AU5682896A - Streptococcal heat shock proteins members of the HSP70 family - Google Patents

Description

STREPTOCOCCAL HEAT SHOCK PROTEINS

MEMBERS OF THE HSP70 FAMILY

TECHNICAL FIELD OF THE INVENTION

This invention relates to novel heat shock proteins of Streptococcus pneumoniae, Streptococcus pyogenes and Streptococcus agalactiae and immunologically related polypeptides, which provide the basis for new immunotherapeutic, prophylactic and diagnostic agents useful in the treatment, prevention and diagnosis of disease. More particularly, this invention relates to heat shock proteins of S. pneumoniae, S. pyogenes and S. agalactiae, members of the HSP70 family which have an apparent molecular mass of 70-72 kilodaltons, to the corresponding nucleotide and derived amino acid sequences, to recombinant DNA methods for the production of

HSP70/HSP72 and immunologically related polypeptides, to antibodies that bind to these HSP's, and to methods and compositions for the diagnosis, prevention and treatment of diseases caused by S. pneumoniae and related bacteria, such as Streptococcus pyogenes and Streptococcus

agalactiae

BACKGROUND OF THE INVENTION

S. pneumoniae is an important agent of disease in humans, especially among infants, the elderly and immunocompromised persons. It is a bacterium frequently isolated from patients with invasive diseases such as bacteraemia/septicaemia, pneumonia, and meningitis with high morbidity and mortality throughout the world.

Although the advent of antimicrobial drugs has reduced the overall mortality from pneumococcal diseases, the presence of resistant pneumococcal organisms has become a major problem in the world today. Effective pneumococcal vaccines could have a major impact on the morbidity and mortality associated with S. pneumoniae disease. Such vaccines would also potentially be useful to prevent otitiε media in infants and young children.

It is clear that a number of pneumococcal factors are potentially important in the pathogenesis of disease [G.J. Boulnois, J. Gen. Microbiol., 138, pp. 249- 259 (1992); C.J. Lee et al., Crit. Rev. Microbiol., 18, pp. 89-114 (1991)]. The capsule of the pneumococcus, despite its lack of toxicity, is considered to be the sine qua non of pneumococcal virulence. More than 80

pneumococcal capsular serotypes are identified on the basis of antigenic differences. Antibodies are the mechanism of protection and the importance of anticapsular antibodies in host defenses against S. pneumoniae is well established [R. Austrian, Am. J . Med., 67, pp. 547-549 (1979)]. Nevertheless, the currently available

pneumococcal vaccine, comprising 23 capsular

polysaccharides that most frequently caused disease, has significant shortcomings such as the poor immunogenicity of capsular polysaccharides, the diversity of the

serotypes and the differences in the distribution of serotypes over time, geographic areas and age groups. In particular, the failure of existing vaccines to protect young children against most serotypes has spurred

evaluation of other S. pneumoniae components. Increasing evidence indicates that certain pneumococcal proteins may play an active role both in terms of protection and pathogenicity [J.C. Paton, Ann. Rev. Microbiol., 47, pp. 89-115 (1993)]. So far, however, only a few S.

pneumoniae proteins have been studied. This might result from the lack of protein-specific antibodies which renders difficult the study of the role of protein antigens in protection and pathogenicity. It is believed that the pneumococcal protein antigens are not very immunogenic and that most antibody responses are to the phόsphocholine and the capsular polysaccharides [L.S. McDaniel et al., J. Exp . Med., 160, pp. 386-397 (1984); R.M. Krause, Adv.

Immunol., 12, pp. 1-56 (1970); D.G. Braun et al., J. Exp. Med., 129, pp. 809-830 (1969)]. In a study using X-linked immunodeficient mice, which respond poorly to carbohydrate antigens and to phosphocholine, but make relatively normal responses to protein antigens, the frequency for obtaining monoclonal antibodies reactive with pneumococcal protein antigens was less than 10%, thus suggesting that S.

pneumoniae proteins are poor immunogens [McDaniel et al., supra].

Streptococcus agalactiae, also called Group B

Streptococcus (GBS), is the most common cause of sepsis

(blood infection) and meningitis in newborns . GBS is also a frequent cause of newborn pneumonia. Approximately 8,000 babies in the United States get GBS disease each year; 5%-15% of these babies die. Babies that survive, particularly those who have meningitis, may have long-term problems, such as hearing or vision loss or learning disabilities. In pregnant women, GBS can cause urinary tract infections, womb infections (amnionitis,

endometritis), and stillbirth. Among women who are not pregnant and men, the most common diseases caused by GBS are blood infections, skin or soft tissue infections, and pneumonia. Approximately 20% of men and nonpregnant women with GBS disease die of the disease. GBS infections in both newborns and adults are usually treated with

antibiotics (e.g., penicillin or ampicillin) given

intravenously. Most GBS disease in newborns can be prevented by giving certain pregnant women antibiotics intravenously during labor. Vaccines to prevent GBS disease are being developed. In the future, it is

expected that women who will be vaccinated will make antibodies that cross the placenta and protect the baby during birth and early infancy.

Since the 1980s, Streptococcus pyogenes, also called Group A Streptococcus (GAS) is reemerging as a cause of severe diseases which would be due to an increase in virulence of the organism. GAS causes pharyngitis, commonly called "strep throat", and skin infections

(impetigo, erysipelas/cellulitis) . "Strep throat" and impetigo can lead to glomerulonephritis (kidney damage). Approximately 3% of "strep throat" infections result into rheumatic fever (migrating arthritis) whose complications include chorea (neurological symptoms) and, in 50% of the cases, rheumatic heart disease (heart valve damage) with endocarditis as a possible long term consequence. It is important to treat impetigo and "strep throat" with antibiotics to prevent the development of complications. Infection with toxin-producing strains can result in scarlet fever (diffuse rash and fever) or in the extremely severe streptococcal toxic shock syndromes (TSS; GAS have been termed 'flesh eating bacteria') which are

characterized by the rapid development of shock and multiple organ system failure. TSS have a 30 to 70% fatality rate in spite of aggressive treatment involving the removing of the focus of bacterial infection and antibiotic therapy. The incidence of TSS is 10 to 20 cases per 100,000. No vaccine against GAS is presently available.

Heat shock or stress proteins ("HSPs") are among the most highly conserved and abundant proteins found in nature [F.C. Neidhardt et al., Ann. Rev. Genet., 18, pp. 295-329 (1984); S. Lindquist, Ann. Rev. Biochem., 55, pp. 1151-1191 (1986)]. They are produced by all cells in response to various physiological and nonphysiological stimuli. The heat shock response, in which a sudden increase in temperature induces the synthesis of HSPs, is the best studied of the stress responses. Other

environmental conditions such as low pH, iron deficiency and hydrogen peroxyde can also induce HSPs. The HSPs have been defined by their size, and members of hsp90, hsp70, and hsp60 families are among the major HSPs found in all prokaryotes and eukaryotes. These proteins fulfill a variety of chaperon functions by aiding protein folding and assembly and assisting translocation across membranes [C. Georgopoulos and W.J. Welch, Ann. Rev. Cell. Biol., 9, pp. 601-634 (1993); D. Ang et al., J. Biol. Chem., 266, pp. 24233-24236 (1991)]. As molecular chaperons and possibly via other mechanisms, HSPs are likely involved in protecting cells from the deleterious effects of stress. The fact that several virulence factors are regulated by environmental conditions suggests a role for HSPs in microbial pathogenicity [J.J. Mekalanos, J. Bacteriol., 174, pp. 1-7 (1992); P.J. Murray and R.A. Young, J.

Bacteriol., 174, pp. 4193-4196 (1992)]. In that respect, recent studies on Salmonella species suggest that the stress response might be critically linked to the ability of intracellular pathogens to initiate and sustain an infection [N.A. Buchmeir and F. Heffron, Science, 248, pp. 730-732 (1990); K.Z. Abshire and F.C. Neidhardt,

J. Bacteriol., 175, pp. 3734-3743 (1993); B.B. Finlay et al., Science, 243, pp. 940-943 (1989)]. Others have demonstrated that lysteriolysin, an essential virulence factor in L . monocytogenes , is induced under heat shock conditions [Z. Sokolovic and W. Goebel, Infect. Immun., 57, pp. 295-298 (1989)].

Evidence is now accumulating that HSPs are major antigens of many pathogens. Members of the hsp60 family, also called GroEL-related proteins for their similarity to the E. coli GroEL protein, are major antigens of a variety of bacterial pathogens including Mycobacterium leprae and Mycobacterium tuberculosis [D. Young et al., Proc. Natl. Acad. Sci. USA, 85, pp. 4267-4270 (1988)], Legionella pneumophila [B.B. Plikaytis et al., J. Clin. Microbiol., 25, pp. 2080-2084 (1987)], Borrelia burgdorferi [B.J. Luft et al., J. Immunol., 146, pp. 2776-2782 (1991)], and

Chlamydia trachomatis [E.A. Wagar et al., J. Infect. Pis., 162, pp. 922-927 (1990)]. This antigen is a homologue of the ubiquitous "common antigen", and is believed to be present in every bacterium [J.E. Thole et al., Microb. Pathogen., 4, pp. 71-83 (1988). Antibodies to the members of the hsp70 family, or DnaK-related proteins, have also been described for several bacterial and parasitic

infections [Young et al., supra; Luft et al., supra; D.M. Engman et al., J. Immunol., 144, pp. 3987-3991 (1990);

N.M. Rothstein et al., Molec. Biochem. Parasitol., 33, pp. 229-235 (1989); V. Nussenzweig and R.S. Nussenzweig, Adv. Immunol., 45, pp. 283-334 (1989)]. HSPs can elicit strong B- and T- cell responses and it was shown that 20% of the CD4⁺ T-lymphocytes from mice inoculated with M.

tuberculosis were reactive to the hsp60 protein alone

[S.H.E. Kaufman et al., Eur. J. Immunol., 17, pp. 351-357 (1987)]. Similarly, 7 out of a collection of 24

monoclonal antibodies to M. leprae proteins recognized determinants on hsp60 [H.D. Engers et al., Infect. Immun., 48, pp. 603-605 (1985)]. It seems that the immune

response to stress proteins might play an important role in protection against infection. Consistent with that is the demonstration that antibodies and T cells reactive with microbial HSPs can exhibit neutralizing and

protective activities [A. Noll et al., Infect. Immun., 62, pp. 2784-2791 (1994); and S.L. Danilition et al., Infect. Immun., 58, pp. 189-196 (1990)]. The immunological properties of stress proteins make them attractive as vaccine components and several HSPs are presently being considered for preventing microbial infection and treating cancer. So far, however, studies have focused on

intracellular pathogens such as Mycobacteria, Salmonella, Chlamydia and several parasites. Information concerning the heat shock protein antigens in extracellular gram- positive bacteria is far less documented. In S.

pneumoniae, S. pyogenes and S. agalactiae, neither the heat shock proteins nor their gene structures have been identified.

DISCLOSURE OF THE INVENTION

The present invention addresses the problems referred to above by providing novel heat shock proteins from S. pneumoniae, S. pyogenes and S. agalactiae, and immunologically related polypeptides. Also provided are DNA sequences that code for the foregoing polypeptides, vectors containing the polypeptides, unicellular hosts transformed with those vectors, and a process for making substantially pure, recombinant polypeptides. Also provided are antibodies specific to the foregoing

polypeptides. The polypeptides, DNA sequences and

antibodies of this invention provide the basis for novel methods and pharmaceutical compositions for the detection, prevention and treatment of disease. Particularly, this invention provides a novel vaccine based on fragments of these polypeptides that are specific to streptococcal strains.

The novel heat shock protein is the

approximately 72 kDa heat shock protein of Streptococcus pneumoniae ("HSP72") (SEQ ID NO:5), the approximately 70 kDa heat shock protein of Streptococcus pyogenes ("HSP70") (SEQ ID NO: 20) and the approximately 70 kDa heat shock protein of Streptococcus agalactiae ("HSP70") (SEQ ID

NO: 22), including analogues, homologues, and derivatives thereof, and fragments of the foregoing polypeptides containing at least one immunogenic epitope. Preferred fragments of HSP70/72 include the C-terminal portion of the HSP70/72 polypeptides. More particularly, it includes the C_terminal 169-residue fragment ("C-169") (residues 439-607, SEQ ID NO:5), the C-terminal 151-residue fragment ("C-151") (residues 457-607, SEQ ID No:5), and smaller fragments consisting of peptide epitopes within the C-169 region. Particularly preferred fragments within the C-169 region of HSP72 include the peptide sequences

GFDAERDAAQAALDD (residues 527-541 of SEQ ID NO:5) and AEGAQATGNAGDDVV (residues 586-600 of SEQ ID NO:5), which are exclusive to HSP72 of Streptococcus pneumoniae . Even more preferred are fragments that elicit an immune

reaction against S. pneumoniae, S. pyogenes and S. agalactiae but do not provoke auto-immune reaction in a human host. Such fragments may be selected from the following peptides: CS870, CS873, CS874, CS875, CS876, CS877, CS878, CS879, CS880, CS882, MAP1, MAP2 , MAP3 and MAP4 (see TABLE 5, supra).

Preferred antibodies of this invention are the F1-Pn3.1, F2-Pn3.2, F2-Pn3.3 and F2-Pn3.4 monoclonal antibodies ("MAbs"), which are specific to HSP72.

More preferred antibodies are the F2-Pn3.2 and F2-Pn3.4 monoclonal anibodies that are specific to both HSP 70 and HSP72. Even more preferred are the F1-Pn3.1 antibodies that are specific for Streptococcus pneumoniae.

The preferred polypeptides and antibodies of this invention provide the basis for novel methods and pharmaceutical compositions for the detection, prevention and treatment of pneumococcal diseases.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 depicts a fluorogram, which shows the effect of heat shock on S. pneumoniae protein synthesis. The cell extracts in panel A are S. pneumoniae type 6 strain 64. The cell extracts in panel B are S. pneumoniae type 4 strain 53. The cell extracts in the odd numbered lanes were incubated at 37°C. The cell extracts in the even numbered lanes were incubated at 45°C for 5 minutes . The cell extracts were then labeled with [³⁵S] methionine for 10 minutes (lanes 1, 2 and 7, 8), 30 minutes (lanes 3, 4 and 9, 10), or 60 minutes (lanes 5, 6). Molecular mass markers in kilodaltons are shown to the left. The

positions of HSP80, HSP72 and HSP62 are shown by arrows at the right-hand side of each panel.

FIG. 2 is a graphical depiction of a comparison of the electrophoretic profiles of [³⁵S]methionine-labeled proteins in S. pneumoniae in the presence (----) or absence (____) of exposure to heat shock. Densitometric tracings were determined by measuring the relative optical density (Y axis) vs. the mobility of labeled protein bands (X axis). The densitometric scans of the SDS PAGE of FIG. 1, lanes 1 and 2, is shown.

FIG. 3 depicts a fluorogram, which shows the S. pneumoniae protein antigens immunoprecipitated by sera from mice immunized with detergent-soluble S. pneumoniae protein extract. [³⁵S]methionine-labeled proteins from S. pneumoniae grown at 37°C and incubated at 37°C (lanes 3, 5, 7 and 9) or heat-shocked at 45°C (lanes 4, 6, 8 and 10) were immunoprecipitated with sera from mouse 1 (lanes 3 to 6) or mouse 2 (lanes 7 to 10) and then analyzed by SDS- PAGE and fluorography . The sera were tested after the first (lanes 3,4 and 7,8) and after the second (lanes 5,6 and 9,10) immunization. Cell lysates from [³⁵S]methionine- labeled non heat-shocked and heat-shocked S. pneumoniae are shown in lanes 1 and 2, respectively. The position of HSPs is indicated by the arrows at the left of the

fluorogram.

FIG. 4 depicts a fluorogram, which shows the S. pneumoniae protein antigens immunoprecipitated by sera from mice immunized with heat-killed S. pneumoniae

bacteria. [³⁵S]methionine-labeled proteins from

S. pneumoniae grown at 37°C and incubated at 37°C (lanes 3, 5 and 7) or heat-shocked at 45°C (lanes 4, 6 and 8) were immunoprecipitated with sera from mouse 1 (lanes 3,4), mouse 2 (lanes 5,6) or mouse 3 (lanes 7, 8) and then analyzed by SDS-PAGE and fluorography. Sera were tested after the second immunization only. Cell lysates from

[³⁵S]methionine-labeled non heat- and heat-shocked

S. pneumoniae are shown in lanes 1 and 2, respectively. The position of HSPs is indicated by the arrows at the left of the fluorogram.

FIG. 5 depicts a photograph, which shows the S. pneumoniae antigens detected by Western blot analysis. Whole cell extracts were probed with sera from 15 mice (lanes 1-15) immunized with heat-killed S. pneumoniae bacteria. Lane 16 shows the HSP72 protein detected by MAb F1-Pn3.1. In panel A, the sera were tested after the second immunization. In panel B, the reactivity of 4 out of 15 sera tested after the first immunization is shown. The positions of 53.5 kDa- and 47 kDa-protein bands are indicated by the bars at the left. The position of HSP72 is shown by the arrows at the right of each panel.

FIG. 6 depicts a fluorogram showing the specificity of MAb F1-Pn3.1 for HSP72. [³⁵S]methionine- labeled proteins of S. pneumoniae in the absence (lanes 1, 3 and 5) or presence (lanes 2, 4 and 6) of exposure to heat shock were immunoprecipitated with IgG2a-control MAb (lane 3,4) or F1-Pn3.1 (lane 5,6) and then analyzed by SDS-PAGE and fluorography. Cell lysates from

[³⁵S]methionine-labeled non heat-shocked and heat-shocked S. pneumoniae are shown in lanes 1 and 2, respectively.

The position of HSPs (all three) is shown by the arrows at the left of the fluorogram.

FIG. 7, panel A, depicts an immunoblot, which shows the reaction of heat-shocked and non heat-shocked [³⁵S]methionine-labelled S. pneumoniae cell extracts with MAb F1-Pn3.1. Lane 1 contains heat-shocked cell lysates (45°C). Lane 2 contains non heat-shocked cell lysates (37°C). Panel B depicts a fluorogram of the immunoblot shown in panel A.

FIG. 8 depicts a Western Blot, which shows subcellular localization of S. pneumoniae HSP72. Sample containing 15 μg protein of membrane fraction (lane 1) and cytoplasmic fraction (lane 2) of S. pneumoniae were electrophoresced on SDS-PAGE transferred to nitrocellulose and probed with MAb F1-Pn3.1.

FIG. 9 is a photograph of an immunoblot showing the reactivity of recombinant fusion proteins containing the C-169 region of S. pneumoniae HSP72 with MAb F1-Pn3.1. Lane 1 contains whole cell extracts from S. pneumoniae strain 64 probed with HSP72-specific MAb F1-Pn3.1.

Lanes 2 and 3 contain phage lysates from E. coli infected with λJBD17 cultured in the presence (+) or absence (-) of IPTG and probed with HSP72-specific MAb F1-Pn3.1. Lanes 4 and 5 contain phage lysates from E. coli infected with λJBD7 cultured in the presence (+) or absence (-) of IPTG and probed with HSP72-specific MAb F1-Pn3.1. Molecular mass markers are shown to the left. The positions of the 74kDa- and 160 kDa-reactive proteins are shown on the left and on the right, respectively.

FIG. 10 is a schematic representation of the restriction map of the HSP72 (DnaK) and Fuc loci and inserts of recombinant clones. The relationships between DNA fragments are shown with respect to each other.

FIGS. 10A and IOC illustrate the restriction map of the HSP72(DnaK) and Fuc loci, respectively. FIG 10B

illustrates the inserts of the various phages and plasmids described in Example 3. H(HindΙII); E(EcoRI); V(EcoRV); P(PstΙ); and X(XhoI) indicate positions of restriction endonuclease sites. DNA fragments on the HSP72/DnaK locus (■); the Fuc locus (///); and fragments used as probes in the Southern blot analyses ( are indicated.

FIG. 11 depicts the SDS-PAGE and Western blot analyses of the recombinant 74 kDa protein. Whole cell extracts from E. coli transformed with plasmids pJBD179 (lane 1), pJBDf51 (lanes 2 and 3) and pJBDf62 (lane 4 and 5) and cultured in presence (+) or absence (-) of IPTG were subjected to 10% polyacrylamide gel electrophoresis. The proteins were then visualized by Coomassie Blue staining (A) or Western blotting (B) using HSP-specific MAb F1-Pn3.1. Molecular mass markers in kilodaltons are shown to the left. The arrow at the left-hand side of each panel marks the 74 kDa protein marker.

FIG. 12 depicts the detection of native and recombinant HSP72 antigens by Western blot analysis.

Whole cell lysates from E. coli transformed with plasmids pJBDk51 (lanes 1 and 3) and pJBD291 (lane 2) and cell lysates from S. pneumoniae strain 64 (lane 4) were subjected to 10% polyacrylamide gel electrophoresis and were electrotransferred to nitrocellulose. The immunoblot was probed with HSP72-specific MAb F1-Pn3.1.

FIGS. 13A-13D depict a comparison of the

predicted amino acid sequence of the S. pneumoniae HSP72 open reading frame (HSP72 SPNEU) with those previously reported for the following HSP70/DnaK proteins: ECOLI, Escherichia coli ; BORBU, Borrelia burgdorferi ; BRUOV, Brucella ovis; CHLPN, Chlamydia pneumonia; BACME, Bacillus megatorium; BACSU, Bacillus subtilis; STAAU,

Staphylococcus aureus ; LACLA, Lactococcus lactis; and

MYCTU, Mycobacterium tuberculosis . Only mismatched amino acids are indicated. Identical and conserved amino acids are boxed and shadowed, respectively.

FIG. 14 depicts a photograph of an SDS-PAGE, which shows the recombinant S. pneumoniae HSP72 purified by affinity chromatography. Supernatant fractions from E. coli (pJBDk51) lysates (lane 2) and 20 μg of

immunoaffinity-purified HSP72_rec (lane 3) were subjected to 10% polyacrylamide gel electrophoresis. The proteins were then visualized by Coomassie Blue staining. Lane 1 shows the migration of molecular mass markers (106 kDa, 80 kDa, 49.5 kDa, 32.5 kDa, 27.5 kDa and 18.5 kDa).

FIG. 15 depicts a photograph of SDS-PAGE, which shows the recombinant S. pneumoniae C-169 fragment

purified by solubilization of inclusion bodies. Various amounts of purified C-169 protein (lane 1, 5 μg; lane 2, 2.5 μg; and lane 3, 1 μg) and whole cell lysates from E. coli transformed with plasmids pDELTAl (lane 4) and pJBDΔ1 (lane 5) were subjected to 10% polyacrylamide gel electrophoresis. The proteins were then visualized by Coomassie Blue staining.

FIG. 16 is a graphical depiction of the survival curve of Balb/c mice protected from S. pneumoniae

infection by immunization with HSP72_rec. Data are

presented as the per cent (%) survival over a period of 14 days for a total of 10 mice per experimental group. FIG. 17 is a graphical depiction of the survival curve of Balb/c mice protected from S. pneuiTioniae

infection by immunization with C-169_rec. Data are

presented as the per cent (%) survival over a period of 14 days for a total of 10 mice per experimental group.

FIG. 18 is a map of plasmid pURV3 containing C- 151_rec, the coding region for the 151 amino acids at the carboxyl end of the HSP72 of S. pneumoniae; Ampi^R,

ampicillin-resistance coding region; ColE1 ori , origin of replication; cI857, bacteriophage λ cI857 temperature- sensitive repressor gene; λ PL, bacteriophage λ

transcription promoter; T1, T1 transcription terminator. The direction of transcription is indicated by the arrows. βglll and BamHI are the restriction sites used to insert the coding region for the C-151_rec of the HSP72 of S.

pneumoniae . FIG. 19 illustrates the

distribution of anti-S. pneumoniae titers in sera from Balb/c mice immunized with HSP72_rec. Sera were collected after the first, second and third injection with 1 μg (O) or 5 μg (•) of HSP72_rec and evaluated individually for anti-S. pneumoniae antibody by ELISA. Titers were defined as the highest dilution at which the A410 values were 0.1 above the background values . Plain lines indicate the median reciprocal of antibody titers for each group of mice while the dashed line indicates the median value for preimmune sera.

FIG. 20 illustrates the distribution of anti-S.

pneumoniae titers in sera from Balb/c mice immunized with C-169_rec. Sera were collected after the first, second and third injection with 1 μg (O) or 5 μg (•) of C-169_rec and evaluated individually for anti-S. pneumoniae antibody by ELISA. Titers were defined as the highest dilution at which the A410 values were 0.1 above the background values. Plain lines indicate the median reciprocal of antibody titers for each group of mice while the dashed line indicates the median value for preimmune sera. FIG. 21 illustrates the distribution of anti-S.

pneumoniae titers in sera from Balb/c mice immunized with C-151_rec. Sera were collected after the first, second and third injection with 0.5 μg of C-151_rec and evaluated individually for anti-S. pneumoniae antibody by ELISA. Titers were defined as the highest dilution at which the A410 values were 0.1 above the background values. Plain lines indicate the median reciprocal of antibody titers for each group of mice while the dashed line indicates the median value for preimmune sera.

FIG. 22 illustrates the antibody response of

cynomolgus monkeys immunized with recombinant HSP72 antigens. Groups of two monkeys were immunized with either HSP72_rec or C-169_rec protein at day 1, day 22 and day 77. Sera were collected regularly during the course of the immunization and evaluated individually for

pneumococcal HSP72 specific antibody by Western blot analysis. Titers were defined as the highest dilution at which the HSP72 band was visualized.

FIG. 23 illustrates the binding of hyperimmune sera to peptides in a solid-phase ELISA. Rabbit, mouse and monkey sera from animals immunized with either HSP72_rec or C-169_rec protein were tested for their reactivity to peptides. Optical density values were obtained with sera tested at a dilution of 1:100 except for the values corresponding to the reactivity of rabbit sera to peptide MAP2 and murine sera to peptides MAP2 and MAP4 which were obtained with sera diluted 1:1000.

FIG. 24 depicts the consensus sequence established from the DNA sequences of the hsplO/dnak open reading frames of Streptococcus pneumoniae (spn-orf),

Streptococcus pyogenes (sga-orf) and Streptococcus

agalactiae (sgb-orf) and indicates the substitutions and insertions of nucleotides specific to each species.

FIG. 25 depicts the consensus sequence established from the protein sequences of the Hsp70 of Streptococcus pneumoniae (spn-prot), Streptococcus pyogenes (sga-prot) and Streptococcus agalactiae (sgb-prot) and indicates the substitutions and insertions of amino acids specific to each species.

FIG. 26 depicts a fluorogram, which shows the effect of heat shock on S. agalactiae protein synthesis and the S. agalactiae protein antigen immunoprecipitated by MAb F2-Pn3.4. Cell lysates from [³⁵S]methionine-labeled proteins from S. agalactiae grown at 37°C and incubated at 37°C (odd numbered lanes) or heat-shocked at 43°C (even numbered lanes) were analysed by SDS-PAGE and

fluorography. Lanes 3 and 4 show the immunoprecipitates obtained using MAb F2-Pn3.4.

DETAILED DESCRIPTION OF THE INVENTION

According to one aspect of the invention, we provide novel heat shock proteins of S. pneumoniae, S. pyogenes and S. agalactiae, and analogues, homologues, derivatives and fragments thereof, containing at least one immunogenic epitope. As used herein, a "heat shock protein" is a naturally occurring protein that exhibits preferential transcription during heat stress conditions. The heat shock protein according to the invention may be of natural origin, or may be obtained through the

application of recombinant DNA techniques, or conventional chemical synthesis techniques.

As used herein, "immunogenic" means having the ability to elicit an immune response. The novel heat shock proteins of this invention are characterized by their ability to elicit a protective immune response against Streptococcal infections, more particularly against lethal S. pneumoniae, S. pyogenes and S.

agalactiae .

The invention particularly provides a Streptoccus pneumoniae heat shock protein of approximately 72 kDa ("HSP72"), having the deduced amino acid sequence of SEQ ID NO: 5, and analogues, homologues, derivatives and fragments thereof, containing at least one immunogenic epitope.

As used herein, "analogues" of HSP72 are those S. pneumoniae proteins wherein one or more amino acid residues in the HSP72 amino acid sequence (SEQ ID NO : 5 ) is replaced by another amino acid residue, providing that the overall functionality and immunogenic properties of the analogue protein are preserved. Such analogues may be naturally occurring, or may be produced synthetically or by recombinant DNA technology, for example, by mutagenesis of the HSP72 sequence. Analogues of HSP72 will possess at least one antigen capable of eliciting antibodies that react with HSP72, e.g. Streptococcus pyogenes and

Streptococcus agalactiae .

As used herein, "homologues" of HSP72 are proteins from Streptococcal species other than pneumoniae, pyogenes or agalactiae, or genera other than Streptococcus wherein one or more amino acid residues in the HSP72 amino acid sequence (SEQ ID NO:5) is replaced by another amino acid residue, providing that the overall functionality and immunogenic properties of the homologue protein are preserved. Such homologues may be naturally occurring, or may be produced synthetically or by recombinant DNA technology. Homologues of HSP72 will possess at least one antigen capable of eliciting antibodies that react with HSP72, e.g. Enterococcus faecalis .

As used herein, a "derivative" is a polypeptide in which one or more physical, chemical, or biological properties has been altered. Such alterations include, but are not limited to: amino acid substitutions,

modifications, additions or deletions; alterations in the pattern of lipidation, glycosylation or phosphorylation; reactions of free amino, carboxyl, or hydroxyl side groups of the amino acid residues present in the polypeptide with other organic and non-organic molecules; and other

alterations, any of which may result in changes in

primary, secondary or tertiary structure. The "fragments" of this invention will have at least one immunogenic epitope. An "immunogenic epitope" is an epitope that is instrumental in eliciting an immune response. The preferred fragments of this invention will elicit an immune response sufficient to prevent or lessen the severity of infection, e.g., S. pneumoniae infection. Preferred fragments of HSP72 include the C-terminal region of the polypeptides. More preferred fragment include the C-terminal 169-residue fragment ("C-169") (SEQ ID NO: 5, residues 439-607), the C-terminal 151-residue ("C-151") (SEQ ID No: 5, residues 457-607) and smaller fragments consisting of peptide epitopes within the C-169 region. Particularly preferred fragments within the C-169 region of HSP72 include the peptide sequences GFDAERDAAQAALDD (residues 527-541 of SEQ ID NO: 5) and AEGAQATGNAGDDVV

(residues 586-600 of SEQ ID N0:5), which are exclusive to HSP72 of Streptococcus pneumoniae, or corresponding degenerate fragments from S. pyogenes or S. agalactiae (see FIG. 25). Even more preferred are fragments that elicit a specific immune reaction against Streptococcal strains . Such fragments may be selected from the

following peptides: CS870, CS873, CS874, CS875, CS876, CS877, CS878, CS879, CS880, CS882, MAP1, MAP2 , MAP3 and MAP4 (see TABLE 5, supra), or homologues thereof.

In a further aspect of the invention, we provide polypeptides that are immunologically related to HSP70/72. As used herein, "immunologically related" polypeptides are characterized by one or more of the following properties:

(a) they are immunologically reactive with

antibodies generated by infection of a mammalian host with Streptococcus pneumoniae cells, which antibodies are immunologically reactive with HSP72 (SEQ ID NO: 5) and HSP70 (SEQ ID NO:20 and SEQ ID NO:22);

(b) they are capable of eliciting antibodies that are immunologically reactive with HSP72 (SEQ ID NO: 5) and HSP70 (SEQ ID NO:20 and SEQ ID NO:22); (c) they are immunologically reactive with

antibodies elicited by immunization of a mammal with HSP72 (SEQ ID NO :5).

By definition, analogues, homologues and

derivatives of HSP70/72 are immunologically related polypeptides. Moreover, all immunologically related polypeptides contain at least one HSP70/72 antigen.

Accordingly, "HSP70/72 antigens" may be found in HSP70/72 itself, or in immunologically related polypeptides.

In a further aspect of the invention, we provide polypeptides that are immunologically related to HSP72. As used herein, "immunologically related" polypeptides are characterized by one or more of the following properties:

(a) they are immunologically reactive with

antibodies generated by infection of a mammalian host with Streptococcus pneumoniae cells, which antibodies are immunologically reactive with HSP72 (SEQ ID NO:5);

(b) they are capable of eliciting antibodies that are immunologically reactive with HSP72 (SEQ ID NO:5);

(c) they are immunologically reactive with

antibodies elicited by immunization of a mammal with HSP72 (SEQ ID NO:5).

By definition, analogues, homologues and

derivatives of HSP72 are immunologically related

polypeptides. Moreover, all immunologically related polypeptides contain at least one HSP72 antigen.

Accordingly, "HSP72 antigens" may be found in HSP72 itself, or in immunologically related polypeptides.

As used herein, "related bacteria" are bacteria that possess antigens capable of eliciting antibodies that react with HSP72. Examples of related bacteria include Streptococcus pneumoniae, Streptococcus pyogenes,

Streptococcus mutans, Streptococcus sanguis, Streptococcus agalactiae and Enterococcus faecalis .

It will be understood that by following the examples of this invention, one of skill in the art may determine without undue experimentation whether a particular analogue, homologue, derivative, immunologically related polypeptide, or fragment would be useful in the diagnosis, prevention or treatment of disease. Useful polypeptides and fragments will elicit antibodies that are immunoreactive with HSP72 (Example 4). Preferably, useful polypeptides and fragments will

demonstrate the ability to elicit a protective immune response against lethal bacterial infection (Example 5).

Also included are polymeric forms of the

polypeptides of this invention. These polymeric forms include, for example, one or more polypeptides that have been crosslinked with crosslinkers such as avidin/biotin, glutaraldehyde or dimethylsuberimidate. Such polymeric forms also include polypeptides containing two or more tandem or inverted contiguous protein sequences, produced from multicistronic mRNAs generated by recombinant DNA technology.

This invention provides substantially pure HSP72 and immunologically related polypeptides. The term

"substantially pure" means that the polypeptides according to the invention, and the DNA sequences encoding them, are substantially free from other proteins of bacterial origin. Substantially pure protein preparations may be obtained by a variety of conventional processes, for example the procedures described in Examples 3 and 5.

In another aspect, this invention provides, for the first time, a DNA sequence coding for a heat shock protein of S. pneumoniae, specifically, HSP72 (SEQ ID NO:4, nucleotides 682-2502).

The DNA sequences of this invention also include

DNA sequences coding for polypeptide analogues and

homologues of HSP72, DNA sequences coding for

immunologically related polypeptides, DNA sequences that are degenerate to any of the foregoing DNA sequences, and fragments of any of the foregoing DNA sequences. It will be readily appreciated that a person of ordinary skill in the art will be able to determine the DNA sequence of any of the polypeptides of this invention, once the

polypeptide has been identified and isolated, using conventional DNA sequencing techniques.

Oligonucleotide primers and other nucleic acid probes derived from the genes encoding the polypeptides of this invention may also be used to isolate and clone other related proteins from S. pneumoniae and related bacteria which may contain regions of DNA bacteria that are

homologous to the DNA sequences of this invention. In addition, the DNA sequences of this invention may be used in PCR reactions to detect the presence of S. pneumoniae or related bacteria in a biological sample.

The polypeptides of this invention may be prepared from a variety of processes, for example by protein fractionation from appropriate cell extracts, using conventional separation techniques such as ion exchange and gel chromatography and electrophoresis, or by the use of recombinant DNA techniques. The use of

recombinant DNA techniques is particularly suitable for preparing substantially pure polypeptides according to the invention.

Thus according to a further aspect of the invention, we provide a process for the production of HSP72, immunologically related polypeptides, and fragments thereof, comprising the steps of (1) culturing a

unicellular host organism transformed with a vector containing a DNA sequence coding for said polypeptide or fragment and one or more expression control sequences operatively linked to the DNA sequence, and (2) recovering a substantially pure polypeptide or fragment.

As is well known in the art, in order to obtain high expression levels of a transfected gene in a host, the gene must be operatively linked to transcriptional and ranslational expression control sequences that are

functional in the chosen expression host. Preferably, the expression control sequences, and the gene of interest, will be contained in an expression vector that further comprises a bacterial selection marker and origin of replication. If the expression host is a eukaryotic cell, the expression vector should further comprise an

expression marker useful in the eukaryotic expression host.

The DNA sequences encoding the polypeptides of this invention may or may not encode a signal sequence. If the expression host is eukaryotic, it generally is preferred that a signal sequence be encoded so that the mature protein is secreted from the eukaryotic host.

An amino terminal methionine may or may not be present on the expressed polypeptides of this invention. If the terminal methionine is not cleaved by the

expression host, it may, if desired, be chemically removed by standard techniques .

A wide variety of expression host/vector

combinations may be employed in expressing the DNA

sequences of this invention. Useful expression vectors for eukaryotic hosts include, for example, vectors

comprising expression control sequences from SV40, bovine papilloma virus, adenovirus, adeno-associated virus, cytomegalovirus, and retroviruses . Useful expression vectors for bacterial hosts include bacterial plasmids, such as those from E. coli , including pBluescript, pGEX2T, pUC vectors, col E1, pCR1, pBR322, pMB9 and their

derivatives, wider host range plasmids, such as RP4, phage DNAs, e.g., the numerous derivatives of phage lambda, e.g. λgt10 and λgt11, NM989, and other DNA phages, such as M13 and filamentous single stranded DNA phages. Useful expression vectors for yeast cells include the 2μ plasmid and derivatives thereof. Useful vectors for insect cells include pVL 941.

In addition, any of a wide variety of expression control sequences may be used in these vectors to express the DNA sequences of this invention. Useful expression control sequences include the expression control sequences associated with structural genes of the foregoing expression vectors. Examples of useful expression control sequences include, for example, the early and late

promoters of SV40 or adenovirus, the lac system, the trp system, the TAC or TRC system, the T3 and T7 promoters the major operator and promoter regions of phage lambda, the control regions of fd coat protein, the promoter for 3- phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters of the yeast alpha-mating system and other constitutive and indueible promoter sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof. The T7 RNA polymerase promoter Φ10 is particularly useful in the expression of HSP72 in E. coli (Example 3).

Host cells transformed with the foregoing vectors form a further aspect of this invention. A wide variety of unicellular host cells are useful in expressing the DNA sequences of this invention. These hosts may include well known eukaryotic and prokaryotic hosts, such as strains of E. coli , Pseudomonas, Bacillus,

Streptomyces, fungi, yeast, insect cells such as

Spodoptera frugiperda (SF9), animal cells such as CHO and mouse cells, African green monkey cells such as COS 1, COS 7, BSC 1, BSC 40, and BMT 10, human cells, and plant cells in tissue culture. Preferred host organisms include bacteria such as E . coli and B . subtilis, and mammalian cells in tissue culture.

It should of course be understood that not all vectors and expression control sequences will function equally well to express the DNA sequences of this

invention. Neither will all hosts function equally well with the same expression system. However, one of skill in the art may make a selection among these vectors,

expression control sequences and hosts without undue experimentation and without departing from the scope of this invention. For example, in selecting a vector, the host must be considered because the vector must replicate in it. The vector's copy number, the ability to control that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered. In selecting an expression control sequence, a variety of factors should also be considered. These include, for example, the relative strength of the sequence, its controllability, and its compatibility with the DNA sequences of this invention, particularly as regards potential secondary structures. Unicellular hosts should be selected by consideration of their compatibility with the chosen vector, the toxicity of the product coded for by the DNA sequences of this invention, their

secretion characteristics, their ability to fold the protein correctly, their fermentation or culture

requirements, and the ease of purification from them of the products coded for by the DNA sequences of this invention. Within these parameters, one of skill in the art may select various vector/expression control

sequence/host combinations that will express the DNA sequences of this invention on fermentation or in large scale animal culture.

The polypeptides encoded by the DNA sequences of this invention may be isolated from the fermentation or cell culture and purified using any of a variety of conventional methods including: liquid chromatography such as normal or reversed phase, using HPLC, FPLC and the like; affinity chromatography (such as with inorganic ligands or monoclonal antibodies); size exclusion

chromatography; immobilized metal chelate chromatography; gel electrophoresis; and the like. One of skill in the art may select the most appropriate isolation and

purification techniques without departing from the scope of this invention.

In addition, the polypeptides of this invention may be generated by any of several chemical techniques. For example, they may be prepared using the solid-phase synthetic technique originally described by R. B. Merrifield, "Solid Phase Peptide Synthesis. I. The

Synthesis Of A Tetrapeptide", J. Am. Chem. Soc., 83, pp. 2149-54 (1963), or they may be prepared by synthesis in solution. A summary of peptide synthesis techniques may be found in E. Gross & H. J. Meinhofer, 4 The

Peptides: Analysis, Synthesis, Biology; Modern Techniques Of Peptide And Amino Acid Analysis, John Wiley & Sons, (1981) and M. Bodanszky, Principles Of Peptide Synthesis, Springer-Verlag (1984).

The preferred compositions and methods of this invention comprise polypeptides having enhanced

immunogenicity. Such polypeptides may result when the native forms of the polypeptides or fragments thereof are modified or subjected to treatments to enhance their immunogenic character in the intended recipient.

Preferred polypeptides are fragments that are specific to Streptococcal species such as fragments selected from the C-terminal portion of thenative polypeptides. Numerous techniques are available and well known to those of skill in the art which may be used, without undue

experimentation, to substantially increase the

immunogenicity of the polypeptides herein disclosed. For example, the polypeptides may be modified by coupling to dinitrophenol groups or arsanilic acid, or by denaturation with heat and/or SDS . Particularly if the polypeptides are small polypeptides synthesized chemically, it may be desirable to couple them to an immunogenic carrier. The coupling of course, must not interfere with the ability of either the polypeptide or the carrier to function

appropriately. For a review of some general

considerations in coupling strategies, see Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, ed. E. Harlow and D. Lane (1988). Useful immunogenic carriers are well known in the art. Examples of such carriers are keyhole limpet hemocyanin (KLH); albumins such as bovine serum albumin (BSA) and ovalbumin, PPD (purified protein derivative of tuberculin); red blood cells; tetanus toxoid; cholera toxoid; agarose beads; activated carbon; or bentonite.

Modification of the amino acid sequence of the polypeptides disclosed herein in order to alter the lipidation state is also a method which may be used to increase their immunogenicity and biochemical properties. For example, the polypeptides or fragments thereof may be expressed with or without the signal sequences that direct addition of lipid moieties.

In accordance with this invention, derivatives of the polypeptides may be prepared by a variety of methods, including by in vi tro manipulation of the DNA encoding the native polypeptides and subsequent expression of the modified DNA, by chemical synthesis of derivatized DNA sequences, or by chemical or biological manipulation of expressed amino acid sequences.

For example, derivatives may be produced by substitution of one or more amino acids with a different natural amino acid, an amino acid derivative or non-native amino acid, conservative substitution being preferred, e.g., 3-methylhistidine may be substituted for histidine, 4-hydroxyproline may be substituted for proline, 5- hydroxylysine may be substituted for lysine, and the like.

Causing amino acid substitutions which are less conservative may also result in desired derivatives, e.g., by causing changes in charge, conformation and other biological properties . Such substitutions would include for example, substitution of a hydrophilic residue for a hydrophobic residue, substitution of a cysteine or proline for another residue, substitution of a residue having a small side chain for a residue having a bulky side chain or substitution of a residue having a net positive charge for a residue having a net negative charge. When the result of a given substitution cannot be predicted with certainty, the derivatives may be readily assayed

according to the methods disclosed herein to determine the presence or absence of the desired characteristics. The polypeptides may also be prepared with the objective of increasing stability or rendering the

molecules more amenable to purification and preparation. One such technique is to express the polypeptides as fusion proteins comprising other S. pneumoniae or non- S. pneumoniae sequences. It is preferred that- the fusion proteins comprising the polypeptides of this invention be produced at the DNA level, e.g., by constructing a nucleic acid molecule encoding the fusion, transforming host cells with the molecule, inducing the cells to express the fusion protein, and recovering the fusion protein from the cell culture. Alternatively, the fusion proteins may be produced after gene expression according to known methods. An example of a fusion protein according to this invention is the FucI/HSP72 (C-169) protein of Example 3, infra.

The polypeptides of this invention may also be part of larger multimeric molecules which may be produced recombinantly or may be synthesized chemically. Such multimers may also include the polypeptides fused or coupled to moieties other than amino acids, including lipids and carbohydrates.

The polypeptides of this invention are particularly well-suited for the generation of antibodies and for the development of a protective response against disease. Accordingly, in another aspect of this

invention, we provide antibodies, or fragments thereof, that are immunologically reactive with HSP72. The

antibodies of this invention are either elicited by immunization with HSP72 or an immunologically related polypeptide, or are identified by their reactivity with HSP72 or an immunologically related polypeptide. It should be understood that the antibodies of this invention are not intended to include those antibodies which are normally elicited in an animal upon infection with

naturally occurring S. pneumoniae and which have not been removed from or altered within the animal in which they were elicited. The antibodies of this invention may be intact immunoglobulin molecules or fragments thereof that contain an intact antigen binding site, including those fragments known in the art as F(v), Fab, Fab' and F(ab')2. The antibodies may also be genetically engineered or

synthetically produced. The antibody or fragment may be of animal origin, specifically of mammalian origin, and more specifically of murine, rat, monkey or human origin. It may be a natural antibody or fragment, or if desired, a recombinant antibody or fragment. The antibody or

antibody fragments may be of polyclonal, or preferably, of monoclonal origin. They may be specific for a number of epitopes but are preferably specific for one.

Specifically preferred are the monoclonal antibodies F1- Pn3.1, F2-Pn3.2, F2-Pn3.3 and F2-Pn3.4 of Example 2,

infra. One of skill in the art may use the polypeptides of this invention to produce other monoclonal antibodies which could be screened for their ability to confer protection against S. pneumoniae , S. pyogenes, S.

agalactiae or other Streptococcal related bacterial infection when used to immunize naive animals. Once a given monoclonal antibody is found to confer protection, the particular epitope that is recognized by that antibody may then be identified. Methods to produce polyclonal and monoclonal antibodies are well known to those of skill in the art. For a review of such methods, see Antibodies, A Laboratory Manual , supra, and D.E. Yelton, et al., Ann. Rev, of Biochem., 50, pp. 657-80 (1981). Determination of immunoreactivity with a polypeptide of this invention may be made by any of several methods well known in the art, including by immunoblot assay and ELISA.

An antibody of this invention may also be a hybrid molecule formed from immunoglobulin sequences from different species (e.g., mouse and human) or from portions of immunoglobulin light and heavy chain sequences from the same species. It may be a molecule that has multiple binding specificities, such as a bifunctional antibody prepared by any one of a number of techniques known to those of skill in the art including: the production of hybrid hybridomas; disulfide exchange; chemical cross- linking; addition of peptide linkers between two

monoclonal antibodies; the introduction of two sets of immunoglobulin heavy and light chains into a particular cell line; and so forth. The antibodies of this invention may also be human monoclonal antibodies, for example those produced by immortalized human cells, by SCID-hu mice or other non-human animals capable of

producing "human" antibodies, or by the expression of cloned human immunoglobulin genes.

In sum, one of skill in the art, provided with the teachings of this invention, has available a variety of methods which may be used to alter the biological properties of the antibodies of this invention including methods which would increase or decrease the stability or half-life, immunogenicity, toxicity, affinity or yield of a given antibody molecule, or to alter it in any other way that may render it more suitable for a particular

application.

The polypeptides, DNA sequences and antibodies of this invention are useful in prophylactic, therapeutic and diagnostic compositions for preventing, treating and diagnosing disease.

Standard immunological techniques may be

employed with the polypeptides and antibodies of this invention in order to use them as immunogens and as vaccines. In particular, any suitable host may be

injected with a pharmaceutically effective amount of polypeptide to generate monoclonal or polyvalent

antibodies or to induce the development of a protective immunological response against disease. Preferably, the polypeptide is selected from the group consisting of

HSP72 (SEQ ID NO:5), HSP70 (SEQ ID NO:20 and SEQ ID NO: 22) or fragments thereof. As used herein, a "pharmaceutically effective amount" of a polypeptide or of an antibody is the amount that, when administered to a patient, elicits an immune response that is effective to prevent or lessen the severity of Streptococcal or related bacterial

infections.

The administration of the polypeptides or antibodies of this invention may be accomplished by any of the methods described in Example 10, infra, or by a variety of other standard procedures. For a detailed discussion of such techniques, see Antibodies, A

Laboratory Manual , Cold Spring Harbor Laboratory, ed.

E. Harlow and D. Lane (1988). Preferably, if a

polypeptide is used, it will be administered with a pharmaceutically acceptable adjuvant, such as complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptides) or ISCOM (immunostimulating complexes). Preferably, the composition will include a water-in-oil emulsion or aluminum hydroxide as adjuvant and will be administered intramuscularly. The vaccine composition may be

administered to the patient at one time or over a series of treatments. The most effective mode of administration and dosage regimen will depend upon the level of

immunogenicity, the particular composition and/or adjuvant used for treatment, the severity and course of the

expected infection, previous therapy, the patient's health status and response to immunization, and the judgment of the treating physician. For example, in an

immunocompetent patient, the more highly immunogenic the polypeptide, the lower the dosage and necessary number of immunizations. Similarly, the dosage and necessary treatment time will be lowered if the polypeptide is administered with an adjuvant.

Generally, the dosage will consist of an initial injection, most probably with adjuvant, of about 0.01 to 10 mg, and preferable 0.1 to 1.0 mg, HSP72 antigen per patient, followed most probably by one or maybe more booster injections. Preferably, boosters will be

administered at about 1 and 6 months after the initial injection.

Any of the polypeptides of this invention may be used in the form of a pharmaceutically acceptable salt. Suitable acids and bases which are capable of forming salts with the polypeptides of the present invention are well known to those of skill in the art, and include inorganic and organic acids and bases.

To screen the polypeptides and antibodies of this invention for their ability to confer protection against diseases caused by S. pneumoniae or related bacteria, or their ability to lessen the severity of such infection, one of skill in the art will recognize that a number of animal models may be used. Any animal that is susceptible to infection with S. pneumoniae or related bacteria may be useful. The Balb/c mice of Example 5, infra, are the preferred animal model for active

immunoprotection screening, and the severe-combined immunodeficient mice of Example 5 are the preferred animal model for passive screening. Thus, by administering a particular polypeptide or antibody to these animal models, one of skill in the art may determine without undue experimentation whether that polypeptide or antibody would be useful in the methods and compositions claimed herein.

According to another embodiment of this invention, we describe a method which comprises the steps of treating a patient with a vaccine comprising a

pharmaceutically effective amount of any of the

polypeptides of this invention in a manner sufficient to prevent or lessen the severity, for some period of time, of Streptococcal or related bacterial infection. Again, the preferred polypeptide for use in such methods is

HSP70/HSP72, or fragments thereof.

The polypeptides, DNA sequences and antibodies of this invention may also form the basis for diagnostic methods and kits for the detection of pathogenic organisms. Several diagnostic methods are possible. For example, this invention provides a method for the

detection of Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae or related bacteria in a biological sample comprising the steps of:

(a) isolating the biological sample from a patient;

(b) incubating an antibody of this invention, or fragment thereof with the biological sample to form a mixture; and

(c) detecting specifically bound antibody or fragment in the mixture which indicates the presence of Streptococcus pneumoniae, Streptococcus pyogenes,

Streptococcus agalactiae or related bacteria. Preferable antibodies for use in this method include monoclonal antibodies F1-Pn3.1, F2-Pn3.2, F2-Pn3.3 and F2-Pn3.4.

Alternatively, this invention provides a method for the detection of antibodies specific to Streptococcus pneumoniae or related bacteria in a biological sample comprising:

(a) isolating the biological sample from a patient;

(b) incubating a polypeptide of this invention or fragment thereof, with the biological sample to form a mixture; and

(c) detecting specifically bound polypeptide in the mixture which indicates the presence of antibodies specific to Streptococcus pneumoniae or related bacteria. HSP72 (SEQ ID NO :5), the C-169 fragment thereof (residues 439-607 of SEQ ID NO:5), the C-151 fragment thereof

(residues 457-607 of SEQ ID NO; 5) and peptide fragments GFDAERDAAQAALDD (residues 527-541 of SEQ ID NO : 5) and AEGAQATGNAGDDVV (residues 586-600 of SEQ ID NO : 5) are the preferred polypeptide and fragments in the above method for the detection of antibodies.

One of skill in the art will recognize that these diagnostic tests may take several forms, including an enzyme-linked immunosorbent assay (ELISA), a

radioimmunoassay or a latex agglutination assay.

The diagnostic agents may be included in a kit which may also comprise instructions for use and other appropriate reagents, preferably a means for detecting when the polypeptide or antibody is bound. For example, the polypeptide or antibody may be labeled with a

detection means that allows for the detection of the polypeptide when it is bound to an antibody, or for the detection of the antibody when it is bound to

S. pneumoniae or related bacteria. The detection means may be a fluorescent labeling agent such as fluorescein isocyanate (FIC), fluorescein isothiocyanate (FITC), and the like, an enzyme, such as horseradish peroxidase (HRP), glucose oxidase or the like, a radioactive element such as ¹²⁵I or ⁵¹Cr that produces gamma ray emissions, or a radioactive element that emits positrons which produce gamma rays upon encounters with electrons present in the test solution, such as ¹¹C , ¹⁵O, or ¹³N. Binding may also be detected by other methods, for example via avidin- biotin complexes. The linking of the detection means is well known in the art. For instance, monoclonal antibody molecules produced by a hybridoma may be metabolically labeled by incorporation of radioisotope-containing amino acids in the culture medium, or polypeptides may be conjugated or coupled to a detection means through

activated functional groups.

The DNA sequences of this invention may be used to design DNA probes for use in detecting the presence of Streptococcus pneumoniae or related bacteria in a

biological sample. The probe-based detection method of this invention comprises the steps of:

(a) isolating the biological sample from a patient;

(b) incubating a DNA probe having a DNA

sequence of this invention with the biological sample to form a mixture; and (c) detecting specifically bound DNA probe in the mixture which indicates the presence of Streptococcus pneumoniae or related bacteria.

The DNA probes of this invention may also be used for detecting circulating nucleic acids in a sample, for example using a polymerase chain reaction, as a method of diagnosing Streptococcus pneumoniae or related

bacterial infections. The probes may be synthesized using conventional techniques and may be immobilized on a solid phase, or may be labeled with a detectable label. A preferred DNA probe for this application is an oligomer having a sequence complementary to at least about 6 contiguous nucleotides of HSP72 (SEQ ID NO : 4, nucleotides 682-2502).

The polypeptides of this invention may also be used to purify antibodies directed against epitopes present on the protein, for example, using immunoaffinity purification of antibodies on an antigen column.

The antibodies or antibody fragments of this invention may be used to prepare substantially pure proteins according to the invention for example, using immunoaffinity purification of antibodies on an antigen column.

EXAMPLES

In order that this invention may be better understood, the following examples are set forth. These examples are for purposes of illustration only, and are not to be construed as limiting the scope of the invention in any manner .

Example 1 describes the identification of HSP72, an immunoreactive heat shock protein according to the invention. Example 2 describes the isolation of

monoclonal antibodies against epitopes of HSP72. Example 3 describes the preparation of recombinant HSP72 and fragments of HSP72 according to the invention. Example 4 describes the antigenic specificity and immunoreactivity of monoclonal antibodies directed against HSP72, and the identification of immunologically related proteins

according to the invention. Example 5 describes processes for obtaining substantially pure HSP72, and the use of HSP72 or antibodies against it to protect against

experimental S. pneumoniae infection. Example 6 describes the preparation of recombinant C-151 fragment of HSP72 according to the invention. Example 7 describes the humoral immune response following the immunization with recombinant HSP72 or fragments of HSP72 according to the invention. Example 8 describes the localization of linear B-cell epitopes on the HSP72. Example 9 describes the hsp70 genes and HSP70 proteins from S. agalactiae and S. pyogenes . Example 10 describes the use of HSP72 antigen in a human vaccine.

EXAMPLE 1 - Identification of Immunoreactive

S. pneumoniae Heat Shock Proteins

A. Procedures

Unless otherwise noted, the following procedures were used throughout the Examples herein. 1. Bacteria

S. pneumoniae strains were provided by the

Laboratoire de la Sante Publique du Quebec, Sainte-Anne de Bellevue. S. pneumoniae strains included type 4 strain 53 and type 6 strain 64. If not specified, S. pneumoniae type 6 strain 64 was used. Bacterial strains were grown overnight at 37°C in 5% CO₂ on chocolate agar plates.

2. Antigen Preparations

Various S. pneumoniae antigens were prepared for immunization and immunoassays . Heat-killed whole cell antigens were obtained by incubating bacterial suspensions in a water bath prewarmed at 56 C for 20 minutes.

Detergent-soluble proteins were extracted from

S. pneumoniae as follows. Heat-killed bacteria were suspended in 10 mM Hepes buffer (4-(2-Hydroxyethyl)-1- piperazinethan-sulfonsaure) (Boehringer Mannheim GmbH,

Germany) at pH 7.4 and sonicated at 20,000 Kz/second, four times for 30 seconds. Intact cells and large debris were removed by centrifugation at 1,700 g for 20 minutes. The supernatant was collected and centrifuged at 100,000 g for 60 minutes. The pellet was resuspended in 1 ml of Hepes buffer, and 1 ml of 2% N-lauroyl sarcosine (Sigma Chemical Co., St. Louis, Mo.) was added. The mixture was incubated for 30 minutes at room temperature and the detergent- soluble fraction was harvested by centrifugation at

100,000 g for 60 minutes.

3. Heat Shock Treatment

S. pneumoniae bacteria (type 4, strain 53 and type 6, strain 64) were resuspended in Eagle's Minimal

Essential Medium lacking methionine (ICN Biomedicals Inc., Costa Mesa, CA) and supplemented with 1% BIO-X® (Quelab Laboratories, Montreal, Canada) for 15 minutes at 37°C and then divided into fractions of equal volume. The samples were incubated at either 37°C or 45°C for 5 minutes and then labeled with 100 μCi/ml [³⁵S] methionine (ICN) for 10, 30, or 60 minutes at37°C. The bacteria were harvested and cell extracts were prepared using Tris-HCl lysis buffer as described above, or SDS-PAGE sample buffer.

4. Immunization Of Mice

Female Balb/c mice (Charles River Laboratories, St-Constant, Quebec, Canada) were immunized with

S. pneumoniae antigens. Immune sera to S. pneumoniae type 6 strain 64 were obtained from mice immunized, at two-week intervals, by subcutaneous injections of 10⁷ heat- killed bacteria or 20 μg of detergent-soluble pneumococcal proteins absorbed to aluminum hydroxide adjuvant

(Alhydrogel®; Cedarlane Laboratories Ltd., Horny, Ontario, Canada). Blood samples were collected prior to

immunization and at seven days following the first and second immunization.

5. SDS-PAGE and Immunoassays

Cell extracts were prepared for SDS-PAGE,

Western blot analysis and radioimmunoprecipitation assay by incubating bacterial suspensions in Tris-HCl lysis buffer (50mM Tris, 150 mM NaCl, 0.1% Na dodecyl sulfate, 0.5% Na deoxycholate, 2% Triton® X-100, 100 μg/ml

phenylmethylsulfonylfluoride, and 2μg/ml aprotinin) at pH 8.0 for 30 minutes on ice. Lysed cells were cleared by centrifugation and the supernatants were aliquoted and kept frozen at -70 C.

SDS-PAGE were performed on a 10% polyacrylamide gel according to the method of Laemmli [Nature, 227, pp. 680-685 (1970)], using the Mini Protean® system (Bio- Rad Laboratories Ltd., Mississauga, Canada). Samples were denatured by boiling for 5 minutes in sample buffer containing 2% 2-mercaptoethanol . Proteins were resolved by staining the polyacrylamide gel with PhastGel Blue® (Pharmacia Biotech Inc., Baie d'Urfe, Canada). The radiolabeled products were visualized by fluorography. Fluorograms were scanned using a laser densitometer.

Immunoblot procedures were performed according to the method of Towbin et al. [Proc. Natl . Acad. Sci. USA, 76, pp. 4350-4354 (1979)]. The detection of antigens reactive with antibodies was performed by an indirect antibody immunoassay using peroxidase-labeled anti-mouse immunoglobulins and the o-dianisidine color substrate.

Radioimmunoprecipitation assays were performed as described by J.A. Wiley et al. [J. Virol., 66,

pp. 5744-5751 (1992)]. Briefly, sera or hybridoma culture supernatants were added to radiolabeled samples containing equal amounts of [³⁵S] methionine. The mixtures were allowed to incubate for 90 minutes at 4 C with constant agitation. The immune complexes were then precipitated with bovine serum albumin-treated protein A Sepharose (Pharmacia) for 1 hour at 4 C. The beads were pelleted and washed three times in Tris buffered saline at pH 8.0, and the antigen complexes were then dissociated by boiling in sample buffer. The antigens were analyzed by

electrophoresis on SDS-PAGE. The gels were fixed,

enhanced for fluorography using Amplify® (Amersham Canada Limited, Oakville, Ontario, Canada), dried, and then exposed to X-ray film.

B. Characterization of the Heat

Shock Response in S. pneumoniae

We studied the heat shock response of S. pneumoniae by examining the pattern of protein

synthesis before and after a shift from 37°C to 45°C.

FIG. 1 shows the results when S. pneumoniae type 6 strain 64 (panel A) and type 4 strain 53 (panel B) were grown at 37°C, incubated at 37°C (lanes 1,3,5,7 and 9) or at 45°C (lanes 2, 4, 6, 8 and 10) for 5 minutes, and then labeled with [³⁵S] methionine for 10 minutes (lanes 1,2 and 7,8), 30 minutes (lanes 3,4 and 9,10), or 60 minutes (lanes 5,6).

The fluorogram derived from SDS-PAGE indicated that the synthesis of at least three proteins was

increased by increasing the temperature (FIG. 1). The most prominent induced protein was about 72 kDa (HSP72), whereas the other two were approximately 80 kDa (HSP80) and 62 kDa (HSP62). Increased protein synthesis was already apparent after 10 minutes of labeling (FIG. 1, lanes 1, 2 and 7, 8) and became more significant when the labeling period was prolonged to 30 minutes (FIG. 1, lanes 3, 4 and 9, 10) and 60 minutes (FIG. 1, lanes 5, 6). The effect of elevated temperature on the protein

synthesis profile of two different S. pneumoniae strains was similar, with HSPs of similar molecular mass being synthesized (compare Panel A (type 6 strain 64) to Panel B (type 4 strain 53) in FIG. 1).

Analysis of the densitometric tracings from scanning the protein synthesis profiles allowed the estimation of the relative amounts of proteins. For example, with respect to heat-shocked S. pneumoniae type 6 strain 64, after 10 minutes of labeling, HSP80 and HSP62 made up 2.9% and 6.8% of the labeled proteins,

respectively, compared to less than 0.1% at 37°C (FIG. 2). Labeled proteins having an apparent molecular mass of 72 kDa were detected at both 37°C and 45°C conditions

(FIG. 2). Radioimmunoprecipitation analysis revealed, however, that HSP72 was undetectable at 37°C (supra; and FIGS. 3, 4 and 6) thus indicating that peak 9 from FIG. 2 corresponds to protein component(s) comigrating with

HSP72. Assuming no variation in the labeling of this material, these results would suggest that the amount of HSP72 represents 8.7% of the total labeled cell protein after heat shock treatment. A comparison of the

densitometric tracings revealed that cellular proteins corresponding to peaks 4, 10, 13, 17, 19, and 21 were synthesized at almost the same rate irrespective of heat shock treatment (FIG. 2). However, the synthesis of several proteins (peaks 1, 2, 3, 15, 20, 22, 24, and 26) declined considerably in response to heat shock (FIG. 2).

C . Immune Responses to S. pneumoniae HSPs

In order to assess the antibody response to pneumococcal HSPs, mouse sera were first assayed by radioimmunoprecipitation. The repertoire of labeled proteins recognized by sera from mice immunized with S. pneumoniae antigen preparations are shown in FIGS. 3 and 4. FIG. 3 relates to detergent soluble protein preparations. FIG. 4 relates to heat-killed bacterial preparation. Although many bands were detected by most antisera, HSP72 was a major precipitation product. The specificity of antibodies for HSP72 was demonstrated by the detection of proteins among heat-shocked products only (FIG. 3, lanes 4, 6, 8 and 10; FIG. 4, lanes 4, 6 and 8 ) . Interestingly, all immunized mice consistently recognized HSP72. The antibodies reactive with the HSP72 were not specific to the strain used during the immunization since strong reactivities were observed with heterologous

S. pneumoniae HSP72. It should be noted that in addition to HSP72, one sera precipitated comigrating product labeled at both 37°C and 45°C (FIG. 4, lane 4). This 72 kDa-product probably corresponds to component from peak 9 in FIG. 2 and was not detected in immunoblots. HSP62 is another immune target which was precipitated by some but not all immune sera (FIG. 3, lane 6 and, FIG. 4, lanes 4 and 6). None of the sera tested reacted with HSP80. No proteins were precipitated when preimmune sera taken from the mice used in this study were tested for the presence of antibodies reactive with the labeled products.

As depicted in FIGS. 3 and 5, antibodies to HSP72 could be detected after one immunization with either detergent-soluble proteins or whole cells extracts of S. pneumoniae . In addition, a marked increase in the antibody response to HSP72 was observed after a second immunization (FIG. 3, compare 4 and 6, and lanes 8 and 10).

The immunoblot patterns of 15 mice immunized with heat-killed S. pneumoniae bacteria were remarkably consistent with the results of the previously described radioimmunoprecipitation. Although antibody response variation occurred to a variety of proteins, HSP72 was a major immunoreactive antigen with 8 (53%) positive sera after the first immunization (FIG. 5). Antibodies to HSP72 were detected in 13 out of 15 (87%) immune sera tested after the second immunization. Two other prominent antigens having apparent molecular mass of 53.5 and 47 kDa were detected in 5 (33%) and 7 (47%) sera, respectively (FIG. 5) . The 72 kDa-reactive band was confirmed as the pneumococcal HSP72 by using recombinant HSP72 antigens

(Example 3, infra) in an immunoblot assay. Preimmune sera failed to detect any pneumococcal proteins.

EXAMPLE 2 - Isolation of Monoclonal Antibodies

Against Epitopes of HSP72

Procedures

1. Immunization of Mice And Fusion

Female Balb/c mice (Charles River Laboratories) were immunized with S. pneumoniae antigens. One set of mice (fusion experiment 1) were immunized by peritoneal injection with 10⁷ formalin-killed whole cell antigen from strain MTL suspended in Freund's complete adjuvant, and were boosted at two-week intervals with the same antigen and then with a sonicate from heat-killed bacteria in Freund's incomplete adjuvant. A second group of mice

(fusion experiment 2) were immunized three times at three- week intervals with 75 μg of detergent-soluble

pneumococcal antigens extracted from strain 64 (type 6) in 25 μg of Quil A adjuvant (Cedarlane Laboratories Ltd., Hornby, Ontario, Canada). Three days before fusion, all mice were injected intraperitoneally with the respective antigen suspended in PBS alone. Hybridomas were produced by fusion of spleen cells with nonsecreting SP2/0 myeloma cells as previously described by J. Hamel et al. [J. Med. Microbiol., 23, pp. 163-170 (1987)]. Specific hybridoma were cloned by sequential limiting dilutions, expanded and frozen in liquid nitrogen. The class, subclass, and light-chain type of MAbs were determined by ELISA as described by D. Martin et al., [Eur. J. Immunol., 18, pp. 601-606 (1988)] using reagents obtained from Southern Biotechnology Associates Inc. (Birmingham, AL). 2. Subcellular Fractionation

Pneumococci were separated into subcellular fractions according to the technique described by Pearce et al. [Mol. Microbiol., 9, pp. 1037-1050 (1993)].

Briefly, S. pneumoniae strain 64 (type 6) was grown in Todd Hewitt broth supplemented with 0.5% (w/v) yeast extract for 6 hours at 37°C and isolated by centrifugation. Cell pellets were resuspended in 25 mM Tris-HCl pH 8.0, 1 mM EDTA, 1 mM phenylmethylsulphonylfluoride (PMSF) and sonicated for 4 minutes with 15 second bursts. Cellular debris were removed by centrifugation. The bacterial membranes and cytoplasmic contents were separated by centrifugation at 98,000 g for 4 hours. The cytoplasmic (supernatant) and the membrane (pellet) fractions were adjusted to 1 mg protein per ml and subjected to SDS-PAGE and immunoblot analyses. B. Identification and Characterization

of MAbs to the HSP72 of S. pneumoniae

Culture supernatants of hybridomas were initially screened by dot enzyme immunoassay using whole cells from S. pneumoniae strain 65 (type 4) according to the procedures described in D. Martin et al. (supra).

Positive hybridomas were then retested by immunoblotting in order to identify the hybridomas secreting MAbs

reactive with the HSP72. Of 26 hybridomas with anti- S. pneumoniae reactivity in immunoblot, four were found to recognize epitopes present on a protein band with an apparent molecular mass of 72 kDa. The four hybridomas were designated F1-Pn3.1 (from fusionn experiment 1) and F2-Pn3.2, F2-Pn3.3 and F2-Pn3.4 (from fusion experiment 2). Isotype analysis revealed that hybridoma F1-Pn3.1

(from fusion experiment 1) secreted IgG_-2ak immunoglobulins, whereas hybridomas F2-Pn3.2, F2-Pn3.3, and F2-Pn3.4 (from fusion experiment 2) all secreted IgG_1k. The specificity of the MAbs for HSP72 was clearly demonstrated by the lack of radioimmunoprecipitation activity against

[³⁵S] methionine-labeled S. pneumoniae proteins obtained from cultures incubated at 37°C and the immunoprecipitation of a 72kDa-protein with heat shock-derived lysates

incubated at 45°C. FIG. 6, (lanes 5 and 6) demonstrates the results obtained for MAb F1-Pn3.1. The same results were obtained with MAbs F2-Pn3.2, F2-Pn3.3 and F2-Pn3.4

[³⁵S]methionine-labelled lysates from nonheat- shocked and heat-shocked S. pneumoniae cells probed with the MAbs were electrophoresed on SDS-PAGE gels and then subjected to Western blot analysis. The resulting

immunoblots revealed the presence of HSP72 antigen in both samples. FIG. 7, panel A, shows the results obtained for MAb F1-Pn3.1. The same results were obtained with MAbs F2-Pn3.2, F2-Pn3.3 and F2-Pn3.4. Accordingly, the heat shock stress did not significantly increase the reactivity of anti-HSP72 monoclonal antibodies. The fluorograph of the immunoblots, however, clearly showed that the heat shock response had occurred (FIG. 7, panel B). These experiments revealed that the rate of synthesis of

S. pneumoniae HSP72 increases in response to heat shock, but that the absolute amounts of HSP72 do not increase after heat shock.

C. Cellular localization of HSP72

In order to investigate the cellular location of HSP72, S. pneumoniae cell lysates were fractionated by differential centrifugation resulting in a soluble

fraction and a particulate fraction, enriched in membrane proteins, supra. Sample containing 15 μg protein of membrane fraction (lane 1) and cytoplasmic fraction (lane 2) of S. pneumoniae were electrophoresed on SDS-PAGE, transferred to nitrocellulose and probed with MAb F1- Pn3.1. In the resulting Western blots, HSP72 was found in both fractions, with the majority of the protein

associated with the cytoplasmic fraction (FIG. 8). EXAMPLE 3 - Molecular Cloning, Sequencing

and Expression of Genes Coding

for HSP72 Antigens

A. Procedures

1. Strains and Plasmids

Strains and plasmids used in this study are listed in Table 1.

E. coli strains were grown in L broth or on L agar at 37°C. When necessary, ampicillin was added to the media at the concentration of 50 μg/ml. Plasmids were isolated by using the Magic/Wizard® Mini-Preps kit

(Promega, Fisher Scientific, Ottawa, Canada).

2. General Recombinant DNA Techniques

Restriction endonucleases, T4 DNA ligase, and DNA molecular weight standards were purchased from

Boehringer Mannheim Canada, Laval, Quebec or Pharmacia Biotech, Uppsala, Sweden. DNA restriction endonuclease digestion and ligation were performed as described by J. Sambrook et al. [Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory Press, N.Y.

(1989)]. Agarose gel electrophoresis of DNA fragments was performed following the procedure of J. Sambrook et al.

(supra) using the TAE buffer (0.04 M Tris-acetate; 0.002 M EDTA) from Boehringer Mannheim. DNA fragments were purified from agarose gel by using the Prep-A-Gene® DNA purification kit (Bio-Rad Laboratories Ltd., Mississauga, Ontario). Transformation was carried out by

electroporation with the Gene Pulser® (Bio-Rad) following the protocol provided by the manufacturer.

3. Construction and Screening

of Genomic Library

A genomic S. pneuznoniae DNA library was

generated in the bacteriophage expression vector λgt11 (λgt11 cloning system, Amersham) according to the procedure provided by the manufacturer. Chromosomal DNA of S. pneumoniae type 6 strain 64 was prepared by

following the procedure of J.C. Paton et al. [Infect.

Immun., 54, pp. 50-55 (1986)]. The S. pneumoniae

chromosomal DNA was partially digested with EcoRI, and the 4- to 7-kb fragments were fractionated and purified from agarose gel. The fragments were ligated into λgt11 arms, packaged, and the resulting phage mixtures used to infect E. coli Y1090. Immunoscreening of plaques expressing recombinant HSP72 antigens was performed using HSP72- specific monoclonal antibody F1-Pn3.1, supra. Plaque clones expressing peptides recognized by MAb F1-Pn3.1 were isolated and purified. Liquid lysates were prepared and DNA was purified from a Promega LambdaSorb phage adsorbent according to the manufacturer's directions followed by conventional DNA purification procedures.

4. Southern Blot Analysis

The nonradioactive DIG DNA Labelling and

Detection kit, obtained from Boehringer Mannheim, was used to perform Southern blot analysis in this example. The DNA fragments selected for use as probes (infra) were purified by agarose gel electrophoresis and then labelled with digoxigenin (DIG)-11-dUTP. Pneumococcal chromosomal DNA was digested with Hindlll and the digests were

separated by electrophoresis on an 0.8% SDS-PAGE gel and transformed onto positive charged nylon membranes

(Boehringer Mannheim) as described by J. Sambrook et al. (supra). The membrane was then blotted with the DIG- labelled DNA probes according to the protocol of the manufacturer.

5. DNA Sequencing and Sequence Analysis

The DNA fragments sequenced in this example were first cloned into plasmid pDELTA 1 (GIBCO BRL Life Technologies, Burlington, Ontario). A series of nested deletions were generated from both strands by in vivo deletion mediated by Tn 1000 transposon transposition (Deletion Factory System, GIBCO BRL) following the

procedures provided by the supplier. These deletions were sized by agarose gel electrophoresis and appropriate deletion derivatives were selected for sequencing by the dideoxynucleotide chain terminating method of F. Sanger et al. [Proc. Natl. Acad. Sci. USA, 74, pp. 5463-5467 (1977)]. To sequence the gaps between deletion templates, oligonucleotides were synthesized by oligonucleotide synthesizer 392 (ABI, Applied Biosystems Inc., Foster City, CA). The sequencing reaction was carried out by PCR (DNA Thermal Cycler 480®, Perkin Elmer) using the Taq DyeDeoxy Terminator Cycle Sequencing kit (ABI), and DNA electrophoresis was performed on automated DNA sequencer 373A (ABI).

6. Expression of Cloned Gene in

E. coli T7 RNA pol/promoter system

High level expression of the cloned gene in this example was achieved by employing the bacteriophage T7 RNA polymerase/promoter system in E. coli. The DNA fragment specifying the recombinant protein was ligated into plasmids pT7-5 or pT7-6 [S. Tabor and C.C. Richardson, Proc. Natl. Acad. Sci. USA, 82, PP. 1074-1078 (1985)], in a proper orientation in which the gene to be expressed was placed under the control of phage T7 RNA polymerase specific promoter Φ10. The resulting plasmid was

transformed into E. coli strain BL21(DE3) [F.W. Studier, and B.A. Moffatt, J. Mol. Biol., 189, pp. 113-130 (1986)] which carries the T7 RNA polymerase structural gene on its chromosome under the control of the inducible lacUV5 promoter. Upon IPTG induction, the T7 RNA polymerase induced in the BL21(DE3) transformants specifically transcribed the gene under the control of T7 promoter Φ10. The overexpressed recombinant proteins were visualized by either Western blotting or Coomassie Blue staining. 7. N-terminal Amino Acid Sequence

Analysis of HSP72

Pneumococcal HSP72 was purified by

immunoprecipitation using MAb F1-Pn3.1 (supra) and samples of cell wall extracts of S. pneumoniae strain 64 prepared as described by L.S. Daniels et al. [Microb. Pathogen., 1, pp. 519-531 (1986)] as antigen. The immune precipitates were resolved by SDS-PAGE and then transferred to

polyvinylidene difluoride (PVDF) membrane by the method of P. Matsudaira [J. Biol. Chem., 262, pp. 10035-10038

(1987)]. PVDF membrane was stained with Coomassie Blue, the HSP72 band excised and then analyzed in an automated protein sequencer (ABI), according to standard procedures. B. Construction of Plasmids Containing

S. pneumoniae HSP72 Gene Fragments

Corresponding to C-169

The λgt11 S. pneumoniae genomic DNA library was screened with the HSP72-specific MAb F1-Pn3.1. Seventeen (17) immunoreactive clones were isolated and purified from a total of 1500 phages tested. To confirm the specificity of the proteins expressed by the recombinant phages,

Western blot analysis of the recombinant phage lysates was performed. Two groups of clones were identified among the 17 positive clones recognized by MAb F1-Pn3.1 and their representatives were designated as λJBD7 and λJBD17 for further characterization. As shown in FIG. 9, whole cell extracts from S. pneumoniae strain 64 (lane 1) and phage lysates from E. coli infected with λJBD17 (lanes 2 and 3) or λJBD7 (lanes 4 and 5) cultured in the presence (+) or absence (-) of IPTG were subjected to 10% polyacrylamide gel electrophoresis and were electrotransferred to

nitrocellulose. The immunoblot was probed with HSP72- specific MAb F1-Pn3.1. Clone λJBD17 had two EcoRI-EcoRI insert fragments of 2.4 kb and 2.3 kb (FIG. 10), and expressed a chimeric recombinant protein having an

apparent molecular mass of 74 kDa on SDS-PAGE gel (FIG. 9, lanes 2 and 3). Clone λJBD7 was found to contain a 2.3 kb EcoRI insert fragment and produced an apparent fusion protein consisting of LacZ and the 74 kDa chimeric .protein expressed from clone λJBD17. The fusion protein had an apparent molecular mass of 160 kDa as estimated by SDS- PAGE (FIG. 9, lane 5). The expression of the chimeric recombinant protein encoded by phage λJBD17 was

independent of IPTG induction (FIG. 9, lanes 2 and 3) while the expression of the recombinant fusion protein encoded by phage λJBD7 was dependent on induction of the lac promoter (FIG. 9, lanes 4 and 5).

In an attempt to subclone the HSP72 gene, the pneumococcal DNA insert from clone λJBD17 was extracted, purified and ligated into a low copy plasmid pWSK29 [R.F. Wang and S.R. Kushner, Gene, 100, pp. 195-199 (1991)] to generate plasmid pJBD171. The insert from pJBD171 was characterized by restriction mapping (Fig. 10B), and a series of subcloning and immunoblotting was carried out to define the boundaries of the gene coding for the antigen reactive with MAb F1-Pn3.1. The region responsible for expression of the 74 kDa chimeric protein was found to localize on the 3.2 kb EcoRI-EcoRV fragment, which

consists of the intact 2.4 kb EcoRI-EcoRI fragment and the 0.8 kb EcoRI-EcoRV portion of the 2.3 kb EcoRI-EcoRI fragment. The plasmid carrying the 3.2 kb EcoRI-EcoRV insert was designated pJBD179. C. Expression and DNA Sequence

Analysis of a Chimeric Gene

Coding for C-169

To further determine the transcriptional

direction of the gene coding for the 74 kDa chimeric protein on the 3.2 kb EcoRI-EcoRV fragment, and to

increase the yield of the 74 kDa chimeric protein for immunological study, we decided to express the 74 kDa chimeric protein in the E. coli T7 RNA and T7 promoter system. The 3.2 kb EcoRI-EcoRV fragment, derived from PJBD179, was ligated into plasmids pT7-5 and pT7-6 in which the multi-cloning sites were placed in opposite orientation with respect to the T7 RNA polymerase specific T7 promoter Φ10. The ligation mixture was used to transform E. coli JM109 and positive transformants

reactive with MAb F1-Pn3.1 were identified by the colony lifting method described by J. Sambrook et al. [supra]. The resulting recombinant plasmids, derived from pT7-5 and pT7-6, were designated pJBDf51 and pJBDf62, respectively. The intact 3.2 kb EcoRI-EcoRV insert in these recombinant plasmids and their orientation was determined by

restriction mapping. To achieve overexpression of the 74 kDa chimeric protein, pJBDf51 and pJBDf62 were

transformed, separately, into E. coli BL21(DE3). The transformants were induced with IPTG (1 mM) for 3 hours at 37°C. The cells were harvested, washed, resuspended in 1% SDS and boiled for 10 minutes. The lysates were then used for SDS-PAGE and immunoblot analysis. As expected, both transformants produced the 74 kDa chimeric protein readily detected by Western blotting with MAb F1-Pn3.1 (FIG. 11). However, under the IPTG induction condition, only transformants BL21 (DE3 ) (pJBDf51) overexpressed the 74 kDa chimeric protein (FIG. 11A and B, lane 2) indicating that the transcriptional direction of the gene on the 3.2 kb EcoRI-EcoRV fragment is from the EcoRI end towards the EcoRV end (FIG. 10A).

The 3.2 kb EcoRI-EcoRV fragment was cloned into plasmid pDELTA 1 to yield plasmid pJBDΔl . A series of overlapping deletions were generated and used as DNA sequencing templates. The DNA sequence of the entire 3.2 kb EcoRI-EcoRV insert is SEQ ID NO:1. Two open reading frames ("ORFs") were found and their orientation is indicated in FIG. 10B ("ORF27" and "FucI-HSP72 (C-169)"). In front of these two ORFs, putative ribosome-binding sites were identified (SEQ ID NO:1, nucleotides 18-21 and 760-763). No obvious -10 and -35 promoter sequences were detected. ORF27 spans nucleotides 30-755 (SEQ ID NO:1) and encodes a protein of 242 amino acids with a calculated molecular weight of 27,066 daltons . The deduced amino acid sequence of this protein is SEQ ID NO:2. We

designated this gene orf27, and compared it to other known sequences. No homologous gene or protein was found. The large ORF (nucleotides 771-2912, SEQ ID NO:1) specifies a protein of 714 amino acids with a predicted molecular mass of 79,238 daltons. The deduced amino acid sequence of this protein is SEQ ID NO:3. This ORF was compared with other known sequences to determine its relationship to other amino acid sequences. This analysis revealed a high degree of similarity of the encoded protein to the

sequence of E. coli fucose isomerase (Fuel) and to several HSP70 gene family members, also known as DnaK genes.

Alignment of SEQ ID NO : 3 and those of the E. coli Fuel and HSP70 (Dnak) proteins indicated that the N-terminal portion corresponding to amino acids 1 to 545 (SEQ ID

NO:3) of the 74 kDa chimeric protein is highly homologous to E. coli Fuel, while the C-terminal portion

corresponding to amino acids 546-714 (SEQ ID NO:3) is similar to HSP70 (DnaK) proteins. It is noteworthy that there is an EcoRI restriction site lying in the junction of these two portions of the gene coding for the 74 kDa protein (SEQ ID NO:1, between nucleotides 2404 and 2405). Other restriction sites exist between nucleotides 971 and 972 (Pst I), nucleotides 1916 and 1917 (Pst I),

nucleotides 1978 and 1979 (Xho I), and nucleotides 3164 and 3165 (EcoRV). From these data we concluded that the 74 kDa protein was a chimeric protein encoded by two pieces of S. pneumoniae chromosomal DNA, a 2.4 kb EcoRI- EcoRI fragment derived from the Fuel homologous gene and a 2.3 kb EcoRI-EcoRI fragment derived from the HSP72 gene.

D. Southern Blot Analysis

Southern blotting was performed in order to confirm that the 74 kDa protein is a chimeric protein and to attempt to clone the entire pneumococcal HSP72 gene. Chromosomal S. pneumoniae DNA was digested with Hindlll to completion, separated on a 0.8% agarose gel, and

transferred onto two positively charged nylon membranes (Boehringer Mannheim). The membranes were then blotted with either the 0.8 kb EcoRI-EcoRV probe, derived from the 2.3 kb EcoRI-EcoRI fragment, or the 1 kb Pstl-PstI probe, obtained from the 2.4 kb EcoRI-EcoRI fragment. Both probes had been previously labelled with digoxigenin-dUTP. These two probes hybridized two individual Hindlll

fragments of different sizes (FIGS. 10B and 10C). The 0.8 kb EcoRI-EcoRV probe recognized the 3.2 kb Hindlll

fragment and the 1 kb Pstl-PstI probe reacted with the 4 kb Hindlll fragment. This result further indicated that the gene responsible for the expression of the 74 kDa chimeric protein was generated by fusion, in frame, of two pieces of EcoRI fragments, one originated from the

fragment containing the 5' portion of the S. pneumoniae Fuel homologue, the other derived from the segment

carrying the C-169 fragment of the pneumococcal HSP72 gene. The fact that the 0.8 kb EcoRI-EcoRV probe

hybridized a single 3.2 kb fragment suggested that there is only a single HSP72 gene copy in S. pneumoniae . E. Production of Recombinant HSP72

A partial pneumococcal genomic library was generated by ligation of the pool of Hindlll digests of chromosomal DNA, with sizes ranging from 2.8 to 3.7 kb, into plasmid pWSK29/Hindlll. The ligation mixture was used to transform E. coli strain JM 109 and the

transformants were screened by hybridization with the 0.8 kb EcoRI-EcoRV probe. One representative plasmid from four positive hybridizing clones was named pJBD291.

Restriction analysis of the insert and Western blot of the cell lysate of transformants were employed to verify that the plasmid pJBD291 indeed carries the 3.2 kb Hindlll fragment containing the HSP72 gene expressing the

recombinant HSP72 protein (FIG. 10B). The HSP72 protein expressed by the transformants (pJBD291) migrated on the SDS-PAGE gel at the same position as the native HSP72 protein (FIG. 12). To sequence the entire HSP72 gene and to overexpress the full-length HSP72 protein, the 3.2 kb Hindlll fragment was isolated from plasmid pJBD291, and subcloned into plasmids pDELTA 1 and pT7-5 to generate pJBDΔ4 and pJBDk51, respectively.

The entire 3.2 kb Hindlll DNA fragment carried on the plasmid pJBDΔ4 and the 2.3 kb EcoRI-EcoRI DNA fragment contained on the plasmid pJBD177 were sequenced. Altogether, the nucleotide sequence comprised 4320 base pairs and revealed two ORFs (SEQ ID NO : 4). The first ORF, starting at nucleotide 682 and ending at nucleotide 2502 (SEQ ID NO:4), was identified as the pneumococcal HSP72 gene, and the second ORF, spanning from nucleotide 3265 to nucleotide 4320 (SEQ ID NO :4), was located 764 base pairs downstream from the HSP72 structural gene and was

identified as the 5' portion of the pneumococcal DnaJ gene. The putative ribosome binding site ("AGGA") was located 9 base pairs upstream from the start codon of the HSP72 structural gene, while the typical ribosome binding site ("AGGA") was found 66 base pairs upstream from the start codon of the DnaJ structural gene. No typical 5' regulatory region was identified in front of these two genes. Restriction sites are located between nucleotides 1 and 2 (Hindlll), nucleotides 1318 and 1319 (EcoRI), nucleotides 1994 and 1995 (EcoRI), nucleotides 3343 and 3344 (Hindlll), and nucleotides 4315 and 4316 (EcoRI).

The gene organization of HSP72 (DnaK) and DnaJ in

S. pneumoniae is similar to that of E. coli [Saito, H. and Uchida, Mol. Gen. Genet. 164, 1-8 (1978)] as well as several other Gram positive bacteria [Wetzstein, M.

et al., J. Bacteriol. 174, 3300-3310 (1992)]. However, the intragenic region of S. pneumoniae is significantly larger and no ORF for the grpE gene was found upstream of the HSP72 (DnaK) structural gene.

The predicted HSP72 protein has 607 amino acids and a calculated molecular mass of 64,755 daltons, as compared to the 72 kDa molecular mass estimated by SDS- PAGE. The predicted HSP72 protein is acidic with an isoelectric point (pi) of 4.35. Automated Edman

degradation of the purified native HSP72 protein extracted from S. pneumoniae strain 64 revealed SKIIGIDLGTTN-AVAVLE as the 19 amino acid N-terminal sequence of the protein. The amino-terminal methionine was not detected, presumably due to in si tu processing which is known to occur in many proteins. No amino acid residue was identified on

position 13. The 19 amino acid N-terminal sequence obtained from the native HSP72 protein is in full

agreement with the 19 amino acid N-terminal sequence deduced from the nucleotide sequence of the recombinant

S. pneumoniae HSP72 gene (SEQ ID NO:5) thus confirming the cloning. This N-terminal sequence showed complete

identity with the DnaK protein from Lactococcus lactis and 68.4% identity with the DnaK protein from Escherichia Coli . Similarly, the alignment of the predicted amino acid sequence of HSP72 (SEQ ID NO:5) with those from other bacterial HSP70 (DnaK) proteins also revealed high homology (FIGS. 13A-13D). For example, HSP72 showed 54% - identity with the E. coli DnaK protein. The highest identity value was obtained from comparison with the Gram positive bacterium Lactococcus lactis , showing 85%

identity with HSP72. Like other HSP70 proteins of Gram positive bacteria, HSP72 misses a stretch of 24 amino acids near the amino terminus when compared with DnaK proteins from Gram negative bacteria (FIGS. 13A-13D).

Although HSP72 shares homology with HSP70 (DnaK) proteins from other organisms, it does possess some unique features. Sequence divergence of the HSP70 (DnaK) proteins is largely localized to two regions (residues 244 to 330 and 510 to 607, SEQ ID NO:5). More specifically, the peptide sequences GFDAERDAAQAALDD (residues 527 to 541, SEQ ID NO:5) and AEGAQATGNAGDDVV (residues 586 to 600, SEQ ID NO:5) are exclusive to HSP72. The fact that the C-terminal portion of HSP72 is highly variable

suggests that this portion carries antigenic determinants specific to S. pneumoniae . Consistent with this

hypothesis, monoclonal antibodies directed against the C- 169 fragment of HSP72 (infra), were not reactive with E. coli and S. aureus , which are known to express DnaK proteins similar to HSP72.

The truncated DnaJ protein of S. pneumoniae (SEQ ID NO: 6) has 352 amino acids, which show a high degree of similarity with the corresponding portions of the L .

lactis DnaJ protein (72% identity) and the E. coli DnaJ protein (51% identity). The predicted truncated DnaJ protein contains high glycine content (15%). Four Gly-, Cys-rich repeats, each with the Cys-X-X-Cys-X-Gly-X-Gly motif characteristic of DnaJ proteins [P.A. Silver and J.C. Way, Cell, 74, pp. 5-6 (1993)], were identified between amino acids 148 and 212 of the S. pneumoniae DnaJ protein (SEQ ID NO :6). Three repeated GGFGG sequences (residues 75-79, 81-85, and 90-94) were found near the N- terminus. F. Reactivity of MAbs Against

Recombinant Antigens

The four HSP72 specific MAbs (F1-Pn3.1, F2-

Pn3.2, F2-Pn3.3 and F2-Pn3.4, supra) were tested for their reactivity against proteins expressed by E. coli infected or transformed with recombinant phages and plasmids containing HSP72 sequences . The four individual MAbs reacted with the lacZ-HSP72 fusion protein expressed by the clone λJBD7, thus localizing the epitopes recognized by these MAbs to the C-terminal 169 residues.

Surprisingly, the proteins encoded by the pneumoccocal inserts in λJBD17 and pJBDΔl were recognized by only 3 of 4 Mabs . These results suggest that although the C-169 fragments synthesized in E. coli infected with λJBD7 and λJBD17 have the same primary structure, they have distinct conformation. The lack of reactivity of MAb F2-Pn3.2 with some recombinant proteins raised the possibility that this particular MAb recognizes a more complex epitope.

Although complex, F2-Pn3.2 epitopes are still recognizable on Western immunoblots. The complete HSP72_rec protein expressed by E. coli containing the recombinant plasmid pJBDΔ4 was reactive with all four MAbs.

EXAMPLE 4 - Antigenic Specificity and

Reactivity of HSP72-Specific

Monoclonal Antibodies The reactivity of MAbs F1-Pn3.1, F2-3.2., F2-

Pn3.3 and F2-Pn3.4 to a collection of bacterial strains including 20 S. pneumoniae strains representing 16

capsular serotypes (types 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 14, 15, 19, 20, and 22) and the 17 non-pneumococcal bacterial strains listed in Table 2, was tested using a dot enzyme immunoassay as described by D. Martin et al. [supra] and immunoblotting. For dot enzyme immunoassay, the bacteria were grown overnight on chocolate agar plates and then suspended in PBS, pH 7.4. A volume of 5 μl of a suspension containing approximately 10⁹ CFU/ml was applied to a nitrocellulose paper, blocked with PBS containing 3% bovine serum albumin, and then incubated sequentially with MAbs and peroxydase-labeled secondary antibody. Whole cell extracts were prepared for Western blot analysis by boiling bacterial suspensions in sample buffer for 5 minutes.

When tested by dot enzyme immunoassay, each MAb reacted with each of the S. pneumoniae strains and none of the non-pneumococcal isolates. These results were

unexpected since comparison studies revealed that HSP72 is very similar to other known bacterial HSP70 (DnaK)

proteins, for example those from E. coli and S. aureus .

Immunoblots were then performed to further investigate the immunoreactivities of our MAbs. As shown in Table 3, each MAb exhibited some reactivity. Although the percent identity of the E. coli amino acid sequence and the HSP72 amino acid sequence (SEQ ID NO:5) is 54%, the four HSP72-specific MAbs did not recognize the E. coli HSP70 (DnaK) protein. Similarly, the HSP72-specific MAbs did not react with the C. trachomatis HSP70 (DnaK)

protein, which has 56% amino acid identity with the amino acid sequence of HSP72. High amino acid sequence homology is observed between HSP72 and the HSP70 (DnaK) proteins from gram positive bacterial species. However, again, none of the HSP72-specific MAbs reacted with S. aureaus or Bacillus gram positive species, which exhibit 74% and 76% amino acid sequence homology, respectively, with HSP72. From these data it is clear that although HSP70 (DnaK) proteins may be structurally related to HSP72, they are immunologically distinct. Among the non-pneumococcal isolates that reacted with at least one MAb, there is S. pyogenes , Enterococcus faecalis , S. mutans and S. sanguis, which all belong to the Streptococcus or Streptococcus- related En terococcus genus. So far, neither the HSP70 protein, nor the gene structure has been identified in these Streptococcus or Enterococcus species. Altogether, these observations indicate that hypervariable amino acid sequences or residues within HSP70 (DnaK) proteins are involved in antigenicity. Interestingly, immunoblotting analysis revealed that there was no significant variation in the molecular mass of the HSP70 (DnaK) proteins among both S. pneumoniae isolates and immunoreactive non- pneumococcal isolates. EXAMPLE 5 - Purification of HSP72 And Its

Use As An Immunogen to Protect

Against Lethal S. Pneumoniae Infection

A. Procedures

1. Preparation of Purified

Recombinant HSP72 Protein

and Recombinant C-169

High level exclusive expression of the HSP72 gene was achieved by employing the bacteriophage T7 RNA polymerase/T7 promoter system in E. coli . The 3.2 kb Hindlll fragment was cloned in both orientations in front of the T7 promoter Φ10 in the plasmid pT7-5. The

resulting plasmid pJBDk51 was then transformed into

E. coli strain BL21 (DE3). Overexpression of the

recombinant HSP72 protein (HSP72_rec) was induced by

culturing in broth supplemented with antibiotics for a 3- hour period after the addition of IPTG to a final

concentration of 1 mM. E. coli expressing high levels of HSP72_rec were concentrated by centrifugation and lysed by mild sonication in 50 mM Tris-Cl (pH 8.0), 1 mM EDTA and 100 mM NaCl lysis buffer containing 0.2 mg/ml lysozyme. The cell lysates were centrifuged at 12,000 g for 15 minutes and the supernatants were collected. HSP72_rec was purified by immunoaffinity using monoclonal antibody F1- Pn3.1 immobilized on sepharose 4B beads (Pharmacia). The purity of eluates was assessed on SDS-PAGE.

The recombinant C-169 protein (C-169_rec) was expressed in the form of insoluble inclusion bodies in E. coli strain JM109 transformed with the plasmid pJBDΔ1. Protein inclusion bodies were recovered from pelleted bacterial cells disrupted by sonication as described before. The pellets were washed in lysis buffer

containing 1 mg/ml of deoxycholate to remove contaminating materials, and the protein inclusion bodies were then solubilized in urea 6 M. The protein solution was centrifuged at 100,000 g and the cleared supernatant collected and dialysed against phosphate-buffered saline. After purification, the protein content was determined by the Bio-Rad protein assay (Bio-Rad Laboratories,

Mississauga, Ontario, Canada).

2. Active Immunoprotection Studies

Two groups of 10 female Balb/c mice (Charles River Laboratories) were immunized subcutaneously three times at two-week intervals with 0.1 ml of purified

HSP72_rec or C-169_rec antigens absorbed to Alhydrogel

adjuvant. Two antigen doses, approximately 1 and 5 μg, were tested. A third group of 10 control mice were immunized identically via the same route with Alhydrogel adjuvant alone. Blood samples were collected from the orbital sinus prior to each immmunization and five to seven days following the third injection. The mice were then challenged with approximately 10⁶ CFU of the type 3 S. pneumoniae strain WU2. Samples of the S. pneumoniae challenge inoculum were plated on chocolate agar plates to determine the CFU and to verify the challenge dose.

Deaths were recorded at 6-hour intervals for the first 3-4 days post-infection and then at 24-hour intervals for a period of 14 days. On days 14 or 15, the surviving mice were sacrificed and blood samples tested for the presence of S. pneumoniae organisms. Antibody responses to the recombinant HSP72 antigens are described in Example 7. 3. Passive Immunoprotection Studies

One NZW rabbit (Charles River Laboratories) was immunized subcutaneously at multiple sites with

approximately 50 μg of the purified C-169_rec protein adsorbed to Alhydrogel adjuvant. The rabbit was boosted three times at two-week intervals with the same antigen and blood samples collected 7 and 14 days following the last immunization. The serum samples were pooled and antibodies were purified by precipitation using 40% saturated ammonium sulfate.

Severe-combined immunodeficient SCID mice were injected intraperitoneally with 0.25 ml of the purified rabbit antibodies 1 hour before intravenous challenge with 5000 or 880 CFU of the type 3 S. pneumoniae strain WU2. Control SCID mice received sterile buffer or antibodies purified from nonimmune rabbit sera. Samples of the

S. pneumoniae challenge inoculum were plated on chocolate agar plates to determine the CFU and to verify the

challenge dose. The SCID mice were chosen because of their high susceptibility to S. pneumoniae infection.

Blood samples (20 μl each) obtained 24 hours post- challenge were plated on chocolate agar and tested for the presence of S. pneumoniae organisms. The level of

detection was 50 CFU/ml. Deaths were recorded at 24-hour intervals for a period of 5 days.

B. Results

The availability of cloned S. pneumoniae DNA inserts encoding the complete or partial (C-169) HSP72 protein and the expression of recombinant proteins in E. coli allowed the obtention of purified proteins useful for the investigation of the vaccinogenic potential of HSP72 protein. Both HSP72_rec and C-169_rec proteins were obtained in a relatively pure state with no contaminants detected on Coomassie Blue-stained SDS polyacrylamide gels (FIGS. 14 and 15, respectively) .

To evaluate the vaccinogenic potential of HSP72, we first examined the ability of HSP72_rec to elicit a protective immune response. Groups of 10 mice were immunized with full-length HSP72_rec (1 μg or 5 μg dose) and challenged with 4.2 million CFU of S. pneumoniae type 3 strain WU2. Eighty percent (80%) of the mice dosed with 1 μg HSP72_rec survived the challenge, as did 50% of the mice dosed with 5 μg HSP72. None of the naive mice immunized with Alhydrogel adjuvant alone without antigen survived the challenge (FIG. 16). No S. pneumoniae organisms were detected in any of the blood samples collected on days 14 or 15 from mice surviving infection. The observation that HSP72_rec elicited protection against type 3 strain WU2 pneumococci indicated that HSP72 derived from DNA

extracted from a type 6 strain contains epitopes capable of eliciting protection against a heterologous strain having a different capsular type.

We further examined the immune response to the HSP72 protein by using recombinant protein fragments expressed from E. coli transformed with a chimeric fucI- HSP72 gene. Mice immunized with purified C-169_rec were protected from fatal pneumococcal challenge, thus

demonstrating that some, if not all, epitopes eliciting protection are present in the C-terminal region of the HSP72 molecule comprising the last 169 residues. Groups of 10 mice were immunized with C-169_rec (1 μg or 5 μg doses) and challenged with 6 million CFU of S. pneumoniae type 3 strain WU2. Sixty percent (60%) of the mice dosed with 1 μg C-169_rec survived the challenge, as did 70% of the mice dosed with 5 μg C-169_rec (FIG. 17). In contrast, all of the naive mice were dead by 2 days post-challenge. Therefore, the C-terminal portion of S. pneumoniae HSP72, which includes the region of maximum divergence among DnaK proteins, is a target for the protective immune response.

As illustrated in Table 4 below, two independent experiments demonstrated that SCID mice passively

transferred with rabbit anti-C-169_rec antibodies were protected from fatal infection with S. pneumoniae WU2. In contrast, none of the 15 control mice survived. The control mice received antibodies from nonimmune rabbit sera or received sterile buffer alone. In addition, all mice from the control groups had positive S. pneumoniae hemoculture 24 hours post-challenge, while S. pneumoniae organisms were detected in only 2 out of a total of 10 immunized SCID mice.

In experiments 1 and 2 (Table 4), mice were challenged with 5000 and 880 CFU of type 3 S. pneumoniae strain WU2 , respectively. Results in Table 4 are

expressed as the number of mice surviving challenge, or testing positive for the presence of S. pneumoniae , compared to the total number of mice in each group.

Demonstration of the anti-HSP72 specificity of the antibody elicited by immunization with recombinant HSP72 or C-169 proteins came from Western Blot analyses using S. pneumoniae cell lysates as antigens. A single band corresponding to HSP72 was detected by all rabbit and mouse antisera tested. These serologic results suggested that the protection following the immunization with recombinant proteins was due to the production of

antibodies reactive with S. pneumoniae HSP72.

EXAMPLE 6 - Heat-Indueible Expression System for High Level Production of the C-151 Terminal Portion of the HSP72 Protein A. Construction of Plasmid pURV3 Containing the C- 151 terminal coding region of the HSP72 of S.

pneumoniae

The DNA region coding for 151 amino acids at the carboxyl end of the HSP72 of S. pneumoniae was

inserted downstream of the promoter λ PL into the

translation vector p629 [H. J. George et al.,

Bio/Technology 5, pp. 600-603 (1987)]. This vector contains a cassette of the bacteriophage λ CI857

temperature sensitive repressor gene from which the functional PR promoter has been deleted. The inactivation of the cl857 repressor by a temperature increase from the ranges of 30-37°C to 37-42°C results in the induction of the gene under the control of λ PL. The induction of gene expression in E. coli cells by a temperature shift is advantageous for large scale fermentation since it can easily be achieved with modern fermenters. However, it should be understood that while E. coli was the

microorganism of choice in the experiments herein

described, other host organisms, such as yeast, are intended to be included within the scope of this

invention.

A fragment of 477 nucleotides, including the region of 457 bases between 2050 to 2506 in HSP72 gene of S. pneumoniae (see SEQ ID NO 4), was amplified by the polymerase chain reaction (PCR) from the S. pneumoniae type 6 strain 64 genomic DNA using the oligonucleotide primers OCRR26 (5'-GGCAGATCTATGAAGGCCAAAGACCTTGGAAC) and OCRR27 (5'-CGCGGATCCTTACTTTTCCGTAAACTCTCCGT).

Chromosomal DNA was prepared from a 90 ml culture of exponentionally growing cells of S. pneumoniae in heart infusion broth using the method of Jayarao et al. [J.

Clin. Microbiol., 29, pp. 2774-2778 (1991)]. DNA

amplification reactions were made using a DNA Thermal Cycler, Perkin Elmer, San Jose, CA. In OCRR26, an ATG start codon is present in frame just upstream of the coding region for the amino-terminus region of the C-15X The primers OCRR26 and OCRR27 contain, respectively, a Bglll (AGATCT) and a BamHI (GGATCC) recognition site in order to facilitate the cloning of the PCR product into the dephosphorylated restriction sites Bglll and BamHI of p629. The PCR product was purified from agarose gels by the method of phenol freeze [S. A. Benson, Biotechniques 2, pp. 67-68 (1984)] and digested with the restriction enzymes Bglll and BamHI. The Bglll-BamHI fragment of 471 base pairs was then ligated into the Bglll. and BamHI recognition sites dephosphorylated of p629. A partial map of the resulting plasmid pURV3 is shown in FIG. 18. This plasmid was transformed by the method of Simanis [Hanahan, D. In D. M. Glover (ed.), DNA Cloning, pp. 109-135,

(1985)] into the E. coli strain XLI Blue MRF' (A(mcrA) 183 A(mcrCB-hsdSMR-mrr) 173 endAl supE44 thi-1 recAl gyrA96 relA1 lac [F' proAB lacI^qZAM15 Tn10 (Tet^r)]^c ) which was obtained from Stratagene, La Jolla, CA. The transformants grown at 37°C were screened by colony immunoblot [J.

Sambrook et al. (supra)] using the MAb F1-Pn3.1 reactive with C-169_rec. Plasmid DNA was purified from a selected transformant and the DNA insert was seguenced by PCR using the Taq Dye Deoxy Terminator Cycle Sequencing kit of

Applied Biosystems Inc. (ABI) and DNA electrophoresis was performed on automated DNA sequencer 373A (ABI). The nucleotide sequence of the insert perfectly matched the nucleotide sequence of the C-151 coding region of the HSP72 gene. (See SEQ ID No: 25 and corresponding amino acid sequence at SEQ ID No: 26.) The plasmid was

transformed into the prototrophic E. coli strain W3110 (ATCC 27325) for the production of C-151_rec.

B . Expression of C-151rec and Antigen

Preparation

The recombinant C-151_rec was synthesized with a methionine residue at its amino end in E. coli strain W3110 harboring the plasmid pURV3. E. coli cells were grown at 30°C in LB broth containing 100 μg of ampicillin- per ml until the A₆₀₀ reached a value of 0.6. The cells were then cultivated at 40°C for 18 hours to induce the production of C-151_rec protein. A semi-purified C-151_rec protein was prepared using the following procedures. The bacterial cells were harvested by centrifugation and the resulting pellet was washed and resuspended in phosphate- buffered saline. Lysozyme was added and the cells were incubated for 15 min on ice before disruption by pulse sonication. The cell lysates were cleared by

centrifugation and the supernatants were collected and subjected to separation using an Amicon's ultrafiltration equipment (stirred cells series 8000, Amicon Canada Ltd. Oakville, Ontario). The ultrafiltrate not retained by a YM30 membrane was recovered, analysed by SDS-PAGE and stained with Coomassie blue R-250. Protein concentrations were estimated by comparing the staining intensity of the C-151_rec protein with those obtained with defined

concentrations of soybean trypsin inhibitor.

C . Reactivity of MAbs Against C-151rec

A panel of 10 monoclonal antibodies selected for their reactivity with the_S. pneumoniae HSP72 protein were tested for their reactivity to C-151_rec by Western blot analysis using YM30-ultrafiltrates prepared as described above. The MAbs included a series of six monoclonal antibodies raised to the HSP72_rec protein (F3- Pn3.5 to F3-Pn3.10) and monoclonal antibodies F1-Pn3.1, F2-Pn3.2, F2-Pn3.3, F2-Pn3.4. The three MAbs F1-Pn3.1, F2- Pn3.3 and F2-Pn3.4 that were reactive with C-169_rec also recognized the C-151_rec fragment. All other MAbs were only reactive with HSP72_rec thus indicating that they may be directed against epitopes present in the amino terminal region of the HSP72 protein. EXAMPLE 7 - Antibody Response of Balb/c Mice and Macaca- Fascicularis (cynomolgus) Monkeys to Recombinant HSP72 Antigens

A. Procedures

1. Immunization of Animals

Groups of 10 female Balb/c mice were immunized subcutaneously with either HSP72 _rec or C-169 _rec as described in Example 5. In order to assess the antibody response to C-151_rec, a group of 6 mice were immunized three times at two-week intervals with 0.5 μg of C-151_rec absorbed to Alhydrogel adjuvant by intraperitoneal

injection. Sera from blood samples collected prior each immunization and four to seven days after the third immunization were tested for antibody reactive with S. pneumoniae by ELISA using plates coated with S. pneumoniae cell wall extracts.

Female cynomolgus monkeys were immunized intramuscularly at Day 1, 22 and 77 with 0.5 ml containing 150 μg of purified HSP72_rec or C-169_rec antigens absorbed to Alhydrogel adjuvant. Blood samples were collected regularly before and after each immunization and the sera were tested for antibody reactive with S. pneumoniae HSP72 antigen by Western blot analysis.

The specificity of the raised antibodies for_S. pneumoniae HSP72 was confirmed by Western blot analyses to S. pneumoniae cell extracts and purified recombinant antigens.

B. Results

The results previously described in Example 5 clearly demonstrate the protective nature of the antibody response elicited following immunization with recombinant HSP72 antigens . Here we monitored the appearance of serum antibody response in mice (FIG. 19, 20 and 21) and in monkeys (FIG. 22) during the immunization schedule. Both species responded strongly to the full-length and

truncated recombinant HSP72 proteins used as immunogens with average titers of 1:64000 after the third injection.- Detailed analysis of individual sera revealed that each animal responded to the immunization in developping antibodies reactive with S. pneumoniae HSP72.

In mice immunized with C-169_rec , the two doses tested, i.e. 1 and 5 μg, were similarly efficient with the induction of similar antibody titers (FIG. 20). A strong boost response was observed after the second injection with C-169_rec with no enhancement in the antibody titers after a third injection. In contrast to this, we observed that the immune response to the HSP72_rec was dose- dependent. Increases in the specific antibody titers were observed after a second and a third injection with either HSP72_rec or C-151_rec (FIG. 19 and 21).

Study of the immune response of monkeys clearly indicated that the immunogenicity of recombinant HSP72 antigens is not restricted to rodents such as rabbit and mouse . The humoral response following the second

injection with either antigen is characterized by a strong increase in HSP72-specific antibody titers that can persist for several weeks without any detectable decrease in their antibody titers (FIG. 22). In addition, specific serum antibodies were detectable in the sera of each monkey after a single injection of recombinant antigens.

EXAMPLE 8 - B-Cell Epitope Mapping of HSP72 Stress

Protein

In Example 3, it was shown that significant variability in the primary sequence of the HSP70 proteins was mainly localized to two regions corresponding to amino acid residues 244 to 330 and 510 to 607 of the S.

pneumoniae HSP72 protein. These variable regions may contain B-cell epitopes responsable for the antigenic heterogeneity reported in Example 4. To investigate this possibility, the reactivity of polyclonal and monoclonal antibodies to S. pneumoniae HSP72 were tested against fourteen peptides selected to cover most of these regions.

A. Procedures

Fourteen peptides of 14 to 30 amino acids residues were synthesized. The peptide sequences and their locations in the protein are summarized in Table 5. Peptides CS870, CS873, CS874, CS875, CS876, CS877, CS878, CS879, CS880 and CS882 were synthesized by Biochem

Immunosystem Inc. (Montreal, Canada) using an automated peptide synthesizer. Peptides MAP1, MAP2 , MAP3 and MAP4 were synthesized onto a branching lysine core as Multiple Antigenic Peptides (MAP) by the Service de Sequence de Peptides de l'Est du Quebec, Centre de recherche du CHUL (Sainte-Foy, Canada). Peptides were purified by reverse- phase high-pressure liquid chromatography. Peptides were solubilized in distilled water except for peptides CS874 and CS876 which were solubilized in a small volume of either 6M guanidine-HCl or dimethyl sulfoxide and then adjusted to 1 mg/ml with distilled water.

Peptide ELISA were performed by coating synthetic peptides onto Immunolon 4 microtitration plates (Dynatech Laboratories, Inc., Chantilly, VA) at a concentration of 50 μg/ml according to the prodedures described in J. Hamel et al. [supra]. To confirm the reactivity of MAbs with peptides, the ability of fluid-phase peptides to inhibit MAb binding to solid HSP72 was determined. For the inhibition assay, microtitration plates were coated with S. pneumoniae cell wall extracts. Hybridoma culture supernatants containing the HSP72-specific MAbs were incubated overnight at 4°C with several concentrations of peptide. Peptide treated and control supernatants were then tested by ELISA as described above.

Immune sera were from animals immunized three times with recombinant HSP72 antigens. One rabbit was immunized with 37.5 μg of purified HSP72_rec according to the immunization protocol described in Example 5. Pool murine sera were from three Balb/c mice immunized with HSP72_rec from Example 5 and monkey pool sera were from groups of two animals immunized with either HSP72_rec or C- 169_rec. TABLE 5: SEQUENCES AND LOCATIONS OF SYNTHETIC PEPTIDES CORRESPONDING TO S. PNEUMONIAE HSP72

AMINO ACID RESIDUES

B. Identification and Localization of Linear B-

Cell Epitopes

The results presented in FIG. 23 revealed that most of the immunological reactivity was observed with the peptides localized within amino acid residues 457 and 607- corresponding to the C-151 fragment of HSP72. Rabbit, mice and monkey sera antibody from animals immunized with either recombinant HSP72_rec of C-169_rec were reactive with both, peptide MAP2 and peptide MAP4. Interestingly, the sequence of peptides MAP2 and MAP4 spans the

hypervariable carboxyl-terminal region containing the sequences GFDAERDAAQAALDD (residues 527 to 541) and

AEGAQATGNAGDDVV (residues 586 to 600) defined as exclusive to S. pneumoniae HSP72 based on the comparison of HSP70 protein sequences available in the data banks. Our data thus revealed that both peptide sequences contain linear B-cell epitopes. In addition, the peptide MAP4 alone was also recognized by the MAb F1-Pn3.1. This reactivity was confirmed by fluid-phase inhibition assays in which 10 μg/ml of MAP4 caused complete inhibition of F1-Pn3.1 binding to HSP72. Polyclonal antisera from animals immunized with the complete HSP72 recombinant protein also recognized B-cell epitopes localized on peptides CS875, MAPI and MAP3. All together these data indicate that the hypervariable C-151 terminal fragment of the HSP72

stimulates B-cell responses and possibly constitutes the immunodominant portion of the HSP72 protein. The lack of reactivity of MAbs F2-Pn3.3 and F2-Pn3.4 with the

synthetic peptides suggest that they react with

conformational determinants present on the C-terminal region of the HSP72. The existence of protective epitopes in the C-151 region was strongly suggested in Example 5 where mice immunized with purified C-169_rec were protected from fatal infection with a virulent strain of S.

pneumoniae thus suggesting that the carboxyl-terminal fragments C-169 or C-151 of_S. pneumoniae HSP72 or even smaller fragments thereof may prove very useful for the development of a future vaccine.

The variable region comprised within the amino acid residues 244 to 330 also constitutes an antigenic domain. Linear epitopes located on overlapping peptides CS877 (amino acids 257 to 271) and CS878 (amino acids 268- to 281), peptides CS880 (amino acis 286-299) and peptides CS882 (amino acids 315-333) were identified by hyperimmune sera.

EXAMPLE 9 - HSP70 (DnaK) from Streptococcus pyogenes and Streptococcus agalactiae : Molecular Cloning and DNA

Sequencing of the hsp70 Genes; Nucleotide and Protein

Sequence Analyses; Antigenic Relatedness to S. pneumoniae; Increased Streptococcus agalactiae HSP70 synthesis "in

response to heat.

A. Procedures

1. Bacterial Strains and Plasmid Vector

The strains of S. pyogenes (Group A

Streptococcus ) and S. agalactiae (Group B Streptococcus) used in this study were provided by the Laboratoire de la Sante Publique du Quebec (LSPQ), Sainte-Anne de Bellevue, Quebec, Canada. S. agalactiae type II strain V8

corresponds to the ATCC strain 12973. S. pyogenes strain Bruno corresponds to the ATCC strain 19615. The E. coli strain XLI Blue MRF' was obtained from Stratagene.

Streptococcal strains were grown at 37°C in a 5 % CO2 incubator. The streptococci were streaked on tryptic soy agar plates containing 5 % sheep blood (Les Laboratoires Quelab, Montreal, Canada), liquid cultures were made in heart infusion broth (Difco Laboratories, Detroit, MI) without agitation. The E. coli strain was grown at 37°C in L-broth with agitation at 250 rpm or on L- agar.

The general cloning phagemid pBluescript KS(-) was purchased from Stratagene.

2. Recombinant DNA Techniques

Restriction enzymes, T4 DNA ligase, and calf intestinal phosphatase were used as recommended by the suppliers (Pharmacia [Canada] Inc., Baie d'Urfe, Canada; and New England Biolabs Ltd., Mississauga, Canada). Preparation of plasmids by equilibrium centrifugation in- CsCl-ethidium bromide gradients, agarose gel

electrophoresis of DNA fragments, Southern hybridization, and colony DNA hybridization were performed as described by J. Sambrook et al. [ supra]. Chromosomal DNA of the streptococcal bacteria was prepared using the procedure of B. M. Jayarao et al. [J. Clin. Microbiol., 29, pp. 2774- 2778 (1991)] adapted for bacterial cultures of 90 ml.

Rapid plasmid preparations were made accordingly to D. Ish-Horowicz et al. [Nucl. Acids Res. 9, pp. 2989-2998 (1981)]. Plasmids used for DNA sequencing were purified using plasmid kits from Qiagen Inc. (Chatsworth, CA). DNA fragments were purified from agarose gels by the method of phenol freeze [S. A. Benson, Biotechniques 2, pp. 67-68 (1984)]. DNA probes were labeled with a³²P-dCTP or digoxigenin (DIG)-11-dUTP using the random primer labeling kits of Boehringer Mannheim (Laval, Canada). Plasmid transformations were carried out by the method of Simanis [Hanahan, D. In D. M. Glover (ed.), DNA Cloning, pp. 109- 135, (1985)]. The sequencing of genomic DNA inserts in plasmids was done using synthetic oligonucleotides. The sequencing reactions were carried out by the polymerase chain reaction (PCR) using the Taq Dye Deoxy Terminator Cycle Sequencing kit (ABI) and DNA electrophoresis was performed on automated DNA sequencer 373A (ABI). The assembly of the DNA sequence was performed using the program Sequencher 3.0 from the Gene Codes Corporation (Ann Arbor, MI). Analysis of the DNA sequences and their predicted polypeptides were performed with the program Gene Works version 2.45 from Intelligenetics, Inc.

(Mountain View, CA). DNA amplification reactions were made using a DNA Thermal Cycler 480, Perkin Elmer.

Oligonucleotides were synthesized by oligonucleotide synthesizer model 394 (ABI). 3. Molecular Cloning of the Genes hsp70/dnak- of S. agalactiae and S. pyogenes

Chromosomal DNA from S. agalactiae and S.

pyogenes was digested to completion with various

restriction enzymes with palindromic hexanucleotide recognition sequences. The digests were analysed by

Southern hybridization using a labeled PCR-amplified DNA probe corresponding to a 782 base-pairs region starting at base 332 downstream from the ATG initiation codon of the HSP72 gene of S. pneumoniae (see SEQ ID NO 4). This DNA region was selected because it is relatively well

conserved among the hsp70 genes of Gram-positive bacteria that have been characterized. The PCR amplification was done on the genomic DNA of S. pneumoniae using the oligonucleotides OCRR2 (5'-AAGCTGTTATCACAGTTCCGG) and OCRR3 (5'-GATACCAAGTGACAATGGCG). Hybridizing genomic restriction fragments of sufficient size to code for a 70- kDa polypeptide (>1.8 kb) were partially purified by extraction of genomic fragments of corresponding size from agarose gel. Verification of the presence of the hsp70 gene among the purified genomic restriction fragments was done by Southern hybridization using the labeled 782-bp S. pneumoniae DNA probe.

The purified genomic DNA restriction fragments were cloned into dephosphorylated compatible restriction sites of pBluescript KS(-) and transformed into the E. coli strain XLI Blue MRF'. The colonies were screened by DNA hybridization using the labeled 782-bp S. pneumoniae DNA probe. Extracted plasmids were digested with various restriction enzymes to evaluate the size of the inserts and to verify the presence of the hsp70 gene by Southern hybridization using the labeled 782-bp S. pneumoniae DNA probe. Plasmid pURV5 contains a 4.2-kb Hindlll insert of the genomic DNA of S. agalactiae . Plasmid pURV4 contains a 3.5-kb Hindlll fragment of the genomic DNA of S.

pyogenes . 4. Heat Shock and Protein Labeling

The stress response of S. agalactiae to an heat shock was assayed by pulse-labeling with [³⁵S]methionme as described before in Example 1. S. agalactiae bacteria grown overnight in SMAM (Methionine assay Medium

supplemented with 1 mg/l methionine, 1% (v/v) Isovitalex and 1 mg/l choline chloride) were pelleted by

centrifugation and then resuspended in the methionine-free SMAM medium. The bacteria were incubated at 37°C for 1 h and then divided into two fractions of equal volume. The samples were either incubated at 37 or 43°C for 10 minutes and then labeled with 100 μCi/ml [³⁵S]methionine for 30 minutes at 37°C. The bacteria were extensively washed with PBS and cell extracts were prepared by treatment with mutanolysine and lysozyme as described for the DNA

isolation (M.Jayarao et al., supra) followed by

sonication.

5. Immunological Characterization

A series of six monoclonal antibodies raised to the HSP72_rec protein (F3-Pn3.5 to F3-Pn3.10) and the monoclonal antibodies F1-Pn3.1, F2-Pn3.2, F2-Pn3.3, F2- Pn3.4 were tested for their reactivity to HSP70 antigens from S. pyogenes and S._agalactiae_by Western blot

analysis. Cell lysates from S. pyogenes and_S. agalactiae were obtained from treatment with mutanolysine and

lysozyme (M.Jayarao et al., supra)., sonication and boiling in SDS-PAGE sample buffer. Cell lysates from E. coli transformed with either pURV4 or pURV6 producing truncated S._pyogenes HSP70 antigens were tested after boiling in SDS-PAGE sample buffer.

B. DNA Sequence Analysis of the hsp70 /dnak Genes of Streptococcus pyogenes, Streptococcus agalactiae and Streptococcus pneumoniae

A region of 2438 bases in the 4.2-kb Hindlll insert of plasmid pURV5 was sequenced. This sequence contains an open reading frame (ORF) of 1830 nucleotides coding for a polypeptide of 609 amino acids with a

molecular weight of 64907 (see SEQ ID NO: 7). The ORF has an ATG start codon beginning at position 248 and TAA stop codon ending at position 2077. The ATG start codon is preceeded by the sequence GAGG, starting at position 237, which is complementary to 16S rRNA and serves as a

ribosome binding site in E. coli [G. D. Stormo et al., Nucleic Acids Res. 10, pp. 2971-2996 (1982)]. The ORF and the polypeptide of the HSP70 of S. agalactiae are,

respectively, identical at 85 and 95 % to the ORF and polypeptide of the HSP72 of S. pneumoniae .

Preliminary sequence comparisons with the HSP72 of S. pneumoniae showed that the 3.5-kb Hindlll insert in plasmid pURV4 lacks the 3'-end coding region of the hsp70 of S. pyogenes . An attempt to clone a 3-kb Sail genomic fragment containing the entire coding region of hsp70 of S. pyogenes yielded plasmid pURV6 containing a 3.1-kb insert lacking the 5'-end coding region of the gene. The assembly of the hsp70 gene regions present in plasmids pURV4 and pURV6 gave a 2183 nucleotide region containing an ORF of 1824 bases coding for a polypeptide of 608 amino acids with a molecular weight of 64847 (see SEQ ID NO:

20). The ATG start codon begins at position 204 and the TAA stop codon extends to position 2030. Similarly to the hsp70 of S. agalactiae, the ATG start codon is preceeded by a putative ribosome binding site sequence GAGG starting at position 193 [G. D. Stormo, supra]. The ORF and the deduced polypeptide of the hsp70 of S. pyogenes are, respectively, identical at 85 and 94 % to the ORF and polypeptide of the HSP72 of S. pneumoniae . The ORF of plasmid pURV4 lacks 125 base pairs coding for 41 amino acids at the carboxyl end of the HSP70 of S. pyogenes ; the ORF thus codes for the 567 amino acids of the amino end of that HSP70 (N-567_rec). The ORF of plasmid pURVδ lacks 114 base pairs coding for 38 amino acids at the amino end of the HSP70 of S. pyogenes ; the ORF thus codes for the 570 amino acids of the carboxyl end of that HSP7θ

(C-570_rec).

The global comparison of the DNA open reading frames (FIG. 24) and amino acid sequences (FIG. 25) of the

HSP70/DnaK of S. pyogenes, S. agalactiae, and S. pneumoniae gave percentages of identity of 82 and 93 %, respectively.

C. Increased Synthesis of HSP70 by S. agalactiae in Response to Heat

One dimensional SDS-polyacrylamide gel electrophoretic analysis of cell extracts of heat-shocked and control S. agalactiae pulse-labeled with

[³⁵S]methionine revealed that the synthesis of a 70 kDa- protein was significantly increased after a thermal stress (FIG. 26, lanes 1 and 2). Radioimmunoprecipitation analysis revealed that the heat inducible 70kDa-protein was easily detected at 43°C using monoclonal antibody F2- Pn3.4 thus indicating that the protein belongs to the heat shock protein 70 (hsp70/DnaK) family (FIG. 26, lanes 3 and 4).

D. Antigenic Relatedness of HSP70 Proteins in S. pneumoniae,_S. pyogenes and S. agalactiae

In this study, a panel of MAbs were used to investigate the antigenic relatedness of S. pyogenes, S. agalactiae and S. pneumoniae HSP70 proteins. Eight of ten MAbs reacted with all three Streptoccocus species thus indicating that some B-cell epitopes are widely

distributed among S. pneumoniae , S. pyogenes and S.

agalactiae . The MAb F1-Pn3.1 which is directed against an epitope located between amino acid residues 584 and 607 of HSP72 from_S. pneumoniae did not react with HSP70

antigens from either S. pyogenes or S. agalactiae .

Comparison of this region among the three Streptococcus species revealed differences in 5 to 8 amino acids located between amino acids 589 and 596. The MAb F2-Pn3.3 which was also directed against epitopes present in the C-151 regiron was reactive with S. agalactiae but not wih S.

pyogenes. These data clearly indicate that HSP70 proteins from Streptococcus species are structurally and

immunologically related. There is however immunological distinction.

Analysis of the reactivity of MAbs F3-Pn3.5, F3- Pn3.6, F3-Pn3.7 and F3-Pn3.10 with truncated recombinant S. pyogenes HSP70 antigens allowed the identification of an antigenic region near the amino-terminal end on the S. pneumoniae HSP72. These MAbs reacted with constructs expressing the N-terminal 567 amino acid residues but failed to react with constructs expressing the C-570 fragment. These data localized the epitopes recognized by the MAbs F3-Pn3.5, F3-Pn3.6, F3-Pn3.7 and F3-Pn3.10 to between residues 1 and 38 of the HSP72 protein.

EXAMPLE 10 - Use of HSP70/HSP72 As A Human Vaccine

To formulate a vaccine for human use, appropriate HSP72 antigens may be selected from the polypeptides described herein. For example, one of skill in the art could design a vaccine around the HSP70/HSP72 polypeptide or fragments thereof containing an immunogenic epitope. The use of molecular biology techniques is particularly well-suited for the preparation of

substantially pure recombinant antigens.

The vaccine composition may take a variety of forms. These include, for example solid, semi-solid and liquid dosage forms, such as powders, liquid solutions or suspensions, and liposomes. Based on our belief that the HSP70/HSP72 antigens of this invention may elicit a protective immune response when administered to a human, the compositions of this invention will be similar to those used for immunizing humans with other proteins and polypeptides, e.g. tetanus and diphtheria. Therefore, the compositions of this invention will preferably comprise a pharmaceutcially acceptable adjuvant such as incomplete Freund's adjuvant, aluminum hydroxide, a muramyl peptide, a water-in oil emulsion, a liposome, an ISCOM or CTB, or a non-toxic B subunit from cholera toxin. Most preferably, the compositions will include a water-in-oil emulsion or aluminum hydroxide as adjuvant.

The composition would be administered to the patient in any of a number of pharmaceutically acceptable forms including intramuscular, intradermal, subcutaneous or topic. Preferrably, the vaccine will be administered intramuscularly.

Generally, the dosage will consist of an initial injection, most probably with adjuvant, of about 0.01 to 10 mg, and preferably 0.1 to 1.0 mg HSP72 antigen per patient, followed most probably by one or more booster injections. Preferably, boosters will be administered at about 1 and 6 months after the initial injection.

An important consideration relating to pneumococcal vaccine development is the question of mucosal immunity. The ideal mucosal vaccine will be safely taken orally or intranasally as one or a few doses and would elicit protective antibodies on the appropriate surfaces along with systemic immunity. The mucosal vaccine composition may include adjuvants, inert

particulate carriers or recombinant live vectors.

The anti-HSP72 antibodies of this invention are useful for passive immunotherapy and immunoprophylaxis of humans infected with S. pneumoniae, S. pyogenes, S.

agalactiae or related bacteria. The dosage forms and regimens for such passive immunization would be similar to those of other passive immunotherapies .

An antibody according to this invention is exemplified by a hybridoma producing MAb F1-Pn3.1

deposited in the American Type Culture Collection in

Rockville, Maryland, USA on July 21, 1995, and identified as Murine Hybridoma Cell Line, F1-Pn3.1. This deposit was assigned accession number HB 11960.

While we have described herein a number of embodiments of this invention, it is apparent that our basic embodiments may be altered to provide other

embodiments that utilize the compositions and processes of this invention. Therefore, it will be appreciated that the scope of this invention includes all alternative embodiments and variations that are defined in the

foregoing specification and by the claims appended hereto; and the invention is not to be limited by the specific embodiments which have been presented herein by way of example.

Claims (101)

We claim:

1. A polypeptide selected from the group consisting of:

(a) the HSP72 polypeptide having the amino acid sequence of SEQ ID NO:5;

(b) the HSP70(DnaK) polypeptide having the amino acid sequence of SEQ ID NO: 20;

(c) the HSP70 (DnaK) polypeptide having the amino acid sequence of SEQ ID NO:22;

(d) polypeptides that are immunologically reactive with antibodies generated by infection of a mammalian host with Streptococcus pneumoniae cells, which antibodies are immunologically reactive with the

polypeptide of paragraph (a), (b), or (c);

(e) polypeptides that are capable of eliciting antibodies that are immunologically reactive with the polypeptide of paragraph (a), (b), or (c);

(f) polypeptides that are immunologically reactive with antibodies elicited by immunization with the polypeptide of paragraph (a), (b), or (c); and

(g) fragments of any of the foregoing polypeptides, either alone or in combination with other polypeptides to form a fusion protein.

2. The polypeptide of claim 1, wherein the polypeptides of paragraph (d) are selected from the group consisting of polypeptides of the genera Streptococcus and Enterococcus .

3. The polypeptide of claim 1, wherein the polypeptides of paragraph (d) are selected from the group consisting of polypeptides of the species Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus pyogenes, Streptococcus mutans, Streptococcus sanguis, and Enterococcus faecalis .

4. The polypeptide of claim 1, wherein the polypeptides of paragraph (d) are selected from the group consisting of polypeptides of the species Streptococcus pneumoniae, Streptococcus agalactiae, and Streptococcus pyogenes .

5. The polypeptide of claim 1, wherein the fragments of paragraph (g) are selected from the group consisting of amino acids 439-607 of SEQ ID NO:5 (C-169), amino acids 457-607 of SEQ ID NO:5 (C-151), amino acids 527-541 of SEQ ID NO:5, and amino acids 586-600 of SEQ ID NO:5.

6. A polypeptide having the amino acid sequence of SEQ ID NO : 5 analogues , homologues and

derivatives thereof .

7. A polypeptide having the amino acid sequence of SEQ ID NO:20, analogues, homologues and derivatives thereof.

8. A polypeptide having the amino acid sequence of SEQ ID NO: 22, analogues, homologues and derivatives thereof.

9. A polypeptide having the amino acid sequence of SEQ ID NO:26, analogues, homologues and derivatives thereof.

10. A polypeptide having the amino acid sequence of SEQ ID NO:7, analogues, homologues and derivatives thereof.

11. A polypeptide having the amino acid sequence of SEQ ID NO:8, analogues, homologues and derivatives thereof.

12. A polypeptide having the amino acid sequence of SEQ ID NO:9, analogues, homologues and derivatives thereof.

13. A polypeptide having the amino acid sequence of SEQ ID NO:10, analogues, homologues and derivatives thereof.

14. A polypeptide having the amino acid sequence of SEQ ID NO:11, analogues, homologues and derivatives thereof.

15. A polypeptide having the amino acid sequence of SEQ ID NO:12, analogues, homologues and derivatives thereof.

16. A polypeptide having the amino acid sequence of SEQ ID NO:13, analogues, homologues and derivatives thereof.

17. A polypeptide having the amino acid sequence of SEQ ID NO:14, analogues, homologues and derivatives thereof.

18. A polypeptide having the amino acid

sequence of SEQ ID NO: 15, analogues, homologues and derivatives thereof.

19. A polypeptide having the amino acid

sequence of SEQ ID NO: 16 analogues, homologues and

derivatives thereof.

20. A polypeptide having the amino acid

sequence of SEQ ID NO: 17, analogues, homologues and derivatives thereof.

21. A polypeptide having the amino acid

sequence of SEQ ID NO: 18, analogues, homologues and derivatives thereof.

22. The polypeptide of any one of claims 1 to 21 and 100-101, wherein said polypeptide elicits an immune reaction that is specific to Streptococcal strains.

23. A polypeptide selected from the group consisting of:

(a) the HSP72 polypeptide having the amino acid sequence of SEQ ID NO :5;

(b) polypeptides that are immunologically reactive with antibodies generated by infection of a mammalian host with Streptococcus pneumoniae cells, which antibodies are immunologically reactive with the HSP72 polypeptide of paragraph (a);

(c) polypeptides that are capable of eliciting antibodies that are immunologically reactive with the HSP72 polypeptide of paragraph (a) ; (d) polypeptides that are immunologically reactive with antibodies elicited by immunization with the HSP72 polypeptide of paragraph (a); and

(e) fragments of any of the foregoing polypeptides, either alone or in combination with other polypeptides to form a fusion protein.

24. The polypeptide of claim 23, wherein the polypeptides of paragraph (b) are selected from the group consisting of polypeptides of the genera Streptococcus and Enterococcus .

25. The polypeptide of claim 23, wherein the polypeptides of paragraph (b) are selected from the group consisting of polypeptides of the species Streptococcus pyogenes, Streptococcus mutans, Streptococcus sanguis, and Enterococcus faecalis .

26. The polypeptide of claim 23, wherein the fragments of paragraph (e) are selected from the group consisting of amino acids 439-607 of SEQ ID NO:5 (C-169); amino acids 527-541 of SEQ ID NO:5, and amino acids 586- 600 of SEQ ID NO: 5.

27. The polypeptide of claim 23, wherein the fusion protein of paragraph (e) is the Fucose Isomerase- HSP72 (C-169) protein having the amino acid sequence of SEQ ID NO:3.

28. A DNA sequence selected from the group consisting of:

(a) the HSP72 DNA sequence of SEQ ID NO:4;

(b) the HSP70 (DnaK) DNA sequence of SEQ ID NO:19; (c) the HSP70 (DnaK) DNA sequence of SEQ ID NO: 21;

(d) DNA sequences encoding polypeptides that are immunologically reactive with antibodies

generated by infection of a mammalian host with

Streptococcus pneumoniae cells, which antibodies are immunologically reactive with the HSP72 polypeptide (SEQ ID NO:5);

(e) DNA sequences encoding polypeptides that are capable of eliciting antibodies that are

immunologically reactive with the HSP72 polypeptide (SEQ ID NO :5);

(f) DNA sequences encoding polypeptides that are immunologically reactive with antibodies elicited by immunization with the HSP72 polypeptide (SEQ ID NO:5);

(g) DNA sequences that are degenerate to any of the foregoing DNA sequences; and

(h) fragments of any of the foregoing DNA sequences, either alone or in combination with other DNA sequences to form a fusion DNA sequence.

29. The DNA sequence of claim 28, wherein the DNA sequences of paragraph (d) are selected from the group consisting of DNA sequences of the genera Streptococcus and Enterococcus .

30. The DNA sequence of claim 28, wherein the DNA sequences of paragraph (d) are selected from the group consisting of DNA sequences of the species Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus pyogenes, Streptococcus mutans, Streptococcus sanguis, and Enterococcus faecalis .

31. The DNA sequence of claim 28, wherein the DNA sequences of paragraph (d) are selected from the group consisting of DNA sequences of the species Streptococcus pneumoniae, Streptococcus agalactiae, and Streptococcus pyogenes .

32. A DNA sequence of the formula of SEQ ID

NO: 4 from nucleotide 682 to nucleotide 2502, or

derivatives thereof, coding for HSP72.

33. A DNA sequence of the formula of SEQ ID NO: 4 from nucleotide 1996 to nucleotide 2502, or

derivatives thereof, coding for the C-169 fragment of

HSP72.

34. A DNA sequence of the formula of SEQ ID NO: 4 from nucleotide 2050 to nucleotide 2502, or

derivatives thereof, coding for the C-151 fragment of HSP72.

35. A DNA sequence of the formula of SEQ ID NO: 4 from nucleotide 2260 to nucleotide 2304, or

derivatives thereof.

36. A DNA sequence of the formula of SEQ ID NO: 4 from nucleotide 2437 to nucleotide 2481, or

derivatives thereof.

37. A DNA sequence of the formula of SEQ ID NO: 19 from nucleotide 204 to nucleotide 2027, or

derivatives thereof, coding for HSP70 of Streptococcus agalactiae .

38. A DNA sequence of the formula of SEQ ID NO: 21 from nucleotide 248 to nucleotide 2074, or derivatives thereof, coding for HSP70 of Streptococcus pyogenes .

39. A DNA sequence of the formula of SEQ ID NO: 25 from nucleotide 4 to nucleotide 456, or derivatives thereof, coding for the C-terminal 151-residue fragment (C-151) of HSP72.

40. A DNA sequence coding for a polypeptide according to any one of claims 1-21 and 100-101.

41. A DNA sequence selected from the group consisting of:

(a) the HSP72 DNA sequence of SEQ ID NO:4; (b) DNA sequences encoding polypeptides that are immunologically reactive with antibodies

generated by infection of a mammalian host with

Streptococcus pneumoniae cells, which antibodies are immunologically reactive with the HSP72 polypeptide (SEQ ID NO:5);

(c) DNA sequences encoding polypeptides that are capable of eliciting antibodies that are

immunologically reactive with the HSP72 polypeptide (SEQ ID NO :5);

(d) DNA sequences encoding polypeptides that are immunologically reactive with antibodies elicited by immunization with the HSP72 polypeptide (SEQ ID NO:5) ;

(e) DNA sequences that are degenerate to any of the foregoing DNA sequences; and

(f) fragments of any of the foregoing DNA sequences, either alone or in combination with other DNA sequences to form a fusion DNA sequence.

42. The DNA sequence of claim 41, wherein the DNA sequences of paragraph (b) are selected from the group consisting of DNA sequences of the genera Streptococcus and Enterococcus .

43. The DNA sequence of claim 41, wherein the DNA sequences of paragraph (b) are selected from the group consisting of DNA sequences of the species Streptococcus pyogenes, Streptococcus mutans, Streptococcus sanguis, and Enterococcus faecalis .

44. The DNA sequence of claim 41, wherein the fragments of paragraph (f) are selected from the group consisting of nucleotide 1996-2502 (amino acids 439-607) of SEQ ID N0:4 (C-169); nucleotide 2260-2304 (amino acids 527-541) of SEQ ID NO:4; and nucleotide 2437-2481 (amino acids 586-600) of SEQ ID NO:4.

45. The DNA sequence of claim 41, wherein the fusion DNA sequence of paragraph (f) is the Fucose

Isomerase-HSP72 (C-169) DNA sequence of SEQ ID NO:1

(nucleotides 771-2912).

46. An expression vector including at least one DNA sequence according to claim 41 operably linked to a promoter.

47. A recombinant DNA molecule comprising a DNA sequence according to any one of claims 28 to 40, and one or more expression control sequence operably linked to the DNA sequence.

48. The recombinant DNA molecule of claim 47, wherein said expression control sequence is an inducible expression vector.

49. The recombinant molecule of claim 48, wherein said expression vector comprises the λ PL

promoter.

50. A recombinant molecule according to claim

47 consisting of a plasmid selected from the group

consisting of: pURV3 , pURV4, pURV5 , pURV6, pJBD291, pJBDΔ4, pJBDk51, pJBD177, pJBD171, pJBD177, pJBD179, pJBDΔ1, pJBDf51, and pJBDf62.

51. A unicellular host transformed with an expression vector of claim 46.

52. A unicellular host transformed with a recombinant DNA molecule of claim 47.

53. A unicellular host according to claim 52, wherein said host is selected from the group consisting of: E.coli strains XLI Blue MRF', W3110, JM109, Y1090 and BL2KDE3).

54. A method for producing a polypeptide or fragment thereof comprising the steps of culturing the unicellular host of claim 51 and isolating said

polypeptide or fragment.

55. An antibody or fragment thereof that specifically binds to a polypeptide of claim 23.

56. An antibody or fragment thereof that specifically binds to the epitope recognized by monoclonal antibody F1-Pn3.1.

57. The antibody or fragment of claim 55, which is a monoclonal antibody.

58. The monoclonal antibody or fragment of claim 57, which is of murine origin.

59. The monoclonal antibody or fragment of claim 58, which is of IgG type.

60. The monoclonal antibody of claim 59, which is selected from the group consisting of F1-Pn3.1, F2- Pn3.2, F2-Pn3.3, and F2-Pn3.4.

61. The monoclonal antibody F1-Pn3.1.

62. A method for isolating the antibody of claim 55 comprising:

(a) introducing a preparation of the polypeptide of claim 23 into a mammal; and

(b) isolating serum from the mammal containing said antibody.

63. A method for isolating the monoclonal antibody of claim 57 comprising:

(a) introducing a preparation of the polypeptide of claim 23 to antibody producing cells of a mammal;

(b) fusing the antibody producing cells with myeloma cells to form hybridoma cells, and

(c) isolating said monoclonal antibody from the hybridoma cells.

64. A pharmaceutical composition comprising a polypeptide of claim 23.

65. The pharmaceutical composition of claim 64, which is a vaccine.

66. The pharmaceutical composition of claim 64, further comprising one or more pharmaceutically acceptable excipients.

67. A method for preventing infection of a patient by Streptococcus pneumoniae or related bacteria comprising the administration of a pharmaceutically effective amount of the vaccine of claim 65.

68. A pharmaceutical composition comprising one or more antibodies or fragments thereof according to claim 55.

69. The pharmaceutical composition of claim 68, which is a vaccine.

70. The pharmaceutical composition of claim 69, further comprising a pharmaceutically acceptable

excipient.

71. The pharmaceutical composition of claim 69, wherein the antibody is selected from the group consisting of F1-Pn3.1, F2-Pn3.2, F2-Pn3.3, and F2-Pn3.4.

72. The pharmaceutical composition of claim 69, wherein the antibody is F1-Pn3.1.

73. A method for treating a patient infected with or suspected of being infected with Streptococcus pneumoniae or related bacteria comprising the administration of a pharmaceutically effective amount of the vaccine of claim 69.

74. A method for the detection of Streptococcus pneumoniae or related bacteria in a biological sample comprising:

(a) isolating the biological sample from a patient;

(b) incubating the antibody or fragment of claim 55 with the biological sample to form a mixture; and

(c) detecting specifically bound antibody or fragment in the mixture which indicates the presence of Streptococcus pneumoniae or related bacteria.

75. The method of claim 74, wherein the

antibody is selected from the group consisting of F1- Pn3.1, F2-Pn3.2, F2-Pn3.3, and F2-Pn3.4.

76. The method of claim 74, wherein the

antibody is F1-Pn3.1.

77. A method for the detection of antibodies specific to Streptococcus pneumoniae or related bacteria in a biological sample comprising:

(a) isolating the biological sample from a patient;

(b) incubating a polypeptide of claim 23 with the biological sample to form a mixture; and

(c) detecting specifically bound

polypeptide in the mixture, which indicates the presence of antibodies specific to Streptococcus pneumoniae or related bacteria.

78. A method for the detection of Streptococcus pneumoniae or related bacteria in a biological sample comprising:

(a) isolating the biological sample from a patient;

(b) incubating a DNA probe having the DNA sequence of claim 41 with the biological sample to form a mixture; and

(c) detecting specifically bound DNA probe in the mixture which indicates the presence of

Streptococcus pneumoniae and related bacteria.

79. The method of claim 78, wherein the DNA probe is an oligomer having a sequence complementary to at least about 6 contiguous nucleotides of a DNA sequence of claim 41.

80. The method of claim 79, which further comprises:

(a) providing a set of oligomers which are primers for a polymerase chain reaction method and which flank the target region; and

(b) amplifying the target region via the polymerase chain reaction method.

81. The use of a pharmaceutically effective amount of the polypeptide of claim 23 for the prevention of Streptococcus pneumoniae or related bacterial

infections in humans.

82. The use of a pharmaceutically effective amount of an antibody specific to HSP72 for the prevention of Streptococcus pneumoniae or related bacterial

infections in humans.

83. A method for producing a polypeptide or fragment thereof comprising the steps of culturing the unicellular host of claim 52 or 53 and isolating said polypeptide or fragment.

84. A polypeptide in substantially pure form as obtained by the method of claim 83.

85. An antibody or fragment thereof that specifically binds to a polypeptide of claim 1 or 22.

86. A method for isolating the antibody of claim 86 comprising:

(a) introducing a preparation of the polypeptide of claim 1 or 22 into a mammal; and

(b) isolating serum from the mammal containing said antibody.

87. A pharmaceutical composition comprising a polypeptide of claim 1 or 22.

88. The pharmaceutical composition of claim 87, which is a vaccine.

89. The pharmaceutical composition of claim 87, further comprising one or more pharmaceutically acceptable excipients.

90. A method for preventing infection of a patient by Streptococcus pneumoniae, Streptococcus

pyogenes or Streptococcus agalactiae comprising the administration of a pharmaceutically effective amount of the vaccine of claim 88.

91. An antibody or fragment thereof that specifically binds to a polypeptide of claim 1 or 22.

92. A method for the detection of Streptococcus pneumoniae, Streptococcus pyogenes ox Streptococcus agalactiae in a biological sample comprising:

(a) isolating the biological sample from a patient;

(b) incubating the antibody or fragment of claim 91 with the biological sample to form a mixture; and

(c) detecting specifically bound antibody or fragment in the mixture which indicates the presence of Streptococcus pneumoniae, Streptococcus pyogenes or

Streptococcus agalactiae.

93. A method for the detection of antibodies specific to Streptococcus pneumoniae, Streptococcus pyogenes or Streptococcus agalactiae in a biological sample comprising:

(a) isolating the biological sample from a patient;

(b) incubating a polypeptide of claim 1 or 22 with the biological sample to form a mixture; and

(c) detecting specifically bound

polypeptide in the mixture, which indicates the presence of antibodies specific to Streptococcus pneumoniae.

Streptococcus pyogenes or Streptococcus agalactiae .

94. A method for the detection of Streptococcus pneumoniae. Streptococcus pyogenes or Streptococcus agalactiae in a biological sample comprising:

(a) isolating the biological sample from a patient; (b) incubating a DNA probe having the DNA sequence of claim 28 with the biological sample to form a mixture; and

(c) detecting specifically bound DNA probe in the mixture which indicates the presence of

Streptococcus pneumoniae, Streptococcus pyogenes or

Streptococcus agalactiae .

95. The method of claim 94, wherein the DNA probe is an oligomer having a sequence complementary to at least about 6 contiguous nucleotides of a DNA sequence of claim 28.

96. The method of claim 95, which further comprises:

(a) providing a set of oligomers which are primers for a polymerase chain reaction method and which flank the target region; and

(b) amplifying the target region via the polymerase chain reaction method.

97. The use of a pharmaceutically effective amount of the polypeptide of claim 1 or 22 for the

prevention of Streptococcus pneumoniae, Streptococcus pyogenes or Streptococcus agalactiae infection in humans.

98. The use of a pharmaceutically effective amount of an antibody specific to HSP72 for the prevention of Streptococcus pneumoniae, Streptococcus pyogenes or Streptococcus agalactiae infection in humans.

99. The use of a pharmaceutically effective amount of a polypeptide according to any one of claims 2 to 21 for the prevention of Streptococcal infections in humans.

100. A polypeptide having the amino acid sequence of SEQ ID NO:23, analogues, homologues, or derivatives thereof.

101. A polypeptide having the amino acid sequence of SEQ ID NO: 24, analogues, homologues or derivatives thereof.