on .DELTA.128 bacteria. As
expected, virtually no shift in fluorescence is observed when
antibodies immunoreactive to M1.sub.--128 are used with the
.DELTA.128 bacteria (FIG. 179 E).
[0797] By contrast, deletion of the M1.sub.--130 gene did not
effect polymerization of M1.sub.--128 (FIG. 163 F). See also FIGS.
177 A-C, which provide Western blot analysis results of the
M1.sub.--130 deleted (.DELTA.130) strain SF370 bacteria using
anti-M1.sub.--130 (FIG. 177 A), anti-M1.sub.--128 (FIG. 177 B), and
anti-M1.sub.--126 antiserum (FIG. 177 C). The anti-M1.sub.--128 and
anti-M1.sub.--126 antiserum both detected the presence of high
molecular weight ladders in the A130 strain SF370 bacteria,
indicating that the A130 bacteria form pili that comprise
M1.sub.--126 and M1.sub.--128 polypeptides in the absence of
M1.sub.--130. As expected, the Western blot probed with antiserum
immunoreactive with M1.sub.--130 did not detect any proteins for
the .DELTA.130 bacteria (FIG. 177A).
[0798] Hence, the composition of the pili in GAS resembles that
previously described for both C. diphtheria (7, 8) and S.
agalactiae (described above) (9) in that each pilus is formed by a
backbone component which abundantly stains the pili in EM and is
essential for the incorporation of the other components.
[0799] Also similar to C. diphtheria, elimination of the srtC1 gene
from the FCT region of M1_SF370 abolished polymerization of all
three proteins and assembly of pili (FIG. 163 G). See also FIGS.
177 A-C, which provide Western blot analysis of the SrtC1 deleted
(.DELTA.srtC1) strain SF370 bacteria using anti-M1.sub.--130 (FIG.
177 A), anti-M1.sub.--128 (FIG. 177 B), and anti-M1.sub.--126
antiserum (FIG. 177 C). None of the three antisera immunoreacted
with high molecular weight structures (pili) in the .DELTA.SrtC1
bacteria. Confirming that deletion of the SrtC1 gene abrogates
pilus assembly in strain SF370, immunoelectron microscopy using
antisera against M1.sub.--128 failed to detect pilus formation on
the bacteria surface. See FIG. 178 C. Although no assembled pili
were detected on .DELTA.SrtC1 SF370, M1.sub.--128 proteins could be
detected on the surface of SF370. Thus, it appeared that SrtC1
deletion prevented pilus assembly on the surface of the SF370
bacteria, but not anchoring of the proteins that comprise pili to
the bacterial cell wall. FACS analysis of the .DELTA.SrtC1 strain
SF370 confirmed that deletion of SrtC1 does not eliminate cell
surface expression of M1.sub.--126, M1.sub.--128 or M1.sub.--130.
See FIG. 179 G-I, which show a shift in fluorescence when
antibodies immunoreactive to M1.sub.--126 (FIG. 179 G),
M1.sub.--128 (FIG. 179 H), and M1.sub.--130 (FIG. 179 I) are used
to detect cell surface protein expression on .DELTA.SrtC1 bacteria.
Thus, SrtC1 deletion prevents pilus formation, but not surface
anchoring of proteins involved in pilus formation on the surface of
bacteria. Another sortase is possibly involved in anchoring of the
proteins to the bacteria surface. Pilus polymerization in C.
diphtheriae is also dependent on particular sortase enzyme whose
gene resides at the same genetic locus as the pilus components (7,
8).
[0800] The LepA signal peptidase, Spy0127, also appears to be
essential for pilus assembly in strain SF370. LepA deletion mutants
(.DELTA.LepA) of strain SF370 fail to assemble pili on the cell
surface. Not only are the .DELTA.LepA mutants unable to assemble
pili, they are also deficient at cell surface M1 expression. See
FIG. 180, which provides a FACS analysis of the wildtype (A) and
.DELTA.LepA mutant (B) SF370 bacteria using M1 antisera. No shift
in fluorescence is observed for the .DELTA.LepA mutant bacteria in
the presence of M1 immune serum. It is possible that these deletion
mutants of LepA will be useful for detecting non-M, non-pili,
surface exposed antigens on the surface of GAS, or any Gram
positive bacteria. These antigens may also be useful in immunogenic
compositions.
[0801] Pili were also observed in M5 strain ISS4882 and M12 strain
20010296. The M5 strain ISS4882 contains genes for four predicted
surface exposed proteins (GAS AI-3). Antisera against three of the
four products of the FCT region (GAS AI-3) of M5_ISS4883 (Cpa,
M5_orf80, M5_orf82) stained high molecular weight ladders in
Western blot analysis (FIG. 163 C). Long pili were visible when
antisera against M5_orf80 was used in immunogold staining followed
by electron microscopy (FIG. 163L).
[0802] The M12 strain 20010296 contains genes for five predicted
surface exposed proteins. (GAS AI-4) Antisera against three of the
five products of the FCT region (GAS AI-4) of M12.sub.--20010296
(Cpa, EftLSL.A, Orf2) stained high molecular weight ladders in
Westen blot analysis (FIG. 163 D). Long pili were visible when
antisera against EftLSL.A were used (FIG. 163M).
[0803] The major pilus forming proteins identified in the four
strains studied by applicants (T6, M1.sub.--128, M5_orf80 and
EftLSL.A) share between 23% and 65% amino acid identity in any
pairwise comparison, indicating that each pilus may represent a
different Lancefield T-antigen. Each pilus is part of a trypsin
resistant structure on the GAS bacteria surface, as is the case for
the Lancefield T antigens. See FIG. 165, which provides a FACS
analysis of bacteria harboring each of the FCT types that had or
had not been treated with trypsin (6). Following treatment, surface
expression of the pilus proteins was assayed by indirect
immunofluorescence and flow cytometry using antibodies specific for
the pilus proteins, the bacteria's respective M proteins, or
surface proteins not associated with the pili (FIG. 165). Staining
the cells with sera specific for proteins associated with the pili
was not effected by trypsin treatment, whereas trypsin treatment
substantially reduced detection of M-proteins or surface proteins
not associated with pili.
[0804] The pili structures identified on the surface of the GAS
bacteria were confirmed to be Lancefield T antigens when
commercially available T-serotyping sera detected the pili on the
surface of bacteria. Western blot analysis was initially performed
to determine if polyvalent serum pools (designated T, U, W, X, and
Y) could detect recombinant proteins for each of the major pilis
components (T6, M1.sub.--128, M5_orf80 and EftLSL.A) identified in
the strains of bacteria discussed above. Pool U, which contains the
T6 serum, recognized the T6 protein specifically (a surface exposed
pilus protein from GAS AI-1) (FIG. 166 B). Pool T specifically
recognized M1.sub.--128 (a surface exposed pilus protein from GAS
AI-2) (FIG. 166 A). Pool W recognized both M5_orf80 and EftLSL.A
(FIG. 166 C). Using monovalent sera representative of each of the
components of each polyvalent pool, applicants confirmed the
specificity of the T6 antigen (corresponding to a surface exposed
pilus protein from GAS AI-1) (FIG. 166 E) and identified
M1.sub.--28 as antigen T1 (corresponding to a surface exposed pilus
protein from GAS AI-2) (FIG. 166 D), EftLSL.A as antigen T12
(corresponding to a surface exposed pilus protein from GAS AI-4)
(FIG. 166 G) and M5_orf80 as a common antigen recognized by the
related sera T5, T27 and T44 (corresponding to a surface exposed
pilus protein from GAS AI-3).
[0805] Confirming applicants observations, discussed above, that
deleting the M1.sub.--128 gene from M1_SF370 abolishes pilus
formation, the pool T sera stained whole M1_SF370 bacteria (FIG.
166 H) but failed to stain M1_SF370 bacteria lacking the
M1.sub.--128 gene (FIG. 166 I).
[0806] As discussed above, Applicants have identified at least four
different Group A Streptococcus Adhesin Islands. While these GAS AI
sequences can be identified in numerous M types, Applicants have
surprisingly discovered a correlation between the four main pilus
subunits from the four different GAS AI types and specific T
classifications. While other trypsin-resistant surface exposed
proteins are likely also implicated in the T classification
designations, the discovery of the role of the GAS adhesin islands
(and the associated hyper-oligomeric pilus like structures) in T
classification and GAS serotype variance has important implications
for prevention and treatment of GAS infections. Applicants have
identified protein components within each of the GAS adhesin
islands which are associated with the pilus formation. These
proteins are believed to be involved in the bacteria's initial
adherence mechanisms. Immunological recognition of these proteins
may allow the host immune response to slow or prevent the
bacteria's transition into the more pathogenic later stages of
infection. In addition, the GAS pili may be involved in formation
of biofilms. Applicants have discovered that the GBS pili
structures appear to be implicated in the formation of biofilms
(populations of bacteria growing on a surface, often enclosed in an
exopolysaccharide matrix). Biofilms are generally associated with
bacterial resistance, as antibiotic treatments and host immune
response are frequently unable to erradicate all of the bacteria
components of the biofilm. Direction of a host immune response
against surface proteins exposed during the first steps of
bacterial attachment (i.e., before complete biofilm formation) is
preferable.
[0807] The invention therefore provides for improved immunogenic
compositions against GAS infection which may target GAS bacteria
during their initial attachment efforts to the host epithelial
cells and may provide protection against a wide range of GAS
serotypes. The immunogenic compositions of the invention include
GAS AI surface proteins which may be formulated in an oligomeric,
or hyperoligomeric (pilus) form. The invention also includes
combinations of GAS AI surface proteins. Combinations of GAS AI
surface proteins may be selected from the same adhesin island or
they may be selected from different GAS adhesin islands.
[0808] The invention comprises compositions comprising a first GAS
AI protein and a second GAS AI protein wherein the first and second
GAS AI proteins are derived from different GAS adhesin islands. For
example, the invention includes a composition comprising at least
two GAS AI proteins wherein the GAS AI proteins are encoded by the
adhesin islands selected from the group consisting of GAS AI-1 and
AI-2; GAS AI-1 and GAS AI-3; GAS AI-1 and GAS AI-4; GAS AI-2 and
GAS AI-3; GAS AI-2 and GAS AI-4; and GAS AI-3 and GAS AI-4.
Preferably the two GAS AI proteins are derived from different
T-types.
[0809] A schematic arrangement of GAS Adhesin Island sequences is
set forth in FIG. 162. In all strains, the AI region is flanked by
the highly conserved open reading frames M1.sub.--123 and M1-136.
Between three and five genes in each locus code for surface
proteins containing LPXTG motifs. These surface proteins also all
belong to the family of genes coding for ECM binding adhesins.
[0810] Adhesin island sequences can be identified in numerous M
types of Group A Streptococcus. Examples of AI sequences within M1,
M6, M3, M5, M12, M18, and M49 serotypes are discussed below.
[0811] GAS Adhesin Islands generally include a series of open
reading frames within a GAS genome that encode for a collection of
surface proteins and sortases. A GAS Adhesin Island may encode for
amino acid sequences comprising at least one surface protein.
Alternatively, a GAS Adhesin Island may encode for at least two
surface proteins and at least one sortase. Preferably, a GAS
Adhesin Island encodes for at least three surface proteins and at
least two sortases. One or more of the surface proteins may include
an LPXTG motif (such as LPXTG (SEQ ID NO: 122)) or other sortase
substrate motif. One or more GAS AI surface proteins may
participate in the formation of a pilus structure on the surface of
the Gram positive bacteria.
[0812] GAS Adhesin Islands of the invention preferably include a
divergently transcribed transcriptional regulator. The
transcriptional regulator may regulate the expression of the GAS AI
operon. Examples of transcriptional regulators found in GAS AI
sequences include RofA and Nra.
[0813] The GAS AI surface proteins may bind or otherwise adhere to
fibrinogen, fibronectin, or collagen. One or more of the GAS AI
surface proteins may comprise a fimbrial structural subunit.
[0814] One or more of the GAS AI surface proteins may include an
LPXTG motif or other sortase substrate motif. The LPXTG motif may
be followed by a hydrophobic region and a charged C terminus, which
are thought to retard the protein in the cell membrane to
facilitate recognition by the membrane-localized sortase. See
Barnett, et al., J. Bacteriology (2004) 186 (17): 5865-5875.
[0815] GAS AI sequences may be generally categorized as Type 1,
Type 2, Type 3, or Type 4, depending on the number and type of
sortase sequences within the island and the percentage identity of
other proteins (with the exception of RofA and cpa) within the
island. FIG. 167 provides a chart indicating the number and type of
sortase sequences identified within the adhesin islands of various
strains and serotypes of GAS. As can be seen in this figure, all
GAS strains and serotypes thus far characterized as an AI-1 have a
SrtB type sortase, all GAS strains and serotypes thus far
characterized as an AI-2 have SrtB and SrtC1 type sortases, all GAS
strains and serotypes thus far characterized as an AI-3 have a
SrtC2 type sortase, and all GAS strains and serotypes thus far
characterized as an AI-4 have SrtB and SrtC2 type sortases. A
comparison of the percentage identity of sequences within the
adhesin islands was presented in Table 45, see above.
[0816] (1) Adhesin Island Sequence within M6: GAS Adhesin Island 1
("GAS AI-1")
[0817] A GAS Adhesin Island within M6 serotype (MGAS10394) is
outlined in Table 4 below. This GAS adhesin island 1 ("GAS AI-1")
comprises surface proteins, a srtB sortase and a rofA divergently
transcribed transcriptional regulator.
[0818] GAS AI-1 surface proteins include Spy0157 (a fibronectin
binding protein), Spy0159 (a collagen adhesion protein) and Spy0160
(a fimbrial structural subunit). Preferably, each of these GAS AI-1
surface proteins includes an LPXTG sortase substrate motif, such as
LPXTG (SEQ ID NO: 122) or LPXSG (SEQ ID NO: 134) (conservative
replacement of threonine with serine).
[0819] GAS AI-1 includes a srtB type sortase. GAS srtB sortases may
preferably anchor surface proteins with an LPSTG motif (SEQ ID NO:
166), particularly where the motif is followed by a serine.
TABLE-US-00011 TABLE 4 GAS AI-1 sequences from M6 isolate
(MGAS10394) Sortase substrate AI-1 sequence sequence or sortase
identifier type functional description M6_Spy0156 Transcriptional
regulator (rofA) M6_Spy0157 LPXTG Fibronectin-binding protein
M6_Spy0158 Reverse transcriptase M6_Spy0159 LPXSG Collagen adhesion
protein M6_Spy0160 LPXTG Fimbrial structural subunit M6_Spy0161
srtB Sortase
[0820] M6_Spy0160 appears to be present on the surface of GAS as
part of oligomeric (pilus) structures. FIGS. 127-132 present
electron micrographs of GAS serotype M6 strain 3650 immunogold
stained for M6 Spy0160 using anti-M6 Spy0160 antiserum. Oligomeric
or hyperoligmeric structures labelled with gold particles can be
seen extending from the surface of the GAS in each of these
figures, indicating the presence of multiple M6_Spy0160
polypeptides in the oligomeric or hyperoligomeric structures. FIG.
176 A-F present electron micrographs of GAS M6 strain 2724
immunogold stained for M6_Spy0160 using anti-M6_Spy0160 antiserum
(FIGS. 176 A-E) or immunogold stained for M6_Spy0159 using
anti-M6_Spy0159 antiserum (FIG. 176 F). Oligomeric or
hyperoligomeric structures labelled with gold particles can again
be seen extending from the surface of the M6 strain 2724 GAS
bacteria immunogold stained for M6_Spy0160. M6_Spy0159 is also
detected on the surface of the M6 strain 2724 GAS.
[0821] FACS analysis has confirmed that the GAS AI-1 surface
proteins spyM6.sub.--0159 and spyM6.sub.--0160 are indeed expressed
on the surface of GAS. FIG. 73 provides the results of FACS
analysis for surface expression of spyM6.sub.--0159 on each of GAS
serotypes M6 2724, M6 3650, and M6 2894. A shift in fluorescence is
observed for each GAS serotype when anti-spyM6.sub.--0159 antiserum
is present, demonstrating cell surface expression. Table 18, below,
quantitatively summarizes the FACS fluorescence values obtained for
each GAS serotype in the presence of pre-immune antiserum,
anti-spyM6.sub.--0159 antiserum, and the difference in fluorescence
value between the pre-immune and anti-spyM6.sub.--0159 antiserum.
TABLE-US-00012 TABLE 18 Summary of FACS values for surface
expression of spyM6_0159 2724 3650 2894 Pre- Anti- Pre- Anti- Pre-
Anti- immune spyM6_0159 Change immune spyM6_0159 Change immune
spyM6_0159 Change 134.84 427.48 293 149.68 712.62 563 193.86 597.8
404
[0822] FIG. 74 provides the results of FACS analysis for surface
expression of spyM6.sub.--0160 on each of GAS serotypes M6 2724, M6
3650, and M6 2894. In the presence of of anti-spyM6.sub.--0160
antiserum, a shift in fluorescence is observed for each GAS
serotype, which demonstrates its cell surface expression. Table 19,
below, quantitatively summarizes the FACS fluorescence values
obtained for each GAS serotype in the presence of pre-immune
antiserum, anti-spyM6.sub.--0160 antiserum, and the change in
fluorescence value between the pre-immune and anti-spyM6.sub.--0160
antiserum. TABLE-US-00013 TABLE 19 Summary of FACS values for
surface expression of spyM6_0160 2724 3650 2894 Pre- Anti- Pre-
Anti- Pre- Anti- immune spyM6_0160 change immune spyM6_0160 change
immune spyM6_0160 change 117.12 443.24 326 128.57 776.39 648 125.87
621.17 495
[0823] Surface expression of M6_Spy0159 and M6_Spy0160 on M6
serotype GAS has also been confirmed by Western blot analysis. FIG.
98 shows that while pre-immune sera (P .alpha.-0159) does not
detect expression of M6_Spy0159 in GAS serotype M6, anti-M6_Spy0159
immune sera (I .alpha.-0159) is able to detect M6_Spy0159 protein
in both total GAS M6 extracts (M6 tot) and GAS M6 fractions
enriched for cell surface proteins (M6 surf prot). The M6_Spy0159
proteins detected in the total GAS M6 extracts or the GAS M6
extracts enriched for surface proteins are also present as high
molecular weight structures, indicating that M6_Spy0159 may be in
an oligomeric (pilus) form.
[0824] FIG. 112 shows that while preimmune sera (Preimmune Anti
106) does not detect expression of M6_Spy0160 in GAS serotype M6
strain 2724, anti-M6_Spy0160 immune sera (Anti 160) does in both
total GAS M6 strain 2724 extracts (M6 2724 tot) and GAS M6 strain
2724 fractions enriched for surface proteins. The M6_Spy0160
proteins detected in the total GAS M6 strain 2724 extracts or the
GAS M6 strain 2724 extracts enriched for surface proteins are also
present as high molecular weight structures, indicating that
M6_Spy0160 may be in an oligomeric (pilus) form.
[0825] FIGS. 110 and 111 both further verify the presence of
M6_Spy0159 and M6_Spy0160 in higher molecular weight structures on
the surface of GAS. FIG. 110 provides a Western blot performed to
detect M6_Spy0159 and M6_Spy0160 in GAS M6 strain 2724 extracts
enriched for surface proteins. Antiserum raised against either
M6_Spy0159 (Anti-159) or M6_Spy0160 (Anti-160) cross-hybridizes
with high molecular weight structures (pili) in these extracts.
FIG. 111 provides a similar Western blot that verifies the presence
of M6_Spy0159 and M6_Spy0160 in high molecular weight structures in
GAS M6 strain 3650 extracts enriched for surface proteins.
[0826] SpyM6.sub.--0157 (a fibronectin-binding protein) may also be
expressed on the surface of GAS serotype M6 bacteria. FIG. 174
shows the results of FACS analysis for surface expression of
spyM6.sub.--0157 on M6 strain 3650. A slight shift in fluorescence
is observed, which demonstrates that some spyM6.sub.--0157 may be
expressed on the GAS cell surface.
Adhesin Island Sequence within M6: GAS Adhesin Island 2 ("GAS
AI-2")
[0827] A GAS Adhesin Island within M1 serotype (SF370) is outlined
in Table 5 below. This GAS adhesin island 2 ("GAS AI-2") comprises
surface proteins, a SrtB sortase, a SrtC1 sortase and a RofA
divergently transcribed transcriptional regulator.
[0828] GAS AI-2 surface proteins include GAS 15 (Cpa), Spy0128
(thought to be a fimbrial protein) and Spy0130 (a hypothetical
protein). Preferably, each of these GAS AI-2 surface proteins
includes an LPXTG sortase substrate motif, such as LPXTG (SEQ ID
NO: 122), VVXTG (SEQ ID NO: 135), or EVXTG (SEQ ID NO: 136).
[0829] GAS AI-2 includes a srtB type sortase and a srtC1 sortase.
As discussed above, GAS SrtB sortases may preferably anchor surface
proteins with an LPSTG (SEQ ID NO: 166) motif, particularly where
the motif is followed by a serine. GAS SrtC1 sortase may
preferentially anchor surface proteins with a V(P/V)PTG (SEQ ID NO:
167) motif. GAS SrtC1 may be differentially regulated by RofA.
[0830] GAS AI-2 may also include a LepA putative signal peptidase I
protein. TABLE-US-00014 TABLE 5 GAS AI-2 sequence from M1 isolate
(SF370) Sortase substrate AI-2 sequence sequence or identifier
sortase type functional description SPy0124 rofA regulatory protein
GAS15(not VVXTG cpa annotated in SF370) SPy0127 LepA putative
signal peptidase I SPy0128 (GAS16) EVXTG hypothetical protein
(fimbrial) SPy0129 (GAS17) srtC1 sortase SPy0130 (GAS18) LPXTG
hypothetical protein SPy0131 conserved hypothetical protein SPy0133
conserved hypothetical protein SPy0135 (GAS20) srtB sortase
(putative fimbrial- associated protein)
[0831] GAS 15, GAS 16, and GAS 18 appear to be present on the
surface of GAS as part of oligomeric (pilus) structures. FIGS.
113-115 present electron micrographs of GAS serotype M1 strain
SF370 immunogold stained for GAS 15 using anti-GAS 15 antiserum.
FIGS. 116-121 provide electron micrographs of GAS serotype M1
strain SF370 immunogold stained for GAS 16 using anti-GAS 16
antiserum. FIGS. 122-125 present electron micrograph of GAS
serotype M1 strain SF370 immunogold stained for GAS 18 using
anti-GAS 18 antiserum. Oligomers of these proteins can be seen on
the surface of SF370 bacteria in the immuno-gold stained
micrographs.
[0832] FIG. 126 reveals a hyperoligomer on the surface of a GAS
serotype M1 strain SF370 bacterium immunogold stained for GAS 18.
This long hyperoliogmeric structure comprising GAS 18 stretches far
out into the supernatant from the surface of the bacteria.
[0833] FACS analysis has confirmed that the GAS AI-2 surface
proteins GAS 15, GAS 16, and GAS 18 are expressed on the surface of
GAS. FIG. 75 provides the results of FACS analysis for surface
expression of GAS 15 on each of GAS serotypes M1 2719, M1 2580, M1
3280, M1 SF370, M1 2913, and M1 3348. A shift in fluorescence is
observed for each GAS serotype when anti-GAS 15 antiserum is
present, demonstrating cell surface expression. Table 20, below,
quantitatively summarizes the FACS fluorescence values obtained for
each GAS serotype in the presence of pre-immune antiserum, anti-GAS
15 antiserum, and the difference in fluorescence value between the
pre-immune and anti-GAS 15 antiserum. TABLE-US-00015 TABLE 20
Summary of FACS values for surface expression of GAS 15 Pre-
Anti-GAS Pre- Anti-GAS Pre- Anti-GAS immune 15 Change immune 15
Change immune 15 Change 2719 2580 3280 159.46 712.71 553 123.9
682.84 559 217.02 639.69 423 SF370 2913 3348 201.93 722.68 521
121.41 600.45 479 152.09 446.41 294
[0834] FIGS. 76 and 79 provide the results of FACS analysis for
surface expression of GAS 16 on each of GAS serotypes M1 2719, M1
2580, M1 3280, M1 SF370, M1 2913, and M1 3348. The FACS data in
FIG. 76 was obtained using antisera was raised against full length
GAS 16. In the presence of this anti-GAS 16 antiserum, a shift in
fluorescence is observed for each GAS serotype, demonstrating its
cell surface expression. Table 21, below, quantitatively summarizes
the FACS fluorescence values obtained for each GAS serotype in the
presence of pre-immune antiserum, anti-GAS 16 antiserum, and the
change in fluorescence value between the pre-immune and anti-GAS 16
antiserum. TABLE-US-00016 TABLE 21 Summary of FACS values for
surface expression of GAS 16 Pre- Anti-GAS Pre- Anti-GAS Pre-
Anti-GAS immune 16 Change immune 16 Change immune 16 Change 2719
2580 3280 233.27 690.09 457 133.82 732.29 598 264.47 649.43 385
SF370 2913 3348 237.2 727.46 490 138.52 588.04 450 180.56 420.93
240
[0835] The FACS data in FIG. 79 was obtained using antisera was
raised against a truncated GAS 16, which is encoded by SEQ ID NO:
179, shown below. TABLE-US-00017 SEQ ID NO: 179:
GCTACAACAGTTCACGGGGAGACTGTTGTAAACGGAGCCAAACTAACAGT
TACAAAAAACCTTGATTTAGTTAATAGCAATGCATTAATTCCAAATACAG
ATTTTACATTTAAAATCGAACCTGATACTACTGTCAACGAAGACGGAAAT
AAGTTTAAAGGTGTAGCTTTGAACACACCGATGACTAAAGTCACTTACAC
CAATTCAGATAAAGGTGGATCAAATACGAAAACTGCAGAATTTGATTTTT
CAGAAGTTACTTTTGAAAAACCAGGTGTTTATTATTACAAAGTAACTGAG
GAGAAGATAGATAAAGTTCCTGGTGTTTCTTATGATACAACATCTTACAC
TGTTCAAGTTCATGTCTTGTGGAATGAAGAGCAACAAAAACCAGTAGCTA
CTTATATTGTTGGTTATAAAGAAGGTAGTAAGGTGCCAATTCAGTTCAAA
AATAGCTTAGATTCTACTACATTAACGGTGAAGAAAAAAGTTTCAGGTAC
CGGTGGAGATCGCTCTAAAGATTTTAATTTTGGTCTGACTTTAAAAGCAA
ATCAGTATTATAAGGCGTCAGAAAAAGTCATGATTGAGAAGACAACTAAA
GGTGGTCAAGCTCCTGTTCAAACAGAGGCTAGTATAGATCAACTCTATCA
TTTTACCTTGAAAGATGGTGAATCAATCAAAGTCACAAATCTTCCAGTAG
GTGTGGATTATGTTGTCACTGAAGACGATTACAAATCAGAAAAATATACA
ACCAACGTGGAAGTTAGTCCTCAAGATGGAGCTGTAAAAAATATCGCAGG
TAATTCAACTGAACAAGAGACATCTACTGATAAAGATATGACCATTACTT
TTACAAATAAAAAAGATTT
[0836] In the presence of this anti-GAS 16 antiserum, a shift in
fluorescence is observed for each GAS serotype, demonstrating its
cell surface expression. Table 22, below, quantitatively summarizes
the FACS fluorescence values obtained for each GAS serotype in the
presence of pre-immune antiserum, anti-GAS 16 antiserum, and the
change in fluorescence value between the pre-immune and anti-GAS 16
antiserum. TABLE-US-00018 TABLE 22 Summary of FACS values for
surface expression of GAS 16 using a second antisera Pre- Anti-GAS
Pre- Anti-GAS Pre- Anti-GAS immune 16 Change immune 16 Change
immune 16 Change 2719 2580 3280 141.55 650.22 509 119.57 672.35 553
209.18 666.71 458 SF370 2913 3348 159.92 719.32 559 115.97 585.9
470 146.1 414.01 268
[0837] FIGS. 77 and 78 provide the results of FACS analysis for
surface expression of GAS 18 on each of GAS serotypes M12719,
M12580, M1 3280, M1 SF370, M12913, and M13348. The antiserum used
to obtain the FACS data in each of FIGS. 77 and 78 was different,
although each was raised against full length GAS 18. In the
presence of each of the anti-GAS 18 antisera, a shift in
fluorescence is observed for each GAS serotype, demonstrating its
cell surface expression. Tables 23 and 24, below, quantitatively
summarizes the FACS fluorescence values obtained for each GAS
serotype in the presence of pre-immune antiserum, first or second
anti-GAS 18 antiserum, and the change in fluorescence value between
the pre-immune and first or second anti-GAS 18 antiserum.
TABLE-US-00019 TABLE 23 Summary of FACS values for surface
expression of GAS 18 Pre- Anti-GAS Pre- Anti-GAS Pre- Anti-GAS
immune 18 Change immune 18 Change immune 18 Change 2719 2580 3280
135.68 327.98 192 116.32 379.41 263 208.12 380.84 173 SF370 2913
3348 185.39 438.23 253 119.95 373.32 253 147.12 266.51 119
[0838] TABLE-US-00020 TABLE 24 Summary of FACS values for surface
expression of GAS 18 using a second antisera Pre- Anti-GAS Pre-
Anti-GAS Pre- Anti-GAS immune 18 Change immune 18 Change immune 18
Change 2719 2580 3280 150.4 250.39 100 139.18 386.38 247 253.38
347.72 94 SF370 2913 3348 188.64 373.11 184 124.94 384.82 260 168.8
213.65 45
[0839] Surface expression of GAS 15, GAS 16, and GAS 18 on M1
serotype GAS has also been confirmed by Western blot analysis. FIG.
89 shows that while pre-immune sera does not detect GAS M1
expression of GAS 15, anti-GAS 15 immune sera is able to detect GAS
15 protein in both total GAS M1 extracts and GAS M1 proteins
enriched for cell surface proteins. The GAS 15 proteins detected in
the M1 extracts enriched for surface proteins are also present as
high molecular weight structures, indicating that GAS 15 may be in
an oligomeric (pilus) form. FIG. 90 also shows the results of
Western blot analysis of M1 serotype GAS using anti-GAS 15
antisera. Again, the lanes that contain GAS M1 extracts enriched
for surface proteins (M1 prot sup) show the presence of high
molecular weight structures that may be oligomers of GAS 15. FIG.
91 provides an additional Western blot identical to that of FIG.
90, but that was probed with pre-immune sera. As expected, no
proteins were detected on this membrane.
[0840] FIG. 92 provides a Western blot that was probed for GAS 16
protein. While pre-immune sera does not detect GAS M1 expression of
GAS 16, anti-GAS 16 immune sera is able to detect GAS 16 protein in
GAS M1 extracts enriched for cell surface proteins. The GAS 16
proteins detected in the M1 extracts enriched for surface proteins
are present as high molecular weight structures, indicating that
GAS 16 may be in an oligomeric (pilus) form. FIG. 93 also shows the
results of Western blot analysis of M1 serotype GAS using anti-GAS
16 antisera. The lanes that contain total GAS M1 protein (M1 tot
new and M1 tot old) and the lane that contains GAS M1 extracts
enriched for surface proteins (M1 prot sup) show the presence of
high molecular weight structures that may be oligomers of GAS 16.
FIG. 94 provides an additional Western blot identical to that of
FIG. 93, but that was probed with pre-immune sera. As expected, no
proteins were detected on this membrane.
[0841] FIG. 95 provides a Western blot that was probed for GAS 18
protein. While pre-immune sera does not detect GAS M1 expression of
GAS 18, anti-GAS 18 immune sera is able to detect GAS 18 protein in
GAS M1 extracts enriched for cell surface proteins. The GAS 18
proteins detected in the M1 extracts enriched for surface proteins
are present as high molecular weight structures, indicating that
GAS 18 may be in an oligomeric (pilus) form. FIG. 96 also shows the
results of Western blot analysis of M1 serotype GAS using anti-GAS
18 antisera. The lane that contains GAS M1 extracts enriched for
surface proteins (M1 prot sup) show the presence of high molecular
weight structures that may be oligomers of GAS 18. FIG. 97 provides
an additional Western blot identical to that of FIG. 96, but that
was probed with pre-immune sera. As expected, no proteins were
detected on this membrane.
[0842] FIGS. 102-106 provide additional Western blots to verify the
presence of GAS 15, GAS 16, and GAS 18 in high molecular weight
structures in GAS. Each Western blot was performed using proteins
from a different GAS M1 strain, 2580, 2913, 3280, 3348, and 2719.
Each Western blot was probed with antisera raised against each of
GAS 15, GAS 16, and GAS 18. As can be seen in FIGS. 102-106, none
of the Western blots shows detection of proteins using pre-immune
serum (P.alpha.-158, P.alpha.-15, P.alpha.-16, or P.alpha.-18),
while each Western blot shows cross-hybridization of the GAS 15
(1.alpha.-15), GAS 16 (1.alpha.-16), and GAS 18 (1.alpha.-18)
antisera to high molecular weight structures. Thus, these Western
blots confirm that GAS 15, GAS 16, and GAS 18 can be present in
pili in GAS M1.
[0843] FIG. 107 provides a similar Western blot performed to detect
GAS 15, GAS 16, and GAS 18 proteins in a GAS serotype M1 strain
SF370 protein fraction enriched for surface proteins. This Western
blot also shows detection of GAS 15 (Anti-15), GAS 16 (Anti-16),
and GAS 18 (Anti-18) as high molecular weight structures.
[0844] (3) Adhesin Island Sequence within M3, M5 and M18: GAS
Adhesin Island 3 ("GAS AI-3")
[0845] GAS Adhesin Island sequences within M3, M5, and M18
serotypes are outlined in Tables 6-8 and 10 below. This GAS adhesin
island 3 ("GAS AI-3") comprises surface proteins, a SrtC2 sortase,
and a Negative transcriptional regulator (Nra) divergently
transcribed transcriptional regulator.
[0846] GAS AI-3 surface proteins within include a collagen binding
protein, a fimbrial protein, a F2 like fibronectin-binding protein.
GAS AI-3 surface proteins may also include a hypothetical surface
protein. Preferably, each of these GAS AI-3 surface proteins
include an LPXTG sortase substrate motif, such as LPXTG (SEQ ID NO:
122), VPXTG (SEQ ID NO: 137), QVXTG (SEQ ID NO: 138) or LPXAG (SEQ
ID NO: 139).
[0847] GAS AI-3 includes a SrtC2 type sortase. GAS SrtC2 type
sortases may preferably anchor surface proteins with a QVPTG (SEQ
ID NO: 140) motif, particularly when the motif is followed by a
hydrophobic region and a charged C terminus tail. GAS SrtC2 may be
differentially regulated by Nra.
[0848] GAS AI-3 may also include a LepA putative signal peptidase I
protein.
[0849] GAS AI-3 may also include a putative multiple sugar
metabolism regulator. TABLE-US-00021 TABLE 6 GAS AI-3 sequences
from M3 isolate (MGAS315) Sortase substrate AI-3 sequence sequence
or identifier sortase type Functional description SpyM3_0097
Negative transcriptional regulator (Nra) SpyM3_0098 VPXTG putative
collagen binding protein (Cpb) SpyM3_0099 LepA putative signal
peptidase I SpyM3_0100 QVXTG conserved hypothetical protein
(fimbrial) SpyM3_0101 SrtC2 sortase SpyM3_0102 LPXAG hypothetical
protein SpyM3_0103 putative multiple sugar metabolism regulator
SpyM3_0104 LPXTG protein F2 like fibronectin-binding protein
[0850] TABLE-US-00022 TABLE 7 GAS AI-3 sequence from M3 isolate
(SSI-1) Sortase Substrate AI-3 sequence sequence or identifier
sortase type Functional description SPs0099 Negative
transcriptional regulator (Nra) SPs0100 VPXTG putative collagen
binding protein (Cpb) SPs0101 LepA putative signal peptidase I
SPs0102 QVXTG conserved hypothetical protein (fimbrial) SPs0103
SrtC2 sortase SPs0104 LPXAG hypothetical protein SPs0105 putative
multiple sugar metabolism regulator SPs0106 LPXTG protein F2 like
fibronectin-binding protein
[0851] TABLE-US-00023 TABLE 10 GAS AI-3 sequences from M5 isolate
(Manfredo) AI-3 Sortase substrate sequence sequence or identifier
sortase type Functional description orf77 Negative transcriptional
regulator (Nra) orf78 VPXTG putative collagen binding protein (Cpb)
orf79 LepA putative signal peptidase I orf80 QVXTG conserved
hypothetical protein (fimbrial) orf81 SrtC2 sortase orf82 LPXAG
hypothetical protein orf83 putative multiple sugar metabolism
regulator orf84 LPXTG protein F2 like fibronectin-binding
protein
[0852] TABLE-US-00024 TABLE 8 GAS AI-3 sequences from M18 isolate
(MGAS8232) Sortase substrate AI-3 sequence sequence or identifier
sortase type Functional description spyM18_0125 Negative
transcriptional regulator (Nra) (N-terminal fragment) spyM18_0126
VPXTG putative collagen binding protein (Cpb) spyM18_0127 LepA
putative signal peptidase I spyM18_0128 QVXTG conserved
hypothetical protein (fimbrial) spyM18_0129 SrtC2 sortase
spyM18_0130 LPXAG hypothetical protein spyM18_0131 putative
multiple sugar metabolism regulator spyM18_0132 LPXTG protein F2
like fibronectin-binding protein
[0853] TABLE-US-00025 TABLE 44 GAS AI-3 sequences from M49 isolate
(591) Sortase substrate AI-3 sequence sequence or identifier
sortase type Functional description SpyoM01000156 Negative
transcriptional regulator (Nra) SpyoM01000155 VPXTG collagen
binding protein (Cpa) SpyoM01000154 LepA putative signal peptidase
I SpyoM01000153 QVXTG conserved hypothetical protein (fimbrial)
SpyoM01000152 SrtC2 sortase SpyoM01000151 LPXAG hypothetical
protein SpyoM01000150 MsmRL SpyoM01000149 LPXTG protein F2 like
fibronectin- binding protein
[0854] A schematic of AI-3 serotypes M3, M5, M 18, and M49 is shown
in FIG. 51A. Each contains an open reading frame encoding a
SrtC2-type sortase of nearly identical amino acid sequence. See
FIG. 52B for an amino acid sequence alignment for each of the SrtC2
amino acid sequences.
[0855] The protein F2-like fibronectin-binding protein of each
these type 3 adhesin islands contains a pilin motif and an E-box.
FIG. 60 indicates the amino acid sequence of the pilin motif and
E-box of each of GAS AI-3 serotype M3 MGAS315
(SpyM3.sub.--0104/21909640), GAS AI-3 serotype M3 SSI
(Sps0106/28895018), GAS AI-3 serotype M18
(SpyM18.sub.--0132/19745307), and GASAI-3 serotype M5 (orf84).
[0856] FACS analysis has confirmed that the GAS AI-3 surface
proteins SpyM3.sub.--0098, SpyM3.sub.--0100, SpyM3.sub.--0102, and
SpyM3.sub.--0104 are expressed on the surface of GAS. FIG. 80
provides the results of FACS analysis for surface expression of
SpyM3.sub.--0098 on each of GAS serotypes M3 2721 and M3 3135. A
shift in fluorescence is observed for each GAS serotype when
anti-SpyM3.sub.--0098 antiserum is present, demonstrating cell
surface expression. Table 25, below, quantitatively summarizes the
FACS fluorescence values obtained for each GAS serotype in the
presence of pre-immune antiserum, anti-SpyM3.sub.--0098 antiserum,
and the difference in fluorescence value between the pre-immune and
anti-SpyM3.sub.--0098 antiserum. TABLE-US-00026 TABLE 25 Summary of
FACS values for surface expression of SpyM3_0098 2721 3135 Pre-
Anti- Pre- Anti- immune spyM3_0098 Change immune spyM3_0098 Change
117.85 249.51 132 99.17 277.21 178
[0857] FIG. 81 provides the results of FACS analysis for surface
expression of SpyM3.sub.--0100 on each of GAS serotypes M3 2721 and
M3 3135. A shift in fluorescence is observed for each GAS serotype
when anti-SpyM3.sub.--0100 antiserum is present, demonstrating cell
surface expression. Table 26, below, quantitatively summarizes the
FACS fluorescence values obtained for each GAS serotype in the
presence of pre-immune antiserum, anti-SpyM3.sub.--0100 antiserum,
and the difference in fluorescence value between the pre-immune and
anti-SpyM3.sub.--0100 antiserum. TABLE-US-00027 TABLE 26 Summary of
FACS values for surface expression of SpyM3_0100 2721 3135 Pre-
Anti- Pre- Anti- immune spyM3_0100 Change immune spyM3_0100 Change
110.31 181.91 72 97.87 250.01 152
[0858] FIG. 82 provides the results of FACS analysis for surface
expression of SpyM3.sub.--0102 on each of GAS serotypes M3 2721 and
M3 3135. A shift in fluorescence is observed for each GAS serotype
when anti-SpyM3.sub.--0102 antiserum is present, demonstrating cell
surface expression. Table 27, below, quantitatively summarizes the
FACS fluorescence values obtained for each GAS serotype in the
presence of pre-immune antiserum, anti-SpyM3.sub.--0102 antiserum,
and the difference in fluorescence value between the pre-immune and
anti-SpyM3.sub.--0102 antiserum. TABLE-US-00028 TABLE 27 Summary of
FACS values for surface expression of SpyM3_0102 in M3 serotypes
2721 3135 Pre- Anti- Pre- Anti- immune spyM3_0102 Change immune
spyM3_0102 Change 109.86 155.26 45 100.02 112.58 13
[0859] FIG. 82 also provides the results of FACS analysis for
surface expression of a pilin antigen that has homology to
SpyM3.sub.--0102 identified in a different GAS serotype, M6. FACS
analysis conducted with the SpyM3.sub.--0102 antisera was able to
detect surface expression of the homologous SpyM3.sub.--0102
antigen on each of GAS serotypes M6 2724, M6 3650, and M6 2894.
Table 28, below, quantitatively summarizes the FACS fluorescence
values obtained for each GAS serotype in the presence of pre-immune
antiserum, anti-SpyM3.sub.--0102 antiserum, and the difference in
fluorescence value between the pre-immune and anti-SpyM3.sub.--0102
antiserum. TABLE-US-00029 TABLE 28 Summary of FACS values for
surface expression of SpyM3_0102 in M6 serotypes 2724 3650 2894
Pre- Anti- Pre- Anti- Pre- Anti- immune spyM3_0102 Change immune
spyM3_0102 Change immune spyM3_0102 Change 146.59 254.03 107 162.56
294.03 131 175.49 313.69 138
[0860] SpyM3.sub.--0102 is also homologous to pilin antigen
19224139 of GAS serotype M12. Antisera raised against
SpyM3.sub.--0102 is able to detect high molecular weight structures
in GAS serotype M12 strain 2728 protein fractions enriched for
surface proteins, which would contain the 19224139 antigen. See
FIG. 109 at the lane labelled M112 2728 surf prot.
[0861] FIG. 83 provides the results of FACS analysis for surface
expression of SpyM3.sub.--0104 on each of GAS serotypes M3 2721 and
M3 3135. A shift in fluorescence is observed for each GAS serotype
when anti-SpyM3.sub.--0104 antiserum is present, demonstrating cell
surface expression. Table 29, below, quantitatively summarizes the
FACS fluorescence values obtained for each GAS serotype in the
presence of pre-immune antiserum, anti-SpyM3.sub.--0104 antiserum,
and the difference in fluorescence value between the pre-immune and
anti-SpyM3.sub.--0104 antiserum. TABLE-US-00030 TABLE 29 Summary of
FACS values for surface expression of SpyM3_0104 in M3 serotypes
2721 3135 Pre- Anti- Pre- Anti- immune spyM3_0104 Change immune
spyM3_0104 Change 128.45 351.65 223 105.1 339.88 235
[0862] FIG. 83 also provides the results of FACS analysis for
surface expression of a pilin antigen that has homology to
SpyM3.sub.--0104 identified in a different GAS serotype, M12. FACS
analysis conducted with the SpyM3.sub.--0104 antisera was able to
detect surface expression of the homologous SpyM3.sub.--0104
antigen on GAS serotype M12 2728. Table 30, below, quantitatively
summarizes the FACS fluorescence values obtained for this GAS
serotype in the presence of pre-immune antiserum,
anti-SpyM3.sub.--0104 antiserum, and the difference in fluorescence
value between the pre-immune and anti-SpyM3.sub.--0104 antiserum.
TABLE-US-00031 TABLE 30 Summary of FACS values for surface
expression of SpyM3_0104 in an M12 serotype 2728 Pre-immune
Anti-spyM3_0104 Change 198.57 288.75 90
[0863] FIG. 84 provides the results of FACS analysis for surface
expression of SPs.sub.--0106 on each of GAS serotypes M3 2721 and
M3 3135. A shift in fluorescence is observed for each GAS serotype
when anti-SPs.sub.--0106 antiserum is present, demonstrating cell
surface expression. Table 31, below, quantitatively summarizes the
FACS fluorescence values obtained for each GAS serotype in the
presence of pre-immune antiserum, anti-SPs.sub.--0106 antiserum,
and the difference in fluorescence value between the pre-immune and
anti-SPs.sub.--0106 antiserum. TABLE-US-00032 TABLE 31 Summary of
FACS values for surface expression of SPs_0106 in M3 serotypes 2721
3135 Anti- Anti- Pre-immune SPs_0106 Change Pre-immune SPs_0106
Change 116 463.28 347 103.02 494.27 391
[0864] FIG. 84 also provides the results of FACS analysis for
surface expression of a pilin antigen that has homology to
SPs.sub.--0106 identified in a different GAS serotype, M12. FACS
analysis conducted with the SPs.sub.--0106 antisera was able to
detect surface expression of the homologous SPs.sub.--0106 antigen
on GAS serotype M12 2728. Table 32, below, quantitatively
summarizes the FACS fluorescence values obtained for each GAS
serotype in the presence of pre-immune antiserum,
anti-SPs.sub.--0106 antiserum, and the difference in fluorescence
value between the pre-immune and anti-SPs.sub.--0106 antiserum.
TABLE-US-00033 TABLE 32 Summary of FACS values for surface
expression of SPs_0106 in an M12 serotype 2728 Pre-immune
Anti-SPs_0106 Change 304.01 254.64 -49
[0865] (4) Adhesin Island Sequence within M12: GAS Adhesin Island 4
("GAS AI-4")
[0866] GAS Adhesin Island sequences within M12 serotype are
outlined in Table 11 below. This GAS adhesin island 4 ("GAS AI-4")
comprises surface proteins, a SrtC2 sortase, and a RofA regulatory
protein.
[0867] GAS AI-4 surface proteins within may include a fimbrial
protein, an F or F2 like fibronectin-binding protein, and a
capsular polysaccharide adhesion protein (Cpa). GAS AI-4 surface
proteins may also include a hypothetical surface protein in an open
reading frame (orf). Preferably, each of the GAS AI-4 surface
proteins include an LPXTG sortase substrate motif, such as LPXTG
(SEQ ID NO: 122), VPXTG (SEQ ID NO: 137), QVXTG (SEQ ID NO: 138) or
LPXAG (SEQ ID NO: 139).
[0868] GAS AI-4 includes a SrtC2 type sortase. GAS SrtC2 type
sortases may preferably anchor surface proteins with a QVPTG (SEQ
ID NO: 140) motif, particularly when the motif is followed by a
hydrophobic region and a charged C terminus tail.
[0869] GAS AI-4 may also include a LepA putative signal peptidase I
protein and a MsmRL protein. TABLE-US-00034 TABLE 11 GAS AI-4
sequences from M12 isolate (A735) Sortase substrate AI-4 sequence
sequence or sortase identifier type Functional description 19224133
RofA regulatory protein 19224134 LPXTG protein F SrtB SrtB (stop
codon*) 19224135 VPXTG Cpa 19224136 LepA 19224137 QVXTG EftLSL.A
(fimbrial) 19224138 SrtC2 EftLSL.B 19224139 LPXAG Orf2 19224140
MsmRL 19224141 LPXTG protein F2
[0870] A schematic of AI-4 serotype M12 is shown in FIG. 51A.
[0871] One of the open reading frames encodes a SrtC2-type sortase
having an amino acid sequence nearly identical to the amino acid
sequence of the SrtC2-type sortase of the AI-3 serotypes described
above. See FIG. 52B for an amino acid sequence alignment for each
of the SrtC2 amino acid sequences.
[0872] Other proteins encoded by the open reading frames of the
AI-4 serotype M12 are homologous to proteins encoded by other known
adhesin islands in S. pyogenes, as well as the GAS AI-3 serotype M5
(Manfredo). FIG. 52 is an amino acid alignment of the capsular
polysaccharide adhesion protein (cpa) of AI-4 serotype M112
(19224135), GAS AI-3 serotype M5 (ORF78), S. pyogenes strain
MGAS315 serotype M3 (21909634), S. pyogenes SSI-1 serotype M3
(28810257), S. pyogenes MGAS8232 serotype M3 (19745301), and GAS
AI-2 serotype M1 (GAS15). The amino acid sequence of the AI-4
serotype M12 cpa shares a high degree of homology with other cpa
proteins.
[0873] FIG. 53 shows that the F-like fibronectin-binding protein
encoded by the AI-4 serotype M12 open frame (19224134) shares
homology with a F-like fibronectin-binding protein found in S.
pyogenes strain MGAS10394 serotype M6 (50913503).
[0874] FIG. 54 is an amino acid sequence alignment that illustrates
that the F2-like fibronectin-binding protein of AI-4 serotype M12
(19224141) shares homology with the F2-like fibronectin-binding
protein of S. pyogenes strain MGAS8232 serotype M3 (19745307), GAS
AI-3 serotype M5 (ORF84), S. pyogenes strain SSI serotype M3
(28810263), and S. pyogenes strain MGAS315 serotype M3
(21909640).
[0875] FIG. 55 is an amino acid sequence alignment that illustrates
that the fimbrial protein of AI-4 serotype M12 (19224137) shares
homology with the fimbrial protein of GAS AI-3 serotype M5 (ORF80),
and the hypothetical protein of S. pyogenes strain MGAS315 serotype
M3 (21909636), S. pyogenes strain SSI serotype M3 (28810259), S.
pyogenes strain MGAS8732 serotype M3 (19745303), and S. pyogenes
strain M1 GAS serotype M1 (13621428).
[0876] FIG. 56 is an amino acid sequence alignment that illustrates
that the hypothetical protein of GAS AI-4 serotype M12 (19224139)
shares homology with the hypothetical protein of S. pyogenes strain
MGAS315 serotype M3 (21909638), S. pyogenes strain SSI-1 serotype
M3 (28810261), GAS AI-3 serotype M5 (ORF82), and S. pyogenes strain
MGAS8232 serotype M3 (19745305).
[0877] The protein F2-like fibronectin-binding protein of the type
4 adhesin island also contains a highly conserved pilin motif and
an E-box. FIG. 60 indicates the amino acid sequence of the pilin
motif and E-box in AI-4 serotype M12.
[0878] FACS analysis has confirmed that the GAS AI-4 surface
proteins 19224134, 19224135, 19224137, and 19224141 are expressed
on the surface of GAS. FIG. 85 provides the results of FACS
analysis for surface expression of 19224134 on GAS serotype M12
2728. A shift in fluorescence is observed when anti-19224134
antiserum is present, demonstrating cell surface expression. Table
33, below, quantitatively summarizes the FACS fluorescence values
obtained for GAS serotype M12 2728 in the presence of pre-immune
antiserum, anti-19224134 antiserum, and the difference in
fluorescence value between the pre-immune and anti-19224134
antiserum. TABLE-US-00035 TABLE 33 Summary of FACS values for
surface expression of 19224134 in an M12 serotype 2728 Pre-immune
Anti-19224134 Change 137.8 485.32 348
[0879] FIG. 85 also provides the results of FACS analysis for
surface expression of a pilin antigen that has homology to 19224134
identified in a different GAS serotype, M6. FACS analysis conducted
with the 19224134 antisera was able to detect surface expression of
the homologous 19224134 antigen on each of GAS serotypes M6 2724,
M6 3650, and M6 2894. Table 34, below, quantitatively summarizes
the FACS fluorescence values obtained for each GAS serotype in the
presence of pre-immune antiserum, anti-19224134 antiserum, and the
difference in fluorescence value between the pre-immune and
anti-19224134 antiserum. TABLE-US-00036 TABLE 34 Summary of FACS
values for surface expression of 19224134 in M6 serotypes 2724 3650
2894 Pre- Anti- Pre- Anti- Pre- Anti- immune 19224134 Change immune
19224134 Change immune 19224134 Change 123.58 264.59 141 140.82
262.64 122 135.4 307.25 172
[0880] FIG. 86 provides the results of FACS analysis for surface
expression of 19224135 on GAS serotype M12 2728. A shift in
fluorescence is observed when anti-19224135 antiserum is present,
demonstrating cell surface expression. Table 35, below,
quantitatively summarizes the FACS fluorescence values obtained for
GAS serotype M12 2728 in the presence of pre-immune antiserum,
anti-19224135 antiserum, and the difference in fluorescence value
between the pre-immune and anti-19224135 antiserum. TABLE-US-00037
TABLE 35 Summary of FACS values for surface expression of 19224135
in an M12 serotype 2728 Pre-immune Anti-19224135 Change 151.38
471.95 321
[0881] FIG. 87 provides the results of FACS analysis for surface
expression of 19224137 on GAS serotype M12 2728. A shift in
fluorescence is observed when anti-19224137 antiserum is present,
demonstrating cell surface expression. Table 36, below,
quantitatively summarizes the FACS fluorescence values obtained for
GAS serotype M12 2728 in the presence of pre-immune antiserum,
anti-19224137 antiserum, and the difference in fluorescence value
between the pre-immune and anti-19224137 antiserum. TABLE-US-00038
TABLE 36 Summary of FACS values for surface expression of 19224137
in an M12 serotype 2728 Pre-immune Anti-19224137 Change 140.44
433.25 293
[0882] FIG. 88 provides the results of FACS analysis for surface
expression of 19224141 on GAS serotype M12 2728. A shift in
fluorescence is observed when anti-19224141 antiserum is present,
demonstrating cell surface expression. Table 37, below,
quantitatively summarizes the FACS fluorescence values obtained for
GAS serotype M12 2728 in the presence of pre-immune antiserum,
anti-19224141 antiserum, and the difference in fluorescence value
between the pre-immune and anti-19224141 antiserum. TABLE-US-00039
TABLE 37 Summary of FACS values for surface expression of 19224141
in an M12 serotype 2728 Pre-immune Anti-19224141 Change 147.02 498
351
[0883] 19224139 (designated as orf2) may also be expressed on the
surface of GAS serotype M12 bacteria. FIG. 175 shows the results of
FACS analysis for surface expression of 19224139 on M12 strain
2728. A slight shift in fluorescence is observed, which
demonstrates that some 19224139 may be expressed on the GAS cell
surface.
[0884] Surface expression of 19224135 on M12 serotype GAS has also
been confirmed by Western blot analysis. FIG. 99 shows that while
pre-immune sera (P .alpha.-4135) does not detect GAS M12 expression
of 19224135, anti-19224135 immune sera (I .alpha.-4135) is able to
detect 19224135 protein in both total GAS M12 extracts (M12 tot)
and GAS M12 fractions enriched for cell surface proteins (M12 surf
prot). The 19224135 proteins detected in the total GAS M12 extracts
or the GAS M12 extracts enriched for surface proteins are also
present as high molecular weight structures, indicating that
19224135 may be in an oligomeric (pilus) form. See also FIG. 108,
which provides a further Western blot showing that anti-19224135
antiserum (Anti-19224135) immunoreacts with high molecular weight
structures in GAS M12 strain 2728 protein extracts enriched for
surface proteins.
[0885] Surface expression of 19224137 on M12 serotype GAS has also
been confirmed by Western blot analysis. FIG. 100 shows that while
pre-immune sera (P .alpha.-4137) does not detect GAS M12 expression
of 19224137, anti-19224137 immune sera (I .alpha.-4137) is able to
detect 19224137 protein in both total GAS M12 extracts (M12 tot)
and GAS M12 fractions enriched for cell surface proteins (M12 surf
prot). The 19224137 proteins detected in the total GAS M12 extracts
or the GAS M12 extracts enriched for surface proteins are also
present as high molecular weight structures, indicating that
19224137 may be in an oligomeric (pilus) form. See also FIG. 108,
which provides a further Western blot showing that anti-19224137
antiserum (Anti-19224137) immunoreacts with high molecular weight
structures in GAS M12 strain 2728 protein extracts enriched for
surface proteins.
Streptococcus pneumoniae
[0886] Adhesin island sequences can be identified in Streptococcus
pneumoniae genomes. Several of these genomes include the publicly
available Streptococcus pneumoniae TIGR4 genome or Streptococcus
pneumoniae strain 670 genome. Examples of these S. pneumoniae AI
sequence are discussed below.
[0887] S. pneumoniae Adhesin Islands generally include a series of
open reading frames within a S. pneumoniae genome that encode for a
collection of surface proteins and sortases. A S. pneumoniae
Adhesin Island may encode for amino acid sequences comprising at
least one surface protein. Alternatively, an S. pneumoniae Adhesin
Island may encode for at least two surface proteins and at least
one sortase. Preferably, a S. pneumoniae Adhesin Island encodes for
at least three surface proteins and at least two sortases. One or
more of the surface proteins may include an LPXTG motif (such as
LPXTG (SEQ ID NO: 122)) or other sortase substrate motif. One or
more S. pneumoniae AI surface proteins may participate in the
formation of a pilus structure on the surface of the S. pneumoniae
bacteria.
[0888] S. pneumoniae Adhesin Islands of the invention preferably
include a divergently transcribed transcriptional regulator. The
transcriptional regulator may regulate the expression of the S.
pneumoniae AI operon.
[0889] The S. pneumoniae AI surface proteins may bind or otherwise
adhere to fibrinogen, fibronectin, or collagen.
[0890] A schematic of the organization of a S. pneumoniae AI locus
is provided in FIG. 137. The locus comprises open reading frames
encoding a transcriptional regulator (rlrA), cell wall surface
proteins (rrgA, rrgB, rrgc), and sortases (srtB, srtC, srtD). FIG.
137 also indicates the S. pneumoniae strain TIGR4 gene name
corresponding to each of these open reading reading frames.
[0891] Tables 9 and 38 identify the genomic location of each of
these open reading frames in S. pneumoniae strains TIGR4 and 670,
respectively. TABLE-US-00040 TABLE 9 S. pneumoniae AI sequences
from TIGR4 Synonym (AI Genomic Location Strand Length PID Sequence
Identifier) Functional description 436302 . . . 437831 - 509
15900377 SP0461 transcriptional regulator 438326 . . . 441007 + 893
15900378 SP0462 cell wall surface anchor family protein 441231 . .
. 443228 + 665 15900379 SP0463 cell wall surface anchor family
protein 443275 . . . 444456 + 393 15900380 SP0464 cell wall surface
anchor family protein 444675 . . . 444806 - 43 15900381 SP0465
hypothetical protein 444857 . . . 445696 + 279 15900382 SP0466
sortase 445791 . . . 446576 + 261 15900383 SP0467 sortase 446563 .
. . 447414 + 283 15900384 SP0468 sortase
[0892] TABLE-US-00041 TABLE 38 S. pneumoniae strain 670 AI
sequences AI Sequence Genomic Location Strand Identifier\
Functional description 4383-5645 - Orf1_670 IS1167, transposase
5910-7439 - Orf2_670 transcriptional regulator, putative 7934-10606
+ Orf3_670 cell wall surface anchor family protein 10839-12773 +
Orf4_670 cell wall surface anchor family protein 12796-14001 +
Orf5_670 cell wall surface anchor family protein 14327-15241 +
Orf6_670 sortase, putative 15336-16121 + Orf7_670 sortase, putative
16108-16959 + Orf8_670 sortase, putative
[0893] The full-length nucleotide sequence of the S. pneumoniae
strain 670 .mu.l is also shown in FIG. 101, as is its translated
amino acid sequence.
[0894] At least eight other S. pneumoniae strains contain an
adhesin island locus described by the locus depicted in FIG. 137.
These strains were identified by an amplification analysis. The
genomes of different S. pneumoniae strains were amplified with
eleven separate sets of primers. The sequence of each of these
primers is provided below in Table 41. TABLE-US-00042 TABLE 41
Sequences of primers used to amplify AI locus Primer Pair Forward
Primer Sequence Reverse Primer Sequence 1 ACTTTCTAATGAGTTGTTTAGGCG
AGCGACAAGCCACTGTATCATATT 2 CTGGTCGATAACTCCTTCAATCTT
GTACGACAAAAGTGTGGCTTGTT 3 GAATGCGATATTCAGGACCAACTA
ATCTCACTGAGTTAATCCGTTCAC 4 TGTATACAAGTGTGTCATTGCCAG
CATCTTCACCTGTTCTCACATTTT 5 GCGGTCTTTAGTCTTCAAAAACA
CAAGAGAAAAACACAGAGCCATAA 6 TTGCTTAAGTAAGAGAGAAAGGAGC
CAGGAGTATAGTGTCCGCTTTCTT 7 GGCAATGTTGACTTTATGAAGGTG
TATCAGCATCCCTTTATCTTCAAAC 8 TGAGATTTTCTCGTTTCTCTTAGC
AATAGACGATGGGTATTGATCATGT 9 CCGACGAACTTTGATGATTTATTG
ACCAACAGACGATGACTGTTAATC 10 AATGACTTTGAGCCTGTCTTGAT
TTCTACAATTTCCTGGCCATTATC 11 GCCATTTGGATCAGCTAAAAGTT
TTTTTCAACCCACTACAGTTGACA
These primers hybridized along the entire length of the AI locus to
generate amplification products representative of sequences
throughout the locus. See FIG. 138, which is a schematic of the
location where each of these primers hybridizes to the S.
pneumoniae AI locus. FIG. 139A provides the set of amplicons
obtained from amplification of the AI locus in S. pneumoniae strain
TIGR4. FIG. 139B provides the length, in base pairs, of each
amplicon in S. pneumoniae strain TIGR4. Amplification of the genome
of S. pneumoniae strains 19A Hungary 6, 6B Finland 12, 6B Spain 2,
9V Spain 3, 14 CSR 10, 19F Taiwan 14, 23F Taiwan 15, and 23F Poland
16 produced a set of eleven amplicons for the eleven primer pairs,
indicating that each of these strains also contained the S.
pneumoniae AI locus.
[0895] The S. pneumoniae strains were also identified as containing
the AI locus by comparative genome hybridization (CGH) analysis.
The genomes of sixteen S. pneumoniae strains were interrogated for
the presence of the AI locus by comparison to unique open reading
frames of strain TIGR4. The AI locus was detected by this method in
strains 19A Hungary 6 (19AHUN), 6B Finland 12 (6BFIN12), 6B Spain 2
(6BSP2), 14CSR10 (14 CSR10), 9V Spain 3 (9VSP3), 19F Taiwan 14
(19FTW14), 23F Taiwan 15 (19FTW15), and 23F Poland 16 (23FP16). See
FIG. 140.
[0896] The AI locus has been sequenced for each of these strains
and the nucleotide and encoded amino acid seqeunce for each orf has
been determined. An alignment of the complete nucleotide sequence
of the adhesin island present in each of the ten strains is
provided in FIG. 196. Aligning the amino acid sequences encoded by
the orfs reveals conservation of many of the AI polypeptide amino
acid sequences. For example, Table 39 provides a comparison of the
percent identities of the polypeptides encoded within the S.
pneumoniae strain 670 and TIGR4 adhesin islands. TABLE-US-00043
TABLE 39 Pecent identity comparison of S. pneumoniae strains AI
sequences S. pneumoniae S. pneumoniae strain 670 from TIGR4
polypeptide polypeptide Shared identity of polypeptides Orf1_670
SP0460 99.3% identity in 422 aa overlap Orf2_670 SP0461 100.0%
identity in 509 aa overlap Orf3_670 SP0462 83.2% identity in 895 aa
overlap Orf4_670 SP0463 47.9% identity in 678 aa overlap Orf5_670
SP0464 99.7% identity in 393 aa overlap Orf6_670 SP0466 100.0%
identity in 279 aa overlap Orf7_670 SP0467 94.2% identity in 260 aa
overlap Orf8_670 SP0468 91.5% identity in 283 aa overlap
FIGS. 141-147 each provide a multiple sequence alignment for the
polypeptides encoded by one of the open reading frames in all ten
AI-positive S. pneumoniae strains. In each of the sequence
alignments, light shading indicates an LPXTG motif and dark shading
indicates the presence of an E-box motif with the conserved
glutamic acid residue of the E-box motif in bold.
[0897] The sequence alignments also revealed that the polypeptides
encoded by most of the open reading frames may be divided into two
groups of homology, S. pneumoniae AI-a and AI-b. S. pneumoniae
strains that comprise AI-a include 14 CSR 10, 19A Hungary 6, 23F
Poland 15, 670, 6B Finland 12, and 6B Spain 2. S. pneumoniae
strains that comprise AI-b include 19F Taiwan 14, 9V Spain 3, 23F
Taiwan 15, and TIGR4. An immunogenic composition of the invention
may comprise one or more polypeptides from within each of S.
pneumoniae AI-a and AI-b. For example, polypeptide RrgB, encoded by
open reading frame 4, may be divided within two such groups of
homology. One group contains the RrgB sequences of six S.
pneumoniae strains and a second group contains the RrgB sequences
of four S. pneumoniae strains. While the amino acid sequence of the
strains within each individual group is 99-100 percent identical,
the amino acid sequence identity of the strains in the first
relative to the second group is only 48%. Table 41 provides the
identity comparisons of the amino acid sequences encoded by each
open reading frame for the ten S. pneumoniae strains.
TABLE-US-00044 TABLE 42 Conservation of amino acid sequences
encoded by the S. pneumoniae AI locus % Identity % Identity
Putative Role Encoded Groups of in Between of Polypeptide by Orf
Homology Group Groups RlrA, 2 1 group (10 strains) 100 --
transcriptional regulator RrgA, cell 3 2 groups (6 + 4) 98-100 83
wall surface protein RrgB, cell 4 2 groups (6 + 4) 99-100 48 wall
surface protein RrgC, cell 5 2 groups (6 + 4) 99-100 97 wall
surface protein SrtB, putative 6 2 groups (7 + 3) 99-100 97 sortase
SrtC, putative 7 2 groups (6 + 4) 95-100 93 sortase SrtD, putative
8 2 groups (6 + 4) 99-100 92 sortase
[0898] The division of homology between the RrgB polypeptide in the
S. pneumoniae strains is due a lack of amino acid sequence identity
in the central amino acid residues. Amino acid residues 1-30 and
617-665 are identical for each of the ten S. pneumoniae strains.
However, amino acid residues 31-616 share between 42 and 100
percent identity between strains. See FIG. 149. The shared N- and
C-terminal regions of identity in the RrgB polypeptides may be
preferred portions of the RrgB polypeptide for use in an
immunogenic composition. Similarly, shared regions of identity in
any of the polypeptides encoded by the S. pneumoniae AI locus may
be preferable for use in immunogenic compositions. One of skill in
the art, using the amino acid alignments provided in FIGS. 141-147,
would readily be able to determine these regions of identity.
[0899] The S. pneumoniae comprising these AI loci do, in fact,
express high molecular weight polymers on their surface, indicating
the presence of pili. See FIG. 182, which shows detection of high
molecular weight structures expressed by S. pneumoniae strains that
comprise the adhesin island locus depicted in FIG. 137, these
strains are indicated as rlrA+. Confirming these findings, electron
microscopy and negative staining detects the presence of pili
extending from the surface of S. pneumoniae. See FIG. 185. To
demonstrate that the adhesin island locus was responsible for the
pili, the rrgA-srtD region of TIGR 4 were deleted. Deletion of this
region of the adhesin island resulted in a loss of pili expression.
See FIG. 186. See also FIG. 235, which provides an electron
micrograph of S. pneumoniae lacking the rrgA-srtD region immunogold
stained using anti-RrgB and anti-RrgC antibodies. No pili can be
seen. Similarly to that described above, a S. pneumoniae bacteria
that lacks a transcriptional repressor, mgrA, of genes in the
adhesin island expresses pili. See FIG. 187. However, and as
expected, a S. pneumoniae bacteria that lacks both the mgrA and
adhesin island genes in the rrgA-srtD region does not express pili.
See FIG. 188.
[0900] These high molecular weight pili structures appear to play a
role in adherence of S. pneumoniae to cells. S. pneumoniae TIGR4
that lack the pilus operon have significantly diminished ability to
adhere to A549 Alveolar cells in vitro. See FIG. 184.
[0901] The Sp0463 (S. pneumoniae TIGR4 rrgB) adhesion island
polypeptide is expressed in oligomeric form. Whole cell extracts
were analyzed by Western blot using a Sp0463 antiserum. The
antiserum cross-hybridized with high molecular weight Sp0463
polymers. See FIG. 156. The antiserum did not cross-hybridize with
polypeptides from D39 or R6 strains of S. pneumoniae, which do not
contain the AI locus depicted in FIG. 137. Immunogold labelling of
S. pneumoniae TIGR 4 using RrgB antiserum confirms the presence of
RrgB in pili. FIG. 189 shows double-labeling of S. pneumoniae TIGR
4 bacteria with immunolabeling for RrgB (5 nm gold particles) and
RrgC (10 nm gold particles) protein. The RrgB protein is detected
as present at intervals along the pilus structure. The RrgC protein
is detected at the tips of the pili. See FIG. 234 at arrows; FIG.
234 is a close up of a pilus in FIG. 189 at the location indicated
by *.
[0902] The RrgA protein appears to be present in and necessary for
formation of high molecular weight structures on the surface of S.
pneumoniae TIGR4. See FIG. 181 which provides the results of
Western blot analysis of TIGR4 S. pneumoniae lacking the gene
encoding RrgA. No high molecular weight structures are detected in
S. pneumoniae that do not express RrgA using antiserum raised
against RrgB. See also FIG. 183.
[0903] A detailed diagram of the amino acid sequence comparions of
the RrgA protein in the ten S. pneumoniae strains is shown in FIG.
148. The diagram reveals the division of the individual S.
pneumoniae strains into the two different homology groups.
[0904] The cell surface polypeptides encoded by the S. pneumoniae
TIGR4 AI, Sp0462 (rrgA), Sp0463 (rrgB), and Sp0464 (rrgC), have
been cloned and expressed. See examples 15-17. A polyacrylamide gel
showing successful recombinant expression of RrgA is provided in
FIG. 190A. Detection of the RrgA protein, which is expressed in
pET21b with a histidine tag, is also shown by Western blot analysis
in FIG. 190B, using an anti-histidine tag antibody.
[0905] Antibodies that detect RrgB and RrgC antibodies have been
produced in mice. See FIGS. 191 and 192, which show detection of
RrgB and RrgC, respectively, using the raised antibodies.
[0906] In addition to the identification of these S. pneumoniae
adhesion islands, coding sequences for SrtB type sortases have been
identified in several S. pneumoniae clinical isolates,
demonstrating conservation of a SrtB type sortase across these
isolates.
Recombinantly Produced AI polypeptides
[0907] It is also an aspect of the invention to alter a non-AI
polypeptide to be expressed as an AI polypeptide. The non-AI
polypeptide may be genetically manipulated to additionally contain
AI polypeptide sequences, e.g., a sortase substrate, pilin, or
E-box motif, which may cause expression of the non-AI polypeptide
as an AI polypeptide. Alternatively the non-AI polypeptide may be
genetically manipulated to replace an amino acid sequence within
the non-AI polypeptide for AI polypeptide sequences, e.g., a
sortase substrate, pilin, or E-box motif, which may cause
expression of the non-AI polypeptide as an AI polypeptide. Any
number of amino acid residues may be added to the non-AI
polypeptide or may be replaced within the non-AI polypeptide to
cause its expression as an AI polypeptide. At least 5, 6, 7, 8, 9,
10, 15, 20, 25, 30, 35, 50, 75, 100, 150, 200, or 250 amino acid
residues may be replaced or added to the non-AI polypeptide amino
acid sequence. GBS 322 may be one such non-AI polypeptide that may
be expressed as an AI polypeptide.
GBS Adhesin Island Sequences
[0908] The GBS AI polypeptides of the invention can, of course, be
prepared by various means (e.g. recombinant expression,
purification from GBS, chemical synthesis etc.) and in various
forms (e.g. native, fusions, glycosylated, non-glycosylated etc.).
They are preferably prepared in substantially pure form (i.e.
substantially free from other streptococcal or host cell proteins)
or substantially isolated form.
[0909] The GBS AI proteins of the invention may include polypeptide
sequences having sequence identity to the identified GBS proteins.
The degree of sequence identity may vary depending on the amino
acid sequence (a) in question, but is preferably greater than 50%
(e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, 99.5% or more). Polypeptides having sequence
identity include homologs, orthologs, allelic variants and
functional mutants of the identified GBS proteins. Typically, 50%
identity or more between two proteins is considered to be an
indication of functional equivalence. Identity between proteins is
preferably determined by the Smith-Waterman homology search
algorithm as implemented in the MPSRCH program (Oxford Molecular),
using an affinity gap search with parameters gap open penalty=]12
and gap extension penalty=1.
[0910] The GBS adhesin island polynucleotide sequences may include
polynucleotide sequences having sequence identity to the identified
GBS adhesin island polynucleotide sequences. The degree of sequence
identity may vary depending on the polynucleotide sequence in
question, but is preferably greater than 50% (e.g. 60%, 65%, 70%,
75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
99.5% or more).
[0911] The GBS adhesin island polynucleotide sequences of the
invention may include polynucleotide fragments of the identified
adhesin island sequences. The length of the fragment may vary
depending on the polynucleotide sequence of the specific adhesin
island sequence, but the fragment is preferably at least 10
consecutive polynucleotides, (e.g. at least 10, 12, 14, 16, 18, 20,
25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200 or more).
[0912] The GBS adhesin island amino acid sequences of the invention
may include polypeptide fragments of the identified GBS proteins.
The length of the fragment may vary depending on the amino acid
sequence of the specific GBS antigen, but the fragment is
preferably at least 7 consecutive amino acids, (e.g. 8, 10, 12, 14,
16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200 or
more). Preferably the fragment comprises one or more epitopes from
the sequence. Other preferred fragments include (1) the N-terminal
signal peptides of each identified GBS protein, (2) the identified
GBS protein without their N-terminal signal peptides, and (3) each
identified GBS protein wherein up to 10 amino acid residues (e.g.
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) are deleted from
the N-terminus and/or the C-terminus e.g. the N-terminal amino acid
residue may be deleted. Other fragments omit one or more domains of
the protein (e.g. omission of a signal peptide, of a cytoplasmic
domain, of a transmembrane domain, or of an extracellular
domain).
GBS 80
[0913] Examples of preferred GBS 80 fragments are discussed below.
Polynucleotide and polypeptide sequences of GBS 80 from a variety
of GBS serotypes and strain isolates are set forth in FIGS. 18 and
22. The polynucleotide and polypeptide sequences for GBS 80 from
GBS serotype V, strain isolate 2603 are also included below as SEQ
ID NOS 1 and 2: TABLE-US-00045 SEQ ID NO.1
ATGAAATTATCGAAGAAGTTATTGTTTTCGGCTGCTGTTTTAACAATGGT
GGCGGGGTCAACTGTTGAACCAGTAGCTCAGTTTGCGACTGGAATGAGTA
TTGTAAGAGCTGCAGAAGTGTCACAAGAACGCCCAGCGAAAACAACAGTA
AATATCTATAAATTACAAGCTGATAGTTATAAATCGGAAATTACTTCTAA
TGGTGGTATCGAGAATAAAGACGGCGAAGTAATATCTAACTATGCTAAAC
TTGGTGACAATGTAAAAGGTTTGCAAGGTGTACAGTTTAAACGTTATAAA
GTCAAGACGGATATTTCTGTTGATGAATTGAAAAAATTGACAACAGTTGA
AGCAGCAGATGCAAAAGTTGGAACGATTCTTGAAGAAGGTGTCAGTCTAC
CTCAAAAAACTAATGCTCAAGGTTTGGTCGTCGATGCTCTGGATTCAAAA
AGTAATGTGAGATACTTGTATGTAGAAGATTTAAAGAATTCACCTTCAAA
CATTACCAAAGCTTATGCTGTACCGTTTGTGTTGGAATTACCAGTTGCTA
ACTCTACAGGTACAGGTTTCCTTTCTGAAATTAATATTTACCCTAAAAAC
GTTGTAACTGATGAACCAAAAACAGATAAAGATGTTAAAAAATTAGGTCA
GGACGATGCAGGTTATACGATTGGTGAAGAATTCAAATGGTTCTTGAAAT
CTACAATCCCTGCCAATTTAGGTGACTATGAAAAATTTGAAATTACTGAT
AAATTTGCAGATGGCTTGACTTATAAATCTGTTGGAAAAATCAAGATTGG
TTCGAAAACACTGAATAGAGATGAGCACTACACTATTGATGAACCAACAG
TTGATAACCAAAATACATTAAAAATTACGTTTAAACCAGAGAAATTTAAA
GAAATTGCTGAGCTACTTAAAGGAATGACCCTTGTTAAAAATCAAGATGC
TCTTGATAAAGCTACTGCAAATACAGATGATGCGGCATTTTTGGAAATTC
CAGTTGCATCAACTATTAATGAAAAAGCAGTTTTAGGAAAAGCAATTGAA
AATACTTTTGAACTTCAATATGACCATACTCCTGATAAAGCTGACAATCC
AAAACCATCTAATCCTCCAAGAAAACCAGAAGTTCATACTGGTGGGAAAC
GATTTGTAAAGAAAGACTCAACAGAAACACAAACACTAGGTGGTGCTGAG
TTTGATTTGTTGGCTTCTGATGGGACAGCAGTAAAATGGACAGATGCTCT
TATTAAAGCGAATACTAATAAAAACTATATTGCTGGAGAAGCTGTTACTG
GGCAACCAATCAAATTGAAATCACATACAGACGGTACGTTTGAGATTAAA
GGTTTGGCTTATGCAGTTGATGCGAATGCAGAGGGTACAGCAGTAACTTA
CAAATTAAAAGAAACAAAAGCACCAGAAGGTTATGTAATCCCTGATAAAG
AAATCGAGTTTACAGTATCACAAACATCTTATAATACAAAACCAACTGAC
ATCACGGTTGATAGTGCTGATGCAACACCTGATACAATTAAAAACAACAA
ACGTCCTTCAATCCCTAATACTGGTGGTATTGGTACGGCTATCTTTGTCG
CTATCGGTGCTGCGGTGATGGCTTTTGCTGTTAAGGGGATGAAGCGTCGT ACAAAAGATAAC SEQ
ID NO: 2 MKLSKKLLFSAAVLTMVAGSTVEPVAQFATGMSIVRAAEVSQERPAKTTV
NIYKLQADSYKSEITSNGGIENKDGEVISNYAKLGDNVKGLQGVQFKRYK
VKTDISVDELKKLTTVEAADAKVGTILEEGVSLPQKTNAQGLVVDALDSK
SNVRYLYVEDLKNSPSNITKAYAVPFVLELPVANSTGTGFLSEINIYPKN
VVTDEPKTDKDVKKLGQDDAGYTIGEEFKWFLKSTIPANLGDYEKFEITD
KFADGLTYKSVGKIKIGSKTLNRDEHYTIDEPTVDNQNTLKITFKPEKFK
EIAELLKGMTLVKNQDALDKATANTDDAAFLEIPVASTINEKAVLGKAIE
NTFELQYDHTPDKADNPKPSNPPRKPEVHTGGKRFVKKDSTETQTLGGAE
FDLLASDGTAVKWTDALIKANTNKNYIAGEAVTGQPIKLKSHTDGTFEIK
GLAYAVDANAEGTAVTYKLKETKAPEGYVIPDKEIEFTVSQTSYNTKPTD
ITVDSADATPDTIKNNKRPSIPNTGGIGTAIFVAIGAAVMAFAVKGMKRR TKDN
[0914] As described above, the compositions of the invention may
include fragments of AI proteins. In some instances, removal of one
or more domains, such as a leader or signal sequence region, a
transmembrane region, a cytoplasmic region or a cell wall anchoring
motif, may facilitate cloning of the gene encoding the protein
and/or recombinant expression of the GBS AI protein. In addition,
fragments comprising immunogenic epitopes of the cited GBS AI
proteins may be used in the compositions of the invention.
[0915] For example, GBS 80 contains an N-terminal leader or signal
sequence region which is indicated by the underlined sequence at
the beginning of SEQ ID NO: 2 above. In one embodiment, one or more
amino acids from the leader or signal sequence region of GBS 80 are
removed. An example of such a GBS 80 fragment is set forth below as
SEQ ID NO: 3: TABLE-US-00046 SEQ ID NO: 3
AEVSQERPAKTTVNIYKLQADSYKSEITSNGGIENKDGEVISNYAKLGDN
VKGLQGVQFKRYKVKTDISVDELKKLTTVEAADAKVGTILEEGVSLPQKT
NAQGLVVDALDSKSNVRYLYVEDLKNSPSNITKAYAVPFVLELPVANSTG
TGFLSEINIYPKNVVTDEPKTDKDVKKLGQDDAGYTIGEEFKWFLKSTIP
ANLGDYEKFEITDKFADGLTYKSVGKIKIGSKTLNRDEHYTIDEPTVDNQ
NTLKITFKPEKFKEIAELLKGMTLVKNQDALDKATANTDDAAFLEIPVAS
TINEKAVLGKAIENTFELQYDHTPDKADNPKPSNPPRKPEVHTGGKRFVK
KDSTETQTLGGAEFDLLASDGTAVKWTDALIKANTNKNYIAGEAVTGQPI
KLKSHTDGTFEIKGLAYAVDANAEGTAVTYKLKETKAPEGYVIPDKEIEF
TVSQTSYNTKPTDITVDSADATPDTIKNNKRPSIPNTGGIGTAIFVAIGA
AVMAFAVKGMKRRTKDN
[0916] GBS 80 contains a C-terminal transmembrane region which is
indicated by the underlined sequence near the end of SEQ ID NO: 2
above. In one embodiment, one or more amino acids from the
transmembrane region and/or a cytoplasmic region are removed. An
example of such a GBS 80 fragment is set forth below as SEQ ID NO:
4: TABLE-US-00047 SEQ ID NO: 4
MKLSKKLLFSAAVLTMVAGSTVEPVAQFATGMSIVRAAEVSQERPAKTTV
NIYKLQADSYKSEITSNGGIENKDGEVISNYAKLGDNVKGLQGVQFKRYK
VKTDISVDELKKLTTVEAADAKVGTILEEGVSLPQKTNAQGLVVDALDSK
SNVRYLYVEDLKNSPSNITKAYAVPFVLELPVANSTGTGFLSEINIYPKN
VVTDEPKTDKDVKKLGQDDAGYTIGEEFKWFLKSTIPANLGDYEKFEITD
KFADGLTYKSVGKIKIGSKTLNRDEHYTIDEPTVDNQNTLKITFKPEKFK
EIAELLKGMTLVKNQDALDKATANTDDAAFLEIPVASTINEKAVLGKAIE
NTFELQYDHTPDKADNPKPSNPPRKPEVHTGGKRFVKKDSTETQTLGGAE
FDLLASDGTAVKWTDALIKANTNKNYIAGEAVTGQPIKLKSHTDGTFEIK
GLAYAVDANAEGTAVTYKLKETKAPEGYVIPDKEIEFTVSQTSYNTKPTD
ITVDSADATPDTIKNNKRPSIPNTG
[0917] GBS 80 contains an amino acid motif indicative of a cell
wall anchor: SEQ ID NO: 5 IPNTG (shown in italics in SEQ ID NO: 2
above). In some recombinant host cell systems, it may be preferable
to remove this motif to facilitate secretion of a recombinant GBS
80 protein from the host cell. Accordingly, in one preferred
fragment of GBS 80 for use in the invention, the transmembrane
and/or cytoplasmic regions and the cell wall anchor motif are
removed from GBS 80. An example of such a GBS 80 fragment is set
forth below as SEQ ID NO: 6. TABLE-US-00048 SEQ ID NO: 6
MKLSKKLLFSAAVLTMVAGSTVEPVAQFATGMSIVRAAEVSQERPAKTTV
NIYKLQADSYKSEITSNGGIENKDGEVISNYAKLGDNVKGLQGVQFKRYK
VKTDISVDELKKLTTVEAADAKVGTILEEGVSLPQKTNAQGLVVDALDSK
SNVRYLYVEDLKNSPSNITKAYAVPFVLELPVANSTGTGFLSEINIYPKN
VVTDEPKTDKDVKKLGQDDAGYTTGEEFKWFLKSTTPANLGDYEKFEITD
KFADGLTYKSVGKIKIGSKTLNRDEHYTIDEPTVDNQNTLKTTFKPEKFK
EIAELLKGMTLVKNQDALDKATANTDDAAELEIPVASTLNEKAVLGKAIE
NTFELQYDHTPDKADNPKPSNPPRKPEVHTGGKRFVKKDSTETQTLGGAE
FDLLASDGTAVKWTDALIKANTNKNYIAGEAVTGQPIKLKSHTDGTFEIK
GLAYAVDANAEGTAVTYKLKETKAPEGYVIPDKEIEFTVSQTSYNTKPTD
ITVDSADATPDTIKNNKRPS
[0918] Alternatively, in some recombinant host cell systems, it may
be preferable to use the cell wall anchor motif to anchor the
recombinantly expressed protein to the cell wall. The extracellular
domain of the expressed protein may be cleaved during purification
or the recombinant protein may be left attached to either
inactivated host cells or cell membranes in the final
composition.
[0919] In one embodiment, the leader or signal sequence region, the
transmembrane and cytoplasmic regions and the cell wall anchor
motif are removed from the GBS 80 sequence. An example of such a
GBS 80 fragment is set forth below as SEQ ID NO: 7. TABLE-US-00049
SEQ ID NO: 7 AEVSQERPAKTTVNIYKLQADSYKSEITSNGGIENKDGEVISNYAKLGDN
VKGLQGVQFKRYKVKTDISVDELKKLTTVEAADAKVGTILEEGVSLPQKT
NAQGLVVDALDSKSNVRYLYVEDLKNSPSNITKAYAVPFVLELPVANSTG
TGFLSEINIYPKNVVTDEPKTDKDVKKLGQDDAGYTIGEEFKWFLKSTIP
ANLGDYEKFEITDKFADGLTYKSVGKIKIGSKTLNRDEHYTIDEPTVDNQ
NTLKITFKPEKFKEIAELLKGMTLVKNQDALDKATANTDDAAFLEIPVAS
TINEKAVLGKAIENTFELQYDHTPDKADNPKPSNPPRKPEVHTGGKRFVK
KDSTETQTLGGAEFDLLASDGTAVKWTDALIKANTNKNYIAGEAVTGQPI
KLKSHTDGTFEIKGLAYAVDANAEGTAVTYKLKETKAPEGYVIPDKEIEF
TVSQTSYNTKPTDITVDSADATPDTIKNNKRPS
[0920] Applicants have identified a particularly immunogenic
fragment of the GBS 80 protein. This immunogenic fragment is
located towards the N-terminus of the protein and is underlined in
the GBS 80 SEQ ID NO: 2 sequence below. The underlined fragment is
set forth below as SEQ ID NO: 8. TABLE-US-00050 SEQ ID NO: 2
MKLSKKLLFSAAVLTMVAGSTVEPVAQFATGMSIVRAAEVSQERPAKTTV
NIYKLQADSYKSEITSNGGIENKDGEVISNYAKLGDNVKGLQGVQFKRYK
VKTDISVDELKKLTTVEAADAKVGTILEEGVSLPQKTNAQGLVVDALDSK
SNVRYLYVEDLKNSPSNITKAYAVPFVLELPVANSTGTGFLSEINIYPKN
VVTDEPKTDKDVKKLGQDDAGYTIGEEFKWFLKSTIPANLGDYEKFEITD
KFADGLTYKSVGKIKIGSKTLNRDEHYTIDEPTVDNQNTLKTTFKPEKFK
EIAELLKGMTLVKNQDALDKATANTDDAAFLEIPVASTINEKAVLGKAIE
NTFELQYDHTPDKADNPKPSNPPRKPEVHTGGKRFVKKDSTETQTLGGAE
FDLLASDGTAVKWTDALIKANTNKNYIAGEAVTGQPIKLKSHTDGTFEIK
GLAYAVDANAEGTAVTYKLKETKAPEGYVIPDKEIEFTVSQTSYNTKPTD
ITVDSADATPDTIKNNKRPSIPNTGGIGTAIFVAIGAAVMAFAVKGMKRR TKDN SEQ ID NO:
8 AEVSQERPAKTTVNIYKLQADSYKSEITSNGGIENKDGEVISNYAKLGDN
VKGLQGVQFKRYKVKTDISVDELKKLTTVEAADAKVGTILEEGVSLPQKT
NAQGLVVDALDSKSNVRYLYVEDLKNSPSNITKAYAVPFVLELPVANSTG
TGFLSEINIYPKNVVTDEPKTDKDVKKLGQDDAGYTIGEEFKWFLKSTIP
ANLGDYEKFEITDKFADGLTYKSVGKIKIGSKTLNRDEHYTIDEPTVDNQ
NTLKITFKPEKFKEIAELLKG
[0921] The immunogenicity of the protein encoded by SEQ ID NO: 7
was compared against PBS, GBS whole cell, GBS 80 (full length) and
another fragment of GBS 80, located closer to the C-terminus of the
peptide (SEQ ID NO: 9, below). TABLE-US-00051 SEQ ID NO: 9
MTLVKNQDALDKATANTDDAAFLEIPVASTINEKAVLGKAIENTFELQYD
HTPDKADNPKPSNPPRKPEVHTGGKRFVKKDSTETQTLGGAEFDLLASDG
TAVKWTDALIKANTNKNYIAGEAVTGQPIKLKSHTDGTFEIKGLAYAVDA
NAEGTAVTYKLKETKAPEGYVIPDKEIEFTVSQTSYNTKPTDITVDSADA TPDTIKNNKRPS
[0922] Both an Active Maternal Immunization Assay and a Passive
Maternal Immunization Assay were conducted on this collection of
proteins.
[0923] As used herein, an Active Maternal Immunization assay refers
to an in vivo protection assay where female mice are immunized with
the test antigen composition. The female mice are then bred and
their pups are challenged with a lethal dose of GBS. Serum titers
of the female mice during the immunization schedule are measured as
well as the survival time of the pups after challenge.
[0924] Specifically, the Active Maternal Immunization assays
referred to herein used groups of four CD-1 female mice (Charles
River Laboratories, Calco Italy). These mice were immunized
intraperitoneally with the selected proteins in Freund's adjuvant
at days 1, 21 and 35, prior to breeding. 6-8 weeks old mice
received 20 .mu.g protein/dose when immunized with a single
antigen, 30-45 .mu.g protein/dose (15 .mu.g each antigen) when
immunized with combination of antigens. The immune response of the
dams was monitored by using serum samples taken on day 0 and 49.
The female mice were bred 2-7 days after the last immunization (at
approximately t=36-37), and typically had a gestation period of 21
days. Within 48 hours of birth, the pups were challenged via I.P.
with GBS in a dose approximately equal to a amount which would be
sufficient to kill 70-90% of unimmunized pups (as determined by
empirical data gathered from PBS control groups). The GBS challenge
dose is preferably administered in 50 .mu.l of THB medium.
Preferably, the pup challenge takes place at 56 to 61 days after
the first immunization. The challenge inocula were prepared
starting from frozen cultures diluted to the appropriate
concentration with THB prior to use. Survival of pups was monitored
for 5 days after challenge.
[0925] As used herein, the Passive Maternal Immunization Assay
refers to an in vivo protection assay where pregnant mice are
passively immunized by injecting rabbit immune sera (or control
sera) approximately 2 days before delivery. The pups are then
challenged with a lethal dose of GBS.
[0926] Specifically, the Passive Maternal Immunization Assay
referred to herein used groups of pregnant CD1 mice which were
passively immunized by injecting 1 ml of rabbit immune sera or
control sera via I.P., 2 days before delivery. Newborn mice (24-48
hrs after birth) are challenged via I.P. with a 70-90% lethal dose
of GBS serotype II COH1. The challenge dose, obtained by diluting a
frozen mid log phase culture, was administered in 50 .mu.l of THB
medium.
For both assays, the number of pups surviving GBS infection was
assessed every 12 hrs for 4 days. Statistical significance was
estimated by Fisher's exact test.
[0927] The results of each assay for immunization with SEQ ID NO:
7, SEQ ID NO: 8, PBS and GBS whole cell are set forth in Tables 1
and 2 below. TABLE-US-00052 TABLE 1 Immunization Antigen
Alive/total % Survival Fisher's exact test PBS (neg control) 13/80
16% GBS (whole cell) 54/65 83% P < 0.00000001 GBS80 (intact)
62/70 88% P < 0.00000001 GBS80 (fragment) 35/64 55% P =
0.0000013 SEQ ID 7 GBS80 (fragment) 13/67 19% P = 0.66 SEQ ID 8
[0928] TABLE-US-00053 TABLE 2 Passive Maternal Immunization Antigen
Alive/total % Survival Fisher's exact test PBS (neg control) 12/42
28% GBS (whole cell) 48/52 92% P < 0.00000001 GBS80 (intact)
48/55 87% P < 0.00000001 GBS80 (fragment) 45/57 79% P =
0.0000006 SEQ ID 7 GBS80 (fragment) 13/54 24% P = 1 SEQ ID 8
[0929] As shown in Tables 1 and 2, immunization with the SEQ ID NO:
7 GBS 80 fragment provided a substantially improved survival rate
for the challenged pups than the comparison SEQ ID NO: 8 GBS 80
fragment. These results indicate that the SEQ ID NO: 7 GBS 80
fragment may comprise an important immunogenic epitope of GBS
80.
[0930] As discussed above, pilin motifs, containing conserved
lysine (K) residues have been identified in GBS 80. The pilin motif
sequences are underlined in SEQ ID NO: 2, below. Conserved lysine
(K) residues are marked in bold, at amino acid residues 199 and 207
and at amino acid residues 368 and 375. The pilin sequences, in
particular the conserved lysine residues, are thought to be
important for the formation of oligomeric, pilus-like structures of
GBS 80. Preferred fragments of GBS 80 include at least one
conserved lysine residue. Preferably, fragments include at least
one pilin sequence. TABLE-US-00054 SEQ ID NO: 2
MKLSKKLLFSAAVLTMVAGSTVEPVAQFATGMSIVRAAEVSQERPAKTTV
NIYKLQADSYKSEITSNGGIENKDGEVISNYAKLGDNVKGLQGVQFKRYK
VKTDISVDELKKLTTVEAADAKVGTILEEGVSLPQKTNAQGLVVDALDSK
SNVRYLYVEDLKNSPSNITKAYAVPFVLELPVANSTGTGFLSEINIYPKN
VVTDEPKTDKDVKKLGQDDAGYTIGEEFKWFLKSTIPANLGDYEKFEITD
KFADGLTYKSVGKIKIGSKTLNRDEHYTIDEPTVDNQNTLKITFKPEKFK
EIAELLKGMTLVKNQDALDKATANTDDAAFLEIPVASTINEKAVLGKAIE
NTFELQYDHTPDKADNPKPSNPPRKPEVHTGGKRFVKKDSTETQTLGGAE
FDLLASDGTAVKWTDALIKANTNKNYIAGEAVTGQPIKLKSHTDGTFEIK
GLAYAVDANAEGTAVTYKLKETKAPEGYVIPDKEIEFTVSQTSYNTKPTD
ITVDSADATPDTIKNNKRPSIPNTGGIGTAIFVAIGAAVMAFAVKGMKRR TKDN
[0931] E boxes containing conserved glutamic residues have also
been identified in GBS 80. The E box motifs are underlined in SEQ
ID NO: 2 below. The conserved glutamic acid (E) residues, at amino
acid residues 392 and 471, are marked in bold. The E box motifs, in
particular the conserved glutamic acid residues, are thought to be
important for the formation of oligomeric pilus-like structures of
GBS 80. Preferred fragments of GBS 80 include at least one
conserved glutamic acid residue. Preferably, fragments include at
least one E box motif. TABLE-US-00055 SEQ ID NO: 2
MKLSKKLLFSAAVLTMVAGSTVEPVAQFATGMSIVRAAEVSQERPAKTTV
NIYKLQADSYKSEITSNGGIENKDGEVISNYAKLGDNVKGLQGVQFKRYK
VKTDISVDELKKLTTVEAADAKVGTILEEGVSLPQKTNAQGLVVDALDSK
SNVRYLYVEDLKNSPSNITKAYAVPFVLELPVANSTGTGFLSEINIYPKN
VVTDEPKTDKDVKKLGQDDAGYTIGEEFKWFLKSTTPANLGDYEKEEITD
KFADGLTYKSVGKIKIGSKTLNRDLHYTIDEPTVDNQNTLKITFKPEKFK
EIAELLKGMTLVKNQDALDKATANTDDAAFLEIPVASTINEKAVLGKAIE
NTFELQYDHTPDKADNPKPSNPPRKPEVHTGGKRFVKKDSTETQTLGGAE
FDLLASDGTAVKWTDALIKANTNKNYIAGEAVTGQPIKLKSHTDGTFEIK
GLAYAVDANAEGTAVTYKLKETKAPEGYVIPDKEIEFTVSQTSYNTKPTD
ITVDSADATPDTIKNNKRPSIPNTGGIGTAIFVAIGAAVMAFAVKGMKRR TKDN
[0932] Similarly, the following offers examples of preferred GBS
104 fragments. Nucleotide and amino acid sequences of GBS 104
sequenced from serotype V isolated strain 2603 are set forth below
as SEQ ID NOS 10 and 11: TABLE-US-00056 SEQ ID NO. 10
ATGAAAAAGAGACAAAAAATATGGAGAGGGTTATCAGTTACTTTACTAAT
CCTGTCCCAAATTCCATTTGGTATATTGGTACAAGGTGAAACCCAAGATA
CCAATCAAGCACTTGGAAAAGTAATTGTTAAAAAAACGGGAGACAATGCT
ACACCATTAGGCAAAGCGACTTTTGTGTTAAAAAATGACAATGATAAGTC
AGAAACAAGTCACGAAACGGTAGAGGGTTCTGGAGAAGCAACCTTTGAAA
ACATAAAACCTGGAGACTACACATTAAGAGAAGAAACAGCACCAATTGGT
TATAAAAAAACTGATAAAACCTGGAAAGTTAAAGTTGCAGATAACGGAGC
AACAATAATCGAGGGTATGGATGCAGATAAAGCAGAGAAACGAAAAGAAG
TTTTGAATGCCCAATATCCAAAATCAGCTATTTATGAGGATACAAAAGAA
AATTACCCATTAGTTAATGTAGAGGGTTCCAAAGTTGGTGAACAATACAA
AGCATTGAATCCAATAAATGGAAAAGATGGTCGAAGAGAGATTGCTGAAG
GTTGGTTATCAAAAAAAATTACAGGGGTCAATGATCTCGATAAGAATAAA
TATAAAATTGAATTAACTGTTGAGGGTAAAACCACTGTTGAAACGAAAGA
ACTTAATCAACCACTAGATGTCGTTGTGCTATTAGATAATTCAAATAGTA
TGAATAATGAAAGAGCCAATATATTCTCAAAGAGCATTAAAAGCTGGGAA
GCAGTTGAAAAGCTGATTGATAAAATTACATCAAATAAAGACAATAGAGT
AGCTCTTGTGACATATGCCTCAACCATTTTTGATGGTACTGAAGCGACCG
TATCAAAGGGAGTTGCCGATCAAAATGGTAAAGCGCTGAATGATAGTGTA
TCATGGGATTATCATAAAACTACTTTTACAGCAACTACACATAATTACAG
TTATTTAAATTTAACAAATGATGCTAACGAAGTTAATATTCTAAAGTCAA
GAATTCCAAAGGAAGCGGAGCATATAAATGGGGATCGCACGCTCTATCAA
TTTGGTGCGACATTTACTCAAAAAGCTCTAATGAAAGCAAATGAAATTTT
AGAGACACAAAGTTCTAATGCTAGAAAAAAACTTATTTTTCACGTAACTG
ATGGTGTCCCTACGATGTCTTATGCCATAAATTTTAATCCTTATATATCA
ACATCTTACCAAAACCAGTTTAATTCTTTTTTAAATAAAATACCAGATAG
AAGTGGTATTCTCCAAGAGGATTTTATAATCAATGGTGATGATTATCAAA
TAGTAAAAGGAGATGGAGAGAGTTTTAAACTGTTTTCGGATAGAAAAGTT
CCTGTTACTGGAGGAACGACACAAGCAGCTTATCGAGTACCGCAAAATCA
ACTCTCTGTAATGAGTAATGAGGGATATGCAATTAATAGTGGATATATTT
ATCTCTATTGGAGAGATTACAACTGGGTCTATCCATTTGATCCTAAGACA
AAGAAAGTTTCTGCAACGAAACAAATCAAAACTCATGGTGAGCCAACAAC
ATTATACTTTAATGGAAATATAAGACCTAAAGGTTATGACATTTTTACTG
TTGGGATTGGTGTAAACGGAGATCCTGGTGCAACTCCTCTTGAAGCTGAG
AAATTTATGCAATCAATATCAAGTAAAACAGAAAATTATACTAATGTTGA
TGATACAAATAAAATTTATGATGAGCTAAATAAATACTTTAAAACAATTG
TTGAGGAAAAACATTCTATTGTTGATGGAAATGTGACTGATCCTATGGGA
GAGATGATTGAATTCCAATTAAAAAATGGTCAAAGTTTTACACATGATGA
TTACGTTTTGGTTGGAAATGATGGCAGTCAATTAAAAAATGGTGTGGCTC
TTGGTGGACCAAACAGTGATGGGGGAATTTTAAAAGATGTTACAGTGACT
TATGATAAGACATCTCAAACCATCAAAATCAATCATTTGAACTTAGGAAG
TGGACAAAAAGTAGTTCTTACCTATGATGTACGTTTAAAAGATAACTATA
TAAGTAACAAATTTTACAATACAAATAATCGTACAACGCTAAGTCCGAAG
AGTGAAAAAGAACCAAATACTATTCGTGATTTCCCAATTCCCAAAATTCG
TGATGTTCGTGAGTTTCCGGTACTAACCATCAGTAATCAGAAGAAAATGG
GTGAGGTTGAATTTATTAAAGTTAATAAAGACAAACATTCAGAATCGCTT
TTGGGAGCTAAGTTTCAACTTCAGATAGAAAAAGATTTTTCTGGGTATAA
GCAATTTGTTCCAGAGGGAAGTGATGTTACAACAAAGAATGATGGTAAAA
TTTATTTTAAAGCACTTCAAGATGGTAACTATAAATTATATGAAATTTCA
AGTCCAGATGGCTATATAGAGGTTAAAACGAAACCTGTTGTGACATTTAC
AATTCAAAATGGAGAAGTTACGAACCTGAAAGCAGATCCAAATGCTAATA
AAAATCAAATCGGGTATCTTGAAGGAAATGGTAAACATCTTATTACCAAC
ACTCCCAAACGCCCACCAGGTGTTTTTCCTAAAACAGGGGGAATTGGTAC
AATTGTCTATATATTAGTTGGTTCTACTTTTATGATACTTACCATTTGTT
CTTTCCGTCGTAAACAATTG SEQ ID NO. 11
MKKRQKIWRGLSVTLLILSQIPFGILVQGETQDTNQALGKVIVKKTGDNA
TPLGKATFVLKNDNDKSETSHETVEGSGEATFENIKPGDYTLREETAPIG
YKKTDKTWKVKVADNGATIIEGMDADKAEKRKEVLNAQYPKSAIYEDTKE
NYPLVNVEGSKVGEQYKALNPINGKDGRREIAEGWLSKKTTGVNDLDKNK
YKIELTVEGKTTVETKELNQPLDVVVLLDNSNSMNNERANNSQRALKAGE
AVEKLIDKTTSNKDNRVALVTYASTIFDGTEATVSKGVADQNGKALNDSV
SWDYHKTTFTATTHNYSYLNLTNDANEVNILKSRIPKEAEHINGDRTLYQ
FGATFTQKALMKANEILETQSSNARKKLIFHVTDGVPTMSYAINFNPYTS
TSYQNQFNSFLNKIPDRSGILQEDFIINGDDYQIVKGDGESFKLFSDRKV
PVTGGTTQAAYRVPQNQLSVMSNEGYAINSGYIYLYWRDYNWVYPFDPKT
KKVSATKQIKTHGEPTTLYFNGNIRPKGYDIFTVGIGVNGDPGATPLEAE
KFMQSISSKTENYTNVDDTNKIYDELNKYFKTIVEEKHSIVDGNVTDPMG
EMIEFQLKNGQSFTHDDYVLVGNDGSQLKNGVALGGPNSDGGILKDVTVT
YDKTSQTIKINHLNLGSGQKVVLTYDVRLKDNYISNKEYNTNNRTTLSPK
SEKEPNTIRDFPIPKIRDVREFPVLTISNQKKMGEVEFIKVNKDKHSESL
LGAKFQLQIEKDFSGYKQFVPEGSDVTTKNDGKIYFKALQDGNYKLYEIS
SPDGYIEVKTKPVVTFTIQNGEVTNLKADPNANKNQIGYLEGNGKHLITN
TPKRPPGVFPKTGGIGTIVYILVGSTFMILTICSFRRKQL
[0933] GBS 104 contains an N-terminal leader or signal sequence
region which is indicated by the underlined sequence at the
beginning of SEQ ID NO 11 above. In one embodiment, one or more
amino acid sequences from the leader or signal sequence region of
GBS 104 are removed. An example of such a GBS 104 fragment is set
forth below as SEQ ID NO 12. TABLE-US-00057 SEQ ID NO: 12
GETQDTNQALGKVIVKKTGDNATPLGKATFVLKNDNDKSETSHETVEGSG
EATFENIKPGDYTLREETAPIGYKKTDKTWKVKVADNGATIIEGMDADKA
EKRKEVLNAQYPKSAIYEDTKENYPLVNVEGSKVGEQYKALNPINGKDGR
REIAEGWLSKKITGVNDLDKNKYKIELTVEGKTTVETKELNQPLDVVVLL
DNSNSMNNERANNSQPALKAGEAVEKLIDKITSNKDNRVALVTYASTIFD
GTEATVSKGVADQNGKALNDSVSWDYHKTTFTATTHNYSYLNLTNDANEV
NILKSRIPKEAEHINGDRTLYQFGATFTQKALMIANEILETQSSNARKKL
IFHVTDGVPTMSYAINFNPYISTSYQNQFNSFLNKIPDRSGILQEDFIIN
GDDYQIVKGDGESFKLFSDRKVPVTGGTTQAAYRVPQNQLSVMSNEGYAT
NSGYIYLYWRDYNWVYPFDPKTKKVSATKQIKTHGEPTTLYPNGNTRPKG
YDIFTVGIGVNGDPGATPLEAEKFMQSISSKTENYTNVDDTNKIYDELNK
YEKTIVEEKHSIVDGNVTDPMGEMIEFQLKNGQSPTHDDYVLVGNDGSQL
KNGVALGGPNSDGGILKDVTVTYDKTSQTIKINHLNLGSGQKVVLTYDVR
LKDNYISNKFYNTNNRTTLSPKSEKEPNTIRDFPIPKIRDVREFPVLTIS
NQKKMGEVEFIKVNKDKHSESLLGAKFQLQIEKDESGYKQFVPEGSDVTT
KNDGKIYFKALQDGNYKLYEISSPDGYIEVKTKPVVTFTIQNGEVTNLKA
DPNANKNQIGYLEGNGKHLITNTPKRPPGVFPKTGGIGTIVYILVGSTFM ILTICSFRRKQL
[0934] GBS 104 contains a C-terminal transmembrane and/or
cytoplasmic region which is indicated by the underlined region near
the end of SEQ ID NO 11 above. In one embodiment, one or more amino
acids from the transmembrane or cytomplasmic regions are removed.
An example of such a GBS 104 fragment is set forth below as SEQ ID
NO 13. TABLE-US-00058 SEQ ID NO: 13
MKKRQKIWRGLSVTLLILSQIPFGILVQGETQDTNQALGKVIVKKTGDNA
TPLGKATFVLKNDNDKSETSHETVEGSGEATFENIKPGDYTLREETAPIG
YKKTDKTWKVKVADNGATIIEGMDADKAEKRKEVLNAQYPKSAIYEDTKE
NYPLVNVEGSKVGEQYKALNPINGKDGRREIAEGWLSKKITGVNDLDKNK
YKIELTVEGKTTVETKELNQPLDVVVLLDNSNSMNNERANNSQRALKAGE
AVEKLIDKITSNKDNRVALVTYASTIFDGTEATVSKGVADQNGKALNDSV
SWDYHKTTFTATTHNYSYLNLTNDANEVNILKSRIPKEAEHINGDRTLYQ
FGATFTQKALMKANEILETQSSNARKKLIFHVTDGVPTMSYAINFNPYIS
TSYQNQFNSFLNKIPDRSGTLQEDFIINGDDYQIVKGDGESFKLFSDRKV
PVTGGTTQAAYRVPQNQLSVMSNEGYAINSGYIYLYWRDYNWVYPFDPKT
KKVSATKQIKTHGEPTTLYFNGNTRPKGYDIFTVGIGVNGDPGATPLEAE
KFMQSISSKTENYTNVDDTNKIYDELNKYFKTIVEEKHSIVDGNVTDPMG
EMIEFQLKNGQSFTHDDYVLVGNDGSQLKNGVALGGPNSDGGILKDVTVT
YDKTSQTIKINHLNLGSGQKVVLTYDVRLKDNYISNKFYNTNNRTTLSPK
SEKEPNTIRDFPIPKIRDVREFPVLTISNQKKMGEVEFIKVNKDKHSESL
LGAKFQLQIEKDFSGYKQFVPEGSDVTTKNDGKTYFKALQDGNYKLYEIS
SPDGYIEVKTKPVVTFTIQNGEVTNLKADPNANKNQIGYLEGNGKHLITN T
[0935] In one embodiment, one or more amino acids from the leader
or signal sequence region and one or more amino acids from the
transmembrane or cytoplasmic regions are removed. An example of
such a GBS 104 fragment is set forth below as SEQ ID NO 14.
TABLE-US-00059 SEQ ID NO: 14
GETQDTNQALGKVIVKKTGDNATPLGKATFVLKNDNDKSETSHETVEGSG
EATFENIKPGDYTLREETAPIGYKKTDKTWKVKVADNGATIIEGMDADKA
EKRKEVLNAQYPKSAIYEDTKENYPLVNVEGSKVGEQYKALNPINGKDGR
REIAEGWLSKKITGVNDLDKNKYKIELTVEGKTTVETKELNQPLDVVVLL
DNSNSMNNERANNSQRALKAGEAVEKLIDKITSNKDNRVALVTYASTIFD
GTEATVSKGVADQNGKALNDSVSWDYHKTTFTATTHNYSYLNLTNDANEV
NILKSRIPKEAEHINGDRTLYQFGATFTQKALMKANEILETQSSNARKKL
IFHVTDGVPTMSYAINFNPYISTSYQNQFNSFLNKIPDRSGILQEDFIIN
GDDYQIVKGDGESFKLFSDRKVPVTGGTTQAAYRVPQNQLSVMSNEGYAI
NSGYIYLYWRDYNWVYPFDPKTKKVSATKQIKTHGEPTTLYFNGNIRPKG
YDIFTVGIGVNGDPGATPLEAEKFMQSISSKTENYTNVDDTNKIYDELNK
YFKTIVEEKHSIVDGNVTDPMGEMTEFQLKNGQSFTHDDYVLVGNDGSQL
KNGVALGGPNSDGGILKDVTVTYDKTSQTIKINHLNLGSGQKVVLTYDVR
LKDNYISNKFYNTNNRTTLSPKSEKEPNTIRDFPIPKIRDVREFPVLTIS
NQKKMGEVEFIKVNKDKHSESLLGAKFQLQIEKDFSGYKQFVPEGSDVTT
KNDGKIYEKALQDGNYKLYEISSPDGYIEVKTKPVVTFTTQNGEVTNLKA
DPNANKNQIGYLEGNGKHLITNT
[0936] GBS 104, like GBS 80, contains an amino acid motif
indicative of a cell wall anchor: SEQ ID NO: 123 FPKTG (shown in
italics in SEQ ID NO: 11 above). In some recombinant host cell
systems, it may be preferable to remove this motif to facilitate
secretion of a recombinant GBS 104 protein from the host cell.
Accordingly, in one preferred fragment of GBS 104 for use in the
invention, only the transmembrane and/or cytoplasmic regions and
the cell wall anchor motif are removed from GBS 104. Alternatively,
in some recombinant host cell systems, it may be preferable to use
the cell wall anchor motif to anchor the recombinantly expressed
protein to the cell wall. The extracellular domain of the expressed
protein may be cleaved during purification or the recombinant
protein may be left attached to either inactivated host cells or
cell membranes in the final composition.
[0937] Two pilin motifs, containing conserved lysine (K) residues,
have been identified in GBS 104. The pilin motif sequences are
underlined in SEQ ID NO: 11, below. Conserved lysine (K) residues
are marked in bold, at amino acid residues 141 and 149 and at amino
acid residues 499 and 507. The pilin sequence, in particular the
conserved lysine residues, are thought to be important for the
formation of oligomeric, pilus-like structures of GBS 104.
Preferred fragments of GBS 104 include at least one conserved
lysine residue. Preferably, fragments include at least one pilin
sequence. TABLE-US-00060 SEQ ID NO. 11
MKKRQKIWRGLSVTLLILSQIPFGILVQGETQDTNQALGKVIVKKTGDNA
TPLGKATFVLKNDNDKSETSHETVEGSGEATFENIKPGDYTLREETAPIG
YKKTDKTWKVKVADNGATIIEGMDADKAEKRKEVLNAQYPKSAIYEDTKE
NYPLVNVEGSKVGEQYKALNPINGKDGRREIAEGWLSKKITGVNDLDKNK
YKIELTVEGKTTVETKELNQPLDVVVLLDNSNSMNNERANNSQRALKAGE
AVEKLIDKITSNKDNRVALVTYASTIFDGTEATVSKGVADQNGKALNDSV
SWDYHKTTFTATTHNYSYLNLTNDANEVNILKSRIPKEAEHINGDRTLYQ
FGATFTQKALMKANEILETQSSNARKKLIFHVTDGVPTMSYAINFNPYIS
TSYQNQFNSFLNKIPDRSGILQEDFIINGDDYQIVKGDGESEKLFSDRKV
PVTGGTTQAAYRVPQNQLSVMSNEGYAINSGYIYLYWRDYNWVYPFDPKT
KKVSATKQIKTHGEPTTLYFNGNIRPKGYDIFTVGIGVNGDPGATPLEAE
KFMQSISSKTENYTNVDDTNKIYDELNKYFKTIVEEKHSIVDGNVTDPMG
EMIEFQLKNGQSFTHDDYVLVGNDGSQLKNGVALGGPNSDGGILKDVTVT
YDKTSQTTKINHLNLGSGQKVVLTYDVRLKDNYISNKFYNTNNRTTLSPK
SEKEPNTIRDFPIPKIRDVREFPVLTISNQKKMGEVEFIKVNKDKHSESL
LGAKFQLQIEKDFSGYKQFVPEGSDVTTKNDGKTYFKALQDGNYKLYEIS
SPDGYIEVKTKPVVTFTIQNGEVTNLKADPNANKNQIGYLEGNGKHLITN
TPKRPPGVFPKTGGIGTIVYILVGSTFMILLTICSFRRKQL
[0938] Two E boxes containing a conserved glutamic residues have
also been identified in GBS 104. The E box motifs are underlined in
SEQ ID NO: 11 below. The conserved glutamic acid (E) residues, at
amino acid residues 94 and 798, are marked in bold. The E box
motifs, in particular the conserved glutamic acid residues, are
thought to be important for the formation of oligomeric pilus-like
structures of GBS 104. Preferred fragments of GBS 104 include at
least one conserved glutamic acid residue. Preferably, fragments
include at least one E box motif. TABLE-US-00061 SEQ ID NO. 11
MKKRQKIWRGLSVTLLILSQIPFGILVQGETQDTNQALGKVIVKKTGDNA
TPLGKATFVLKNDNDKSETSHETVEGSGEATFENIKPGDYTLREETAPIG
YKKTDKTWKVKVADNGATIIEGMDADKAEKRKEVLNAQYPKSAIYEDTKE
NYPLVNVEGSKVGEQYKALNPINGKDGRREIAEGWLSKKITGVNDLDKNK
YKIELTVEGKTTVETKELNQPLDVVVLLDNSNSMNNERANNSQRALKAGE
AVEKLIDKITSNKDNRVALVTYASTIFDGTEATVSKGVADQNGKALNDSV
SWDYHKTTFTATTHNYSYLNLTNDANEVNILKSRIPKEAEHINGDRTLYQ
FGATFTQKALMKANEILETQSSNARKKLIFHVTDGVPTMSYAINFNPYIS
TSYQNQFNSFLNKIPDRSGILQEDFIINGDDYQIVKGDGESEKLFSDRKV
PVTGGTTQAAYRVPQNQLSVMSNEGYAINSGYIYLYWRDYNWVYPFDPKT
KKVSATKQIKTHGEPTTLYFNGNIRPKGYDIFTVGIGVNGDPGATPLEAE
KFMQSISSKTENYTNVDDTNKIYDELNKYFKTIVEEKHSIVDGNVTDPMG
EMIEFQLKNGQSFTHDDYVLVGNDGSQLKNGVALGGPNSDGGILKDVTVT
YDKTSQTTKINHLNLGSGQKVVLTYDVRLKDNYISNKFYNTNNRTTLSPK
SEKEPNTIRDFPIPKIRDVREFPVLTISNQKKMGEVEFIKVNKDKHSESL
LGAKFQLQIEKDFSGYKQFVPEGSDVTTKNDGKTYFKALQDGNYKLYEIS
SPDGYIEVKTKPVVTFTIQNGEVTNLKADPNANKNQIGYLEGNGKHLITN
TPKRPPGVFPKTGGIGTIVYILVGSTFMILLTICSFRRKQL
GBS 067
[0939] The following offers examples of preferred GBS 067
fragments. Nucleotide and amino acid sequence of GBS 067 sequences
from serotype V isolated strain 2603 are set forth below as SEQ ID
NOS: 15 and 16. TABLE-US-00062 SEQ ID NO: 15
ATGAGAAAATACCAAAAATTTTCTAAAATATTGACGTTAAGTCTTTTTTG
TTTGTCGCAAATACCGCTTAATACCAATGTTTTAGGGGAAAGTACCGTAC
CGGAAAATGGTGCTAAAGGAAAGTTAGTTGTTAAAAAGACAGATGACCAG
AACAAACCACTTTCAAAAGCTACCTTTGTTTTAAAAACTACTGCTCATCC
AGAAAGTAAAATAGAAAAAGTAACTGCTGAGCTAACAGGTGAAGCTACTT
TTGATAATCTCATACCTGGAGATTATACTTTATCAGAAGAAACAGCGCCC
GAAGGTTATAAAAAGACTAACCAGACTTGGCAAGTTAAGGTTGAGAGTAA
TGGAAAAACTACGATACAAAATAGTGGTGATAAAAATTCCACAATTGGAC
AAAATCAGGAAGAACTAGATAAGCAGTATCCCCCCACAGGAATTTATGAA
GATACAAAGGAATCTTATAAACTTGAGCATGTTAAAGGTTCAGTTCCAAA
TGGAAAGTCAGAGGCAAAAGCAGTTAACCCATATTCAAGTGAAGGTGAGC
ATATAAGAGAAATTCCAGAGGGAACATTATCTAAACGTATTTCAGAAGTA
GGTGATTTAGCTCATAATAAATATAAAATTGAGTTAACTGTCAGTGGAAA
AACCATAGTAAAACCAGTGGACAAACAAAAGCCGTTAGATGTTGTCTTCG
TACTCGATAATTCTAACTCAATGAATAACGATGGCCCAAATTTTCAAAGG
CATAATAAAGCCAAGAAAGCTGCCGAAGCTCTTGGGACCGCAGTAAAAGA
TATTTTAGGAGCAAACAGTGATAATAGGGTTGCATTAGTTACCTATGGTT
CAGATATTTTTGATGGTAGGAGTGTAGATGTCGTAAAAGGATTTAAAGAA
GATGATAAATATTATGGCCTTCAAACTAAGTTCACAATTCAGACAGAGAA
TTATAGTCATAAACAATTAACAAATAATGCTGAAGAGATTATAAAAAGGA
TTCCGACAGAAGCTCCTAAAGCTAAGTGGGGATCTACTACCAATGGATTA
ACTCCAGAGCAACAAAAGGAGTACTATCTTAGTAAAGTAGGAGAAACATT
TACTATGAAAGCCTTCATGGAGGCAGATGATATTTTGAGTCAAGTAAATC
GAAATAGTCAAAAAATTATTGTTCATGTAACTGATGGTGTTCCTACGAGA
TCATATGCTATTAATAATTTTAAACTGGGTGCATCATATGAAAGCCAATT
TGAACAAATGAAAAAAAATGGATATCTAAATAAAAGTAATTTTCTACTTA
CTGATAAGCCCGAGGATATAAAAGGAAATGGGGAGAGTTACTTTTTGTTT
CCCTTAGATAGTTATCAAACACAGATAATCTCTGGAAACTTACAAAAACT
TCATTATTTAGATTTAAATCTTAATTACCCTAAAGGTACAATTTATCGAA
ATGGACCAGTGAAAGAACATGGAACACCAACCAAACTTTATATAAATAGT
TTAAAACAGAAAAATTATGACATTTTTAATTTTGGTATCGATATATCTGG
TTTTAGACAAGTTTATAATGAGGAGTATAAGAAAAATCAAGATGGTACTT
TTCAAAAATTGAAAGAGGAAGCTTTTAAACTTTCAGATGGAGAAATCACA
GAACTAATGAGGTCGTTCTCTTCCAAACCTGAGTACTACACCCCTATCGT
AACTTCAGCCGATACATCTAACAATGAAATTTTATCTAAAATTCAGCAAC
AATTTGAAACGATTTTAACAAAAGAAAACTCAATTGTTAATGGAACTATC
GAAGATCCTATGGGTGATAAAATCAATTTACAGCTTGGTAATGGACAAAC
ATTACAGCCAAGTGATTATACTTTACAGGGAAATGATGGAAGTGTAATGA
AGGATGGTATTGCAACTGGTGGGCCTAATAATGATGGTGGAATACTTAAG
GGGGTTAAATTAGAATACATCGGAAATAAACTCTATGTTAGAGGTTTGAA
TTTAGGAGAAGGTCAAAAAGTAACACTCACATATGATGTGAAACTAGATG
ACAGTTTTATAAGTAACAAATTCTATGACACTAATGGTAGAACAACATTG
AATCCTAAGTCAGAGGATCCTAATACACTTAGAGATTTTCCAATCCCTAA
AATTCGTGATGTGAGAGAATATCCTACAATAACGATTAAAAACGAGAAGA
AGTTAGGTGAAATTGAATTTATAAAAGTTGATAAAGATAATAATAAGTTG
CTTCTCAAAGGAGCTACGTTTGAACTTCAAGAATTTAATGAAGATTATAA
ACTTTATTTACCAATAAAAAATAATAATTCAAAAGTAGTGACGGGAGAAA
ACGGCAAAATTTCTTACAAAGATTTGAAAGATGGCAAATATCAGTTAATA
GAAGCAGTTTCGCCGGAGGATTATCAAAAAATTACTAATAAACCAATTTT
AACTTTTGAAGTGGTTAAAGGATCGATAAAAAATATAATAGCTGTTAATA
AACAGATTTCTGAATATCATGAGGAAGGTGACAAGCATTTAATTACCAAC
ACGCATATTCCACCAAAAGGAATTATTCCTATGACAGGTGGGAAAGGAAT
TCTATCTTTCATTTTAATAGGTGGAGCTATGATGTCTATTGCAGGTGGAA
TTTATATTTGGAAAAGGTATAAGAAATCTAGTGATATGTCCATCAAAAAA GAT SEQ ID NO:
16 MRKYQKFSKILTLSLFCLSQIPLNTNVLGESTVPENGAKGKLVVKKTDDQ
NKPLSKATFVLKTTAHPESKIEKVTAELTGEATFDNLIPGDYTLSEETAP
EGYKKTNQTWQVKVESNGKTTIQNSGDKNSTIGQNQEELDKQYPPTGIYE
DTKESYKLEHVKGSVPNGKSEAKAVNPYSSEGEHIREIPEGTLSKRISEV
GDLAHNKYKIELTVSGKTIVKPVDKQKPLDVVFVLDNSNSMNNDGPNFQR
HNKAKKAAEALGTAVKDILGANSDNRVALVTYGSDIFDGRSVDVVKGFKE
DDKYYGLQTKFTIQTENYSHKQLTNNAEEIIKRIPTEAPKAKWGSTTNGL
TPEQQKEYYLSKVGETFTMKAFMEADDILSQVNRNSQKIIVHVTDGVPTR
SYAINNFKLGASYESQFEQMKKNGYLNKSNFLLTDKPEDIKGNGESYFLF
PLDSYQTQIISGNLQKLHYLDLNLNYPKGTIYRNGPVKEHGTPTKLYINS
LKQKNYDIFNFGIDISGFRQVYNEEYKKNQDGTFQKLKEEAFKLSDGEIT
ELMRSFSSKPEYYTPIVTSADTSNNEILSKIQQQFETILTKENSIVNGTI
EDPMGDKTNLQLGNGQTLQPSDYTLQGNDGSVMKDGIATGGPNNDGGILK
GVKLEYIGNKLYVRGLNLGEGQKVTLTYDVKLDDSFTSNKFYDTNGRTTL
NPKSEDPNTLRDFPIPKIRDVREYPTITIKNEKKLGEIEFIKVDKDNNKL
LLKGATFELQEFNEDYRLYLPIKNNNSKVVTGENGKISYKDLKDGKYQLI
EAVSPEDYQKITNKPILTFEVVKGSIKNIIAVNKQISEYHEEGDKHLITN
THIPPKGIIPMTGGKGILSFILIGGAMMSIAGGIYIWKRYKKSSDMSIKK D
[0940] GBS 067 contains a C-terminus transmembrane region which is
indicated by the underlined region closest to the C-terminus of SEQ
ID NO: 16 above. In one embodiment, one or more amino acids from
the transmembrane region is removed and or the amino acid is
truncated before the transmembrane region. An example of such a GBS
067 fragment is set forth below as SEQ ID NO: 17. TABLE-US-00063
SEQ ID NO: 17 MRKYQKFSKILTLSLFCLSQIPLNTNVLGESTVPENGAKGKLVVKKTDDQ
NKPLSKATFVLKTTAHPESKIEKVTAELTGEATPDNLIPGDYTLSEETAP
EGYKKTNQTWQVKVESNGKTTIQNSGDKNSTIGQNQEELDKQYPPTGIYE
DTKESYKLEHVKGSVPNGKSEAKAVNPYSSEGEHIRETPEGTLSKRISEV
GDLAHNKYKTELTVSGKTIVKPVDKPKPLDVVFVLDNSNSMNNDGPNFQR
HNKAKKAAEALGTAVKDILGANSDNRVALVTYGSDIFDGRSVDVVKGFKE
DDKYYGLQTKETIQTENYSHKQLTNNAEEIIKRIPTEAPKAKWGSTTNGL
TPEQQKEYYLSKVGETFTMKAFMEADDILSQVNRNSQKIIVHVTDGVPTR
SYAINNFKLGASYESQFEQMKKNGYLNKSNFLLTDKPEDIKGNGESYFLF
PLDSYQTQIISGNLQKLHYLDLNLNYPKGTIYRNGPVKEHGTPTKLYINS
LKQKNYDIFNFGTDISGFRQVYNEEYKKNQDGTFQKLKEEAFKLSDGEIT
ELMRSESSKPEYYTPIVTSADTSNNEILSKIQQQFETILTKENSIVNGTI
EDPMGDKINLQLGNGQTLQPSDYTLQGNDGSVMKDGIATGGPNNDGGILK
GVKLEYIGNKLYVRGLNLGEGQKVTLTYDVKLDDSFISNKFYDTNGRTTL
NPKSEDPNTLRDFPIPKIRDVREYPTITIKNEKKLGEIEFIKVDKDNNKL
LLKGATFELQEFNEDYKLYLPIKNNNSKVVTGENGKISYKDLKDGKYQLI
EAVSPEDYQKITNKPILTFEVVKGSIKNIIAVNKQISEYHEEGDKHLITN
THIPPKGIIPMTGGKGILS
[0941] GBS 067 contains an amino acid motif indicative of a cell
wall anchor (an LPXTG (SEQ ID NO: 122) motif): SEQ ID NO: 18 IPMTG.
(shown in italics in SEQ ID NO: 16 above). In some recombinant host
cell systems, it may be preferable to remove this motif to
facilitate secretion of a recombinant GBS 067 protein from the host
cell. Accordingly, in one preferred fragment of GBS 067 for use in
the invention, the transmembrane and the cell wall anchor motif are
removed from GBS 67. An example of such a GBS 067 fragment is set
forth below as SEQ ID NO: 19. TABLE-US-00064 SEQ ID NO: 19
MRKYQKFSKILTLSLFCLSQIPLNTNVLGESTVPENGAKGKLVVKKTDDQ
NKPLSKATFVLKTTAHPESKIEKVTAELTGEATFDNLIPGDYTLSEETAP
EGYKKTNQTWQVKVESNGKTTIQNSGDKNSTIGQNQEELDKQYPPTGIYE
DTKESYKLEHVKGSVPNGKSEAKAVNPYSSEGEHIREIPEGTLSKRTSEV
GDLAHNKYKIELTVSGKTIVKPVDKQKPLDVVFVLDNSNSMNNDGPNFQR
HNKAKKPAEALGTAVKDILGANSDNRVALVTYGSDIFDGRSVDVVKGFKE
DDKYYGLQTKFTIQTENYSHKQLTNNAEEIIKRIPTEAPKAKWGSTTNGL
TPEQQKEYYLSKVGETFTMKAFMEADDILSQVNRNSQKIIVHVTDGVPTR
SYAINNFKLGASYESQFEQMKKNGYLNKSNFLLTDKPEDIKGNGESYFLF
PLDSYQTQIISGNLQKLHYLDLNLNYPKGTIYRNGPVKEHGTPTKLYINS
LKQKNYDIFNFGIDISGERQVYNEEYKKNQDGTFQKLKEEAFKLSDGEIT
ELMRSFSSKPEYYTPIVTSADTSNNEILSKIQQQFETILTKENSIVNGTI
EDPMGDKINLQLGNGQTLQPSDYTLQGNDGSVMKDGIATGGPNNDGGILK
GVKLEYIGNKLYVRGLNLGEGQKVTLTYDVKLDDSFTSNKFYDTNGRTTL
NPKSEDPNTLRDFPIPKIRDVREYPTITIKNEKKLGEIEFIKVDKDNNKL
LLKGATFELQEFNEDYKLYLPIKNNNSKVVTGENGKISYKDLKDGKYQLI
EAVSPEDYQKITNKPILTFEVVKGSIKNIIAVNKQISEYHEEGDKHLITN THIPPKGI
[0942] Alternatively, in some recombinant host cell systems, it may
be preferable to use the cell wall anchor motif to anchor the
recombinantly expressed protein to the cell wall. The extracellular
domain of the expressed protein may be cleaved during purification
or the recombinant protein may be left attached to either
inactivated host cells or cell membranes in the final
composition.
[0943] Three pilin motifs, containing conserved lysine (K) residues
have been identified in GBS 67. The pilin motif sequences are
underlined in SEQ ID NO: 16, below. Conserved lysine (K) residues
are marked in bold, at amino acid residues 478 and 488, at amino
acid residues 340 and 342, and at amino acid residues 703 and 717.
The pilin sequences, in particular the conserved lysine residues,
are thought to be important for the formation of oligomeric,
pilus-like structures of GBS 67. Preferred fragments of GBS 67
include at least one conserved lysine residue. Preferably,
fragments include at least one pilin sequence. TABLE-US-00065 SEQ
ID NO: 16 MRKYQKFSKILTLSLFCLSQIPLNTNVLGESTVPENGAKGKLVVKKTDDQ
NKPLSKATFVLKTTAHPESKIEKVTAELTGEATFDNLIPGDYTLSEETAP
EGYKKTNQTWQVKVESNGKTTIQNSGDKNSTIGQNQEELDKQYPPTGIYE
DTKESYKLEHVKGSVPNGKSEAKAVNPYSSEGEHIREIPEGTLSKRISEV
GDLAHNKYKIELTVSGKTIVKPVDKQKPLDVVFVLDNSNSMNNDGPNFQR
HNKAKKAAEALGTAVKDILGANSDNRVALVTYGSDIFDGRSVDVVKGFKE
DDKYYGLQTKFTIQTENYSHKQLTNNAEEIIKRIPTEAPKAKWGSTTNGL
TPEQQKEYYLSKVGETFTMKAFMEADDILSQVNRNSQKIIVHVTDGVPTR
SYAINNFKLGASYESQFEQMKKNGYLNKSNFLLTDKPEDIKGNGESYFLF
PLDSYQTQIISGNLQKLHYLDLNLNYPKGTIYRNGPVKEHGTPTKLYINS
LKQKNYDIFNFGIDISGFRQVYNEEYKKNQDGTFQKLKEEAFKLSDGEIT
ELMRSFSSKPEYYTPIVTSADTSNNEILSKIQQQFETILTKENSIVNGTI
EDPMGDKINLQLGNGQTLQPSDYTLQGNDGSVMKDGIATGGPNNDGGILK
GVKLEYIGNKLYVRGLNLGEGQKVTLTYDVKLDDSFISNKFYDTNGRTTL
NPKSEDPNTLRDFPIPKIRDVREYPTITIKNEKKLGEIEFIKVDKDNNKL
LLKGATFELQEFNEDYKLYLPIKNNNSKVVTGENGKISYKDLKDGKYQLI
EAVSPEDYQKITNKPILTFEVVKGSIKNIIAVNKQISEYHEEGDKHLITN
THIPPKGIIPMTGGKGILSFILIGGAMMSIAGGIYIWKRYKKSSDMSIKK D
[0944] Two E boxes containing conserved glutamic residues have also
been identified in GBS 67. The E box motifs are underlined in SEQ
ID NO: 16 below. The conserved glutamic acid (E) residues, at amino
acid residues 96 and 801, are marked in bold. The E box motifs, in
particular the conserved glutamic acid residues, are thought to be
important for the formation of oligomeric pilus-like structures of
GBS 67. Preferred fragments of GBS 67 include at least one
conserved glutamic acid residue. Preferably, fragments include at
least one E box motif. TABLE-US-00066 SEQ ID NO: 16
MRKYQKFSKILTLSLFCLSQIPLNTNVLGESTVPENGAKGKLVVKKTDDQ
NKPLSKATFVLKTTAHPESKIEKVTAELTGEARFDNLIPGDYTLSEETAP
EGYKKTNQTWQVKVESNGKTTIQNSGDKNSTIGQNQEELDKQYPPTGIYE
DTKESYKLEHVKGSVPNGKSEAKAVNPYSSEGEHIREIPEGTLSKRISEV
GDLAHNKYKIELTVSGKTIVKPVDKQKPLDVVFVLDNSNSMNNDGPNFQR
HNKAKKAAEALGTAVKDILGANSDNRVALVTYGSDIFDGRSVDVVKGFKE
DDKYYGLQTKFTIQTENYSHKQLTNNAEEIIKRIPTEAPKAKWGSTTNGL
TPEQQKEYYLSKVGETFTMKAFMEADDILSQVNRNSQKIIVHVTDGVPTR
SYAINNFKLGASYESQFEQMKKNGYLNKSNFLLTDKPEDIKGNGESYFLF
PLDSYQTQIISGNLQKLHYLDLNLNYPKGTIYRNGPVKEHGTPTKLYINS
LKQKNYDIFNFGIDISGFRQVYNEEYKKNQDGTFQKLKEEAFKLSDGEIT
ELMRSFSSKPEYYTPIVTSADTSNNEILSKIQQQFETILTKENSIVNGTI
EDPMGDKINLQLGNGQTLQPSDYTLQGNDGSVMKDGIATGGPNNDGGILK
GVKLEYIGNKLYVRGLNLGEGQKVTLTYDVKLDDSFISNKFYDTNGRTTL
NPKSEDPNTLRDFPIPKIRDVREYPTITIKNEKKLGEIEFIKVDKDNNKL
LLKGATFELQEFNEDYKLYLPIKNNNSKVVTGENGKISYKDLKDGKYQLI
EAVSPEDYQKITNKPILTFEVVKGSIKNIIAVNKQISEYHEEGDKHLITN
THIPPKGIIPMTGGKGILSFILIGGAMMSIAGGIYIWKRYKKSSDMSIKK D
[0945] Predicted secondary structure for the GBS 067 amino acid
sequence is set forth in FIG. 33. As shown in this figure, GBS 067
contains several regions predicted to form alpha helical
structures. Such alpha helical regions are likely to form
coiled-coil structures and may be involved in oligomerization of
GBS 067.
[0946] The amino acid sequence for GBS 067 also contains a region
which is homologous to the Cna_B domain of the Staphylococcus
aureus collagen-binding surface protein (pfam05738). Although the
Cna_B region is not thought to mediate collagen binding, it is
predicted to form a beta sandwich structure. In the Staph aureus
protein, this beta sandwich structure is through to form a stalk
that presents the ligand binding domain away from the bacterial
cell surface. This same amino acid sequence region is also
predicted to be an outer membrane protein involved in cell envelope
biogenesis.
[0947] The amino acid sequence for GBS 067 contains a region which
is homologous to a von Willebrand factor (vWF) type A domain. The
vWF type A domain is present at amino acid residues 229-402 of GBS
067 as shown in SEQ ID NO: 16. This type of sequence is typically
found in extracellular proteins such as integrins and it thought to
mediate adhesion, including adhesion to collagen, fibronectin, and
fibrinogen, discussed above.
[0948] Because applicants have identified GBS 67 as a surface
exposed protein on GBS and because GBS 67 may be involved in GBS
adhesion, the immunogenicity of the GBS 67 protein was examined in
mice. The results of an immunization assay with GBS 67 are set
forth in Table 48, below. TABLE-US-00067 TABLE 48 GBS 67 Protects
Mice in an Immunization Assay Challenge GBS 67 immungen GBS strain
dead/ PBS immunogen FACS (serotype) treated % survival dead/treated
% survival .DELTA.mean 3050 (II) 0/30 100 29/49 41 460 CJB111 (V)
76/185 59 143/189 24 481 7357 b (Ib) 34/56 39 65/74 12 316
[0949] As shown in Table 48, immunization with GBS 67 provides a
substantially improved survival rate for challenged mice relative
to negative control, PBS, immunized mice. These results indicate
that GBS 67 may comprise an immunogenic composition of the
invention.
GBS 59
[0950] The following offers examples of GBS 59 fragments.
Nucleotide and amino acid sequences of GBS 59 sequenced from
serotype V isolated strain 2603 are set forth below as SEQ ID NOS:
125 and 126. The GBS 59 polypeptide of SEQ ID NO: 126 is referred
to as SAG1407. TABLE-US-00068 SEQ ID NO: 125
ttaagcttcctttgattggcgtcttttcatgataactactgctccaagca
taatgcttaaaccaataattgtgaaaagaattgtaccaataccacctgtt
tgtgggattgttacctttttattttctacacgtgtcgcatctttttggtt
gctgttagcaacgtagtcaatgttaccacctgttatgtatgacccttgat
taactacaaacttaatattacctgccaacttagcaaatcctgctggagca
agtgtttcttcaaggttgtaagtaccgtctgcaagacctgtaacttcaaa
ttgaccttgatcgtttgaagtgtaggtaatggctctagccttatctgtta
tccactcataagctgtacgagcctcaatgaaggctgcatcgtaatctgct
tgtttagttttgataagttcttttgcagtaattcctttttcacctttttg
gtctgttgcagacaacttgttataagcagcgatagcttcatctaaagcta
ttttcttagcagctaaagttttttgaccttctgattgatctgctttaaga
gcaaggtatttacctgctgagtttttcacaacgaattgtgcaccagccaa
acggtcaccttgttcattagttttgacaaatttcttaccatgagtttcaa
cttttggttcagttgggttcaatggtgttgggttatcagaatctttggta
ttggtaatggttactttaccattttctagatttattgcacttccgtaacc
agaaacacgttctgagatcatgtatgatttgttttctagaccagtgaatt
tacccgagaagttaccagatacttcaaatttgataccatttccaaggtcg
attgtacctttagatgtttttgtcaatgatactgaagcaacagttttatc
tttatctttcaatgtgtaaacaacgtttacaccatcaggtgcaattccgt
cagaccaagttttagcaactgttacttcaccctttgaaggtgtaacagga
agttcagtcaagtctttacctggtttgttaccatacgacaatttgatatc
attggattctggattatcaataattgcttgaccattaacagtagcactat
aagtcaatgtaaattcaatatcagctgttttagctgctttttccaatttg
cccaatccatcagctgtgaattttaatgtgaaaccacgggcatcaatgct
aagttcatagtctgtatccttagcaaaagtttctgtagttcctgaagctt
taaggctaacagttgaacccattgtcaaaccatttgacattatatctgtc
caaaccaagttttcgtatttagaacctttgtgaatttttgttttaacttc
ataaggaacaactttaccgatttcagcagtagcagttgctttgtcacgtg
cataattaccataatttgcgccagctgtcaaaagtctattaacatctgtc
aatgctgtcaaatcgtttgttttagcaaagtttttatcaatttctggttt
ttcttcagtgttctttggataaacatgggcatcagcaacaacaccatctt
catttaccaatggaagagtgatgttaactggaaccgcttttgaagcagcc
aggagggaaccattattgttgtaagtagattttgatttaacttcaacaat
tttaaactcgcctttcaatcctttggtgttgaaaacaagtccagtatctc
cctctggtgtcaatccagacacggcctcatcaatatttactgttatttca
ggagtaccatctttattaattaaggctggtgttaatttgttaccttcttt
tgccttaacatattgcactttaccacttttatcttctttcaaagctaaag
caaagaacgcaccttcgatttctttagatccctcgccaaagtaaccagca
aggtcagaaatagctccacctttgtagtcttttccgttaagacctgtagt
tcctgggaagttacttttgttaagatttgattcggtttgcaaaatcttgt
gcaaagtcactgtattagttgttgcttcatccgcaaacgctggtgcaact
gagagcaatgacgttaaagtcagtaacaatgccgagaacattgcaaaata
tttgttgattcttttcat SEQ ID NO: 126
MKRINKYFAMFSALLLTLTSLLSVAPAFADEATTNTVTLHKILQTESNLN
KSNFPGTTGLNGKDYKGGAISDLAGYFGEGSKEIEGAFFALALKEDKSGK
VQYVKAKEGNKLTPALINKDGTPEITVNIDEAVSGLTPEGDTGLVFNTKG
LKGEFKIVEVKSKSTYNNNGSLLAASKAVPVNITLPLVNEDGVVADAHVY
PKNTEEKPEIDKNFAKTNDLTALTDVNRLLTAGANYGNYARDKATATAEI
GKVVPYEVKTKIHKGSKYENLVWTDIMSNGLTMGSTVSLKASGTTETFAK
DTDYELSIDARGFTLKFTADGLGKLEKAAKTADIEFTLTYSATVNGQAII
DNPESNDIKLSYGNKPGKDLTELPVTPSKGEVTVAKTWSDGIAPDGVNVV
YTLKDKDKTVASVSLTKTSKGTIDLGNGIKPEVSGNFSGKETGLENKSYM
ISERVSGYGSAINLENGKVTITNTKDSDNPTPLNPTEPKVETHGKKFVKT
NEQGDRLAGAQFVVKNSAGKYLALKADQSEGQKTLAAKKIALDEAIAAYN
KLSATDQKGEKGITAKELIKTKQADYDAAFIEARTAYEWITDKARAITYT
SNDQGQFEVTGLADGTYNLEETLAPAGFAKLAGNIKFVVNQGSYITGGNI
DYVANSNQKDATRVENKKVTIPQTGGIGTILFTIIGLSIMLGAVVIMKRR QSKEA
[0951] Nucleotide and amino acid sequences of GBS 59 sequenced from
serotype V isolated strain CJB111 are set forth below as SEQ ID
NOS: 127 and 128. The GBS 59 polypeptide of SEQ ID NO: 128 is
referred to as BO1575. TABLE-US-00069 SEQ ID NO: 127
ATGAAAAAAATCAACAAATGTCTTACAATGTTCTCGACACTGCTATTGAT
CTTAACGTCACTATTCTCAGTTGCACCAGCGTTTGCGGACGACGCAACAA
CTGATACTGTGACCTTGCACAAGATTGTCATGCCACAAGCTGCATTTGAT
AACTTTACTGAAGGTACAAAAGGTAAGAATGATAGCGATTATGTTGGTAA
ACAAATTAATGACCTTAAATCTTATTTTGGCTCAACCGATGCTAAAGAAA
TCAAGGGTGCTTTCTTTGTTTTCAAAAATGAAACTGGTACAAAATTCATT
ACTGAAAATGGTAAGGAAGTCGATACTTTGGAAGCTAAAGATGCTGAAGG
TGGTGCTGTTCTTTCAGGGTTAACAAAAGACAATGGTTTTGTTTTTAACA
CTGCTAAGTTAAAAGGAATTTACCAAATCGTTGAATTGAAAGAAAAATCA
AACTACGATAACAACGGTTCTATCTTGGCTGATTCAAAAGCAGTTCCAGT
TAAAATCACTCTGCCATTGGTAAACAACCAAGGTGTTGTTAAAGATGCTC
ACATTTATCCAAAGAATACTGAAACAAAACCACAAGTAGATAAGAACTTT
GCAGATAAAGATCTTGATTATACTGACAACCGAAAAGACAAAGGTGTTGT
CTCAGCGACAGTTGGTGACAAAAAAGAATACATAGTTGGAACAAAAATTC
TTAAAGGCTCAGACTATAAGAAACTGGTTTGGACTGATAGCATGACTAAA
GGTTTGACGTTCAACAACAACGTTAAAGTAACATTGGATGGTGAAGATTT
TCCTGTTTTAAACTACAAACTCGTAACAGATGACCAAGGTTTCCGTCTTG
CCTTGAATGCAACAGGTCTTGCAGCAGTAGCAGCAGCTGCAAAAGACAAA
GATGTTGAAATCAAGATCACTTACTCAGCTACGGTGAACGGCTCCACTAC
TGTTGAAATTCCAGAAACCAATGATGTTAAATTGGACTATGGTAATAACC
CAACGGAAGAAAGTGAACCACAAGAAGGTACTCCAGCTAACCAAGAAATT
AAAGTCATTAAAGACTGGGCAGTAGATGGTACAATTACTGATGCTAATGT
TGCAGTTAAAGCTATCTTTACCTTGCAAGAAAAACAAACGGATGGTACAT
GGGTGAACGTTGCTTCACACGAAGCAACAAAACCATCACGCTTTGAACAT
ACTTTCACAGGTTTGGATAATGCTAAAACTTACCGCGTTGTCGAACGTGT
TAGCGGCTACACTCCAGAATACGTATCATTTAAAAATGGTGTTGTGACTA
TCAAGAACAACAAAAACTCAAATGATCCAACTCCAATCAACCCATCAGAA
CCAAAAGTGGTGACTTATGGACGTAAATTTGTGAAAACAAATCAAGCTAA
CACTGAACGCTTGGCAGGAGCTACCTTCCTCGTTAAGAAAGAAGGCAAAT
ACTTGGCACGTAAAGCAGGTGCAGCAACTGCTGAAGCAAAGGCAGCTGTA
AAAACTGCTAAACTAGCATTGGATGAAGCTGTTAAAGCTTATAACGACTT
GACTAAAGAAAAACAAGAAGGCCAAGAAGGTAAAACAGCATTGGCTACTG
TTGATCAAAAACAAAAAGCTTACAATGACGCTTTTGTTAAAGCTAACTAC
TCATATGAATGGGTTGCAGATAAAAAGGCTGATAATGTTGTTAAATTGAT
CTCTAACGCCGGTGGTCAATTTGAAATTACTGGTTTGGATAAAGGCACTT
ATGGCTTGGAAGAAACTCAAGCACCAGCAGGTTATGCGACATTGTCAGGT
GATGTAAACTTTGAAGTAACTGCCACATCATATAGCAAAGGGGCTACAAC
TGACATCGCATATGATAAAGGCTCTGTAAAAAAAGATGCCCAAGAAGTTC
AAAACAAAAAAGTAACCATCCCACAAACAGGTGGTATTGGTACAATTCTT
TTCACAATTATTGGTTTAAGCATTATGCTTGGAGCAGTAGTTATCATGAA
AAAACGTCAATCAGAGGAAGCTTAA SEQ ID NO: 128
MKKINKCLTMFSTLLLILTSLFSVAPAFADDATTDTVTLHKIVMPQAAFD
NFTEGTKGKNDSDYVGKQINDLKSYFGSTDAKEIKGAFFVFKNETGTKFI
TENGKEVDTLEAKDAEGGAVLSGLTKDNGFVFNTAKLKGIYQIVELKEKS
NYDNNGSILADSKAVPVKITLPLVNNQGVVKDAHIYPKNTETKPQVDKNF
ADKDLDYTDNRKDKGVVSATVGDKKEYIVGTKILKGSDYKKLVWTDSMTK
GLTFNNNVKVTLDGEDFPVLNYKLVTDDQGFRLALNATGLAAVAAAAKDK
DVEIKITYSATVNGSTTVEIPETNDVKLDYGNNPTEESEPQEGTPANQEI
KVIKDWAVDGTITDANVAVKAIFTLQEKQTDGTWVNVASHEATKPSRFEH
TFTGLDNAKTYRVVERVSGYTPEYVSFKNGVVTIKNNKNSNDPTPINPSE
PKVVTYGRKFVKTNQANTERLAGATFLVKKEGKYLARKAGAATAEAKAAV
KTAKLALDEAVKAYNDLTKEKQEGQEGKTALATVDQKQKAYNDAFVKANY
SYEWVADKKADNVVKLISNAGGQFEITGLDKGTYGLEETQAPAGYATLSG
DVNFEVTATSYSKGATTDIAYDKGSVKKDAQQVQNKKVTIPQTGGIGTIL
FTIIGLSIMLGAVVIMKKRQSEEA
[0952] The GBS 59 polypeptides contain an amino acid motif
indicative of a cell wall anchor: SEQ ID NO: 129 IPQTG (shown in
italics in SEQ ID NOs: 126 and 128 above). In some recombinant host
cell systems, it may be preferable to remove this motif to
facilitate secretion of a recombinant GBS 59 protein from the host
cell. Alternatively, in some recombinant host cell systems, it may
be preferable to use the cell wall anchor motif to anchor the
recombinantly expressed protein to the cell wall. The extracellular
domain of the expressed protein may be cleaved during purification
or the recombinant protein may be left attached to either
inactivated host cells or cell membranes in the final
composition.
[0953] Pilin motifs, containing conserved lysine (K) residues have
been identified in the GBS 59 polypeptides. The pilin motif
sequences are underlined in each of SEQ ID NOs: 126 and 128, below.
Conserved lysine (K) residues are marked in bold. The conserved
lysine (K) residues are located at amino acid residues 202 and 212
and amino acid residues 489 and 495 of SEQ ID NO: 126 and at amino
acid residues 188 and 198 of SEQ ID NO: 128. The pilin sequences,
in particular the conserved lysine residues, are thought to be
important for the formation of oligomeric, pilus-like structures of
GBS 59. Preferred fragments of GBS 59 include at least one
conserved lysine residue. Preferably, fragments include at least
one pilin sequence. TABLE-US-00070 SEQ ID NO: 126
MKRINKYFAMFSALLLTLTSLLSVAPAFADEATTNTVTLHKILQTESNLN
KSNFPGTTGLNGKDYKGGATSDLAGYFGEGSKSIEGAFFALALKEDKSGK
VQYVKAKEGNKLTPALINKDGTPEITVNIDEAVSGLTPEGDTGLVFNTKG
LKGEFKIVEVKSKSTYNNNGSLLAASKAVPVNITLPLVNEDGVVADAHVY
PKNTEEKPEIDKNFAKTNDLTALTDVNRLLTAGANYGNYARDKATATAET
GKVVPYEVKTKIHKGSKYENLVWTDIMSNGLTMGSTVSLKASGTTETFAK
DTDYELSIDARGFTLKFTADGLGKLEKAAKTADIEFTLTYSATVNGQAII
DNPESNDIKLSYGNKPGKDLTELPVTPSKGEVTVAKTWSDGIAPDGVNVV
YTLKDKDKTVASVSLTKTSKGTIDLGNGIKEEVSGNFSGKFTGLENKSYM
ISERVSGYGSAINLENGKVTITNTKDSDNPTPLNPTEPKVETHGKKFVKT
NEQGDRLAGAQFVVKNSAGKYLALKADQSEGQKTLAAKKIALDEAIAAYN
KLSATDQKGEKGITAKELIKTKQADYDAAFIEARTAYEWITDKARAITYT
SNDQGQFEVTGLADGTYNLEETLAPAGFAKLAGNIKFVVNQGSYITGGNI
DYVANSNQKDATRVENKKVTIPQTGGIGTILFTIIGLSIMLGAVVIMKRR QSKEA SEQ ID NO:
128 MKKINKCLTMFSTLLLILTSLFSVAPAFADDATTDTVTLHKIVMPQPAFD
NFTEGTKGKNDSDYVGKQINDLKSYFGSTDAKEIKGAFFVFKNETGTKFI
TENGKEVDTLEAKDAEGGAVLSGLTKDNGFVFNTAKLKGIYQIVELKEKS
NYDNNGSILADSKAVPVKITLPLVNNQGVVKDAHIYPKNTETKPQVDKNF
ADKDLDYTDNRKDKGVVSATVGDKKEYIVGTKILKGSDYKKLVWTDSMTK
GLTFNNNVKVTLDGEDFPVLNYKLVTDDQGFRLALNATGLAAVAAAAKDK
DVEIKITYSATVNGSTTVEIPETNDVKLDYGNNPTEESEPQEGTPANQEI
KVIKDWAVDGTITDANVAVKAIFTLQEKQTDGTWVNVASHEATKPSRFEH
TFTGLDNAKTYRVVERVSGYTPEYVSFKNGVVTIKNNKNSNDPTPINPSE
PKVVTYGRKFVKTNQANTERLAGATFLVKKEGKYLARKAGAATAEAKAAV
KTAKLALDEAVKAYNDLTKEKQEGQEGKTALATVDQKQKAYNDAFVKANY
SYEWVADKKADNVVKLISNAGGQFEITGLDKGTYGLEETQAPAGYATLSG
DVNFEVTATSYSKGATTDIAYDKGSVKKDAQQVQNKKVTIPQTGGIGTIL
FTIIGLSIMLGAVVIMKKRQSEEA
[0954] An E box containing a conserved glutamic residue has also
been identified in each of the GBS 59 polypeptides. The E box motif
is underlined in each of SEQ ID NOs: 126 and 128 below. The
conserved glutamic acid (E) is marked in bold at amino acid residue
621 in SEQ ID NO: 126 and at amino acid residue 588 in SEQ ID NO:
128. The E box motif, in particular the conserved glutamic acid
residue, is thought to be important for the formation of oligomeric
pilus-like structures of GBS 59. Preferred fragments of GBS 59
include the conserved glutamic acid residue. Preferably, fragments
include the E box motif. TABLE-US-00071 SEQ ID NO: 126
MKRINKYFAMFSALLLTLTSLLSVAPAFADEATTNTVTLHKILQTESNLN
KSNFPGTTGLNGKDYKGGAISDLAGYFGEGSKEIEGAFFALALKEDKSGK
VQYVKAKEGNKLTPALINKDGTPEITVNIDEAVSGLTPEGDTGLVFNTKG
LKGEFKIVEVKSKSTYNNNGSLLAASKAVPVNITLPLVNEDGVVADAHVY
PKNTEEKPEIDKNFAKTNDLTALTDVNRLLTAGANYGNYARDKATATAEI
GKVVPYEVKTKIHKGSKYENLVWTDIMSNGLTMGSTVSLKASGTTETFAK
DTDYELSIDARGFTLKFTADGLGKLEKAAKTADIEFTLTYSATVNGQAII
DNPESNDIKLSYGNKPGKDLTELPVTPSKGEVTVAKTWSDGIAPDGVNVV
YTLKDKDKTVASVSLTKTSKGTIDLGNGIKFEVSGNFSGKFTGLENKSYM
ISERVSGYGSAINLENGKVTITNTKDSDNPTPLNPTEPKVETHGKKEVKT
NEQGDRLAGAQFVVKNSAGKYLALKADQSEGQKTLAAKKIALDEAIAAYN
KLSATDQKGEKGITAKELIKTKQADYDAAFIEARTAYEWITDKARAITYT
SNDQGQFEVTGLADGTYNLEETLAPAGFAKLAGNIKEVVNQGSYITGGNI
DYVANSNQKDATRVENKKVTIPQTGGIGTILFTIIGLSIMLGAVVIMKRR QSKEA SEQ ID NO:
128 MKKINKCLTMFSTLLLILTSLFSVAPAFADDATTDTVTLHKIVMPQAAFD
NFTEGTKGKNDSDYVGKQINDLKSYFGSTDAKEIKGAFFVFKNETGTKFI
TENGKEVDTLEAKDAEGGAVLSGLTKDNGFVFNTAKLKGIYQIVELKEKS
NYDNNGSILADSKAVPVKITLPLVNNQGVVKDAHIYPKNTETKPQVDKNF
ADKDLDYTDNRKDKGVVSATVGDKKEYTVGTKILKGSDYKKLVWTDSMTK
GLTFNNNVKVTLDGEDFPVLNYKLVTDDQGFRLALNATGLAAVAAAAKDK
DVEIKITYSATVNGSTTVEIPETNDVKLDYGNNPTEESEPQEGTPANQEI
KVIKDWAVDGTITDANVAVKAIFTLQEKQTDGTWVNVASHEATKPSRFEH
TFTGLDNAKTYRVVERVSGYTPEYVSFKNGVVTIKNNKNSNDPTPINPSE
PKVVTYGRKFVKTNQANTERLAGATFLVKKEGKYLARKAGAATAEAKAAV
KTAKLALDEAVKAYNDLTKEKQEGQEGKTALATVDQKQKAYNDAFVKANY
SYEWVADKKADNVVKLISNAGGQFEITGLDKGTYGLEETQAPAGYATLSG
DVNFEVTATSYSKGATTDIAYDKGSVKKKAQQVQNKKVTIPQTGGIGTIL
FTIIGLSIMLGAVVIMKKRQSEEA
[0955] Female mice were immunized with either SAG1407 (SEQ ID NO:
126) or BO1575 (SEQ ID NO: 128) in an active maternal immunization
assay. Pups bred from the immunized female mice survived GBS
challenge better than control (PBS) treated mice. Results of the
active maternal immunization assay using the GBS 59 immunogenic
compositions are shown in Table 17, below. TABLE-US-00072 TABLE 17
Active maternal immunization assay for GBS 59 Challenge GBS 59 PBS
GBS strain Survival Survival (serotype) Dead/treated (%)
Dead/treated (%) FACS CJB111 (V)* 7/20 65 41/49 16 493 18RS21
(II)** 18/30 40 39/40 2.5 380 *immunized with BO1575 **immunized
with SAG1407
[0956] Opsonophagocytosis assays also demonstrated that antibodies
against BO 1575 are opsonic for GBS serotype V, strain CJB111. See
FIG. 67.
GBS 52
[0957] Examples of polynucleotide and amino acid sequences for GBS
52 are set forth below. SEQ ID NO: 20 and 21 represent GBS 52
sequences from GBS serotype V, strain isolate 2603. TABLE-US-00073
SEQ ID NO: 20 ATGAAACAAACATTAAAACTTATGTTTTCTTTTCTGTTGATGTTAGGGAC
TATGTTTGGAATTAGCCAAACTGTTTTAGCGCAAGAAACTCATCAGTTGA
CGATTGTTGATCTTGAAGCAAGGGATATTGATCGTCCAAATCCACAGTTG
GAGATTGCCCCTAAAGAAGGGACTCCAATTGAAGGAGTACTCTATCAGTT
GTACCAATTAAAATCAACTGAAGATGGGGATTTGTTGGCAGATTGGAATT
CCCTAACTATCACAGAATTGAAAAAACAGGCGCAGCAGGTTTTTGAAGCG
ACTACTAATCAACAAGGAAAGGCTACATTTAACCAACTACCAGATGGAAT
TTATTATGGTCTGGCGGTTAAAGCCGGTGAAAAAAATCGTAATGTCTCAG
CTTTCTTGGTTGACTTGTCTGAGGATAAAGTGATTTATCCTAAAATCATC
TGGTCCACAGGTGAGTTGGACTTGCTTAAAGTTGGTGTGGATGGTGATAC
CAAAAAACCACTAGCAGGCGTTGTCTTTGAACTTTATGAAAAGAATGGTA
GGACTCCTATTCGTGTGAAAAATGGGGTGCATTCTCAAGATATTGACGCT
GCAAAACATTTAGAAACAGATTCATCAGGGCATATCAGAATTTCCGGGCT
CATCCATGGGGACTATGTCTTAAAAGAAATCGAGACACAGTCAGGATATC
AGATCGGACAGGCAGAGACTGCTGTGACTATTGAAAAATCAAAAACAGTA
ACAGTAACGATTGAAAATAAAAAAGTTCCGACACCTAAAGTGCCATCTCG
AGGAGGTCTTATTCCCAAAACAGGTGAGCAACAGGCAATGGCACTTGTAA
TTATTGGTGGTATTTTAATTGCTTTAGCCTTACGATTACTATCAAAACAT
CGGAAACATCAAAATAAGGAT SEQ ID NO: 21
MKQTLKLMFSFLLMLGTMFGISQTVLAQETHQLTIVHLEARDIDRPNPQL
EIAPKEGTPIEGVLYQLYQLKSTEDGDLLAHWNSLTITELKKQAQQVFEA
TTNQQGKATFNQLPDGIYYGLAVKAGEKNRNVSAFLVDLSEDKVIYPKII
WSTGELDLLKVGVDGDTKKPLAGVVFELYEKNGRTPTRVKNGVHSQDIDA
AKHLETDSSGHIRISGLIHGDYVLKEIETQSGYQIGQAETAVTIEKSKTV
TVTIENKKVPTPKVPSRGGLIPKTGEQQAMALVIIGGILIALALRLLSKH RKHQNKD
[0958] GBS 52 contains an amino acid motif indicative of a cell
wall anchor: SEQ ID NO: 124 IPKTG (shown in italics in SEQ ID NO:
21, above). In some recombinant host cell systems, it may be
preferable to remove this motif to facilitate secretion of a
recombinant GBS 52 protein from the host cell. Alternatively, in
other recombinant host cell systems, it may be preferable to use
the cell wall anchor motif to anchor the recombinantly expressed
protein to the cell wall. The extracellular domain of the expressed
protein may be cleaved during purification or the recombinant
protein may be left attached to either inactivated host cells or
cell membranes in the final composition.
[0959] A pilin motif, discussed above, containing a conserved
lysine (K) residue has also been identified in GBS 52. The pilin
motif sequence is underlined in SEQ ID NO: 21, below. Conserved
lysine (K) residues are also marked in bold, at amino acid residues
148 and 160. The pilin sequence, in particular the conserved lysine
residues, are thought to be important for the formation of
oligomeric, pilus-like structures. Preferred fragments of GBS 52
include at least one conserved lysine residue. Preferably,
fragments include the pilin sequence. TABLE-US-00074 SEQ ID NO: 21
MKQTLKLMFSFLLMLGTMFGISQTVLAQETHQLTIVHLEARDIDRPNPQL
EIAPKEGTPIEGVLYQLYQLKSTEDGDLLAHWNSLTITELKKQAQQVFEA
TTNQQGKATFNQLPDGIYYGLAVKAGEKNRNVSAFLVDLSEDKVIYPKII
WSTGELDLLKVGVDGDTKKPLAGVVFELYEKNGRTPTRVKNGVHSQDIDA
AKHLETDSSGHIRISGLIHGDYVLKEIETQSGYQIGQAETAVTIEKSKTV
TVTIENKKVPTPKVPSRGGLIPKTGEQQAMALVIIGGILIALALRLLSKH RKHQNKD
[0960] An E box containing a conserved glutamic residue has been
identified in GBS 52. The E-box motif is underlined in SEQ ID NO:
21, below. The conserved glutamic acid (E), at amino acid residue
226, is marked in bold. The E box motif, in particular the
conserved glutamic acid residue, is thought to be important for the
formation of oligomeric pilus-like structures of GBS 52. Preferred
fragments of GBS 52 include the conserved glutamic acid residue.
Preferably, fragments include the E box motif. TABLE-US-00075 SEQ
ID NO: 21 MKQTLKLMFSFLLMLGTMFGISQTVLAQETHQLTIVHLEARDIDRPNPQL
EIAPKEGTPIEGVLYQLYQLKSTEDGDLLAHWNSLTITELKKQAQQVFEA
TTNQQGKATFNQLPDGIYYGLAVKAGEKNRNVSAFLVDLSEDKVIYPKII
WSTGELDLLKVGVDGDTKKPLAGVVFELYEKNGRTPTRVKNGVHSQDIDA
AKHLETDSSGHIRISGLIHGDYVLKEIETQSGYQIGQAETAVTIEKSKTV
TVTIENKKVPTPKVPSRGGLIPKTGEQQAMALVIIGGILIALALRLLSKH RKHQNKD
SAG0647
[0961] Examples of polynucleotide and amino acid sequences for
SAG0647 are set forth below. SEQ ID NO: 22 and 23 represent SAG0647
sequences from GBS serotype V, strain isolate 2603. TABLE-US-00076
SEQ ID NO: 22 ATGGGACAAAAATCAAAAATATCTCTAGCTACGAATATTCGTATATGGAT
TTTTCGTTTAATTTTCTTAGCGGGTTTCCTTGTTTTGGCATTTCCCATCG
TTAGTCAGGTCATGTACTTTCAAGCCTCTCACGCCAATATTAATGCTTTT
AAAGAAGGTGTTACCAAGATTGACCGGGTGGAGATTAATCGGCGTTTAGA
ACTTGCTTATGCTTATAAGGCCAGTATAGCAGGTGCCAAAACTAATGGCG
AATATCCAGCGCTTAAAGACCCCTACTCTGGTGAACAAAAGCAGGCAGGG
GTCGTTGAGTACGCCCGCATGCTTGAAGTCAAAGAACAAATAGGTCATGT
GATTATTCCAAGAATTAATCAGGATATCCCTATTTACGCTGGCTCTGCTG
AAGAAAATCTTCAGAGGGGCGTTGGACATTTAGAGGGGACCAGTCTTCCA
GTCGGTGGTGAGTCAACTCATGCCGTTGTAACTGCCCATCGAGGGCTACC
AACGGCCAAGCTATTTACGAATTTAGACAAGGTAACAGTAGGTGACCGTT
TTTACATTGAAGACATCGGCGGAAAGATTGCTTATCAGGTAGACCAAATC
AAAGTTATCGGCCCTGATCAGTTAGAGGATTTGTACGTGATTCAAGGAGA
AGATCACGTCACCCTATTAACTTGCACAGCTTATATGATAAATAGTCATC
GCCTCCTCGTTCGAGGCAAGCGAATTCCTTATGTGGAAAAAACAGTGCAG
AAAGATTCAAAGACCTTCAGGCAACAACAATACCTAACCTATGCTATGTG
GGTAGTCGTTGGACTTATGTTGCTGTCGCTTCTCATTTGGTTTAAAAAGA
CGAAACAGAAAAAGCGGAGAAAGAATGAAAAAGCGGCTAGTCAAAATAGT
CACAATAATTCGAAATAA SEQ ID NO: 23
MGQKSKISLATNIRIWIERLIFLAGFLVLAFPIVSQVMYFQASHANINAF
KEAVTKIDRVEINRRLELAYAYNASIAGAKTNGEYPALKDPYSAEQKQAG
VVEYARMLEVKEQIGHVIIPRINQDIPIYAGSAEENLQRGVGHLEGTSLP
VGGESTHAVLTAHRGLPTAKLFTNLDKVTVGDRFYIEHIGGKIAYQVDQI
KVIAPDQLEDLYVIQGEDHVTLLTCTPYMINSHRLLVRGKRIPYVEKTVQ
KDSKTFRQQQYLTYAMWVVVGLILLSLLIWFKKTKQKKRRKNEKAASQNS HNNSK
SAG0648
[0962] Examples of polynucleotide and amino acid sequences for
SAG0648 are set forth below. SEQ ID NO: 24 and 25 represent SAG0648
sequences from GBS serotype V, strain isolate 2603. TABLE-US-00077
SEQ ID NO: 24 ATGGGAAGTCTGATTCTCTTATTTCCGATTGTGAGCCAGGTAAGTTACTA
CCTTGCTTCGCATCAAAATATTAATCAATTTAAGCGGGAAGTCGCTAAGA
TTGATACTAATACGGTTGAACGACGCATCGCTTTAGCTAATGCTTACAAT
GAGACGTTATCAAGGAATCCCTTGCTTATAGACCCTTTTACCAGTAAGCA
AAAAGAAGGTTTGAGAGAGTATGCTCGTATGCTTGAAGTTCATGAGCAAA
TAGGTCATGTGGCAATCCCAAGTATTGGGGTTGATATTCCAATTTATGCT
GGAACATCCGAAACTGTGCTTCAGAAAGGTAGTGGGCATTTGGAGGGAAC
CAGTCTTCCAGTGGGAGGTTTGTCAACCCATTCAGTACTAACTGCCCACC
GTGGCTTGCCAACAGCTAGGCTATTTACCGACTTAAATAAAGTTAAAAAA
GGCCAGATTTTCTATGTGACGAACATCAAGGAAACACTTGCCTACAAAGT
CGTGTCTATCAAAGTTGTGGATCCAACAGCTTTAAGTGAGGTTAAGATTG
TCAATGGTAAGGATTATATAACCTTGCTGACTTGCACACCTTACATGATC
AATAGTCATGGTCTCTTGGTAAAAGGAGAGCGTATTCCTTATGATTCTAC
CGAGGCGGAAAAGCACAAAGAACAAACGGTACAAGATTATCGTTTGTCAC
TAGTGTTGAAGATACTACTAGTATTATTAATTGGACTCTTCATCGTGATA
ATGATGAGAAGATGGATGGAACATCGTCAATAA SEQ ID NO: 25
MGSLILLFPIVSQVSYYLASHQNINQFKREVAKIDTNTVERRIALANAYN
ETLSRNPLLIDPFTSKQKEGLREYARMLEVHEQTGHVATPSIGVDIPIYA
GTSETVLQKGSGHLEGTSLPVGGLSTHSVLTAHRGLPTARLFTDLNKVKK
GQIFYVTNIKETLAYKVVSIKVVDPTALSEVKIVNGKDYITLLTCTPYMI
NSHRLLVKGERIPYDSTEAEKHKEQTVQDYRLSLVLKILLVLLIGLFIVI MMRRWMQHRQ
GBS 150
[0963] Examples of polynucleotide and amino acid sequences for GBS
150 are set forth below. SEQ ID NO: 26 and 27 represent GBS 150
sequences from GBS serotype V, strain isolate 2603. TABLE-US-00078
SEQ ID NO: 26 ATGAAAAAGATTAGAAAAAGTTTAGGACTTCTAGTATGTTGGTTTTTAGG
ATTGGTACAATTAGCGTTTTTTTCGGTAGGCAGTGTAAATGCTGATACCC
CTAATCAACTAACAATCACAGAGATAGGACTTCAGCGAAATACTACAGAG
GAGGGGATTTCTTATGGTTTATGGACTGTGACTGACAAGTTAAAAGTTGA
TTTATTGAGGCAAATGACAGATAGCGAATTGAAGGAGAAGTATAAGAGTA
TGTTGACTTCTCCTAGTGATACTAATGGTCAGACAAAGATAGGACTGGGA
AATGGTTCGTACTTTGGTCGTGCTTATAAAGCTGATGAAAGGGTTTCAAC
AATAGTACCTTTTTATATTGAATTAGGAGATGATAAGTTATGAAATCAAT
TACAGATAAATCGTAAGCGAAAAGTTGAAACAGGCGGATTAAAACTTATT
AAATATACAAAAGAAGGAAAGATAAAGAAAAGGCTATCCGGAGTAATATT
TGTATTATACGATAACCAGAATGAGGGAGTTCGCTTTAAAAATGGACGAT
TTACGACGGATCAAGATGGGATTAGTTGATTAGTAACTGATGATAAGGGA
GAAATTGAGGTTGAAGGTTTATTACGTGGTAAGTATATTTTTCGAGAAGC
AAAAGCACTAACTGGTTACCGTATATGTATGAAGGATGGTGTAGTTGCTG
TAGTTGCTAATAAAACACAGGAAGTAGAGGTAGAAAAcGAAAAAGAAACT
CCTCCACCAACAAATCCTAAACCATCACAACCGCTTTTTGCACAATCATT
TCTTCCTAAAACAGGAATGATTATTGGTGGAGGACTGACAATTCTTGGTT
GTATTATTTTGGGAATTTTGTTTATCTTTTTAAGAAAAACTAAAAATAGC
AAATCTGAAAGAAACGATACAGTA SEQ ID NO: 27
MKKIRKSLGLLLCCFLGLVQLAFESVASVNADTPNQLTITQIGLQPNTTE
EGISYRLWTVTDNLKVDLLSQMTDSELNQKYKSILTSPTDTNGQTKIALP
NGSYFGRAYKADQSVSTIVPEYIELPDDKLSNQLQINPKRKVETGRLKLI
KYTKEGKIKKRLSGVIFVLYDNQNQPVRFKNGRFTTDQDGITSLVTDDKG
EIEVEGLLPGKYIFREAKALTGYRISMKDAVVAVVANKTQEVEVENEKET
PPPTNPKPSQPLFPQSFLPKTGMIIGGGLTILGCIILGILFIFLRKTKNS KSERNDTV
[0964] GBS 150 contains an amino acid motif indicative of a cell
wall anchor: SEQ ID NO: 130 LPKTG (shown in italics in SEQ ID NO:
27 above). In some recombinant host cell systems, it may be
preferable to remove this motif to facilitate secretion of a
recombinant GBS 150 protein from the host cell. Alternatively, in
other recombinant host cell systems, it may be preferable to use
the cell wall anchor motif to anchor the recombinantly expressed
protein to the cell wall. The extracellular domain of the expressed
protein may be cleaved during purification or the recombinant
protein may be left attached to either inactivated host cells or
cell membranes in the final composition.
[0965] As discussed above, a pilin motif, containing a conserved
lysine (K) residue has been identified in GBS 150. The pilin motif
sequence is underlined in SEQ ID NO: 27, below. Conserved lysine
(K) residues are marked in bold, at amino acid residues 139 and
148. The pilin sequence, in particular the conserved lysine
residues, are thought to be important for the formation of
oligomeric, pilus-like structures of GBS 150. Preferred fragments
of GBS 150 include a conserved lysine residue. Preferably,
fragments include the pilin sequence. TABLE-US-00079 SEQ ID NO: 27
MKKIRKSLGLLLCCFLGLVQLAFFSVASVNADTPNQLTITQIGLQPNTTE
EGISYRLWTVTDNLKVDLLSQMTDSELNQKYKSILTSPTDTNGQTKIALP
NGSYFGRAYKADQSVSTIVPFYIELPDDKLSNQLQINPKRKVETGRLKLI
KYTKEGKIKKRLSGVIFVLYDNQNQPVRFKNGRFTTDQDGITSLVTDDKG
EIEVEGLLPGKYIFREAKALTGYRISMKDAVVAVVANKTQEVEVENEKET
PPPTNPKPSQPLFPQSFLPKTGMIIGGGLTILGCTTLGILFIFLRKTKNS KSERNDTV
[0966] An E box containing a conserved glutamic residue has also
been identified in GBS 150. The E box motif is underlined in SEQ ID
NO: 27 below. The conserved glutamic acid (E), at amino acid
residue 216, is marked in bold. The E box motif, in particular the
conserved glutamic acid residue, is thought to be important for the
formation of oligomeric pilus-like structures of GBS 150. Preferred
fragments of GBS 150 include the conserved glutamic acid residue.
Preferably, fragments include the E box motif. TABLE-US-00080 SEQ
ID NO: 27 MKKIRKSLGLLLCCFLGLVQLAFFSVASVNADTPNQLTITQIGLQPNTTE
EGISYRLWTVTDNLKVDLLSQMTDSELNQKYKSILTSPTDTNGQTKIALP
NGSYFGRAYKADQSVSTIVPFYIELPDDKLSNQLQINPKRKVETGRLKLI
KYTKEGKIKKRLSGVIFVLYDNQNQPVRFKNGRFTTDQDGITSLVTDDKG
EIEVEGLLPGKYIFREAKALTGYRISMKDAVVAVVANKTQEVEVENEKET
PPPTNPKPSQPLFPQSFLPKTGMIIGGGLTILGCIILGILFIPLRKTKNS KSERNDTV
[0967] Examples of polynucleotide and amino acid sequences for
SAG1405 are set forth below. SEQ ID NO: 28 and 29 represent SAG1405
sequences from GBS serotype V, strain isolate 2603. TABLE-US-00081
SEQ ID NO: 28 ATGGGAGGAAAATTTCAGAAAAACCTTAAGAAATCGGTCGTTTTAAATCG
ATGGATGAATGTAGGCTTGATACTATTGTTCTTAGTTGGTCTTTTGATAA
CCTCATATCCTTTTATTTCAAATTGGTACTATAATATTAAAGCTAATAAT
CAAGTAACTAACTTTGATAATCAAACCCAAAAATTAAATACTAAAGAGAT
TAATAGACGATTTGAGTTAGCAAAAGCTTATAATAGAACACTGGACCCAA
GCCGCCTATCAGATCCCTATACTGAAAAAGAAAAAAAAGGTATTGCTGAA
TACGCCCACATGCTTGAGATTGCTGAAATGATTGGATATATTGATATACC
GTCTATCAAGCAAAAATTACCTATCTATGCGGGGACTACCAGTAGTGTTC
TTGAAAAAGGAGCAGGACACCTTGAAGGAACCTCCTTGCCAATTGGTGGA
AAAAGTTCACATACTGTTATCACAGCTCATCGCGGCTTACCTAAAGCTAA
GTTATTTACAGATTTAGATAAACTTAAAAAAGGAAAAATTTTTTATATTC
ATAATATCAAAGAAGTTTTAGCCTATAAGGTTGATCAAATAAGTGTTGTA
AAGCCAGATAATTTTTCTAAATTATTGGTTGTTAAAGGTAAGGATTATGC
GACTTTGCTAACATGTACACCTTATTCGATTAATTCACATCGTTTACTAG
TTAGAGGGCATCGAATCAAGTATGTACCTCCTGTTAAAGAAAAGAACTAT
TTAATGAAAGAATTGCAAACACACTATAAACTTTATTTCCTCTTATCAAT
CCTAGTTATTCTTATATTAGTCGCTTTACTATTATATTTAAAACGAAAAT
TTAAAGAGAGAAAGAGAAAGGGAAATCAAAAATGA SEQ ID NO: 29
MGGKFQKNLKKSVVLNRWMNVGLILLFLVGLLITSYPFISNWYYNIKANN
QVTNFDNQTQKLNTKEINRRFELAKAYNRTLDPSRLSDPYTEKEKKGIAE
YAHMLEIAEMIGYIDIPSIKQKLPIYAGTTSSVLEKGAGHLEGTSLPIGG
KSSHTVITAHRGLPKAKLFTDLDKLKKGKIFYIHNIKEVLAYKVDQISVV
KPDNFSKLLVVKGKDYATLLTCTPYSINSHRLLVRGHRIKYVPPVKEKNY
LMKELQTRYKLYFLLSILVILILVALLLYLKRKPKERKRKGNQK
SAG1406
[0968] Examples of polynucleotide and amino acid sequences for
SAG1405 are set forth below. SEQ ID NO: 30 and 31 represent SAG1405
sequences from GBS serotype V, strain isolate 2603. TABLE-US-00082
SEQ ID NO: 30 GTGAAGACTAAAAAAATCATCAAAAAAACAAAAAAAAAGAAGAAGTCAAA
TCTTCCTTTTATCATTCTTTTTCTAATAGGTCTATCTATTTTATTGTATC
CAGTGGTATCACGTTTTTACTATACGATAGAATCTAATAATCAAACACAG
GATTTTGAGAGAGCTGCTAAAAAACTTAGTCAGAAAGAAATCAATCGACG
TATGGCTCTAGCACAAGCTTATAATGATTCTTTAAATAATGTCCATCTTG
AAGATCCTTATGAGAAAAAACGAATTCAAAAGGGGGTAGCAGAGTACGCC
CGTATGTTAGAGGTAAGTGAAAAAATCGGAACAATTTCAGTTCCTAAGAT
AGGTCAAAAACTCCCTATATTTGCAGGTTCAAGTCAAGAAGTTCTATCTA
AAGGAGCAGGGCATTTAGAAGGTACCTCTCTTCCAATTGGGGGCAATAGT
ACACATACTGTTATAACAGCGCATTCAGGAATTCCAGATAAAGAACTCTT
TTCTAACCTTAAAAAGTTAAAAAAAGGAGATAAGTTTTATATTCAAAACA
TAAAAGAAACGATAGCATATCAAGTAGATCAGATAAAAGTCGTTACACCC
GATAACTTTTCAGATTTGTTGGTTGTTCCTGGACATGATTATGCAACCTT
ATTGACTTGCACCCCGATTATGATCAATACACACAGACTTTTAGTAAGGG
GACATCGTATCCCTTATAAAGGTCCTATTGATGAAAAATTAATAAAAGAC
GGTCATTTAAACACGATTTATAGATATCTATTCTATATATCTTTAGTTAT
TATTGCTTGGTTACTTTGGTTAATAAAACGTCAACGTCAAAAAAATCGTT
TAGCAAGTGTTAGAAAAGGAATTGAATCATAA SEQ ID NO: 31
MKTKKIIKKTKKKKKSNLPFIILFLIGLSILLYPVVSRFYYTIESNNQTQ
DFERAAKKLSQKEINRRMALAQAYNDSLNNVHLEDPYEKKRIQKGVAEYA
RMLEVSEKIGTISVPKIGQKLPIFAGSSQEVLSKGAGHLEGTSLPIGGNS
THTVITAHSGIPDKELFSNLKKLKKGDKFYIQNIKETIAYQVDQIKVVTP
DNFSDLLVVPGHDYATLLTCTPTMINTHRLLVRGHRIPYKGPIDEKLIKD
GHLNTIYRYLFYISLVIIAWLLWLIKRQRQKNRLASVRKGIES
01520
[0969] An example of an amino acid sequence for 01520 is set forth
below. SEQ ID NO: 32 represents a 01520 sequence from GBS serotype
III, strain isolate COH1. TABLE-US-00083 SEQ ID NO: 32
MIRRYSANFLAILGIILVSSGIYWGWYNINQAHQADLTSQHIVKVLDKSI
THQVKGSENGELPVKKLDKTDYLGTLDIPNLKLHLPVAANYSFEQLSKTP
TRYYGSYLTNNMVICAHNFPYHFDALKNVDMGTDVYFTTTTGQIYHYKIS
NREIIEPTAIEKVYKTATSDNDWDLSLFTCTKAGVARVLVRCQLIDVKN
01521
[0970] An example of an amino acid sequence for 01521 is set forth
below. SEQ ID NO: 33 represents a 01521 sequence from GBS serotype
III, strain isolate COH1. TABLE-US-00084 SEQ ID NO: 33
MIYKKILKITLLLLFSLSTQLVSADTNDQMKTGSITIQNKYNNQGIAGGN
LLVYQVAQAKDVDGNQVFTLTTPFQGIGIKDDDLTQVNLDSNQAKYVNLL
TKAVHKTQPLQTFDNLPAEGIVANNLPQGIYLFIQTKTAQGYELMSPFIL
SIPKDGKYDITAFEKMSPLNAKPKKEETITPTVTHQTKGKLPFTGQVWWP
IPILIMSGLLCLIIALKWRRRRD
[0971] 01521 contains an amino acid motif indicative of a cell wall
anchor: SEQ ID NO: 132 LPFTG (shown in italics in SEQ ID NO: 33
above). In some recombinant host cell systems, it may be preferable
to remove this motif to facilitate secretion of a recombinant 01521
protein from the host cell. Alternatively, it may be preferable to
use the cell wall anchor motif to anchor the recombinantly
expressed protein to the cell wall. The extracellular domain of the
expressed protein may be cleaved during purification or the
recombinant protein may be left attached to either inactivated host
cells or cell membranes in the final composition.
[0972] Two pilin motifs, containing conserved lysine (K) residues
have been identified in 01521. The pilin motif sequences are
underlined in SEQ ID NO: 33, below. Conserved lysine (K) residues
are marked in bold, at amino acid residues 154 and 165 and at amino
acid residues 174 and 188. The pilin sequences, in particular the
conserved lysine residues, are thought to be important for the
formation of oligomeric, pilus-like structures of 01521. Preferred
fragments of 01521 include at least one conserved lysine residue.
Preferably, fragments include at least one pilin sequence.
TABLE-US-00085 SEQ ID NO: 33
MIYKKILKITLLLLFSLSTQLVSADTNDQMKTGSITIQNKYNNQGIAGGN
LLVYQVAQAKDVDGNQVFTLTTPFQGIGIKDDDLTQVNLDSNQAKYVNLL
TKAVHKTQPLQTFDNLPAEGIVANNLPQGIYLFIQTKTAQGYELMSPFIL
SIPKDGKYDITAFEKMSPLNAKPKKEETITPTVTHQTKGKLPFTGQVWWP
TPILIMSGLLCLIIALKWRRRRD
[0973] An E box containing a conserved glutamic residue has also
been identified in 01521. The E box motif is underlined in SEQ ID
NO: 33 below. The conserved glutamic acid (E), at amino acid
residue 177, is marked in bold. The E box motif, in particular the
conserved glutamic acid residue, is thought to be important for the
formation of oligomeric pilus-like structures of 01521. Preferred
fragments of 01521 include the conserved glutamic acid residue.
Preferably, fragments include the E box motif. TABLE-US-00086 SEQ
ID NO: 33 MIYKKILKTTLLLLFSLSTQLVSADTNDQMKTGSTTIQNKYNNQGIAGGN
LLVYQVAQAKDVDGNQVFTLTTPFQGIGIKDDDLTQVNLDSNQAKYVNLL
TKAVHKTQPLQTFDNLPAEGIVANNLPQGIYLFIQTKTAQGYELMSPFTL
SIPKDGKYDITAFEKMSPLNAKPKKEETITPTVTHQTKGKLPFTGQWPIP
ILIMSGLLCLIIALKWRRRRD
01522
[0974] An example of an amino acid sequence for 01522 is set forth
below. SEQ ID NO: 34 represents a 01522 sequence from GBS serotype
III, strain isolate COH1. TABLE-US-00087 SEQ ID NO: 34
MAYPSLANYWNSFHQSRAIMDYQDRVTHMDENDYKKITNRAKEYNKQFKT
SGMKWHMTSQERLDYNSQLAIDKTGNMGYISIPKINIKLPLYHGTSEKVL
QTSIGHLEGSSLPIGGDSTHSILSGHRGLPSSRLFSDLDKLKVGDHWTVS
ILNETYTYQVDQIRTVKPDDLRDLQIVKGKDYQTLVTCTPYGVNTHRLLV
RGHRVPNDNGNALVVAEAIQIEPIYIAPFIAIFLTLILLLISLEVTRRAR
QRKKILKQAMRKEENNDL
01523
[0975] An example of an amino acid sequence for 01523 is set forth
below. SEQ ID NO: 35 represents a 01523 sequence from GBS serotype
III, strain isolate COH1. TABLE-US-00088 SEQ ID NO: 35
MKKKMIQSLLVASLAFGMAVSPVTPIAFAAETGTITVQDTQKGATYKAYK
VFDAEIDNANVSDSNKDGASYLIPQGKEAEYKASTDFNSLFTTTTNGGRT
YVTKKDTASANEIATWAKSISANTTPVSTVTESNNDGTEVINVSQYGYYY
VSSTVNNGAVIMVTSVTPNATIHEKNTDATWGDGGGKTVDQKTYSVGDTV
KYTITYKNAVNYHGTEKVYQYVIKDTMPSASVVDLNEGSYEVTITDGSGN
ITTLTQGSEKATGKYNLLEENNNFTITIPWAATNTPTGNTQNGANDDFFY
KGINTITVTYTGVLKSGAKPGSADLPENTNIATINPNTSNDDPGQKVTVR
DGQITIKKIDGSTKASLQGAIFVLKNATGQFLNFNDTNNVEWGTEANATE
YTTGADGIITITGLKEGTYYLVEKKAPLGYNLLDNSQKVILGDGATDTTN
SDNLLVNPTVENNKGTELPSTGGIGTTIEYIIGAILVIGAGIVLVARRRL RS
[0976] 01523 contains an amino acid motif indicative of a cell wall
anchor: SEQ ID NO: 131 LPSTG (shown in italics in SEQ ID NO: 35
above). In some recombinant host cell systems, it may be preferable
to remove this motif to facilitate secretion of a recombinant 01523
protein from the host cell. Alternatively, it may be preferable to
use the cell wall anchor motif to anchor the recombinantly
expressed protein to the cell wall. The extracellular domain of the
expressed protein may be cleaved during purification or the
recombinant protein may be left attached to either inactivated host
cells or cell membranes in the final composition.
[0977] An E box containing a conserved glutamic residue has also
been identified in 01523. The E box motif is underlined in SEQ ID
NO: 35 below. The conserved glutamic acid (E), at amino acid
residue 423, is marked in bold. The E box motif, in particular the
conserved glutamic acid residue, is thought to be important for the
formation of oligomeric pilus-like structures of 01523. Preferred
fragments of 01523 include the conserved glutamic acid residue.
Preferably, fragments include the E box motif. TABLE-US-00089 SEQ
ID NO: 35 MKKKMIQSLLVASLAFGMAVSPVTPIAFAAETGTITVQDTQKGATYKAYK
VFDAEIDNANVSDSNKDGASYLIPQGKEAEYKASTDFNSLFTTTTNGGRT
YVTKKDTASANEIATWAKSISANTTPVSTVTESNNDGTEVINVSQYGYYY
VSSTVNNGAVIMVTSVTPNATIHEKNTDATWGDGGGKTVDQKTYSVGDTV
KYTITYKNAVNYHGTEKVYQYVIKDTMPSASVVDLNEGSYEVTITDGSGN
ITTLTQGSEKATGKYNLLEENNNFTITIPWAATNTPTGNTQNGANDDFFY
KGINTITVTYTGVLKSGAKPGSADLPENTNIATINPNTSNDDPGQKVTVR
DGQITIKKIDGSTKASLQGAIFVLKNATGQFLNFNDTNNVEWGTEANATE
YTTGADGIITITGLKEGTYYLVEKKAPLGYNLLDNSQKVILGDGATDTTN
SDNLLVNPTVENNKGTELPSTGGIGTTIEYIIGAILVIGAGIVLVARRRL RS
01524
[0978] An example of an amino acid sequence for 01524 is set forth
below. SEQ ID NO: 36 represents a 01524 sequence from GBS serotype
m, strain isolate COH1. TABLE-US-00090 SEQ ID NO: 36
MLKKCQTFIIESLKKKKHPKEWKIIMWSLMILTTFLTTYFLILPAITVEE
TKTDDVGITLENKNSSQVTSSTSSSQSSVEQSKPQTPASSVTETSSSEEA
AYREEPLMFRGADYTVTVTLTKEAKIPKNADLKVTELKDNSATFKDYKKK
ALTEVAKQDSEIKNFKLYDITIESNGKEAEPQAPVKVEVNYDKPLEASDE
NLKVVHFKDDGQTEVLKSKDTAETKNTSSDVAFKTDSFSTYAIVQEDNTE
VPRLTYHFQNNDGTDYDPLTASGMQVHHQIIKDGESLGEVGIPTIKAGEH
FNGWYTYDPTTGKYGDPVKEGEPITVTETKEICVRPFMSKVATVTLYDDS
AGKSILERYQVPLDSSGNGTADLSSFKVSPPTSTLLFVGWSKTQNGAPLS
ESEIQALPVSSDISLYPVFKESYGVEFNTGDLSTGVTYIAPRRVLTGQPA
STIKPNDPTRPGYTFAGWYTAASGGAAFDFNQVLTKDTTLYAHWSPAQTT
YTINYWQQSATDNKNATDAQKTYEYAGQVTRSGLSLSNQTLTQQDINDKL
PTGFKVNNTRTETSVMIKDDGSSVVNVYYDRKLITIKFAKYGGYSLPEYY
YSYNWSSDADTYTGLYGTTLAANGYQWKTGAWGYLANVGNNQVGTYGMSY
LGEFILPNDTVDSDVIKLFPKGNIVQTYRFTKQGLDGTYSLADTGGGAGA
DEFTFTEKYLGFNVKYYQRLYPDNYLFDQYASQTSAGVKVPISDEYYDRY
GAYHKDYLNLVVWYERNSYKIKYLDPLDNTELPNFPVKDVLYEQNLSSYA
PDTTTVQPKPSRPGYVWDGKWYKDQAQTQVFDFNTTMPPHDVKVYAGWQK
VTYRVNIDPNGGRLSKTDDTYLDLHYGDRIPDYTDITRDYIQDPSGTYYY
KYDSRDKDPDSTKDAYYTTDTSLSNVDTTTKYKYVKDAYKLVGWYYVNPD
GSIRPYNFSGAVTQDINLRAIWRKAGDYHIIYSNDAVGTDGKPALDASGQ
QLQTSNEPTDPDSYDDGSHSALLRRPTMPDGYRFRGWWYNGKIYNPYDSI
DIDAHLADANKNITIKPVIIPVGDIKLEDTSIKYNGNGGTRVENGNVVTQ
VETPRMELNSTTTIPENQYFTRTGYNLIGWHHDKDLADTGRVEFTAGQSI
GIDNNPDATNTLYAVWQPKEYTVRVSKTVVGLDEDKTKDFLFNPSETLQQ
ENFPLRDGQTKEEKVPYGTSISIDEQAYDEFKVSESITEKNLATGEADKT
YDATGLQSLTVSGDVDISFTNTRIKQKVRLQKVNVENDNNFLAGAVFDIY
ESDANGNKASHPMYSGLVTNDKGLLLVDANNYLSLPVGKYYLTETKAPPG
YLLPKNDISVLVISTGVTFEQNGNNATPIKENLVDGSTVYTFKITNSKGT
ELPSTGGIGTHIYILVGLALALPSGLILYYRKKI
[0979] 01524 contains an amino acid motif indicative of a cell wall
anchor: SEQ ID NO: 131 LPSTG (shown in italics in SEQ ID NO: 36
above). In some recombinant host cell systems, it may be preferable
to remove this motif to facilitate secretion of a recombinant 01524
protein from the host cell. Alternatively, it may be preferable to
use the cell wall anchor motif to anchor the recombinantly
expressed protein to the cell wall. The extracellular domain of the
expressed protein may be cleaved during purification or the
recombinant protein may be left attached to either inactivated host
cells or cell membranes in the final composition.
[0980] Three pilin motifs, containing conserved lysine (K) residues
have been identified in 01524. The pilin motif sequences are
underlined in SEQ ID NO: 36, below. Conserved lysine (K) residues
are marked in bold, at amino acid residues 128 and 138, amino acid
residues 671 and 682, and amino acid residues 809 and 820. The
pilin sequences, in particular the conserved lysine residues, are
thought to be important for the formation of oligomeric, pilus-like
structures of 01524. Preferred fragments of 01524 include at least
one conserved lysine residue. Preferably, fragments include at
least one pilin sequence. TABLE-US-00091 SEQ ID NO: 36
MLKKCQTFIIESLKKKKHPKEWKIIMWSLMILTTFLTTYFLILPAITVEE
TKTDDVGITLENKNSSQVTSSTSSSQSSVEQSKPQTPASSVTETSSSEEA
AYREEPLMFRGADYTVTVTLTKEAKIPKNADLKVTELKDNSATFKDYKKK
ALTEVAKQDSEIKNFKLYDITIESNGKEAEPQAPVKVEVNYDKPLEASDE
NLKVVHFKDDGQTEVLKSKDTAETKNTSSDVAFKTDSFSIYAIVQEDNTE
VPRLTYHFQNNDGTDYDPLTASGMQVHHQIIKDGESLGEVGIPTIKAGEH
FNGWYTYDPTTGKYGDPVKFGEPITVTETKEICVRPFMSKVATVTLYDDS
AGKSILERYQVPLDSSGNGTADLSSFKVSPPTSTLLFVGWSKTQNGAPLS
ESEIQALPVSSDISLYPVFKESYGVEFNTGDLSTGVTYIAPRRVLTGQPA
STIKPNDPTRPGYTFAGWYTAASGGAAFDFNQVLTKDTTLYAHWSPAQTT
YTINYWQQSATDNKNATDAQKTYEYAGQVTRSGLSLSNQTLTQQDINDKL
PTGFKVNNTRTETSVMIKDDGSSVVNVYYDRKLITIKFAKYGGYSLPEYY
YSYNWSSDADTYTGLYGTTLAANGYQWKTGAWGYLANVGNNQVGTYGMSY
LGEFILPNDTVDSDVIKLFPKGNIVQTYRFFKQGLDGTYSLADTGGGAGA
DEFTFTEKYLGFNVKYYQRLYPDNYLFDQYASQTSAGVKVPISDEYYDRY
GAYHKDYLNLVVWYERNSYKIKYLDPLDNTELPNFPVKDVLYEQNLSSYA
PDTTTVQPKPSRPGYVWDGKWYKDQAQTQVFDFNTTMPPHDVKVYAGWQK
VTYRVNIDPNGGRLSKTDDTYLDLHYGDRIPDYTDITRDYIQDPSGTYYY
KYDSRDKDPDSTKDAYYTTDTSLSNVDTTTKYKYVKDAYKLVGWYYVNPD
GSIRPYNFSGAVTQDINLRAIWRKAGDYHIIYSNDAVGTDGKPALDASGQ
QLQTSNEPTDPDSYDDGSHSALLRRPTMPDGYRFRGWWYNGKIYNPYDSI
DIDAHLADANKNITIKPVIIPVGDIKLEDTSIKYNGNGGTRVENGNVVTQ
VETPRMELNSTTTIPENQYFTRTGYNLIGWHHDKDLADTGRVEFTAGQSI
GIDNNPDATNTLYAVWQPKEYTVRVSKTVVGLDEDKTKDFLFNPSETLQQ
ENFPLRDGQTKEEKVPYGTSISIDEQAYDEFKVSESITEKNLATGEADKT
YDATGLQSLTVSGDVDISFTNTRIKQKVRLQKVNVENDNNFLAGAVFDIY
ESDANGNKASHPMYSGLVTNDKGLLLVDANNYLSLPVGKYYLTETKAPPG
YLLPKNDISVLVISTGVTFEQNGNNATPIKENLVDGSTVYTFKITNSKGT
ELPSTGGIGTHIYILVGLALALPSGLILYYRKKI
[0981] An E box containing a conserved glutamic residue has also
been identified in 01524. The E box motif is underlined in SEQ ID
NO: 36 below. The conserved glutamic acid (E), at amino acid
residue 1344, is marked in bold. The E box motif, in particular the
conserved glutamic acid residue, is thought to be important for the
formation of oligomeric pilus-like structures of 01524. Preferred
fragments of 01524 include the conserved glutamic acid residue.
Preferably, fragments include the E box motif. TABLE-US-00092 SEQ
ID NO: 36 MLKKCQTFIIESLKKKKHPKEWKIIMWSLMILTTFLTTYFLILPAITVEE
TKTDDVGITLENKNSSQVTSSTSSSQSSVEQSKPQTPASSVTETSSSEEA
AYREEPLMFRGADYTVTVTLTKEAKIPKNADLKVTELKDNSATFKDYKKK
ALTEVAKQDSEIKNFKLYDITIESNGKEAEPQAPVKVEVNYDKPLEASDE
NLKVVHFKDDGQTEVLKSKDTAETKNTSSDVAFKTDSFSTYAIVQEDNTE
VPRLTYHFQNNDGTDYDPLTASGMQVHHQIIKDGESLGEVGIPTIKAGEH
FNGWYTYDPTTGKYGDPVKEGEPITVTETKEICVRPFMSKVATVTLYDDS
AGKSILERYQVPLDSSGNGTADLSSFKVSPPTSTLLFVGWSKTQNGAPLS
ESEIQALPVSSDISLYPVFKESYGVEFNTGDLSTGVTYIAPRRVLTGQPA
STIKPNDPTRPGYTFAGWYTAASGGAAFDFNQVLTKDTTLYAHWSPAQTT
YTINYWQQSATDNKNATDAQKTYEYAGQVTRSGLSLSNQTLTQQDINDKL
PTGFKVNNTRTETSVMIKDDGSSVVNVYYDRKLITIKFAKYGGYSLPEYY
YSYNWSSDADTYTGLYGTTLAANGYQWKTGAWGYLANVGNNQVGTYGMSY
LGEFILPNDTVDSDVIKLFPKGNIVQTYRFTKQGLDGTYSLADTGGGAGA
DEFTFTEKYLGFNVKYYQRLYPDNYLFDQYASQTSAGVKVPISDEYYDRY
GAYHKDYLNLVVWYERNSYKIKYLDPLDNTELPNFPVKDVLYEQNLSSYA
PDTTTVQPKPSRPGYVWDGKWYKDQAQTQVFDFNTTMPPHDVKVYAGWQK
VTYRVNIDPNGGRLSKTDDTYLDLHYGDRIPDYTDITRDYIQDPSGTYYY
KYDSRDKDPDSTKDAYYTTDTSLSNVDTTTKYKYVKDAYKLVGWYYVNPD
GSIRPYNFSGAVTQDINLRAIWRKAGDYHIIYSNDAVGTDGKPALDASGQ
QLQTSNEPTDPDSYDDGSHSALLRRPTMPDGYRFRGWWYNGKIYNPYDSI
DIDAHLADANKNITIKPVIIPVGDIKLEDTSIKYNGNGGTRVENGNVVTQ
VETPRMELNSTTTIPENQYFTRTGYNLIGWHHDKDLADTGRVEFTAGQSI
GIDNNPDATNTLYAVWQPKEYTVRVSKTVVGLDEDKTKDFLFNPSETLQQ
ENFPLRDGQTKEEKVPYGTSISIDEQAYDEFKVSESITEKNLATGEADKT
YDATGLQSLTVSGDVDISFTNTRIKQKVRLQKVNVENDNNFLAGAVFDIY
ESDANGNKASHPMYSGLVTNDKGLLLVDANNYLSLPVGKYYLTETKAPPG
YLLPKNDISVLVISTGVTFEQNGNNATPIKENLVDGSTVYTFKITNSKGT
ELPSTGGIGTHIYILVGLALALPSGLILYYRKKI
01525
[0982] An example of an amino acid sequence for 01525 is set forth
below. SEQ ID NO: 37 represents a 01525 sequence from GBS serotype
III, strain isolate COH1. TABLE-US-00093 SEQ ID NO: 37
MKRQISSDKLSQELDRVTYQKRFWSVIKNTIYILMAVASIAILIAVLWLP
VLRIYGHSMNKTLSAGDVVFTVKGSNFKTGDVVAFYYNNKVLVKRVTAES
GDWVNIDSQGDVYVNQHKLKEPYVIHKALGNSNIKYPYQVPDKKIFVLGD
NRKTSIDSRSTSVGDVSEEQIVGKTSFRIWPLGKISSIN
GBS 322
[0983] GBS 322 refers to a surface immunogenic protein, also
referred to as "sip". Nucleotide and amino acid sequences of GBS
322 sequenced from serotype V isolated strain 2603 V/R are set
forth in Ref. 3 as SEQ ID 8539 and SEQ OD 8540. These sequences are
set forth below as SEQ ID NOS 38 and 39: TABLE-US-00094 SEQ ID
NO.38 ATGAATAAAAAGGTACTATTGACATCGACAATGGCAGCTTCGCTATTATC
AGTCGCAAGTGTTCAAGCACAAGAAACAGATACGACGTGGACAGCACGTA
CTGTTTCAGAGGTAAAGGCTGATTTGGTAAAGCAAGACAATAAATCATCA
TATACTGTGAAATATGGTGATACACTAAGCGTTATTTCAGAAGCAATGTC
AATTGATATGAATGTCTTAGCAAAAATAAATAACATTGCAGATATCAATC
TTATTTATCCTGAGACAACACTGACAGTAACTTACGATCAGAAGAGTCAT
ACTGCCACTTCAATGAAAATAGAAACACCAGCAACAAATGCTGCTGGTCA
AACAACAGCTACTGTGGATTTGAAAACCAATCAAGTTTCTGTTGCAGACC
AAAAAGTTTCTCTCAATACAATTTCGGAAGGTATGACACCAGAAGCAGCA
ACAACGATTGTTTGGCCAATGAAGACATATTCTTCTGCGCCAGCTTTGAA
ATCAAAAGAAGTATTAGCACAAGAGCAAGCTGTTAGTCAAGCAGCAGCTA
ATGAACAGGTATCACGAGCTCCTGTGAAGTCGATTACTTCAGAAGTTCCA
GCAGCTAAAGAGGAAGTTAAACCAACTCAGACGTCAGTCAGTCAGTCAAC
AACAGTATCACCAGCTTCTGTTGCCGCTGAAACACCAGCTCCAGTAGCTA
AAGTAGCACCGGTAAGAACTGTAGCAGCCCCTAGAGTGGCAAGTGTTAAA
GTAGTCACTCCTAAAGTAGAAACTGGTGCATCACCAGAGCATGTATCAGC
TCCAGCAGTTCCTGTGACTACGACTTCACCAGCTACAGACAGTAAGTTAC
AAGCGACTGAAGTTAAGAGCGTTCCGGTAGCACAAAAAGCTCCAACAGCA
ACACCGGTAGCACAACCAGCTTCAACAACAAATGCAGTAGCTGCACATCC
TGAAAATGCAGGGCTCCAACCTCATGTTGCAGCTTATAAAGAAAAAGTAG
CGTCAACTTATGGAGTTAATGAATTCAGTACATACCGTGCGGGAGATCCA
GGTGATCATGGTAAAGGTTTAGCAGTTGACTTTATTGTAGGTACTAATCA
AGCACTTGGTAATAAAGTTGCACAGTACTCTACACAAAATATGGCAGCAA
ATAACATTTCATATGTTATCTGGCAACAAAAGTTTTACTCAAATACAAAC
AGTATTTATGGACCTGCTAATACTTGGAATGCAATGCCAGATCGTGGTGG
CGTTACTGCCAACCACTATGACCACGTTCACGTATCATTTAACAAATAAT
ATAAAAAAGGAAGCTATTTGGCTTCTTTTTTATATGCCTTGAATAGACTT
TCAAGGTTCTTATATAATTTTTATTA SEQ ID NO.39
MNKKVLLTSTMAASLLSVASVQAQETDTTWTARTVSEVKADLVKQDNKSS
YTVKYGDTLSVISEAMSIDMNVLAKINNIADINLIYPETTLTVTYDQKSH
TATSMKIETPATNAAGQTTATVDLKTNQVSVADQKVSLNTISEGMTPEAA
TTIVSPMKTYSSAPALKSKEVLAQEQAVSQAAANEQVSPAPVKSITSEVP
AAKEEVKPTQTSVSQSTTVSPASVAAETPAPVAKVAPVRTVAAPRVASVK
VVTPKVETGASPEHVSAPAVPVTTTSPATDSKLQATEVKSVPVAQKAPTA
TPVAQPASTTNAVAAHPENAGLQPHVAAYKEKVASTYGVNEFSTYRAGDP
GDHGKGLAVDFIVGTNQALGNKVAQYSTQNMAANNISYVIWQQKFYSNTN
SIYGPANTWNAMPDRGGVTANHYDHVHVSFNK
[0984] GBS 322 contains an N-terminal leader or signal sequence
region which is indicated by the underlined sequence near the
beginning of SEQ ID NO: 39. In one embodiment, one or more amino
acids from the leader or signal sequence region of GBS 322 are
removed. An example of such a GBS 322 fragment is set forth below
as SEQ ID NO: 40. TABLE-US-00095 SEQ ID NO: 40
DLVKQDNKSSYTVKYGDTLSVISEAMSIDMNVLAKINNIADINLIYPETT
LTVTYDQKSHTATSMKIETPATNAAGQTTATVDLKTNQVSVADQKVSLNT
ISEGMTPEAATTIVSPMKTYSSAPALKSKEVLAQEQAVSQAAANEQVSPA
PVKSITSENPAAKEEVKPTQTSVSQSTTVSPASVAAETPAPVAKVAPVRT
VAAPRVASVKVVTPKVETGASPEHVSAPAVPVTTTSPATDSKLQATEVKS
VPVAQKAPTATPVAQPASTTNAVAAHPENAGLQPHVAAYKEKVASTYGVN
EFSTYRAGDPGDHGKGLAVDFIVGTNQALGNKVAQYSTQNMAANNISYVI
WQQKFYSNTNSIYGPANTWNAMPDRGGVTANHYDHVHVSFNK
[0985] Additional preferred fragments of GBS 322 comprise the
immunogenic epitopes identified in WO 03/068813, each of which are
specifically incorporated by reference herein.
[0986] There may be an upper limit to the number of GBS proteins
which will be in the compositions of the invention. Preferably, the
number of GBS proteins in a composition of the invention is less
than 20, less than 19, less than 18, less than 17, less than 16,
less than 15, less than 14, less than 13, less than 12, less than
11, less than 10, less than 9, less than 8, less than 7, less than
6, less than 5, less than 4, or less than 3. Still more preferably,
the number of GBS proteins in a composition of the invention is
less than 6, less than 5, or less than 4. Still more preferably,
the number of GBS proteins in a composition of the invention is
3.
[0987] The GBS proteins and polynucleotides used in the invention
are preferably isolated, i.e., separate and discrete, from the
whole organism with which the molecule is found in nature or, when
the polynucleotide or polypeptide is not found in nature, is
sufficiently free of other biological macromolecules so that the
polynucleotide or polypeptide can be used for its intended
purpose.
Group A Streptococcus Adhesin Island Sequences
[0988] The GAS AI polypeptides of the invention can, of course, be
prepared by various means (e.g. recombinant expression,
purification from GAS, chemical synthesis etc.) and in various
forms (e.g. native, fusions, glycosylated, non-glycosylated etc.).
They are preferably prepared in substantially pure form (i.e.
substantially free from other streptococcal or host cell proteins)
or substantially isolated form.
[0989] The GAS AI proteins of the invention may include polypeptide
sequences having sequence identity to the identified GAS proteins.
The degree of sequence identity may vary depending on the amino
acid sequence (a) in question, but is preferably greater than 50%
(e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, 99.5% or more). Polypeptides having sequence
identity include homologs, orthologs, allelic variants and
functional mutants of the identified GBS proteins. Typically, 50%
identity or more between two proteins is considered to be an
indication of functional equivalence. Identity between proteins is
preferably determined by the Smith-Waterman homology search
algorithm as implemented in the MPSRCH program (Oxford Molecular),
using an affinity gap search with parameters gap open penalty=12
and gap extension penalty=1.
[0990] The GAS adhesin island polynucleotide sequences may include
polynucleotide sequences having sequence identity to the identified
GAS adhesin island polynucleotide sequences. The degree of sequence
identity may vary depending on the polynucleotide sequence in
question, but is preferably greater than 50% (e.g. 60%, 65%, 70%,
75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
99.5% or more).
[0991] The GAS adhesin island polynucleotide sequences of the
invention may include polynucleotide fragments of the identified
adhesin island sequences. The length of the fragment may vary
depending on the polynucleotide sequence of the specific adhesin
island sequence, but the fragment is preferably at least 10
consecutive polynucleotides, (e.g. at least 10, 12, 14, 16, 18, 20,
25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200 or more).
[0992] The GAS adhesin island amino acid sequences of the invention
may include polypeptide fragments of the identified GAS proteins.
The length of the fragment may vary depending on the amino acid
sequence of the specific GAS antigen, but the fragment is
preferably at least 7 consecutive amino acids, (e.g. 8, 10, 12, 14,
16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200 or
more). Preferably the fragment comprises one or more epitopes from
the sequence. Other preferred fragments include (1) the N-terminal
signal peptides of each identified GAS protein, (2) the identified
GAS protein without their N-terminal signal peptides, and (3) each
identified GAS protein wherein up to 10 amino acid residues (e.g.
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) are deleted from
the N-terminus and/or the C-terminus e.g. the N-terminal amino acid
residue may be deleted. Other fragments omit one or more domains of
the protein (e.g. omission of a signal peptide, of a cytoplasmic
domain, of a transmembrane domain, or of an extracellular
domain).
[0993] GAS AI-1 Sequences
[0994] As discussed above, a GAS AI-1 sequence is present in an M6
strain isolate (MGAS10394). Examples of GAS AI-1 sequences from M6
strain isolate MGAS10394 are set forth below.
[0995] M6_Spy0156: Spy0156 is a rofA transcriptional regulator. An
example of an amino acid sequence for M6_Spy0156 is set forth in
SEQ ID NO: 41. TABLE-US-00096 SEQ ID NO: 41
MIEKYLESSIESKCQLVVLEFKTSYLPITEVAEKTGLTFLQLNHYCEELN
AFFPDSLSMTIQKRMISCQFTHPFKETYLYQLYASSNVLQLLAFLIKNGS
HSRPLTDFARSHFLSNSSAYRMREALIPLLRNFELKLSKNKIVGEEYRIR
YLIALLYSKFGIKVYDLTQQDKNTIHSFLSHSSTHLKTSPWLSESFSFYD
ILLALSWKRHQFSVTIPQTRIFQQLKKLFIYDSLKKSSRDIIETYCQLNF
SAGDLDYLYLIYITANNSFASLQWTPEHIRQCCQLFEENDTFRLLLKPII
TLLPNLKEQKPSLVKALMFESKSFLENLQHFIPETNLFVSPYYKGNQKLY
TSLKLIVEEWLAKLPGKRYLNHKHFHLFCHYVEQILRNIQPPLVVVFVAS
NFINAHLLTDSFPRYFSDKSIDFHSYIAR
[0996] M6_Spy0157: M6_Spy0157 is a fibronectin binding protein. It
contains a sortase substrate motif LPXTG (SEQ ID NO: 122), shown in
italics in the amino acid sequence SEQ ID NO: 42. TABLE-US-00097
SEQ ID NO: 42 MVSSYMFVRGEKMNNKIFLNKEASFLAHTKRKRRFAVTLVGVFFMLLACA
GAIGFGQVAYAADEKTVPSHSSPNPEFPWYGYDAYGKEYPGYNIWTRYHD
LRVNLNGSRSYQVYCFNIQSNYPSQKNSEIKNWFKKIEGNGKSFVDYAHT
TKLGKEELEQRLLSLLYNAYPNDANGYMKGLEHLNAITVTQYAVWHYSDN
SQYQFETLWESEAKEGKISRSQVTLMREALKKLIDPNLEATAVNKIPSGY
RLNIFESENEAYQNLLSAEYVPDDPPKPGETSEHNPKTPELDGTPIPEDP
KHPDDNLEPTLPPVMLDGEEVPEVPSESLEPALPPLMPELDGQEVPEKPS
IDLPIEVPRYEFNNKDQSPLAGESGETEYITEVYGNQQNPVDIDKKLPNE
TGFSGNMVETEDTKEPEVLMGGQSESVEFTKDTQTGMSGQTTPQVETEDT
KEPEVLMGGQSESVEFTKDTQTGMSGQTTPQIETEDTKEPEVLMGGQSES
VEFTKDTQTGMSGQTTPQIETEDTKEPEVLMGGQSESVEFTKDTQTGMSG
FSETATVVEDTRPKLVFHFDNNEPKVEENREKPTKNITPILPATGDIENV
LAFLGILILSVLSIFSLLKNKQSNKKV
[0997] M6_Spy0157 contains an amino acid motif indicative of a cell
wall anchor: SEQ ID NO: 180 LPATG (shown in italics in SEQ ID NO:
42, above). In some recombinant host cell systems, it may be
preferable to remove this motif to facilitate secretion of a
recombinant M6_Spy0157 protein from the host cell. Alternatively,
in other recombinant host cell systems, it may be preferable to use
the cell wall anchor motif to anchor the recombinantly expressed
protein to the cell wall. The extracellular domain of the expressed
protein may be cleaved during purification or the recombinant
protein may be left attached to either inactivated host cells or
cell membranes in the final composition.
[0998] A pilin motif, discussed above, containing a conserved
lysine (K) residue has also been identified in M6_Spy0157. The
pilin motif sequence is underlined in SEQ ID NO: 42, below.
Conserved lysine (K) residues are also marked in bold, at amino
acid residues 277, 287, and 301. The pilin sequence, in particular
the conserved lysine residues, are thought to be important for the
formation of oligomeric, pilus-like structures. Preferred fragments
of M6_Spy0157 include at least one conserved lysine residue.
Preferably, fragments include the pilin sequence. TABLE-US-00098
SEQ ID NO: 42 MVSSYMFVRGEKMNNKTFLNKEASFLAHTKRKRRFAVTLVGVFFMLLACA
GAIGFGQVAYAADEKTVPSHSSPNPEFPWYGYDAYGKEYPGYNIWTRYHD
LRVNLNGSRSYQVYCFNIQSNYPSQKNSFIKNWFKKIEGNGKSFVDYAHT
TKLGKEELEQRLLSLLYNAYPNDANGYMKGLEHLNAITVTQYAVWHYSDN
SQYQFETLWESEAKEGKISRSQVTLMREALKKLIDPNLEATAVNKIPSGY
RLNIFESENEAYQNLLSAEYVPDDPPKPGETSEHNPKTPELDGTPTPEDP
KHPDDNLEPTLPPVMLDGEEVPEVPSESLEPALPPLMPELDGQEVPEKPS
TDLPIEVPRYEFNNKDQSPLAGESGETEYITEVYGNQQNPVDIDKKLPNE
TGFSGNMVETEDTKEPEVLMGGQSESVEFTKDTQTGMSGQTTPQVETEDT
KEPEVLMGGQSESVEFTKDTQTGMSGQTTPQIETEDTKEPEVLMGGQSES
VEFTKDTQTGMSGQTTPQIETEDTKEPEVLMGGQSESVEFTKDTQTGMSG
ESETATVVEDTRPKLVFHFDNNEPKVEENREKPTKNITPILPATGDIENV
LAFLGILILSVLSIFSLLKNKQSNKKV
[0999] A repeated series of four E boxes containing a conserved
glutamic residue have been identified in M6_Spy0157. The E-box
motifs are underlined in SEQ ID NO: 42, below. The conserved
glutamic acid (E) residues, at amino acid residues 415, 452, 489,
and 526 are marked in bold. The E box motif, in particular the
conserved glutamic acid residue, is thought to be important for the
formation of oligomeric pilus-like structures of M6_Spy0157.
Preferred fragments of M6_Spy0157 include at least one conserved
glutamic acid residue. Preferably, fragments include at least one E
box motif. TABLE-US-00099 SEQ ID NO: 42
MVSSYMFVRGEKMNNKIFLNKEASFLAHTKRKRRFAVTLVGVFFMLLACA
GAIGFGQVAYAADEKTVPSHSSPNPEFPWYGYDAYGKEYPGYNIWTRYHD
LRVNLNGSRSYQVYCFNIQSNYPSQKNSFIKNWFKKIEGNGKSFVDYAHT
TKLGKEELEQRLLSLLYNAYPNDANGYMKGLEHLNAITVTQYAVWHYSDN
SQYQFETLWESEAKEGKISRSQVTLMREALKKLIDPNLEATAVNKIPSGY
RLNIFESENEAYQNLLSAEYVPDDPPKPGETSEHNPKTPELDCTPIPEDP
KHPDDNLEPTLPPVMLDGEEVPEVPSESLEPALPPLMPELDGQEVPEKPS
IDLPIEVPRYEFNNKDQSPLAGESGETEYITEVYGNQQNPVDIDKKLPNE
TGFSGNMVETEDTKEPEVLMGGQSESVEFTKDTQTGMSGQTTPQVETEDT
KEPEVLMGGQSESVEFTKDTQTGMSGQTTPQIETEDTKEPEVLMGGQSES
VEFTKDTQTGMSGQTTPQIETEDTKEPEVLMGGQSESVEFTKDTQTGMSG
FSETATVVEDTRPKLVFHFDNNEPKVEENREKPTKNITPILPATGDIENV
LAFLGILILSVLSIFSLLKNKQSNKKV
[1000] M6_Spy0158: M6_Spy0158 is a reverse transcriptase. An
example of Spy0158 is shown in the amino acid sequence SEQ ID NO
43.
SEQ ID NO: 43
MSLRHQNKKGIRKEGWKSRPQSRWSDHCQLVAQKSVLKQAISKTVLAERGLFSCLDDYLERHALKVN
[1001] M6_Spy0159: M6_Spy0159 is a collagen adhesion protein. It
contains a sortase substrate motif LPXSG, shown in italics in the
amino acid sequence SEQ ID NO: 44. TABLE-US-00100 SEQ ID NO: 44
MYSRLKRELVIVINRKKKYKLIRLMVTVGLIFSQLVLPIRRLGLQMISTQ
TKVIPQEIVTQTETQGTQVVATKQKLESENSSLKVALKRESGFEHNATID
ASLDTESQGDNSQRSVTQAIVTMALELRKQGLSIVDTKIVRIQSSTNQRN
DITTTLTFKNGLSLEGASTEANDPNVRVGIVNPNDTVQTITPTIKQDADG
KVKNLVFTGRLGKQVIIVSTTRLKEEQTISLDSYGELVIDGAVGLSQKDR
PPYSKPITVNILKPKLSSIESSLDSKDFETVKTIDNLYTWDDQFYLLDFI
SKQYEVLKTDYQSAKDSTPQTRKILFGEYTVEPLVMNKGHNNTINIYIRS
TRPLGLKPIGAAPALIQPRSFRSLTPRSTRMKRSAPVEKFEGELEHHKRI
DYLGDNQNNPDTTIDDKEDEHDTSDLYRLYLDMTGKKNPLDILVVVDKSG
SMQEGIGSVQRYRYYAQRWDDYYSQWVYHGTFDYSSYQGESFNRGQIHYR
YRGIVSVSDGIRRDDAVKNSLLGVNGLLQRFVNINPENKLSVIGFQGSAD
YHAGKWYPDQSPRGGFYQPNLNNSRDAELLKGWSTNSLLDPNTLTALHNN
GTNYHAALLKAKEILNEVKDDGRRKIMIFISDGVPTEYFGEDGYRSGNGS
SNDRNNVTRSQEGSKLAIDEFKARYPNLSIYSLGVSKDINSDTASSSPVV
LKYLSGEEHYYGITDTAELEKTLNKIVEDSKLSQLGISDSLSQYVDYYDK
QPDVLVTRKSKVNDETEILYQKDQVQEAGKDIIDKVVFTPKTTSQPKGKV
TLTFKSDYKVDDEYTYTLSFNVKASDEAYEKYKDNEGRYSEMGDSDTDYG
TNQTSSGKGGLPSNSDASVNYMADGREQKLPYKHPVIQVKTVPITFTKVD
ADNNQKKLAGVEFELRKEDKKIVWEKGTTGSNGQLNFKYLQKGKTYYLYE
TKAKLGYTLPENPWEVAVANNGDIKVKHPIEGELKSKDGSYMIKNYKIYQ
LPSSGGRGSQIFIIVGSMTATVALLFYRRQHRKKQY
[1002] M6_Spy0159 contains an amino acid motif indicative of a cell
wall anchor: SEQ ID NO: 181 LPSSG (shown in italics in SEQ ID NO:
44, above). In some recombinant host cell systems, it may be
preferable to remove this motif to facilitate secretion of a
recombinant M6_Spy0159 protein from the host cell. Alternatively,
in other recombinant host cell systems, it may be preferable to use
the cell wall anchor motif to anchor the recombinantly expressed
protein to the cell wall. The extracellular domain of the expressed
protein may be cleaved during purification or the recombinant
protein may be left attached to either inactivated host cells or
cell membranes in the final composition.
[1003] A pilin motif, discussed above, containing a conserved
lysine (K) residue has also been identified in M6_Spy0159. The
pilin motif sequence is underlined in SEQ ID NO: 44, below.
Conserved lysine (K) residues are also marked in bold, at amino
acid residues 265 and 276. The pilin sequence, in particular the
conserved lysine residues, are thought to be important for the
formation of oligomeric, pilus-like structures. Preferred fragments
of M6_Spy0159 include at least one conserved lysine residue.
Preferably, fragments include the pilin sequence. TABLE-US-00101
SEQ ID NO: 44 MYSRLKRELVIVINRKKKYKLIRLMVTVGLIFSQLVLPIRRLGLQMISTQ
TKVIPQEIVTQTETQGTQVVATKQKLESENSSLKVALKRESGFEHNATID
ASLDTESQGDNSQRSVTQAIVTMALELRKQGLSIVDTKIVRIQSSTNQRN
DITTTLTFKNGLSLEGASTEANDPNVRVGIVNPNDTVQTITPTIKQDADG
KVKNLVFTGRLGKQVIIVSTTRLKEEQTISLDSYGELVIDGAVGLSQKDR
PPYSKPITVNILKPKLSSIESSLDSKDFETVKTIDNLYTWDDQFYLLDFI
SKQYEVLKTDYQSAKDSTPQTRKILFGEYTVEPLVMNKGHNNTINIYIRS
TRPLGLKPIGAAPALIQPRSFRSLTPRSTRMKRSAPVEKFEGELEHHKRI
DYLGDNQNNPDTTIDDKEDEHDTSDLYRLYLDMTGKKNPLDILVVVDKSG
SMQEGIGSVQRYRYYAQRWDDYYSQWVYHGTFDYSSYQGESFNRGQIHYR
YRGIVSVSDGIRRDDAVKNSLLGVNGLLQRFVNINPENKLSVIGFQGSAD
YHAGKWYPDQSPRGGFYQPNLNNSRDAELLKGWSTNSLLDPNTLTALHNN
GTNYHAALLKAKEILNEVKDDGRRKIMIFISDGVPTEYFGEDGYRSGNGS
SNDRNNVTRSQEGSKLAIDEFKARYPNLSIYSLGVSKDINSDTASSSPVV
LKYLSGEEHYYGITDTAELEKTLNKIVEDSKLSQLGISDSLSQYVDYYDK
QPDVLVTRKSKVNDETEILYQKDQVQEAGKDIIDKVVFTPKTTSQPKGKV
TLTFKSDYKVDDEYTYTLSFNVKASDEAYEKYKDNEGRYSEMGDSDTDYG
TNQTSSGKGGLPSNSDASVNYMADGREQKLPYKHPVIQVKTVPITFTKVD
ADNNQKKLAGVEFELRKEDKKIVWEKGTTGSNGQLNFKYLQKGKTYYLYE
TKAKLGYTLPENPWEVAVANNGDIKVKHPIEGELKSKDGSYMIKNYKIYQ
LPSSGGRGSQIFIIVGSMTATVALLFYRRQHRKKQY
[1004] An E box containing a conserved glutamic residue has been
identified in M6_Spy0159. The E-box motif is underlined in SEQ ID
NO: 44, below. The conserved glutamic acid (E), at amino acid
residue 950, is marked in bold. The E box motif, in particular the
conserved glutamic acid residue, is thought to be important for the
formation of oligomeric pilus-like structures of M6_Spy0159.
Preferred fragments of M6_Spy0159 include the conserved glutamic
acid residue. Preferably, fragments include the E box motif.
TABLE-US-00102 SEQ ID NO: 44
MYSRLKRELVIVINRKKKYKLIRLMVTVGLIFSQLVLPIRRLGLQMISTQ
TKVIPQEIVTQTETQGTQVVATKQKLESENSSLKVALKRESGFEHNATID
ASLDTESQGDNSQRSVTQAIVTMALELRKQGLSIVDTKIVRIQSSTNQRN
DITTTLTFKNGLSLEGASTEANDPNVRVGIVNPNDTVQTITPTIKQDADG
KVKNLVFTGRLGKQVIIVSTTRLKEEQTISLDSYGELVIDGAVGLSQKDR
PPYSKPITVNILKPKLSSIESSLDSKDFETVKTIDNLYTWDDQFYLLDFI
SKQYEVLKTDYQSAKDSTPQTRKILFGEYTVEPLVMNKGHNNTINIYIRS
TRPLGLKPIGAAPALIQPRSFRSLTPRSTRMKRSAPVEKFEGELEHHKRI
DYLGDNQNNPDTTIDDKEDEHDTSDLYRLYLDMTGKKNPLDILVVVDKSG
SMQEGIGSVQRYRYYAQRWDDYYSQWVYHGTFDYSSYQGESFNRGQIHYR
YRGIVSVSDGIRRDDAVKNSLLGVNGLLQRFVNINPENKLSVIGFQGSAD
YHAGKWYPDQSPRGGFYQPNLNNSRDAELLKGWSTNSLLDPNTLTALHNN
GTNYHAALLKAKEILNEVKDDGRRKIMIFISDGVPTEYFGEDGYRSGNGS
SNDRNNVTRSQEGSKLAIDEFKARYPNLSIYSLGVSKDINSDTASSSPVV
LKYLSGEEHYYGITDTAELEKTLNKIVEDSKLSQLGISDSLSQYVDYYDK
QPDVLVTRKSKVNDETEILYQKDQVQEAGKDIIDKVVFTPKTTSQPKGKV
TLTFKSDYKVDDEYTYTLSFNVKASDEAYEKYKDNEGRYSEMGDSDTDYG
TNQTSSGKGGLPSNSDASVNYMADGREQKLPYKHPVIQVKTVPITFTKVD
ADNNQKKLAGVEFELRKEDKKIVWEKGTTGSNGQLNFKYLQKGKTYYLYE
TKAKLGYTLPENPWEVAVANNGDIKVKHPIEGELKSKDGSYMIKNYKIYQ
LPSSGGRGSQIFIIVGSMTATVALLFYRRQHRKKQY
[1005] M6_Spy0160: M6_Spy0160 is a fimbrial structural subunit. It
contains a sortase substrate motif LPXTG (SEQ ID NO: 122), shown in
italics in amino acid sequence SEQ ID NO: 45. TABLE-US-00103 SEQ ID
NO: 45 MTNRRETVREKILITAKKLMLACLATLAVVGLGMTRVSALSKDDTAQLKI
TNIEGGPTVTLYKIGEGVYNTNGDSFINFKYAEGVSLTETGPTSQEITTI
ANGINTGKIKPFSTENVSISNGTATYNARGASVYIALLTGATDGRTYNPI
LLAASYNGEGNLVTKNIDSKSNYLYGQTSVAKSSLPSITKKVTGTIDDVN
KKTTSLGSVLSYSLTFELPSYTKEAVNKTVYVSDNMSEGLTFNFNSLTVE
WKGKMANITEDGSVMVENTKIGIAKEVNNGFNLSFIYDSLESISPNISYK
AVVNNKAIVGEEGNPNKAEFFYSNNPTKGNTYDNLDKKPDKGNGITSKED
SKIVYTYQIAFRKVDSVSKTPLTGAIFGVYDTSNKLIDIVTTNKNGYAIS
TQVSSGKYKIKELKAPKGYSLNTETYEITANWVTATVKTSANSKSTTYTS
DKNKATDNSEQVGWLKNGIFYSIDSRPTGNDVKEAYIESTKALTDGTTFS
KSNEGSGTVLLETDIPNTKLGELPSTGSIGTYLEKAIGSAAMIGAIGIYI VKRRKA
[1006] M6_Spy0160 contains an amino acid motif indicative of a cell
wall anchor: SEQ ID NO: 131 LPSTG (shown in italics in SEQ ID NO:
45, above). In some recombinant host cell systems, it may be
preferable to remove this motif to facilitate secretion of a
recombinant M6_Spy0160 protein from the host cell. Alternatively,
in other recombinant host cell systems, it may be preferable to use
the cell wall anchor motif to anchor the recombinantly expressed
protein to the cell wall. The extracellular domain of the expressed
protein may be cleaved during purification or the recombinant
protein may be left attached to either inactivated host cells or
cell membranes in the final composition.
[1007] An E box containing a conserved glutamic residue has been
identified in M6_Spy0160. The E-box motif is underlined in SEQ ID
NO: 45, below. The conserved glutamic acid (E), at amino acid
residue 412, is marked in bold. The E box motif, in particular the
conserved glutamic acid residue, is thought to be important for the
formation of oligomeric pilus-like structures of M6_Spy0160.
Preferred fragments of M6_Spy0160 include the conserved glutamic
acid residue. Preferably, fragments include the E box motif.
TABLE-US-00104 SEQ ID NO: 45
MTNRRETVREKILITAKKLMLACLATLAVVGLGMTRVSALSKDDTAQLKI
TNIEGGPTVTLYKIGEGVYNTNGDSFINFKYAEGVSLTETGPTSQEITTI
ANGINTGKIKPFSTENVSISNGTATYNARGASVYIALLTGATDGRTYNPI
LLAASYNGEGNLVTKNIDSKSNYLYGQTSVAKSSLPSITKKVTGTIDDVN
KKTTSLGSVLSYSLTFELPSYTKEAVNKTVYVSDNMSEGLTFNFNSLTVE
WKGKMANITEDGSVMVENTKIGIAKEVNNGFNLSFIYDSLESISPNISYK
AVVNNKAIVGEEGNPNKAEFFYSNNPTKGNTYDNLDKKPDKGNGITSKED
SKIVYTYQIAFRKVDSVSKTPLTGAIFGVYDTSNKLIDIVTTNKNGYAIS
TQVSSGKYKIKELKAPKGYSLNTETYEITANWVTATVKTSANSKSTTYTS
DKNKATDNSEQVGWLKNGIFYSIDSRPTGNDVKEAYIESTKALTDGTTFS
KSNEGSGTVLLETDIPNTKLGELPSTGSIGTYLFKAIGSAAMIGAIGIYI VKRRKA
[1008] M6_Spy0161 is a srtB type sortase. An example of an amino
acid sequence of M6_Spy-161 is shown in SEQ ID NO: 46.
TABLE-US-00105 SEQ ID NO: 46
MTERLKNLGILLLFLLGTAIFLYPTLSSQWNAYRDRQLLSTYHKQVIQKK
PSEMEEVWQKAKAYNARLGIQPVPDAFSFRDGIHDKNYESLLQIENNDIM
GYVEVPSIKVTLPIYHYTTDEVLTKGAGHLFGSALPVGGDGTHTVISAHR
GLPSAEMFTNLNLVKKGDTFYFRVLNKVLAYKVDQILIVEPDQATSLSGV
MGKDYATLVTCTPYGVNTKRLLVRGHRIAYHYKKYQQAKKAMKLVDKSRM
WAEVVCAAFGVVIAIILVFMYSRVSAKKSK
[1009] As discussed above, applicants have also determined the
nucleotide and encoded amino acid sequence of fimbrial structural
subunits in several other GAS AI-1 strains of bacteria. Examples of
sequences of these fimbrial structural subunits are set forth
below.
[1010] M6 strain isolate CDC SS 410 is a GAS AI-1 strain of
bacteria. CDC SS 410_fimbrial is thought to be a fimbrial
structural subunit of M6 strain isolate CDC SS 410. An example of a
nucleotide sequence encoding the CDC SS 410_fimbrial protein (SEQ
ID NO: 267) and a CDC SS 410_fimbrial protein amino acid sequence
(SEQ ID NO: 268) are set forth below. TABLE-US-00106 SEQ ID NO: 267
aaagatgatactgcacaactaaagataacaaatattgaaggtgggccaac
agtaacactttataaaataggagaaggtgtttacaacactaatggtgatt
cttttattaactttaaatatgctgagggggtttctttaactgaaacagga
cctacatcacaagaaattactactattgcaaatggtattaatacgggtaa
aataaagccttttagtactgaaaacgttagtatttctaatggaacagcaa
cttataatgcgagaggtgcatctgtttatattgcattattaacaggtgcg
acagatggccgtacctacaatcctattttattagctgcatcttataatgg
tgagggaaatttagttactaaaaatattgattccaaatctaattatttat
atggacaaacaagtgttgcaaaatcatcattaccatctattacaaagaaa
gtaaccgggacaatagatgacgtgaataaaaagactacctcgttaggaag
tgtattgtcttattcgctgacatttgaattaccaagttataccaaagaag
cagtcaataaaacagtatatgtttctgataatatgtcggaaggtcttact
tttaactttaatagtcttacagtagaatggaaaggtaagatggctaatat
tactgaagatggttcagtaatggtagaaaatacaaaaatcggaatagcta
aggaggttaataacggttttaatttaagttttatttatgatagtttagaa
tctatatcaccaaatataagttataaagctgttgtaaacaataaagctat
tgttggtgaagagggtaatcctaataaagctgaattcttctattcaaata
atccaacaaaaggtaatacatacgataatttagataagaagcctgataaa
gggaatggtattacatccaaagaagattctaaaattgtttatacttatca
aatagcgtttagaaaagttgatagtgttagtaagaccccacttattggtg
caatttttggagtttatgatactagtaataaattaattgatattgttaca
accaataaaaatggatatgctatttcaacacaagtatcttcaggaaaata
taaaattaaggaattaaaagctcctaaaggttattcattgaatacagaaa
cttatgaaattacggcaaattgggtaactgctacagtcaagacaagtgct
aattcaaaaagtactacttatacatctgataaaaataaggcgacagataa
ttcagagcaagtaggatggttaaaaaatggtatattctattctatagata
gtagacctacaggaaatgatgttaaagaggcttatattgaatctactaag
gctttaactgatggaacaactttctcaaaatcgaatgaaggttcaggtac
agtattattagaaactgacatccctaacaccaagctaggtgaactc SEQ ID NO: 268
KDDTAQLKTTNIEGGPTVTLYKIGEGVYNTNGDSFINFKYAEGVSLTETG
PTSQEITTIANGINTGKIKPFSTENVSISNGTATYNARGASVYIALLTGA
TDGRTYNPILLAASYNGEGNLVTKNIDSKSNYLYGQTSVAKSSLPSITKL
VTGTIDDVNKKTTSLGSVLSYSLTFELPSYTKEAVNKTVYVSDNMSEGLT
FNFNSLTVEWKKGMANITEDGSVMVENTKIGIAKEVNNGFNLSFIYDSLE
SISPNISYKAVVNNKAIVGEEGNPNKAEFFYSNNPTKGNTYDNLDKKPDK
GNGITSKEDSKIVYTYQIAFRKVDSVSKTPLIGAIFGVYDTSNKLIDIVT
TNKNGYAISTQVSSGKYKIKELKAPKGYSLNTETYEITANWVTATVKTSA
NSKSTTYTSDKNKATDNSEQVGWLKNGIFYSIDSRPTGNDVKEAYTESTK
ALTDGTTFSKSNEGSGTVLLETDIPNTKLGEL
[1011] M6 strain isolate ISS 3650 is a GAS AI-1 strain of bacteria.
ISS3650_fimbrial is thought to be a fimbrial structural subunit of
M6 strain isolate ISS 3650. An example of a nucleotide sequence
encoding the ISS3650_fimbrial protein (SEQ ID NO: 269) and an
ISS3650_fimbrial protein amino acid sequence (SEQ ID NO: 270) are
set forth below. TABLE-US-00107 SEQ ID NO: 269
gaatggaaaggtaagatggctaatattactgaagatggttcagtaatggt
agaaaatacaaaaatcggaatagctaaggaggttaataacggttttaatt
taagttttatttatgatagtttagaatctatatcaccaaatataagttat
aaagctgttgtaaacaataaagctattgttggtgaagagggtaatcctaa
taaagctgaattcttctattcaaataatccaacaaaaggtaatacatacg
ataatttagataagaagcctgataaagggaatggtattacatccaaagaa
gattctaaaattgtttatacttatcaaatagcgtttagaaaagttgatag
tgttagtaagaccccacttattggtgcaatttttggagtttatgatacta
gtaataaattaattgatattgttacaaccaataaaaatggatatgctatt
tcaacacaagtatcttcaggaaaatataaaattaaggaattaaaagctcc
taaaggttattcattgaatacagaaacttatgaaattacggcaaattggg
taactgctacagtcaagacaagtgctaattcaaaaagtactacttataca
tctgataaaaataaggcgacagataattcagagcaagtaggatggttaaa
aaatggtatattctattctatagatagtagacctacaggaaatgatgtta
aagaggcttatattgaatctactaaggctttaactgatggaacaactttc
tcaaaatcgaatgaaggttcaggtacagtattattagaaactgacatcc SEQ ID NO: 270
EWKGKMANITEDGSVMVENTKIGIAKEVNNGFNLSFIYDSLESISPNISY
KAVVNNKAIVGEEGNPNKAEFFYSNNPTKGNTYDNLDKKPDKGNGITSKE
DSDIVYTYQIAFRKVDSVSKTPLIGAIFGVYDTSNKLIDIVTTNKNGYAI
STQVSSGKYKIKELKAPKGYSLNTETYEITANWVTATVKTSANSKSTTYT
SDKNKATDNSEQVGWLKNGIFYSIDSRPTGNDVKEAYIESTKALTDGTTF
SKSNEGSGTVLLETDI
[1012] M23 strain isolate DSM2071 is a GAS AI-1 strain of bacteria.
DSM2071_fimbrial is thought to be a fimbrial structural subunit of
M23 strain DSM2071. An example of a nucleotide sequence encoding
the DSM2071_fimbrial protein (SEQ ID NO: 251) and a
DSM2071_fimbrial protein amino acid sequence (SEQ ID NO: 252) are
set forth below. TABLE-US-00108 SEQ ID NO: 251
atgagagagaaaatattaatagcagcaaaaaaactaatgctagcttgttt
agctatcttagctgtagtagggcttggaatgacaagagtatcagctttat
caaaagatgataaggcggagttgaagataacaaatatcgaaggtaaaccg
accgtgacactgtataaaattggtgatggaaaatacagtgagcgagggga
ttcttttattggatttgagttaaagcaaggtgtggagctaaataaggcaa
aacctacatctcaagaaataaataaaatcgctaatggtattaataaaggt
agtgttaaggctgaagtagttaatataaaagaacatgctagtacaactta
tagttatacaacaactggtgcaggtatttacttggctatattgactggag
ctactgatggacgtgcctataatcctatcttactgacagcttcttacaat
gaggaaaatccacttaagggagggcagattgacgcaactagtcattatct
ttttggagaagaagcagttgctaaatctagccaaccaacaattagcaagt
caattacaaaatccacaaaagatggtgataaagatacagcatctgtaggt
gaaaaagttgattacaaattaactgttcagttaccaagttattcgaaaga
tgctatcaataaaacggtgtttatcactgacaaattgtctcagggactta
ctttccttccaaaaagtttaaagattatctggaatggtcaaacgttaaca
aaggtgaatgaagaatttaaagctggagataaggtaattgctcaacttaa
ggttgaaaataatggatttaatctgaactttaattatgataaccttgata
atcatgccccagaagttaactatagtgctctactaaatgaaaacgcagtt
gttggtaaaggtggtaatgacaataatgtagactattactattcaaataa
tccgaataaaggagagacccataaaacaactgagaagcctaaagagggtg
aaggtactggtatcactaaaaagacggataaaaaaaccgtctacacctat
cgtgtagcctttaagaaaacaggcaaagatcatgccccactagctggtgc
tgttttcggtatctattcagataaggaagcgaaacaattagtcgatattg
ttgtgacaaatgcacagggttatgcagcatcaagcgaagttgggaaaggg
acttattacattaaagaaattaaatcccctaagggttactctttaaatac
aaatatttatgaagtggaaacttcatgggaaaaagctacaacgacttcta
caactaatcgtttagagacaatttatacaacagatgataatcaaaagtct
ccaggaactaatacagttggttggttggaagatggtgtcttttacaaaga
aaatccaggtggtgatgctaaacttgcctatatcaaacaatcaacagagg
agacttctacaactatagaagtcaaagaaaatcaagctgaaggttcaggt
acggtattattagaaactgaaattcctaacaccaaattaggtgaattacc
ttcgacaggtagcattggtacttacctctttaaagctattggttcggctg
ctatgatcggtgcaattggtatttatattgttaaacgtcgtaaagcttaa SEQ ID NO: 252
MREKILIAAKKLMLACLATLAVVGLGMTRVSALSKDDKAELKITNIEGKP
TVTLYKIGDGKYSERGDSFIGFELKQGVELNKAKPTSQEINKIANGINKG
SVKAEVVNIKEHASTTYSYTTTGAGIYLAILTGATDGRAYNPILLTASYN
EENPLKGGQIDATSHYLFGEEAVAKSSQPTISKSITKSTKDGDKDTASVG
EKVDYKLTVQLPSYSKDAINKTVFITDKLSQGLTFLPKSLKIIWNGQTLT
KVNEEFKAGDKVIAQLKVENNGFNLNFNYDNLDNHAPEVNYSALLNENAV
VGKGGNDNNVDYYYSNNPNKGETHKTTEKPKEGEGTGITKKTDKKTVYTY
RVAFKKTGKDHAPLAGAVFGIYSDKEAKQLVDIVVTNAQGYAASSEVGKG
TYYIKEIKSPKGYSLNTNIYEVETSWEKATTTSTTNRLETIYTTDDNQKS
PGTNTVGWLEDGVFYKENPGGDAKLAYIKQSTEETSTTIEVKENQAEGSG
TVLLETEIPNTKLGELPSTGSIGTYLFKAIGSAAMIGAIGIYIVKRRKA
[1013] GAS AI-2 sequences
[1014] As discussed above, a GAS AI-2 sequence is present in an M1
strain isolate (SF370). Examples of GAS AI-2 sequences from M1
strain isolate SF370 are set forth below.
[1015] Spy0124 is a rofA transcriptional regulator. An example of
an amino acid sequence for
[1016] Spy0124 is set forth in SEQ ID NO:47. TABLE-US-00109 SEQ ID
NO: 47 MIEKYLESSIESKGQLIVLFFKTSYLPITEVAEKTGLTFLQLNHYCEELN
AFFPGSLSMTIQKRMISCQFTHPFKETYLYQLYASSNVLQLLAFLIKNGS
HSRPLTDPARSHFLSNSSAYRMREALIPLLRNFELKLSKNKIVGEEYRIR
YLIALLYSKFGIKVYDLTQQDKNTIHSFLSHSSTHLKTSPWLSESESFYD
ILLALSWKRHQFSVTIPQTRIFQQLKKLFVYDSLKKSSHDIIETYGQLNF
SAGDLDYLYLIYITANNSFASLQWTPEHIRQYCQLFEENDTFRLLLNPII
TLLPNLKEQKASLVKALMFFSKSFLFNLQHFIPETNLFVSPYYKGNQKLY
TSLKLIVEEWMAKLPGKRDLNHKHFHLFCHYVEQSLRNIQPPLVVVFVAS
NFINAHLLTDSFPRYFSDKSIDFHSYYLLQDNVYQIPDLKPDLVITHSQL
IPFVHHELTKGIAVAEISFDESILSIQELMYQVKEEKEQADLTKQLT
[1017] GAS 015 is also referred to as Cpa. It contains a sortase
substrate motif VVXTG (SEQ ID NO: 135), shown in italics in SEQ ID
NO: 48. TABLE-US-00110 SEQ ID NO: 48
LRGEKMKKTRFPNKLNTLNTQRVLSKNSKRFTVTLVGVFLMIEALVTSMV
GAKTVFGLVESSTPNAINPDSSSEYRWYGYESYVRGHPYYKQFRVAHDLR
VNLEGSRSYQVYCFNLKKAFPLGSDSSVKKWYKKHDGISTKFEDYAMSPR
ITGDELNQKLRAVMYNGHPQNANGIMEGLEPLNAIRVTQEAVWYYSDNAP
ISNPDESFKRESESNLVSTSQLSLMRQALKQLIDPNLATKMPKQVPDDFQ
LSIFESEDKGDKYNKGYQNLLSGGLVPTKPPTPGDPPMPPNQPQTTSVLI
RKYAIGDYSKLLEGATLQLTGDNVNSFQARVFSSNDIGERIELSDGTYTL
TELNSPAGYSIAEPITFKVEAGKVYTIIDGKQIENPNKEIVEPYSVEAYN
DFEEFSVLTTQNYAKFYYAKNKNGSSQVVYCFNADLKSPPDSEDGGKTMT
PDFTTGEVKYTHIAGRDLFKYTVKPRDTDPDTFLKHIKKVIEKGYREKGQ
AIEYSGLTETQLRAATQLATYYFTDSAELDKDKLKDYHGFGDMNDSTLAV
AKILVEYAQDSNPPQLTDLDFFIPNNNKYQSLIGTQWHPEDLVDTIRMED
KKEVIPVTHNLTLRKTVTGLAGDRTKDFHFEIELKNNKQELLSQTVKTDK
TNLEFKDGKATINLKHGESLTLQGLPEGYSYLVKETDSEGYKVKVNSQEV
ANATVSKTGITSDETLAFENNKEPVVPTGVDQKINGYLALIVIAGISLGI WGIHTIRIRKHD
[1018] GAS 015 contains an amino acid motif indicative of a cell
wall anchor: SEQ ID NO: 182 VVPTG (shown in italics in SEQ ID NO:
48, above). In some recombinant host cell systems, it may be
preferable to remove this motif to facilitate secretion of a
recombinant GAS 015 protein from the host cell. Alternatively, in
other recombinant host cell systems, it may be preferable to use
the cell wall anchor motif to anchor the recombinantly expressed
protein to the cell wall. The extracellular domain of the expressed
protein may be cleaved during purification or the recombinant
protein may be left attached to either inactivated host cells or
cell membranes in the final composition.
[1019] A pilin motif, discussed above, containing a conserved
lysine (K) residue has also been identified in GAS 015. The pilin
motif sequence is underlined in SEQ ID NO: 48, below. Conserved
lysine (K) residues are also marked in bold, at amino acid residue
243. The pilin sequence, in particular the conserved lysine
residues, are thought to be important for the formation of
oligomeric, pilus-like structures. Preferred fragments of GAS 015
include the conserved lysine residue. Preferably, fragments include
the pilin sequence. TABLE-US-00111 SEQ ID NO: 48
LRGEKMKKTRFPNKLNTLNTQRVLSKNSKRFTVTLVGVFLMIFALVTSMV
GAKTVFGLVESSTPNAINPDSSSEYRWYGYESYVRGHPYYKQFRVAHDLR
VNLEGSRSYQVYCFNLKKAFPLGSDSSVKKWYKKHDGISTKFEDYANSPR
ITGDELNQKLRAVMYNGHPQNANGIMEGLEPLNAIRVTQEAVWYYSDNAP
ISNPDESFKRESESNLVSTSQLSLMRQALKQLIDPNLATKMPKQVPDDFQ
LSIFESEDKGDKYNKGYQNLLSGGLVPTKPPTPGDPPMPPNQPQTTSVLI
RKYAIGDYSKLLEGATLQLTGDNVNSFQARVFSSNDIGERIELSDGTYTL
TELNSPAGYSIAEPITFKVEAGKVYTIIDGKQIENPNKETVEPYSVEAYN
DFEEFSVLTTQNYAKFYYAKNKNGSSQVVYCFNADLKSPPDSEDGGKTMT
PDFTTGEVKYTHIAGRDLFKYTVKPRDTDPDTFLKHIKKVIEKGYREKGQ
AIEYSGLTETQLRAATQLAIYYFTDSAELDKDKLKDYHGFGDMNDSTLAV
AKILVEYAQDSNPPQLTDLDFFIPNNNKYQSLIGTQWHPEDLVDTIRMED
KKEVIPVTHNLTLRKTVTGLAGDRTKDFHPEIELKNNKQELLSQTVKTDK
TNLEFKDGKATINLKHGESLTLQGLPEGYSYLVKETDSEGYKVKVNSQEV
ANATVSKTGTTSDETLAFENNKEPVVPTGVDQKINGYLALIVIAGISLGI WGIHTIRIRKHD
[1020] An E box containing a conserved glutamic residue has been
identified in GAS 015. The E-box motif is underlined in SEQ ID NO:
48, below. The conserved glutamic acid (E), at amino acid residue
352, is marked in bold. The E box motif, in particular the
conserved glutamic acid residue, is thought to be important for the
formation of oligomeric pilus-like structures of GAS 015. Preferred
fragments of GAS 015 include the conserved glutamic acid residue.
Preferably, fragments include the E box motif. TABLE-US-00112 SEQ
ID NO: 48 LRGEKMKKTRFPNKLNTLNTQRVLSKNSKRFTVTLVGVFLMIFALVTSMV
GAKTVFGLVESSTPNAINPDSSSEYRWYGYESYVRGHPYYDQFRVAHDLR
VNLEGSRSYQVYCFNLKKAFPLGSDSSVKKWYKKHDGISTKFEDYAMSPR
ITGDELNQKLRAVMYNGHPQNANGIMEGLEPLNAIRVTQEAVWYYSDNAP
ISNPDESFKRESESNLVSTSQLSLMRQALKQLIDPNLATKMPKQVPDDFQ
LSIFESEDKGDKYNKGYQNLLSGGLVPTKPPTPGDPPMPPNQPQTTSVLI
RKYAIGDYSKLLEGATLQLTGDNVNSFQARVFSSNDIGERIELSDGTYTL
TELNSPAGYSIAEPITFKVEAGKVYTIIDGKQIENPNKEIVEPYSVEAYN
DFEEFSVLTTQNYAKFYYAKNKNGSSQVVYCPNADLKSPPDSEDGGKTMT
PDFTTGEVKYTHIAGRDLFKYTVKPRDTDPDTFLKHIKKVIEKGYREKGQ
AIEYSGLTETQLRAATQLAIYYFTDSAELDKDKLKDYHGFGDMNDSTLAV
AKILVEYAQDSNPPQLTDLDFFIPNNNKYQSLIGTQWHPEDLVDIIRMED
KKEVIPVTHNLTLRKTVTGLAGDRTKDFHFEIELKNNKQELLSQTVKTDK
TNLEFKDGKATINLKHGESLTLQGLPEGYSYLVKETDSEGYKVKVNSQEV
ANATVSKTGITSDETLAFENNKEPVVPTGVDQKINGYLALIVIAGISLGI WGIHTIRIRKHD
[1021] Spy0127 is a LepA putative signal peptidase. An example of
an amino acid sequence for Spy0127 is set forth in SEQ ID NO: 49.
TABLE-US-00113 SEQ ID NO: 49
MIIKRNDMAPSVKAGDAILFYRLSQTYKVEEAVVYEDSKTSITKVGRIIA
QAGDEVDLTEQGELKINGHIQNEGLTFIKSREANYPYRIADNSYLTLNDY
YSQESENYLQDAIAKDAIKGTINTLIRLRNH
[1022] Spy0128 is thought to be a fibrial protein. It contains a
sortase substrate motif EVXTG (SEQ ID NO: 136) shown in italics in
SEQ ID NO: 50. TABLE-US-00114 SEQ ID NO: 50
MKLRHLLLTGAALTSFAATTVHGETVVNGAKLTVTKNLDLVNSNALIPNT
DFTFKIEPDTTVNEDGNKFKGVALNTPMTKVTYTNSDKGGSNTKTAEFDF
SEVTFEKPGVYYYKVTEEKIDKVPGVSYDTTSYTVQVHVLWNEEQQKPVA
TYIVGYKEGSKVPIQFKNSLDSTTLTVKKKVSGTGGDRSKDFNFGLTLKA
NQYYKASEKVMIEKTTKGGQAPVQTEASIDQLYHFTLKDGESIKVTNLPV
GVDYVVTEDDYKSEKYTTNVEVSPQDGAVKNIAGNSTEQETSTDKDMTIT
FTNKKDFEVPTGVAMTVAPYIALGIVAVGGALYFVKKKNA
[1023] Spy0128 contains an amino acid motif indicative of a cell
wall anchor: SEQ ID NO: 183 EVPTG (shown in italics in SEQ ID NO:
50, above). In some recombinant host cell systems, it may be
preferable to remove this motif to facilitate secretion of a
recombinant Spy0128 protein from the host cell. Alternatively, in
other recombinant host cell systems, it may be preferable to use
the cell wall anchor motif to anchor the recombinantly expressed
protein to the cell wall. The extracellular domain of the expressed
protein may be cleaved during purification or the recombinant
protein may be left attached to either inactivated host cells or
cell membranes in the final composition.
[1024] Two E boxes containing a conserved glutamic residue have
been identified in Spy0128. The E-box motifs are underlined in SEQ
ID NO: 50, below. The conserved glutamic acid (E) residues, at
amino acid residues 271 and 290, are marked in bold. The E box
motifs, in particular the conserved glutamic acid residues, are
thought to be important for the formation of oligomeric pilus-like
structures of Spy0128. Preferred fragments of Spy0128 include at
least one conserved glutamic acid residue. Preferably, fragments
include at least one E box motif. TABLE-US-00115 SEQ ID NO: 50
MKLRHLLLTGAALTSFAATTVHGETVVNGAKLTVTKNLDLVNSNALIPNT
DFTFKIEPDTTVNEDGNKFKGVALNTPMTKVTYTNSDKGGSNTKTAEFDF
SEVTPEKPGVYYYKVTEEKIDKVPGVSYDTTSYTVQVHVLWNEEQQKPVA
TYIVGYKEGSKVPIQFKNSLDSTTLTVKKKVSGTGGDRSKDFNFGLTLKA
NQYYKASEKVMIEKTTKGGQAPVQTEASTDQLYHFTLKDGESIKVTNLPV
GVDYVVTEDDYKSEKYTTNVEVSPQDGAVKNIAGNSTEQETSTDKDMTIT
FTNKKDFEVPTGVAMTVAPYIALGIVAVGGALYFVKKKNA
[1025] Spy0129 is a srtC1 type sortase. An example of an amino acid
sequence for Spy0129 is set forth in SEQ ID NO: 51. TABLE-US-00116
SEQ ID NO: 51 MIVRLIKLLDKLINVIVLCFFFLCLLIAALGIYDALTVYQGANATNYQQY
KKKGVQFDDLLAINSDVMAWLTVKGTHIDYPIVQGENNLEYINKSVEGEY
SLSGSVFLDYRNKVTFEDKYSLIYAHHMAGNVMFGELPNFRKKSFFNKHK
EESIETKTKQKLKINIFACIQTDAFDSLLFNPIDVDISSKNEFLNHIKQK
SVQYREILTTNESRFVALSTCEDMTTDGRIIVIGQIE''
[1026] Spy0130 is referred to as a hypothetical protein. It
contains a sortase substrate motif LPXTG (SEQ ID NO: 122), shown in
italics in SEQ ID NO: 52. TABLE-US-00117 SEQ ID NO: 52
MKKSILRILAIGYLLMSFCLLDSVEAENLTASINIEVINQVDVATNKQSS
DIDETFMPVIEALDKESPLPNSVTTSVKGNGKTSFEQLTPSEVGQYHYKI
HQLLGKNSQYHYDETVYEVVIYVLYNEQSGALETNLVSNKLGETEKSELI
FKQEYSEKTPEPHQPDTTEKEKPQKKRNGILPSTGEMVSYVSALGIVLVA
TITLYSIYKKLKTSK
[1027] Spy0130 contains an amino acid motif indicative of a cell
wall anchor: SEQ ID NO: 131 LPSTG (shown in italics in SEQ ID NO:
52, above). In some recombinant host cell systems, it may be
preferable to remove this motif to facilitate secretion of a
recombinant Spy0130 protein from the host cell. Alternatively, in
other recombinant host cell systems, it may be preferable to use
the cell wall anchor motif to anchor the recombinantly expressed
protein to the cell wall. The extracellular domain of the expressed
protein may be cleaved during purification or the recombinant
protein may be left attached to either inactivated host cells or
cell membranes in the final composition.
[1028] Two E boxes containing conserved glutamic residues have been
identified in Spy0130. The E-box motifs are underlined in SEQ ID
NO: 52, below. The conserved glutamic acid (E) residues, at amino
acid residues 118 and 148, are marked in bold. The E box motifs, in
particular the conserved glutamic acid residues, are thought to be
important for the formation of oligomeric pilus-like structures of
Spy0130. Preferred fragments of Spy0130 include at least one
conserved glutamic acid residue. Preferably, fragments include at
least one E box motif. TABLE-US-00118 SEQ ID NO:52
MKKSILRILAIGYLLMSFCLLDSVEAENLTASINIEVINQVDVATNKQSS
DTDETFMFVIEALDKESPLPNSVTTSVKGNGKTSFEQLTFSEVGQYHYKI
HQLLGKNSQYHYDETVYEVVIYVLYNEQSGALETNLVSNKLGETEKSELI
FKQEYSEKTPEPHQPDTTEKEKPQKKRNGILPSTGEMVSYVSALGIVLVA
TITLYSIYKKLKTSK
[1029] Spy0131 is referred to as a conserved hypothetical protein.
An example of an amino acid sequence of Spy0131 is set forth in SEQ
ID NO: 53 TABLE-US-00119 SEQ ID NO: 53
MTRTNYQKKRMTCPVETEDITYRRKKIKGRRQAILAQFEPELVHHELIGD
SCTCPDCHGTLTEIGSVVQRQELVFIPAQLKRINHVQHAYKCQTCSDNSL
SDKIIKAPVPKAPLAHSLGSASIIAHTVHQKFTLKVPNYRQEEDWNKLGL
SISRKEIANWHIKSSQYYFEPLYDLLRDILLSQEVIHADETSYRVLESDT
QLTYYWTFLSGKHEKKGITLYHHDKRRSGLVTQEVLGDYSGYVHCDMHGA
YRQLEHAKLVGCWAHVRRKFFEATPKQADKTSLGRKGLVYCDKLFALEAE
WCELPPQERLVKRKEILTPLMTTFFDWCREQVVLSGSKLGLAIAYSLKHE
RTFRTVLEDGHIVLSNNMAERAIKSLVMGRKNWLFSQSFEGAKAAAIIMS
LLETAKRHGLNSEKYISYLLDRLPNEETLAKREVLEAYLPWAKKVQTNCQ
[1030] Spy0133 is referred to as a conserved hypothetical protein.
An example of an amino acid sequence of Spy0133 is set forth in SEQ
ID NO: 54. TABLE-US-00120 SEQ ID NO: 54
MTIRLNDLGQVYLVCGKTDMRQGIDSLAYLVKSQHELDLFSGAVYLFCGG
RTRDRFKALYWDGQGFWLLYKRFENGKLAWPRNRDEVKCLTAVQVDWLMK
GFFISPNIKISKSHDFY
[1031] Spy0135 is a SrtB type sortase. It is also referred to as a
putative fibria-associated protein. An example of an amino acid
sequence of Spy0135 is set forth in SEQ ID NO: 55. TABLE-US-00121
SEQ ID NO: 55 MECYRDRQLLSTYHKQVTQKKPSEMEEVWQKAKAYNARLGIQPVPDAFSF
RDGIHDKNYESLLQIENNDIMGYVEVPSIKVTLPIYHYTTDEVLTKGAGH
LFGSALPVGGDGTHTVISAHRGLPSAEMETNLNLVKKGDTFYFRVLNKVL
AYKVDQILTVEPDQVTSLSGVMGKDYATLVTCTPYGVNTKRLLVRGHRIA
YHYKKYQQAKKAMKLVDKSRMWAEVVCAAFGVVIAIILVFMYSRVSAKKS K
[1032] GAS AI-3 Sequences
[1033] As discussed above, a GAS AI-3 sequence is present in a M3,
M18 and M5 strain isolates. Examples of GAS AI-3 sequences from M3
strain isolate MGAS315 are set forth below.
[1034] SpyM30097 is as a negative transcriptional regulator (Nra).
An example of an amino acid sequence of SpyM30097 is set forth in
SEQ ID NO: 56. TABLE-US-00122 SEQ ID NO: 56
MPYVKKKKDSFLVETYLEQSIRDKSELVLLLFKSPTIIFSHVAKQTGLTA
VQLKYYCKELDDFFGNNLDTTIKKGKIICCFVKPVKEFYLHQLYDTSTIL
KLLVFFIKNGTSSQPLIKFSKKYFLSSSSAYRLRESLIKLLREFGLRVSK
NTIVGEEYRIRYLIAMLYSKFGIVIYPLDHLDNQIIYRFLSQSATNLRTS
PWLEEPFSFYNMLLALSWKRHQFAVSIPQTRIFRQLKKLFIYDCLTRSSR
QVIENAFSLTFSQGDLEYLFLIYITTNNSFASLQWTPQHIETCCHIFEKN
DTFRLLLEPILKRLPQLNHSKQDLIKALMYFSKSFLFNLQHFVIEIPSFS
LPTYTGNSNLYKALKNIVNQWLAQLPGKRHLNEKHLQLFCSHIEQILKNK
QPALTVVLISSNFINAKLLTDTIPRYPSDKGIHFYSFYLLRDDIYQIPSL
KPDLVITHSRLIPFVKNDLVKGVTVAEFSFDNPDYSIASIQNLTYQLKDK KYQDFLNEQLQ
[1035] SpyM30098 is thought to be a collagen binding protein (Cpb).
It contains a sortase substrate motif VPXTG (SEQ ID NO: 137) shown
in italics in SEQ ID NO: 57. TABLE-US-00123 SEQ ID NO: 57
MQKRDKTNYGSANNKRRQTTIGLLKVFLTFVALIGIVGFSIRAFGAEEQS
VPNKQSSVQDYPWYGYDSYSKGYPDYSPLKTYHNLKVNLDGSKEYQAYCF
NLTKHFPSKSDSVRSQWYKKLEGTNENFIKLADKPRIEDGQLQQNILRIL
YNGYPNDRNGIMKGIDPLNAILVTQNAIWYYTDSSYISDTSKAFQQEETD
LKLDSQQLQLMRNALKRLINPKEVESLPNQVPANYQLSIFQSSDKTFQNL
LSAEYVPDTPPKPGEEPPAKTEKTSVIIRKYAEGDYSKLLEGATLKLAQI
EGSGFQEKIFDSNKSGEKVELPNGTYVLSELKPPQGYGVATPITFKVAAE
KVLIKNKEGQFVENQNKEIAEPYSVTAFNDEEEIGYLSDFNNYGKFYYAK
NTNGTNQVVYCFNADLHSPPDSYDHGANIDPDVSESKEIKYTHVSGYDLY
KYAATPRDKDADFFLKHIKKILDKGYKKKGDTYKTLTEAQFRAATQLAIY
YYTDSADLTTLKTYNDNKGYHGFDKLDDATLAVVHELITYAEDVTLPMTQ
NLDEFVPNSSRYQALIGTQYHPNELIDVISMEDKQAPIIPITHKLTISKT
VTGTIADKKKEFNFEIHLKSSDGQAISGTYPTNSGELTVTDGKATFTLKD
GESLIVEGLPSGYSYEITETGASDYEVSVNGKNAPDGKATKASVKEDETV
AFENRKDLVPPTGLTTDGAIYLWLLLLVPFGLLVWLFGRKGTKK
[1036] SpyM30098 contains an amino acid motif indicative of a cell
wall anchor: SEQ ID NO: 184 VPPTG (shown in italics in SEQ ID NO:
57, above). In some recombinant host cell systems, it may be
preferable to remove this motif to facilitate secretion of a
recombinant SpyM30098 protein from the host cell. Alternatively, in
other recombinant host cell systems, it may be preferable to use
the cell wall anchor motif to anchor the recombinantly expressed
protein to the cell wall. The extracellular domain of the expressed
protein may be cleaved during purification or the recombinant
protein may be left attached to either inactivated host cells or
cell membranes in the final composition.
[1037] A pilin motif, discussed above, containing a conserved
lysine (K) residue has also been identified in SpyM30098. The pilin
motif sequence is underlined in SEQ ID NO: 57, below. Conserved
lysine (K) residues are also marked in bold, at amino acid residues
262 and 270. The pilin sequence, in particular the conserved lysine
residues, are thought to be important for the formation of
oligomeric, pilus-like structures. Preferred fragments of SpyM30098
include at least one conserved lysine residue. Preferably,
fragments include the pilin sequence. TABLE-US-00124 SEQ ID NO: 57
MQKRDKTNYGSANNKRRQTTIGLLKVFLTFVALIGIVGFSIRAFGAEEQS
VPNKQSSVQDYPWYGYDSYSKGYPDYSPLKTYHNLKVNLDGSKEYQAYCF
NLTKHFPSKSDSVRSQWYKKLEGTNENFIKLADKPRIEDGQLQQNILRIL
YNGYPNDRNGIMKGTDPLNATLVTQNAIWYYTDSSYISDTSKAFQQEETD
LKLDSQQLQLMRNALKRLINPKEVESLPNQVPANYQLSIFQSSDKTFQNL
LSAEYVPDTPPKPGEEPPAKTEKTSVIIRKYAEGDYSKLLEGATLKLAQI
EGSGFQEKIFDSNKSGEKVELPNGTYVLSELKPPQGYGVATPITFKVAAE
KVLIKNKEGQFVENQNKEIAEPYSVTAFNDFEEIGYLSDFNNYGKFYYAK
NTNGTNQVVYCFNADLHSPPDSYDHGANIDPDVSESKEIKYTHVSGYDLY
KYAATPRDKDADFFLKHIKKILDKGYKKKGDTYKTLTEAQFRAATQLAIY
YYTDSADLTTLKTYNDNKGYHGFDKLDDATLAVVHELITYAEDVTLPMTQ
NLDFFVPNSSRYQALIGTQYHPNELIDVISMEDKQAPIIPITHKLTISKT
VTGTIADKKKEFNFEIHLKSSDGQAISGTYPTNSGELTVTDGKATFTLKD
GESLIVEGLPSGYSYEITETGASDYEVSVNGKNAPDGKATKASVKEDETV
AFENRKDLVPPTGLTTDGAIYLWLLLLVPFGLLVWLFGRKGTKK
[1038] An E box containing a conserved glutamic residue has been
identified in SpyM30098. The E-box motif is underlined in SEQ ID
NO: 57, below. The conserved glutamic acid (E), at amino acid
residue 330, is marked in bold. The E box motif, in particular the
conserved glutamic acid residue, is thought to be important for the
formation of oligomeric pilus-like structures of SpyM30098.
Preferred fragments of SpyM30098 include the conserved glutamic
acid residue. Preferably, fragments include the E box motif.
TABLE-US-00125 SEQ ID NO: 57
MQKRDKTNYGSANNKRRQTTIGLLKVFLTFVALIGIVGFSIRAFGAEEQS
VPNKQSSVQDYPWYGYDSYSKGYPDYSPLKTYHNLKVNLDGSKEYQAYCF
NLTKHFPSKSDSVRSQWYKKLEGTNENFIKLADKPRIEDGQLQQNILRIL
YNGYPNDRNGIMKGTDPLNATLVTQNAIWYYTDSSYISDTSKAFQQEETD
LKLDSQQLQLMRNALKRLINPKEVESLPNQVPANYQLSIFQSSDKTFQNL
LSAEYVPDTPPKPGEEPPAKTEKTSVIIRKYAEGDYSKLLEGATLKLAQI
EGSGFQEKIFDSNKSGEKVELPNGTYVLSELKPPQGYGVATPITFKVAAE
KVLIKNKEGQFVENQNKEIAEPYSVTAFNDFEEIGYLSDFNNYGKFYYAK
NTNGTNQVVYCFNADLHSPPDSYDHGANIDPDVSESKEIKYTHVSGYDLY
KYAATPRDKDADFFLKHIKKILDKGYKKKGDTYKTLTEAQFRAATQLAIY
YYTDSADLTTLKTYNDNKGYHGFDKLDDATLAVVHELITYAEDVTLPMTQ
NLDFFVPNSSRYQALIGTQYHPNELIDVISMEDKQAPIIPITHKLTISKT
VTGTIADKKKEFNFEIHLKSSDGQAISGTYPTNSGELTVTDGKATFTLKD
GESLIVEGLPSGYSYEITETGASDYEVSVNGKNAPDGKATKASVKEDETV
AFENRKDLVPPTGLTTDGAIYLWLLLLVPFGLLVWLFGRKGTKK
[1039] SpyM30099 is referred to as LepA. An example of an amino
acid sequence of SpyM30099 is set forth in SEQ ID) NO: 58.
TABLE-US-00126 SEQ ID NO: 58
MTNYLNRLNENPLLKAFIRLVLKISTIGFLGYILFQYVFGVMIVNTNQMS
PAVSAGDGVLYYRLTDRYHINDVVVYEVDDTLKVGRIAAQAGDVENFTQE
GGLLINGHPPEKEVPYLTYPHSSGPNFPYKVPTGTYFILNDYREERLDSR
YYGALPINQIKGKISTLLRVRGI
[1040] SpyM30100 is thought to be a fimbrial protein. An example of
an amino acid sequence of SpyM30100 is set forth in SEQ ID NO: 59.
TABLE-US-00127 SEQ ID NO: 59
MKKNKLLLATAILATALGTASLNQNVKAETAGVSENAKLIVKKTFDSYTD
NEVLMPKADYTFKVEADSTASGKTKDGLEIKPGIVNGLTEQIISYTNTDK
PDSKVKSTEFDFSKVVFPGIGVYRYTVSEKQGDVEGITYDTKKWTVDVYV
GNKEGGGFEPKFIVSKEQGTDVKKPVNFNNSFATTSLKVKKNVSGNTGEL
QKEFDFTLTLNESTNFKKDQIVSLQKGNEKFEVKIGTPYKFKLKNGESIQ
LDKLPVGITYKVNEMEANKDGYKTTASLKEGDGQSKMYQLDMEQKTDESA
DEIVVTNKRDTQVPTGVVGTLAPFAVLSIVAIGGVIYITKRKKA
[1041] SpyM30100 contains an amino acid motif indicative of a cell
wall anchor: SEQ ID NO: 140 QVPTG (shown in italics in SEQ ID NO:
59, above). In some recombinant host cell systems, it may be
preferable to remove this motif to facilitate secretion of a
recombinant SpyM30100 protein from the host cell. Alternatively, in
other recombinant host cell systems, it may be preferable to use
the cell wall anchor motif to anchor the recombinantly expressed
protein to the cell wall. The extracellular domain of the expressed
protein may be cleaved during purification or the recombinant
protein may be left attached to either inactivated host cells or
cell membranes in the final composition.
[1042] Two pilin motifs, discussed above, containing conserved
lysine (K) residues have also been identified in SpyM30100. The
pilin motif sequences are underlined in SEQ ID NO: 59, below.
Conserved lysine (K) residues are also marked in bold, at amino
acid residues 57 and 63 and at amino acid residues 161 and 166. The
pilin sequences, in particular the conserved lysine residues, are
thought to be important for the formation of oligomeric, pilus-like
structures. Preferred fragments of SpyM30100 include at least one
conserved lysine residue. Preferably, fragments include at least
one pilin sequence. TABLE-US-00128 SEQ ID NO: 59
MKKNKLLLATAILATALGTASLNQNVKAETAGVSENAKLIVKKTFDSYTD
NEVLMPKADYTFKVEADSTASGKTKDGLEIKPGIVNGLTEQIISYTNTDK
PDSKVKSTEFDFSKVVFPGIGVYRYTVSEKQGDVEGITYDTKKWTVDVYV
GNKEGGGFEPKFIVSKEQGTDVKKPVNFNNSFATTSLKVKKNVSGNTGEL
QKEFDFTLTLNESTNFKKDQIVSLQKGNEKFEVKIGTPYKFKLKNGESIQ
LDKLPVGITYKVNEMEANKDGYKTTASLKEGDGQSKMYQLDMEQKTDESA
DEIVVTNKRDTQVPTGVVGTLAPFAVLSIVAIGGVIYITKRKKA
[1043] Two E boxes, each containing a conserved glutamic residue,
have been identified in SpyM30100. The E-box motifs are underlined
in SEQ ID NO: 59, below. The conserved glutamic acid (E) residues,
at amino acid residues 232 and 264, are marked in bold. The E box
motifs, in particular the conserved glutamic acid residues, are
thought to be important for the formation of oligomeric pilus-like
structures of SpyM30100. Preferred fragments of SpyM30100 include
at least one conserved glutamic acid residue. Preferably, fragments
include at least one E box motif. TABLE-US-00129 SEQ ID NO: 59
MKKNKLLLATAILATALGTASLNQNVKAETAGVSENAKLIVKKTFDSYTD
NEVLMPKADYTFKVEADSTASGKTKDGLEIKPGIVNGLTEQIISYTNTDK
PDSKVKSTEFDFSKVVFPGIGVYRYTVSEKQGDVEGITYDTKKWTVDVYV
GNKEGGGFEPKFIVSKEQGTDVKKPVNFNNSFATTSLKVKKNVSGNTGEL
QKEFDFTLTLNESTNFKKDQIVSLQKGNEKFEVKIGTPYKFKLKNGESIQ
LDKLPVGITYKVNEMEANKDGYKTTASLKEGDGQSKMYQLDMEQKTDESA
DEIVVTNKRDTQVPTGVVGTLAPFAVLSIVAIGGVIYITKRKKA
[1044] SpyM30101 is a SrtC2 type sortase. An example of an amino
acid sequence of SpyM30101 is set forth in SEQ ID NO: 60.
TABLE-US-00130 SEQ ID NO: 60
MTIVQVINKAIDTLILIFCLVVLFLAGFGLWDSYHLYQQADASNPKKFKT
AQQQPKFEDLLALNEDVIGWLNIPGTHIDYPLVQGKTNLEYINKAVDGSV
ANSGSLFLDTRNHNDFTDDYSLIYGHHMAGNAMFGEIPKFLKKDFPSKHN
KATIETKERKKLTVTIFACLKTDAFNQLVFNPNAITNQDQQRQLVDYISK
RSKQFKPVKLKHHTKFVAFSTCENFSTDNRVIVVGTTQE
[1045] SpyM30102 is referred to as a hypothetical protein. An
example of an amino acid sequence of SpyM30102 is set forth in SEQ
ID NO: 61. TABLE-US-00131 SEQ ID NO: 61
MILTMLAFNQTVLAKDSTVQTSISVENVLERAGDSTPFSIALESIDAMKT
IEEITIAGSGKASFSPLTFTTVGQYTYRVYQKPSQNKDYQASTTVFDVLV
YVTYDEDGTLVAKVISRRAGDEEKSAITFKPKWLVKPIPPRQPNIPKTPL
PLAGEVKSLLGILSIVLLGLLVLLYVKKLKSRL
[1046] SpyM30102 contains an amino acid motif indicative of a cell
wall anchor: SEQ ID NO: 185 LPLAG (shown in italics in SEQ ID NO:
61, above). In some recombinant host cell systems, it may be
preferable to remove this motif to facilitate secretion of a
recombinant SpyM30102 protein from the host cell. Alternatively, in
other recombinant host cell systems, it may be preferable to use
the cell wall anchor motif to anchor the recombinantly expressed
protein to the cell wall. The extracellular domain of the expressed
protein may be cleaved during purification or the recombinant
protein may be left attached to either inactivated host cells or
cell membranes in the final composition.
[1047] A pilin motif, discussed above, containing a conserved
lysine (K) residue has also been identified in SpyM30102. The pilin
motif sequence is underlined in SEQ ID NO: 61, below. The conserved
lysine (K) residue is also marked in bold, at amino acid residue
132. The pilin sequence, in particular the conserved lysine
residues, are thought to be important for the formation of
oligomeric, pilus-like structures. Preferred fragments of SpyM30102
include the conserved lysine residue. Preferably, fragments include
the pilin sequence. TABLE-US-00132 SEQ ID NO: 61
MILTMLAFNQTVLAKDSTVQTSISVENVLERAGDSTPFSIALESIDAMKT
IEEITIAGSGKASFSPLTFTTVGQYTYRVYQKPSQNKDYQADTTVFDVLV
YVTYDEDGTLVAKVISRRAGDEEKSAITFKPKWLVKPIPPRQPNIPKTPL
PLAGEVKSLLGILSIVLLGLLVLLYVKKLKSRL
[1048] Two E boxes containing conserved glutamic residues have been
identified in SpyM30102. The E-box motifs are underlined in SEQ ID
NO: 61, below. The conserved glutamic acid (E) residues, at amino
acid residues 52 and 122, are marked in bold. The E box motifs, in
particular the conserved glutamic acid residues, are thought to be
important for the formation of oligomeric pilus-like structures of
SpyM30102. Preferred fragments of SpyM30102 include at least one
conserved lysine residue. Preferably, fragments include at least
one pilin sequence. TABLE-US-00133 SEQ ID NO: 61
MILTMLAFNQTVLAKDSTVQTSISVENVLERAGDSTPFSIALESIDAMKT
IEEITIAGSGKASFSPLTFTTVGQYTYRVYQKPSQNKDYQASTTVFDVLV
YVTYDEDGTLVAKVISRRAGDEEKSAITFKPKWLVKPIPPRQPNIPKTPL
PLAGEVKSLLGILSIVLLGLLVLLYVKKLKSRL
[1049] SpyM30103 is referred to as a putative multiple sugar
metabolism regulator. An example of an amino acid sequence for
SpyM3103 is set forth in SEQ ID NO: 62. TABLE-US-00134 SEQ ID NO:
62 MVRFDLKHVQTLHSLSQLPISVMSQDKALIQVYGNDDYLLCYYQFLKHLA
IPQAAQDVIFYEGLFEESFMIFPLCHYIIAIGPGYPYSLNKDYQEQLANN
CLKHSSHRSKEELLSYMALVPHFPINNVRNLLIAIDAFFDTQFETTCQQT
IHQLLQHSKQMTADPDIIHRLKHISKASSQLPPVLEHLNHTMDLVKLGNP
QLLKQETNRIPLSSITSSSISALRAEKNLTVIYLTRLLEFSFVENTDVAK
HYSLVKYYMALNEEASDLLKVLRIRCAAIIHFSESLTNKSISDKRQMYNS
VLHYVDSHLYSKLKVSDIAKRLYVSESHLRSVFKKYSNVSLQHYILSTKI
KEAQLLLKRGIPVGEVAKSLYFYDTTHFHKIFKKYTGISSKDYLAKYRDN I
[1050] SpyM30104 is thought to be a F2 like fibronectic binding
protein. An example of an amino acid sequence for SpyM30104 is set
forth in SEQ ID NO: 63. TABLE-US-00135 SEQ ID NO: 63
MSSSDEETLKQYASKYTSNRRGDTSGNLKKQIAKVLTEGYPTNKSDWLNG
LTENEKIEVTQDAIWYFTETTVPADRSYTNRNVNSQKMKEVYQKLIDTTD
IDKYEDVQFDLFVPQDTNLQAVISVEPVIESLPWTSLKPIAQKDITAKKI
WVDAPKEKPIIYFKLYRQLPGEKEVAVDDAELKQINSEGQQEISVTWTNQ
LVTDEKGMAYIYSVKEVDKNGELLEPKDYIKKEDGLTVTNTYVKPTSGHY
DIEVTFGNGHIDTTEDTTPDIVSGENQMKQIEGEDSKPIDEVTENNLIEF
GKNTMPGEEDGTNSNKYEEVEDSRPVDTLSGLSSEQGQSGDMTIEEDSAT
HIKFSKRDIDGKELAGATMELRDSSGKTISTWISDGQVKDFYLMPGKYTF
VETAAPDGYEVATAITFTVNEQGQVTVNGKATKGKAHIVMVDAYKPTKGS
GQVIDIEEKLPDEQGHSGSTTEIEDSKSSDVIIGGQGEVVDTTEDTQSGM
TGHSGSTTEIEDSKSSDVIIGGQGEVVDTTEDTQSGMTGHSGSTTKIEDS
KSSDVIVGGQGQIVETTEDTQTGMHGDSGRKTEVEDTKLVQSFHEDNKEP
ESNSEIPKKDKSKSNTSLPATGEKQHNKFFWMVTSCSLISSVFVISLKSK KRLSSC
[1051] SpyM30104 contains an amino acid motif indicative of a cell
wall anchor: SEQ ID NO: 180 LPATG (shown in italics in SEQ ID NO:
63, above). In some recombinant host cell systems, it may be
preferable to remove this motif to facilitate secretion of a
recombinant SpyM30104 protein from the host cell. Alternatively, in
other recombinant host cell systems, it may be preferable to use
the cell wall anchor motif to anchor the recombinantly expressed
protein to the cell wall. The extracellular domain of the expressed
protein may be cleaved during purification or the recombinant
protein may be left attached to either inactivated host cells or
cell membranes in the final composition.
[1052] Two pilin motifs, discussed above, containing conserved
lysine (K) residues have also been identified in SpyM30104. The
pilin motif sequences are underlined in SEQ ID NO: 63, below.
Conserved lysine (K) residues are also marked in bold, at amino
acid residues 156 and 227. The pilin sequences, in particular the
conserved lysine residues, are thought to be important for the
formation of oligomeric, pilus-like structures. Preferred fragments
of SpyM30104 include at least one conserved lysine residue.
Preferably, fragments include at least one pilin sequence.
TABLE-US-00136 SEQ ID NO: 63
MSSSDEETLKQYASKYTSNRRGDTSGNLKKQIAKVLTEGYPTNKSDWLNG
LTENEKIEVTQDAIWYFTETTVPADRSYTNRNVNSQKMKEVYQKLIDTTD
IDKYEDVQFDLFVPQDTNLQAVISVEPVIESLPWTSLKPIAQKDITAKKI
WVDAPKEKPIIYFKLYRQLPGEKEVAVDDAELKQINSEGQQEISVTWTNQ
LVTDEKGMAYIYSVKEVDKNGELLEPKDYIKKEDGLTVTNTYVKPTSGHY
DIEVTFGNGHIDTTEDTTPDIVSGENQMKQIEGEDSKPIDEVTENNLIEF
GKNTMPGEEDGTNSNKYEEVEDSRPVDTLSGLSSEQGQSGDMTIEEDSAT
HIKFSKRDIDGKELAGATMELRDSSGKTISTWISDGQVKDFYLMPGKYTF
VETAAPDGYEVATAITFTVNEQGQVTVNGKATKGKAHIVMVDAYKPTKGS
GQVIDIEEKLPDEQGHSGSTTEIEDSKSSDVIIGGQGEVVDTTEDTQSGM
TGHSGSTTEIEDSKSSDVIIGGQGEVVDTTEDTQSGMTGHSGSTTKIEDS
KSSDVIVGGQGQIVETTEDTQTGMHGDSGRKTEVEDTKLVQSFHEDNKEP
ESNSEIPKKDKSKSNTSLPATGEKQHNKFFWMVTSCSLISSVFVISLKSK KRLSSC
[1053] An E box containing a conserved glutamic residue has been
identified in SpyM30104. The E-box motif is underlined in SEQ ID
NO: 63, below. The conserved glutamic acid (E), at amino acid
residue 402, is marked in bold. The E box motif, in particular the
conserved glutamic acid residue, is thought to be important for the
formation of oligomeric pilus-like structures of SpyM30104.
Preferred fragments of SpyM30104 include the conserved glutamic
acid residue. Preferably, fragments include the E box motif.
TABLE-US-00137 SEQ ID NO: 63
MSSSDEETLKQYASKYTSNRRGDTSGNLKKQIAKVLTEGYPTNKSDWLNG
LTENEKIEVTQDAIWYFTETTVPADRSYTNRNVNSQKMKEVYQKLIDTTD
IDKYEDVQFDLFVPQDTNLQAVISVEPVIESLPWTSLKPIAQKDITAKKI
WVDAPKEKPIIYFKLYRQLPGEKEVAVDDAELKQINSEGQQEISVTWTNQ
LVTDEKGMAYIYSVKEVDKNGELLEPKDYIKKEDGLTVTNTYVKPTSGHY
DIEVTFGNGHIDITEDTTPDIVSGENQMKQIEGEDSKPIDEVTENNLIEF
GKNTMPGEEDGTNSNKYEEVEDSRPVDTLSGLSSEQGQSGDMTIEEDSAT
HIKFSKRDIDGKELAGATMELRDSSGKTISTWISDGQVKDFYLMPGKYTF
VETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHIVMVDAYKPTKGS
GQVIDTEEKLPDEQGHSGSTTEIEDSKSSDVIIGGQGEVVDTTEDTQSGM
TGHSGSTTEIEDSKSSDVIIGGQGEVVDTTEDTQSGMTGHSGSTTKIEDS
KSSDVIVGGQGQIVETTEDTQTGMHGDSGRKTEVEDTKLVQSFHFDNKEP
ESNSEIPKKDKSKSNTSLPATGEKQHNKFFWMVTSCSLISSVFVISLKSK KRLSSC
[1054] Examples of GAS AI-3 sequences from M3 strain isolate SSI-1
are set forth below.
[1055] Sps0099 is a negative transcriptional regulator (Nra). An
example of an amino acid sequence for Sps0099 is set forth in SEQ
ID NO: 64. TABLE-US-00138 SEQ ID NO: 64
MPYVKKKKDSPLVETYLEQSIRDKSELVLLLFKSPTIIFSHVAKQTGLTA
VQLKYYCKELDDFFGNNLDITIKKGKIICCFVKPVKEFYLHQLYDTSTIL
KLLVFFIKNGTSSQPLIKFSKKYFLSSSSAYRLRESLIKLLREFGLRVSK
NTIVGEEYRIRYLIAMLYSKFGIVIYPLDHLDNQIIYRFLSQSATNLRTS
PWLEEPFSFYNMLLALSWKRHQFAVSIPQTRIFRQLKKLFIYDCLTRSSR
QVIENAFSLTESQGDLEYLFLIYITTNNSFASLQWTPQHIETCCHIFEKN
DTFRLLLEPILKRLPQLNHSKQDLIKALMYFSKSFLFNLQHFVIEIPSFS
LPTYTGNSNLYKALKNIVNQWLAQLPGKRHLNEKHLQLFCSHIEQILKNK
QPALTVVLTSSNFINAKLLTDTIPRYFSDKGIHFYSFYLLRDDIYQIPSL
KPDLVTTHSRLIPFVKNDLVKGVTVAEFSFDNPDYSIASIQNLIYQLKDK KYQDFLNEQLQ
[1056] Sps0100 is thought to be a collagen binding protein (Cbp).
It contains a sortase substrate motif VPXTG shown in italics in SEQ
ID NO: 65. TABLE-US-00139 SEQ ID NO: 65
MQKRDKTNYGSANNKRRQTTIGLLKVFLTFVALIGIVGFSIRAFGAEEQS
VPNKQSSVQDYPWYGYDSYSKGYPDYSPLKTYHNLKVNLDGSKEYQAYCF
NLTKHFPSKSDSVRSQWYKKLEGTNENFIKLADKPRIEDGQLQQNILRIL
YNGYPNDRNGIMKGIDPLNAILVTQNAIWYYTDSSYISDTSKAFQQEETD
LKLDSQQLQLMRNALKRLINPKEVESLPNQVPANYQLSIFQSSDKTFQNL
LSAEYVPDTPPKPGEEPPAKTEKTSVIIRKYAEGDYSKLLEGATLKLAQI
EGSGFQEKIFDSNKSGEKVELPNGTYVLSELKPPQGYGVATPITFKVAAE
KVLIKNKEGQFVENQNKETAEPYSVTAFNDFEEIGYLSDFNNYGKFYYAK
NTNGTNQVVYCFNADLHSPPDSYDHGANIDPDVSESKEIKYTHVSGYDLY
KYAATPRDKDADFFLKHIKKILDKGYKKKGDTYKTLTEAQFRAATQLAIY
YYTDSADLTTLKTYNDNKGYHGFDKLDDATLAVVHELITYAEDVTLPMTQ
NLDFFVPNSSRYQALIGTQYHPNELIDVISMEDKQAPIIPITHKLTISKT
VTGTIADKKKEFNFEIHLKSSDGQAISGTYPTNSGELTVTDGKATFTLKD
GESLIVEGLPSGYSYEITETGASDYEVSVNGKNAPDGKATKASVKEDETV
AFENRKDLVPPTGLTTDGAIYLWLLLLVPFGLLVWLFGRKGTKK
[1057] Sps0101 is referred to as a LepA protein. An example of an
amino acid sequence of Sps0101 is set forth as SEQ ID NO: 66
TABLE-US-00140 SEQ ID NO: 66
MTNYLNRLNENPLLKAFIRLVLKISIIGFLGYILFQYVFGVMIVNTNQMS
PAVSAGDGVLYYRLTDRYHINDVVVYEVDDTLKVGRIAAQAGDEVNFTQE
GGLLINGHPPEKEVPYLTYPHSSGPNFPYKVPTGTYFILNDYREERLDSR
YYGALPINQIKGKISTLLRVRGI
[1058] Sps0102 is thought to be a fimbrial protein. It contains a
sortase substrate motif QVXTG shown in italics in SEQ ID NO: 67.
TABLE-US-00141 SEQ ID NO: 67
MEREKMKKNKLLLATAILATALGTASLNQNVKAETAGVSENAKLIVKKTF
DSYTDNEVLMPKADYTFKVEADSTASGKTKDGLEIKPGIVNGLTEQIISY
TNTDKPDSKVKSTEFDFSKVVFPGIGVYRYTVSEKQGDVEGITYDTKKWT
VDVYVGNKEGGGFEPKFIVSKEQGTDVKKPVNFNNSFATTSLKVKKNVSG
NTGELQKEFDFTLTLNESTNFKKDQIVSLQKGNEKFEVKIGTPYKFKLKN
GESIQLDKLPVGITYKVNEMEANKDGYKTTASLKEGDGQSKMYQLDMEQK
TDESADEIVVTNKRDTQVPTGVVGTLAPFAVLSIVAIGGVIYITKRKKA
[1059] Sps0103 is a SrtC2 type sortase. An example of Sps0103 is
set forth in SEQ ID NO: 68. TABLE-US-00142 SEQ ID NO: 68
MVMTIVQVINKATDTLILTFGLVVLFLAGFGLWDSYHLYQQADASNFKKF
KTAQQQPKFEDLLALNEDVIGWLNIPGTHIDYPLVQGKTNLEYINKAVDG
SVAMSGSLFLDTRNHNDFTDDYSLTYGHHMAGNAMFGEIPKFLKKDFFSK
HNKAIIETKERKKLTVTIFACLKTDAFNQLVFNPNAITNQDQQRQLVDYI
SKRSKQFKPVKLKHHTKFVAFSTCENFSTDNRVTVVGTIQE
[1060] Sps0104 is referred to as a hypothetical protein. It
contains a sortase substrate motif LPXAG shown in italics in SEQ ID
NO: 69. TABLE-US-00143 SEQ ID NO: 69
MLFSVVMILTMLAFNQTVLAKDSTVQTSISVENVLERAGDSTPFSIALES
IDAMKTIEEITIAGSGKASFSPLTFTTVGQYTYRVYQKPSQNKDYQADTT
VFDVLVYVTYDEDGTLVAKVISRRAGDEEKSAITFKPKWLVKPIPPRQPN
IPKTPLPLAGEVKSLLGILSIVLLGLLVLLYVKKLKSRL
[1061] Sps0105 is referred to as a putative multiple sugar
metabolism regulator. An example of Sps0105 is set forth in SEQ ID
NO: 70. TABLE-US-00144 SEQ ID NO: 70
MALVPHFPINNVRNLLIAIDAFFDTQFETTCQQTIHQLLQHSKQMTADPD
ITHRLKHISKASSQLPPVLEHLNHIMDLVKLGNPQLLKQEINRIPLSSIT
SSSISALRAEKNLTVIYLTRLLEPSFVENTDVAKHYSLVKYYMALNEEAS
DLLKVLRIRCAAIIHFSESLTNKSISDKRQMYNSVLHYVDSHLYSKLKVS
DIAKRLYVSESHLRSVEKKYSNVSLQHYTLSTKIKEAQLLLKRGIPVGEV
AKSLYFYDTTHFHKIFKKYTGISSKDYLAKYRDNI
[1062] Sps0106 is thought to be a F2 like fibronectic binding
protein. It contains a sortase substrate LPXTG (SEQ ID NO: 122)
shown in italics in SEQ ID NO: 71. TABLE-US-00145 SEQ ID NO: 71
MTQKNSYKLSFLLSLTGFILGLLLVFIGLSGVSVGHAETRNGANKQGAFE
IKKNKSQEEYNYEVYDNRNILQDGEHKLEIKRVDGTGKTYQGFCFQLTKN
FPTAQGVSKKLYKKLSSSDEETLKQYASKYTSNRRGDTSGNLKKQIAKVL
TEGYPTNKSDWLNGLTENEKIEVTQDATWYFTETTVPADRSYTNRNVNSQ
KMKEVYQKLIDTTDIDKYEDVQFDLFVPQDTNLQAVISVEPVIESLPWTS
LKPIAQKDITAKKIWVDAPKEKPTIYFKLYRQLPGEKEVAVDDAELKQTN
SEGQQEISVTWTNQLVTDEKGMAYIYSVKEVDKNGELLEPKDYIKKEDGL
TVTNTYVKPTSGHYDIEVTFGNGHIDITEDTTPDIVSGENQMKQIEGEDS
KPIDEVTENNLIEFGKNTMPGEEDGTNSNKYEEVEDSRPVDTLSGLSSEQ
GQSGDMTTEEDSATHIKFSKRDIDGKELAGATMELRDSSGKTISTWISDG
QVKDFYLMPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDA
HIVMVDAYKPTKGSGQVIDIEEKLPDEQGHSGSTTEIEDSKSSDVIIGGQ
GEVVDTTEDTQSGMTGHSGSTTKIEDSKSSDVIVGGQGQIVETTEDTQTG
MHGDSGRKTEVEDTKLVQSFHFDNKEPESNSEIPKKDKSKSNTSLPATGE
KQHNKFFWMVTSCSLISSVFVISLKSKKRLSSC
[1063] Examples of GAS AI-3 sequences from M5 isolate Manfredo are
set forth below.
[1064] Orf 77 encodes a negative transcription regulator (Nra). An
example of the nucleotide sequence encoding Nra (SEQ ID NO: 88) and
an Nra amino acid sequence (SEQ ID NO: 89) are set forth below.
TABLE-US-00146 SEQ ID NO: 88
ATGCCTTATGTCAAAAAGAAAAAGGATAGTTTCTTAGTAGAAACATATCT
TGAACAGTCTATTAGAGATAAAAGTGAATTAGTCTTACTGTTATTTAAAT
CGCCTACTATCATTTTTTCTCATGTTGCTAAACAAACTGGTCTGACGGCT
GTACAATTAAAATATTACTGTAAAGAACTTGATGACTTTTTTGGAAATAA
TTTAGACATTACCATTAAAAAGGGCAAAATAATATGTTGTTTTGTCAAAC
CTGTTAAGGAATTCTACCTTCATCAACTCTATGACACATCAACAATATTA
AAATTATTAGTTTTCTTTATTAAAAATGGAACGTCATCACAACCTCTGAT
TAAATTTTCAAAAAAGTATTTTCTATCAAGCTCCTCAGCTTATCGACTAC
GGGAATCGCTGATCAAATTACTACGGGAATTTGGCTTGAGAGTCTCAAAA
AATACAATTGTCGGAGAGGAATATCGTATTCGCTATCTTATTGCCATGCT
ATATAGTAAATTTGGCATTGTCATCTATCCGTTAGATCATCTAGACAATC
AAATTATTTATCGCTTCTTATCACAAAGTGCAACCAATTTAAGAACATCG
CCCTGGCTAGAGGAACCTTTTTCTTTTTATAATATGTTACTTGCCTTGTC
ATGGAAACGTCACCAATTTGCAGTTAGCATTCCTCAAACACGTATTTTTC
GACAATTAAAAAAGCTTTTTATCTATGATTGTTTAACTCGAAGCAGTCGA
CAAGTAATCGAAAATGCTTTTTCGTTAATGTTCTCACAAGGAGATCTCGA
TTATCTTTTTTTAATTTATATTACCACCAATAATTCCTTTGCCAGCCTAC
AATGGACTCCACAGCATATTGAAACTTGCTGCCATATTTTTGAAAAAAAT
GACACATTTCGGTTATTGTTAGAGCCCATTCTTAAACGTTTACCGCAATT
AAACCATTCTAAACAAGACCTTATTAAAGCCCTTATGTATTTTTCAAAAT
CTTTTCTATTTAACCTCCAACATTTCGTCATCGAGATTCCTTCTTTTTCC
TTGCCGACCTATACAGGCAACTCTAATCTTTACAAAGCTTTAAAAAATAT
TGTAAATCAGTGGCTTGCTCAATTACCCGGAAAGCGTCATCTTAACGAAA
AGCATCTCCAACTTTTTTGCTCTCATATTGAACAAATCTTAAAAAATAAA
CAACCTGCTTTAACTGTCGTTTTAATATCTAGTAACTTTATAAATGCTAA
ACTCCTTACAGATACTATCCCACGATATTTTTCTGATAAAGGAATTCATT
TTTATTCTTTTTACTTATTAAGAGATGATATCTATCAAATTCCAAGCTTA
AAACCAGATTTAGTTATCACTCATAGCCGATTAATTCCTTTTGTTAAGAA
TGATCTGGTCAAAGGTGTTACTGTTGCTGAATTTTCTTTTGATAACCCTG
ACTACTCTATTGCTTCAATTCAAAACTTGATATATCAGCTCAAAGATAAA
AAATATCAAGATTTTCTAAACGAGCAATTACAA SEQ ID NO: 89
MPYVKKKKDSFLVETYLEQSIRDKSELVLLLFKSPTIIFSHVAKQTGLTA
VQLKYYGKELDDFPGNNLDITIKKGKIICCFVKPVKEFYLHQLYDTSTIL
KLLVFFIKNGTSSQPLIKFSKKYFLSSSSAYRLRESLIKLLREFGLRVSK
NTIVGEEYRIRYLIAMLYSKFGIVIYPLDHLDNQIIYRFLSQSATNLRTS
PWLEEPFSFYNMLLALSWKRHQFAVSIPQTRIFRQLKKLFIYDCLTRSSR
QVIENAFSLMFSQGDLDYLFLIYITTNNSFASLQWTPQHIETCCHIFEKN
DTFRLLLEPILKRLPQLNHSKQDLIKALMYFSKSFLFNLQHFVIEIPSFS
LPTYTGNSNLYKALKNIVNQWLAQLPGKRHLNEKHLQLFCSHIEQILKNK
QPALTVVLISSNFINAKLLTDTIPRYFSDKGIHFYSEYLLRDDIYQIPSL
KPDLVITHSRLIPFVKNDLVKGVTVAEFSFDNPDYSIASIQNLIYQLKDK KYQDFLNEQLQ
[1065] Orf 78 is thought to be a collagen binding protein (Cbp). An
example of the nucleotide sequence encoding Cbp (SEQ ID NO: 90) and
a Cbp amino acid sequence (SEQ ID NO: 91) are set forth below.
TABLE-US-00147 SEQ ID NO: 90
TTGCAAAAGAGGGATAAAACCAATTATGGAAGCGCTAACAACAAACGACG
ACAAACGACGATCGGATTACTGAAAGTATTTTTGACGTTTGTAGCTCTGA
TAGGAATAGTAGGGTTTTCTATCAGAGCGTTCGGAGCTGAAGAAAAATCT
ACTGAAACTAAAAAAACGTCAGTCATTATTAGAAAATATGCTGAAGGTGA
CTACTCTAAACTTCTAGAGGGAGCAACTTTGCGTTTAACAGGGGAAGATA
TCCCAGATTTTCAAGAAAAAGTCTTCCAAAGTAATGAACAGGAGAAAAAG
ATTGAATTATCAAATGGGACTTATACCTTAACAGAAACATCATCTCCAGA
TGGATATAAAATTACGGAGCCGATTAAGTTTAGAGTAGTGAATAAAAAAG
TATTTATCGTCCAAAAAGATGGTTCTCAAGTGGAAAACCCAAACAAAGAA
CTAGGTTCTCCATATACTATAGAGGCATACAATGATTTTGATGAATTTGG
CTTACTGTCAACACAAAATTATGCGAAATTTTATTATGGAAAAAACTATG
ATGGCAGTTCACAAATTGTTTATTGCTTCAATGCCAACTTGAAATCTCCA
CCTGACTCGGAAGATCATGGTGCTACAATAAATCCTGACTTTACGACTGG
TGATATTAGGTACAGTCATATTGCTGGTTCAGATTTGATAAAATACGCTA
ATACAGCTAGGGATGAAGATCCTCAATTATTTTTAAAACACGTAAAAAAA
GTAATTGAAAATGGGTATCATAAAAAAGGTCAAGCTATTCCATATAACGG
TCTGACTGAGGCACAGTTTCGTGCGGCTACTCAACTGGCAATTTATTATT
TTACAGATAGTGTTGACTTAACTAAGGATAGATTGAAAGACTTCCATGGA
TTTGGAGATATGAATGATCAAACTTTGGGTGTAGCTAAAAAAATTGTAGA
ATACGCTTTGAGTGATGAAGATTCAAAACTAACAAATCTTGATTTCTTCG
TACCTAATAATAGCAAATACCAATCTCTTATTGGGACAGAATACCATCCA
GATGATTTGGTTGACGTGATTCGTATGGAAGATAAAAAGCAAGAAGTTAT
TCCAGTAACTCATAGTTTGACGGTGCAAAAAACAGTAGTCGGTGAGTTGG
GAGATAAGACTAAAGGCTTTCAATTTGAACTTGAGTTGAAAGATAAAACT
GGACAGCCTATTGTTAACACTCTAAAAACTAATAATCAAGATTTAGTAGC
TAAAGATGGGAAATATTCATTTAATCTAAAGCATGGTGACACCATAAGAA
TAGAAGGATTACCGACGGGATATTCTTATACCCTGAAAGAGACTGAAGCT
AAGGATTATATAGTAACTGTTGATAACAAAGTTAGTCAAGAAGCTCAATC
AGCAAGTGAGAATGTCACAGCAGACAAAGAAGTCACTTTTGAAAACCGAA
AAGATCTTGTCCCACCAACTGGTTTGACAACAGATGGGGCTATCTATCTT
TGGTTATTACTACTTGTTCCATTTGGGTTATTGGTTTGGCTATTTGGTCG
TAAAGGGTTAAAAAATGAC SEQ ID NO: 91
MQKRDKTNYGSANNKRRQTTIGLLKVFLTFVALIGIVGFSIRAFGAEEKS
TETKKTSVIIRKYAEGDYSKLLEGATLRLTGEDIPDFQEKVFQSNGTGEK
IELSNGTYTLTETSSPDGYKITEPEKFRVVNKKVFIVQKDGSQVENPNKE
LGSPYTIEAYNDFDEFGLLSTQNYAKFYYGKNYDGSSQIVYCFNANLKSP
PDSEDHGATINPDFTTGDIRYSHIAGSDLIKYANTARDEDPQLFLKHVKK
VIENGYHKKGQATPYNGLTEAQFRAATQLAIYYETDSVDLTKDRLKDPHG
FGDMNDQTLGVAKKIVEYALSDEDSKLTNLDFFVPNNSKYQSLIGTEYHP
DDLVDVIRMEDKKQEVTPVTHSLTVQKTVVGELGDKTKGFQFELELKDKT
GQPIVNTLKTNNQDLVAKDGKYSFNLKHGDTIRIEGLPTGYSYTLKETEA
KDYIVTVDNKVSQEAQSASENVTADKEVTFENRKDLVPPTGLTTDGAIYL
WLLLLVPFGLLVWLFGRKGLKND
[1066] Orf 78 contains an amino acid motif indicative of a cell
wall anchor: SEQ ID NO: 184 VPPTG (shown in italics in SEQ ID NO:
91, above). In some recombinant host cell systems, it may be
preferable to remove this motif to facilitate secretion of a
recombinant Orf 78 protein from the host cell. Alternatively, in
other recombinant host cell systems, it may be preferable to use
the cell wall anchor motif to anchor the recombinantly expressed
protein to the cell wall. The extracellular domain of the expressed
protein may be cleaved during purification or the recombinant
protein may be left attached to either inactivated host cells or
cell membranes in the final composition.
[1067] Three E boxes containing conserved glutamic residues have
been identified in Orf 78. The E-box motifs are underlined in SEQ
ID NO: 91, below. The conserved glutamic acid (E) residues, at
amino acid residues 112, 395, and 447, are marked in bold. The E
box motifs, in particular the conserved glutamic acid residues, are
thought to be important for the formation of oligomeric pilus-like
structures of Orf 78. Preferred fragments of Orf 78 include at
least one conserved glutamic acid residue. Preferably, fragments
include at least one E box motif. TABLE-US-00148 SEQ ID NO: 91
MQKRDKTNYGSANNKRRQTTIGLLKVFLTFVALIGIVGFSIRAFGAEEKS
TETKKTSVIIRKYAEGDYSKLLEGATLRLTGEDIPDFQEKVFQSNGTGEK
IELSNGTYTLTETSSPDGYKITEPEKFRVVNKKVFIVQKDGSQVENPNKE
LGSPYTIEAYNDFDEFGLLSTQNYAKFYYGKNYDGSSQIVYCFNANLKSP
PDSEDHGATINPDFTTGDIRYSHIAGSDLIKYANTARDEDPQLFLKHVKK
VIENGYHKKGQATPYNGLTEAQFRAATQLAIYYETDSVDLTKDRLKDPHG
FGDMNDQTLGVAKKIVEYALSDEDSKLTNLDFFVPNNSKYQSLIGTEYHP
DDLVDVIRMEDKKQEVTPVTHSLTVQKTVVGELGDKTKGFQFELELKDKT
GQPIVNTLKTNNQDLVAKDGKYSFNLKHGDTIRIEGLPTGYSYTLKETEA
KDYIVTVDNKVSQEAQSASENVTADKEVTFENRKDLVPPTGLTTDGAIYL
WLLLLVPFGLLVWLFGRKGLKND
[1068] Orf 79 is thought to be a LepA signal peptidase I. An
example of the nucleotide sequence encoding a LepA signal peptidase
I (SEQ ID NO: 92) and a LepA signal peptidase I amino acid sequence
(SEQ ID NO: 93) are set forth below. TABLE-US-00149 SEQ ID NO: 92
ATGACTAATTACCTAAATCGTTTAAATGAGAATTCACTATTTAAAGCTTT
CATACGGTTAGTACTTAAGATTTCTATTATTGGGTTTCTAGGTTACATTC
TATTTCAGTATGTTTTTGGTGTTATGATTATTAACACTAATGATATGAGT
CCTGCTTTAAGTGCAGGTGACGGTGTTTTATATTATCGTTTGACTGATCG
CTATCATATTAATGATGTGGTGGTCTATGAGGTTGATAACACTTTGAAAG
TTGGTCGAATTGTCGCTCAAGCTGGCGATGAGGTTAGTTTTACGCAAGAA
GGAGGACTGTTGATTAATGGGCATCCACCAGAAAAAGAGGTCCCTTACCT
GACGTATCCTCACTCAAGTGGCCCAAACTTTCCCTATAAAGTTCCTACGG
GTAAGTATTTCATATTGAATGATTATCGTGAAGAACGTTTGGACAGTCGT
TATTATGGGGCGTTACCCGTCAATCAAATAAAAGGGAAAATCTCAACTCT
ATTAAGAGTGAGAGGAATT SEQ ID NO: 93
MTNYLNRLNENSLFKAFIRLVLKISIIGFLGYTLFQYVFGVMIINTNDMS
PALSAGDGVLYYRLTDRYHINDVVVYEVDNTLKVGRTVAQAGDEVSFTQE
GGLLINGHPPEKEVPYLTYPHSSGPNFPYKTPTGKYFILNDYREERLDSR
YYGALPVNQIKGKISTLLRVRGI
[1069] Orf 80 is thought to to be a fimbrial protein. An example of
the nucleotide sequence encoding the fimbrial protein (SEQ ID NO:
94) and a fimbrial protein amino acid sequence (SEQ ID NO: 95) are
set forth below. TABLE-US-00150 SEQ ID NO: 94
TTGGAGAGAGAAAAAATGAAAAAAAACAAATTATTACTTGCTACTGCAAT
CTTAGCAACTGCTTTAGGAACAGCTTCTTTAAATCAAAACGTAAAAGCTG
AGACGGCAGGGGTTGTAACAGGAAAATCACTACAAGTTACAAAGACAATG
ACTTATGATGATGAAGAGGTGTTAATGCCCGAAACCGCCTTTACTTTTAC
TATAGAGCCTGATATGACTGCAAGTGGAAAAGAAGGCAGCCTAGATATTA
AAAATGGAATTGTAGAAGGCTTAGACAAACAAGTAACAGTAAAATATAAG
AATACAGATAAAACATCTCAAAAAACTAAAATAGCACAATTTGATTTTTC
TAAGGTTAAATTTCCAGCTATAGGTGTTTACCGCTATATGGTTTCAGAGA
AAAACGATAAAAAAGACGGAATTACGTACGATGATAAAAAGTGGACTGTA
GATGTTTATGTTGGGAATAAGGCCAATAACGAAGAAGGTTTCGAAGTTCT
ATATATTGTATCAAAAGAAGGTACTTCTAGTACTAAAAAACCAATTGAAT
TTACAAACTCTATTAAAACTACTTCCTTAAAAATTGAAAAACAAATAACT
GGCAATGCAGGAGATCGTAAAAAATCATTCAACTTCACATTAACATTACA
ACCAAGTGAATATTATAAAACTGGATCAGTTGTGAAAATCGAACAGGATG
GAAGTAAAAAAGATGTGACGATAGGAACGCCTTACAAATTTACTTTGGGA
CACGGTAAGAGTGTCATGTTATCGAAATTACCAATTGGTATCAATTACTA
TCTTAGTGAAGACGAAGCGAATAAAGACGGCTACACTACAACGGCAACAT
TAAAAGAACAAGGCAAAGAAAAGAGTTCCGATTTCACTTTGAGTACTCAA
AACCAGAAAACAGACGAATCTGCTGACGAAATCGTTGTCACAAATAAGCG
TGACACTCAAGTTCCAACTGGTGTTGTAGGGACCCTTGCTCCATTTGCAG
TTCTTAGCATTGTGGCTATTGGTGGAGTTATCTATATTACAAAACGTAAA AAAGCT SEQ ID
NO: 95 MEREKMKKNKLLLATAILATALGTASLNQNVKAETAGVVTGKSLQVTKTM
TYDDEEVLMPETAFTPTIEPDMTASGKEGSLDIKNGIVEGLDKQVTVKYK
NTDKTSQKTKIAQFDFSKVKFPAIGVYRYMVSEKNDKKDGITYDDKKWTV
DVYVGNKANNEEGPEVLYIVSKEGTSSTKKPIEFTNSIKTTSLKIEKQIT
GNAGDRKKSFNFTLTLQPSEYYKTGSVVKIEQDGSKKDVTIGTPYKFTLG
HGKSVMLSKLPIGINYYLSEDEANKDGYTTTATLKEQGKEKSSDFTLSTQ
NQKTDESADEIVVTNKRDTQVPTGVVGTLAPFAVLSIVAIGGVIYITKRK KA
[1070] Orf 82 contains an amino acid motif indicative of a cell
wall anchor: SEQ ID NO: 140 QVPTG (shown in italics in SEQ ID NO:
95, above). In some recombinant host cell systems, it may be
preferable to remove this motif to facilitate secretion of a
recombinant Orf 82 protein from the host cell. Alternatively, in
other recombinant host cell systems, it may be preferable to use
the cell wall anchor motif to anchor the recombinantly expressed
protein to the cell wall. The extracellular domain of the expressed
protein may be cleaved during purification or the recombinant
protein may be left attached to either inactivated host cells or
cell membranes in the final composition.
[1071] An E box containing a conserved glutamic residue has been
identified in Orf 80. The E-box motif is underlined in SEQ ID NO:
95, below. The conserved glutamic acid (E), at amino acid residue
270, is marked in bold. The E box motif, in particular the
conserved glutamic acid residue, is thought to be important for the
formation of oligomeric pilus-like structures of Orf 80. Preferred
fragments of Orf 80 include at least one conserved glutamic acid
residue. Preferably, fragments include at least one E box motif.
TABLE-US-00151 SEQ ID NO: 95
MEREKMKKNKLLLATAILATALGTASLNQNVKAETAGVVTGKSLQVTKTM
TYDDEEVLMPETAFTPTIEPDMTASGKEGSLDIKNGIVEGLDKQVTVKYK
NTDKTSQKTKIAQFDFSKVKFPAIGVYRYMVSEKNDKKDGITYDDKKWTV
DVYVGNKANNEEGPEVLYIVSKEGTSSTKKPIEFTNSIKTTSLKIEKQIT
GNAGDRKKSFNFTLTLQPSEYYKTGSVVKIEQDGSKKDVTIGTPYKFTLG
HGKSVMLSKLPIGINYYLSEDEANKDGYTTTATLKEQGKEKSSDFTLSTQ
NQKTDESADEIVVTNKRDTQVPTGVVGTLAPFAVLSIVAIGGVIYITKRK KA
[1072] Orf 81 is thought to to be a SrtC2 type sortase. An example
of the nucleotide sequence encoding the SrtC2 sortase (SEQ ID NO:
96) and a SrtC2 sortase amino acid sequence (SEQ ID NO: 97) are set
forth below. TABLE-US-00152 SEQ ID NO: 96
GTGATTAGTCAAAGAATGATGATGACAATTGTACAGGTTATCAATAAAGC
CATTGATACTCTCATTCTTATCTTTTGTTTAGTCGTACTATTTTTAGCTG
GTTTTGGTTTGTGGGATTCTTATCATCTCTATCAACAAGCAGACGCTTCT
AATTTCAAAAAATTTAAAACAGCTCAACAACAGCCTAAATTTGAAGACTT
GTTAGCTTTGAATGAGGATGTCATTGGTTGGTTAAATATCCCAGGGACTC
ATATTGATTATCCTCTAGTTCAGGGAAAAACGAATTTAGAGTATATTAAT
AAAGCAGTTGATGGCAGTGTTGCCATGTCTGGTAGTTTATTTTTAGATAC
ACGGAATCATAATGATTTTACGGACGATTACTCTCTGATTTATGGCCATC
ATATGGCAGGTAATGCCATGTTTGGCGAAATTCCAAAATTTTTAAAAAAG
GATTTTTTCAACAAACATAATAAAGCTATCATTGAAACAAAAGAGAGAAA
AAAACTAACCGTCACTATTTTTGCTTGTCTCAAGACAGATGCCTTTGACC
AGTTAGTTTTTAATCCTAATGCTATTACCAATCAAGACCAACAAAAGCAG
CTCGTTGATTATATCAGTAAAAGATCAAAACAATTTAAACCTGTTAAATT
GAAGCATCATACAAAGTTCGTTGCTTTTTCAACGTGTGAAAATTTTTCTA
CTGACAATCGTGTTATCGTTGTCGGTACTATTCAAGAA SEQ ID NO: 97
MISQRMMMTIVQVINKAIDTLILIFCLVVLFLAGFGLWDSYHLYQQADAS
NFKKFKTAQQQPKFEDLLALNEDVIGWLNIPGTHIDYPLVQGKTNLEYIN
KAVDGSVAMSGSLFLDTRNHNDFTDDYSLIYGHHMAGNAMFGEIPKFLKK
DFFNKHNKAIIETKERKKLTVTIFACLKTDAFDQLVFNPNAITNQDQQKQ
LVDYISKRSKQFKPVKLKHHTKFVAFSTCENFSTDNRVIVVGTIQE
[1073] Orf 82 is referred to as a hypothetical protein. It contains
a sortase substrate motif LPXAG shown in italics in SEQ ID NO: 99.
An example of the nucleotide sequence encoding the hypothetical
protein (SEQ ID NO: 98) and a hypothetical protein amino acid
sequence (SEQ ID NO: 99) are set forth below. TABLE-US-00153 SEQ ID
NO: 98 TTGCTTTTTCAACGTGTGAAAATTTTTCTACTGACAATCGTGTTATCGTT
GTCGGTACTATTCAAGAATAACGAAAGGAGGAGACTTTTGAGAAAATATT
GGAAAATGTTATTTTCTGTCGTAATGATATTAACCATGCTGGCCTTTAAT
CAGACTGTTTTAGCAAAAGACAGCACTGTTCAAACTAGCATTAGTGTCGA
AAATGTCTTAGAGAGAGCAGGCGATAGTACCCCATTTTCGGTTGCATTAG
AATCAATTGATGCGATGAAAACAATAGACGAAATAACAATTGCTGGTTCT
GGAAAAGCAAGCTTTTCCCCTCTGACCTTCACAACAGTTGGGCAATATAC
TTATCGTGTTTATCAGAAGCCTTCACAAAATAAAGATTATCAAGCAGATA
CTACTGTATTTGACGTTCTTGTCTATGTGACCTATGATGAAGATGGGACT
CTAGTCGCAAAAGTTATTTCTCGAAGGGCTGGAGACGAAGAAAAATCAGC
GATTACTTTTAAGCCCAAACGGTTAGTAAAACCAATACCGCCTAGACAAC
CTAACATCCCTAAAACCCCATTACCATTAGCTGGTGAAGTAAAAAGTTTA
TTGGGTATCTTAAGTATCGTATTACTGGGGTTACTAGTTCTTCTTTATGT
TAAAAAACTGAAGAGTAGGCTA SEQ ID NO: 99
MLFQRVKIFLLTIVLSLSVLFKNNERRRLLRKYWKMLFSVVMILTMLAFN
QTVLAKDSTVQTSISVENVLEPAGDSTPFSVALESIDAMKTIDEITIAGS
GKASFSPLTFTTVGQYTYRVYQKPSQNKDYQADTTVFDVLVYVTYDEDGT
LVAKVISRRAGDEEKSAITFKPKRLVKPIPPRQPNIPKTPLPLAGEVKSL
LGILSTVLLGLLVLLYVKKLKSRL
[1074] Orf 82 contains an amino acid motif indicative of a cell
wall anchor: SEQ ID NO: 185 LPLAG (shown in italics in SEQ ID NO:
99, above). In some recombinant host cell systems, it may be
preferable to remove this motif to facilitate secretion of a
recombinant Orf 82 protein from the host cell. Alternatively, in
other recombinant host cell systems, it may be preferable to use
the cell wall anchor motif to anchor the recombinantly expressed
protein to the cell wall. The extracellular domain of the expressed
protein may be cleaved during purification or the recombinant
protein may be left attached to either inactivated host cells or
cell membranes in the final composition.
[1075] A pilin motif, discussed above, containing a conserved
lysine (K) residue has also been identified in Orf 82. The pilin
motif sequence is underlined in SEQ ID NO: 99, below. Conserved
lysine (K) residues are also marked in bold, at amino acid residues
173 and 188. The pilin sequence, in particular the conserved lysine
residues, are thought to be important for the formation of
oligomeric, pilus-like structures. Preferred fragments of Orf 82
include at least one conserved lysine residue. Preferably,
fragments include the pilin sequence. TABLE-US-00154 SEQ ID NO: 99
MLFQRVKIFLLTIVLSLSVLFKNNERRRLLRKYWKMLFSVVMILTMLAFN
QTVLAKDSTVQTSISVENVLERAGDSTPFSVALESIDAMKTIDEITTAGS
GKASFSPLTFTTVGQYTYRVYQKPSQNKDYQADTTVFDVLVYVTYDEDGT
LVAKVISRRAGDEEKSAITFKPKRLVKPIPPRQPNIPKTPLPLAGEVKSL
LGILSIVLLGLLVLLYVKKLKSRL
[1076] An E box containing a conserved glutamic residue has been
identified in Orf 82. The E-box motif is underlined in SEQ ID NO:
99, below. The conserved glutamic acid (E), at amino acid residue
163, is marked in bold. The E box motif, in particular the
conserved glutamic acid residue, is thought to be important for the
formation of oligomeric pilus-like structures of Orf 82. Preferred
fragments of Orf 82 include the conserved glutamic acid residue.
Preferably, fragments include the E box motif. TABLE-US-00155 SEQ
ID NO: 99 MLFQRVKIFLLTIVLSLSVLFKNNERRRLLRKYWKMLFSVVMILTMLAFN
QTVLAKDSTVQTSISVENVLEPAGDSTPFSVALESIDAMKTIDEITIAGS
GKASFSPLTFTTVGQYTYRVYQKPSQNKDYQADTTVFDVLVYVTYDEDGT
LVAKVISRRAGDEEKSAITFKPKRLVKPIPPRQPNIPKTPLPLAGEVKSL
LGILSTVLLGLLVLLYVKKLKSRL
[1077] Orf 83 is thought to to be a multiple sugar metabolism
regulator protein. An example of a nucleotide sequence encoding the
sugar metabolism regulator protein (SEQ ID NO: 100) and a sugar
metabolism regulator protein amino acid sequence (SEQ ID NO: 101)
are set forth below. TABLE-US-00156 SEQ ID NO: 100
ATGATACAACTAAGGATGGGGGCAATCTATCAAATGGTTATATTCGATTT
AAAACATGTGCAAACATTACACAGCTTGTCTCAATTACCTATTTCAGTGA
TGTCACAAGATAAGGCACTTATTCAAGTATATGGTAATGACGACTATTTA
TTATGTTACTATCAATTTTTAAAGCATCTAGCTATTCCTCAAGCTGCACA
AGATGTTATTTTTTATGAGGGTTTATTTGAAGAGTCCTTTATGATTTTTC
CTCTTTGTCACTACATTATTGCCATTGGACCTTTCTATCCTTATTCACTT
AATAAAGACTATCAGGAACAATTAGCTAATAATTTTTTAAAACATTCTTC
TCATCGTAGCAAAGAAGAGCTCTTGTCCTATATGGCACTTGTCCCACATT
TTCCAATTAATAATGTGCGGAACCTTTTGATAGCTATTGACGCTTTTTTT
GACACACAATTTGAGACGACTTGCCAACAAACGATTCATCAATTGTTGCA
GCATTCAAAACAGATGACTGCTGATCCTGATATCATTCATCGCCTTAAGC
ATATTAGCAAAGCATCTAGCCAATTACCGCCTGTTTTAGAGCACCTAAAT
CATATTATGGATCTGGTAAAGCTAGGCAATCCACAATTGCTCAAGCAAGA
AATCAATCGCATCCCCTTATCAAGTATCACCTCATCTTCTATTTCTGCTC
TAAGGGCGGAAAGAACCTCACTGTTATCTATTTCAACTAGGTTACTGGAA
TTCAGTTTTGTAGAAAATACTGACGTAGCAAAGCATTATAGCCTTGTCAA
ATACTACATGGCCTTAAATGAAGAAGCGAGTGACTTGCTCAAAGTTTTGA
GAATTCGCTGTGCAGCTATCATCCATTTTTCCGAATCATTAACCAATAAA
AGTATTTCTGATAAACGTCAAATGTACAATAGTGTGCTTCATTATGTCGA
TAGTCACCTGTATTCCAAATTAAAGGTATCTGATATCGCTAAGCGCCTAT
ATGTTTCCGAATCTCACTTACGTTCAGTCTTTAAAAAATACTCAAATGTT
TCCTTACAACATTATATTCTAAGTACAAAAATCAAAGAAGCTCAACTACT
CTTAAAACGAGGAATTCCTGTTGGAGAAGTGGCTAAAAGCTTATATTTTT
ATGACACTACCCATTTTCATAAAATCTTTAAAAAATACACGGGTATTTCT
TCAAAAGACTATCTTGCTAAATACCGAGATAATATT SEQ ID NO: 101
MIQLRMGAIYQMVIFDLKHVQTLHSLSQLPISVMSQDKALIQVYGNDDYL
LCYYQFLKHLAIPQAAQDVIFYEGLFEESFMIFPLCHYIIAIGPFYPYSL
NKDYQEQLANNFLKHSSHRSKEELLSYMALVPHFPINNVRNLLIAIDAFF
DTQFETTCQQTIHQLLQHSKQMTADPDIIHRLKHISKASSQLPPVLEHLN
HIMDLVKLGNPQLLKQEINRIPLSSITSSSISALRAEKNLTVIYLTRLLE
FSFVENTDVAKHYSLVKYYMALNEEASDLLKVLRIRCAAIIHFSESLTNK
SISDKRQMYNSVLHYVDSHLYSKLKVSDIAKRLYVSESHLRSVFKKYSNV
SLQHYILSTKIKEAQLLLKRGIPVGEVAKSLYFYDTTHFHKIFKKYTGTS SKDYLAKYRDNI
[1078] Orf 84 is thought to to be a F2-like fibronectin-binding
protein. An example of a nucleotide sequence encoding the F2-like
fibronectin-binding protein (SEQ ID NO: 102) and a F2-like
fibronectin-binding protein amino acid sequence (SEQ ID NO: 103)
are set forth below. TABLE-US-00157 SEQ ID NO: 102
ATGACACAAAAAAATAGCTATAAGTTAAGCTTCCTGTTATCCCTAACAGG
ATTTATTTTAGGTTTATTATTGGTTTTTATAGGATTGTCCGGAGTATCAG
TAGGACATGCGGAAACAAGAAATGGAGCAAACAAACAAGGAGCTTTTGAA
ATCAAGAAAAATAAAAGTCAAGAAGAATATAATTATGAAGTTTATGATAA
CAGAAACATACTTCAGGATGGGGAACATAAACTTGAAATAAAAAGAGTTG
ATGGGACAGGTAAAACTTATCAAGGTTTTTGCTTTCAGTTAACGAAAAAT
TTTCCCACTGCTCAAGGTGTAAGTAAAAAGCTGTATAAAAAATTGAGTAG
TAGTGATGAAGAAACACTAAAGCAATATGCCTCTAAGTATACAAGTAATA
GGAGAGGAGATACTAGTGGTAATCTTAAAAAGCAAATTGCTAAGGTTCTG
ACAGAAGGTTACCCAACTAACAAAAGTGATTGGTTAAATGGATTGACTGA
AAACGAAAAAATAGAAGTAACCCAGGATGCAATTTGGTATTTTACAGAAA
CGACAGTTCCGGCTGATAGAAGTTATACGAATCGCAACGTAAATAGTCAA
AAAATGAAAGAAGTGTATCAAAAGCTAATTGATACAACAGATATAGATAA
ATATGAAGATGTACAATTTGATTTATTTGTGCCACAAGATACAAACTTAC
AGGCAGTAATTAGTGTAGAGCCTGTTATCGAAAGCCTTCCTTGGACATCG
TTGAAGCCAATAGCCCAGAAGGATATCACTGCCAAAAAAATCTGGGTAGA
TGCACCTAAAGAAAAACCAATTATTTATTTTAAGCTATATAGACAGCTGC
CTGGAGAAAAGGAAGTAGCAGTGGATGACGCTGAGCTAAAACAGATAAAT
AGTGAAGGTCAACAAGAAATATCAGTAACTTGGACAAATCAACTTGTTAC
AGATGAAAAAGGAATGGCTTACATTTATTCTGTAAAAGAAGTAGATAAAA
ATGGCGAGTTACTTGAGCCAAAAGATTATATCAAGAAGGAAGATGGACTT
ACAGTTACTAATACTTATGTAAAGCCAACTAGTGGGCACTATGATATAGA
AGTGACATTTGGAAATGGACATATTGATATTACAGAAGATACTACACCAG
ATATTGTTTCAGGTGAAAACCAAATGAAGCAAATAGAGGGAGAAGATAGT
AAGCCTATTGATGAAGTAACGGAAAATAATTTAATTGAATTTGGTAAAAA
CACGATGCCAGGTGAAGAAGATGGCACAAATTCTAATAAGTATGAAGAAG
TCGAAGACTCACGCCCAGTTGATACCTTGTCAGGTTTATCAAGTGAGCAA
GGTCAGTCCGGTGATATGACAATTGAAGAAGATAGTGCTACCCATATTAA
ATTCTCAAAACGTGATATTGACGGCAAAGAGTTAGCTGGTGCAACTATGG
AGTTGCGTGATTCATCTGGTAAAACTATTAGTACATGGATTTCAGATGGA
CAAGTGAAAGATTTCTACCTGATGCCAGGAAAATATACATTTGTCGAAAC
CGCAGCACCAGACGGTTATGAGATAGCAACTGCTATTACCTTTACAGTTA
ATGAGCAAGGTCAGGTTACTGTAAATGGCAAAGCAACTAAAGGTGACGCT
CATATTGTCATGGTTGATGCTTACAAGCCAACTAAGGGTTCAGGTCAGGT
TATTGATATTGAAGAAAAGCTTCCAGACGAGCAGGGCCATTCTGGCTCAA
CTACTGAAATAGAAGATAGCAAGTCTTCAGACGTTATCATTGGTGGTCAG
GGGCAGATTGTCGAGACAACAGAGGATACCCAAACTGGCATGCACGGGGA
TTCTGGTTGTAAAACGGAAGTCGAAGATACTAAACTAGTACAATCCTTCC
ACTTTGATAACAAGGAATCAGAAAGTAACTCTGAGATTCCTAAAAAAGAT
AAGCCAAAGAGTAATACTAGTTTACCAGCAACTGGTGAGAAGCAACATAA
TATGTTCTTTTGGATGGTTACTTCTTGCTCACTTATTAGTAGTGTTTTTG
TAATATCACTAAAAACTAAAAAACGCCTATCATCATGT SEQ ID NO: 103
MTQKNSYKLSFLLSLTGFILGLLLVFIGLSGVSVGHAETRNGANKQGAFE
IKKNKSQEEYNYEVYDNRNILQDGEHKLEIKRVDGTGKTYQGFCFQLTKN
FPTAQGVSKKLYKKLSSSDEETLKQYASKYTSNRRGDTSGNLKKQIAKVL
TEGYPTNKSDWLNGLTENEKIEVTQDAIWYFTETTVPADRSYTNRNVNSQ
KMKEVYQKLIDTTDIDKYEDVQFDLFVPQDTNLQAVISVEPVIESLPWTS
LKPIAQKDITAKKIWVDAPKEKPIIYFKLYRQLPGEKEVAVDDAELKQIN
SEGQQEISVTWTNQLVTDEKGMAYIYSVKEVDKNGELLEPKDYIKKEDGL
TVTNTYVKPTSGHYDIEVTFGNGHIDITEDTTPDIVSGENQMKQIEGEDS
KPIDEVTENNLIEFGKNTMPGEEDGTNSNKYEEVEDSRPVDTLSGLSSEQ
GQSGDMTIEEDSATHIKFSKRDIDGKELAGATMELRDSSGKTISTWISDG
QVKDFYLMPGKYTFVETAAPDGYEIATAITFTVNEQGQVTVNGKATKGDA
HIVMVDAYKPTKGSGQVIDIEEKLPDEQGHSGSTTEIEDSKSSDVIIGGQ
GQIVETTEDTQTGMHGDSGCKTEVEDTKLVQSFHFDNKESESNSEIPKKD
KPKSNTSLPATGEKQHNMFFWMVTSCSLISSVFVISLKTKKRLSSC
[1079] Orf 84 contains an amino acid motif indicative of a cell
wall anchor: SEQ ID NO: 181 LPATG (shown in italics in SEQ ID NO:
103, above). In some recombinant host cell systems, it may be
preferable to remove this motif to facilitate secretion of a
recombinant Orf 84 protein from the host cell. Alternatively, in
other recombinant host cell systems, it may be preferable to use
the cell wall anchor motif to anchor the recombinantly expressed
protein to the cell wall. The extracellular domain of the expressed
protein may be cleaved during purification or the recombinant
protein may be left attached to either inactivated host cells or
cell membranes in the final composition.
[1080] A pilin motif, discussed above, containing a conserved
lysine (K) residue has also been identified in Orf 84. The pilin
motif sequence is underlined in SEQ ID NO: 103, below. A conserved
lysine (K) residue is also marked in bold, at amino acid residue
270. The pilin sequence, in particular the conserved lysine
residue, is thought to be important for the formation of
oligomeric, pilus-like structures. Preferred fragments of Orf 84
include the conserved lysine residue. Preferably, fragments include
the pilin sequence. TABLE-US-00158 SEQ ID NO: 103
MTQKNSYKLSFLLSLTGFILGLLLVFIGLSGVSVGHAETRNGANKQGAFE
IKKNKSQEEYNYEVYDNRNILQDGEHKLEIKRVDGTGKTYQGFCFQLTKN
FPTAQGVSKKLYKKLSSSDEETLKQYASKYTSNRRGDTSGNLKKQIAKVL
TEGYPTNKSDWLNGLTENEKIEVTQDAIWYFTETTVPADRSYTNRNVNSQ
KMKEVYQKLIDTTDIDKYEDVQFDLFVPQDTNLQAVTSVEPVIESLPWTS
LKPIAQKDITAKKIWVDAPKEKPIIYFKLYRQLPGEKEVAVDDAELKQIN
SEGQQEISVTWTNQLVTDEKGMAYIYSVKEVDKNGELLEPKDYIKKEDGL
TVTNTYVKPTSGHYDIEVTFGNGHIDITEDTTPDIVSGENQMKQIEGEDS
KPIDEVTENNLIEPGKNTMPGEEDGTNSNKYEEVEDSRPVDTLSGLSSEQ
GQSGDMTIEEDSATHIKFSKRDIDGKELAGATMELRDSSGKTISTWISDG
QVKDFYLMPGKYTFVETAAPDGYEIATAITFTVNEQGQVTVNGKATKGDA
HIVMVDAYKPTKGSGQVIDIEEKLPDEQGHSGSTTEIEDSKSSDVIIGGQ
GQIVETTEDTQTGMHGDSGCKTEVEDTKLVQSFHFDNKESESNSEIPKKD
KPKSNTSLPATGEKQHNMFEWMVTSCSLISSVFVISLKTKKRLSSC
[1081] An E box containing a conserved glutamic residue has been
identified in Orf 84. The E-box motif is underlined in SEQ ID NO:
103, below. The conserved glutamic acid (E), at amino acid residue
516, is marked in bold. The E box motif, in particular the
conserved glutamic acid residue, is thought to be important for the
formation of oligomeric pilus-like structures of Orf 84. Preferred
fragments of Orf 84 include the conserved glutamic acid residue.
Preferably, fragments include the E box motif. TABLE-US-00159 SEQ
ID NO: 103 MTQKNSYKLSFLLSLTGFILGLLLVFIGLSGVSVGHAETRNGANKQGAFE
IKKNKSQEEYNYEVYDNRNILQDGEHKLEIKRVDGTGKTYQGFCFQLTKN
FPTAQGVSKKLYKKLSSSDEETLKQYASKYTSNRRGDTSGNLKKQIAKVL
TEGYPTNKSDWLNGLTENEKIEVTQDAIWYFTETTVPADRSYTNRNVNSQ
KMKEVYQKLIDTTDIDKYEDVQFDLFVPQDTNLQAVTSVEPVIESLPWTS
LKPIAQKDITAKKIWVDAPKEKPIIYFKLYRQLPGEKEVAVDDAELKQIN
SEGQQEISVTWTNQLVTDEKGMAYIYSVKEVDKNGELLEPKDYIKKEDGL
TVTNTYVKPTSGHYDIEVTFGNGHIDITEDTTPDIVSGENQMKQIEGEDS
KPIDEVTENNLIEPGKNTMPGEEDGTNSNKYEEVEDSRPVDTLSGLSSEQ
GQSGDMTIEEDSATHIKFSKRDIDGKELAGATMELRDSSGKTISTWISDG
QVKDFYLMPGKYTFVETAAPDGYEIATAITFTVNEQGQVTVNGKATKGDA
HIVMVDAYKPTKGSGQVIDIEEKLPDEQGHSGSTTEIEDSKSSDVIIGGQ
GQIVETTEDTQTGMHGDSGCKTEVEDTKLVQSFHFDNKESESNSEIPKKD
KPKSNTSLPATGEKQHNMFEWMVTSCSLISSVFVISLKTKKRLSSC
[1082] Examples of GAS AI-3 sequences from M18 strain isolate MGAS
8232 are set forth below.
[1083] SpyM18.sub.--0125 is a negative transcriptional regulator
(Nra). An example of SpyM18.sub.--0125 is set forth in SEQ ID NO:
72. TABLE-US-00160 SEQ ID NO: 72
MPYVKKKKDSFLVETYLEQSIRDKSELVLLLFKSPTIIFSHVAKQTGLTA
VQLKYYCKELDDFFGNNLDITIKKGKIICCFVKPVKEFYLHQLYDTSTIL
KLLVFFIKNGTTSQPLIKFSKKYFLSSSSAYRLRESLIKLLREFGLRVSK
NTIVGEEYRIRYLIAMLYSKFGIVIYPLDHLDNQIIYRFLSQSATNLRTS
PWLEEPFSFYNMLLALS
[1084] SpyM18.sub.--0126 is thought to be a collagen binding
protein (CBP). An example of SpyM18.sub.--0126 is set forth in SEQ
ID NO: 73. TABLE-US-00161 SEQ ID NO: 73
MQKRDKTNYGSANNKRRQTTIGLLKVFLTFVALIGIVGFSIRAFGAEEQS
TETKKTSVIIRKYAEGDYSKLLEGATLKLAQIEGSGFQEQSFESSTSGQK
LQLSDGTYILTETKSPQGYEIAEPITFKVTAGKVFIKGKDGQFVENQNKE
VAEPYSVTAYNDFDDSGFINPKTFTPYGKFYYAKNANGTSQVVYCFNVDL
HSPPDSLDKGETIDPDFNEGKEIKYTHILGADLFSYANNPRASTNDELLS
QVKKVLEKGYRDDSTTYANLTSVEFRAATQLAIYYFTDSVDLDNLADYHG
FGALTTEALNATKEIVAYAEDRANLPNISNLDFYVPNSNKYQSLIGTQYH
PESLVDIIRMEDKQAPIIPITHKLTISKTVTGTIADKKKEFNFEIHLKSS
DGQAISGTYPTNSGELTVTDGKATFTLKDGESLIVEGLPSGYSYEITETG
ASDYEVSVNGKNAPDGKATKASVKEDETITFENRKDLVPPTGLTTDGAIY
LWLLLLVLLGLWVWLIGRKGLKND
[1085] SpyM18.sub.--0126 contains an amino acid motif indicative of
a cell wall anchor: SEQ II ID NO: 184 VPPTG (shown in italics in
SEQ ID NO: 73, above). In some recombinant host cell systems, it
may be preferable to remove this motif to facilitate secretion of a
recombinant SpyM18.sub.--0126 protein from the host cell.
Alternatively, in other recombinant host cell systems, it may be
preferable to use the cell wall anchor motif to anchor the
recombinantly expressed protein to the cell wall. The extracellular
domain of the expressed protein may be cleaved during purification
or the recombinant protein may be left attached to either
inactivated host cells or cell membranes in the final
composition.
[1086] A pilin motif, discussed above, containing a conserved
lysine (K) residue has also been identified in SpyM18.sub.--0126.
The pilin motif sequence is underlined in SEQ ID NO: 73, below.
Conserved lysine (K) residues are also marked in bold, at amino
acid residues 172 and 179. The pilin sequence, in particular the
conserved lysine residues, are thought to be important for the
formation of oligomeric, pilus-like structures. Preferred fragments
of SpyM18.sub.--0126 include at least one conserved lysine residue.
Preferably, fragments include the pilin sequence. TABLE-US-00162
SEQ ID NO: 73 MQKRDKTNYGSANNKRRQTTIGLLKVFLTFVALIGIVGFSIRAFGAEEQS
TETKKTSVIIRKYAEGDYSKLLEGATLKLAQIEGSGFQEQSFESSTSGQK
LQLSDGTYILTETKSPQGYEIAEPITFKVTAGKVFIKGKDGQFVENQNKE
VAEPYSVTAYNDFDDSGFINPKTFTPYGKFYYAKNANGTSQVVYCFNVDL
HSPPDSLDKGETIDPDFNEGKEIKYTHILGADLFSYANNPRASTNDELLS
QVKKVLEKGYRDDSTTYANLTSVEFRAATQLAIYYFTDSVDLDNLADYHG
FGALTTEALNATKEIVAYAEDRANLPNISNLDFYVPNSNKYQSLIGTQYH
PESLVDIIRMEDKQAPIIPITHKLTISKTVTGTIADKKKEFNFEIHLKSS
DGQAISGTYPTNSGELTVTDGKATFTLKDGESLIVEGLPSGYSYEITETG
ASDYEVSVNGKNAPDGKATKASVKEDETITFENRKDLVPPTGLTTDGAIY
LWLLLLVLLGLWVWLIGRKGLKND
[1087] Three E boxes containing conserved glutamic residues have
been identified in SpyM18.sub.--0126. The E-box motifs are
underlined in SEQ ID NO: 73, below. The conserved glutamic acid (E)
residues, at amino acid residues 112, 257, and 415, are marked in
bold. The E box motifs, in particular the conserved glutamic acid
residues, are thought to be important for the formation of
oligomeric pilus-like structures of SpyM18.sub.--0126. Preferred
fragments of SpyM18.sub.--0126 include at least one conserved
glutamic acid residue. Preferably, fragments include at least one E
box motif. TABLE-US-00163 SEQ ID NO: 73
MQKRDKTNYGSANNKRRQTTIGLLKVFLTFVALIGIVGFSIRAFGAEEQS
TETKKTSVIIRKYAEGDYSKLLEGATLKLAQIEGSGFQEQSFESSTSGQK
LQLSDGTYILTETKSPQGYEIAEPITFKVTAGKVFIKGKDGQFVENQNKE
VAEPYSVTAYNDFDDSGFINPKTFTPYGKFYYAKNANGTSQVVYCFNVDL
HSPPDSLDKGETIDPDFNEGKEIKYTHILGADLFSYANNPRASTNDELLS
QVKKVLEKGYRDDSTTYANLTSVEFRAATQLAIYYFTDSVDLDNLADYHG
FGALTTEALNATKEIVAYAEDRANLPNISNLDFYVPNSNKYQSLIGTQYH
PESLVDIIRMEDKQAPIIPITHKLTISKTVTGTIADKKKEFNFEIHLKSS
DGQAISGTYPTNSGELTVTDGKATFTLKDGESLIVEGLPSGYSYEITETG
ASDYEVSVNGKNAPDGKATKASVKEDETITFENRKDLVPPTGLTTDGAIY
LWLLLLVLLGLWVWLIGRKGLKND
[1088] SpyM18.sub.--0127 is a LepA protein. An example of
SpyM18.sub.--0127 is shown in SEQ ID NO: 74. TABLE-US-00164 SEQ ID
NO: 74 MTNYLNRLNENPLEKAFIRLVLKISIIGFLGYILFQYIFGVMIINTNVMS
PALSAGDGILYYRLTDRYHINDVVVYEVDNTLKVGRIVAQAGDEVSFTQE
GGLLINGHPPEKEVPYLTYPHSSGPNFPYKVPTGTYFILNDYREERLDSR
YYGALPINQIKGKISTLLRVRGI
[1089] SpyM18.sub.--0128 is thought to be a fimbrial protein. An
example of SypM18.sub.--0128 is shown in SEQ ID NO: 75.
TABLE-US-00165 SEQ ID NO: 75
MKKNKLLLATAILATALGTASLNQNVKAETAGVIDGSTLVVKKTFPSYTD
DKVLMPKADYTFKVEADDNAKGKTKDGLDIKPGVTDGLENTKTIHYGNSD
KTTAKEKSVNFDFANVKFPGVGVYRYTVSEVNGNKAGIAYDSQQWTVDVY
VVNREDGGFEAKYIVSTEGGQSDKKPVLFKNFFDTTSLKVTKKVTGNTGE
HQRSFSFTLLLTPNECFEKGQVVNILQGGETKKVVIGEEYSFTLKDKESV
TLSQLPVGIEYKVTEEDVTKDGYKTSATLKDGDVTDGYNLGDSKTTDKST
DEIVVTNKRDTQVPTGVVGTLAPFAVLSIVATGGVIYITKRKKA
[1090] SpyM18.sub.--0128 contains an amino acid motif indicative of
a cell wall anchor: SEQ ID NO: 140 QVPTG (shown in italics in SEQ
ID NO: 75, above). In some recombinant host cell systems, it may be
preferable to remove this motif to facilitate secretion of a
recombinant SpyM18.sub.--0128 protein from the host cell.
Alternatively, in other recombinant host cell systems, it may be
preferable to use the cell wall anchor motif to anchor the
recombinantly expressed protein to the cell wall. The extracellular
domain of the expressed protein may be cleaved during purification
or the recombinant protein may be left attached to either
inactivated host cells or cell membranes in the final
composition.
[1091] A pilin motif, discussed above, containing a conserved
lysine (K) residue has also been identified in SpyM18.sub.--0128.
The pilin motif sequence is underlined in SEQ ID NO: 75, below. A
conserved lysine (K) residue is also marked in bold, at amino acid
residue 57. The pilin sequence, in particular the conserved lysine
residue, is thought to be important for the formation of
oligomeric, pilus-like structures. Preferred fragments of
SpyM18.sub.--0128 include the conserved lysine residue. Preferably,
fragments include at least one pilin sequence. TABLE-US-00166 SEQ
ID NO: 75 MKKNKLLLATAILATALGTASLNQNVKAETAGVIDGSTLVVKKTFPSYTD
DKVLMPKADYTFKVEADDNAKGKTKDGLDIKPGVIDGLENTKTIHYGNSD
KTTAKEKSVNFDFANVKFPGVGVYRYTVSEVNGNKAGIAYDSQQWTVDVY
VVNREDGGFEAKYIVSTEGGQSDKKPVLFKNFFDTTSLKVTKKVTGNTGE
HQRSFSFTLLLTPNECFEKGQVVNILQGGETKKVVIGEEYSFTLKDKESV
TLSQLPVGIEYKVTEEDVTKDGYKTSATLKDGDVTDGYNLGDSKTTDKST
DEIVVTNKRDTQVPTGVVGTLAPFAVLSIVAIGGVIYITKRKKA
[1092] An E box containing a conserved glutamic residue has been
identified in SpyM18.sub.--0128. The E-box motif is underlined in
SEQ ID NO: 75, below. The conserved glutamic acid (E), at amino
acid residue 266, is marked in bold. The E box motif, in particular
the conserved glutamic acid residue, is thought to be important for
the formation of oligomeric pilus-like structures of
SpyM18.sub.--0128. Preferred fragments of SpyM18.sub.--0128 include
the conserved glutamic acid residue. Preferably, fragments include
the E box motif. TABLE-US-00167 SEQ ID NO: 75
MKKNKLLLATAILATALGTASLNQNVKAETAGVIDGSTLVVKKTFPSYTD
DKVLMPKADYTFKVEADDNAKGKTKDGLDIKPGVIDGLENTKTIHYGNSD
KTTAKEKSVNFDFANVKFPGVGVYRYTVSEVNGNKAGIAYDSQQWTVDVY
VVNREDGGFEAKYIVSTEGGQSDKKPVLFKNFFDTTSLKVTKKVTGNTGE
HQRSFSFTLLLTPNECFEKGQVVNILQGGETKKVVIGEEYSFTLKDKESV
TLSQLPVGIEYKVTEEDVTKDGYKTSATLKDGDVTDGYNLGDSKTTDKST
DEIVVTNKRDTQVPTGVVGTLAPFAVLSIVAIGGVIYITKRKKA
[1093] SpyM18.sub.--0129 is a SrtC2 type sortase. An example of
SpyM18.sub.--0129 is shown in SEQ ID NO: 76 TABLE-US-00168 SEQ ID
NO: 76 MISQRMMMTIVQVINKAIDTLILIFCLVVLFLAGFGLWDSYHLYQQADAS
NFKKFKTAQQQPKFEDLLALNEDVIGWLNIPGTHMDYPLVQGKTNLEYIN
KAVDGSVAMSGSLFLDTRNHNDFTDDYSLIYGHHMAGNAMFGEIPKFLKK
DFFNKHNKAIIETKERKKLTVTIFACLKTDAFDQLVFNPNAITNQDQQRQ
LVDYISKRSKQFKPVKLKHHTKFVAFSTCENFSTDNRVIVVGTIQE
[1094] SpyM18.sub.--0130 is referred to as a hypothetical protein.
An example of SpyM18.sub.--0130 is shown in SEQ ID NO: 77.
TABLE-US-00169 SEQ ID NO: 77
MRKYWKMLFSVVMILTMLAFNQTVLAKDSTVQTSISVENVLERAGDSTSF
SVALESIDAMKTIDEITIAGSGKASFSPLTFTTVGQYTYRVYQKPSQNKD
YQADTTVFDVLVYVTYDEDGTLVAKVISRRAGDEEKSAITFKPKRLVKPI
PPRQPDIPKTPLPLAGEVKSLLGILSIVLLGLLVLLYVKKLKSRL
[1095] SpyM18.sub.--0130 contains an amino acid motif indicative of
a cell wall anchor: SEQ ID NO: 185 LPLAG (shown in italics in SEQ
ID NO: 77, above). In some recombinant host cell systems, it may be
preferable to remove this motif to facilitate secretion of a
recombinant SpyM18.sub.--0130 protein from the host cell.
Alternatively, in other recombinant host cell systems, it may be
preferable to use the cell wall anchor motif to anchor the
recombinantly expressed protein to the cell wall. The extracellular
domain of the expressed protein may be cleaved during purification
or the recombinant protein may be left attached to either
inactivated host cells or cell membranes in the final
composition.
[1096] A pilin motif, discussed above, containing a conserved
lysine (K) residue has also been identified in SpyM18.sub.--0130.
The pilin motif sequence is underlined in SEQ ID NO: 77, below.
Conserved lysine (K) residues are also marked in bold, at amino
acid residues 144, 159, and 169. The pilin sequence, in particular
the conserved lysine residues, are thought to be important for the
formation of oligomeric, pilus-like structures. Preferred fragments
of SpyM18.sub.--0130 include at least one conserved lysine residue.
Preferably, fragments include the pilin sequence. TABLE-US-00170
SEQ ID NO: 77 MRKYWKMLFSVVMILTMLAFNQTVLAKDSTVQTSISVENVLERAGDSTSF
SVALESIDAMKTIDEITIAGSGKASFSPLTFTTVGQYTYRVYQKPSQNKD
YQADTTVFDVLVYVTYDEDGTLVAKVISRRAGDEEKSAITFKPKRLVKPI
PPRQPDIPKTPLPLAGEVKSLLGTLSIVLLGLLVLLYVKKLKSRL
[1097] An E box containing a conserved glutamic residue has been
identified in SpyM18.sub.--0130. The E-box motif is underlined in
SEQ ID NO: 77, below. The conserved glutamic acid (E), at amino
acid residue 134, is marked in bold. The E box motif, in particular
the conserved glutamic acid residue, is thought to be important for
the formation of oligomeric pilus-like structures of
SpyM18.sub.--0130. Preferred fragments of SpyM18.sub.--0130 include
the conserved glutamic acid residue. Preferably, fragments include
the E box motif. TABLE-US-00171 SEQ ID NO: 77
MRKYWKMLFSVVMILTMLAFNQTVLAKDSTVQTSISVENVLERAGDSTSF
SVALESIDAMKTIDEITIAGSGKASFSPLTFTTVGQYTYRVYQKPSQNKD
YQADTTVFDVLVYVTYDEDGTLVAKVISRRAGDEEKSAITFKPKRLVKPI
PPRQPDIPKTPLPLAGEVKSLLGTLSIVLLGLLVLLYVKKLKSRL
[1098] SpyM18.sub.--0131 is referred to as a putative multiple
sugar metabolism regulator. An example of SpyM18.sub.--0131 is set
forth in SEQ ID NO: 78. TABLE-US-00172 SEQ ID NO: 78
MAIFDLKHVQTLHSLSQLPISVMSQDKALIQVYGNDDYLLCYYQFLKHLA
IPQAAQDVIFYEGLFEESFMIFPLCHYIIAIGPFYPYSLNKDYQEQLANN
CLKHSSHRSKEELLSYMALVPHFPINNVRNLLIAIDAFFDTQFETTCQQT
IHQLLQHSKQMTADPDIIHRLKHISKASSQLPPVLEHLNHIMDLVKLGNP
QLLKQEINRIPLSSITSSSISALRAEKNLTVIYLTRLLEFSFVENTDVAK
HYSLVKYYMALNEEASDLLKVLRIRCAAIIHFSESLTNKSISDKRQMYNS
VLHYVDSHLYSKLKVSDTAKRLYVSESHLRSVFKKYSNVSLQHYTLSTKT
KEAQLLLKRGIPVGEVAKSLYFYDTTHFHKIFKKYTGISSKDYLAKYRDN I
[1099] SpyM18.sub.--0132 is a F2 like fibronectic-binding protein.
An example of SpyM18.sub.--0132 is set forth in SEQ ID NO: 79.
TABLE-US-00173 SEQ ID NO: 79
MTQKNSYKLSFLLSLTGFILGLLLVFIGLSGVSVGHAETRNGANKQGAFE
IKKNKSQEEYNYEVYDNRNILQDGEHKLEIKRVDGTGKTYQGFCFQLTKN
FPTAQGVSKKLYKKLSSSDEETLKQYASKYTSNRRGDTSGNLKKQIAKVL
TEGYPTNKSDWLNGLTENEKIEVTQDAIWYFTETTVPADRSYTNRNVNSQ
KMKEVYQKLIDTTDIDKYEDVQFDLFVPQDTNLQAVISVEPVIESLPWTS
LKPIAQKDITAKKIWVDAPKEKPIIYFKLYRQLPGEKEVAVDDAELKQIN
SEGQQEISVTWTNQLVTDEKGMAYIYSVKEVDKNGELLEPKDYIKKEDGL
TVTNTYVKPTSGHYDIEVTFGNGHIDITEDTTPDIVSGENQMKQIEGEDS
KPIDEVTENNLIEPGKNTMPGEEDGTNSNKYEEVEDSRPVDTLSGLSSEQ
GQSGDMTIEEDSATHIKFSKRDIDGKELAGATMELRDSSGKTISTWISDG
QVKDFYLMPGKYTFVETAAPDGYEIATAITFTVNEQGQVTVNGKATKGDA
HIVMVDAYKPTKGSGQVIDIEEKLPDEQGHSGSTTEIEDSKSSDVIIGGQ
GQIVETTEDTQTGMHGDSGCKTEVEDTKLVQSFHFDNKESESNSEIPKKD
KPKSNTSLPATGEKQHNMFFWMVTSCSLISSVPVISLKTKKRLSSC
[1100] SpyM18.sub.--0132 contains an amino acid motif indicative of
a cell wall anchor: SEQ ID NO: 180 LPATG (shown in italics in SEQ
ID NO: 79, above). In some recombinant host cell systems, it may be
preferable to remove this motif to facilitate secretion of a
recombinant SpyM18.sub.--0132 protein from the host cell.
Alternatively, in other recombinant host cell systems, it may be
preferable to use the cell wall anchor motif to anchor the
recombinantly expressed protein to the cell wall. The extracellular
domain of the expressed protein may be cleaved during purification
or the recombinant protein may be left attached to either
inactivated host cells or cell membranes in the final
composition.
[1101] A pilin motif, discussed above, containing a conserved
lysine (K) residue has also been identified in SpyM18.sub.--0132.
The pilin motif sequence is underlined in SEQ ID NO: 79, below. A
conserved lysine (K) residue is also marked in bold, at amino acid
residue 270. The pilin sequence, in particular the conserved lysine
residue, is thought to be important for the formation of
oligomeric, pilus-like structures. Preferred fragments of
SpyM18.sub.--0132 include the conserved lysine residue. Preferably,
fragments include the pilin sequence. TABLE-US-00174 SEQ ID NO: 79
MTQKNSYKLSFLLSLTGFILGLLLVFIGLSGVSVGHAETRNGANKQGAFE
IKKNKSQEEYNYEVYDNRNILQDGEHKLEIKRVDGTGKTYQGFCFQLTKN
FPTAQGVSKKLYKKLSSSDEETLKQYASKYTSNRRGDTSGNLKKQIAKVL
TEGYPTNKSDWLNGLTENEKIEVTQDAIWYFTETTVPADRSYTNRNVNSQ
KMKEVYQKLIDTTDIDKYEDVQFDLFVPQDTNLQAVISVEPVIESLPWTS
LKPIAQKDITAKKIWVDAPKEKPIIYFKLYRQLPGEKEVAVDDAELKQIN
SEGQQEISVTWTNQLVTDEKGMAYIYSVKEVDKNGELLEPKDYIKKEDGL
TVTNTYVKPTSGHYDIEVTFGNGHIDITEDTTPDIVSGENQMKQIEGEDS
KPIDEVTENNLIEPGKNTMPGEEDGTNSNKYEEVEDSRPVDTLSGLSSEQ
GQSGDMTIEEDSATHIKFSKRDIDGKELAGATMELRDSSGKTISTWISDG
QVKDFYLMPGKYTFVETAAPDGYEIATAITFTVNEQGQVTVNGKATKGDA
HIVMVDAYKPTKGSGQVIDIEEKLPDEQGHSGSTTEIEDSKSSDVIIGGQ
GQIVETTEDTQTGMHGDSGCKTEVEDTKLVQSFHFDNKESESNSEIPKKD
KPKSNTSLPATGEKQHNMFFWMVTSCSLISSVPVISLKTKKRLSSC
[1102] An E box containing a conserved glutamic residue has been
identified in SpyM18.sub.--0132. The E-box motif is underlined in
SEQ ID NO: 79, below. The conserved glutamic acid (E), at amino
acid residue 516, is marked in bold. The E box motif, in particular
the conserved glutamic acid residue, is thought to be important for
the formation of oligomeric pilus-like structures of
SpyM18.sub.--0132. Preferred fragments of SpyM18.sub.--0132 include
the conserved glutamic acid residue. Preferably, fragments include
the E box motif. TABLE-US-00175 SEQ ID NO: 79
MTQKNSYKLSFLLSLTGFILGLLLVFIGLSGVSVGHAETRNGANKQGAFE
IKKNKSQEEYNYEVYDNRNILQDGEHKLEIKRVDGTGKTYQGFCFQLTKN
FPTAQGVSKKLYKKLSSSDEETLKQYASKYTSNRRGDTSGNLKKQIAKVL
TEGYPTNKSDWLNGLTENEKIEVTQDAIWYFTETTVPADRSYTNRNVNSQ
KMKEVYQKLIDTTDIDKYEDVQFDLFVPQDTNLQAVISVEPVIESLPWTS
LKPIAQKDITAKKIWVDAPKEKPIIYFKLYRQLPGEKEVAVDDAELKQIN
SEGQQEISVTWTNQLVTDEKGMAYIYSVKEVDKNGELLEPKDYIKKEDGL
TVTNTYVKPTSGHYDIEVTFGNGHIDITEDTTPDIVSGENQMKQIEGEDS
KPIDEVTENNLIEPGKNTMPGEEDGTNSNKYEEVEDSRPVDTLSGLSSEQ
GQSGDMTIEEDSATHIKFSKRDIDGKELAGATMELRDSSGKTISTWISDG
QVKDFYLMPGKYTFVETAAPDGYEIATAITFTVNEQGQVTVNGKATKGDA
HIVMVDAYKPTKGSGQVIDIEEKLPDEQGHSGSTTEIEDSKSSDVIIGGQ
GQIVETTEDTQTGMHGDSGCKTEVEDTKLVQSFHFDNKESESNSEIPKKD
KPKSNTSLPATGEKQHNMFFWMVTSCSLISSVPVISLKTKKRLSSC
[1103] Examples of GAS AI-3 sequences from M49 strain isolate 591
are set forth below.
[1104] SpyoM01000156 is a negative transcriptional regulator (Nra).
An example of SpyoM01000156 is set forth in SEQ ID NO: 243.
TABLE-US-00176 SEQ ID NO: 243
MPYVKKKKDSFLVETYLEQSIRDKSELVLLLFKSPTIIFSHVAKQTGLTA
VQLKYYCKELDDEFGNNLDITIKKGKIIGCFVKPVKEFYLHQLYDTSTIL
KLLVFFIKNGTSSQPLIKFSKKYFLSSSSAYRLRESLIKLLREFGLRVSK
NTIVGEEYRIRYLIAMLYSKFGIVIYPLDHLDNQIIYRFLSQSATNLRTS
PWLEEPFSFYNMLLALSWKRHQFAVSIPQTRIFRQLKKLFIYDCLTRSSR
QVIENAFSLTFSQGDLDYLELIYITTNNSEASLQWTPQHIETCCHIFEKN
DTERLLLEPILKRLPQLNHSKQDLIKALMYFSKSFLFNLQHEVIEIPSFS
LPTYTGNSNLYKALKNIVNQWLAQLPGKRHLNEKHLQLFCSHIEQILKNK
QPALTVVLISSNFINAKLLTDTIPRYFSDKGIHFYSFYLLRDDTYQIPSL
KPDLVITHSRLTPFVKNDLVKGVTVAEFSFDNPDYSIASIQNLIYQLKDK KYQDFLNEQLQ
[1105] SpyoM01000155 is thought to be a collagen binding protein
(CPA). An example of SpyoM01000155 is set forth in SEQ ID NO: 244.
TABLE-US-00177 SEQ ID NO: 244
MQKRDKTNYGSANNKRRQTTIGLLKVFLTFVALIGIVGESIRAFGAEEQS
VPNRQSSIQDYPWYGYDSYPKGYPDYSPLKTYHNLKVNLEGSKDYQAYCF
NLTKHFPSKSDSVRSQWYKKLEGTNENFIKLADKPRIEDGQLQQNILRIL
YNGYPNNRNGIMKGIDPLNAILVTQNAIWYYTDSAQINPDESFKTEARSN
GINDQQLGLMRKALKELIDPNLGSKYSNKTPSGYRLNVFESHDKTFQNLL
SAEYVPDTPPKPGEEPPAKTEKTSVIIRKYAEGDYSKLLEGATLKLSQIE
GSGFQEKDFQSNSLGETVELPNGTYTLTETSSPDGYKIAEPIKFRVENKK
VETVQKDGSQVENPNKEVAEPYSVEAYNDFMDEEVLSGFTPYGKFYYAKN
KDKSSQVVYCPNADLHSPPDSYDSGETTNPDTSTMKEVKYTHTAGSDLFK
YALRPRDTNPEDFLKHIKKVIEKGYKKKGDSYNGLTETQFRAATQLAIYY
FTDSADLKTLKTYNNGKGYHGFESMDEKTLAVTKELITYAQNGSAPQLTN
LDFFVPNNSKYQSLIGTEYHPDDLVDVTRMEDKKQEVIPVTHSLTVKKTV
VGELGDKTKGFQFELELKDKTGQPIVNTLKTNNQDLVAKDGKYSFNLKHG
DTIRIEGLPTGYSYTLKETEAKDYIVTVDNKVSQEAQSVGKDITEDKKVT
FENRKDLVPPTGLTTDGAIYLWLLLLVPLGLLVWLFGRKGLKND
[1106] SpyoM01000155 contains an amino acid motif indicative of a
cell wall anchor: SEQ ID NO: 184 VPPTG (shown in italics in SEQ ID
NO: 244, above). In some recombinant host cell systems, it may be
preferable to remove this motif to facilitate secretion of a
recombinant SpyoM1000155 protein from the host cell. Alternatively,
in other recombinant host cell systems, it may be preferable to use
the cell wall anchor motif to anchor the recombinantly expressed
protein to the cell wall. The extracellular domain of the expressed
protein may be cleaved during purification or the recombinant
protein may be left attached to either inactivated host cells or
cell membranes in the final composition.
[1107] Two pilin motifs, discussed above, containing conserved
lysine (K) residues have also been identified in SpyoM01000155. The
pilin motif sequence is underlined in SEQ ID NO: 244, below.
Conserved lysine (K) residues are also marked in bold, at amino
acid residues 71 and 261. The pilin sequences, in particular the
conserved lysine residues, are thought to be important for the
formation of oligomeric, pilus-like structures. Preferred fragments
of SpyoM01000155 include at least one conserved lysine residue.
Preferably, fragments include at least one pilin sequence.
TABLE-US-00178 SEQ ID NO: 244
MQKRDKTNYGSANNKRRQTTIGLLKVFLTFVALIGIVGFSIRAFGAEEQS
VPNRQSSIQDYPWYGYDSYPKGYPDYSPLKTYHNLKVNLEGSKDYQAYCF
NLTKHFPSKSDSVRSQWYKKLEGTNENFIKLADKPRIEDGQLQQNILRIL
YNGYPNNRNGIMKGIDPLNAILVTQNAIWYYTDSAQINPDESFKTEARSN
GINDQQLGLMRKALKELIDPNLGSKYSNKTPSGYRLNVFESHDKTFQNLL
SAEYVPDTPPKPGEEPPAKTEKTSVIIRKYAEGDYSKLLEGATLKLSQIE
GSGFQEKDFQSNSLGETVELPNGTYTLTETSSPDGYKIAEPIKFRVENKK
VFIVQKDGSQVENPNKEVAEPYSVEAYNDFMDEEVLSGFTPYGKFYYAKN
KDKSSQVVYCFNADLHSPPDSYDSGETINPDTSTMKEVKYTHTAGSDLFK
YALRPRDTNPEDFLKHIKKVIEKGYKKKGDSYNGLTETQFRAATQLAIYY
FTDSADLKTLKTYNNGKGYHGFESMDEKTLAVTKELITYAQNGSAPQLTN
LDFFVPNNSKYQSLIGTEYHPDDLVDVIRMEDKKQEVIPVTHSLTVKKTV
VGELGDKTKGFQFELELKDKTGQPIVNTLKTNNQDLVAKDGKYSFNLKHG
DTIRIEGLPTGYSYTLKETEAKDYIVTVDNKVSQEAQSVGKDITEDKKVT
FENRKDLVPPTGLTTDGAIYLWLLLLVPLGLLVWLFGRKGLKND
[1108] Two E boxes containing conserved glutamic residues have been
identified in SpyoM1000155. The E-box motifs are underlined in SEQ
ID NO: 244, below. The conserved glutamic acid (E) residues, at
amino acid residues 329 and 668, are marked in bold. The E box
motifs, in particular the conserved glutamic acid residues, are
thought to be important for the formation of oligomeric pilus-like
structures of SpyoM01000155. Preferred fragments of SpyoM01000155
include at least one conserved glutamic acid residue. Preferably,
fragments include at least one E box motif. TABLE-US-00179 SEQ ID
NO: 244 MQKRDKTNYGSANNKRRQTTIGLLKVFLTFVALIGIVGESIRAFGAEEQS
VPNRQSSIQDYPWYGYDSYPKGYPDYSPLKTYHNLKVNLEGSKDYQAYCF
NLTKHFPSKSDSVRSQWYKKLEGTNENFIKLADKPRIEDGQLQQNILRIL
YNGYPNNRNGIMKGIDPLNAILVTQNAIWYYTDSAQINPDESFKTEARSN
GINDQQLGLMRKALKELIDPNLGSKYSNKTPSGYRLNVFESHDKTFQNLL
SAEYVPDTPPKPGEEPPAKTEKTSVIIRKYAEGDYSKLLEGATLKLSQIE
GSGFQEKDFQSNSLGETVELPNGTYTLTETSSPDGYKIAEPIKFRVENKK
VETVQKDGSQVENPNKEVAEPYSVEAYNDFMDEEVLSGFTPYGKFYYAKN
KDKSSQVVYCPNADLHSPPDSYDSGETTNPDTSTMKEVKYTHTAGSDLFK
YALRPRDTNPEDFLKHIKKVIEKGYKKKGDSYNGLTETQFRAATQLAIYY
FTDSADLKTLKTYNNGKGYHGFESMDEKTLAVTKELITYAQNGSAPQLTN
LDFFVPNNSKYQSLIGTEYHPDDLVDVTRMEDKKQEVIPVTHSLTVKKTV
VGELGDKTKGFQFELELKDKTGQPIVNTLKTNNQDLVAKDGKYSFNLKHG
DTIRIEGLPTGYSYTLKETEAKDYIVTVDNKVSQEAQSVGKDITEDKKVT
FENRKDLVPPTGLTTDGAIYLWLLLLVPLGLLVWLFGRKGLKND
[1109] SpyoM01000154 is a LepA protein. An example of SpyoM01000154
is shown in SEQ ID NO: 245. TABLE-US-00180 SEQ ID NO: 245
MTNYLNRLNENSLFKAFIRLVLKISTIGFLGYILFQYVFGVMIINTNDMS
PALSAGDGVLYYRLADRSHINDVVVYEVDNTLKVGRIAAQAGDEVNFTQE
GGLLINGHPPEKEVPYLTYPHSSGPNFPYKVPTGTYFILNDYREERLDSR
YYGALPINQIKGKISTLLRVRGI
[1110] SpyoM01000153 is thought to be a fimbrial protein. An
example of SpyoM01000153 is shown in SEQ ID NO: 246. TABLE-US-00181
SEQ ID NO: 246 MKKNKLLLATAILATALGMASMSQNIKAETAGVIDGSTLVVKKTFPSYTD
DNVLMPKADYSFKVEADDNAKGKTKDGLDIKPGVIDGLENTKTIRYSNSD
KITAKEKSVNFEFANVKFPGVGVYRYTVAEVNGNKAGITYDSQQWTVDVY
VVNKEGGGFEVKYIVSTEVGQSEKKPVLFKNSFDTTSLKIEKQVTGNTGE
HQRLFSFTLLLTPNECFEKGQVVNILQGGETKKVVIGEEYSFTLKDKESV
TLSQLPVGIEYKLTEEDVTKDGYKTSATLKDGEQSSTYELGKDHKTDKSA
DEIVVTNKRDTQVPTGVVGTLAPFAVLSIVAIGGVIYITKRKKA
[1111] SpyoM01000153 contains an amino acid motif indicative of a
cell wall anchor: SEQ ID NO: 140 QVPTG (shown in italics in SEQ ID
NO: 246, above). In some recombinant host cell systems, it may be
preferable to remove this motif to facilitate secretion of a
recombinant SpyoM01000153 protein from the host cell.
Alternatively, in other recombinant host cell systems, it may be
preferable to use the cell wall anchor motif to anchor the
recombinantly expressed protein to the cell wall. The extracellular
domain of the expressed protein may be cleaved during purification
or the recombinant protein may be left attached to either
inactivated host cells or cell membranes in the final
composition.
[1112] A pilin motif, discussed above, containing a conserved
lysine (K) residue has also been identified in SpyoM01000153. The
pilin motif sequence is underlined in SEQ ID NO: 246, below. A
conserved lysine (K) residue is also marked in bold, at amino acid
residue 57. The pilin sequence, in particular the conserved lysine
residue, is thought to be important for the formation of
oligomeric, pilus-like structures. Preferred fragments of
SpyoM01000153 include the conserved lysine residue. Preferably,
fragments include the pilin sequence. TABLE-US-00182 SEQ ID NO: 246
MKKNKLLLATAILATALGMASMSQNIKAETAGVIDGSTLVVKKTFPSYTD
DNVLMPKADYSFKVEADDNAKGKTKDGLDIKPGVIDGLENTKTIRYSNSD
KITAKEKSVNFEFANVKFPGVGVYRYTVAEVNGNKAGITYDSQQWTVDVY
VVNKEGGGEEVKYIVSTEVGQSEKKPVLFKNSFDTTSLKIEKQVTGNTGE
HQRLFSFTLLLTPNECFEKGQVVNILQGGETKKVVIGEEYSFTLKDKESV
TLSQLPVGIEYKLTEEDVTKDGYKTSATLKDGEQSSTYELGKDHKTDKSA
DEIVVTNKRDTQVPTGVVGTLAPEAVLSIVAIGGVIYITKRKKA
[1113] An E box containing a conserved glutamic residue has been
identified in SpyoM01000153. The E-box motif is underlined in SEQ
ID NO: 246, below. The conserved glutamic acid (E), at amino acid
residue 265, is marked in bold. The E box motif, in particular the
conserved glutamic acid residue, is thought to be important for the
formation of oligomeric pilus-like structures of SpyoM01000153.
Preferred fragments of SpyoM01000153 include the conserved glutamic
acid residue. Preferably, fragments include the E box motif.
TABLE-US-00183 SEQ ID NO: 246
MKKNKLLLATAILATALGMASMSQNIKAETAGVIDGSTLVVKKTFPSYTD
DNVLMPKADYSFKVEADDNAKGKTKDGLDIKPGVIDGLENTKTIRYSNSD
KITAKEKSVNFEFANVKFPGVGVYRYTVAEVNGNKAGITYDSQQWTVDVY
VVNKEGGGFEVKYIVSTEVGQSEKKPVLFKNSFDTTSLKIEKQVTGNTGE
HQRLFSFTLLLTPNECFEKGQVVNILQGGETKKVVIGEEYSFTLKDKESV
TLSQLPVGIEYKLTEEDVTKDGYKTSATLKDGEQSSTYELGKDHKTDKSA
DEIVVTNKRDTQVPTGVVGTLAPFAVLSIVAIGGVIYITKRKKA
[1114] SpyoM01000152 is a SrtC2 type sortase. An example of
SpyoM01000152 is shown in SEQ ID NO: 247 TABLE-US-00184 SEQ ID NO:
247 MMMTIVQVINKAIDTLILIFCLVVLFLAGFGLWDSYHLYQQADASNFKKF
KTAQQQPKFEDLLALNEDVIGWLNIPGTHIDYPLVQGKTNLEYINKAVDG
SVAMSGSLFLDTRNHNDFTDDYSLIYGHHMAGNAMFGEIPKFLKKNFFNK
HNKAIIETKERKKLTVTIFACLKTDAFDQLVFNPNAITNQDQQRQLVDYI
SKRSKQFKPVKLKHHTKFVAPSTCENFSTDNRVTVVGTIQE
[1115] SpyoM01000151 is referred to as a hypothetical protein. An
example of SpyoM01000151 is shown in SEQ ID NO: 248. TABLE-US-00185
SEQ ID NO: 248 MLFSVVMMLTMLAFNQTVLAKDSTVQTSISVENVLERAGDSTPFSIALES
IDAMKTIEEITIAGSGKASFSPLTFTTVGQYTYRVYQKPSQNKDYQADTT
VFDVLVYVTYDEDGTLVAKVISRRAGDEEKSAITFKPKRLVKPIPPRQPD
IPKTPLPLAGEVKSLLGILSIVLLGLLVLLYVKKLKSRL
[1116] SpyoM01000151 contains an amino acid motif indicative of a
cell wall anchor: SEQ ID NO: 185 LPLAG (shown in italics in SEQ ID
NO: 248, above). In some recombinant host cell systems, it may be
preferable to remove this motif to facilitate secretion of a
recombinant SpyoM01000151 protein from the host cell.
Alternatively, in other recombinant host cell systems, it may be
preferable to use the cell wall anchor motif to anchor the
recombinantly expressed protein to the cell wall. The extracellular
domain of the expressed protein may be cleaved during purification
or the recombinant protein may be left attached to either
inactivated host cells or cell membranes in the final
composition.
[1117] A pilin motif, discussed above, containing a conserved
lysine (K) residue has also been identified in SpyoM01000151. The
pilin motif sequence is underlined in SEQ ID NO: 248, below.
Conserved lysine (K) residues are also marked in bold, at amino
acid residue 138. The pilin sequence, in particular the conserved
lysine residue, is thought to be important for the formation of
oligomeric, pilus-like structures. Preferred fragments of
SpyoM01000151 include the conserved lysine residue. Preferably,
fragments include the pilin sequence. TABLE-US-00186 SEQ ID NO: 248
MLFSVVMMLTMLAFNQTVLAKDSTVQTSISVENVLERAGDSTPFSIALES
IDAMKTIEEITIAGSGKASFSPLTFTTVGQYTYRVYQKPSQNKDYQADTT
VFDVLVYVTYDEDGTLVAKVISRRAGDEEKSAITFKPKRLVKPIPPRQPD
IPKTPLPLAGEVKSLLGILSTVLLGLLVLLYVKKLKSRL
[1118] Two E boxes containing conserved glutamic residues have been
identified in SpyoM01000151. The E-box motifs are underlined in SEQ
ID NO: 248, below. The conserved glutamic acid (E) residues, at
amino acid residues 58 and 128, are marked in bold. The E box
motifs, in particular the conserved glutamic acid residues, are
thought to be important for the formation of oligomeric pilus-like
structures of SpyoM01000151. Preferred fragments of SpyoM01000151
include at least one conserved glutamic acid residue. Preferably,
fragments include at least one E box motif. TABLE-US-00187 SEQ ID
NO: 248 MLFSVVMMLTMLAFNQTVLAKDSTVQTSISVENVLERAGDSTPFSIALES
IDAMKTIEEITIAGSGKASFSPLTFTTVGQYTYRVYQKPSQNKDYQADTT
VFDVLVYVTYDEDGTLVAKVISRRAGDEEKSAITFKPKRLVKPIPPRQPD
IPKTPLPLAGEVKSLLGILSIVLLGLLVLLYVKKLKSRL
[1119] SpyoM01000150 is referred to as a putative MsmRL. An example
of SpyoM01000150 is set forth in SEQ ID NO: 249. TABLE-US-00188 SEQ
ID NO: 249 MVIFDLKHVQTLHSLSQLPISVMSQDKALIQVYGNDDYLLCYYQFLKHLA
IPQAAQDVIFYEGLFEESFMIFPLCHYIIAIGPFYPYSLNKDYQEQLANN
FLKHSSHRSKEELLSYMALVPHFPINNVRNLLIAIDAFFDTQFETTCQQT
THQLLQHSKQMTADPDIIHRLKHISKASSQLPPVLEHLNHIMDLVKLGNP
QLLKQEINRIPLSSITSSSISALRAEKNLTVIYLTRLLEFSFVENTDVAK
HYSLVKYYMALNEEASDLLKVLRIRCAAIIHFSESLTNKSISDKRQMYNS
VLHYVDSHLYSKLKVSDIAKRLYVSESHLRSVFKKYSNVSLQHYILSTKI
KEAQLLLKRGIPVGEVAKSLYFYDTTHFHKIFKKYTGISSKDYLAKYRDN I
[1120] SpyoM01000149 is a F2 like fibronectin-binding protein. An
example of SpyoM01000149 is set forth in SEQ ID NO: 250.
TABLE-US-00189 SEQ ID NO: 250
MTQKNSYKLSFLLSLTGFILGLLLVFIGLSGVSVGHAETRNGANKQGYFE
IKKVDQNNKPLSGATFSLTPKDGKGKPVQTFTSSEEGIIDAQNLQPGTYT
LKEETAPDGYDKTSRTWTVTVYENGYTKLVENPYNGEIISKAGSKDVSSS
LQLENPKMSVVSKYGEQEKTSNSADFYRNHAAYFKMSFELKQKDKSETIN
PGDTFVLQLDRRLNPKGISQDIPKIIYDSENSPLAIGKYDAKTHQLTYTE
TNYIAGLDKVQLSAELSLFLENKEVLENTNISDFKSTIGGQEITYKGTVN
VLYGNESTKESNYITNGLSNVGGSIESYNTETGEFVWYVYVNPNRTNIPY
AVLNLWGFAKRTAQGENDNSSVSSAQLTGYDIYEVPHNYRLPTSYGVDIS
RLNLRKDLEAKLPQGSTQGANKRLRIDFGENLQGKAFVVKVTGKADQSGK
ELIVQSHLSSPNNWGSYKTLRPNSHVSETNEIALSPSKGSGSGTSEETKP
AITVANLKRVAQLRFKKVSTDNVPLPEAAFELRSSNGNSQKLEASSNTQG
EIHPKDLTSGTYDLYETKAPKGYQQVTEKLATVTVDTTKPAEQMVKWEKP
HSFVKVEANKEVTIVNHKETLTFSGKKIWENDRPDQRPAKIQVQLLQNGQ
KMPNQIQEVTKDNDWSYHFKDLPKYDAKNQEYKYSVEEVKVPDGYKVSYL
GNDIFNTRETEFVFEQNNENLEFGNAEIKGQSGSKIIDEDTLTSFKGKKI
WKNDTAENRPQAIQVQLYADGVAVEGQTKFISGSGNEWSFEFKNLKKYNG
TGNDIIYSVKEVTVPTGYDVTYSANDIINTKREVITQQGPNLEIEETLPL
ESGASGGTTTVEDSRSVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDI
DGKELAGATMELRDSSGKTISTWISDGQVKDFYLMPGKYTFVETAAPDGY
EIATAITFTVNEQGQVTVNGKATKGDAHIVMVDAYKPTKGSGQVIDIEEK
LPDEQGHSGSTTEIEDSKPSDVIIGGQGEVVDTTEDTQSGMTGHSGSTTE
IEDSKSSDVIIGGQGQVVETTEDTQTGMHGDSGCKTEVEDTKLVQFFHFD
NKEPESNSEIPKKDKPKSNTSLPATGEKQHNKFFWMVTSCSLISSVFVIS LKSKKRLLSC
[1121] SpyoM01000149 contains an amino acid motif indicative of a
cell wall anchor: SEQ ID NO: 180 LPATG (shown in italics in SEQ ID
NO: 250, above). In some recombinant host cell systems, it may be
preferable to remove this motif to facilitate secretion of a
recombinant SpyoM01000149 protein from the host cell.
Alternatively, in other recombinant host cell systems, it may be
preferable to use the cell wall anchor motif to anchor the
recombinantly expressed protein to the cell wall. The extracellular
domain of the expressed protein may be cleaved during purification
or the recombinant protein may be left attached to either
inactivated host cells or cell membranes in the final
composition.
[1122] Two pilin motifs, discussed above, containing conserved
lysine (K) residues have also been identified in SpyoM01000149. The
pilin motif sequences are underlined in SEQ ID NO: 250, below.
Conserved lysine (K) residues are also marked in bold, at amino
acid residues 157 and 163, and 216 and 224. The pilin sequences, in
particular the conserved lysine residues, are thought to be
important for the formation of oligomeric, pilus-like structures.
Preferred fragments of SpyoM01000149 include at least one conserved
lysine residue. Preferably, fragments include at least one pilin
sequence. TABLE-US-00190 SEQ ID NO: 250
MTQKNSYKLSFLLSLTGFILGLLLVFIGLSGVSVGHAETRNGANKQGYFE
IKKVDQNNKPLSGATFSLTPKDGKGKPVQTFTSSEEGIIDAQNLQPGTYT
LKEETAPDGYDKTSRTWTVTVYENGYTKLVENPYNGEIISKAGSKDVSSS
LQLENPKMSVVSKYGEQEKTSNSADFYRNHAAYFKMSFELKQKDKSETIN
PGDTFVLQLDRRLNPKGISQDIPKIIYDSENSPLAIGKYDAKTHQLTYTE
TNYIAGLDKVQLSAELSLFLENKEVLENTNISDFKSTIGGQEITYKGTVN
VLYGNESTKESNYITNGLSNVGGSIESYNTETGEFVWYVYVNPNRTNIPY
AVLNLWGFAKRTAQGENDNSSVSSAQLTGYDIYEVPHNYRLPTSYGVDIS
RLNLRKDLEAKLPQGSTQGANKRLRIDFGENLQGKAFVVKVTGKADQSGK
ELIVQSHLSSPNNWGSYKTLRPNSHVSETNEIALSPSKGSGSGTSEETKP
AITVANLKRVAQLRFKKVSTDNVPLPEAAFELRSSNGNSQKLEASSNTQG
EIHPKDLTSGTYDLYETKAPKGYQQVTEKLATVTVDTTKPAEQMVKWEKP
HSFVKVEANKEVTIVNHKETLTFSGKKIWENDRPDQRPAKIQVQLLQNGQ
KMPNQIQEVTKDNDWSYHFKDLPKYDAKNQEYKYSVEEVKVPDGYKVSYL
GNDIFNTRETEFVFEQNNENLEFGNAEIKGQSGSKIIDEDTLTSFKGKKI
WKNDTAENRPQAIQVQLYADGVAVEGQTKFISGSGNEWSFEFKNLKKYNG
TGNDIIYSVKEVTVPTGYDVTYSANDIINTKREVITQQGPNLEIEETLPL
ESGASGGTTTVEDSRSVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDI
DGKELAGATMELRDSSGKTISTWISDGQVKDFYLMPGKYTFVETAAPDGY
EIATAITFTVNEQGQVTVNGKATKGDAHIVMVDAYKPTKGSGQVIDIEEK
LPDEQGHSGSTTEIEDSKPSDVIIGGQGEVVDTTEDTQSGMTGHSGSTTE
IEDSKSSDVIIGGQGQVVETTEDTQTGMHGDSGCKTEVEDTKLVQFFHFD
NKEPESNSEIPKKDKPKSNTSLPATGEKQHNKFFWMVTSCSLISSVFVIS LKSKKRLLSC
[1123] Two E boxes containing conserved glutamic residues have been
identified in SpyoM01000149. The E-box motifs are underlined in SEQ
ID NO: 250, below. The conserved glutamic acid (E) residues, at
amino acid residues 329 and 668, are marked in bold. The E box
motifs, in particular the conserved glutamic acid residues, are
thought to be important for the formation of oligomeric pilus-like
structures of SpyoM01000149. Preferred fragments of SpyoM01000149
include at least one conserved glutamic acid residue. Preferably,
fragments include at least one E box motif. TABLE-US-00191 SEQ ID
NO: 250 MTQKNSYKLSFLLSLTGFILGLLLVFIGLSGVSVGHAETRNGANKQGYFE
IKKVDQNNKPLSGATFSLTPKDGKGKPVQTFTSSEEGIIDAQNLQPGTYT
LKEETAPDGYDKTSRTWTVTVYENGYTKLVENPYNGEIISKAGSKDVSSS
LQLENPKMSVVSKYGEQEKTSNSADFYRNHAAYFKMSFELKQKDKSETIN
PGDTFVLQLDRRLNPKGISQDIPKIIYDSENSPLAIGKYDAKTHQLTYTE
TNYIAGLDKVQLSAELSLFLENKEVLENTNISDFKSTIGGQEITYKGTVN
VLYGNESTKESNYITNGLSNVGGSIESYNTETGEFVWYVYVNPNRTNIPY
AVLNLWGFAKRTAQGENDNSSVSSAQLTGYDIYEVPHNYRLPTSYGVDIS
RLNLRKDLEAKLPQGSTQGANKRLRIDFGENLQGKAFVVKVTGKADQSGK
ELIVQSHLSSPNNWGSYKTLRPNSHVSETNEIALSPSKGSGSGTSEETKP
AITVANLKRVAQLRFKKVSTDNVPLPEAAFELRSSNGNSQKLEASSNTQG
EIHPKDLTSGTYDLYETKAPKGYQQVTEKLATVTVDTTKPAEQMVKWEKP
HSFVKVEANKEVTIVNHKETLTFSGKKIWENDRPDQRPAKIQVQLLQNGQ
KMPNQIQEVTKDNDWSYHFKDLPKYDAKNQEYKYSVEEVKVPDGYKVSYL
GNDIFNTRETEFVFEQNNENLEFGNAEIKGQSGSKIIDEDTLTSFKGKKI
WKNDTAENRPQAIQVQLYADGVAVEGQTKFISGSGNEWSFEFKNLKKYNG
TGNDIIYSVKEVTVPTGYDVTYSANDIINTKREVITQQGPNLEIEETLPL
ESGASGGTTTVEDSRSVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDI
DGKELAGATMELRDSSGKTISTWISDGQVKDFYLMPGKYTFVETAAPDGY
EIATAITFTVNEQGQVTVNGKATKGDAHIVMVDAYKPTKGSGQVIDIEEK
LPDEQGHSGSTTEIEDSKPSDVIIGGQGEVVDTTEDTQSGMTGHSGSTTE
IEDSKSSDVIIGGQGQVVETTEDTQTGMHGDSGCKTEVEDTKLVQFFHFD
NKEPESNSEIPKKDKPKSNTSLPATGEKQHNKFFWMVTSCSLISSVFVIS LKSKKRLLSC
[1124] As discussed above, applicants have also determined the
nucleotide and encoded amino acid sequence of fimbrial structural
subunits in several other GAS AI-3 strains of bacteria. Examples of
sequences of these fimbrial structural subunits are set forth
below.
[1125] M3 strain isolate ISS 3040 is a GAS AI-3 strain of bacteria.
ISS3040_fimbrial is thought to be a fimbrial structural subunit of
M3 strain isolate ISS 3040. An example of a nucleotide sequence
encoding the ISS3040_fimbrial protein (SEQ ID NO: 263) and an
ISS3040_fimbrial protein amino acid sequence (SEQ ID NO: 264) are
set forth below. TABLE-US-00192 SEQ ID NO: 263
gagacggcaggagtgtccgaaaatgcaaaattaatagtaaaaaagacatt
tgactcttatacagacaatgaagttttaatgccaaaagctgattatactt
ttaaagtagaggcagatagtacagctagtggcaaaacgaaagacggttta
gagattaagccaggtattgttaatggtttaacagaacagattaccagcta
tactaatactgataaaccagatagtaaagttaaaagtacagagtttgatt
tttcaaaagtagtattccctggtattggtgtttaccgctatactgtttca
gaaaaacaaggtgatgttgaaggaattacctacgatactaagaagtggac
agtagatgtttatgttggaaacaaagaaggtggtggttttgaacctaagt
ttattgtatctaaggaacaaggaacagacgtcaaaaaaccagttaatttt
aacaactcgtttgcaactacttcgttaaaagttaagaagaatgtatcggg
gaatactggagaattgcaaaaagaatttgactttacattgacgcttaatg
aaagcacgaattttaaaaaagatcaaattgtttctttacaaaaaggaaac
gagaaatttgaagttaagattggtactccctacaagtttaaactcaaaaa
tggggaatctattcaactagacaagttaccagttggtattacttataaag
tcaatgaaatggaagctaataaagatgggtataaaacaacagcatccttg
aaagagggagatggtcaatctaaaatgtatcaattggatatggaacaaaa
aacagacgaatctgctgacgaaatcgttgtcacaaataagcgtgacactc
aagttccaactggtgttgtaggcacccttgctccatttgcagttcttagc SEQ ID NO: 264
ETAGVSENAKLIVKKTFDSYTDNEVLMPKADYTFKVEADSTASGKTKDGL
EIKPGIVNGLTEQIISYTNTDKPDSKVKSTEFDFSKVVFPGIGVYRTVSE
KQGDVEGITYDTKKWTVDVYVGNKEGGGFEPKFIVSKEQGTDVKKPVNFN
NSFATTSLKVKKNVSGNTGELQKEFDFTLTLNESTNFKKDQIVSLQKGNE
KFEVKIGTPYKFKLKNGESIQLDKLPVGITYKVNEMEANKDGYKTTASLK
EGDGQSKMYQLDMEQKTDESADEIVVTNKRDTQVPTGVVGTLAPFAVLS
[1126] M44 strain isolate ISS 3776 is a GAS AI-3 strain of
bacteria. ISS3776_fimbrial is thought to be a fimbrial structural
subunit of M44 isolate ISS 3776. An example of a nucleotide
sequence encoding the ISS3776_fimbrial protein (SEQ ID NO: 253) and
an ISS3776_fimbrial protein amino acid sequence (SEQ ID NO: 254)
are set forth below. TABLE-US-00193 SEQ ID NO: 253
ttggagagagaaaaaatgaaaaaaaacaaattattacttgctactgcaat
cttagcaactgctttaggaacagcttctttaaatcaaaacgtaaaagctg
agacggcaggggttgtaacaggaaaatcactacaagttacaaagacaatg
acttatgatgatgaagaggtgttaatgcccgaaaccgcctttacttttac
tatagagcctgatatgactgcaagtggaaaagaaggcagcctagatatta
aaaatggaattgtagaaggcttagacaaacaagtaacagtaaaatataag
aatacagataaaacatctcaaaaaactaaaatagcacaatttgatttttc
taaggttaaatttccagctataggtgtttaccgctatatggtttcagaga
aaaacgataaaaaagacggaattacgtacgatgataaaaagtggactgta
gatgtttatgttgggaataaggccaataacgaagaaggtttcgaagttct
atatattgtatcaaaagaaggtacttctagtactaaaaaaccaattgaat
ttacaaactctattaaaactacttccttaaaaattgaaaaacaaataact
ggcaatgcaggagatcgtaaaaaatcattcaacttcacattaacattaca
accaagtgaatattataaaactggatcagttgtgaaaatcgaacaggatg
gaagtaaaaaagatgtgacgataggaacgccttacaaatttactttggga
cacggtaagagtgtcatgttatcgaaattaccaattggtatcaattacta
tcttagtgaagacgaagcgaataaagacggctacactacaacggcaacat
taaaagaacaaggcaaagaaaagagttccgatttcactttgagtactcaa
aaccagaaaacagacgaatctgctgacgaaatcgttgtcacaaataagcg
tgacactcaagttccaactggtgttgtagggacccttgctccatttgcag
ttcttagcattgtggctattggtggagttatctatattacaaaacgtaaa aaagcttaa SEQ ID
NO: 254 MEREKMKKNKLLLATAILATALGTASLNQNVKAETAGVVTGKSLQVTKTM
TYDDEEVLMPETAFTFTIEPDMTASGKEGSLDIKNGIVEGLDKQVTVKYK
NTDKTSQKTKIAQFDFSKVKFPAIGVYRYMVSEKNDKKDGTTYDDKKWTV
DVYVGNKANNEEGFEVLYIVSKEGTSSTKKPIEFTNSIKTTSLKIEKQIT
GNAGDRKKSFNFTLTLQPSEYYKTGSVVKIEQDGSKKDVTIGTPYKFTLG
HGKSVMLSKLPIGINYYLSEDEANKDGYTTTATLKEQGKEKSSDFTLSTQ
NQKTDESADEIVVTNKRDTQVPTGVVGTLAPFAVLSIVAIGGVIYTTKRK KA
[1127] M77 strain isolate ISS4959 is a GAS AI-3 strain of bacteria.
ISS4959_fimbrial is thought to be a fimbrial structural subunit of
M77 strain ISS 4959. An example of a nucleotide sequence encoding
the ISS4959_fimbrial protein (SEQ ID NO: 271) and an
ISS4959_fimbrial protein amino acid sequence (SEQ ID NO: 272) are
set forth below. TABLE-US-00194 SEQ ID NO: 271
gtaacagtaaaatataagaatacagataaaacatctcaaaaaactaaaat
agcacaatttgatttttctaaggttaaatttccagctataggtgtttacc
gctatatggtttcagagaaaaacgataaaaaagacggaattacgtacgat
gataaaaagtggacngtagatgtttatgttgggaataaggccaataacga
agaaggtttcgaagttctatatattgtatcaaaagaaggtacttctagtn
ctaaaaaaccaattgaatttacaaactctattaaaactacttccttaaaa
attgaaaaacaaataactggcaatgcaggagatcgtaaaaaatcattcaa
cttcacattnacattacanccaagtgaatattataaaactggatcagttg
tgaaaatcgaacaggatggaagtaaaaaagatgtgacgataggaacgcct
tacaaatttactttgggacacggtaagagtgtcatgttatcgaaattncc
aattggtatcaattactatcttagtgaagacgaagcgaataaagacggnt
acactacancggcaacattaaaagaacaaggcaaagaaaagagttccgat
ttcactttgagtactcaaaaccagaaaacagacgaatctgctg SEQ ID NO: 272
VTVKYKNTDKTSQKTKIAQFDFSKVKFPAIGVYRYMVSEKNDKKDGITYD
DKKWTVDVYVGNKANNEEGREVLYIVSKEGTSSXKKPIEFTNSIKTTSLK
IEKQITGNAGDRKKSFNFTXTLXPSEYYKTGSVVKIEQDGSKKDVTIGTP
YKFTLGHGKSVNLSKXPIGINYYLSEDEANKDGYTTXATLKEQGKEKSSD
FTLSTQNQKTDESA
[1128] Examples of GAS AI-4 sequences from M12 strain isolate A735
are set forth below.
[1129] 19224133 is thought to be a RofA regulatory protein. An
example of a nucleotide sequence encoding the RofA regulatory
protein (SEQ ID NO: 104) and a RofA regulatory protein amino acid
sequence (SEQ ID NO: 105) are set forth below. TABLE-US-00195 SEQ
ID NO: 104 ATGACCATCCAAAAAAGGATGATATCTTGCCAATTTACACATCCTTCTAA
AGAAACTTATCTTTACCAACTCTATGCATCATCTAATGTCTTACAATTAC
TAGCGTTTTTAATAAAAAATGGTTCCCACTCTCGTCCCCTTACGGATTTT
GCAAGAAGTCATTTTTTATCAAACTCCTCAGCTTATCGGATGCGCGAAGC
ATTGATTCCTTTATTAAGAAACTTTGAATTAAAACTCTCTAAGAACAAGA
TTGTCGGTGAGGAATATCGTATCCGTTACCTCATCGCTCTGCTATATAGT
AAGTTTGGCATTAAAGTTTATGACTTGACGCAGCAAGACAAAAACATTAT
TCATAGCTTTTTATCCCATAGTTCCACCCACCTTAAAACTTCTCCTTGGT
TATCGGAATCGTTTTCTTTCTATGACATTTTATTAGCTTTATCGTGGAAG
CGGCATCAATTTTCGGTAACTATTCCCCAAACCAGAATTTTTCAACAATT
AAAAAAACTTTTTGTCTACGATTCTTTGAAAAAAAGTAGCCGTGATATTA
TCGAAACTTACTGCCAACTAAACTTTTCAGCAGGAGATTTGGACTACCTC
TATTTAATTTATATCACCGCTAATAATTCTTTTGCGAGCTTACAATGGAC
ACCTGAGCATATCAGACAATGTTGTCAACTTTTTGAAGAAAATGATACTT
TTCGCCTGCTTTTAAATCCTATCATCACTCTTTTACCTAACCTAAAAGAG
CAAAAGGCTAGTTTAGTAAAAGCTCTTATGTTTTTTTCAAAATCATTCTT
GTTTAATCTGCAACATTTTATTCCTACAGATTCTTTCCCAAGGTATTTCT
CGGATAAAAGCATTGATTTTCATTCCTATTATCTATTGCAAGATAATGTT
TATCAAATTCCTGATTTAAAGCCAGATTTGGTCATCACTCACAGTCAACT
GATTCCTTTTGTTCACCATGAACTTACAAAAGGAATTGCTGTTGCTGAAA
TATCTTTTGATGAATCGATTCTGTCTATCCAAGAATTGATGTATCAAGTT
AAAGAGGAAAAATTCCAAGCTGATTTAACCAAACAATTAACATAA SEQ ID NO: 105
MTIQKRMISCQFTHPSKETYLYQLYASSNVLQLLAFLIKNGSHSRPLTDF
ARSHFLSNSSAYRMREALIPLLRNFELKLSKNKIVGEEYRIRYLIALLYS
KFGIKVYDLTQQDKNIIHSFLSHSSTHLKTSPWLSESFSFYDILLALSWK
RHQFSVTIPQTRIFQQLKKLFVYDSLKKSSRDIIETYCQLNFSAGDLDYL
YLIYITANNSFASLQWTPEHIRQCCQLFEENDTFRLLLNPIITLLPNLKE
QKASLVKALMFFSKSFLFNLQHFIPETNLFVSPYYKGNQKLYTSLKLIVE
EWMAKLPGKRYLNHKHFHLFCHYVEQILRNIQPPLVVVFVASNFINAHLL
TDSFPRYFSDKSIDFHSYYLLQDNVYQIPDLKPDLVITHSQLIPFVHHEL
TKGTAVAEISFDESTLSIQELMYQVKEEKFQADLTKQLT
[1130] 19224134 is thought to be a protein F fibronectin binding
protein. An example of a nucleotide sequence encoding the protein F
fibronectin binding protein (SEQ ID NO: 106) and a protein F
fibronectin binding protein amino acid sequence (SEQ ID NO: 107)
are set forth below. TABLE-US-00196 SEQ ID NO: 106
ATGGTAAGCTCATATATGTTTGCGAGAGGAGAGAAAATGAATAACAAAAT
GTTTTTGAACAAAGAAGCCGGTTTTTTGGTACACACAAAAAGAAAAAGGC
GATTTGCTGTCACTTTAGTGGGAGTCTTTTTTCTGCTTTTGGCATGTGCG
GGTGCTATCGGTTTTGGTCAAGTAGCCTATGCTGCGGATGAGAAGACTGT
GCCGAATTTTAAAAGCCCAGATCCAGATTATCCCTGGTATGGTTATGATT
CGTATAGAGGAATATTTGCAAGATATCACAATTTAAAAGTAAATCTAAAA
GGAAGTAAGGAGTATCAAGCGTATTGTTTTAACCTAACAAAATACTTTCC
TCGCCCCACTTATAGTACTACAAATAATTTTTACAAGAAAATTGATGGGA
GTGGATCAGCGTTCAAATCTTATGCAGCGAATCCTAGGGTTTTAGATGAG
AATTTAGATAAATTAGAAAAAAATATACTGAATGTAATTTATAATGGATA
TAAAAGTAATGCAAATGGTTTTATGAATGGTATAGAAGATCTTAATGCTA
TACTAGTAACTCAAAACGCTATTTGGTACTATTCAGATAGTGCTCCATTA
AATGATGTTAATAAAATGTGGGAAAGAGAGGTTCGGAATGGGGAGATTAG
TGAGTCACAAGTTACTTTAATGCGTGAGGCATTGAAAAAACTAATTGATC
CCAATTTAGAAGCTACTGCAGCTAATAAAATCCCATCAGGATATCGTTTA
AATATCTTTAAGTCTGAAAATGAAGATTACCAAAATCTTTTAAGTGCTGA
ATATGTACCTGATGATCCCCCTAAACCTGGTGATACGTCAGAACATAATC
CTAAAACTCCCGAGTTGGATGGCACTCCAATTCCCGAGGACCCAAAACGT
CCAGATGAGAGTTCAGAACCTGCGCTTCCCCCATTAATGCCAGAGCTAGA
TGGTGAAGAAGTCCCAGAAGTTCCAAGCGAGAGCTTAGAACCTGCGCTTC
CCCCATTGATGCCAGAGCTAGATGGTGAAGAAGTCCCAGAAGTTCCAAGC
GAGAGCTTAGAACCTGCGCTTCCCCCATTGATGCCAGAGCTAGATGGTGA
AGAAGTCCCAGAAGTTCCAAGCGAGAGCTTAGAACCTGCGCTTCCCCCAT
TAATGCCAGAGCTAGATGGTGAAGAAGTCCCAGAAGTTCCAAGCGAGAGC
TTAGAACCTGCGCTTCCCCCATTGATGCCAGAGTTAGATGGTGAAGAAGT
CCCTGAAAAACCTAGTGTTGACTTACCTATTGAAGTTCCTCGTTATGAGT
TTAACAATAAAGACCAGTCACCTCTAGCGGGTGAGTCTGGTGAGACGGAG
TATATTACCGAAGTCTATGGAAATCAACAGAACCCTGTTGATATTGATAA
AAAACTTCCGAATGAAACAGGTTTTTCAGGAAATATGGTTGAGACAGAAG
ATACGAAAGAGCCAGAAGTGTTGATGGGAGGTCAAAGTGAGTCTGTTGAA
TTTACTAAAGACACTCAAACAGGCATGAGTGGTCAAACAACTCCTCAGGT
TGAGACAGAAGATACGAAAGAGCCAGAAGTGTTGATGGGAGGTCAAAGTG
AGTCTGTTGAATTTACTAAAGACACTCAAACAGGCATGAGTGGTCAAACA
ACTCCTCAGGTTGAGACAGAAGATACGAAAGAGCCAGGAGTGTTGATGGG
AGGCCAAAGTGAGTCTGTTGAATTTACTAAAGACACTCAAACAGGCATGA
GTGGTCAAACAACTCCTCAGGTTGAGACAGAAGACACGAAAGAGCCAGGA
AATCGGGAAAAGCCTACAAAAAATATAACACCTATCCTTCCTGCAACAGG
AGATATTGAGAATGTTTTGGCCTTTCTTGGAATCCTTATTTTGTCAGTAC
TTTCTATTTTTAGCCTTTTAAAAACAAACAAAACAATAAAGTCTGA SEQ ID NO: 107
MVSSYMFARGEKMNNKMFLNKEAGFLVHTKRKRRFAVTLVGVFFLLLACA
GAIGFGQVAYAADEKTVPNFKSPDPDYPWYGYDSYRGIFARYHNLKVNLK
GSKEYQAYCFNLTKYFPRPTYSTTNNFYKKIDGSGSAFKSYAANPRVLDE
NLDKLEKNILNVIYNGYKSNANGFMNGIEDLNAILVTQNAIWYYSDSAPL
NDVNKMWEREVRNGEISESQVTLMREALKKLTDPNLEATAANKIPSGYRL
NIFKSENEDYQNLLSAEYVPDDPPKPGDTSEHNPKTPELDGTPIPEDPKR
PDESSEPALPPLMPELDGEEVPEVPSESLEPALPPLMPELDGEEVPEVPS
ESLEPALPPLMPELDGEEVPEVPSESLEPALPPLMPELDGEEVPEVPSES
LEPALPPLMPELDGEEVPEKPSVDLPIEVPRYEFNNKDQSPLAGESGETE
YITEVYGNQQNPVDIDKKLPNETGFSGNMVETEDTKEPEVLMGGQSESVE
FTKDTQTGMSGQTTPQVETEDTKEPEVLMGGQSESVEETKDTQTGMSGQT
TPQVETEDTKEPGVLMGGQSESVEFTKDTQTGMSGQTTPQVETEDTKEPG
VLMGGQSESVEFTKDTQTGMSFFSETVTIVEDTRPKLVFHFDNNEPKVEE
NREKPTKNITPILPATGDIENVLAFLGILILSVLSIFSLLKNKQNNKV
[1131] 19224134 contains an amino acid motif indicative of a cell
wall anchor: SEQ ID NO: 181 LPATG (shown in italics in SEQ ID NO:
107, above). In some recombinant host cell systems, it may be
preferable to remove this motif to facilitate secretion of a
recombinant 19224134 protein from the host cell. Alternatively, in
other recombinant host cell systems, it may be preferable to use
the cell wall anchor motif to anchor the recombinantly expressed
protein to the cell wall. The extracellular domain of the expressed
protein may be cleaved during purification or the recombinant
protein may be left attached to either inactivated host cells or
cell membranes in the final composition.
[1132] A pilin motif, discussed above, containing a conserved
lysine (K) residue has also been identified in 19224134. The pilin
motif sequence is underlined in SEQ ID NO: 107, below. Conserved
lysine (K) residues are also marked in bold, at amino acid residues
275, 285, and 299. The pilin sequence, in particular the conserved
lysine residues, are thought to be important for the formation of
oligomeric, pilus-like structures. Preferred fragments of 19224134
include at least one conserved lysine residue. Preferably,
fragments include the pilin sequence. TABLE-US-00197 SEQ ID NO: 107
MVSSYMFARGEKMNNKMFLNKEAGFLVHTKRKRRFAVTLVGVFFLLLACA
GAIGFGQVAYAADEKTVPNFKSPDPDYPWYGYDSYRGIFARYHNLKVNLK
GSKEYQAYCFNLTKYFPRPTYSTTNNFYKKIDGSGSAFKSYAANPRVLDE
NLDKLEKNTLNVIYNGYKSNANGFMNGIEDLNAILVTQNAIWYYSDSAPL
NDVNKMWEREVRNGEISESQVTLMREALKKLIDPNLEATAANKIPSGYRL
NIFKSENEDYQNLLSAEYVPDDPPKPGDTSEHNPKTPELDGTPIPEDPKR
PDESSEPALPPLMPELDGEEVPEVPSESLEPALPPLMPELDGEEVPEVPS
ESLEPALPPLMPELDGEEVPEVPSESLEPALPPLMPELDGEEVPEVPSES
LEPALPPLMPELDGEEVPEKPSVDLPIEVPRYEFNNKDQSPLAGESGETE
YITEVYGNQQNPVDIDKKLPNETGFSGNMVETEDTKEPEVLMGGQSESVE
FTKDTQTGMSGQTTPQVETEDTKEPEVLMGGQSESVEFTKDTQTGMSGQT
TPQVETEDTKEPGVLMGGQSESVEFTKDTQTGMSGQTTPQVETEDTKEPG
VLMGGQSESVEFTKDTQTGMSGFSETVTIVEDTRPKLVEHFDNNEPKVEE
NREKPTKNITPILPATGDIENVLAFLGILILSVLSIPSLLKNKQNNKV
[1133] Two E boxes containing conserved glutamic residues have been
identified in 19224134. The E-box motifs are underlined in SEQ ID
NO: 107, below. The conserved glutamic acid (E) residues, at amino
acid residues 487 and 524, are marked in bold. The E box motifs, in
particular the conserved glutamic acid residues, are thought to be
important for the formation of oligomeric pilus-like structures of
19224134. Preferred fragments of 19224134 include at least one
conserved glutamic acid residue. Preferably, fragments include at
least one E box motif. TABLE-US-00198 SEQ ID NO: 107
MVSSYMFARGEKMNNKMFLNKEAGFLVHTKRKRRFAVTLVGVFFLLLACA
GAIGFGQVAYAADEKTVPNFKSPDPDYPWYGYDSYRGIFARYHNLKVNLK
GSKEYQAYCFNLTKYFPRPTYSTTNNFYKKIDGSGSAFKSYAANPRVLDE
NLDKLEKNTLNVIYNGYKSNANGFMNGIEDLNAILVTQNAIWYYSDSAPL
NDVNKMWEREVRNGEISESQVTLMREALKKLIDPNLEATAANKIPSGYRL
NIFKSENEDYQNLLSAEYVPDDPPKPGDTSEHNPKTPELDGTPIPEDPKR
PDESSEPALPPLMPELDGEEVPEVPSESLEPALPPLMPELDGEEVPEVPS
ESLEPALPPLMPELDGEEVPEVPSESLEPALPPLMPELDGEEVPEVPSES
LEPALPPLMPELDGEEVPEKPSVDLPIEVPRYEFNNKDQSPLAGESGETE
YITEVYGNQQNPVDIDKKLPNETGFSGNMVETEDTKEPEVLMGGQSESVE
FTKDTQTGMSGQTTPQVETEDTKEPEVLMGGQSESVEFTKDTQTGMSGQT
TPQVETEDTKEPGVLMGGQSESVEFTKDTQTGMSGQTTPQVETEDTKEPG
VLMGGQSESVEFTKDTQTGMSGFSETVTIVEDTRPKLVEHFDNNEPKVEE
NREKPTKNITPILPATGDIENVLAFLGILILSVLSIPSLLKNKQNNKV
[1134] 19224135 is thought to be a capsular polysaccharide adhesin
(Cpa) protein. An example of a nucleotide sequence encoding the Cpa
protein (SEQ ID NO: 108) and a Cpa protein amino acid sequence (SEQ
ID NO: 109) are set forth below. TABLE-US-00199 SEQ ID NO: 108
ATGAATAACAAAAAATTGCAAAAGAAGCAAGATGCTCCTCGGGTATCAAA
CAGAAAGCCAAAACAATTAACTGTCACTTTAGTGGGAGTATTTTTAATGT
TTTTGACCTTGGTAAGTTCCATGAGAGGTGCTCAAAGCATATTTGGAGAG
GAAAAGAGAATTGAAGAAGTCAGTGTTCCTAAAATAAAAAGTCCAGATGA
TGCCTACCCTTGGTATGGCTATGATTCATATGACTCTAGTCATCCTTACT
ATGAACGTTTTAAAGTAGCACATGATTTAAGGGTTAATTTAAATGGAAGT
AAGAGCTACCAAGTATATTGCTTTAATATCAATTCTCATTATCCGAATAG
AAAAAATGCTTTTTCTAAACAATGGTTTAAGAGAGTTGATGGGACAGGTG
ATGTGTTCACAAATTATGCTCAGACACCTAAGATTCGTGGAGAATCATTG
AATAATAAACTTTTAAGTATTATGTACAACGCTTATCCTAAAAATGCTAA
TGGCTATATGGATAAGATAGAACCATTAAATGCTATTTTAGTAACTCAAC
AAGCTGTTTGGTACTATTCTGACAGTTCTTATGGTAATATAAAAACGTTA
TGGGCATCTGAGCTTAAAGACGGAAAAATAGATTTTGAACAAGTAAAATT
AATGCGTGAAGCTTACTCAAAACTAATTAGTGATGATTTAGAAGAAACAT
CTAAAAATAAGCTACCTCAAGGATCTAAACTGAATATTTTTGTTCCGCAA
GATAAATCTGTTCAAAATTTATTAAGTGCAGAGTACGTGCCTGAATCCCC
TCCGGCACCAGGTCAGTCTCCAGAACCGCCAGTGCAAACAAAAAAAACAT
CAGTCATTATCAGAAAATATGCGGAAGGTGACTACTCTAAACTTCTAGAG
GGAGCAACTTTGCGTTTAACAGGGGAAGATATCCTAGATTTTCAAGAAAA
AGTCTTCCAAAGTAATGGAACAGGAGAAAAGATTGAATTATCAAATGGGA
CTTATACCTTAACAGAAACATCATCTCCAGATGGATATAAAATTGCGGAG
CCGATTAAGTTTAGAGTAGTGAATAAAAAAGTATTTATCGTCCAAAAAGA
TGGTTCTCAAGTGGAAAATCCAAACAAAGAAGTAGCAGAGCCATACTCAG
TGGAAGCGTACAGCGATATGCAAGATAGTAACTATATTAATCCAGAAACG
TTCACTCCTTATGGGAAATTTTATTACGCTAAAAATAAGGATAAAAGTTC
ACAAGTTGTCTACTGTTTTAATGCTGATTTACACTCTCCACCTGAATCAG
AGGATGGGGGAGGAACTATAGATCCTGATATTAGTACGATGAAAGAAGTC
AAGTACACACATACGGCAGGTAGTGATTTGTTTAAATACGCGCTAAGACC
GAGAGATACAAATCCAGAAGACTTCTTAAAGCACATTAAAAAAGTAATTG
AAAAAGGCTACAATAAAAAAGGTGATAGCTATAATGGATTAACAGAAACA
CAGTTTCGCGCGGCTACTCAGCTTGCTATCTATTACTTTACAGACAGCAC
TGACTTAAAAACCTTAAAAACTTATAACAATGGGAAAGGTTACCATGGAT
TTGAATCTATGGATGAAAAAACCCTAGCTGTAACAAAAGAATTAATTAAT
TACGCTCAAGATAATAGTGCCCCTCAACTAACAAATCTTGATTTCTTCGT
ACCTAATAATAGCAAATACCAATCTCTTATTGGGACAGAATACCATCCAG
ATGATTTGGTTGACGTGATTCGTATGGAAGATAAAAAGCAAGAAGTTATT
CCAGTAACTCACAGTTTGACAGTGAAAAAAACAGTAGTCGGTGAGTTGGG
AGATAAAACTAAAGGCTTCCAATTTGAACTTGAGTTGAAAGATAAAACTG
GACAGCCTATTGTTAACACTCTAAAAACTAATAATCAAGATTTAGTAGCT
AAAGATGGGAAATATTCATTTAATCTAAAGCATGGTGACACCATAAGAAT
AGAAGGATTACCGACGGGATATTCTTATACTCTGAAAGAGACTGAAGCTA
AGGATTATATAGTAACCGTTGATAACAAAGTTAGTCAAGAAGCTCAATCA
GCAAGTGAGAATGTCACAGCAGACAAAGAAGTCACTTTTGAAAACCGTAA
AGATCTTGTCCCACCAACTGGTTTTATTACTGATGGTGGAACCTATGTGT
GGTTATTATTGCTTGTCGCATTTGGTTTGTTAGTGTGGTTCTTTGGTGGT
AAAGGACTAAAAAATGACTAA SEQ ID NO: 109
MNNKKLQKKQDAPRVSNRKPKQLTVTLVGVFLMELTLVSSMRGAQSIFGE
EKRIEEVSVPKIKSPDDAYPWYGYDSYDSSHPYYERFKVAHDLRVNLNGS
KSYQVYCFNINSHYPNRKNAFSKQWFKRVDGTGDVFTNYAQTPKIRGESL
NNKLLSIMYNAYPKNANGYMDKIEPLNAILVTQQAVWYYSDSSYGNIKTL
WASELKDGKIDFEQVKLMREAYSKLISDDLEETSKNKLPQGSKLNIFVPQ
DKSVQNLLSAEYVPESPPAPGQSPEPPVQTKKTSVIIRKYAEGDYSKLLE
GATLRLTGEDILDFQEKVFQSNGTGEKIELSNGTYTLTETSSPDGYKIAE
PIKFRVVNKKVFIVQKDGSQVENPNKEVAEPYSVEAYSDMQDSNYINPET
FTPYGKFYYAKNKDKSSQVVYCFNADLHSPPESEDGGGTIDPDISTMKEV
KYTHTAGSDLFKYALRPRDTNPEDFLKHIKKVTEKGYNKKGDSYNGLTET
QFRAATQLAIYYFTDSTDLKTLKTYNNGKGYHGFESMDEKTLAVTKELIN
YAQDNSAPQLTNLDFFVPNNSKYQSLIGTEYHPDDLVDVIRMEDKKQEVI
PVTHSLTVKKTVVGELGDKTKGFQFELELKDKTGQPIVNTLKTNNQDLVA
KDGKYSFNLKHGDTIRIEGLPTGYSYTLKETEAKDYIVTVDNKVSQEAQS
ASENVTADKEVTFENRKDLVPPTGFITDGGTYLWLLLLVPFGLLVWFFGR KGLKND
[1135] 19224135 contains an amino acid motif indicative of a cell
wall anchor: SEQ ID NO: 184 VPPTG (shown in italics in SEQ ID NO:
109, above). In some recombinant host cell systems, it may be
preferable to remove this motif to facilitate secretion of a
recombinant 19224135 protein from the host cell. Alternatively, in
other recombinant host cell systems, it may be preferable to use
the cell wall anchor motif to anchor the recombinantly expressed
protein to the cell wall. The extracellular domain of the expressed
protein may be cleaved during purification or the recombinant
protein may be left attached to either inactivated host cells or
cell membranes in the final composition.
[1136] A pilin motif, discussed above, containing a conserved
lysine (K) residue has also been identified in 19224135. The pilin
motif sequence is underlined in SEQ ID NO: 109, below. Conserved
lysine (K) residues are also marked in bold, at amino acid residues
164 and 172. The pilin sequence, in particular the conserved lysine
residues, are thought to be important for the formation of
oligomeric, pilus-like structures. Preferred fragments of 19224135
include at least one conserved lysine residue. Preferably,
fragments include the pilin sequence. TABLE-US-00200 SEQ ID NO: 109
MNNKKLQKKQDAPRVSNRKPKQLTVTLVGVFLMELTLVSSMRGAQSIFGE
EKRIEEVSVPKIKSPDDAYPWYGYDSYDSSHPYYERFKVAHDLRVNLNGS
KSYQVYCFNINSHYPNRKNAFSKQWFKRVDGTGDVFTNYAQTPKIRGESL
NNKLLSIMYNAYPKNANGYMDKIEPLNAILVTQQAVWYYSDSSYGNIKTL
WASELKDGKIDFEQVKLMREAYSKLISDDLEETSKNKLPQGSKLNIFVPQ
DKSVQNLLSAEYVPESPPAPGQSPEPPVQTKKTSVIIRKYAEGDYSKLLE
GATLRLTGEDILDFQEKVFQSNGTGEKIELSNGTYTLTETSSPDGYKIAE
PIKFRVVNKKVFIVQKDGSQVENPNKEVAEPYSVEAYSDMQDSNYINPET
FTPYGKFYYAKNKDKSSQVVYCFNADLHSPPESEDGGGTIDPDISTMKEV
KYTHTAGSDLFKYALRPRDTNPEDFLKHIKKVTEKGYNKKGDSYNGLTET
QFRAATQLAIYYFTDSTDLKTLKTYNNGKGYHGFESMDEKTLAVTKELIN
YAQDNSAPQLTNLDFFVPNNSKYQSLIGTEYHPDDLVDVIRMEDKKQEVI
PVTHSLTVKKTVVGELGDKTKGFQFELELKDKTGQPIVNTLKTNNQDLVA
KDGKYSFNLKHGDTIRIEGLPTGYSYTLKETEAKDYIVTVDNKVSQEAQS
ASENVTADKEVTFENRKDLVPPTGFITDGGTYLWLLLLVPFGLLVWFFGR KGLKND
[1137] An E box containing a conserved glutamic residue has been
identified in 19224135. The E-box motif is underlined in SEQ ID NO:
109, below. The conserved glutamic acid (E), at amino acid residue
339, is marked in bold. The E box motif, in particular the
conserved glutamic acid residue, is thought to be important for the
formation of oligomeric pilus-like structures of 19224135.
Preferred fragments of 19224135 include the conserved glutamic acid
residue. Preferably, fragments include the E box motif.
TABLE-US-00201 SEQ ID NO: 109
MNNKKLQKKQDAPRVSNRKPKQLTVTLVGVFLMELTLVSSMRGAQSIFGE
EKRIEEVSVPKIKSPDDAYPWYGYDSYDSSHPYYERFKVAHDLRVNLNGS
KSYQVYCFNINSHYPNRKNAFSKQWFKRVDGTGDVFTNYAQTPKIRGESL
NNKLLSIMYNAYPKNANGYMDKIEPLNAILVTQQAVWYYSDSSYGNIKTL
WASELKDGKIDFEQVKLMREAYSKLISDDLEETSKNKLPQGSKLNIFVPQ
DKSVQNLLSAEYVPESPPAPGQSPEPPVQTKKTSVIIRKYAEGDYSKLLE
GATLRLTGEDILDFQEKVFQSNGTGEKIELSNGTYTLTETSSPDGYKIAE
PIKFRVVNKKVFIVQKDGSQVENPNKEVAEPYSVEAYSDMQDSNYINPET
FTPYGKFYYAKNKDKSSQVVYCFNADLHSPPESEDGGGTIDPDISTMKEV
KYTHTAGSDLFKYALRPRDTNPEDFLKHIKKVTEKGYNKKGDSYNGLTET
QFRAATQLAIYYFTDSTDLKTLKTYNNGKGYHGFESMDEKTLAVTKELIN
YAQDNSAPQLTNLDFFVPNNSKYQSLIGTEYHPDDLVDVIRMEDKKQEVI
PVTHSLTVKKTVVGELGDKTKGFQFELELKDKTGQPIVNTLKTNNQDLVA
KDGKYSFNLKHGDTIRIEGLPTGYSYTLKETEAKDYIVTVDNKVSQEAQS
ASENVTADKEVTFENRKDLVPPTGFITDGGTYLWLLLLVPFGLLVWFFGR KGLKND
[1138] 19224136 is thought to be a LepA protein. An example of a
nucleotide sequence encoding the LepA protein (SEQ ID NO: 110) and
a LepA protein amino acid sequence (SEQ ID NO: 111) are set forth
below. TABLE-US-00202 SEQ ID NO: 110
ATGACTAATTACCTAAATCGCTTAAATGAGAATCCACTATTTAAAGCTTT
CATACGGTTAGTACTTAAGATTTCTATTATTGGATTTCTAGGTTACATTC
TATTTCAGTATGTTTTTGGCGTCATGATTGTTAACACAAATCAGATGAGT
CCTGCTGTAAGTGCTGGTGATGGAGTCTTATATTATCGTTTGACTGATCG
CTATCATATTAATGATGTGGTGGTCTATGAGGTTGATAACACTTTGAAAG
TTGGTCGAATTGCCGCTCAAGCTGGCGATGAGGTTAGTTTTACGCAAGAA
GGAGGACTGTTGATTAATGGGCATCCACCAGAAAAAGAGGTCCCTTACCT
GACGTATCCTCACTCAAGTGGTCCAAACTTTCCCTATAAAGTTCCTACGG
GTACGTATTTCATATTGAATGATTATCGTGAAGAACGTTTGGACAGTCGT
TATTATGGGGCGTTACCCATCAATCAAATCAAAGGGAAAATCTCAACTCT
ATTAAGAGTGAGAGGAATTTAA SEQ ID NO: 111
MTNYLNRLNENPLFKAFIRLVLKISIIGFLGYILFQYVFGVMIVNTNQMS
PAVSAGDGVLYYRLTDRYHINDVVVYEVDNTLKVGRIAAQAGDEVSFTQE
GGLLINGHPPEKEVPYLTYPHSSGPNFPYKVPTGTYFILNDYREERLDSR
YYGALPINQIKGKISTLLRVRGI
[1139] 19224137 is thought to be a fimbrial protein. An example of
a nucleotide sequence encoding the fimbrial protein (SEQ ID NO:
112) and a fimbrial protein amino acid sequence (SEQ ID NO: 113)
are set forth below. TABLE-US-00203 SEQ ID NO: 112
ATGAAAAAAAATAAATTATTACTTGCTACTGCAATCTTAGCAACTGCTTT
AGGAACAGCTTCTTTAAATCAAAACGTAAAAGCTGAGACGGCAGGGGTTG
TTAGCAGTGGTCAATTAACAATAAAAAAATCAATTACAAATTTTAATGAT
GATACACTTTTGATGCCTAAGACAGACTATACTTTTAGCGTTAATCCGGA
TAGTGCGGCTACAGGTACTGAAAGTAATTTACCAATTAAACCAGGTATTG
CTGTTAACAATCAAGATATTAAGGTTTCTTATTCTAATACTGATAAGACA
TCAGGTAAAGAAAAACAAGTTGTTGTTGACTTTATGAAAGTTACTTTTCC
TAGCGTTGGTATTTACCGTTATGTTGTTACCGAGAATAAAGGGACAGCAG
AAGGAGTTACATATGATGATACAAAATGGTTAGTTGACGTCTATGTTGGT
AATAATGAAAAGGGAGGTCTTGAACCAAAGTATATTGTATCTAAAAAAGG
AGATTCTGCTACTAAAGAACCAATCCAGTTTAATAATTCATTCGAAACAA
CGTCATTAAAAATTGAAAAGGAAGTTACTGGTAATACAGGAGATCATAAA
AAAGCATTTACCTTTACATTAACATTGCAACCAAATGAATACTATGAGGC
AAGTTCGGTTGTGAAAATTGAAGAGAACGGACAAACGAAAGATGTGAAAA
TTGGGGAGGCATATAAGTTTACTTGAACGAATAGTCAGAGTGTGATATTG
TCTAAATTACCAGTTGGTATTAATTATAAAGTTGAAGAAGCAGAAGCTAA
TCAAGGTGGATATACTACAACAGCAACTTTAAAAGATGGAGAAAAGTTAT
CTACTTATAACTTAGGTCAGGAACATAAAACAGACAAGACTGCTGATGAA
ATCGTTGTCACAAATAACCGTGACACTCAAGTTCCAACTGGTGTTGTAGG
CACCCTTGCTCCATTTGCAGTTCTTAGCATTGTGGCTATTGGTGGAGTTA
TCTATATTACAAAACGTAAAAAAGCTTAA SEQ ID NO: 113
MKKNKLLLATAILATALGTASLNQNVKAETAGVVSSGQLTIKKSITNFND
DTLLMPKTDYTFSVNPDSAATGTESNLPIKPGIAVNNQDIKVSYSNTDKT
SGKEKQVVVDFMKVTFPSVGIYRYVVTENKGTAEGVTYDDTKWLVDVYVG
NNEKGGLEPKYIVSKKGDSATKEPIQFNNSFETTSLKIEKEVTGNTGDHK
KAFTFTLTLQPNEYYEASSVVKIEENGQTKDVKIGEAYKFTLNDSQSVIL
SKLPVGINYKVEEAEANQGGYTTTATLKDGEKLSTYNLGQEHKTDKTADE
IVVTNNRDTQVPTGVVGTLAPFAVLSIVAIGGVIYITKRKKA
[1140] 19224137 contains an amino acid motif indicative of a cell
wall anchor: SEQ ID NO: 140 QVPTG (shown in italics in SEQ ID NO:
113, above). In some recombinant host cell systems, it may be
preferable to remove this motif to facilitate secretion of a
recombinant 19224137 protein from the host cell. Alternatively, in
other recombinant host cell systems, it may be preferable to use
the cell wall anchor motif to anchor the recombinantly expressed
protein to the cell wall. The extracellular domain of the expressed
protein may be cleaved during purification or the recombinant
protein may be left attached to either inactivated host cells or
cell membranes in the final composition.
[1141] A pilin motif, discussed above, containing a conserved
lysine (K) residue has also been identified in 19224137. The pilin
motif sequence is underlined in SEQ ID NO: 113, below. A conserved
lysine (K) residue is also marked in bold, at amino acid residue
160. The pilin sequence, in particular the conserved lysine
residues, are thought to be important for the formation of
oligomeric, pilus-like structures. Preferred fragments of 19224137
include the conserved lysine residue. Preferably, fragments include
the pilin sequence. TABLE-US-00204 SEQ ID NO: 113
MKKNKLLLATAILATALGTASLNQNVKAETAGVVSSGQLTIKKSITNFND
DTLLMPKTDYTFSVNPDSAATGTESNLPIKPGIAVNNQDIKVSYSNTDKT
SGKEKQVVVDFMKVTFPSVGIYRYVVTENKGTAEGVTYDDTKWLVDVYVG
NNEKGGLEPKYIVSKKGDSATKEPIQFNNSFETTSLKIEKEVTGNTGDHK
KAFTFTLTLQPNEYYEASSVVKIEENGQTKDVKIGEAYKFTLNDSQSVIL
SKLPVGINYKVEEAEANQGGYTTTATLKDGEKLSTYNLGQEHKTDKTADE
IVVTNNRDTQVPTGVVGTLAPFAVLSIVAIGGVIYITKRKKA
[1142] An E box containing a conserved glutamic residue has been
identified in 19224137. The E-box motif is underlined in SEQ ID NO:
113, below. The conserved glutamic acid (E), at amino acid residue
263, is marked in bold. The E box motif, in particular the
conserved glutamic acid residue, is thought to be important for the
formation of oligomeric pilus-like structures of 19224137.
Preferred fragments of 19224137 include the conserved glutamic acid
residue. Preferably, fragments include the E box motif.
TABLE-US-00205 SEQ ID NO: 113
MKKNKLLLATAILATALGTASLNQNVKAETAGVVSSGQLTIKKSITNFND
DTLLMPKTDYTFSVNPDSAATGTESNLPIKPGIAVNNQDIKVSYSNTDKT
SGKEKQVVVDFMKVTFPSVGIYRYVVTENKGTAEGVTYDDTKWLVDVYVG
NNEKGGLEPKYIVSKKGDSATKEPIQFNNSFETTSLKIEKEVTGNTGDHK
KAFTFTLTLQPNEYYEASSVVKIEENGQTKDVKIGEAYKFTLNDSQSVIL
SKLPVGINYKVEEAEANQGGYTTTATLKDGEKLSTYNLGQEHKTDKTADE
IVVTNNRDTQVPTGVVGTLAPFAVLSIVAIGGVIYITKRKKA
[1143] 19224138 is thought to be a SrtC2-type sortase. An example
of a nucleotide sequence encoding the SrtC2 sortase (SEQ ID NO:
114) and a SrtC2 sortase amino acid sequence (SEQ ID NO: 115) are
set forth below. TABLE-US-00206 SEQ ID NO: 114
ATGATGATGACAATTGTACAGGTTATCAATAAAGCCATTGATACTCTCAT
TCTTATCTTTTGTTTAGTCGTACTATTTTTAGCTGGTTTTGGTTTGTGGG
ATTCTTATCATCTCTATCAACAAGCAGACGCTTCTAATTTCAAAAAATTT
AAAACAGCTCAACAACAGCCTAAATTTGAAGACTTGTTAGCTTTGAATGA
GGATGTCATTGGTTGGTTAAATATCCCGGGGACTCATATTGATTATCCTC
TAGTTCAGGGAAAAACGAATTTAGAGTATATTAATAAAGCAGTTGATGGC
AGTGTTGCCATGTCTGGTAGTTTATTTTTAGATACACGGAATCATAATGA
TTTTACGGACGATTACTCTCTGATTTATGGCCATCATATGGCAGGTAATG
CCATGTTTGGCGAAATTCCAAAATTTTTAAAAAAGGATTTTTTCAACAAA
CATAATAAAGCTATCATTGAAACAAAAGAGAGAAAAAAACTAACCGTCAC
TATTTTTGCTTGTCTCAAGACAGATGCCTTTGACCAGTTAGTTTTTAATC
CTAATGCTATTACCAATCAAGACCAACAAAGGCAGCTCGTTGATTATATC
AGTAAAAGATCAAAACAATTTAAACCTGTTAAATTGAAGCATCATACAAA
GTTCGTTGCTTTTTCAACGTGTGAAAATTTTTCTACTGACAATCGTGTTA
TCGTTGTCGGTACTATTCAAGAATAA SEQ ID NO: 115
MMMTIVQVINKAIDTLILIFCLVVLFLAGFGLWDSYHLYQQADASNFKKF
KTAQQQPKFEDLLALNEDVIGWLNIPGTHIDYPLVQGKTNLEYINKAVDG
SVAMSGSLFLDTRNHNDFTDDYSLIYGHHMAGNAMFGEIPKFLKKDFFNK
HNKAIIETKERKKLTVTIFACLKTDAFDQLVFNPNAITNQDQQRQLVDYI
SKRSKQFKPVKLKHHTKFVAFSTCENFSTDNRVIVVGTIQE
[1144] 19224139 is an open reading frame that encodes a sortase
substrate motif LPXAG shown in italics in SEQ ID NO: 117. An
example of a nucleotide sequence of the open reading frame (SEQ ID
NO: 116) and the amino acid sequence encoded by the open reading
frame (SEQ ID NO: 117) are set forth below. TABLE-US-00207 SEQ ID
NO: 116 ATGTTATTTTCTGTCGTAATGATATTAACCATGCTGGCCTTTAATCAGAC
TGTTTTAGCAAAAGACAGCACTGTTCAAACTAGCATTAGTGTCGAAAATG
TCTTAGAGAGAGCAGGCGATAGTACCCCATTTTCGATTGCATTAGAATCA
ATTGATGCGATGAAAACAATAGAAGAAATAACAATTGCTGGTTCTGGAAA
AGCAAGCTTTTCCCCTCTGACCTTCACAACAGTTGGGCAATATACTTATC
GTGTTTATCAGAAGCCTTCACAAAATAAAGATTATCAAGCAGATACTACT
GTATTTGACGTTCTTGTCTATGTGACCTATGATGAAGATGGGACTCTAGT
CGCAAAAGTTATTTCTCGAAGGGCTGGAGACGAAGAAAAATCAGCGATTA
CTTTTAAGCCCAAACGGTTAGTAAAACCAATACCGCCTAGACAACCTAAC
ATCCCTAAAACCCCATTACCATTAGCTGGTGAAGTAAAAAGTTTATTGGG
TATCTTAAGTATCGTATTACTGGGGTTACTAGTTCTTCTTTATGTTAAAA AACTGAAGAG SEQ
ID NO: 117 MLFSVVMILTMLAFNQTVLAKDSTVQTSISVENVLERAGDSTPFSIALES
IDAMKTIEEITIAGSGKASFSPLTFTTVGQYTYRVYQKPSQNKDYQADTT
VFDVLVYVTYDEDGTLVAKVISRRAGDEEKSAITFKPKRLVKPIPPRQPN
IPKTPLPLAGEVKSLLGILSIVLLGLLVLLYVKKLKSKL
[1145] 19224139 contains an amino acid motif indicative of a cell
wall anchor: SEQ ID NO: 185 LPLAG (shown in italics in SEQ ID NO:
117, above). In some recombinant host cell systems, it may be
preferable to remove this motif to facilitate secretion of a
recombinant 19224139 protein from the host cell. Alternatively, in
other recombinant host cell systems, it may be preferable to use
the cell wall anchor motif to anchor the recombinantly expressed
protein to the cell wall. The extracellular domain of the expressed
protein may be cleaved during purification or the recombinant
protein may be left attached to either inactivated host cells or
cell membranes in the final composition.
[1146] A pilin motif, discussed above, containing a conserved
lysine (K) residue has also been identified in 19224139. The pilin
motif sequence is underlined in SEQ ID NO: 117, below. A conserved
lysine (K) residue is also marked in bold, at amino acid residue
138. The pilin sequence, in particular the conserved lysine
residue, is thought to be important for the formation of
oligomeric, pilus-like structures. Preferred fragments of 19224139
include the conserved lysine residue. Preferably, fragments include
the pilin sequence. TABLE-US-00208 SEQ ID NO: 117
MLFSVVMILTMLAFNQTVLAKDSTVQTSISVENVLERAGDSTPFSIALES
IDAMKTIEEITIAGSGKASFSPLTFTTVGQYTYRVYQKPSQNKDYQADTT
VFDVLVYVTYDEDGTLVAKVISRRAGDEEKSAITFKPKRLVKPIPPRQPN
IPKTPLPLAGEVKSLLGILSIVLLGLLVLLYVKKLKSKL
[1147] Two E boxes containing conserved glutamic residues have been
identified in 19224139. The E-box motifs are underlined in SEQ ID
NO: 117, below. The conserved glutamic acid (E) residues, at amino
acid residues 58 and 128, are marked in bold. The E box motifs, in
particular the conserved glutamic acid residues, are thought to be
important for the formation of oligomeric pilus-like structures of
19224139. Preferred fragments of 19224139 include at least one
conserved glutamic acid residue. Preferably, fragments include at
least one E box motif. TABLE-US-00209 SEQ ID NO: 117
MLFSVVMILTMLAFNQTVLAKDSTVQTSISVENVLERAGDSTPFSIALES
IDAMKTIEEITIAGSGKASFSPLTFTTVGQYTYRVYQKPSQNKDYQADTT
VFDVLVYVTYDEDGTLVAKVISRRAGDEEKSAITFKPKRLVKPIPPRQPN
IPKTPLPLAGEVKSLLGILSIVLLGLLVLLYVKKLKSKL
[1148] 19224140 is thought to be a MsmRL protein. An example of a
nucleotide sequence encoding the MsmRL protein (SEQ ID NO: 118) and
a MsmRL protein amino acid sequence (SEQ ID NO: 119) are set forth
below. TABLE-US-00210 SEQ ID NO: 118
ATGGTTATATTCGATTTAAAACATGTGCAAACATTACACAGCTTGTCTCA
ATTACCTATTTCAGTGATGTCACAAGATAAGGCACTTATTCAAGTATATG
GTAATGACGACTATTTATTATGTTACTATCAATTTTTAAAGCATCTAGCT
ATTCCTCAAGCTGCACAAGATGTTATTTTTTATGAGGGTTTATTTGAAGA
GTCCTTTATGATTTTTCCTCTTTGTCACTACATTATTGCCATTGGACCTT
TCTACCCTTATTCACTTAATAAAGACTATCAGGAACAATTAGCTAATAAT
TTTTTAAAACATTCTTCTCATCGTAGCAAAGAAGAGCTCTTATCCTATAT
GGCACTTGTCCCACATTTTCCAATTAATAATGTGCGGAACCTTTTGATAG
CTATTGACGCTTTTTTTGACACACAATTTGAGACGACTTGCCAACAAACA
ATTCATCAATTGTTGCAGCATTCAAAACAGATGACTGCTGATCCTGATAT
CATTCATCGCCTTAAGCATATTAGCAAAGCATCTAGCCAACTACCGCCTG
TTTTAGAGCACCTAAATCATATTATGGATCTGGTAAAGCTAGGCAATCCA
CAATTGCTCAAGCAAGAAATCAATCGCATCCCCTTATCAAGTATCACCTC
ATCTTCTATTTCTGCTCTAAGGGCGGAAAAGAACCTCACTGTTATCTATT
TAACTAGGTTACTGGAATTCAGTTTTGTAGAAAATACTGACGTAGCAAAG
CATTATAGCCTTGTCAAATACTACATGGCCTTAAATGAAGAAGCGAGTGA
CTTGCTCAAAGTTTTGAGAATTCGCTGTGCAGCCATCATCCATTTTTCCG
AATCATTAACCAATAAAAGTATTTCTGATAAACGTCAAATGTACAATAGT
GTGCTTCATTATGTCGATAGTCACCTGTATTCCAAATTAAAGGTATCTGA
TATCGCTAAGCGCCTATATGTTTCCGAATCTCACTTACGTTCAGTCTTTA
AAAAATACTCAAATGTTTCCTTACAACATTATATTCTAAGTACAAAAATC
AAAGAAGCTCAACTACTCTTAAAACGAGGAATTCCTGTTGGAGAAGTGGC
TAAAAGCTTATATTTTTATGACACTACCCATTTTCATAAAATCTTTAAAA
AATACACGGGTATTTCTTCAAAAGACTATCTTGCTAAATACCGAGATAAT ATTTAA SEQ ID
NO: 119 MVIFDLKHVQTLHSLSQLPISVMSQDKALIQVYGNDDYLLCYYQFLKHLA
TPQAAQDVIFYEGLFEESFMIFPLCHYIIAIGPFYPYSLNKDYQEQLANN
FLKHSSHRSKEELLSYMALVPHFPINNVRNLLIAIDAFFDTQFETTCQQT
IHQLLQHSKQMTADPDIIHRLKHISKASSQLPPVLEHLNHIMDLVKLGNP
QLLKQEINRIPLSSITSSSISALRAEKNLTVIYLTRLLEFSFVENTDVAK
HYSLVKYYMALNEEASDLLKVLRIRCAAIIHFSESLTNKSISDKRQMYNS
VLHYVDSHLYSKLKVSDIAKRLYVSESHLRSVFKKYSNVSLQHYILSTKI
KEAQLLLKRGIPVGEVAKSLYFYDTTHFHKIFKKYTGISSKDYLAKYRDN I
[1149] 19224141 is thought to be a protein F2 fibronectin binding
protein. An example of a nucleotide sequence encoding the protein
F2 fibronectin binding protein (SEQ ID NO: 120) and a protein F2
fibronectin binding protein amino acid sequence (SEQ ID NO: 121)
are set forth below. TABLE-US-00211 SEQ ID NO: 120
ATGACACAAAAAAATAGCTATAAGTTAAGCTTCCTGTTATCCCTAACAGG
ATTTATTTTAGGTTTATTATTGGTTTTTATAGGATTGTCCGGAGTATCAG
TAGGACATGCGGAAACAAGAAATGGAGCAAACAAACAAGGATCTTTTGAA
ATCAAGAAAGTCGAGCAAAAGAATAAGGCTTTACCGGGAGCAAGTTTTTC
AGTGACATCAAAGGATGGCAAGGGAACATGTGTTCAAAGGTTGACTTGAA
ATGATAAAGGTATTGTAGATGGTCAAAATCTCGAACCAGGGACTTATAGC
TTAAAAGAAGAAACAGCACCAGATGGTTATGATAAAACCAGCCGGAGTTG
GACAGTGACTGTTTATGAGAACGGCTATAGCAAGTTGGTTGAAAATCCCT
ATAATGGGGAAATCATCAGTAAAGCAGGGTCAAAAGATGTTAGTAGTTCT
TTACAGTTGGAAAATCCGAAAATGTCAGTTGTTTCTAAATATGGGAAAAC
AGAGGTTAGTAGTGGCGCAGCGGATTTCTAGCGGAAGGATGCCGCCTATT
TTAAAATGTGTTTTGAGTTGAAACAAAAGGATAAATCTGAAACAATCAAC
CCAGGTGATACCTTTGTGTTACAGCTGGATAGACGTCTGAATCCTAAAGG
TATCAGTCAAGATATCCCTAAAATCATTTACGACAGTGGAAATAGTCGGG
TTGCGATTGGAAAATACCATGGTGAGAACCATCAACTTATCTATACTTTC
ACAGATTATATTGCGGGTTTAGATAAAGTCCAGTTGTCTGCAGAATTGAG
CTTATTCCTAGAGAATAAGGAAGTGTTGGAAAATACTAGTATGTCAAATT
TTAAGAGTAGCATAGGTGGGCAGGAGATCAGCTATAAAGGAACGGTTAAT
GTTCTTTATGGAAATGAGAGCACTAAAGAAAGCAATTATATTAGTAATGG
ATTGAGGAATGTGGGTGGGAGTATTGAAAGCTACAACACCGAAACGGGAG
AATTTGTCTGGTATGTTTATGTCAATCCAAACCGTACCAATATTCCTTAT
GCGACGATGAATTTATGGGGATTTGGAAGGGCTCGTTCAAATACAAGCGA
CTTAGAAAACGACGCTAATACAAGTAGTGCTGAGCTTGGAGAGATTCAGG
TCTATGAAGTACCTGAAGGAGAAAAATTACCATCAAGTTATGGGGTTGAT
GTTAGAAAAGTTACTTTAAGAACGGATATCACAGCAGGCCTAGGAAATGG
TTTTCAAATGAGCAAACGTCAGCGAATTGACTTTGGAAATAATATCCAAA
ATAAAGCATTTATCATCAAAGTAACAGGGAAAACAGACCAATCTGGTAAG
CCATTGGTTGTTCAATCCAATTTGGCAAGTTTTCGTGGTGCTTCTGAATA
TGCTGCTTTTACTCCAGTTGGAGGAAATGTCTACTTCCAAAACGAAATTG
CCTTGTCTCCTTCTAAGGGTAGTGGTTCTGGGAAAAGTGAATTTACTAAG
CCCTCTATTACAGTAGCAAATCTAAAACGAGTGGCTCAGCTTCGCTTTAA
GAAAATGTCAACTGAGAATGTGCCATTGCCAGAAGCGGCTTTTGAGCTGC
GTTCATCAAATGGTAATAGTCAGAAATTAGAAGCCAGTTCAAACACACAA
GGAGAGGTTCACTTTAAGGACCTGACCTCGGGCACATATGACCTGTATGA
AACAAAAGCGCCAAAAGGTTATCAGCAGGTGACAGAGAAATTGGCGACCG
TTACTGTTGATACTACCAAACCTGCTGAGGAAATGGTCACTTGGGGAAGC
CCACATTCGTCTGTAAAAGTAGAAGCTAACAAAGAAGTCACGATTGTCAA
CCATAAAGAAACCCTTACGTTTTCAGGGAAGAAAATTTGGGAGAATGACA
GACCAGATCAACGCCCAGCAAAGATTCAAGTGCAACTGTTGCAAAATGGT
CAAAAGATGCCTAACCAGATTCAAGAAGTAACGAAGGATAACGATTGGTC
TTATCACTTCAAAGACTTGCCTAAGTACGATGCCAAGAATCAGGAGTATA
AGTACTCAGTTGAAGAAGTAAATGTTCCAGACGGCTACAAGGTGTCGTAT
TTAGGAAATGATATATTTAACACCAGAGAAACAGAATTTGTGTTTGAACA
GAATAACTTTAACCTTGAATTTGGAAATGCTGAAATAAAAGGTCAATCTG
GGTCAAAAATCATTGATGAAGACACGCTAACGTCTTTCAAAGGTAAGAAA
ATTTGGAAAAATGATACGGCAGAAAATCGTCCCCAAGCCATTCAAGTGCA
GCTTTATGCTGATGGAGTGGCTGTGGAAGGTCAAACCAAATTTATTTCTG
GCTCAGGTAATGAGTGGTCATTTGAGTTTAAAAACTTGAAGAAGTATAAT
GGAACAGGTAATGACATCATTTACTCAGTTAAAGAAGTAACTGTTCCAAC
AGGTTATGATGTGACTTACTCAGCTAATGATATTATTAATACCAAACGTG
AGGTTATTACACAACAAGGACCGAAACTAGAGATTGAAGAAACGCTTCCG
CTAGAATCAGGTGCTTCAGGCGGTACCACTACTGTCGAAGACTCACGCCC
AGTTGATACCTTATCAGGTTTATCAAGTGAGCAAGGTCAGTCCGGTGATA
TGACAATTGAAGAAGATAGTGCTACCCATATTAAATTCTCAAAACGTGAT
ATTGACGGCAAAGAGTTAGCTGGTGCAACTATGGAGTTGCGTGATTCATC
TGGTAAAACTATTAGTACATGGATTTCAGATGGACAAGTGAAAGATTTCT
ACCTGATGCCAGGAAAATATACATTTGTCGAAACCGCAGCACCAGACGGT
TATGAGATAGCAACTGCTATTACCTTTACAGTTAATGAGCAAGGTCAGGT
TACTGTAAATGGCAAAGCAACTAAAGGTGACACTCATATTGTCATGGTTG
ATGCTTACAAGCCAACTAAGGGTTCAGGTCAGGTTATTGATATTGAAGAA
AAGCTTCCAGACGAGCAAGGTCATTCTGGTTCAACTACTGAAATAGAAGA
CAGTAAATCTTCAGACCTTATCATTGGCGGTCAAGGTGAAGTTGTTGACA
CAACAGAAGACACACAAAGTGGTATGACGGGCCATTCTGGCTCAACTACT
GAAATAGAAGATAGCAAGTCTTCAGACCTTATCATTGGTGGTCAGGGGCA
GGTTGTCGAGACAACAGAGGATACCCAAACTGGCATGTACGGGGATTCTG
GTTGTAAAACGGAAGTCGAAGATACTAAACTAGTACAATCCTTCCACTTT
GATAACAAGGAACCAGAAAGTAACTCTGAGATTCCTAAAAAAGATAAGCC
AAAGAGTAATAGTAGTTTACCAGCAACTGGTGAGAAGCAACATAATATGT
TCTTTTGGATGGTTACTTCTTGCTCACTTATTAGTAGTGTTTTTGTAATA
TCACTAAAATCCAAAAAACGCCTATCATCATGTTAA SEQ ID NO: 121
MTQKNSYKLSFLLSLTGFILGLLLVFIGLSGVSVGHAETRNGANKQGSFE
IKKVDQNNKPLPGATFSLTSKDGKGTSVQTFTSNDKGIVDAQNLQPGTYT
LKEETAPDGYDKTSRTWTVTVYENGYTKLVENPYNGEIISKAGSKDVSSS
LQLENPKMSVVSKYGKTEVSSGAADFYRNHAAYFKMSFELKQKDKSETIN
PGDTFVLQLDRRLNPKGISQDIPKIIYDSANSPLAIGKYHAENHQLIYTF
TDYIAGLDKVQLSAELSLFLENKEVLENTSISNFKSTIGGQEITYKGTVN
VLYGNESTKESNYITNGLSNVGGSIESYNTETGEFVWYVYVNPNRTNTPY
ATMNLWGFGRARSNTSDLENDANTSSAELGEIQVYEVPEGEKLPSSYGVD
VTKLTLRTDITAGLGNGFQMTKRQRIDFGNNIQNKAFIIKVTGKTDQSGK
PLVVQSNLASERGASEYAAFTPVGGNVYFQNEIALSPSKGSGSGKSEFTK
PSITVANLKRVAQLRFKKMSTDNVPLPEAAFELRSSNGNSQKLEASSNTQ
GEVHFKDLTSGTYDLYETKAPKGYQQVTEKLATVTVDTTKPAEEMVTWGS
PHSSVKVEANKEVTIVNHKETLTFSGKKIWENDRPDQRPAKIQVQLLQNG
QKMPNQIQEVTKDNDWSYHFKDLPKYDAKNQEYKYSVEEVNVPDGYKVSY
LGNDIFNTRETEFVFEQNNFNLEFGNAETKGQSGSKIIDEDTLTSFKGKK
IWKNDTAENRPQAIQVQLYADGVAVEGQTKFISGSGNEWSFEFKNLKKYN
GTGNDTIYSVKEVTVPTGYDVTYSANDIINTKREVITQQGPKLEIEETLP
LESGASGGTTTVEDSRPVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRD
IDGKELAGATMELRDSSGKTISTWISDGQVKDFYLMPGKYTFVETAAPDG
YEIATAITFTVNEQGQVTVNGKATKGDTHTVMVDAYKPTKGSGQVIDIEE
KLPDEQGHSGSTTEIEDSKSSDLIIGGQGEVVDTTEDTQSGMTGHSGSTT
EIEDSKSSDVIIGGQGQVVETTEDTQTGMYGDSGCKTEVEDTKLVQSFHF
DNKEPESNSEIPKKDKPKSNTSLPATGEKQHNMFFWMVTSCSLISSVFVI SLKSKKRLSSC
[1150] 19224141 contains an amino acid motif indicative of a cell
wall anchor: SEQ ID NO: 181 LPATG (shown in italics in SEQ ID NO:
121, above). In some recombinant host cell systems, it may be
preferable to remove this motif to facilitate secretion of a
recombinant 19224141 protein from the host cell. Alternatively, in
other recombinant host cell systems, it may be preferable to use
the cell wall anchor motif to anchor the recombinantly expressed
protein to the cell wall. The extracellular domain of the expressed
protein may be cleaved during purification or the recombinant
protein may be left attached to either inactivated host cells or
cell membranes in the final composition.
[1151] Two pilin motifs, discussed above, containing conserved
lysine (K) residues have also been identified in 19224141. The
pilin motif sequences are underlined in SEQ ID NO: 121, below.
Conserved lysine (K) residues are also marked in bold, at amino
acid residues 157 and 163 and at amino acid residues 216, 224, and
238. The pilin sequence, in particular the conserved lysine
residues, are thought to be important for the formation of
oligomeric, pilus-like structures. Preferred fragments of 19224141
include at least one conserved lysine residue. Preferably,
fragments include at least one pilin sequence. TABLE-US-00212 SEQ
ID NO: 121 MTQKNSYKLSFLLSLTGFILGLLLVFIGLSGVSVGHAETRNGANKQGSFE
IKKVDQNNKPLPGATFSLTSKDGKGTSVQTFTSNDKGIVDAQNLQPGTYT
LKEETAPDGYDKTSRTWTVTVYENGYTKLVENPYNGEIISKAGSKDVSSS
LQLENPKMSVVSKYGKTEVSSGAADFYRNHAAYFKMSFELKQKDKSETIN
PGDTFVLQLDRRLNPKGISQDIPKIIYDSANSPLAIGKYHAENHQLIYTF
TDYIAGLDKVQLSAELSLFLENKEVLENTSISNFKSTIGGQEITYKGTVN
VLYGNESTKESNYITNGLSNVGGSIESYNTETGEFVWYVYVNPNRTNTPY
ATMNLWGFGRARSNTSDLENDANTSSAELGEIQVYEVPEGEKLPSSYGVD
VTKLTLRTDITAGLGNGFQMTKRQRIDFGNNIQNKAFIIKVTGKTDQSGK
PLVVQSNLASERGASEYAAFTPVGGNVYFQNEIALSPSKGSGSGKSEFTK
PSITVANLKRVAQLRFKKMSTDNVPLPEAAFELRSSNGNSQKLEASSNTQ
GEVHFKDLTSGTYDLYETKAPKGYQQVTEKLATVTVDTTKPAEEMVTWGS
PHSSVKVEANKEVTIVNHKETLTFSGKKIWENDRPDQRPAKIQVQLLQNG
QKMPNQIQEVTKDNDWSYHFKDLPKYDAKNQEYKYSVEEVNVPDGYKVSY
LGNDIFNTRETEFVFEQNNFNLEFGNAETKGQSGSKIIDEDTLTSFKGKK
IWKNDTAENRPQAIQVQLYADGVAVEGQTKFISGSGNEWSFEFKNLKKYN
GTGNDTIYSVKEVTVPTGYDVTYSANDIINTKREVITQQGPKLEIEETLP
LESGASGGTTTVEDSRPVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRD
IDGKELAGATMELRDSSGKTISTWISDGQVKDFYLMPGKYTFVETAAPDG
YEIATAITFTVNEQGQVTVNGKATKGDTHTVMVDAYKPTKGSGQVIDIEE
KLPDEQGHSGSTTEIEDSKSSDLIIGGQGEVVDTTEDTQSGMTGHSGSTT
EIEDSKSSDVIIGGQGQVVETTEDTQTGMYGDSGCKTEVEDTKLVQSFHF
DNKEPESNSEIPKKDKPKSNTSLPATGEKQHNMFFWMVTSCSLISSVFVI SLKSKKRLSSC
[1152] Two E boxes containing conserved glutamic residues have been
identified in 19224141. The E-box motifs are underlined in SEQ ID
NO: 121, below. The conserved glutamic acid (E) residues, at amino
acid residues 567 and 944, are marked in bold. The E box motifs, in
particular the conserved glutamic acid residues, are thought to be
important for the formation of oligomeric pilus-like structures of
19224141. Preferred fragments of 19224141 include at least one
conserved glutamic acid residue. Preferably, fragments include at
least one E box motif. TABLE-US-00213 SEQ ID NO: 121
MTQKNSYKLSFLLSLTGFILGLLLVFIGLSGVSVGHAETRNGANKQGSFE
IKKVDQNNKPLPGATFSLTSKDGKGTSVQTFTSNDKGIVDAQNLQPGTYT
LKEETAPDGYDKTSRTWTVTVYENGYTKLVENPYNGEIISKAGSKDVSSS
LQLENPKMSVVSKYGKTEVSSGAADFYRNHAAYFKMSFELKQKDKSETIN
PGDTFVLQLDRRLNPKGISQDIPKIIYDSANSPLAIGKYHAENHQLIYTF
TDYIAGLDKVQLSAELSLFLENKEVLENTSISNFKSTIGGQEITYKGTVN
VLYGNESTKESNYITNGLSNVGGSIESYNTETGEFVWYVYVNPNRTNTPY
ATMNLWGFGRARSNTSDLENDANTSSAELGEIQVYEVPEGEKLPSSYGVD
VTKLTLRTDITAGLGNGFQMTKRQRIDFGNNIQNKAFIIKVTGKTDQSGK
PLVVQSNLASERGASEYAAFTPVGGNVYFQNEIALSPSKGSGSGKSEFTK
PSITVANLKRVAQLRFKKMSTDNVPLPEAAFELRSSNGNSQKLEASSNTQ
GEVHFKDLTSGTYDLYETKAPKGYQQVTEKLATVTVDTTKPAEEMVTWGS
PHSSVKVEANKEVTIVNHKETLTFSGKKIWENDRPDQRPAKIQVQLLQNG
QKMPNQIQEVTKDNDWSYHFKDLPKYDAKNQEYKYSVEEVNVPDGYKVSY
LGNDIFNTRETEFVFEQNNFNLEFGNAETKGQSGSKIIDEDTLTSFKGKK
IWKNDTAENRPQAIQVQLYADGVAVEGQTKFISGSGNEWSFEFKNLKKYN
GTGNDTIYSVKEVTVPTGYDVTYSANDIINTKREVITQQGPKLEIEETLP
LESGASGGTTTVEDSRPVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRD
IDGKELAGATMELRDSSGKTISTWISDGQVKDFYLMPGKYTFVETAAPDG
YEIATAITFTVNEQGQVTVNGKATKGDTHTVMVDAYKPTKGSGQVIDIEE
KLPDEQGHSGSTTEIEDSKSSDLIIGGQGEVVDTTEDTQSGMTGHSGSTT
EIEDSKSSDVIIGGQGQVVETTEDTQTGMYGDSGCKTEVEDTKLVQSFHF
DNKEPESNSEIPKKDKPKSNTSLPATGEKQHNMFFWMVTSCSLISSVFVI SLKSKKRLSSC
[1153] As discussed above, applicants have also determined the
nucleotide and encoded amino acid sequence of fimbrial structural
subunits in several other GAS AI-4 strains of bacteria. Examples of
sequences of these fimbrial structural subunits are set forth
below.
[1154] M12 strain isolate 20010296 is a GAS AI-4 strain of
bacteria. 20010296_fimbrial is thought to be a fimbrial structural
subunit of M12 strain isolate 20010296. An example of a nucleotide
sequence encoding the 20010296_fimbrial protein (SEQ ID NO: 257)
and a 20010296_fimbrial protein amino acid sequence (SEQ ID NO:
258) are set forth below. TABLE-US-00214 SEQ ID NO: 257
agcagtggtcaattaacaataaaaaaatcaattacaaattttaatgatga
tacacttttgatgcctaagacagactatacttttagcgttaatccggata
gtgcggctacaggtactgaaagtaatttaccaattaaaccaggtattgct
gttaacaatcaagatattaaggtttcttattctaatactgataagacatc
aggtaaagaaaaacaagttgttgttgactttatgaaagttacttttccta
gcgttggtatttaccgttatgttgttaccgagaataaagggacagcagaa
ggagttacatatgatgatacaaaatggttagttgacgtctatgttggtaa
taatgaaaagggaggtcttgaaccaaagtatattgtatctaaaaaaggag
attctgctactaaagaaccaatccagtttaataattcattcgaaacaacg
tcattaaaaattgaaaaggaagttactggtaatacaggagatcataaaaa
agcatttaactttacattaacattgcaaccaaatgaatactatgaggcaa
gttcggttgtgaaaattgaagagaacggacaaacgaaagatgtgaaaatt
ggggaggcatataagtttactttgaacgatagtcagagtgtgatattgtc
taaattaccagttggtattaattataaagttgaagaagcagaagctaatc
aaggtggatatactacaacagcaactttaaaagatggagaaaagttatct
acttataacttaggtcaggaacataaaacagacaagactgctgatgaaat cgt SEQ ID NO:
258 SSGQLTIKKSITNFNDDTLLMPKTDYTFSVNPDSAATGTESNLPIKPGIA
VNNQDIKVSYSNTDKTSGKEKQVVVDFMKVTFPSVGIYRYVVTENKGTAE
GVTYDDTKWLVDVYVGNNEKGGLEPKYIVSKKGDSATKEPIQFNNSFETT
SLKIEKEVTGNTGDHKKAFNFTLTLQPNEYYEASSVVKIEENGQTKDVKI
GEAYKFTLNDSQSVILSKLPVGINYKVEEAEANQGGYTTTATLKDGEKLS
TYNLGQEHKTDKTADEIV
[1155] M12 strain isolate 20020069 is a GAS AI-4 strain of
bacteria. 20020069_fimbrial is thought to be a fimbrial structural
subunit of M12 strain isolate 20020069. An example of a nucleotide
sequence encoding the 20020069_fimbrial protein (SEQ ID NO: 259)
and a 20020069_fimbrial protein amino acid sequence (SEQ ID NO:
260) are set forth below. TABLE-US-00215 SEQ ID NO: 259
agcagtggtcaattaacaataaaaaaatcaattacaaattttaatgatga
tacacttttgatgcctaagacagactatacttttagcgttaatccggata
gtgcggctacaggtactgaaagtaatttaccaattaaaccaggtattgct
gttaacaatcaagatattaaggtttcttattctaatactgataagacatc
aggtaaagaaaaacaagttgttgttgactttatgaaagttacttttccta
gcgttggtatttaccgttatgttgttaccgagaataaagggacagcagaa
ggagttacatatgatgatacaaaatggttagttgacgtctatgttggtaa
taatgaaaagggaggtcttgaaccaaagtatattgtatctaaaaaaggag
attctgctactaaagaaccaatccagtttaataattcattcgaaacaacg
tcattaaaaattgaaaaggaagttactggtaatacaggagatcataaaaa
agcatttaactttacattaacattgcaaccaaatgaatactatgaggcaa
gttcggttgtgaaaattgaagagaacggacaaacgaaagatgtgaaaatt
ggggaggcatataagtttactttgaacgatagtcagagtgtgatattgtc
taaattaccagttggtattaattataaagttgaagaagcagaagctaatc
aaggtggatatactacaacagcaactttaaaagatggagaaaagttatct
acttataacttaggtcaggaacataaaacagacaagactgctgatgaaat cgt SEQ ID NO:
260 SSGQLTIKKSITNFNDDTLLMPKTDYTFSVNPDSAATGTESNLPIKPGIA
VNNQDIKVSYSNTDKTSGKEKQVVVDEMKVTFPSVGIYRYVVTENKGTAE
GVTYDDTKWLVDVYVGNNEKGGLEPKYIVSKKGDSATKEPIQFNNSEETT
SLKIEKEVTGNTGDHKKAFNFTLTLQPNEYYEASSVVKIEENGQTKDVKI
GEAYKFTLNDSQSVILSKLPVGINYKVEEAEANQGGYTTTATLKDGEKLS
TYNLGQEHKTDKTADEIV
[1156] M12 strain isolate CDC SS 635 is a GAS AI-4 strain of
bacteria. CDC SS 635_fimbrial is thought to be a fimbrial
structural subunit of M12 strain isolate CDC SS 635. An example of
a nucleotide sequence encoding the CDC SS 635_fimbrial protein (SEQ
ID NO: 261) and a CDC SS 635_fimbrial protein amino acid sequence
(SEQ ID NO: 262) are set forth below. TABLE-US-00216 SEQ ID NO: 261
gagacggcaggggttgttagcagtggtcaattaacaataaaaaaatcaat
tacaaattttaatgatgatacacttttgatgcctaagacagactatactt
ttagcgttaatccggatagtgcggctacaggtactgaaagtaatttacca
attaaaccaggtattgctgttaacaatcaagatattaaggtttcttattc
taatactgataagacatcaggtaaagaaaaacaagttgttgttgacttta
tgaaagttacttttcctagcgttggtatttaccgttatgttgttaccgag
aataaagggacagcagaaggagttacatatgatgatacaaaatggttagt
tgacgtctatgttggtaataatgaaaagggaggtcttgaaccaaagtata
ttgtatctaaaaaaggagattctgctactaaagaaccaatccagtttaat
aattcattcgaaacaacgtcattaaaaattgaaaaggaagttactggtaa
tacaggagatcataaaaaagcatttaactttacattaacattgcaaccaa
atgaatactatgaggcaagttcggttgtgaaaattgaagagaacggacaa
acgaaagatgtgaaaattggggaggcatataagtttactttgaacgatag
tcagagtgtgatattgtctaaattaccagttggtattaattataaagttg
aagaagcagaagctaatcaaggtggatatactacaacagcaactttaaaa
gatggagaaaagttatctacttataacttaggtcaggaacataaaacaga
caagactgctgatgaaatcgttgtcacaaataaccgtgacact SEQ ID NO: 262
ETAGVVSSGQLTTKKSITNFNDDTLLMPKTDYTFSVNPDSAATGTESNLP
IKPGIAVNNQDIKVSYSNTDKTSGKEKQVVVDPMKVTFPSVGIYRYVVTE
NKGTAEGVTYDDTKWLVDVYVGNNEKGGLEPKYIVSKKGDSATKEPIQFN
NSFETTSLKIEKEVTGNTGDHKKAFNFTLTLQPNEYYEASSVVKIEENGQ
TKDVKIGEAYKFTLNDSQSVILSKLPVGINYKVEEAEANQGGYTTTATLK
DGEKLSTYNLGQEHKTDKTADEIVVTNNRDT
[1157] M5 strain isolate ISS 4883 is a GAS AI-4 strain of bacteria.
ISS4883_fimbrial is thought to be a fimbrial structural subunit of
M5 strain isolate ISS 4883. An example of a nucleotide sequence
encoding the ISS4883_fimbrial protein (SEQ ID NO: 265) and an
ISS4883_fimbrial protein amino acid sequence (SEQ ID NO: 266) are
set forth below. TABLE-US-00217 SEQ ID NO: 265
gagacggcaggggttgtaacaggaaaatcactacaagttacaaagacaat
gacttatgatgatgaagaggtgttaatgcccgaaaccgcctttactttta
ctatagagcctgatatgactgcaagtggaaaagaaggcgacctagatatt
aaaaatggaattgtagaaggcttagacaaacaagtaacagtaaaatataa
gaatacagataaaacatctcaaaaaactaaaatagcacaatttgattttt
ctaaggttaaatttccagctataggtgtttaccgctatatggtttcagag
aaaaacgataaaaaagacggaattaggtacgatgataaaaagtggactgt
agatgtttatgttgggaataaggccaataacgaagaaggtttcgaagttc
tatatattgtatcaaaagaaggtacttctagtactaaaaaaccaattgaa
tttacaaactctattaaaactacttccttaaaaattgaaaaacaaataac
tggcaatgcaggagatcgtaaaaaatcattcaacttcacattaacattac
aaccaagtgaatattataaaaccggatcagttgtgaaaatcgaacaggat
ggaagtaaaaaagatgtgacgataggaacgccttacaaatttactttggg
acacggtaagagtgtcatgttatcgaaattaccaattggtatcaattact
atcttagtgaagacgaagcgaataaagacggttacactacaacggcaaca
ttaaaagaacaaggcaaagaaaagagttccgatttcactttgagtactca
aaaccagaaaacagacgaatctgctgacgaaatcgttgtcacaaataagc gtgacactctcgag
SEQ ID NO: 266 ETAGVVTGKSLQVTKTMTYDDEEVLMPETAFTFTIEPDMTASGKEGDLDI
KNGIVEGLDKQVTVKYKNTDKTSQKTKIAQFDFSKVKFPAIGVYRYMVSE
KNDKKDGIRYDDKKWTVDVYVGNKANNEEGFEVLYIVSKEGTSSTKKPIE
FTNSIKTTSLKIEKQITGNAGDRKKSFNFTLTLQPSEYYKTGSVVKIEQD
GSKKDVTIGTPYKFTLGHGKSVMLSKLPIGINYYLSEDEANKDGYTTTAT
LKEQGKEKSSDFTLSTQNQKTDESADEIVVTNKRDTLE
[1158] M50 strain isolate ISS4538 is a GAS AI-4 strain of bacteria.
ISS4538_fimbrial is thought to be a fimbrial structural subunit of
M50 strain ISS 4538. An example of a nucleotide sequence encoding
the ISS4538_fimbrial protein (SEQ ID NO: 255) and an
ISS4538_fimbrial protein amino acid sequence (SEQ ID NO: 256) are
set forth below. TABLE-US-00218 SEQ ID NO: 255
atgaaaaaaaataaattattacttgctactgcaatcttagcaactgcttt
aggaacagcttctttaaatcaaaacgtaaaagctgagacggcaggggttg
ttagcagtggtcaattaacaataaaaaaatcaattacaaattttaatgat
gatacacttttgatgcctaagacagactatacttttagcgttaatccgga
tagtgcggctacaggtactgaaagtaatttaccaattaaaccaggtattg
ctgttaacaatcaagatattaaggtttcttattctaatactgataagaca
tcaggtaaagaaaaacaagttgttgttgactttatgaaagttacttttcc
tagcgttggtatttaccgttatgttgttaccgagaataaagggacagcag
aaggagttacatatgatgatacaaaatggttagttgacgtctatgttggt
aataatgaaaagggaggtcttgaaccaaagtatattgtatctaaaaaagg
agattctgctactaaagaaccaatccagtttaataattcattcgaaacaa
cgtcattaaaaattgaaaagaaagttactggtaatacaggagatcataaa
aaagcatttaactttacattaacattgcaaccaaatgaatactatgaggc
aagttcggttgtgaaaattgaagagaacggacaaacgaaagatgtgaaaa
ttggggaggcatataagtttactttgaacgatagtcagagtgtgatattg
tctaaattaccagttggtattaattataaagttgaagaagcagaagctaa
tcaaggtggatatactacaacagcaactttaaaagatggagaaaagttat
ctacttataacttaggtcaggaacataaaacagacaagactgctgatgaa
atcgttgtcacaaataancgngacactcnagttccaacnggtgtngtagg
caccccncctccattcncagttcttancattgnggctantggtggngtna
tntatnttacaaaacgnaaaaaagnataa SEQ ID NO: 256
MKKNKLLLATAILATALGTASLNQNVKAETAGVVSSGQLTIKKSITNFND
DTLLMPKTDYTFSVNPDSAATGTESNLPIKPGIAVNNQDIKVSYSNTDKT
SGKEKQVVVDFMKVTFPSVGIYRYVVTENKGTAEGVTYDDTKWLVDVYVG
NNEKGGLEPKYIVSKKGDSATKEPIQFNNSFETTSLKIEKKVTGNTGDHK
KAFNFTLTLQPNEYYEASSVVKTEENGQTKDVKIGEAYKFTLNDSQSVIL
SKLPVGINYKVEEAEANQGGYTTTATLKDGEKLSTYNLGQEHKTDKTADE
IVVTNXRDTXVPTGVVGTPPPFXVLXIXAXGGVXYXTKRKKX
[1159] There may be an upper limit to the number of GAS proteins
which will be in the compositions of the invention. Preferably, the
number of GAS proteins in a composition of the invention is less
than 20, less than 19, less than 18, less than 17, less than 16,
less than 15, less than 14, less than 13, less than 12, less than
11, less than 10, less than 9, less than 8, less than 7, less than
6, less than 5, less than 4, or less than 3. Still more preferably,
the number of GAS proteins in a composition of the invention is
less than 6, less than 5, or less than 4. Still more preferably,
the number of GAS proteins in a composition of the invention is
3.
[1160] The GAS proteins and polynucleotides used in the invention
are preferably isolated, i.e., separate and discrete, from the
whole organism with which the molecule is found in nature or, when
the polynucleotide or polypeptide is not found in nature, is
sufficiently free of other biological macromolecules so that the
polynucleotide or polypeptide can be used for its intended
purpose.
Examples Other Gram Positive Bacterial Adhesin Island Sequences
[1161] The Gram positive bacteria AI polypeptides of the invention
can, of course, be prepared by various means (e.g. recombinant
expression, purification from a gram positive bacteria, chemical
synthesis etc.) and in various forms (e.g. native, fusions,
glycosylated, non-glycosylated etc.). They are preferably prepared
in substantially pure form (i.e. substantially free from other
streptococcal or host cell proteins) or substantially isolated
form.
[1162] The Gram positive bacteria AI proteins of the invention may
include polypeptide sequences having sequence identity to the
identified Gram positive bacteria proteins. The degree of sequence
identity may vary depending on the amino acid sequence (a) in
question, but is preferably greater than 50% (e.g. 60%, 65%, 70%,
75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
99.5% or more). Polypeptides having sequence identity include
homologs, orthologs, allelic variants and mutants of the identified
Gram positive bacteria proteins. Typically, 50% identity or more
between two proteins is considered to be an indication of
functional equivalence. Identity between proteins is preferably
determined by the Smith-Waterman homology search algorithm as
implemented in the MPSRCH program (Oxford Molecular), using an
affinity gap search with parameters gap open penalty=12 and gap
extension penalty=1.
[1163] The Gram positive bacteria adhesin island polynucleotide
sequences may include polynucleotide sequences having sequence
identity to the identified Gram positive bacteria adhesin island
polynucleotide sequences. The degree of sequence identity may vary
depending on the polynucleotide sequence in question, but is
preferably greater than 50% (e.g. 60%, 65%, 70%, 75%, 80%, 85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or
more).
[1164] The Gram positive bacteria adhesin island polynucleotide
sequences of the invention may include polynucleotide fragments of
the identified adhesin island sequences. The length of the fragment
may vary depending on the polynucleotide sequence of the specific
adhesin island sequence, but the fragment is preferably at least 10
consecutive polynucleotides, (e.g. at least 10, 12, 14, 16, 18, 20,
25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200 or more).
[1165] The Gram positive bacteria adhesin island amino acid
sequences of the invention may include polypeptide fragments of the
identified Gram positive bacteria proteins. The length of the
fragment may vary depending on the amino acid sequence of the
specific Gram positive bacteria antigen, but the fragment is
preferably at least 7 consecutive amino acids, (e.g. 8, 10, 12, 14,
16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200 or
more). Preferably the fragment comprises one or more epitopes from
the sequence. The fragment may comprise at least one T-cell or,
preferably, a B-cell epitope of the sequence. T- and B-cell
epitopes can be identified empirically (e.g., using PEPSCAN [Geysen
et al. (1984) PNAS USA 81:39984002; Carter (1994) Methods Mol.
Biol. 36:207-223, or similar methods], or they can be predicted
(e.g., using the Jameson-Wolf antigenic index [Jameson, B A et al.
1988, CABIOS 4 (1):1818-186], matrix-based approaches [Raddrizzani
and Hammer (2000) Brief Bioinform. 1(2):179-189], TEPITOPE [De
Lalla et al. (199) J. Immunol. 163:1725-1729], neural networks
[Brusic et al. (1998) Bioinformatics 14(2):121-130], OptiMer &
EpiMer [Meister et al. (1995) Vaccine 13(6):581-591; Roberts et al.
(1996) AIDS Res. Hum. Retroviruses 12(7):593-610], ADEPT [Maksyutov
& Zagrebelnaya (1993) Comput. Appl. Biosci. 9(3):291-297],
Tsites [Feller & de la Cruz (1991) Nature 349(6311):720-721],
hydrophilicity [Hopp (1993) Peptide Research 6:183-190], antigenic
index [Welling et al. (1985) FEBS Lett. 188:215-218] or the methods
disclosed in Davenport et al. (1995) Immunogenetics 42:392-297,
etc. Other preferred fragments include (1) the N-terminal signal
peptides of each identified Gram positive bacteria protein, (2) the
identified Gram positive bacteria protein without their N-terminal
signal peptides, (3) each identified Gram positive bacteria protein
wherein up to 10 amino acid residues (e.g. 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 15, 20, 25 or more) are deleted from the N-terminus and/or
the C-terminus e.g. the N-terminal amino acid residue may be
deleted. Other fragments omit one or more domains of the protein
(e.g. omission of a signal peptide, of a cytoplasmic domain, of a
transmembrane domain, or of an extracellular domain), and (4) the
polypeptides, but without their N-terminal amino acid residue.
[1166] As indicated in the above text, nucleic acids and
polypeptides of the invention may include sequences that: [1167]
(a) are identical (i.e., 100% identical) to the sequences disclosed
in the sequence listing; [1168] (b) share sequence identity with
the sequences disclosed in the sequence listing; [1169] (c) have 1,
2, 3, 4, 5, 6, 7, 8, 9, or 10 single nucleotide or amino acid
alterations (deletions, insertions, substitutions), which may be at
separate locations or may be contiguous, as compared to the
sequences of (a) or (b); [1170] (d) when aligned with a particular
sequence from the sequence listing using a pairwise alignment
algorithm, a moving window of x monomers (amino acids or
nucleotides) moving from start (N-terminus or 5') to end
(C-terminus or 3'), such that for an alignment that extends top
monomers (where p>x) there are p-x+1 such windows, each window
has at least xy identical aligned monomers, where: x is slected
from 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200; y
is selected from 0.50, 0.60, 0.70, 0.75, 0.80, 0.85, 0.90, 0.91,
0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99; and if xy is not an
integer then it is rounded up to the nearest integer. The preferred
pairwise alignment algorithm is the Needleman-Wunsch global
alignment algorithm [Needlman &Wunsch (1970) J. Mol. Biol. 48,
443-453], using default parameters (e.g., with Gap opening
penalty=10.0, and with Gap extension penalty=0.5, using the
EBLOSUM62 scoring matrix). This algorithm is conveniently
implemented in the needle tool in the EMBOSS package [Rice et al.
(2000) Trends Genet. 16:276-277].
[1171] The nucleic acids and polypeptides of the inention may
additionally have further sequences to the N-terminus/5' and/or
C-terminus/3' of these sequences (a) to (d).
[1172] All of the Gram positive bacterial sequences referenced
herein are publicly available through PubMed on GenBank.
Streptococcus pneumoniae Adhesin Island Sequences
[1173] As discussed above, a S. pneumoniae AI sequence is present
in the TIGR4 S. pneumoniae genome. Examples of S. pneumoniae AI
sequences are set forth below.
[1174] SrtD (Sp0468) is a sortase. An example of an amino acid
sequence of SrtD is set forth in SEQ ID NO: 80. TABLE-US-00219 SEQ
ID NO: 80 MSRTKLRALLGYLLMLVACLIPIYCFGQMVLQSLGQVKGHATFVKSMTTE
MYQEQQNHSLAYNQRLASQNRTVDPFLAEGYEVNYQVSDDPDAVYGYLSI
PSLEIMEPVYLGADYHHLGMGLAHVDGTPLPLDGTGIRSVIAGHRAEPSH
VFFRHLDQLKVGDALYYDNGQEIVEYQMMDTEIILPSEWEKLESVSSKNI
MTLITCDPIPTFNKRLLVNFERVAVYQKSDPQTAAVARVAFTKEGQSVSR
VATSQWLYRGLVVLAFLGILFVLWKLARLLRGK
[1175] SrtC (Sp0467) is a sortase. An example of an amino acid
sequence of SrtC is set forth in SEQ ID NO: 81. TABLE-US-00220 SEQ
ID NO: 81 MSRYYYRIESNEVIKEFDETVSQMDKAELEERWRLAQAENATLKPSEILD
PFTEQEKKKGVSEYANMLKVHERIGYVEIPAIDQEIPMYVGTSEDILQKG
AGLLEGASLPVGGENTHTVITAHRGLPTAELFSQLDKMKKGDIFYLHVLD
QVLAYQVDQIVTVEPNDFEPVLIQHGEDYATLLTCTPYMINSHRLLVRGK
RIPYTAPIAERNRAVRERGQFWLWLLLGAMAVILLLLYRVYRNRRIVKGL EKQLEGRHVKD
[1176] SrtB (SP0466) is a sortase. An example of an amino acid
sequence of SrtB is set forth in SEQ ID NO: 82. TABLE-US-00221 SEQ
ID NO: 82 MAVMAYPLVSRLYYRVESNQQIADFDKEKATLDEADIDERMKLAQAFNDS
LNNVVSGDPWSEEMKKKGRAEYARMLEIHERMGHVEIPVIDVDLPVYAGT
AEEVLQQGAGHLEGTSLPIGGNSTHAVITAHTGLPTAKMFTDLTKLKVGD
KFYVHNIKEVMAYQVDQVKVIEPTNFDDLLIVPGHDYVTLLTCTPYMINT
HRLLVRGHRIPYVAEVEEEFIAANKLSHLYRYLFYVAVCLIVTLLWITRR
LRKKKKQPEKALKALKAARKEVKVEDGQQ
[1177] Sp0465 is a hypothetical protein. An example of an amino
acid sequence of Sp0465 is set forth in SEQ ID NO: 83.
TABLE-US-00222 SEQ ID NO: 83
MFLPFLSASLYLQTHHFIAFPNRQSYLLRETRKSHFFLIHHPF
[1178] RrgC (SP0464) is a cell wall surface anchor family protein.
RrgC contains a sortase substrate motif VPXTG (SEQ ID NO: 137),
shown in italics in SEQ ID NO: 84. TABLE-US-00223 SEQ ID NO: 84
MISRIFFVMALCFSLVWGAHAVQAQEDHTLVLQLENYQEVVSQLPSRDGH
RLQVWKLDDSYSYDDRVQIVRDLHSWDENKLSSFKKTSFEMTFLENQIEV
SHIPNGLYYVRSIIQTDAVSYPAEFLFEMTDQTVEPLVIVAKKTDTMTTK
VKLIKVDQDHNRLEGVGFKLVSVARDVSEKEVPLIGEYRYSSSGQVGRTL
YTDKNGEIFVTNLPLGNYRFKEVEPLAGYAVTTLDTDVQLVDHQLVTITV
VNQKLPRGNVDFMKVDGRTNTSLQGAMFKVMKEESGHYTPVLQNGKEVVV
TSGKDGRFRVEGLEYGTYYLWELQAPTGYVQLTSPVSFTIGKDTRKELVT
VVKNNKRPRIDVPDTGEETLYILMLVAILLFGSGYYLTKKPNN
[1179] RrgB (Sp0463) is a cell wall surface anchor protein. RrgB
contains a sortase substrate motif IPXTG (SEQ ID NO: 133), shown in
italics in SEQ ID NO: 85. TABLE-US-00224 SEQ ID NO: 85
MKSINKFLTMLAALLLTASSLFSAATVFAAGTTTTSVTVHKLLATDGDMD
KIANELETGNYAGNKVGVLPANAKEIAGVMFVWTNTNNEIIDENGQTLGV
NIDPQTFKLSGAMPATAMKKLTEAEGAKFNTANLPAAKYKIYEIHSLSTY
VGEDGATLTGSKAVPIEIELPLNDVVDAHVYPKNTEAKPKIDKDFKGKAN
PDTPRVDKDTPVNHQVGDVVEYEIVTKIPALANYATANWSDRMTEGLAFN
KGTVKVTVDDVALEAGDYALTEVATGFDLKLTDAGLAKVNDQNAEKTVKI
TYSATLNDKAIVEVPESNDVTFNYGNNPDHGNTPKPNKPNENGDLTLTKT
WVDATGAPIPAGAEATFDLVNAQTGKVVQTVTLTTDKNTVTVNGLDKNTE
YKFVERSIKGYSADYQEITTAGEIAVKNWKDENPKPLDPTEPKVVTYGKK
FVKVNDKDNRLAGAEFVIANADNAGQYLARKADKVSQEEKQLVVTTKDAL
DRAVAAYNALTAQQQTQQEKEKVDKAQAAYNAAVIAANNAFEWVADKDNE
NVVKLVSDAQGRFEITGLLAGTYYLEETKQPAGYALLTSRQKFEVTATSY
SATGQGIEYTAGSGKDDATKVVNKKITIPQTGGIGTIIFAVAGAAIMGIA
VYAYVKNNKDEDQLA
[1180] RrgA (Sp0462) is a cell wall surface anchor protein. RrgA
contains a sortase substrate motif YPXTG (SEQ ID NO: 186),
indicated in italics in SEQ ID NO: 86. TABLE-US-00225 SEQ ID NO: 86
MLNRETHMKKVRKIFQKAVAGLCCISQLTAFSSIVALAETPETSPAIGKV
VIKETGEGGALLGDAVFELKNNTDGTTVSQRTEAQTGEAIFSNIKPGTYT
LTEAQPPVGYKPSTKQWTVEVEKNGRTTVQGEQVENREEALSDQYPQTGT
YPDVQTPYQIIKVDGSEKNGQHKALNPNPYERVIPEGTLSKRIYQVNNLD
DNQYGIELTVSGKTVYEQKDKSVPLDVVILLDNSNSMSNIRNKNARRAER
AGEATRSLIDKITSDSENRVALVTYASTIFDGTEFTVEKGVADKNGKRLN
DSLFWNYDQTSFTTNTKDYSYLKLTNDKNDIVELKNKVPTEAEDHDGNRL
MYQFGATFTQKALMKADEILTQQARQNSQKVIFHITDGVPTMSYPINFNH
ATFAPSYQNQLNAFFSKSPNKDGILLSDFITQATSGEHTIVRGDGQSYQM
FTDKTVYEKGAPAAFPVKPEKYSEMKAAGYAVIGDPINGGYIWLNWRESI
LAYPFNSNTAKITNHGDPTRWYYNGNIAPDGYDVFTVGIGINGDPGTDEA
TATSFMQSISSKPENYTNVTDTTKILEQLNRYFHTIVTEKKSIENGTITD
PMGELIDLQLGTDGRFDPADYTLTANDGSRLENGQAVGGPQNDGGLLKNA
KVLYDTTEKRIRVTGLYLGTDEKVTLTYNVRLNDEFVSNKFYDTNGRTTL
HPKEVEQNTVRDFPIPKIRDVRKYPEITISKEKKLGDIEFIDVNDNDKKP
LRGAVFSLQKQHPDYPDIYGAIDQNGTYQNVRTGEDGKLTFKNLSDGKYR
LFENSEPAGYKPVQNKPIVAFQIVNGEVRDVTSIVPQDIPAGYEFTNDKH
YITNEPIPPKREYPRTGGIGMLPFYLIGCMMMGGVLLYTRKHP
[1181] RlrA (Sp0461) is a transcriptional regulator. An example of
an amino acid sequence for RlrA is set forth in SEQ ID NO: 87.
TABLE-US-00226 SEQ ID NO: 87
MLNKYIEKRITDKITILNILLDIRSIELDELSTLTSLQSKSLLSILQELQ
ETFEEELTFNLDTQQVQLIEHHSHQTNYYFHQLYNQSTILKILRFFLLQG
NQSFNEFTQKEYISIATGYRVRQKCGLLLRSVGLDLVKNQVVGPEYRIRF
LIALLQFHFGTEIYDLNDGSMDWVTHMIVQSNSQLSHELLEITPDEYVHF
SILVALTWKRREFPLEFPESKEFEKLKNLFMYPILMEHCQTYLEPHANMT
FTQEELDYIFLVYCSANSSFSKDKWNQEKKTHTIQLILQHTRGKHLLSKE
KNILGNDISNSLSFLTALTFLTRTFLFGLQNLVPYYNYYEHYGIESDKPL
YHISKAIVQEWMTEQKIEGVIDQHRLYLESLYLTETIFSSLPAIPIKIIL
NNQADVNLTKSIILRNFTDKVASVTGYNILISPPPSEEHLTEPLIIITTK
EYLPYVKKQYPKGKHHFLTIALDLHVSQQRLIYQTIVDIRKEAFDKRVAM IAKKAHYLL
[1182] As discussed above, a S. pneumoniae AI sequence is present
in the S. pneumoniae strain 670 genome. Examples of S. pneumoniae
AI sequences are set forth below.
[1183] Orf1.sub.--670 is a transposase. An example of an amino acid
sequence of orf1.sub.--670 is set forth in SEQ ID NO: 171.
TABLE-US-00227 SEQ ID NO: 171
MEHINHTTLLIGIKDKNITLNKAIQHDTHIEVFATLDYHPPKCKHCKGKQ
IKYDFQKPSKIPFIEIGGFPSLIHLKKRRFQCKSCRKVTVAETTLVQKNC
QISEMVRQKIAQLLLNREALTHIASKLAISTSTSTVYRKLKQFHFQEDYT
TLPEILSWDEFSYQKGKLAFIAQDFNTKKIMTILDNRRQTTIRNHFFKYS
KEARKKVKVVTVDMSGSYIPLIKKLFPNAKIVLDRFHIVQHMSRALNQTR
INIMKQFDDKSLEYRALKYYWKFILKDSRKLSLKPFYARTFRETLTPREC
LKKTFTLVPELKDYYDLYQLLLFHLQEKNTDQFWGLIQDTLPHLNRTFKT
TLSTFICYKNYITNAIELPYSNAKLEATNKLIKDIKRNAFGFRNFENFKK
RIFIALNTKKERTKFVLSRA
[1184] Orf2.sub.--670 is a transcriptional regulator. An example of
an amino acid sequence of Orf2.sub.--670 is set forth in SEQ ID NO:
172. TABLE-US-00228 SEQ ID NO: 172
MLNKYIEKRITDKITILNILLDIRSIELDELSTLTSLQSKSLLSILQELQ
ETFEEELTFNLDTQQVQLIEHHSHQTNYYFHQLYNQSTILKILRFFLLQG
NQSFNEFTQKEYISIATGYRVRQKCGLLLRSVGLDLVKNQVVGPEYRIRF
LIALLQFHFGIEIYDLNDGSMDWVTHMIVQSNSQLSHELLEITPDEYVHF
SILVALTWKRREFPLEFPESKEFEKLKNLFMYPILMEHCQTYLEPHANMT
FTQEELDYIFLVYCSANSSFSKDKWNQEKKTHTIQLILQHTRGKHLLSKF
KNILGNDISNSLSFLTALTFLTRTFLFGLQNLVPYYNYYEHYGIESDKPL
YHISKAIVQEWMTEQKIEGVIDQHRLYLFSLYLTETIFSSLPAIPIFIIL
NNQADVNLIKSIILRNETDKVASVTGYNILISPPPSEEHLTEPLIIITTK
EYLPYVKKQYPKGKHHFLTIALDLHVSQQRLIYQTIVDIRKEAFDKRVAM IAKKAHYLL
[1185] Orf3.sub.--670 is a cell wall surface anchor family proten.
An example of an amino acid sequence of Orf3.sub.--670 is set forth
in SEQ ID NO: 173. TABLE-US-00229 SEQ ID NO: 173
MLNRETHMKKVRKIFQKAVAGLCCISQLTAFSSIVALAETPETSPAIGKV
VIKETGEGGALLGDAVFELKNNTDGTTVSQRTEAQTGEAIFSNIKPGTYT
LTEAQPPVGYKPSTKQWTVEVEKNGRTTVQGEQVENREEALSDQYPQTGT
YPDVQTPYQIIKVDGSEKNGQHKALNPNPYERVIPEGTLSKRIYQVNNLD
DNQYGIELTVSGKTTVETKEASTPLDVVILLDNSNSMSNIRHNHAHRAEK
AGEATPALVDKITSNPDNRVALVTYGSTIFDGSEATVEKGVADANGKILN
DSALWTEDRTTFTAKTYNYSFLNLTSDPTDIQTIKDRIPSDAEELNKDKL
MYQFGATFTQKALMTADDILTKQARPNSKKVIFHITDGVPTMSYPINFKY
TGTTQSYRTQLNNFKAKTPNSSGILLEDFVTWSADGEHKIVRGDGESYQM
FTKKPVTDQYGVHQILSITSMEQRAKLVSAGYRFYGTDLYLYWRDSILAY
PFNSSTDWITNHGDPTTWYYNGNMAQDGYDVFTVGVGVNGDPGTDEATAT
RFMQSISSSPDNYTNVADPSQILQELNRYFYTIVNEKKSIENGTITDPMG
ELIDFQLGADGRFDPADYTLTANDGSSLVNNVPTGGPQNDGGLLKNAKVF
YDTTEKRIRVTGLYLGTGEKVTLTYNVRLNDQFVSNKFYDTNGRTTLHPK
EVEKNTVRDFPIPKIRDVRKYPEITIPKEKKLGEIEFIKINKNDKKPLRD
AVFSLQKQHPDYPDIYGAIDQNGTYQNVRTGEDGKLTFKNLSDGKYRLFE
NSEPAGYKPVQNKPIVAFQIVNGEVRDVTSIVPQDIPAGYEFTNDKHYIT
NEPIPPKREYPRTGGIGMLPFYLIGCMMMGGVLLYTRKHP
[1186] Orf4.sub.--670 is a cell wall surface anchor family protein.
An example of an amino acid sequence of orf4.sub.--670 is set forth
in SEQ ID NO: 174. TABLE-US-00230 SEQ ID NO: 174
MKSINKFLTMLAALLLTASSLFSAATVFAADNVSTAPDAVTKTLTIHKLL
LSEDDLKTWDTNGPKGYDGTQSSLKDLTGVVAEEIPNVYFELQKYNLTDG
KEKENLKDDSKWTTVHGGLTTKDGLKIETSTLKGVYRIREDRTKTTYVGP
NGQVLTGSKAVPALVTLPLVNNNGTVIDAHVFPKNSYNKPVVDKRIADTL
NYNDQNGLSIGTKIPYVVNTTIPSNATFATSFWSDEMTEGLTYNEDVTIT
LNNVAMDQADYEVTKGNNGFNLKLTEAGLAKINGKDADQKIQITYSATLN
SLAVADIPESNDITYHYGNHQDHGNTPKPTKPNNGQITVTKTWDSQPAPE
GVKATVQLVNAKTGEKVGAPVELSENNWTYTWSGLDNSIEYKVEEEYNGY
SAEYTVESKGKLGVKNWKDNNPAPINPEEPRVKTYGKKFVKVDQKDTRLE
NAQFVVKKADSNKYIAFKSTAQQAADEKAAATAKQKLDAAVAAYTNAADK
QAAQALVDQAQQEYNVAYKEAKFGYVEVAGKDEAMVLTSNTDGQFQISGL
AAGTYKLEEIKAPEGFAKIDDVEFVVGAGSWNQGEFNYLKDVQKNDATKV
VNKKITIPQTGGIGTIIFAVAGAAIMGIAVYAYVKNNKDEDQLA
[1187] Orf5.sub.--670 is a cell wall surface anchor family protein.
An example of an amino acid sequence of orf5.sub.--670 is set forth
in SEQ ID NO: 175. TABLE-US-00231 SEQ ID NO: 175
MTMQKMQKMISRIFFVMALCFSLVWGAHAVQAQEDHTLVLQLENYQEVVS
QLPSRDGHRLQVWKLDDSYSYDDRVQIVRDLHSWDENKSLLFKKTSFEMT
FLENQIEVSHIPNGLYYVRSIIQTDAVSYPAEFLFEMTDQTVEPLVIVAK
KTDTMTTKVKLIKVDQDHNRLEGVGFKLVSVARDGSEKEVPLIGEYRYSS
SGQVGRTLYTDKNGEIFVTNLPLGNYRFKEVEPLAGYAVTTLDTDVQLVD
HQLVTTTVVNQKLPRGNVDFMKVDGRTNTSLQGAMFKVMKEESGHYTPVL
QNGKEVVVTSGKDGRFRVEGLEYGTYYLWELQAPTGYVQLTSPVSFTIGK
DTRKELVTVVKNNKRPRIDVPDTGEETLYILMLVAILLFGSGYYLTKKPN N
[1188] Orf6.sub.--670 is a sortase. An example of an amino acid
sequence of orf6.sub.--670 is set forth in SEQ ID NO: 176.
TABLE-US-00232 SEQ ID NO: 176
MLIKMVKTKKQKRNNLLLGVVFFIGMAVMAYPLVSRLYYRVESNQQIADF
DKEKATLDEADIDERMKLAQAFNDSLNNVVSGDPWSEEMKKKGRAEYARM
LEIHERMGHVEIPVIDVDLPVYAGTAEEVLQQGAGHLEGTSLPIGGNSTH
AVITAHTGLPTAKMFTDLTKLKVGDKFYVHNIKEVMAYQVDQVKVIEPTN
FDDLLIVPGHDYVTLLTCTPYMINTHRLLVRGHRIPYVAEVEEEFIAANK
LSHLYRYLFYVAVGLIVILLWIIRRLRKKKKQPEKALKALKAARKEVKVE DGQQ
[1189] Orf7.sub.--670 is a sortase. An example of an amino acid
sequence of orf7.sub.--670 is set forth in SEQ ID NO: 177.
TABLE-US-00233 SEQ ID NO: 177
VSRYYYRIESNEVIKEFDETVSQMDKAELEERWRLAQAFNATLKPSEILD
PFTEQEKKKGVSEYANMLKVHERIGYVEIPAIDQEIPMYVGTSEEILQKG
AGLLEGASLPVGGENTHTVVTAHRGLPTAELFSQLDKMKKGDVFYLHVLD
QVLAYQVDQILTVEPNDEEPVLIQHGEDYATLLTCTPYMINSHRLLVRGK
RIPYTAPTAERNRAVRERGQFWLWLLLAALVMILVLSYGVYRHRRIVKGL EKQLEEHHVKG
[1190] Orf8.sub.--670 is a sortase. An example of an amino acid
sequence of orf8.sub.--670 is set forth in SEQ ID NO: 178.
TABLE-US-00234 SEQ ID NO: 178
MSKAKLQKLLGYLLMLVALVIPVYCEGQMVLQSLGQVKGHEIFSESVTAD
SYQEQLQRSLDYNQRLDSQNRIVDPFLAEGYEVNYQVSDDPDAVYGYLSI
PSLEIMEPVYLGADYHHLAMGLAHVDGTPLPVEGKGIRSVIAGHRAEPSH
VFFRHLDQLKVGDALYYDNGQEIVEYQMMDTEIILPSEWEKLESVSSKNI
MTLITCDPIPTFNKRLLVNFERVAVYQKSDPQTAAVARVAFTKEGQSVSR
VATSQWLYRGLVVLAFLGILFVLWKLARLLRGK
[1191] As discussed above, a S. pneumoniae AI sequence is present
in the 19A Hungary 6 S. pneumoniae genome. Examples of S.
pneumoniae AI sequences from 19A Hungary 6 are set forth below.
[1192] ORF2.sub.--19AH is a transcriptional regulator. An example
of an amino acid sequence of ORF2.sub.--19AH is set forth in SEQ ID
NO: 187. TABLE-US-00235 SEQ ID NO: 187
MLNKYIEKRITDKITILNILLDIRSIELDELSTLTSLQSKSLLSILQELQ
ETFEEELTFNLDTQQVQLIEHHSHQTNYYFHQLYNQSTILKILRFFLLQG
NQSFNEFTQKEYISIATGYRVRQKCGLLLRSVGLDLVKNQVVGPEYRIRF
LIALLQFHFGIEIYDLNDGSMDWVTHMIVQSNSQLSHELLEITPDEYVHF
SILVALTWKRREFPLEFPESKEFEKLKNLFMYPILMEHCQTYLEPHANMT
FTQEELDYIFLVYCSANSSFSKDKWNQEKKTHTIQLILQHTRGKHLLSKF
KNILGNDISNSLSFLTALTFLTRTFLFGLQNLVPYYNYYEHYGIESDKPL
YHISKAIVQEWMTEQKIEGVTDQHRLYLFSLYLTETIFSSLPAIPIFIIL
NNQADVNLIKSTILRNFTDKVASVTGYNILISPPPSEEHLTEPLIIITTK
EYLPYVKKQYPKGKHHFLTIALDLHVSQQRLIYQTIVDIRKEAFDKRVAM IAKKAHYLL
[1193] ORF3.sub.--19AH is a cell wall surface protein. An example
of an amino acid sequence of ORF3.sub.--19AH is set forth in SEQ ID
NO: 188. TABLE-US-00236 SEQ ID NO: 188
MKKVRKIFQKAVAGLCCISQLTAFSSIVALAETPETSPAIGKVVIKETGE
GGALLGDAVFELKNNTDGTTVSQRTEAQTGEAIFSNIKPGTYTLTEAQPP
VGYKPSTKQWTVEVEKNGRTTVQGEQVENREEALSDQYPQTGTYPDVQTP
YQIIKVDGSEKNGQHKALNPNPYERVIPEGTLSKRIYQVNNLDDNQYGIE
LTVSGKTTVETKEASTPLDVVILLDNSNSMSNTRHNHAHRAEKAGEATRA
LVDKITSNPDNRVALVTYGSTIFDGSEATVEKGVADANGKILNDSALWTF
DRTTFTAKTYNYSFLNLTSDPTDIQTIKDRIPSDAEELNKDKLMYQFGAT
FTQKALMTADDILTKQARPNSKKVIFHITDGVPTMSYPINFKYTGTTQSY
RTQLNNFKAKTPNSSGILLEDFVTWSADGEHKIVRGDGESYQMFTKKPVT
DQYGVHQILSITSMEQRAKLVSAGYRFYGTDLYLYWRDSILAYPFNSSTD
WITNHGDPTTWYYNGNMAQDGYDVFTVGVGVNGDPGTDEATATRFMQSIS
SSPDNYTNVADPSQILQELNRYFYTTVNEKKSIENGTITDPMGELIDFQL
GADGRFDPADYTLTANDGSSLVNNVPTGGPQNDGGLLKNAKVFYDTTEKR
IRVTGLYLGTGEKVTLTYNVRLNDQFVSNKFYDTNGRTTLHPKEVEKNTV
RDFPIPKIRDVRKYPEITIPKEKKLGETEFIKINKNDKKPLRDAVFSLQK
QHPDYPDIYGAIDQNGTYQNVRTGEDGKLTFKNLSDGKYRLFENSEPAGY
KPVQNKPIVAFQIVNGEVRDVTSIVPQDIPAGYEFTNDKHYITNEPIPPK
REYPRTGGIGMLPFYLIGCMMMGGVLLYTRKNP
[1194] ORF4.sub.--19AH is a cell wall surface protein. An example
of an amino acid sequence of ORF4.sub.--19AH is set forth in SEQ ID
NO: 189. TABLE-US-00237 SEQ ID NO: 189
MKSINKFLTMLAALLLTASSLFSAATVFAADNVSTAPDAVTKTLTIHKLL
LSEDDLKTWDTNGPKGYDGTQSSLKDLTGVVAEEIPNVYFELQKYNLTDG
KEKENLKDDSKWTTVHGGLTTKDGLKIETSTLKGVYRIREDRTKTTYVGP
NGQVLTGSKAVPALVTLPLVNNNGTVTDAHVFPKNSYNKPVVDKRIADTL
NYNDQNGLSIGTKTPYVVNTTIPSNATFATSFWSDEMTEGLTYNEDVTIT
LNNVAMDQADYEVTKGXNGFNLKLTEAGLAKINGKDADQKIQITYSATLN
SLAVADIPESNDITYHYGNHQDHGNTPKPTKPNNGQITVTKTWDSQPAPE
GVKATVQLVNAKTGEKVGAPVELSENNWTYTWSGLDNSIEYKVEEEYNGY
SAEYTVESKGKLGVKNWKDNNPAPINPEEPRVKTYGKKFVKVDQKDTRLE
NAQFVVKKADSNKYIAFKSTAQQAADEKAAATAKQKLDAAVAAYTNAADK
QAAQALVDQAQQEYNVAYKEAKEGYVEVAGKDEAMVLTSNTDGQFQISGL
AAGTYKLEEIKAPEGFAKIDDVEFVVGAGSWNQGEFNYLKDVQKNDATKV
VNKKITIPQTGGIGTIIFAVAGAAIMGIAVYAYVKNNKDEDQLA
[1195] ORF5.sub.--19AH is a cell wall surface protein. An example
of an amino acid sequence of ORF5.sub.--19AH is set forth in SEQ ID
NO: 190. TABLE-US-00238 SEQ ID NO: 190
MTMQKMQKMISRIFFVMALCPSLVWGAHAVQAQEDHTLVLQLENYQEVVS
QLPSRDGHRLQVWKLDDSYSYDDRVQIVRDLHSWDENKLSSEKKTSFEMT
FLENQIEVSHIPNGLYYVRSIIQTDAVSYPAEELFEMTDQTVEPLVIVAK
KTDTMTTKVKLIKVDQDHNRLEGVGFKLVSVARDGSEKEVPLIGEYRYSS
SGQVGRTLYTDKNGEIFVTNLPLGNYRFKEVEPLAGYAVTTLDTDVQLVD
HQLVTITVVNQKLPRGNVDEMKVDGRTNTSLQGAMFKVMKEESGHYTPVL
QNGKEVVVTSGKDGRFRVEGLEYGTYYLWELQAPTGYVQLTSPVSETIGK
DTRKELVTVVKNNKRPRTDVPDTGEETLYILMLVAILLFGSGYYLTKKPN N
[1196] ORF6.sub.--19AH is a putative sortase. An example of an
amino acid sequence of ORF6.sub.--19AH is set forth in SEQ ID NO:
191. TABLE-US-00239 SEQ ID NO: 191
MLIKMVKTKKQKRNNLLLGVVFFIGMAVMAYPLVSRLYYRVESNQQTADF
DKEKATLDEADIDERMKLAQAFNDSLNNVVSGDPWSEEMKKKGRAEYARM
LEIHERMGHVEIPVIDVDLPVYAGTAEEVLQQGAGHLEGTSLPIGGNSTH
AVITAHTGLPTAKMFTDLTKLKVGDKFYVHNIKEVMAYQVDQVKVIEPTN
FDDLLIVPGHDYVTLLTCTPYMINTHRLLVRGHRIPYVAEVEEEFIAANK
LSHLYRYLFYVAVGLIVILLWIIRRLRKKKKQPEKALKALKAARKEVKVE DGQQ
[1197] ORF7.sub.--19AH is a putative sortase. An example of an
amino acid sequence of ORF7.sub.--19AH is set forth in SEQ ID NO:
192. TABLE-US-00240 SEQ ID NO: 192
MDNSRRSRKKGTKKKKHPLILLLIFLVGFAVAIYPLVSRYYYRIESNEVI
KEFDETVSQMDKAELEERWRLAQAFNATLKPSEILDPFTEQEKKKGVSEY
ANMLKVHERIGYVEIPAIDQEIPMYVGTSEEILQKGAGLLEGASLPVGGE
NTHTVVTAHRGLPTAELFSQLDKMKKGDVFYLHVLDQVLAYQVDQILTVE
PNDFEPVLIQHGEDYATLLTCTPYMINSHRLLVRGKRIPYTAPIAERNRA
VRERGQFWLWLLLAALVMILVLSYGVYRHRRIVKGLEKQLEEHHVKG
[1198] ORF8.sub.--19AH is a putative sortase. An example of an
amino acid sequence of ORF8.sub.--19AH is set forth in SEQ ID NO:
193. TABLE-US-00241 SEQ ID NO: 193
MSKAKLQKLLGYLLMLVALVIPVYCFGQMVLQSLGQVKGHEIFSESVTAD
SYQEQLQRSLDYNQRLDSQNRIVDPFLAEGYEVNYQVSDDPDAVYGYLSI
PSLEIMEPVYLGADYHHLAMGLAHVDGTPLPVEGKGIRSVIAGHPAEPSH
VFFRHLDQLKVGDALYYDNGQEIVEYQMMDTEIILPSEWEKLESVSSKNI
MTLITCDPIPTFNKRLLVNFERVAVYQKSDPQTAAVARVAFTKEGQSVSR
VATSQWLYRGLVVLAFMGILFVLWKLARLLRGK
[1199] As discussed above, a S. pneumoniae AI sequence is present
in the 6B Finland 12 S. pneumoniae genome. Examples of S.
pneumoniae AI sequences from 6B Finland 12 are set forth below.
[1200] ORF2.sub.--6BF is a transcriptional regulator. An example of
an amino acid sequence of ORF2.sub.--6BF is set forth in SEQ ID NO:
194. TABLE-US-00242 SEQ ID NO: 194
MLNKYIEKRITDKITILNILLDIRSIELDELSTLTSLQSKSLLSILQELQ
ETFEEELTFNLDTQQVQLIEHHSHQTNYYFHQLYNQSTILKILRFFLLQG
NQSENEFTQKEYISIATGYRVRQKCGLLLRSVGLDLVKNQVVGPEYRIRF
LIALLQFHFGIEIYDLNDGSMDWVTHMIVQSNSQLSHELLEITPDEYVHF
SILVALTWKRREFPLEFPESKEFEKLKNLFMYPILMEHGQTYLEPHANMT
FTQEELDYIFLVYCSANSSFSKDKWNQEKKTHTIQLILQHTRGKHLLSKF
KNILGNDISNSLSFLTALTFLTRTFLFGLQNLVPYYNYYEHYGIESDKPL
YHISKAIVQEWMTEQKIEGVIDQHRLYLFSLYLTETIFSSLPAIPIFIIL
NNQADVNLIKSIILRNFTDKVASVTGYNILISPPPSEEHLTEPLIIITTK
EYLPYVKKQYPKGKHHFLTIALDLHVSQQRLIYQTIVDIRKEAFDKRVAM IAKKAHYLL
[1201] ORF3.sub.--6BF is a cell wall surface protein. An example of
an amino acid sequence of ORF3.sub.--6BF is set forth in SEQ ID NO:
195. TABLE-US-00243 SEQ ID NO: 195
MKKVRKIFQKAVAGLCCISQLTAFSSIVALAETPETSPAIGKVVIKETGE
GGALLGDAVFELKNNTDGTTVSQRTEAQTGEAIFSNIKPGTYTLTEAQPP
VGYKPSTKQWTVEVEKNGRTTVQGEQVENREEALSDQYPQTGTYPDVQTP
YQIIKVDGSEKNGQHKALNPNPYERVIPEGTLSKRTYQVNNLDDNQYGIE
LTVSGKTTVETKEASTPLDVVILLDNSNSMSNIRHNHAHRAEKAGEATRA
LVDKITSNPDNRVALVTYGSTIFDGSEATVEKGVADANGKILNDSALWTF
DRTTFTAKTYNYSFLNLTSDPTDIQTIKDRIPSDAEELNKDKLMYQFGAT
FTQKALMTADDILTKQARPNSKKVIFHITDGVPTMSYPINFKYTGTTQSY
RTQLNNFKAKTPNSSGILLEDFVTWSADGEHKIVRGDGESYQMFTKKPVT
DQYGVHQILSITSMEQRAKLVSAGYRFYGTDLYLYWRDSILAYPFNSSTD
WITNHGDPTTWYYNGNMAQDGYDVFTVGVGVNGDPGTDEATATRFMQSIS
SSPDNYTNVADPSQILQELNRYFYTIVNEKKSIENGTITDPMGELIDFQL
GADGRFDPADYTLTANDGSSLVNNVPTGGPQNDGGLLKNAKVFYDTTEKR
IRVTGLYLGTGEKVTLTYNVRLNDQFVSNKEYDTNGRTTLHPKEVEKNTV
RDFPIPKIRDVRKYPEITTPKEKKLGEIEFIKINKNDKKPLRDAVFSLQK
QHPDYPDIYGAIDQNGTYQNVRTGEDGKLTFKNLSDGKYRLFENSEPAGY
KPVQNKPIVAFQIVNGEVRDVTSIVPQDIPAGYEFTNDKHYITNEPIPPK
REYPRTGGIGMLPEYLIGCMMMGGVLLYTRKHP
[1202] ORF4.sub.--6BF is a cell wall surface protein. An example of
an amino acid sequence of ORF4.sub.--6BF is set forth in SEQ ID NO:
196. TABLE-US-00244 SEQ ID NO: 196
MKSINKFLTMLAALLLTASSLFSAATVFAADNVSTAPDAVTKTLTIHKLL
LSEDDLKTWDTNGPKGYDGTQSSLKDLTGVVAEEIPNVYFELQKYNLTDG
KEKENLKDDSKWTTVHGGLTTKDGLKIETSTLKGVYRIREDRTKTTYVGP
NGQVLTGSKAVPALVTLPLVNNNGTVIDAHVFPKNSYNKPVVDKRIADTL
NYNDQNGLSIGTKIPYVVNTTIPSNATFATSFWSDEMTEGLTYNEDVTIT
LNNVAMDQADYEVTKGNNGFNLKLTEAGLAKINGKDADQKIQITYSATLN
SLAVADIPESNDITYHYGNHQDHGNTPKPTKPNNGQITVTKTWDSQPAPE
GVKATVQLVNAKTGEKVGAPVELSENNWTYTWSGLDNSIEYKVEEEYNGY
SAEYTVESKGKLGVKNWKDNNPAPINPEEPRVKTYGKKFVKVDQKDTRLE
NAQFVVKKADSNKYIAFKSTAQQAADEKAAATAKQKLDAAVAAYTNAADK
QAAQALVDQAQQEYNVAYKEAKFGYVEVAGKDEAMVLTSNTDGQFQISGL
AAGTYKLEEIKAPEGFAKIDDVEFVVGAGSWNQGEFNYLKDVQKNDATKV
VNKKITIPQTGGIGTIIEAVAGAAIMGIAVYAYVKNNKDEDQLA
[1203] ORF5.sub.--6BF is a cell wall surface protein. An example of
an amino acid sequence of ORF5.sub.--6BF is set forth in SEQ ID NO:
197. TABLE-US-00245 SEQ ID NO: 197
MTMQKMQKMISRIFFVMALCFSLVWGAHAVQAQEDHTLVLQLENYQEVVS
QLPSRDGHRLQVWKLDDSYSYDDRVQIVRDLHSWDENKLSSFKKTSFEMT
FLENQIEVSHIPNGLYYVRSIIQTDAVSYPAEFLFEMTDQTVEPLVIVAK
KTDTMTTKVKLIKVDQDHNRLEGVGFKLVSVARDGSEKEVPLIGEYRYSS
SGQVGRTLYTDKNGEIFVTNLPLGNYRFKEVEPLAGYAVTTLDTDVQLVD
HQLVTITVVNQKLPRGNVDFMKVDGRTNTSLQGAMFKVMKEESGHYTPVL
QNGKEVVVTSGKDGRFRVEGLEYGTYYLWELQAPTGYVQLTSPVSFTIGK
DTRKELVTVVKNNKRPRIDVPDTGEETLYILMLVAILLFGSGYYLTKKPN N
[1204] ORF6.sub.--6BF is a putative sortase. An example of an amino
acid sequence of ORF6.sub.--6BF is set forth in SEQ ID NO: 198.
TABLE-US-00246 SEQ ID NO: 198
MLIKMVKTKKQKRNNLLLGVVFFIGMAVMAYPLVSRLYYRVESNQQIADF
DKEKATLDEADIDERMKLAQAFNDSLNNVVSGDPWSEEMKKKGRAEYARM
LEIHERMGHVEIPVIDVDLPVYAGTAEEVLQQGAGHLEGTSLPTGGNSTH
AVITAHTGLPTAKMFTDLTKLKVGDKFYVHNIKEVMAYQVDQVKVIEPTN
FDDLLIVPGHDYVTLLTCTPYMINTHRLLVRGHRIPYVAEVEEEFIAANK
LSHLYRYLFYVAVGLIVILLWIIRRLRKKKKQPEKALKALKAARKEVKVE DGQQ
[1205] ORF7.sub.--6BF is a putative sortase. An example of an amino
acid sequence of ORF7.sub.--6BF is set forth in SEQ ID NO: 199.
TABLE-US-00247 SEQ ID NO: 199
MDNSRRSRKKGTKKKKHPLILLLIFLVGFAVAIYPLVSRYYYRIESNEVI
KEFDETVSQMDKAELEERWRLAQAFNATLKPSEILDPFTEQEKKKGVSEY
ANMLKVHERIGYVEIPAIDQEIPMYVGTSEEILQKGAGLLEGASLPVGGE
NTHTVVTAHRGLPTAELFSQLDKMKKGDVFYLHVLDQVLAYQVDQILTVE
PNDFEPVLIQHGEDYATLLTCTPYMINSHRLLVRGKRIPYTAPIAERNRA
VRERGQFWLWLLLAALVMILVLSYGVYRHRRIVKGLEKQLEEHHVKG
[1206] ORF8.sub.--6BF is a putative sortase. An example of an amino
acid sequence of ORF8.sub.--6BF is set forth in SEQ ID NO: 200.
TABLE-US-00248 SEQ ID NO: 200
MSKAKLQKLLGYLLMLVALVTPVYCFGQMVLQSLGQVKGHEIFSESVTAD
SYQEQLQRSLDYNQRLDSQNRIVDPFLAEGYEVNYQVSDDPDAVYGYLSI
PSLETMEPVYLGADYHHLAMGLAHVDGTPLPVEGKGIRSVIAGHRAEPSH
VFFRHLDQLKVGDALYYDNGQEIVEYQMMDTEIILPSEWEKLESVSSKNI
MTLITCDPIPTFNKRLLVNFERVAVYQKSDPQTAAVARVAPTKEGQSVSR
VATSQWLYRGLVVLAFLGILFVLWKLARLLRGK
[1207] As discussed above, a S. pneumoniae AI sequence is present
in the 6B Spain 2 S. pneumoniae genome. Examples of S. pneumoniae
AI sequences from 6B Spain 2 are set forth below.
[1208] ORF2.sub.--6BSP is a transcriptional regulator. An example
of an amino acid sequence of ORF2.sub.--6BSP is set forth in SEQ ID
NO: 201. TABLE-US-00249 SEQ ID NO: 201
MLNKYIEKRITDKITILNILLDIRSIELDELSTLTSLQSKSLLSTLQELQ
ETFEEELTFNLDTQQVQLTEHHSHQTNYYFHQLYNQSTILKILRFFLLQG
NQSFNEFTQKEYISIATGYRVRQKCGLLLRSVGLDLVKNQVVGPEYRIRF
LIALLQFHFGIEIYDLNDGSMDWVTHMIVQSNSQLSHELLEITPDEYVHF
SILVALTWKRREFPLEFPESKEFEKLKNLFMYPILMEHCQTYLEPHANMT
FTQEELDYIFLVYCSANSSFSKDKWNQEKKTHTIQLILQHTRGKHLLSKF
KNILGNDISNSLSPLTALTFLTRTFLFGLQNLVPYYNYYEHYGIESDKPL
YHISKAIVQEWMTEQKIEGVIDQHRLYLFSLYLTETIFSSLPAIPIFITL
NNQADVNLTKSIILRNFTDKVASVTGYNILISPPPSEEHLTEPLIIITTK
EYLPYVKKQYPKGKHHFLTIALDLHVSQQRLIYQTIVDTRKEAFDKRVAM IAKKAHYLL
[1209] ORF3.sub.--6BSP is a cell wall surface protein. An example
of an amino acid sequence of ORF3.sub.--6BSP is set forth in SEQ ID
NO: 202. TABLE-US-00250 SEQ ID NO: 202
MKKVRKIFQKAVAGLCCISQLTAFSSIVALAETPETSPAIGKVVIKETGE
GGALLGDAVFELKNNTDGTTVSQRTEAQTGEAIFSNIKPGTYTLTEAQPP
VGYKPSTKQWTVEVEKNGRTTVQGEQVENREEALSDQYPQTGTYPDVQTP
YQIIKVDGSEKNGQHKALNPNPYERVIPEGTLSKRIYQVNNLDDNQYGIE
LTVSGKTTVETKEASTPLDVVILLDNSNSMSNIRHNHAHRAEKAGEATRA
LVDKITSNPDNRVALVTYGSTIFDGSEATVEKGVADANGKILNDSALWTF
DRTTFTAKTYNYSFLNLTSDPTDIQTIKDRIPSDAEELNKDKLMYQFGAT
FTQKALMTAKKILTKQARPNSKKVIFHITDGVPTMSYPINFKYTGTTQSY
RTQLNNFKAKTPNSSGILLEDFVTWSADGEHKIVRGDGESYQMFTKKPVT
DQYGVHQILSITSMEQRAKLVSAGYRFYGTDLYLYWRDSILAYPFNSSTD
WITNHGDPTTWYYNGNMAQDGYDVFTVGVGVNGDPGTDEATATRFMQSIS
SSPDNYTNVADPSQILQELNRYEYTIVNEKKSIENGTITDPMGELIDFQL
GADGRFDPADYTLTANDGSSLVNNVPTGGPQNDGGLLKNAKVFYDTTEKR
TRVTGLYLGTGEKVTLTYNVRLNDQFVSNKFYDTNGRTTLHPKEVEKNTV
RDFPIPKIRDVRKYPEITIPKEKKLGEIEFIKINKNDKKPLRDAVFSLQK
QHPDYPDIYGAIDQNGTYQNVRTGEDGKLTFKNLSDGKYRLFENSEPAGY
KPVQNKPIVAFQIVNGEVRDVTSIVPQDIPAGYEFTNDKHYITNEPIPPK
REYPRTGGIGMLPFYLIGCMMMGGVLLYTRKHP
[1210] ORF4.sub.--6BSP is a cell wall surface protein. An example
of an amino acid sequence of ORF4.sub.--6BSP is set forth in SEQ ID
NO: 203. TABLE-US-00251 SEQ ID NO: 203
MKSINKFLTMLAALLLTASSLFSAATVFAADNVSTAPDAVTKTLTIHKLL
LSEDDLKTWDTNGPKGYDGTQSSLKDLTGVVAEEIPNVYFELQKYNLTDG
KEKENLKDDSKWTTVHGGLTTKDGLKIETSTLKGVYRTREDRTKTTYVGP
NGQVLTGSKAVPALVTLPLVNNNGTVTDAHVFPKNSYNKPVVDKRIADTL
NYNDQNGLSIGTKIPYVVNTTIPSNATFATSFWSDEMTEGLTYNEDVTIT
LNNVAMDQADYEVTKGNNGFNLKLTEAGLAKINGKDADQKIQITYSATLN
SLAVADIPESNDTTYHYGNHQDHGNTPKPTKPNNGQITVTKTWDSQPAPE
GVKATVQLVNAKTGEKVGAPVELSENNWTYTWSGLDNSIEYKVEEEYNGY
SAEYTVESKGKLGVKNWKDNNPAPINPEEPRVKTYGKKFVKVDQKDTRLE
NAQFVVKKADSNKYIAFKSTAQQAADEKAAATAKQKLDAAVAAYTNAADK
QAAQALVDQAQQEYNVAYKEAKPGYVEVAGKDEAMVLTSNTDGQEQISGL
AAGTYKLEEIKAPEGFAKIDDVEFVVGAGSWNQGEFNYLKDVQKNDATKV
VNKKITIPQTGGIGTIIFAVAGAAIMGIAVYAYVKNNKDEDQLA
[1211] ORF5.sub.--6BSP is a cell wall surface protein. An example
of an amino acid sequence of ORF5.sub.--6BSP is set forth in SEQ ID
NO: 204. TABLE-US-00252 SEQ ID NO: 204
MTMQKMQKMISRIFFVMALCFSLVWGAHAVQAQEDHTLVLQLENYQEVVS
QLPSRDGHRLQVWKLDDSYSYDDRVQIVRDLHSWDENKLSSFKKTSFEMT
FLENQIEVSHIPNGLYYVRSIIQTDAVSYPAEFLFEMTDQTVEPLVIVAK
KTDTMTTKVKLIKVDQDHNRLEGVGFKLVSVARDGSEKEVPLIGEYRYSS
SGQVGRTLYTDKNGEIFVTNLPLGNYRFKEVEPLAGYAVTTLDTDVQLVD
HQLVTITVVNQKLPRGNVDFMKVDGRTNTSLQGAMFKVMKEESGHYTPVL
QNGKEVVVTSGKDGRFRVEGLEYGTYYLWELQAPTGYVQLTSPVSFTIGK
DTRKELVTVVKNNKRPRIDVPDTGEETLYILMLVAILLFGSGYYLTKKPN N
[1212] ORF6.sub.--6BSP is a putative sortase. An example of an
amino acid sequence of ORF6.sub.--6BSP is set forth in SEQ ID NO:
205. TABLE-US-00253 SEQ ID NO: 205
MLIKMVKTKKQKRNNLLLGVVFFIGMAVMAYPLVSRLYYRVESNQQIADF
DKEKATLDEADIDERMKLAQAFNDSLNNVVSGDPWSEEMKKKGRAEYARM
LEIHERMGHVETPVIDVDLPVYAGTAEEVLQQGAGHLEGTSLPIGGNSTH
AVITAHTGLPTAKMFTDLTKLKVGDKFYVHNIKEVMAYQVDQVKVIEPTN
FDDLLIVPGHDYVTLLTCTPYMINTHRLLVRGHRIPYVAEVEEEFTAANK
LSHLYRYLFYVAVGLIVILLWIIRRLRKKKKQPEKALKALKAARKEVKVE DGQQ
[1213] ORF7.sub.--6BSP is a putative sortase. An example of an
amino acid sequence of ORF7.sub.--6BSP is set forth in SEQ ID NO:
206. TABLE-US-00254 SEQ ID NO: 206
MDNSRRSRKKGTKKKKHPLILLLIFLVGFAVAIYPLVSRYYYRIESNEVI
KEFDETVSQMDKAELEERWRLAQAFNATLKPSEILDPFTEQEKKKGVSEY
ANMLKVHERIGYVEIPAIDQEIPMYVGTSEEILQKGAGLLEGASLPVGGE
NTHTVVTAHRGLPTAELFSQLDKMKKGDVFYLHVLDQVLAYQVDQILTVE
PNDFEPVLIQHGEDYATLLTCTPYMINSHRLLVRGKRIPYTAPIAERNRA
VRERGQFWLWLLLAALVMILVLSYGVYRHRRIVKGLEKQLEEHHVKG
[1214] ORF8.sub.--6BSP is a putative sortase. An example of an
amino acid sequence of ORF8.sub.--6BSP is set forth in SEQ ID NO:
207. TABLE-US-00255 SEQ ID NO: 207
MSKAKLQKLLGYLLMLVALVIPVYCFGQMVLQSLGQVKGHEIFSESVTAD
SYQEQLQRSLDYNQRLDSQNRIVDPFLAEGYEVNYQVSDDPDAVYGYLSI
PSLETMEPVYLGADYHHLAMGLAHVDGTPLPVEGKGIRSVIAGHRAEPSH
VFFRHLDQLKVGDALYYDNGQEIVEYQMMDTEIILPSEWEKLESVSSKNI
MTLITCDPTPTFNKRLLVNFERVAVYQKSDPQTAAVARVAFTKEGQSVSR
VATSQWLYRGLVVLAFLGILFVLWKLARLLRGK
[1215] As discussed above, a S. pneumoniae AI sequence is present
in the 9V Spain 3 S. pneumoniae genome. Examples of S. pneumoniae
AI sequences from 9V Spain 3 are set forth below.
[1216] ORF2.sub.--9VSP is a transcriptional regulator. An example
of an amino acid sequence of ORF2.sub.--9VSP is set forth in SEQ ID
NO: 208. TABLE-US-00256 SEQ ID NO: 208
MLNKYIEKRITDKITILNILLDIRSIELDELSTLTSLQSKSLLSILQELQ
ETFEEELTFNLDTQQVQLIEHHSHQTNYYFHQLYNQSTILKILREELLQG
NQSFNEFTQKEYISIATGYRVRQKCGLLLRSVGLDLVKNQVVGPEYRIRF
LIALLQFHFGIEIYDLNDGSMDWVTHMTVQSNSQLSHELLEITPDEYVHF
SILVALTWKRREFPLEFPESKEFEKLKNLFMYPILMEHCQTYLEPHANMT
FTQEELDYIFLVYCSANSSFSKDKWNQEKKTHTIQLILQHTRGKHLLSKF
KNILGNDISNSLSFLTALTFLTRTFLFGLQNLVPYYNYYEHYGIESDKPL
YHISKAIVQEWMTEQKIEGVIDQHRLYLFSLYLTETIFSSLPAIPIFIIL
NNQADVNLIKSIILRNFTDKVASVTGYNILILSPPPSEEHLTEPLIITTK
EYLPYVKKQYPKGKHHFLTTALDLHVSQQRLIYQTIVDIRKEAFDKRVAM IAKKAHYLL
[1217] ORF3.sub.--9VSP is a cell wall surface protein. An example
of an amino acid sequence of ORF3.sub.--9VSP is set forth in SEQ ID
NO: 209. TABLE-US-00257 SEQ ID NO: 209
MKKVRKIFQKAVAGLCCISQLTAFSSIVALAETPETSPAIGKVVIKETGE
GGALLGDAVFELKNNTNGTTVSQRTEAQTGEAIFSNIKPGTYTLTEAQPP
VGYKPSTKQRTVEVEKNGRTTVQGEQVENREEALSDQYPQTGTYPDVQTP
YQIIKVDGSEKNGQHKALNPNPYERVIPEGTLSKRIYQVNNLDDNQYGIE
LTVSGKTVYERKDKSVPLDVVILLDNSNSMSNIRNKNARRAERAGEATRS
LIDKITSDPENRVALVTYASTIFDGTEFTVEKGVADKNGKRLNDSLFWNY
DQTSFTTNTKDYSYLKLTNDKNDIVELKNKVPTEAEDHDGNRLMYQFGAT
FTQKALMKADEILTQQARQNSQKVIFHITDGVPTMSYPINFNHATFAPSY
QNQLNAFFSKSPNKDGILLSDFITQATSGEHTIVRGDGQSYQMFTDKTVY
EKGAPAAFPVKPEKYSEMKAVGYAVIGDPINGGYIWLNWRESILAYPFNS
NTAKITNHGDPTRWYYNGNIAPDGYDVFTVGIGINGDPGTDEATATSFMQ
SISSKPENYTNVTDTTKILEQLNRYFHTIVTEKKSIENGTITDPMGELID
LQLGTDGRFDPADYTLTANDGSRLENGQAVGGPQNDGGLLKNAKVFYDTT
EKRIRVTGLYLGTGEKVTLTYNVRLNDQFVSNKFYDTNGRTTLHPKEVEK
NTVRDFPIPKIRDVRKYPAITIAKEKKLGEIEFIKINKNDKKPLRDAVFS
LQKQHPDYPDIYGAIDQNGTYQNVRTGEDGKLTFKNLSDGKYRLFENSEP
AGYKPVQNKPIVAFQIVNGEVRDVISTVPQDIPAGYEFTNDKHYITNEPI
PPKREYPRTGGIGMLLFYLIGCMMMGGVLLYTRKHP
[1218] ORF4.sub.--9VSP is a cell wall surface protein. An example
of an amino acid sequence of ORF4.sub.--9VSP is set forth in SEQ ID
NO: 210. TABLE-US-00258 SEQ ID NO: 210
MKSINKFLTMLAALLLTASSLFSAATVFAAGTTTTSVTVHKLLATDGDMD
KIANELETGNYAGNKVGVLPANAKEIAGVMFVWTNTNNEIIDENGQTLGV
NIDPQTFKLSGAMPATAMKKLTEAEGAKFNTANLPAAKYKIYEIHSLSTY
VGEDGATLTGSKAVPIEIELPLNDVVDAHVYPKNTEAKPKIDKDFKGKAN
PDTPRVDKDTPVNHQVGDVVEYEIVTKIPALANYATANWSDRMTEGLAFN
KGTVKVTVDDVALEAGDYALTEVATGFDLKLTDAGLAKVNDQNAEKTVKI
TYSATLNDKAIVEVPESNDVTFNYGNNPDHGNTPKPNKPNENGDLTLTKT
WVDATGAPIPAGAEATFDLVNAQTGDVVQTVTLTTDKNTVTVNGLDKNTE
YKFVERSIKGYSADYQEITTAGEIAVKNWKDENPKPLDPTEPKVVTYGKK
FVKVNDKDNRLAGAEFVIANADNAGQYLARKADKVSQEEKQLVVTTKDAL
DRAVAAYNALTAQQQTQQEKEKVDKAQAAYNAAVTAANNAFEWVADKDNE
NVVKLVSDAQGRFEITGLLAGTYYLEETKQPAGYALLTSRQKFEVTATSY
SATGQGIEYTAGSGKDDATKVVNKKITIPQTGGIGTIIFAVAGAVIMGIA
VYAYVKNNKDEDQLA
[1219] ORF5.sub.--9VSP is a cell wall surface protein. An example
of an amino acid sequence of ORF5.sub.--9VSP is set forth in SEQ ID
NO: 211. TABLE-US-00259 SEQ ID NO: 211
MTMQKMQKMQKMQKMQKMQKMISRIFFVMALCFSLVWGAHAVQAQEDHTL
VLQLENYQEVVSQLPSRDGHRLQVWKLDDSYSYDNRVQTVRDLHSWDENK
LSSFKKTSFEMTFLENQIEVSHIPNGLYYVRSIIQTDAVSYPAEFLFEMT
DQTVEPLVIVAKKADTVTTKVKLIKVDQDHNRLEGVGFKLVSVARDGSEK
EVPLIGEYRYSSSGQVGRTLYTDKNGEIVVTNLPLGTYRFKEVEPLAGYT
VTTMDTDVQLVDHQLVTITVVNQKLPRGNVDFMKVDGRTNTSLQGAMFKV
MKEENGHYTPVLQNGKEVVVASGKDGRFRVEGLEYGTYYLWELQAPTGYV
QLTSPVSFTIGKDTRKELVTVVKNNKRPRIDVPDTGEETLYILMLVAILL
FGSGYYLTKKTNN
[1220] ORF6.sub.--9VSP is a putative sortase. An example of an
amino acid sequence of ORF6.sub.--9VSP is set forth in SEQ ID NO:
212. TABLE-US-00260 SEQ ID NO: 212
MLIKMAKTKKQKRNNLLLGVVFFIGIAVMAYPLVSRLYYRVESNQQIADF
DKEKATLDEADIDERMKLAQAFNDSLNNVVSGDPWSEEMKKKGRAEYARM
LEIHERMGHVEIPAIDVDLPVYAGTAEEVLQQGAGHLEGTSLPIGGNSTH
AVITAHTGLPTAKMFTDLTKLKVGDKFYVHNIKEVMAYQVDQVKVIEPTN
FDDLLIVPGHDYVTLLTCTPYMINTHRLLVRGHRIPYVAEVEEEFIAANK
LSHLYRYLFYVAVGLIVILLWIIRRLRKKKRQSERALKALKEATKEVKVE DE
[1221] ORF7.sub.--9VSP is a putative sortase. An example of an
amino acid sequence of ORF7.sub.--9VSP is set forth in SEQ ID NO:
213. TABLE-US-00261 SEQ ID NO: 213
MSKSRYSRKKSVKKKKNPFILLLIFLVGLAVANYPLVSRYYYRIESNEVI
KEFDETVSQMDKAELEERWRLAQAFNATLKPSEILDPFTEQEKKKGVSEY
ANMLKVHERTGYVEIPAIDQEIPMYVGTSEEILQKGAGLLEGASLPVGGE
NTHTVVTAHRGLPTAELFSQLDKMKKGDIEYLHVLDQVLAYQVDQIVTVE
PNDFEPVLIQHGEDYATLLTCTPYMINSHRLLVRGKRIPYTAPIAERNRA
VRERGQFWLWLLLGAMAVILLLLYRVYRNRRIVKGLEKQLEGRHVKD
[1222] ORF8.sub.--9VSP is a putative sortase. An example of an
amino acid sequence of ORF8.sub.--9VSP is set forth in SEQ ID NO:
214. TABLE-US-00262 SEQ ID NO: 214
MSRTKLRALLGYLLMLVACLIPIYGFGQMVLQSLGQVKGHATFVKSMTTE
MYQEQQNHSLAYNQRLASQNRIVDPELAEGYEVNYQVSDDPDAVYGYLSI
PSLEIMEPVYLGADYHHLGMGLAHVDGTPLPLDGTGIRSVIAGHPAEPSH
VFTRHLDQLKVGDALYYDNGQEIVEYQMMDTEIILPSEWEKLESVSSKNI
MTLITCDPIPTFNKRLLVNEERVAVYQKSDPQTAAVARVAFTKEGQSVSR
VATSQWLYRGLVVLAFLGILFVLWKLARLLRGK
[1223] As discussed above, a S. pneumoniae AI sequence is present
in the 14 CSR 10 S. pneumoniae genome. Examples of S. pneumoniae AI
sequences from 14 CSR 10 are set forth below.
[1224] ORF2.sub.--14CSR is a transcriptional regulator. An example
of an amino acid sequence of ORF2.sub.--14CSR is set forth in SEQ
ID NO: 215. TABLE-US-00263 SEQ ID NO: 215
MLNKYIEKRITDKITILNILLDIRSIELDELSTLTSLQSKSLLSILQELQ
ETFEEELTFNLDTQQVQLIEHHSHQTNYYFHQLYNQSTILKTLRFFLLQG
NQSFNEFTQKEYISIATGYRVRQKCGLLLRSVGLDLVKNQVVGPEYRIRF
LIALLQFHFGIEIYDLNDGSMDWVTHMIVQSNSQLSHELLEITPDEYVHF
SILVALTWKRREFPLEFPESKEFEKLKNLFMYPILMEHCQTYLEPHANMT
FTQEELDYIFLVYCSANSSFSKDKWNQEKKTHTTQLILQHTRGKHLLSKF
KNILGNDISNSLSFLTALTFLTRTFLFGLQNLVPYYNYYEHYGIESDKPL
YHTSKATVQEWMTEQKIEGVTDQHRLYLFSLYLTETIFSSLPAIPIFIIL
NNQADVNLIKSIILRNFTDKVASVTGYNILISPPPSEEHLTEPLIIITTK
EYLPYVKKQYPKGKHHFLTIALDLHVSQQRLIYQTIVDIRKEAFDKRVAM IAKKAHYLL
[1225] ORF3.sub.--14CSR is a cell wall surface protein. An example
of an amino acid sequence of ORF3.sub.--14CSR is set forth in SEQ
ID NO: 216. TABLE-US-00264 SEQ ID NO: 216
MKKVRKIFQKAVAGLCCISQLTAFSSIVALAETPETSPAIGKVVIKETGE
GGALLGDAVFELKNNTDGTTVSQRTEAQTGEAIFSNIKPGTYTLTEAQPP
VGYKPSTKQWTVEVEKNGRTTVQGEQVENREEALSDQYPQTGTYPDVQTP
YQIIKVDGSEKNGQHKALNPNPYERVIPEGTLSKRIYQVNNLDDNQYGIE
LTVSGKTTVETKEASTPLDVVTLLDNSNSMSNIRHNHAHRAEKAGEATRA
LVDKITSNPDNRVALVTYGSTIFDGSEATVEKGVADANGKILNDSALWTF
DRTTFTAKTYNYSFLNLTSDPTDIQTIKDRIPSDAEELNKDKLMYQFGAT
FTQKALMTADDILTKQARPNSKKVIFHITDGVPTMSYPINFKYTGTTQSY
RTQLNNFKAKTPNSSGILLEDFVTWSADGEHKIVRGDGESYQMFTKKPVT
DQYGVHQILSITSMEQRAKLVSAGYRFYGTDLYLYWRDSILAYPFNSSTD
WITNHGDPTTWYYNGNMAQDGYDVFTVGVGVNGDPGTDEARARTFMQSIS
SSPDNYTNVADPSQILQELNRYFYTIVNEKKSIENGTITDPMGELIDFQL
GADGRFDPADYTLTANDGSSLVNNVPTGGPQNDGGLLKNAKVFYDTTEKR
IRVTGLYLGTGEKVTLTYNVRLNDQFVSNKFYDTNGRTTLHPKEVEKNTV
RDFPIPKIRDVRKYPEITIPKEKKLGEIEFIKINKNDKKPLRDAVPSLQK
QHPDYPDIYGAIDQNGTYQNVRTGEDGKLTEKNLSDGKYRLFENSEPAGY
KPVQNKPIVAFQIVNGEVRDVTSIVPQDIPAGYEFTNDKHYITNEPTPPK
REYPRTGGIGMLPFYLTGCMMMGGVLLYTRKHP
[1226] ORF4.sub.--14CSR is a cell wall surface protein. An example
of an amino acid sequence of ORF4.sub.--14CSR is set forth in SEQ
ID NO: 217. TABLE-US-00265 SEQ ID NO: 217
MKSINKFLTMLAALLLTASSLFSAATVFAADNVSTAPDAVTKTLTIHKLL
LSEDDLKTWDTNGPKGYDGTQSSLKDLTGVVAEEIPNVYFELQKYNLTDG
KEKENLKDDSKWTTVHGGLTTKDGLKIETSTLKGVYRIREDRTKTTYVGP
NGQVLTGSKAVPALVTLPLVNNNGTVIDAHVFPKNSYNKPVVDKRTADTL
NYNDQNGLSIGTKIPYVVNTTIPSNATFATSFWSDEMTEGLTYNEDVTIT
LNNVAMDQADYEVTKGNNGFNLKLTEAGLAKINGKDADQKIQITYSATLN
SLAVADIPESNDITYHYGNHQDHGNTPKPTKPNNGQITVTKTWDSQPAPE
GVKATVQLVNAKTGEKVGAPVELSENNWTYTWSGLDNSIEYKVEEEYNGY
SAEYTVESKGKLGVKNWKDNNPAPINPEEPRVKTYGKKFVKVDQKDTRLE
NAQFVVKKADSNKYIAFKSTAQQAADEKAAATAKQKLDAAVAAYTNAADK
QAAQALVDQAQQEYNVAYKEAKFGYVEVAGKDEAMVLTSNTDGQFQISGL
AAGTYKLEEIKAPEGFAKIDDVEFVVGAGSWNQGEFNYLKDVQKNDATKV
VNKKITIPQTGGIGTIIFAVAGAAIMGIAVYAYVKNNKDEDQLA
[1227] ORF5.sub.--14CSR is a cell wall surface protein. An example
of an amino acid sequence of ORF5.sub.--14CSR is set forth in SEQ
ID NO: 218. TABLE-US-00266 SEQ ID NO: 218
MTMQKMQKMISRIFFVMALCFSLVWGAHAVQAQEDHTLVLQLENYQEVVS
QLPSRDGHRLQVWKLDDSYSYDDRVQIVRDLHSWDENKLSSFKKTSFEMT
FLENQIEVSHIPNGLYYVRSIIQTDAVSYPAEFLFEMTDQTVEPLVIVAK
KTDTMTTKVKLIKVDQDHNRLEGVGFKLVSVARDGSEKEVPLIGEYRYSS
SGQVGRTLYTDKNGETFVTNLPLGNYRFKEVEPLAGYAVTTLDTDVQLVD
HQLVTITVVNQKLPRGNVDFMKVDGRTNTSLQGAMFKVMKEESGHYTPVL
QNGKEVVVTSGKDGRFRVEGLEYGTYYLWELQAPTGYVQLTSPVSFTIGK
DTRKELVTVVKNNKRPRIDVPDTGEETLYILMLVAILLFGSGYYLTKKPN N
[1228] ORF6.sub.--14CSR is a putative sortase. An example of an
amino acid sequence of ORF6.sub.--14CSR is set forth in SEQ ID NO:
219. TABLE-US-00267 SEQ ID NO: 219
MLIKMVKTKKQKRNNLLLGVVFFIGMAVMAYPLVSRLYYRVESNQQIADF
DKEKATLDEADIDERMKLAQAFNDSLNNVVSGDPWSEEMKKKGRAEYARM
LEIHERMGHVEIPVIDVDLPVYAGTAEEVLQQGAGHLEGTSLPIGGNSTH
AVITAHTGLPTAKMFTDLTKLKVGDKFYVHNIKEVMAYQVDQVKVIEPTN
FDDLLIVPGHDYVTLLTCTPYMINTHRLLVRGHRIPYVAEVEEEFIAANK
LSHLYRYLFYVAVGLIVILLWIIRRLRKKKKQPEKALKALKAARKEVKVE DGQQ
[1229] ORF7.sub.--14CSR is a putative sortase. An example of an
amino acid sequence of ORF7.sub.--14CSR is set forth in SEQ ID NO:
220. TABLE-US-00268 SEQ ID NO: 220
MDNSRRSRKKGTKKKKHPLILLLIFLVGFAVAIYPLVSRYYYRIESNEVI
KEFDETVSQMDKAELEERWRLAQAFNATLKPSEILDPFTEQEKKKGVSEY
ANMLKVHERIGYVEIPAIDQEIPMYVGTSEEILQKGAGLLEGASLPVGGE
NTHTVVTAHRGLPTAELFSQLDKMKKGDVFYLHVLDQVLAYQVDQILTVE
PNDFEPVLIQHGEDYATLLTCTPYMINSHRLLVRGKRIPYTAPIAERNRA
VRERGQEWLWLLLAALVMILVLSYGVYRHRRIVKGLEKQLEEHHVKG
[1230] ORF8.sub.--14CSR is a putative sortase. An example of an
amino acid sequence of ORF8.sub.--14CSR is set forth in SEQ ID NO:
221. TABLE-US-00269 SEQ ID NO: 221
MSKAKLQKLLGYLLMLVALVIPVYCFGQMVLQSLGQVKGHEIFSESVTAD
SYQEQLQRSLDYNQRLDSQNRIVDPFLAEGYEVNYQVSDDPDAVYGYLSI
PSLEIMEPVYLGADYHHLAMGLAHVDGTPLPVEGKGIRSVIAGHPAEPSH
VFFRHLDQLKVGDALYYDNGQEIVEYQMMDTEIILPSEWEKLESVSSKNI
MTLITCDPIPTPNKRLLVNFERVAVYQKSDPQTAAVARVAFTKEGQSVSR
VATSQWLYRGLVVLAFLGTLFVLWKLARLLRGK
[1231] As discussed above, a S. pneumoniae AI sequence is present
in the 19F Taiwan 14 S. pneumoniae genome. Examples of S.
pneumoniae AI sequences from 19F Taiwan 14 are set forth below.
[1232] ORF2.sub.--19FTW is a transcriptional regulator. An example
of an amino acid sequence of ORF2.sub.--19FTW is set forth in SEQ
ID NO: 222. TABLE-US-00270 SEQ ID NO: 222
MLNKYIEKRITDKITILNILLDIRSTELDELSTLTSLQSKSLLSILQELQ
ETFEEELTFNLDTQQVQLIEHHSHQTNYYFHQLYNQSTILKTLRFFLLQG
NQSENEFTQKEYISIATGYRVRQKCGLLLRSVGLDLVKNQVVGPEYRIRF
LTALLQFHFGTEIYDLNDGSMDWVTHMIVQSNSQLSHELLEITPDEYVHE
STLVALTWKRREFPLEFPESKEFEKLKNLFMYPILMEHCQTYLEPHANMT
FTQEELDYIFLVYCSANSSFSKDKWNQEKKTHTIQLILQHTRGKHLLSKF
KNILGNDISNSLSFLTALTFLTRTFLFGLQNLVPYYNYYEHYGIESDKPL
YHISKAIVQEWMTEQKIEGVIDQHRLYLFSLYLTETIFSSLPAIPIFIIL
NNQADVNLIKSIILRNFTDKVASVTGYNILTSPPPSEEHLTEPLIIITTK
EYLPYVKKQYPKGKHHFLTIALDLHVSQQRLIYQTIVDIRKEAFDKRVAM IAKKAHYLL
[1233] ORF3.sub.--19FTW is a cell wall surface protein. An example
of an amino acid sequence of ORF3.sub.--19FTW is set forth in SEQ
ID NO: 223. TABLE-US-00271 SEQ ID NO: 223
MKKVRKIFQKAVAGLCCISQLTAFSSIVALAETPETSPAIGKVVIKETGE
GGALLGDAVFELKNNTDGTTVSQRTEAQTGEAIESNIKPGTYTLTEAQPP
VGYKPSTKQWTVEVEKNGRTTVQGEQVENREEALSDQYPQTGTYPDVQTP
YQIIKVDGSEKNGQHKALNPNPYERVTPEGTLSKRIYQVNNLDDNQYGIE
LTVSGKTVYERKDKSVPLDVVILLDNSNSMSNIRNKNARRAERAGEATRS
LIDKITSDPENRVALVTYASTIPDGTEFTVEKGVADKNGKRLNDSLFWNY
DQTSFTTNTKDYSYLKLTNDKNDIVELKNKVPTEAEDHDGNRLMYQFGAT
FTQKALMKADEILTQQARQNSQKVIFHITDGVPTMSYPINFNHATFAPSY
QNQLNAFFSKSPNKDGILLSDFITQATSGEHTIVRGDGQSYQMFTDKTVY
EKGAPAAFPVKPEKYSEMKAVGYAVIGDPINGGYIWLNWRESILAYPFNS
NTAKITNHGAPTRWYYNGNIAPDGYDVFTVGIGINGDPGTDEATATSFMQ
SISSKPENYTNVTDTTKILEQLNRYFHTIVTEKKSIENGTITDPMGELID
LQLGTDGRFDPADYTLTANDGSRLENGQAVGGPQNDGGLLKNAKVFYDTT
EKRIRVTGLYLGTGEKVTLTYNVRLNDQEVSNKFYDTNGRTTLHPKEVEK
NTVRDFPIPKIRDVRKYPAITIAKEKKLGEIEFIKINKNDKKPLRDAVFS
LQKQHPDYPDIYGAIDQNGTYQNVRTGEDGKLTFKNLSDGKYRLFENSEP
AGYKPVQNKPIVAFQIVNGEVRDVTSIVPQDIPAGYEFTNDKHYITNEPI
PPKREYPRTGGIGMLPFYLIGCMMMGGVLLYTRKHP
[1234] ORF4.sub.--19FTW is a cell wall surface protein. An example
of an amino acid sequence of ORF4.sub.--19FTW is set forth in SEQ
ID NO: 224. TABLE-US-00272 SEQ ID NO: 224
MKSINKFLTMLAALLLTASSLFSAATVFAAGTTTTSVTVHKLLATDGDMD
KIANELETGNYAGNKVGVLPANAKEIAGVMFVWTNTNNEIIDENGQTLGV
NIDPQTFKLSGAMPATAMKKLTEAEGAKFNTANLPAAKYKIYEIHSLSTY
VGEDGATLTGSKAVPIEIELPLNDVVDAHVYPKNTEAKPKIDKDFKGKAN
PDTPRVDKDTPVNHQVGDVVEYEIVTKIPALANYATANWSDRMTEGLAFN
KGTVKVTVDDVALEAGDYALTEVATGFDLKLTDAGLAKVNDQNAEKTVKI
TYSATLNDKAIVEVPESNDVTFNYGNNPDHGNTPKPNKPNENGDLTLTKT
WVDATGAPIPAGAEATFDLVNAQTGKVVQTVTLTTDKNTVTVNGLDKNTE
YKFVERSIKGYSADYQEITTAGEIAVKNWKDENPKPLDPTEPKVVTYGKK
FVKVNDKDNRLAGAEFVTANADNAGQYLARKADKVSQEEKQLVVTTKDAL
DRAVAAYNALTAQQQTQQEKEKVDKAQAAYNAAVIAANNAFEWVADKDNE
NVVKLVSDAQGRFEITGLLAGTYYLEETKQPAGYALLTSRQKFEVTATSY
SATGQGIEYTAGSGKDDATKVVNKKITIPQTGGIGTIIFAVAGAVIMGIA
VYAYVKNNKDEDQLA
[1235] ORF5.sub.--19FTW is a cell wall surface protein. An example
of an amino acid sequence of ORF5.sub.--19FTW is set forth in SEQ
ID NO: 225. TABLE-US-00273 SEQ ID NO: 225
MTMQKMQKMTSRIFFVMALCFSLVWGAHAVQAQEDHTLVLQLENYQEVVS
QLPSRDGHRLQVWKLDDSYSYDNRVQIVRDLHSWDENKLSSFKKTSFEMT
FLENQIEVSHIPNGLYYVRSIIQTDAVSYPAEFLFEMTDQTVEPLVIVAK
KADTVTTKVKLIKVDQDHNRLEGVGFKLVSVARDGSEKEVPLIGEYRYSS
SGQVGRTLYTDKNGEIVVTNLPLGTYRFKEVEPLAGYTVTTMDTDVQLVD
HQLVTITVVNQKLPRGNVDFMKVDGRTNTSLQGAMFKVMKEENGHYTPVL
QNGKEVVVASGKDGRFRVEGLEYGTYYLWELQAPTGYVQLTSPVSFTIGK
DTRKELVTVVKNNKRPRIDVPDTGEETLYILMLVAILLFGSGYYLTKKTN N
[1236] ORF6.sub.--19FTW is a putative sortase. An example of an
amino acid sequence of ORF6.sub.--19FTW is set forth in SEQ ID NO:
226. TABLE-US-00274 SEQ ID NO: 226
MLIKMAKTKKQKRNNLLLGVVFFIGMAVMAYPLVSRLYYRVESNQQIADF
DKEKATLDEADTDERMKLAQAFNDSLNNVVSGDPWSEEMKKKGRAEYARM
LEIHERMGHVEIPAIDVDLPVYAGTAEEVLQQGAGHLEGTSLPIGGNSTH
AVITAHTGLPTAKMFTDLTKLKVGDKFYVHNIKEVMAYQVDQVKVIEPTN
FDDLLIVPGHDYVTLLTCTPYMINTHRLLVRGHRIPYVAEVEEEFIAANK
LSHLYRYLFYVAVGLIVILLWIIRRLRKKKRQSERALKALKEATKEVKVE DE
[1237] ORF7.sub.--19FTW is a putative sortase. An example of an
amino acid sequence of ORF7.sub.--19FTW is set forth in SEQ ID NO:
227. TABLE-US-00275 SEQ ID NO: 227
MSKSRYSRKKSVKKKKNPFILLLIFLVGLAVAMYPLVSRYYYRIESNEVT
KEFDETVSQMDKAELEERWRLAQAFNATLKPSEILDPFTDQEKKQGVSEY
ANMLKVHERIGYVEIPAIEQEIPMYVGTSEDILQKGAGLLEGASLPVGGE
NTHTVITAHRGLPTAELFSQLDKMKKGDIFYLHVLDQVLAYQVDQIVTVE
PNDFEPVLIQHGQDYATLLTCTPYMLNSHRLLVRGKRTPYTAPIAERNRA
VRERGQFWLWLLLGAMAVILLLLYRVYRNRRTVKGLEKQLEGRHVKD
[1238] ORF8.sub.--19FTW is a putative sortase. An example of an
amino acid sequence of ORF8.sub.--19FTW is set forth in SEQ ID NO:
228. TABLE-US-00276 SEQ ID NO: 228
MSRTKLRALLGYLLMLVACLIPIYCFGQMVLQSLGQVKGHATFVKSMTTE
MYQEQQNHSLAYNQRLASQNRIVDPFLAEGYEVNYQVSDDPDAVYGYLSI
PSLEIMEPVYLGADYHHLGMGLAHVDGTPLPLDGTGIRSVIAGHRAEPSH
VFFRHLDQLKVGDALYYDNGQEIVEYQMMDTEIILPSEWEKLESVSSKNI
MTLITCDPIPTFNKRLLVNFERVAVYQKSDPQTAAVARVAFTKEGQSVSR
VATSQWLYRGLVVLAFLGILFVLWKLARLLRGK
[1239] As discussed above, a S. pneumoniae AI sequence is present
in the 23F Taiwan 15 S. pneumoniae genome. Examples of S.
pneumoniae AI sequences from 23F Taiwan 15 are set forth below.
[1240] ORF2.sub.--23FTW is a transcriptional regulator. An example
of an amino acid sequence of ORF2.sub.--23FTW is set forth in SEQ
ID NO: 229. TABLE-US-00277 SEQ ID NO: 229
MLNKYIEKRITDKITILNILLDIRSIELDELSTLTSLQSKSLLSILQELQ
ETFEEELTPNLDTQQVQLIEHHSHQTNYYFHQLYNQSTILKILRFFLLQG
NQSFNEFTQKEYISIATGYRVRQKCGLLLRSVGLDLVKNQVVGPEYRIRF
LIALLQFHFGIEIYDLNDGSMDWVTHMIVQSNSQLSHELLETTPDEYVHF
SILVALTWKRREFPLEFPESKEFEKLKNLFMYPILMEHCQTYLEPHANMT
FTQEELDYIFLVYCSANSSFSKDKWNQEKKTHTIQLILQHTRGKHLLSKF
KNILGNDISNSLSFLTALTFLTRTFLFGLQNLVPYYNYYEHYGIESDKPL
YHISKAIVQEWMTEQKIEGVIDQHRLYLFSLYLTETIFSSLPAIPIFIIL
NNQADVNLIKSIILRNFTDKVASVTGYNILISPPPSEEHLTEPLITITTK
EYLPYVKKQYPKGKHHFLTIALDLHVSQQRLIYQTIVDIRKEAFDKRVAM IAKKAHYLL
[1241] ORF3.sub.--23FTW is a cell wall surface protein. An example
of an amino acid sequence of ORF3.sub.--23FTW is set forth in SEQ
ID NO: 230. TABLE-US-00278 SEQ ID NO: 230
MKKVRKIFQKAVAGLCCISQLTAFSSIVALAETPETSPAIGKVVIKETGE
GGALLGDAVFELKNNTDGTTVSQRTEAQTGEAIFSNIKPGTYTLTEAQPP
VGYKPSTKQWTVEVEKNGRTTVQGEQVENREEALSDQYPQTGTYPDVQTP
YQIIKVDGSEKNGQHKALNPNPYERVIPEGTLSKRIYQVNNLDDNQYGIE
LTVSGKTVYEQKDKSVPLDVVILLDNSNSMSNIRNKNARRAERAGEATRS
LIDKITSDPENRVALVTYASTIFDGTEFTVEKGVADKNGKRLNDSLFWNY
DQTSFTTNTKDYSYLKLTNDKNDIVELKNKVPTFAEDHDGNRLMYQFGAT
FTQKALMKADEILTQQARQNSQKVIFHITDGVPTMSYPINFNHATFAPSY
QNQLNAETSKSPNKDGILLSDFITQATSGEHTIVRGDGQSYQMFTDKTVY
EKGAPAAFPVKPEKYSEMKAAGYAVIGDPINGGYIWLNWRESILAYPFNS
NTAKITNHGDPTRWYYNGNIAPDGYDVFTVGIGINGDPGTDEATATSFMQ
SISSKPENYTNVTDTTKILEQLNRYFHTIVTEKKSIENGTITDPMGELID
LWLGTDGRFDPADYTLTANDGSRLENGQAVGGPQNDGGLLKNAKVLYDTT
EKRIRVTGLYLGTKEKVTLTYNVRLNDEFVSNKFYDTNGRTTLHPKEVEQ
NTVRDFPIPKIRDVRKYPEITISKEKKLGDIEFIKVNKNDKKPLRDAVFS
LQKQHPDYPDIYGAIDQNGTYQNVRTGEDGKLTFKNLSDGKYRLFENSEP
AGYKPVQNKPIVAFQIVNGEVRDVTSIVPQDIPAGYEFTNDKHYITNEPI
PPKREYPRTGGIGMLPFYLIGCMMMGGVLLYTRKHP
[1242] ORF4.sub.--23FTW is a cell wall surface protein. An example
of an amino acid sequence of ORF4.sub.--23FTW is set forth in SEQ
ID NO: 231. TABLE-US-00279 SEQ ID NO: 231
MKSINKFLTILAALLLTVSSLFSAATVFAAEQKTKTLTVHKLLMTDQELD
AWNSDAITTAGYDGSQNFEQFKQLQGVPQGVTEISGVAFELQSYTGPQGK
EQENLTNDAVWTAVNKGVTTETGVKFDTEVLQGTYRLVEVRKESTYVGPN
GKVLTGMKAVPALITLPLVNQNGVVENAHVYPKNSEDKPTATKTFDTAAG
FVDPGEKGLAIGTKVPYIVTTTIPKNSTLATAPWSDEMTEGLDYNGDVVV
NYNGQPLDNSHYTLEAGHNGFILKLNEKGLEAINGKDAEATITLKYTATL
NALAVADVPEANDVTFHYGNNPGHGNTPKPNKPKNGELTITKTWADAKDA
PIAGVEVTFDLVNAQTGEVVKVPGHETGIVLNQTNNWTFTATGLDNNTEY
KFVERTIKGYSADYQTITETGKIAVKNWKDENPEPINPEEPRVKTYGKKF
VKVDQKDERLKEAQFVVKNEQGKYLALKSAAQQAVNEKAAAEAKQALDAA
IAAYTNAADKNAAQAVVDAAQKTYNDNYRAARFGYVEVERKEDALVLTSN
TDGQFQISGLAAGSYTLEETKAPEGFAKLGDVKFEVGAGSWNQGDFNYLK
DVQKNDATKVVNKKITIPQTGGIGTIIFAVAGAVIMGIAVYAYVKNNKDE DQLA
[1243] ORF5.sub.--23FTW is a cell wall surface protein. An example
of an amino acid sequence of ORF5.sub.--23FTW is set forth in SEQ
ID NO: 232. TABLE-US-00280 SEQ ID NO: 232
MTMQKMQKMISRIFFVMALCFSLVWGAHAVQAQEDHTLVLQLENYQEVVS
QLPSRDGHRLQVWKLDDSYSYDNRVQIVRDLHSWDENKLSSFKKTSEEMT
FLENQIEVSHIPNGLYYVRSIIQTDAVSYPAEFLFEMTDQTVEPLVIVAK
KADTVTTKVKLIKVDQDHNRLEGVGFKLVSVARDGSEKEVPLIGEYRYSS
SGQVGRTLYTDKNGEIVVTNLPLGTYRFKEVEPLAGYTVTTMDTDVQLVD
HQLVTITVVNQKLPRGNVDFMKVDGRTNTSLQGAMFKVMKEENGHYTPVL
QNGKEVVVASGKDGRFRVEGLEYGTYYLWELQAPTGYVQLTSPVSFTIGK
DTRKELVTVVKNNKRPRIDVPDTGEETLYILMLVAILLFGSGYYLTKKTN N
[1244] ORF6.sub.--23FTW is a putative sortase. An example of an
amino acid sequence of ORF6.sub.--23FTW is set forth in SEQ ID NO:
233. TABLE-US-00281 SEQ ID NO: 233
MLIKMVKTKKQKRNNLLLGVVFFIGMAVMAYPLVSRLYYRVESNQQIADF
DKEKATLDEADIDERMKLAQAFNDSLNNVVSGDPWSEEMKKKGRAEYARM
LEIHERMGHVEIPVIDVDLPVYAGTAEEVLQQGAGQLEGTSLPIGGNSTH
AVITAHTGLPTAKMFTDLTKLKVGDKFYVHNIKEVMAYQVDQVKVIEPTN
FDDLLIVPGHDYVTLLTCTPYMINTHRLLVRGHRIPYVAEVEEEFIAANK
LSHLYRYLFYVAVGLIVILLWIIRRLRKKKKQPEKALKALKAARKEVKVE DGQQ
[1245] ORF7.sub.--23FTW is a putative sortase. An example of an
amino acid sequence of ORF7.sub.--23FTW is set forth in SEQ ID NO:
234. TABLE-US-00282 SEQ ID NO: 234
MDNSRRSRKKGTKKKKHPLILLLIFLVGFAVAIYPLVSRYYYRIESNEVI
KEFDETVSQMDKAELEERWRLAQAFNATLKPSEILDPFThQEKKKGVSEY
ANMLKVHERIGYVEIPAIDQEIPMYVGTSEEILQKGAGLLEGASLPVGGE
NTHTVVTAHRGLPTAELFSQLDKMKKGDVFYLHVLDQVLAYQVDQILTVE
PNDFEPVLIQHGKDYATLLTCTPYMINSHRLLVRGKRIPYTAPIAERNRA
VRERGQFWLWLLLAALVMILVLSYGVYRHRRIVKGLEKQLEEHHVKG
[1246] ORF8.sub.--23FTW is a putative sortase. An example of an
amino acid sequence of ORF8.sub.--23FTW is set forth in SEQ ID NO:
235. TABLE-US-00283 SEQ ID NO: 235
MSKAKLQKLLGYLLMLVALVIPVYCFGQMVLQSLGQVKGHETFSESVTAD
SYQEQLQRSLDYNQRLDSQNRIVDPFLAEGYEVNYQVSDDPDAVYGYLSI
PSLEIMEPVYLGADYHHLAMGLAHVDGTPLPVEGKGTRSVIAGHPAEPSH
VFFRHLDQLKVGDALYYDNGQEIVEYQMMDTEIILPSEWEKLESVSSKNI
MTLITCDPIPTFNKRLLVNFERVAVYQKSDPQTAAVARVAFTKEGQSVSR
VATSQWLYRGLVVLAFLGILFVLWKLARLLRGK
[1247] As discussed above, a S. pneumoniae AI sequence is present
in the 23F Poland 16 S. pneumoniae genome. Examples of S.
pneumoniae AI sequences from 23F Poland 16 are set forth below.
[1248] ORF2.sub.--23FP is a transcriptional regulator. An example
of an amino acid sequence of ORF2.sub.--23FP is set forth in SEQ ID
NO: 236. TABLE-US-00284 +TR, SEQ ID NO: 236
MLNKYIEKRITDKITILNILLDIRSIELDELSTLTSLQSKSLLSILQELQ
ETFEEELTFNLDTQQVQLIEHHSHQTNYYFHQLYNQSTILKILRFFLLQG
NQSFNEFTQKEYISIATGYRVRQKCGLLLRSVGLDLVKNQVVGPEYRIRF
LIALLQFHFGIEIYDLNDGSMDWVTHMIVQSNSQLSHELLEITPDEYVHF
SILVALTWKRREFPLEFPESKEFEKLKNLFMYPILMEHCQTYLEPHANMT
FTQEELDYIFLVYCSANSSFSKDKWNQEKKTHTIQLILQHTRGKHLLSKF
KNILGNDISNSLSFLTALTFLTRTFLFGLQNLVPYYNYYEHYGIESDKPL
YHISKATVQEWMTEQKIEGVIDQHRLYLFSLYLTETIFSSLPAIPIFIIL
NNQADVNLIKSIILRNFTDKVASVTGYNILISPPPSEEHLTEPLIIITTK
EYLPYVKKQYPKGKHHELTIALDLHVSQQRLIYQTIVDIRKEAFDKRVAN IAKKAHYLL
[1249] ORF3.sub.--23FP is a cell wall surface protein. An example
of an amino acid sequence of ORF3.sub.--23FP is set forth in SEQ ID
NO: 237. TABLE-US-00285 SEQ ID NO: 237
MKKVRKIFQKAVAGLCCISQLTAFSSIVALAETPETSPAIGKVVIKETGE
GGALLGDAVFELKNNTDGTTVSQRTEAQTGEAIFSNIKPGTYTLTEAQPP
VGYKPSTKQWTVEVEKNGRTTVQGEQVENREEALSDQYPQTGTYPDVQTP
YQIIKVDGSEKNGQHKALNPNPYERVIPEGTLSKRIYQVNNLDDNQYGIE
LTVSGKTTVETKEASTPLDVVTLLDNSNSMSNIRHNHAHRAEKAGEATRA
LVDKITSNPDNRVALVTYGSTIFDGSEATVEKGVADANGKTLNDSALWTE
DRTTFTAKTYNYSFLNLTSDPTDIQTIKDRIPSDAEELNKDKLMYQFGAT
FTQKALMTADDILTKQARPNSKKVIFHITDGVPTMSYPINFKYTGTTQSY
RTQLNNFKAKTPNSSGILLEDFVTWSADGEHKIVRGDGESYQMFTKKPVT
DQYGVHQILSITSMEQRAKLVSAGYRFYGTDLYLYWRDSILAYPFNSSTD
WITNHGDPTTWYYNGNMAQDGYDVFTVGVGVNGDPGTDEATATRFMQSIS
SSPDNYTNVADPSQILQELNRYFYTIVNEKKSIENGTITDPMGELIDFQL
GADGRFDPADYTLTANDGSSLVNNVPTGGPQNDGGLLKNAKVFYDTTEKR
IRVTGLYLGTGEKVTLTYNVRLNDQFVSNKFYDTNGRTTLHPKEVEKNTV
RDFPIPKIRDVRKYPEITIPKEKKLGEIEFIKINKNDKKPLRDAVFSLQK
QHPDYPDIYGAIDQNGTYQNVRTGEDGKLTFKNLSDGKYRLFENSEPAGY
KPVQNKPIVAFQIVNGEVRDVTSIVPQDIPAGYEETNDKHYITNEPIPPK
REYPRTGGIGMLPFYLIGCMMMGGVLLYTRKNP
[1250] ORF4.sub.--23FP is a cell wall surface protein. An example
of an amino acid sequence of ORF4.sub.--23FP is set forth in SEQ ID
NO: 238. TABLE-US-00286 SEQ ID NO: 238
MKSINKFLTMLAALLLTASSLFSAATVFAADNVSTAPDAVTKTLTIHKLL
LSEDDLKTWDTNGPKGYDGTQSSLKDLTGVVAEEIPNVYFELQKYNLTDG
KEKENLKDDSKWTTVHGGLTTKDGLKIETSTLKGVYRIREDRTKTTYVGP
NGQVLTGSKAVPALVTLPLVNNNGTVIDAHVEPKNSYNKPVVDKRIADTL
NYNDQNGLSIGTKIPYVVNTTIPSNATFATSFWSDEMTEGLTYNEDVTIT
LNNVAMDQADYEVTKGINGFNLKLTEAGLAKINGKDADQKIQITYSATLN
SLAVADIPESNDITYHYGNHQDHGNTPKPTKPNNGQITVTKTWDSQPAPE
GVKATVQLVNAKTGEKVGAPVELSENNWTYTWSGLDNSIEYKVEEEYNGY
SAEYTVESKGKLGVKNWKDNNPAPINLEEPRVKTYGKKFVKVDQKDTRLE
NAQFVVKKADSNKYIAFKSTAQQAADEKAAATAKQKLDAAVAAYTNAADK
QAAQALVDQAQQEYNVAYKEAKFGYVEVAGKDEAMVLTSNTDGQFQISGL
AAGTYKLEEIKAPEGFAKIDDVEFVVGAGSWNQGEFNYLKDVQKNDATKV
VNKKITIPQTGGIGTIIFAVAGAVIMGIAVYAYVKNNKDEDQLA
[1251] ORF5.sub.--23FP is a cell wall surface protein. An example
of an amino acid sequence of ORF5.sub.--23FP is set forth in SEQ ID
NO: 239. TABLE-US-00287 SEQ ID NO: 239
MTMQKMQKMISRIFFVMALCFSLVWGAHAVQAQEDHTLVLQLENYQEVVS
QLPSRDGHRLQVWKLDDSYSYDNRVQIVRDLHSWDENKLSSFKKTSFEMT
FLSNQIEVSHIPNGLYYVRSIIQTDAVSYPAEFLFEMTDQTVEPLVIVAK
KADTVTTKVKLIKVDQDHNRLEGVGFKLVSVARDGSEKEVPLIGEYRYSS
SGQVGRTLYTDKNGEIVVTNLPLGTYRFKEVEPLAGYAVTTMDTDVQLVD
HQLVTITVVNQKLPRGNVDFMKVDGRTNTSLQGAMFKVMKEENGHYTPVL
QNGKEVVVASGKDGRFRVEGLEYGTYYLWELQAPTGYVQLTSPVSFTIGK
DTRKELVTVVKNNKRPRTDVPDTGEETLYILMLVAILLFGSGYYLTKKTN N
[1252] ORF6.sub.--23FP is a putative sortase. An example of an
amino acid sequence of ORF6.sub.--23FP is set forth in SEQ ID NO:
240. TABLE-US-00288 SEQ ID NO: 240
MLIKMAKTKKQKRNNLLLGVVFFIGIAVMAYPLVSRLYYRVESNQQIADF
DKEKATLDEADIDERMKLAQAFNDSLNNVVSGDPWSEEMKKKGRAEYARM
LEIHERMGHVETPAIDVDLPVYAGTAEEVLQQGAGHLEGTSLPIGGNSTH
AVITAHTGLPTAKMFTDLTKLKVGDKFYVHNIKEVMAYQVDQVKVIEPTN
FDDLLIVPGHDYVTLLTCTPYMINTHRLLVRGHRIPYVAEVEEEFIAANK
LSHLYRYLFYVAVGLIVILLWIIRRLRKKKRQSERALKALKEATKEVKVE DE
[1253] ORF7.sub.--23FP is a putative sortase. An example of an
amino acid sequence of ORF7.sub.--23FP is set forth in SEQ ID NO:
241. TABLE-US-00289 SEQ ID NO: 241
MSKSRYSRKKSVKKKKNPFILLLIFLVGLAVAMYPLVSRYYYRIESNEVI
KEFDETVSQMDKAELEERWRLAQAFNATLKPSEILDPFTEQEKKKGVSEY
ANMLKVHERIGYVEIPAIDQEIPMYVGTSEETLQKGAGLLEGASLPVGGE
NTHTVVTAHRGLPTAELFSQLDKMKKGDIFYLHVLDQVLAYQVDQIVTVE
PNDFEPVLIQHGEDYATLLTCTPYMINSHRLLVRGKRIPYTAPIAERNRA
VRERGQFWLWLLLGAMAVILLLLYRVYRNRRIVKGLEKQLEGRHVKD
[1254] ORF8.sub.--23FP is a putative sortase. An example of an
amino acid sequence of ORF8.sub.--23FP is set forth in SEQ ID NO:
242. TABLE-US-00290 SEQ ID NO: 242
MSKSRYSRKKSVKKKKNPFILLLIFLVGLAVAMYPLVSRYYYRIESNEVI
KEFDETVSQMDKAELEERWRLAQAFFLAEGYEVNYQVSDDPDAVYGYLSI
PSLEIMEPVYLGADYHHLGMGLAHVDGTPLPLDGTGIRSVIAGHPAEPSH
VEFRHLDQLKVGDALYYDNGQEIVEYQMMDTEIILPSEWEKLESVSSKNI
MTLITCDPIPTFNKRLLVNFERVAVYQKSDPQTAAVARVAFTKEGQSVSR
VATSQWLYRGLVVLAFLGILFVLWKLARLLRGK
[1255] Immunogenic compositions of the invention comprising AI
antigens may further comprise one or more antigenic agents.
Preferred antigens include those listed below. Additionally, the
compositions of the present invention may be used to treat or
prevent infections caused by any of the below-listed microbes.
Antigens for use in the immunogenic compositions include, but are
not limited to, one or more of the following set forth below, or
antigens derived from one or more of the following set forth
below:
[1256] Bacterial Antigens
[1257] N. meningitides: a protein antigen from N. meningitides
serogroup A, C, W135, Y, and/or B (1-7); an outer-membrane vesicle
(OMV) preparation from N. meningitides serogroup B. (8, 9, 10, 11);
a saccharide antigen, including LPS, from N. meningitides serogroup
A, B, C W135 and/or Y, such as the oligosaccharide from serogroup C
(see PCT/US99/09346; PCT IB98/01665; and PCT IB99/00103);
[1258] Streptococcus pneumoniae: a saccharide or protein antigen,
particularly a saccharide from Streptooccus pneumoniae;
[1259] Streptococcus agalactiae: particularly, Group B
streptococcus antigens;
[1260] Streptococcus pyogenes: particularly, Group A streptococcus
antigens;
[1261] Enterococcus faecalis or Enterococcus faecium: Particularly
a trisaccharide repeat or other Enterococcus derived antigens
provided in U.S. Pat. No. 6,756,361;
[1262] Helicobacter pylori. including: Cag, Vac, Nap, HopX, HopY
and/or urease antigen;
[1263] Bordetella pertussis: such as petussis holotoxin (PT) and
filamentous haemagglutinin (FHA) from B. pertussis, optionally also
combination with pertactin and/or agglutinogens 2 and 3
antigen;
[1264] Staphylococcus aureus: including S. aureus type 5 and 8
capsular polysaccharides optionally conjugated to nontoxic
recombinant Pseudomonas aeruginosa exotoxin A, such as
StaphVAX.TM., or antigens derived from surface proteins, invasins
(leukocidin, kinases, hyaluronidase), surface factors that inhibit
phagocytic engulfment (capsule, Protein A), carotenoids, catalase
production, Protein A, coagulase, clotting factor, and/or
membrane-damaging toxins (optionally detoxified) that lyse
eukaryotic cell membranes (hemolysins, leukotoxin, leukocidin);
[1265] Staphylococcus epidermis: particularly, S. epidermidis
slime-associated antigen (SAA);
[1266] Staphylococcus saprophyticus: (causing urinary tract
infections) particularly the 160 kDa hemagglutinin of S.
saprophyticus antigen;
[1267] Pseudomonas aeruginosa. particularly, endotoxin A, Wzz
protein, P. aeruginosa LPS, more particularly LPS isolated from
PAO1 (O5 serotype), and/or Outer Membrane Proteins, including Outer
Membrane Proteins F (OprF) (Infect Immun. 2001 May; 69(5):
3510-3515);
[1268] Bacillus anthracis (anthrax): such as B. anthracis antigens
(optionally detoxified) from A-components (lethal factor (LF) and
edema factor (EF)), both of which can share a common B-component
known as protective antigen (PA);
[1269] Moraxella catarrhalis. (respiratory) including outer
membrane protein antigens (HMW-OMP), C-antigen, and/or LPS;
[1270] Yersinia pestis (plague): such as F1 capsular antigen
(Infect Immun. 2003 January; 71(1)): 374-383, LPS (Infect Immun.
1999 October; 67(10): 5395), Yersinia pestis V antigen (Infect
Immun. 1997 November; 65(11): 4476-4482);
[1271] Yersinia enterocolitica (gastrointestinal pathogen):
particularly LPS (Infect Immun. 2002 August; 70(8): 4414);
[1272] Yersinia pseudotuberculosis: gastrointestinal pathogen
antigens;
[1273] Mycobacterium tuberculosis: such as lipoproteins, LPS, BCG
antigens, a fusion protein of antigen 85B (Ag85B) and/or ESAT-6
optionally formulated in cationic lipid vesicles (Infect Immun.
2004 October; 72(10): 6148), Mycobacterium tuberculosis (Mtb)
isocitrate dehydrogenase associated antigens (Proc Natl Acad Sci
USA. 2004 Aug. 24; 101(34): 12652), and/or MPT51 antigens (Infect
Immun. 2004 July; 72(7): 3829);
[1274] Legionella pneumophila (Legionnairs' Disease): L.
pneumophila antigens--optionally derived from cell lines with
disrupted asd genes (Infect Immun. 1998 May; 66(5): 1898);
[1275] Rickettsia: including outer membrane proteins, including the
outer membrane protein A and/or B (OmpB) (Biochim Biophys Acta.
2004 Nov. 1; 1702(2): 145), LPS, and surface protein antigen (SPA)
(J Autoimmun. 1989 June; 2 Suppl:81);
[1276] E. coli: including antigens from enterotoxigenic E. coli
(ETEC), enteroaggregative E. coli (EAggEC), diffusely adhering E.
coli (DAEC), enteropathogenic E. coli (EPEC), and/or
enterohemorrhagic E. coli (EHEC);
[1277] Vibrio cholerae: including proteinase antigens, LPS,
particularly lipopolysaccharides of Vibrio cholerae II, O1 Inaba
O-specific polysaccharides, V. cholera O139, antigens of IEM108
vaccine (Infect Immun. 2003 October; 71(10):5498-504), and/or
Zonula occludens toxin (Zot);
[1278] Salmonella typhi (typhoid fever): including capsular
polysaccharides preferably conjugates (Vi, i.e. vax-TyVi);
[1279] Salmonella typhimurium (gastroenteritis): antigens derived
therefrom are contemplated for microbial and cancer therapies,
including angiogenesis inhibition and modulation of flk;
[1280] Listeria monocytogenes (sytemic infections in
immunocompromised or elderly people, infections of fetus): antigens
derived from L. monocytogenes are preferably used as
carriers/vectors for intracytoplasmic delivery of
conjugates/associated compositions of the present invention;
[1281] Porphyromonas gingivalis: particularly, P. gingivalis outer
membrane protein (OMP);
[1282] Tetanus: such as tetanus toxoid (TT) antigens, preferably
used as a carrier protein in conjunction/conjugated with the
compositions of the present invention;
[1283] Diphtheria: such as a diphtheria toxoid, preferably
CRM.sub.197, additionally antigens capable of modulating,
inhibiting or associated with ADP ribosylation are contemplated for
combination/co-administration/conjugation with the compositions of
the present invention, the diphtheria toxoids are preferably used
as carrier proteins;
[1284] Borrelia burgdorferi (Lyme disease): such as antigens
associated with P39 and P13 (an integral membrane protein, Infect
Immun. 2001 May; 69(5): 3323-3334), VlsE Antigenic Variation
Protein (J Clin Microbiol. 1999 December; 37(12): 3997);
[1285] Haemophilis influenzae B: such as a saccharide antigen
therefrom;
[1286] Klebsiella: such as an OMP, including OMP A, or a
polysaccharide optionally conjugated to tetanus toxoid;
[1287] Neiserria gonorrhoeae: including, a Por (or porin) protein,
such as PorB (see Zhu et al., Vaccine (2004) 22:660-669), a
transferring binding protein, such as ThpA and TbpB (See Price et
al., Infection and Immunity (2004) 71(1):277-283), a opacity
protein (such as Opa), a reduction-modifiable protein (Rmp), and
outer membrane vesicle (OMV) preparations (see Plante et al., J
Infectious Disease (2000) 182:848-855), also see e.g. WO99/24578,
WO99/36544, WO99/57280, WO02/079243);
[1288] Chlamydia pneumoniae. particularly C. pneumoniae protein
antigens;
[1289] Chlamydia trachomatis: including antigens derived from
serotypes A, B, Ba and C are (agents of trachoma, a cause of
blindness), serotypes L.sub.1, L.sub.2 & L.sub.3 (associated
with Lymphogranuloma venereum), and serotypes, D-K;
[1290] Treponema pallidum (Syphilis): particularly a TmpA antigen;
and
[1291] Haemophilus ducreyi (causing chancroid): including outer
membrane protein (DsrA).
[1292] Where not specifically referenced, further bacterial
antigens of the invention may be capsular antigens, polysaccharide
antigens or protein antigens of any of the above. Further bacterial
antigens may also include an outer membrane vesicle (OMV)
preparation. Additionally, antigens include live, attenuated,
split, and/or purified versions of any of the aforementioned
bacteria. The bacterial or microbial derived antigens of the
present invention may be gram-negative or gram-positive and aerobic
or anaerobic.
[1293] Additionally, any of the above bacterial-derived saccharides
(polysaccharides, LPS, LOS or oligosaccharides) can be conjugated
to another agent or antigen, such as a carrier protein (for example
CRM.sub.197). Such conjugation may be direct conjugation effected
by reductive amination of carbonyl moieties on the saccharide to
amino groups on the protein, as provided in U.S. Pat. No. 5,360,897
and Can J Biochem Cell Biol. 1984 May;62(5):270-5. Alternatively,
the saccharides can be conjugated through a linker, such as, with
succinamide or other linkages provided in Bioconjugate Techniques,
1996 and CRC, Chemistry of Protein Conjugation and Cross-Linking,
1993.
[1294] Viral Antigens
[1295] Influenza: including whole viral particles (attenuated),
split, or subunit comprising hemagglutinin (HA) and/or
neuraminidase (NA) surface proteins, the influenza antigens may be
derived from chicken embryos or propogated on cell culture, and/or
the influenza antigens are derived from influenza type A, B, and/or
C, among others;
[1296] Respiratory syncytial virus (RSV): including the F protein
of the A2 strain of RSV (J Gen Virol. 2004 November; 85(Pt
11):3229) and/or G glycoprotein;
[1297] Parainfluenza virus (PIV): including PIV type 1, 2, and 3,
preferably containing hemagglutinin, neuraminidase and/or fusion
glycoproteins;
[1298] Poliovirus: including antigens from a family of
picornaviridae, preferably poliovirus antigens such as OPV or,
preferably IPV;
[1299] Measles: including split measles virus (MV) antigen
optionally combined with the Protollin and or antigens present in
MMR vaccine;
[1300] Mumps: including antigens present in MMR vaccine;
[1301] Rubella: including antigens present in MMR vaccine as well
as other antigens from Togaviridae, including dengue virus;
[1302] Rabies: such as lyophilized inactivated virus
(RabAvert.TM.);
[1303] Flaviviridae viruses: such as (and antigens derived
therefrom) yelow fever virus, Japanese encephalitis virus, dengue
virus (types 1, 2, 3, or 4), tick borne encephalitis virus, and
West Nile virus;
[1304] Caliciviridae; antigens therefrom;
[1305] HIV: including HIV-1 or HIV-2 strain antigens, such as gag
(p24gag and p55gag), env (gp160 and gp41), pol, tat, nef, rev vpu,
miniproteins, (preferably p55 gag and gp140v delete) and antigens
from the isolates HIV.sub.IIb, HIV.sub.SF2, HIV.sub.LAV,
HIV.sub.LA1, HUV.sub.MN, HIV-1.sub.CM235, HIV-1.sub.US4, HIV-2;
simian immunodeficiency virus (SIV) among others;
[1306] Rotavirus: including VP4, VP5, VP6, VP7, VP8 proteins
(Protein Expr Purif. 2004 December; 38(2):205) and/or NSP4;
[1307] Pestivirus: such as antigens from classical porcine fever
virus, bovine viral diarrhoea virus, and/or border disease
virus;
[1308] Parvovirus: such as parvovirus B19;
[1309] Coronavirus: including SARS virus antigens, particularly
spike protein or proteases therefrom, as well as antigens included
in WO 04/92360;
[1310] Hepatitis A virus: such as inactivated virus;
[1311] Hepatitis B virus: such as the surface and/or core antigens
(sAg), as well as the presurface sequences, pre-S1 and pre-S2
(formerly called pre-S), as well as combinations of the above, such
as sAg/pre-S1, sAg/pre-S2, sAg/pre-S1/pre-S2, and pre-S1/pre-S2,
(see, e.g., AHBV Vaccines--Human Vaccines and Vaccination, pp.
159-176; and U.S. Pat. Nos. 4,722,840, 5,098,704, 5,324,513; Beames
et al., J. Virol. (1995) 69:6833-6838, Birnbaum et al., J. Virol.
(1990) 64:3319-3330; and Zhou et al., J. Virol. (1991)
65:5457-5464);
[1312] Hepatitis C virus: such as E1, E2, E1/E2 (see, Houghton et
al., Hepatology (1991) 14:381), NS345 polyprotein, NS 345-core
polyprotein, core, and/or peptides from the nonstructural regions
(International Publication Nos. WO 89/04669; WO 90/11089; and WO
90/14436);
[1313] Delta hepatitis virus (HDV): antigens derived therefrom,
particularly .delta.-antigen from HDV (see, e.g., U.S. Pat. No.
5,378,814);
[1314] Hepatitis E virus (HEV); antigens derived therefrom;
[1315] Hepatitis G virus (HGV), antigens derived therefrom;
[1316] Varcicella zoster virus: antigens derived from varicella
zoster virus (VZV) (J. Gen. Virol. (1986) 67:1759);
[1317] Epstein-Barr virus: antigens derived from EBV (Baer et al.,
Nature (1984) 310:207);
[1318] Cytomegalovirus: CMV antigens, including gB and gH
(Cytomegaloviruses (J. K. McDougall, ed., Springer-Verlag 1990) pp.
125-169);
[1319] Herpes simplex virus: including antigens from HSV-1 or HSV-2
strains and glycoproteins gB, gD and gH (McGeoch et al., J. Gen.
Virol. (1988)69:1531 and U.S. Pat. No. 5,171,568);
[1320] Human Herpes Virus: antigens derived from other human
herpesviruses such as HHV6 and HHV7; and
[1321] HPV: including antigens associated with or derived from
human papillomavirus (HPV), for example, one or more of E1-E7, L1,
L2, and fusions thereof, particularly the compositions of the
invention may include a virus-like particle (VLP) comprising the L1
major capsid protein, more particular still, the HPV antigens are
protective against one or more of HPV serotypes 6, 11, 16 and/or
18.
[1322] Further provided are antigens, compostions, methods, and
microbes included in Vaccines, 4.sup.th Edition (Plotkin and
Orenstein ed. 2004); Medical Microbiology 4.sup.th Edition (Murray
et al. ed. 2002); Virology, 3rd Edition (W. K. Joklik ed. 1988);
Fundamental Virology, 2nd Edition (B. N. Fields and D. M. Knipe,
eds. 1991), which are contemplated in conjunction with the
compositions of the present invention.
[1323] Additionally, antigens include live, attenuated, split,
and/or purified versions of any of the aforementioned viruses.
[1324] Fungal Antigens
[1325] Fungal antigens for use herein, associated with vaccines
include those described in: U.S. Pat. Nos. 4,229,434 and 4,368,191
for prophylaxis and treatment of trichopytosis caused by
Trichophyton mentagrophytes; U.S. Pat. Nos. 5,277,904 and 5,284,652
for a broad spectrum dermatophyte vaccine for the prophylaxis of
dermatophyte infection in animals, such as guinea pigs, cats,
rabbits, horses and lambs, these antigens comprises a suspension of
killed T. equinum, T. mentagrophytes (var. granulare), M. canis
and/or M gypseum in an effective amount optionally combined with an
adjuvant; U.S. Pat. Nos. 5,453,273 and 6,132,733 for a ringworm
vaccine comprising an effective amount of a homogenized,
formaldehyde-killed fungi, i.e., Microsporum canis culture in a
carrier; U.S. Pat. No. 5,948,413 involving extracellular and
intracellular proteins for pythiosis. Additional antigens
identified within antifungal vaccines include Ringvac bovis LTF-130
and Bioveta.
[1326] Further, fungal antigens for use herein may be derived from
Dermatophytres, including: Epidermophyton floccusum, Microsporum
audouini, Microsporum canis, Microsporum distortum, Microsporum
equinum, Microsporum gypsum, Microsporum nanum, Trichophyton
concentricum, Trichophyton equinum, Trichophyton gallinae,
Trichophyton gypseum, Trichophyton megnini, Trichophyton
mentagrophytes, Trichophyton quinckeanum, Trichophyton rubrum,
Trichophyton schoenleini, Trichophyton tonsurans, Trichophyton
verrucosum, T. verrucosum var. album, var. discoides, var.
ochraceum, Trichophyton violaceum, and/or Trichophyton
faviforme.
[1327] Fungal pathogens for use as antigens or in derivation of
antigens in conjunction with the compositions of the present
invention comprise Aspergillus fumigatus, Aspergillus flavus,
Aspergillus niger, Aspergillus nidulans, Aspergillus terreus,
Aspergillus sydowi, Aspergillus flavatus, Aspergillus glaucus,
Blastoschizomyces capitatus, Candida albicans, Candida enolase,
Candida tropicalis, Candida glabrata, Candida krusei, Candida
parapsilosis, Candida stellatoidea, Candida kusei, Candida
parakwsei, Candida lusitaniae, Candida pseudotropicalis, Candida
guilliermondi, Cladosporium carrionii, Coccidioides immitis,
Blastomyces dermatidis, Cryptococcus neoformans, Geotrichum
clavatum, Histoplasma capsulatum, Klebsiella pneumoniae,
Paracoccidioides brasiliensis, Pneumocystis carinii, Pythiumn
insidiosum, Pityrosporum ovale, Sacharomyces cerevisae,
Saccharomyces boulardii, Saccharomyces pombe, Scedosporium
apiosperum, Sporothrix schenckii, Trichosporon beigelii, Toxoplasma
gondii, Penicillium marneffei, Malassezia spp., Fonsecaea spp.,
Wangiella spp., Sporothrix spp., Basidiobolus spp., Conidiobolus
spp., Rhizopus spp, Mucor spp, Absidia spp, Mortierella spp,
Cunninghamella spp, and Saksenaea spp.
[1328] Other fungi from which antigens are derived include
Alternaria spp, Curvularia spp, Helminthosporium spp, Fusarium spp,
Aspergillus spp, Penicillium spp, Monolinia spp, Rhizoctonia spp,
Paecilomyces spp, Pithomyces spp, and Cladosporium spp.
[1329] Processes for producing a fungal antigens are well known in
the art (see U.S. Pat. No. 6,333,164). In a preferred method a
solubilized fraction extracted and separated from an insoluble
fraction obtainable from fungal cells of which cell wall has been
substantially removed or at least partially removed, characterized
in that the process comprises the steps of: obtaining living fungal
cells; obtaining fungal cells of which cell wall has been
substantially removed or at least partially removed; bursting the
fungal cells of which cell wall has been substantially removed or
at least partially removed; obtaining an insoluble fraction; and
extracting and separating a solubilized fraction from the insoluble
fraction.
[1330] STD Antigens
[1331] In particular embodiments, microbes (bacteria, viruses
and/or fungi) against which the present compositions and methods
can be implement include those that cause sexually transmitted
diseases (STDs) and/or those that display on their surface an
antigen that can be the target or antigen composition of the
invention. In a preferred embodiment of the invention, compositions
are combined with antigens derived from a viral or bacterial STD.
Antigens derived from bacteria or viruses can be administered in
conjunction with the compositions of the present invention to
provide protection against at least one of the following STDs,
among others: chlamydia, genital herpes, hepatitis (particularly
HCV), genital warts, gonorrhoea, syphilis and/or chancroid (See,
WO00/15255).
[1332] In another embodiment the compositions of the present
invention are co-administered with an antigen for the prevention or
treatment of an STD.
[1333] Antigens derived from the following viruses associated with
STDs, which are described in greater detail above, are preferred
for co-administration with the compositions of the present
invention: hepatitis (particularly HCV), HPV, HIV, or HSV.
[1334] Additionally, antigens derived from the following bacteria
associated with STDs, which are described in greater detail above,
are preferred for co-administration with the compositions of the
present invention: Neiserria gonorrhoeae, Chlamydia pneumoniae,
Chlamydia trachomatis, Treponema pallidum, or Haemophilus
ducreyi.
[1335] Respiratory Antigens
[1336] The antigen may be a respiratory antigen and could further
be used in an immunogenic composition for methods of preventing
and/or treating infection by a respiratory pathogen, including a
virus, bacteria, or fungi such as respiratory syncytial virus
(RSV), PIV, SARS virus, influenza, Bacillus anthracis, particularly
by reducing or preventing infection and/or one or more symptoms of
respiratory virus infection. A composition comprising an antigen
described herein, such as one derived from a respiratory virus,
bacteria or fungus is administered in conjunction with the
compositions of the present invention to an individual which is at
risk of being exposed to that particular respiratory microbe, has
been exposed to a respiratory microbe or is infected with a
respiratory virus, bacteria or fungus. The composition(s) of the
present invention is/are preferably co-administered at the same
time or in the same formulation with an antigen of the respiratory
pathogen. Administration of the composition results in reduced
incidence and/or severity of one or more symptoms of respiratory
infection.
[1337] Pediatric/Geriatric Antigens
[1338] In one embodiment the compositions of the present invention
are used in conjunction with an antigen for treatment of a
pediatric population, as in a pediatric antigen. In a more
particular embodiment the pediatric population is less than about 3
years old, or less than about 2 years, or less than about 1 years
old. In another embodiment the pediatric antigen (in conjunction
with the composition of the present invention) is administered
multiple times over at least 1, 2, or 3 years.
[1339] In another embodiment the compositions of the present
invention are used in conjunction with an antigen for treatment of
a geriatric population, as in a geriatric antigen.
[1340] Other Antigens
[1341] Other antigens for use in conjunction with the compositions
of the present include hospital acquired (nosocomial) associated
antigens.
[1342] In another embodiment, parasitic antigens are contemplated
in conjunction with the compositions of the present invention.
Examples of parasitic antigens include those derived from organisms
causing malaria and/or Lyme disease.
[1343] In another embodiment, the antigens in conjunction with the
compositions of the present invention are associated with or
effective against a mosquito bom illness. In another embodiment,
the antigens in conjunction with the compositions of the present
invention are associated with or effective against encephalitis. In
another embodiment the antigens in conjunction with the
compositions of the present invention are associated with or
effective against an infection of the nervous system.
[1344] In another embodiment, the antigens in conjunction with the
compositions of the present invention are antigens transmissible
through blood or body fluids.
[1345] Antigen Formulations
[1346] In other aspects of the invention, methods of producing
microparticles having adsorbed antigens are provided. The methods
comprise: (a) providing an emulsion by dispersing a mixture
comprising (i) water, (ii) a detergent, (iii) an organic solvent,
and (iv) a biodegradable polymer selected from the group consisting
of a poly(.alpha.-hydroxy acid), a polyhydroxy butyric acid, a
polycaprolactone, a polyorthoester, a polyanhydride, and a
polycyanoacrylate. The polymer is typically present in the mixture
at a concentration of about 1% to about 30% relative to the organic
solvent, while the detergent is typically present in the mixture at
a weight-to-weight detergent-to-polymer ratio of from about
0.00001:1 to about 0.1:1 (more typically about 0.0001:1 to about
0.1:1, about 0.001:1 to about 0.1:1, or about 0.005:1 to about
0.1:1); (b) removing the organic solvent from the emulsion; and (c)
adsorbing an antigen on the surface of the microparticles. In
certain embodiments, the biodegradable polymer is present at a
concentration of about 3% to about 10% relative to the organic
solvent.
[1347] Microparticles for use herein will be formed from materials
that are sterilizable, non-toxic and biodegradable. Such materials
include, without limitation, poly(.alpha.-hydroxy acid),
polyhydroxybutyric acid, polycaprolactone, polyorthoester,
polyanhydride, PACA, and polycyanoacrylate. Preferably,
microparticles for use with the present invention are derived from
a poly(.alpha.-hydroxy acid), in particular, from a poly(lactide)
("PLA") or a copolymer of D,L-lactide and glycolide or glycolic
acid, such as a poly(D,L-lactide-co-glycolide) ("PLG" or "PLGA"),
or a copolymer of D,L-lactide and caprolactone. The microparticles
may be derived from any of various polymeric starting materials
which have a variety of molecular weights and, in the case of the
copolymers such as PLG, a variety of lactide:glycolide ratios, the
selection of which will be largely a matter of choice, depending in
part on the coadministered macromolecule. These parameters are
discussed more fully below.
[1348] Further antigens may also include an outer membrane vesicle
(OMV) preparation.
[1349] Additional formulation methods and antigens (especially
tumor antigens) are provided in U.S. patent Ser. No.
09/581,772.
[1350] Antigen References
[1351] The following references include antigens useful in
conjunction with the compositions of the present invention: [1352]
1 International patent application WO99/24578 [1353] 2
International patent application WO99/36544. [1354] 3 International
patent application WO99/57280. [1355] 4 International patent
application WO00/22430. [1356] 5 Tettelin et al. (2000) Science
287:1809-1815. [1357] 6 International patent application
WO96/29412. [1358] 7 Pizza et al. (2000) Science 287:1816-1820.
[1359] 8 PCT WO 01/52885. [1360] 9 Bjune et al. (1991) Lancet
338(8775). [1361] 10 Fuskasawa et al. (1999) Vaccine 17:2951-2958.
[1362] 11 Rosenqist et al. (1998) Dev. Biol. Strand 92:323-333.
[1363] 12 Constantino et al. (1992) Vaccine 10:691-698. [1364] 13
Constantino et al. (1999) Vaccine 17:1251-1263. [1365] 14 Watson
(2000) Pediatr Infect Dis J 19:331-332. [1366] 15 Rubin (20000)
Pediatr Clin North Am 47:269-285, v. [1367] 16 Jedrzejas (2001)
Microbiol Mol Biol Rev 65:187-207. [1368] 17 International patent
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[1416] The contents of all of the above cited patents, patent
applications and journal articles are incorporated by reference as
if set forth fully herein.
[1417] There may be an upper limit to the number of Gram positive
bacterial proteins which will be in the compositions of the
invention. Preferably, the number of Gram positive bacterial
proteins in a composition of the invention is less than 20, less
than 19, less than 18, less than 17, less than 16, less than 15,
less than 14, less than 13, less than 12, less than 11, less than
10, less than 9, less than 8, less than 7, less than 6, less than
5, less than 4, or less than 3. Still more preferably, the number
of Gram positive bacterial proteins in a composition of the
invention is less than 6, less than 5, or less than 4. Still more
preferably, the number of Gram positive bacterial proteins in a
composition of the invention is 3.
[1418] The Gram positive bacterial proteins and polynucleotides
used in the invention are preferably isolated, i.e., separate and
discrete, from the whole organism with which the molecule is found
in nature or, when the polynucleotide or polypeptide is not found
in nature, is sufficiently free of other biological macromolecules
so that the polynucleotide or polypeptide can be used for its
intended purpose.
Fusion Proteins: GBS AI Sequences
[1419] The GBS AI proteins used in the invention may be present in
the composition as individual separate polypeptides, but it is
preferred that at least two (i.e. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, or 18) of the antigens are expressed as a
single polypeptide chain (a "hybrid" or "fusion" polypeptide). Such
fusion polypeptides offer two principal advantages: first, a
polypeptide that may be unstable or poorly expressed on its own can
be assisted by adding a suitable fusion partner that overcomes the
problem; second, commercial manufacture is simplified as only one
expression and purification need be employed in order to produce
two polypeptides which are both antigenically useful.
[1420] The fusion polypeptide may comprise one or more AI
polypeptide sequences. Preferably, the fusion comprises an AI
surface protein sequence. Preferably, the fusion polypeptide
includes one or more of GBS 80, GBS 104, and GBS 67. Most
preferably, the fusion peptide includes a polypeptide sequence from
GBS 80. Accordingly, the invention includes a fusion peptide
comprising a first amino acid sequence and a second amino acid
sequence, wherein said first and second amino acid sequences are
selected from a GBS AI surface protein or a fragment thereof.
Preferably, the first and second amino acid sequences in the fusion
polypeptide comprise different epitopes.
[1421] Hybrids (or fusions) consisting of amino acid sequences from
two, three, four, five, six, seven, eight, nine, or ten GBS
antigens are preferred. In particular, hybrids consisting of amino
acid sequences from two, three, four, or five GBS antigens are
preferred.
[1422] Different hybrid polypeptides may be mixed together in a
single formulation. Within such combinations, a GBS antigen may be
present in more than one hybrid polypeptide and/or as a non-hybrid
polypeptide. It is preferred, however, that an antigen is present
either as a hybrid or as a non-hybrid, but not as both.
[1423] Hybrid polypeptides can be represented by the formula
NH.sub.2-A-{-X-L-}.sub.n-B--COOH, wherein: X is an amino acid
sequence of a GBS AI protein or a fragment thereof; L is an
optional linker amino acid sequence; A is an optional N-terminal
amino acid sequence; B is an optional C-terminal amino acid
sequence; and n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or
15.
[1424] If a --X-- moiety has a leader peptide sequence in its
wild-type form, this may be included or omitted in the hybrid
protein. In some embodiments, the leader peptides will be deleted
except for that of the --X-- moiety located at the N-terminus of
the hybrid protein i.e. the leader peptide of X.sub.1 will be
retained, but the leader peptides of X.sub.2 . . . X.sub.n will be
omitted. This is equivalent to deleting all leader peptides and
using the leader peptide of X.sub.1 as moiety -A-.
[1425] For each n instances of {-X-L-}, linker amino acid sequence
-L- may be present or absent. For instance, when n=2 the hybrid may
be NH.sub.2--X.sub.1-L.sub.1-X.sub.2-L.sub.2-COOH,
NH.sub.2--X.sub.1--X.sub.2--COOH,
NH.sub.2--X.sub.1-L.sub.1-X.sub.2--COOH,
NH.sub.2--X.sub.1--X.sub.2-L.sub.2-COOH, etc. Linker amino acid
sequence(s) -L- will typically be short (e.g. 20 or fewer amino
acids i.e. 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5,
4, 3, 2, 1). Examples comprise short peptide sequences which
facilitate cloning, poly-glycine linkers (i.e. comprising Gly.sub.n
where n=2, 3, 4, 5, 6, 7, 8, 9, 10 or more), and histidine tags
(i.e. His.sub.n where n=3, 4, 5, 6, 7, 8, 9, 10 or more). Other
suitable linker amino acid sequences will be apparent to those
skilled in the art. A useful linker is GSGGGG, with the Gly-Ser
dipeptide being formed from a BamHI restriction site, thus aiding
cloning and manipulation, and the (Gly).sub.4 tetrapeptide being a
typical poly-glycine linker.
[1426] -A- is an optional N-terminal amino acid sequence. This will
typically be short (e.g. 40 or fewer amino acids i.e. 39, 38, 37,
36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20,
19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1).
Examples include leader sequences to direct protein trafficking, or
short peptide sequences which facilitate cloning or purification
(e.g. histidine tags i.e. His.sub.n where n=3, 4, 5, 6, 7, 8, 9, 10
or more). Other suitable N-terminal amino acid sequences will be
apparent to those skilled in the art. If X.sub.1 lacks its own
N-terminus methionine, -A- is preferably an oligopeptide (e.g. with
1, 2, 3, 4, 5, 6, 7 or 8 amino acids) which provides a N-terminus
methionine.
[1427] --B-- is an optional C-terminal amino acid sequence. This
will typically be short (e.g. 40 or fewer amino acids i.e. 39, 38,
37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21,
20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2,
1). Examples include sequences to direct protein trafficking, short
peptide sequences which facilitate cloning or purification (e.g.
comprising histidine tags i.e. His.sub.n, where n=3, 4, 5, 6, 7, 8,
9, 10 or more), or sequences which enhance protein stability. Other
suitable C-terminal amino acid sequences will be apparent to those
skilled in the art.
[1428] Most preferably, n is 2 or 3.
Fusion Proteins: Gram Positive Bacteria AI Sequences
[1429] The Gram positive bacteria AI proteins used in the invention
may be present in the composition as individual separate
polypeptides, but it is preferred that at least two (i.e. 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18) of the
antigens are expressed as a single polypeptide chain (a "hybrid" or
"fusion" polypeptide). Such fusion polypeptides offer two principal
advantages: first, a polypeptide that may be unstable or poorly
expressed on its own can be assisted by adding a suitable fusion
partner that overcomes the problem; second, commercial manufacture
is simplified as only one expression and purification need be
employed in order to produce two polypeptides which are both
antigenically useful.
[1430] The fusion polypeptide may comprise one or more AI
polypeptide sequences. Preferably, the fusion comprises an AI
surface protein sequence. Accordingly, the invention includes a
fusion peptide comprising a first amino acid sequence and a second
amino acid sequence, wherein said first and second amino acid
sequences are selected from a Gram positive bacteria AI protein or
a fragment thereof. Preferably, the first and second amino acid
sequences in the fusion polypeptide comprise different
epitopes.
[1431] Hybrids (or fusions) consisting of amino acid sequences from
two, three, four, five, six, seven, eight, nine, or ten Gram
positive bacteria antigens are preferred. In particular, hybrids
consisting of amino acid sequences from two, three, four, or five
Gram positive bacteria antigens are preferred.
[1432] Different hybrid polypeptides may be mixed together in a
single formulation. Within such combinations, a Gram positive
bacteria AI sequence may be present in more than one hybrid
polypeptide and/or as a non-hybrid polypeptide. It is preferred,
however, that an antigen is present either as a hybrid or as a
non-hybrid, but not as both.
[1433] Hybrid polypeptides can be represented by the formula
NH.sub.2-A-{-X-L-}.sub.n-B--COOH, wherein: X is an amino acid
sequence of a Gram positive bacteria AI sequence or a fragment
thereof; L is an optional linker amino acid sequence; A is an
optional N-terminal amino acid sequence; B is an optional
C-terminal amino acid sequence; and n is 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14 or 15.
[1434] If a --X-- moiety has a leader peptide sequence in its
wild-type form, this may be included or omitted in the hybrid
protein. In some embodiments, the leader peptides will be deleted
except for that of the --X-- moiety located at the N-terminus of
the hybrid protein i.e. the leader peptide of X.sub.1 will be
retained, but the leader peptides of X.sub.2 . . . X.sub.n will be
omitted. This is equivalent to deleting all leader peptides and
using the leader peptide of X.sub.1 as moiety -A-.
[1435] For each n instances of {--X-L-}, linker amino acid sequence
-L- may be present or absent. For instance, when n=2 the hybrid may
be NH.sub.2--X.sub.1-L.sub.1-X.sub.2-L.sub.2-COOH,
NH.sub.2--X.sub.1--X.sub.2--COOH,
NH.sub.2--X.sub.1-L.sub.1-X.sub.2--COOH,
NH.sub.2--X.sub.1--X.sub.2-L.sub.2-COOH, etc. Linker amino acid
sequence(s) -L- will typically be short (e.g. 20 or fewer amino
acids i.e. 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5,
4, 3, 2, 1). Examples comprise short peptide sequences which
facilitate cloning, poly-glycine linkers (i.e. comprising
Gly.sub.n, where n=2, 3, 4, 5, 6, 7, 8, 9, 10 or more), and
histidine tags (i.e. His.sub.n where n=3, 4, 5, 6, 7, 8, 9, 10 or
more). Other suitable linker amino acid sequences will be apparent
to those skilled in the art. A useful linker is GSGGGG, with the
Gly-Ser dipeptide being formed from a BamHI restriction site, thus
aiding cloning and manipulation, and the (Gly).sub.4 tetrapeptide
being a typical poly-glycine linker.
[1436] -A- is an optional N-terminal amino acid sequence. This will
typically be short (e.g. 40 or fewer amino acids i.e. 39, 38, 37,
36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20,
19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1).
Examples include leader sequences to direct protein trafficking, or
short peptide sequences which facilitate cloning or purification
(e.g. histidine tags i.e. His, where n=3, 4, 5, 6, 7, 8, 9, 10 or
more). Other suitable N-terminal amino acid sequences will be
apparent to those skilled in the art. If X.sub.1 lacks its own
N-terminus methionine, -A- is preferably an oligopeptide (e.g. with
1, 2, 3, 4, 5, 6, 7 or 8 amino acids) which provides a N-terminus
methionine.
[1437] --B-- is an optional C-terminal amino acid sequence. This
will typically be short (e.g. 40 or fewer amino acids i.e. 39, 38,
37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21,
20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2,
1). Examples include sequences to direct protein trafficking, short
peptide sequences which facilitate cloning or purification (e.g.
comprising histidine tags i.e. His.sub.n, where n=3, 4, 5, 6, 7, 8,
9, 10 or more), or sequences which enhance protein stability. Other
suitable C-terminal amino acid sequences will be apparent to those
skilled in the art.
[1438] Most preferably, n is 2 or 3.
Antibodies: GBS AI Sequences
[1439] The GBS AI proteins of the invention may also be used to
prepare antibodies specific to the GBS AI proteins. The antibodies
are preferably specific to the an oligomeric or hyper-oligomeric
form of an AI protein. The invention also includes combinations of
antibodies specific to GBS AI proteins selected to provide
protection against an increased range of GBS serotypes and strain
isolates. For example, a combination may comprise a first and
second antibody, wherein said first antibody is specific to a first
GBS AI protein and said second antibody is specific to a second GBS
AI protein. Preferably, the nucleic acid sequence encoding said
first GBS AI protein is not present in a GBS genome comprising a
polynucleotide sequence encoding for said second GBS AI protein.
Preferably, the nucleic acid sequence encoding said first and
second GBS AI proteins are present in the genomes of multiple GBS
serotypes and strain isolates.
[1440] The GBS specific antibodies of the invention include one or
more biological moieties that, through chemical or physical means,
can bind to or associate with an epitope of a GBS polypeptide. The
antibodies of the invention include antibodies which specifically
bind to a GBS AI protein. The invention includes antibodies
obtained from both polyclonal and monoclonal preparations, as well
as the following: hybrid (chimeric) antibody molecules (see, for
example, Winter et al. (1991) Nature 349: 293-299; and U.S. Pat.
No. 4,816,567; F(ab').sub.2 and F(ab) fragments; F.sub.v molecules
(non-covalent heterodimers, see, for example, Inbar et al. (1972)
Proc Natl Acad Sci USA 69:2659-2662; and Ehrlich et al. (1980)
Biochem 19:4091-4096); single-chain Fv molecules (sFv) (see, for
example, Huston et al. (1988) Proc Natl Acad Sci USA 85:5897-5883);
dimeric and trimeric antibody fragment constructs; minibodies (see,
e.g., Pack et al. (1992) Biochem 31:1579-1584; Cumber et al. (1992)
J Immunology 149B: 120-126); humanized antibody molecules (see, for
example, Riechmann et al. (1988) Nature 332:323-327; Verhoeyan et
al. (1988) Science 239:1534-1536; and U.K. Patent Publication No.
GB 2,276,169, published 21 Sep. 1994); and, any functional
fragments obtained from such molecules, wherein such fragments
retain immunological binding properties of the parent antibody
molecule. The invention further includes antibodies obtained
through non-conventional processes, such as phage display.
[1441] Preferably, the GBS specific antibodies of the invention are
monoclonal antibodies. Monoclonal antibodies of the invention
include an antibody composition having a homogeneous antibody
population. Monoclonal antibodies of the invention may be obtained
from murine hybridomas, as well as human monoclonal antibodies
obtained using human rather than murine hybridomas. See, e.g.,
Cote, et al. Monoclonal Antibodies and Cancer Therapy, Alan R.
Liss, 1985, p 77.
[1442] The antibodies of the invention may be used in diagnostic
applications, for example, to detect the presence or absence of GBS
in a biological sample. The antibodies of the invention may also be
used in the prophylactic or therapeutic treatment of GBS
infection.
Antibodies: Gram Positive Bacteria AI Sequences
[1443] The Gram positive bacteria AI proteins of the invention may
also be used to prepare antibodies specific to the Gram positive
bacteria AI proteins. The antibodies are preferably specific to the
an oligomeric or hyper-oligomeric form of an AI protein. The
invention also includes combinations of antibodies specific to Gram
positive bacteria AI proteins selected to provide protection
against an increased range of Gram positive bacteria genus,
species, serotypes and strain isolates.
[1444] For example, a combination may comprise a first and second
antibody, wherein said first antibody is specific to a first Gram
positive bacteria AI protein and said second antibody is specific
to a second Gram positive bacteria AI protein. Preferably, the
nucleic acid sequence encoding said first Gram positive bacteria AI
protein is not present in a Gram positive bacterial genome
comprising a polynucleotide sequence encoding for said second Gram
positive bacteria AI protein. Preferably, the nucleic acid sequence
encoding said first and second Gram positive bacteria AI proteins
are present in the genomes of multiple Gram positive bacteria
genus, species, serotypes or strain isolates.
[1445] As an example of an instance where the combination of
antibodies provides protection against an increased range of
bacteria serotypes, the first antibody may be specific to a first
GAS AI protein and the second antibody may be specific to a second
GAS AI protein. The first GAS AI protein may comprise a GAS AI-1
surface protein, while the second GAS AI protein may comprise a GAS
AI-2 or AI-3 surface protein.
[1446] As an example of an instance where the combination of
antibodies provides protection against an increased range of
bacterial species, the first antibody may be specific to a GBS AI
protein and the second antibody may be specific to a GAS AI
protein. Alternatively, the first antibody may be specific to a GAS
AI protein and the second antibody may be specific to a S.
pneumoniae AI protein.
[1447] The Gram positive specific antibodies of the invention
include one or more biological moieties that, through chemical or
physical means, can bind to or associate with an epitope of a Gram
positive bacteria AI polypeptide. The antibodies of the invention
include antibodies which specifically bind to a Gram positive
bacteria AI protein. The invention includes antibodies obtained
from both polyclonal and monoclonal preparations, as well as the
following: hybrid (chimeric) antibody molecules (see, for example,
Winter et al. (1991) Nature 349: 293-299; and U.S. Pat. No.
4,816,567; F(ab').sub.2 and F(ab) fragments; F.sub.v molecules
(non-covalent heterodimers, see, for example, Inbar et al. (1972)
Proc Natl Acad Sci USA 69:2659-2662; and Ehrlich et al. (1980)
Biochem 19:4091.sub.--4096); single-chain Fv molecules (sFv) (see,
for example, Huston et al. (1988) Proc Natl Acad Sci USA
85:5897-5883); dimeric and trimeric antibody fragment constructs;
minibodies (see, e.g., Pack et al. (1992) Biochem 31:1579-1584;
Cumber et al. (1992) J Immunology 149B: 120-126); humanized
antibody molecules (see, for example, Riechmann et al. (1988)
Nature 332:323-327; Verhoeyan et al. (1988) Science 239:1534-1536;
and U.K. Patent Publication No. GB 2,276,169, published 21 Sep.
1994); and, any functional fragments obtained from such molecules,
wherein such fragments retain immunological binding properties of
the parent antibody molecule. The invention further includes
antibodies obtained through non-conventional processes, such as
phage display.
[1448] Preferably, the Gram positive specific antibodies of the
invention are monoclonal antibodies. Monoclonal antibodies of the
invention include an antibody composition having a homogeneous
antibody population. Monoclonal antibodies of the invention may be
obtained from murine hybridomas, as well as human monoclonal
antibodies obtained using human rather than murine hybridomas. See,
e.g., Cote, et al. Monoclonal Antibodies and Cancer Therapy, Alan
R. Liss, 1985, p 77.
[1449] The antibodies of the invention may be used in diagnostic
applications, for example, to detect the presence or absence of
Gram positive bacteria in a biological sample. The antibodies of
the invention may also be used in the prophylactic or therapeutic
treatment of Gram positive bacteria infection.
Nucleic Acids
[1450] The invention provides nucleic acids encoding the Gram
positive bacteria sequences and/or the hybrid fusion polypeptides
of the invention. The invention also provides nucleic acid encoding
the GBS antigens and/or the hybrid fusion polypeptides of the
invention. Furthermore, the invention provides nucleic acid which
can hybridise to these nucleic acids, preferably under "high
stringency" conditions (e.g. 65.degree. C. in a 0.1.times.SSC, 0.5%
SDS solution).
[1451] Polypeptides of the invention can be prepared by various
means (e.g. recombinant expression, purification from cell culture,
chemical synthesis, etc.) and in various forms (e.g. native,
fusions, non-glycosylated, lipidated, etc.). They are preferably
prepared in substantially pure form (i.e. substantially free from
other GAS or host cell proteins).
[1452] Nucleic acid according to the invention can be prepared in
many ways (e.g. by chemical synthesis, from genomic or cDNA
libraries, from the organism itself, etc.) and can take various
forms (e.g. single stranded, double stranded, vectors, probes,
etc.). They are preferably prepared in substantially pure form
(i.e. substantially free from other GBS or host cell nucleic
acids).
[1453] The term "nucleic acid" includes DNA and RNA, and also their
analogues, such as those containing modified backbones (e.g.
phosphorothioates, etc.), and also peptide nucleic acids (PNA),
etc. The invention includes nucleic acid comprising sequences
complementary to those described above (e.g. for antisense or
probing purposes).
[1454] The invention also provides a process for producing a
polypeptide of the invention, comprising the step of culturing a
host cell transformed with nucleic acid of the invention under
conditions which induce polypeptide expression.
[1455] The invention provides a process for producing a polypeptide
of the invention, comprising the step of synthesising at least part
of the polypeptide by chemical means.
[1456] The invention provides a process for producing nucleic acid
of the invention, comprising the step of amplifying nucleic acid
using a primer-based amplification method (e.g. PCR).
[1457] The invention provides a process for producing nucleic acid
of the invention, comprising the step of synthesising at least part
of the nucleic acid by chemical means.
Purification and Recombinant Expression
[1458] The Gram positive bacteria AI proteins of the invention may
be isolated from the native Gram positive bacteria, or they may be
recombinantly produced, for instance in a heterologous host. For
example, the GAS, GBS, and S. pneumoniae antigens of the invention
may be isolated from Streptococcus agalactiae, S. pyogenes, S.
pneumoniae, or they may be recombinantly produced, for instance, in
a heterologous host. Preferably, the GBS antigens are prepared
using a heterologous host.
[1459] The heterologous host may be prokaryotic (e.g. a bacterium)
or eukaryotic. It is preferably E. coli, but other suitable hosts
include Bacillus subtilis, Vibrio cholerae, Salmonella typhi,
Salmonella typhimurium, Neisseria lactamica, Neisseria cinerea,
Mycobacteria (e.g. M. tuberculosis), S. gordonii, L. lactis,
yeasts, etc.
[1460] Recombinant production of polypeptides is facilitated by
adding a tag protein to the Gram positive bacteria AI sequence to
be expressed as a fusion protein comprising the tag protein and the
Gram positive bacteria antigen. For example, recombinant production
of polypeptides is facilitated by adding a tag protein to the GBS
antigen to be expressed as a fusion protein comprising the tag
protein and the GBS antigen. Such tag proteins can facilitate
purification, detection and stability of the expressed protein. Tag
proteins suitable for use in the invention include a polyarginine
tag (Arg-tag), polyhistidine tag (His-tag), FLAG-tag, Strep-tag,
c-myc-tag, S-tag, calmodulin-binding peptide, cellulose-binding
domain, SBP-tag, chitin-binding domain, glutathione
S-transferase-tag (GST), maltose-binding protein, transcription
termination anti-terminiantion factor (NusA), E. coli thioredoxin
(TrxA) and protein disulfide isomerase I (DsbA). Preferred tag
proteins include His-tag and GST. A full discussion on the use of
tag proteins can be found at Terpe et al., "Overview of tag protein
fusions: from molecular and biochemical fundamentals to commercial
systems", Appl Microbiol Biotechnol (2003) 60:523-533.
[1461] After purification, the tag proteins may optionally be
removed from the expressed fusion protein, i.e., by specifically
tailored enzymatic treatments known in the art. Commonly used
proteases include enterokinase, tobacco etch virus (TEV), thrombin,
and factor X.sub.a.
GBS Polysaccharides
[1462] The compositions of the invention may be further improved by
including GBS polysaccharides. Preferably, the GBS antigen and the
saccharide each contribute to the immunological response in a
recipient. The combination is particularly advantageous where the
saccharide and polypeptide provide protection from different GBS
serotypes.
[1463] The combined antigens may be present as a simple combination
where separate saccharide and polypeptide antigens are administered
together, or they may be present as a conjugated combination, where
the saccharide and polypeptide antigens are covalently linked to
each other.
[1464] Thus the invention provides an immunogenic composition
comprising (i) one or more GBS AI proteins and (ii) one or more GBS
saccharide antigens. The polypeptide and the polysaccharide may
advantageously be covalently linked to each other to form a
conjugate.
[1465] Between them, the combined polypeptide and saccharide
antigens preferably cover (or provide protection from) two or more
GBS serotypes (e.g. 2, 3, 4, 5, 6, 7, 8 or more serotypes). The
serotypes of the polypeptide and saccharide antigens may or may not
overlap. For example, the polypeptide might protect against
serogroup II or V, while the saccharide protects against either
serogroups Ia, Ib, or III. Preferred combinations protect against
the following groups of serotypes: (1) serotypes Ia and Ib, (2)
serotypes Ia and H, (3) serotypes Ia and III, (4) serotypes Ia and
IV, (5) serotypes Ia and V, (6) serotypes Ia and VI, (7) serotypes
Ia and VII, (8) serotypes Ia and VIII, (9) serotypes Ib and II,
(10) serotypes Ib and III, (11) serotypes Ib and IV, (12) serotypes
Ib and V, (13) serotypes Ib and VI, (14) serotypes Ib and VII, (15)
serotypes Ib and VIII, 16) serotypes II and III, (17) serotypes II
and IV, (18) serotypes II and V, (19) serotypes II and VI, (20)
serotypes H and VII, (21) serotypes II and VII, (22) serotypes III
and IV, (23) serotypes III and V, (24) serotypes III and VI, (25)
serotypes III and VII, (26) serotypes III and VIII, (27) serotypes
IV and V, (28) serotypes IV and VI, (29) serotypes IV and VII, (30)
serotypes IV and VIII, (31) serotypes V and VI, (32) serotypes V
and VII, (33) serotypes V and VIII, (34) serotypes VI and VII, (35)
serotypes VI and VIII, and (36) serotypes VII and VIII.
[1466] Still more preferably, the combinations protect against the
following groups of serotypes: (1) serotypes Ia and II, (2)
serotypes Ia and V; (3) serotypes Ib and II, (4) serotypes Ib and
V, (5) serotypes III and II, and (6) serotypes III and V. Most
preferably, the combinations protect against serotypes III and
V.
[1467] Protection against serotypes II and V is preferably provided
by polypeptide antigens. Protection against serotypes Ia, Ib and/or
III may be polypeptide or saccharide antigens.
Immunogenic Compositions and Medicaments
[1468] Compositions of the invention are preferably immunogenic
compositions, and are more preferably vaccine compositions. The pH
of the composition is preferably between 6 and 8, preferably about
7. The pH may be maintained by the use of a buffer. The composition
may be sterile and/or pyrogen-free. The composition may be isotonic
with respect to humans.
[1469] Vaccines according to the invention may either be
prophylactic (i.e. to prevent infection) or therapeutic (i.e. to
treat infection), but will typically be prophylactic. Accordingly,
the invention includes a method for the therapeutic or prophylactic
treatment of a Gram positive bacteria infection in an animal
susceptible to such gram positive bacterial infection comprising
administering to said animal a therapeutic or prophylactic amount
of the immunogenic composition of the invention. For example, the
invention includes a method for the therapeutic or prophylactic
treatment of a Streptococcus agalactiae, S. pyogenes, or S.
pneumoniae infection in an animal susceptible to streptococcal
infection comprising administering to said animal a therapeutic or
prophylactic amount of the immunogenic compositions of the
invention.
[1470] The invention also provides a composition of the invention
for use of the compositions described herein as a medicament. The
medicament is preferably able to raise an immune response in a
mammal (i.e. it is an immunogenic composition) and is more
preferably a vaccine.
[1471] The invention also provides the use of the compositions of
the invention in the manufacture of a medicament for raising an
immune response in a mammal. The medicament is preferably a
vaccine.
[1472] The invention also provides kits comprising one or more
containers of compositions of the invention. Compositions can be in
liquid form or can be lyophilized, as can individual antigens.
Suitable containers for the compositions include, for example,
bottles, vials, syringes, and test tubes. Containers can be formed
from a variety of materials, including glass or plastic. A
container may have a sterile access port (for example, the
container may be an intravenous solution bag or a vial having a
stopper pierceable by a hypodermic injection needle). The
composition may comprise a first component comprising one or more
Gram positive bacteria AI proteins. Preferably, the AI proteins are
surface AI proteins. Preferably, the AI surface proteins are in an
oligomeric or hyperoligomeric form. For example, the first
component comprises a combination of GBS antigens or GAS antigens,
or S. pneumoniae antigens. Preferably said combination includes GBS
80. Preferably GBS 80 is present in an oligomeric or
hyperoligomeric form.
[1473] The kit can further comprise a second container comprising a
pharmaceutically-acceptable buffer, such as phosphate-buffered
saline, Ringer's solution, or dextrose solution. It can also
contain other materials useful to the end-user, including other
buffers, diluents, filters, needles, and syringes. The kit can also
comprise a second or third container with another active agent, for
example an antibiotic.
[1474] The kit can also comprise a package insert containing
written instructions for methods of inducing immunity against S
agalactiae and/or S. pyogenes and/or S pneumoniae or for treating S
agalactiae and/or S. pyogenes and/or S pneumoniae infections. The
package insert can be an unapproved draft package insert or can be
a package insert approved by the Food and Drug Administration (FDA)
or other regulatory body.
[1475] The invention also provides a delivery device pre-filled
with the immunogenic compositions of the invention.
[1476] The invention also provides a method for raising an immune
response in a mammal comprising the step of administering an
effective amount of a composition of the invention. The immune
response is preferably protective and preferably involves
antibodies and/or cell-mediated immunity. This immune response will
preferably induce long lasting (e.g., neutralising) antibodies and
a cell mediated immunity that can quickly respond upon exposure to
one or more GBS and/or GAS and/or S. pneumoniae antigens. The
method may raise a booster response.
[1477] The invention provides a method of neutralizing GBS, GAS, or
S. pneumoniae infection in a mammal comprising the step of
administering to the mammal an effective amount of the immunogenic
compositions of the invention, a vaccine of the invention, or
antibodies which recognize an immunogenic composition of the
invention.
[1478] The mammal is preferably a human. Where the vaccine is for
prophylactic use, the human is preferably a female (either of child
bearing age or a teenager). Alternatively, the human may be elderly
(e.g., over the age of 50, 55, 60, 65, 70 or 75) and may have an
underlying disease such as diabetes or cancer. Where the vaccine is
for therapeutic use, the human is preferably a pregnant female or
an elderly adult.
[1479] These uses and methods are preferably for the prevention
and/or treatment of a disease caused by Streptococcus agalactiae,
or S. pyogenes, or S. pneumoniae. The compositions may also be
effective against other streptococcal bacteria. The compositions
may also be effective against other Gram positive bacteria.
[1480] One way of checking efficacy of therapeutic treatment
involves monitoring Gram positive bacterial infection after
administration of the composition of the invention. One way of
checking efficacy of prophylactic treatment involves monitoring
immune responses against the Gram positive bacterial antigens in
the compositions of the invention after administration of the
composition.
[1481] One way of checking efficacy of therapeutic treatment
involves monitoring GBS infection after administration of the
composition of the invention. One way of checking efficacy of
prophylactic treatment involves monitoring immune responses against
the GBS antigens in the compositions of the invention after
administration of the composition.
[1482] A way of assessing the immunogenicity of the component
proteins of the immunogenic compositions of the present invention
is to express the proteins recombinantly and to screen patient sera
or mucosal secretions by immunoblot. A positive reaction between
the protein and the patient serum indicates that the patient has
previously mounted an immune response to the protein in
question--that is, the protein is an immunogen. This method may
also be used to identify immunodominant proteins and/or
epitopes.
[1483] Another way of checking efficacy of therapeutic treatment
involves monitoring GBS or GAS or S pneumoniae infection after
administration of the compositions of the invention. One way of
checking efficacy of prophylactic treatment involves monitoring
immune responses both systemically (such as monitoring the level of
IgG1 and IgG2a production) and mucosally (such as monitoring the
level of IgA production) against the GBS and/or GAS and/or S
pneumoniae antigens in the compositions of the invention after
administration of the composition. Typically, GBS and/or GAS and/or
S pneumoniae serum specific antibody responses are determined
post-immunization but pre-challenge whereas mucosal GBS and/or GAS
and/or S pneumoniae specific antibody body responses are determined
post-immunization and post-challenge.
[1484] The vaccine compositions of the present invention can be
evaluated in in vitro and in vivo animal models prior to host,
e.g., human, administration.
[1485] The efficacy of immunogenic compositions of the invention
can also be determined in vivo by challenging animal models of GBS
and/or GAS and/or S pneumoniae infection, e.g., guinea pigs or
mice, with the immunogenic compositions. The immunogenic
compositions may or may not be derived from the same serotypes as
the challenge serotypes. Preferably the immunnogenic compositions
are derivable from the same serotypes as the challenge serotypes.
More preferably, the immunogenic composition and/or the challenge
serotypes are derivable from the group of GBS and/or GAS and/or S
pneumoniae serotypes.
[1486] In vivo efficacy models include but are not limited to: (i)
A murine infection model using human GBS and/or GAS and/or S
pneumoniae serotypes; (ii) a murine disease model which is a murine
model using a mouse-adapted GBS and/or GAS and/or S pneumoniae
strain, such as those strains outlined above which is particularly
virulent in mice and (iii) a primate model using human GBS or GAS
or S pneumoniae isolates.
[1487] The immune response may be one or both of a TH1 immune
response and a TH2 response.
[1488] The immune response may be an improved or an enhanced or an
altered immune response.
[1489] The immune response may be one or both of a systemic and a
mucosal immune response.
[1490] Preferably the immune response is an enhanced system and/or
mucosal response.
[1491] An enhanced systemic and/or mucosal immunity is reflected in
an enhanced TH1 and/or TH2 immune response. Preferably, the
enhanced immune response includes an increase in the production of
IgG1 and/or IgG2a and/or IgA Preferably the mucosal immune response
is a TH2 immune response. Preferably, the mucosal immune response
includes an increase in the production of IgA.
[1492] Activated TH2 cells enhance antibody production and are
therefore of value in responding to extracellular infections.
Activated TH2 cells may secrete one or more of IL-4, IL-5, IL-6,
and IL-10. A TH2 immune response may result in the production of
IgG1, IgE, IgA and memory B cells for future protection.
[1493] A TH2 immune response may include one or more of an increase
in one or more of the cytokines associated with a TH2 immune
response (such as IL-4, IL-5, IL-6 and IL-10), or an increase in
the production of IgG1, IgE, IgA and memory B cells. Preferably,
the enhanced TH2 immune resonse will include an increase in IgG1
production.
[1494] A TH1 immune response may include one or more of an increase
in CTLs, an increase in one or more of the cytokines associated
with a TH1 immune response (such as IL-2, IFN.gamma., and
TNF.beta., an increase in activated macrophages, an increase in NK
activity, or an increase in the production of IgG2a. Preferably,
the enhanced TH1 immune response will include an increase in IgG2a
production.
[1495] Immunogenic compositions of the invention, in particular,
immunogenic composition comprising one or more GAS antigens of the
present invention may be used either alone or in combination with
other GAS antigens optionally with an immunoregulatory agent
capable of eliciting a Th1 and/or Th2 response.
[1496] Compositions of the invention will generally be administered
directly to a patient. Certain routes may be favored for certain
compositons, as resulting in the generation of a more effective
immune response, preferably a CMI response, or as being less likely
to induce side effects, or as being easier for administration.
Direct delivery may be accomplished by parenteral injection (e.g.
subcutaneously, intraperitoneally, intradermally, intravenously,
intramuscularly, or to the interstitial space of a tissue), or by
rectal, oral (e.g. tablet, spray), vaginal, topical, transdermal
(e.g. see WO 99/27961) or transcutaneous (e.g. see WO 02/074244 and
WO 02/064162), intranasal (e.g. see WO03/028760), ocular, aural,
pulmonary or other mucosal administration.
[1497] The invention may be used to elicit systemic and/or mucosal
immunity.
[1498] In one particularly preferred embodiment, the immunogenic
composition comprises one or more GBS or GAS or S pneumoniae
antigen(s) which elicits a neutralising antibody response and one
or more GBS or GAS or S pneumoniae antigen(s) which elicit a cell
mediated immune response. In this way, the neutralising antibody
response prevents or inhibits an initial GBS or GAS or S pneumoniae
infection while the cell-mediated immune response capable of
eliciting an enhanced Th1 cellular response prevents further
spreading of the GBS or GAS or S pneumoniae infection. Preferably,
the immunogenic composition comprises one or more GBS or GAS or S
pneumoniae surface antigens and one or more GBS or GAS or S
pneumoniae cytoplasmic antigens. Preferably the immunogenic
composition comprises one or more GBS or GAS or S pneumoniae
surface antigens or the like and one or other antigens, such as a
cytoplasmic antigen capable of eliciting a Th1 cellular
response.
[1499] Dosage treatment can be a single dose schedule or a multiple
dose schedule. Multiple doses may be used in a primary immunisation
schedule and/or in a booster immunisation schedule. In a multiple
dose schedule the various doses may be given by the same or
different routes e.g. a parenteral prime and mucosal boost, a
mucosal prime and parenteral boost, etc.
[1500] The compositions of the invention may be prepared in various
forms. For example, the compositions may be prepared as
injectables, either as liquid solutions or suspensions. Solid forms
suitable for solution in, or suspension in, liquid vehicles prior
to injection can also be prepared (e.g. a lyophilised composition).
The composition may be prepared for topical administration e.g. as
an ointment, cream or powder. The composition may be prepared for
oral administration e.g. as a tablet or capsule, as a spray, or as
a syrup (optionally flavoured). The composition may be prepared for
pulmonary administration e.g. as an inhaler, using a fine powder or
a spray. The composition may be prepared as a suppository or
pessary. The composition may be prepared for nasal, aural or ocular
administration e.g. as drops. The composition may be in kit form,
designed such that a combined composition is reconstituted just
prior to administration to a patient. Such kits may comprise one or
more antigens in liquid form and one or more lyophilised
antigens.
[1501] Immunogenic compositions used as vaccines comprise an
immunologically effective amount of antigen(s), as well as any
other components, such as antibiotics, as needed. By
`immunologically effective amount`, it is meant that the
administration of that amount to an individual, either in a single
dose or as part of a series, is effective for treatment or
prevention, or increases a measurable immune response or prevents
or reduces a clinical symptom. This amount varies depending upon
the health and physical condition of the individual to be treated,
age, the taxonomic group of individual to be treated (e.g.
non-human primate, primate, etc.), the capacity of the individual's
immune system to synthesise antibodies, the degree of protection
desired, the formulation of the vaccine, the treating doctor's
assessment of the medical situation, and other relevant factors. It
is expected that the amount will fall in a relatively broad range
that can be determined through routine trials.
Further Components of the Composition
[1502] The composition of the invention will typically, in addition
to the components mentioned above, comprise one or more
`pharmaceutically acceptable carriers`, which include any carrier
that does not itself induce the production of antibodies harmful to
the individual receiving the composition. Suitable carriers are
typically large, slowly metabolised macromolecules such as
proteins, polysaccharides, polylactic acids, polyglycolic acids,
polymeric amino acids, amino acid copolymers, and lipid aggregates
(such as oil droplets or liposomes). Such carriers are well known
to those of ordinary skill in the art. The vaccines may also
contain diluents, such as water, saline, glycerol, etc.
Additionally, auxiliary substances, such as wetting or emulsifying
agents, pH buffering substances, and the like, may be present. A
thorough discussion of pharmaceutically acceptable excipients is
available in Gennaro (2000) Remington: The Science and Practice of
Pharmacy. 20th ed., ISBN: 0683306472.
Adjuvants
[1503] Vaccines of the invention may be administered in conjunction
with other immunoregulatory agents. In particular, compositions
will usually include an adjuvant. Adjuvants for use with the
invention include, but are not limited to, one or more of the
following set forth below:
[1504] A. Mineral Containing Compositions
[1505] Mineral containing compositions suitable for use as
adjuvants in the invention include mineral salts, such as aluminum
salts and calcium salts. The invention includes mineral salts such
as hydroxides (e.g. oxyhydroxides), phosphates (e.g.
hydroxyphosphates, orthophosphates), sulfates, etc. (e.g. see
chapters 8 & 9 of Vaccine Design . . . (1995) eds. Powell &
Newman. ISBN: 030644867X. Plenum.), or mixtures of different
mineral compounds (e.g. a mixture of a phosphate and a hydroxide
adjuvant, optionally with an excess of the phosphate), with the
compounds taking any suitable form (e.g. gel, crystalline,
amorphous, etc.), and with adsorption to the salt(s) being
preferred. The mineral containing compositions may also be
formulated as a particle of metal salt (WO 00/23105).
[1506] Aluminum salts may be included in vaccines of the invention
such that the dose of Al.sup.3+ is between 0.2 and 1.0 mg per
dose.
[1507] B. Oil-Emulsions
[1508] Oil-emulsion compositions suitable for use as adjuvants in
the invention include squalene-water emulsions, such as MF59 (5%
Squalene, 0.5% Tween 80, and 0.5% Span 85, formulated into
submicron particles using a microfluidizer). See WO90/14837. See
also, Podda, "The adjuvanted influenza vaccines with novel
adjuvants: experience with the MF59-adjuvanted vaccine", Vaccine
(2001) 19: 2673-2680; Frey et al., "Comparison of the safety,
tolerability, and immunogenicity of a MF59-adjuvanted influenza
vaccine and a non-adjuvanted influenza vaccine in non-elderly
adults", Vaccine (2003) 21:42344237. MF59 is used as the adjuvant
in the FLUAD.TM. influenza virus trivalent subunit vaccine.
[1509] Particularly preferred adjuvants for use in the compositions
are submicron oil-in-water emulsions. Preferred submicron
oil-in-water emulsions for use herein are squalene/water emulsions
optionally containing varying amounts of MTP-PE, such as a
submicron oil-in-water emulsion containing 4-5% w/v squalene,
0.25-1.0% w/v Tween 80.TM. (polyoxyelthylenesorbitan monooleate),
and/or 0.25-1.0% Span85.TM. (sorbitan trioleate), and, optionally,
N-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(1'-2'-dipalmitoyl-s-
n-glycero-3-huydroxyphosphophoryloxy)-ethylamine (MTP-PE), for
example, the submicron oil-in-water emulsion known as "MF59"
(International Publication No. WO 90/14837; U.S. Pat. Nos.
6,299,884 and 6,451,325, incorporated herein by reference in their
entireties; and Ott et al., "MF59--Design and Evaluation of a Safe
and Potent Adjuvant for Human Vaccines" in Vaccine Design: The
Subunit and Adjuvant Approach (Powell, M. F. and Newman, M. J.
eds.) Plenum Press, New York, 1995, pp. 277-296). MF59 contains
4-5% w/v Squalene (e.g. 4.3%), 0.25-0.5% w/v Tween 80.TM., and 0.5%
w/v Span 85.TM. and optionally contains various amounts of MTP-PE,
formulated into submicron particles using a microfluidizer such as
Model 110Y microfluidizer (Microfluidics, Newton, Mass.). For
example, MTP-PE may be present in an amount of about 0-500
.mu.g/dose, more preferably 0-250 .mu.g/dose and most preferably,
0-100 .mu.g/dose. As used herein, the term "MF59-0" refers to the
above submicron oil-in-water emulsion lacking MTP-PE, while the
term MF59-MTP denotes a formulation that contains MTP-PE. For
instance, "MF59-100" contains 100 .mu.g MTP-PE per dose, and so on.
MF69, another submicron oil-in-water emulsion for use herein,
contains 4.3% w/v squalene, 0.25% w/v Tween 80.TM., and 0.75% w/v
Span 85.TM. and optionally MTP-PE. Yet another submicron
oil-in-water emulsion is MF75, also known as SAF, containing 10%
squalene, 0.4% Tween 80.TM., 5% pluronic-blocked polymer L121, and
thr-MDP, also microfluidized into a submicron emulsion. MF75-MTP
denotes an MF75 formulation that includes MTP, such as from 100-400
.mu.g MTP-PE per dose.
[1510] Submicron oil-in-water emulsions, methods of making the same
and immunostimulating agents, such as muramyl peptides, for use in
the compositions, are described in detail in International
Publication No. WO 90/14837 and U.S. Pat. Nos. 6,299,884 and
6,451,325, incorporated herein by reference in their
entireties.
[1511] Complete Freund's adjuvant (CFA) and incomplete Freund's
adjuvant (IFA) may also be used as adjuvants in the invention.
[1512] C. Saponin Formulations
[1513] Saponin formulations, may also be used as adjuvants in the
invention. Saponins are a heterologous group of sterol glycosides
and triterpenoid glycosides that are found in the bark, leaves,
stems, roots and even flowers of a wide range of plant species.
Saponin from the bark of the Quillaia saponaria Molina tree have
been widely studied as adjuvants. Saponin can also be commercially
obtained from Smilax ornata (sarsaprilla), Gypsophilla paniculata
(brides veil), and Saponaria officianalis (soap root). Saponin
adjuvant formulations include purified formulations, such as QS21,
as well as lipid formulations, such as ISCOMs.
[1514] Saponin compositions have been purified using High
Performance Thin Layer Chromatography (HP-LC) and Reversed Phase
High Performance Liquid Chromatography (RP-HPLC). Specific purified
fractions using these techniques have been identified, including
QS7, QS17, QS18, QS21, QH-A, QH-B and QH-C. Preferably, the saponin
is QS21. A method of production of QS21 is disclosed in U.S. Pat.
No. 5,057,540. Saponin formulations may also comprise a sterol,
such as cholesterol (see WO96/33739).
[1515] Combinations of saponins and cholesterols can be used to
form unique particles called Immunostimulating Complexs (ISCOMs).
ISCOMs typically also include a phospholipid such as
phosphatidylethanolamine or phosphatidylcholine. Any known saponin
can be used in ISCOMs. Preferably, the ISCOM includes one or more
of Quil A, QHA and QHC. ISCOMs are further described in EP0109942,
WO 96/11711 and WO 96/33739. Optionally, the ISCOMS may be devoid
of additional detergent. See WO 00/07621.
[1516] A review of the development of saponin based adjuvants can
be found at Barr, et al., "ISCOMs and other saponin based
adjuvants", Advanced Drug Delivery Reviews (1998) 32:247-271. See
also Sjolander, et al., "Uptake and adjuvant activity of orally
delivered saponin and ISCOM vaccines", Advanced Drug Delivery
Reviews (1998) 32:321-338.
[1517] D. Virosomes and Virus Like Particles (VLPs)
[1518] Virosomes and Virus Like Particles (VLPs) can also be used
as adjuvants in the invention. These structures generally contain
one or more proteins from a virus optionally combined or formulated
with a phospholipid. They are generally non-pathogenic,
non-replicating and generally do not contain any of the native
viral genome. The viral proteins may be recombinantly produced or
isolated from whole viruses. These viral proteins suitable for use
in virosomes or VLPs include proteins derived from influenza virus
(such as HA or NA), Hepatitis B virus (such as core or capsid
proteins), Hepatitis E virus, measles virus, Sindbis virus,
Rotavirus, Foot-and-Mouth Disease virus, Retrovirus, Norwalk virus,
human Papilloma virus, HIV, RNA-phages, Q.beta.-phage (such as coat
proteins), GA-phage, fr-phage, AP205 phage, and Ty (such as
retrotransposon Ty protein p1). VLPs are discussed further in WO
03/024480, WO 03/024481, and Niikura et al., "Chimeric Recombinant
Hepatitis E Virus-Like Particles as an Oral Vaccine Vehicle
Presenting Foreign Epitopes", Virology (2002) 293:273-280; Lenz et
al., "Papillomarivurs-Like Particles Induce Acute Activation of
Dendritic Cells", Journal of Immunology (2001) 5246-5355; Pinto, et
al., "Cellular Immune Responses to Human Papillomavirus (HPV)-16 L1
Healthy Volunteers Immunized with Recombinant HPV-16 L1 Virus-Like
Particles", Journal of Infectious Diseases (2003) 188:327-338; and
Gerber et al., "Human Papillomavrisu Virus-Like Particles Are
Efficient Oral Immunogens when Coadministered with Escherichia coli
Heat-Labile Entertoxin Mutant R192G or CpG", Journal of Virology
(2001) 75(10):4752-4760. Virosomes are discussed further in, for
example, Gluck et al., "New Technology Platforms in the Development
of Vaccines for the Future", Vaccine (2002) 20:B10-B16.
Immunopotentiating reconstituted influenza virosomes (IRIV) are
used as the subunit antigen delivery system in the intranasal
trivalent IFLEXAL.TM. product {Mischler & Metcalfe (2002)
Vaccine 20 Suppl 5:B17-23} and the INFLUVAC PLUS.TM. product.
[1519] E. Bacterial or Microbial Derivatives
[1520] Adjuvants suitable for use in the invention include
bacterial or microbial derivatives such as:
[1521] (1) Non-Toxic Derivatives of Enterobacterial
Lipopolysaccharide (LPS)
[1522] Such derivatives include Monophosphoryl lipid A (MPL) and
3-O-deacylated MPL (3dMPL). 3dMPL is a mixture of 3 De-O-acylated
monophosphoryl lipid A with 4, 5 or 6 acylated chains. A preferred
"small particle" form of 3 De-O-acylated monophosphoryl lipid A is
disclosed in EP 0 689 454. Such "small particles" of 3dMPL are
small enough to be sterile filtered through a 0.22 micron membrane
(see EP 0 689 454). Other non-toxic LPS derivatives include
monophosphoryl lipid A mimics, such as aminoalkyl glucosaminide
phosphate derivatives e.g. RC-529. See Johnson et al. (1999) Bioorg
Med Chem Lett 9:2273-2278.
[1523] (2) Lipid A Derivatives
[1524] Lipid A derivatives include derivatives of lipid A from
Escherichia coli such as OM-174. OM-174 is described for example in
Meraldi et al., "OM-174, a New Adjuvant with a Potential for Human
Use, Induces a Protective Response with Administered with the
Synthetic C-Terminal Fragment 242-310 from the circumsporozoite
protein of Plasmodium berghei", Vaccine (2003) 21:2485-2491; and
Pajak, et al., "The Adjuvant OM-174 induces both the migration and
maturation of murine dendritic cells in vivo", Vaccine (2003)
21:836-842.
[1525] (3) Immunostimulatory Oligonucleotides
[1526] Immunostimulatory oligonucleotides suitable for use as
adjuvants in the invention include nucleotide sequences containing
a CpG motif (a sequence containing an unmethylated cytosine
followed by guanosine and linked by a phosphate bond). Bacterial
double stranded RNA or oligonucleotides containing palindromic or
poly(dG) sequences have also been shown to be
immunostimulatory.
[1527] The CpG's can include nucleotide modifications/analogs such
as phosphorothioate modifications and can be double-stranded or
single-stranded. Optionally, the guanosine may be replaced with an
analog such as 2'-deoxy-7-deazaguanosine. See Kandimalla, et al.,
"Divergent synthetic nucleotide motif recognition pattern: design
and development of potent immunomodulatory oligodeoxyribonucleotide
agents with distinct cytokine induction profiles", Nucleic Acids
Research (2003) 31(9): 2393-2400; WO02/26757 and WO99/62923 for
examples of possible analog substitutions. The adjuvant effect of
CpG oligonucleotides is further discussed in Krieg, "CpG motifs:
the active ingredient in bacterial extracts?", Nature Medicine
(2003) 9(7): 831-835; McCluskie, et al., "Parenteral and mucosal
prime-boost immunization strategies in mice with hepatitis B
surface antigen and CpG DNA", FEMS Immunology and Medical
Microbiology (2002) 32:179-185; WO98/40100; U.S. Pat. No.
6,207,646; U.S. Pat. No. 6,239,116 and U.S. Pat. No. 6,429,199.
[1528] The CpG sequence may be directed to TLR9, such as the motif
GTCGTT or TTCGTT. See Kandimalla, et al., "Toll-like receptor 9:
modulation of recognition and cytokine induction by novel synthetic
CpG DNAs", Biochemical Society Transactions (2003) 31 (part 3):
654-658. The CpG sequence may be specific for inducing a Th1 immune
response, such as a CpG-A ODN, or it may be more specific for
inducing a B cell response, such a CpG-B ODN. CpG-A and CpG-B ODNs
are discussed in Blackwell, et al., "CpG-A-Induced Monocyte
IFN-gamma-Inducible Protein-10 Production is Regulated by
Plasmacytoid Dendritic Cell Derived IFN-alpha", J. Immunol. (2003)
170(8):4061-4068; Krieg, "From A to Z on CpG", TRENDS in Immunology
(2002) 23(2): 64-65 and WO01/95935. Preferably, the CpG is a CpG-A
ODN.
[1529] Preferably, the CpG oligonucleotide is constructed so that
the 5' end is accessible for receptor recognition. Optionally, two
CpG oligonucleotide sequences may be attached at their 3' ends to
form "immunomers". See, for example, Kandimalla, et al., "Secondary
structures in CpG oligonucleotides affect immunostimulatory
activity", BBRC (2003) 306:948-953; Kandimalla, et al., "Toll-like
receptor 9: modulation of recognition and cytokine induction by
novel synthetic GpG DNAs", Biochemical Society Transactions (2003)
31(part 3):664-658; Bhagat et al., "CpG penta- and
hexadeoxyribonucleotides as potent immunomodulatory agents" BBRC
(2003) 300:853-861 and WO 03/035836.
[1530] (4) ADP-Ribosylating Toxins and Detoxified Derivatives
Thereof.
[1531] Bacterial ADP-ribosylating toxins and detoxified derivatives
thereof may be used as adjuvants in the invention. Preferably, the
protein is derived from E. coli (i.e., E. coli heat labile
enterotoxin "LT), cholera ("CT"), or pertussis ("PT"). The use of
detoxified ADP-ribosylating toxins as mucosal adjuvants is
described in WO95/17211 and as parenteral adjuvants in WO98/42375.
Preferably, the adjuvant is a detoxified LT mutant such as LT-K63,
LT-R72, and LTR192G. The use of ADP-ribosylating toxins and
detoxified derivaties thereof, particularly LT-K63 and LT-R72, as
adjuvants can be found in the following references, each of which
is specifically incorporated by reference herein in their entirety:
Beignon, et al., "The LTR72 Mutant of Heat-Labile Enterotoxin of
Escherichia coli Enahnces the Ability of Peptide Antigens to Elicit
CD4+ T Cells and Secrete Gamma Interferon after Coapplication onto
Bare Skin", Infection and Immunity (2002) 70(6):3012-3019; Pizza,
et al., "Mucosal vaccines: non toxic derivatives of LT and CT as
mucosal adjuvants", Vaccine (2001) 19:2534-2541; Pizza, et al.,
"LTK63 and LTR72, two mucosal adjuvants ready for clinical trials"
Int. J. Med. Microbiol (2000) 290(4-5):455-461; Scharton-Kersten et
al., "Transcutaneous Immunization with Bacterial ADP-Ribosylating
Exotoxins, Subunits and Unrelated Adjuvants", Infection and
Immunity (2000) 68(9):5306-5313; Ryan et al., "Mutants of
Escherichia coli Heat-Labile Toxin Act as Effective Mucosal
Adjuvants for Nasal Delivery of an Acellular Pertussis Vaccine:
Differential Effects of the Nontoxic AB Complex and Enzyme Activity
on Th1 and Th2 Cells" Infection and Immunity (1999)
67(12):6270-6280; Partidos et al., "Heat-labile enterotoxin of
Escherichia coli and its site-directed mutant LTK63 enhance the
proliferative and cytotoxic T-cell responses to intranasally
co-immunized synthetic peptides", Immunol. Lett. (1999)
67(3):209-216; Peppoloni et al., "Mutants of the Escherichia coli
heat-labile enterotoxin as safe and strong adjuvants for intranasal
delivery of vaccines", Vaccines (2003) 2(2):285-293; and Pine et
al., (2002) "Intranasal immunization with influenza vaccine and a
detoxified mutant of heat labile enterotoxin from Escherichia coli
(LTK63)" J. Control Release (2002) 85(1-3):263-270. Numerical
reference for amino acid substitutions is preferably based on the
alignments of the A and B subunits of ADP-ribosylating toxins set
forth in Domenighini et al., Mol. Microbiol (1995) 15(6):1165-1167,
specifically incorporated herein by reference in its entirety.
[1532] F. Bioadhesives and Mucoadhesives
[1533] Bioadhesives and mucoadhesives may also be used as adjuvants
in the invention. Suitable bioadhesives include esterified
hyaluronic acid microspheres (Singh et al. (2001) J. Cont. Rele.
70:267-276) or mucoadhesives such as cross-linked derivatives of
poly(acrylic acid), polyvinyl alcohol, polyvinyl pyrollidone,
polysaccharides and carboxymethylcellulose. Chitosan and
derivatives thereof may also be used as adjuvants in the invention.
E.g. WO99/27960.
[1534] G. Microparticles
[1535] Microparticles may also be used as adjuvants in the
invention. Microparticles (i.e. a particle of .about.100 nm to
.about.150 .mu.m in diameter, more preferably .about.200 nm to
.about.30 .mu.m in diameter, and most preferably .about.500 nm to
.about.10 .mu.m in diameter) formed from materials that are
biodegradable and non-toxic (e.g. a poly(.alpha.-hydroxy acid), a
polyhydroxybutyric acid, a polyorthoester, a polyanhydride, a
polycaprolactone, etc.), with poly(lactide-co-glycolide) are
preferred, optionally treated to have a negatively-charged surface
(e.g. with SDS) or a positively-charged surface (e.g. with a
cationic detergent, such as CTAB).
[1536] H. Liposomes
[1537] Examples of liposome formulations suitable for use as
adjuvants are described in U.S. Pat. No. 6,090,406, U.S. Pat. No.
5,916,588, and EP 0 626 169.
[1538] I. Polyoxyethylene Ether and Polyoxyethylene Ester
Formulations
[1539] Adjuvants suitable for use in the invention include
polyoxyethylene ethers and polyoxyethylene esters. WO99/52549. Such
formulations further include polyoxyethylene sorbitan ester
surfactants in combination with an octoxynol (WO01/21207) as well
as polyoxyethylene alkyl ethers or ester surfactants in combination
with at least one additional non-ionic surfactant such as an
octoxynol (WO 01/21152).
[1540] Preferred polyoxyethylene ethers are selected from the
following group: polyoxyethylene-9-lauryl ether (laureth 9),
polyoxyethylene-9-steoryl ether, polyoxytheylene-8-steoryl ether,
polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether,
and polyoxyethylene-23-lauryl ether.
[1541] J. Polyphosphazene (PCPP)
[1542] PCPP formulations are described, for example, in Andrianov
et al., "Preparation of hydrogel microspheres by coacervation of
aqueous polyphophazene solutions", Biomaterials (1998)
19(1-3):109-115 and Payne et al., "Protein Release from
Polyphosphazene Matrices", Adv. Drug. Delivery Review (1998)
31(3):185-196.
[1543] K. Muramyl Peptides
[1544] Examples of muramyl peptides suitable for use as adjuvants
in the invention include N-acetyl-muramyl-L-threonyl-D-isoglutamine
(thr-MDP), N-acetyl-normuramyl-1-alanyl-d-isoglutamine (nor-MDP),
and
N-acetylmuramyl-1-alanyl-d-isoglutaminyl-1-alanine-2-(1'-2'-dipalmitoyl-s-
n-glycero-3-hydroxyphosphoryloxy)-ethylamine MTP-PE).
[1545] L. Imidazoquinolone Compounds.
[1546] Examples of imidazoquinolone compounds suitable for use
adjuvants in the invention include Imiquamod and its homologues,
described further in Stanley, "Imiquimod and the imidazoquinolones:
mechanism of action and therapeutic potential" Clin Exp Dermatol
(2002) 27(7):571-577 and Jones, "Resiquimod 3M", Curr Opin Investig
Drugs (2003) 4(2):214-218.
[1547] The invention may also comprise combinations of aspects of
one or more of the adjuvants identified above. For example, the
following adjuvant compositions may be used in the invention:
(1) a saponin and an oil-in-water emulsion (WO 99/11241);
(2) a saponin (e.g., QS21)+a non-toxic LPS derivative (e.g. 3dMPL)
(see WO 94/00153);
(3) a saponin (e.g., QS21)+a non-toxic LPS derivative (e.g.
3dMPL)+a cholesterol;
(4) a saponin (e.g. QS21)+3dMPL+IL-12 (optionally+a sterol) (WO
98/57659);
(5) combinations of 3dMPL with, for example, QS21 and/or
oil-in-water emulsions (See European patent applications 0835318,
0735898 and 0761231);
(6) SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-block
polymer L121, and thr-MDP, either microfluidized into a submicron
emulsion or vortexed to generate a larger particle size
emulsion.
[1548] (7) Ribi.TM. adjuvant system (RAS), (Ribi Immunochem)
containing 2% Squalene, 0.2% Tween 80, and one or more bacterial
cell wall components from the group consisting of
monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell
wall skeleton (CWS), preferably MPL+CWS (Detox.TM.);
(8) one or more mineral salts (such as an aluminum salt)+a
non-toxic derivative of LPS (such as 3dPML).
(9) one or more mineral salts (such as an aluminum salt)+an
immunostimulatory oligonucleotide (such as a nucleotide sequence
including a CpG motif). Combination No. (9) is a preferred adjuvant
combination.
[1549] M. Human Immunomodulators
[1550] Human immunomodulators suitable for use as adjuvants in the
invention include cytokines, such as interleukins (e.g. IL-1, IL-2,
IL-4, IL-5, 1L-6, IL-7, IL-12, etc.), interferons (e.g.
interferon-.gamma.), macrophage colony stimulating factor, and
tumor necrosis factor.
[1551] Aluminum salts and MF59 are preferred adjuvants for use with
injectable influenza vaccines. Bacterial toxins and bioadhesives
are preferred adjuvants for use with mucosally-delivered vaccines,
such as nasal vaccines.
[1552] The immunogenic compositions of the present invention may be
administed in combination with an antibiotic treatment regime. In
one embodiment, the antibiotic is administered prior to
administration of the antigen of the invention or the composition
comprising the one or more of the antigens of the invention.
[1553] In another embodiment, the antibiotic is administered
subsequent to the adminstration of the one or more antigens of the
invention or the composition comprising the one or more antigens of
the invention. Examples of antibiotics suitable for use in the
treatment of the Steptococcal infections of the invention include
but are not limited to penicillin or a derivative thereof or
clindamycin or the like.
Further Antigens
[1554] The compositions of the invention may further comprise one
or more additional Gram positive bacterial antigens which are not
associated with an AI. Preferably, the Gram positive bacterial
antigens that are not associated with an AI can provide protection
across more than one serotype or strain isolate. For example, a
first non-AI antigen, in which the first non-AI antigen is at least
90% (i.e., at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or
100%) homologous to the amino acid sequence of a second non-AI
antigen, wherein the first and the second non-AI antigen are
derived from the genomes of different serotypes of a Gram positive
bacteria, may be further included in the compositions. The first
non-AI antigen may also be homologous to the amino acid sequence of
a third non-AI antigen, such that the first non-AI antigen, the
second non-AI antigen, and the third non-AI antigen are derived
from the genomes of different serotypes of a Gram positive
bacteria. The first non-AI antigen may also be homologous to the
amino acid sequence of a fourth non-AI antigen, such that the first
non-AI antigen, the second non-AI antigen, the third non-AI
antigen, and the fourth non-AI antigen are derived from the genomes
of different serotypes of a Gram positive bacteria.
[1555] The first non-AI antigen may be GBS 322. The amino acid
sequence of GBS 322 across GBS strains from serotypes Ia, Ib, II,
III, V, and VIII is greater than 90%. Alternatively, the first
non-AI antigen may be GBS 276. The amino acid sequence of GBS 276
across GBS strain from serotypes Ia, Ib, II, III, V, and VIII is
greater than 90%. Table 13 provides the percent amino acid sequence
identity of GBS 322 and GBS 276 across different GBS strains and
serotypes. TABLE-US-00291 TABLE 13 Conservation of GBS 322 and GBS
276 amino acid sequences GBS 322 GBS 276 Serotype Strains cGH % AA
identity cGH % AA identity Ia 090 + 98.60 + 97.90 A909 + 98.30 +
97.90 515 + 98.80 + 97.50 DK1 + + DK8 + + Davis + + Ib 7357b + +
H36B + 98.30 + 97.80 II 18RS21 + 100.00 + 99.90 DK21 + + III NEM316
+ 100.00 + 97.00 COH31 + + D136 + + M732 + 98.00 + 100.00 COH1 +
98.30 + 100.00 M781 + 98.30 + 99.60 No type CJB110 + 98.60 + 97.90
1169NT + 97.40 + 97.90 V CJB111 + 100.00 + 2603 + 100.00 + 100.00
VIII JM130013 + 100.00 + 97.90 SMU014 + + total 22/22 98.28 +/- 0.4
22/22 98.44 +/- 1.094
[1556] As an example, inclusion of a non-AI protein, GBS 322, in
combination with AI antigens GBS 67, GBS 80, and GBS 104 provided
protection to newborn mice in an active maternal immunization
assay. TABLE-US-00292 TABLE 14 Active maternal immunization assay
for a combination of fragments from GBS 322, GBS 80, GBS 104, and
GBS 67 MIX = 322 + FACS (.DELTA. Mean) 80 + 104 + 67 PBS GBS
strains Type GBS 80 GBS 67 GBS 322 alive/treated % protection
alive/treated % protection 515 Ia 0 409 227 39/40 97 6/40 15 7357b-
Ib 91 316 102 19/30 63 1/30 3 DK21 II 0 331 416 25/34 73 17/48 35
5401 II 170 618 135 35/40 87 3/37 8 3050 II 43 460 188 48/48 100
1/30 3 COH1 III 305 0 130 36/36 100 7/40 17 M781 III 65 0 224 30/40
75 4/39 10 2603 V 125 105 313 27/33 82 10/35 28 CJB111 V 370 481 63
25/28 89 4/46 9 JM9130013 VIII 597 83 143 37/39 95 5/40 12 JMU071
VIII 556 79 170 44/50 88 18/50 36 NT1169 NT 0 443 213 12/32 37
11/35 31
[1557] In fact, the non-AI GBS 322 antigen may itself provide
protection to newborn mice in an active maternal immunization
assay. TABLE-US-00293 TABLE 16 Active maternal immunization assay
for each of GBS 80 and GBS 322 antigens GBS 80 GBS 322 Protection
Protection FACS (% survival) FACS (% survival) GBS strains Type
.DELTA. Mean antigen ctrl- .DELTA. Mean antigen ctrl- CJB111 V 370
72% 40% 63 57% 40% COH1 III 305 76% 10% 130 3% 10% 2603 V 82 22%
34% 313 83% 34% 7357b- Ib 91 36% 34% 102 43% 34% 18RS21 II 0 15%
24% 268 84% 24% DK21 II 0 10% 21% 416 67% 25% A909 Ia 0 0% 14% 090
Ia 0 0% 0% H36B Ib 105 34% 32%
Thus, inclusion of a non-AI protein in an immunogenic composition
of the invention may provide increased protection a mammal.
[1558] The immunogenic compositions comprising S. pneumonaie AI
polypeptides may further secondary SP protein antigens which
include (a) any of the SP protein antigens disclosed in WO
02/077021 or U.S. provisional application ______, filed Apr. 20,
2005 (Attorney Docket Number 002441.00154), (2) immunogenic
portions of the antigens comprising at least 7 contiguous amino
acids, (3) proteins comprising amino acid sequences which retain
immunogenicity and which are at least 95% identical to these SP
protein antigens (e.g., 95%, 96%, 97%, 98%, 99%, or 99.5%
identical), and (4) fusion proteins, including hybrid SP protein
antigens, comprising (1)-(3).
[1559] Alternatively, the invention may include an immunogenic
composition comprising a first and a second Gram positive bacteria
non-AI protein, wherein the polynucleotide sequence encoding the
sequence of the first non-AI protein is less than 90% (i.e., less
than 90, 88, 86, 84, 82, 81, 78, 76, 74, 72, 70, 65, 60, 55, 50,
45, 40, 35, or 30 percent) homologous than the corresponding
sequence in the genome of the second non-AI protein.
[1560] The compositions of the invention may further comprise one
or more additional non-Gram positive bacterial antigens, including
additional bacterial, viral or parasitic antigens. The compositions
of the invention may further comprise one or more additional
non-GBS antigens, including additional bacterial, viral or
parasitic antigens.
[1561] In another embodiment, the GBS antigen combinations of the
invention are combined with one or more additional, non-GBS
antigens suitable for use in a vaccine designed to protect elderly
or immunocomprised individuals. For example, the GBS antigen
combinations may be combined with an antigen derived from the group
consisting of Enterococcus faecalis, Staphylococcus aureus,
Staphylococcus epidermis, Pseudomonas aeruginosa, Legionella
pneumophila, Listeria monocytogenes, Neisseria meningitides,
influenza, and Parainfluenza virus (`PIV`).
[1562] Where a saccharide or carbohydrate antigen is used, it is
preferably conjugated to a carrier protein in order to enhance
immunogenicity {e.g. Ramsay et al. (2001) Lancet 357(9251):195-196;
Lindberg (1999) Vaccine 17 Suppl 2:S28-36; Buttery & Moxon
(2000) J R Coll Physicians Lond 34:163-168; Ahmad & Chapnick
(1999) Infect Dis Clin North Am 13:113-133, vii.; Goldblatt (1998)
J. Med. Microbiol. 47:563-567; European patent 0 477 508; U.S. Pat.
No. 5,306,492; International patent application WO98/42721;
Conjugate Vaccines (eds. Cruse et al.) ISBN 3805549326,
particularly vol. 10:48-114; and Hermanson (1996) Bioconjugate
Techniques ISBN: 0123423368 or 012342335X}. Preferred carrier
proteins are bacterial toxins or toxoids, such as diphtheria or
tetanus toxoids. The CRM.sub.197 diphtheria toxoid is particularly
preferred {Research Disclosure, 453077 (January 2002)}. Other
carrier polypeptides include the N. meningitidis outer membrane
protein (EP-A-0372501), synthetic peptides (EP-A-0378881;
EP-A-0427347), heat shock proteins (WO 93/17712; WO 94/03208),
pertussis proteins (WO 98/58668; EP A 0471177), protein D from H.
influenzae (WO 00/56360), cytokines (WO 91/01146), lymphokines,
hormones, growth factors, toxin A or B from C. difficile
(WO00/61761), iron-uptake proteins (WO01/72337), etc. Where a
mixture comprises capsular saccharides from both serogroups A and
C, it may be preferred that the ratio (w/w) of MenA saccharide:MenC
saccharide is greater than 1 (e.g. 2:1, 3:1, 4:1, 5:1, 10:1 or
higher). Different saccharides can be conjugated to the same or
different type of carrier protein. Any suitable conjugation
reaction can be used, with any suitable linker where necessary.
[1563] Toxic protein antigens may be detoxified where necessary
e.g. detoxification of pertussis toxin by chemical and/or genetic
means.
[1564] Where a diphtheria antigen is included in the composition it
is preferred also to include tetanus antigen and pertussis
antigens. Similarly, where a tetanus antigen is included it is
preferred also to include diphtheria and pertussis antigens.
Similarly, where a pertussis antigen is included it is preferred
also to include diphtheria and tetanus antigens.
[1565] Antigens in the composition will typically be present at a
concentration of at least 1 .mu.g/ml each. In general, the
concentration of any given antigen will be sufficient to elicit an
immune response against that antigen.
[1566] As an alternative to using protein antigens in the
composition of the invention, nucleic acid encoding the antigen may
be used {e.g. refs. Robinson & Torres (1997) Seminars in
Immunology 9:271-283; Donnelly et al. (1997) Annu Rev Immunol
15:617-648; Scott-Taylor & Dalgleish (2000) Expert Opin
Investig Drugs 9:471480; Apostolopoulos & Plebanski (2000) Curr
Opin Mol Ther 2:441-447; Ilan (I 999) Curr Opin Mol Ther 1:116-120;
Dubensky et al. (2000) Mol Med 6:723-732; Robinson & Pertmer
(2000) Adv Virus Res 55:1-74; Donnelly et al. (2000) Am J Respir
Crit Care Med 162(4 Pt 2):S 190-193; and Davis (1999) Mt. Sinai J.
Med. 66:84-90}. Protein components of the compositions of the
invention may thus be replaced by nucleic acid (preferably DNA e.g.
in the form of a plasmid) that encodes the protein.
Definitions
[1567] The term "comprising" means "including" as well as
"consisting" e.g. a composition "comprising" X may consist
exclusively of X or may include something additional e.g. X+Y.
[1568] The term "about" in relation to a numerical value x means,
for example, x.+-.10%.
[1569] References to a percentage sequence identity between two
amino acid sequences means that, when aligned, that percentage of
amino acids are the same in comparing the two sequences. This
alignment and the percent homology or sequence identity can be
determined using software programs known in the art, for example
those described in section 7.7.18 of Current Protocols in Molecular
Biology (F. M. Ausubel et al., eds., 1987) Supplement 30. A
preferred alignment is determined by the Smith-Waterman homology
search algorithm using an affine gap search with a gap open penalty
of 12 and a gap extension penalty of 2, BLOSUM matrix of 62. The
Smith-Waterman homology search algorithm is disclosed in Smith
& Waterman (1981) Adv. Appl. Math. 2: 482-489.
[1570] The invention is further illustrated, without limitation, by
the following examples.
EXAMPLE 1
Binding of an Adhesin Island Surface Protein, GBS 80, to Fibrinogen
and Fibronectin
[1571] This example demonstrates that an Adhesin Island surface
protein, GBS 80 can bind to fibrinogen and fibronectin.
[1572] An enzyme-linked immunosorbent assay (ELISA) was used to
analyse the in vitro binding ability of recombinant GBS 80 to
immobilized extra-cellular matrix (ECM) proteins but not to bovine
serum albumin (BSA). Microtiter plates were coated with ECM
proteins (fibrinogen, fibronectin, laminin, collagen type IV) and
binding assessed by adding varying concentrations of a recombinant
form of GBS 80, over-expressed and purified from E. coli (FIG. 5A).
Plates were then incubated sequentially with a) mouse anti-GBS 80
primary antibody; b) rabbit anti-mouse AP-conjugated secondary
antibody; c) pNPP colorimetric substrate. Relative binding was
measured by monitoring absorbance at 405 nm, using 595 nm as a
reference wavelength. FIG. 5b shows binding of recombinant GBS 80
to immobilized ECM proteins (1 .mu.g) as a function of
concentration of GBS 80. BSA was used as a negative control. Data
points represent the means of OD.sub.405 values.+-.standard
deviation for 3 wells.
[1573] Binding of GBS 80 to the tested ECM proteins was found to be
concentration dependent and exhibited saturation kinetics. As is
also evident from FIG. 5, binding of GBS 80 to fibronectin and
fibrinogen was greater than binding to laminin and collagen type IV
at all the concentrations tested.
EXAMPLE 2
GBS 80 is Required for Surface Localization of GBS 104
[1574] This example demonstrates that co-expression of GBS 80 is
required for surface localization of GBS 104.
[1575] The polycistronic nature of the Adhesin Island I mRNA was
investigated through reverse transcriptase-PCR (RT-PCR) analysis
employing primers designed to detect transcripts arising from
contiguous genes. Total RNA was isolated from GBS cultures grown to
an optical density at 600 nm (OD.sub.600) of 0.3 in THB
(Todd-Hewitt broth) by the RNeasy Total RNA isolation method
(Qiagen) according to the manufacturer's instructions. The absence
of contaminating chromosomal DNA was confirmed by failure of the
gene amplification reactions to generate a product detectable by
agarose gel electrophoresis, in the absence of reverse
transcriptase. RT-PCR analysis was performed with the Access RT-PCR
system (Promega) according to the manufacturer's instructions,
employing PCR cycling temperatures of 60.degree. C. for annealing
and 70.degree. C. for extension. Amplification products were
visualized alongside 100-bp DNA markers in 2% agarose gels after
ethidium bromide staining.
[1576] FIG. 5 shows that all the genes are co-transcribed as an
operon. A schematic of the AI-1 operon is shown above the agarose
gel analysis of the RT-PCR products. Large rectangular arrows
indicate the predicted transcript direction. Primer pairs were
selected such as "1-4" cross the 3'finish-5'start of successive
genes and overlap each gene by at least 200 bp. Additionally, "1"
crosses a putative rho-independent transcriptional terminator. "5"
is an internal GBS 80 control and "6" is an unrelated control from
a highly expressed gene. Lanes: "a": RNA plus RTase enzyme; "b" RNA
without RTase; "c": genomic DNA control.
[1577] In the effort to elucidate the functions of the AI-1
proteins, in frame deletions of all of the genes within the operon
have been constructed and the resulting mutants characterized with
respect to surface exposure of the encoded antigens (see FIG.
8).
[1578] Each in-frame deletion mutation was constructed by splice
overlap extension PCR(SOE-PCR) essentially as decribed by Horton et
al. [Horton R. M., Z. L. Cai, S. N. Ho, L. R. Pease (1990)
Biotechniques 8:528-35] using suitable primers and cloned into the
temperature sensitive shuttle vector pJRS233 to replace the wild
type copy by allelic exchange [Perez-Casal, J., J. A. Price, et al.
(1993) Mol Microbiol 8(5): 809-19.]. All plasmid constructions
utilized standard molecular biology techniques, and the identities
of DNA fragments generated by PCR were verified by sequencing.
Following SOE-PCR, the resulting mutant DNA fragments were digested
with XhoI and EcoRI, and ligated into a similarly digested pJRS233.
The resuting vectors were introduced by electroporation into the
chromosome of 2603 and COH1 GBS strains in a three-step process,
essentially as described in Framson et al. [Framson, P. E., A.
Nittayajarn, J. Merry, P. Youngman, and C. E. Rubens. (1997) Appl.
Environ. Microbiol. 63(9):353947]. Briefly, the vector pJRS233
contains an erm gene encoding erythromycin resistance and a
temperature-sensitive gram-positive replicon that is active at
30.degree. C. but not at 37.degree. C. Initially, the constructs
are electroporated into GBS electro-competent cells prepared as
described by Frameson et al., and transformants containing free
plasmid are selected by their ability to grow at 30.degree. C. on
Todd-Hewitt Broth (THB) agar plates containing 1 .mu.g/ml
erythromycin. The second step includes a selection step for strains
in which the plasmid has integrated into the chromosome via a
single recombination event over the homologous plasmid insert and
chromosome sequence by their ability to grow at 37.degree. C. on
THB agar medium containing 1 mg/ml erythromycin. In the third step,
GBS cells containing the plasmid integrated within the chromosome
(integrants) are serially passed in broth culture in the absence of
antibiotics at 30.degree. C. Plasmid excision from the chromosome
via a second recombination event over the duplicated target gene
sequence either completed the allelic exchange or reconstituted the
wild-type genotype. Subsequent loss of the plasmid in the absence
of antibiotic selection pressure resulted in an
erythromycin-sensitive phenotype. In order to assess gene
replacement a screening of erythromycin-sensitive colonies was
performed by analysis of the target gene PCR amplicons.
[1579] FIG. 7 reports a schematic of the IS-1 operon for each
knock-out strain generated, along with the deletion position within
the amino acidic sequence. Most data presented here concern the
COH1 deletion strains, in which the expression of each of the
antigens is higher by DNA microarray analysis (data not shown) as
well as detectable by FACS analysis (see FIG. 8). The double mutant
in 2603 .DELTA.80, .DELTA.104 double mutant was constructed by
sequential allelic exchanges of the shown alleles.
[1580] Immunization Protocol
[1581] Immune sera for FACS experiments were obtained as
follows.
[1582] Groups of 4 CD-1 outbred female mice 6-7 weeks old (Charles
River Laboratories, Calco Italy) were immunized with the selected
GBS antigens, (20 .mu.g of each recombinant GBS antigen), suspended
in 100 .mu.l of PBS. Each group received 3 doses at days 0, 21 and
35. Immunization was performed through intra-peritoneal injection
of the protein with an equal volume of Complete Freund's Adjuvant
(CFA) for the first dose and Incomplete Freund's Adjuvant (IFA) for
the following two doses. In each immunization scheme negative and
positive control groups are used. Immune response was monitored by
using serum samples taken on day 0 and 49.
[1583] FACS Analysis
[1584] Preparation of paraformaldehyde treated GBS cells and their
FACS analysis were carried out as follows.
[1585] GBS serotype COH1 strain cells were grown in Todd Hewitt
Broth (THB; Difco Laboratories, Detroit, Mich.) to OD600 nm=0.5.
The culture was centrifuged for 20 minutes at 5000 rpm and bacteria
were washed once with PBS, resuspended in PBS containing 0.05%
paraformaldehyde, and incubated for 1 hours at 37.degree. C. and
then overnight at 4.degree. C. 50 .mu.l of fixed bacteria (OD600
0.1) were washed once with PBS, resuspended in 20 .mu.l of Newborn
Calf Serum, (Sigma) and incubated for 20 min. at room temperature.
The cells were then incubated for 1 hour at 4.degree. C. in 100
.mu.l of preimmune or immune sera, diluted 1:200 in dilution buffer
(PBS, 20% Newborn Calf Serum, 0.1% BSA). After centrifugation and
washing with 200 .mu.l of washing buffer (0.1% BSA in PBS), samples
were incubated for 1 hour at 4.degree. C. with 50 .mu.l of
R-Phicoerytrin conjugated F(ab)2 goat anti-mouse IgG (Jackson
ImmunoResearch Laboratories; Inc.), diluted 1:100 in dilution
buffer. Cells were washed with 200 .mu.l of washing buffer and
resuspended in 200 .mu.l of PBS. Samples were analysed using a FACS
Calibur apparatus (Becton Dickinson, Mountain View, Calif.) and
data were analyzed using the Cell Quest Software (Becton
Dickinson). A shift in mean fluorescence intensity of >75
channels compared to preimmune sera from the same mice was
considered positive. This cutoff was determined from the mean plus
two standard deviations of shifts obtained with control sera raised
against mock purified recombinant proteins from cultures of E. coli
carrying the empty expression vector and included in every
experiment. Artifacts due to bacterial lysis were excluded using
antisera raised against 6 different known cytoplasmic proteins all
of which were negative
[1586] FACS data on COH1 single KO mutants for GBS 104 and GBS 80
indicated that GBS 80 is required for surface localization of GBS
104.
[1587] As shown in FIG. 8, GBS 104 is not surface exposed in the
.DELTA.80 strain (second column, bottom), but is present in the
whole protein extracts (see FIG. 10). Mean shift values suggest
that GBS 104 is partially responsible for GBS 80 surface exposure
(Mean shift of GBS 80 is reduced to .about.60% wild-type levels in
.DELTA.104), and that GBS 80 is over-expressed in the complemented
strain (mean shift value .about.200% wild-type level). The
.DELTA.80/pGBS 80 strain contains the GBS 80 orf cloned in the
shuttle-vector pAM401 (Wirth, R., F. Y. An, et al. (1986). J
Bacteriol 165(3): 831-6). The vector alone does not alter the
secretion pattern of GBS 104 (right column). FACS was performed on
mid-log fixed bacteria with mouse polyclonal antibodies as
indicated at left. Black peak is pre-immune sera, colored peaks are
sera from immunized animals.
EXAMPLE 3
Deletion of GBS 80 Causes Attenuation In Vivo
[1588] This example demonstrates that deletion of GBS 80 causes
attenuation in vivo, suggesting that this protein contributes to
bacterial virulence.
[1589] By using a mouse animal model, we studied the role of GBS 80
and GBS 104 in the virulence of S. agalactiae.
[1590] Groups of ten outbred female mice 5-6 week weeks old
(Charles River Laboratories, Calco Italy) were inoculated
intraperitoneally with different dilutions of the mutant strains
and LD50 (lethal dose 50) were calculated according to the method
of Reed and Muench [Reed, L. J. and H. Muench (1938). The American
Journal of Hygiene 27(3): 493-7]. As presented in the table below
the number of colony forming units (cfu) counted for both the
.DELTA.80 and the .DELTA.80, .DELTA.104 double mutants is about 10
fold higher when compared to the wild type strain suggesting that
inactivation of GBS 80 but not GBS 104 is responsible for an
attenuation in virulence. This finding indicates that GBS 80 gene
in the AI-1 might contribute to virulence. TABLE-US-00294 TABLE
Lethal dose 50% analysis of AI-1 mutants in the 2603 strain
background. LD.sub.50s were performed by IP injection of female CD1
mice at an age of 5-6 weeks. LD.sub.50s were calculated by the
method of Reed and Muench (8). GBS strain LD.sub.50, cfu Number of
Experiments Wild Type 2603 2 .times. 10.sup.8 4 .DELTA.104 mutant
.about.2 .times. 10.sup.8 1 .DELTA.80 mutant 2.6 .times. 10.sup.9 3
.DELTA.80, .DELTA.104 double mutant .about.2 .times. 10.sup.9 1
EXAMPLE 4
Effect of Adhesin Island Sortase Deletions on Surface Antigen
Presentation
[1591] This example demonstrates the effect of adhesin island
sortase deletions on surface antigen presentation.
[1592] FACS analysis results set forth in FIG. 9 show that a
deletion in sortase SAG0648 prevented GBS 104 from reaching the
surface and slightly reduced the surface exposure of GBS 80 (fourth
panel; mean shift value .about.60% wild-type COH1). In the double
sortase knock-out strain, neither antigen was surface exposed (far
right panel). Either sortase alone was sufficient for GBS 80 to
arrive at the bacterial surface (third and fourth columns, top). No
effect was seen on surface exposure of antigens GBS 80 or GBS 104
in the .DELTA.GBS 52 strain. Antibodies derived from purified GBS
52 were either non-specific or were FACS negative for GBS 52 (data
not shown). FACS analysis was performed as described above (see
EXAMPLE 2).
[1593] As shown in FIG. 10, inactivation of GBS 80 has no effect on
GBS 104 expression as much as GBS 104 knock out doesn't change the
total amount GBS 80 expressed. The Western blot of whole protein
extracts (strains noted above lanes) probed with anti-GBS 80
antisera is shown in panel A. Arrow indicates expected size of GBS
80 (60 kDa). GBS 80 antibodies recognize a doublet, the lower band
is not present in .DELTA.GBS 80 strains. Panel B shows a Western
blot of whole protein extracts probed with anti-GBS 104 antisera.
Arrow indicates expected size of GBS 104 (99.4 kDa). Protein
extracts were prepared from the same bacterial cultures used for
FACS (FIGS. 8 and 9). In conclusion, although GBS 104 does not
arrive at the surface in the .DELTA.80 strain by FACS (FIG. 8,
second column), it is present at approximately wild-type levels in
the whole protein preps (B, second lane). Approximately 20 .mu.g of
each protein extract was loaded per lane.
[1594] Western-Blot Analysis
[1595] Aliquots of total protein extract mixed with SDS loading
buffer (1.times.:60 mM TRIS-HCl pH 6.8, 5% w/v SDS, 10% v/v
glycerin, 0.1% Bromophenol Blue, 100 mM DTT) and boiled 5 minutes
at 95.degree. C., were loaded on a 12.5% SDS-PAGE precast gel
(Biorad). The gel is run using a SDS-PAGE running buffer containing
250 mM TRIS, 2.5 mM Glycine and 0.1% SDS. The gel is electroblotted
onto nitrocellulose membrane at 200 mA for 60 minutes. The membrane
is blocked for 60 minutes with PBS/0.05% Tween-20 (Sigma), 10%
skimmed milk powder and incubated O/N at 4.degree. C. with
PBS/0.05% Tween 20, 1% skimmed milk powder, with the appropriate
dilution of the sera. After washing twice with PBS/0.05% Tween, the
membrane is incubated for 2 hours with peroxidase-conjugated
secondary anti-mouse antibody (Amersham) diluted 1:4000. The
nitrocellulose is washed three times for 10 minutes with PBS/0.05%
Tween and once with PBS and thereafter developed by Opti-4CN
Substrate Kit (Biorad).
EXAMPLE 5
Binding of Adhesin Island Proteins to Epithelial Cells and Effect
of Adhesin Island Proteins on Capacity of GBS to Adhere to
Epithelial Cells
[1596] This example illustrates the binding of AI proteins to
epithelial cells and the effect of AI proteins on the capacity of
GBS to adhere to epithelial cells.
[1597] Applicants analysed whether recombinant AI surface proteins
GBS 80 or GBS 104 would demonstrate binding to various epithelial
cells in a FACS analysis. Applicants also analysed whether deletion
of AI surface proteins GBS 80 or GBS 104 would effect the capacity
of GBS to adhere to and invade ME180 cervical epithelial cells.
[1598] As shown in FIG. 28, deletion of GBS 80 sequence from GBS
strain isolate 2603 (serotype V) did not affect the capacity of the
mutated GBS to adhere to and invade ME180 cervical epithelial
cells. Here ME180 cervical carcinoma epithelial cells were infected
with wild type GBS 2603 or GBS 2603 .DELTA.80 isogenic mutant.
After two hours of infection, non-adherent bacteria were washed off
and infection prolonged for a further two hours and four hours. In
invasion experiments, after each time point, was followed by a two
hour antibiotic treatment. Cells were then lysed with 1% saponin
and lysates platedon TSA plates. As shown in FIG. 28, there was
little difference between the percent invasion or percent adhesion
of wild type and mutant strains up to the four hour time point.
[1599] FIG. 30 repeats this experiment with both .DELTA.104 and
.DELTA.80 mutants from a different strain isolate. Here, ME180
cervical carcinoma epithelial cells were infected with GBS strain
isolate COH (serotype III) wild type or COH1 .DELTA.GBS 104 or COH1
.DELTA.80 isogenic mutant. After one hour of infection,
non-adherent bacteria were washed off and the cells were lysed with
1% saponin. The lysates were plated on TSA plates. As shown in FIG.
30, while there was little difference in the percent invasion,
there was a significant decrease in the percent association of the
.DELTA.104 mutant compared to both the wild type and .DELTA.80
mutant.
[1600] The affect of AI surface proteins on the ability of GBS to
translocate through an epithelial monolayer was also analysed. As
shown in FIG. 31, a GBS 80 knockout mutant strain partially loses
the ability to translocate through an epithelial monolayer. Here
epithelial monolayers were inoculated with wildtype or knockout
mutant in the apical chamber of a transwell system for two hours
and then non-adherent bacteria were washed off. Infection was
prolonged for a further two and four hours. Samples were taken from
the media of the basolateral side and the number of colony forming
unties measured. Transepithelial electrical resistance measured
prior to and after infection gave comparable values, indicating the
maintenance of the integrity of the monolayer. By the six hour time
point, the .DELTA.80 mutants demonstrated a reduced percent
transcytosis.
[1601] A similar experiment was conducted with GBS 104 knock out
mutants. Here, as shown in FIG. 22, the .DELTA.104 mutants also
demonstrated a reduced percent transcytosis, indicating that the
mutant strains translocate through an epithelial monolayer less
efficiently than their isogenic wild type counterparts.
[1602] Applicants also studied the effect of AI proteins on the
capacity of a GBS strain to invade J774 macrophage-like cells.
Here, J774 cells were infected with GBS COH1 wild type or COH1
.DELTA.GBS 104 or COH1 .DELTA.GBS80 isogenic mutants. After one
hour of infection, non-adherent bacteria were washed off and
intracellular bacteria were recovered at two, four and six hours
post antibiotic treatment. At each time point, cells were lysed
with 0.25% Triton X-100 and lysates plated on TSA plates. As shown
in FIG. 32, the .DELTA.104 mutant demonstrated a significantly
reduced percent invasion compared to both the wild type and
.DELTA.80 mutant.
EXAMPLE 6
Hyperoligomeric Structures Comprising AI Surface Proteins GBS 80
and GBS 104
[1603] This example illustrates hyperoligomeric structures
comprising AI surface proteins GBS 80 and GBS 104. A GBS isolate
COH1 (serotype III) was adapted to increase expression of GBS 80.
FIG. 34 presents a regular negative stain electron micrograph of
this mutant; no pilus or hyperoligomeric structures are
distinguishable on the surface of the bacteria. When the EM stain
is based on anti-GBS 80 antibodies labelled with 10 or 20 nm gold
particles, the presence of GBS 80 throughout the hyperoligomeric
structure is clearly indicated (FIGS. 36, 37 and 38). EM staining
against GBS 104 (anti-GBS 104 antibodies labelled with 10 nm gold
particles) also reveals the presence of GBS 104 primarily on or
near the surface of the bacteria or potentially associated with
bacterial peptidoglycans (FIG. 39). Analysis of this same strain
(over-expressing GBS 80) with a combination of both anti-GBS 80
(using 20 nm gold particles) and anti-GBS 104 (using 10 nm gold
particles) reveals the presence of GBS 104 on the surface and
within the hyperoligomeric structures (see FIGS. 40 and 41).
EXAMPLE 7
GBS 80 is Necessary for Polymer Formation and GBS 104 and Sortase
SAG0648 are Necessary for Efficient Pili Assembly
[1604] This example demonstrates that GBS 80 is necessary for
formation of polymers and that GBS 104 and sortase SAG0648 are
necessary for efficient pili assembly. GBS 80 and GBS 104 polymeric
assembly was systematically analyzed in Coh1 strain single knock
out mutants of each of the relevant coding genes in AI-1 (GBS 80,
GBS 104, GBS 52, sag0647, and sag0648). FIG. 41 provides Western
blots of total protein extracts (strains noted above lanes) probed
with either anti-GBS 80 (left panel) sera or anti-GBS 104 sera
(right panel) for each of these Coh1 and Coh1 knock out strains.
(Coh1, wild type Coh1; .DELTA.80, Coh1 with GBS 80 knocked out;
.DELTA.104, Coh1 with GBS 104 knocked out; .DELTA.52, Coh1 with GBS
52 knocked out; A647, Coh1 with SAG0647 knocked out; A648, Coh1
with SAG0648 knocked out, A647-8, Coh1 with SAG0647 and SAG0648
knocked out; .DELTA.80/pGBS80, Coh1 with GBS 80 knocked out but
complemented with a high copy number plasmid expressing GBS 80.
Asterisks identify the monomer of GBS 80 and GBS 104.)
[1605] The smear of immunoreactive material observed in the wild
type strain, along with its disappearance in .DELTA.80 and
.DELTA.104 mutants, is consistent with the notion that such high
molecular weight structures are composed of covalently linked
(SDS-resistant) GBS 80 and GBS 104 subunits. The immunoblotting
with both anti-GBS 80 (.alpha.-GBS 80) and anti-GBS 104
(.alpha.-GBS 104) revealed that deletion of sortase SAG0648 also
interferes with the assembly of high molecular weight species,
whereas the knock out mutant of the second sortase (SAG0647), even
if somehow reduced, still maintains the ability to form polymeric
structures.
[1606] Total extracts form GBS were prepared as follows. Bacteria
were grown in 50 ml of Todd-Hewitt broth (Difco) to an OD.sub.600nm
of 0.5-0.6 and successively pelleted. After two washes in PBS the
pellet was resuspended and incubated 3 hours at 37.degree. C. with
mutanolisin. Cells were then lysed with at least three
freezing-thawing cycles in dry ice and a 37.degree. C. bath. The
lysate was then centrifuged to eliminate the cellular debris and
the supernatant was quantified. Approximately 40 .mu.g of each
protein extract was separated on SDS-PAGE. The gel was then
subjected to immunoblotting with mice antisera and detected with
chemiluminescence.
EXAMPLE 8
GBS 80 is Polymerized by an AI-2 Sortase
[1607] This example illustrates that GBS 80 can be polymerized not
only by AI-1 sortases, but also by AI-2 sortases. FIG. 42 shows
total cell extract immunoblots of GBS 515 strain, which lacks AI-1.
The left panel, where an anti-GBS 67 sera was used, shows that GBS
67 from AI-2 is assembled into high-molecular weight-complexes,
suggesting the formation of a second type of pilus. The same high
molecular structure is observed when GBS 80 is highly expressed by
reintroducing the gene within a plasmid (pGBS 80). By using
anti-GBS 80 (right panel) sera on the same extracts, again it is
observed that, with GBS 80 over expression (515/pGBS 80), a
high-molecular weight structure is assembled. This implies that, in
the absence of AI-1 sortases, AI-2 sortases (SAG1405 and SAG1406)
can complement the lacking function, still being able to assemble
GBS 80 in a pilus structure.
EXAMPLE 9
Coh1 Produces a High Molecular Weight Molecule, the GBS 80
Pilin
[1608] This example illustrates that Coh1 produces a high molecular
weight molecule, greater than 1000 kDa, which is the GBS 80 pilin.
FIG. 43 provides silver-stained electrophoretic gels that show that
Coh1 produces two macromolecules. One of these macromolecules
disappears in the Coh1 GBS 80 knock out cells, but does not
disappear in the Coh1 GBS 52 knock out mutant cells. The last two
lanes on the right were loaded with 15 times the amount loaded in
the other lanes. This was done in order to be able to count the
bands. By doing this, a conservative size estimate of the top bands
was calculated by starting at 240 kDa and considering each of 14
higher bands as the result of consecutive additions of a GBS 80
monomer.
[1609] Coh1, wild type Coh1; .DELTA.80, Coh1 cells with GBS 80
knocked out; .DELTA.52, Coh1 cells with GBS 52 knocked out;
.DELTA.80/pGBS 80, Coh1 cells with GBS 80 knocked out and
complemented with a high copy number construct expressing GBS
80.
EXAMPLE 10
GBS 52 is a Minor Component of the GBS Pilus
[1610] This example illustrates that GBS 52 is present in the GBS
pilus and is a minor component of the pilus. FIG. 45 shows an
immunoblot of total cell extracts from a GBS Coh1 strain and a GBS
Coh1 strain knocked out for GBS 52 (.DELTA.52). The total cell
extracts were immunoblotted anti-GBS 80 antisera (left) and
anti-GBS 52 antisera (right). Immunoblotting was performed using a
3-8% Tris-acetate polyacrylamide gel (Invitrogen) which provided
excellent separation of large molecular weight proteins (see FIG.
41). When the gel was incubated with anti-GBS 80 sera, the bands
from the Coh1 wild-type strain appeared shifted when compared to
the .DELTA.52 mutant. This observation indicated a different size
of the pilus polymeric components in the two strains. When the same
gel was stripped and incubated with anti-GBS 52 sera the
high-molecular subunits in the Coh1 wild-type strain showed similar
molecular size of those in the correspondent lane in the left
panel. These findings confirmed that GBS 52 is indeed associated
with GBS 80 macro-molecular structures but represents a minor
component of the GBS pilus.
EXAMPLE 11
Pilus Structures are Present in the Supernatant of GBS Bacterial
Cultures
[1611] This example illustrates that the pilus structure assembled
in Coh1 GBS is present in the supernatant of a bacterial cell
culture. FIG. 46 shows an immunoblot where the protein extract of
the supernatant from cultures of different GBS mutant strains
(117=Coh1 GBS 80 knockout; 159=Coh1 GBS 104 knockout; 202=Coh1 GBS
52 knockout; 206=Coh1 GBS sag0647 knockout; 208=Coh1 GBS sag0648
knockout; 197=Coh1 GBS sag0647/sag0648 knockout; 179=Coh1 GBS 80
knockout complemented with a high copy plasmid expressing GBS 80).
GBS 80 antisera detects the presence of pilus structures in the
appropriate Coh1 strains.
[1612] The protein extract was prepared as follows. Bacteria were
grown in THB to an OD.sub.600nm of 0.5-0.6 and the supernatant was
separated from the cells by centrifugation. The supernatant was
then filtered (527 0.2 .mu.m) and 1 ml was added with 60% TCA for
protein precipitation.
[1613] GBS pili were also extracted from the fraction of
surface-exposed proteins in Coh1 strain and its GBS 80 knock out
mutant as described hereafter. Bacteria were grown to an
OD.sub.600nm of 0.6 in 50 ml of THB at 37.degree. C. Cells were
washed once with PBS and the pellet was then resuspended in 0.1 M
KPO4 pH 6.2, 40% sucrose, 10 mM MgCl2, 400 U/ml mutanolysin and
incubated 3 hours at 37.degree. C. Protoplasts were separated by
centrifugation and the supernatant was recovered and its protein
content measured.
[1614] In order to study the dynamics of pilus production during
different growth phases, 1 ml supernatant of a culture at different
OD.sub.600nm was TCA precipitated and loaded onto a 3-8% SDS-PAGE
as described before. FIG. 47 shows the corresponding Western blot
with GBS 80 anti-sera. The first group of lanes (left five sample
lanes) refer to a Coh1 strain growth (OD.sub.600nm are noted above
the lanes) whereas the second group of lanes (right five samples)
are from a GBS 80 knock out strain over expressing GBS 80. The
experiment shows that pilus macromolecular structures can be found
in the supernatant in all of the growth phases tested.
EXAMPLE 12
In GBS Strain Coh1, only GBS 80 and a Sortase (sag0647 or sag0648)
is Required for Polymerization
[1615] This example describes requirements for pilus formation in
Coh1. FIG. 48 shows a Western blot of total protein extracts
(prepared as described before) using anti-GBS 80 sera on Coh1
clones. (Coh1, wild type Coh1; .DELTA.104, Coh1 knocked out for GBS
104, .DELTA.647, Coh1 knocked out for sag0647, .DELTA.648, Coh1
knocked for sag0648, .DELTA.647-8, Coh1 knocked out for sag0647 and
sag0648; 515, wild type bacterial strain 515, which lacks an AI-1;
p80 a high copy number plasmid which expresses GBS 80.) The data
show that only the double sortase mutant is unable to polymerize
GBS 80 indicating that the `conditio sine qua non` for pilus
polymerization is the co-existence of GBS 80 with at least one
sortase. This result leads to a reasonable assumption that SAG1405
and SAG1406 are responsible for polymerization in this strain.
EXAMPLE 13
GBS 80 can be Expressed in L. lactis Under its Own Promoter and
Terminator Sequences
[1616] This example demonstrates that L. lactis, a non-pathogenic
bacterium, can express GBS AI polypeptides such as GBS 80. L.
lactis M1363 (J. Bacteriol. 154 (1983):1-9) was transformed with a
construct encoding GBS 80. Briefly, the construct was prepared by
cloning a DNA fragment containing the gene coding for GBS 80 under
its own promoter and terminator sequences into plasmid pAM401 (a
shuttle vector for E. coli and other Gram positive bacteria; J.
Bacteriol. 163 (1986):831-836). Total extracts of the transformed
bacteria in log phase were separated on SDS-PAGE, transferred to
membranes, and incubated with antiserum against GBS 80. A
polypeptide corresponding to the molecular weight of GBS 80 was
detected in the lanes containing total extracts of L. lactis
transformed with the GBS 80 construct. See FIGS. 133A and 133B,
lanes 6 and 7. This same polypeptide was not detected in the lane
containing total extracts of L. lactis not transformed with the GBS
80 construct, lane 9. This example shows that L. lactis can express
GBS 80 under its own promoter and terminator.
EXAMPLE 14
L. lactis Modified to Express GBS AI-1 Under the GBS 80 Promoter
and Terminator Sequences Expresses GBS 80 in Polymeric
Structures
[1617] This example demonstrates the ability of L. lactis to
express GBS AI-1 polypeptides and to incorporate at least some of
the polypeptides into oligomers. L. lactis was transformed with a
construct containing the genes encoding GBS AI-1 polypeptides.
Briefly, the construct was prepared by cloning a DNA fragment
containing the genes for GBS 80, GBS 52, SAG0647, SAG0648, and GBS
104 under the GBS 80 promoter and terminator sequences into
construct pAM401. The construct was transformed into L. lactis
M1363. Total extracts of log phase transformed bacteria were
separated on reducing SDS-PAGE, transferred to membranes, and
incubated with antiserum against GBS 80. A polypeptide with a
molecular weight corresponding to the molecular weight of GBS 80
was detected in the lanes containing L. lactis transformed with the
GBS AI-1 encoding construct. See FIG. 134, lane 2. In addition, the
same lane also showed immunoreactivity of polypeptides having
higher molecular weights than the polypeptide having the molecular
weight of GBS 80. These higher molecular weight polypeptides are
likely oligomers of GBS 80. Oligomers of similar molecular weights
were also observed on a Western blot of the culture supernatant of
the transformed L. lactis. See lane 4 of FIG. 135. Thus, this
example shows that L. lactis transformed to express GBS AI-1 can
efficiently polymerize GBS 80 in the form of a pilus. This pilus
structure can likely be purified from either the cell culture
supernatant or cell extracts.
EXAMPLE 15
Cloning and Expression of S. pneumoniae Sp0462
[1618] This example describes the production of a clone encoding a
Sp0462 polypeptide and expression of the clone. To produce a clone
encoding Sp0462, the open reading frame encoding Sp0462 was
amplified using primers that annealed within the full-length Sp0462
open reading frame sequence. FIG. 150A provides a 893 amino acid
sequence of Sp0462. The primers used to produce a clone encoding
the Sp0462 polypeptide are shown in FIG. 150B. These primers
annealed to the nucleotide sequences encoding the amino acid
residues indicated by underlining in FIG. 150A. Amplification of
the open reading frame encoding Sp0462 using these primers produced
the amplicon shown at lane 2 of the agarose gel provided in FIG.
160. The Sp0462 clone encodes amino acid residues 38-862 of the 893
amino acid residue Sp0462 protein; the italicized residues in FIG.
150A were eliminated. FIG. 151A provides a schematic depiction of
the recombinant Sp0462 polypeptide. FIG. 151B shows a schematic
depiction of the full-length Sp0462 polypeptide. Both the
recombinant Sp0462 encoded by the clone and the full-length Sp0462
protein have two collagen binding protein type B (Cna B) domains
and a von Hillebrand factor A (vWA) domain. The cloned recombinant
Sp0462 lacks the LPXTG motif present in the full-length Sp0462
protein. Western blot analysis for expression of the Sp0462 clone
did not result in detection of polypeptides with serum obtained
from S. pneumoniae-infected patients (FIG. 152A) or GBS 80
antiserum (FIG. 152B).
EXAMPLE 16
Cloning and Expression of S. pneumoniae Sp0463
[1619] This example describes the production of a clone encoding a
Sp0463 polypeptide and detection of recombinant Sp0463 polypeptide
expressed from the clone. To produce a clone encoding Sp0463, the
open reading frame encoding Sp0463 was amplified using primers that
annealed within the full-length Sp0463 open reading frame sequence.
FIG. 153A provides a 665 amino acid sequence of Sp0463. The primers
used to produce the clone encoding Sp0463 polypeptide are shown in
FIG. 153B. These primers annealed to the nucleotide sequences
encoding the amino acid residues indicated by underlining in FIG.
153A. Amplification of the open reading frame encoding Sp0463 using
these primers produced the amplicon shown at lane 3 of the agarose
gel provided in FIG. 160. The Sp0463 clone encodes amino acid
residues 23-627 of the 665 amino acid residue Sp0463 protein; the
italicized residues in FIG. 153A were eliminated. FIG. 154A
provides a schematic depiction of the recombinant Sp0463
polypeptide. FIG. 154B shows a schematic depiction of the
full-length Sp0463 polypeptide. Both the recombinant Sp0463 encoded
by the clone and the full-length Sp0463 protein have a Cna B domain
and an E box motif. The cloned recombinant Sp0463 lacks the LPXTG
motif present in the full-length Sp0463 protein. Expression of the
Sp0463 clone resulted in the detection of a 60 kD polypeptide, the
expected molecular weight of the recombinant Sp0463 polypeptide, by
Western blot analysis. See FIG. 155.
EXAMPLE 17
Cloning and Expression of S. pneumoniae Sp0464
[1620] This example describes the production of a clone encoding a
Sp0464 polypeptide and detection of recombinant Sp0464 polypeptide
expressed from the clone. To produce a clone encoding Sp0464, the
open reading frame encoding Sp0464 was amplified using primers that
annealed either within the full-length Sp0464 open reading frame
sequence. FIG. 157A provides a 393 amino acid sequence of Sp0464.
The primers used to produce a clone encoding the Sp0464 polypeptide
are shown in FIG. 157B. These primers annealed to the nucleotide
sequences encoding the amino acid residues indicated by underlining
in FIG. 157A. Amplification of the open reading frame encoding
Sp0464 using these primers produced the amplicon shown at lane 4 of
the agarose gel provided in FIG. 160. The Sp0464 clone encodes
amino acid residues 19-356 of the 393 amino acid residue Sp0464
protein; the italicized residues in FIG. 157A were eliminated. FIG.
158A provides a schematic depiction of the recombinant Sp0464
polypeptide. FIG. 158B shows a schematic depiction of the
full-length Sp0464 polypeptide. Both the recombinant Sp0464 encoded
by the clone and the full-length Sp0464 protein have two Cna B
domains. The cloned recombinant Sp0464 lacks the LPXTG motif
present in the full-length Sp0464 protein. Expression of the Sp0464
clone resulted in the detection of a 38 kD polypeptide, the
expected molecular weight of the recombinant Sp0464 polypeptide, by
Western blot analysis. See FIG. 159.
EXAMPLE 18
Intranasal Immunization of Mice with Recombinant L. lactis
Expressing GBS 80 and Subsequent Challenge
[1621] This example describes a method of intranasally immunizing
mice using L. lactis that express GBS 80. Intranasal immunization
consisted of 3 doses at days 0, 14 and 28, each dose administered
in three consecutive days. Each day, groups of 3 CD-1 outbred
female mice 6-7 weeks old (Charles River Laboratories, Calco Italy)
were immunized intranasally with 10.sup.9 or 10.sup.10 CFU of the
recombinant Lactococcus lactis suspended in 20 .mu.l of PBS. In
each immunization scheme negative (wild-type L. lactis) and
positive (recombinant GBS80) control groups were used. The immune
response of the dams was monitored by using serum samples taken on
day 0 and 49. The female mice were bred 2-7 days after the last
immunization (at approximately t=36-37), and typically had a
gestation period of 21 days. Within 48 hours of birth, the pups
were challenged via I.P. with GBS in a dose approximately equal to
an amount which would be sufficient to kill 90% of immunized pups
(as determined by empirical data gathered from PBS control groups).
The GBS challenge dose is preferably administered in 50 ml of THB
medium. Preferably, the pup challenge takes place at 56 to 61 days
after the first immunization. The challenge inocula were prepared
starting from frozen cultures diluted to the appropriate
concentration with THB prior to use. Survival of pups was monitored
for 5 days after challenge.
EXAMPLE 19
Subcutaneous Immunization of Mice with Recombinant L. lactis
Expressing GBS 80 and Subsequent Challenge
[1622] This example describes a method of subcutaneous immunization
mice using L. lactis that express GBS 80. Subcutaneous immunization
consists of 3 doses at days 0, 14 and 28. Groups of 3 CD-1 outbred
female mice 6-7 weeks old (Charles River Laboratories, Calco Italy)
were injected subcutaneously with 10.sup.9 or 10.sup.10 CFU of the
recombinant Lactococcus lactis suspended in 100 .mu.l of PBS. In
each immunization scheme, negative (wild-type L. lactis) and
positive (recombinant GBS80) control groups were used. The immune
response of the dams was monitored by using serum samples taken on
day 0 and 49. The female mice were bred 2-7 days after the last
immunization (at approximately t=36-37), and typically had a
gestation period of 21 days. Within 48 hours of birth, the pups
were challenged via I.P. with GBS in a dose approximately equal to
an amount which would be sufficient to kill 90% of immunized pups
(as determined by empirical data gathered from PBS control groups).
The GBS challenge dose is preferably administered in 50 ml of THB
medium. Preferably, the pup challenge takes place at 56 to 61 days
after the first immunization. The challenge inocula were prepared
starting from frozen cultures diluted to the appropriate
concentration with THB prior to use. Survival of pups was monitored
for 5 days after challenge.
EXAMPLE 20
Immunization of Mice with GAS AI Polypeptides and Subsequent
Intranasal Challenge
[1623] This example describes a method of immunizing mice with GAS
AI polypeptides and subsequently intranasally challenging the mice
with GAS bacteria. Groups of 10 CD1 female mice aged between 6 and
7 weeks are immunized with a combination of GAS antigens of the
invention GAS 15, GAS 16, and GAS 18, (15 .mu.g of each recombinant
antigen, derived from M1 strain SF370) or L. lactis expressing the
M1 strain SF370 adhesin island, suspended in 100 .mu.l of suitable
solution. Each group receives 3 doses at days 0, 21 and 45.
Immunization is performed through subcutaneous or intraperitoneal
injection for the GAS 15, GAS 16, GAS 18 protein combination. The
protein combination is administered with an equal volume of
Complete Freund's Adjuvant (CFA) for the first dose and Incomplete
Freund's Adjuvant (IFA) for the following two doses. Immunization
is performed intranasally for the L. lactis expressing the M1
strain SF370 adhesin island. In each immunization scheme negative
and positive control groups are used.
[1624] The negative control group for the mice immunized with the
GAS 15, GAS 16, GAS 18 protein combination included mice immunized
with PBS. The negative control group for the mice immunized with L.
lactis expressing the M1 strain SF370 adhesin island, included mice
immunized with either wildtype L. lactis or L. lactis transformed
with the pAM401 expression vector lacking any cloned adhesin island
sequence.
[1625] The positive control groups included mice immunized with
purified M1 strain SF370 M protein.
[1626] Immunized mice are then anaesthetized with Zoletil and
challenged intranasally with a 25 .mu.L suspension containing
1.2.times.10.sup.6 or 1.2.times.10.sup.8 CFU of ISS 3348 in THB.
Animals are observed daily and checked for survival.
EXAMPLE 21
Active Maternal Immunization Assay
[1627] As used herein, an Active Maternal Immunization assay refers
to an in vivo protection assay where female mice are immunized with
the test antigen composition. The female mice are then bred and
their pups are challenged with a lethal dose of GBS. Serum titers
of the female mice during the immunization schedule are measured as
well as the survival time of the pups after challenge.
Mouse Immunization
[1628] Specifically, groups of 4 CD-1 outbred female mice 6-8 weeks
old (Charles River Laboratories, Calco Italy) are immunized with
one or more GBS antigens, (20 .mu.g of each recombinant GBS
antigen), suspended in 100 .mu.l of PBS. Each group receives 3
doses at days 0, 21 and 35. Immunization is performed through
intra-peritoneal injection of the protein with an equal volume of
Complete Freund's Adjuvant (CFA) for the first dose and Incomplete
Freund's Adjuvant (IFA) for the following two doses. In each
immunization scheme negative and positive control groups are
used.
Immune response is monitored by using serum samples taken on day 0
and 49. The sera are analyzed as pools from each group of mice.
Active Maternal Immunization
[1629] A maternal immunization/neonatal pup challenge model of GBS
infection was used to verify the protective efficacy of the
antigens in mice. The mouse protection study was adapted from
Rodewald et al. (Rodewald et al. J. Infect. Diseases 166, 635
(1992)). In brief, CD-1 female mice (6-8 weeks old) were immunized
before breeding, as described above. The mice received 20 .mu.g of
protein per dose when immunized with a single antigen and 60 .mu.g
of protein per dose (15 .mu.g of each antigen) when immunized with
the combination of antigens. Mice were bred 2-7 days after the last
immunization. Within 48 h of birth, pups were injected
intraperitoneally with 50 .mu.l of GBS culture. Challenge inocula
were prepared starting from frozen cultures diluted to the
appropriate concentration with THB before use. In preliminary
experiments (not shown), the challenge doses per pup for each
strain tested were determined to cause 90% lethality. Survival of
pups was monitored for 2 days after challenge. Protection was
calculated as (percentage deadControl minus percentage deadVaccine)
divided by percentage deadControl multiplied by 100. Data were
evaluated for statistical significance by Fisher's exact test.
EMBODIMENTS OF THE INVENTION
[1630] The invention encompasses, but is not limited to, the
embodiments enumerated below.
[1631] 1. An immunogenic composition comprising a purified Group B
Streptococcus (GBS) adhesin island (AI) polypeptide in oligomeric
form.
[1632] 2. The immunogenic composition of embodiment 1 wherein the
GBS AI polypeptide is selected from a GBS AI-1.
[1633] 3. The immunogenic composition of embodiment 1 wherein the
GBS AI polypeptide is selected from a GBS AI-2.
[1634] 1. An immunogenic composition comprising a purified Group B
Streptococcus (GBS) adhesin island (AI) polypeptide in oligomeric
form.
[1635] 2. The immunogenic composition of embodiment 1 wherein the
GBS AI polypeptide is selected from a GBS AI-1.
[1636] 3. The immunogenic composition of embodiment 1 wherein the
GBS AI polypeptide is selected from a GBS AI-2.
[1637] 4. The immunogenic composition of any of embodiments 1-3
wherein the GBS AI polypeptide comprises a sortase substrate
motif.
[1638] 5. The immunogenic composition of embodiment 4 wherein the
sortase substrate motif is an LPXTG motif.
[1639] 6. The immunogenic composition of embodiment 5 wherein the
LPXTG motif is represented by the amino acid sequence XPXTG,
wherein the X at amino acid position 1 is an L, an I, or an F and
the X at amino acid position 3 is any amino acid residue.
[1640] 7. The immunogenic composition of any one of embodiments 1-3
wherein the GBS AI polypeptide affects the ability of GBS bacteria
to adhere to epithelial cells.
[1641] 8. The immunogenic composition of any one of embodiments 1-3
wherein the GBS AI polypeptide affects the ability of GBS bacteria
to invade epithelial cells.
[1642] 9. The immunogenic composition of any one of embodiments 1-3
wherein the GBS AI polypeptide affects the ability of GBS bacteria
to translocate through an epithelial cell layer.
[1643] 10. The immunogenic composition of any one of embodiments
1-3 wherein the GBS AI polypeptide is capable of associating with
an epithelial cell surface.
[1644] 11. The immunogenic composition of embodiment 10 wherein the
associating with an epithelial cell surface is binding to the
epithelial cell surface.
[1645] 12. The immunogenic composition of any of embodiments 1-3
wherein the GBS AI polypeptide is a full-length GBS AI protein.
[1646] 13. The immunogenic composition of any of embodiments 1-3
wherein the GBS AI polypeptide is a fragment of a full-length GBS
AI protein.
[1647] 14. The immunogenic composition of embodiment 13 wherein the
fragment comprises at least 7 contiguous amino acid residues of the
GBS AI protein.
[1648] 15. The immunogenic composition of embodiment 2 wherein the
GBS AI polypeptide is selected from the group consisting of GBS 80,
GBS 104, GBS 52, and fragments thereof.
[1649] 16. The immunogenic composition of embodiment 3 wherein the
GBS AI polypeptide is selected from the group consisting of GBS 59,
GBS 67, GBS 150, 01521, 01523, 01524, and fragments thereof.
[1650] 17. The immunogenic composition of embodiment 15 wherein the
GBS AI polypeptide is GBS 80.
[1651] 18. The immunogenic composition of any of embodiments 1-3 or
15-17 wherein the oligomeric form is a hyperoligomer.
[1652] 19. The immunogenic composition of any of embodiments 1-3,
or 15-17 further comprising a Gram positive bacterium antigen not
associated with an AI.
[1653] 20. The immunogenic composition of embodiment 19 wherein the
antigen is selected from the group consisting of GBS 322 and GBS
276.
[1654] 21. The immunogenic composition of embodiment 20 wherein the
antigen is GBS 322.
[1655] 22. An immunogenic composition comprising a purified Gram
positive bacteria adhesin island (AI) polypeptide in an oligomeric
form.
[1656] 23. The immunogenic composition of embodiment 22 wherein the
Gram positive bacteria is of a genus selected from the group
consisting of Streptococcus, Enterococcus, Staphylococcus, or
Listeria.
[1657] 24. The immunogenic composition of embodiment 23 wherein the
Gram positive bacteria is of the genus Streptococcus.
[1658] 25. The immunogenic composition of any of embodiments 22-24
wherein the Gram positive bacteria AI polypeptide comprises a
sortase substrate motif.
[1659] 26. The immunogenic composition of embodiment 25 wherein the
sortase substrate motif is an LPXTG motif.
[1660] 27. The immunogenic composition of any one of embodiments
22-24 wherein the Gram positive bacteria AI polypeptide affects the
ability of Gram positive bacteria to adhere to epithelial
cells.
[1661] 28. The immunogenic composition of any one of embodiments
22-24 wherein the Gram positive bacteria AI polypeptide affects the
ability of Gram positive bacteria to invade epithelial cells.
[1662] 29. The immunogenic composition of any one of embodiments
22-24 wherein the Gram positive bacteria AI polypeptide affects the
ability of Gram positive bacteria to translocate through an
epithelial cell layer.
[1663] 30. The immunogenic composition of any one of embodiments
22-24 wherein the Gram positive bacteria AI polypeptide is capable
of associating with an epithelial cell surface.
[1664] 31. The immunogenic composition of embodiment 30 wherein the
associating with an epithelial cell surface is binding to the
epithelial cell surface.
[1665] 32. The immunogenic composition of any of embodiments 22-24
wherein the Gram positive bacteria AI polypeptide is a full-length
Gram positive bacteria AI protein.
[1666] 33. The immunogenic composition of any of embodiments 22-24
wherein the Gram positive bacteria AI polypeptide is a fragment of
a full-length Gram positive bacteria AI protein.
[1667] 34. The immunogenic composition of embodiment 33 wherein the
fragment comprises at least 7 contiguous amino acid residues of the
Gram positive bacteria AI protein.
[1668] 35. The immunogenic composition of embodiment 24 wherein the
genus Streptococcus bacteria is Group A Streptococcus (GAS)
bacteria and the Gram positive bacteria AI polypeptide is a GAS AI
polypeptide.
[1669] 36. The immunogenic composition of embodiment 35 wherein the
GAS AI polypeptide is selected from a GAS AI-1.
[1670] 37. The immunogenic composition of embodiment 35 wherein the
GAS AI polypeptide is selected from a GAS AI-2.
[1671] 38. The immunogenic composition of embodiment 35 wherein the
GAS AI polypeptide is selected from a GAS AI-3.
[1672] 39. The immunogenic composition of embodiment 35 wherein the
GAS AI polypeptide is selected from a GAS AI-4.
[1673] 40. The immunogenic composition of any of embodiments 35-39
wherein the GAS AI polypeptide comprises a sortase substrate
motif.
[1674] 41. The immunogenic composition of embodiment 40 wherein the
sortase substrate motif is an LPXTG motif.
[1675] 42. The immunogenic composition of embodiment 41 wherein the
LPXTG motif is represented by XXXXG, wherein the X at the first
amino acid position is an L, a V, an E, or a Q, wherein the X at
the second amino acid position is P if the X at the first amino
acid position is an L, the X at the second amino acid position is a
V if the X at the first amino acid position is an E or a Q, or the
X at the second amino acid position is a V or a P if the X at the
first amino acid position is a V, wherein the X at the third amino
acid position is any amino acid residue, and wherein the X at the
fourth amino acid position is a T if the X at the first amino acid
position is a V, an E, or a Q, or the X at the fourth amino acid
position is a T, an S, or an A if the X at the first amino acid
position is an L.
[1676] 43. The immunogenic composition of any one of embodiments
35-39 wherein the GAS AI polypeptide affects the ability of GAS
bacteria to adhere to epithelial cells.
[1677] 44. The immunogenic composition of any one of embodiments
35-39 wherein the GAS AI polypeptide affects the ability of GAS
bacteria to invade epithelial cells.
[1678] 45. The immunogenic composition of any one of embodiments
35-39 wherein the GAS AI polypeptide affects the ability of GAS
bacteria to translocate through an epithelial cell layer.
[1679] 46. The immunogenic composition of any one of embodiments
35-39 wherein the GAS AI polypeptide is capable of associating with
an epithelial cell surface.
[1680] 47. The immunogenic composition of embodiment 46 wherein the
associating with an epithelial cell surface is binding to the
epithelial cell surface.
[1681] 48. The immunogenic composition of any of embodiments 35-39
wherein the GAS AI polypeptide is a full-length GAS AI protein.
[1682] 49. The immunogenic composition of any of embodiments 35-39
wherein the GAS AI polypeptide is a fragment of a full-length GAS
AI protein.
[1683] 50. The immunogenic composition of embodiment 49 wherein the
fragment comprises at least 7 contiguous amino acid residues of the
GAS AI protein.
[1684] 51. The immunogenic composition of embodiment 36 wherein the
GAS AI-1 polypeptide is selected from the group consisting of
M6_Spy0157, M6_Spy0159, M6_Spy0160, CDC SS 410_fimbrial,
ISS3650_fimbrial, DSM2071_fimbrial, and fragments thereof.
[1685] 52. The immunogenic composition of embodiment 37 wherein the
GAS AI-2 polypeptide is selected from the group consisting of GAS
15, GAS 16, GAS 18, and fragments thereof.
[1686] 53. The immunogenic composition of embodiment 38 wherein the
GAS AI-3 polypeptide is selected from the group consisting of
SpyM3.sub.--0098, SpyM3.sub.--0100, SpyM3.sub.--0102,
SpyM3.sub.--0104, SPs0100, SPs0102, SPs0104, SPs0106, orf78, orf80,
orf82, orf84, spyM18.sub.--0126, spyM18.sub.--0128,
spyM18.sub.--0130, spyM18.sub.--0132, SpyoM01000156, SpyoM01000155,
SpyoM01000154, SpyoM01000153, SpyoM01000152, SpyoM01000151,
SpyoM01000150, SpyoM01000149, ISS3040_fimbrial, ISS3776_fimbrial,
ISS4959_fimbrial, and fragments thereof.
[1687] 53. The immunogenic composition of embodiment 39 wherein the
GAS AI-4 polypeptide is selected from the group consisting of
19224134, 19224135, 19224137, 19224139, 19224141,
20010296_fimbrial, 20020069_fimbrial, CDC SS 635_fimbrial,
ISS4883_fimbrial, ISS4538_fimbrial, and fragments thereof.
[1688] 54. The immunogenic composition of embodiment 24 wherein the
Streptococcus bacteria is Streptococcus pneumoniae and the Gram
positive bacteria AI polypeptide is a S. pneumoniae AI
polypeptide.
[1689] 55. The immunogenic composition of embodiment 54 wherein the
S. pneumoniae AI polypeptide comprises a sortase substrate
motif.
[1690] 56. The immunogenic composition of embodiment 55 wherein the
sortase substrate motif is an LPXTG motif.
[1691] 57. The immunogenic composition of embodiment 54 wherein the
S. pneumoniae AI polypeptide affects the ability of S. pneumoniae
to adhere to epithelial cells.
[1692] 58. The immunogenic composition of embodiment 54 wherein the
S. pneumoniae AI polypeptide affects the ability of S. pneumoniae
to invade epithelial cells.
[1693] 59. The immunogenic composition of embodiment 54 wherein the
S. pneumoniae AI polypeptide affects the ability of S. pneumoniae
to translocate through an epithelial cell layer.
[1694] 60. The immunogenic composition of embodiment 54 wherein the
S. pneumoniae AI polypeptide is capable of associating with an
epithelial cell surface.
[1695] 61. The immunogenic composition of embodiment 60 wherein the
associating with an epithelial cell surface is binding to the
epithelial cell surface.
[1696] 62. The immunogenic composition of embodiment 54 wherein the
S. pneumoniae AI polypeptide is a full-length S. pneumoniae AI
protein.
[1697] 63. The immunogenic composition of embodiment 54 wherein the
S. pneumoniae AI polypeptide is a fragment of a full-length S.
pneumoniae AI protein.
[1698] 64. The immunogenic composition of embodiment 63 wherein the
fragment comprises at least 7 contiguous amino acid residues of the
S. pneumoniae AI protein.
[1699] 65. The immunogenic composition of embodiment 54 wherein the
S. pneumoniae AI polypeptide is selected from the group consisting
of SP0462, SP0463, SP0464, orf3.sub.--670, orf4.sub.--670,
orf5.sub.--670, ORF3.sub.--14CSR, ORF4.sub.--14CSR,
ORF5.sub.--14CSR, ORF3.sub.--19AH, ORF4.sub.--19AH,
ORF5.sub.--19AH, ORF3.sub.--19FTW, ORF4.sub.--19FTW,
ORF5.sub.--19FTW, ORF3.sub.--23FP, ORF4.sub.--23FP,
ORF5.sub.--23FP, ORF3.sub.--23FTW, ORF4.sub.--23FTW,
ORF5.sub.--23FTW, ORF3.sub.--6BF, ORF4.sub.--6BF, ORF5.sub.--6BF,
ORF3.sub.--6BSP, ORF4.sub.--6BSP, ORF5.sub.--6BSP, ORF3.sub.--9VSP,
ORF4.sub.--9VSP, ORF5.sub.--9VSP, and fragments thereof.
[1700] 66. The immunogenic composition of any one of embodiments
22-24, 35-39, 51-54, or 65 wherein the oligomeric form is a
hyperoligomer.
[1701] 67. The immunogenic composition of any one of embodiments
22-24, 35-39, 51-54, or 65 further comprising a Gram positive
bacteria antigen not associated with an AI.
[1702] 68. The immunogenic composition of embodiment 67 wherein the
antigen is selected from the group consisting of GBS 322 and GBS
276.
[1703] 69. An immunogenic composition comprising a first and a
second Group B Streptococcus (GBS) adhesin island (AI)
polypeptide.
[1704] 70. The immunogenic composition of embodiment 69 wherein a
full-length polynucleotide sequence encoding for the first GBS AI
polypeptide is not present in a GBS bacteria genome comprising a
polynucleotide sequence encoding for the second GBS AI
polypeptide.
[1705] 71. The immunogenic composition of embodiment 69 wherein
polynucleotides encoding the first and the second GBS AI
polypeptide are each present in genomes of more than one GBS
serotype and strain isolate.
[1706] 72. The immunogenic composition of embodiment 69 wherein the
first GBS AI polypeptide is encoded by a GBS AI-1.
[1707] 73. The immunogenic composition of embodiment 69 wherein the
first GBS AI polypeptide is encoded by a GBS AI-2.
[1708] 74. The immunogenic composition of embodiment 72 wherein the
second GBS AI polypeptide is encoded by a GBS AI-2.
[1709] 75. The immunogenic composition of embodiment 73 wherein the
second GBS AI polypeptide is encoded by a GBS AI-2.
[1710] 76. The immunogenic composition of embodiment 72 wherein the
second GBS AI polypeptide is encoded by a GBS AI-1.
[1711] 77. The immunogenic composition of embodiment 73 wherein the
second GBS AI polypeptide is encoded by a GBS AI-1.
[1712] 78. The immunogenic composition of embodiment 72 wherein the
first GBS AI polypeptide is selected from the group consisting of
GBS 80, GBS 104, GBS 52, and fragments thereof.
[1713] 79. The immunogenic composition of embodiment 73 wherein the
first GBS AI polypeptide is selected from the group consisting of
GBS 59, GBS 67, GBS 150, 01521, 01523, 01524, and fragments
thereof.
[1714] 80. The immunogenic composition of embodiment 74 or 75
wherein the second GBS AI polypeptide is selected from the group
consisting of GBS 59, GBS 67, GBS 150, 01521, 01523, 01524, and
fragments thereof, and wherein the first and the second GBS AI
polypeptide are not the same polypeptide.
[1715] 81. The immunogenic composition of embodiment 76 or 77
wherein the second GBS AI polypeptide is selected from the group
consisting of GBS 80, GBS 104, GBS 52, and fragments thereof, and
wherein the first and the second GBS AI polypeptide are not the
same polypeptide.
[1716] 82. The immunogenic composition of any one of embodiments
69-77 wherein the first GBS AI polypeptide comprises a sortase
substrate motif.
[1717] 83. The immunogenic composition of embodiment 82 wherein the
sortase substrate motif is an LPXTG motif.
[1718] 84. The immunogenic composition of embodiment 83 wherein the
LPXTG motif is represented by the sequence XPXTG, wherein the X at
amino acid position 1 is an L, an I, or an F and the X at amino
acid position 3 is any amino acid residue.
[1719] 85. The immunogenic composition of any one of embodiments
69-77 wherein the first GBS AI polypeptide affects the ability of
GBS bacteria to adhere to epithelial cells.
[1720] 86. The immunogenic composition of any one of embodiments
69-77 wherein the first GBS AI polypeptide affects the ability of
GBS bacteria to invade epithelial cells.
[1721] 87. The immunogenic composition of any one of embodiments
69-77 wherein the first GBS AI polypeptide affects the ability of
GBS bacteria to translocate through an epithelial cell layer.
[1722] 88. The immunogenic composition of any one of embodiments
69-77 wherein the first GBS AI polypeptide is capable of
associating with an epithelial cell surface.
[1723] 89. The immunogenic composition of embodiment 88 wherein the
associating with an epithelial cell surface is binding to the
epithelial cell surface.
[1724] 90. The immunogenic composition of any of embodiments 69-77
wherein the first GBS AI polypeptide is a full-length GBS AI
protein.
[1725] 91. The immunogenic composition of any of embodiments 69-77
wherein the first GBS AI polypeptide is a fragment of a full-length
GBS AI protein.
[1726] 92. The immunogenic composition of embodiment 91 wherein the
fragment comprises at least 7 contiguous amino acid residues of the
first GBS AI protein.
[1727] 93. The immunogenic composition of any one of embodiments
69-79 wherein the first GBS AI polypeptide is in oligomeric
form.
[1728] 94. The immunogenic composition of any one of embodiments
69-77 wherein the second GBS AI polypeptide is in oligomeric
form.
[1729] 95. The immunogenic composition of any one of embodiments
69-79 wherein the first and the second GBS AI polypeptide are
associated in a single oligomeric form.
[1730] 96. The immunogenic composition of embodiment 95 wherein the
first and the second GBS AI polypeptides are chemically
associated.
[1731] 97. The immunogenic composition of embodiment 95 wherein the
first and the second GBS AI polypeptides are physically
associated.
[1732] 98. The immunogenic composition of embodiment 93 wherein the
oligomeric form is a hyperoligomer.
[1733] 99. The immunogenic composition of embodiment 94 wherein the
oligomeric form is a hyperoligomer.
[1734] 100. The immunogenic composition of embodiment 76 wherein
the first GBS AI polypeptide is GBS 80 and the second GBS AI
polypeptide is GBS 104.
[1735] 101. The immunogenic composition of embodiment 74 wherein
the first GBS AI polypeptide is GBS 80 and the second GBS AI
polypeptide is GBS 67.
[1736] 102. The immunogenic composition of any one of embodiments
69-79, 100, or 101 further comprising a GBS polypeptide not
associated with an AI.
[1737] 103. The immunogenic composition of embodiment 102 wherein
the GBS polypeptide not associated with an AI is selected from the
group consisting of GBS 322 and GBS 276.
[1738] 104. The immunogenic composition of embodiment 103 wherein
the GBS polypeptide not associated with an AI is GBS 322.
[1739] 105. An immunogenic composition comprising a first and a
second Gram positive bacteria adhesin island (AI) polypeptide.
[1740] 106. The immunogenic composition of embodiment 105 wherein a
full length polynucleotide sequence encoding for the first Gram
positive bacteria AI polypeptide is not present in a genome of a
Gram positive bacteria comprising a full length polynucleotide
sequence encoding for the second Gram positive bacteria AI
polypeptide.
[1741] 107. The immunogenic composition of embodiment 105 wherein
polynucleotides encoding the first and the second Gram positive
bacteria AI polypeptide are each present in genomes of more than
one Gram positive bacteria serotype and strain isolate.
[1742] 108. The immunogenic composition of embodiment 105 wherein
the first and the second Gram positive bacteria AI polypeptides are
of different Gram positive bacteria species.
[1743] 109. The immunogenic composition of embodiment 105 wherein
the first and the second Gram positive bacteria AI polypeptides are
of the same Gram positive bacteria species.
[1744] 110. The immunogenic composition of embodiment 105 wherein
the first and the second Gram positive bacteria AI polypeptides are
from different AI subtypes.
[1745] 111. The immunogenic composition of embodiment 105 wherein
the first and the second Gram positive bacteria AI polypeptides are
from the same AI subtype.
[1746] 112. The immunogenic composition of embodiment 105 wherein
the first Gram positive bacteria AI polypeptide has detectable
surface exposure on a first Gram positive bacteria strain or
serotype but not a second Gram positive bacteria strain or subtype
and the second Gram positive bacteria AI polypeptide has detectable
surface exposure on the second Gram positive bacteria strain or
serotype but not the first Gram positive bacteria strain or
serotype.
[1747] 113. The immunogenic composition of embodiment 105 wherein
the Gram positive bacteria is S. pneumonaie, S. mutans, E.
faecalis, E. faecium, C. difficile, L. monocytogenes, or C.
diphtheriae.
[1748] 114. The immunogenic composition of any of embodiments
105-113 wherein the first and the second Gram positive bacteria AI
polypeptides comprise a sortase substrate motif.
[1749] 115. The immunogenic composition of embodiment 114 wherein
the sortase substrate motif is an LPXTG motif.
[1750] 116. The immunogenic composition of embodiment 115 wherein
the LPXTG motif is represented by XXXXG, wherein the X at amino
acid position 1 is an L, a V, an E, an I, an F, or a Q, wherein X
at amino acid position 2 is a P if X at amino acid position 1 is an
L, an I, or an F, wherein X at amino acid position 2 is a V if X at
amino acid position 1 is a E or a Q, wherein X at amino acid
position 2 is a V or a P if X at amino acid position 1 is a V,
wherein X at amino acid position 3 is any amino acid residue,
wherein X at amino acid position 4 is a T if X at amino acid
position 1 is a V, E, I, F, or Q, and wherein X at amino acid
position 4 is a T, S, or A if X at amino acid position 1 is an
L.
[1751] 117. The immunogenic composition of embodiment 105 wherein
the first Gram positive bacteria AI polypeptide is a first Group A
Streptococcus (GAS) AI polypeptide.
[1752] 118. The immunogenic composition of embodiment 117 wherein
the first GAS AI polypeptide comprises a sortase substrate
motif.
[1753] 119. The immunogenic composition of embodiment 118 wherein
the sortase substrate motif is an LPXTG motif.
[1754] 120. The immunogenic composition of embodiment 119 wherein
the LPXTG motif is represented by XXXXG, wherein the X at the first
amino acid position is an L, a V, an E, or a Q, wherein the X at
the second amino acid position is P if the X at the first amino
acid position is an L, the X at the second amino acid position is a
V if the X at the first amino acid position is an E or a Q, or the
X at the second amino acid position is a V or a P if the X at the
first amino acid position is a V, wherein the X at the third amino
acid position is any amino acid residue, and wherein the X at the
fourth amino acid position is a T if the X at the first amino acid
position is a V, an E, or a Q, or the X at the fourth amino acid
position is a T, an S, or an A if the X at the first amino acid
position is an L.
[1755] 121. The immunogenic composition of embodiment 117 wherein
the first GAS AI polypeptide affects the ability of GAS bacteria to
adhere to epithelial cells.
[1756] 122. The immunogenic composition of embodiment 117 wherein
the first GAS AI polypeptide affects the ability of GAS bacteria to
invade epithelial cells.
[1757] 123. The immunogenic composition of embodiment 117 wherein
the first GAS AI polypeptide affects the ability of GAS bacteria to
translocate through an epithelial cell layer.
[1758] 124. The immunogenic composition of embodiment 117 wherein
the first GAS AI polypeptide is capable of associating with an
epithelial cell surface.
[1759] 125. The immunogenic composition of embodiment 117 wherein
the associating with an epithelial cell surface is binding to the
epithelial cell surface.
[1760] 126. The immunogenic composition of embodiment 117 wherein
the first GAS AI polypeptide is a full-length GAS AI protein.
[1761] 127. The immunogenic composition of embodiment 117 wherein
the first GAS AI polypeptide is a fragment of a full-length GAS AI
protein.
[1762] 128. The immunogenic composition of embodiment 127 wherein
the fragment comprises at least 7 contiguous amino acid residues of
the GAS AI protein.
[1763] 129. The immunogenic composition of embodiment 117 wherein
the first GAS AI polypeptide is a first GAS AI-1 polypeptide.
[1764] 130. The immunogenic composition of embodiment 117 wherein
the first GAS AI polypeptide is a first GAS AI-2 polypeptide.
[1765] 131. The immunogenic composition of embodiment 117 wherein
the first GAS AI polypeptide is a first GAS AI-3 polypeptide.
[1766] 132. The immunogenic composition of embodiment 117 wherein
the first GAS AI polypeptide is a first GAS AI-4 polypeptide.
[1767] 133. The immunogenic composition of any one of embodiments
117 or 129-132 wherein the second Gram positive bacteria AI
polypeptide is a second GAS AI polypeptide.
[1768] 134. The immunogenic composition of embodiment 133 wherein
the second GAS AI polypeptide is a second GAS AI-1 polypeptide.
[1769] 135. The immunogenic composition of embodiment 133 wherein
the second GAS AI polypeptide is a second GAS AI-2 polypeptide.
[1770] 136. The immunogenic composition of embodiment 133 wherein
the second GAS AI polypeptide is a second GAS AI-3 polypeptide.
[1771] 137. The immunogenic composition of embodiment 133 wherein
the second GAS AI polypeptide is a second GAS AI-4 polypeptide.
[1772] 138. The immunogenic composition of embodiment 129 wherein
the first GAS AI-1 polypeptide is selected from the group
consisting of M6_Spy0157, M6_Spy0159, M6_Spy0160, CDC SS
410_fimbrial, ISS3650_fimbrial, DSM2071_fimbrial, and fragments
thereof.
[1773] 139. The immunogenic composition of embodiment 130 wherein
the first GAS AI-2 polypeptide is selected from the group
consisting of GAS 15, GAS 16, GAS 18, and fragments thereof.
[1774] 140. The immunogenic composition of embodiment 131 wherein
the first GAS AI-3 polypeptide is selected from the group
consisting of SpyM3.sub.--0098, SpyM3.sub.--0100, SpyM3.sub.--0102,
SpyM3.sub.--0104, SPs0100, SPs0102, SPs0104, SPs0106, orf78, orf80,
orf82, orf84, spyM18.sub.--0126, spyM18.sub.--0128,
spyM18.sub.--0130, spyM18.sub.--0132, SpyoM01000156, SpyoM01000155,
SpyoM01000154, SpyoM01000153, SpyoM01000152, SpyoM01000151,
SpyoM01000150, SpyoM01000149, ISS3040_fimbrial, ISS3776_fimbrial,
ISS4959_fimbrial, and fragments thereof.
[1775] 141. The immunogenic composition of embodiment 132 wherein
the first GAS AI-4 polypeptide is selected from the group
consisting of 19224134, 19224135, 19224137, 19224139, 19224141,
20010296_fimbrial, 20020069_fimbrial, CDC SS 635_fimbrial,
ISS4883_fimbrial, ISS4538_fimbrial, and fragments thereof.
[1776] 142. The immunogenic composition of embodiment 134 wherein
the second GAS AI-1 polypeptide is selected from the group
consisting of M6_Spy0157, M6_Spy0159, M6_Spy0160, CDC SS
410_fimbrial, ISS3650_fimbrial, DSM2071_fimbrial, and fragments
thereof.
[1777] 143. The immunogenic composition of embodiment 135 wherein
the second GAS AI-2 polypeptide is selected from the group
consisting of GAS15, GAS16, GAS18, and fragments thereof.
[1778] 144. The immunogenic composition of embodiment 136 wherein
the second GAS AI-3 polypeptide is selected from the group
consisting of SpyM3.sub.--0098, SpyM3.sub.--0100, SpyM3.sub.--0102,
SpyM3.sub.--0104, SPs0100, SPs0102, SPs0104, SPs0106, orf78, orf80,
orf82, orf84, spyM18.sub.--0126, spyM18.sub.--0128,
spyM18.sub.--0130, spyM18.sub.--0132, SpyoM01000156, SpyoM01000155,
SpyoM01000154, SpyoM01000153, SpyoM01000152, SpyoM01000151,
SpyoM01000150, SpyoM01000149, ISS3040_fimbrial, ISS3776_fimbrial,
ISS4959_fimbrial, and fragments thereof.
[1779] 145. The immunogenic composition of embodiment 137 wherein
the second GAS AI-4 polypeptide is selected from the group
consisting of 19224134, 19224135, 19224137, 19224139, 19224141,
20010296_fimbrial, 20020069_fimbrial, CDC SS 635_fimbrial,
ISS4883_fimbrial, ISS4538_fimbrial, and fragments thereof.
[1780] 146. The immunogenic composition of any one of embodiments
117-132 or 138-141 wherein the second Gram positive bacteria AI
polypeptide is a Group B Streptococcus (GBS) AI polypeptide.
[1781] 147. The immunogenic composition of embodiment 146 wherein
the GBS AI polypeptide comprises a sortase substrate motif.
[1782] 148. The immunogenic composition of embodiment 147 wherein
the sortase substrate motif is an LPXTG motif.
[1783] 149. The immunogenic composition of embodiment 148 wherein
the LPXTG motif is represented by the amino acid sequence XPXTG,
wherein the X at amino acid position 1 is an L, an I, or an F and
the X at amino acid position 3 is any amino acid residue.
[1784] 150. The immunogenic composition of embodiment 146 wherein
the GBS AI polypeptide affects the ability of GBS bacteria to
adhere to epithelial cells.
[1785] 151. The immunogenic composition of embodiment 146 wherein
the GBS AI polypeptide affects the ability of GBS bacteria to
invade epithelial cells.
[1786] 152. The immunogenic composition of embodiment 146 wherein
the GBS AI polypeptide affects the ability of GBS bacteria to
translocate through an epithelial cell layer.
[1787] 153. The immunogenic composition of embodiment 146 wherein
the GBS AI polypeptide is capable of associating with an epithelial
cell surface.
[1788] 154. The immunogenic composition of embodiment 146 wherein
the associating with an epithelial cell surface is binding to the
epithelial cell surface.
[1789] 155. The immunogenic composition of embodiment 146 wherein
the GBS AI polypeptide is a full-length GBS AI protein.
[1790] 156. The immunogenic composition of embodiment 146 wherein
the GBS AI polypeptide is a fragment of a full-length GBS AI
protein.
[1791] 157. The immunogenic composition of embodiment 156 wherein
the fragment comprises at least 7 contiguous amino acid residues of
the GBS AI protein.
[1792] 158. The immunogenic composition of embodiment 146 wherein
the GBS AI polypeptide is a GBS AI-1 polypeptide.
[1793] 159. The immunogenic composition of embodiment 146 wherein
the GBS AI polypeptide is a GBS AI-2 polypeptide.
[1794] 160. The immunogenic composition of embodiment 158 wherein
the GBS AI-1 polypeptide is selected from the group consisting of
GBS 80, GBS 104, GBS 52, and fragments thereof.
[1795] 161. The immunogenic composition of embodiment 159 wherein
the GBS AI-2 polypeptide is selected from the group consisting of
GBS 59, GBS 67, GBS 150, 01521, 01523, 01524, and fragments
thereof.
[1796] 162. The immunogenic composition of any one of embodiments
117-132 or 138-141 wherein the second Gram positive bacteria AI
polypeptide is a Streptococcus pneumoniae AI polypeptide.
[1797] 163. The immunogenic composition of embodiment 162 wherein
the S. pneumoniae AI polypeptide comprises a sortase substrate
motif.
[1798] 164. The immunogenic composition of embodiment 163 wherein
the sortase substrate motif is an LPXTG motif.
[1799] 165. The immunogenic composition of embodiment 162 wherein
the S. pneumoniae AI polypeptide affects the ability of S.
pneumoniae to adhere to epithelial cells.
[1800] 166. The immunogenic composition of embodiment 162 S.
pneumoniae AI polypeptide affects the ability of S. pneumoniae to
invade epithelial cells.
[1801] 167. The immunogenic composition of embodiment 162 wherein
the S. pneumoniae AI polypeptide affects the ability of S.
pneumoniae to translocate through an epithelial cell layer.
[1802] 168. The immunogenic composition of embodiment 162 wherein
the S. pneumoniae AI polypeptide is capable of associating with an
epithelial cell surface.
[1803] 169. The immunogenic composition of embodiment 168 wherein
the associating with an epithelial cell surface is binding to the
epithelial cell surface.
[1804] 170. The immunogenic composition of embodiment 162 wherein
the S. pneumoniae AI polypeptide is a full-length S. pneumoniae AI
protein.
[1805] 171. The immunogenic composition of embodiment 162 wherein
the S. pneumoniae AI polypeptide is a fragment of a full-length S.
pneumoniae AI protein.
[1806] 172. The immunogenic composition of embodiment 162 wherein
the fragment comprises at least 7 contiguous amino acid residues of
the S. pneumoniae AI protein.
[1807] 173. The immunogenic composition of embodiment 162 wherein
the S. pneumoniae AI polypeptide is selected from the group
consisting of SP0462, SP0463, SP0464, orf3.sub.--670,
orf4.sub.--670, orf5.sub.--670, ORF3.sub.--14CSR, ORF4.sub.--14CSR,
ORF5.sub.--14CSR, ORF3.sub.--19AH, ORF4.sub.--19AH,
ORF5.sub.--19AH, ORF3.sub.--19FTW, ORF4.sub.--19FTW,
ORF5.sub.--19FTW, ORF3.sub.--23FP, ORF4.sub.--23FP,
ORF5.sub.--23FP, ORF3.sub.--23FTW, ORF4.sub.--23FTW,
ORF5.sub.--23FTW, ORF3.sub.--6BF, ORF4.sub.--6BF, ORF5.sub.--6BF,
ORF3.sub.--6BSP, ORF4.sub.--6BSP, ORF5.sub.--6BSP, ORF3.sub.--9VSP,
ORF4.sub.--9VSP, ORF5.sub.--9VSP, and fragments thereof.
[1808] 174. The immunogenic composition of any one of embodiments
105-117 wherein the first Gram positive bacteria AI polypeptide is
in oligomeric form.
[1809] 175. The immunogenic composition of embodiment 174 wherein
the oligomeric form is a hyperoligomer.
[1810] 176. The immunogenic composition of embodiment 174 wherein
the second Gram positive bacteria AI polypeptide is in oligomeric
form.
[1811] 177. The immunogenic composition of embodiment 176 wherein
the oligomeric form is a hyperoligomer.
[1812] 178. The immunogenic composition of embodiment 176 wherein
the first and the second Gram positive bacteria AI polypeptide are
associated in a single oligomeric form.
[1813] 179. The immunogenic composition of embodiment 178 wherein
the first and the second Gram positive bacteria AI polypeptide are
chemically associated.
[1814] 180. The immunogenic composition of embodiment 178 wherein
the first and the second Gram positive bacteria AI polypeptide are
physically associated.
[1815] 181. The immunogenic composition of any one of embodiments
105-117 further comprising a Gram positive bacteria polypeptide not
associated with an AI.
[1816] 182. The immunogenic composition of embodiment 181 wherein
the Gram positive bacteria polypeptide not associated with an AI is
selected from the group consisting of GBS 322 and GBS 276.
[1817] 183. The immunogenic composition of embodiment 182 wherein
the Gram positive bacteria polypeptide not associated with an AI is
GBS 322.
[1818] 184. A modified Gram positive bacterium adapted to produce
increased levels of AI surface protein.
[1819] 185. The modified Gram positive bacterium of embodiment 184
wherein the AI surface protein is in oligomeric form.
[1820] 186. The modified Gram positive bacterium of embodiment 185
wherein the oligomeric form is a hyperoligomer.
[1821] 187. The modified Gram positive bacterium of any one of
embodiments 184-186 which is a Group B Streptococcus bacterium.
[1822] 188. The modified Gram positive bacterium of any one of
embodiments 184-186 which is a Group A Streptococcus bacterium.
[1823] 189. The modified Gram positive bacterium of any one of
embodiments 184-186 which is a non-pathogenic Gram positive
bacterium.
[1824] 190. The modified Gram positive bacterium of embodiment 189
wherein the non-pathogenic Gram positive bacterium is Streptococus
gordonii.
[1825] 191. The modified Gram positive bacterium of embodiment 189
wherein the non-pathogenic Gram positive bacterium is Lactococcus
lactis.
[1826] 192. The modified Gram positive bacterium of any one of
embodiments 184-186 which has been inactivated and wherein the AI
surface protein is exposed on the surface of the Gram positive
bacterium.
[1827] 193. The modified Gram positive bacterium of any one of
embodiments 184-186 which has been attenuated and wherein the AI
surface protein is exposed on the surface of the Gram positive
bacterium.
[1828] 194. The modified GBS bacterium of embodiment 187 which has
been inactivated and wherein the AI surface protein is exposed on
the surface of the GBS bacterium.
[1829] 195. The modified GBS bacterium of embodiment 187 which has
been attenuated and wherein the AI surface protein is exposed on
the surface of the GBS bacterium.
[1830] 196. The modified GAS bacterium of embodiment 188 which has
been inactivated and wherein the AI surface protein is exposed on
the surface of the GAS bacterium.
[1831] 197. The modified GAS bacterium of embodiment 188 which has
been attenuated and wherein the AI surface protein is exposed on
the surface of the GAS bacterium.
[1832] 198. The modified non-pathogenic bacterium of embodiment 189
which has been inactivated and wherein the AI surface protein is
exposed on the surface of the non-pathogenic Gram positive
bacterium.
[1833] 199. The modified non-pathogenic bacterium of embodiment 189
which has been attenuated and wherein the AI surface protein is
exposed on the surface of the non-pathogenic Gram positive
bacterium.
[1834] 200. A method for manufacturing an oligomeric adhesin island
(AI) surface antigen comprising:
[1835] culturing a Gram positive bacterium that expresses an
oligomeric AI surface antigen and
[1836] isolating the expressed oligomeric AI surface antigen.
[1837] 201. The method of embodiment 200 wherein the step of
isolating is performed by collecting said oligomeric AI surface
antigen from Gram positive bacterium secretions in the Gram
positive bacterium culture.
[1838] 202. The method of embodiment 200 further comprising a step
of purifying.
[1839] 203. The method of embodiment 202 wherein the oligomeric AI
surface antigen is purified from the Gram positive bacterium cell
surface.
[1840] 204. The method of embodiment 200 wherein the Gram positive
bacterium is adapted for increased AI protein expression.
[1841] 205. The method of any one of embodiments 200-204 wherein
the Gram positive bacterium is a Group A Streptococcus
bacterium.
[1842] 206. The method of any one of embodiments 200-204 wherein
the Gram positive bacterium is a Group B Streptococcus
bacterium.
[1843] 207. The method of any one of embodiments 200-204 wherein
the oligomeric AI surface antigen is in hyperoligomeric form.
[1844] 208. The method of embodiment 200 wherein the Gram positive
bacterium expresses the oligomeric AI surface antigen
recombinantly.
[1845] 209. The method of embodiment 208 wherein the Gram positive
bacterium further manipulated expresses at least 1 AI sortase.
[1846] 210. The modified Gram positive bacterium of any one of
embodiments 184-186 which is a S. pneumoniae bacterium.
[1847] 211. The method of any one of embodiments 200-204 wherein
the Gram positive bacterium is S. pneumoniae.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20060165716A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20060165716A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References