AU700080B2 - Streptococcal heat shock proteins members of the HSP70 family - Google Patents

Description

WO 96/40928 PCT/CA96/00322 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. pneunoniae disease. Such I I ~LLI L -re~s s WO 96/40928 PCT/CA96/00322 vaccines would also potentially be useful to prevent otitis media in infants and young children.

It is clear that a number of pneumococcal factors are potentially important in the pathogenesis of disease 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 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 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 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 phosphocholine and the capsular polysaccharides 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.

I

WO 96/40928 PCT/CA96/00322 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 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 plac.mta and protect th? 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 WO 96/40928 PCT/CA96/00322 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 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 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 Neidhardt et al., Ann. Rev. Genet., 18, pp. 295-329 (1984); S. Lindquist, Ann. Rev. Biochem., 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, and hsp60 families are among the major HSPs found in all prokaryotes and eukaryotes. These proteins fulfill a WO 96/40928 PCT/CA96/00322 variety of chaperon functions by aiding protein folding and assembly and assisting translocation across membranes 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 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 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 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 Young et al., Proc. Natl.

Acad. Sci. USA, 85, pp. 4267-4270 (1988)], Legionella pneumophila Plikaytis et al., J. Clin. Microbiol., pp. 2080-2084 (1987)], Borrelia burgdorferi Luft et al., J. Immunol., 146, pp. 2776-2782 (1991)], and Chlamydia trachomatis Wagar et al., J. Infect. Dis., 162, pp. 922-927 (1990)]. This antigen is a homologue of the ubiquitous "common antigen", and is believed to be present in every bacterium Thole et al., Microb.

I WO 96/40928 PCT/CA96/00322 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 of the CD4' T-lymphocytes from mice inoculated with M.

tuberculosis were reactive to the hsp60 protein alone 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 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 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 grampositive 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 ~e WO 96/40928 PCT/CA96/00322 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 kDa heat shock protein of Streptococcus pyogenes (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 (residues 439-607, SEQ ID NO:5), the C-terminal 151-residue fragment (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 AEGAQATGNAGDDW (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.

C

WO 96/40928 PCT/CA96/00322 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 Fl-Pn3.1, F2-Pn3.2, F2-Pn3.3 and F2-Pn3.4 monoclonal antibodies 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 Fl-Pn3.1 antibodies that are specific for Streptococcus pneumoniae.

The preferred polypeptides and antibodies of this invention provide the basis for novel mrethods 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 0 C. The cell extracts in the even numbered lanes were incubated at 45 0 C for 5 minutes.

The cell extracts were then labeled with 35 S]methionine for 10 minutes (lanes 1, 2 and 7, 30 minutes (lanes 3, 4 and 9, 10), or 60 minutes (lanes 5, 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 cf the electrophoretic profiles of 35 S]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 WO 96/40928 PCT/CA96!00322 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. 35 S]methionine-labeled proteins from S. pneumoniae grown at 37 0 C and incubated at 37 0 C (lanes 3, 7 and 9) or heat-shocked at 45 0 C (lanes 4, 6, 8 and 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 3 S]methioninelabeled 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 tle 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. 35 S]methiunine-labeled proteins from S. pneumoniae grown at 37 0 C and incubated at 37 0 C (lanes 3, and 7) or heat-shocked at 45 0 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 35 S]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 ~--~IIIPSk-P _I--dl b~ II I WO 96/40928 PCT/CA96/00322 Fl-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 pan.l.

FIG. 6 depicts a fluorogram showing the specificity of MAb Fl-Pn3.1 for HSP72. ["3S]methioninelabeled 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 Fi-Pn3.1 (lane 5,6) and then analyzed by SDS-PAGE and fluorography. Cell lysates from 35 S]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 35 S]methionine-labelled S. pneumoniae cell extracts with MAb Fl-Pn3.1. Lane 1 contains heat-shocked cell lysates 0 C) Lane 2 contains non heat-shocked cell lysates (37 0 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 pg 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 Fl-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 Fl-Pn3.1.

Lane 1 contains whole cell extracts from S. pneumoniae strain 64 prcbed with HSP72-specific MAb Fl-Pn3.1.

Lanes 2 and 3 contain phage lysates from E. coli infected with XJBD17 cultured in the presence or absence of I I I II 4 =~sl WO 96/40928 PCT/CA96/00322 IPTG and probed with HSP72-specific MAb Fl-Pn3.1. Lanes 4 and 5 contain phage lysates from E. coli infected with XJBD7 cultured in the presence or absence of IPTG and probed with HSP72-specific MAb Fl-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 10C illustrate the restriction map of the HSP72(DnaK) and Fuc loci, respectively. FIG illustrates the inserts of the various phages and plasmids described in Example 3. H(HindIII); E(EcoRI); V(EcoRV); P(PstI); 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 ce.

extracts from E. coli transformed with plasmids pJBD179 (lane pJBDf51 (lanes 2 and 3) and pJBDf62 (lane 4 and and cultured in presence or absence of IPTG were subjected to 10% polyacrylamide gel electrophoresis.

The proteins were then visualized by Coomassie Blue staining or Western blotting using HSP-specific MAb Fl-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 were subjected to 10% polyacrylamide gel electrophoresis and WO 96/40928 PCT/CA96/O0322 were electrotransferred to nitrocellulose. The immunoblot.

was probed with HSP72-specific MAb Fl-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 pg of immunoaffinity-purified HSP72rc (lane 3) were subjected to polyacrylamide gel electrophoresis. The proteins were then visualized by Coomassie Blue staining. Lane 1 I 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 pg; lane 2, ug; and lane 3, 1 pg) and whole cell lysates from E. coli transformed with plasmids pDELTAl (lane 4) and pJBDAl (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 HSP72rec. Data are presented as the per cent survival over a period of 14 days for a total of 10 mice per experimental group.

WO 96/40928 PCT/CA96/00322 FIG. 17 is a graphical depiction of the survival curve of Balb/c mice protected from S. pneumoniae infection by immunization with C-169rac. 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; AmpiR, ampicillin-resistance coding region; ColE1 ori, origin of replication; c1857, bacteriophage X c1857 temperaturesensitive repressor gene; X PL, bacteriophage X transcription promoter; Tl, T1 transcription terminator.

The direction of transcription is indicated by the arrows.

BglII and BamHI are the restriction sites used to insert the coding region for the C-151rec of the HSP72 of S.

pneumoniae. FIG. 19 illustrates the distribution of anti-S. pneumoniae titers in sera from Balb/c mice immunized with HSP 72 rec. Sera were collected after the first, second and third injection with 1 pg (0) )r 5 pg of HSP 7 2 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-169rec. Sera were collected after the first, second and third injection with 1 pg or 5 pg of C-169rec 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.

WO 96/40928 PCT/CA96/00322 FIG. 21 illustrates the distribution of anti-S.

pneumoniae titers in sera from Balb/c mice immunized with C-151rec. Sera were collected after the first, second and third injection with 0.5 pg of C-151rec 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 HSP 72 rec or C-169rec 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 HSP 72 rec or C-169rec 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 hsp70/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) WO 96/40928 PCT/CA96/00322 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 35 S]methionine-labeled proteins from S. agalactiae grown at 37 0 C and incubated at 37 0 C (odd numbered lanes) or heat-shocked at 43 0 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 WO 96/40928 PCT/CA96/00322 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 m'v 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.

WO 96/40928 PCT/CA96/00322 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, 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 (SEQ ID 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 NO: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: 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 (SEQ ID NO:20 and SEQ ID NO:22); they are capable of eliciting antibodies that are immunologically reactive with HSP72 (SEQ ID NO:5) and (SEQ ID NO:20 and SEQ ID NO:22); WO 96/40928 PCT/CA96/00322 they are immunologically reactive with antibodies elicited by immunization of a mammal with HSP72 (SEQ ID 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: 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 they are capable of eliciting antibodies that are immunologically reactive with HSP72 (SEQ ID they are immunologically reactive with antibodies elicited by immunization of a mammal with HSP72 (SEQ ID 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 WO 96/40928 PCT/CA96/00322 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 i-mmunoreactive with HSP72 (Example 4).

Preferably, useful polypeptides and fragments will demonstrate the ability to elicit a protective immune response against lethal bacterial infection (Example Also included are polymeric forms of the polypeptides of this invention. These polymeric f6rms 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 mult .istronic mRNAs generated by recombinant DNA technology.

This invention provides substantially pure HSP72 and immunologically related polypeptides. T.e term "substantially pure" means that the polypep .%:,ccording to the invention, and the DNA sequences encocii, 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 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 WO 96/40928 PCT/CA96/00322 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 reconbinant DNA techniques. The use of recombinant DNA techniques is particularly suitable for preparing substantially pure polypeptides accord .ig to the invention.

Thus according to a further aspect of uhe invention, we provide a process for the pr.-cuctLon of HSP72, immunologically related polypeptides, and fagments thereof, comprising the steps of 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 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 WO 96/40928 PCT/CA96/00322 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 e .pression 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, i. may, if desired, be chemically removed by standard techniques.

A wide variety of expression host/vector combinations may be enmloyed 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 El, pCR1, pBR322, pMB9 and their derivatives, wider host range plasmids, such as RP4, phage DNAs, the numerous derivatives of phage lambda, e.g.

Xgtl0 and Xgtll, NM989, and other DNA phages, such as M13 and filamentous single stranded DNA phages. Useful expression vectors for yeast cells include the 2p 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 WO 96/40928 PCT/CA96/00322 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 T? and T7 promoters the major operator and promoter regions of phage lambda, the control regions of fd coat protein, the promoter for 3phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, Pho5, the promoters of the yeast alpha-mating system and other constitutive and inducible 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 (10 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 WO 96/40928 PCT/CA96/00322 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 lid-phase synthetic technique originally described by R. B.

WO 96/40928 PCT/CA96/00322 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 WO 96/40928 PCT/CA96/00322 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 polvpeptides 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 vitro 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, 3-methylhistidine may be substituted for histidine, 4-hydroxyproline may be substituted for proline, 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 h'.-drophilic esidue 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.

WO 96/40928 PCT/CA96/003"2 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 fusior 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, 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 cll culture. Alternatively, the fusion proteins may be produced after gene expression according to known methods.

An example of a fusion protein according to tl:is invention is the FucI/HSP72 (C-169) protein of Example 3, infra.

The polypeptides of this invention may also be par.: 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 id 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, v' provide antibodies, or fragments thereof, that are immunologically rea"tive 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.

WO 96/40928 PCT/CA96/00322 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 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 Fl- 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 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 WO 96/40928 PCT/CA96/00322 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 crosslinking; 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 axpression 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 invent..on 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.

WO 96/40928 PCT/CA96/00322 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 trea\:ments. 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 mg, and preferable 0.1 to 1.0 mg, HSP72 antigen per patient, followed most probably by one or maybe more WO 96/40928 PCT/CA96/00322 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 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 WO 96/40928 PCT/CA96/00322 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: isolating the biological sample from a patient; incubating an antibody of this invention, or fragment thereof with the biological sample to form a mixture; and 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 Fl-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: isolating the biological sample from a patient; incubating a polypeptide of this invention or fragment thereof, with the biological sample to form a mixture; and 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), che 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 AEGAQATGNAGDDW (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 WO 96/40928 PCT/CA96/00322 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 51 Cr 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 1C, 150, or 1N. Binding may also be detected by other methods, for example via avidinbiotin 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: isolating the biological sample from a patient; incubating a DNA probe having a DNA sequence of this invention with the biological sample to form a mixture; and WO 96/40928 PCT/CA96/00322 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 WO 96/40928 PCT/CA96/00322 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 HSP7? 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 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 0 C in 5% CO 2 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 WO 96/40928 PCT/CA96/00322 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)-1piperazinethan-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 detergentsoluble 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 0 C and then divided into fractions of equal volume. The samples were incubated at either 37 0 C or 45 0 C for 5 minutes and then labeled with 100 pCi/ml 3 S]methionine (ICN) for or 60 minutes at37 0 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 10 7 heatkilled bacteria or 20 pg of detergent-soluble pneumococcal WO 96/40928 PCT/CA96/00322 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.

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 NaC1, 0.1% Na dodecyl sulfate, Na deoxycholate, 2% Triton® X-100, 100 pg/ml phenylmethylsulfonylfluoride, and 2pg/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 perforned as described by J.A. Wiley et al. Virol., 66, pp. 5744-5751 (1992)]. Briefly, sera or hybridoma culture supernatants were added to radiolabeled samples containing WO 96/40928 PCT/CA96/00322 equal amounts of [3S]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 (Pharma.cia) for 1 hour at 4 C. The beads were pelleted and washed three times in Tris buffered saline at pH 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 studiel the heat shock response of S. pneumoniae by examining the pattern of protein synthesis before and after a shift from 37 0 C to 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 0 C, incubated at 37°C (lanes 1,3,5,7 and 9) or at (lanes 2, 4, 6, 8 and 10) for 5 minutes, and then labeled with 3 S]methionine for 10 minutt.s (lanes 1,2 and 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. The most prominent induced protein was about 72 kDa (HSP72), whereas the other two were approximately 80 kDa 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 WO 96/40928 PCT/CA96/00322 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).

Ana'v 13 of the densitometric tracings from scanning the piStnc 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 0 C (FIG. 2) Labeled proteins having an apparent molecular mass of 72 kDa were detected at both 37 0 C and 45 0 C conditions (FIG. Radioimmunoprecipitation analysis revealed, however, that HSP72 was undetectable at 37 0 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. 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 1 I WO 96/40928 PCT/CA96/00322 specificity of antibodies for HSP72 was demonstrated by the detection of proteins among heat-.aocked 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 0 C and 45 0 C (FIG. 4, lr-'e This 72 kDa-product probably corresponds to component from peak 9 in FIG. 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 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 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 positive sera after the first immunization (FIG. Antibodies to HSP72 were detected in 13 out of 15 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 and 7 sera, respectively i WO 96/40928 PCT/CA96/00322 (FIG. 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 A. Procedures 1. Immunization of Mice And Fusion Female Dalb/c mice (Charles River Laboratories) were immunized with S. pneumoniae antigens. Cne set of mice (fusion experiment 1) were immunized 'by peritoneal injection with 107 formalin-kiJled whole cell antigen from strain MTL suspended in Freund's cmplete 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) wer- immunized three times at threeweek intervals with 75 p detergent-soluble pneumococcal antigens extracted from strain 64 (type 6) in pg of Quil A adjuvant (Cedarlane Laboratorie. 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. 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 Scuthern Biotechnology Associates Inc. (Birmingham, AL).

WO 96/40928 PCT/CA96/00322 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% yeast extract for 6 hours at 37C 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 Fl-Pn3.1 (from fusion experiment 1) and F2-Pn3.2, F2-Pn3.3 and F2-Pn3.4 (from fusion experiment Isotype analysis revealed that hybridoma Fl-Pn3.1 (from fusion experiment 1) secreted IgG-2ak immunoglobulins, whereas hybridomas F2-Pn3.2, F2-Pn3.3, and F2-Pn3.4 (from WO 96/40928 PCT/CA96/00322 fusion experiment 2) all secreted IgGik. The specificity of the MAbs for HSP72 was clearly demonstrated by the lack of radioimmunoprecipitation activity against [asS]methionine-labeled S. pneumoniae proteins obtained from cultures incubated at 37 0 C and the immunoprecipitation of a 72kDa-protein with heat shock-derived lysates incubated at 45 0 C. FIG. 6, (lanes 5 and 6) demonstrates the results obtained for MAb Fl-Pn3.1. The same results were obtained with MAbs F2-Pn3.2, F2-Pn3.3 and F2-Pn3.4 3 5 S]methionine-labelled lysates from nonheatshocked 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 Fl-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 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 wer\ fractionated by diffeiential centrifugation resulting in a soluble fraction and a particulate fraction, enriched in membrane proteins, supra. Sample containing 15 pg protein of membrane fraction (lane 1) and cytoplasmic fraction (lane 2) cf S. pneumoniae were electrophoresed on SDS-PAGE, transferred to nitrocellulose and probed with MIAb Fl- WO 96/40928 PCT/CA96/00322 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.

WO 96/40928 PTC9/02 PCT/CA96/00322 TABLE 1: BACTERIAL STR~AINS, PHAGES AND PLASMIDS Strain, Phage Plasmid E. coli Strai-ns JM1 09 Relevant Characteristics Reference or Source Yl1090 BL21 (DE3) Phages Xgtl XJBD7 XJBD17 A (lac-proAB) [F'traD proAB rk-Mk- ion supF [p14C9] lacUV5-T7 RNA polymerase c1857 S100 cloning vector LacZ-HSP72 fusion; 2.3 kb EcoRI fragment in Xgtll FucI-HSP72 chimeric; 2.4 kb EcoRI and 2.3 kb EcoRI fragments in ?Xgtil Studier et al. (infra)

BRL

Amers ham Amersham This study This study Plasmids pWSK29 pJBDl7 1 pJBD177 pJBDl79 pT7-6 pJBDf 51 pjBDf 62 pDELTAl pJBDAl Ampr; low copy number cloning vector same as pWSK29 but opposite multi cloning site same as )XJBDl7 but in pWSK29 2.8 kb XhoI-EcoRT fragment in no recombinant HSP72 protein expressed FucI-HSP72 fusion; 2 4 kb EcoRI and 0.8 kb EcoRI- EcoRV fragments in pWSK29 Amp'; T7 promoter 0 same as pT*7-5 but opposite multi cloning site same as pjBDl79 but in same as pJBDl79 but in pT7-6 .AMpr; Tn 1000 same as pJBDl79 but in p DELTA 1 Wang et al.

(infra) Wang et al.

(infra) This study This study This study Tabor et al.

(infra) Tabor et al.

(infra) This study This study

BRL

This study WO 96/40928 PCT/CA96/00322 pJBD291 HSP72; 3.2 kb HindIII This study fragment in pWSK29 pJBDk51 same as pJBD291 but in This study pJBDA4 same as pJBD291 but in This study pDELTAl E. coli strains were grown in L broth or on L agar at 37 0 C. When necessary, ampicillin was added to the media at the concentration of 50 pg/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. pneumoniae DNA library was generated in the bacteriophage expression vector Xgtll (Xgtll clonirg system, Amersham) according to the WO 96/40928 PCT/CA96/00322 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 Igtll arms, packaged, and the resulting phage mixtures used to infect E. coli Y1090. Immunoscreening of plaques expressing recombinant HSP72 antigens was performed using HSP72specific monoclonal antibody Fl-Pn3.1, supra. Plaque clones expressing peptides recognized by MAb Fl-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 HindIII 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 DIGlabelled DNA probes according to the protocol of the manufacturer.

DNA Sequencing and Sequence Analysis The DNA fragments sequenced in this example were first cloned into plasmid pDELTA 1 (GIBCO BRL Life WO 96/40928 PCT/CA96/00322 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 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 010. The resulting plasmid was transformed into E. coli strain BL21(DE3) 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 promoter. Upon IPTG induction, the T7 RNA polymerase induced in the BL21(DE3) transformants specifically WO 96/40928 PCT/CA96/00322 transcribed the gene under the control of T7 promoter 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 Fl-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 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 Xgtll S. pneumoniae genomic DNA library was screened with the HSP72-specific MAb Fl-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 Fl-Pn3.1 and their representatives were designated as kJBD7 and XJBD17 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 kJBD17 (lanes 2 and 3) or XJBD7 (lanes 4 and 5) cultured in the presence or absence of IPTG were subjected to 10% polyacrylamide WO 96/40928 PCT/CA9/00322 gel electrophoresis and were electrotransferred to nitrocellulose. The immunoblot was probed with HSP72specific MAb Fl-Pn3.1. Clone XJBD17 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 Clone XJBD7 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 XJBD17. The fusion protein had an apparent molecular mass of 160 kDa as estimated by SDS- PAGE (FIG. 9, lane The expression of the chimeric recombinant protein encoded by phage XJBD17 was independent of IPTG induction (FIG. 9, lanes 2 and 3) while the expression of the recombinant fusion protein encoded by phage XJBD7 was dependent on induction of the lac promoter (FIG. 9, lanes 4 and In an attempt to subclone the HSP72 gene, the pneumococcal DNA insert from clone XJBD17 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 Fl-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.

WO 96/40928 PCT/CA96/00322 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 010. The ligation mixture was used to transform E. coli JM109 and positive transformants reactive with MAb Fl-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 0 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 FI-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 WO 96/40928 PCT/CA96/00322 kb EcoRI-EcoRV fragment is from the EcoRI end towards the EcoRV end (FIG. The 3.2 kb EcoRI-EcoRV fragment was cloned in,o plasmid pDELTA 1 to yield plasmid pJBDA1. 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 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 (FucI) and to several gene family members, also known as DnaK genes.

Alignment of SEQ ID NO:3 and those of the E. coli FucI and (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 FucI, 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).

WO 96/40928 PCT/CA96/00322 Other restriction sites exist between nucleotides 971 and 972 (Pst nucleotides 1916 and 1917 (Pst I), nucleotides 1978 and 1979 (Xho 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 FucI 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 HindIII 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 PstI-PstI probe, obtained from the 2.4 kb EcoRI-EcoRI fragment. Both probes had been previously labelled wih digoxigenin-dUTP.

These two probes hybridized two individual HindIII fragments of different sizes (FIGS. 10B and 10C). The 0.8 kb EcoRI-EcoRV probe recognized the 3.2 kb HindIII fragment and the 1 kb PstI-PstI probe reacted with the 4 kb HindIII 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 FucI 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.

WO 96/40928 PCT/CA96/00322 E. Production of Recombinant HSP72 A partial pneumococcal genomic library was generated by ligation of the pool of HindIII digests of chromosomal DNA, with sizes ranging from 2.8 to 3.7 kb, into plasmid pWSK29/HindIII. 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 HindIII 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 HindIII fragment was isolated from plasmid pJBD291, and subcloned into plasmids pDELTA 1 and pT7-5 to generate pJBDA4 and pJBDk51, respectively.

The entire 3.2 kb HindIII DNA fragment carried on the plasmid pJBDA4 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 I _L WO 96/40928 PCT/CA96/00322 site ("AGGA") was found 66 base pairs upstream from the start codon of the DnaJ structural gene. No typical regulatory region was identified in front of these two gencs. Restriction sites are located between nucleotides 1 and 2 (HindIII), nucleotides 1318 and 1319 (EcoRI), nucleotides 1994 and 1995 (EcoRI), nucleotides 3343 and 3344 (HindIII), 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, Mcj. Gen. Genet. 164, 1-8 (1978)] as well as several other Gram positive bacteria [Watzstein, 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 t, 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 .itu 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 st I WO 96/40928 PCT/CA96/00322 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 identity with HSP72. Like other HSP70 proteins of Gram positive bacteria, HSP72 miss=s a stretch of 24 amino acids near the amino terminus when compared with DnaK proteins from Grain negative bacteria (FIGS. 13A-13D).

Although HFP72 shares homology with HSP70 (DnaK) proteins from other organisms, it does possess som6 unique features. Sequence divergence of the HSP70 (DnaK) pr,-eins 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 AEGAQATGNAGDDW (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 deteraminants 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 Four Gly-, Cys-rich repeats, each with the Cys-X-X-Cys-X-Gly-X-Gly motif characteristic of DnaJ proteins 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 Nterminus.

-II

I WO 96/40928 PCT/CA96/00322 F. Reactivity of MAbs Against Recombinant Antigens The four HSP72 specific MAbs (Fl-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 kJBD7, thus localizing the epitopes recognized by these MAbs to the C-terminal 169 residues.

Surprisingly, the proteins encoded by the pneumoccocal inserts in kJBD17 and pJBDAl were recognized by only 3 of 4 Mabs. These results suggest that although the C-169 fragments synthesized in E. coli infected with kJBD7 and kJBD17 have the same primary structure, they have distinct conformation. The lack of reactivity of MAb F2-Pn3.2 with some recombinant proteins raised the possibili:y that this particular MAb recognizes a more complex epitope.

Although complex, F2-Pn3.2 epitopes are still recognizable on Western immunoblots. The complete HSP 7 2rc protein expressed by E. coli containing the recombinant plasmid pJBDA4 was reactive with all four MAbs.

EXAMPLE 4 Antigenic Specificity and Reactivity of HSP72-Specific Monoclonal Antibodies The reactivity of MAbs Fl-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

I

I-C P WO 96/40928 PCT/CA96/00322 and then suspended in PBS, pH 7.4. A volume of 5 pl 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 extrz-ts were prepared for Western blot analysis by boiling bacterial suspensions in sample buffer for minutes.

TABLE 2:LIST OF NON-PNEUMOCOCCAL ISOLATES TESTED BY DOT ENZYME IMMUNOASSAY Strain Designation C-2 C-3 C-7 C-9 C-14 C-19 C-21 C-22 C-23 C-24 C-27 C-33 C-36 Genus species Streptococcus pyogenes Streptococcus agalactiae Enterococcus faecalis Streptococcus bovis Streptococcus mutans Streptococcus salivarius Streptococcus sanguis Streptococcus sanguis Streptococcus sanguis Streptococcus sanguis Streptococcus sanguis Streptococcus sanguis Streptococcus sanguis Gemella morbillorum Staphylococcus aureus Bacillus Escherichia coli group or type group A group B group D group D

I

I

I

II

II

II

II

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 -p e~I WO 96/40928 PCT/CA96/00322 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 (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 Streptococcusrelated Enterococcus genus. So far, neither the 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 nonpneumocuccal isolates.

I

WO 96/40928 WO 9640928PCT/CA96/00322 TABLE 3: REACTIVITY OF MABS WITH NON-PEUMOCOCCAL ISOLATES IN WESTERN IMKNOBLCTY'iING Bacterial Strain MAbs Designation C-2 C3 C-7 C-9 C-14 C-19 C-21 C-23 C-24 C-27 C-33 genus /species Strep to coccus pyogen es Streptococcus agala ctia e En terococcus faecalis Streptococcus bovi s Streptococcus mu tans Strepto coccus salivarius Strep to coccus san gui Strep to coccus sanguis Strept-ococcus san gui 5 Streptococcus san gui Streptococcus sangui s Streptococcus san gui s Strepto coccus san gui Gemella morbil11orurn Staphylococcus aureus Bacillus E:scheric' a coli fbi arnyd: a Lrachoma r.stype group A group B group D group D Fl- PN3 1 F2 F2 PN-3 3 F2 Pn3 .4 C-36

C-RP

a indicates a weak signal compared to Lne reactivity observed with S. pnenoriiae antigens b C. trachomatis purified elementary bodies were tested.

WO 96/40928 PCT/CA96/00322 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 HindIII fragment was cloned in both orientations in front of the T7 promoter 010 in the plasmid pT7-5. The resulting plasmid pJBDk51 was then transformed into E. coli strain BL21 (DE3). Overexpression of the recombinant HSP72 protein (HSP72roc) was induced by culturing in broth supplemented with antibiotics for a 3hour period after the addition of IPTG to a final concentration of 1 mM. E. coli expressing high levels of HSP72rc were concentrated by centrifugation and lysed by mild sonication in 50 mM Tris-Cl (pH 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 minutes and the supernatants were collected. HSP72rc was purified by immunoaffinity using monoclonal antibody Fl- Pn3.1 immobilized on sepharose 4B beads (Pharmacia). The purity of eluates was assessed on SDS-PAGE.

The recombinant C-169 protein (C-169rec) was expressed in the form of insoluble inclusion bodies in E. coli strain JM109 transformed with the plasmid pJBDAl.

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 pro- -in solution was WO 96/40928 PCT/CA96/00322 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 HSP72rec or C-169r.c antigens absorbed to Alhydrogel adjuvant. Two antigen doses, approximately 1 and 5 pg, 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 ug of the purified C-169rc 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 WO 96/40928 PCT/CA96/00322 last immunization. The serum samples were pooled and antibodies were purified by precipitation using 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 pl each) obtained 24 hours postchallenge 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 HSP72rac and C-169rc 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 HSP72rac to elicit a protective immune response. Groups of 10 mice were immunized with full-length HSP72rc (1 pg or 5 pg dose) and challenged with 4.2 million CFU of S. pneumoniae type 3 strain WU2. Eighty percent of the mice dosed with 1 pg HSP72r.c survived the challenge, as did 50% of the mice WO 96/40928 PCT/CA96/00322 dosed with 5 pg 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 HSP72r.c 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 prcLein fragments expressed from E. coli transformed with a chimeric fucl- HSP72 gene. Mice immunized with purified C-169ec 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-169r.c (1 ug or 5 pg doses) and challenged with 6 million CFU of S. pneumoniae type 3 strain WU2. Sixty percent of the mice dosed with 1 pg C-169roc survived the challenge, as did 70% of the mice dosed with 5 pg C-169roc (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-169rec 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 2? hours post-challenge, while S. pneumoniae WO 96/40928 PCT/CA96/00322 organisms were detected in only 2 out of a total of immunized SCID mice.

TABLE 4: PASSIVE IMMUNIZATION STUDIES SHOWING PROTECTION OF SCID MICE FROM EXPERIMENTAL S. PNEUMONIAE INFECTION BY ANTI-C-169rac RABBIT ANTIBODIES Experiment Injection No. of Mice No. of Mice Surviving Testing Challenge Positive for after 5 days the Presence of S. pneumoniae 1 sterile 0/5 buffer anti-C-169rc 4/5 control 0/5 antibodies 2 sterile 0/5 buffer anti-C-169rc 5/5 In experiments 1 and 2 (Table 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-Inducible Expression System for High Level Production of the C-151 Terminal Portion of the HSP72 Protein 64 WO 96/40928 PCT/CA96/00322 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 X PL into the translation vector p629 J. George et al., Bio/Technology 5, pp. 600-603 (1987)]. This vector contains a cassette of the bacteriophage X c1857 temperature sensitive repressor gene from which the functional PR promoter has been deleted. The inactivation of the c1857 repressor by a temperature increase from the ranges of 30-37C to 37-42 0 C results in the induction of the gene under the control of X 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 :TQ ID NO was amplified by the polymerase chain reaction (PCR) from the S. pneumoniae type 6 strain 64 genomic DNA using the oligonucleotide primers OCRR26 and OCRR27 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 WO 96/40928 PCT/CA96/00322 coding region for the amino-terminus region of the C-151.

The primers OCRR26 and OCRR27 contain, respectively, a BglII (AGATCT) and a BamHI (GGATCC) recognition site in order to facilitate the cloning of the PCR product into the dephosphorylated restriction sites BglII and BamHI of p629. The PCR product was purified from agarose gels by the method of phenol freeze A. Benson, Biotechniques 2, pp. 67-68 (1984)] and digested with the restriction enzymes BglII and BamHI. The BglII-BamHI fragment of 471 base pairs was then ligated into the BglII 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 DNA Cloning, pp. 109-135, (1985)] into the E. coli strain XLI Blue MRF' (A(mcrA)183 A(mcrCB-hsdSMR-mxr')173 endAl supE44 thi-1 recAl gyrA96 relAl lac proAB laclqZAM15 TnlO (Tetr)]c which was obtained from Stratagene, La Jolla, CA. The transformants grown at 37 0 C were screened by colony immunoblot [J.

Sambrook et al. (supra)] using the MAb Fl-Pn3.1 reactive with C-1 69 rec. Plasmid DNA was purified from a selected transformant and the DNA insert was sequenced 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-151rec.

B. Expression of C-151rec and Antigen Preparation The recombinant C-151rec was synthesized with a methionine residue at its amino end in E. coli strain W3110 harboring the plasmid pURV3. E. coli cells were "1 e dl I- s WO 96/40928 PCT/CA96/00322 grown at 300C in LB broth containing 100 pg of ampicillin per ml until the A 600 reached a value of 0.6. The cells were then cultivated at 400C 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 phosphatebuffered 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 teactivity to C-151rec by Western blot analysis using YM30-ultrafiltrates prepared as described above. The MAbs included a series of six monoclonal antibodies raised to the HSP 72 rec protein (F3to F3-Pn3.10) and monoclonal antibodies Fl-Pn3.1, F2-Pn3.2, F2-Pn3.3, F2-Pn3.4. The three MAbs Fl-Pn3.1, F2- Pn3.3 and F2-Pn3.4 that were reactive with C- 169 rec also recognized the C-151rec fragment. All other MAbs were only reactive with HSP 72 rec thus indicating that they may be directed against epitopes present in the amino terminal region of the HSP72 protein.

it C WO 96/40928 PCT/CA96/00322 EXAMPLE 7 Antibody Response of Balb/c Mice and Macaca- Fascicularis (cynomolg' 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 'o C-151rec, a group of 6 mice were immunized three times at two-week intervals w4th 0.5 pg of C-151rec 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.

pnei moniae 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 pg of purified HSP 7 2 rec or C-169rec 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 clearly demonstrate tLe 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. 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 WO 96/40928 PCT/CA96/00322 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-169rec, the two doses tested, i.e. 1 and 5 pg, were similarly efficient with the induction of similar antibody titers (FIG. 20). A strong boost response was observed after the second injection with C-169rec with no enhancement in the antibody titers after a third injection. In contrast to this, we observed that the immune response to the HSP 72 rec was dosedependent. Increases in the specific antibody titers were observed after a second and a third injection with either

HSP

72 rec or C-1Slrec (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 co:: ain B-cell epitopes responsable for the antigenic h-terogeneity reported in Example 4. To investigate this )o :"ibility, the reactivity of polyclonal and monoclonal WO 96/40928 PCT/CA96/00322 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 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 reversephase 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 ug/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 0 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 pg of purified HSP 7 2 rec according to the immunization protocol described in Example 5. Pool murine sera were from three Balb/c mice immunized with WO 96/40928 PCT'/CA96/00322

HSP

7 2 rec from Example 5 and monkey pool sera were from groups of two animals immunized with either HSP 72 rec or C- 169 rec- TABLE 5: SEQUENCES AND LOCATIONS OF SYNTHETIC PEPTIDES CORRESPONDING TO S. PNEUNONIAE HSP72 AMINO ACID RESIDUES Peptide Location Sequence Sequenlce 11) No.

CS876 247-261 TSTQISLPFITAGEA 7 CS877 257-271 TAGEAGPLHLEMTLT 8 CS878 268-281 MTLTPAKFDDLTRD 9 CS879 1276-290 DDLTRDLVERTIVPV CS880 286-299 TKVPVRQALSDAGL 11 CS882 315-333 RIPAVVEAVKAETGKEPNK 23 CS873 457-471 KAKDLGTQKEQTIVI 12 CS874 467-481 QTIVIQSNSGLTDEE 24 CS875 477-491 LTDEIDRMMKDAEA 13 MAP 1 487-510 KDAEANAESDKKRKEEVDL,rfVV*. 14 CS870 507-521 NEVDQAIFATEKTIK 151 MAP 2 517-544 EKTIKETEGKGFDAERDAAQAALD 16

DLKK

MAP 3 .544-573 KAQEDNNLDDMKAKLEALNEKAQG 17

_______LAVKLY

MAP 4 .583-607 QEGAEGAQATGNAGDDWVDGEFTE 18 1 i KI B Cell Epito Identification and Localization of Linear B- )es The results presented in FIG. 23 revealed that most of the immunological reactivity was observed with the WO 96/40928 PCT/CA96/00322 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 HSP 72 rec of C-169rec 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 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 Fl-Pn3.1. This reactivity was confirmed by fluid-phase inhibition assays in which pg/ml of MAP4 caused complete inhibition of Fl-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, MAP1 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 where mice immunized with purified C-169rec 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 WO 96/40928 PCT/CA96/00322 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 Sant6 Publique du Quebec (LSPQ), Sainte-Anne de Bellevue, Qu6bec, 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 0 C in a

CO

2 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 0 C in L-broth with agitation at 250 rpm or on Lagar.

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).

I

WO 96/40928 PCT/CA96/00322 Preparation of plasmids by equilibrium centrifugation in CsCl-ethidium bromide gradients, agarose gel lectrophoresis 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. 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 A. Benson, Biotechniques 2, pp. 67-68 (1984)]. DNA probes were labeled with a 32 P-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 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).

~L

WO 96/40928 PCT/CA96/00322 3. Molecular Cloning of the Genes 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. pneunoniae (see SEQ ID NO 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 kDa polypeptide kb) were partially purified by extraction of genomic fragments of corresponding size from agarose gel. Verification of the presence of the 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 and transformed into the E.

coli strain XL. 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 HindIII insert of the genomic DNA of S. agalactiae. Plasmid pURV4 contains a 3.5-kb HindIII fragment of the genomic DNA of S.

pyogenes.

WO 96/40928 PCT/CA96/00322 4. Heat Shock and Protein Labeling The stress response of S. agalactiae to an heat shock was assayed by pulse-labeling with 35 S]methionine as described before in Example 1. S. agalactiae bacteria grown overnight in SMAM (Methionine assay Medium supplemented with 1 mg/l methionine, 1% 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 0 C for 1 h and then divided into two fractions of equal volume. The samples were either incubated at 37 or 43 0 C for 10 minutes and then labeled with 100 pCi/ml 35 S]methionine for minutes at 37 0 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.

Immunological Characterization A series of six monoclonal antibodies raised to the HSP 72 rec protein (F3-Pn3.5 to F3-Pn3.10) and the monoclonal antibodies Fl-Pn3.1, F2-Pn3.2, F2-Pn3.3, F2- Pn3.4 were tested for their reactivity to HSP70 antigens from S. pyogenes and S. agalactiaeby 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 HindIII insert of plasmid pURV5 was sequenced. This sequence WO 96/40928 PCT/CA96/00322 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: 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 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 HindIII insert in plasmid pURV4 lacks the 3'-end coding region of the of S. pyogenes. An attempt to clone a 3-kb SalI 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: The ATG start codon begins at position 204 and the TAA stop codon extends to position 2030. Similarly to the 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-567rec). The ORF of plasmid pURV6 lacks 114 base pairs coding for 38 amino acids at the amino end of the HSP70 of S. pyogenes the ORF thus codes WO 96/40928 PCT/CA96/00322 for the 570 amino acids of the carboxyl end of that (C-570rec)- 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 cel-. extracts of heat-shocked and control S. agalactiae pulse-labeled with 3 5 S]methionine revealed that the synthesis of a 70 kDaprotein was significantly increased after a thermal stress (FIG. 26, lanes 1 and Radioimmunoprecipitation analysis revealed that the heat inducible was easily detected at 43 0 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 Fl-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 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 ,I WO 96/40928 PCT/CA96/00322 was also directed against epitopes present in the C-151 region 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 St ra IPI WO 96/40928 PCT/CA96/00322 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 Fl-Pn3.1 deposited in the American Type Culture Collection in Rockville, Maryland, USA on July 21, 1995, and identified -r I WO 96/40928 PCT/CA96/00322 as Murine Hybridoma Cell Line, Fl-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.

I I~ C- es 1 WO 96/40928 PCT/CA96/00322 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: Hamel, Josee Brodeur, Bernard R Martin, Denis Rioux, Clement (ii) TITLE OF INVENTION: STREPTOCOCCAL HEAT SHOCK PROTEINS MEMBERS OF THE HSP70 FAMILY (iii) NUMBER OF SEQUENCES: 26 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Goudreau Gage Dubuc Martineau Walker STREET: 800 Place Victoria, Suite 3400, Stock Exchange Tower CITY: Montreal STATE: Quebec COUNTRY: CANADA ZIP: H4ZlE9 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE:

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 08/472,534 FILING DATE: 07-JUN-1995 (vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: US (PROVIS)60/001,805 FILING DATE: 04-AUG-1995 (viii) ATTORNEY/AGENT INFORMATION: NAME: Leclerc/Dubuc/Prince, Alain/Jean/Gaetan REFERENCE/DOCKET NUMBER: BIOVAC2-PCT (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: :514) 397-7400 TELEFAX: (514) 397-4382 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 3167 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Streptococcus pneumoniae (ix) FEATURE: NAME/KEY: CDS 4 -1 L~~ WO 96/40928 PCT/CA9600322 LOCATION: 30..755 (ix) FEATURE: NAME/KEY: CDS LOCATION: 771..2912 OTHER INFORMATION: /product= "FucI/-SP72 (C-169) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: GAACTTCATT TTTAGMAGG AGTGAGTTT ATG Met

CGT

Arg

GTT

Val

ATT

Ile

AAG

Lys

GAT

CAG

Gin

GCT

Ala

GCA

Ala

GTG

Val

TGT

Asp Tyr Cys

GAA

Glu

ACA

Thr 105

GAG

Glu

CCG

Pro

CAT

His

GAT

Asp

AAG

Lys 185

GAG

Glu Glu ATT TGT GAT Ile Cys Asp AAC GAT GGG Asn Asp Gly 30 ACA CCT ACT Thr Pro Thr 45 AAG TTA AAT Lys Leu Asn CCT TCT AGT Pro Ser Ser GAT GTT CGT Asp Val Arg GCT CTT GCA Ala Leu Ala 110 ATT GTG GTT Ile Val Val 125 ATG GAA GTG Met Giu Val 140 ATG CTA TTA Met Leu Leu ACA GCA TAC Thr Ala Tyr TC CAC GGA Phe His Gly 190 ATT GCT CGT Ile Ala Arg 205 MG GTT ACA Lys Val Thr 220 OTT TGT CAT Val C~s His MT GTA TC'2 Ajm Vai Ser GGT ATC AGC Gly Ile Ser CTT AAA GGA Leu Lys Gly 65 GAA ATT AAA Glu Ile Lys 80 TCA OTT OTT Ser Val Val CAC ATT CCT His lie Pro GGG GCA ATT Gly Ala Ile CCA GAA GCA Pro Giu Ala 145 GM AAT CAT Glu Asn His 160 TAC COT ATG Tyr Arg Met 175 AGA ATG TTA Arg Met Leu CCG ACT TA Pro Thr Leu GGT CGT CAC Gly Arg His TCT CM OAT Ser Gin Asp AAG ATG TGG Lys Met Trp OTT CGA TTA Val Arg Le,.

35 AMA AGT TTT Lys Ser Phe 50 GAG ATT TTA Glu Ile Leu ATO CAC ATT Met His Ile CAC GCG CAT His Ala His 100 TTA GAr" ACT Leu Asp Thr 115 CCT ATT ACC Pro lie Thr 130 ATT ACA CCT Ile Thr Pro GGA GCT CTG Gly Ala Leu GAA ACT TTA Glu Thr Leu 180 CTT TCT ACA Leu Ser Thr 195 AA CGT CTA Glu Arg Leu 210 CCA GOC TAC Pro Gly Tyr GMA AM Glu Lys CAA CTT Gin Leu GAT GAG Asp Glu ATT ACA Ile Thr GAA GCA Glu Ala CGG TGC Arg Cys CCA CCG Pro Pro TAT TCA Tyr Ser CCA TTT Pro Phe TAT CTG Tyr Leu 150 ACT GTC Thr Val 165 GM TTA Glu Leu AAG GGC Lys Gly ITC TCA Phe Ser :GT AAA Arg Lys 230

TTA

Leu

GGT

Gly

OAT

Asp

CCA

Pro

GAA

Glu

TAC

Tyr

ATT

Ile

CTA

Leu

GGA

Gly 135

CCC

Pro

GGA

Gly

GTC

Val

ATT

Ile

ATG

Met 215

TAT

Lyr

ATT

Ile

TGG

Trp

ACC

Thr

GAA

Glu

GGT

Gly

GAA

Glu

GCA

Ala

ATT

Ile 120

OTA

Val

OAT

Asp

AGC

Ser

GCA

Ala

GAG

Glu 200

CGA

Arg

MAT

Asn 53 101 149 197 245 293 341 389 437 485 533 581 629 677 725 225 I ~~_LIL~ WO 96/40928 GGC GAT C Gly Asp C PCTICA96/00322 AAA GAA ACA AAA AAA TAAGAGGAAA GTAT ATG ATC 776 ;GT AGT ATA 'ly Ser Ile Lys Giu Thr Lys Lys 240 Met Ile CAA CAT CCA CGT ATT GGG ATT CGT CCG ACT ATT GAT GGT CGT CGT CAA Gin His Pro Arg Ile Gly Ile Arg Pro Thr Ile Asp Arg Arg Gin GGT GTA Gly Vai GTG GCA Val Ala GTG GAA Val Giu GCA GCT Ala Ala ACA GTT Thr Val CCA GAT Pro Asp 100 GGA GCT I Gly Ala 115 ATT CCA Ile Pro ACA GCT Thr Ala GCA GTT Ala Vai GGT AGT C Gly Ser N 180 TTC CAA C Phe Gin C 195 TTC ACG C Phe Thr P CGT GCG C Arg Ala L AAC CGT C

CGC

Arg

GAT

Asp

TGT

Cys

TCC

Ser

ACA

Thr

ATT

Ile 3TC Val

GCC

Ala

ATT

Ile

:TT

Leu

TT

lal

.AA

lu

:GC

~rg

TC

reu

'AA

GAA TCA CTT Glu Ser Leu *TG ATT TCA Leu Ile Ser 40 GTG ATT TCT Val Ile Ser 55 CAT GAG TTG His Glu Leu CCA TGC TGG Pro Cys Trp CCT CAT GCT Pro His Ala TAT CTT GCA I Tyr Leu Ala 120 TIT GGG ATT Phe Gly Ile 135 CCA GAA GAT Pro Glu Asp 150 GCA ACT GGC Ala Thr Gly I TCG ATG GGG 2 Ser Met Gly I 1 TAC TTA GGA I Tyr Leu Gly t 200 CGT ATG GAC C Arg Met Asp E 215 AAA TGG GTG A Lys Trp Val I 230 GAC CTT GTT I

GA

G1 2'

AG(

Sei CC2 Prc

TT

Phe

TG

Cys

ATT

Ile 105

GCT

Ala

TAT

ITyr

GTC

Jal ErG LeU

.TT

[le .85

'TG

et

GT

Lrg

AA

,ys 7AA

GTA

u Val 5

ACA

r Thr k TCT Ser

SAAA

Lys

TAT

Tyr 90

TGG

Trp G OTA Val

GGT

Gly

AAA(

Lys

ATG

Met 170

GGTC

Gly CGA 2 Arg I GGT I Gly I GAA P Glu 2

AGC

Ser 2 250

CAA

Gin

TTG

Leu

ACC

Thr

AAA

Lys 75

GGT

Gly

GGA

Gly

CTA

Leu

AGAA

Arg

GAA

lu L55

AGA

;GT

gly

AT

\sn

TT

:1e

AC

Is !35

ACA

Thr

AAA

Lys

ATT

Ile 60

TCA

Ser

AGT

Ser

ITT

Phe

GCT

Ala

GAT

Asp 140

AAA

Lys

GAC

Asp

TCT

Ser

GAA

Glu

TAC

Tyr 220

GTA

Val ATG AAC Met Asn TAT CCA Tyr Pro 45 *GGT CGT Gly Arg A.AT GTT Asn Val GAA ACT Glu Thr AAT GGG Asn Gly 110 TCA CAT Ser His 125 GTT CAG Val Gin CTT TTA Leu Leu ACT GCT Thr Ala ATT GTA Ile Val 190 TCG GTA Ser Val 205 GAC CCT Asp Pro C AAA GAA C Lys Glu C ATG GCq Met Ale GAT GC Asp Gly GTT CCA Val Pro TOC GCA Cys Ala ATG GAT Met Asp ACA GAA Thr Glu ACT CAA Thr Gin GAA GCT Glu Ala CGT TAT Arg Tyr 160 TAC CTA Tyr Leu 175 !AT CCA Asn Pro GAT ATG Asp Met 3AA GAG 1lu Glu ;GA TTC 'ly Phe 240 AAA AGT Lys Ser GAA CCT Olu Pro GAG GCT Glu Ala ACA ATT Thr Ile ATG TCT Met Ser CGC CCA Arg Pro AAA GGG Lys Gly 130 AAT GAT Asn Asp 145 GCG CGG Ala Arg TCA ATG Ser Met GAT TTC Asp Phe ACG GAG Thr Glu 210 TTC GAA Phe Glu 225 GAC CAT Asp His 872 920 968 1016 1064 1112 1160 1208 1256 1304 1352 1400 1448 1496 1544 ,T GAA GAA AAA GAT AGA CAA TG Asn Arg Glu 245 Asp Leu Val Leu rg Glu Glu Lys Asp Arg Gin Trp 255 -L I _1 I WO 96/40928 GAA TT GTI Glu Phe Val 260 AAC CCA AGA Asn Pro Arg 275 ATT AAG ATG TTC ATG ATT Ile Lys Met Phe Met Ile 265 CTT GCT GAA CTT GGT TTT GGA CGT Gly Arg GAG GAA ATG GTT GGT Met Val Gly GTT GGT CAC CT/CA96/00322 1592 1640 Leu Ala Glu 280 Leu Giy Phe Glu Glu Glu Ala Val Gly His

CAT

His

TTT

Phe

TGG

Trp

CTA

Leu

CAA

Gin 355

CGT

Arg

CAT

His ACT I Thr

AGT

Ser CGC C Arg 435 GGG C Gly I GGT C Gly I GAT C Asp ACT Thr T TAT C Tyr A 515 GCT TTA GTA GCT GGT TTC CAA Ala Leu Val Gly Phe Gin

CCA

Prc

AAT

Asn

AAT

Asn 340

ATC

Ile

GTA

Val

CTA

Leu

CGA

Arg

GAA

Glu 420

,AA

lu

AT

sp

:CA

?ro

;TT

al 'rGG 'rp ;00

,AC

AAT

Asn

GGT

Gly 325

GGT

Gly

TTT

Phe

ACA

Thr

ATC

Ile

GAT

Asp 405

GTA

Val

TAC

Tyr

ATG

Met

GTG

Vai I

CAC

His 1 485 TTT C Phe I GTC I

GGC

Gl 31C

ATT

Ile

GTG

Val

GCT

Ala

GGA

Gly

AAC

Asn 390

GGC

Giy

CAG

G1n

ITC

Phe

CCA

Pro

CTA

Leu 170

:AT

is

;CT

la

\TG

GAC TTT ATG GMA Asp Phe Met Glu

CGA

Arg

TCT

Ser

GAT

Asp

TAT

Tyr 375

TCT

Ser

AAA

Lys

GCT

Ala I

CGT

Arg

GTA

Val 455

CAA

Gin ACT Thr I CCA C Pro P

AAT

AAA

Lys

ATG

Met

GTG

Val 360

ACT

Thr

GGA

Gly

CCT

Pro

ATG

Met

GGA

Gly 440 kCA Thr kTT Ile

ETA

~eu

GT

~rg

CCA

Pro

CTC

Leu 345

CGT

Arg

TTA

Leu

TCT

Ser

GTT

Val

CTT

Leu 425

GGA

Gly

ATG

Met

GCA

Ala C GAT 2 Asp 2 4

TTG

Leu J 505

TTT

Phe 330

TTT

Phe

ACT

Thr

GAG

Glu

TGT

Cys

ATG

Met 410

GAA

Glu

GGA

Gly 3TA Ial

"AA

3lu

AT

Lsn 190

~CA

.hr GGT CAA Gly Gin 300 ACT TTC Thr Phe 315 GTA TTT Val Phe AAT TAT Asn Tyr TAT TGG Tyr Trp GGT CGT Gly Arg 380 ACA TTO Thr Leu 395 AAA CCA Lys Pro AAT ACA Asn Thr 2 TTC TCA I Phe Ser 4 CGT CTC 2 Arg Leu 2 460 GGT TAC P Gly Tyr rj 475 CGT ACA C Arg Thr A GGA MA C Gly Lys C

CG

Are

CT

Let

GCC

Al

CTI

Leu

AGT

Ser 365

GCT

Ala

GAT

Asp

ITC

Phe

GAC

%sp

~CT

1hr 145

AT

~sn

~CA

.hr

AT

sp

GT

ly 9 Gin

AAT

u Asn 3 ACA i Thr

'ITA

Leu 350

CCA

Pro

GCA

Ala

GGT

Gly

TGG

Trp

TTC

Phe I 430

CGT

Arg I CTT Leu I CTT C Leu C CCA C Pro G 4 GCT I Ala P 510 Trr

ACT

Thr

GAG

Glu 335

ACA

Thr

GAG

Glu

GCT

Ala

ACA

Thr

GAG

lu 41S

CCA

?ro

ETC

Phe

ETA

.eu

'AA

;lu

;GA

;ly

TC

he Thr

CAG

Gin 320

AAT

Asn

AAT

Asn

GCT

Ala

GGA

Gly

GGT

Gly 400

TIG

Leu

CCA

Pro

TTG

Leu

AAA

Lys

CTT

Leu 480

TGG

Trp

MG

Lys r CAG TGG ACA GAC CAT *Asp His 305 TT GAC Phe Asp GAT TCA Asp Ser ACT CCA Thr Pro GTT GMA Val Glu 370 TTC TTA Phe Leu 385 CAA GCT Gin Ala GAT GAA Asp Glu GCA AAC Ala Asn ACG MAG Thr Lys 450 GGG GTT Gly Val 465 CCT GAA Pro Glu CCA ACT Pro Thr TCT GTC Ser Val 1688 1736 1784 1832 1880 1928 1976 2024 2072 2120 2168 2216 2264 2312 2360 ~sp Val Met Asn A 5 LAT TGG GGA GCT AAT CAC GGA GCC ATA ACA TAT ~sn Trp Gly Ala Asn His Giy Ala Ile Thr Tyr 20 525 530 I s, WO 96/40928 GGA CAC k.TT GGA GCA GAC FI'G ATT ACC ITG GCT TCT ATG TI'G AGA AT

P(

e Gly His Ile Gly Ala Asp 535 Leu Ile Thr Ala Ser Met Leu Arg Il 545 CCT CA.A Pro Gin GTT AAG Vai Lys CAA TCG Gin Ser 580 GAT GCA Asp Aia 595 GAC CTT Asp Leu ATC AAG Ile Lys CAA GCT Gin Aia GAC GAC Asp Asp 660 GTT GCT Leu Ala 675 GAA GGA Glu Gly GTA GAG Val Asp

ATC

Ile

GCC

Ala 565

AAC

Asn

GAA

Giu

CGT

Arg

GAA

GiU

GCC

Ala 645

ATG

Met

GTT

Val

GCA

Al a

GGA

Gly GAA GTA Giu Val 550 AAA GAC Lys Asp TCA GGT Ser Gly GCA AAC Ala Asn AAT GAA Asn Giu 615 ACT GAA Thr Giu 630 CTT GAT Leu Asp AAA GCA Lys Ala AAA GTG Lys Leu GAA GGC Giu Gly 695 GAG TT Giu Phe

ACA

Thr

CTT

Leu

TTG

Leu

GGT

Al a 600

GTG

Val

GGT

Gly

GAG

Asp

AAA

Lys

TAG

Tyr 680

GGA

Ala

AG

TTT GAG Phe Asp GGA AGT Gly Thr 570 ACT GAG Thr Asp 585 GAA TGG Glu Ser GAG CAA Asp Gin AAA GGG Lys Gly GTT AAG Leu Lys 650 GTT GAA Leu Glu 665 GAA CAA Giu Gin GAkA GGA Gin Ala GAA AAG ATG GAG AAG AAG GOT ATG GTG TGT Ile Asp Lys Asn Gly Ilie Val Ser 555 560 CAA JAAA GAA CAA ACT ATT GTG ATG Gin Lys Giu Gin Thr Ile Val Ile 575 GAA GAA ATG GAG CG ATG ATG AAA Glu Giu Ile Asp Arg Met Met Lys 590 GAT AAG AAA GGT AAA GAA GAA GTA Asp Lys Lys Arg Lys Giu Giu Val 605 610 GGA ATG TI'T GCG ACT GAA AAG ACA Ala Ile Phe Ala Thr Giu Lys Thr 620 625 TI'G GAG GGA GAA CGT GAG GGT GGG Phe Asp Aia Glu Arg Asp Ala Ala 635 640 AAA GGT CAA GAA GAG MGC AAG rTG Lys Ala Gin Glu Asp Asn Asn Leu 655 GGA TTIG AAG GAA AAA GGT CAA GGA Ala Leu Asn Glu Lys Ala Gin Gly 670 GGG GGA GGA GGG CAA GAA GGT CAA Aia Ala Ala Ala Gin Gin Ala Gin 685 690 AGA GGA MGC GGA GG GAT GAG GTG Thr Gly Asn Ala Gly Asp Asp Vai 700 705 TAAGATGAGT GTATTGGATG AAGAGTATGT T/CA96/00322 2408 2456 2504 2552 2600 2648 2696 2744 2792 2840 2888 2942 Thr Giu Lys AAAAAATACA CGAAM-GTTT ATAATGATTT TTIGTAATGAA GGTGATAACT ATAGAACA.

AAAAGATTIT A'ITGATAATA TI'GCAATAGA ATAITTAGCT AGATATAGAG AAAITATA~ AGGTGAGCAT GATAGITGTG TCAAAAATGA TGAAGCGGTA AGGAATT 1G TTAGCTCAC ATTG'ITGTCT GCATI'TGTAT CGGCGATGGT ATGAGGTATG A' ATC INFORMATION FOR SEQ ID NO:2: Wi SEQUENCE CHARACTERISTICS: LENGTH: 242 amino acids TYPE: amino acid TOPOLOGY: iinear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Ser Gin Asp Glu Lys Leu Ile Arg Giu Gin Ile Gys Asp Val Gys 1 5 10 86

['T

3002 3062 3122 3167 WO 96/40928 WO 9640928PCTICA96/00322 His Ser Met Trp Arg Leu Gin Leu Asp Glu Gly Trp Asp Thr Ala Ala Leu Ala Asp Gly Pro Thr Asn Val Gly Ile 40 Ser Gly Ser Phe Ile Thr Pro Glu Lys Leu Val Ile Leu Giu Gly Asp Ty'r Lys Met His Ile Val Pro Ile Ala 145 His Met Leu Leu His 225 Lys Arg Pro Tyr Pro Tyr Thr 165 Glu Cys Tyr Giu Giu Lys Pro Asp Ala Ile Met 140 Met Thr Phe Leu Asn Leu Lys Ser Val Leu Val 125 Glu Leu Al a His Giu Ser His Gly Pro Glu Tyr 175 Arg I Thr Lys Gly Ile GlU Giu Gin Glu Ile Ala 200 205 Arg Pro Thr Ser Met 215 Lys Tyr 230 Arg Glu Asn Gly Lys Val Thr 220 Ser Ile Lys Giy Arg Giu Thr 240 INFORMATION FOR SEQ ID NO:3: r,(iW SEQUENCE CHARACTERISTICS: LENGTH: 714 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Met Ile Gin His Pro Arg Ile Gly Ile Arg Pro Thr Ile Asp Gly Arg 1 5 10 Arg Gin Gly Val Arg Giu Ser Leu Giu Val Gin Thr Met Asn Met Ala 25 Lys Ser Val Ala Asp Leu Ile Ser Ser Thr Leu Lys Tyr Pro Asp Gly 40 Giu Pro Val Giu Cys Val Ile Ser Pro Ser Thr Ile Gly Arg Val Pro 55 WO 96/40928 Glu Ala PCT/CA96/00322 Ala Ala Ser His Glu Leu Phe Lys Lys Ser Asn Val CYs Ala 70 75 Thr Ile Thr Val Thr Pro Cys Trp Cys Tyr Gly Ser Glu Thr 90 Met Asp Met Ser Pro Arg Pro Gly 115 Pro His Ala Ile 105 Tyr Leu Ala Ala Trp Gly Phe Asn Gly Thr Glu 110 Val Leu Ala Ser His Thr Gin Lys Gly Ile Pro Ala Phe 4 4 6 6 130 Asn Asp 145 Ala Arg Ser Met Asp Phe Thr Glu 210 Phe Gu 225 Asp His Gin Trp 00 Val Gly Gly His r5 290 Asp His 305 0 Phe Asp Asp Ser I Thr Pro C Vai Glu 2 0 370 Phe Leu 1 385 5 Gin Ala Thr Ala Gly Phe 195 Phe Arg Asn Glu Asn 275 His Phe rrp Leu 31n 355 rg Iis 7hr Ala Val Ser 180 Gin Thr Ala Arg Phe 260 Pro Ala Pro Asn Asn 340 Ile Val Leu Arg Ile Leu 165 Val Glu Arg Leu Glu 245 Val Arg Leu Asn Gly 325 Gly Phe 2 Thr C Ile 3 Asp C 405 Pro 150 Ala Ser Tyr Arg Lys 230 Asp Ile Leu Val ly 310 Tie 2 Jai dla I Uy I 3 ~sn S 90 ;iy L Gl) 13E Glu Thr Met Leu Met 215 Trp Leu Lys Ala a 295 %sp krg ;er Isp ,yr '75 er ,ys 120 r Ile Ty: Asp Va.

Gly Let Gly liE 18E Gly Met 200 Asp Arg Val Lys Val Leu Met Phe 265 Glu Leu 280 Gly Phe Phe Met Lys Pro Met Leu 345 Val Arg 360 Thr Leu Gly Ser Pro Val r Gly Arg Asp 140 1 Lys Giu Lys 155 i Met Arg Asp 170 Gly Gly Ser Arg Asn Glu Gly Ile Ty r 220 Glu Asn Val 235 Ser Arg Glu 250 Met Ile Gly Gly Phe Glu Gin Gly Gin 300 Glu Thr Phe 315 Phe Val Phe 330 Phe Asn Tyr Thr Tyr Trp Glu Gly Arg 380 Cys Thr Leu 2 395 Met Lys Pro 410 Glu Asn Thr I 125 Va1 Leu Thr Ile Ser 205 Asp Lys Glu Arg Glu 285 Arg Leu Ala Leu I Ser I 365 k1a I ksp C Phe T1 tsp P 4 Gir Le Ala Val 190 Vai Pro Glu Lys Asp 270 Glu GIn A.sn Thr eu 350 ?ro 1 a ily 'rp he 1 Giu Arg Tyr 175 Asn Asp Glu Gly Asp 255 Leu Ala Trp Thr Glu 2 335 Thr I Glu I Ala C Thr G 4 Glu L 415 Pro P Ala Tyr 160 Leu Pro Met Glu Phe 240 Arg Met Vral Ihr 31n 320 ksn s n l a 1 y ;1y 00 eu ro Asp Glu Ser Glu 420 Vai Gin Ala Met Leu 425 II g C ~A WO 96/40928 PCT/CA96/00322 Ala Asn Arg Glu Tyr Phe Arg 435 Gly Gly Gly Phe 440 Ser Thr Arg Phe Leu 445 Thr Lys 450 Gly Val 465 Pro Glu Pro Thr Ser Val Thr Tyr 530 Arg Ile 545 Val Ser Val Ile Met Lys Glu Val 610 Lys Thr 625 Ala Ala Asn Leu Gin Gly I Ala Gin C 690 Asp Val 705 Gly Asp Thr Tyr 515 Gly Pro Val Gin Asp 595 Asp Ile Gln Asp Leu 675 ;lu Val Pro Val Trp 500 Asp His Gin Lys Ser 580 Ala Leu Lys Ala Asp 660 Ala Gly Asp Val His 485 Phe Val Ile Ile Ala 565 Asn Glu Arg Glu Ala 645 Met I Val I Ala C Gly C 7 Gly Asp Met Pro Val Thr Met Val Arg Leu Asn Leu Leu Lys Leu 470 His Ala Met Gly Glu 550 Lys Ser Ala Asn Thr 530 Leu Lys Lys ;lu 455 SGin Thr Pro Asn Ala 535 Val Asp Gly Asn Glu 615 Glu Asp Ala Leu Gly 695 Ile Ala Leu Asp Arg Leu 505 Asn Trp 520 Asp Leu Thr Phe Leu Gly Leu Thr 585 Ala Glu 600 Val Asp Gly Lys Asp Leu Lys Leu 665 Tyr Glu 680 Ala Gin I Glu Asn 490 Thr Gly lie Asp Thr 570 Asp Ser Gln Gly Lys 650 3lu G1n Ala 460 Gly Tyr 475 Arg Thr Gly Lys Ala Asn Thr Leu 540 Ile Asp 555 Gin Lys Glu Glu Asp Lys Ala Ile 620 Phe Asp 635 Lys Ala Ala Leu Ala Ala Thr Gly 700 Thr Asp Gly His 525 Ala Lys Glu Ile Lys 605 Phe Ala Gin Asn Ala 685 Asn Leu Pro Ala 510 Gly Ser Asn Gin Asp 590 Arg Ala Glu Glu Glu 670 Ala Ala Glu Gly 495 Phe Ala Met Gly Thr 575 Arg Lys Thr Arg Asp 655 Lys Gin Gly Leu 480 Trp Lys Ile Leu Ile 560 Ile Met Glu Glu Asp 640 Asn Ala Gln Asp ;lu Phe Thr Glu Lys INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 4320 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Streptococcus pneumoniae 89 II -1 WO 96/40928 WO 9640928PCT/CA96/00322 (ix) FEATURE: NAM~E/KEY: CDS LOCATION: 682. .2502 OTHER INFORMATION: /product= ""Heat-shock protein 72"" (ix) FEATURE: NAME/KEY: CDS LOCATI ON: 3 2 65. .4 32 0 OTHER INFORMATION: /product= "NH2-terminai portion of DNA J" (ix) FEATURE: NAME/KEY: mat..peptide LOCATION: 682. .2502 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: AAGCTIGATT CACGCTTTGA AAGAAGAAGG TGACCATMAC TACCATATGG CCATCCAAAC TACCATCGCC CAAGTCTTTC AAAAAGGCTA AATGGTAGTG GTGTATAACT AAGATACAAA GATTGACGAA GTGTCGATG AACACAAGAA GTGTTCGA'IT CGGCAATTCT GACGGTAGCT ATGGCGTITTG TCTAGC'I CC 'ITACTMACTC CGTGTCGCAA TTI'ACATAAT AGAAAACTG TAAAATATGT TTGGCT1TGT AATAGTGAGC TGCGCTAFT TGCGCAAATT TTGAGACCTI' AAGTCAAGCT CTGACGGCGT CGCCACTTAA AATTGAAGAA ATCGCAGCAG ATGGCGAATT

TCTCCCAGCA

CAAACTCCAT

GCCCGTAAAA

AATCTATCTT

AMAGCAACTC

GTCGTCGAA.A

TCCGAAACGA

GAAGCGAACC

AGGCTCAAAG

GAAGAGTATC

GACGATGAAC ACCCAGTAGA GACCGCATCC TACGCCCAGC AGCTCGCAGT AAAAATAGGA 'ITTTACTCAG AGC FIAGGGC GTCAGAAAAC GGCAGTCGCT TAAA-ATCGAT TTCGACTCTI' CAATAAACTA TGAAGAAAGA AAAGACGATA CTCTTCGCTG 'ITTAGTCAAA GAGATTGACA AAAAAGAAAA ATAGAAAATT AACTAACAAG GAGMMAACA C ATG TCT AAA ATT ATC GGT ATT GAC TTA GGT Met Ser Lys Ile Ile Gly Ile Asp Leu Gly ACA ACA MAC TCA GCA GTT GCA Thr Thr Asn Ser Ala Val Ala ATC GCA AAC CCA GAA GGA AAC Ile Ala Asn Pro Giu Gly Asn GTT CTT GAA GGA Val Leu Giu Gly 20 CGC ACA ACT CCA Arg '1h r Thr Pro 35 GGT GAT GCT GCA Gly Asp Ala Ala 50 ACT GAA AGC AAA ATC Thr Giu Ser Lys Ile TCT GTA GTC TCA TI'C Ser Val Val Ser Phe AAA AAC GGA Lys Asn Gly ACA AAC CCA Thr Asn Pro GAA AAA GTT Giu Lys Val GAA ATC ATC GTT Gu Ile Ile Val GAT ACA GTT ATC Asp Thr Val Ile 65 TCT .3CA MAT GGA Ser Ala Asn Gly 80 AMA CGT Lys Arg TCT ATC AMA TCT MAG ATG Ser Ile Lys Ser Lys Met CMA GCA GTT Gin Aia Val GGA ACT TCT Gly Thr Ser GMA ATC TCA Giu Ile Ser AMA GMA TAC Lys Giu Tyr ACT CCA CMA Thr Pro Gin 85 GCT ATG ATC CTT CMA TAC 'PIG AMA GGC TAC GCT GMA GAC TAC CTT GGT Ala Met Ile Leu Gin Tyr Leu Lys Gly Tyr Ala Giu Asp Tyr Leu Gly 100 105 WO 96/40928 GAG AAA GTA ACC Glu Lys Val Thr 110 GCT CAA CGT CAA Ala Gin Arg Gin 125 GTA GAA CGT ATT Val Glu Arg Ile 140 TTG GAC AAG ACT PCT/CA96/00322 C 1047 GCT Ala

ACA

Thr

AAC

Asn

AAA

Lys 160 ACA GTT CCG GCT TAC Thr Val Pro Ala Tyr 115 GCT GGT AAA ATT GCT Ala Gly Lys Ile Ala 135 ACT GCA GCA GCT CTT Thr Ala Ala Ala Leu 150 AAA ATC TTG GTA MTT Leu 155 Asp Lys Thr Asp Glu Glu Lys Ile Leu Val Phe Asp Leu Gly GGT GGT Gly Gly GAC GTA Asp Val GAC CAA Asp Gin GGT ATC Gly Ile 220 GCG GCT Ala Ala 235 ATC AGC Ile Ser GAA ATG Glu Met GTT GAA Val Glu TTG AGC Leu Ser 300 CGT ATC Arg Ile 315 CCA AAC Pro Asn ATC CAA Ile Gin

ACI

Thi

TTG

Leu A2 Lys 205

GAC

Asp

GMA

Glu

TTG

Leu

ACT

Thr

CGT

Arg 285

TTG

Eeu

:CT

Pro Lys 3GT ,ly ITC GAC Phe Asp 175 TCA ACT Ser Thr 190 ATC ATT Ile Ile TTG TCT Leu Ser AAA GCG Lys Ala CCA TTT Pro Phe 255 TTA ACT Leu Thr 270 ACA AAA Thr Lys TCA GAA Ser Glu GCC GTT Ala Val TCA GTA Ser Val 2 335 GGT GTG 2 Gly Vai 350 GTC TCT ATC Val Ser IlE

GCA

Ala

GAC

Asp

ACT

Thr

AAG

Lys 240

ATC

Ile

CGT

Arg 3TT Val

ATC

Ile

GTT

Val 320 kAC ksn kTT Ile GGG GAC Gly Asp CAC TI'G His Leu 210 GAC AAG Asp Lys 225 AAA GAC Lys Asp ACT GCA Thr Ala GCG AAA Ala Lys CCA GTT Pro Val 290 GAC GAA Asp Glu 305 GAA GCT Glu Ala CCT GAT Pro Asp ACT GGT Thr Gly CTT GAA ITG Leu Giu Leu 180 AAC AAA CTT p Asn Lys Leu 195 GTA GCA GAA Val Ala Glu ATG GCA ATG Met Ala Met CTT TCT GGT Leu Ser Gly 245 GGT GAG GCT Gly Glu Ala 260 TTT GAT GAT Phe Asp Asp 275 CGT CAA GCC Arg Gin Ala GTT ATC CTT Val Ile Leu GTT AAA GCT Val Lys Ala 325 GAA GTA GTT Glu Val Val j40 GAT GTC AAG Asp Val Lys 355

GGT

Gly

GGT

Gly

TITC

Phe

CAA

Gin 230

GTA

Val

GGA

Gly

TTG

Leu

CTT

Leu

GTT

Val 310

GAA

Glu

GCT

Ala

GAT

Asp

GA

Asl

GG

G11

AAC

Lys 215

CGT

Arg

ACT

Thr

CCT

Pro

ACT

Thr

TCA

Ser 295

GGT

Gly

ACT

Thr

ATG

Met

GTT

Val C GGT GTC Gly Val 185 r GAC GAC Asp Asp 200 AAA GAA Lys Glu TTG AA Leu Lys TCA ACA Ser Thr CTT CAC Leu His 265 CGT GAC Arg Asp 280 GAT GCA Asp Ala GGT TCA Gly Ser GGT AAA Gly Lys GGT GCG Gly Ala 345 GTC CTT Val Leu I 360

TC

Phe

TTT

Phe

AAC

Asn

GAT

Asp

CAM

Gin 250

ITG

Leu

CTT

Leu

GGT

Gly

ACT

Thr

GAA

Glu 330

GCT

Ala

CTT

Leu 1095 1143 1191 1239 '287 1335 1383 1431 1479 1527 1575 1623 1671 1719 1767 1815 GAT GTA ACG CCA TTG TCA CTT GOT ATC GAA ACA ATG GOT GGA GTA ITT Asp Val Thr 365 Pro Leu Ser Leu Gly 370 Ile Glu TJhr Met Gly 375 Gly Val Phe ~111~ -4 1 1_ WO 96/40928 ACA AMA CTT ATC GAT CGC Thr Lys Leu Ile Asp Arg 380 AAC ACT ACA ATC Asn Thr Thr Ile 385 CCA ACA TCT AAA TCA CAA Pro Thr 390 Ser Lys Ser G.

GTC

Val 39

CT]

Lei.

TTC

Phe

GAA

Glu

AAA

Lys

TCA

Ser 475

GCA

Ala

AAT

Asn

ACT

Thr

CTT

Leu

AAA

Lys 555

A

Lys

GAA

Glu

GAG

Giu TITC TC, *Phe Se: CAA GG'.

IGin G1, CAA TC *Gin Lei *GTA AC; Val Thi 44E GAC CT] Asp LeL 460 GGT TTG- Gly Leu AAC GCT Asn Ala GAA GTG Giu Val GAA GGT Giu Gly 525 GAT GAC Asp Asp 540 GCA AAA Ala Lys CTC TAC Leu Tyr GGC GCA Gly Ala TTT ACG Phe Thr kACA GC; r- Thr AlE r' GMA CGC Giu Arg 415 ACT GA7 Thffr Asp 430 TTT GAC Phe Asp GGA ACT Gly Thr ACT GAC Thr Asp GAA TCC Glu Ser 495 GAC CAA Asp Gin 510 AAA GGC Lys Gly CTT AAG Leu Lys CTT GAA Leu Glu GAP. CAA Giu Gin 575 CAA GCA Gin Ala 590 GAA AAG Giu Lys GCA GAC .AAC CAA CCA GCC GTT GAT ATC CAC GTT Ile Pro Ala Pro Arg Gly ATC GAC Ile Asp CAA A Gin Lys 465 GAA GAA Giu Giu 480 GAT MAG Asp Lys GCA ATC Ala Ile TT GAC Phe Asp MAA GCT Lys Ala 545 GCA 'ITG Ala Leu 560 GCC GCA Ala Ala ACA GGA

MAG

Lys 450

GMA

Gi u

ATC

Ile

AMA

Lys Phe

GCA

Al a 53 0 Gin

MAC

Asn

GCA

Al a

MAC

MAC GGT Asn Gly CMA ACT Gin Th-r GAC CGC Asp Arg CGT AMA Arg Lys 500 GCG ACT Ala Thr 515 GM. CGT Giu Arg GMA GAC Giu Asp GMA AMA Giu Lys GCG CMA Ala Gin 580 GCA GGC

ATC

Ile

ATT

Ile

ATG

Met 485

GMA

Giu

GMA

Giu

GAC

Asp

MAC

Asn

GCT

Ala 565

CMA

Gin

GAT

GTG

Val

GTC

Val 470

ATG

Met

GMA

Glu

MAG

Lys

GCT

Ala

AAC

Asn 550 Sin

GCT

Al a

GAC

Asr

AC]

AT]

Ile

TCT

Ser 455

ATC

Ile

AAA

Lys

GTA

Val

ACA

Thr

GCC

Al a 535

TI'G

Leu

GGA

Gly

CMA

Gin

GTC

Ile His Val 410 CTT GGA CGC Leu Gly Arg 425 CCT CMA ATC Pro Gin Ile 440 GTT MAG GCC Val Lys Ala CMA TCG MAC Gin Ser Asn GAT GCA GMA Asp Ala Giu 490 GAC CTT CGT Asp Leu Arg 505 ATC MAG GMA Ile Lys Giu 520 CMA GCT GCC Gin Ala Ala GAC GAC ATG Asp Asp Met CTT GCT GTT Leu Ala Val 570 GMA GGA GCA Glu Gly Ala 585 GTA GAC GGA Val Asp Gly CT/CA 96/00322 1863 1911 1959 2007 2055 2103 2151 "199 2247 2295 2343 2391 2439 2487 2542 Thr Gly Asn Ala Gly Asp Asp Val TMAGATGAGT GTATTGGATG MAGAGTATCT AAAAXATACA 605 CGAAMAGTrI' ATM.ATGA'ITT TI'GTMTCMA GCTGATMACT ATAGMACATC AAMAGAT TTT ATTGATMATA TTCCM-TAGA ATATTTAGCT AGATATAGAG MAATTATAFI AGCTGAGCAT GATAGTTGTG TCAAAPATGA TGAAGCGGTA AGGMATrTTG, TACCTCAGT ATTGTTGTCT GCATTTGTAT CGGCGATGGT ATCAGCTATIG ATATCATTAC '\?\ATACAMAC ATATAMFT GTMATACCGT TCATMATTGG TATGATITGG ACAGTAGTI'G TAFI'TCTI'AT GATCMATTGG 2602 2662 2722 2782 2842

I

w 0 96/40928 PCT AATTATATAG GCAAATACTA AGAAGAGACA AAA.ATATATA AATATTTCTG TACTTATAGG ATAT'ITAA.AA TCCAA.ATAAA GTTAATTTAC TTATTTGCAG AGGTTGCAAC CCAGCCTCTG TIT1'TCGATA MAAAGGGACG G.AATCTCATT TGT'ITGGGTT TTGTCTCATC AATAGAAAGG A.ACAAAGAGT GITCGTAACT GAACACGGGT TTCAGAATI'T CTTACTAMAT ATAAAAGAAA GGAA'FTGAAC CCGACCTAA.A TGGTGGTTCG AITCAGAACA TCAATAGAAA GGAATAAGGG TGITCGTAAC TGAACACGGG CTACGGACTG TGCCAAAAAG ATAGTTTTT CTAGGA-GTA AGCGTCCGTC GTCAAMACTC CTAGATGGCT GTGTCCGTTT GACGCCCTTT GTATCTTGMA TT? ATO AAC AAT ACT GAA TT1' TA~T GAT CGT CTG GGG GTA TCC AAA AAC Met Asn Asn Thr Glu Phe Tyr Asp Arg Leu Gly Val Ser Lys Asn 2. 5 10 GCT TCG GCA GAC GAA ATC AJA AAG GCT TAT CGT MAG CTT TCC AAA AAA /CA96/00322 2902 2 962 3022 1082 3142 3202 3262 3309 3357 3405 3453 3501 3549 Ala Ser Ala Asp Giu Ile Lys Lys TAT CAC CCA GAT ATC Tyr His Pro Asp Ile A.AC AAG GAG Asn Lys Glu Ala Tyr Arg Lys Leu CCT GGT GCT GAG GAC Pro Gly Ala Giu Asp 40 TJG AGT GAC GAC CA.

Leu Ser Asp Asp Gin Ser Lys Lys AAG TAC AAG Lys Tyr Lys AAA CGT GCT Lys Arg Ala TTT GGT GGk.

Phe Gly Gly GA-k GTT CA.A GAA GCC TAT Giu Val Gin Giu Ala Tyr GAG ACT Giu Thr GCC TAT Ala Tyr GCT GOT Ala Gly s0 GAC CAG TAT GGT Asp Gin TEyr Gly GCA GGC GCC Ala Giy Ala AAT GGG GCA Asn Gly Ala AAT GGT GGT Asn Gly Gly GGT ITC GGC Gly Phe Gly GGC TTC GGT GGT Gly Phe Gly Gly GAG GAT ATT TTC Glu Asp Ile Phe AGT TTC ITC GGC Ser Phe Phe Gly GGC GGT TCT TCG Gly Gly Ser Ser CGC MAT Arg Asn 110 CCA AAC GCT Pro Asn Al a ACC TTT GAA Thr Phe Glu 130 CGT GAA GCT Arg Giu Ala CAA GGA GAT Gin Gly Asp GAT CTC CAG Asp Leu Gin 120 TAT CGT Tyr Arg GCT ATC TTC Ala Ile Phe

ACT

Thr

AAT

Asn GGC TGT CGT Gly Cys Arg GAG AAG GAA GTT Giu Lys Giu Val 140 GGA TCT CST GCT Gly Ser Gly Ala 155 CAT GGC GCT GGT His Gly Ala Giy 170 GTC AAT TI'G Val Asn Leu 125 AAG TAT CAT Lys Tyr His AAG CCA GGG Lys Pro Gly GTC ATT MAC Val Ile Asn 175 145 ACA AGT Thr Ser 160 3597 3645 3693 3741 3789 3837 3885 3933 CCA GTC ACT Pro Val Thr CGC TGT Arg Cys GTC GAT ACG CAG ACT Val Asp Thr Gin Thr 180 CCT CTT GGT Pro Leu Gly ATG ATG Met Met 185 GMA ATC Glu Ile 200 CGT CGC CMA GTA ACC TGT Arg Arg Gin Val Thr Cys 190 AAA TAT CCA TGT ACA ACC Lys Tyr Pro Cys Thr Thr 205 GAT GTC TGT CAC GGT CGA GGA AAA Asp Val Cys His Gly Arg Gly Lys 195 TGT CAT GGA ACA GGT CAT GAG AMA CYS His Gly Thr Gly {-is Glu Lys 210 215 CMA GCT CAT AGC GTA CAT GTG MA Gin Ala His Ser Val His Val Lys 220 WO 96/40928 ATC CCT GCT Ile Pro Ala 225 GGT GAA GCA Gly Giu Ala 240 GTT TCT GTG Val Ser Val.

TrTC TAC AAT Phe Ty r Asn GTA GAT ATT Val Asp Ilie 290 GGA ACT CAG Gly Thr Gin 305 AGC CTT CGT Ser Leu Arg 320 GTA ACA CCG Val Thr Pro

TTC

Phe PCTICA96/00322 kA 3981 *GTG GAA ACA Vai Giu Thr 230 TTT MAC GGT Phe Asn Giy 245 GCT AGT GAC Ala Ser Asp 260 AAC CTC MAC Asn Leu Asn ACT GTT CAC Thr Val His GGT MAG MAA Giy Lys Lys 310 GGT GCA GTT Gly Aia Val 325 GGC 'FIG MAC Gly Leu Asn 340 GGT CMA CMA Gly Gin Gin GGA CCT TAT Giy Pro Tyr MAG 'FIT GMA Lys Phe Giu 265 TT7 GTC CMA Phe Val Gin 280 GGT GAT GTT Giy Asp Vai 295 TTC CGC CTA Phe Arg Leu GGT GA C CMA Giy Asp Gin GAC CGC CMA Asp Arg Gin 345 ATT CGC CTC Ile Arg Leu 235 GGT GAC TTG Gly Asp Leu 250 CGT GMA GGA Arg Giu Giy GCG GCT CTT Aia Ala Leu GM 'FIG GTT Giu Leu Vai 300 CGT AGT MAG Arg Ser Lys 315 TAC GTT ACT Tyr Vai Thr 330 AAA GTA GCC Lys Val Aia GCT GGT Ala Giy TAT GTA Tyr Val ACG ACT Thr Thr 270 GGT GAT Giy Asp 285 ATT CCA Ile Pro GGG GCA Giy Aia GTT MAT Val Asn 'FIG A Leu Lys 350 4029 4077 4125 4173 4221 4269 4317 4320 INFORMATION FO)R SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 607 amino acids fB) TYPE: amino acid TOPOLOGY: iinear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Met Ser Lys Ile Ilie Gly Ilie Asp Leu Gly 1 5 Aia Val Leu Giu Giy Thr Giu Ser Lys Ile Asn Arg Thr Thr Pro Ser Val Vai Ser Phe Val Gly Asp Ala Ala Ly-, Arg Gin Ala Vai 50 55 Ile Ser Ilie Lys Ser Lys Met Gly Thr Ser 70 Gly Lys Giu Tyr Thr Pro Gin Giu Ilie Ser Leu Lys Gly Tyr Ala Glu Asp Tyr Leu Gly 100 105 NO0: Thr Thr Ile Ala Lys Asn flir Asn Glu Lys Ala Met Glu Lys WO 96/40928 'ral Ile Thr Val Pro Ala Tyr Phe Asn Asp Ala Gin 115 120 PCT/CA96/00322 Arg Gin Ala Thr 125 Lys Asp Ala Gly Lys 130 Glu Pro Thr Ala AlaI Ile Ala Gly Leu Glu Val Glu 1 145 Glu Ser Gly ,0 His Asp 225 Lys Thr Ala Pro Asp 305 Glu Pro Thr Leu Asn 385 Asp Met I Pro 1 Asp I 4 Lys G 465 Glu Ile Asp Leu 210 Lys Asp Ala Lys Val 290 Glu Ala Asp Gly Gly 370 Thr \sn Ala Ula Lys 150 Lys Leu Asn 195 Val Met Leu Gly Phe 275 Arg Val Val Glu Asp 355 Ile Thr Gin Ala Ala 435 Asn Ile Glu 130 Lys Ala Ala Ser Glu 260 Asp Gin Ile Lys Val 340 Val Glu Ile Pro Asp 420 Pro Gly Leu 165 Leu Leu Glu Met Gly 245 Ala Asp Ala Leu Ala 325 Val Lys Thr Pro Ala 405 Asn I Arg G Ile V Al 15 Va.

GlI G12 Gly Phe Glr 230 Val Gly Leu Leu Val 310 Glu Ala Asp let Thr 90 Val ys ;ly 'al 135 a Leu 0 1 Phe y Asp SGly Lys 215 1 Arg Thr Pro Thr Ser Gly Thr Met Val Gly 375 Ser I Asp Thr I Ile F 4 Ser V 455 Al Asp Gly Asp 200 Lys Leu Ser Leu Arg 280 Asp Gly Gly Gly Val 360 Gly .ys :le Leu Pro 40 'al a Tyr SLeu Val 185 SAsp Glu Lys Thr His 265 Asp Ala Ser Lys Ala 345 Leu Val Ser His 4 Gly 1 425 Gin I Lys Gl Gl 17 Ph Ph Asi Asi Glr 250 Leu Leu Gly Thr Glu 330 Ala Leu Phe 3ln Val 110 Arg :le ila y Leu 155 y Gly 0 a Asp a Asp I Gly Ala 235 Ile SGlu Val SLeu Arg 315 Pro Ile Asp Thr Val 395 Leu Phe Glu Lys 1 4 Ser G 475 140 Asp Gly Val Gin Ile 220 Ala Ser Met Glu Ser 300 Ile Asn 1n Val Lys I 380 Phe S 31n C Gln L Val 4 Asp L 160 ;ly L Ar Lys Thr Leu Lys 205 Asp Glu Leu Thr Arg 285 Leu Pro Lys Gly Thr 365 Leu Ser Gly Leu 'hr eu eu g Ile Val Asn SThr Asp Lys 160 Phe Asp Val 175 SSer Thr Ala 190 Ile Ile Asp Leu Ser Thr Lys Ala Lys 240 Pro Phe lie 255 Leu Thr Arg 270 Thr Lys Val Ser Glu Ile Ala Val Val 320 Ser Val Asn 335 Gly Val Ile 350 Pro Leu Ser Ile Asp Arg Thr Ala Ala 400 Glu Arg Pro 415 Thr Asp Ile 430 Phe Asp Ile Gly Thr Gin Thr Asp Glu 480 lu Gin Thr Ile Val 470 Ile Gin Ser Asn

L

I I_ WO 96/40928 Glu Ile Asp Arg Met Met Lys Asp Ala Glu Ala 485 490 Lys Lys Arg Lys Glu Glu Val Asp Leu Arg Asn 500 505 Ile Phe Ala Thr Glu Lys Thr Ile Lys Glu Thr 515 520 Asp Ala Glu Arg Asp Ala Ala Gin Ala Ala Leu 530 535 Ala Gin Glu Asp Asn Asn Leu Asp Asp Met Lys 545 550 555 Leu Asn Glu Lys Ala Gin Gly Leu Ala Val Lys 565 570 Ala Ala Ala Gin Gin Ala Gin Glu Gly Ala Glu 580 585 Gly Asn Ala Gly Asp Asp Val Val Asp Gly Glu 595 600 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 352 amino acids TYPE: amino acid TOPOLOGY: linear PCT/CA96/00322 Ser Asp 495 Gin Ala Gly Phe Lys Lys Glu Ala 560 Gin Ala 575 Ala Thr Lys iii) MOLECULE TYPE: protein (xi) SEQUENCE Met Asn Asn Thr Glu 1 Ser Ala Asp Glu Ile His Pro Asp Ile Asn Val Gin Glu Ala Tyr Tyr Asp Gin Tyr Gly Gly Gly Phe Gly Gly Asp Ile Phe Ser Ser 100 Asn Ala Pro Arg Gin 115 Phe Glu Glu Ala Ile 130 Glu Ala Gly Cys Arg 145 Ser Pro Val Thr Cys 165 DESCRIPTION: SEQ ID NO:6: Phe Tyr Asp Arg Leu Gly Val Ser Lys Asn Ala Tyr Arg Gly Ala Ser Asp Ala Asn Ala Gly Gly Gly 105 Leu Gin Glu Lys Gly Ser His Gly 170 I ~1 I WO 96/40928 Asp Thr PCT/CA96/00322 Gin Thr Pro Leu Gly Met Met Arg Arg Gin Val Thr Cys Asp 180 185 190 Val Cys His Gly 195 His Gly Thr Gly 210 Pro Ala Gly Val 225 Glu Ala Gly Phe Ser Val Glu Ala 260 Tyr Asn Leu Asn 275 Asp Ile Pro Thr 290 Thr Gin Thr Gly 305 Leu Arg Gly Gly Arg Gly Lys Glu Ile 200 His Glu Lys Gin Ala 215 Glu Thr Gly Gin Gin 230 Asn Gly Gly Pro Tyr 245 Ser Asp Lys Phe Glu 265 Leu Asn Phe lal Gin 280 Val His Gly Asp Val 295 Lys Lys Phe Arg Leu 310 Ala Val Gly Asp Gin 325 Lys Tyr Pro His Ile Gly 250 Arg Ala Glu Arg Tyr' 330 Thr Thr Cys Val Lys Ile Gly Gln Gly 240 Val Val Val 255 Thr Ile Phe 270 Asp Thr Val Pro Glu Gly Ala Pro Ser 320 Asn Val Val 335 Lys Glu Phe 350 Thr Pro Thr Gly Leu 340 Asn Asp Arg Lys Val Ala Leu INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Thr Ser Thr Gin Ile Ser Leu Pro Phe Ile Thr Ala Gly Glu Ala 1 5 10 INIFORMATION FOR SEQ ID N0:8: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Thr Ala Gly Gli Ala Gly Pro Leu His Leu Glu Met Thr Leu Thr 1 5 10 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 1' amino acids c WO 96/40928 PCT/CA96/00322 TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: Met Thr Leu Thr Arg Ala Lys Phe Asp Asp Leu Thr Arg Asp 1 5 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID Asp Asp Leu Thr Arg Asp Leu Val Glu Arg Thr Lys Val Pro Val 1 5 10 INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: LENGTH: 14 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: Thr Lys Val Pro Val Arg Gln Ala Leu Ser Asp Ala Gly Leu 1 5 INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: Lys Ala Lys Asp Leu Gly Thr Gln Lys Glu Gln Thr Ile Val Ile 1 5 10 INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENGTH: 14 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide WO 96/40928 PCT/CA96/00322 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: Leu Thr Asp Glu Ile Asp Arg Met Met Lys Asp Ala Glu Ala 1 5 INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 24 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: Lys Asp Ala Glu Ala Asn Ala Glu Ser Asp Lys Lys Arg Lys Glu Glu 1 5 10 Val Asp Leu Arg Asn Glu Val Asp INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID Asn Glu Val Asp Gln Ala Ile Phe Ala Thr Glu Lys Thr Ile Lys 1 5 10 INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 28 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: Glu Lys Thr Ile Lys Glu Thr Glu Gly Lys Gly Phe Asp Ala Glu Arg 1 5 10 Asp Ala Ala Gin Ala Ala Leu Asp Asp Leu Lys Lys INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 30 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: WO 96/40928 PCT/CA96/00322 Lys Ala Gin Glu Asp Asn Asn Leu Asp Asp Met Lys Ala Lys Leu Glu 1 5 10 Ala Leu Asn Glu Lys Ala Gin Gly Leu Ala Val Lys Leu Tyr 25 INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 25 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: Gin Glu Gly Ala Glu Gly Ala Gin Ala Thr Gly Asn Ala Gly Asp Asp 1 5 10 Val Val Asp Gly Glu Phe Thr Glu Lys 20 INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 2183 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Streptococcus pyogenes (ix) FEATURE: NAME/KEY: CDS LOCATION: 204..2030 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: CAGCGATGGT AGTTGTTTAT AACTAAGGTA AATGAGTTTT CGTTTTTGTC CGTAATGACA GTAAACTAGA TAGCAAGTTA GAAGCTATTT CGCTTGCTGA TTAAACTATA GTGATTGCTT AGAATTGGAA GTAAAATAAT TCGAGTGCTT ACTAAGATAA ATTGAAATAA AAAGTAATAA AGTATAAAAT AAGAGGTATT AAC ATG TCT AAA ATT AZT GGT ATT GAC TTA Met Ser Lys Ile Ile Gly Ile Asp Leu 1 GGT ACA ACA AAC TCA GCA GTA GCA GTT CTT GAA GGG ACT GAA TCA AAA Gly Thr Thr Asn Ser Ala Val Ala Val Leu Glu Gly Thr Glu Ser Lys 15 20 ATC ATT GCT AAC CCA GAA GGC AAT CGT ACA ACT CCT TCA GTA GTA TCA Ile Ile Ala Asn Pro Glu Gly Asn Arg Thr Thr Pro Ser Val Val Ser 35 TTC AAA AAT GGT GAA ATT ATC GTG GGT GAT GCT GCA AAA CGC CAA GCA Phe Lys Asn Gly Glu Ile Ile Val Gly Asp Ala Ala Lys Arg Gin Ala 50 WO 96/40928 GTG ACA AAC CCA GAA ACA GTA ATC TCT ATT AAA TCT ALA ATO GGA ACT Val Thr Asn Pro Giu Thr Val Ile Ser Ile Lys Ser Lys Met Gly Thr 65 TCT GAA AAA Ser Giu Lys TCA GCA ATO Ser Ala Met OGA GAA AAA GLy Giu Lys OAT OCA CAA Asp Ala Gin GAA GTA GAA Glu Val Glu 140 GGT ATG GAC Gly Met Asp 155 GOT GOT GGT Gly Oly Gly 170 TTC GAC GTT Phe Asp Val TTT GAC CAA Phe Asp Gin AAT GOT ATT Asn Oly Ile 220 OAT OCT OCT Asp Ala Ala 235 CAA ATT TCA Gin Ile Ser 250 TTA GAG ATG Leu Giu Met CTT GTT GAA Leu Val Glu OGA TG TCA Gly Leu Ser 300 OTT TCT OCA Vai Ser Ala ATT CTT CAA Ile Leu Gin 95 GTA OAA AAA Val Glu Lys 110 CGT CAA OCA Arg Gin Ala 125 CGT ATC OTT Arg Ile Val AAO ACT GAC Lys Thr Asp ACA TTT GAC Thr Phe Asp 175 CTT OCA ACA Leu Ala Thr 190 AAA ATT ATT Lys Ile Ile 205 GAC TA TCA Asp Leu Ser GAA AAA GCT Glu Lys Ala ITA CCG TTC Leu Pro Phe 255 AGC TTA TCT Ser Leu Ser 270 0GT ACO AAA Arg Thr Lys 285 ITO TCA GAA 2 Leu Ser Glu

AA

As 8[

TAC

Tyl GC1 Ala

ACI

Thr

AAI

Asn

AAG

Lys 160

OTA

Val

OCA

Ala

OAT

Asp

CAA

Gin

AAA

Lys 240

ATC

Ile

:GT

Arg

ACT

T'hr kTT Ile T GOT I Gly

CTT

Leu

OTT

Vai

AAA

Lys

GAA

Glu 145

OAT

Asp

TCA.

Ser

GGT

Gly

TTC

Phe OAT 2 Asp I 225

AA.C

Lys 2 ACT C Thr OCT Ala I CCA C Pro V 2 OAT C Asp G 305

AAA

Lys

AAA

Lys

ATT

Ile

GAC

Asp 130

CCA

Pro

GAM

Glu iTC Ile

GAT

Asp

TTA

Leu 210 G4 Lys

AT

ksp

CT

la LkA ,ys

;TT

tal 90

'AA

;lu GAA TAT ACT CC9 Glu Tyr Thr Prc GGT TAT Oly Tyr 100 ACT OTT Thr Val 115 OCT GGT Ala Gly ACA OCA Thr Ala AAA ATC Lys Ile CTT OAA Leu Olu 180 AAC AA Asn Lys 195 GTG GCT Val Ala ATG GCA Met Ala CTT TCA Leu Ser I GGT TCT Oly Ser 260 TTT GAC Phe Asp 275 OCT GAA Ala Glu CCA GCI Pro Ala AAA ATT Lys Ile OCT OCA Ala Ala 150 TTA OTT Leu Val 165 TTA GGT Ieu Gly CTT GGT Leu Oly GAA TTT Glu Phe CTT CAA Leu Gin 230 GOT GTG Gly Val 245 GCT GGT Ala Oly GAT CTC Asp Leu CM-A GM.A ATT Gin Giu Ile GAC TAT CTT Asp Tyr Leu 105 TAT 'TC AAC Tyr Phe Asn 120 OCA GOT CTT Ala Gly Leu 135 CTT OCT TAT Leu Ala Tyr TTT GAC CTT Phe Asp Leu OAT GOT GTC Asp Gly Val 185 GGT GAC GAC Oly Asp Asp 200 AAG AAA 3MA Lys Lys Glu 215 COC TTG AAA Arg Leu Lys ACA CM.A ACA Thr Gin Thr CCT CTT CAC Pro Leu His 265 ACT COT GAC Thr Arg Asp 280 TCA GAT OCA CT/CA96/00322 422 470 518 566 614 662 710 758 806 854 902 950 998 1046 1094 CGT CAA GCT CTT Arg Gin Ala Leu Ser Asp Ala GTT ATC Val Ile 295 CTT OTT GOT GOA TCA Leu Val Oly Oly Ser 310 1142 1190 ACT CGT Thr Arg 315 ATC CCA OCA OTT GTC GAA GCT OTA AAA GCT GMA ACT GOT AAA Ile Pro Ala Val Val Olu Ala Val Lys Ala Glu Thr Gly Lys I L I= WO 96/40928 PCT/CA96/00322 GAA CCA AAT AAA TCT GTA AAC CCT GAT GAA GTG GTT GCT ATG GGT GCT 1238 Glu Pro Asn Lys Ser Val Asn Pro Asp Glu Val Val Ala Met Gly Ala 330 335 340 345 GCT ATC CAA GGT GGG GTT ATC ACT GGG GAT GTG AAA GAC GTT GTC CTT 1286 Ala Ile Gin Gly Gly Val Ile Thr Gly Asp Val Lys Asp Val Val Leu 350 355 360 CTT GAC GTA ACA CCA TTG TCA CTT GGT ATT GAA ACA ATG GGT GGT GTC 1334 Leu Asp Val Thr Pro Leu Ser Leu Gly Ile Glu Thr Met Gly Gly Val 365 370 375 TTC ACT AAA TTG ATC GAC CGC AAT ACA ACT ATC CCA ACA TCT AAA TCA 1382 Phe Thr Lys Leu Ile Asp Arg Asn Thr Thr Ile Pro Thr Ser Lys Ser 380 385 390 CAA GTC TTIC TCA ACA GCA GCA GAC AAC CAA CCA GCC GTT GAT ATC CAT 1430 Gin Val Phe Ser Thr Ala Ala Asp Asn Gin Pro Ala Val Asp Ile His 395 400 405 GTT CTT CAA GGT GAA CGC CCA ATG GCA GCA GAT AAC AAG ACT CTT GGT 1478 Val Leu Gin Gly Glu Arg Pro Met Ala Ala Asp Asn Lys Thr Leu Gly 410 415 420 425 CGC TTC CAA TTG A. GAT ATC CCA GCT GCA CCT CGT GGA ATC CCA CAA 1526 Arg Phe Gin Leu Asp Ile Pro Ala Ala Pro Arg Gly Ile Pro Gin 430 435 440 ATT GAA GTA ACA TTT GAT ATC GAT AAA AAC GGT ATT GTT TCT GTA AAA 1574 Ile Glu Val Thr Phe Asp Ile Asp Lys Asn Gly Ile Val Ser Val Lys 445 450 455 GCT AAA GAC CTT GGT ACG CAA AAG GAA CAA CAC ATC GTT ATC AAA TCA 2 Ala Lys Asp Leu Gly Thr Gin Lys Glu Gin His Ile Val Ile Lys Ser 460 465 470 AAC GAC GGA CTT TCT GAA GAA GAA ATT GAT CGC ATG ATG AAA GAC GCT 1670 Asn Asp Gly Leu Ser Glu Glu Glu Ile Asp Arg Met Met Lys Asp Ala 475 480 485 GAA GCT AAT GCC GAA GCC GAT GCG AAA CGT AAA GAA GAA GTT GAC CTT 1718 Glu Ala Asn Ala Glu Ala Asp Ala Lys Arg Lys Glu Glu Val Asp Leu 490 495 500 505 AAA AAC GAA GTT GAC CAA GCT ATC TTT GCT ACT GAA AAA ACA ATC AAA 1766 Lys Asn Glu Val Asp Gin Ala Ile Phe Ala Thr Glu Lys Thr Ile Lys 510 515 520 GAA ACT GAA GGT AAA GGC TTT GAC ACA GAA CGC GAT GCA GCG CAA TCA 1814 Glu Thr Glu Gly Lys Gly Phe Asp Thr Glu Arg Asp Ala Ala Gin Ser 525 530 535 GCT CTT GAC GAG TTA AAA GCT GCG CAA GAA TCT GGC AAC CTT GAC GAC 1862 Ala Leu Asp Glu Leu Lys Ala Ala Gin Glu Ser Gly Asn Leu Asp Asp 540 545 550 ATG AAA GCT AAA CTT GAA GCA TTA AAT GAA AAA GCG CAA GCT TTG GCT 1910 Met Lys Ala Lys Leu Glu Ala Leu Asn Glu Lys Ala Gin Ala Leu Ala 555 560 565 GTT AAA ATG TAC GAG CAA GCT GCA GCA GCT CAA CAA GCA GCA CAA GGT 1958 Val Lys Met Ty- Glu Gin Ala Ala Ala Ala Gin Gin Ala Ala Gin Gly 570 575 580 585 GCA GAA GGT GCA CAA GCT AAT GAT TCA GCA AAT AAT GAT GAT GTT GTA 2006 Ala Glu Gly Ala Gin Ala Asn Asp Ser Ala Agn Asn Asp Asp Val Val 590 595 600 WO 96/40928 PCT/CA96/00322 GAT GGC GAA 'rTT ACA GAA AAG TAATGATTTA GTTATCTAGT AACATTAATA 2057 Asp Gly Glu Phe Thr Glu LYS 605 TCCGAATTCA GAGGT TGTAC CAAACCTCTG TTTTTGGCTA AATAAAATGT AAAAA-4CTG 2117 ACGTCAAAAT ATTTTAAZGAA AGGAATACAA GTTCGATTAT TCGA.ACACAG GCTAAAGCGT 2177 GTAAAG 2183 INFOR~MATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 608 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Met Ser Lys Ile Ile Gly Ile Asp Leu 1 Al a Asn Val Ile Gly Leu Val Lys Gi u 145 Asp Ser Gly Phe Asp 225 Lys Gly Pro Ala Ser Thr 85 Al a Pro Lys Al a Leu 165 Leu Leu Gi u Leu Gly 245 Gly Thr Ile Ile Phe Lys Val Thr Ser Glu Ser Ala Gly Glu Asp Ala Giu Val Gly Met 155 Gly Gly 170 Phe Asp Phe Asp Asn Gly Asp Ala 235 Gin Ile 250 Thr Asn Ser Ala Val Gi u Ile Thr Ala

GI

Ala Val Asp Asp 175 Thr Ile Ser Al a Phe 255 WO 96/40928 Thr Ala Gly Ser 260 Ala Lys Phe Asp 275 Pro Val Arg Gin 290 Asp Glu Val Ilie 305 Glu Ala Val Lys Pro Asp Glu Val 340 Thr Gly Asp Val 355 Leu Gly Ile Glu 370 Asn Thr Thar Ilie 385 Asp Asn Gin Pro Met Ala Ala Asp 420 Pro Ala Ala Pro 435 Asp Lys Asn Gly 450 Lys Glu Gin His 465 Glu Ile Asp Arg Ala Lys Arg Lys 500 Ile Phe Ala Thr 515 Asp Thr Glu Arg 530 Ala Gin Glu Ser 545 Leu Asn Glu Lys Ala Ala Ala Gin 580 Asp Ser Ala Asn 595 Al a Asp Al a Leu Al a 325 Val Lys Thr Pro a 405 A~n Arg Ile Ilie Met 485 Glu Giu Asp Gly Al a 565 Gin Asn Gly Leu Leu Val 310 Giu Ala Asp Met Thr 390 Val Lys Gly Val Val 470 Met Giu Lys Al a Asn 550 Gin Al a Asp Pro Thr Ser 295 Gly Thr Met Val Gly 375 Ser Asp Thr Ile Ser 455 Ilie Lys Val Thr Al a 535 Leu Al a Al a Asp Leu Arg 280 Asp Gly Gly Gly Val 360 C-'Ily Lys Ile Leu Pro 440 Val Lys Asp Asp Ilie 520 Gin Asp Leu Gin Val 600 His 265 Asp Ala Ser Lys Ala 345 Leu Val Ser His Gly 425 Gin Lys Ser Al a Leu 505 Lys Ser Asp Al a Gly 585 Val Leu *Leu Gly Thr Glu 330 Ala Leu Phe Gin Val 410 Arg Ile Al a As n Giu 490 Lys Gi U Ala Met Val 570 hl a Asp Ser Arg 285 Leu Pro Lys Gly Thr 365 Leu Ser Gly Leu Thr 445 Leu Leu Al a Val Gly 525 Giu Lys Tyr Al' a Phe 605 Leu Ser 270 Thr Lys Ser Giu Ala Val Ser Val 335 Gly Val 350 Pro Leu Ile Asp Thr Ala Giu Arg 415 Thr Asp 430 Phe Asp Gly Thr Ser Giu Giu Aia 495 Asp Gin 510 Lys Gly Leu Lys Leu Giu Giu Gin 575 Gin Ala 590 Thr Glu PCT/CA96/00322 Ar g Th-r .Ie Val1 320 Asn Ile Ser Arg Al a 400 Pro Ile Ile Gin Giu 480 Asp Al a Phe Al a Al a 560 Al a Asn Lys WO 96/40928 INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 2438 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Streptococcus agalactiae (ix) FEATURE: NAME/KEY: CDS LOCATION: 248..2077 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: CTTTCAAAAG GGATATAAAT TGCACGAGCG 1'CTGCTAAGA CCAGC TAACTAAGGT AAATGAGTTT TCGTTTTTGT CCGTAATGAC AGTAA AGAAGCTATT CAGCTTGCTG ATTAAACTAT AGTGATTGCT TAGAA TTCGAGTGCT TACTAAGATA AATTGAAATA AAAAGTAATA AAGTA TATTAAC ATG TCT AAA ATT ATT GGT ATT GAC TTA GGT A PCT/CA96/00322 :GATGG TAGTTGTCTA ACTAG ATAGCAAGTT TTGGA AGTAAAATAA ,TTATA AAATAAGAGG CA ACA AAC TCA Met Ser Lys Ile Ile Gly Ile Asp Leu Gly Thr Thr Asn Ser

GCA

Ala 15

GAA

Glu

ATT

Ile

ACT

Thr

GCA

Ala

CAA

Gin 95

AAA

Lys

GCA

Ala

GTT

Val

CGT

Arg

GGT

Gly

TCT

Ser

AAA

Lys

AAA

Lys

ATT

Ile

GAC

Asp 130 CTT GAA Leu Glu 20 ACA ACT Thr Thr GAT GCT Asp Ala ATC AAA Ile Lys GAA TAT Glu Tyr GGT TAT Gly Tyr 100 ACT GTT Thr Val 115 GCT GGT Ala Gly GGG ACT Gly Thr CCT TCA Pro Ser GCA AAA Ala Lys TCA AAG Ser Lys ACT CCT Thr Pro GCT GAA Ala Glu CCA GCT Pro Ala AAA ATT Lys Ile

GAA

Glu

GTA

Val

CGT

Arg 55

ATG

Met

CAA

Gln

GAC

Asp

TAC

Tyr

GCA

Ala 135 TCA AAA Ser Lys 25 GTA TCA Val Ser 40 CAA GCG Gin Ala GGA ACT Gly Thr GAA ATT Glu Ile TAT CTT Tyr Leu 105 ITC AAC Phe Asn 120 GGT CTT Gly Leu ATT GCT Ile Ala AAA AAT Lys Asn ACA AAT Thr Asn GAA AAA Glu Lys GCA ATG Ala Met GAA AAA Glu Lys GCA CAA Ala Gin GTA GAA Val Glu 140 AAC CCA Asn Pro GGT GAA Gly Glu CCA GAT Pro Asp GTT TCT Val Ser ATT CTT Ile Leu GTA GAA Val Glu 110 CGT CAG Arg 125 CGT ATC Arg Ile 120 160 240 289 337 385 433 481 529 577 625 673 WO 96/40928 GTT AAC GAA Val Asn Glu PCT/CA96/00322 CCA ACA GCA GCC GCA CTT GCT TAT GGT ATG GAC AAG ACT 721 Pro Thr Ala Ala Ala Leu Ala Tyr Gly Met Asp Lys Thr 145 150 155

GAC

Asp

GAC

Asp 175

ACA

Thr

ATT

Ile

TCT

Ser

GCT

Ala TTc Phe 255

TCA

Ser

AAA

Lys

GAA

Glu

GTT

Val

GTT

Val 335

GTT

Val

TTG

Leu

GAC

Asp

GCA

Ala

AAG

Lys 160

GTA

Val

GCA

Ala

GAT

Asp

CAA

Gln

AAA

Lys 240

ATC

Ile

CGT

Arg

ACT

Thr

ATT

Ile

GTT

Val 320

AAC

Asn

ATC

Ile

TCA

Ser

CGC

Arg

GCA

Ala 400 GAT GAA AAA Asp Glu Lys TCA ATC CTT Ser Ile Leu GGT GAT AAC Gly Asp Asn 195 TTC TTG GTA Phe Leu Val 210 GAC AAA ATG Asp Lys Met 225 AAA GAC CTT Lys Asp Leu ACT GCT GGT Thr Ala Gly GCT AAA TTT Ala Lys Phe 275 CCA GTT CGT Pro Val Arg 290 GAT GAA GTT Asp Glu Val 305 GAA GCT GTA Glu Ala Val CCT GAT GAA Pro Asp Glu ACT GGG GAT Thr Gly Asp 355 CTT GGT ATT Leu Gly Ile 370 AAC ACA ACT Asn Thr Thr 385 GAC AAC CAA Asp Asn Gin

ATC

Ile

GAA

Glu 180

AAA

Lys

GAA

Glu

GCT

Ala

TCA

Ser

TCT

Ser 260

GAC

Asp

CA

Gin

ATC

Ile

AAA

Lys

GTG

Val 340

GTG

Val

GA

31u

ATC

lie

CCA

Pro TTA GTT Leu Val 165 TTA GGT Leu Gly CTT GGT Leu Gly GAA TTC Glu Phe CTT CAA Leu Gin 230 GGT GTA Gly Val 245 GCT GGT Ala Gly GAT CTC Asp Leu GCT CTT Ala Leu CTC GTT Leu Val 310 GCT GAA Ala Glu 325 GTT GCC Val Ala AAA GAC Lys Asp ACA ATG Thr Met CCA ACA Pro Thr 390 GCC GTT Ala Val 405 TTT GAC Phe Asp GAT GGT Asp Gly GGT GAC Gly Asp 200 AAG AAA Lys Lys 215 CGC TTG Arg Leu ACT CAA Thr Gin CCT CTT Pro Leu ACT CGT Thr Arg 280 TCA GAT Ser Asp 295 GGT GGA Gly Gly ACT GGT Thr Gly ATG GGT Met Gly GTT GTA Val Val 360 GGT GGT G1y Gly 375 TCT AAA Ser Lys GAT ATC Asp Ile

CTT

Leu

GTC

Val 185

GAC

Asp

GAA

Glu

AAA

Lys

ACT

Thr

CAC

His 265

GAC

Asp

GCA

Ala

TCA

Ser

AAA

Lys

GCT

Ala 345

CTT

Leu

GTC

Val

TCA

Ser

CAT

His GGT GGT GGT Gly Gly Gly 170 TTC GAC GTT Phe Asp Val 'ITT GAC CAG Phe Asp Gin AAT GGT ATT Asn Gly Ile 220 GAT GCT GCT Asp Ala Ala 235 CAA ATT TCA Gin Ile Ser 250 TTG GAG ATG Leu Giu Met CTT GTT GAA Leu Val Glu GGC TTG TCA Gly Leu Ser 300 ACA CGT ATC Thr Arg Ile 315 GAA CCA AAT Glu Pro Asn 330 GCT ATC CAA Ala Ile Gin CTT GAC GTA Leu Asp Val TTC ACT AAA Phe Thr Lys 380 CAA GTC TTC Gin Val Phe 395 GTT CTT CAA Val Leu Gin 410

ACA'

Thr

CTT

Leu

AAA

Lys 205

GAT

Asp

GAA

Glu

ITA

Leu

AGC

Ser

CGT.

Arg 285

TTG

Leu

CCA

Pro AAA Lys

GGT

Gly

ACA

Thr 365 Leu

TCA

Ser

GGT

Gly 769 817 865 913 961 1109 1057 1105 1153 1201 1249 1297 1345 1393 1441 1489 s WO 96/40928 CGC CCA ATG GCA GCA Arg Pro Met Ala Ala 415 GAT ATC CCA GCT GCA Asp Ile Pro Ala Ala 435 GAT ATC GAT AAA AAT Asp Ile Asp Lys Asn 450 ACT CAA AAA GAA CAA Thr Gin Lys Giu Gin 465 GAT GAA GAA ATT GAT Asp Giu Glu Ile Asp 480 GCA GAT GCA AA CGT Ala Asp Ala Lys Arg 495 CAA GCC ATC TTT GCA Gln Ala Ile Phe Ala 515 GGT TTT GAT ACA GAA Gly Phe Asp Thr Glu 530 AAA AAA GCT CAA GAA Lys Lys Ala Gin Glu 545 GAA GCT CTT AAC GAA Glu Aia Leu Asn Glu 560 CAA GCG GCT GCA GCA Gin Ala Ala Ala Ala 575 TCA GCT GAT TCA TCA PCT/CA96/00322 :T 1537 GAT AAC AAA ACA Asp Asn Lys Thr 420 CCT CGT GGA ATC Pro Arg Gly Ile GGT ATT Gly Ile CAC ATT His Ile AAA ATG Lys Met 485 AAA GAA Lys Glu 500 ACA GAA Thr Glu CGC GAT Arg Asp TCA GGT Ser Gly AAA GCA Lys Ala 565 CAA CAA Glm Gin 580 AGC AAG

GTA

Va1

GTT

Val 470

ATG

Met

GAA

Glu

AAA

Lys

GCA

Ala

AAC

Asn 550

CAA

Gin

GCA

Ala

GGT

TCT

Ser 455

ATC

Ile

AAA

Lys

GTT

Val

ACT

Thr

GCG

Ala 535

CTT

Leu

GCT

Ala

GCT

Ala

GAT

CTC GGT Leu Gly 425 CCA CAA Pro Gin 440 GTT AAA Val Lys CAA TCT Gin Ser GAT GCT Asp Ala GAT CTT Asp Leu 505 ATT AAA Ile Lys 520 CAA TCA Gin Ser GAC GAC Asp Asp CTT GCA Leu Ala CAA GGG Gin Gly 585 GAT GTT CGC TTC Arg Phe ATT GAAk Ile Glu GCT AAA Ala Lys AAT TCA Asn Ser 475 GAA GCA Glu Ala 490 AAA AAT Lys Asn GAA ACT Glu Thr GCA CTT Ala Leu ATG AAA Met Lys 555 GTT AAA Val Lys 570 GCT GAA Ala Glu GTA GAT

CAA

Gin

GTA

Val

GAT

Asp 460

GGA

Gly

AAT

Asn

GAA

Glu

GAA

Glu

GAT

Asp 540

GCT

Ala

CTT

Leu

GGT

Gly

GGC

TTG

Leu

ACA

Thr 445

CTC

Leu

TTA

Leu

GCT

Ala

GTT

Val

GGC

Gly 525

GAG

Glu

AAA

Lys

TAC

Tyr

GCA

Ala

GAA

1585 1633 1681 1729 1777 1825 1873 1921 1969 2017 2065 2114 2174 2234 2294 2354 2414 2438 Ser Ala Asp Ser Ser Ser Lys Gly Asp Asp Val Val Asp Gly Giu Phe 595 600 605 ACT GAG AAA TAATTATAA TATTGITCAG ATTCATTTGA ATATAAGCAT Thr Giu Lys 610 GAAAACTATA CTAGCATAGT AAAGTICTTC GTGATAGGGA GTTTCAGATT ACATAAGCTA ATTTCGCTAT CACTAAATAA GGCGGGGCGC CTCGCTCCGT CTGTTTTATI AAGTGTCATA CTGTAACTGG GCAAGAATAA TITGTTAATCT CTTCAAGTGT AGATTAGAT AATGAACAAT ACAGAATTTT ATGATCGTCT CTCAGGACGA AATAAAAAAA GCTT INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 609 amino acids TYPE: amino acid WGCTCAATA ATCTAGATAA AAACATATTA ATAATAAATA TATATGTTAA CTATTTAGAG AGTATATGAA CAAAATATAA TGGCGTTTCA AAAGATGCTT WO 96/40928 PCT/CA96/00322 TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: Met Ser Lys Ile Ile Gly Ile Asp Leu Gly Thr Thr Asn Ser Ala Val 1 5 10 Ala Val Leu Glu Gly Thr Glu Ser Lys Ile Ile Ala Asn Pro Glu Gly ?5 Asn Arg Thr Thr Pro Ser Val Val Ser Phe Lys Asn Gly Glu Ile Ile 40 Val Gly Asp Ala Ala Lys Arg Gin Ala Val Thr Asn Pro Asp Thr Val 55 Ile Ser Ile Lys Ser Lys Met Gly Thr Ser Glu Lys Val Ser Ala Asn 65 70 75 Gly Lys Glu Tyr Thr Pro Gin Glu Ile Ser Ala Met Ile Leu Gin Tyr 90 Leu Lys Gly Tyr Ala Glu Asp Tyr Leu Gly Glu Lys Val Glu Lys Ala 100 105 110 Val Ile Thr Val Pro Ala Tyr Phe Asn Asp Ala Gin Arg Gin Ala Thr 115 120 125 Lys Asp Ala Gly Lys Ile Ala Gly Leu Glu Val Glu Arg Ile Val Asn 130 135 140 Glu Pro Thr Ala Ala Ala Leu Ala Tyr Gly Met Asp Lys Thr Asp Lys 145 150 155 160 Asp Glu Lys Ile Leu Val Phe Asp Leu G,1y Gly Gly Thr Phe Asp Val 165 170 175 Ser Ile Leu Glu Leu Gly As- Gly Val Phe Asp Val Leu Ala Thr Ala 180 185 190 Gly Asp Asn Lys Leu Gly Gly Asp Asp Phe Asp Gin Lys Ile Ile Asp 195 200 205 Phe Leu Val Glu Glu Phe Lys Lys Glu Asn Gly Ile Asp Leu Ser Gin 210 215 220 Asp Lys Met Ala Leu Gin Arg Leu Lys Asp Ala Ala Glu Lys Ala Lys 225 230 235 24L Lys Asp Leu Ser Gly Val Thr Gin Thr Gin Ile Ser Leu Pro Phe Ile 245 250 255 Thr Ala Gly Ser Ala Gly &ro Leu His Leu Glu Met Ser Leu Ser Arg 260 265 270 Ala Lys Phe Asp Asp Leu Thr Arg Asp Leu Val Glu Arg Thr Lys Thr 275 280 285 Pro Val Arg Gin Ala Leu Ser Asp Ala Gly Leu Ser Leu Ser Glu Ile 290 295 300 Asp Glu Val Ile Leu Val Gly Gly Ser Thr Arg Ile Pro Ala Val Val 305 310 315 320 Glu Ala Val Lys Ala Glu Thr Gly Lys Glu Pro Asn Lys Ser Val Asn 325 330 335 Pro Asp Glu Val Val Ala Met Gly Ala Ala Ile Gin Gly Gly Val Ile 340 345 350 WO 96/40928 Thr Gly Asp 355 PCT/CA96/00322 Val Lys Asp Val Val Leu Leu Asp Val. Thr Pro Leu Ser 360 Leu Asn 365 Asp Met Pro Asp Lys 465 Giu Ala ILie Asp Ala 545 Leu Ala Asp Lys Ile Thr Gin Ala Ala 435 Asn Gin A ~p Arg Ala 515 Giu Giu Giu Ala Ser 595 Met Thr 390 Val Lys Gly Val Val 470 Met Gi; Lys Ala Asn 550 Gin Ala Gi" Gly Lys Ile Leu Pro 440 Val Gin Asp Asp 11 e 520 Gin Asp~ Leu Gin Asp 600 Phe Gin Val 410 Arg Ile Ala Asn Glu 490 Lys Glu Ala Met V~q 3 570 Ala Val 365 Leu Ile Asp Arg Ser Thr Ala Ala 400 Gly Glu Arg Pro 415 Leu Thr Asp Ile 430 Thr Phe Asp Ile 445 Leu Gly Thr Gin Leu Thr Asp Giu 480 Ala Giu Ala Asp 495 Val Asp Gin Ala 510 Gly Lys Gly Phe 525 Glu Leu Lys Lys Lys Leu Giu Ala 560 Tyr Giu Gin Ala 575 Gin Ser Ala 590 Giu Phe Thr Giu 605 2) INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 19 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: Arg Ile Pro Ala Val Val Giu Ala V).1 Lys Ala Giu Tbhr Gly Lys Glu 1 5 10 Pro Asn Lys WO 96/40928 PCT/CA96/00322 INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: Gin Thr Ile Val Ile Gin Ser Asn Ser Gly Leu Thr Asp Glu Glu 1 5 10 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 460 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Streptococcus pneumoniae (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..456 OTHER INFORM?1TION: /product= "C-terminal 151-residue fragment (C-151) of HSP72" (xi) SEQUENCE DESCRIPTION: SEQ ID ATG AAG GCC AAA GAC CTT GGA ACT CAA AAA GAA CAA ACT ATT GTC ATC 48 Met Lys Ala Lys Asp Leu Gly Thr Gin Lys Glu Gin Thr Ile Val Ile 1 5 10 CAA TCG AAC TCA GGT TTG ACT GAC GAA GAA ATC GAC CGC ATG ATG AAA 96 Gin Ser Asn Ser Gly Leu Thr Asp Glu Glu Ile Asp Arg Met Met Lys 20 25 GAT GCA GAA GCA AAC GCT GAA TCC GAT AAG AAA CGT AAA GAA GAA GTA 144 Asp Ala Glu Ala Asn Ala Glu Ser Asp Lys Lys Arg Lys Glu Glu Val 40 GAC CTT CGT AAT GAA GTG GAC CAA GCA ATC TTT GCG ACT GAA AAG ACA 192 Asp Leu Arg Asn Glu Val Asp Gln Ala Ile Phe Ala Thr Glu Lys Thr 55 ATC AAG GAA ACT GAA GGT AAA GGC TTC GAC GCA GAA CGT GAC GCT GCC 240 Ile Lys Glu Thr Glu Gly Lys Gly Phe Asp Ala Glu Arg Asp Ala Ala 70 75 CAA GCT GCC CTT GAT GAC CTT AAG AAA GCT CAA GAA GAC AAC AAC TTG 288 Gin Ala Ala Leu Asp Asp Leu Lys Lys Ala Gin Glu Asp Asn Asn Leu 90 GAC GAC ATG AAA GCA AAA CTT GAA GCA TTG AAC GAA AAA GCT CAA GO'A 336 Asp Asp Met Lys Ala Lys Leu Glu Ala Leu Asn Glu Lys Ala Gin Gly 100 105 110 WO 96/40928 PCT/CA96/00322 CTT GCT GTT AAA CTC TAC GAA CAA GCC GCA GCA GCG CAA CAA GCT CAA 384 Leu Ala Val Lys Leu Tyr Glu Gin Ala Ala Ala Ala Gn Gin Ala Gin 115 120 125 GAA GGA GCA GAA GGC GCA CAA GCA ACA GGA AAC GCA GGC GAT GAC GTC 432 Glu Gly Ala Glu Gly Ala Gin Ala Thr Gly Azn Ala Gly Asp Asp Val 130 135 140 GTA GAC GGA GAG TTT ACG GAA AAG TAAG 460 Val Asp Gly Glu Phe Thr Glu Lys 145 150 INFORMATION FOR SEQ ID NO:26: SEQUENCE CHARACTERISTICS: LENGTH: 152 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: Met Lys Ala Lys Asp Leu Gly Thr Gin Lys Glu Gin Thr Ile Val Ile 1 5 10 Gin Ser Asn Ser Gly Leu Thr Asp Glu Glu Ile Asp Arg Met Met Lys 25 Asp Ala Glu Ala Asn Ala Glu Ser Asp Lys Lys Arg Lys Glu Glu Val 40 Asp Leu Arg Asn Glu Val Asp Gin Ala Ile Phe Ala Thr Glu Lys Thr 50 55 Ile Lys Glu Thr Glu Gly Lys Gly Phe Asp Ala Glu Arg Asp Ala Ala 70 75 Gin Ala Ala Leu Asp Asp Leu Lys Lys Ala Gin Glu Asp Asn Asn Leu 90 Asp Asp Met Lys Ala Lys Leu Glu Ala Leu Asn Glu Lys Ala Gin Gly 100 105 110 Leu Ala Val Lys Leu Tyr Glu Gin Ala Ala Ala Ala Gin Gin Ala Gin 115 120 125 Glu Gly Ala Glu Gly Ala Gin Ala Thr Gly Asn Ala Gly Asp Asp Val 130 135 140 Val Asp Gly Glu Phe Thr Glu Lys 145 150

Claims (18)

1. 2. The polypentide of claim 1, wherein the fragments of paragraph are selected from the group consisting of amino acids 439-607 of SEQ ID NO:5 (C-169) amiro acids 457-607 Df SEQ ID NO:5 (C-151), amino acids 527-541 of SEQ ID NO:5, and amino acids 586-600 of SEQ ID
2. 3. A polypepcide according mo claim 1 .aving the amino acid sequence of SEQ ID NO:5, or analcgusa or derivatives thereof.
3. 4. A polypeptide according to claim 1 having the amino acid sequence of SEQ ID NO:20, or analogues or derivatives thereof. 112 AMENDED SHEET HAij 4' 4I N I INtAU 0hN''4 Q I I I .1 i w t I 1 11 L-1 A4 FaY14Y S. A polypeptide accordirzg to claim 1 having the amino ac-id sequence Of SEQ ID NO:22, or analogues or dorivatiYes thcrcof. S. A polypeptide according to claim 1 havring =he amino acid sequence of SEQ ID NC:26, or analogues or derivatives thereof.
4. 7. A poJlypeptide according to claim 1 having the amino acid sequience of SEQ ID NO:7, or analogues or derivatives thereof.
5. 8. A polypeptide according to claim I having the amino acid sequence of SEQ ID N0:8, or analog-ues or derivatives thereof.
6. 9. A polypeptide according to claim having the amino acid sequence of SEQ ID INO:9, or analogues or derivatives thereof. A poly-peptide according to Clair, 1 having the amino acid sequence of SEQ 1D N0:10, or analcoguna or derivatives thereof.
7. 11. A polypeptide according to claim I havi.ng the amino acid sequence of SEQ ID NO:1l, or analogues or derivatives thereof'. 113 RA4 I' Ar AMENDED SHEET I I M I I I i I I I I j e_ U U e 1 4 4-kIAXI INtAU ALkH A WG.c j s 1 4-W-4J3U ii!1 -1 ,j h I'4 J 'ayeU 1.2. A polypeptide according to claim I. having :he amino ac .d mequence off SEQ ID NO;!2, or an-aloguee or derivativ.es thereof.
8. 12. A polypeptide according to cl-aimn i having the amino acid oaquarica of SEQ ID NO:13, or anal.oguaQ or derivatives thereof. 1~4. A pol.ypeptide according to claim I having the amino acid sequence of SEQ ID NO!14, or analogues or derivatives thereoff, A poJlypeptide according to claim 2- having the amino acid sequence of SEQ ID NO:15, or analogues or derivatives chereof.
9. 16. A polypeptide according to claim hain he amino acid sequence of SEQ ID NC:16, or analogues or derivatives chereof,
10. 17. A poly-peptide according to claim i having the amino acid sequence of SEQ ID NO:17, or analogues or derivativea hereof.
11. 18. A poly-peptide according to claim having the amino acid sequence of SEQ ID NO:18, or analogues or derivatives hereof, 114 '1 '.12DSnH 1. I I i I I I I I I 14w 1
12. 19. A polypeptide according to claim I having t. amino ac-id sequence of SEQ ID NO:23, or analogues or derivat ivr-o thereof, A polypeptide. according to claim having the amino acid seguence of SRQ ID NO:24, or analogues or derivatives thereof.
13. 21. The polypeptide of any one of claims 1 zo wherein said polypeptide elicits an immune reaction that is specific t~o Streptaococcal strains,
14. 22. A polypeptide according to claim 1 selected froma the group consisting of: che 1HSP72 polypeptide having the am~.no acid sequence of SEQ ID NO;5; an-d fragments off the foregoing poly-peptide, either alone or in combination with other Dolypepcides to formt a fusion protein.
15. 23. The polypeptide of claim 22, wherein- the fragments of paragraph are selected from the group consisting of amino acids 439-607 of SEQ ID NO-S IC-169) amino acids 527-541 of SEQ ID NO:5, and amino acids 586-60C of SEQ ID .NC
16. 24. The polypeptide of claim 22, wherein the fusion protein of paragraph is the Fucose :aomerase-HS?72 (C-169) protein having the amino acid sequence of SEQ ID NO:]. .ED SHEET M AX:J Uo- I M II ZAU R'5 A 'JL. 4 4 4 h-j 4 J 1 A DNA sequence selected from the group consisting of: the HSP72 DNA qcquence of SEQ ID NO:4; the HSP70 (DnaK) DNA sequence of SEQ ID NO: 19; the HSP70 (DnaK) DNA sequence of SEQ ID NO:21; DNA sequences that are degenerate to any of the foregoing DNA sequences; and fragments of any of the foregoing DNA sequences, either alone or in combination with other DNA sequences to form a fusion DNA sequence.
17. 26. A DNA sequence according to claim 25 comprising the formula of SEQ ID NO:4 from nucleotide 682 to nucleotide
18. 2502. 27. A DNA sequence according to claim 25 comprising the formula of SEQ 1D NO:4 from nucleotide 1996 to nucleocide 2502. 28. A DNA sequence according to claim 25 comprising the formula of SEQ ID NO:4 from nucleotide 2C50 to nucleotide 2502, 29. A DNA sequence according to claim 25 comprising the formula of SEQ ID NO:4 from nucleotide 2260 to nucleotide 2304. 116 ED SHEET I A DNA sequence dccarirg to Claim 25 compris.inq the forTvua of SEQ ID NO:4 from nuclteotide 2437 co nlucleotide 2481. 31. A DNA sequence according to claim 25 coipr-'Ing the formula of SEQ ID NO:19 from nucleotide 204 to nuclectide 2027. 32, A DNA sequence according to claim 25 com-orising th~e formula of SEQ ID NO:21 from nucleotide 248 to nucleotide 2074. 33. A DNA sequence according to claim 25 comprising the formula of SEQ 1D NO:25 from nucleotide 4 to nucleotide 456, 34. A DNA sequence coding for a polypeptide according co any one of claims 1-20. A DNA auquance according o claim 25 select-ed from the group consisting of: r-he 14SP7-2 DNA oequence of SRQ :D ,TO:4; DNA siequancea that are degenerate to the foregoing DNA sequence; and fragments of any of the foregoing DNA sequences, either alone or in combination with other DNA sequences to form a fusion DNA seq~uence. 117 L:;rJSHEET e~ud~ J U-~AIv INtAU MAhN ASWU.t~ t- 1'n 'ave4d 36. The DNA sequence of claim 35, wherein t).he fragments off paragraph are salectad from then group COnaistizng Of nucleotide 1996-2502 (amino acids 439-0-07) of 9FQ ID NO:4 (C-169); nucleotide 2260-2304 (amino acids 527-54:) of SEQ ID NO:4; and nucleotide 2437-2481 (amino acids 58 8-600) of SEQ ID NO:4. 37, The DNA sequence of claim 35, wherein the fusion DNA sequence of paragraph is the Fucose Isomerase-HS?72 (C- 169) DNA sequence of SEQ ID NO:l (nucleocides 771-2912). 38. AnP expression vector including at least one DNA sequence according to claim 35 operably linked to a promoter.- 39. A recombinant IDNA molecule comprising a DNA sequence according to any one of claims 25 to 34, and one or more expression control sequence operably linked to the DNA sequence, The recombinant rDNA molecule of clalm 39, wherein said expression control sequence is an inducible expression vector. 41. The recombinant molecule of claim 40, w-erein said expression vector comprises the X PL promoter. 42. A recomhinant molecule according to claim 39 comprising a plasmid selected from the group consistingc of: AENDED SHEET -rN 41-U-VI e tJb UaPMNIIMAU MtAkXl MAIU.b t--3J 14 el 4 aL1 'ptRV3, PU?.V4, ptJRVS, puRVx6, PJBD-191, pJ2DA4, PJBD--k~l, FCBD1.71, pJBD177, pJBD179, PJBDA1, PJBDf51, and pj~f2 43. A unicellular hosc transformed with an e-xPressiOn vector of claim 38. 44. A Urlicellular host transformed with a recombinant; DNA molecule of claim 39, A unicellular host according to claim 44, wherein said host is selected from the group consisting of; E.coZi strains XLI Blue M4.jFt, W3110, w5M109, Y1090 and BL21(DE3). 46. A method for producing a oolypeptide or fragment thereof comprising Che stePs of cultu-ring nhe unicellular host of any one of4 claims 43-45 and isolating said polypeptide or fragment. 47. A polypantide in subatan=4 ally pure form as obtained by the method of clai;m 46, 48. Az~ antibody or fragment thariaof that specifically binds to a polypePtide of any one of claims 1-20, 49. An antibody or fragment thereof t:hat specifically binds to the epicope recognized by mor4oclona- antibody F1-Pn3.l. 119 I' CN II-UU-,I I, Ub UVtA-M I1MAU RALAtN ASWULIh 1-I-i-44U t-j( 1 I Ira/1 1 The antibody or fragment of claim 48, which is a monocloral antibody. 51, The monoclonal antibody or fragment of claim which is of murine origin, 52. The moncclonal antibody or fragment of claim 51, which is of IgG type. 53, The monoclonal antibody FI-Pn3,1. 54. A method for isolating the antibody of claim 48 comprising: introducing a preparation of the polypeptide of any one of claims 1-20 intc a mammal; and isolating serum from the mammal containing said antibody. A method for isolating the monoclonal antibody of claim 50 comprising: introducing a preparation of the polypeptide of any one of claims 1-20 to antibody producing cells of a mammal; fusing the antibody producing cells with myeloma cells to form hybridoma cells, and isolating said monoclonal antibody from the hybridoma cells. 120 I, ,t II I \I I i I I t) MI I 1 11 I J I I l: 56. A pharmaceutical composition comprising a polypeptide of any one of claims 1-20. 57. The pharmaceutical composition of claim 56, which is a vaccine. 58. The pharmaceutical composition of claim further comprising one or more pharmaceutically acceptable excipients. 59. A pharmaceutical composition comprising one or more antibodies or fragments thereof according to claim 4. The pharmaceutical composition of claim 59, which is a vaccine. 61. The pharmaceutical composition of claim further comprising a pharmaceutically acceptable excipient. G2. The pharmaceutical composition of claim 60 or 61, wherein the antibody is Fl-Pn3.1. 63. 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 57, 60 or 61. 64, A mechod for preventing infection of a patient by Streptococcus pneumaniae, Streptococcus pyogenes or ,I StrePtococcusO agalacriao compriirjS the ddmin-tratjo oZ a pharmace.utically effective amount of the V'accine o:E Claim 57, or 91. A method for treating a patient infected with or suspected of being infected with St-rep cOcoccua pneumoniae or related bacteria comprising the administration of a pharmaceutically effective amount of the vaccine ,f claim 60 or 61. 66. A method for the detection of Stzxeptococcus pne.umon.ae or related bacteria in a biological sample comprising: incubating the antibody or fragment of claim 48 with the biological sample to form a mixture; and detecting specifically bound antibody or fragment in the mixture which indicatea the presence of Stretocccu~pneumonia.e vzr related bacteria. 67. The method of claim 66, wherein the antibody is FI-Pn3 .1. 68, A method for the detection of antibodia& -)ecific to ScreptococcuB pnewnonlae or related bactaxia in a biological sample comprisi4ng! incubating a polypeptide of claim 2 or with the biological sample to form a mixture; and 122 ~-uw eju Ufl-ORIMAU MAER~ ASWUCO+-AJ-SUh31 4/I rra1 4 detecting specifioaily bound poly-peptide in the mix~ture, tihich inidicates the presence of antibodies specific to S'treptccoccuBs pnewnor iae or related bacteria. 69. A method f or the detecti~on of Screptococcus pneumonlae or related bacc~aria in a biological sample Compriaing; incubating a DNA probe having the EmA sequence of claim 35 with the biological sample to form a mixture; and detecting specifically bound DNA probe in the mixture whl-.h indicates t.'e prelience of Streptococcus pneurnoniae and reiated bacteria. The method of cli- 69, wherein the DNA probe is an oligorner having a sequence cotr-lementary to at least about 6 contiguous nucleot~ides of a DNA sequence of claim 71. The method of clai~m 70, which further rcmpvises: pr'oviding a set of ollgomers which. are primers "-or a polymerase chain reaction method an~d which flank the target region;, and amplifying the target region via the po~ymerase chain reaction method. 72. A method for the detection of Screptcco~vcua .pzeuzoniae, $treptococcus pyogernes or Streptococcus agala--tiae in a biological sample comprising: incubating the antibody or ffragmeac of claim 4E8with the biological Oarnple Lo form a mixture, and deteccir, specifically bound antibody o~r fragjment in the mixture which indicates the presence of Streptococcus pneu-monlae, Streptococcua pyogeies or Streptococcus agalactiae. 73, A method for the detection of antibodiea specific to Streptococcua prneumoniae, Streptococcus pyogenes or Streptococcus agralacclae in a biological sample comprising: incubating a polypeptide of claim I. or 21 with the biological sample to form a mixture; and detecting specifically bound polypeptide in the mixture, which indicates the presence of antibodies specific- to StZ-Cpt;QCCCUS pneumoniae, Strep tococcus pyogeines or. Screptococcus agalact.iae. 74. A Method for the detectio~n off Streptococcus pneuxnoniae, Streptococcus pycgenes or Straptococcus ayalactiae 'n biological sample comprising; incubating a DNA probe having t.-e DNP, sequence of claim 25 or 34 with the biological sample to form a mixturel and detecting specifically bound IDNA probe in -he mfixrure wh~ch indicates the presence of Strepcococcua pneunioniae, Streptococcus pyogenes or Strepto~coccus agalactiae. The method of claim '74, wherein the DNA probe is an oligomer hav$,ng a maquenco complementary to at least about 6 contiguous nucleotides ol a DNA caquance of claim 2s or 34. 76. The method of claim 75, which further cornpi-isaa: providing a set of oligomers which are primers for a polymerase chain reaction method and which flank the target region; and amplifying the target region via the polymerase chain reaction method. Dated this 30th day of June 1998. BIOCHEM VACCINES INC By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent Attorneys of Australia .125