U.S. patent application number 11/106649 was filed with the patent office on 2005-08-18 for streptococcus pneumoniae antigens and vaccines.
This patent application is currently assigned to Human Genome Sciences, Inc.. Invention is credited to Barash, Steven C., Choi, Gil H., Dillon, Patrick J., Dougherty, Brian, Fannon, Michael R., Kunsch, Charles A., Rosen, Craig A..
Application Number | 20050181439 11/106649 |
Document ID | / |
Family ID | 21851789 |
Filed Date | 2005-08-18 |
United States Patent
Application |
20050181439 |
Kind Code |
A1 |
Choi, Gil H. ; et
al. |
August 18, 2005 |
Streptococcus pneumoniae antigens and vaccines
Abstract
The present invention relates to novel vaccines for the
prevention or attenuation of infection by Streptococcus pneumoniae.
The invention further relates to isolated nucleic acid molecules
encoding antigenic polypeptides of Streptococcus pneumoniae.
Antigenic polypeptides are also provided, as are vectors, host
cells and recombinant methods for producing the same. The invention
additionally relates to diagnostic methods for detecting
Streptococcus nucleic acids, polypeptides and antibodies in a
biological sample.
Inventors: |
Choi, Gil H.; (Rockville,
MD) ; Kunsch, Charles A.; (Norcross, GA) ;
Barash, Steven C.; (Rockville, MD) ; Dillon, Patrick
J.; (Carlsbad, CA) ; Dougherty, Brian;
(Killingworth, CT) ; Fannon, Michael R.; (Silver
Spring, MD) ; Rosen, Craig A.; (Laytonsville,
MD) |
Correspondence
Address: |
HUMAN GENOME SCIENCES INC
INTELLECTUAL PROPERTY DEPT.
14200 SHADY GROVE ROAD
ROCKVILLE
MD
20850
US
|
Assignee: |
Human Genome Sciences, Inc.
Intellectual Property Dept. 14200 Shady Grove Road
Rockville
MD
20850
|
Family ID: |
21851789 |
Appl. No.: |
11/106649 |
Filed: |
April 15, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11106649 |
Apr 15, 2005 |
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08961083 |
Oct 30, 1997 |
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11106649 |
Apr 15, 2005 |
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09765271 |
Jan 22, 2001 |
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6887663 |
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09765271 |
Jan 22, 2001 |
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09536784 |
Mar 28, 2000 |
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6573082 |
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09536784 |
Mar 28, 2000 |
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08961083 |
Oct 30, 1997 |
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09765271 |
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08961083 |
Oct 30, 1997 |
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60029960 |
Oct 31, 1996 |
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60029960 |
Oct 31, 1996 |
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60029960 |
Oct 31, 1996 |
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Current U.S.
Class: |
435/6.15 ;
435/252.3; 435/320.1; 435/69.3; 530/395; 536/23.7 |
Current CPC
Class: |
C07K 14/315 20130101;
A61P 31/04 20180101; A61K 39/00 20130101; A61P 37/04 20180101; A61K
38/00 20130101; C07K 14/3156 20130101 |
Class at
Publication: |
435/006 ;
435/069.3; 435/252.3; 435/320.1; 530/395; 536/023.7 |
International
Class: |
C12Q 001/68; C07H
021/04; C07K 014/31; C12N 001/21; C12N 015/74 |
Claims
What is claimed is:
1. An isolated nucleic acid molecule comprising a polynucleotide
having a nucleotide sequence at least 95% identical to a sequence
selected from the group consisting of: (a) a nucleotide sequence
encoding any of the amino acid sequences of the polypeptides shown
in Table 1; and (b) a nucleotide sequence complementary to any of
the nucleotide sequences in (a).
2. An isolated nucleic acid molecule comprising a polynucleotide
which hybridizes under stringent hybridization conditions to a
polynucleotide having a nucleotide sequence identical to a
nucleotide sequence in (a) or (b) of claim 1 wherein said
polynucleotide which hybridizes does not hybridize under stringent
hybridization conditions to a polynucleotide having a nucleotide
sequence consisting of only A residues or of only T residues.
3. An isolated nucleic acid molecule comprising a polynucleotide
which encodes the amino acid sequence of an epitope-bearing portion
of a polypeptide having an amino acid sequence in (a) of claim
1.
4. The isolated nucleic acid molecule of claim 3, wherein said
epitope-bearing portion of a polypeptide has an amino acid sequence
listed in Table 2.
5. A method for making a recombinant vector comprising inserting an
isolated nucleic acid molecule of claim 1 into a vector.
6. A recombinant vector produced by the method of claim 5.
7. A method of making a recombinant host cell comprising
introducing the recombinant vector of claim 6 into a host cell.
8. A recombinant host cell produced by the method of claim 7.
9. A method of producing a polypeptide encoded by said isolated
nucleic acid molecule comprising culturing the host cell of claim 8
under conditions favoring expressing the heterologous
polypeptide.
10. A polypeptide produced according to the method of claim 9.
11. An isolated polypeptide comprising an amino acid sequence at
least 70% identical to a sequence selected from the group
consisting of an amino acid sequence of any of the polypeptides
described in Table 1.
12. An isolated polypeptide antigen comprising an amino acid
sequence of an S. pneumoniae epitope shown in Table 2.
13. An isolated nucleic acid molecule comprising a polynucleotide
with a nucleotide sequence encoding a polypeptide of claim 9.
14. An isolated antibody that binds specifically to a polypeptide
of claim 11.
15. A hybridoma which produces an antibody according to claim
14.
16. A vaccine, comprising: (1) one of more S. pneumoniae
polypeptides selected from the group consisting of a polypeptide
comprising an amino acid sequence identified in Table 1, or a
fragment thereof; and (2) a pharmaceutically acceptable diluent,
carrier, or excipient; wherein said polypeptide is present, in an
amount effective to elicit protective antibodies in an animal to a
member of the Streptococcus genus.
17. A method of preventing or attenuating an infection caused by a
member of the Streptococcus genus in an animal, comprising
administering to said animal a polypeptide of claim 11, wherein
said polypeptide is administered in an amount effective to prevent
or attenuate said infection.
18. A method of detecting Streptococcus nucleic acids in a
biological sample obtained from an animal involving assaying for
one or more nucleic acid sequences encoding Streptococcus
polypeptides in a sample comprising: (a) contacting the sample with
one or more of the above-described nucleic acid probes, under
conditions such that hybridization occurs, and (b) detecting
hybridization of said one or more probes to the one or more
Streptococcus nucleic acid sequences present in the biological
sample.
19. A method of detecting Streptococcus nucleic acids in a
biological sample obtained from an animal, comprising: (a)
amplifying one or more Streptococcus nucleic acid sequences in said
sample using polymerase chain reaction, and (b) detecting said
amplified Streptococcus nucleic acid.
20. A kit for detecting Streptococcus antibodies in a biological
sample obtained from an animal, comprising (a) a polypeptide of
claim 12 attached to a solid support; and (b) detecting means.
21. A method of detecting Streptococcus antibodies in a biological
sample obtained from an animal, comprising (a) contacting the
sample with a polypeptide of claim 12; and (b) detecting
antibody-antigen complexes.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 08/961,083, filed Oct. 30, 1997, which claims benefit of U.S.
Provisional Application No. 60/029,960, filed Oct. 31, 1996; this
application is also a divisional of U.S. application Ser. No.
09/765,271, filed Jan. 22, 2001, which is a continuation of U.S.
application Ser. No. 09/536,784, filed Mar. 28, 2000 (now U.S. Pat.
No. 6,573,082, issued Jun. 3, 2003), which is a continuation of
U.S. application Ser. No. 08/961,083, filed Oct. 30, 1997, which
claims benefit of U.S. Provisional Application No. 60/029,960,
filed Oct. 31, 1996; U.S. application Ser. No. 09/765,271 is also a
continuation of U.S. application Ser. No. 08/961,083, filed Oct.
30, 1997, which claims benefit of U.S. Provisional Application No.
60/029,960, filed Oct. 31, 1996.
REFERENCE TO SEQUENCE LISTING ON COMPACT DISC
[0002] This application refers to a "Sequence Listing" which is
provided as an electronic document on two identical compact discs
(CD-R), labeled "Copy 1" and "Copy 2". These compact discs each
contain one file designated "PB340P2C3D1.ST25.txt" (537,687 bytes,
created on Apr. 12, 2005), which is incorporated by reference in
its entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to novel Streptococcus
pneumoniae antigens for the detection of Streptococcus and for the
prevention or attenuation of disease caused by Streptococcus. The
invention further relates to isolated nucleic acid molecules
encoding antigenic polypeptides of S. pneumoniae. Antigenic
polypeptides are also provided, as are vectors, host cells and
recombinant methods for producing the same. The invention
additionally relates to diagnostic methods for detecting
Streptococcus gene expression.
BACKGROUND OF THE INVENTION
[0004] Streptococcus pneumoniae has been one of the most
extensively studied microorganisms since its first isolation in
1881. It was the object of many investigations that led to
important scientific discoveries. In 1928, Griffith observed that
when heat-killed encapsulated pneumococci and live strains
constitutively lacking any capsule were concomitantly injected into
mice, the nonencapsulated could be converted into encapsulated
pneumococci with the same capsular type as the heat-killed strain.
Years later, the nature of this "transforming principle," or
carrier of genetic information, was shown to be DNA. (Avery, O. T.,
et al., J. Exp. Med., 79:137-157 (1944)).
[0005] In spite of the vast number of publications on S. pneumoniae
many questions about its virulence are still unanswered, and this
pathogen remains a major causative agent of serious human disease,
especially community-acquired pneumonia. (Johnston, R. B., et al.,
Rev. Infect. Dis. 13(Suppl. 6):S509-517 (1991)). In addition, in
developing countries, the pneumococcus is responsible for the death
of a large number of children under the age of 5 years from
pneumococcal pneumonia. The incidence of pneumococcal disease is
highest in infants under 2 years of age and in people over 60 years
of age. Pneumococci are the second most frequent cause (after
Haemophilus influenzae type b) of bacterial meningitis and otitis
media in children. With the recent introduction of conjugate
vaccines for H. influenzae type b, pneumococcal meningitis is
likely to become increasingly prominent. S. pneumoniae is the most
important etiologic agent of community-acquired pneumonia in adults
and is the second most common cause of bacterial meningitis behind
Neisseria meningitidis.
[0006] The antibiotic generally prescribed to treat S. pneumoniae
is benzylpenicillin, although resistance to this and to other
antibiotics is found occasionally. Pneumococcal resistance to
penicillin results from mutations in its penicillin-binding
proteins. In uncomplicated pneumococcal pneumonia caused by a
sensitive strain, treatment with penicillin is usually successful
unless started too late. Erythromycin or clindamycin can be used to
treat pneumonia in patients hypersensitive to penicillin, but
resistant strains to these drugs exist. Broad spectrum antibiotics
(e.g., the tetracyclines) may also be effective, although
tetracycline-resistant strains are not rare. In spite of the
availability of antibiotics, the mortality of pneumococcal
bacteremia in the last four decades has remained stable between 25
and 29%. (Gillespie, S. H., et al., J. Med. Microbiol. 28:237-248
(1989).
[0007] S. pneumoniae is carried in the upper respiratory tract by
many healthy individuals. It has been suggested that attachment of
pneumococci is mediated by a disaccharide receptor on fibronectin,
present on human pharyngeal epithelial cells. (Anderson, B. J., et
al., J. Immunol. 142:2464-2468 (1989). The mechanisms by which
pneumococci translocate from the nasopharynx to the lung, thereby
causing pneumonia, or migrate to the blood, giving rise to
bacteremia or septicemia, are poorly understood. (Johnston, R. B.,
et al., Rev. Infect. Dis. 13(Suppl. 6):S509-517 (1991).
[0008] Various proteins have been suggested to be involved in the
pathogenicity of S. pneumoniae, however, only a few of them have
actually been confirmed as virulence factors. Pneumococci produce
an IgA 1 protease that might interfere with host defense at mucosal
surfaces. (Kornfield, S. J., et al., Rev. Inf. Dis. 3:521-534
(1981). S. pneumoniae also produces neuraminidase, an enzyme that
may facilitate attachment to epithelial cells by cleaving sialic
acid from the host glycolipids and gangliosides. Partially purified
neuraminidase was observed to induce meningitis-like symptoms in
mice; however, the reliability of this finding has been questioned
because the neuraminidase preparations used were probably
contaminated with cell wall products. Other pneumococcal proteins
besides neuraminidase are involved in the adhesion of pneumococci
to epithelial and endothelial cells. These pneumococcal proteins
have as yet not been identified. Recently, Cundell et al., reported
that peptide permeases can modulate pneumococcal adherence to
epithelial and endothelial cells. It was, however, unclear whether
these permeases function directly as adhesions or whether they
enhance adherence by modulating the expression of pneumococcal
adhesions. (De Velasco, E. A., et al., Micro. Rev. 59:591-603
(1995). A better understanding of the virulence factors determining
its pathogenicity will need to be developed to cope with the
devastating effects of pneumococcal disease in humans.
[0009] Ironically, despite the prominent role of S. pneumoniae in
the discovery of DNA, little is known about the molecular genetics
of the organism. The S. pneumoniae genome consists of one circular,
covalently closed, double-stranded DNA and a collection of
so-called variable accessory elements, such as prophages, plasmids,
transposons and the like. Most physical characteristics and almost
all of the genes of S. pneumoniae are unknown. Among the few that
have been identified, most have not been physically mapped or
characterized in detail. Only a few genes of this organism have
been sequenced. (See, for instance current versions of GENBANK and
other nucleic acid databases, and references that relate to the
genome of S. pneumoniae such as those set out elsewhere herein.)
Identification of in vivo-expressed, and broadly protective,
antigens of S. pneumoniae has remained elusive.
SUMMARY OF THE INVENTION
[0010] The present invention provides isolated nucleic acid
molecules comprising polynucleotides encoding the S. pneumoniae
polypeptides described in Table 1 and having the amino acid
sequences shown as SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and so on
through SEQ ID NO:226. Thus, one aspect of the invention provides
isolated nucleic acid molecules comprising polynucleotides having a
nucleotide sequence selected from the group consisting of: (a) a
nucleotide sequence encoding any of the amino acid sequences of the
polypeptides shown in Table 1; and (b) a nucleotide sequence
complementary to any of the nucleotide sequences in (a).
[0011] Further embodiments of the invention include isolated
nucleic acid molecules that comprise a polynucleotide having a
nucleotide sequence at least 90% identical, and more preferably at
least 95%, 96%, 97%, 98% or 99% identical, to any of the nucleotide
sequences in (a) or (b) above, or a polynucleotide which hybridizes
under stringent hybridization conditions to a polynucleotide in (a)
or (b) above. This polynucleotide which hybridizes does not
hybridize under stringent hybridization conditions to a
polynucleotide having a nucleotide sequence consisting of only A
residues or of only T residues. Additional nucleic acid embodiments
of the invention relate to isolated nucleic acid molecules
comprising polynucleotides which encode the amino acid sequences of
epitope-bearing portions of an S. pneumoniae polypeptide having an
amino acid sequence in (a) above.
[0012] The present invention also relates to recombinant vectors,
which include the isolated nucleic acid molecules of the present
invention, and to host cells containing the recombinant vectors, as
well as to methods of making such vectors and host cells and for
using these vectors for the production of S. pneumoniae
polypeptides or peptides by recombinant techniques.
[0013] The invention further provides isolated S. pneumoniae
polypeptides having an amino acid sequence selected from the group
consisting of an amino acid sequence of any of the polypeptides
described in Table 1.
[0014] The polypeptides of the present invention also include
polypeptides having an amino acid sequence with at least 70%
similarity, and more preferably at least 75%, 80%, 85%, 90%, 95%,
96%, 97%, 98%, or 99% similarity to those described in Table 1, as
well as polypeptides having an amino acid sequence at least 70%
identical, more preferably at least 75% identical, and still more
preferably 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to
those above; as well as isolated nucleic acid molecules encoding
such polypeptides.
[0015] The present invention further provides a vaccine, preferably
a multi-component vaccine comprising one or more of the S.
pneumoniae polynucleotides or polypeptides described in Table 1, or
fragments thereof, together with a pharmaceutically acceptable
diluent, carrier, or excipient, wherein the S. pneumoniae
polypeptide(s) are present in an amount effective to elicit an
immune response to members of the Streptococcus genus in an animal.
The S. pneumoniae polypeptides of the present invention may further
be combined with one or more immunogens of one or more other
streptococcal or non-streptococcal organisms to produce a
multi-component vaccine intended to elicit an immunological
response against members of the Streptococcus genus and,
optionally, one or more non-streptococcal organisms.
[0016] The vaccines of the present invention can be administered in
a DNA form, e.g., "naked" DNA, wherein the DNA encodes one or more
streptococcal polypeptides and, optionally, one or more
polypeptides of a non-streptococcal organism. The DNA encoding one
or more polypeptides may be constructed such that these
polypeptides are expressed fusion proteins.
[0017] The vaccines of the present invention may also be
administered as a component of a genetically engineered organism.
Thus, a genetically engineered organism which expresses one or more
S. pneumoniae polypeptides may be administered to an animal. For
example, such a genetically engineered organism may contain one or
more S. pneumoniae polypeptides of the present invention
intracellularly, on its cell surface, or in its periplasmic space.
Further, such a genetically engineered organism may secrete one or
more S. pneumoniae polypeptides.
[0018] The vaccines of the present invention may be co-administered
to an animal with an immune system modulator (e.g., CD86 and
GM-CSF).
[0019] The invention also provides a method of inducing an
immunological response in an animal to one or more members of the
Streptococcus genus, preferably one or more isolates of the S.
pneumoniae genus, comprising administering to the animal a vaccine
as described above.
[0020] The invention further provides a method of inducing a
protective immune response in an animal, sufficient to prevent or
attenuate an infection by members of the Streptococcus genus,
preferably at least S. pneumoniae, comprising administering to the
animal a composition comprising one or more of the polynucleotides
or polypeptides described in Table 1, or fragments thereof.
Further, these polypeptides, or fragments thereof, may be
conjugated to another immunogen and/or administered in admixture
with an adjuvant.
[0021] The invention further relates to antibodies elicited in an
animal by the administration of one or more S. pneumoniae
polypeptides of the present invention and to methods for producing
such antibodies.
[0022] The invention also provides diagnostic methods for detecting
the expression of genes of members of the Streptococcus genus in an
animal. One such method involves assaying for the expression of a
gene encoding S. pneumoniae peptides in a sample from an animal.
This expression may be assayed either directly (e.g., by assaying
polypeptide levels using antibodies elicited in response to amino
acid sequences described in Table 1) or indirectly (e.g., by
assaying for antibodies having specificity for amino acid sequences
described in Table 1). An example of such a method involves the use
of the polymerase chain reaction (PCR) to amplify and detect
Streptococcus nucleic acid sequences.
[0023] The present invention also relates to nucleic acid probes
having all or part of a nucleotide sequence described in Table 1
(shown as SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, and so on through
SEQ ID NO:225) which are capable of hybridizing under stringent
conditions to Streptococcus nucleic acids. The invention further
relates to a method of detecting one or more Streptococcus nucleic
acids in a biological sample obtained from an animal, said one or
more nucleic acids encoding Streptococcus polypeptides, comprising:
(a) contacting the sample with one or more of the above-described
nucleic acid probes, under conditions such that hybridization
occurs, and (b) detecting hybridization of said one or more probes
to the Streptococcus nucleic acid present in the biological
sample.
[0024] The invention also includes immunoassays, including an
immunoassay for detecting Streptococcus, preferably at least
isolates of the S. pneumoniae genus, comprising incubation of a
sample (which is suspected of being infected with Streptococcus)
with a probe antibody directed against an antigen/epitope of S.
pneumoniae, to be detected under conditions allowing the formation
of an antigen-antibody complex; and detecting the antigen-antibody
complex which contains the probe antibody. An immunoassay for the
detection of antibodies which are directed against a Streptococcus
antigen comprising the incubation of a sample (containing
antibodies from a mammal suspected of being infected with
Streptococcus) with a probe polypeptide including an epitope of S.
pneumoniae, under conditions that allow the formation of
antigen-antibody complexes which contain the probe epitope
containing antigen.
[0025] Some aspects of the invention pertaining to kits are those
for: investigating samples for the presence of polynucleotides
derived from Streptococcus which comprise a polynucleotide probe
including a nucleotide sequence selected from Table 1 or a fragment
thereof of approximately 15 or more nucleotides, in an appropriate
container; analyzing the samples for the presence of antibodies
directed against a Streptococcus antigen made up of a polypeptide
which contains a S. pneumoniae epitope present in the polypeptide,
in a suitable container; and analyzing samples for the presence of
Streptococcus antigens made up of an anti-S. pneumoniae antibody,
in a suitable container.
DETAILED DESCRIPTION
[0026] The present invention relates to recombinant antigenic S.
pneumoniae polypeptides and fragments thereof. The invention also
relates to methods for using these polypeptides to produce
immunological responses and to confer immunological protection to
disease caused by members of the genus Streptococcus, at least
isolates of the S. pneumoniae genus. The invention further relates
to nucleic acid sequences which encode antigenic S. pneumoniae
polypeptides and to methods for detecting S. pneumoniae nucleic
acids and polypeptides in biological samples. The invention also
relates to S. pneumoniae-specific antibodies and methods for
detecting such antibodies produced in a host animal.
[0027] Definitions
[0028] The following definitions are provided to clarify the
subject matter which the inventors consider to be the present
invention.
[0029] As used herein, the phrase "pathogenic agent" means an agent
which causes a disease state or affliction in an animal. Included
within this definition, for examples, are bacteria, protozoans,
fungi, viruses and metazoan parasites which either produce a
disease state or render an animal infected with such an organism
susceptible to a disease state (e.g., a secondary infection).
Further included are species and strains of the genus Streptococcus
which produce disease states in animals.
[0030] As used herein, the term "organism" means any living
biological system, including viruses, regardless of whether it is a
pathogenic agent.
[0031] As used herein, the term "Streptococcus" means any species
or strain of bacteria which is members of the genus Streptococcus.
Such species and strains are known to those of skill in the art,
and include those that are pathogenic and those that are not.
[0032] As used herein, the phrase "one or more S. pneumoniae
polypeptides of the present invention" means polypeptides
comprising the amino acid sequence of one or more of the S.
pneumoniae polypeptides described in Table 1 and disclosed as SEQ
ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and so on through SEQ ID NO:226.
These polypeptides may be expressed as fusion proteins wherein the
S. pneumoniae polypeptides of the present invention are linked to
additional amino acid sequences which may be of streptococcal or
non-streptococcal origin. This phrase further includes polypeptide
comprising fragments of the S. pneumoniae polypeptides of the
present invention.
[0033] Additional definitions are provided throughout the
specification.
[0034] Explanation of Table 1
[0035] Table 1, below, provides information describing 113 open
reading frames (ORFs) which encode potentially antigenic
polypeptides of S. pneumoniae of the present invention. The table
lists the ORF identifier which consists of the letters SP, which
denote S. pneumoniae, followed immediately by a three digit numeric
code, which arbitrarily number the potentially antigenic
polypeptides of S. pneumoniae of the present invention. The table
further correlates the ORF identifier with a sequence
identification number (SEQ ID NO:). The actual nucleotide or amino
acid sequence of each ORF identifier is shown in the Sequence
Listing under the corresponding SEQ ID NO.
[0036] Thus, for example, the designation "SP126" refers to both
the nucleotide and amino acid sequences of S. pneumoniae
polypeptide number 126 of the present invention. Further, "SP126"
correlates with the nucleotide sequence shown as SEQ ID NO:223 and
with the amino acid sequence shown as SEQ ID NO:224 as is described
in Table 1.
[0037] The open reading frame within each "ORF" begins with the
second nucleotide shown. Thus, the first codon for each nucleotide
sequence shown is bases 2-4, the second 5-7, the third 8-10, and so
on.
[0038] Explanation of Table 2
[0039] Table 2 lists the antigenic epitopes present in each of the
S. pneumoniae polypeptides described in Table 1 as predicted by the
inventors. Each S. pneumoniae polypeptide shown in Table 1 has one
or more antigenic epitopes described in Table 2. It will be
appreciated that depending on the analytical criteria used to
predict antigenic determinants, the exact address of the
determinant may vary slightly. The exact location of the antigenic
determinant may shift by about 1 to 5 residues, more likely 1 to 2
residues, depending on the criteria used. Thus, the first antigenic
determinant described in Table 2, "Lys-1 to Ile-10" of SP100,
represents a peptide comprising the lysine at position 1 in SEQ ID
NO:2 through and including the isoleucine at position 10 in SEQ ID
NO:2, but may include more or fewer residues than those 10. It will
also be appreciated that, generally speaking, amino acids can be
added to either terminus of a peptide or polypeptide containing an
antigenic epitope without affecting its activity, whereas removing
residues from a peptide or polypeptide containing only the
antigenic determinant is much more likely to destroy activity. It
will be appreciated that the residues and locations shown described
in Table 2 correspond to the amino acid sequences for each ORF
shown in Table 1 and in the Sequence Listing.
[0040] Explanation of Table 3
[0041] Table 3 shows PCR primers designed by the inventors for the
amplification of polynucleotides encoding polypeptides of the
present invention according to the method of Example 1. PCR primer
design is routine in the art and those shown in Table 3 are
provided merely for the convenience of the skilled artisan. It will
be appreciated that others can be used with equal success. For each
primer, the table lists the corresponding ORF designation from
Table 1 followed by either an "A" or a "B". The "A" primers are the
5' primers and the "B" primers 3'. A restriction enzyme site was
built into each primer to allow ease of cloning. The restriction
enzyme which will recognize and cleave a sequence within each
primer is shown in Table 3, as well, under the heading "RE" for
restriction enzyme. Finally the sequence identifier is shown in
Table 3 for each primer for easy correlation with the Sequence
Listing.
[0042] Selection of Nucleic Acid Sequences Encoding Antigenic S.
pneumoniae Polypeptides
[0043] The present invention provides a select number of ORFs from
those presented in the fragments of the S. pneumoniae genome which
may prove useful for the generation of a protective immune
response. The sequenced S. pneumoniae genomic DNA was obtained from
a sub-cultured isolate of S. pneumoniae Strain 7/87 14.8.91, which
has been deposited at the American Type Culture Collection, as a
convenience to those of skill in the art. The S. pneumoniae isolate
was deposited on Oct. 10, 1996 at the ATCC.TM., 10801 University
Boulevard, Manassas, Va. 20110-2209 (present address), and given
accession number 55840. A genomic library constructed from DNA
isolated from the S. pneumoniae isolate was also deposited at the
ATCC.TM. on Oct. 11, 1996 and given ATCC.TM. Deposit No. 97755. A
more complete listing of the sequence obtained from the S.
pneumoniae genome may be found in co-pending U.S. Provisional
Application Ser. No. 60/029,960, filed Oct. 31, 1996, incorporated
herein by reference in its entirety. Some ORFs contained in the
subset of fragments of the S. pneumoniae genome disclosed herein
were derived through the use of a number of screening criteria
detailed below.
[0044] The selected ORFs do not consist of complete ORFs. Although
a polypeptide representing a complete ORF may be the closest
approximation of a protein native to an organism, it is not always
preferred to express a complete ORF in a heterologous system. It
may be challenging to express and purify a highly hydrophobic
protein by common laboratory methods. Thus, the polypeptide vaccine
candidates described herein may have been modified slightly to
simplify the production of recombinant protein. For example,
nucleotide sequences which encode highly hydrophobic domains, such
as those found at the amino terminal signal sequence, have been
excluded from some constructs used for in vitro expression of the
polypeptides. Furthermore, any highly hydrophobic amino acid
sequences occurring at the carboxy terminus have also been excluded
from the recombinant expression constructs. Thus, in one
embodiment, a polypeptide which represents a truncated or modified
ORF may be used as an antigen.
[0045] While numerous methods are known in the art for selecting
potentially immunogenic polypeptides, many of the ORFs disclosed
herein were selected on the basis of screening all theoretical S.
pneumoniae ORFs for several aspects of potential immunogenicity.
One set of selection criteria are as follows:
[0046] 1. Type I signal sequence: An amino terminal type I signal
sequence generally directs a nascent protein across the plasma and
outer membranes to the exterior of the bacterial cell. Experimental
evidence obtained from studies with Escherichia coli suggests that
the typical type I signal sequence consists of the following
biochemical and physical attributes (Izard, J. W. and Kendall, D.
A. Mol. Microbiol. 13:765-773 (1994)). The length of the type I
signal sequence is approximately 15 to 25 primarily hydrophobic
amino acid residues with a net positive charge in the extreme amino
terminus. In addition, the central region of the signal sequence
adopts an alpha-helical conformation in a hydrophobic environment.
Finally, the region surrounding the actual site of cleavage is
ideally six residues long, with small side-chain amino acids in the
-1 and -3 positions.
[0047] 2. Type IV signal sequence: The type IV signal sequence is
an example of the several types of functional signal sequences
which exist in addition to the type I signal sequence detailed
above. Although functionally related, the type IV signal sequence
possesses a unique set of biochemical and physical attributes
(Strom, M. S. and Lory, S., J. Bacteriol. 174:7345-7351 (1992)).
These are typically six to eight amino acids with a net basic
charge followed by an additional sixteen to thirty primarily
hydrophobic residues. The cleavage site of a type IV signal
sequence is typically after the initial six to eight amino acids at
the extreme amino terminus. In addition, type IV signal sequences
generally contain a phenylalanine residue at the +1 site relative
to the cleavage site.
[0048] 3. Lipoprotein: Studies of the cleavage sites of twenty-six
bacterial lipoprotein precursors has allowed the definition of a
consensus amino acid sequence for lipoprotein cleavage. Nearly
three-fourths of the bacterial lipoprotein precursors examined
contained the sequence L-(A,S)-(G,A)-C (SEQ ID NO:453) at positions
-3 to +1, relative to the point of cleavage (Hayashi, S. and Wu, H.
C., J. Bioenerg. Biomembr. 22:451-471 (1990)).
[0049] 4. LPXTG motif: It has been experimentally determined that
most anchored proteins found on the surface of gram-positive
bacteria possess a highly conserved carboxy terminal sequence. More
than fifty such proteins from organisms such as S. pyogenes, S.
mutans, E. faecalis, S. pneumoniae, and others, have been
identified based on their extracellular location and carboxy
terminal amino acid sequence (Fischetti, V. A., ASM News 62:405-410
(1996)). The conserved region consists of six charged amino acids
at the extreme carboxy terminus coupled to 15-20 hydrophobic amino
acids presumed to function as a transmembrane domain. Immediately
adjacent to the transmembrane domain is a six amino acid sequence
conserved in nearly all proteins examined. The amino acid sequence
of this region is L-P-X-T-G-X (SEQ ID NO:454), where X is any amino
acid.
[0050] An algorithm for selecting antigenic and immunogenic S.
pneumoniae polypeptides including the foregoing criteria was
developed. Use of the algorithm by the inventors to select
immunologically useful S. pneumoniae polypeptides resulted in the
selection of a number of the disclosed ORFs. Polypeptides
comprising the polypeptides identified in this group may be
produced by techniques standard in the art and as further described
herein.
[0051] Nucleic Acid Molecules
[0052] The present invention provides isolated nucleic acid
molecules comprising polynucleotides encoding the S. pneumoniae
polypeptides having the amino acid sequences described in Table 1
and shown as SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and so on
through SEQ ID NO:226, which were determined by sequencing the
genome of S. pneumoniae and selected as putative immunogens.
[0053] Unless otherwise indicated, all nucleotide sequences
determined by sequencing a DNA molecule herein were determined
using an automated DNA sequencer (such as the Model 373 from
Applied Biosystems, Inc.), and all amino acid sequences of
polypeptides encoded by DNA molecules determined herein were
predicted by translation of DNA sequences determined as above.
Therefore, as is known in the art for any DNA sequence determined
by this automated approach, any nucleotide sequence determined
herein may contain some errors. Nucleotide sequences determined by
automation are typically at least about 90% identical, more
typically at least about 95% to at least about 99.9% identical to
the actual nucleotide sequence of the sequenced DNA molecule. The
actual sequence can be more precisely determined by other
approaches including manual DNA sequencing methods well known in
the art. As is also known in the art, a single insertion or
deletion in a determined nucleotide sequence compared to the actual
sequence will cause a frame shift in translation of the nucleotide
sequence such that the predicted amino acid sequence encoded by a
determined nucleotide sequence will be completely different from
the amino acid sequence actually encoded by the sequenced DNA
molecule, beginning at the point of such an insertion or
deletion.
[0054] Unless otherwise indicated, each "nucleotide sequence" set
forth herein is presented as a sequence of deoxyribonucleotides
(abbreviated A, G, C and T). However, by "nucleotide sequence" of a
nucleic acid molecule or polynucleotide is intended, for a DNA
molecule or polynucleotide, a sequence of deoxyribonucleotides, and
for an RNA molecule or polynucleotide, the corresponding sequence
of ribonucleotides (A, G, C and U), where each thymidine
deoxyribonucleotide (T) in the specified deoxyribonucleotide
sequence is replaced by the ribonucleotide uridine (U). For
instance, reference to an RNA molecule having a sequence described
in Table 1 set forth using deoxyribonucleotide abbreviations is
intended to indicate an RNA molecule having a sequence in which
each deoxyribonucleotide A, G or C described in Table 1 has been
replaced by the corresponding ribonucleotide A, G or C, and each
deoxyribonucleotide T has been replaced by a ribonucleotide U.
[0055] Nucleic acid molecules of the present invention may be in
the form of RNA, such as mRNA, or in the form of DNA, including,
for instance, cDNA and genomic DNA obtained by cloning or produced
synthetically. The DNA may be double-stranded or single-stranded.
Single-stranded DNA or RNA may be the coding strand, also known as
the sense strand, or it may be the non-coding strand, also referred
to as the anti-sense strand.
[0056] By "isolated" nucleic acid molecule(s) is intended a nucleic
acid molecule, DNA or RNA, which has been removed from its native
environment. For example, recombinant DNA molecules contained in a
vector are considered isolated for the purposes of the present
invention. Further examples of isolated DNA molecules include
recombinant DNA molecules maintained in heterologous host cells or
purified (partially or substantially) DNA molecules in solution.
Isolated RNA molecules include in vivo or in vitro RNA transcripts
of the DNA molecules of the present invention. Isolated nucleic
acid molecules according to the present invention further include
such molecules produced synthetically.
[0057] Isolated nucleic acid molecules of the present invention
include DNA molecules comprising a nucleotide sequence described in
Table 1 and shown as SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and so
on through SEQ ID NO:225; DNA molecules comprising the coding
sequences for the polypeptides described in Table 1 and shown as
SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and so on through SEQ ID
NO:226; and DNA molecules which comprise sequences substantially
different from those described above but which, due to the
degeneracy of the genetic code, still encode the S. pneumoniae
polypeptides described in Table 1. Of course, the genetic code is
well known in the art. Thus, it would be routine for one skilled in
the art to generate such degenerate variants.
[0058] The invention also provides nucleic acid molecules having
sequences complementary to any one of those described in Table 1.
Such isolated molecules, particularly DNA molecules, are useful as
probes for detecting expression of Streptococcal genes, for
instance, by Northern blot analysis or the polymerase chain
reaction (PCR).
[0059] The present invention is further directed to fragments of
the isolated nucleic acid molecules described herein. By a fragment
of an isolated nucleic acid molecule having a nucleotide sequence
described in Table 1, is intended fragments at least about 15 nt,
and more preferably at least about 17 nt, still more preferably at
least about 20 nt, and even more preferably, at least about 25 nt
in length which are useful as diagnostic probes and primers as
discussed herein. Of course, larger fragments 50-100 nt in length
are also useful according to the present invention as are fragments
corresponding to most, if not all, of a nucleotide sequence
described in Table 1. By a fragment at least 20 nt in length, for
example, is intended fragments which include 20 or more contiguous
bases of a nucleotide sequence as described in Table 1. Since the
nucleotide sequences identified in Table 1 are provided as SEQ ID
NO:1, SEQ ID NO:3, SEQ ID NO:5, and so on through SEQ ID NO:225,
generating such DNA fragments would be routine to the skilled
artisan. For example, such fragments could be generated
synthetically.
[0060] Preferred nucleic acid fragments of the present invention
also include nucleic acid molecules comprising nucleotide sequences
encoding epitope-bearing portions of the S. pneumoniae polypeptides
identified in Table 1. Such nucleic acid fragments of the present
invention include, for example, nucleotide sequences encoding
polypeptide fragments comprising from about the amino terminal
residue to about the carboxy terminal residue of each fragment
shown in Table 2. The above referred to polypeptide fragments are
antigenic regions of the S. pneumoniae polypeptides identified in
Table 1.
[0061] In another aspect, the invention provides isolated nucleic
acid molecules comprising polynucleotides which hybridize under
stringent hybridization conditions to a portion of a polynucleotide
in a nucleic acid molecule of the invention described above, for
instance, a nucleic acid sequence identified in Table 1. By
"stringent hybridization conditions" is intended overnight
incubation at 42.degree. C. in a solution comprising: 50%
formamide, 5.times.SSC (150 mM NaCl, 15 mM trisodium citrate), 50
mM sodium phosphate (pH 7.6), 5.times. Denhardt's solution, 10%
dextran sulfate, and 20 g/ml denatured, sheared salmon sperm DNA,
followed by washing the filters in 0.1.times.SSC at about
65.degree. C.
[0062] By polynucleotides which hybridize to a "portion" of a
polynucleotide is intended polynucleotides (either DNA or RNA)
which hybridize to at least about 15 nucleotides (nt), and more
preferably at least about 17 nt, still more preferably at least
about 20 nt, and even more preferably about 25-70 nt of the
reference polynucleotide. These are useful as diagnostic probes and
primers as discussed above and in more detail below.
[0063] Of course, polynucleotides hybridizing to a larger portion
of the reference polynucleotide, for instance, a portion 50-100 nt
in length, or even to the entire length of the reference
polynucleotide, are also useful as probes according to the present
invention, as are polynucleotides corresponding to most, if not
all, of a nucleotide sequence as identified in Table 1. By a
portion of a polynucleotide of "at least 20 nt in length," for
example, is intended 20 or more contiguous nucleotides from the
nucleotide sequence of the reference polynucleotide (e.g., a
nucleotide sequences as described in Table 1). As noted above, such
portions are useful diagnostically either as probes according to
conventional DNA hybridization techniques or as primers for
amplification of a target sequence by PCR, as described in the
literature (for instance, in Molecular Cloning, A Laboratory
Manual, 2nd. edition, Sambrook, J., Fritsch, E. F. and Maniatis,
T., eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y. (1989), the entire disclosure of which is hereby incorporated
herein by reference).
[0064] Since nucleic acid sequences encoding the S. pneumoniae
polypeptides of the present invention are identified in Table 1 and
provided as SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and so on
through SEQ ID NO:225, generating polynucleotides which hybridize
to portions of these sequences would be routine to the skilled
artisan. For example, the hybridizing polynucleotides of the
present invention could be generated synthetically according to
known techniques.
[0065] As indicated, nucleic acid molecules of the present
invention which encode S. pneumoniae polypeptides of the present
invention may include, but are not limited to those encoding the
amino acid sequences of the polypeptides by themselves; and
additional coding sequences which code for additional amino acids,
such as those which provide additional functionalities. Thus, the
sequences encoding these polypeptides may be fused to a marker
sequence, such as a sequence encoding a peptide which facilitates
purification of the fused polypeptide. In certain preferred
embodiments of this aspect of the invention, the marker amino acid
sequence is a hexa-histidine peptide, such as the tag provided in a
pQE vector (Qiagen, Inc.), among others, many of which are
commercially available. As described by Gentz and colleagues (Proc.
Natl. Acad. Sci. USA 86:821-824 (1989)), for instance,
hexa-histidine provides for convenient purification of the
resulting fusion protein.
[0066] Thus, the present invention also includes genetic fusions
wherein the S. pneumoniae nucleic acid sequences coding sequences
identified in Table 1 are linked to additional nucleic acid
sequences to produce fusion proteins. These fusion proteins may
include epitopes of streptococcal or non-streptococcal origin
designed to produce proteins having enhanced immunogenicity.
Further, the fusion proteins of the present invention may contain
antigenic determinants known to provide helper T-cell stimulation,
peptides encoding sites for post-translational modifications which
enhance immunogenicity (e.g., acylation), peptides which facilitate
purification (e.g., histidine "tag"), or amino acid sequences which
target the fusion protein to a desired location (e.g., a
heterologous leader sequence).
[0067] In all cases of bacterial expression, an N-terminal
methionine residues is added. In many cases, however, the
N-terminal methionine residues is cleaved off post-translationally.
Thus, the invention includes polypeptides shown in Table 1 with,
and without an N-terminal methionine.
[0068] The present invention thus includes nucleic acid molecules
and sequences which encode fusion proteins comprising one or more
S. pneumoniae polypeptides of the present invention fused to an
amino acid sequence which allows for post-translational
modification to enhance immunogenicity. This post-translational
modification may occur either in vitro or when the fusion protein
is expressed in vivo in a host cell. An example of such a
modification is the introduction of an amino acid sequence which
results in the attachment of a lipid moiety.
[0069] Thus, as indicated above, the present invention includes
genetic fusions wherein a S. pneumoniae nucleic acid sequence
identified in Table 1 is linked to a nucleotide sequence encoding
another amino acid sequence. These other amino acid sequences may
be of streptococcal origin (e.g., another sequence selected from
Table 1) or non-streptococcal origin.
[0070] The present invention further relates to variants of the
nucleic acid molecules of the present invention, which encode
portions, analogs or derivatives of the S. pneumoniae polypeptides
described in Table 1. Variants may occur naturally, such as a
natural allelic variant. By an "allelic variant" is intended one of
several alternate forms of a gene occupying a given locus on a
chromosome of an organism (Genes II, Lewin, B., ed., John Wiley
& Sons, New York (1985)). Non-naturally occurring variants may
be produced using art-known mutagenesis techniques.
[0071] Such variants include those produced by nucleotide
substitutions, deletions or additions. The substitutions, deletions
or additions may involve one or more nucleotides. These variants
may be altered in coding regions, non-coding regions, or both.
Alterations in the coding regions may produce conservative or
non-conservative amino acid substitutions, deletions or additions.
Especially preferred among these are silent substitutions,
additions and deletions, which do not alter the properties and
activities of the S. pneumoniae polypeptides disclosed herein or
portions thereof. Silent substitution are most likely to be made in
non-epitopic regions. Guidance regarding those regions containing
epitopes is provided herein, for example, in Table 2. Also
especially preferred in this regard are conservative
substitutions.
[0072] Further embodiments of the invention include isolated
nucleic acid molecules comprising a polynucleotide having a
nucleotide sequence at least 90% identical, and more preferably at
least 95%, 96%, 97%, 98% or 99% identical to: (a) a nucleotide
sequence encoding any of the amino acid sequences of the
polypeptides identified in Table 1; and (b) a nucleotide sequence
complementary to any of the nucleotide sequences in (a) above.
[0073] By a polynucleotide having a nucleotide sequence at least,
for example, 95% "identical" to a reference nucleotide sequence
encoding a S. pneumoniae polypeptide described in Table 1, is
intended that the nucleotide sequence of the polynucleotide is
identical to the reference sequence except that the polynucleotide
sequence may include up to five point mutations per each 100
nucleotides of the reference nucleotide sequence encoding the
subject S. pneumoniae polypeptide. In other words, to obtain a
polynucleotide having a nucleotide sequence at least 95% identical
to a reference nucleotide sequence, up to 5% of the nucleotides in
the reference sequence may be deleted or substituted with another
nucleotide, or a number of nucleotides up to 5% of the total
nucleotides in the reference sequence may be inserted into the
reference sequence. These mutations of the reference sequence may
occur at the 5' or 3' terminal positions of the reference
nucleotide sequence or anywhere between those terminal positions,
interspersed either individually among nucleotides in the reference
sequence or in one or more contiguous groups within the reference
sequence.
[0074] Certain nucleotides within some of the nucleic acid
sequences shown in Table 1 were ambiguous upon sequencing.
Completely unknown sequences are shown as an "N". Other unresolved
nucleotides are known to be either a purine, shown as "R", or a
pyrimidine, shown as "Y". Accordingly, when determining identity
between two nucleotide sequences, identity is met where any
nucleotide, including an "R", "Y" or "N", is found in a test
sequence and at the corresponding position in the reference
sequence (from Table 1). Likewise, an A, G or "R" in a test
sequence is identical to an "R" in the reference sequence; and a T,
C or "Y" in a test sequence is identical to a "Y" in the reference
sequence.
[0075] As a practical matter, whether any particular nucleic acid
molecule is at least 90%, 95%, 96%, 97%, 98% or 99% identical to,
for instance, a nucleotide sequence described in Table 1 can be
determined conventionally using known computer programs such as the
Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for
Unix, Genetics Computer Group, University Research Park, 575
Science Drive, Madison, Wis. 53711). Bestfit uses the local
homology algorithm of Smith and Waterman (Advances in Applied
Mathematics 2:482-489 (1981)), to find the best segment of homology
between two sequences. When using Bestfit or any other sequence
alignment program to determine whether a particular sequence is,
for instance, 95% identical to a reference sequence according to
the present invention, the parameters are set, of course, such that
the percentage of identity is calculated over the full length of
the reference nucleotide sequence and that gaps in homology of up
to 5% of the total number of nucleotides in the reference sequence
are allowed.
[0076] The present application is directed to nucleic acid
molecules at least 90%, 95%, 96%, 97%, 98% or 99% identical to a
nucleic acid sequences described in Table 1. One of skill in the
art would still know how to use the nucleic acid molecule, for
instance, as a hybridization probe or a polymerase chain reaction
(PCR) primer. Uses of the nucleic acid molecules of the present
invention include, inter alia, (1) isolating Streptococcal genes or
allelic variants thereof from either a genomic or cDNA library and
(2) Northern Blot or PCR analysis for detecting Streptococcal mRNA
expression.
[0077] Of course, due to the degeneracy of the genetic code, one of
ordinary skill in the art will immediately recognize that a large
number of nucleic acid molecules having a sequence at least 90%,
95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence
identified in Table 1 will encode the same polypeptide. In fact,
since degenerate variants of these nucleotide sequences all encode
the same polypeptide, this will be clear to the skilled artisan
even without performing the above described comparison assay.
[0078] It will be further recognized in the art that, for such
nucleic acid molecules that are not degenerate variants, a
reasonable number will also encode proteins having antigenic
epitopes of the S. pneumoniae polypeptides of the present
invention. This is because the skilled artisan is fully aware of
amino acid substitutions that are either less likely or not likely
to significantly effect the antigenicity of a polypeptide (e.g.,
replacement of an amino acid in a region which is not believed to
form an antigenic epitope). For example, since antigenic epitopes
have been identified which contain as few as six amino acids (see
Harlow, et al., Antibodies: A Laboratory Manual, 2nd Ed.; Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988),
page 76), in instances where a polypeptide has multiple antigenic
epitopes the alteration of several amino acid residues would often
not be expected to eliminate all of the antigenic epitopes of that
polypeptide. This is especially so when the alterations are in
regions believed to not constitute antigenic epitopes.
[0079] Vectors and Host Cells
[0080] The present invention also relates to vectors which include
the isolated DNA molecules of the present invention, host cells
which are genetically engineered with the recombinant vectors, and
the production of S. pneumoniae polypeptides or fragments thereof
by recombinant techniques.
[0081] Recombinant constructs may be introduced into host cells
using well known techniques such as infection, transduction,
transfection, transvection, electroporation and transformation. The
vector may be, for example, a phage, plasmid, viral or retroviral
vector. Retroviral vectors may be replication competent or
replication defective. In the latter case, viral propagation
generally will occur only in complementing host cells.
[0082] The polynucleotides may be joined to a vector containing a
selectable marker for propagation in a host. Generally, a plasmid
vector is introduced in a precipitate, such as a calcium phosphate
precipitate, or in a complex with a charged lipid. If the vector is
a virus, it may be packaged in vitro using an appropriate packaging
cell line and then transduced into host cells.
[0083] Preferred are vectors comprising cis-acting control regions
to the polynucleotide of interest. Appropriate trans-acting factors
may be supplied by the host, supplied by a complementing vector or
supplied by the vector itself upon introduction into the host.
[0084] In certain preferred embodiments in this regard, the vectors
provide for specific expression, which may be inducible and/or cell
type-specific. Particularly preferred among such vectors are those
inducible by environmental factors that are easy to manipulate,
such as temperature and nutrient additives.
[0085] Expression vectors useful in the present invention include
chromosomal-, episomal- and virus-derived vectors, e.g., vectors
derived from bacterial plasmids, bacteriophage, yeast episomes,
yeast chromosomal elements, viruses such as baculoviruses, papova
viruses, vaccinia viruses, adenoviruses, fowl pox viruses,
pseudorabies viruses and retroviruses, and vectors derived from
combinations thereof, such as cosmids and phagemids.
[0086] The DNA insert should be operatively linked to an
appropriate promoter, such as the phage lambda PL promoter, the E.
coli lac, trp and tac promoters, the SV40 early and late promoters
and promoters of retroviral LTRs, to name a few. Other suitable
promoters will be known to the skilled artisan. The expression
constructs will further contain sites for transcription initiation,
termination and, in the transcribed region, a ribosome binding site
for translation. The coding portion of the mature transcripts
expressed by the constructs will preferably include a translation
initiating site at the beginning and a termination codon (UAA, UGA
or UAG) appropriately positioned at the end of the polypeptide to
be translated.
[0087] As indicated, the expression vectors will preferably include
at least one selectable marker. Such markers include dihydrofolate
reductase or neomycin resistance for eukaryotic cell culture and
tetracycline or ampicillin resistance genes for culturing in E.
coli and other bacteria. Representative examples of appropriate
hosts include, but are not limited to, bacterial cells, such as E.
coli, Streptomyces and Salmonella typhimurium cells; fungal cells,
such as yeast cells; insect cells such as Drosophila S2 and
Spodoptera Sf9 cells; animal cells such as CHO, COS and Bowes
melanoma cells; and plant cells. Appropriate culture mediums and
conditions for the above-described host cells are known in the
art.
[0088] Among vectors preferred for use in bacteria include pQE70,
pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript
vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A
available from Stratagene; pET series of vectors available from
Novagen; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available
from Pharmacia. Among preferred eukaryotic vectors are pWLNEO,
pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3,
pBPV, pMSG and pSVL available from Pharmacia. Other suitable
vectors will be readily apparent to the skilled artisan.
[0089] Among known bacterial promoters suitable for use in the
present invention include the E. coli lacI and lacZ promoters, the
T3 and T7 promoters, the gpt promoter, the lambda PR and PL
promoters and the trp promoter. Suitable eukaryotic promoters
include the CMV immediate early promoter, the HSV thymidine kinase
promoter, the early and late SV40 promoters, the promoters of
retroviral LTRs, such as those of the Rous sarcoma virus (RSV), and
metallothionein promoters, such as the mouse metallothionein-I
promoter.
[0090] Introduction of the construct into the host cell can be
effected by calcium phosphate transfection, DEAE-dextran mediated
transfection, cationic lipid-mediated transfection,
electroporation, transduction, infection or other methods. Such
methods are described in many standard laboratory manuals (for
example, Davis, et al., Basic Methods In Molecular Biology
(1986)).
[0091] Transcription of DNA encoding the polypeptides of the
present invention by higher eukaryotes may be 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 to increase transcriptional activity of a promoter in a given
host cell-type. Examples of enhancers include the SV40 enhancer,
which is located on the late side of the replication origin at bp
100 to 270, the cytomegalovirus early promoter enhancer, the
polyoma enhancer on the late side of the replication origin, and
adenovirus enhancers.
[0092] For secretion of the translated polypeptide into the lumen
of the endoplasmic reticulum, into the periplasmic space or into
the extracellular environment, appropriate secretion signals may be
incorporated into the expressed polypeptide. The signals may be
endogenous to the polypeptide or they may be heterologous
signals.
[0093] The polypeptide may be expressed in a modified form, such as
a fusion protein, and may include not only secretion signals, but
also additional heterologous functional regions. For instance, a
region of additional amino acids, particularly charged amino acids,
may be added to the N-terminus of the polypeptide to improve
stability and persistence in the host cell, during purification, or
during subsequent handling and storage. Also, peptide moieties may
be added to the polypeptide to facilitate purification. Such
regions may be removed prior to final preparation of the
polypeptide. The addition of peptide moieties to polypeptides to
engender secretion or excretion, to improve stability and to
facilitate purification, among others, are familiar and routine
techniques in the art. A preferred fusion protein comprises a
heterologous region from immunoglobulin that is useful to
solubilize proteins. For example, EP-A-O 464 533 (Canadian
counterpart 2045869) discloses fusion proteins comprising various
portions of constant region of immunoglobin molecules together with
another human protein or part thereof. In many cases, the Fc part
in a fusion protein is thoroughly advantageous for use in therapy
and diagnosis and thus results, for example, in improved
pharmacokinetic properties (EP-A 0232 262). On the other hand, for
some uses it would be desirable to be able to delete the Fc part
after the fusion protein has been expressed, detected and purified
in the advantageous manner described. This is the case when Fc
portion proves to be a hindrance to use in therapy and diagnosis,
for example when the fusion protein is to be used as antigen for
immunizations. In drug discovery, for example, human proteins, such
as, hIL5-receptor has been fused with Fc portions for the purpose
of high-throughput screening assays to identify antagonists of
hIL-5. See Bennett, D. et al., J. Molec. Recogn. 8:52-58 (1995) and
Johanson, K. et al., J. Biol. Chem. 270 (16):9459-9471 (1995).
[0094] The S. pneumoniae polypeptides can be recovered and purified
from recombinant cell cultures by well-known methods including
ammonium sulfate or ethanol precipitation, acid extraction, anion
or cation exchange chromatography, phosphocellulose chromatography,
hydrophobic interaction chromatography, affinity chromatography,
hydroxylapatite chromatography, lectin chromatography and high
performance liquid chromatography ("HPLC") is employed for
purification. Polypeptides of the present invention include
naturally purified products, products of chemical synthetic
procedures, and products produced by recombinant techniques from a
prokaryotic or eukaryotic host, including, for example, bacterial,
yeast, higher plant, insect and mammalian cells.
[0095] Polypeptides and Fragments
[0096] The invention further provides isolated polypeptides having
the amino acid sequences described in Table 1, and shown as SEQ ID
NO:2, SEQ ID NO:4, SEQ ID NO:6, and so on through SEQ ID NO:226,
and peptides or polypeptides comprising portions of the above
polypeptides. The terms "peptide" and "oligopeptide" are considered
synonymous (as is commonly recognized) and each term can be used
interchangeably as the context requires to indicate a chain of at
least two amino acids coupled by peptidyl linkages. The word
"polypeptide" is used herein for chains containing more than ten
amino acid residues. All oligopeptide and polypeptide formulas or
sequences herein are written from left to right and in the
direction from amino terminus to carboxy terminus.
[0097] Some amino acid sequences of the S. pneumoniae polypeptides
described in Table 1 can be varied without significantly effecting
the antigenicity of the polypeptides. If such differences in
sequence are contemplated, it should be remembered that there will
be critical areas on the polypeptide which determine antigenicity.
In general, it is possible to replace residues which do not form
part of an antigenic epitope without significantly effecting the
antigenicity of a polypeptide. Guidance for such alterations is
given in Table 2 wherein epitopes for each polypeptide is
delineated.
[0098] The polypeptides of the present invention are preferably
provided in an isolated form. By "isolated polypeptide" is intended
a polypeptide removed from its native environment. Thus, a
polypeptide produced and/or contained within a recombinant host
cell is considered isolated for purposes of the present invention.
Also intended as an "isolated polypeptide" is a polypeptide that
has been purified, partially or substantially, from a recombinant
host cell. For example, recombinantly produced versions of the S.
pneumoniae polypeptides described in Table 1 can be substantially
purified by the one-step method described by Smith and Johnson
(Gene 67:31-40 (1988)).
[0099] The polypeptides of the present invention include: (a) an
amino acid sequence of any of the polypeptides described in Table
1; and (b) an amino acid sequence of an epitope-bearing portion of
any one of the polypeptides of (a); as well as polypeptides with at
least 70% similarity, and more preferably at least 75%, 80%, 85%,
90%, 95%, 96%, 97%, 98%, or 99% similarity to those described in
(a) or (b) above, as well as polypeptides having an amino acid
sequence at least 70% identical, more preferably at least 75%
identical, and still more preferably 80%, 85%, 90%, 95%, 96%, 97%,
98%, or 99% identical to those above.
[0100] By "% similarity" for two polypeptides is intended a
similarity score produced by comparing the amino acid sequences of
the two polypeptides using the Bestfit program (Wisconsin Sequence
Analysis Package, Version 8 for Unix, Genetics Computer Group,
University Research Park, 575 Science Drive, Madison, Wis. 53711)
and the default settings for determining similarity. Bestfit uses
the local homology algorithm of Smith and Waterman (Advances in
Applied Mathematics 2:482-489 (1981)) to find the best segment of
similarity between two sequences.
[0101] By a polypeptide having an amino acid sequence at least, for
example, 95% "identical" to a reference amino acid sequence of a S.
pneumoniae polypeptide is intended that the amino acid sequence of
the polypeptide is identical to the reference sequence except that
the polypeptide sequence may include up to five amino acid
alterations per each 100 amino acids of the reference amino acid
sequence. In other words, to obtain a polypeptide having an amino
acid sequence at least 95% identical to a reference amino acid
sequence, up to 5% of the amino acid residues in the reference
sequence may be deleted or substituted with another amino acid, or
a number of amino acids up to 5% of the total amino acid residues
in the reference sequence may be inserted into the reference
sequence. These alterations of the reference sequence may occur at
the amino or carboxy terminal positions of the reference amino acid
sequence or anywhere between those terminal positions, interspersed
either individually among residues in the reference sequence or in
one or more contiguous groups within the reference sequence.
[0102] The amino acid sequences shown in Table 1 may have on or
more "X" residues. "X" represents unknown. Thus, for purposes of
defining identity, if any amino acid is present at the same
position in a reference amino acid sequence (shown in Table 1)
where an X is shown, the two sequences are identical at that
position.
[0103] As a practical matter, whether any particular polypeptide is
at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%
identical to, for instance, an amino acid sequence shown in Table
1, can be determined conventionally using known computer programs
such the Bestfit program (Wisconsin Sequence Analysis Package,
Version 8 for Unix, Genetics Computer Group, University Research
Park, 575 Science Drive, Madison, Wis. 53711). When using Bestfit
or any other sequence alignment program to determine whether a
particular sequence is, for instance, 95% identical to a reference
sequence according to the present invention, the parameters are
set, of course, such that the percentage of identity is calculated
over the full length of the reference amino acid sequence and that
gaps in homology of up to 5% of the total number of amino acid
residues in the reference sequence are allowed.
[0104] As described below, the polypeptides of the present
invention can also be used to raise polyclonal and monoclonal
antibodies, which are useful in assays for detecting Streptococcal
protein expression.
[0105] In another aspect, the invention provides peptides and
polypeptides comprising epitope-bearing portions of the S.
pneumoniae polypeptides of the invention. These epitopes are
immunogenic or antigenic epitopes of the polypeptides of the
invention. An "immunogenic epitope" is defined as a part of a
protein that elicits an antibody response when the whole protein or
polypeptide is the immunogen. These immunogenic epitopes are
believed to be confined to a few loci on the molecule. On the other
hand, a region of a protein molecule to which an antibody can bind
is defined as an "antigenic determinant" or "antigenic epitope."
The number of immunogenic epitopes of a protein generally is less
than the number of antigenic epitopes (Geysen, et al., Proc. Natl.
Acad. Sci. USA 81:3998-4002 (1983)). Predicted antigenic epitopes
are shown in Table 2, below.
[0106] As to the selection of peptides or polypeptides bearing an
antigenic epitope (i.e., that contain a region of a protein
molecule to which an antibody can bind), it is well known in that
art that relatively short synthetic peptides that mimic part of a
protein sequence are routinely capable of eliciting an antiserum
that reacts with the partially mimicked protein (for instance,
Sutcliffe, J., et al., Science 219:660-666 (1983)). Peptides
capable of eliciting protein-reactive sera are frequently
represented in the primary sequence of a protein, can be
characterized by a set of simple chemical rules, and are confined
neither to immunodominant regions of intact proteins (i.e.,
immunogenic epitopes) nor to the amino or carboxyl terminals.
Peptides that are extremely hydrophobic and those of six or fewer
residues generally are ineffective at inducing antibodies that bind
to the mimicked protein; longer, peptides, especially those
containing proline residues, usually are effective (Sutcliffe, et
al., supra, p. 661). For instance, 18 of 20 peptides designed
according to these guidelines, containing 8-39 residues covering
75% of the sequence of the influenza virus hemagglutinin HA1
polypeptide chain, induced antibodies that reacted with the HA1
protein or intact virus; and 12/12 peptides from the MuLV
polymerase and 18/18 from the rabies glycoprotein induced
antibodies that precipitated the respective proteins.
[0107] Antigenic epitope-bearing peptides and polypeptides of the
invention are therefore useful to raise antibodies, including
monoclonal antibodies, that bind specifically to a polypeptide of
the invention. Thus, a high proportion of hybridomas obtained by
fusion of spleen cells from donors immunized with an antigen
epitope-bearing peptide generally secrete antibody reactive with
the native protein (Sutcliffe, et al., supra, p. 663). The
antibodies raised by antigenic epitope-bearing peptides or
polypeptides are useful to detect the mimicked protein, and
antibodies to different peptides may be used for tracking the fate
of various regions of a protein precursor which undergoes
post-translational processing. The peptides and anti-peptide
antibodies may be used in a variety of qualitative or quantitative
assays for the mimicked protein, for instance in competition assays
since it has been shown that even short peptides (e.g., about 9
amino acids) can bind and displace the larger peptides in
immunoprecipitation assays (for instance, Wilson, et al., Cell
37:767-778 (1984) p. 777). The anti-peptide antibodies of the
invention also are useful for purification of the mimicked protein,
for instance, by adsorption chromatography using methods well known
in the art.
[0108] Antigenic epitope-bearing peptides and polypeptides of the
invention designed according to the above guidelines preferably
contain a sequence of at least seven, more preferably at least nine
and most preferably between about 15 to about 30 amino acids
contained within the amino acid sequence of a polypeptide of the
invention. However, peptides or polypeptides comprising a larger
portion of an amino acid sequence of a polypeptide of the
invention, containing about 30 to about 50 amino acids, or any
length up to and including the entire amino acid sequence of a
polypeptide of the invention, also are considered epitope-bearing
peptides or polypeptides of the invention and also are useful for
inducing antibodies that react with the mimicked protein.
Preferably, the amino acid sequence of the epitope-bearing peptide
is selected to provide substantial solubility in aqueous solvents
(i.e., the sequence includes relatively hydrophilic residues and
highly hydrophobic sequences are preferably avoided); and sequences
containing proline residues are particularly preferred.
[0109] Non-limiting examples of antigenic polypeptides or peptides
that can be used to generate Streptococcal-specific antibodies
include portions of the amino acid sequences identified in Table 1.
More specifically, Table 2 discloses antigenic fragments of
polypeptides of the present invention, which antigenic fragments
comprise amino acid sequences from about the first amino acid
residues indicated to about the last amino acid residue indicated
for each fragment. The polypeptide fragments disclosed in Table 2
are believed to be antigenic regions of the S. pneumoniae
polypeptides described in Table 1. Thus the invention further
includes isolated peptides and polypeptides comprising an amino
acid sequence of an epitope shown in Table 2 and polynucleotides
encoding said polypeptides.
[0110] The epitope-bearing peptides and polypeptides of the
invention may be produced by any conventional means for making
peptides or polypeptides including recombinant means using nucleic
acid molecules of the invention. For instance, an epitope-bearing
amino acid sequence of the present invention may be fused to a
larger polypeptide which acts as a carrier during recombinant
production and purification, as well as during immunization to
produce anti-peptide antibodies. Epitope-bearing peptides also may
be synthesized using known methods of chemical synthesis. For
instance, Houghten has described a simple method for synthesis of
large numbers of peptides, such as 10-20 mg of 248 different 13
residue peptides representing single amino acid variants of a
segment of the HA1 polypeptide which were prepared and
characterized (by ELISA-type binding studies) in less than four
weeks (Houghten, R. A. Proc. Natl. Acad. Sci. USA 82:5131-5135
(1985)). This "Simultaneous Multiple Peptide Synthesis (SMPS)"
process is further described in U.S. Pat. No. 4,631,211 to Houghten
and coworkers (1986). In this procedure the individual resins for
the solid-phase synthesis of various peptides are contained in
separate solvent-permeable packets, enabling the optimal use of the
many identical repetitive steps involved in solid-phase methods. A
completely manual procedure allows 500-1000 or more syntheses to be
conducted simultaneously (Houghten, et al., supra, p. 5134).
[0111] Epitope-bearing peptides and polypeptides of the invention
are used to induce antibodies according to methods well known in
the art (for instance, Sutcliffe, et al., supra; Wilson, et al.,
supra; Chow, M., et al., Proc. Natl. Acad. Sci. USA 82:910-914; and
Bittle, F. J., et al., J. Gen. Virol. 66:2347-2354 (1985)).
Generally, animals may be immunized with free peptide; however,
anti-peptide antibody titer may be boosted by coupling of the
peptide to a macromolecular carrier, such as keyhole limpet
hemacyanin (KLH) or tetanus toxoid. For instance, peptides
containing cysteine may be coupled to carrier using a linker such
as m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other
peptides may be coupled to carrier using a more general linking
agent such as glutaraldehyde. Animals such as rabbits, rats and
mice are immunized with either free or carrier-coupled peptides,
for instance, by intraperitoneal and/or intradermal injection of
emulsions containing about 100 .mu.g peptide or carrier protein and
Freund's adjuvant. Several booster injections may be needed, for
instance, at intervals of about two weeks, to provide a useful
titer of anti-peptide antibody which can be detected, for example,
by ELISA assay using free peptide adsorbed to a solid surface. The
titer of anti-peptide antibodies in serum from an immunized animal
may be increased by selection of anti-peptide antibodies, for
instance, by adsorption to the peptide on a solid support and
elution of the selected antibodies according to methods well known
in the art.
[0112] Immunogenic epitope-bearing peptides of the invention, i.e.,
those parts of a protein that elicit an antibody response when the
whole protein is the immunogen, are identified according to methods
known in the art. For instance, Geysen, et al., supra, discloses a
procedure for rapid concurrent synthesis on solid supports of
hundreds of peptides of sufficient purity to react in an
enzyme-linked immunosorbent assay. Interaction of synthesized
peptides with antibodies is then easily detected without removing
them from the support. In this manner a peptide bearing an
immunogenic epitope of a desired protein may be identified
routinely by one of ordinary skill in the art. For instance, the
immunologically important epitope in the coat protein of
foot-and-mouth disease virus was located by Geysen et al. supra
with a resolution of seven amino acids by synthesis of an
overlapping set of all 208 possible hexapeptides covering the
entire 213 amino acid sequence of the protein. Then, a complete
replacement set of peptides in which all 20 amino acids were
substituted in turn at every position within the epitope were
synthesized, and the particular amino acids conferring specificity
for the reaction with antibody were determined. Thus, peptide
analogs of the epitope-bearing peptides of the invention can be
made routinely by this method. U.S. Pat. No. 4,708,781 to Geysen
(1987) further describes this method of identifying a peptide
bearing an immunogenic epitope of a desired protein.
[0113] Further still, U.S. Pat. No. 5,194,392, to Geysen (1990),
describes a general method of detecting or determining the sequence
of monomers (amino acids or other compounds) which is a topological
equivalent of the epitope (i.e., a "mimotope") which is
complementary to a particular paratope (antigen binding site) of an
antibody of interest. More generally, U.S. Pat. No. 4,433,092, also
to Geysen (1989), describes a method of detecting or determining a
sequence of monomers which is a topographical equivalent of a
ligand which is complementary to the ligand binding site of a
particular receptor of interest. Similarly, U.S. Pat. No. 5,480,971
to Houghten, R. A. et al. (1996) discloses linear
C.sub.1-C.sub.7-alkyl peralkylated oligopeptides and sets and
libraries of such peptides, as well as methods for using such
oligopeptide sets and libraries for determining the sequence of a
peralkylated oligopeptide that preferentially binds to an acceptor
molecule of interest. Thus, non-peptide analogs of the
epitope-bearing peptides of the invention also can be made
routinely by these methods.
[0114] The entire disclosure of each document cited in this section
on "Polypeptides and Fragments" is hereby incorporated herein by
reference.
[0115] As one of skill in the art will appreciate, the polypeptides
of the present invention and the epitope-bearing fragments thereof
described above can be combined with parts of the constant domain
of immunoglobulins (IgG), resulting in chimeric polypeptides. These
fusion proteins facilitate purification and show an increased
half-life in vivo. This has been shown, e.g., for chimeric proteins
consisting of the first two domains of the human CD4-polypeptide
and various domains of the constant regions of the heavy or light
chains of mammalian immunoglobulins (EPA 0,394,827; Traunecker et
al., Nature 331:84-86 (1988)). Fusion proteins that have a
disulfide-linked dimeric structure due to the IgG part can also be
more efficient in binding and neutralizing other molecules than a
monomeric S. pneumoniae polypeptide or fragment thereof alone
(Fountoulakis et al., J. Biochem. 270:3958-3964 (1995)).
[0116] Diagnostic Assays
[0117] The present invention further relates to a method for
assaying for Streptococcal infection in an animal via detecting the
expression of genes encoding Streptococcal polypeptides (e.g., the
polypeptides described Table 1). This method comprises analyzing
tissue or body fluid from the animal for Streptococcus-specific
antibodies or Streptococcal nucleic acids or proteins. Analysis of
nucleic acid specific to Streptococcus can be done by PCR or
hybridization techniques using nucleic acid sequences of the
present invention as either hybridization probes or primers (cf
Molecular Cloning: A Laboratory Manual, second edition, edited by
Sambrook, Fritsch, & Maniatis, Cold Spring Harbor Laboratory,
1989; Eremeeva et al., J. Clin. Microbiol. 32:803-810 (1994) which
describes differentiation among spotted fever group Rickettsiae
species by analysis of restriction fragment length polymorphism of
PCR-amplified DNA). Methods for detecting B. burgdorferi nucleic
acids via PCR are described, for example, in Chen et al., J. Clin.
Microbiol. 32:589-595 (1994).
[0118] Where diagnosis of a disease state related to infection with
Streptococcus has already been made, the present invention is
useful for monitoring progression or regression of the disease
state whereby patients exhibiting enhanced Streptococcus gene
expression will experience a worse clinical outcome relative to
patients expressing these gene(s) at a lower level.
[0119] By "assaying for Streptococcal infection in an animal via
detection of genes encoding Streptococcal polypeptides" is intended
qualitatively or quantitatively measuring or estimating the level
of one or more Streptococcus polypeptides or the level of nucleic
acid encoding Streptococcus polypeptides in a first biological
sample either directly (e.g., by determining or estimating absolute
protein level or nucleic level) or relatively (e.g., by comparing
to the Streptococcus polypeptide level or mRNA level in a second
biological sample). The Streptococcus polypeptide level or nucleic
acid level in the second sample used for a relative comparison may
be undetectable if obtained from an animal which is not infected
with Streptococcus. When monitoring the progression or regression
of a disease state, the Streptococcus polypeptide level or nucleic
acid level may be compared to a second sample obtained from either
an animal infected with Streptococcus or the same animal from which
the first sample was obtained but taken from that animal at a
different time than the first. As will be appreciated in the art,
once a standard Streptococcus polypeptide level or nucleic acid
level which corresponds to a particular stage of a Streptococcus
infection is known, it can be used repeatedly as a standard for
comparison.
[0120] By "biological sample" is intended any biological sample
obtained from an animal, cell line, tissue culture, or other source
which contains Streptococcus polypeptide, mRNA, or DNA. Biological
samples include body fluids (such as plasma and synovial fluid)
which contain Streptococcus polypeptides, and muscle, skin, and
cartilage tissues. Methods for obtaining tissue biopsies and body
fluids are well known in the art.
[0121] The present invention is useful for detecting diseases
related to Streptococcus infections in animals. Preferred animals
include monkeys, apes, cats, dogs, cows, pigs, mice, horses,
rabbits and humans. Particularly preferred are humans.
[0122] Total RNA can be isolated from a biological sample using any
suitable technique such as the single-step
guanidinium-thiocyanate-phenol- -chloroform method described in
Chomczynski and Sacchi, Anal. Biochem. 162:156-159 (1987). mRNA
encoding Streptococcus polypeptides having sufficient homology to
the nucleic acid sequences identified in Table 1 to allow for
hybridization between complementary sequences are then assayed
using any appropriate method. These include Northern blot analysis,
S 1 nuclease mapping, the polymerase chain reaction (PCR), reverse
transcription in combination with the polymerase chain reaction
(RT-PCR), and reverse transcription in combination with the ligase
chain reaction (RT-LCR).
[0123] Northern blot analysis can be performed as described in
Harada et al., Cell 63:303-312 (1990). Briefly, total RNA is
prepared from a biological sample as described above. For the
Northern blot, the RNA is denatured in an appropriate buffer (such
as glyoxal/dimethyl sulfoxide/sodium phosphate buffer), subjected
to agarose gel electrophoresis, and transferred onto a
nitrocellulose filter. After the RNAs have been linked to the
filter by a UV linker, the filter is prehybridized in a solution
containing formamide, SSC, Denhardt's solution, denatured salmon
sperm, SDS, and sodium phosphate buffer. A S. pneumoniae
polypeptide DNA sequence shown in Table 1 labeled according to any
appropriate method (such as the .sup.32P-multiprimed DNA labeling
system (Amersham)) is used as probe. After hybridization overnight,
the filter is washed and exposed to x-ray film. DNA for use as
probe according to the present invention is described in the
sections above and will preferably at least 15 bp in length.
[0124] S 1 mapping can be performed as described in Fujita et al.,
Cell 49:357-367 (1987). To prepare probe DNA for use in S 1
mapping, the sense strand of an above-described S. pneumoniae DNA
sequence of the present invention is used as a template to
synthesize labeled antisense DNA. The antisense DNA can then be
digested using an appropriate restriction endonuclease to generate
further DNA probes of a desired length. Such antisense probes are
useful for visualizing protected bands corresponding to the target
mRNA (i.e., mRNA encoding Streptococcus polypeptides).
[0125] Preferably, levels of mRNA encoding Streptococcus
polypeptides are assayed using the RT-PCR method described in
Makino et al., Technique 2:295-301 (1990). By this method, the
radioactivities of the "amplicons" in the polyacrylamide gel bands
are linearly related to the initial concentration of the target
mRNA. Briefly, this method involves adding total RNA isolated from
a biological sample in a reaction mixture containing a RT primer
and appropriate buffer. After incubating for primer annealing, the
mixture can be supplemented with a RT buffer, dNTPs, DTT, RNase
inhibitor and reverse transcriptase. After incubation to achieve
reverse transcription of the RNA, the RT products are then subject
to PCR using labeled primers. Alternatively, rather than labeling
the primers, a labeled dNTP can be included in the PCR reaction
mixture. PCR amplification can be performed in a DNA thermal cycler
according to conventional techniques. After a suitable number of
rounds to achieve amplification, the PCR reaction mixture is
electrophoresed on a polyacrylamide gel. After drying the gel, the
radioactivity of the appropriate bands (corresponding to the mRNA
encoding the Streptococcus polypeptides)) is quantified using an
imaging analyzer. RT and PCR reaction ingredients and conditions,
reagent and gel concentrations, and labeling methods are well known
in the art. Variations on the RT-PCR method will be apparent to the
skilled artisan.
[0126] Assaying Streptococcus polypeptide levels in a biological
sample can occur using any art-known method. Preferred for assaying
Streptococcus polypeptide levels in a biological sample are
antibody-based techniques. For example, Streptococcus polypeptide
expression in tissues can be studied with classical
immunohistological methods. In these, the specific recognition is
provided by the primary antibody (polyclonal or monoclonal) but the
secondary detection system can utilize fluorescent, enzyme, or
other conjugated secondary antibodies. As a result, an
immunohistological staining of tissue section for pathological
examination is obtained. Tissues can also be extracted, e.g., with
urea and neutral detergent, for the liberation of Streptococcus
polypeptides for Western-blot or dot/slot assay (Jalkanen, M., et
al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, M., et al., J.
Cell. Biol. 105:3087-3096 (1987)). In this technique, which is
based on the use of cationic solid phases, quantitation of a
Streptococcus polypeptide can be accomplished using an isolated
Streptococcus polypeptide as a standard. This technique can also be
applied to body fluids.
[0127] Other antibody-based methods useful for detecting
Streptococcus polypeptide gene expression include immunoassays,
such as the enzyme linked immunosorbent assay (ELISA) and the
radioimmunoassay (RIA). For example, a Streptococcus
polypeptide-specific monoclonal antibodies can be used both as an
immunoabsorbent and as an enzyme-labeled probe to detect and
quantify a Streptococcus polypeptide. The amount of a Streptococcus
polypeptide present in the sample can be calculated by reference to
the amount present in a standard preparation using a linear
regression computer algorithm. Such an ELISA for detecting a tumor
antigen is described in lacobelli et al., Breast Cancer Research
and Treatment 11:19-30 (1988). In another ELISA assay, two distinct
specific monoclonal antibodies can be used to detect Streptococcus
polypeptides in a body fluid. In this assay, one of the antibodies
is used as the immunoabsorbent and the other as the enzyme-labeled
probe.
[0128] The above techniques may be conducted essentially as a
"one-step" or "two-step" assay. The "one-step" assay involves
contacting the Streptococcus polypeptide with immobilized antibody
and, without washing, contacting the mixture with the labeled
antibody. The "two-step" assay involves washing before contacting
the mixture with the labeled antibody. Other conventional methods
may also be employed as suitable. It is usually desirable to
immobilize one component of the assay system on a support, thereby
allowing other components of the system to be brought into contact
with the component and readily removed from the sample.
[0129] Streptococcus polypeptide-specific antibodies for use in the
present invention can be raised against an intact S. pneumoniae
polypeptide of the present invention or fragment thereof. These
polypeptides and fragments may be administered to an animal (e.g.,
rabbit or mouse) either with a carrier protein (e.g., albumin) or,
if long enough (e.g., at least about 25 amino acids), without a
carrier.
[0130] As used herein, the term "antibody" (Ab) or "monoclonal
antibody" (Mab) is meant to include intact molecules as well as
antibody fragments (such as, for example, Fab and F(ab').sub.2
fragments) which are capable of specifically binding to a
Streptococcus polypeptide. Fab and F(ab').sub.2 fragments lack the
Fc fragment of intact antibody, clear more rapidly from the
circulation, and may have less non-specific tissue binding of an
intact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)).
Thus, these fragments are preferred.
[0131] The antibodies of the present invention may be prepared by
any of a variety of methods. For example, the S. pneumoniae
polypeptides identified in Table 1, or fragments thereof, can be
administered to an animal in order to induce the production of sera
containing polyclonal antibodies. In a preferred method, a
preparation of a S. pneumoniae polypeptide of the present invention
is prepared and purified to render it substantially free of natural
contaminants. Such a preparation is then introduced into an animal
in order to produce polyclonal antisera of high specific
activity.
[0132] In the most preferred method, the antibodies of the present
invention are monoclonal antibodies. Such monoclonal antibodies can
be prepared using hybridoma technology (Kohler et al., Nature
256:495 (1975); Kohler et al., Eur. J. Immunol. 6:511 (1976);
Kohler et al., Eur. J. Immunol. 6:292 (1976); Hammerling et al.,
In: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y.,
(1981) pp. 563-681). In general, such procedures involve immunizing
an animal (preferably a mouse) with a S. pneumoniae polypeptide
antigen of the present invention. Suitable cells can be recognized
by their capacity to bind anti-Streptococcus polypeptide antibody.
Such cells may be cultured in any suitable tissue culture medium;
however, it is preferable to culture cells in Earle's modified
Eagle's medium supplemented with 10% fetal bovine serum
(inactivated at about 56.degree. C.), and supplemented with about
10 g/l of nonessential amino acids, about 1,000 U/ml of penicillin,
and about 100 .mu.g/ml of streptomycin. The splenocytes of such
mice are extracted and fused with a suitable myeloma cell line. Any
suitable myeloma cell line may be employed in accordance with the
present invention; however, it is preferable to employ the parent
myeloma cell line (SP.sub.2O), available from the American Type
Culture Collection, Rockville, Md. After fusion, the resulting
hybridoma cells are selectively maintained in HAT medium, and then
cloned by limiting dilution as described by Wands et al.
(Gastroenterology 80:225-232 (1981)). The hybridoma cells obtained
through such a selection are then assayed to identify clones which
secrete antibodies capable of binding the Streptococcus polypeptide
antigen administered to immunized animal.
[0133] Alternatively, additional antibodies capable of binding to
Streptococcus polypeptide antigens may be produced in a two-step
procedure through the use of anti-idiotypic antibodies. Such a
method makes use of the fact that antibodies are themselves
antigens, and that, therefore, it is possible to obtain an antibody
which binds to a second antibody. In accordance with this method,
Streptococcus polypeptide-specific antibodies are used to immunize
an animal, preferably a mouse. The splenocytes of such an animal
are then used to produce hybridoma cells, and the hybridoma cells
are screened to identify clones which produce an antibody whose
ability to bind to the Streptococcus polypeptide-specific antibody
can be blocked by a Streptococcus polypeptide antigen. Such
antibodies comprise anti-idiotypic antibodies to the Streptococcus
polypeptide-specific antibody and can be used to immunize an animal
to induce formation of further Streptococcus polypeptide-specific
antibodies.
[0134] It will be appreciated that Fab and F(ab').sub.2 and other
fragments of the antibodies of the present invention may be used
according to the methods disclosed herein. Such fragments are
typically produced by proteolytic cleavage, using enzymes such as
papain (to produce Fab fragments) or pepsin (to produce
F(ab').sub.2 fragments). Alternatively, Streptococcus
polypeptide-binding fragments can be produced through the
application of recombinant DNA technology or through synthetic
chemistry.
[0135] Of special interest to the present invention are antibodies
to Streptococcus polypeptide antigens which are produced in humans,
or are "humanized" (i.e., non-immunogenic in a human) by
recombinant or other technology. Humanized antibodies may be
produced, for example by replacing an immunogenic portion of an
antibody with a corresponding, but non-immunogenic portion (i.e.,
chimeric antibodies) (Robinson, R. R. et al., International Patent
Publication PCT/US86/02269; Akira, K. et al., European Patent
Application 184,187; Taniguchi, M., European Patent Application
171,496; Morrison, S. L. et al., European Patent Application
173,494; Neuberger, M. S. et al., PCT Application WO 86/01533;
Cabilly, S. et al., European Patent Application 125,023; Better, M.
et al., Science 240:1041-1043 (1988); Liu, A. Y. et al., Proc.
Natl. Acad. Sci. USA 84:3439-3443 (1987); Liu, A. Y. et al., J.
Immunol. 139:3521-3526 (1987); Sun, L. K. et al., Proc. Natl. Acad.
Sci. USA 84:214-218 (1987); Nishimura, Y. et al., Canc. Res.
47:999-1005 (1987); Wood, C. R. et al., Nature 314:446-449 (1985));
Shaw et al., J. Natl. Cancer Inst. 80:1553-1559 (1988). General
reviews of "humanized" chimeric antibodies are provided by
Morrison, S. L. (Science, 229:1202-1207 (1985)) and by Oi, V. T. et
al., BioTechniques 4:214 (1986)). Suitable "humanized" antibodies
can be alternatively produced by CDR or CEA substitution (Jones, P.
T. et al., Nature 321:552-525 (1986); Verhoeyan et al., Science
239:1534 (1988); Beidler, C. B. et al., J. Immunol. 141:4053-4060
(1988)).
[0136] Suitable enzyme labels include, for example, those from the
oxidase group, which catalyze the production of hydrogen peroxide
by reacting with substrate. Glucose oxidase is particularly
preferred as it has good stability and its substrate (glucose) is
readily available. Activity of an oxidase label may be assayed by
measuring the concentration of hydrogen peroxide formed by the
enzyme-labeled antibody/substrate reaction. Besides enzymes, other
suitable labels include radioisotopes, such as iodine (.sup.125I,
.sup.121I), carbon (.sup.14C), sulphur (35S), tritium (.sup.3H),
indium (.sup.112In), and technetium (.sup.99mTc), and fluorescent
labels, such as fluorescein and rhodamine, and biotin.
[0137] Further suitable labels for the Streptococcus
polypeptide-specific antibodies of the present invention are
provided below. Examples of suitable enzyme labels include malate
dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase,
yeast-alcohol dehydrogenase, alpha-glycerol phosphate
dehydrogenase, triose phosphate isomerase, peroxidase, alkaline
phosphatase, asparaginase, glucose oxidase, beta-galactosidase,
ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase,
glucoamylase, and acetylcholine esterase.
[0138] Examples of suitable radioisotopic labels include .sup.3H,
.sup.111In, .sup.125I, .sup.131I, .sup.32P, .sup.35S, .sup.14C,
.sup.51Cr, .sup.57To, .sup.58Co, .sup.59Fe, .sup.75Se, .sup.152Eu,
.sup.90Y, .sup.67Cu, .sup.217Ci, .sup.211At, .sup.212Pb, .sup.47Sc,
.sup.109Pd, etc. .sup.111In is a preferred isotope where in vivo
imaging is used since its avoids the problem of dehalogenation of
the .sup.125I, or .sup.131I-labeled monoclonal antibody by the
liver. In addition, this radionucleotide has a more favorable gamma
emission energy for imaging (Perkins et al., Eur. J. Nucl. Med.
10:296-301 (1985); Carasquillo et al., J. Nucl. Med. 28:281-287
(1987)). For example, .sup.111In coupled to monoclonal antibodies
with 1-(P-isothiocyanatobenzyl)-DPTA has shown little uptake in
non-tumorous tissues, particularly the liver, and therefore
enhances specificity of tumor localization (Esteban et al., J.
Nucl. Med. 28:861-870 (1987)).
[0139] Examples of suitable non-radioactive isotopic labels include
.sup.157Gd, .sup.55Mn, .sup.162Dy, .sup.52Tr, and .sup.56Fe.
[0140] Examples of suitable fluorescent labels include an
.sup.152Eu label, a fluorescein label, an isothiocyanate label, a
rhodamine label, a phycoerythrin label, a phycocyanin label, an
allophycocyanin label, an o-phthaldehyde label, and a fluorescamine
label.
[0141] Examples of suitable toxin labels include diphtheria toxin,
ricin, and cholera toxin.
[0142] Examples of chemiluminescent labels include a luminal label,
an isoluminal label, an aromatic acridinium ester label, an
imidazole label, an acridinium salt label, an oxalate ester label,
a luciferin label, a luciferase label, and an aequorin label.
[0143] Examples of nuclear magnetic resonance contrasting agents
include heavy metal nuclei such as Gd, Mn, and iron.
[0144] Typical techniques for binding the above-described labels to
antibodies are provided by Kennedy et al., Clin. Chim. Acta 70:1-31
(1976), and Schurs et al., Clin. Chim. Acta 81:1-40 (1977).
Coupling techniques mentioned in the latter are the glutaraldehyde
method, the periodate method, the dimaleimide method, the
m-maleimidobenzyl-N-hydroxy- -succinimide ester method, all of
which methods are incorporated by reference herein.
[0145] In a related aspect, the invention includes a diagnostic kit
for use in screening serum containing antibodies specific against
S. pneumoniae infection. Such a kit may include an isolated S.
pneumoniae antigen comprising an epitope which is specifically
immunoreactive with at least one anti-S. pneumoniae antibody. Such
a kit also includes means for detecting the binding of said
antibody to the antigen. In specific embodiments, the kit may
include a recombinantly produced or chemically synthesized peptide
or polypeptide antigen. The peptide or polypeptide antigen may be
attached to a solid support.
[0146] In a more specific embodiment, the detecting means of the
above-described kit includes a solid support to which said peptide
or polypeptide antigen is attached. Such a kit may also include a
non-attached reporter-labeled anti-human antibody. In this
embodiment, binding of the antibody to the S. pneumoniae antigen
can be detected by binding of the reporter labeled antibody to the
anti-S. pneumoniae antibody.
[0147] In a related aspect, the invention includes a method of
detecting S. pneumoniae infection in a subject. This detection
method includes reacting a body fluid, preferably serum, from the
subject with an isolated S. pneumoniae antigen, and examining the
antigen for the presence of bound antibody. In a specific
embodiment, the method includes a polypeptide antigen attached to a
solid support, and serum is reacted with the support. Subsequently,
the support is reacted with a reporter-labeled anti-human antibody.
The support is then examined for the presence of reporter-labeled
antibody.
[0148] The solid surface reagent employed in the above assays and
kits is prepared by known techniques for attaching protein material
to solid support material, such as polymeric beads, dip sticks,
96-well plates or filter material. These attachment methods
generally include non-specific adsorption of the protein to the
support or covalent attachment of the protein, typically through a
free amine group, to a chemically reactive group on the solid
support, such as an activated carboxyl, hydroxyl, or aldehyde
group. Alternatively, streptavidin coated plates can be used in
conjunction with biotinylated antigen(s).
[0149] Therapeutics and Modes of Administration
[0150] The present invention also provides vaccines comprising one
or more polypeptides of the present invention. Heterogeneity in the
composition of a vaccine may be provided by combining S. pneumoniae
polypeptides of the present invention. Multi-component vaccines of
this type are desirable because they are likely to be more
effective in eliciting protective immune responses against multiple
species and strains of the Streptococcus genus than single
polypeptide vaccines. Thus, as discussed in detail below, a
multi-component vaccine of the present invention may contain one or
more, preferably 2 to about 20, more preferably 2 to about 15, and
most preferably 3 to about 8, of the S. pneumoniae polypeptides
identified in Table 1, or fragments thereof.
[0151] Multi-component vaccines are known in the art to elicit
antibody production to numerous immunogenic components. Decker, M.
and Edwards, K., J. Infect. Dis. 174:S270-275 (1996). In addition,
a hepatitis B, diphtheria, tetanus, pertussis tetravalent vaccine
has recently been demonstrated to elicit protective levels of
antibodies in human infants against all four pathogenic agents.
Aristegui, J. et al., Vaccine 15:7-9 (1997).
[0152] The present invention thus also includes multi-component
vaccines. These vaccines comprise more than one polypeptide,
immunogen or antigen. An example of such a multi-component vaccine
would be a vaccine comprising more than one of the S. pneumoniae
polypeptides described in Table 1. A second example is a vaccine
comprising one or more, for example 2 to 10, of the S. pneumoniae
polypeptides identified in Table 1 and one or more, for example 2
to 10, additional polypeptides of either streptococcal or
non-streptococcal origin. Thus, a multi-component vaccine which
confers protective immunity to both a Streptococcal infection and
infection by another pathogenic agent is also within the scope of
the invention.
[0153] As indicated above, the vaccines of the present invention
are expected to elicit a protective immune response against
infections caused by species and strains of Streptococcus other
than strain of S. pneumoniae deposited with that ATCC.TM..
[0154] Further within the scope of the invention are whole cell and
whole viral vaccines. Such vaccines may be produced recombinantly
and involve the expression of one or more of the S. pneumoniae
polypeptides described in Table 1. For example, the S. pneumoniae
polypeptides of the present invention may be either secreted or
localized intracellular, on the cell surface, or in the periplasmic
space. Further, when a recombinant virus is used, the S. pneumoniae
polypeptides of the present invention may, for example, be
localized in the viral envelope, on the surface of the capsid, or
internally within the capsid. Whole cells vaccines which employ
cells expressing heterologous proteins are known in the art. See,
e.g., Robinson, K. et al., Nature Biotech. 15:653-657 (1997);
Sirard, J. et al., Infect. Immun. 65:2029-2033 (1997); Chabalgoity,
J. et al., Infect. Immun. 65:2402-2412 (1997). These cells may be
administered live or may be killed prior to administration.
Chabalgoity, J. et al., supra, for example, report the successful
use in mice of a live attenuated Salmonella vaccine strain which
expresses a portion of a platyhelminth fatty acid-binding protein
as a fusion protein on its cells surface.
[0155] A multi-component vaccine can also be prepared using
techniques known in the art by combining one or more S. pneumoniae
polypeptides of the present invention, or fragments thereof, with
additional non-streptococcal components (e.g., diphtheria toxin or
tetanus toxin, and/or other compounds known to elicit an immune
response). Such vaccines are useful for eliciting protective immune
responses to both members of the Streptococcus genus and
non-streptococcal pathogenic agents.
[0156] The vaccines of the present invention also include DNA
vaccines. DNA vaccines are currently being developed for a number
of infectious diseases. Boyer, J et al., Nat. Med. 3:526-532
(1997); reviewed in Spier, R., Vaccine 14:1285-1288 (1996). Such
DNA vaccines contain a nucleotide sequence encoding one or more S.
pneumoniae polypeptides of the present invention oriented in a
manner that allows for expression of the subject polypeptide. The
direct administration of plasmid DNA encoding B. burgdorferi OspA
has been shown to elicit protective immunity in mice against
borrelial challenge. Luke, C. et al., J. Infect. Dis. 175:91-97
(1997).
[0157] The present invention also relates to the administration of
a vaccine which is co-administered with a molecule capable of
modulating immune responses. Kim, J. et al., Nature Biotech.
15:641-646 (1997), for example, report the enhancement of immune
responses produced by DNA immunizations when DNA sequences encoding
molecules which stimulate the immune response are co-administered.
In a similar fashion, the vaccines of the present invention may be
co-administered with either nucleic acids encoding immune
modulators or the immune modulators themselves. These immune
modulators include granulocyte macrophage colony stimulating factor
(GM-CSF) and CD86.
[0158] The vaccines of the present invention may be used to confer
resistance to streptococcal infection by either passive or active
immunization. When the vaccines of the present invention are used
to confer resistance to streptococcal infection through active
immunization, a vaccine of the present invention is administered to
an animal to elicit a protective immune response which either
prevents or attenuates a streptococcal infection. When the vaccines
of the present invention are used to confer resistance to
streptococcal infection through passive immunization, the vaccine
is provided to a host animal (e.g., human, dog, or mouse), and the
antisera elicited by this antisera is recovered and directly
provided to a recipient suspected of having an infection caused by
a member of the Streptococcus genus.
[0159] The ability to label antibodies, or fragments of antibodies,
with toxin molecules provides an additional method for treating
streptococcal infections when passive immunization is conducted. In
this embodiment, antibodies, or fragments of antibodies, capable of
recognizing the S. pneumoniae polypeptides disclosed herein, or
fragments thereof, as well as other Streptococcus proteins, are
labeled with toxin molecules prior to their administration to the
patient. When such toxin derivatized antibodies bind to
Streptococcus cells, toxin moieties will be localized to these
cells and will cause their death.
[0160] The present invention thus concerns and provides a means for
preventing or attenuating a streptococcal infection resulting from
organisms which have antigens that are recognized and bound by
antisera produced in response to the polypeptides of the present
invention. As used herein, a vaccine is said to prevent or
attenuate a disease if its administration to an animal results
either in the total or partial attenuation (i.e., suppression) of a
symptom or condition of the disease, or in the total or partial
immunity of the animal to the disease.
[0161] The administration of the vaccine (or the antisera which it
elicits) may be for either a "prophylactic" or "therapeutic"
purpose. When provided prophylactically, the compound(s) are
provided in advance of any symptoms of streptococcal infection. The
prophylactic administration of the compound(s) serves to prevent or
attenuate any subsequent infection. When provided therapeutically,
the compound(s) is provided upon or after the detection of symptoms
which indicate that an animal may be infected with a member of the
Streptococcus genus. The therapeutic administration of the
compound(s) serves to attenuate any actual infection. Thus, the S.
pneumoniae polypeptides, and fragments thereof, of the present
invention may be provided either prior to the onset of infection
(so as to prevent or attenuate an anticipated infection) or after
the initiation of an actual infection.
[0162] The polypeptides of the invention, whether encoding a
portion of a native protein or a functional derivative thereof, may
be administered in pure form or may be coupled to a macromolecular
carrier. Example of such carriers are proteins and carbohydrates.
Suitable proteins which may act as macromolecular carrier for
enhancing the immunogenicity of the polypeptides of the present
invention include keyhole limpet hemacyanin (KLH) tetanus toxoid,
pertussis toxin, bovine serum albumin, and ovalbumin. Methods for
coupling the polypeptides of the present invention to such
macromolecular carriers are disclosed in Harlow et al., Antibodies:
A Laboratory Manual, 2nd Ed.; Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y. (1988), the entire disclosure of which is
incorporated by reference herein.
[0163] A composition is said to be "pharmacologically acceptable"
if its administration can be tolerated by a recipient animal and is
otherwise suitable for administration to that animal. Such an agent
is said to be administered in a "therapeutically effective amount"
if the amount administered is physiologically significant. An agent
is physiologically significant if its presence results in a
detectable change in the physiology of a recipient patient.
[0164] While in all instances the vaccine of the present invention
is administered as a pharmacologically acceptable compound, one
skilled in the art would recognize that the composition of a
pharmacologically acceptable compound varies with the animal to
which it is administered. For example, a vaccine intended for human
use will generally not be co-administered with Freund's adjuvant.
Further, the level of purity of the S. pneumoniae polypeptides of
the present invention will normally be higher when administered to
a human than when administered to a non-human animal.
[0165] As would be understood by one of ordinary skill in the art,
when the vaccine of the present invention is provided to an animal,
it may be in a composition which may contain salts, buffers,
adjuvants, or other substances which are desirable for improving
the efficacy of the composition. Adjuvants are substances that can
be used to specifically augment a specific immune response. These
substances generally perform two functions: (1) they protect the
antigen(s) from being rapidly catabolized after administration and
(2) they nonspecifically stimulate immune responses.
[0166] Normally, the adjuvant and the composition are mixed prior
to presentation to the immune system, or presented separately, but
into the same site of the animal being immunized. Adjuvants can be
loosely divided into several groups based upon their composition.
These groups include oil adjuvants (for example, Freund's complete
and incomplete), mineral salts (for example, AlK(SO.sub.4).sub.2,
AlNa(SO.sub.4).sub.2, AlNH.sub.4(SO.sub.4), silica, kaolin, and
carbon), polynucleotides (for example, poly IC and poly AU acids),
and certain natural substances (for example, wax D from
Mycobacterium tuberculosis, as well as substances found in
Corynebacterium parvum, or Bordetella pertussis, and members of the
genus Brucella. Other substances useful as adjuvants are the
saponins such as, for example, Quil A. (Superfos A/S, Denmark).
Preferred adjuvants for use in the present invention include
aluminum salts, such as AlK(SO.sub.4).sub.2, AlNa(SO.sub.4).sub.2,
and AlNH.sub.4(SO.sub.4). Examples of materials suitable for use in
vaccine compositions are provided in Remington's Pharmaceutical
Sciences (Osol, A, Ed, Mack Publishing Co, Easton, Pa., pp.
1324-1341 (1980), which reference is incorporated herein by
reference).
[0167] The therapeutic compositions of the present invention can be
administered parenterally by injection, rapid infusion,
nasopharyngeal absorption (intranasopharangeally), dermoabsorption,
or orally. The compositions may alternatively be administered
intramuscularly, or intravenously. Compositions for parenteral
administration include sterile aqueous or non-aqueous solutions,
suspensions, and emulsions. Examples of non-aqueous solvents are
propylene glycol, polyethylene glycol, vegetable oils such as olive
oil, and injectable organic esters such as ethyl oleate. Carriers
or occlusive dressings can be used to increase skin permeability
and enhance antigen absorption. Liquid dosage forms for oral
administration may generally comprise a liposome solution
containing the liquid dosage form. Suitable forms for suspending
liposomes include emulsions, suspensions, solutions, syrups, and
elixirs containing inert diluents commonly used in the art, such as
purified water. Besides the inert diluents, such compositions can
also include adjuvants, wetting agents, emulsifying and suspending
agents, or sweetening, flavoring, or perfuming agents.
[0168] Therapeutic compositions of the present invention can also
be administered in encapsulated form. For example, intranasal
immunization of mice against Bordetella pertussis infection using
vaccines encapsulated in biodegradable microsphere composed of
poly(DL-lactide-co-glycolide) has been shown to stimulate
protective immune responses. Shahin, R. et al., Infect. Immun.
63:1195-1200 (1995). Similarly, orally administered encapsulated
Salmonella typhimurium antigens have also been shown to elicit
protective immunity in mice. Allaoui-Attarki, K. et al., Infect.
Immun. 65:853-857 (1997). Encapsulated vaccines of the present
invention can be administered by a variety of routes including
those involving contacting the vaccine with mucous membranes (e.g.,
intranasally, intracolonicly, intraduodenally).
[0169] Many different techniques exist for the timing of the
immunizations when a multiple administration regimen is utilized.
It is possible to use the compositions of the invention more than
once to increase the levels and diversities of expression of the
immunoglobulin repertoire expressed by the immunized animal.
Typically, if multiple immunizations are given, they will be given
one to two months apart.
[0170] According to the present invention, an "effective amount" of
a therapeutic composition is one which is sufficient to achieve a
desired biological effect. Generally, the dosage needed to provide
an effective amount of the composition will vary depending upon
such factors as the animal's or human's age, condition, sex, and
extent of disease, if any, and other variables which can be
adjusted by one of ordinary skill in the art.
[0171] The antigenic preparations of the invention can be
administered by either single or multiple dosages of an effective
amount. Effective amounts of the compositions of the invention can
vary from 0.01-1,000 .mu.g/ml per dose, more preferably 0.1-500
.mu.g/ml per dose, and most preferably 10-300 .mu.g/ml per
dose.
[0172] Having now generally described the invention, the same will
be more readily understood through reference to the following
example which is provided by way of illustration, and is not
intended to be limiting of the present invention, unless
specified.
EXAMPLES
Example 1
[0173] Expression and Purification of S. pneumoniae Polypeptides in
E. coli
[0174] The bacterial expression vector pQE10 (QIAGEN, Inc., 9259
Eton Avenue, Chatsworth, Calif., 91311) is used in this example for
cloning of the nucleotide sequences shown in Table 1 and for
expressing the polypeptides identified in Table 1. The components
of the pQE10 plasmid are arranged such that the inserted DNA
sequence encoding a polypeptide of the present invention expresses
the polypeptide with the six His residues (i.e., a "6.times.His
tag")) covalently linked to the amino terminus.
[0175] The DNA sequences encoding the desired portions of the
polypeptides of Table 1 are amplified using PCR oligonucleotide
primers from either a DNA library constructed from S. pneumoniae,
such as the one deposited by the inventors at the ATCC.TM. for
convenience, ATCC.TM. Deposit No. 97755, or from DNA isolated from
the same organism such as the S. pneumoniae strain deposited with
the ATCC.TM. as Deposit No. 55840. A list of PCR primers which can
be used for this purpose is provided in Table 3, below. The PCR
primers anneal to the nucleotide sequences encoding both the amino
terminal and carboxy terminal amino acid sequences of the desired
portion of the polypeptides of Table 1. Additional nucleotides
containing restriction sites to facilitate cloning in the pQE10
vector were added to the 5' and 3' primer sequences, respectively.
Such restriction sites are listed in Table 3 for each primer. In
each case, the primer comprises, from the 5' end, 4 random
nucleotides to prevent "breathing" during the annealing process, a
restriction site (shown in Table 3), and approximately 15
nucleotides of S. pneumoniae ORF sequence (the complete sequence of
each cloning primer is shown as SEQ ID NO:227 through SEQ ID
NO:452).
[0176] For cloning the polypeptides of Table 1, the 5' and 3'
primers were selected to amplify their respective nucleotide coding
sequences. One of ordinary skill in the art would appreciate that
the point in the protein coding sequence where the 5' primer begins
may be varied to amplify a DNA segment encoding any desired portion
of the complete amino acid sequences described in Table 1.
Similarly, one of ordinary skill in the art would further
appreciate that the point in the protein coding sequence where the
3' primer begins may also be varied to amplify a DNA segment
encoding any desired portion of the complete amino acid sequences
described in Table 1.
[0177] The amplified DNA fragment and the pQE10 vector are digested
with the appropriate restriction enzyme(s) and the digested DNAs
are then ligated together. The ligation mixture is transformed into
competent E. coli cells using standard procedures such as those
described in Sambrook et al., Molecular Cloning: a Laboratory
Manual, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. (1989). Transformants are identified by their ability
to grow under selective pressure on LB plates. Plasmid DNA is
isolated from resistant colonies and the identity of the cloned DNA
confirmed by restriction analysis, PCR and DNA sequencing.
[0178] Clones containing the desired constructs are grown overnight
("O/N") in liquid culture under selection. The O/N culture is used
to inoculate a large culture, at a dilution of approximately 1:25
to 1:250. The cells are grown to an optical density at 600 nm
("OD600") of between 0.4 and 0.6.
Isopropyl-b-D-thiogalactopyranoside ("IPTG") is then added to a
final concentration of 1 mM to induce transcription from the lac
repressor sensitive promoter, by inactivating the lacI repressor.
Cells subsequently are incubated further for 3 to 4 hours. Cells
are then harvested by centrifugation.
[0179] The cells are stirred for 3-4 hours at 4 C in 6M
guanidine-HCl, pH 8. The cell debris is removed by centrifugation,
and the supernatant containing the protein of interest is loaded
onto a nickel-nitrilo-tri-acetic acid ("NiNTA") affinity resin
column (available from QIAGEN, Inc., supra). Proteins with a
6.times.His tag bind to the NI-NTA resin with high affinity and can
be purified in a simple one-step procedure (for details see: The
QIAexpressionist, 1995, QIAGEN, Inc., supra). Briefly, the
supernatant is loaded onto the column in 6 M guanidine-HCl, pH8,
the column is first washed with 10 volumes of 6 M guanidine-HCl,
pH8, then washed with 10 volumes of 6 M guanidine-HCl pH6, and
finally the polypeptide is eluted with 6 M guanidine-HCl, pH
5.0.
[0180] The purified protein is then renatured by dialyzing it
against phosphatebuffered saline (PBS) or 50 mM Na-acetate, pH 6
buffer plus 200 mM NaCl. Alternatively, the protein can be
successfully refolded while immobilized on the Ni-NTA column. The
recommended conditions are as follows: renature using a linear
6M-1M urea gradient in 500 mM NaCl, 20% glycerol, 20 mM Tris/HCl
pH7.4, containing protease inhibitors. The renaturation should be
performed over a period of 1.5 hours or more. After renaturation
the proteins can be eluted by the addition of 250 mM imidazole.
Imidazole is removed by a final dialyzing step against PBS or 50 mM
sodium acetate pH6 buffer plus 200 mM NaCl. The purified protein is
stored at 4.degree. C. or frozen at -80.degree. C.
[0181] The DNA sequences encoding the amino acid sequences of Table
1 may also be cloned and expressed as fusion proteins by a protocol
similar to that described directly above, wherein the pET-32b(+)
vector (Novagen, 601 Science Drive, Madison, Wis. 53711) is
preferentially used in place of pQE10.
[0182] Each of the polynucleotides shown in Table 1, was
successfully amplified and subcloned into pQE10 as described above
using the PCR primers shown in Table 3. These pQE10 plasmids
containing the DNAs of Table 1, except SP023, SP042, SP054, SP063,
SP081, SP092, SP114, SP122, SP123, SP126, and SP127, were deposited
with the ATCC.TM. as a pooled deposit as a convenience to those of
skill in the art. This pooled deposit was deposited on Oct. 16,
1997 and given ATCC.TM. Deposit No. 209369. Those of ordinary skill
in the art appreciate that isolating an individual plasmid from the
pooled deposit is trivial provided the information and reagents
described herein. Each of the deposited clones is capable of
expressing its encoded S. pneumoniae polypeptide.
Example 2
[0183] Immunization and Detection of Immune Responses Methods
[0184] Growth of Bacterial Innoculum, Immunization of Mice and
Challenge with S pneumoniae.
[0185] Propagation and storage of, and challenge by S. pneumoniae
are preformed essentially as described in Aaberge, I. S. et al.,
Virulence of Streptococcus pneumoniae in mice: a standardized
method for preparation and frozen storage of the experimental
bacterial inoculum, Microbial Pathogenesis, 18:141 (1995),
incorporated herein by reference.
[0186] Briefly, Todd Hewitt (TH) broth (Difco laboratories,
Detroit, Mich.) with 17% FCS, and horse blood agar plates are used
for culturing the bacteria. Both broth and blood plates are
incubated at 37.degree. C. in a 5% CO.sub.2 atmosphere. Blood
plates are incubated for 18 hr. The culture broth is regularly
10-fold serially diluted in TH broth kept at room temperature and
bacterial suspensions are kept at room temperature until challenge
of mice.
[0187] For active immunizations C3H/HeJ mice (The Jackson
Laboratory, Bar Harbor, Me.) are injected intraperitoneally (i.p.)
at week 0 with 20 g of recombinant streptococcal protein, or
phosphate-buffered saline (PBS), emulsified with complete Freund's
adjuvant (CFA), given a similar booster immunization in incomplete
Freund's adjuvant (IFA) at week 4, and challenged at week 6. For
challenge S. pneumoniae are diluted in TH broth from
exponentially-growing cultures and mice are injected subcutaneously
(s.c.) at the base of the tail with 0.1 ml of these dilutions
(serial dilutions are used to find medium infectious dose).
Streptococci used for challenge are passaged fewer than six times
in vitro. To assess infection, blood samples are obtained from the
distal part of the lateral femoral vein into heparinized capillary
tubes. A 25 ul blood sample is serially 10-fold diluted in TH
broth, and 25 ul of diluted and undiluted blood is plated onto
blood agar plates. The plates are incubated for 18 hr. and colonies
are counted.
[0188] Other methods are known in the art, for example, see
Langermann, S. et al., J. Exp. Med., 180:2277 (1994), incorporated
herein by reference.
[0189] Immunoassays
[0190] Several immunoassay formats are used to quantify levels of
streptococcal-specific antibodies (ELISA and immunoblot), and to
evaluate the functional properties of these antibodies (growth
inhibition assay). The ELISA and immunoblot assays are also used to
detect and quantify antibodies elicited in response to
streptococcal infection that react with specific streptococcal
antigens. Where antibodies to certain streptococcal antigens are
elicited by infection this is taken as evidence that the
streptococcal proteins in question are expressed in vivo. Absence
of infection-derived antibodies (seroconversion) following
streptococcal challenge is evidence that infection is prevented or
suppressed. The immunoblot assay is also used to ascertain whether
antibodies raised against recombinant streptococcal antigens
recognize a protein of similar size in extracts of whole
streptococci. Where the natural protein is of similar, or
identical, size in the immunoblot assay to the recombinant version
of the same protein, this is taken as evidence that the recombinant
protein is the product of a full-length clone of the respective
gene.
[0191] Enzyme-Linked Immunosorbant Assay (ELISA).
[0192] The ELISA is used to quantify levels of antibodies reactive
with streptococcus antigens elicited in response to immunization
with these streptococcal antigens. Wells of 96 well microtiter
plates (Immunlon 4, Dynatech, Chantilly, Va., or equivalent) are
coated with antigen by incubating 50 l of 1 g/ml protein antigen
solution in a suitable buffer, typically 0.1 M sodium carbonate
buffer at pH 9.6. After decanting unbound antigen, additional
binding sites are blocked by incubating 100 l of 3% nonfat milk in
wash buffer (PBS, 0.2% Tween 20, pH 7.4). After washing, duplicate
serial two-fold dilutions of sera in PBS, Tween 20, 1% fetal bovine
serum, are incubated for 1 hr, removed, wells are washed three
times, and incubated with horseradish peroxidase-conjugated goat
anti-mouse IgG. After three washes, bound antibodies are detected
with H.sub.2O.sub.2 and 2,2'-azino-di-(3-ethylbenzthiazoline
sulfonate) (Schwan, T. G., et al., Proc. Natl. Acad. Sci. USA
92:2909-2913 (1985)) (ABTS.RTM., Kirkegaard & Perry Labs.,
Gaithersburg, Md.) and A.sub.405 is quantified with a Molecular
Devices, Corp. (Menlo Park, Calif.) Vmax.TM. plate reader. IgG
levels twice the background level in serum from naive mice are
assigned the minimum titer of 1:100.
[0193] Sodiumdodecylsulfate-Polyacrylamide Gel Electrophoresis
(SDS-PAGE) and Immunoblotting
[0194] Using a single well format, total streptococcal protein
extracts or recombinant streptococcal antigen are boiled in
SDS/2-ME sample buffer before electrophoresis through 3% acrylamide
stacking gels, and resolving gels of higher acrylamide
concentration, typically 10-15% acrylamide monomer. Gels are
electro-blotted to nitrocellulose membranes and lanes are probed
with dilutions of antibody to be tested for reactivity with
specific streptococcal antigens, followed by the appropriate
secondary antibody-enzyme (horseradish peroxidase) conjugate. When
it is desirable to confirm that the protein had transferred
following electro-blotting, membranes are stained with Ponceau S.
Immunoblot signals from bound antibodies are detected on x-ray film
as chemiluninescence using ECL.TM. reagents (Amersham Corp.,
Arlington Heights, Ill.).
Example 3
[0195] Detection of Streptococcus mRNA Expression
[0196] Northern blot analysis is carried out using methods
described by, among others, Sambrook et al., supra. to detect the
expression of the S. pneumoniae nucleotide sequences of the present
invention in animal tissues. A cDNA probe containing an entire
nucleotide sequence shown in Table 1 is labeled with .sup.32p using
the rediprime.TM. DNA labeling system (Amersham Life Science),
according to manufacturer's instructions. After labeling, the probe
is purified using a CHROMA SPIN-100.TM. column (Clontech
Laboratories, Inc.), according to manufacturer's protocol number
PT1200-1. The purified labeled probe is then used to detect the
expression of Streptococcus mRNA in an animal tissue sample.
[0197] Animal tissues, such as blood or spinal fluid, are examined
with the labeled probe using ExpressHyb.TM. hybridization solution
(Clontech) according to manufacturer's protocol number PT 1190-1.
Following hybridization and washing, the blots are mounted and
exposed to film at -70 C overnight, and films developed according
to standard procedures.
[0198] It will be clear that the invention may be practiced
otherwise than as particularly described in the foregoing
description and examples.
[0199] Numerous modifications and variations of the present
invention are possible in light of the above teachings and,
therefore, are within the scope of the appended claims.
[0200] The entire disclosure of all publications (including
patents, patent applications, journal articles, laboratory manuals,
books, or other documents) cited herein are hereby incorporated by
reference.
1 TABLE 1 Description SEQ ID NO: SP001 nucleotide 1 SP001 AMINO
ACID 2 SP004 nucleotide 3 SP004 amino acid 4 SP006 nucleotide 5
SP006 amino acid 6 SP007 nucleotide 7 SP007 amino acid 8 SP008
nucleotide 9 SP008 amino acid 10 SP009 nucleotide 11 SP009 amino
acid 12 SP010 nucleotide 13 SP010 amino acid 14 SP011 nucleotide 15
SP011 amino acid 16 SP012 nucleotide 17 SP012 nucleotide 18 SP013
nucleotide 19 SP013 amino acid 20 SP014 nucleotide 21 SP014 amino
acid 22 SP015 nucleotide 23 SP015 amino acid 24 SP016 nucleotide 25
SP016 amino acid 26 SP017 nucleotide 27 SP017 amino acid 28 SP019
nucleotide 29 SP019 amino acid 30 SP020 nucleotide 31 SP020 amino
acid 32 SP021 nucleotide 33 SP021 amino acid 34 SP022 nucleotide 35
SP022 amino acid 36 SP023 nucleotide 37 SP023 amino acid 38 SP025
nucleotide 39 SP025 amino acid 40 SP028 nucleotide 41 SP028 amino
acid 42 SP030 nucleotide 43 SP030 amino acid 44 SP031 nucleotide 45
SP031 amino acid 46 SP032 nucleotide 47 SP032 amino acid 48 SP033
nucleotide 49 SP033 amino acid 50 SP034 nucleotide 51 SP034 amino
acid 52 SP035 nucleotide 53 SP035 amino acid 54 SP036 nucleotide 55
SP036 amino acid 56 SP038 nucleotide 57 SP038 amino acid 58 SP039
nucleotide 59 SP039 amino acid 60 SP040 nucleotide 61 SP040 amino
acid 62 SP041 nucleotide 63 SP041 amino acid 64 SP042 nucleotide 65
SP042 amino acid 66 SP043 nucleotide 67 SP043 amino acid 68 SP044
nucleotide 69 SP044 amino acid 70 SP045 nucleotide 71 SP045
nucleotide 72 SP046 nucleotide 73 SP046 amino acid 74 SP048
nucleotide 75 SP048 amino acid 76 SP049 nucleotide 77 SP049 amino
acid 78 SP050 nucleotide 79 SP050 amino acid 80 SP051 nucleotide 81
SP051 amino acid 82 SP052 nucleotide 83 SP052 amino acid 84 SP053
nucleotide 85 SP053 amino acid 86 SP054 nucleotide 87 SP054 amino
acid 88 SP055 nucleotide 89 SP055 amino acid 90 SP056 nucleotide 91
SP056 amino acid 92 SP057 nucleotide 93 SP057 amino acid 94 SP058
nucleotide 95 SP058 amino acid 96 SP059 nucleotide 97 SP059 amino
acid 98 SP060 nucleotide 99 SP060 amino acid 100 SP062 nucleotide
101 SP062 amino acid 102 SP063 nucleotide 103 SP063 amino acid 104
SP064 nucleotide 105 SP064 amino acid 106 SP065 nucleotide 107
SP065 amino acid 108 SP067 nucleotide 109 SP067 amino acid 110
SP068 nucleotide 111 SP068 amino acid 112 SP069 nucleotide 113
SP069 amino acid 114 SP070 nucleotide 115 SP070 amino acid 116
SP071 nucleotide 117 SP071 amino acid 118 SP072 nucleotide 119
SP072 amino acid 120 SP073 nucleotide 121 SP073 amino acid 122
SP074 nucleotide 123 SP074 amino acid 124 SP075 nucleotide 125
SP075 amino acid 126 SP076 nucleotide 127 SP076 amino acid 128
SP077 nucleotide 129 SP077 amino acid 130 SP078 nucleotide 131
SP078 amino acid 132 SP079 nucleotide 133 SP079 amino acid 134
SP080 nucleotide 135 SP080 amino acid 136 SP081 nucleotide 137
SP081 amino acid 138 SP082 nucleotide 139 SP082 amino acid 140
SP083 nucleotide 141 SP083 amino acid 142 SP084 nucleotide 143
SP084 amino acid 144 SP085 nucleotide 145 SP085 amino acid 146
SP086 nucleotide 147 SP086 amino acid 148 SP087 nucleotide 149
SP087 amino acid 150 SP088 nucleotide 151 SP088 amino acid 152
SP089 nucleotide 153 SP089 amino acid 154 SP090 nucleotide 155
SP090 amino acid 156 SP091 nucleotide 157 SP091 amino acid 158
SP092 nucleotide 159 SP092 amino acid 160 SP093 nucleotide 161
SPO93 amino acid 162 SP094 nucleotide 163 SP094 amino acid 164
SP095 nucleotide 165 SP095 amino acid 166 SP096 nucleotide 167
SP096 amino acid 168 SP097 nucleotide 169 SP097 amino acid 170
SP098 nucleotide 171 SP098 amino acid 172 SP099 nucleotide 173
SP099 amino acid 174 SP100 nucleotide 175 SP100 amino acid 176
SP101 nucleotide 177 SP101 amino acid 178 SP102 nucleotide 179
SP102 amino acid 180 SP103 nucleotide 181 SP103 amino acid 182
SP105 nucleotide 183 SP105 amino acid 184 SP106 nucleotide 185
SP106 amino acid 186 SP107 nucleotide 187 SP107 amino acid 188
SP108 nucleotide 189 SP108 amino acid 190 SP109 nucleotide 191
SP109 amino acid 192 SP110 nucleotide 193 SP110 amino acid 194
SP111 nucleotide 195 SP111 amino acid 196 SP0112 nucleotide 197
SP0112 amino acid 198 SP113 nucleotide 199 SP113 amino acid 200
SP114 nucleotide 201 SP114 amino acid 202 SP115 nucleotide 203
SP115 amino acid 204 SP117 nucleotide 205 SP117 amino acid 206
SP118 nucleotide 207 SP118 amino acid 208 SP119 nucleotide 209
SP119 amino acid 210 SP120 nucleotide 211 SP120 amino acid 212
SP121 nucleotide 213 SP121 amino acid 214 SP122 nucleotide 215
SP122 amino acid 216 SP123 nucleotide 217 SP123 amino acid 218
SP124 amino acid 219 SP124 amino acid 220 SP125 nucleotide 221
SP125 amino acid 222 SP126 nucleotide 223 SP126 amino acid 224
SP127 nucleotide 225 SP127 amino acid 226
[0201]
2TABLE 2 S. pneumoniae Antigenic Epitopes Sp001 Lys-1 to Ile-10;
Leu-13 to Lys-32; Arg-41 to Ile- 51; Ser-85 to Glu-97; Ala-159 to
His-168; Val-309 to Thr-318; Val-341 to Asn-352; Asn-415 to Met-
430; Phe-454 to Asn-464; Ser-573 to Gly-591; Asn- 597 to Thr-641;
and Asn-644 to Ala-664. SP004 Thr-9 to Thr-24; Ile-29 to Ala-48;
Thr-49 to Val- 56; Val-286 to Val-312; Pro-316 to Glu-344; Val- 345
to Ile-367; Gln-368 to Val-399; Ser-400 to Glu-431; Asn-436 to
Ala-457; Ile-467 to Ala-498; and Thr-499 to Glu-540. SP006 Glu-1 to
Lys-13; Pro-24 to Gly-36; Val-104 to Thr- 112; Ala-118 to Asn-130;
Trp-137 to Ala-146; Ser- 151 to Ile-159; Ile-181 to Leu-188; and
Pro-194 to Tyr-202. SP007 Gly-1 to Asn-7; Tyr-24 to Gln-34; His-47
to Phe- 55; Ser-60 to Ala-67; Ala-122 to Leu-129; Leu-221 to
Lys-230; Val-236 to Phe-256; and Asp-271 to Gly-283; and Leu-291 to
Asp-297. SP008 Leu-4 to Lys-17; Gln-24 to Leu-32; Asp-60 to Ser-
66; Ser-70 to Asp-76; Ala-276 to Lys-283; Asn-304 to Lys-311; and
Thr-429 to Pro-437. SP009 Thr-4 to Glu-11; Leu-50 to Asp-60;
Ile-102 to Trp- 123; and Ser-138 to Ile-157. SP010 Phe-34 to
Gly-41; Asp-44 to Lys-50; Leu-172 to Val-186; Leu-191 to Val-198;
Ser-202 to Ile-209; and Val-213 to Leu-221. Sp011 Asn-2 to Thr-10;
Asp-87 to Ala-102; Tyr-125 to Glu-132; Thr-181 to Tyr-189; Arg-217
to Thr-232; Asn-257 to Lys-264; Pro-271 to Ser-278; Tyr-317 to
Ala-325; Glu-327 to Pro-337; and Thr-374 to Val- 381. SP012 Gly-1
to Lys-19; Phe-34 to Tyr-41; Leu-109 to Lys- 126; and Leu-231 to
Glu-247. SP013 Ala-1 to Lys-12; Ile-42 to Pro-53; Leu-138 to Lys-
146; Ile-205 to Lys-217; Ser-235 to Ile-251; and Ser-261 to
Tyr-272. SP014 Gly-1 to Val-16; Leu-35 to Leu-44; Asp-73 to Asp-
81; Ile-83 to Asp-92; Glu-145 to Ile-153; Phe-188 to Asn-196;
Ser-208 to Phe-215; Ile-224 to Leu- 231; and Asn-235 to Ala-243.
SP015 Ser-1 to Pro-16; Asn-78 to Glu-88; Ala-100 to Val- 108;
Ala-122 to Thr-129; Thr-131 to Ser-137; Leu- 201 to Ser-220; and
Gly-242 to Val-251. SP016 Gly-1 to Glu-20; Thr-30 to Val-38; Gln-94
to Asn- 105; Lys-173 to Pro-182; Gly-189 to Arg-197; Ser- 207 to
Val-224; Pro-288 to Leu-298; Ala-327 to Ala-342; and Ser-391 to
Ala-402. SP017 Ser-1 to Thr-12; Ala-36 to Tyr-45; Gln-48 to Ile-
54; Lys-59 to Lys-76; Tyr-113 to Leu-138; and Phe- 212 to Asp-219.
SP019 Val-97 to Glu-117; Asp-163 to Leu-169; Thr-182 to Thr-191;
and Lys-241 to Ser-250. SP020 Asn-18 to Lys-25; Thr-47 to Glu-60;
Trp-75 to Val- 84; Gly-102 to Val-110; Pro-122 to Ala-131; and
Glu-250 to Pro-258. SP021 Ser1 to Asp-8; Val-44 to Asp-54; Ala-117
to Val- 125; Thr-165 to Thr-173; and Glu-180 to Pro-189. SP022
Phe-5 to Lys-13; Thr-20 to Ser-36; Glu-59 to Lys- 81; Tyr-85 to
Gly-93; Trp-94 to Trp-101; and Thr- 195 to Trp-208. SP023 Gln-45 to
Glu-59; Asp-69 to Pro-85; Lys-111 to Asn-121; Pro-218 to Ala-228;
and Glu-250 to Asn- 281. SP025 Gln-14 to Thr-20; Gly-27 to Phe-33;
Gly-63 to Glu- 71; and Ile-93 to Phe-102. SP028 Asp-171 to Pro-179;
Tyr-340 to Glu-350; Pro-455 to Tyr-463; and Asp-474 to Pro-480.
SP030 Leu-22 to Leu-37; Trp-81 to Ala-90; Phe-101 to Ala-106;
Thr-124 to Tyr-130; and Asn-138 to Glu- 144. SP031 Asp-8 to Val-16;
Gly-27 to Thr-35; Gly-178 to Asp- 195; Thr-200 to Asp209; Trp-218
to Leu-224; and Lys-226 to Asp-241. SP032 Ser-9 to Asp-28; Phe-31
to Val-40; Gly-42 to Arg- 50; Ile-52 to Leu-60; Asp-174 to Phe-186;
Leu-324 to Met-333; and Thr-340 to Asn-347. SP033 Gln-2 to Ile-13;
Phe-46 to Ile-53; and Asp-104 to Thr-121. SP034 Glu-36 to Gly-43;
Ala-188 to Asp-196; Trp-313 to Gly-320; and Leu-323 to Leu-329.
SP035 Arg-19 to Asp-36; Asp-47 to Val-57; Asn-134 to Thr-143;
Asp-187 to Arg-196; and Glu-222 to Ser- 230. SP036 Arg-10 to
Arg-17; Lys-29 to Ser-39; Ser-140 to Ala-153; Arg-158 to Tyr-169;
Asp-175 to Ala-183; Gly-216 to Asn-236; Ala-261 to Leu-270; Arg-282
to Phe-291; and Thr-297 to Ala-305; Pro-342 to Gln- 362; Phe-455 to
Asp-463; His-497 to Thr-511; Ala- 521 to Gly-529; Ile-537 to
Val-546; Ile-556 to Ala-568; Pro-581 to Ser-595; Glu-670 to
Ala-685; Ser-696 to Ala-705 and Leu-782 to Ser-791. SP038 Glu-61 to
Pro-69; Phe-107 to Ala-115; Leu-130 to Tyr-141; Ala-229 to Glu-237;
Ser-282 to Asn-287; Ala-330 to Glu-338; and Tyr-387 to Glu-393.
SP039 Ser-28 to Asp-35; Pro-88 to Pro-96; Leu-125 to Arg-135;
Phe-149 to Leu-157; Gln-246 to Val-254; Ala-357 to Thr-362; Gly-402
to Lys-411; and Leu- 440 to Pro-448. SP040 Thr-21 to Ile-30; His-54
to Gln-68; Arg-103 to Leu-117; and Thr-127 to Leu-136. SP041 Gly-36
to Asp-49; Leu-121 to Val-128; and Ala-186 to Ile-196. SP042 Gly-11
to Arg-19; Ile-23 to Lys-31; His-145 to Asn-151; Gln-159 to
Asp-166; Ile-175 to Asp-181; Gly-213 to Tyr-225; Ile-283 to
Val-291; Pro-329 to Glu-364; Arg-372 to Ser-386; Thr-421 to
Phe-430; Leu-445 to Val-453; Ile-486 to Ala-497; Asp-524 to
Ala-535; His-662 to Gly-674; and His-679 to Gln- 702. SP043 Lys-2
to Asp-12; Val-58 to Asn-68; Ser-87 to Asp- 95; and Asp-102 to
Lys-117. SP044 Gln-3 to Lys-11; Asp-37 to Tyr-52; Glu-171 to Leu-
191; His-234 to Asn-247; and Asn-283 to Ala-291. SP045 Tyr-52 to
Ile-63; Asp-212 to Gln-227; Ser-315 to Thr-332; Leu-345 to Phe-354;
Asp-362 to Val-370; Thr-518 to Asn-539; Ala-545 to Lys-559; and
Val- 601 to Pro-610. SP046 Gln-9 to Ala-18; Glu-179 to Lys-186;
Lys-264 to Glu-271; Gly-304 to Glu-17; Ser-503 to Asn-511; Asn-546
to Thr-553; and Asn-584 to Asp-591. SP048 Tyr-4 to Asp-25; Lys-33
to Val-70; Asp-151 to Thr- 170; Asp-222 to Val-257; Thr-290 to
Phe-301; and Gly-357 to Val-367. SP049 Ala-23 to Arg-37; Tyr-85 to
Gln-95; Glu-106 to Ile-118; Arg-131 to ILE-144; Gly-150 to Ser-162;
and Ala-209 to Asp-218. SP050 Asp-95 to Glu-113; Gly-220 to
Gly-228; Asn-284 to Glu-295; Thr-298 to Val-315. SP051 Lys-16 to
Glu-50; Lys-57 to Asn-104; Ser-158 to Trp-173; Asp-265 to Pro-279;
Val-368 to Tyr-386; Glu-420 to Ile-454; Pro-476 to Ile-516; Phe-561
to Gly-581; Thr-606 to Gly-664; and Glu-676 to Val-696. SP052
Asn-41 to Tyr-60; Phe-80 to Glu-103; Ala-117 to Val-139; Ile-142 to
Leu-155; Val-190 to Lys-212; Glu-276 to Phe-283; Arg-290 to
Ser-299; Leu-328 to Val-351; Gly-358 to Thr-388; Glu-472 to
Ala-483; Val-533 to Asn-561; Asp-595 to Val-606; Glu-609 to
Val-620; Glu-672 to Ser 691. SP053 Ala-62 to Val-101; Thr-147 to
Leu-174; Lys-204 to Val-216; Gln-228 to Val-262; Ser-277 to
Gly-297; Thr-341 to Glyn-368; Thr-385 to Ala-409; Thr-414 to
Ser-453; Asn-461 to Leu-490; Glu-576 to Thr- 625; Gly-630 to
Arg-639; and Asp-720 to Leu-740. SP054 Glu-7 to Val-28; and Tyr-33
to Glu-44. SP055 Pro-3 to Val-18; Thr-21 to Lys-53; Val-84 to Lys-
99; Ile-162 to Val-172; and Val-204 to Ser 241. SP056 Val-34 to
Tyr-41; Leu-47 to Glu-55; and Pro-57 to Gln-66. SP057 Asp-1 to
Val-25; Pro-29 to Ile-80; Asn-96 to Val- 145; and Pro-150 to
Glu-172. SP058 Ala-64 to Thr-70; Leu-82 to His-138; and Val-228 to
Asn-236. SP059 Val-10 to Thr-24; Ser-76 to Pro-102; Ser-109 to
Ile-119; Ser-124 to Val-130; Thr-186 to Ile-194; and Asn-234 to
Ser-243. SP060 Leu-70 to Arg-76; and Val-79 to Ile-88. SP062 Glu-14
to Lys-28; Ser-32 to Lys-46; and Glu-66 to Thr-74. SP063 Ile-10 to
Val-25; Val-30 to Thr-40; Asp-44 to Pro- 54; Asn-57 to Val-63;
Pro-71 to Val-100; and Thr- 105 to Thr-116. SP064 Pro-12 to Leu-32;
Val-40 to Leu-68; Asp-95 to Ala- 125; Ser-164 to Glu-184; Ser-314
to Glu-346; Asn- 382 to Val-393; Leu-463 to Gln-498; Asn-534 to
Lys-548; and Lys-557 to Gly-605. SP065 Asn-2 to Ile-12; Ala-39 to
Thr-61; and His-135 to Ala-155. SP067 Gly-1 to Thr-13; Asp-203 to
Asn-218; and Gly-240 to Asp-253. SP068 Ser-2 to Ser-12; Val-17 to
Gln-26; and Lys-54 to Cys-67. SP069 Ser-32 to Thr-41; Pro-66 to
Glu-80; Thr-110 to Val-122; and Val-147 to Thr-180. SP070 Lys-6 to
Tyr-16; Gln-19 to Ile-27; Arg-50 to Ala- 58; Leu-112 to Val-128;
Ile-151 to Asn-167; Leu- 305 to Phe-321. SP071 Gln-92 to Asn-158;
Gln-171 to Gln-188; Val-204 to Val-240; Thr-247 to Ala-273; Glu-279
to Thr-338; Pro-345 to Glu-368; Asn-483 to Lys-539; Val-552 to
Ala-568; Glu-575 to Ser-591; Ser-621 to Gly-640; Gln-742 to
Gly-758. SP072 Val-68 to Tyr-81; Tyr-86 to Val-121; Leu-127 to
Gly-140; Gly-144 to Ala-155; Gln-168 to Val-185; Asp-210 to
Try-241; Glu-246 to Thr-269; Lys-275 to Tyr-295; Gly-303 to
Pro-320; Arg-327 to Ile-335; Thr-338 to Thr-364; Tyr-478 to
Phe-495; and Tyr- 499 to Arg-521. SP073 Glu-37 to Val-45; Glu-55 to
Val-68; Thr-104 to Thr-119; Ile-127 to Tyr-135; Asn-220 to Ile-232;
Thr-237 to Ala-250; Ser-253 to Ala-263; Glu-284 to Ile-297; and
Met-438 to Asn-455. SP074 Gly-2 to Ala-12; Gly-96 to Ile-110; and
Thr-220 to Phe-239. SP075 Phe-33 to Tyr-42; Gln-93 to Gly-102; and
Val-196 to Asp-211. SP076 Ser-64 to Leu-76; and Phe-81 to Ala-101.
SP077 Asp-1 to Glu-12; Tyr-26 to Val-36; and Val-51 to Try-62.
SP078 Ala-193 to Ile-208; Tyr-266 to Asn-275; Glu-356 to Leu-369;
Ala-411 to Gly-422; Ser-437 to Pro-464; Thr-492 to Glu-534; and
Glu-571 to Gln-508. SP079 Gly-11 to Leu-20; Lys-39 to Leu-48;
Leu-72 to Val- 85; Asn-147 to Ser-158; Ile-178 to Asp-187; Tyr- 189
to Gln-201; and Leu-203 to Ala-216 SP080 Ser-2 to Glu-12; Gln-42 to
Ala-51; Ala-116 to Ser- 127; Phe-131 to Asp-143; and Ile-159 to
Ile-171. SP081 Gln-2 to Leu-9; Gln-49 to Cys-57; Ile-108 to Val-
131; Gly-134 to Leu-145; and Trp-154 to Cys 162. SP082 Ile-101 to
Ser-187; Gly-191 to Asn-221; Arg-225 to Arg-236; Tyr-239 to
Leu-255; and Gly-259 to Arg- 268. SP083 Ser-28 to Asp-70. SP084
Leu-42 to Gln-66; Thr-69 to Lys-81; Glu-83 to Arg- 92; and Gly-98
to Asn-110. SP085 Gln-2 to Val-22; and Ser-45 to Glu-51. SP086
Leu-18 to Gln-65; and Lys-72 to Val-83. SP087 Ser-45 to Leu-53; and
Thr-S5 to Gln-63 SP088 Pro-8 to Ile-16; Leu-25 to Trp-33; Tyr-35 to
Gln- 43; Leu-51 to Val-59; Val-59 to Arg-67; Thr-55 to Tyr-63;
Asn-85 to Gly-93; Thr-107 to Leu-115; Leu- 115 to Trp-123; Ala-121
to Thr-129; Tyr-153 to Ala-161; His-176 to Gly-184; Tyr-194 to
Ala-202; Ala-217 to Gly-225; and Asn-85 to Gly-93. SP089 Trp-43 to
Ala-51; Gln-68 to Phe-76; Val-93 to Gln- 101; Phe-106 to Phe-114;
Lys-117 to Lys-125; Trp- 148 to Phe-156; Glu-168 to Gln-176;
Ile-193 to Tyr-201; Lys-203 to Lys-211; Glu-212 to Gln-220; Ile-237
to Tyr-245; Lys-247 to Lys-255; Glu-256 to Gln-264; Met-275 to
Gly-283; Lys-286 to Gly-294; Trp-292 to Glu-300; Asp-289 to
Thr-297; Tyr-315 to Ser-323; Asp-334 to Lys-342; Pro-371 to
Arg-379; Arg-485 to Asn-493; Lys-527 to Arg-535; Phe-537 to
Met-545; and Tyr-549 to Glu-557. SP090 Phe-2 to Gln-10; Gln-13 to
Lys-21; Tyr-19 to Glu- 27; Tyr-39 to Met-47; Pro-65 to Leu-73;
Tyr-121 to His-129; Lys-147 to Ile-155; Gly-161 to Lys-169; Gly-218
to Trp-226; Asp-230 to Thr-238; Tyr-249 to Ala-257; and Ala-272 to
Gly-280. SP091 Ser-19 to Ser-27; Asn-25 to Thr-33; Val-51 to Gln-
59; Asn-75 to Asn-83; Ile-103 to Trp-111; Tyr-113 to Ala-121;
Leu-175 to Asn-183; Glu-185 to Trp- 193; Ala-203 to Tyr-211;
Val-250 to Phe-258; Asn- 260 to Thr-268; Ser-278 to Asp-286;
Tyr-305 to Leu-313; Asn-316 to Gly-324; Asn-374 to Asp-382; Asn-441
to Gly-449; and Ser-454 to Gln-462. SP092 Arg-95 to Glu-103;
Ala-216 to Val-224; Leu-338 to Glu-346; Pro-350 to Ala-358; Pro-359
to Ala-367; Pro-368 to Ala-376; Pro-377 to Ala-385; Pro-386 to
Ala-394; Pro-395 to Ala-403; Pro-350 to Ala-358; Gln-414 to
Lys-422; Pro-421 to Asn-429; Trp-465 to Tyr-473; Phe-487 to
Tyr-495; Asn-517 to Gly-525; Trp-586 to Tyr-594; Phe-608 to
Tyr-616; and Asp- 630 to Gly-638. SP093 Gln-30 to Ile-38; Gln-52 to
Val-60; Ala-108 to His-116; Tyr-133 to Glu-141; Tyr-192 to Ala-200;
and Phe-207 to Ser-215. SP094 Ala-87 to Val-95; Leu-110 to Cys-118;
Gln-133 to Leu-141; Ser-185 to Leu-193; Ile-195 to Gly-203; Asp-206
to Gln-214; Ser-211 to Gly-219; Ile-241 to Thr-249. SP095 Arg-1 to
Gln-9; Phe-7 to Asn-15; Thr-21 to Asn-30; Leu-46 to Phe-54; and
Ser-72 to Met-80. SP096 Gly-29 to Ile-37; Glu-52 to Ser-60; and
Leu-64 to Gly-72. SP097 Ala-11 to Thr-19; Glu-53 to Glu-61; Ser-91
to Lys- 99; Thr-123 to Gln-131; and Gly-209 to Lys-217. SP098 Thr-3
to Ser-11; Gly-38 to Phe-46; Tyr-175 to Asn- 183; Met-187 to
Cys-195; Gln-197 to Leu-205; Tyr- 307 to Gln-315; Gly-318 to
Tyr-326; Asn-348 to Val-356; Lys-377 to Pro-385; and Leu-415 to
Val- 423. SP099 Arg-19 to Gly-27; Asp-76 to Ser-84; Val-90 to Lys-
98; Phe-165 to Val-173; Leu-237 to Pro-245. SP100 His-111 to
Gln-119; Ser-141 to His-149; Asp-154 to Ser-162; Gln-158 to
Gln-166; Asp-154 to Gln-166; Lys-180 to Gln-188; and Ser-206 to
Gln-214. SP101 Glu-23 to Glu-31; Glu-40 to Val-48; Gln-50 to Ser-
58; Thr-61 to Ile-69; Leu-82 to Ile-90; Ala-108 to Leu-116; Gln-121
to Pro-129; and Leu-130 to Thr- 138. SP102 Asp-32 to His-40; Arg-48
to Lys-56; and Asp-102 to Thr-110. SP103 Arg-5 to Gln-13; Gln-22 to
Leu-30; Arg-151 to Gln- 159; Arg-167 to Gln-175; Pro-189 to
Glu-197; Gly- 207 to Leu-215; Ser-219 to Gln-227; Ser-233 to
Ser-241; Pro-255 to Asp-264; Lys-272 to Gly-280; Ser-318 to
Val-326; Thr-341 to Asp-351; Asn-356 to Thr-364; Val-370 to
Tyr-378; Ile-379 to Gln-387; and Met-435 to Tyr-443. SP105 Asn-28
to Pro-36; Thr-77 to Phe-85; Arg-88 to Val- 96; Gly-107 to Phe-115;
Asp-169 to Asp-177; His- 248 to Ser-256; and Ser-274 to Ala-282.
SP106 Val-10 to Thr-18; Ile-62 to Tyr-70; Ile-71 to Pro- 79; Lys-86
to Gln-94; Lys-100 to Thr-108; Phe-132 to Leu-140; and Asp-145 to
Arg-153. SP107 Asp-33 to Val-41; and Arg-63 to Gln-71. SP108 Lys-9
to Gln-17; Leu-44 to Ser-52; Ser-63 to Phe- 71; Tyr-109 to Ser-117;
Ile-183 to Ile-191; Pro- 194 to Leu-202; Gly-257 to Gln-265;
Ala-323 to Thr-331; and Leu-381 to Tyr-389. SP109 Asn-2 to Gln-10;
Ala-65 to Lys-73; Leu-76 to Glu- 84; Thr-111 to Asp-119; Gln-116 to
Tyr-124; Tyr- 130 to Val-138; Asp-173 to Gly-181; Asp-196 to
Ser-204; Asn-231 to Ser-239; Phe-252 to Ser-260; Phe-270 to
Tyr-278; Val-291 to His-299; Asp-306 to Leu-314; and Pro-327 to
Gly-335. SP110 Ser-8 to Glu-16; Ile-37 to Val-45; Ala-107 to Val-
115; and Gly-122 to Thr-130. SP111 Asp-19 to Glu-28; Leu-43 to
Ala-51; Asn-102 to Phe-110; Gln-133 to Ser-141; Phe-162 to Asp-170;
Tyr-194 to Met-202; and Asp-273 to Ser-281. SP112 Asp-3 to Gln-11;
Gly-21 to Ile-29; Ala-46 to Arg- 54; Arg-98 to Arg-106; Thr-114 to
Val-122; Gln-133 to Asn-141; and Leu-223 to Thr-231. SP113 Asn-19
to Gly-27; Arg-54 to Ser-62; Val-69 to Gln- 77; Ser-117 to Asn-125;
Gly-164 to Leu-172; Tyr- 193 to Ser-201; Cys-303 to Phe-311;
His-315 to Ile-323; Arg-341 to Cys-349; Ile-347 to Ser-355; Arg-403
to Phe-4111; Gln-484 to Pro-492; Ser-499 to Leu-507; Ile-541 to
Thr-549 Asn-622 to Ile-630; and Glu-645 to Gly-653. SP114 Gly-17 to
Leu-25; His-40 to Gln-48; Arg-49 to Arg- 57; Ile-65 to Pro-73;
Asn-101 to Asp-111; Gly-128 to Cys-136; Phe-183 to Thr-191; and
Pro-268 to Ile-276. SP115 Met-8 to Ser-16; Tyr-24 to Leu-32; Cys-68
to Leu- 76; Ser-100 to Pro-108; Thr-193 to Thr-201; Gly- 238 to
Pro-250; Thr-280 to Phe-288; Pro-303 to Asn-312; Trp-319 to
Leu-328; Leu-335 to Leu-344; Lys-395 to Ala-403; Asn-416 to
Gln-424; Tyr-430 to Ser-438;
Val-448 to Leu-456; Leu-460 to Thr-468; Pro-502 to Thr-510; Lys-515
to Ile-524; Gln-523 to His-532; Tyr-535 to Thr-543; Ser-559 to
Pro-567; Thr-572 to Asn-580; Val-594 to Arg-602; Arg-603 to
Asn-611; Thr-620 to Trp-628; and Tyr-644 to Arg- 653. SP117 Ala-6
to Gly-14; Ile-19 to Thr-27; Thr-99 to Leu- 107; Ser-117 to
Asp-125; His-131 to Val-139; Ile- 193 to Gly-201; and Val-241 to
Gln-249. SP118 Ser-8 to Trp-23; His-46 to Ala-54; Asn-93 to Gly-
101; Val-100 to Ser-108; Arg-155 to Asp-163; and His-192 to
Leu-200. SP119 Tyr-46 to Lys-54; Ser-93 to Ser-101; Trp-108 to
Asn-116; Val-121 to Glu-129; and Tyr-131 to Gln- 139. SP120 Ala-57
to Lys-65; Leu-68 to Glu-76; Thr-103 to Tyr-116; Tyr-122 to
Val-130; His-163 to Gly-173; Asp-188 to Ser-196; Ser-222 to
Ser-231; Phe-244 to Ser-252; Pro-262 to Tyr-270; Val-283 to
His-291; and Asp-298 to Leu-306. SP121 Ser-3 to Ala-11; Asp-13 to
Leu-21; Ser-36 to Val- 44; and Gln-136 to Met-144. SP122 Asn-28 to
Lys-36; Glu-39 to Thr-50; Val-54 to Lys- 62; Asn-106 to Leu-114;
Phe-159 to Gly-167; Asn- 172 to Arg-180; Glu-199 to Asn-207;
Lys-230 to His-241; Asn-252 to Gly-263; Met-278 to Ala-287; Thr-346
to Asp-354; Lys-362 to Thr-370; Asp-392 to Asn-405; Asp-411 to
Ala-424; Gly-434 to Gly-443; Tyr-484 to Glu-492; Ile-511 to
Leu-519; Asn-524 to Asp-538; Glu-552 to Ile-567; Val-605 to
Lys-613; Phe-697 to Ala-705; Phe-722 to Leu-730; Leu-753 to
Leu-761; Asp-787 to Gln-795; Leu-858 to Asn-866; Ala-892 to
Thr-901; Gly-903 to Ile-913; Ile-921 to Asn-931; Asn-938 to
Pro-951; Gly-960 to Lys-970; Leu-977 to Asp-985; and Leu-988 to
Pro-996. SP123 Val-4 to Asn-12; Glu-47 to Leu-55; Lys-89 to Glu-
100; Ser-165 to Thr-173; Lys-234 to Val-242; Ser- 258 to Ser-266;
Glu-284 to Asn-292; Tyr-327 to Leu-335; Tyr-457 to Thr-465; Tyr-493
to Glu-501; Thr-506 to Tyr-514; Lys-517 to Thr-525; Asn-532 to
Gly-540; and Arg-556 to Glu-564. SP124 rg-16 to Glu-24; Gln-52 to
Arg-60; Asn-69 to Tyr- 77; Glu-121 to Asn-129; Ala-134 to Val-142;
Thr- 151 to Ala-159; Asn-164 to Glu-172; His-181 to His-189;
Thr-210 to Ala-218; Ser-244 to Val-252; Phe-287 to Tyr-297; Ser-312
to Thr-323; His-433 to Tyr-441; Ser-445 to Asn-453; Asn-469 to
Thr-477; Asn-501 to Asn-509; Gln-536 to Ala-547; and Gln- 608 to
Asp-621. SP125 Ser-9 to Asp-21; Ala-28 to Leu-36; Asn-49 to Phe-
57; Val-137 to Arg-145; Asn-155 to Leu-163; Glu- 183 to Asp-191;
Gly-202 to Tyr-210; Pro-221 to Asp-229; Phe-263 to Ala-271; Phe-300
to Gln-308; Asp-313 to Glu-321; Asn-324 to Asp-332; Ile-346 to
Asn-354; Asp-362 to Lys-370; Met-402 to Gly-410; Gly-437 to
Gly-445; Ser-471 to Glu-483; Gly-529 to Asp-537; Gln-555 to
Val-563; and Leu-579 to Lys- 587. SP126 Leu-22 to Thr-30; Val-65 to
Leu-73; and Thr-75 to Asp-83. SP127 Glu-2 to Ala-12; Asp-28 to
Thr-36; Val-105 to Thr- 113; Lys-121 to Thr-129; Trp-138 to
Pro-146; Ser- 152 to Ile-160; Lys-180 to Asp-188; Leu-194 to
Asn-202; and Gly-228 to Thr-236.
[0202]
3TABLE 3 S. pneumoniae ORF Cloning Primers Primer Name SEQ ID NO:
RE SP001A 227 Bam HI SP001B 228 Sal I SP004A 229 Bam HI SP004B 230
Sal I SP006A 231 Bam HI SP006B 232 Hind III SP007A 233 Bam HI
SP007B 234 Hind III SP008A 235 Bgl II SP008B 236 Hind III SP009A
237 Bam HI SP009B 238 Hind III SP010A 239 Bam HI SP010B 240 Hind
III SP011A 241 Bgl II SP011B 242 Pst I SP012A 243 Bam HI SP012B 244
Pst I SP013A 245 Bam HI SP013B 246 Hind III SP014A 247 Bgl II
SP014B 248 Pst I SP015A 249 Bam HI SP015B 250 Pst I SP016A 251 Bam
HI SP016B 252 Hind III SP017A 253 Bam HI SP017B 254 Hind III SP019A
255 Bam HI SP019B 256 Hind III SP020A 257 Bam HI SP020B 258 Hind
III SP021A 259 Bam HI SP021B 260 Hind III SP022A 261 Bam HI SP022B
262 Hind III SP023A 263 Bam HI SP023B 264 Hind III SP025A 265 Bam
HI SP025B 266 Sal I SP028A 267 Bam HI SP028B 268 Pst I SP030A 269
Bam HI SP030B 270 Hind III SP031A 271 Bam HI SP031B 272 Hind III
SP032A 273 Bam HI SP032B 274 Pst I SP033A 275 Bgl II SP033B 276
Hind III SP034A 277 Bam HI SP034B 278 Hind III SP035A 279 Bam HI
SP035B 280 Hind III SP036A 281 Bam HI SP036B 282 Hind III SP038A
283 Bam HI SP038B 284 Xho I SP039A 285 Bam HI SP039B 286 Hind III
SP040A 287 Bam HI SP040B 288 Hind III SP041A 289 Bam HI SP041B 290
Hind III SP042A 291 Bam HI SP042B 292 Hind III SP043A 293 Bam HI
SP043B 294 Hind III SP044A 295 Bam HI SP044B 296 Hind III SP045A
297 Bam HI SP045B 298 Sal I SP046A 299 Bam HI SP046B 300 Pst I
SP048A 301 Bam HI SP048B 302 Hind III SP049A 303 Bam HI SP049B 304
Hind III SP050A 305 Bam HI SP050B 306 Hind III SP051A 307 Bam HI
SP051B 308 Sal I SP052A 309 Bam HI SP052B 310 Hind III SP053A 311
Bam HI SP053B 312 Sal I SP054A 313 Bam HI SP054B 314 Hind III
SP055A 315 Bam HI SP055B 316 Hind III SP056A 317 Bam HI SP056B 318
Hind III SP057A 319 Bam HI SP057B 320 Hind III SP058A 321 Bam HI
SP058B 322 Sal I SP059A 323 Bam HI SP059B 324 Pst I SP060A 325 Bam
HI SP060B 326 Hind III SP062A 327 Bam HI SP062B 328 Pst I SP063A
329 Bam HI SP063B 330 Hind III SP064A 331 Bam HI SP064B 332 Pst I
SP065A 333 Bam HI SP065B 334 Hind III SP067A 335 Bam HI SP067B 336
Sal I SP068A 337 Bam HI SP068B 338 Sal I SP069A 339 Bam HI SP069B
340 Hind III SP070A 341 Bam HI SP070B 342 Hind III SP071A 343 Bgl
II SP071B 344 Hind III SP072A 345 Bgl II SP072B 346 Hind III SP073A
347 Sal I SP073B 348 Hind III SP074A 349 Bam HI SP074B 350 Pst I
SP075A 351 Bam HI SP075B 352 Hind III SP076A 353 Bam HI SP076B 354
Hind III SP077A 355 Bgl II SP077B 356 Hind III SP078A 357 Bam HI
SP078B 358 Sal I SP079A 359 Bam HI SP079B 360 Pst I SP080A 361 Bam
HI SP080B 362 Hind III SP081A 363 Bam HI SP081B 364 Hind III SP082A
365 Bam HI SP082B 366 Hind III SP083A 367 Bam HI SP083B 368 Bgl II
SP084A 369 Bam HI SP084B 370 Hind III SP085A 371 Bam HI SP085B 372
Hind III SP086A 373 Bam HI SP086B 374 Hind III SP087A 375 Bam HI
SP087B 376 Hind III SP088A 377 Bam HI SP088B 378 Hind III SP089A
379 Bam HI SP089B 380 Pst I SP090A 381 Bam HI SP090B 382 Pst I
SP091A 383 Bam HI SP091B 384 Hind III SP092A 385 Bgl II SP092B 386
Hind III SP093A 387 Bam HI SP093B 388 Hind III SP094A 389 Bam HI
SP094B 390 Hind III SP095A 391 Bam HI SP095B 392 Hind III SP096A
393 Bam HI SP096B 394 Hind III SP097A 395 Bam HI SP097B 396 Hind
III SP098A 397 Bam HI SP098B 398 Hind III SP099A 399 Bam HI SP099B
400 Hind III SP100A 401 Bam HI SP100B 402 Pst I SP101A 403 Bam HI
SP101B 404 Hind III SP102A 405 Bam HI SP102B 406 Hind III SP103A
407 Sal I SP103B 408 Pst I SP105A 409 Bam HI SP105B 410 Hind III
SP106A 411 Sal I SP106B 412 Hind III SP107A 413 Bam HI SP107B 414
Hind III SP108A 415 Bam HI SP108B 416 Hind III SP109A 417 Bam HI
SP109B 418 Hind III SP110A 419 Bam HI SP110B 420 Hind III SP111A
421 Bam HI SP111B 422 Hind III SP112A 423 Sal I SP112B 424 Hind III
SP113A 425 Bam HI SP113B 426 Hind III SP114A 427 Bam HI SP114B 428
Hind III SP115A 429 Bam HI SP115B 430 Hind III SP117A 431 Bam HI
SP117B 432 Sal I SP118A 433 Sal I SP118B 434 Pst I SP119A 435 Bam
HI SP119B 436 Hind III SP120A 437 Bam HI SP120B 438 Hind III SP121A
439 Bam HI SP121B 440 Hind III SP122A 441 Bam HI SP122B 442 Sal I
SP123A 443 Bam HI SP123B 444 Hind III SP124A 445 Bam HI SP124B 446
Sal I SP125A 447 Bam HI SP125B 448 Sal I SP126A 449 Bam HI SP126B
450 Hind III SP127A 451 Bam HI SP127B 452 Hind III
* * * * *