U.S. patent application number 10/387783 was filed with the patent office on 2004-01-08 for vaccine compositions comprising streptococcus pneumoniae polypeptides having selected structural motifs.
Invention is credited to Adamou, John E., Johnson, Leslie S..
Application Number | 20040005331 10/387783 |
Document ID | / |
Family ID | 22347343 |
Filed Date | 2004-01-08 |
United States Patent
Application |
20040005331 |
Kind Code |
A1 |
Johnson, Leslie S. ; et
al. |
January 8, 2004 |
Vaccine compositions comprising Streptococcus pneumoniae
polypeptides having selected structural motifs
Abstract
A vaccine composition is disclosed that comprises polypeptides
and fragments of polypeptides containing histidine triad residues
or coiled-coil regions, some of which polypeptides or fragments lie
between 80 and 680 residues in length. Also disclosed are processes
for preventing infection caused by S. pneumoniae comprising
administering of vaccine compositions.
Inventors: |
Johnson, Leslie S.;
(Germantown, MD) ; Adamou, John E.; (Rockville,
MD) |
Correspondence
Address: |
CARELLA, BYRNE, BAIN, GILFILLAN,
CECCHI, STEWART & OLSTEIN
6 Becker Farm Road
Roseland
NJ
07068
US
|
Family ID: |
22347343 |
Appl. No.: |
10/387783 |
Filed: |
March 13, 2003 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10387783 |
Mar 13, 2003 |
|
|
|
09468656 |
Dec 21, 1999 |
|
|
|
6582706 |
|
|
|
|
60113048 |
Dec 21, 1998 |
|
|
|
Current U.S.
Class: |
424/190.1 ;
530/350; 536/23.7 |
Current CPC
Class: |
C07K 14/3156 20130101;
C07K 16/1275 20130101; A61P 31/04 20180101; A61K 39/092 20130101;
A61K 2039/505 20130101; A61P 31/10 20180101 |
Class at
Publication: |
424/190.1 ;
530/350; 536/23.7 |
International
Class: |
A61K 039/02; C07H
021/04; C07K 014/315 |
Claims
What is claimed is:
1. A composition comprising a polypeptide at least 90% identical to
a member selected from the group consisting of (i) amino acids
21-480 of SEQ ID NO: 6, (ii) amino acids 20-819 of SEQ ID NO: 8,
and (iii) amino acids 20-819 of SEQ ID NO: 10, wherein said member
is in a pharmaceutically acceptable carrier in an amount effective
to elicit production of an antibody that binds to S. pneumoniae
when said composition is administered to a mammal.
2. The composition of claim 1 wherein said percent identity is at
least 95%.
3. The composition of claim 1 wherein said percent identity is at
least 97%.
4. The composition of claim 1 wherein said polypeptide has the
sequence of amino acids 21-480 of SEQ ID NO: 6.
5. The composition of claim 1 wherein said polypeptide has the
sequence of amino acids 20-819 of SEQ ID NO: 8.
6. The composition of claim 1 wherein said polypeptide has the
sequence of amino acids 20-819 of SEQ ID NO: 10,
7. A composition comprising an active fragment of a polypeptide
selected from the group consisting of: (i) amino acids 21-480 of
SEQ ID NO: 6, (ii) amino acids 20-819 of SEQ ID NO: 8, and (iii)
amino acids 20-819 of SEQ ID NO: 10, wherein said active fragment
comprises at least about 10% and no more than about 85% of said
polypeptide, wherein said active fragment comprises at least one
histidine triad and at least one coiled coil region and wherein
said active fragment is in a pharmaceutically acceptable carrier in
an amount effective to elicit production of an antibody that binds
to S. pneumoniae when said composition is administered to a
mammal.
8. The composition of claim 7 wherein said active fragment further
comprises at least 2 histidine triad regions.
9. The composition of claim 7 wherein said active fragment further
comprises at least 2 coiled coil regions.
10. A vaccine comprising a polypeptide containing the sequence of
amino acids 21-480 of SEQ ID NO: 6 in a pharmaceutically acceptable
carrier in an amount effective to protect against pneumococcal
infection when administered to a mammal.
11. A vaccine comprising a polypeptide containing the sequence of
amino acids 20-819 of SEQ ID NO: 8 in a pharmaceutically acceptable
carrier in an amount effective to protect against pneumococcal
infection when administered to a mammal.
12. A vaccine comprising a polypeptide containing the sequence of
amino acids 20-819 of SEQ ID NO: 10 in a pharmaceutically
acceptable carrier in an amount effective to protect against
pneumococcal infection when administered to a mammal.
13. A process for preventing infection caused by S. pneumoniae
comprising administering the vaccine of claim 10.
14. A process for preventing infection caused by S. pneumoniae
comprising administering the vaccine of claim 11.
15. A process for preventing infection caused by S. pneumoniae
comprising administering the vaccine of claim 12.
16. A process for preventing infection caused by S. pneumoniae
comprising administering the composition of claim 1.
17. An isolated polypeptide at least 95% identical to a member
selected from the group consisting of (i) amino acids 21-480 of SEQ
ID NO: 6, (ii) amino acids 20-819 of SEQ ID NO: 8, and (iii) amino
acids 20-819 of SEQ ID NO: 10, wherein said polypeptide binds to an
antibody that binds to S. pneumoniae.
18. The isolated polypeptide of claim 17 wherein said percent
identity is at least 95%.
19. The isolated polypeptide of claim 17 wherein said percent
identity is at least 97%.
20. The isolated polypeptide of claim 17 wherein said isolated
polypeptide has the amino acid sequence of said member.
21. A composition comprising at least one antibody that binds to a
polypeptide comprising an amino acid sequence at least 90%
identical to a member selected from the group consisting of: (i)
amino acids 20-838 of SEQ ID NO: 4, (ii) amino acids 21-480 of SEQ
ID NO: 6, (iii) amino acids 20-819 of SEQ ID NO: 8, and (iv) amino
acids 20-819 of SEQ ID NO: 10, wherein said antibody is in a
pharmaceutically acceptable carrier.
22. The composition of claim 21 wherein the antibody is the
antibody against the polypeptide in (i).
23. The composition of claim 21 wherein the antibody is the
antibody against the fragment in (ii).
24. The composition of claim 21 wherein the antibody is the
antibody against the polypeptide in (iii).
25. The composition of claim 21 wherein the antibody is the
antibody against the polypeptide in (iv).
26. A composition comprising at least one antibody that binds to an
active fragment of a polypeptide comprising a member selected from
the group consisting of: (i) amino acids 20-838 of SEQ ID NO: 4,
(ii) amino acids 21-480 of SEQ ID NO: 6, (iii) amino acids 20-819
of SEQ ID NO: 8, and (iv) amino acids 20-819 of SEQ ID NO: 10,
wherein said active fragment comprises at least about 10% and no
more than about 85% of said polypeptide, wherein said active
fragment comprises at least one histidine triad and at least one
coiled coil region and wherein said active fragment is in a
pharmaceutically acceptable carrier in an amount effective to
elicit production of an antibody that binds to S. pneumoniae when
said composition is administered to a mammal.
27. A process for treating or preventing infection caused by S.
pneumoniae comprising administering to a mammal afflicted with said
ifection, or at risk of said infection, an effective amount of the
composition of claim 21.
28. A process for treating or preventing infection caused by S.
pneumoniae comprising administering to a mammal afflicted with said
ifection, or at risk of said infection, an effective amount of the
composition of claim 27.
Description
[0001] This application is a divisional of U.S. application Ser.
No. 09/468,656, filed Dec. 21, 1999, which is based on U.S.
Provisional Application No. 60/113,048, filed Dec. 21, 1998, the
disclosures of which are hereby incorporated in their entirety.
FIELD OF THE INVENTION
[0002] This invention relates generally to the field of bacterial
antigens and their use, for example, as immunogenic agents in
humans and animals to stimulate an immune response. More
specifically, it relates to the vaccination of mammalian species
with a polypeptide comprising at least one conserved histidine
triad residue (HxxHxH--SEQ ID NO: 12) and at least one
helix-forming polypeptide obtained from Streptococcus pneumoniae as
a mechanism for stimulating production of antibodies that protect
the vaccine recipient against infection by a wide range of
serotypes of pathogenic S. pneumoniae. Further, the invention
relates to antibodies against such polypeptides useful in diagnosis
and passive immune therapy with respect to diagnosing and treating
such pneumococcal infections.
[0003] In a particular aspect, the present invention relates to the
prevention and treatment of pneumococcal infections such as
infections of the middle ear, nasopharynx, lung and bronchial
areas, blood, CSF, and the like, that are caused by pneumococcal
bacteria.
BACKGROUND OF THE INVENTION
[0004] Streptococcus pneumoniae is a gram positive bacteria which
is a major causative agent in invasive infections in animals and
humans, such as sepsis, meningitis, otitis media and lobar
pneumonia (Tuomanen et al. New Engl. J. Med. 322:1280-1284 (1995)).
As part of the infective process, pneumococci readily bind to
non-inflamed human epithelial cells of the upper and lower
respiratory tract by binding to eukaryotic carbohydrates in a
lectin-like manner (Cundell et al., Micro. Path. 17:361-374
(1994)). Conversion to invasive pneumococcal infections for bound
bacteria may involve the local generation of inflammatory factors
which may activate the epithelial cells to change the number and
type of receptors on their surface (Cundell et al., Nature,
377:435-438 (1995)). Apparently, one such receptor, platelet
activating factor (PAF) is engaged by the pneumococcal bacteria and
within a very short period of time (minutes) from the appearance of
PAF, pneumococci exhibit strongly enhanced adherence and invasion
of tissue. Certain soluble receptor analogs have been shown to
prevent the progression of pneumococcal infections
(Idanpaan-Heikkila et al., J. Inf. Dis., 176:704-712 (1997)). A
number of various other proteins have been suggested as being
involved in the pathogenicity of S. pneumoniae. There remains a
need for identifying polypeptides having epitopes in common from
various strains of S. pneumoniae in order to utilize such
polypeptides as vaccines to provide protection against a wide
variety of S. pneumoniae.
SUMMARY OF INVENTION
[0005] In accordance with the present invention, there is provided
vaccines and vaccine compositions that include polypeptides
obtained from S. pneumoniae and/or variants of said polypeptides
and/or active fragments of such polypeptides.
[0006] The active fragments, as hereinafter defined, include a
histidine triad residue(s) and/or coiled coil regions of such
polypeptides.
[0007] The term "percent identity" or "percent identical," when
referring to a sequence, means that a sequence is compared to a
claimed or described sequence from an alignment of the sequence to
be compared (the "Compared Sequence") with the described or claimed
sequence (the "Reference Sequence"). The percent identity is
determined as follows:
Percent Identity=[1-(C/R)]100
[0008] wherein C is the number of differences between the Reference
Sequence and the Compared Sequence over the length of the alignment
between the Compared Sequence and the Reference Sequence wherein
(i) each base or amino acid in the Reference Sequence that does not
have an aligned base or amino acid in the Compared Sequence and
(ii) each gap in the Reference Sequence and (iii) each aligned base
or amino acid in the Reference Sequence that is different from an
aligned base or amino acid in the Compared Sequence, each being a
difference; and R is the number of bases or amino acids in the
Reference Sequence over the length of the alignment with the
Compared Sequence with any gap created in the Reference Sequence
also being counted as a base or amino acid.
[0009] If an alignment exists between the Compared Sequence and the
Reference Sequence in which the Percent Identity as calculated
above is about equal to or greater than a specified minimum Percent
Identity then the Compared Sequence has the specified minimum
Percent Identity to the Reference Sequence even though alignments
may exist in which the hereinabove calculated Percent Identity is
less than the specified Percent Identity.
[0010] "Isolated" in the context of the present invention with
respect to polypeptides and/or polynucleotides means that the
material is removed from its original environment (e.g., the
natural environment if it is naturally occurring). For example, a
naturally-occurring polynucleotide or polypeptide present in a
living organism is not isolated, but the same polynucleotide or
polypeptide, separated from some or all of the co-existing
materials in the natural system, is isolated. Such polynucleotides
could be part of a vector and/or such polynucleotides or
polypeptides could be part of a composition, and still be isolated
in that such vector or composition is not part of its natural
environment. The polypeptides and polynucleotides of the present
invention are preferably provided in an isolated form, and
preferably are purified to homogeneity.
BRIEF DESCRIPTION OF DRAWINGS
[0011] FIGS. 1A-1C, respectively, report the results of three
experiments using different preparations of SP36. The results
demonstrate that active immunization with recombinant SP36 derived
from pneumococcal strain Norway serotype 4 is able to protect mice
from death in a model of pneumococcal sepsis using a heterologous
strain, SJ2 (serotype 6B). In each of the three experiments shown,
one hundred percent of the mice immunized with SP36 survived for
the 14-day observation period following challenge with
approximately 500 cfu of pneumococci, while eighty to one hundred
percent of sham-immunized mice (injected with PBS and adjuvant)
died during the same period.
[0012] FIGS. 2A-2B show that passive administration of rabbit
antiserum raised against Sp36 derived from Norway type 4 was able
to protect mice in the pneumococcal sepsis model using two
heterologous strains. FIG. 2A shows that one hundred percent of the
mice immunized with the SP36 antiserum survived the 21-day
observation period after challenge with 172 CFU of strain SJ2
(serotype 6B). Eighty percent of the mice immunized with a control
serum (rabbit anti-FimC) died by day 8, and ninety percent died by
day 12. FIG. 2B shows that 90 percent of the mice immunized with
the Sp36 antiserum survived the 8-day observation after challenge
with 862 CFU of strain EF6796 (serotype 6A). Ninety percent of the
mice immunized with a control serum (collected before immunization)
died by day 5.
[0013] FIG. 3 is a western blot demonstrating the ability of
antisera raised against recombinant Sp36 derived from strain Norway
type 4 to react with Sp36 of heterologous strains. Total cell
lysates were immunoblotted with mouse antisera to Sp36. A band
representing Sp36 protein was detected in all 23 S. pneumoniae
strains tested, which included isolates from each of the 23
pneumococcal serotypes represented in the current polysaccharide
vaccine.
[0014] FIG. 4 is a Southern blot showing that the Sp36 gene from
Norway type 4 hybridizes with genomic DNA from 24 other
pneumococcal strains, indicating the presence of similar sequences
in all these strains.
[0015] FIG. 6 is an amino acid alignment comparison of four related
pneumococcal proteins, namely Sp36A (PhtA; SEQ ID NO:8), Sp36B
(PhtB; SEQ ID NO:10), Sp36D (PhtD; SEQ ID NO:4), Sp36E (PhtE; SEQ
ID NO:6), respectively. Dashes in a sequence indicate gaps
introduced to maximize the sequence similarity. Amino acid residues
that match are boxed.
[0016] FIG. 7 is a nucleotide alignment comparison of four related
pneumococcal genes, namely Sp36A (PhtA; SEQ ID NO:9), Sp36B (PhtB;
SEQ ID NO:11), Sp36D (PhtD; SEQ ID NO:5), Sp36E (PhtE; SEQ ID
NO:7), respectively. Dashes in a sequence indicate gaps introduced
to maximize the sequence similarity.
[0017] FIG. 8 shows the results of immunization of mice with PhtD
recombinant protein, which leads to protection from lethal sepsis.
C3H/HeJ (Panel A and B) or Balb/cByJ (Panel C) mice were immunized
subcutaneously with PhtD protein (15 .mu.g in 50 .mu.l PBS
emulsified in 50 .mu.l complete Freund's adjuvant (CFA)). The
recombinant PhtD protein used in protection experiments consisted
of 819 amino acid residues, starting with the cysteine (residue
20). A group of 10 sham-immunized mice received PBS with adjuvant.
A second immunization of 15 .mu.g protein with incomplete Freund's
adjuvant (IFA) was administered 3 weeks later; the sham group
received PBS with IFA. Blood was drawn (retro-orbital bleed) at
week 7; and sera from each group was pooled for analysis of
anti-PhtD antibody by ELISA. Mice were challenged at week 8 by an
intraperitonial (i.p.) injection of approximately 550 CFU S.
pneumoniae strain SJ2, serotype 6B (Panel A), 850 CFU of strain
EF6796, serotype 6A (Panel B) or 450 CFU of strain EF5668, serotype
4 (Panel C). In preliminary experiments, the LD.sub.50 for strain
SJ2 and EF6796 were determined to be approximately 10 CFU for both
strains. The LD.sub.50 for strain EF5668 was determined to be <5
CFU. Survival was determined in all groups over the course of 15
days following challenge. Data are presented as the percent
survival for a total of 10 mice per experimental group. Two-sample
Log-rank test was used for statistical analysis comparing
recombinant Pht immunized mice to sham-immunized mice.
SUMMARY OF THE INVENTION
[0018] In accordance with one aspect of the present invention,
there is provided a vaccine, generally in the form of a
composition, that includes at least one polypeptide that is at
least 90% identical to (c) a polypeptide sequence comprising amino
acids 20-838 of SEQ ID NO:4 or (ii) a polypeptide sequence
comprising amino acids 21-480 of SEQ ID NO:6 or an active fragment
of the foregoing.
[0019] In accordance with another aspect of the present invention,
there is provided a vaccine, generally in the form of a
composition, that includes an active fragment of a polypeptide that
is at least 90% identical to (i) a polypeptide comprising amino
acids 20-819 of SEQ ID NO:8 or (ii) a polypeptide comprising amino
acids 20-819 of SEQ ID NO:10.
[0020] The term "active fragment" means a fragment that includes
one or more histidine triad residues and/or one or more coiled coil
regions. A "histidine triad residue" is the portion of the
polypeptide that has the sequence HxxHxH (SEQ ID NO: 12) wherein H
is histidine and x is an amino acid other than histidine
[0021] A coiled coil region is the region predicted by "Coils"
algorithm: Lupas, A., Van Dyke, M., and Stock, J. (1991) Predicting
Coiled Coils from Protein Sequences, Science 252:1162-1164.
[0022] In accordance with one embodiment, the active fragment
includes both one or more histidine triad residues and at least one
coiled coil region of the applicable polypeptide sequence. In
accordance with another embodiment, the active fragment includes at
least two histidine triad residues.
[0023] In another embodiment, the active fragment that includes at
least one histidine triad residue or at least one coiled-coil
region of the applicable polypeptide includes at least about ten
percent of the applicable polypeptide and no more than about 85% of
the applicable polypeptide.
[0024] The polypeptide of SEQ ID NO:4 includes five histidine triad
residues, as follows:
[0025] amino acids 83-88, 207-212, 315-320, 560-565, and
644-649.
[0026] The polypeptide of SEQ ID NO:6 includes five histidine triad
residues, as follows:
[0027] amino acids 83-88, 205-210, 309-314, 396-401 and 461-466
[0028] In addition, the polypeptide of SEQ ID NO:4 includes two
coiled-coil regions (amino acids 139-159 and amino acids 769-791)
and the polypeptide of SEQ ID NO:6 includes one coiled-coil region
(amino acids 139-172).
[0029] The polypeptide of SEQ ID NO: 8 includes the following
regions:
[0030] HxxHxH (SEQ ID NO: 12): amino acids 82-87, 208-213, 328-333,
569-574 and 653-658.
[0031] Coiled-coils: amino acids 137-164, 425-453, 481-512, and
743-770.
[0032] In accordance with a further aspect of the invention, a
vaccine of the type hereinabove described is administered for the
purpose of preventing or treating infection caused by S.
pneumoniae.
[0033] A vaccine, or vaccine composition, in accordance with the
present invention may include one or more of the hereinabove
described polypeptides or active fragments thereof. When employing
more than one polypeptide or active fragment, such two or more
polypeptides and/or active fragments may be used as a physical
mixture or as a fusion of two or more polypeptides or active
fragments. The fusion fragment or fusion polypeptide may be
produced, for example, by recombinant techniques or by the use of
appropriate linkers for fusing previously prepared polypeptides or
active fragments.
[0034] In an embodiment of the invention, there is provided (a) a
polypeptide that is at least 95% identical or at least 97%
identical or 100% identical to (i) a polypeptide sequence
comprising amino acids 20-838 of SEQ ID NO:4 or (ii) a polypeptide
sequence comprising amino acids 21-480 of SEQ ID NO:6; or (b) an
active fragment of the polypeptide of (a).
[0035] In the case where the polypeptide is a variant of the
polypeptide comprising the mature polypeptide of SEQ ID NO:4 or SEQ
ID NO:6, or any of the active fragments of the invention, the
variation in the polypeptide or fragment is generally in a portion
thereof other than the histidine triad residues and the coiled-coil
region, although variations in one or more of these regions may be
made.
[0036] In many cases, the variation in the polypeptide or active
fragment is a conservative amino acid substitution, although other
substitutions are within the scope of the invention.
[0037] In accordance with the present invention, a polypeptide
variant includes variants in which one or more amino acids are
substituted and/or deleted and/or inserted.
[0038] In another aspect, the invention relates to passive immunity
vaccines formulated from antibodies against a polypeptide or active
fragment of a polypeptide of the present invention. Such passive
immunity vaccines can be utilized to prevent and/or treat
pneumococcal infections in patients. In this manner, according to a
further aspect of the invention, a vaccine can be produced from a
synthetic or recombinant polypeptide of the present invention or an
antibody against such polypeptide.
[0039] In still another aspect the present invention relates to a
method of using one or more antibodies (monoclonal, polyclonal or
sera) to the polypeptides of the invention as described above for
the prophylaxis and/or treatment of diseases that are caused by
pneumococcal bacteria. In particular, the invention relates to a
method for the prophylaxis and/or treatment of infectious diseases
that are caused by S. pneumoniae. In a still further preferred
aspect, the invention relates to a method for the prophylaxis
and/or treatment of otitis media, nasopharyngeal, bronchial
infections, and the like in humans by utilizing a vaccine of the
present invention.
[0040] Generally, vaccines are prepared as injectables, in the form
of aqueous solutions or suspensions. Vaccines in an oil base are
also well known such as for inhaling. Solid forms which are
dissolved or suspended prior to use may also be formulated.
Pharmaceutical carriers are generally added that are compatible
with the active ingredients and acceptable for pharmaceutical use.
Examples of such carriers include, but are not limited to, water,
saline solutions, dextrose, or glycerol. Combinations of carriers
may also be used.
[0041] Vaccine compositions may further incorporate additional
substances to stabilize pH, or to function as adjuvants, wetting
agents. or emulsifying agents, which can serve to improve the
effectiveness of the vaccine.
[0042] Vaccines are generally formulated for parental
administration and are injected either subcutaneously or
intramuscularly. Such vaccines can also be formulated as
suppositories or for oral administration, using methods known in
the art.
[0043] The amount of vaccine sufficient to confer immunity to
pathogenic bacteria is determined by methods well known to those
skilled in the art. This quantity will be determined based upon the
characteristics of the vaccine recipient and the level of immunity
required. Typically, the amount of vaccine to be administered will
be determined based upon the judgment of a skilled physician. Where
vaccines are administered by subcutaneous or intramuscular
injection, a range of 50 to 500 .mu.g purified protein may be
given.
[0044] The present invention is also directed to a vaccine in which
a polypeptide or active fragment of the present invention is
delivered or administered in the form of a polynucleotide encoding
the polypeptide or active fragment, whereby the polypeptide or
active fragment is produced in vivo. The polynucleotide may be
included in a suitable expression vector and combined with a
pharmaceutically acceptable carrier.
[0045] In addition, the polypeptides of the present invention can
be used as immunogens to stimulate the production of antibodies for
use in passive immunotherapy, for use as diagnostic reagents, and
for use as reagents in other processes such as affinity
chromatography.
[0046] In another aspect the present invention provides
polynucleotides which encode the hereinabove described polypeptides
and active fragments of the invention. The polynucleotide of the
present invention may be in the form of RNA or in the form of DNA,
which DNA includes cDNA, genomic DNA, and synthetic DNA. The DNA
may be double-stranded or single-stranded, and if single stranded
may be the coding strand or non-coding (anti-sense) strand.
[0047] In accordance with another aspect of the present invention,
there is provided
[0048] (A) an isolated polynucleotide that is at least 90%
identical to a polynucleotide sequence encoding (i) a polypeptide
comprising amino acids 20-838 of SEQ ID NO:4 or (ii) a polypeptide
comprising amino acids 21-480 of SEQ ID NO:6, or
[0049] (B) a fragment of the polynucleotide of (A) that encodes an
active polypeptide fragment or
[0050] (C) a polynucleotide that is at least 90% identical to a
polynucleotide sequence encoding an active fragment of (i) a
polypeptide comprising amino acids 20-819 of SEQ ID NO:8 or (ii) a
polypeptide comprising amino acids 20-819 of SEQ ID NO:10.
[0051] In specific embodiments, the polynucleotide is at least 95%
identical, preferably at least 97% identical, and even 100%
identical to such polynucleotide sequence.
[0052] The term "polynucleotide encoding a polypeptide" encompasses
a polynucleotide which includes only coding sequence for the
polypeptide as well as a polynucleotide which includes additional
coding and/or non-coding sequence.
[0053] The present invention further relates to variants of
polynucleotides. The variants of the polynucleotides may be a
naturally occurring allelic variant of the polynucleotides or a
non-naturally occurring variant of the polynucleotides. The
variants include variants in which one or more bases are
substituted, deleted or inserted. Complements to such coding
polynucleotides may be utilized to isolate polynucleotides encoding
the same or similar polypeptides. In particular, such procedures
are useful to obtain native immunogenic portions of polypeptides
from different serotypes of S. pneumoniae, which is especially
useful in the production of "chain" polypeptide vaccines containing
multiple immunogenic segments.
[0054] SEQ ID NO:5 is a representative example of a polynucleotide
encoding the polypeptide of SEQ ID NO:4 and SEQ ID NO:7 is a
representative example of a polynucleotide encoding the polypeptide
of SEQ ID NO:6. SEQ ID NO:9 is a representative example of a
polynucleotide encoding the polypeptide of SEQ ID NO:8, and SEQ ID
NO:11 is a representative example of a polynucleotide encoding the
polypeptide of SEQ ID NO:10. As a result of the known degeneracy of
the genetic code, other polynucleotides that encode the
polypeptides of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 and SEQ ID
NO:10 should be apparent to those skilled in the art from the
teachings herein.
[0055] The polynucleotides encoding the immunogenic polypeptides
described above may also have the coding sequence fused in frame to
a marker sequence which allows for purification of the polypeptides
of the present invention. The marker sequence may be, for example,
a hexa-histidine tag supplied by a pQE-9 vector to provide for
purification of the mature polypeptides fused to the marker in the
case of a bacterial host, or, for example, the marker sequence may
be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7
cells, is used. The HA tag corresponds to an epitope derived from
the influenza hemagglutinin protein (Wilson, I., et al., Cell,
37:767 (1984)).
[0056] The present invention also relates to vectors which include
polynucleotides encoding one or more of the polypeptides of the
invention, host cells which are genetically engineered with vectors
of the invention and the production of such immunogenic
polypeptides by recombinant techniques in an isolated and
substantially immunogenically pure form.
[0057] Host cells are genetically engineered (transduced or
transformed or transfected) with the vectors comprising a
polynucleotide encoding a polypeptide of the invention. The vector
may be, for example, in the form of a plasmid, a viral particle, a
phage, etc. The engineered host cells can be cultured in
conventional nutrient media modified as appropriate for activating
promoters, selecting transformants or amplifying the
polynucleotides which encode such polypeptides. The culture
conditions, such as temperature, pH and the like, are those
previously used with the host cell selected for expression, and
will be apparent to the ordinarily skilled artisan.
[0058] Vectors include chromosomal, nonchromosomal and synthetic
DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage
DNA; baculovirus; yeast plasmids; vectors derived from combinations
of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus,
fowl pox virus, and pseudorabies. However, any other vector may be
used as long as it is replicable and viable in the host.
[0059] The appropriate DNA sequence may be inserted into the vector
by a variety of procedures. In general, the DNA sequence is
inserted into an appropriate restriction endonuclease site(s) by
procedures known in the art. Such procedures and others are deemed
to be within the scope of those skilled in the art.
[0060] The DNA sequence in the expression vector is operatively
linked to an appropriate expression control sequence(s) (promoter)
to direct mRNA synthesis. As representative examples of such
promoters, there may be mentioned: LTR or SV40 promoter, the E.
coli. lac or trp, the phage lambda P.sub.L promoter and other
promoters known to control expression of genes in prokaryotic or
eukaryotic cells or their viruses. The expression vector also
contains a ribosome binding site for translation initiation and a
transcription terminator. The vector may also include appropriate
sequences for amplifying expression.
[0061] In addition, the expression vectors preferably contain one
or more selectable marker genes to provide a phenotypic trait for
selection of transformed host cells such as dihydrofolate reductase
or neomycin resistance for eukaryotic cell culture, or such as
tetracycline or ampicillin resistance in E. coli.
[0062] The vector containing the appropriate DNA sequence as
hereinabove described, as well as an appropriate promoter or
control sequence, may be employed to transform an appropriate host
to permit the host to express the proteins.
[0063] As representative examples of appropriate hosts, there may
be mentioned: bacterial cells, such as E. coli, Streptomyces,
Salmonella typhimurium; fungal cells, such as yeast; insect cells
such as Drosophila S2 and Spodoptera Sf9; animal cells such as CHO,
COS or Bowes melanoma; adenoviruses; plant cells, etc. The
selection of an appropriate host is deemed to be within the scope
of those skilled in the art from the teachings herein.
[0064] More particularly, the present invention also includes
recombinant constructs comprising one or more of the sequences as
broadly described above. The constructs comprise a vector, such as
a plasmid or viral vector, into which a sequence of the invention
has been inserted, in a forward or reverse orientation. In a
preferred aspect of this embodiment, the construct further
comprises regulatory sequences, including, for example, a promoter,
operably linked to the sequence. Large numbers of suitable vectors
and promoters are known to those of skill in the art, and are
commercially available. The following vectors are provided by way
of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen, Inc.), pbs,
pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a,
pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540,
pRIT5 (Pharmacia). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG
(Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any
other plasmid or vector may be used as long as they are replicable
and viable in the host.
[0065] Promoter regions can be selected from any desired gene using
CAT (chloramphenicol transferase) vectors or other vectors with
selectable markers. Two appropriate vectors are pKK232-8 and pCM7.
Particular named bacterial promoters include lacl, lacZ, T3, T7,
gpt, lambda P.sub.R, P.sub.L and TRP. Eukaryotic promoters include
CMV immediate early, HSV thymidine kinase, early and late SV40,
LTRs from retrovirus, and mouse metallothionein-l. Selection of the
appropriate vector and promoter is well within the level of
ordinary skill in the art.
[0066] In a further embodiment, the present invention relates to
host cells containing the above-described constructs. The host cell
can be a higher eukaryotic cell, such as a mammalian cell, or a
lower eukaryotic cell, such as a yeast cell, or the host cell can
be a prokaryotic cell, such as a bacterial cell. Introduction of
the construct into the host cell can be effected by calcium
phosphate transfection, DEAE-Dextran mediated transfection, or
electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods
in Molecular Biology, (1986)).
[0067] The constructs in host cells can be used in a conventional
manner to produce the gene product encoded by the recombinant
sequence. Alternatively, the polypeptides of the invention can be
synthetically produced by conventional peptide synthesizers.
[0068] Mature proteins can be expressed in mammalian cells, yeast,
bacteria, or other cells under the control of appropriate
promoters. Cell-free translation systems can also be employed to
produce such proteins using RNAs derived from the DNA constructs of
the present invention. Appropriate cloning and expression vectors
for use with prokaryotic and eukaryotic hosts are described by
Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second
Edition, Cold Spring Harbor, N.Y., (1989), the disclosure of which
is hereby incorporated by reference.
[0069] Transcription of the DNA encoding the polypeptides of the
present invention by higher eukaryotes is increased by inserting an
enhancer sequence into the vector. Enhancers are cis-acting
elements of DNA, usually about from 10 to 300 bp that act on a
promoter to increase its transcription. Examples including the SV40
enhancer on the late side of the replication origin bp 100 to 270,
a cytomegalovirus early promoter enhancer, the polyoma enhancer on
the late side of the replication origin, and adenovirus
enhancers.
[0070] Generally, recombinant expression vectors will include
origins of replication and selectable markers permitting
transformation of the host cell, e.g., the ampicillin resistance
gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived
from a highly-expressed gene to direct transcription of a
downstream structural sequence. Such promoters can be derived from
operons encoding glycolytic enzymes such as 3-phosphoglycerate
kinase (PGK), .alpha.-factor, acid phosphatase, or heat shock
proteins, among others. The heterologous structural sequence is
assembled in appropriate phase with translation initiation and
termination sequences. Optionally, the heterologous sequence can
encode a fusion protein including an N-terminal identification
peptide imparting desired characteristics, e.g., stabilization or
simplified purification of expressed recombinant product.
[0071] Useful expression vectors for bacterial use are constructed
by inserting a structural DNA sequence encoding a desired protein
together with suitable translation initiation and termination
signals in operable reading phase with a functional promoter. The
vector will comprise one or more phenotypic selectable markers and
an origin of replication to ensure maintenance of the vector and
to, if desirable, provide amplification within the host. Suitable
prokaryotic hosts for transformation include E. coli, Bacillus
subtilis, Salmonella typhimurium and various species within the
genera Pseudomonas, Streptomyces, and Staphylococcus, although
others may also be employed as a matter of choice.
[0072] As a representative but nonlimiting example, useful
expression vectors for bacterial use can comprise a selectable
marker and bacterial origin of replication derived from
commercially available plasmids comprising genetic elements of the
well known cloning vector pBR322 (ATCC 37017). Such commercial
vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals,
Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis., USA).
These pBR322 "backbone" sections are combined with an appropriate
promoter and the structural sequence to be expressed.
[0073] Following transformation of a suitable host strain and
growth of the host strain to an appropriate cell density, the
selected promoter is induced by appropriate means (e.g.,
temperature shift or chemical induction) and cells are cultured for
an additional period.
[0074] Cells are typically harvested by centrifugation, disrupted
by physical or chemical means, and the resulting crude extract
retained for further purification.
[0075] Microbial cells employed in expression of proteins can be
disrupted by any convenient method, including freeze-thaw cycling,
sonication, a french press, mechanical disruption, or use of cell
lysing agents, such methods are well know to those skilled in the
art. However, preferred are host cells which secrete the
polypeptide of the invention and permit recovery of the polypeptide
from the culture media.
[0076] Various mammalian cell culture systems can also be employed
to express recombinant protein. Examples of mammalian expression
systems include the COS-7 lines of monkey kidney fibroblasts,
described by Gluzman, Cell, 23:175 (1981), and other cell lines
capable of expressing a compatible vector, for example, the C127,
3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors
will comprise an origin of replication, a suitable promoter and
enhancer, and also any necessary ribosome binding sites,
polyadenylation site, splice donor and acceptor sites,
transcriptional termination sequences, and 5' flanking
nontranscribed sequences. DNA sequences derived from the SV40
splice, and polyadenylation sites may be used to provide the
required nontranscribed genetic elements.
[0077] The polypeptides can be recovered and/or purified from
recombinant cell cultures by well-known protein recovery and
purification methods. Such methodology may include ammonium sulfate
or ethanol precipitation, acid extraction, anion or cation exchange
chromatography, phosphocellulose chromatography, hydrophobic
interaction chromatography, affinity chromatography,
hydroxylapatite chromatography and lectin chromatography. Protein
refolding steps can be used, as necessary, in completing
configuration of the mature protein. In this respect, chaperones
may be used in such a refolding procedure. Finally, high
performance liquid chromatography (HPLC) can be employed for final
purification steps.
[0078] The polypeptides that are useful as immunogens in the
present invention may be a naturally purified product, or a product
of chemical synthetic procedures, or produced by recombinant
techniques from a prokaryotic or eukaryotic host (for example, by
bacterial, yeast, higher plant, insect and mammalian cells in
culture). Depending upon the host employed in a recombinant
production procedure, the polypeptides of the present invention may
be glycosylated or may be non- glycosylated.
[0079] Procedures for the isolation of the individually expressed
polypeptides may be isolated by recombinant expression/isolation
methods that are well-known in the art. Typical examples for such
isolation may utilize an antibody to a conserved area of the
protein or to a His tag or cleavable leader or tail that is
expressed as part of the protein structure.
[0080] The polypeptides, their fragments or other derivatives, or
analogs thereof, or cells expressing them can be used as an
immunogen to produce antibodies thereto. These antibodies can be,
for example, polyclonal or monoclonal antibodies. The present
invention also includes chimeric, single chain, and humanized
antibodies, as well as Fab fragments, or the product of an Fab
expression library. Various procedures known in the art may be used
for the production of such antibodies and fragments.
[0081] Antibodies generated against the polypeptides corresponding
to a sequence of the present invention can be obtained by direct
injection of the polypeptides into an animal.
[0082] For preparation of monoclonal antibodies, any technique
which provides antibodies produced by continuous cell line cultures
can be used. Examples include the hybridoma technique (Kohler and
Milstein, 1975, Nature, 256:495-497), the trioma technique, the
human B-cell hybridoma technique (Kozbor et al., 1983, Immunology
Today 4:72), and the EBV-hybridoma technique to produce human
monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies
and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
[0083] Techniques described for the production of single chain
antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce
single chain antibodies to immunogenic polypeptide products of this
invention. Also, transgenic mice may be used to express humanized
antibodies to immunogenic polypeptide products of this
invention.
[0084] The invention will be further described with respect to the
following examples; however, the scope of the invention is not
limited thereby:
EXAMPLE 1
[0085] Active Protection with Anti-Sp36
[0086] A. Cloning, Expression, and Purification of SP36
[0087] The genomic DNA used as target for amplification was
isolated from S. pneumoniae Norway strain (serotype 4), the same
strain used for genomic sequencing. The complete sequence of the
Sp36 gene (SEQ ID NO:9), and its predicted amino acid sequence (SEQ
ID NO:8), are given in the Sequence Listing appended hereto. It was
noted that the predicted amino acid sequence included a hydrophobic
leader sequence followed by a sequence (LSVC-SEQ ID NO: 13) similar
to the consensus sequence for Type II signal peptidase (LxxC (SEQ
ID NO: 14) in which both x's typically represent small amino
acids). Primers (listed as SEQ ID NOS:1-3) were designed that would
amplify the Sp36 gene and allow its cloning into pQE10 and
expression as a histidine-tagged protein lacking the signal
sequence for purification by nickel-affinity chromatography.
Cloning of the fragment amplified by SEQ ID Nos 1 and 3 would
result in a protein containing amino acids 21 through 819 of Sp36;
cloning of the fragment amplified by SEQ ID Nos 2 and 3 would
result in a protein containing amino acids 26 through 819 of Sp36
(amino acid numbers refer to SEQ ID NO:8).
[0088] B. Active Protection with Sp36 Vaccination
[0089] In each of the three experiments shown in FIGS. 1A-1C,
C3H/HeJ mice (10/group) were immunized intraperitoneally (i.p.)
with Sp36 protein (15 .mu.g in 50 .mu.l PBS emulsified in 50 .mu.l
complete Freund's adjuvant (CFA)). A group of 10 sham-immunized
mice received PBS with adjuvant. A second immunization of 15 .mu.g
protein with incomplete Freund's adjuvant (IFA) was administered 4
weeks later; the sham group received PBS with IFA. Blood was drawn
(retro-orbital bleed) at weeks 3, 6, and 9; and sera from each
group were pooled for analysis of anti-Sp36 antibody by ELISA. Mice
were challenged at week 10 by an i.p. injection of approximately
500 CFU S. pneumoniae strain SJ2 (serotype 6B; provided by P.
Flynn, St. Jude Children's Research Hospital, Memphis, Tenn.). In
preliminary experiments, the LD.sub.50 of this strain was
determined to be approximately 10 CFU. Mice were monitored for 14
days for survival.
[0090] The three experiments shown in FIGS. 1A-1C used slightly
different preparations of recombinant Sp36. The experiments shown
in FIGS. 1A and 1B both used Sp36 containing amino acids 20-815,
but different batches of protein were used in the two experiments.
The experiment shown in FIG. 1C used Sp36 containing amino acids
25-815.
[0091] In the experiment shown in FIG. 1A, 9-week sera collected
from the ten mice immunized with Sp36 (first batch) had an endpoint
ELISA titer of 1:4,096,000. No anti-Sp36 antibody was detected in
sera from sham-immunized mice. One hundred percent of the mice
immunized with Sp36 protein survived the challenge (520 cfu of
pneumococci) for 14 days. Eighty percent of sham-immunized mice
were dead by day 4, and the remainder survived.
[0092] In the experiment shown in FIG. 1B, 9-week sera collected
from the ten mice immunized with Sp36 (second batch) had an
endpoint ELISA titer of >1:4,096,000. No anti-Sp36 antibody was
detected in sera from sham-immunized mice. One hundred percent of
the mice immunized with Sp36 protein survived the challenge (510
cfu of pneumococci) for 14 days. Of the sham-immunized mice, eighty
percent were dead by day 4, and all died by day 9.
[0093] In the experiment shown in FIG. 1C, 9-week sera collected
from the ten mice immunized with Sp36 (containing amino acids 25-
815) had an endpoint ELISA titer of 1:4,096,000. No anti-Sp36
antibody was detected in sera from sham-immunized mice. One hundred
percent of the mice immunized with Sp36 protein survived the
challenge (510 cfu of pneumococci) for 14 days. Of the
sham-immunized mice, ninety percent died by day 4, and all died by
day 12. These data demonstrate that immunization of mice with
recombinant Sp36 proteins elicits a response capable of protecting
against systemic pneumococcal infection and death. This protection
was not strain-specific: the recombinant pneumococcal protein was
cloned from a serotype 4 strain, while the challenge was with a
heterologous strain, SJ2 (serotype 6B).
EXAMPLE 2
[0094] Passive Protection with Anti-Sp36 Antisera
[0095] A. Generation of Rabbit Immune Sera
[0096] Following collection of preimmune serum, a New Zealand White
rabbit was immunized with 250 .mu.g of Sp36 (containing amino acids
20-815) in CFA. The rabbit was given two boosts of 125 .mu.g Sp36
in IFA on days 29 and 50 and bled on days 39 and 60. A second
rabbit was immunized with a control antigen, E. coli FimC.
[0097] B. Passive Protection in Mice
[0098] C3H/HeJ mice (10 mice/group) were passively immunized by two
i.p. injections of 100 .mu.l of rabbit serum. The first injection
was administered twenty-four hours before challenge with 172 cfu of
S. pneumoniae strain SJ2, and the second injection was given four
hours after challenge. FIG. 2 shows the survival of mice after
infection with two different strains of pneumococci.
[0099] FIG. 2A shows that of mice injected with 172 cfu of strain
SJ2 (FIG. 2A), one hundred percent of the mice immunized with
rabbit immune serum raised against Sp36 protein survived the 21-day
observation period. Of the mice immunized with the control serum
(anti-FimC), eighty percent died by day 8, and ninety percent died
by day 12. FIG. 2B shows that of mice injected with 862 cfu of
strain EF6796, ninety percent of the mice immunized with rabbit
immune serum raised against Sp36 protein survived the 8-day
observation period. Of those given a control serum (collected from
a rabbit before immunization), ninety percent died by day 8.
[0100] These data indicate that the protection against pneumococcal
infection resulting from immunization with Sp36 is
antibody-mediated, since mice can be protected by passive transfer
of serum from a hyperimmunized rabbit. As seen in the mouse active
challenge experiments described above, serum directed against
recombinant Sp36 protein cloned from a serotype 4 strain was
protective against challenge with heterologous strains.
Example 3
[0101] Conservation of Sp36 among Strains of S. pneumoniae
[0102] A. Western Blotting
[0103] The 23 pneumococcal strains used in this experiment were
obtained from the American Type Culture Collection (Rockville, Md.)
and include one isolate each of the 23 serotypes in the multivalent
pneumococcal vaccine. For total cell lysates, pneumococci were
grown to mid-logarithmic phase (optical density at 620 nm, 0.4 to
0.6) in 2 ml Todd-Hewitt broth with 0.5% yeast extract (Difco,
Detroit, Me.) at 37.degree.C. Bacteria were harvested by
centrifugation and washed twice with water. Pellets were
resuspended in 200 .mu.l lysis buffer (0.01% sodium dodecyl
sulfate, 0.15 M sodium citrate and 0.1% sodium deoxycholate) and
incubated at 37.degree. C. for 30 min, then diluted in an equal
volume 2.times.SSC (0.3 M sodium chloride, 0.03 M sodium citrate).
Lysates were separated by SDS-PAGE, transferred to nitrocellulose
membranes (Bio-Rad Laboratories, Hercules, Calif.), and probed with
antibody in a standard Western blotting procedure. Sera from ten
C3H/HeJ mice immunized with Sp36 (as described in Example 1) were
pooled and used at a dilution of 1:3000. Bound antibody was
detected with peroxidase-conjugated sheep anti- mouse lgG using the
chemiluminescence kit from Amersham, Inc. (Cambridge, Mass.).
[0104] The mouse anti-Sp36 sera detected two major bands with
apparent molecular weights of 97 and 100 kDa in all 23 pneumococcal
lysates tested (shown in FIG. 3). The Sp36 signals obtained from S.
pneumoniae serotypes 1, 5, 17F and 22F were lower, indicating
either that the level of Sp36 expression is reduced in these
strains, or that Sp36 in these strains is antigenically
different.
[0105] These data show that Sp36 is antigenically conserved among
strains of the 23 pneumococcal serotypes represented in the current
polysaccharide vaccine.
[0106] B. Southern Blotting
[0107] Genomic DNA was prepared from each of the 23 pneumococcal
strains listed in the previous section and also from strain SJ2.
DNA was digested with PvuII and BamHI, electrophoresed in an
agarose gel and transferred to a nylon membrane. A probe was
prepared by amplifying the Sp36 gene from Norway type 4 DNA (as in
Example 1) and labeling the amplified fragment with fluorescein by
the random-priming method, using a kit from Amersham.
Hybridization, washing, and exposure of film were carried out as in
the protocol supplied by Amersham. FIG. 4 shows that the Sp36 probe
hybridized with DNA from each of the 24 strains studied. The lane
marked "M" contained DNA from lambda phage, digested with HindIII
and labeled with fluorescein, as molecular weight markers.
EXAMPLE 4
[0108] Immunogenicity of Sp36 in Humans
[0109] In order to determine whether Sp36 is immunogenic during
human pneumococcal infection, sera from patients with
culture-proven pneumococcal bacteremia were used in Western blots
containing recombinant Sp36 protein. In the experiment shown in
FIG. 5, sera from five patients (indicated as 1 through 5) were
diluted 1:3000 and used to probe blots containing full-length Sp36,
the N-terminal half of Sp36 (preceding the proline-rich region), or
the C-terminal half of Sp36 (following the proline-rich region).
Lanes labeled A (acute) were probed with serum collected shortly
after diagnosis of pneumococcal infection; lanes C (convalescent)
were probed with serum collected either one month later (patients
1, 2, and 3) or eight days after the first serum collection
(patients 4 and 5). For patients 2, 3 and 5, reactivity of the
convalescent serum with Sp36 was stronger that that of the
corresponding acute serum. The difference between the acute and
convalescent sera was particularly evident for reactivity with the
C-terminal half of the protein.
[0110] In additional experiments (not shown), convalescent sera
from 23 patients with pneumococcal infections were tested
individually for reactivity with full-length Sp36: 20 of the 23
sera were found to bind Sp36 on a Western blot.
[0111] These experiments indicate that Sp36 is recognized by the
human immune system and suggest that antibodies able to bind the
Sp36 protein may be produced during natural S. pneumoniae infection
in humans. Since the patients were infected with a variety of
pneumococcal strains, these data also support the idea that Sp36 is
antigenically conserved.
EXAMPLE 5
[0112] Table 1 provides the percent identity between the various
sequences.
[0113] Alignment of the predicted amino acid sequences of PhtA,
PhtB, PhtD, and PhtE using the MEGALIGN program of Lasergene showed
strong N-terminal homology with substantial divergence of the
C-termini (FIG. 6). The alignment of the nucleotide sequences of
the same genes is shown in FIG. 7. Amino acid and nucleotide
sequences were compared using the identity weighting in a
Lipman-Pearson pairwise alignment, in which the number of matching
residues is divided by the total of matching residues plus the
number of mismatched residues plus the number of residues in gaps.
In the table below, the percent identity between each pair of
sequences is shown at the intersection of the corresponding row and
column.
EXAMPLE 6
[0114] Active Protection with PhtD Vaccination.
[0115] Mice immunized with recombinant PhtD derived from strain N4
generated potent antibody titers (reciprocal endpoint titers
ranging from 2,048,00 to 4,096,000). Mice immunized with PhtD were
protected against death following intraperitoneal injection with
either of three heterologous strains, SJ2 (serotype 6B; provided by
P. Flynn, St. Jude Children's Research Hospital, Memphis, Tenn.),
EF6796 (serotype 6A) or EF5668 (serotype 4; both strains provided
by D. Briles, University of Alabama, Birmingham). In the experiment
shown in FIG. 8 (Panel A), all ten of the sham-immunized mice died
within 10-days after challenge with virulent pneumococci (strain
SJ2), while eighty percent of the PhtD-immunized mice survived the
15-day observation period. Immunization with PhtD also protected
against a serotype 6A strain, EF6796 (Panel B) and a serotype 4
strain, EF5668 (Panel C). In the experiment shown in FIG. 8 (Panel
B), all ten of the sham-immunized mice died within 7-days after
challenge with virulent pneumococci (strain EF6796), while ninety
percent of the PhtD-immunized mice survived the 15-day observation
period. In the experiment shown in FIG. 8 (Panel C), all ten of the
sham-immunized mice died within 6-days after challenge with
virulent pneumoccoci (strain EF5668), while eight of nine mice
immunized with PhtD survived the 15-day observation period.
1TABLE 1 Percent Identities PhtA PhtB PhtD PhtE Percent Identity
Between Amino Acid Sequences PhtA -- 66.4 63.9 49.5 PhtB -- 87.2
49.5 PhtD -- 49.8 PhtE -- Percent Identity Between Nucleotide
Sequences PhtA -- 58.3 59.3 47.9 PhtB -- 86.4 47.4 PhtD -- 47.9
PhtE --
[0116]
Sequence CWU 1
1
14 1 36 DNA Artificial Sequence Description of Artificial Sequence
Forward primer used in amplification of the Sp36 gene sequence. 1
atcggatcct tcttacgagt tgggactgta tcaagc 36 2 35 DNA Artificial
Sequence Description of Artificial Sequence Forward primer used in
amplification of the Sp36 gene sequence. 2 atcggatcca ctgtatcaag
ctagaacggt taagg 35 3 40 DNA Artificial Sequence Description of
Artificial Sequence Reverse primer used in amplification of the
Sp36 gene sequence. 3 agtcaagctt gtttattttt tccttactta cagatgaagg
40 4 838 PRT Streptococcus pneumoniae 4 Met Lys Ile Asn Lys Lys Tyr
Leu Ala Gly Ser Val Ala Val Leu Ala 1 5 10 15 Leu Ser Val Cys Ser
Tyr Glu Leu Gly Arg His Gln Ala Gly Gln Val 20 25 30 Lys Lys Glu
Ser Asn Arg Val Ser Tyr Ile Asp Gly Asp Gln Ala Gly 35 40 45 Gln
Lys Ala Glu Asn Leu Thr Pro Asp Glu Val Ser Lys Arg Glu Gly 50 55
60 Ile Asn Ala Glu Gln Ile Val Ile Lys Ile Thr Asp Gln Gly Tyr Val
65 70 75 80 Thr Ser His Gly Asp His Tyr His Tyr Tyr Asn Gly Lys Val
Pro Tyr 85 90 95 Asp Ala Ile Ile Ser Glu Glu Leu Leu Met Lys Asp
Pro Asn Tyr Gln 100 105 110 Leu Lys Asp Ser Asp Ile Val Asn Glu Ile
Lys Gly Gly Tyr Val Ile 115 120 125 Lys Val Asp Gly Lys Tyr Tyr Val
Tyr Leu Lys Asp Ala Ala His Ala 130 135 140 Asp Asn Ile Arg Thr Lys
Glu Glu Ile Lys Arg Gln Lys Gln Glu His 145 150 155 160 Ser His Asn
His Gly Gly Gly Ser Asn Asp Gln Ala Val Val Ala Ala 165 170 175 Arg
Ala Gln Gly Arg Tyr Thr Thr Asp Asp Gly Tyr Ile Phe Asn Ala 180 185
190 Ser Asp Ile Ile Glu Asp Thr Gly Asp Ala Tyr Ile Val Pro His Gly
195 200 205 Asp His Tyr His Tyr Ile Pro Lys Asn Glu Leu Ser Ala Ser
Glu Leu 210 215 220 Ala Ala Ala Glu Ala Tyr Trp Asn Gly Lys Gln Gly
Ser Arg Pro Ser 225 230 235 240 Ser Ser Ser Ser Tyr Asn Ala Asn Pro
Ala Gln Pro Arg Leu Ser Glu 245 250 255 Asn His Asn Leu Thr Val Thr
Pro Thr Tyr His Gln Asn Gln Gly Glu 260 265 270 Asn Ile Ser Ser Leu
Leu Arg Glu Leu Tyr Ala Lys Pro Leu Ser Glu 275 280 285 Arg His Val
Glu Ser Asp Gly Leu Ile Phe Asp Pro Ala Gln Ile Thr 290 295 300 Ser
Arg Thr Ala Arg Gly Val Ala Val Pro His Gly Asn His Tyr His 305 310
315 320 Phe Ile Pro Tyr Glu Gln Met Ser Glu Leu Glu Lys Arg Ile Ala
Arg 325 330 335 Ile Ile Pro Leu Arg Tyr Arg Ser Asn His Trp Val Pro
Asp Ser Arg 340 345 350 Pro Glu Gln Pro Ser Pro Gln Ser Thr Pro Glu
Pro Ser Pro Ser Pro 355 360 365 Gln Pro Ala Pro Asn Pro Gln Pro Ala
Pro Ser Asn Pro Ile Asp Glu 370 375 380 Lys Leu Val Lys Glu Ala Val
Arg Lys Val Gly Asp Gly Tyr Val Phe 385 390 395 400 Glu Glu Asn Gly
Val Ser Arg Tyr Ile Pro Ala Lys Asp Leu Ser Ala 405 410 415 Glu Thr
Ala Ala Gly Ile Asp Ser Lys Leu Ala Lys Gln Glu Ser Leu 420 425 430
Ser His Lys Leu Gly Ala Lys Lys Thr Asp Leu Pro Ser Ser Asp Arg 435
440 445 Glu Phe Tyr Asn Lys Ala Tyr Asp Leu Leu Ala Arg Ile His Gln
Asp 450 455 460 Leu Leu Asp Asn Lys Gly Arg Gln Val Asp Phe Glu Ala
Leu Asp Asn 465 470 475 480 Leu Leu Glu Arg Leu Lys Asp Val Pro Ser
Asp Lys Val Lys Leu Val 485 490 495 Asp Asp Ile Leu Ala Phe Leu Ala
Pro Ile Arg His Pro Glu Arg Leu 500 505 510 Gly Lys Pro Asn Ala Gln
Ile Thr Tyr Thr Asp Asp Glu Ile Gln Val 515 520 525 Ala Lys Leu Ala
Gly Lys Tyr Thr Thr Glu Asp Gly Tyr Ile Phe Asp 530 535 540 Pro Arg
Asp Ile Thr Ser Asp Glu Gly Asp Ala Tyr Val Thr Pro His 545 550 555
560 Met Thr His Ser His Trp Ile Lys Lys Asp Ser Leu Ser Glu Ala Glu
565 570 575 Arg Ala Ala Ala Gln Ala Tyr Ala Lys Glu Lys Gly Leu Thr
Pro Pro 580 585 590 Ser Thr Asp His Gln Asp Ser Gly Asn Thr Glu Ala
Lys Gly Ala Glu 595 600 605 Ala Ile Tyr Asn Arg Val Lys Ala Ala Lys
Lys Val Pro Leu Asp Arg 610 615 620 Met Pro Tyr Asn Leu Gln Tyr Thr
Val Glu Val Lys Asn Gly Ser Leu 625 630 635 640 Ile Ile Pro His Tyr
Asp His Tyr His Asn Ile Lys Phe Glu Trp Phe 645 650 655 Asp Glu Gly
Leu Tyr Glu Ala Pro Lys Gly Tyr Thr Leu Glu Asp Leu 660 665 670 Leu
Ala Thr Val Lys Tyr Tyr Val Glu His Pro Asn Glu Arg Pro His 675 680
685 Ser Asp Asn Gly Phe Gly Asn Ala Ser Asp His Val Arg Lys Asn Lys
690 695 700 Val Asp Gln Asp Ser Lys Pro Asp Glu Asp Lys Glu His Asp
Glu Val 705 710 715 720 Ser Glu Pro Thr His Pro Glu Ser Asp Glu Lys
Glu Asn His Ala Gly 725 730 735 Leu Asn Pro Ser Ala Asp Asn Leu Tyr
Lys Pro Ser Thr Asp Thr Glu 740 745 750 Glu Thr Glu Glu Glu Ala Glu
Asp Thr Thr Asp Glu Ala Glu Ile Pro 755 760 765 Gln Val Glu Asn Ser
Val Ile Asn Ala Lys Ile Ala Asp Ala Glu Ala 770 775 780 Leu Leu Glu
Lys Val Thr Asp Pro Ser Ile Arg Gln Asn Ala Met Glu 785 790 795 800
Thr Leu Thr Gly Leu Lys Ser Ser Leu Leu Leu Gly Thr Lys Asp Asn 805
810 815 Asn Thr Ile Ser Ala Glu Val Asp Ser Leu Leu Ala Leu Leu Lys
Glu 820 825 830 Ser Gln Pro Ala Pro Ile 835 5 2531 DNA
Streptococcus pneumoniae 5 atgaaaatta ataaaaaata tctagcaggt
tcagtggcag tccttgccct aagtgtttgt 60 tcctatgaac ttggtcgtca
ccaagctggt caggttaaga aagagtctaa tcgagtttct 120 tatatagatg
gtgatcaggc tggtcaaaag gcagaaaact tgacaccaga tgaagtcagt 180
aagagggagg ggatcaacgc cgaacaaatc gtcatcaaga ttacggatca aggttatgtg
240 acctctcatg gagaccatta tcattactat aatggcaagg tcccttatga
tgccatcatc 300 agtgaagagc tcctcatgaa agatccgaat tatcagttga
aggattcaga cattgtcaat 360 gaaatcaagg gtggttatgt tatcaaggta
gatggaaaat actatgttta ccttaaggat 420 gcagctcatg cggataatat
tcggacaaaa gaagagatta aacgtcagaa gcaggaacac 480 agtcataatc
acgggggtgg ttctaacgat caagcagtag ttgcagccag agcccaagga 540
cgctatacaa cggatgatgg ttatatcttc aatgcatctg atatcattga ggacacgggt
600 gatgcttata tcgttcctca cggcgaccat taccattaca ttcctaagaa
tgagttatca 660 gctagcgagt tagctgctgc agaagcctat tggaatggga
agcagggatc tcgtccttct 720 tcaagttcta gttataatgc aaatccagct
caaccaagat tgtcagagaa ccacaatctg 780 actgtcactc caacttatca
tcaaaatcaa ggggaaaaca tttcaagcct tttacgtgaa 840 ttgtatgcta
aacccttatc agaacgccat gtggaatctg atggccttat tttcgaccca 900
gcgcaaatca caagtcgaac cgccagaggt gtagctgtcc ctcatggtaa ccattaccac
960 tttatccctt atgaacaaat gtctgaattg gaaaaacgaa ttgctcgtat
tattcccctt 1020 cgttatcgtt caaaccattg ggtaccagat tcaagaccag
aacaaccaag tccacaatcg 1080 actccggaac ctagtccaag tccgcaacct
gcaccaaatc ctcaaccagc tccaagcaat 1140 ccaattgatg agaaattggt
caaagaagct gttcgaaaag taggcgatgg ttatgtcttt 1200 gaggagaatg
gagtttctcg ttatatccca gccaaggatc tttcagcaga aacagcagca 1260
ggcattgata gcaaactggc caagcaggaa agtttatctc ataagctagg agctaagaaa
1320 actgacctcc catctagtga tcgagaattt tacaataagg cttatgactt
actagcaaga 1380 attcaccaag atttacttga taataaaggt cgacaagttg
attttgaggc tttggataac 1440 ctgttggaac gactcaagga tgtcccaagt
gataaagtca agttagtgga tgatattctt 1500 gccttcttag ctccgattcg
tcatccagaa cgtttaggaa aaccaaatgc gcaaattacc 1560 tacactgatg
atgagattca agtagccaag ttggcaggca agtacacaac agaagacggt 1620
tatatctttg atcctcgtga tataaccagt gatgaggggg atgcctatgt aactccacat
1680 atgacccata gccactggat taaaaaagat agtttgtctg aagctgagag
agcggcagcc 1740 caggcttatg ctaaagagaa aggtttgacc cctccttcga
cagaccatca ggattcagga 1800 aatactgagg caaaaggagc agaagctatc
tacaaccgcg tgaaagcagc taagaaggtg 1860 ccacttgatc gtatgcctta
caatcttcaa tatactgtag aagtcaaaaa cggtagttta 1920 atcatacctc
attatgacca ttaccataac atcaaatttg agtggtttga cgaaggcctt 1980
tatgaggcac ctaaggggta tactcttgag gatcttttgg cgactgtcaa gtactatgtc
2040 gaacatccaa acgaacgtcc gcattcagat aatggttttg gtaacgctag
cgaccatgtt 2100 cgtaaaaata aggtagacca agacagtaaa cctgatgaag
ataaggaaca tgatgaagta 2160 agtgagccaa ctcaccctga atctgatgaa
aaagagaatc acgctggttt aaatccttca 2220 gcagataatc tttataaacc
aagcactgat acggaagaga cagaggaaga agctgaagat 2280 accacagatg
aggctgaaat tcctcaagta gagaattctg ttattaacgc taagatagca 2340
gatgcggagg ccttgctaga aaaagtaaca gatcctagta ttagacaaaa tgctatggag
2400 acattgactg gtctaaaaag tagtcttctt ctcggaacga aagataataa
cactatttca 2460 gcagaagtag atagtctctt ggctttgtta aaagaaagtc
aaccggctcc tatatagtaa 2520 aagcttaagc c 2531 6 484 PRT
Streptococcus pneumoniae 6 Met Lys Phe Ser Lys Lys Tyr Ile Ala Ala
Gly Ser Ala Val Ile Val 1 5 10 15 Ser Leu Ser Leu Cys Ala Tyr Ala
Leu Asn Gln His Arg Ser Gln Glu 20 25 30 Asn Lys Asp Asn Asn Arg
Val Ser Tyr Val Asp Gly Ser Gln Ser Ser 35 40 45 Gln Lys Ser Glu
Asn Leu Thr Pro Asp Gln Val Ser Gln Lys Glu Gly 50 55 60 Ile Gln
Ala Glu Gln Ile Val Ile Lys Ile Thr Asp Gln Gly Tyr Val 65 70 75 80
Thr Ser His Gly Asp His Tyr His Tyr Tyr Asn Gly Lys Val Pro Tyr 85
90 95 Asp Ala Leu Phe Ser Glu Glu Leu Leu Met Lys Asp Pro Asn Tyr
Gln 100 105 110 Leu Lys Asp Ala Asp Ile Val Asn Glu Val Lys Gly Gly
Tyr Ile Ile 115 120 125 Lys Val Asp Gly Lys Tyr Tyr Val Tyr Leu Lys
Asp Ala Ala His Ala 130 135 140 Asp Asn Val Arg Thr Lys Asp Glu Ile
Asn Arg Gln Lys Gln Glu His 145 150 155 160 Val Lys Asp Asn Glu Lys
Val Asn Ser Asn Val Ala Val Ala Arg Ser 165 170 175 Gln Gly Arg Tyr
Thr Thr Asn Asp Gly Tyr Val Phe Asn Pro Ala Asp 180 185 190 Ile Ile
Glu Asp Thr Gly Asn Ala Tyr Ile Val Pro His Gly Gly His 195 200 205
Tyr His Tyr Ile Pro Lys Ser Asp Leu Ser Ala Ser Glu Leu Ala Ala 210
215 220 Ala Lys Ala His Leu Ala Gly Lys Asn Met Gln Pro Ser Gln Leu
Ser 225 230 235 240 Tyr Ser Ser Thr Ala Ser Asp Asn Asn Thr Gln Ser
Val Ala Lys Gly 245 250 255 Ser Thr Ser Lys Pro Ala Asn Lys Ser Glu
Asn Leu Gln Ser Leu Leu 260 265 270 Lys Glu Leu Tyr Asp Ser Pro Ser
Ala Gln Arg Tyr Ser Glu Ser Asp 275 280 285 Gly Leu Val Phe Asp Pro
Ala Lys Ile Ile Ser Arg Thr Pro Asn Gly 290 295 300 Val Ala Ile Pro
His Gly Asp His Tyr His Phe Ile Pro Tyr Ser Lys 305 310 315 320 Leu
Ser Ala Leu Glu Glu Lys Ile Ala Arg Met Val Pro Ile Ser Gly 325 330
335 Thr Gly Ser Thr Val Ser Thr Asn Ala Lys Pro Asn Glu Val Val Ser
340 345 350 Ser Leu Gly Ser Leu Ser Ser Asn Pro Ser Ser Leu Thr Thr
Ser Lys 355 360 365 Glu Leu Ser Ser Ala Ser Asp Gly Tyr Ile Phe Asn
Pro Lys Asp Ile 370 375 380 Val Glu Glu Thr Ala Thr Ala Tyr Ile Val
Arg His Gly Asp His Phe 385 390 395 400 His Tyr Ile Pro Lys Ser Asn
Gln Ile Gly Gln Pro Thr Leu Pro Asn 405 410 415 Asn Ser Leu Ala Thr
Pro Ser Pro Ser Leu Pro Ile Asn Pro Gly Thr 420 425 430 Ser His Glu
Lys His Glu Glu Asp Gly Tyr Gly Phe Asp Ala Asn Arg 435 440 445 Ile
Ile Ala Glu Asp Glu Ser Gly Phe Val Met Ser His Gly Asp His 450 455
460 Asn His Tyr Phe Phe Lys Lys Asp Leu Thr Glu Glu Gln Ile Lys Val
465 470 475 480 Arg Lys Asn Ile 7 1455 DNA Streptococcus pneumoniae
7 atgaaattta gtaaaaaata tatagcagct ggatcagctg ttatcgtatc cttgagtcta
60 tgtgcctatg cactaaacca gcatcgttcg caggaaaata aggacaataa
tcgtgtctct 120 tatgtggatg gcagccagtc aagtcagaaa agtgaaaact
tgacaccaga ccaggttagc 180 cagaaagaag gaattcaggc tgagcaaatt
gtaatcaaaa ttacagatca gggctatgta 240 acgtcacacg gtgaccacta
tcattactat aatgggaaag ttccttatga tgccctcttt 300 agtgaagaac
tcttgatgaa ggatccaaac tatcaactta aagacgctga tattgtcaat 360
gaagtcaagg gtggttatat catcaaggtc gatggaaaat attatgtcta cctgaaagat
420 gcagctcatg ctgataatgt tcgaactaaa gatgaaatca atcgtcaaaa
acaagaacat 480 gtcaaagata atgagaaggt taactctaat gttgctgtag
caaggtctca gggacgatat 540 acgacaaatg atggttatgt ctttaatcca
gctgatatta tcgaagatac gggtaatgct 600 tatatcgttc ctcatggagg
tcactatcac tacattccca aaagcgattt atctgctagt 660 gaattagcag
cagctaaagc acatctggct ggaaaaaata tgcaaccgag tcagttaagc 720
tattcttcaa cagctagtga caataacacg caatctgtag caaaaggatc aactagcaag
780 ccagcaaata aatctgaaaa tctccagagt cttttgaagg aactctatga
ttcacctagc 840 gcccaacgtt acagtgaatc agatggcctg gtctttgacc
ctgctaagat tatcagtcgt 900 acaccaaatg gagttgcgat tccgcatggc
gaccattacc actttattcc ttacagcaag 960 ctttctgcct tagaagaaaa
gattgccaga atggtgccta tcagtggaac tggttctaca 1020 gtttctacaa
atgcaaaacc taatgaagta gtgtctagtc taggcagtct ttcaagcaat 1080
ccttcttctt taacgacaag taaggagctc tcttcagcat ctgatggtta tatttttaat
1140 ccaaaagata tcgttgaaga aacggctaca gcttatattg taagacatgg
tgatcatttc 1200 cattacattc caaaatcaaa tcaaattggg caaccgactc
ttccaaacaa tagtctagca 1260 acaccttctc catctcttcc aatcaatcca
ggaacttcac atgagaaaca tgaagaagat 1320 ggatacggat ttgatgctaa
tcgtattatc gctgaagatg aatcaggttt tgtcatgagt 1380 cacggagacc
acaatcatta tttcttcaag aaggacttga cagaagagca aattaaggtg 1440
cgcaaaaaca tttag 1455 8 819 PRT Streptococcus pneumoniae 8 Met Lys
Ile Asn Lys Lys Tyr Leu Val Gly Ser Ala Ala Ala Leu Ile 1 5 10 15
Leu Ser Val Cys Ser Tyr Glu Leu Gly Leu Tyr Gln Ala Arg Thr Val 20
25 30 Lys Glu Asn Asn Arg Val Ser Tyr Ile Asp Gly Lys Gln Ala Thr
Gln 35 40 45 Lys Thr Glu Asn Leu Thr Pro Asp Glu Val Ser Lys Arg
Glu Gly Ile 50 55 60 Asn Ala Glu Gln Ile Val Ile Lys Ile Thr Asp
Gln Gly Tyr Val Thr 65 70 75 80 Ser His Gly Asp His Tyr His Tyr Tyr
Asn Gly Lys Val Pro Tyr Asp 85 90 95 Ala Ile Ile Ser Glu Glu Leu
Leu Met Lys Asp Pro Asn Tyr Lys Leu 100 105 110 Lys Asp Glu Asp Ile
Val Asn Glu Val Lys Gly Gly Tyr Val Ile Lys 115 120 125 Val Asp Gly
Lys Tyr Tyr Val Tyr Leu Lys Asp Ala Ala His Ala Asp 130 135 140 Asn
Val Arg Thr Lys Glu Glu Ile Asn Arg Gln Lys Gln Glu His Ser 145 150
155 160 Gln His Arg Glu Gly Gly Thr Pro Arg Asn Asp Gly Ala Val Ala
Leu 165 170 175 Ala Arg Ser Gln Gly Arg Tyr Thr Thr Asp Asp Gly Tyr
Ile Phe Asn 180 185 190 Ala Ser Asp Ile Ile Glu Asp Thr Gly Asp Ala
Tyr Ile Val Pro His 195 200 205 Gly Asp His Tyr His Tyr Ile Pro Lys
Asn Glu Leu Ser Ala Ser Glu 210 215 220 Leu Ala Ala Ala Glu Ala Phe
Leu Ser Gly Arg Gly Asn Leu Ser Asn 225 230 235 240 Ser Arg Thr Tyr
Arg Arg Gln Asn Ser Asp Asn Thr Ser Arg Thr Asn 245 250 255 Trp Val
Pro Ser Val Ser Asn Pro Gly Thr Thr Asn Thr Asn Thr Ser 260 265 270
Asn Asn Ser Asn Thr Asn Ser Gln Ala Ser Gln Ser Asn Asp Ile Asp 275
280 285 Ser Leu Leu Lys Gln Leu Tyr Lys Leu Pro Leu Ser Gln Arg His
Val 290 295 300 Glu Ser Asp Gly Leu Val Phe Asp Pro Ala Gln Ile Thr
Ser Arg Thr 305 310 315 320 Ala Arg Gly Val Ala Val Pro His Gly Asp
His Tyr His Phe Ile Pro 325 330 335 Tyr Ser Gln Met Ser Glu Leu Glu
Glu Arg Ile Ala Arg Ile Ile Pro 340 345 350
Leu Arg Tyr Arg Ser Asn His Trp Val Pro Asp Ser Arg Pro Glu Gln 355
360 365 Pro Ser Pro Gln Pro Thr Pro Glu Pro Ser Pro Gly Pro Gln Pro
Ala 370 375 380 Pro Asn Leu Lys Ile Asp Ser Asn Ser Ser Leu Val Ser
Gln Leu Val 385 390 395 400 Arg Lys Val Gly Glu Gly Tyr Val Phe Glu
Glu Lys Gly Ile Ser Arg 405 410 415 Tyr Val Phe Ala Lys Asp Leu Pro
Ser Glu Thr Val Lys Asn Leu Glu 420 425 430 Ser Lys Leu Ser Lys Gln
Glu Ser Val Ser His Thr Leu Thr Ala Lys 435 440 445 Lys Glu Asn Val
Ala Pro Arg Asp Gln Glu Phe Tyr Asp Lys Ala Tyr 450 455 460 Asn Leu
Leu Thr Glu Ala His Lys Ala Leu Phe Glu Asn Lys Gly Arg 465 470 475
480 Asn Ser Asp Phe Gln Ala Leu Asp Lys Leu Leu Glu Arg Leu Asn Asp
485 490 495 Glu Ser Thr Asn Lys Glu Lys Leu Val Asp Asp Leu Leu Ala
Phe Leu 500 505 510 Ala Pro Ile Thr His Pro Glu Arg Leu Gly Lys Pro
Asn Ser Gln Ile 515 520 525 Glu Tyr Thr Glu Asp Glu Val Arg Ile Ala
Gln Leu Ala Asp Lys Tyr 530 535 540 Thr Thr Ser Asp Gly Tyr Ile Phe
Asp Glu His Asp Ile Ile Ser Asp 545 550 555 560 Glu Gly Asp Ala Tyr
Val Thr Pro His Met Gly His Ser His Trp Ile 565 570 575 Gly Lys Asp
Ser Leu Ser Asp Lys Glu Lys Val Ala Ala Gln Ala Tyr 580 585 590 Thr
Lys Glu Lys Gly Ile Leu Pro Pro Ser Pro Asp Ala Asp Val Lys 595 600
605 Ala Asn Pro Thr Gly Asp Ser Ala Ala Ala Ile Tyr Asn Arg Val Lys
610 615 620 Gly Glu Lys Arg Ile Pro Leu Val Arg Leu Pro Tyr Met Val
Glu His 625 630 635 640 Thr Val Glu Val Lys Asn Gly Asn Leu Ile Ile
Pro His Lys Asp His 645 650 655 Tyr His Asn Ile Lys Phe Ala Trp Phe
Asp Asp His Thr Tyr Lys Ala 660 665 670 Pro Asn Gly Tyr Thr Leu Glu
Asp Leu Phe Ala Thr Ile Lys Tyr Tyr 675 680 685 Val Glu His Pro Asp
Glu Arg Pro His Ser Asn Asp Gly Trp Gly Asn 690 695 700 Ala Ser Glu
His Val Leu Gly Lys Lys Asp His Ser Glu Asp Pro Asn 705 710 715 720
Lys Asn Phe Lys Ala Asp Glu Glu Pro Val Glu Glu Thr Pro Ala Glu 725
730 735 Pro Glu Val Pro Gln Val Glu Thr Glu Lys Val Glu Ala Gln Leu
Lys 740 745 750 Glu Ala Glu Val Leu Leu Ala Lys Val Thr Asp Ser Ser
Leu Lys Ala 755 760 765 Asn Ala Thr Glu Thr Leu Ala Gly Leu Arg Asn
Asn Leu Thr Leu Gln 770 775 780 Ile Met Asp Asn Asn Ser Ile Met Ala
Glu Ala Glu Lys Leu Leu Ala 785 790 795 800 Leu Leu Lys Gly Ser Asn
Pro Ser Ser Val Ser Lys Glu Lys Ile Asn 805 810 815 Lys Leu Asn 9
2451 DNA Streptococcus pneumoniae misc_feature (1)..(2451) n = a,
c, t or g 9 atgaaaatta ataagaaata ccttgttggt tctgcggcag ctttgatttt
aagtgtttgt 60 tcttacgagt tgggactgta tcaagctaga acggttaagg
aaaataatcg tgtttcctat 120 atagatggaa aacaagcgac gcaaaaaacg
gagaatttga ctcctgatga ggttagcaag 180 cgtgaaggaa tcaatgctga
gcaaatcgtc atcaagataa cagaccaagg ctatgtcact 240 tcacatggcg
accactatca ttattacaat ggtaaggttc cttatgacgc tatcatcagt 300
gaagaattac tcatgaaaga tccaaactat aagctaaaag atgaggatat tgttaatgag
360 gtcaagggtg gatatgttat caaggtagat ggaaaatact atgtttacct
taaggatgct 420 gcccacgcgg ataacgtccg tacaaaagag gaaatcaatc
gacaaaaaca agagcatagt 480 caacatcgtg aaggtggaac tccaagaaac
gatggtgctg ttgccttggc acgttcgcaa 540 ggacgctata ctacagatga
tggttatatc tttaatgctt ctgatatcat agaggatact 600 ggtgatgctt
atatcgttcc tcatggagat cattaccatt acattcctaa gaatgagtta 660
tcagctagcg agttggctgc tgcagaagcc ttcctatctg gtcgaggaaa tctgtcaaat
720 tcaagaacct atcgccgaca aaatagcgat aacacttcaa gaacaaactg
ggtaccttct 780 gtaagcaatc caggaactac aaatactaac acaagcaaca
acagcaacac taacagtcaa 840 gcaagtcaaa gtaatgacat tgatagtctc
ttgaaacagc tctacaaact gcctttgagt 900 caacgacatg tagaatctga
tggccttgtc tttgatccag cacaaatcac aagtcgaaca 960 gctagaggtg
ttgcagtgcc acacggagat cattaccact tcatccctta ctctcaaatg 1020
tctgaattgg aagaacgaat cgctcgtatt attccccttc gttatcgttc aaaccattgg
1080 gtaccagatt caaggccaga acaaccaagt ccacaaccga ctccggaacc
tagtccaggc 1140 ccgcaacctg caccaaatct taaaatagac tcaaattctt
ctttggttag tcagctggta 1200 cgaaaagttg gggaaggata tgtattcgaa
gaaaagggca tctctcgtta tgtctttgcg 1260 aaagatttac catctgaaac
tgttaaaaat cttgaaagca agttatcaaa acaagagagt 1320 gtttcacaca
ctttaactgc taaaaaagaa aatgttgctc ctcgtgacca agaattttat 1380
gataaagcat ataatctgtt aactgaggct cataaagcct tgtttgnaaa taagggtcgt
1440 aattctgatt tccaagcctt agacaaatta ttagaacgct tgaatgatga
atcgactaat 1500 aaagaaaaat tggtagatga tttattggca ttcctagcac
caattaccca tccagagcga 1560 cttggcaaac caaattctca aattgagtat
actgaagacg aagttcgtat tgctcaatta 1620 gctgataagt atacaacgtc
agatggttac atttttgatg aacatgatat aatcagtgat 1680 gaaggagatg
catatgtaac gcctcatatg ggccatagtc actggattgg aaaagatagc 1740
ctttctgata aggaaaaagt tgcagctcaa gcctatacta aagaaaaagg tatcctacct
1800 ccatctccag acgcagatgt taaagcaaat ccaactggag atagtgcagc
agctatttac 1860 aatcgtgtga aaggggaaaa acgaattcca ctcgttcgac
ttccatatat ggttgagcat 1920 acagttgagg ttaaaaacgg taatttgatt
attcctcata aggatcatta ccataatatt 1980 aaatttgctt ggtttgatga
tcacacatac aaagctccaa atggctatac cttggaagat 2040 ttgtttgcga
cgattaagta ctacgtagaa caccctgacg aacgtccaca ttctaatgat 2100
ggatggggca atgccagtga gcatgtgtta ggcaagaaag accacagtga agatccaaat
2160 aagaacttca aagcggatga agagccagta gaggaaacac ctgctgagcc
agaagtccct 2220 caagtagaga ctgaaaaagt agaagcccaa ctcaaagaag
cagaagtttt gcttgcgaaa 2280 gtaacggatt ctagtctgaa agccaatgca
acagaaactc tagctggttt acgaaataat 2340 ttgactcttc aaattatgga
taacaatagt atcatggcag aagcagaaaa attacttgcg 2400 ttgttaaaag
gaagtaatcc ttcatctgta agtaaggaaa aaataaacta a 2451 10 819 PRT
Streptococcus pneumoniae 10 Met Lys Ile Asn Lys Lys Tyr Leu Ala Gly
Ser Val Ala Val Leu Ala 1 5 10 15 Leu Ser Val Cys Ser Tyr Glu Leu
Gly Arg Tyr Gln Ala Gly Gln Asp 20 25 30 Lys Lys Glu Ser Asn Arg
Val Ala Tyr Ile Asp Gly Asp Gln Ala Gly 35 40 45 Gln Lys Ala Glu
Asn Leu Thr Pro Asp Glu Val Ser Lys Arg Glu Gly 50 55 60 Ile Asn
Ala Glu Gln Ile Val Ile Lys Ile Thr Asp Gln Gly Tyr Val 65 70 75 80
Thr Ser His Gly Asp His Tyr His Tyr Tyr Asn Gly Lys Val Pro Tyr 85
90 95 Asp Ala Ile Ile Ser Glu Glu Leu Leu Met Lys Asp Pro Asn Tyr
Gln 100 105 110 Leu Lys Asp Ser Asp Ile Val Asn Glu Ile Lys Gly Gly
Tyr Val Ile 115 120 125 Lys Val Asn Gly Lys Tyr Tyr Val Tyr Leu Lys
Asp Ala Ala His Ala 130 135 140 Asp Asn Ile Arg Thr Lys Glu Glu Ile
Lys Arg Gln Lys Gln Glu Arg 145 150 155 160 Ser His Asn His Asn Ser
Arg Ala Asp Asn Ala Val Ala Ala Ala Arg 165 170 175 Ala Gln Gly Arg
Tyr Thr Thr Asp Asp Gly Tyr Ile Phe Asn Ala Ser 180 185 190 Asp Ile
Ile Glu Asp Thr Gly Asp Ala Tyr Ile Val Pro His Gly Asp 195 200 205
His Tyr His Tyr Ile Pro Lys Asn Glu Leu Ser Ala Ser Glu Leu Ala 210
215 220 Ala Ala Glu Ala Tyr Trp Asn Gly Lys Gln Gly Ser Arg Pro Ser
Ser 225 230 235 240 Ser Ser Ser Tyr Asn Ala Asn Pro Ala Gln Pro Arg
Leu Ser Glu Asn 245 250 255 His Asn Leu Thr Val Thr Pro Thr Tyr His
Gln Asn Gln Gly Glu Asn 260 265 270 Ile Ser Ser Leu Leu Arg Glu Leu
Tyr Ala Lys Pro Leu Ser Glu Arg 275 280 285 His Val Glu Ser Asp Gly
Leu Ile Phe Asp Pro Ala Gln Ile Thr Ser 290 295 300 Arg Thr Ala Arg
Gly Val Ala Val Pro His Gly Asn His Tyr His Phe 305 310 315 320 Ile
Pro Tyr Glu Gln Met Ser Glu Leu Glu Lys Arg Ile Ala Arg Ile 325 330
335 Ile Pro Leu Arg Tyr Arg Ser Asn His Trp Val Pro Asp Ser Arg Pro
340 345 350 Glu Glu Pro Ser Pro Gln Pro Thr Pro Glu Pro Ser Pro Ser
Pro Gln 355 360 365 Pro Ala Pro Ser Asn Pro Ile Asp Gly Lys Leu Val
Lys Glu Ala Val 370 375 380 Arg Lys Val Gly Asp Gly Tyr Val Phe Glu
Glu Asn Gly Val Ser Arg 385 390 395 400 Tyr Ile Pro Ala Lys Asp Leu
Ser Ala Glu Thr Ala Ala Gly Ile Asp 405 410 415 Ser Lys Leu Ala Lys
Gln Glu Ser Leu Ser His Lys Leu Gly Thr Lys 420 425 430 Lys Thr Asp
Leu Pro Ser Ser Asp Arg Glu Phe Tyr Asn Lys Ala Tyr 435 440 445 Asp
Leu Leu Ala Arg Ile His Gln Asp Leu Leu Asp Asn Lys Gly Arg 450 455
460 Gln Val Asp Phe Glu Ala Leu Asp Asn Leu Leu Glu Arg Leu Lys Asp
465 470 475 480 Val Ser Ser Asp Lys Val Lys Leu Val Glu Asp Ile Leu
Ala Phe Leu 485 490 495 Ala Pro Ile Arg His Pro Glu Arg Leu Gly Lys
Pro Asn Ala Gln Ile 500 505 510 Thr Tyr Thr Asp Asp Glu Ile Gln Val
Ala Lys Leu Ala Gly Lys Tyr 515 520 525 Thr Ala Glu Asp Gly Tyr Ile
Phe Asp Pro Arg Asp Ile Thr Ser Asp 530 535 540 Glu Gly Asp Ala Tyr
Val Thr Pro His Met Thr His Ser His Trp Ile 545 550 555 560 Lys Lys
Asp Ser Leu Ser Glu Ala Glu Arg Ala Ala Ala Gln Ala Tyr 565 570 575
Ala Glu Glu Lys Gly Leu Thr Pro Pro Ser Thr Asp His Gln Asp Ser 580
585 590 Gly Asn Thr Glu Ala Lys Gly Ala Glu Ala Ile Tyr Asn Arg Val
Lys 595 600 605 Ala Ala Lys Lys Val Pro Leu Asp Arg Met Pro Tyr Asn
Leu Gln Tyr 610 615 620 Thr Val Glu Val Lys Asn Gly Ser Leu Ile Ile
Pro His Tyr Asp His 625 630 635 640 Tyr His Asn Ile Lys Phe Glu Trp
Phe Asp Glu Gly Leu Tyr Glu Ala 645 650 655 Pro Lys Gly Tyr Thr Leu
Glu Asp Leu Leu Ala Thr Val Lys Tyr Tyr 660 665 670 Val Glu His Pro
Asn Glu Arg Pro His Ser Asp Asn Gly Phe Gly Asn 675 680 685 Ala Ser
Asp His Val Gln Arg Asn Lys Asn Gly Gln Ala Asp Thr Asn 690 695 700
Gln Thr Glu Lys Pro Ser Glu Glu Lys Pro Gln Thr Glu Lys Pro Glu 705
710 715 720 Glu Glu Thr Pro Arg Glu Glu Lys Pro Gln Ser Glu Lys Pro
Glu Ser 725 730 735 Pro Lys Pro Thr Glu Glu Pro Glu Glu Ser Pro Glu
Glu Ser Glu Glu 740 745 750 Pro Gln Val Glu Thr Glu Lys Val Glu Glu
Lys Leu Arg Glu Ala Glu 755 760 765 Asp Leu Leu Gly Lys Ile Gln Asp
Pro Ile Ile Lys Ser Asn Ala Lys 770 775 780 Glu Thr Leu Thr Gly Leu
Lys Asn Asn Leu Leu Phe Gly Thr Gln Asp 785 790 795 800 Asn Asn Thr
Ile Met Ala Glu Ala Glu Lys Leu Leu Ala Leu Leu Lys 805 810 815 Glu
Ser Lys 11 2531 DNA Streptococcus pneumoniae 11 atgaaaatta
ataaaaaata tctagcaggt tcagtggcag tccttgccct aagtgtttgt 60
tcctatgagc ttggacgtta ccaagctggt caggataaga aagagtctaa tcgagttgct
120 tatatagatg gtgatcaggc tggtcaaaag gcagaaaact tgacaccaga
tgaagtcagt 180 aagagggagg ggatcaacgc cgaacaaatt gttatcaaga
ttacggatca aggttatgtg 240 acctctcatg gagaccatta tcattactat
aatggcaagg ttccttatga tgccatcatc 300 agtgaagagc tcctcatgaa
agatccgaat tatcagttga aggattcaga cattgtcaat 360 gaaatcaagg
gtggttatgt cattaaggta aacggtaaat actatgttta ccttaaggat 420
gcrgctcatg cggataatat tcggacaaaa gaagagatta aacgtcagaa gcaggaacgc
480 agtcataatc ataactcaag agcagataat gctgttgctg cagccagagc
ccaaggacgt 540 tatacaacgg atgatgggta tatcttcaat gcatctgata
tcattgagga cacgggtgat 600 gcttatatcg ttcctcacgg cgaccattac
cattacattc ctaagaatga gttatcagct 660 agcgagttag ctgctgcaga
agcctattgg aatgggaagc agggatctcg tccttcttca 720 agttctagtt
ataatgcaaa tccagctcaa ccaagattgt cagagaacca caatctgact 780
gtcactccaa cttatcatca aaatcaaggg gaaaacattt caagcctttt acgtgaattg
840 tatgctaaac ccttatcaga acgccatgtg gaatctgatg gccttatttt
cgacccagcg 900 caaatcacaa gtcgaaccgc cagaggtgta gctgtccctc
atggtaacca ttaccacttt 960 atcccttatg aacaaatgtc tgaattggaa
aaacgaattg ctcgtattat tccccttcgt 1020 tatcgttcaa accattgggt
accagattca agaccagaag aaccaagtcc acaaccgact 1080 ccagaaccta
gtccaagtcc gcaaccagct ccaagcaatc caattgatgg gaaattggtc 1140
aaagaagctg ttcgaaaagt aggcgatggt tatgtctttg aggagaatgg agtttctcgt
1200 tatatcccag ccaaggatct ttcagcagaa acagcagcag gcattgatag
caaactggcc 1260 aagcaggaaa gtttatctca taagctagga actaagaaaa
ctgacctccc atctagtgat 1320 cgagaatttt acaataaggc ttatgactta
ctagcaagaa ttcaccaaga tttacttgat 1380 aataaaggtc gacaagttga
ttttgaggct ttggataacc tgttggaacg actcaaggat 1440 gtctcaagtg
ataaagtcaa gttagtggaa gatattcttg ccttcttagc tccgattcgt 1500
catccagaac gtttaggaaa accaaatgcg caaattacct acactgatga tgagattcaa
1560 gtagccaagt tggcaggcaa gtacacagca gaagacggtt atatctttga
tcctcgtgat 1620 ataaccagtg atgaggggga tgcctatgta actccacata
tgacccatag ccactggatt 1680 aaaaaagata gtttgtctga agctgagaga
gcggcagccc aggcttatgc traagagaaa 1740 ggtttgaccc ctccttcgac
agaccatcag gattcaggaa atactgaggc aaaaggagca 1800 gaagctatct
acaaccgmgt gaaagcagct aagaaggtgc cacttgatcg tatgccttac 1860
aatcttcaat atactgtaga agtcaaaaac ggtagtttaa tcatacctca ttatgaccat
1920 taccataaca tcaaatttga gtggtttgac gaaggccttt atgaggcacc
taaggggtat 1980 actcttgagg atcttttggc gactgtcaag tactatgtcg
aacatccaaa cgaacgtccg 2040 cattcagata atggttttgg taacgctagc
gaccatgttc aaagaaacaa aaatggtcaa 2100 gctgatacca atcaaacgga
aaaaccaagc gaggagaaac ctcagacaga aaaacctgag 2160 gaagaaaccc
ctcgagaaga gaaaccgcaa agcgagaaac cagagtctcc aaaaccaaca 2220
gaggaaccag aagaatcacc agaggaatca gaagaacctc aggtcgagac tgaaaaggtt
2280 gaagaaaaac tgagagaggc tgaagattta cttggaaaaa tccaggatcc
aattatcaag 2340 tccaatgcca aagagactct cacaggatta aaaaataatt
tactatttgg cacccaggac 2400 aacaatacta ttatggcaga agctgaaaaa
ctattggctt tattaaagga gagtaagtaa 2460 aggtagaagc ttaagggcga
atttggcacc caggacaaca atactattat ggcagaagct 2520 gaaaaactat t 2531
12 6 PRT Artificial Sequence VARIANT (1)..(6) Xaa = any amino acid
12 His Xaa Xaa His Xaa His 1 5 13 4 PRT Artificial Sequence
Description of Artificial Sequence Substrate sequence for Type II
Signal Peptidase. 13 Leu Ser Val Cys 1 14 4 PRT Artificial Sequence
VARIANT (2)..(3) Xaa = any amino acid 14 Leu Xaa Xaa Cys 1
* * * * *