EP0832238A1 - Streptococcal heat shock proteins members of the hsp70 family - Google Patents
Streptococcal heat shock proteins members of the hsp70 familyInfo
- Publication number
- EP0832238A1 EP0832238A1 EP96914821A EP96914821A EP0832238A1 EP 0832238 A1 EP0832238 A1 EP 0832238A1 EP 96914821 A EP96914821 A EP 96914821A EP 96914821 A EP96914821 A EP 96914821A EP 0832238 A1 EP0832238 A1 EP 0832238A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- ala
- polypeptide
- seq
- glu
- gly
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
- C07K16/1275—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Streptococcus (G)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
- C07K14/3156—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci from Streptococcus pneumoniae (Pneumococcus)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Definitions
- 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
- S. pneumoniae is an important agent of disease in humans, especially among infants, the elderly and immunocompromised persons . It is a bacterium frequently isolated from patients with invasive diseases such as bacteraemia/septicaemia, pneumonia, and meningitis with high morbidity and mortality throughout the world. Although the advent of antimicrobial drugs has reduced the overall mortality from pneumococcal diseases, the presence of resistant pneumococcal organisms has become a major problem in the world today. Effective pneumococcal vaccines could have a major impact on the morbidity and mortality associated with S. pneumoniae disease. Such vaccines would also potentially be useful to prevent otiti ⁇ media in infants and young children.
- 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.
- the failure of existing vaccines to protect young children against most serotypes has spurred evaluation of other S. pneumoniae components.
- certain pneumococcal proteins may play an active role both in terms of protection and pathogenicity [J.C. Paton, Ann. Rev. Microbiol., 47, pp.
- Streptococcus agalactiae also called Group B Streptococcus (GBS)
- GBS Group B Streptococcus
- GBS blood infection
- meningitis 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.
- GBS can cause urinary tract infections, womb infections (amnionitis, endometritis) , and stillbirth.
- the most common diseases caused by GBS are blood infections, skin or soft tissue infections, and pneumonia. Approximately 20% of men and nonpregnant women with GBS disease die of the disease.
- GBS infections in both newborns and adults are usually treated with antibiotics (e.g., penicillin or ampicillin) given intravenously.
- antibiotics e.g., penicillin or ampicillin
- Most GBS disease in newborns can be prevented by giving certain pregnant women antibiotics intravenously during labor.
- Vaccines to prevent GBS disease are being developed. In the future, it is expected that women who will be vaccinated will make antibodies that cross the placenta and protect the baby during birth and early infancy.
- Streptococcus pyogenes also called Group A Streptococcus (GAS) is reemerging as a cause of severe diseases which would be due to an increase in virulence of the organism.
- GAS causes pharyngitis, commonly called “strep throat”, and skin infections (impetigo, erysipelas/cellulitis) .
- strep throat skin infections
- impetigo can lead to glomerulonephritis (kidney damage) .
- TSS extremely severe streptococcal toxic shock syndromes
- HSPs Heat shock or stress proteins
- HSPs have been defined by their size, and members of hsp90, hsp70, and hsp60 families are among the major HSPs found in all prokaryotes and eukaryotes . These proteins fulfill a variety of chaperon functions by aiding protein folding and assembly and assisting translocation across membranes [C. Georgopoulos and .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.
- HSPs are major antigens of many pathogens.
- HSPs can elicit strong B- and T- cell responses and it was shown that 20% of the CD4 * T-lymphocytes from mice inoculated with M. tuberculosis were reactive to the hsp60 protein alone [S.H.E. Kaufman et al.
- the present invention addresses the problems referred to above by providing novel heat shock proteins from S. pneumoniae, S. pyogenes and S. agalactiae, and immunologically related polypeptides .
- DNA sequences that code for the foregoing polypeptides are also provided, vectors containing the polypeptides, unicellular hosts transformed with those vectors, and a process for making substantially pure, recombinant polypeptides.
- antibodies specific to the foregoing polypeptides are also provided.
- 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.
- the novel heat shock protein is the approximately 72 kDa heat shock protein of Streptococcus pneumoniae ("HSP72") (SEQ ID NO:5), the approximately 70 kDa heat shock protein of Streptococcus pyogenes (“HSP70”) (SEQ ID NO:20) and the approximately 70 kDa heat shock protein of Streptococcus agalactiae (“HSP70”) (SEQ ID NO:
- HSP70/72 include the C-terminal portion of the HSP70/72 polypeptides. More particularly, it includes the C_terminal 169-residue fragment ("C-169") (residues 439-607, SEQ ID NO: 5) , the C-terminal 151-residue fragment (“C-151”) (residues 457-607, SEQ ID No:5) , and smaller fragments consisting of peptide epitopes within the C-169 region.
- C-169 C_terminal 169-residue fragment
- C-151 C-terminal 151-residue fragment
- 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. 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, MAPI, 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 (“MAbs”), which are specific to HSP72.
- More preferred antibodies are the F2-Pn3.2 and F2-Pn3.4 monoclonal anibodies that are specific to both HSP 70 and HSP72. Even more preferred are the Fl-Pn3.1 antibodies that are specific for Streptococcus pneumoniae .
- the preferred polypeptides and antibodies of this invention provide the basis for novel methods and pharmaceutical compositions for the detection, prevention and treatment of pneumococcal diseases .
- FIG. 1 depicts a fluorogram, which shows the effect of heat shock on S. pneumoniae protein synthesis.
- the cell extracts in panel A are S. pneumoniae type 6 strain 64.
- the cell extracts in panel B are S. pneumoniae type 4 strain 53.
- the cell extracts in the odd numbered lanes were incubated at 37°C.
- the cell extracts in the even numbered lanes were incubated at 45°C for 5 minutes .
- the cell extracts were then labeled with [ 35 S]methionine for 10 minutes (lanes 1, 2 and 7, 8) , 30 minutes (lanes 3, 4 and 9, 10) , or 60 minutes (lanes 5, 6) .
- Molecular mass markers in kilodaltons are shown to the left.
- the positions of HSP80, H ⁇ P72 and HSP62 are shown by arrows at the right-hand side of each panel.
- FIG. 2 is a graphical depiction of a comparison of the electrophoretic profiles of [ 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 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°C and incubated at 37°C (lanes 3, 5, 7 and 9) or heat-shocked at 45°C (lanes 4, 6, 8 and 10) were immunoprecipitated with sera from mouse 1 (lanes 3 to 6) or mouse 2 (lanes 7 to 10) and then analyzed by SDS- PAGE and fluorography. The sera were tested after the first (lanes 3,4 and 7,8) and after the second (lanes 5,6 and 9,10) immunization.
- 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]methionine-labeled proteins from S. pneumoniae grown at 37°C and incubated at 37°C (lanes 3, 5 and 7) or heat-shocked at 45°C (lanes 4, 6 and 8) were immunoprecipitated with sera from mouse 1 (lanes 3,4), mouse 2 (lanes 5,6) or mouse 3 (lanes 7, 8) and then analyzed by SDS-PAGE and fluorography. Sera were tested after the second immunization only.
- 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 Fl-Pn3.1.
- panel A the sera were tested after the second immunization.
- panel B the reactivity of 4 out of 15 sera tested after the first immunization is shown.
- the positions of 53.5 kDa- and 47 kDa-protein bands are indicated by the bars at the left.
- the position of HSP72 is shown by the arrows at the right of each panel.
- FIG. 6 depicts a fluorogram showing the specificity of MAb Fl-Pn3.1 for HSP72.
- [ 35 S]methionine- labeled proteins of S. pneumoniae in the absence (lanes 1, 3 and 5) or presence (lanes 2, 4 and 6) of exposure to heat shock were immunoprecipitated with IgG2a-control MAb (lane 3,4) or Fl-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.
- HSPs All three
- 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 (45°C) .
- Lane 2 contains non heat-shocked cell lysates (37°C) .
- Panel B depicts a fluorogram of the immunoblot shown in panel A.
- FIG. 8 depicts a Western Blot, which shows subcellular localization of S. pneumoniae HSP72. Sample containing 15 ⁇ g 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 probed with HSP72-specific MAb Fl-Pn3.1.
- Lanes 2 and 3 contain phage lysates from E. coli infected with ⁇ JBDl7 cultured in the presence (+) or absence (-) of IPTG and probed with HSP72-specific MAb Fl-Pn3.1.
- Lanes 4 and 5 contain phage lysates from E. coli infected with ⁇ JBD7 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 IOC illustrate the restriction map of the HSP72(DnaK) and Fuc loci, respectively.
- FIG 10B illustrates the inserts of the various phages and plasmids described in Example 3.
- H(Hind ⁇ II) ; E(EcoRI) ; V(EcoRV) ; P(Pst ⁇ ) ; and X(XhoI) indicate positions of restriction endonuclease sites.
- FIG. 11 depicts the SDS-PAGE and Western blot analyses of the recombinant 74 kDa protein.
- Whole cell extracts from E. coli transformed with plasmids pJBD179 (lane 1) , pJBDf51 (lanes 2 and 3) and pJBDf62 (lane 4 and 5) and cultured in presence (+) or absence (-) of IPTG were subjected to 10% polyacrylamide gel electrophoresis.
- the proteins were then visualized by Coomassie Blue staining (A) or Western blotting (B) using HSP-specific MAb 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 4) were subjected to 10% polyacrylamide gel electrophoresis and 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 ⁇ g of immunoaffinity-purified HSP72 r ⁇ c (lane 3) were subjected to 10% polyacrylamide gel electrophoresis. The proteins were then visualized by Coomassie Blue staining.
- Lane 1 shows the migration of molecular mass markers (106 kDa, 80 kDa, 49.5 kDa, 32.5 kDa, 27.5 kDa and 18.5 kDa) .
- FIG. 15 depicts a photograph of SDS-PAGE, which shows the recombinant S. pneumoniae C-169 fragment purified by solubilization of inclusion bodies.
- Various amounts of purified C-169 protein (lane 1, 5 ⁇ g; lane 2, 2.5 ⁇ g; and lane 3, 1 ⁇ g) and whole cell lysates from E. coli transformed with plasmids pDELTAl (lane 4) and pJBD ⁇ l (lane 5) were subjected to 10% polyacrylamide gel electrophoresis. The proteins were then visualized by Coomassie Blue staining.
- FIG. 16 is a graphical depiction of the survival curve of Balb/c mice protected from S. pneumoniae infection by immunization with HSP72 r ⁇ c . Data are presented as the per cent (%) survival over a period of 14 days for a total of 10 mice per experimental group.
- FIG. 17 is a graphical depiction of the survival curve of Balb/c mice protected from S. pneuiTioniae infection by immunization with C-169 r ⁇ c . Data are presented as the per cent (%) survival over a period of 14 days for a total of 10 mice per experimental group.
- FIG. 18 is a map of plasmid pURV3 containing C- 151 rec , the coding region for the 151 amino acids at the carboxyl end of the HSP72 of S. pneumoniae; Ampi R , ampicillin-resistance coding region; ColEl ori , origin of replication; cI857, bacteriophage ⁇ cI857 temperature- sensitive repressor gene; ⁇ PL, bacteriophage ⁇ transcription promoter; Tl, Tl transcription terminator. The direction of transcription is indicated by the arrows. ⁇ glll and BamHI are the restriction sites used to insert the coding region for the C-151 rec of the HSP72 of S. pneumoniae .
- FIG. 18 is a map of plasmid pURV3 containing C- 151 rec , the coding region for the 151 amino acids at the carboxyl end of the HSP72 of S. pneumoniae; Ampi R , ampicillin-resistance coding region; Col
- FIG. 19 illustrates the distribution of anti-S. pneumoniae titers in sera from Balb/c mice immunized with HSP72 rec .
- Sera were collected after the first, second and third injection with 1 ⁇ g (O) or 5 ⁇ g (•) of HSP72 rec and evaluated individually for anti-S. pneumoniae antibody by ELISA.
- Titers were defined as the highest dilution at which the A410 values were 0.1 above the background values .
- Plain lines indicate the median reciprocal of antibody titers for each group of mice while the dashed line indicates the median value for preimmune sera.
- FIG. 20 illustrates the distribution of anti-S. pneumoniae titers in sera from Balb/c mice immunized with C-169 rec .
- Sera were collected after the first, second and third injection with 1 ⁇ g (O) or 5 ⁇ g (•) of C-169 rec and evaluated individually for anti-S. pneumoniae antibody by ELISA.
- Titers were defined as the highest dilution at which the A410 values were 0.1 above the background values .
- Plain lines indicate the median reciprocal of antibody titers for each group of mice while the dashed line indicates the median value for preimmune sera.
- FIG. 21 illustrates the distribution of anti-S. pneumoniae titers in sera from Balb/c mice immunized with C-151 rec .
- Sera were collected after the first, second and third injection with 0.5 ⁇ g of C-151 rec and evaluated individually for anti-S. pneumoniae antibody by ELISA. Titers were defined as the highest dilution at which the A410 values were 0.1 above the background values. Plain lines indicate the median reciprocal of antibody titers for each group of mice while the dashed line indicates the median value for preimmune sera.
- FIG. 22 illustrates the antibody response of cynomolgus monkeys immunized with recombinant HSP72 antigens.
- Groups of two monkeys were immunized with either HSP72 rec or C-169 rec protein at day 1, day 22 and day 77.
- Sera were collected regularly during the course of the immunization and evaluated individually for pneumococcal HSP72 specific antibody by Western blot analysis . Titers were defined as the highest dilution at which the HSP72 band was visualized.
- FIG. 23 illustrates the binding of hyperimmune sera to peptides in a solid-phase ELISA.
- Rabbit, mouse and monkey sera from animals immunized with either HSP72 rec or C-169 rec protein were tested for their reactivity to peptides .
- Optical density values were obtained with sera tested at a dilution of 1:100 except for the values corresponding to the reactivity of rabbit sera to peptide MAP2 and murine sera to peptides MAP2 and MAP4 which were obtained with sera diluted 1:1000.
- FIG. 24 depicts the consensus sequence established from the DNA sequences of the hsplO/dnak open reading frames of Streptococcus pneumoniae (spn-orf) , Streptococcus pyogenes (sga-orf) and Streptococcus agalactiae (sgb-orf) and indicates the substitutions and insertions of nucleotides specific to each species.
- spn-orf Streptococcus pneumoniae
- sga-orf Streptococcus pyogenes
- sgb-orf Streptococcus agalactiae
- spn-prot Streptococcus pneumoniae
- sga-prot Streptococcus pyogenes
- sgb-prot Streptococcus agalactiae
- 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°C and incubated at 37°C (odd numbered lanes) or heat-shocked at 43°C (even numbered lanes) were analysed by SDS-PAGE and fluorography. Lanes 3 and 4 show the immunoprecipitates obtained using MAb F2-Pn3.4.
- heat shock proteins of S. pneumoniae, S. pyogenes and S. agalactiae and analogues, homologues, derivatives and fragments thereof, containing at least one immunogenic epitope.
- 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.
- immunogenic means having the ability to elicit an immune response.
- novel heat shock proteins of this invention are characterized by their ability to elicit a protective immune response against Streptococcal infections, more particularly against lethal S. pneumoniae , S. pyogenes and S. agalactiae .
- the invention particularly provides a Streptoccus pneumoniae heat shock protein of approximately 72 kDa (“HSP72”), having the deduced amino acid sequence of SEQ ID NO: 5, and analogues, homologues, derivatives and fragments thereof, containing at least one immunogenic epitope .
- HSP72 Streptoccus pneumoniae heat shock protein of approximately 72 kDa
- analogues of HSP72 are those S. pneumoniae proteins wherein one or more amino acid residues in the HSP72 amino acid sequence (SEQ ID NO: 5) is replaced by another amino acid residue, providing that the overall functionality and immunogenic properties of the analogue protein are preserved.
- Such analogues may be naturally occurring, or may be produced synthetically or by recombinant DNA technology, for example, by mutagenesis of the HSP72 sequence.
- Analogues of HSP72 will possess at least one antigen capable of eliciting antibodies that react with HSP72, e.g. Streptococcus pyogenes and Streptococcus agalactiae .
- 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 .
- a "derivative" is a polypeptide in which one or more physical, chemical, or biological properties has been altered. Such alterations include, but are not limited to: amino acid substitutions, modifications, additions or deletions; alterations in the pattern of lipidation, glycosylation or phosphorylation; reactions of free amino, carboxyl, or hydroxyl side groups of the amino acid residues present in the polypeptide with other organic and non-organic molecules; and other alterations, any of which may result in changes in primary, secondary or tertiary structure.
- the "fragments” of this invention will have at _ least one immunogenic epitope.
- An “immunogenic epitope” is an epitope that is instrumental in eliciting an immune response.
- Preferred fragments of HSP72 include the C-terminal region of the polypeptides. More preferred fragment include the C-terminal 169-residue fragment ("C-169") (SEQ ID NO:5, residues 439-607), the C-terminal 151-residue ("C-151”) (SEQ ID No:5, residues 457-607) and smaller fragments consisting of peptide epitopes within the C-169 region. Particularly preferred fragments within the C-169 region of HSP72 include the peptide sequences GFDAERDAAQAALDD (residues 527-541 of SEQ ID NO:5) and AEGAQATGNAGDDW
- fragments 586-600 of SEQ ID N0:5 which are exclusive to HSP72 of Streptococcus pneumoniae , or corresponding degenerate fragments from S. pyogenes or S. agalactiae (see FIG. 25) .
- 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, MAPI, MAP2, MAP3 and MAP4 (see TABLE 5, supra), or homologues thereof.
- polypeptides that are immunologically related to HSP70/72 As used herein, "immunologically related" polypeptides are characterized by one or more of the following properties:
- 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 :
- 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.
- related bacteria are bacteria that possess antigens capable of eliciting antibodies that react with HSP72.
- related bacteria include Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus mutans, Streptococcus sanguis, Streptococcus agalactiae and Enterococcus faecalis . It will be understood that by following the examples of this invention, one of skill in the art may determine without undue experimentation whether a particular analogue, homologue, derivative, immunologically related polypeptide, or fragment would be useful in the diagnosis, prevention or treatment of disease.
- polypeptides and fragments will elicit antibodies that are immunoreactive with HSP72 (Example 4) .
- useful polypeptides and fragments will demonstrate the ability to elicit a protective immune response against lethal bacterial infection (Example 5) .
- polymeric forms of the polypeptides of this invention include, for example, one or more polypeptides that have been crosslinked with crosslinkers such as avidin/biotin, glutaraldehyde or dimethylsuberimidate.
- Such polymeric forms also include polypeptides containing two or more tandem or inverted contiguous protein sequences, produced from multicistronic mRNAs generated by recombinant DNA technology.
- substantially pure HSP72 and immunologically related polypeptides This invention provides substantially pure HSP72 and immunologically related polypeptides .
- the term "substantially pure” means that the polypeptides according to the invention, and the DNA sequences encoding them, are substantially free from other proteins of bacterial origin. Substantially pure protein preparations may be obtained by a variety of conventional processes, for example the procedures described in Examples 3 and 5.
- this invention provides, for the first time, a DNA sequence coding for a heat shock protein of S. pneumoniae , specifically, HSP72 (SEQ ID NO:4, nucleotides 682-2502) .
- the DNA sequences of this invention also include
- DNA sequences coding for polypeptide analogues and homologues of HSP72 DNA sequences coding for immunologically related polypeptides, DNA sequences that are degenerate to any of the foregoing DNA sequences, and fragments of any of the foregoing DNA sequences. It will be readily appreciated that a person of ordinary skill in the art will be able to determine the DNA sequence of any of the polypeptides of this invention, once the polypeptide has been identified and isolated, using conventional DNA sequencing techniques .
- Oligonucleotide primers and other nucleic acid probes derived from the genes encoding the polypeptides of this invention may also be used to isolate and clone other related proteins from S. pneumoniae and related bacteria which may contain regions of DNA bacteria that are homologous to the DNA sequences of this invention.
- 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.
- polypeptides of this invention may be prepared from a variety of processes, for example by protein fractionation from appropriate cell extracts, using conventional separation techniques such as ion exchange and gel chromatography and electrophoresis, or by the use of recombinant DNA techniques.
- the use of recombinant DNA techniques is particularly suitable for preparing substantially pure polypeptides according to the invention.
- HSP72 immunologically related polypeptides, and fragments thereof, comprising the steps of (1) culturing a unicellular host organism transformed with a vector containing a DNA sequence coding for said polypeptide or fragment and one or more expression control sequences operatively linked to the DNA sequence, and (2) recovering a substantially pure polypeptide or fragment.
- the gene 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.
- the expression control sequences, and the gene of interest will be contained in an expression vector that further comprises a bacterial selection marker and origin of replication. If the expression host is a eukaryotic cell, the expression vector should further comprise an expression marker useful in the eukaryotic expression host.
- the DNA sequences encoding the polypeptides of this invention may or may not encode a signal sequence. If the expression host is eukaryotic, it generally is preferred that a signal sequence be encoded so that the mature protein is secreted from the eukaryotic host.
- amino terminal methionine may or may not be present on the expressed polypeptides of this invention. If the terminal methionine is not cleaved by the expression host, it may, if desired, be chemically removed by standard techniques .
- 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, pCRl, pBR322, pMB9 and their derivatives, wider host range plasmids, such as RP4, phage DNAs, e.g., the numerous derivatives of phage lambda, e.g. ⁇ gtlO and ⁇ gtll, NM989, and other DNA phages, such as M13 and filamentous single stranded DNA phages.
- Useful expression vectors for yeast cells include the 2 ⁇ plasmid and derivatives thereof.
- Useful vectors for insect cells include pVL 941.
- any of a wide variety of expression control sequences may be used in these vectors to express the DNA sequences of this invention.
- Useful expression control sequences include the expression control sequences associated with structural genes of the foregoing expression vectors .
- useful expression control sequences include, for example, the early and late promoters of SV40 or adenovirus, the lac system, the trp system, the TAC or TRC system, the T3 and T7 promoters the major operator and promoter regions of phage lambda, the control regions of fd coat protein, the promoter for 3- phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters of the yeast alpha-mating system and other constitutive and indueible promoter sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.
- the T7 RNA polymerase promoter ⁇ 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.
- 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.
- 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.
- 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.
- 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 such as with inorganic ligands or monoclonal antibodies
- immobilized metal chelate chromatography immobilized metal chelate chromatography
- gel electrophoresis and the like.
- polypeptides of this invention may be generated by any of several chemical techniques. For example, they may be prepared using the solid-phase synthetic technique originally described by R. B. Merrifield, "Solid Phase Peptide Synthesis. I. The Synthesis Of A Tetrapeptide” , J. Am. Chem. Soc . , 83, pp. 2149-54 (1963), or they may be prepared by synthesis in solution. A summary of peptide synthesis techniques may be found in E. Gross & H. J. Meinhofer, 4 The
- compositions and methods of this invention comprise polypeptides having enhanced immunogenicity.
- 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.
- the polypeptides may be modified by coupling to dinitrophenol groups or arsanilic acid, or by denaturation with heat and/or SDS.
- 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.
- Bind immunogenic carriers are well known in the art.
- examples of such carriers are keyhole limpet hemocyanin (KLH) ; albumins such as bovine serum albumin (BSA) and ovalbumin, PPD (purified protein derivative of tuberculin); red blood cells; tetanus toxoid; cholera toxoid; agarose beads; activated carbon; or bentonite.
- KLH keyhole limpet hemocyanin
- BSA bovine serum albumin
- PPD purified protein derivative of tuberculin
- red blood cells tetanus toxoid
- cholera toxoid agarose beads
- activated carbon or bentonite.
- Modification of the amino acid sequence of the polypeptides disclosed herein in order to alter the lipidation state is also a method which may be used to increase their immunogenicity and biochemical properties.
- the polypeptides or fragments thereof may be expressed with or without the signal sequences that direct addition of lipid moieties.
- derivatives of the polypeptides may be prepared by a variety of methods, including by in vi tro manipulation of the DNA encoding the native polypeptides and subsequent expression of the modified DNA, by chemical synthesis of derivatized DNA sequences, or by chemical or biological manipulation of expressed amino acid sequences.
- derivatives may be produced by substitution of one or more amino acids with a different natural amino acid, an amino acid derivative or non-native amino acid, conservative substitution being preferred, e.g., 3-methylhistidine may be substituted for histidine, 4-hydroxyproline may be substituted for proline, 5- hydroxylysine may be substituted for lysine, and the like. Causing amino acid substitutions which are less conservative may also result in desired derivatives, e.g., by causing changes in charge, conformation and other biological properties .
- substitutions would include for example, substitution of a hydrophilic residue for a hydrophobic residue, substitution of a cysteine or proline for another residue, substitution of a residue having a small side chain for a residue having a bulky side chain or substitution of a residue having a net positive charge for a residue having a net negative charge.
- the derivatives may be readily assayed according to the methods disclosed herein to determine the presence or absence of the desired characteristics.
- the polypeptides may also be prepared with the objective of increasing stability or rendering the molecules more amenable to purification and preparation.
- One such technique is to express the polypeptides as fusion proteins comprising other S. pneumoniae or non- S. pneumoniae sequences.
- the fusion proteins comprising the polypeptides of this invention be produced at the DNA level, e.g., by constructing a nucleic acid molecule encoding the fusion, transforming host cells with the molecule, inducing the cells to express the fusion protein, and recovering the fusion protein from the cell culture.
- the fusion proteins may be produced after gene expression according to known methods.
- An example of a fusion protein according to this invention is the FucI/HSP72 (C-169) protein of Example 3, infra.
- polypeptides of this invention may also be part of larger multimeric molecules which may be produced recombinantly or may be synthesized chemically. Such multimers may also include the polypeptides fused or coupled to moieties other than amino acids, including lipids and carbohydrates .
- the polypeptides of this invention are particularly well-suited for the generation of antibodies and for the development of a protective response against disease. Accordingly, in another aspect of this invention, we provide antibodies, or fragments thereof, that are immunologically reactive with HSP72.
- the antibodies of this invention are either elicited by immunization with HSP72 or an immunologically related polypeptide, or are identified by their reactivity with HSP72 or an immunologically related polypeptide. It should be understood that the antibodies of this invention are not intended to include those antibodies which are normally elicited in an animal upon infection with naturally occurring S. pneumoniae and which have not been removed from or altered within the animal in which they were elicited.
- the antibodies of this invention may be intact immunoglobulin molecules or fragments thereof that contain an intact antigen binding site, including those fragments known in the art as F(v), Fab, Fab' and F(ab')2.
- the antibodies may also be genetically engineered or synthetically produced.
- the antibody or fragment may be of animal origin, specifically of mammalian origin, and more specifically of murine, rat, monkey or human origin. It may be a natural antibody or fragment, or if desired, a recombinant antibody or fragment.
- the antibody or antibody fragments may be of polyclonal, or preferably, of monoclonal origin. They may be specific for a number of epitopes but are preferably specific for one.
- the monoclonal antibodies Fl- Pn3.1, F2-Pn3.2, F2-Pn3.3 and F2-Pn3.4 of Example 2, infra are preferred.
- 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.
- Determination of immunoreactivity with a polypeptide of this invention may be made by any of several methods well known in the art, including by immunoblot assay and ELISA.
- An antibody of this invention may also be a hybrid molecule formed from immunoglobulin sequences from different species (e.g., mouse and human) or from portions of immunoglobulin light and heavy chain sequences from the same species . It may be a molecule that has multiple binding specificities, such as a bifunctional antibody prepared by any one of a number of techniques known to those of skill in the art including: the production of hybrid hybridomas; disulfide exchange; chemical cross- linking; addition of peptide linkers between two monoclonal antibodies; the introduction of two sets of immunoglobulin heavy and light chains into a particular cell line; and so forth.
- the antibodies of this invention may also be human monoclonal antibodies, for example those produced by immortalized human cells, by SCID-hu mice or other non-human animals capable of producing "human” antibodies, or by the expression of cloned human immunoglobulin genes.
- polypeptides, DNA sequences and antibodies of this invention are useful in prophylactic, therapeutic and diagnostic compositions for preventing, treating and diagnosing disease.
- Standard immunological techniques may be employed with the polypeptides and antibodies of this invention in order to use them as immunogens and as vaccines.
- 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.
- 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.
- 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 .
- 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) .
- a polypeptide if used, it will be administered with a pharmaceutically acceptable adjuvant, such as complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptides) or ISCOM (im unostimulating complexes) .
- the composition will include a water-in-oil emulsion or aluminum hydroxide as adjuvant and will be administered intramuscularly.
- the vaccine composition may be administered to the patient at one time or over a series of treatments.
- the most effective mode of administration and dosage regimen will depend upon the level of immunogenicity, the particular composition and/or adjuvant used for treatment, the severity and course of the expected infection, previous therapy, the patient's health status and response to immunization, and the judgment of the treating physician. For example, in an immunocompetent patient, the more highly immunogenic the polypeptide, the lower the dosage and necessary number of immunizations. Similarly, the dosage and necessary treatment time will be lowered if the polypeptide is administered with an adjuvant.
- the dosage will consist of an initial injection, most probably with adjuvant, of about 0.01 to 10 mg, and preferable 0.1 to 1.0 mg, HSP72 antigen per patient, followed most probably by one or maybe more booster injections.
- 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.
- mice of Example 5 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.
- 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.
- 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.
- the preferred polypeptide for use in such methods is HSP70/HSP72, or fragments thereof.
- the polypeptides, DNA sequences and antibodies of this invention may also form the basis for diagnostic methods and kits for the detection of pathogenic organisms. Several diagnostic methods are possible. For example, this invention provides a method for the detection of Streptococcus pneumoniae , Streptococcus pyogenes , Streptococcus agalactiae or related bacteria in a biological sample comprising the steps of:
- Preferable antibodies for use in this method include monoclonal antibodies Fl-Pn3.1, F2-Pn3.2, F2-Pn3.3 and F2-Pn3.4.
- this invention provides a method for the detection of antibodies specific to Streptococcus pneumoniae or related bacteria in a biological sample comprising:
- diagnostic tests may take several forms, including an enzyme-linked immunosorbent assay (ELISA) , a radioimmunoassay or a latex agglutination assay.
- ELISA enzyme-linked immunosorbent assay
- radioimmunoassay radioimmunoassay
- latex agglutination assay 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.
- 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
- 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 125 I 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 X1 , 15 0, or 13 N. Binding may also be detected by other methods, for example via avidin- biotin complexes.
- the linking of the detection means is well known in the art.
- 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:
- 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.
- Example 1 describes the identification of HSP72, an immunoreactive heat shock protein according to the invention.
- Example 2 describes the isolation of monoclonal antibodies against epitopes of HSP72.
- Example 3 describes the preparation of recombinant HSP72 and fragments of HSP72 according to the invention.
- Example 4 describes the antigenic specificity and immunoreactivity of monoclonal antibodies directed against HSP72, and the identification of immunologically related proteins according to the invention.
- Example 5 describes processes for obtaining substantially pure HSP72, and the use of HSP72 or antibodies against it to protect against experimental S. pneumoniae infection.
- Example 6 describes the preparation of recombinant C-151 fragment of HSP72 according to the invention.
- Example 7 describes the humoral immune response following the immunization with recombinant HSP72 or fragments of HSP72 according to the invention.
- Example 8 describes the localization of linear B-cell epitopes on the HSP72.
- Example 9 describes the hsp70 genes and HSP70 proteins from S. agalactiae and S. pyogenes .
- Example 10 describes the use of HSP72 antigen in a human vaccine.
- S. pneumoniae strains were provided by the Laboratoire de la Sante Publique du Quebec, Sainte-Anne de Bellevue. S. pneumoniae strains included type 4 strain 53 and type 6 strain 64. If not specified, S. pneumoniae type 6 strain 64 was used. Bacterial strains were grown overnight at 37°C in 5% C0 2 on chocolate agar plates.
- S. pneumoniae antigens were prepared for immunization and immunoassays .
- Heat-killed whole cell antigens were obtained by incubating bacterial suspensions in a water bath prewarmed at 56 C for 20 minutes.
- Detergent-soluble proteins were extracted from S. pneumoniae as follows. Heat-killed bacteria were suspended in 10 mM Hepes buffer (4- (2-Hydroxyethyl) -1- piperazinethan-sulfonsaure) (Boehringer Mannheim GmbH,
- S. pneumoniae bacteria (type 4, strain 53 and type 6, strain 64) were resuspended in Eagle's Minimal
- BIO-X® Quelab Laboratories, Montreal, Canada
- the samples were incubated at either 37°C or 45°C for 5 minutes and then labeled with 100 ⁇ Ci/ml [ 35 S]methionine (ICN) for 10, 30, or 60 minutes at37°C.
- the bacteria were harvested and cell extracts were prepared using Tris-HCl lysis buffer as described above, or SDS-PAGE sample buffer.
- 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 heat- killed bacteria or 20 ⁇ g of detergent-soluble pneumococcal proteins absorbed to aluminum hydroxide adjuvant (Alhydrogel®; Cedarlane Laboratories Ltd., Horny, Ontario, Canada) . Blood samples were collected prior to immunization and at seven days following the first and second immunization.
- Cell extracts were prepared for SDS-PAGE, Western blot analysis and radioimmunoprecipitation assay by incubating bacterial suspensions in Tris-HCl lysis buffer (50mM Tris, 150 mM NaCl, 0.1% Na dodecyl sulfate, 0.5% Na deoxycholate, 2% Triton® X-100, 100 ⁇ g/ml phenylmethylsulfonylfluoride, and 2 ⁇ g/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.
- Tris-HCl lysis buffer 50mM Tris, 150 mM NaCl, 0.1% Na dodecyl sulfate, 0.5% Na deoxycholate, 2% Triton® X-100, 100 ⁇ g/ml phenylmethylsulfonylfluoride, and 2 ⁇ g
- SDS-PAGE were performed on a 10% polyacrylamide gel according to the method of Laemmli [Nature, 227, pp. 680-685 (1970)], using the Mini Protean® system (Bio- Rad Laboratories Ltd. , Mississauga, Canada) . Samples were denatured by boiling for 5 minutes in sample buffer containing 2% 2-mercaptoethanol. Proteins were resolved by staining the polyacrylamide gel with PhastGel Blue® (Pharmacia Biotech Inc., Baie d'Urfe, Canada) . The radiolabeled products were visualized by fluorography. Fluorograms were scanned using a laser densitometer.
- Immunoblot procedures were performed according to the method of Towbin et al . [Proc. Natl . Acad. Sci. USA, 76, pp. 4350-4354 (1979)] .
- the detection of antigens reactive with antibodies was performed by an indirect antibody immunoassay using peroxidase-labeled anti-mouse immunoglobulins and the o-dianisidine color substrate.
- Radioimmunoprecipitation assays were performed as described by J.A. Wiley et al. [J. Virol. , 66, pp. 5744-5751 (1992)] . Briefly, sera or hybridoma culture supernatants were added to radiolabeled samples containing equal amounts of [ 35 S]methionine. The mixtures were allowed to incubate for 90 minutes at 4 C with constant agitation. The immune complexes were then precipitated with bovine serum albumin-treated protein A Sepharose (Pharmacia) for 1 hour at 4 C. The beads were pelleted and washed three times in Tris buffered saline at pH 8.0, and the antigen complexes were then dissociated by boiling in sample buffer.
- the antigens were analyzed by electrophoresis on SDS-PAGE.
- the gels were fixed, enhanced for fluorography using Amplify® (Amersham '" Canada Limited, Oakville, Ontario, Canada) , dried, and then exposed to X-ray film.
- FIG. 1 shows the results when S. pneumoniae type 6 strain 64 (panel A) and type 4 strain 53 (panel B) were grown at 37°C, incubated at 37°C (lanes 1,3,5,7 and 9) or at 45°C (lanes 2, 4, 6, 8 and 10) for 5 minutes, and then labeled with [ 35 S]methionine for 10 minutes (lanes 1,2 and 7,8), 30 minutes (lanes 3,4 and 9,10), or 60 minutes (lanes 5,6) .
- the most prominent induced protein was about 72 kDa (HSP72), whereas the other two were approximately 80 kDa (HSP80) and 62 kDa (HSP62) .
- Increased protein synthesis was already apparent after 10 minutes of labeling (FIG. 1, lanes 1, 2 and 7, 8) and became more significant when the labeling period was prolonged to 30 minutes (FIG. 1, lanes 3, 4 and 9, 10) and 60 minutes (FIG. 1, lanes 5, 6) .
- the effect of elevated temperature on the protein synthesis profile of two different S. pneumoniae strains was similar, with HSPs of similar molecular mass being synthesized (compare Panel A (type 6 strain 64) to Panel B (type 4 strain 53) in FIG. 1) .
- FIGS. 3 and 4 relate to detergent soluble protein preparations.
- FIG. 4 relates to heat-killed bacterial preparation. Although many bands were detected by most antisera, HSP72 was a major precipitation product. The specificity of antibodies for HSP72 was demonstrated by the detection of proteins among heat-shocked products only (FIG. 3, lanes 4, 6, 8 and 10; FIG. 4, lanes 4, 6 and 8 ) .
- HSP72 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.
- This 72 kDa-product probably corresponds to component from peak 9 in FIG. 2 and was not detected in immunoblots .
- HSP62 is another immune target which was precipitated by some but not all immune sera (FIG. 3, lane 6 and, FIG. 4, lanes 4 and 6) . None of the sera tested reacted with HSP80. No proteins were precipitated when preimmune sera taken from the mice used in this study were tested for the presence of antibodies reactive with the labeled products.
- antibodies to HSP72 could be detected after one immunization with either detergent-soluble proteins or whole cells extracts of S. pneumoniae .
- a marked increase in the antibody response to HSP72 was observed after a second immunization (FIG. 3, compare 4 and 6, and lanes 8 and 10) .
- HSP72 was a major immunoreactive antigen with 8 (53%) positive sera after the first immunization (FIG. 5) .
- Antibodies to HSP72 were detected in 13 out of 15 (87%) immune sera tested after the second immunization.
- Two other prominent antigens having apparent molecular mass of 53.5 and 47 kDa were detected in 5 (33%) and 7 (47%) sera, respectively (FIG. 5) .
- the 72 kDa-reactive band was confirmed as the - pneumococcal HSP72 by using recombinant HSP72 antigens
- Example 3 infra
- Preimmune sera failed to detect any pneumococcal proteins.
- mice Female Balb/c mice (Charles River Laboratories) were immunized with S. pneumoniae antigens.
- One set of mice (fusion experiment 1) were immunized by peritoneal injection with 10 7 formalin-killed whole cell antigen from strain MTL suspended in Freund's complete adjuvant, and were boosted at two-week intervals with the same antigen and then with a sonicate from heat-killed bacteria in Freund's incomplete adjuvant.
- fusion experiment 1 mice were immunized by peritoneal injection with 10 7 formalin-killed whole cell antigen from strain MTL suspended in Freund's complete adjuvant, and were boosted at two-week intervals with the same antigen and then with a sonicate from heat-killed bacteria in Freund's incomplete adjuvant.
- fusion experiment 2 were immunized three times at three- week intervals with 75 ⁇ g of detergent-soluble pneumococcal antigens extracted from strain 64 (type 6) in 25 ⁇ g of Quil A adjuvant (Cedarlane Laboratories Ltd. , Hornby, Ontario, Canada) .
- Quil A adjuvant Cedarlane Laboratories Ltd. , Hornby, Ontario, Canada
- mice were injected intraperitoneally with the respective antigen suspended in PBS alone.
- Hybridomas were produced by fusion of spleen cells with nonsecreting SP2/0 myeloma cells as previously described by J. Hamel et al. [J. Med. Microbiol. , 23, pp. 163-170 (1987)] .
- Pneumococci were separated into subcellular fractions according to the technique described by Pearce et al. [Mol. Microbiol., 9, pp. 1037-1050 (1993)] . Briefly, S. pneumoniae strain 64 (type 6) was grown in Todd Hewitt broth supplemented with 0.5% (w/v) yeast extract for 6 hours at 37°C and isolated by centrifugation. Cell pellets were resuspended in 25 mM Tris-HCl pH 8.0, 1 mM EDTA, 1 mM phenylmethylsulphonylfluoride (PMSF) and sonicated for 4 minutes with 15 second bursts. Cellular debris were removed by centrifugation.
- S. pneumoniae strain 64 type 6
- PMSF phenylmethylsulphonylfluoride
- 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 .
- hybridomas 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 2) . Isotype analysis revealed that hybridoma Fl-Pn3.1
- HSP72 S. pneumoniae cell lysates were fractionated by differential centrifugation resulting in a soluble fraction and a particulate fraction, enriched in membrane proteins, supra.
- HSP72 was found in both fractions, with the majority of the protein associated with the cytoplasmic fraction (FIG. 8) .
- E. coli strains were grown in L broth or on L agar at 37°C. When necessary, ampicillin was added to the media at the concentration of 50 ⁇ g/ml. Plasmids were isolated by using the Magic/Wizard® Mini-Preps kit (Promega, Fisher Scientific, Ottawa, Canada) . 2. General Recombinant DNA Techniques
- a genomic S. pneuznoniae DNA library was generated in the bacteriophage expression vector ⁇ gtll ( ⁇ gtll cloning system, Amersham) according to the procedure provided by the manufacturer.
- Chromosomal DNA _ of S. pneumoniae type 6 strain 64 was prepared by following the procedure of J.C. Paton et al . [Infect . Immun. , 54, pp. 50-55 (1986)] .
- the S. pneumoniae chromosomal DNA was partially digested with EcoRI, and the 4- to 7-kb fragments were fractionated and purified from agarose gel. The fragments were ligated into ⁇ gtll arms, packaged, and the resulting phage mixtures used to infect E. coli Y1090.
- 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 .
- DIG digoxigenin
- Pneumococcal chromosomal DNA was digested with Hindlll and the digests were separated by electrophoresis on an 0.8% SDS-PAGE gel and transformed onto positive charged nylon membranes (Boehringer Mannheim) as described by J. Sambrook et al . (supra) .
- the membrane was then blotted with the DIG- labelled DNA probes according to the protocol of the manufacturer .
- the DNA fragments sequenced in this example were first cloned into plasmid pDELTA 1 (GIBCO BRL Life Technologies, Burlington, Ontario) .
- plasmid pDELTA 1 GIBCO BRL Life Technologies, Burlington, Ontario
- a series of nested deletions were generated from both strands by in vivo deletion mediated by Tn 1000 transposon transposition (Deletion Factory System, GIBCO BRL) following the procedures provided by the supplier.
- These deletions were sized by agarose gel electrophoresis and appropriate deletion derivatives were selected for sequencing by the dideoxynucleotide chain terminating method of F. Sanger et al. [Proc. Natl . Acad. Sci. USA, 74, pp. 5463-5467 (1977)] .
- 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) .
- High level expression of the cloned gene in this example was achieved by employing the bacteriophage T7 RNA polymerase/promoter system in E. coli .
- the DNA fragment specifying the recombinant protein was ligated into plasmids pT7-5 or pT7-6 [S. Tabor and C.C. Richardson, Proc. Natl. Acad. Sci. USA, 82, PP. 1074-1078 (1985)], in a proper orientation in which the gene to be expressed was placed under the control of phage T7 RNA polymerase specific promoter ⁇ 10.
- the resulting plasmid was transformed into E. coli strain BL21(DE3) [F.W. Studier, and B.A.
- 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 [J. Biol. Chem., 262, pp. 10035-10038
- PVDF membrane was stained with Coomassie Blue, the HSP72 band excised and then analyzed in an automated protein sequencer (ABI), according to standard procedures.
- the ⁇ gtll 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 ⁇ JBD7 and ⁇ JBD17 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 ⁇ JBD17 (lanes 2 and 3) or ⁇ JBD7 (lanes 4 and 5) cultured in the presence (+) or absence (-) of IPTG were subjected to 10% polyacrylamide gel electrophoresis and were electrotransferred to nitrocellulose.
- the immunoblot was probed with HSP72- specific MAb Fl-Pn3.1.
- Clone ⁇ JBDl7 had two EcoRI-EcoRI insert fragments of 2.4 kb and 2.3 kb (FIG. 10), and expressed a chimeric recombinant protein having an apparent molecular mass of 74 kDa on SDS-PAGE gel (FIG. 9, lanes 2 and 3) .
- Clone ⁇ JBD7 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 ⁇ JBD17.
- the fusion protein had an apparent molecular mass of 160 kDa as estimated by SDS- PAGE (FIG. 9, lane 5) .
- the expression of the chimeric recombinant protein encoded by phage ⁇ JBDl7 was independent of IPTG induction (FIG. 9, lanes 2 and 3) while the expression of the recombinant fusion protein encoded by phage ⁇ JBD7 was dependent on induction of the lac promoter (FIG. 9, lanes 4 and 5) .
- the pneumococcal DNA insert from clone ⁇ JBD17 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.
- 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 intact 3.2 kb EcoRI-EcoRV insert in these recombinant plasmids and their orientation was determined by restriction mapping.
- pJBDf51 and pJBDf62 were transformed, separately, into E. coli BL21(DE3) .
- the 3.2 kb EcoRI-EcoRV fragment was cloned into plasmid pDELTA 1 to yield plasmid pJBD ⁇ l. 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 N0:1.
- Two open reading frames (“ORFs") were found and their orientation is indicated in FIG. 10B ("ORF27" and "FucI-HSP72 (C-169)" .
- ORFs open reading frames
- putative ribosome-binding sites were identified (SEQ ID N0:1, nucleotides 18-21 and 760-763) . No obvious -10 and -35 promoter sequences were detected.
- ORF27 spans nucleotides 30-755 (SEQ ID NO:l) 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 or£21 and compared it to other known sequences. No homologous gene or protein was found.
- the large ORF (nucleotides 771-2912, SEQ ID NO:l) 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 N0:3. This ORF was compared with other known sequences to determine its relationship to other amino acid sequences.
- the 74 kDa protein was a chimeric protein encoded by two pieces of S. pneumoniae chromosomal DNA, a 2.4 kb EcoRI- EcoRI fragment derived from the Fuel homologous gene and a 2.3 kb EcoRI-EcoRI fragment derived from the HSP72 gene. D. Southern Blot Analysis
- Southern blotting was performed in order to confirm that the 74 kDa protein is a chimeric protein and to attempt to clone the entire pneumococcal HSP72 gene.
- Chromosomal S. pneumoniae DNA was digested with Hindlll to completion, separated on a 0.8% agarose gel, and transferred onto two positively charged nylon membranes (Boehringer Mannheim) . The membranes were then blotted with either the 0.8 kb EcoRI-EcoRV probe, derived from the 2.3 kb EcoRI-EcoRI fragment, or the 1 kb Pstl-PstI probe, obtained from the 2.4 kb EcoRI-EcoRI fragment.
- Both probes had been previously labelled with digoxigenin-dUTP. These two probes hybridized two individual Hindlll fragments of different sizes (FIGS. 10B and IOC) .
- the 0.8 kb EcoRI-EcoRV probe recognized the 3.2 kb Hindlll fragment and the 1 kb Pstl-PstI probe reacted with the 4 kb Hindlll fragment. This result further indicated that the gene responsible for the expression of the 74 kDa chimeric protein was generated by fusion, in frame, of two pieces of EcoRI fragments, one originated from the fragment containing the 5 ' portion of the S.
- a partial pneumococcal genomic library was generated by ligation of the pool of Hindlll digests of chromosomal DNA, with sizes ranging from 2.8 to 3.7 kb, into plasmid pWSK29/Hindlll.
- the ligation mixture was used to transform E. coli strain JM 109 and the transformants were screened by hybridization with the 0.8 kb EcoRI-EcoRV probe.
- One representative plasmid from four positive hybridizing clones was named pJBD291.
- the 3.2 kb Hindlll fragment was isolated from plasmid pJBD291, and subcloned into plasmids pDELTA 1 and pT7-5 to generate pJBD ⁇ 4 and pJBDk51, respectively.
- 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 H ⁇ P72 structural gene and was identified as the 5 ' portion of the pneumococcal DnaJ gene.
- the putative ribosome binding site was located 9 base pairs upstream from the start codon of the HSP72 structural gene, while the typical ribosome binding site (“AGGA”) was found 66 base pairs upstream from the - start codon of the DnaJ structural gene.
- the predicted HSP72 protein has 607 amino acids and a calculated molecular mass of 64,755 daltons, as compared to the 72 kDa molecular mass estimated by SDS- PAGE.
- the predicted HSP72 protein is acidic with an isoelectric point (pi) of 4.35.
- Automated Edman degradation of the purified native HSP72 protein extracted from S. pneumoniae strain 64 revealed SKIIGIDLGTTN-AVAVLE as the 19 amino acid N-terminal sequence of the protein.
- the amino-terminal methionine was not detected, presumably due to in si tu processing which is known to occur in many proteins. No amino acid residue was identified on position 13.
- the 19 amino acid N-terminal sequence obtained from the native HSP72 protein is in full agreement with the 19 amino acid N-terminal sequence deduced from the nucleotide sequence of the recombinant
- HSP72 S. pneumoniae H ⁇ P72 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 .
- the alignment of the predicted amino acid sequence of HSP72 (SEQ ID NO:5) with those from other bacterial HSP70 (DnaK) proteins also revealed high homology (FIGS. 13A-13D) .
- HSP72 showed 54% - identity with the E. coli DnaK protein.
- the highest identity value was obtained from comparison with the Gram positive bacterium Lactococcus lactis , showing 85% identity with HSP72.
- HSP72 misses a stretch of 24 amino acids near the amino terminus when compared with DnaK proteins from Gram negative bacteria (FIGS. 13A-13D) .
- HSP72 shares homology with HSP70 (DnaK) proteins from other organisms, it does possess some unique features. Sequence divergence of the HSP70 (DnaK) proteins is largely localized to two regions (residues 244 to 330 and 510 to 607, SEQ ID N0:5) . 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 determinants specific to S. pneumoniae .
- the truncated DnaJ protein of S. pneumoniae (SEQ ID NO: 6) has 352 amino acids, which show a high degree of similarity with the corresponding portions of the L . lactis DnaJ protein (72% identity) and the E. coli DnaJ protein (51% identity) .
- the predicted truncated DnaJ protein contains high glycine content (15%) .
- coli infected with ⁇ JBD7 and ⁇ JBD17 have the same primary structure, they have distinct conformation.
- the lack of reactivity of MAb F2-Pn3.2 with some recombinant proteins raised the possibility that this particular MAb recognizes a more complex epitope.
- 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.
- dot enzyme immunoassay the bacteria were grown overnight on chocolate agar plates and then suspended in PBS, pH 7.4.
- a volume of 5 ⁇ l of a suspension containing approximately 10 9 CFU/ml was applied to a nitrocellulose paper, blocked with PBS containing 3% bovine serum albumin, and then incubated sequentially with MAbs and peroxydase-labeled secondary antibody.
- Whole cell extracts were prepared for Western blot analysis by boiling bacterial suspensions in sample buffer for 5 minutes.
- HSP70 (DnaK) proteins may be structurally related to HSP72, they are immunologically distinct.
- S. pyogenes Enterococcus faecalis
- S. mutans S. sanguis
- ⁇ indicates a weak signal compared to the reactivity observed with S. pneumoniae antigens b C. trachomatis purified elementary bodies were tested.
- High level exclusive expression of the HSP72 gene was achieved by employing the bacteriophage T7 RNA polymerase/T7 promoter system in E. coli .
- the 3.2 kb Hindlll fragment was cloned in both orientations in front of the T7 promoter ⁇ 10 in the plasmid pT7-5.
- the resulting plasmid pJBDk51 was then transformed into E. coli strain BL21 (DE3) .
- Overexpression of the recombinant HSP72 protein (HSP72 r ⁇ c ) was induced by culturing in broth supplemented with antibiotics for a 3- hour period after the addition of IPTG to a final concentration of 1 mM.
- HSP72 r ⁇ c expressing high levels of HSP72 r ⁇ c were concentrated by centrifugation and lysed by mild sonication in 50 mM Tris-Cl (pH 8.0) , 1 mM EDTA and 100 mM NaCl lysis buffer containing 0.2 mg/ml lysozyme. The cell lysates were centrifuged at 12,000 g for 15 minutes and the supernatants were collected. HSP72 r ⁇ c 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-169 r ⁇ o) was expressed in the form of insoluble inclusion bodies in E. coli strain JM109 transformed with the plasmid pJBD ⁇ l.
- Protein inclusion bodies were recovered from pelleted bacterial cells disrupted by sonication as described before. The pellets were washed in lysis buffer containing 1 mg/ml of deoxycholate to remove contaminating materials, and the protein inclusion bodies were then solubilized in urea 6 M. The protein solution was centrifuged at 100,000 g and the cleared supernatant collected and dialysed against phosphate-buffered saline. After purification, the protein content was determined by the Bio-Rad protein assay (Bio-Rad Laboratories, Mississauga, Ontario, Canada) .
- mice Two groups of 10 female Balb/c mice (Charles River Laboratories) were immunized subcutaneously three times at two-week intervals with 0.1 ml of purified HSP72 r ⁇ c or C-169 r ⁇ c antigens absorbed to Alhydrogel adjuvant. Two antigen doses, approximately 1 and 5 ⁇ g, 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 10 6 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.
- NZW rabbit (Charles River Laboratories) was immunized subcutaneously at multiple sites with approximately 50 ⁇ g of the purified C-169 r ⁇ c protein adsorbed to Alhydrogel adjuvant. The rabbit was boosted three times at two-week intervals with the same antigen and blood samples collected 7 and 14 days following the last immunization. The serum samples were pooled and antibodies were purified by precipitation using 40% saturated ammonium sulfate.
- Severe-combined immunodeficient SCID mice were injected intraperitoneally with 0.25 ml of the purified rabbit antibodies 1 hour before intravenous challenge with 5000 or 880 CFU of the type 3 S. pneumoniae strain WU2.
- Control SCID mice received sterile buffer or antibodies purified from nonimmune rabbit sera.
- Samples of the S. pneumoniae challenge inoculum were plated on chocolate agar plates to determine the CFU and to verify the challenge dose. The SCID mice were chosen because of their high susceptibility to S. pneumoniae infection.
- Blood samples (20 ⁇ l each) obtained 24 hours post- challenge were plated on chocolate agar and tested for the presence of S. pneumoniae organisms. The level of detection was 50 CFU/ml. Deaths were recorded at 24-hour intervals for a period of 5 days.
- HSP72 r ⁇ c and C-169 r ⁇ c proteins were obtained in a relatively pure state with no contaminants detected on Coomassie Blue-stained SDS polyacrylamide gels (FIGS. 14 and 15, respectively) .
- HSP72 r ⁇ c vaccinogenic potential of HSP72.
- Groups of 10 mice were immunized with full-length HSP72 r ⁇ o (1 ⁇ g or 5 ⁇ g dose) and challenged with 4.2 million CFU of S. pneumoniae type 3 strain WU2. Eighty percent (80%) of the mice dosed with 1 ⁇ g HSP72 r ⁇ c survived the challenge, as did 50% of the mice dosed with 5 ⁇ g HSP72. None of the naive mice immunized with Alhydrogel adjuvant alone without antigen survived the challenge (FIG. 16) . No S.
- HSP72 r ⁇ 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.
- mice immunized with purified C-169 r ⁇ c were protected from fatal pneumococcal challenge, thus demonstrating that some, if not all, epitopes eliciting protection are present in the C-terminal region of the HSP72 molecule comprising the last 169 residues.
- Groups of 10 mice were immunized with C-169 r ⁇ c (1 ⁇ g or 5 ⁇ g doses) and challenged with 6 million CFU of S. pneumoniae type 3 strain WU2.
- mice dosed with 1 ⁇ g C-169 r ⁇ c survived the challenge, as did 70% of the mice dosed with 5 ⁇ g C-169 r ⁇ c (FIG. 17) .
- 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.
- mice passively transferred with rabbit anti-C-169 r ⁇ c antibodies were protected from fatal infection with S. pneumoniae WU2.
- the control mice received antibodies from nonimmune rabbit sera or received sterile buffer alone.
- all mice from the control groups had positive S. pneumoniae hemoculture 24 hours post-challenge, while S. pneumoniae organisms were detected in only 2 out of a total of 10 immunized SCID mice.
- 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.
- EXAMPLE 6 Heat-Indueible Expression System for High Level Production of the C-151 Terminal Portion of the HSP72 Protein A. Construction of Plasmid pURV3 Containing the C- 151 terminal coding region of the HSP72 of S. pneumoniae The DNA region coding for 151 amino acids at the carboxyl end of the HSP72 of S. pneumoniae was inserted downstream of the promoter ⁇ PL into the translation vector p629 [H. J. George et al., Bio/Technology 5, pp. 600-603 (1987)] . This vector contains a cassette of the bacteriophage ⁇ CI857 temperature sensitive repressor gene from which the functional PR promoter has been deleted.
- 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.
- PCR product was purified from agarose gels by the method of phenol freeze [S. A. Benson, Biotechniques 2, pp.
- FIG. 18 A partial map of the resulting plasmid pURV3 is shown in FIG. 18. This plasmid was transformed by the method of Simanis [Hanahan, D. In D. M. Glover (ed.), DNA Cloning, pp. 109-135, (1985)] into the E.
- Plasmid DNA was purified from a selected transformant and the DNA insert was seguenced by PCR using the Taq Dye Deoxy Terminator Cycle Sequencing kit of Applied Biosystems Inc. (ABI) and DNA electrophoresis was performed on automated DNA sequencer 373A (ABI) .
- the nucleotide sequence of the insert perfectly matched the nucleotide sequence of the C-151 coding region of the HSP72 gene. (See SEQ ID No: 25 and corresponding amino acid sequence at SEQ ID No : 26. )
- the plasmid was transformed into the prototrophic E . coli strain W3110 (ATCC 27325) for the production of C-151 rec .
- C-151 rec was synthesized with a methionine residue at its amino end in E. coli strain W3110 harboring the plasmid pURV3.
- E . coli cells were grown at 30°C in LB broth containing 100 ⁇ g of ampicillin- per ml until the AgQO reached a value of 0.6. The cells were then cultivated at 40°C for 18 hours to induce the production of C-151 rec protein.
- a semi-purified C-151 rec protein was prepared using the following procedures. The bacterial cells were harvested by centrifugation and the resulting pellet was washed and resuspended in phosphate- buffered saline.
- Lysozyme was added and the cells were incubated for 15 min on ice before disruption by pulse sonication.
- the cell lysates were cleared by centrifugation and the supernatants were collected and subjected to separation using an Amicon's ultrafiltration equipment (stirred cells series 8000, Amicon Canada Ltd. Oakville, Ontario) .
- the ultrafiltrate not retained by a YM30 membrane was recovered, analysed by SDS-PAGE and stained with Coomassie blue R-250. Protein concentrations were estimated by comparing the staining intensity of the C-151 rec protein with those obtained with defined concentrations of soybean trypsin inhibitor.
- mice Groups of 10 female Balb/c mice were immunized subcutaneously with either HSP72 rec or C-169 rec as described in Example 5.
- C-151 rec a group of 6 mice were immunized three times at two-week intervals with 0.5 ⁇ g of C-151 rec absorbed to Alhydrogel adjuvant by intraperitoneal injection.
- Sera from blood samples collected prior each immunization and four to seven days after the third immunization were tested for antibody reactive with S. pneumoniae by ELISA using plates coated with S. pneumoniae cell wall extracts.
- 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) .
- specific serum antibodies were detectable in the sera of each monkey after a single injection of recombinant antigens.
- Example 3 it was shown that significant variability in the primary sequence of the HSP70 proteins was mainly localized to two regions corresponding to amino acid residues 244 to 330 and 510 to 607 of the S. pneumoniae HSP72 protein. These variable regions may contain B-cell epitopes that are responsible for the antigenic heterogeneity reported in Example 4. To investigate this possibility, the reactivity of polyclonal and monoclonal antibodies to S. pneumoniae HSP72 were tested against fourteen peptides selected to cover most of these regions.
- Peptides were purified by reverse- phase high-pressure liquid chromatography. Peptides were solubilized in distilled water except for peptides CS874 and CS876 which were solubilized in a small volume of either 6M guanidine-HCl or dimethyl sulfoxide and then adjusted to 1 mg/ml with distilled water. Peptide ELISA were performed by coating synthetic peptides onto Immunolon 4 microtitration plates (Dynatech Laboratories, Inc., Chantilly, VA) at a concentration of 50 ⁇ g/ml according to the prodedures described in J. Hamel et al . [supra] .
- Immune sera were from animals immunized three times with recombinant HSP72 antigens.
- One rabbit was immunized with 37.5 ⁇ g of purified HSP72 rec according to the immunization protocol described in Example 5.
- Pool murine sera were from three Balb/c mice immunized with HSP72 rec from Example 5 and monkey pool sera were from groups of two animals immunized with either HSP72 rec or C- 169 ',rec •
- variable region comprised within the amino acid residues 244 to 330 also constitutes an antigenic domain.
- Linear epitopes located on overlapping peptides CS877 (amino acids 257 to 271) and CS878 (amino acids 268- to 281), peptides CS880 (amino acis 286-299) and peptides CS882 (amino acids 315-333) were identified by hyperimmune sera.
- EXAMPLE 9 - HSP70 (DnaK) from Streptococcus pyogenes and Streptococcus agalactiae Molecular Cloning and DNA Sequencing of the hsp70 Genes; Nucleotide and Protein Sequence Analyses; Antigenic Relatedness to S. pneumoniae; Increased Streptococcus agalactiae HSP70 synthesis"in response to heat.
- S. agalactiae type II strain V8 corresponds to the ATCC strain 12973.
- S. pyogenes strain Bruno corresponds to the ATCC strain 19615.
- the E. coli strain XLI Blue MRF' was obtained from Stratagene.
- Streptococcal strains were grown at 37°C in a 5 % CO2 incubator. The streptococci were streaked on tryptic soy agar plates containing 5 % sheep blood (Les Laboratoires Quelab, Montreal, Canada), liquid cultures were made in heart infusion broth (Difco Laboratories, Detroit, MI) without agitation. The E. coli strain was grown at 37°C in L-broth with agitation at 250 rpm or on L- agar.
- the general cloning phagemid pBluescript KS(-) was purchased from Stratagene.
- 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. Jn D. M. Glover (ed. ) , DNA Cloning, pp. 109- 135, (1985)] . The sequencing of genomic DNA inserts in plasmids was done using synthetic oligonucleotides .
- the sequencing reactions were carried out by the polymerase chain reaction (PCR) using the Taq Dye Deoxy Terminator Cycle Sequencing kit (ABI) and DNA electrophoresis was performed on automated DNA sequencer 373A (ABI) .
- the assembly of the DNA sequence was performed using the program Sequencher 3.0 from the Gene Codes Corporation (Ann Arbor, MI) . Analysis of the DNA sequences and their predicted polypeptides were performed with the program Gene Works version 2.45 from Intelligenetics, Inc.
- Chromosomal DNA from S. agalactiae and S. pyogenes was digested to completion with various restriction enzymes with palindromic hexanucleotide recognition sequences.
- the digests were analysed by Southern hybridization using a labeled PCR-amplified DNA probe corresponding to a 782 base-pairs region starting at base 332 downstream from the ATG initiation codon of the HSP72 gene of S. pneumoniae (see SEQ ID NO 4) .
- This DNA region was selected because it is relatively well conserved among the hsp70 genes of Gram-positive bacteria that have been characterized.
- the PCR amplification was done on the genomic DNA of S.
- oligonucleotides OCRR2 (5 ' -AAGCTGTTATCACAGTTCCGG) and OCRR3 (5 '-GATACCAAGTGACAATGGCG) .
- Hybridizing genomic restriction fragments of sufficient size to code for a 70- kDa polypeptide (>1.8 kb) were partially purified by extraction of genomic fragments of corresponding size from agarose gel. Verification of the presence of the hsp70 gene among the purified genomic restriction fragments was done by Southern hybridization using the labeled 782-bp S. pneumoniae DNA probe.
- the purified genomic DNA restriction fragments were cloned into dephosphorylated compatible restriction sites of pBluescript KS(-) and transformed into the E. coli strain XLI Blue MRF' .
- the colonies were screened by DNA hybridization using the labeled 782-bp S. pneumoniae DNA probe.
- Extracted plasmids were digested with various restriction enzymes to evaluate the size of the inserts and to verify the presence of the hsp70 gene by Southern hybridization using the labeled 782-bp S. pneumoniae DNA probe.
- Plasmid pURV5 contains a 4.2-kb Hindlll insert of the genomic DNA of S. agalactiae .
- Plasmid pURV4 contains a 3.5-kb Hindlll fragment of the genomic DNA of S. pyogenes . 4. Heat Shock and Protein Labeling The stress response of S. agalactiae to an heat shock was assayed by pulse-labeling with [35s] me t ion e as described before in Example 1. S. agalactiae bacteria grown overnight in SMAM (Methionine assay Medium supplemented with 1 mg/1 methionine, 1% (v/v) Isovitalex and 1 mg/1 choline chloride) were pelleted by centrifugation and then resuspended in the methionine-free SMAM medium.
- SMAM Methionine assay Medium supplemented with 1 mg/1 methionine, 1% (v/v) Isovitalex and 1 mg/1 choline chloride
- the bacteria were incubated at 37°C for 1 h and then divided into two fractions of equal volume. The samples were either incubated at 37 or 43°C for 10 minutes and then labeled with 100 ⁇ Ci/ml [ ⁇ 5 S] ethionine for 30 minutes at 37°C. The bacteria were extensively washed with PBS and cell extracts were prepared by treatment with mutanolysine and lysozyme as described for the DNA isolation (M.Jayarao et al. , supra) followed by sonication.
- a region of 2438 bases in the 4.2-kb Hindlll insert of plasmid pURV5 was sequenced.
- This sequence contains an open reading frame (ORF) of 1830 nucleotides- coding for a polypeptide of 609 amino acids with a molecular weight of 64907 (see SEQ ID NO: 7) .
- the ORF has an ATG start codon beginning at position 248 and TAA stop codon ending at position 2077.
- the ATG start codon is preceeded by the sequence GAGG, starting at position 237, which is complementary to 16S rRNA and serves as a ribosome binding site in E. coli [G. D. Stormo et al. , Nucleic Acids Res. 10, pp.
- 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 .
- the assembly of the hsp70 gene regions present in plasmids pURV4 and pURV6 gave a 2183 nucleotide region containing an ORF of 1824 bases coding for a polypeptide of 608 amino acids with a molecular weight of 64847 (see SEQ ID NO: 20) .
- the ATG start codon begins at position 204 and the TAA stop codon extends to position 2030.
- the ATG start codon is preceeded by a putative ribosome binding site sequence GAGG starting at position 193 [G. D. Stormo, supra] .
- pyogenes are, respectively, identical at 85 and 94 % to the ORF and polypeptide of the HSP72 of S. pneumoniae .
- the ORF of plasmid pURV4 lacks 125 base pairs coding for 41 amino acids at the carboxyl end of the HSP70 of S. pyogenes ; the ORF thus codes for the 567 amino acids of the amino end of that HSP70 (N-567 rec ) .
- the ORF of plasmid pURV ⁇ lacks 114 base pairs coding for 38 amino acids at the amino end of the HSP70 of S. pyogenes ; the ORF thus codes for the 570 amino acids of the carboxyl end of that HSP7 ⁇
- HSP72 antigens may be selected from the polypeptides described herein.
- 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.
- 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.
- 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.
- the compositions will include a water-in-oil emulsion or aluminum hydroxide as adjuvant.
- composition would be administered to the patient in any of a number of pharmaceutically acceptable forms including intramuscular, intradermal, subcutaneous or topic.
- the vaccine will be administered intramuscularly.
- 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
- MOLECULE TYPE DNA (genomic)
- ORGANISM Streptococcus pneumoniae
- FEATURE FEATURE
- MOLECULE TYPE peptide ( i ) SEQUENCE DESCRIPTION : SEQ ID NO : 13 :
- ORGANISM Streptococcus pyogenes
- GGT ATG GAC AAG ACT GAC AAG GAT GAA AAA ATC TTA GTT TTT GAC CTT 710
- GCT AAA GAC CTT GGT ACG CAA AAG GAA CAA CAC ATC GTT ATC AAA TCA 1622 Ala Lys Asp Leu Gly Thr Gin Lys Glu Gin His He Val He Lys Ser 460 465 470
- 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
- ORGANISM Streptococcus agalactiae
- GAA GGC AAT CGT ACA ACT CCT TCA GTA GTA TCA TTC AAA AAT GGT GAA 385
- GCA ACT AAA GAC GCT GGT AAA ATT GCA GGT CTT GAA GTA GAA CGT ATC 673 Ala Thr Lys Asp Ala Gly Lys He Ala Gly Leu Glu Val Glu Arg He 130 135 140 GTT AAC GAA CCA ACA GCA GCC GCA CTT GCT TAT GGT ATG GAC AAG ACT 721
- CAA GCG GCT GCA GCA CAA CAA GCA GCT CAA GGG GCT GAA GGT GCA CAA 2017 Gin Ala Ala Ala Ala Gin Gin Ala Ala Gin Gly Ala Glu Gly Ala Gin 575 580 585 590
- MOLECULE TYPE protein
- MOLECULE TYPE DNA (genomic)
- ORGANISM Streptococcus pneumoniae
- FEATURE FEATURE
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Abstract
Novel heat shock proteins (HSPs) of Streptococcus pneumoniae, Streptococcus pyogenes and Streptococcus agalactiae having apparent molecular masses of 70-72 kDa, immunologically related polypeptides, the nucleotide and derived amino acid sequences of HSP72 of S. pneumoniae (SEQ ID NO:4; SEQ ID NO:5), the nucleotide and derived amino acid sequences of HSP70 of S. pyogenes (SEQ ID NO:19; SEQ ID NO:20), the nucleotide and derived amino acid sequences of HSP 70 of S. agalactiae (SEQ ID NO:21; SEQ ID NO:22), antibodies that bind to the HSPs, and recombinant DNA methods for the production of the HSPs and immunologically related polypeptides are described. The polypeptides, DNA sequences and antibodies of this invention provide new means for the diagnosis, prevention and/or treatment of Streptococcal disease.
Description
STREPTOCOCCAL HEAT SHOCK PROTEINS MEMBERS OF THE HSP70 FAMILY TECHNICAL FIELD OF THE INVENTION
This invention relates to novel heat shock proteins of Streptococcus pneumoniae, Streptococcus pyogenes and Streptococcus agalactiae and immunologically related polypeptides, which provide the basis for new immunotherapeutic, prophylactic and diagnostic agents useful in the treatment, prevention and diagnosis of disease. More particularly, this invention relates to heat shock proteins of S. pneumoniae, S. pyogenes and S. agalactiae, members of the HSP70 family which have an apparent molecular mass of 70-72 kilodaltons, to the corresponding nucleotide and derived amino acid sequences, to recombinant DNA methods for the production of HSP70/HSP72 and immunologically related polypeptides, to antibodies that bind to these HSP's, and to methods and compositions for the diagnosis, prevention and treatment of diseases caused by S. pneumoniae and related bacteria, such as Streptococcus pyogenes and Streptococcus agalactiae
BACKGROUND OF THE INVENTION
S. pneumoniae is an important agent of disease in humans, especially among infants, the elderly and immunocompromised persons . It is a bacterium frequently isolated from patients with invasive diseases such as bacteraemia/septicaemia, pneumonia, and meningitis with high morbidity and mortality throughout the world. Although the advent of antimicrobial drugs has reduced the overall mortality from pneumococcal diseases, the presence of resistant pneumococcal organisms has become a major problem in the world today. Effective pneumococcal vaccines could have a major impact on the morbidity and mortality associated with S. pneumoniae disease. Such
vaccines would also potentially be useful to prevent otitiε media in infants and young children.
It is clear that a number of pneumococcal factors are potentially important in the pathogenesis of disease [G.J. Boulnois, J. Gen. Microbiol. , 138, pp. 249- 259 (1992); CJ. Lee et al. , Crit. Rev. Microbiol., 18, pp. 89-114 (1991)] . The capsule of the pneumococcus, despite its lack of toxicity, is considered to be the sine qua non of pneumococcal virulence. More than 80 pneumococcal capsular serotypes are identified on the basis of antigenic differences. Antibodies are the mechanism of protection and the importance of anticapsular antibodies in host defenses against S. pneumoniae is well established [R. Austrian, Am. J. Med. , 67, pp. 547-549 (1979)] . Nevertheless, the currently available pneumococcal vaccine, comprising 23 capsular polysaccharides that most frequently caused disease, has significant shortcomings such as the poor immunogenicity of capsular polysaccharides, the diversity of the serotypes and the differences in the distribution of serotypes over time, geographic areas and age groups. In particular, the failure of existing vaccines to protect young children against most serotypes has spurred evaluation of other S. pneumoniae components. Increasing evidence indicates that certain pneumococcal proteins may play an active role both in terms of protection and pathogenicity [J.C. Paton, Ann. Rev. Microbiol., 47, pp. 89-115 (1993)] . So far, however, only a few S. pneumoniae proteins have been studied. This might result from the lack of protein-specific antibodies which renders difficult the study of the role of protein antigens in protection and pathogenicity. It is believed that the pneumococcal protein antigens are not very immunogenic and that most antibody responses are to the phόsphocholine and the capsular polysaccharides [L.S. McDaniel et al . , J. Exp. Med. , 160, pp. 386-397 (1984); R.M. Krause, Adv. Immunol . , 12, pp. 1-56 (1970); D.G. Braun et al. , J. Exp.
Med. , 129, pp. 809-830 (1969)] . In a study using X-linked immunodeficient mice, which respond poorly to carbohydrate antigens and to phosphocholine, but make relatively normal responses to protein antigens, the frequency for obtaining monoclonal antibodies reactive with pneumococcal protein antigens was less than 10%, thus suggesting that S. pneumoniae proteins are poor immunogens [McDaniel et al . , supra] .
Streptococcus agalactiae, also called Group B Streptococcus (GBS), is the most common cause of sepsis
(blood infection) and meningitis in newborns. GBS is also a frequent cause of newborn pneumonia. Approximately 8,000 babies in the United States get GBS disease each year; 5%-15% of these babies die. Babies that survive, particularly those who have meningitis, may have long-term problems, such as hearing or vision loss or learning disabilities. In pregnant women, GBS can cause urinary tract infections, womb infections (amnionitis, endometritis) , and stillbirth. Among women who are not pregnant and men, the most common diseases caused by GBS are blood infections, skin or soft tissue infections, and pneumonia. Approximately 20% of men and nonpregnant women with GBS disease die of the disease. GBS infections in both newborns and adults are usually treated with antibiotics (e.g., penicillin or ampicillin) given intravenously. Most GBS disease in newborns can be prevented by giving certain pregnant women antibiotics intravenously during labor. Vaccines to prevent GBS disease are being developed. In the future, it is expected that women who will be vaccinated will make antibodies that cross the placenta and protect the baby during birth and early infancy.
Since the 1980s, Streptococcus pyogenes, also called Group A Streptococcus (GAS) is reemerging as a cause of severe diseases which would be due to an increase
in virulence of the organism. GAS causes pharyngitis, commonly called "strep throat", and skin infections (impetigo, erysipelas/cellulitis) . "Strep throat" and impetigo can lead to glomerulonephritis (kidney damage) . Approximately 3% of "strep throat" infections result into rheumatic fever (migrating arthritis) whose complications include chorea (neurological symptoms) and, in 50% of the cases, rheumatic heart disease (heart valve damage) with endocarditis as a possible long term consequence. It is important to treat impetigo and "strep throat" with antibiotics to prevent the development of complications . Infection with toxin-producing strains can result in scarlet fever (diffuse rash and fever) or in the extremely severe streptococcal toxic shock syndromes (TSS; GAS have been termed 'flesh eating bacteria') which are characterized by the rapid development of shock and multiple organ system failure. TSS have a 30 to 70% fatality rate in spite of aggressive treatment involving the removing of the focus of bacterial infection and antibiotic therapy. The incidence of TSS is 10 to 20 cases per 100,000. No vaccine against GAS is presently available.
Heat shock or stress proteins ("HSPs") are among the most highly conserved and abundant proteins found in nature [F.C. Neidhardt et al . , Ann. Rev. Genet., 18, pp. 295-329 (1984) ; S. Lindquist, Ann. Rev. Biochem. , 55, pp. 1151-1191 (1986)] . They are produced by all cells in response to various physiological and nonphysiological stimuli. The heat shock response, in which a sudden increase in temperature induces the synthesis of HSPs, is the best studied of the stress responses. Other environmental conditions such as low pH, iron deficiency and hydrogen peroxyde can also induce HSPs. The HSPs have been defined by their size, and members of hsp90, hsp70, and hsp60 families are among the major HSPs found in all prokaryotes and eukaryotes . These proteins fulfill a
variety of chaperon functions by aiding protein folding and assembly and assisting translocation across membranes [C. Georgopoulos and .J. Welch, Ann. Rev. Cell. Biol. , 9, pp. 601-634 (1993); D. Ang et al. , J. Biol. Chem., 266, pp. 24233-24236 (1991)]. As molecular chaperons and possibly via other mechanisms, HSPs are likely involved in protecting cells from the deleterious effects of stress. The fact that several virulence factors are regulated by environmental conditions suggests a role for HSPs in microbial pathogenicity [J.J. Mekalanos, J. Bacteriol. , 174, pp. 1-7 (1992); P.J. Murray and R.A. Young, J. Bacteriol. , 174, pp. 4193-4196 (1992)] . In that respect, recent studies on Salmonella species suggest that the stress response might be critically linked to the ability of intracellular pathogens to initiate and sustain an infection [N.A. Buchmeir and F. Heffron, Science, 248, pp. 730-732 (1990); K.Z. Abshire and F.C. Neidhardt, J. Bacteriol., 175, pp. 3734-3743 (1993); B.B. Finlay et al., Science, 243, pp. 940-943 (1989)]. Others have demonstrated that lysteriolysin, an essential virulence factor in L . monocytogenes , is induced under heat shock conditions [Z. Sokolovic and W. Goebel, Infect. Immun. , 57, pp. 295-298 (1989) ] .
Evidence is now accumulating that HSPs are major antigens of many pathogens. Members of the hsp60 family, also called GroEL-related proteins for their similarity to the E. coli GroEL protein, are major antigens of a variety of bacterial pathogens including Mycobacterium leprae and Nycojbacterium tuberculosis [D. Young et al. , Proc . Natl. Acad. Sci. USA, 85, pp. 4267-4270 (1988)], Legionella pneumophila [B.B. Plikaytis et al. , J. Clin. Microbiol., 25, pp. 2080-2084 (1987)], Borrelia burgdorferi [B.J. Luft et al., J. Immunol. , 146, pp. 2776-2782 (1991)], and Chlamydia trachomatis [E.A. Wagar et al. , J. Infect. Pis. , 162, pp. 922-927 (1990)] . This antigen is a homologue of the ubiquitous "common antigen", and is believed to be present in every bacterium [J.E. Thole et al. , Microb.
Pathogen. , 4, pp. 71-83 (1988) . Antibodies to the members of the hsp70 family, or DnaK-related proteins, have also been described for several bacterial and parasitic infections [Young et al . , supra; Luft et al. , supra; D.M. Engman et al. , J. Immunol., 144, pp. 3987-3991 (1990) ; N.M. Rothstein et al. , Molec. Biochem. Parasitol., 33, pp. 229-235 (1989) ; V. Nussenzweig and R.S. Nussenzweig, Adv. Immunol., 45, pp. 283-334 (1989)] . HSPs can elicit strong B- and T- cell responses and it was shown that 20% of the CD4* T-lymphocytes from mice inoculated with M. tuberculosis were reactive to the hsp60 protein alone [S.H.E. Kaufman et al. , Eur. J. Immunol., 17, pp. 351-357 (1987)] . Similarly, 7 out of a collection of 24 monoclonal antibodies to M. leprae proteins recognized determinants on hspδO [H.D. Engers et al. , Infect. Immun. , 48, pp. 603-605 (1985)] . It seems that the immune response to stress proteins might play an important role in protection against infection. Consistent with that is the demonstration that antibodies and T cells reactive with microbial HSPs can exhibit neutralizing and protective activities [A. Noll et al. , Infect. Immun., 62, pp. 2784-2791 (1994) ; and S.L. Danilition et al . , Infect. Immun. , 58, pp. 189-196 (1990)] . The immunological properties of stress proteins make them attractive as vaccine components and several HSPs are presently being considered for preventing microbial infection and treating cancer. So far, however, studies have focused on intracellular pathogens such as Mycobacteria, Salmonella, Chlamydia and several parasites. Information concerning the heat shock protein antigens in extracellular gram- positive bacteria is far less documented. In S. pneumoniae, S. pyogenes and S. agalactiae, neither the heat shock proteins nor their gene structures have been identified.
DISCLOSURE OF THE INVENTION
The present invention addresses the problems referred to above by providing novel heat shock proteins
from S. pneumoniae, S. pyogenes and S. agalactiae, and immunologically related polypeptides . Also provided are DNA sequences that code for the foregoing polypeptides, vectors containing the polypeptides, unicellular hosts transformed with those vectors, and a process for making substantially pure, recombinant polypeptides. Also provided are antibodies specific to the foregoing polypeptides. The polypeptides, DNA sequences and antibodies of this invention provide the basis for novel methods and pharmaceutical compositions for the detection, prevention and treatment of disease. Particularly, this invention provides a novel vaccine based on fragments of these polypeptides that are specific to streptococcal strains . The novel heat shock protein is the approximately 72 kDa heat shock protein of Streptococcus pneumoniae ("HSP72") (SEQ ID NO:5), the approximately 70 kDa heat shock protein of Streptococcus pyogenes ("HSP70") (SEQ ID NO:20) and the approximately 70 kDa heat shock protein of Streptococcus agalactiae ("HSP70") (SEQ ID
NO:22), including analogues, homologues, and derivatives thereof, and fragments of the foregoing polypeptides containing at least one immunogenic epitope. Preferred fragments of HSP70/72 include the C-terminal portion of the HSP70/72 polypeptides. More particularly, it includes the C_terminal 169-residue fragment ("C-169") (residues 439-607, SEQ ID NO: 5) , the C-terminal 151-residue fragment ("C-151") (residues 457-607, SEQ ID No:5) , and smaller fragments consisting of peptide epitopes within the C-169 region. Particularly preferred fragments within the C-169 region of HSP72 include the peptide sequences GFDAERDAAQAALDD (residues 527-541 of SEQ ID NO:5) and 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.
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, MAPI, 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 ("MAbs"), which are specific to HSP72.
More preferred antibodies are the F2-Pn3.2 and F2-Pn3.4 monoclonal anibodies that are specific to both HSP 70 and HSP72. Even more preferred are the Fl-Pn3.1 antibodies that are specific for Streptococcus pneumoniae .
The preferred polypeptides and antibodies of this invention provide the basis for novel methods and pharmaceutical compositions for the detection, prevention and treatment of pneumococcal diseases .
BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 depicts a fluorogram, which shows the effect of heat shock on S. pneumoniae protein synthesis. The cell extracts in panel A are S. pneumoniae type 6 strain 64. The cell extracts in panel B are S. pneumoniae type 4 strain 53. The cell extracts in the odd numbered lanes were incubated at 37°C. The cell extracts in the even numbered lanes were incubated at 45°C for 5 minutes . The cell extracts were then labeled with [35S]methionine for 10 minutes (lanes 1, 2 and 7, 8) , 30 minutes (lanes 3, 4 and 9, 10) , or 60 minutes (lanes 5, 6) . Molecular mass markers in kilodaltons are shown to the left. The positions of HSP80, HΞP72 and HSP62 are shown by arrows at the right-hand side of each panel.
FIG. 2 is a graphical depiction of a comparison of the electrophoretic profiles of [35S]methionine-labeled proteins in S. pneumoniae in the presence ( ) or absence ( ) of exposure to heat shock. Densitometric tracings were determined by measuring the relative optical
density (Y axis) vs. the mobility of labeled protein bands (X axis) . The densitometric scans of the SDS PAGE of FIG. 1, lanes 1 and 2, is shown.
FIG. 3 depicts a fluorogram, which shows the S. pneumoniae protein antigens immunoprecipitated by sera from mice immunized with detergent-soluble S. pneumoniae protein extract. [35S]methionine-labeled proteins from S. pneumoniae grown at 37°C and incubated at 37°C (lanes 3, 5, 7 and 9) or heat-shocked at 45°C (lanes 4, 6, 8 and 10) were immunoprecipitated with sera from mouse 1 (lanes 3 to 6) or mouse 2 (lanes 7 to 10) and then analyzed by SDS- PAGE and fluorography. The sera were tested after the first (lanes 3,4 and 7,8) and after the second (lanes 5,6 and 9,10) immunization. Cell lysates from [35S]methionine- labeled non heat-shocked and heat-shocked S. pneumoniae are shown in lanes 1 and 2, respectively. The position of HSPs is indicated by the arrows at the left of the fluorogram.
FIG. 4 depicts a fluorogram, which shows the S. pneumoniae protein antigens immunoprecipitated by sera from mice immunized with heat-killed S. pneumoniae bacteria. [35S]methionine-labeled proteins from S. pneumoniae grown at 37°C and incubated at 37°C (lanes 3, 5 and 7) or heat-shocked at 45°C (lanes 4, 6 and 8) were immunoprecipitated with sera from mouse 1 (lanes 3,4), mouse 2 (lanes 5,6) or mouse 3 (lanes 7, 8) and then analyzed by SDS-PAGE and fluorography. Sera were tested after the second immunization only. Cell lysates from [35S]methionine-labeled non heat- and heat-shocked S. pneumoniae are shown in lanes 1 and 2, respectively. The position of HSPs is indicated by the arrows at the left of the fluorogram.
FIG. 5 depicts a photograph, which shows the S. pneumoniae antigens detected by Western blot analysis. Whole cell extracts were probed with sera from 15 mice (lanes 1-15) immunized with heat-killed S. pneumoniae bacteria. Lane 16 shows the HSP72 protein detected by MAb
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 panel. FIG. 6 depicts a fluorogram showing the specificity of MAb Fl-Pn3.1 for HSP72. [35S]methionine- labeled proteins of S. pneumoniae in the absence (lanes 1, 3 and 5) or presence (lanes 2, 4 and 6) of exposure to heat shock were immunoprecipitated with IgG2a-control MAb (lane 3,4) or Fl-Pn3.1 (lane 5,6) and then analyzed by SDS-PAGE and fluorography. Cell lysates from [35S]methionine-labeled non heat-shocked and heat-shocked S. pneumoniae are shown in lanes 1 and 2, respectively.
The position of HSPs (all three) is shown by the arrows at the left of the fluorogram.
FIG. 7, panel A, depicts an immunoblot, which shows the reaction of heat-shocked and non heat-shocked [35S]methionine-labelled S. pneumoniae cell extracts with MAb Fl-Pn3.1. Lane 1 contains heat-shocked cell lysates (45°C) . Lane 2 contains non heat-shocked cell lysates (37°C) . Panel B depicts a fluorogram of the immunoblot shown in panel A. FIG. 8 depicts a Western Blot, which shows subcellular localization of S. pneumoniae HSP72. Sample containing 15 μg protein of membrane fraction (lane 1) and cytoplasmic fraction (lane 2) of S. pneumoniae were electrophoresced on SDS-PAGE transferred to nitrocellulose and probed with MAb 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 probed with HSP72-specific MAb Fl-Pn3.1.
Lanes 2 and 3 contain phage lysates from E. coli infected with λJBDl7 cultured in the presence (+) or absence (-) of
IPTG and probed with HSP72-specific MAb Fl-Pn3.1. Lanes 4 and 5 contain phage lysates from E. coli infected with λJBD7 cultured in the presence (+) or absence (-) of IPTG and probed with HSP72-specific MAb 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 IOC illustrate the restriction map of the HSP72(DnaK) and Fuc loci, respectively. FIG 10B illustrates the inserts of the various phages and plasmids described in Example 3. H(HindΙII) ; E(EcoRI) ; V(EcoRV) ; P(PstΙ) ; and X(XhoI) indicate positions of restriction endonuclease sites. DNA fragments on the HSP72/DnaK locus (■) ; the Fuc locus (///); and fragments used as probes in the Southern blot analyses (;§: ) are indicated. FIG. 11 depicts the SDS-PAGE and Western blot analyses of the recombinant 74 kDa protein. Whole cell extracts from E. coli transformed with plasmids pJBD179 (lane 1) , pJBDf51 (lanes 2 and 3) and pJBDf62 (lane 4 and 5) and cultured in presence (+) or absence (-) of IPTG were subjected to 10% polyacrylamide gel electrophoresis. The proteins were then visualized by Coomassie Blue staining (A) or Western blotting (B) using HSP-specific MAb 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 4) were subjected to 10% polyacrylamide gel electrophoresis and
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 μg of immunoaffinity-purified HSP72rβc (lane 3) were subjected to 10% polyacrylamide gel electrophoresis. The proteins were then visualized by Coomassie Blue staining. Lane 1 shows the migration of molecular mass markers (106 kDa, 80 kDa, 49.5 kDa, 32.5 kDa, 27.5 kDa and 18.5 kDa) .
FIG. 15 depicts a photograph of SDS-PAGE, which shows the recombinant S. pneumoniae C-169 fragment purified by solubilization of inclusion bodies. Various amounts of purified C-169 protein (lane 1, 5 μg; lane 2, 2.5 μg; and lane 3, 1 μg) and whole cell lysates from E. coli transformed with plasmids pDELTAl (lane 4) and pJBDΔl (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 HSP72rβc. Data are presented as the per cent (%) survival over a period of 14 days for a total of 10 mice per experimental group.
FIG. 17 is a graphical depiction of the survival curve of Balb/c mice protected from S. pneuiTioniae infection by immunization with C-169rβc. 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- 151rec, the coding region for the 151 amino acids at the carboxyl end of the HSP72 of S. pneumoniae; AmpiR, ampicillin-resistance coding region; ColEl ori , origin of replication; cI857, bacteriophage λ cI857 temperature- sensitive repressor gene; λ PL, bacteriophage λ transcription promoter; Tl, Tl transcription terminator. The direction of transcription is indicated by the arrows. βglll and BamHI are the restriction sites used to insert the coding region for the C-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 HSP72rec. Sera were collected after the first, second and third injection with 1 μg (O) or 5 μg (•) of HSP72rec 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 μg (O) or 5 μg (•) 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.
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 μg 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 HSP72rec 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 HSP72rec 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 hsplO/dnak open reading frames of Streptococcus pneumoniae (spn-orf) , Streptococcus pyogenes (sga-orf) and Streptococcus agalactiae (sgb-orf) and indicates the substitutions and insertions of nucleotides specific to each species. FIG. 25 depicts the consensus sequence established from the protein sequences of the Hsp70 of Streptococcus pneumoniae (spn-prot) , Streptococcus pyogenes (sga-prot)
and Streptococcus agalactiae (sgb-prot) and indicates the _ substitutions and insertions of amino acids specific to each species.
FIG. 26 depicts a fluorogram, which shows the effect of heat shock on S. agalactiae protein synthesis and the S. agalactiae protein antigen immunoprecipitated by MAb F2-Pn3.4. Cell lysates from [35S]methionine-labeled proteins from S. agalactiae grown at 37°C and incubated at 37°C (odd numbered lanes) or heat-shocked at 43°C (even numbered lanes) were analysed by SDS-PAGE and fluorography. Lanes 3 and 4 show the immunoprecipitates obtained using MAb F2-Pn3.4.
DETAILED DESCRIPTION OF THE INVENTION
According to one aspect of the invention, we provide novel heat shock proteins of S. pneumoniae, S. pyogenes and S. agalactiae, and analogues, homologues, derivatives and fragments thereof, containing at least one immunogenic epitope. As used herein, a "heat shock protein" is a naturally occurring protein that exhibits preferential transcription during heat stress conditions. The heat shock protein according to the invention may be of natural origin, or may be obtained through the application of recombinant DNA techniques, or conventional chemical synthesis techniques.
As used herein, "immunogenic" means having the ability to elicit an immune response. The novel heat shock proteins of this invention are characterized by their ability to elicit a protective immune response against Streptococcal infections, more particularly against lethal S. pneumoniae , S. pyogenes and S. agalactiae .
The invention particularly provides a Streptoccus pneumoniae heat shock protein of approximately 72 kDa ("HSP72"), having the deduced amino acid sequence of SEQ ID NO: 5, and analogues, homologues, derivatives and
fragments thereof, containing at least one immunogenic epitope .
As used herein, "analogues" of HSP72 are those S. pneumoniae proteins wherein one or more amino acid residues in the HSP72 amino acid sequence (SEQ ID NO: 5) is replaced by another amino acid residue, providing that the overall functionality and immunogenic properties of the analogue protein are preserved. Such analogues may be naturally occurring, or may be produced synthetically or by recombinant DNA technology, for example, by mutagenesis of the HSP72 sequence. Analogues of HSP72 will possess at least one antigen capable of eliciting antibodies that react with HSP72, e.g. Streptococcus pyogenes and Streptococcus agalactiae . As used herein, "homologues" of HSP72 are proteins from Streptococcal species other than pneumoniae , pyogenes or agalactiae, or genera other than Streptococcus wherein one or more amino acid residues in the HSP72 amino acid sequence (SEQ ID NO:5) is replaced by another amino acid residue, providing that the overall functionality and immunogenic properties of the homologue protein are preserved. Such homologues may be naturally occurring, or may be produced synthetically or by recombinant DNA technology. Homologues of HSP72 will possess at least one antigen capable of eliciting antibodies that react with HSP72, e.g. Enterococcus faecalis .
As used herein, a "derivative" is a polypeptide in which one or more physical, chemical, or biological properties has been altered. Such alterations include, but are not limited to: amino acid substitutions, modifications, additions or deletions; alterations in the pattern of lipidation, glycosylation or phosphorylation; reactions of free amino, carboxyl, or hydroxyl side groups of the amino acid residues present in the polypeptide with other organic and non-organic molecules; and other alterations, any of which may result in changes in primary, secondary or tertiary structure.
The "fragments" of this invention will have at _ least one immunogenic epitope. An "immunogenic epitope" is an epitope that is instrumental in eliciting an immune response. The preferred fragments of this invention will elicit an immune response sufficient to prevent or lessen the severity of infection, e.g., S. pneumoniae infection. Preferred fragments of HSP72 include the C-terminal region of the polypeptides. More preferred fragment include the C-terminal 169-residue fragment ("C-169") (SEQ ID NO:5, residues 439-607), the C-terminal 151-residue ("C-151") (SEQ ID No:5, residues 457-607) and smaller fragments consisting of peptide epitopes within the C-169 region. Particularly preferred fragments within the C-169 region of HSP72 include the peptide sequences GFDAERDAAQAALDD (residues 527-541 of SEQ ID NO:5) and AEGAQATGNAGDDW
(residues 586-600 of SEQ ID N0:5), which are exclusive to HSP72 of Streptococcus pneumoniae , or corresponding degenerate fragments from S. pyogenes or S. agalactiae (see FIG. 25) . Even more preferred are fragments that elicit a specific immune reaction against Streptococcal strains . Such fragments may be selected from the following peptides: CS870, CS873, CS874, CS875, CS876, CS877, CS878, CS879, CS880, CS882, MAPI, MAP2, MAP3 and MAP4 (see TABLE 5, supra), or homologues thereof. In a further aspect of the invention, we provide polypeptides that are immunologically related to HSP70/72. As used herein, "immunologically related" polypeptides are characterized by one or more of the following properties:
(a) they are immunologically reactive with antibodies generated by infection of a mammalian host with Streptococcus pneumoniae cells, which antibodies are immunologically reactive with HSP72 (SEQ ID NO:5) and HSP70 (SEQ ID NO:20 and SEQ ID NO:22);
(b) they are capable of eliciting antibodies that are immunologically reactive with HSP72 (SEQ ID NO:5) and HSP70 (SEQ ID NO:20 and SEQ ID NO:22);
(c) they are immunologically reactive with antibodies elicited by immunization of a mammal with HSP72 (SEQ ID NO: 5) .
By definition, analogues, homologues and derivatives of HSP70/72 are immunologically related polypeptides. Moreover, all immunologically related polypeptides contain at least one HSP70/72 antigen. Accordingly, "HSP70/72 antigens" may be found in HSP70/72 itself, or in immunologically related polypeptides. In a further aspect of the invention, we provide polypeptides that are immunologically related to HSP72. As used herein, "immunologically related" polypeptides are characterized by one or more of the following properties :
(a) they are immunologically reactive with antibodies generated by infection of a mammalian host with Streptococcus pneumoniae cells, which antibodies are immunologically reactive with HSP72 (SEQ ID NO:5);
(b) they are capable of eliciting antibodies that are immunologically reactive with HSP72 (SEQ ID NO:5); (c) they are immunologically reactive with antibodies elicited by immunization of a mammal with HSP72 (SEQ ID NO:5) .
By definition, analogues, homologues and derivatives of HSP72 are immunologically related polypeptides. Moreover, all immunologically related polypeptides contain at least one HSP72 antigen. Accordingly, "HSP72 antigens" may be found in HSP72 itself, or in immunologically related polypeptides.
As used herein, "related bacteria" are bacteria that possess antigens capable of eliciting antibodies that react with HSP72. Examples of related bacteria include Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus mutans, Streptococcus sanguis, Streptococcus agalactiae and Enterococcus faecalis . It will be understood that by following the examples of this invention, one of skill in the art may determine without undue experimentation whether a
particular analogue, homologue, derivative, immunologically related polypeptide, or fragment would be useful in the diagnosis, prevention or treatment of disease. Useful polypeptides and fragments will elicit antibodies that are immunoreactive with HSP72 (Example 4) . Preferably, useful polypeptides and fragments will demonstrate the ability to elicit a protective immune response against lethal bacterial infection (Example 5) . Also included are polymeric forms of the polypeptides of this invention. These polymeric forms include, for example, one or more polypeptides that have been crosslinked with crosslinkers such as avidin/biotin, glutaraldehyde or dimethylsuberimidate. Such polymeric forms also include polypeptides containing two or more tandem or inverted contiguous protein sequences, produced from multicistronic mRNAs generated by recombinant DNA technology.
This invention provides substantially pure HSP72 and immunologically related polypeptides . The term "substantially pure" means that the polypeptides according to the invention, and the DNA sequences encoding them, are substantially free from other proteins of bacterial origin. Substantially pure protein preparations may be obtained by a variety of conventional processes, for example the procedures described in Examples 3 and 5.
In another aspect, this invention provides, for the first time, a DNA sequence coding for a heat shock protein of S. pneumoniae , specifically, HSP72 (SEQ ID NO:4, nucleotides 682-2502) . The DNA sequences of this invention also include
DNA sequences coding for polypeptide analogues and homologues of HSP72, DNA sequences coding for immunologically related polypeptides, DNA sequences that are degenerate to any of the foregoing DNA sequences, and fragments of any of the foregoing DNA sequences. It will be readily appreciated that a person of ordinary skill in the art will be able to determine the DNA sequence of any
of the polypeptides of this invention, once the polypeptide has been identified and isolated, using conventional DNA sequencing techniques .
Oligonucleotide primers and other nucleic acid probes derived from the genes encoding the polypeptides of this invention may also be used to isolate and clone other related proteins from S. pneumoniae and related bacteria which may contain regions of DNA bacteria that are homologous to the DNA sequences of this invention. In addition, the DNA sequences of this invention may be used in PCR reactions to detect the presence of S. pneumoniae or related bacteria in a biological sample.
The polypeptides of this invention may be prepared from a variety of processes, for example by protein fractionation from appropriate cell extracts, using conventional separation techniques such as ion exchange and gel chromatography and electrophoresis, or by the use of recombinant DNA techniques. The use of recombinant DNA techniques is particularly suitable for preparing substantially pure polypeptides according to the invention.
Thus according to a further aspect of the invention, we provide a process for the production of HSP72, immunologically related polypeptides, and fragments thereof, comprising the steps of (1) culturing a unicellular host organism transformed with a vector containing a DNA sequence coding for said polypeptide or fragment and one or more expression control sequences operatively linked to the DNA sequence, and (2) recovering a substantially pure polypeptide or fragment.
As is well known in the art, in order to obtain high expression levels of a transfected gene in a host, the gene must be operatively linked to transcriptional and ranslational expression control sequences that are functional in the chosen expression host. Preferably, the expression control sequences, and the gene of interest, will be contained in an expression vector that further
comprises a bacterial selection marker and origin of replication. If the expression host is a eukaryotic cell, the expression vector should further comprise an expression marker useful in the eukaryotic expression host.
The DNA sequences encoding the polypeptides of this invention may or may not encode a signal sequence. If the expression host is eukaryotic, it generally is preferred that a signal sequence be encoded so that the mature protein is secreted from the eukaryotic host.
An amino terminal methionine may or may not be present on the expressed polypeptides of this invention. If the terminal methionine is not cleaved by the expression host, it may, if desired, be chemically removed by standard techniques .
A wide variety of expression host/vector combinations may be employed in expressing the DNA sequences of this invention. Useful expression vectors for eukaryotic hosts include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus, adeno-associated virus, cytomegalovirus, and retroviruses. Useful expression vectors for bacterial hosts include bacterial plasmids, such as those from E. coli , including pBluescript, pGEX2T, pUC vectors, col El, pCRl, pBR322, pMB9 and their derivatives, wider host range plasmids, such as RP4, phage DNAs, e.g., the numerous derivatives of phage lambda, e.g. λgtlO and λgtll, NM989, and other DNA phages, such as M13 and filamentous single stranded DNA phages. Useful expression vectors for yeast cells include the 2μ plasmid and derivatives thereof. Useful vectors for insect cells include pVL 941.
In addition, any of a wide variety of expression control sequences may be used in these vectors to express the DNA sequences of this invention. Useful expression control sequences include the expression control sequences associated with structural genes of the foregoing
expression vectors . Examples of useful expression control sequences include, for example, the early and late promoters of SV40 or adenovirus, the lac system, the trp system, the TAC or TRC system, the T3 and T7 promoters the major operator and promoter regions of phage lambda, the control regions of fd coat protein, the promoter for 3- phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters of the yeast alpha-mating system and other constitutive and indueible promoter sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof. The T7 RNA polymerase promoter Φ10 is particularly useful in the expression of HSP72 in E. coli (Example 3) . Host cells transformed with the foregoing vectors form a further aspect of this invention. A wide variety of unicellular host cells are useful in expressing the DNA sequences of this invention. These hosts may include well known eukaryotic and prokaryotic hosts, such as strains of E. coli , Pseudomonas, Bacillus,
Streptomyces , fungi, yeast, insect cells such as Spodoptera frugiperda (SF9), animal cells such as CHO and mouse cells, African green monkey cells such as COS 1, COS 7, BSC 1, BSC 40, and BMT 10, human cells, and plant cells in tissue culture. Preferred host organisms include bacteria such as E . coli and B . subtilis, and mammalian cells in tissue culture.
It should of course be understood that not all vectors and expression control sequences will function equally well to express the DNA sequences of this invention. Neither will all hosts function equally well with the same expression system. However, one of skill in the art may make a selection among these vectors, expression control sequences and hosts without undue experimentation and without departing from the scope of this invention. For example, in selecting a vector, the host must be considered because the vector must replicate
in it. The vector's copy number, the ability to control that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered. In selecting an expression control sequence, a variety of factors should also be considered. These include, for example, the relative strength of the sequence, its controllability, and its compatibility with the DNA sequences of this invention, particularly as regards potential secondary structures. Unicellular hosts should be selected by consideration of their compatibility with the chosen vector, the toxicity of the product coded for by the DNA sequences of this invention, their secretion characteristics, their ability to fold the protein correctly, their fermentation or culture requirements, and the ease of purification from them of the products coded for by the DNA sequences of this invention. Within these parameters, one of skill in the art may select various vector/expression control sequence/host combinations that will express the DNA sequences of this invention on fermentation or in large scale animal culture.
The polypeptides encoded by the DNA sequences of this invention may be isolated from the fermentation or cell culture and purified using any of a variety of conventional methods including: liquid chromatography such as normal or reversed phase, using HPLC, FPLC and the like; affinity chromatography (such as with inorganic ligands or monoclonal antibodies) ; size exclusion chromatography; immobilized metal chelate chromatography; gel electrophoresis; and the like. One of skill in the art may select the most appropriate isolation and purification techniques without departing from the scope of this invention.
In addition, the polypeptides of this invention may be generated by any of several chemical techniques. For example, they may be prepared using the solid-phase synthetic technique originally described by R. B.
Merrifield, "Solid Phase Peptide Synthesis. I. The Synthesis Of A Tetrapeptide" , J. Am. Chem. Soc . , 83, pp. 2149-54 (1963), or they may be prepared by synthesis in solution. A summary of peptide synthesis techniques may be found in E. Gross & H. J. Meinhofer, 4 The
Peptides: Analysis, Synthesis, Biology; Modern Techniques Of Peptide And Amino Acid Analysis, John Wiley & Sons, (1981) and M. Bodanszky, Principles Of Peptide Synthesis, Springer-Verlag (1984) . The preferred compositions and methods of this invention comprise polypeptides having enhanced immunogenicity. Such polypeptides may result when the native forms of the polypeptides or fragments thereof are modified or subjected to treatments to enhance their immunogenic character in the intended recipient.
Preferred polypeptides are fragments that are specific to Streptococcal species such as fragments selected from the C-terminal portion of thenative polypeptides. Numerous techniques are available and well known to those of skill in the art which may be used, without undue experimentation, to substantially increase the immunogenicity of the polypeptides herein disclosed. For example, the polypeptides may be modified by coupling to dinitrophenol groups or arsanilic acid, or by denaturation with heat and/or SDS. Particularly if the polypeptides are small polypeptides synthesized chemically, it may be desirable to couple them to an immunogenic carrier. The coupling of course, must not interfere with the ability of either the polypeptide or the carrier to function appropriately. For a review of some general considerations in coupling strategies, see Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, ed. E. Harlow and D. Lane (1988) . Useful immunogenic carriers are well known in the art. Examples of such carriers are keyhole limpet hemocyanin (KLH) ; albumins such as bovine serum albumin (BSA) and ovalbumin, PPD (purified protein derivative of tuberculin); red blood cells; tetanus
toxoid; cholera toxoid; agarose beads; activated carbon; or bentonite.
Modification of the amino acid sequence of the polypeptides disclosed herein in order to alter the lipidation state is also a method which may be used to increase their immunogenicity and biochemical properties. For example, the polypeptides or fragments thereof may be expressed with or without the signal sequences that direct addition of lipid moieties. In accordance with this invention, derivatives of the polypeptides may be prepared by a variety of methods, including by in vi tro manipulation of the DNA encoding the native polypeptides and subsequent expression of the modified DNA, by chemical synthesis of derivatized DNA sequences, or by chemical or biological manipulation of expressed amino acid sequences.
For example, derivatives may be produced by substitution of one or more amino acids with a different natural amino acid, an amino acid derivative or non-native amino acid, conservative substitution being preferred, e.g., 3-methylhistidine may be substituted for histidine, 4-hydroxyproline may be substituted for proline, 5- hydroxylysine may be substituted for lysine, and the like. Causing amino acid substitutions which are less conservative may also result in desired derivatives, e.g., by causing changes in charge, conformation and other biological properties . Such substitutions would include for example, substitution of a hydrophilic residue for a hydrophobic residue, substitution of a cysteine or proline for another residue, substitution of a residue having a small side chain for a residue having a bulky side chain or substitution of a residue having a net positive charge for a residue having a net negative charge. When the result of a given substitution cannot be predicted with certainty, the derivatives may be readily assayed according to the methods disclosed herein to determine the presence or absence of the desired characteristics.
The polypeptides may also be prepared with the objective of increasing stability or rendering the molecules more amenable to purification and preparation. One such technique is to express the polypeptides as fusion proteins comprising other S. pneumoniae or non- S. pneumoniae sequences. It is preferred that- the fusion proteins comprising the polypeptides of this invention be produced at the DNA level, e.g., by constructing a nucleic acid molecule encoding the fusion, transforming host cells with the molecule, inducing the cells to express the fusion protein, and recovering the fusion protein from the cell culture. Alternatively, the fusion proteins may be produced after gene expression according to known methods. An example of a fusion protein according to this invention is the FucI/HSP72 (C-169) protein of Example 3, infra.
The polypeptides of this invention may also be part of larger multimeric molecules which may be produced recombinantly or may be synthesized chemically. Such multimers may also include the polypeptides fused or coupled to moieties other than amino acids, including lipids and carbohydrates .
The polypeptides of this invention are particularly well-suited for the generation of antibodies and for the development of a protective response against disease. Accordingly, in another aspect of this invention, we provide antibodies, or fragments thereof, that are immunologically reactive with HSP72. The antibodies of this invention are either elicited by immunization with HSP72 or an immunologically related polypeptide, or are identified by their reactivity with HSP72 or an immunologically related polypeptide. It should be understood that the antibodies of this invention are not intended to include those antibodies which are normally elicited in an animal upon infection with naturally occurring S. pneumoniae and which have not been removed from or altered within the animal in which they were elicited.
The antibodies of this invention may be intact immunoglobulin molecules or fragments thereof that contain an intact antigen binding site, including those fragments known in the art as F(v), Fab, Fab' and F(ab')2. The antibodies may also be genetically engineered or synthetically produced. The antibody or fragment may be of animal origin, specifically of mammalian origin, and more specifically of murine, rat, monkey or human origin. It may be a natural antibody or fragment, or if desired, a recombinant antibody or fragment. The antibody or antibody fragments may be of polyclonal, or preferably, of monoclonal origin. They may be specific for a number of epitopes but are preferably specific for one. Specifically preferred are the monoclonal antibodies 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 (e.g., mouse and human) or from portions of immunoglobulin light and heavy chain sequences from the same species . It may be a molecule that has multiple binding specificities, such as a bifunctional antibody
prepared by any one of a number of techniques known to those of skill in the art including: the production of hybrid hybridomas; disulfide exchange; chemical cross- linking; addition of peptide linkers between two monoclonal antibodies; the introduction of two sets of immunoglobulin heavy and light chains into a particular cell line; and so forth. The antibodies of this invention may also be human monoclonal antibodies, for example those produced by immortalized human cells, by SCID-hu mice or other non-human animals capable of producing "human" antibodies, or by the expression of cloned human immunoglobulin genes.
In sum, one of skill in the art, provided with the teachings of this invention, has available a variety of methods which may be used to alter the biological properties of the antibodies of this invention including methods which would increase or decrease the stability or half-life, immunogenicity, toxicity, affinity or yield of a given antibody molecule, or to alter it in any other way that may render it more suitable for a particular application.
The polypeptides, DNA sequences and antibodies of this invention are useful in prophylactic, therapeutic and diagnostic compositions for preventing, treating and diagnosing disease.
Standard immunological techniques may be employed with the polypeptides and antibodies of this invention in order to use them as immunogens and as vaccines. In particular, any suitable host may be injected with a pharmaceutically effective amount of polypeptide to generate monoclonal or polyvalent antibodies or to induce the development of a protective immunological response against disease. Preferably, the polypeptide is selected from the group consisting of HSP72 (SEQ ID NO: 5) , HSP70 (SEQ ID NO:20 and SEQ ID NO:22) or fragments thereof.
As used herein, a "pharmaceutically effective amount" of a polypeptide or of an antibody is the amount that, when administered to a patient, elicits an immune response that is effective to prevent or lessen the severity of Streptococcal or related bacterial infections .
The administration of the polypeptides or antibodies of this invention may be accomplished by any of the methods described in Example 10, infra, or by a variety of other standard procedures. For a detailed discussion of such techniques, see Antibodies, A Laboratory Manual , Cold Spring Harbor Laboratory, ed. E. Harlow and D. Lane (1988) . Preferably, if a polypeptide is used, it will be administered with a pharmaceutically acceptable adjuvant, such as complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptides) or ISCOM (im unostimulating complexes) . Preferably, the composition will include a water-in-oil emulsion or aluminum hydroxide as adjuvant and will be administered intramuscularly. The vaccine composition may be administered to the patient at one time or over a series of treatments. The most effective mode of administration and dosage regimen will depend upon the level of immunogenicity, the particular composition and/or adjuvant used for treatment, the severity and course of the expected infection, previous therapy, the patient's health status and response to immunization, and the judgment of the treating physician. For example, in an immunocompetent patient, the more highly immunogenic the polypeptide, the lower the dosage and necessary number of immunizations. Similarly, the dosage and necessary treatment time will be lowered if the polypeptide is administered with an adjuvant.
Generally, the dosage will consist of an initial injection, most probably with adjuvant, of about 0.01 to 10 mg, and preferable 0.1 to 1.0 mg, HSP72 antigen per patient, followed most probably by one or maybe more
booster injections. Preferably, boosters will be administered at about 1 and 6 months after the initial injection.
Any of the polypeptides of this invention may be used in the form of a pharmaceutically acceptable salt. Suitable acids and bases which are capable of forming salts with the polypeptides of the present invention are well known to those of skill in the art, and include inorganic and organic acids and bases. To screen the polypeptides and antibodies of this invention for their ability to confer protection against diseases caused by S. pneumoniae or related bacteria, or their ability to lessen the severity of such infection, one of skill in the art will recognize that a number of animal models may be used. Any animal that is susceptible to infection with S. pneumoniae or related bacteria may be useful. The Balb/c mice of Example 5, infra, are the preferred animal model for active immunoprotection screening, and the severe-combined immunodeficient mice of Example 5 are the preferred animal model for passive screening. Thus, by administering a particular polypeptide or antibody to these animal models, one of skill in the art may determine without undue experimentation whether that polypeptide or antibody would be useful in the methods and compositions claimed herein. According to another embodiment of this invention, we describe a method which comprises the steps of treating a patient with a vaccine comprising a pharmaceutically effective amount of any of the polypeptides of this invention in a manner sufficient to prevent or lessen the severity, for some period of time, of Streptococcal or related bacterial infection. Again, the preferred polypeptide for use in such methods is HSP70/HSP72, or fragments thereof. The polypeptides, DNA sequences and antibodies of this invention may also form the basis for diagnostic methods and kits for the detection of pathogenic
organisms. Several diagnostic methods are possible. For example, this invention provides a method for the detection of Streptococcus pneumoniae , Streptococcus pyogenes , Streptococcus agalactiae or related bacteria in a biological sample comprising the steps of:
(a) isolating the biological sample from a patient;
(b) incubating an antibody of this invention, or fragment thereof with the biological sample to form a mixture; and
(c) detecting specifically bound antibody or fragment in the mixture which indicates the presence of Streptococcus pneumoniae , Streptococcus pyogenes, Streptococcus agalactiae or related bacteria. Preferable antibodies for use in this method include monoclonal antibodies 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:
(a) isolating the biological sample from a patient;
(b) incubating a polypeptide of this invention or fragment thereof, with the biological sample to form a mixture; and
(c) detecting specifically bound polypeptide in the mixture which indicates the presence of antibodies specific to Streptococcus pneumoniae or related bacteria. HΞP72 (SEQ ID NO: 5) , the C-169 fragment thereof (residues 439-607 of SEQ ID NO: 5) , the C-151 fragment thereof
(residues 457-607 of SEQ ID NO;5) and peptide fragments GFDAERDAAQAALDD (residues 527-541 of SEQ ID NO:5) and 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
an enzyme-linked immunosorbent assay (ELISA) , a radioimmunoassay or a latex agglutination assay.
The diagnostic agents may be included in a kit which may also comprise instructions for use and other appropriate reagents, preferably a means for detecting when the polypeptide or antibody is bound. For example, the polypeptide or antibody may be labeled with a detection means that allows for the detection of the polypeptide when it is bound to an antibody, or for the detection of the antibody when it is bound to
S. pneumoniae or related bacteria. The detection means may be a fluorescent labeling agent such as fluorescein isocyanate (FIC) , fluorescein isothiocyanate (FITC) , and the like, an enzyme, such as horseradish peroxidase (HRP) , glucose oxidase or the like, a radioactive element such as 125I or 51Cr that produces gamma ray emissions, or a radioactive element that emits positrons which produce gamma rays upon encounters with electrons present in the test solution, such as X1 , 150, or 13N. Binding may also be detected by other methods, for example via avidin- biotin complexes. The linking of the detection means is well known in the art. For instance, monoclonal antibody molecules produced by a hybridoma may be metabolically labeled by incorporation of radioisotope-containing amino acids in the culture medium, or polypeptides may be conjugated or coupled to a detection means through activated functional groups.
The DNA sequences of this invention may be used to design DNA probes for use in detecting the presence of Streptococcus pneumoniae or related bacteria in a biological sample. The probe-based detection method of this invention comprises the steps of:
(a) isolating the biological sample from a patient ; (b) incubating a DNA probe having a DNA sequence of this invention with the biological sample to form a mixture ; and
(c) detecting specifically bound DNA probe in the mixture which indicates the presence of Streptococcus pneumoniae or related bacteria.
The DNA probes of this invention may also be used for detecting circulating nucleic acids in a sample, for example using a polymerase chain reaction, as a method of diagnosing Streptococcus pneumoniae or related bacterial infections. The probes may be synthesized using conventional techniques and may be immobilized on a solid phase, or may be labeled with a detectable label. A preferred DNA probe for this application is an oligomer having a sequence complementary to at least about 6 contiguous nucleotides of HSP72 (SEQ ID NO : 4 , nucleotides 682-2502) . The polypeptides of this invention may also be used to purify antibodies directed against epitopes present on the protein, for example, using immunoaffinity purification of antibodies on an antigen column.
The antibodies or antibody fragments of this invention may be used to prepare substantially pure proteins according to the invention for example, using immunoaffinity purification of antibodies on an antigen column. EXAMPLES
In order that this invention may be better understood, the following examples are set forth. These examples are for purposes of illustration only, and are not to be construed as limiting the scope of the invention in any manner .
Example 1 describes the identification of HSP72, an immunoreactive heat shock protein according to the invention. Example 2 describes the isolation of monoclonal antibodies against epitopes of HSP72. Example 3 describes the preparation of recombinant HSP72 and fragments of HSP72 according to the invention. Example 4 describes the antigenic specificity and immunoreactivity
of monoclonal antibodies directed against HSP72, and the identification of immunologically related proteins according to the invention. Example 5 describes processes for obtaining substantially pure HSP72, and the use of HSP72 or antibodies against it to protect against experimental S. pneumoniae infection. Example 6 describes the preparation of recombinant C-151 fragment of HSP72 according to the invention. Example 7 describes the humoral immune response following the immunization with recombinant HSP72 or fragments of HSP72 according to the invention. Example 8 describes the localization of linear B-cell epitopes on the HSP72. Example 9 describes the hsp70 genes and HSP70 proteins from S. agalactiae and S. pyogenes . Example 10 describes the use of HSP72 antigen in a human vaccine.
EXAMPLE 1 - Identification of Immunoreactive S. pneumoniae Heat Shock Proteins A. Procedures
Unless otherwise noted, the following procedures were used throughout the Examples herein.
1. Bacteria
S. pneumoniae strains were provided by the Laboratoire de la Sante Publique du Quebec, Sainte-Anne de Bellevue. S. pneumoniae strains included type 4 strain 53 and type 6 strain 64. If not specified, S. pneumoniae type 6 strain 64 was used. Bacterial strains were grown overnight at 37°C in 5% C02 on chocolate agar plates.
2. Antigen Preparations
Various S. pneumoniae antigens were prepared for immunization and immunoassays . Heat-killed whole cell antigens were obtained by incubating bacterial suspensions
in a water bath prewarmed at 56 C for 20 minutes. Detergent-soluble proteins were extracted from S. pneumoniae as follows. Heat-killed bacteria were suspended in 10 mM Hepes buffer (4- (2-Hydroxyethyl) -1- piperazinethan-sulfonsaure) (Boehringer Mannheim GmbH,
Germany) at pH 7.4 and sonicated at 20,000 Kz/second, four times for 30 seconds. Intact cells and large debris were removed by centrifugation at 1,700 g for 20 minutes. The supernatant was collected and centrifuged at 100,000 g for 60 minutes. The pellet was resuspended in 1 ml of Hepes buffer, and 1 ml of 2% N-lauroyl sarcosine (Sigma Chemical Co., St. Louis, Mo.) was added. The mixture was incubated for 30 minutes at room temperature and the detergent- soluble fraction was harvested by centrifugation at 100,000 g for 60 minutes.
3. Heat Shock Treatment
S. pneumoniae bacteria (type 4, strain 53 and type 6, strain 64) were resuspended in Eagle's Minimal
Essential Medium lacking methionine (ICN Biomedicals Inc., Costa Mesa, CA) and supplemented with 1% BIO-X® (Quelab Laboratories, Montreal, Canada) for 15 minutes at 37°C and then divided into fractions of equal volume. The samples were incubated at either 37°C or 45°C for 5 minutes and then labeled with 100 μCi/ml [35S]methionine (ICN) for 10, 30, or 60 minutes at37°C. The bacteria were harvested and cell extracts were prepared using Tris-HCl lysis buffer as described above, or SDS-PAGE sample buffer.
4. Immunization Of Mice
Female Balb/c mice (Charles River Laboratories, St-Constant, Quebec, Canada) were immunized with S. pneumoniae antigens. Immune sera to S. pneumoniae type 6 strain 64 were obtained from mice immunized, at two-week intervals, by subcutaneous injections of 107 heat- killed bacteria or 20 μg of detergent-soluble pneumococcal
proteins absorbed to aluminum hydroxide adjuvant (Alhydrogel®; Cedarlane Laboratories Ltd., Horny, Ontario, Canada) . Blood samples were collected prior to immunization and at seven days following the first and second immunization.
5. SDS-PAGE and Immunoassays
Cell extracts were prepared for SDS-PAGE, Western blot analysis and radioimmunoprecipitation assay by incubating bacterial suspensions in Tris-HCl lysis buffer (50mM Tris, 150 mM NaCl, 0.1% Na dodecyl sulfate, 0.5% Na deoxycholate, 2% Triton® X-100, 100 μg/ml phenylmethylsulfonylfluoride, and 2μg/ml aprotinin) at pH 8.0 for 30 minutes on ice. Lysed cells were cleared by centrifugation and the supernatants were aliquoted and kept frozen at -70 C.
SDS-PAGE were performed on a 10% polyacrylamide gel according to the method of Laemmli [Nature, 227, pp. 680-685 (1970)], using the Mini Protean® system (Bio- Rad Laboratories Ltd. , Mississauga, Canada) . Samples were denatured by boiling for 5 minutes in sample buffer containing 2% 2-mercaptoethanol. Proteins were resolved by staining the polyacrylamide gel with PhastGel Blue® (Pharmacia Biotech Inc., Baie d'Urfe, Canada) . The radiolabeled products were visualized by fluorography. Fluorograms were scanned using a laser densitometer.
Immunoblot procedures were performed according to the method of Towbin et al . [Proc. Natl . Acad. Sci. USA, 76, pp. 4350-4354 (1979)] . The detection of antigens reactive with antibodies was performed by an indirect antibody immunoassay using peroxidase-labeled anti-mouse immunoglobulins and the o-dianisidine color substrate.
Radioimmunoprecipitation assays were performed as described by J.A. Wiley et al. [J. Virol. , 66, pp. 5744-5751 (1992)] . Briefly, sera or hybridoma culture supernatants were added to radiolabeled samples containing
equal amounts of [35S]methionine. The mixtures were allowed to incubate for 90 minutes at 4 C with constant agitation. The immune complexes were then precipitated with bovine serum albumin-treated protein A Sepharose (Pharmacia) for 1 hour at 4 C. The beads were pelleted and washed three times in Tris buffered saline at pH 8.0, and the antigen complexes were then dissociated by boiling in sample buffer. The antigens were analyzed by electrophoresis on SDS-PAGE. The gels were fixed, enhanced for fluorography using Amplify® (Amersham'"Canada Limited, Oakville, Ontario, Canada) , dried, and then exposed to X-ray film.
B. Characterization of the Heat Shock Response in S. pneumoniae
We studied the heat shock response of S. pneumoniae by examining the pattern of protein synthesis before and after a shift from 37°C to 45°C. FIG. 1 shows the results when S. pneumoniae type 6 strain 64 (panel A) and type 4 strain 53 (panel B) were grown at 37°C, incubated at 37°C (lanes 1,3,5,7 and 9) or at 45°C (lanes 2, 4, 6, 8 and 10) for 5 minutes, and then labeled with [35S]methionine for 10 minutes (lanes 1,2 and 7,8), 30 minutes (lanes 3,4 and 9,10), or 60 minutes (lanes 5,6) .
The fluorogram derived from SDS-PAGE indicated that the synthesis of at least three proteins was increased by increasing the temperature (FIG. 1) . The most prominent induced protein was about 72 kDa (HSP72), whereas the other two were approximately 80 kDa (HSP80) and 62 kDa (HSP62) . Increased protein synthesis was already apparent after 10 minutes of labeling (FIG. 1, lanes 1, 2 and 7, 8) and became more significant when the labeling period was prolonged to 30 minutes (FIG. 1, lanes 3, 4 and 9, 10) and 60 minutes (FIG. 1, lanes 5, 6) . The effect of elevated temperature on the protein synthesis profile of two different S. pneumoniae strains
was similar, with HSPs of similar molecular mass being synthesized (compare Panel A (type 6 strain 64) to Panel B (type 4 strain 53) in FIG. 1) .
Analysis of the densitometric tracings from scanning the protein synthesis profiles allowed the estimation of the relative amounts of proteins. For example, with respect to heat-shocked S. pneumoniae type 6 strain 64, after 10 minutes of labeling, HSP80 and HSP62 made up 2.9% and 6.8% of the labeled proteins, respectively, compared to less than 0.1% at 37°C (FIG. 2) . Labeled proteins having an apparent molecular mass of 72 kDa were detected at both 37°C and 45°C conditions (FIG. 2) . Radioimmunoprecipitation analysis revealed, however, that HSP72 was undetectable at 37°C (supra; and FIGS. 3, 4 and 6) thus indicating that peak 9 from FIG. 2 corresponds to protein component (s) comigrating with HSP72. Assuming no variation in the labeling of this material, these results would suggest that the amount of HSP72 represents 8.7% of the total labeled cell protein after heat shock treatment. A comparison of the densitometric tracings revealed that cellular proteins corresponding to peaks 4, 10, 13, 17, 19, and 21 were synthesized at almost the same rate irrespective of heat shock treatment (FIG. 2) . However, the synthesis of several proteins (peaks 1, 2, 3, 15, 20, 22, 24, and 26) declined considerably in response to heat shock (FIG. 2) .
C . Immune Responses to S. pneumoniae HSPs In order to assess the antibody response to pneumococcal HSPs, mouse sera were first assayed by radioimmunoprecipitation. The repertoire of labeled proteins recognized by sera from mice immunized with S. pneumoniae antigen preparations are shown in FIGS. 3 and 4. FIG. 3 relates to detergent soluble protein preparations. FIG. 4 relates to heat-killed bacterial preparation. Although many bands were detected by most antisera, HSP72 was a major precipitation product. The
specificity of antibodies for HSP72 was demonstrated by the detection of proteins among heat-shocked products only (FIG. 3, lanes 4, 6, 8 and 10; FIG. 4, lanes 4, 6 and 8 ) . Interestingly, all immunized mice consistently recognized HSP72. The antibodies reactive with the HSP72 were not specific to the strain used during the immunization since strong reactivities were observed with heterologous S. pneumoniae HSP72. It should be noted that in addition to HSP72, one sera precipitated co igrating product labeled at both 37°C and 45°C (FIG. 4, lane 4) . This 72 kDa-product probably corresponds to component from peak 9 in FIG. 2 and was not detected in immunoblots . HSP62 is another immune target which was precipitated by some but not all immune sera (FIG. 3, lane 6 and, FIG. 4, lanes 4 and 6) . None of the sera tested reacted with HSP80. No proteins were precipitated when preimmune sera taken from the mice used in this study were tested for the presence of antibodies reactive with the labeled products.
As depicted in FIGS. 3 and 5, antibodies to HSP72 could be detected after one immunization with either detergent-soluble proteins or whole cells extracts of S. pneumoniae . In addition, a marked increase in the antibody response to HSP72 was observed after a second immunization (FIG. 3, compare 4 and 6, and lanes 8 and 10) .
The immunoblot patterns of 15 mice immunized with heat-killed S. pneumoniae bacteria were remarkably consistent with the results of the previously described radioimmunoprecipitation. Although antibody response variation occurred to a variety of proteins, HSP72 was a major immunoreactive antigen with 8 (53%) positive sera after the first immunization (FIG. 5) . Antibodies to HSP72 were detected in 13 out of 15 (87%) immune sera tested after the second immunization. Two other prominent antigens having apparent molecular mass of 53.5 and 47 kDa were detected in 5 (33%) and 7 (47%) sera, respectively
(FIG. 5) . The 72 kDa-reactive band was confirmed as the - pneumococcal HSP72 by using recombinant HSP72 antigens
(Example 3, infra) in an immunoblot assay. Preimmune sera failed to detect any pneumococcal proteins.
EXAMPLE 2 - Isolation of Monoclonal Antibodies Against Epitopes of HSP72
Procedures
1. Immunization of Mice And Fusion
Female Balb/c mice (Charles River Laboratories) were immunized with S. pneumoniae antigens. One set of mice (fusion experiment 1) were immunized by peritoneal injection with 107 formalin-killed whole cell antigen from strain MTL suspended in Freund's complete adjuvant, and were boosted at two-week intervals with the same antigen and then with a sonicate from heat-killed bacteria in Freund's incomplete adjuvant. A second group of mice
(fusion experiment 2) were immunized three times at three- week intervals with 75 μg of detergent-soluble pneumococcal antigens extracted from strain 64 (type 6) in 25 μg of Quil A adjuvant (Cedarlane Laboratories Ltd. , Hornby, Ontario, Canada) . Three days before fusion, all mice were injected intraperitoneally with the respective antigen suspended in PBS alone. Hybridomas were produced by fusion of spleen cells with nonsecreting SP2/0 myeloma cells as previously described by J. Hamel et al. [J. Med. Microbiol. , 23, pp. 163-170 (1987)] . Specific hybridoma were cloned by sequential limiting dilutions, expanded and frozen in liquid nitrogen. The class, subclass, and light-chain type of MAbs were determined by ELISA as described by D. Martin et al . , [Eur. J. Immunol., 18, pp. 601-606 (1988)] using reagents obtained from Southern Biotechnology Associates Inc. (Birmingham, AL) .
2. Subcellular Fractionation
Pneumococci were separated into subcellular fractions according to the technique described by Pearce et al. [Mol. Microbiol., 9, pp. 1037-1050 (1993)] . Briefly, S. pneumoniae strain 64 (type 6) was grown in Todd Hewitt broth supplemented with 0.5% (w/v) yeast extract for 6 hours at 37°C and isolated by centrifugation. Cell pellets were resuspended in 25 mM Tris-HCl pH 8.0, 1 mM EDTA, 1 mM phenylmethylsulphonylfluoride (PMSF) and sonicated for 4 minutes with 15 second bursts. Cellular debris were removed by centrifugation. The bacterial membranes and cytoplasmic contents were separated by centrifugation at 98,000 g for 4 hours. The cytoplasmic (supernatant) and the membrane (pellet) fractions were adjusted to 1 mg protein per ml and subjected to SDS-PAGE and immunoblot analyses .
B. Identification and Characterization of MAbs to the HSP72 of S. pneumoniae
Culture supernatants of hybridomas were initially screened by dot enzyme immunoassay using whole cells from S. pneumoniae strain 65 (type 4) according to the procedures described in D. Martin et al. (supra) . Positive hybridomas were then retested by immunoblotting in order to identify the hybridomas secreting MAbs reactive with the HSP72. Of 26 hybridomas with anti- S. pneumoniae reactivity in immunoblot, four were found to recognize epitopes present on a protein band with an apparent molecular mass of 72 kDa. The four hybridomas were designated Fl-Pn3.1 (from fusion experiment 1) and F2-Pn3.2, F2-Pn3.3 and F2-Pn3.4 (from fusion experiment 2) . 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
fusion experiment 2) all secreted IgGi*. The specificity_ of the MAbs for HSP72 was clearly demonstrated by the lack of radioimmunoprecipitation activity against [35S]methionine-labeled S. pneumoniae proteins obtained from cultures incubated at 37°C and the immunoprecipitation of a 72kDa-protein with heat shock-derived lysates incubated at 45°C. FIG. 6, (lanes 5 and 6) demonstrates the results obtained for MAb Fl-Pn3.1. The same results were obtained with MAbs F2-Pn3.2, F2-Pn3.3 and F2-Pn3.4 [35S]methionine-labelled lysates from nonheat- shocked and heat-shocked S. pneumoniae cells probed with the MAbs were electrophoresed on SDS-PAGE gels and then subjected to Western blot analysis. The resulting immunoblots revealed the presence of HSP72 antigen in both samples. FIG. 7, panel A, shows the results obtained for MAb 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 B) . These experiments revealed that the rate of synthesis of S. pneumoniae HSP72 increases in response to heat shock, but that the absolute amounts of HSP72 do not increase after heat shock.
C. Cellular localization of HSP72
In order to investigate the cellular location of HSP72, S. pneumoniae cell lysates were fractionated by differential centrifugation resulting in a soluble fraction and a particulate fraction, enriched in membrane proteins, supra. Sample containing 15 μg protein of membrane fraction (lane 1) and cytoplasmic fraction (lane 2) of S. pneumoniae were electrophoresed on SDS-PAGE, transferred to nitrocellulose and probed with MAb Fl-
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.
TABLE 1: BACTERIAL STRAINS, PHAGES AND PLASMIDS
Strain, Relevant Characteristics Reference Phage Plasmid or Source
E. coli Strains JM109 A ( lac-proAB) [F ' traD proAB BRL lacl* ZΔM15]
Y1090 rfc-m*- Ion supF [pMC9] Amersham BL2KDE3) lacUV5-T7 RNA polymerase Studier et al. (infra)
Phages λgtll CJ857 SI00 cloning vector Amersham λJBD7 LacZ-HSP72 fusion; 2.3 kb This study
EcoRI fragment in λgtll λJBD17 FucI-HSP72 chimeric; 2.4 This study kb EcoRI and 2.3 kb EcoRI fragments in λgtll
Plasmids pWSK29 Ampr; low copy number Wang et al. cloning vector (infra)
PWKS30 same as pWSK29 but Wang et al . opposite multi cloning (infra) .site
PJBD171 same as λJBDl7 but in This study pWSK29 PJBD177 2.8 kb XhoI-EcoRI This study fragment in pWKS30 no recombinant HSP72 protein expressed
PJBD179 FucI-HSP72 fusion; 2.4 kb This study EcoRI and 0.8 kb EcoRI- EcoRV fragments in pWSK29 pT7-5 Ampr; T7 promoter ΦlO Tabor et al. (infra) pT7-6 same as pT7-5 but Tabor et al. opposite multi cloning (infra) site pJBDf51 same as pJBD17 but in This study pT7-5 pJBDf62 same as pJBD179 but in This study pT7-6 pDELTAl Ampr; Tn 1000 BRL pJBDΔl same as pJBD179 but in This study pDELTAl
PJBD291 HSP72; 3.2 kb Hindlll This study fragment in pWSK29 pJBDk51 same as pJBD291 but in This study pT7-5 pJBDΔ4 same as pJBD2 1 but in This study pDELTAl
E. coli strains were grown in L broth or on L agar at 37°C. When necessary, ampicillin was added to the media at the concentration of 50 μg/ml. Plasmids were isolated by using the Magic/Wizard® Mini-Preps kit (Promega, Fisher Scientific, Ottawa, Canada) . 2. General Recombinant DNA Techniques
Restriction endonucleases, T4 DNA ligase, and DNA molecular weight standards were purchased from Boehringer Mannheim Canada, Laval, Quebec or Pharmacia Biotech, Uppsala, Sweden. DNA restriction endonuclease digestion and ligation were performed as described by J. Sambrook et al . [Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory Press, N.Y. (1989)] . Agarose gel electrophoresis of DNA fragments was performed following the procedure of J. Sambrook et al .
(supra) using the TAE buffer (0.04 M Tris-acetate; 0.002 M EDTA) from Boehringer Mannheim. DNA fragments were purified from agarose gel by using the Prep-A-Gene® DNA purification kit (Bio-Rad Laboratories Ltd., Mississauga, Ontario) . Transformation was carried out by electroporation with the Gene Pulser® (Bio-Rad) following the protocol provided by the manufacturer.
3. Construction and Screening of Genomic Library
A genomic S. pneuznoniae DNA library was generated in the bacteriophage expression vector λgtll (λgtll cloning system, Amersham) according to the
procedure provided by the manufacturer. Chromosomal DNA _ of S. pneumoniae type 6 strain 64 was prepared by following the procedure of J.C. Paton et al . [Infect . Immun. , 54, pp. 50-55 (1986)] . The S. pneumoniae chromosomal DNA was partially digested with EcoRI, and the 4- to 7-kb fragments were fractionated and purified from agarose gel. The fragments were ligated into λgtll arms, packaged, and the resulting phage mixtures used to infect E. coli Y1090. Immunoscreening of plaques expressing recombinant HSP72 antigens was performed using HΞP72- specific 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 Hindlll and the digests were separated by electrophoresis on an 0.8% SDS-PAGE gel and transformed onto positive charged nylon membranes (Boehringer Mannheim) as described by J. Sambrook et al . (supra) . The membrane was then blotted with the DIG- labelled DNA probes according to the protocol of the manufacturer .
5. DNA Sequencing and Sequence Analysis
The DNA fragments sequenced in this example were first cloned into plasmid pDELTA 1 (GIBCO BRL Life
Technologies, Burlington, Ontario) . A series of nested deletions were generated from both strands by in vivo deletion mediated by Tn 1000 transposon transposition (Deletion Factory System, GIBCO BRL) following the procedures provided by the supplier. These deletions were sized by agarose gel electrophoresis and appropriate deletion derivatives were selected for sequencing by the dideoxynucleotide chain terminating method of F. Sanger et al. [Proc. Natl . Acad. Sci. USA, 74, pp. 5463-5467 (1977)] . To sequence the gaps between deletion templates, oligonucleotides were synthesized by oligonucleotide synthesizer 392 (ABI, Applied Biosystems Inc., Foster City, CA) . The sequencing reaction was carried out by PCR (DNA Thermal Cycler 480®, Perkin Elmer) using the Taq DyeDeoxy Terminator Cycle Sequencing kit (ABI), and DNA electrophoresis was performed on automated DNA sequencer 373A (ABI) .
6. Expression of Cloned Gene in E. coli T7 RNA pol/promoter system
High level expression of the cloned gene in this example was achieved by employing the bacteriophage T7 RNA polymerase/promoter system in E. coli . The DNA fragment specifying the recombinant protein was ligated into plasmids pT7-5 or pT7-6 [S. Tabor and C.C. Richardson, Proc. Natl. Acad. Sci. USA, 82, PP. 1074-1078 (1985)], in a proper orientation in which the gene to be expressed was placed under the control of phage T7 RNA polymerase specific promoter Φ10. The resulting plasmid was transformed into E. coli strain BL21(DE3) [F.W. Studier, and B.A. Moffatt, J. Mol. Biol., 189, pp. 113-130 (1986)] which carries the T7 RNA polymerase structural gene on its chromosome under the control of the inducible lacUV5 promoter. Upon IPTG induction, the T7 RNA polymerase induced in the BL21(DE3) transformants specifically
transcribed the gene under the control of T7 promoter Φ1Q.. 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 [J. Biol. Chem., 262, pp. 10035-10038
(1987)] . PVDF membrane was stained with Coomassie Blue, the HSP72 band excised and then analyzed in an automated protein sequencer (ABI), according to standard procedures.
B. Construction of Plasmids Containing S. pneumoniae HSP72 Gene Fragments Corresponding to C-169
The λgtll 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 λJBD7 and λJBD17 for further characterization. As shown in FIG. 9, whole cell extracts from S. pneumoniae strain 64 (lane 1) and phage lysates from E. coli infected with λJBD17 (lanes 2 and 3) or λJBD7 (lanes 4 and 5) cultured in the presence (+) or absence (-) of IPTG were subjected to 10% polyacrylamide
gel electrophoresis and were electrotransferred to nitrocellulose. The immunoblot was probed with HSP72- specific MAb Fl-Pn3.1. Clone λJBDl7 had two EcoRI-EcoRI insert fragments of 2.4 kb and 2.3 kb (FIG. 10), and expressed a chimeric recombinant protein having an apparent molecular mass of 74 kDa on SDS-PAGE gel (FIG. 9, lanes 2 and 3) . Clone λJBD7 was found to contain a 2.3 kb EcoRI insert fragment and produced an apparent fusion protein consisting of LacZ and the 74 kDa chimeric .protein expressed from clone λJBD17. The fusion protein had an apparent molecular mass of 160 kDa as estimated by SDS- PAGE (FIG. 9, lane 5) . The expression of the chimeric recombinant protein encoded by phage λJBDl7 was independent of IPTG induction (FIG. 9, lanes 2 and 3) while the expression of the recombinant fusion protein encoded by phage λJBD7 was dependent on induction of the lac promoter (FIG. 9, lanes 4 and 5) .
In an attempt to subclone the HSP72 gene, the pneumococcal DNA insert from clone λJBD17 was extracted, purified and ligated into a low copy plasmid pWSK29 [R.F. Wang and S.R. Kushner, Gene, 100, pp. 195-199 (1991)] to generate plasmid pJBD171. The insert from pJBD171 was characterized by restriction mapping (Fig. 10B) , and a series of subcloning and immunoblotting was carried out to define the boundaries of the gene coding for the antigen reactive with MAb 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.
C. Expression and DNA Sequence Analysis of a Chimeric Gene Coding for C-169
To further determine the transcriptional direction of the gene coding for the 74 kDa chimeric protein on the 3.2 kb EcoRI-EcoRV fragment, and to increase the yield of the 74 kDa chimeric protein for immunological study, we decided to express the 74 kDa chimeric protein in the E. coli T7 RNA and T7 promoter system. The 3.2 kb EcoRI-EcoRV fragment, derived from PJBD179, was ligated into plasmids pT7-5 and pT7-6 in which the multi-cloning sites were placed in opposite orientation with respect to the T7 RNA polymerase specific T7 promoter Φ10. The ligation mixture was used to transform E. coli JM109 and positive transformants reactive with MAb 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°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 Fl-Pn3.1 (FIG. 11) . However, under the IPTG induction condition, only transformants BL21 (DE3 ) (pJBDf51) overexpressed the 74 kDa chimeric protein (FIG. 11A and B, lane 2) indicating that the transcriptional direction of the gene on the 3.2
kb EcoRI-EcoRV fragment is from the EcoRI end towards the_ EcoRV end (FIG. 10A) .
The 3.2 kb EcoRI-EcoRV fragment was cloned into plasmid pDELTA 1 to yield plasmid pJBDΔl. A series of overlapping deletions were generated and used as DNA sequencing templates. The DNA sequence of the entire 3.2 kb EcoRI-EcoRV insert is SEQ ID N0:1. Two open reading frames ("ORFs") were found and their orientation is indicated in FIG. 10B ("ORF27" and "FucI-HSP72 (C-169)") . In front of these two ORFs, putative ribosome-binding sites were identified (SEQ ID N0:1, nucleotides 18-21 and 760-763) . No obvious -10 and -35 promoter sequences were detected. ORF27 spans nucleotides 30-755 (SEQ ID NO:l) 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 or£21 , and compared it to other known sequences. No homologous gene or protein was found. The large ORF (nucleotides 771-2912, SEQ ID NO:l) 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 N0:3. This ORF was compared with other known sequences to determine its relationship to other amino acid sequences. This analysis revealed a high degree of similarity of the encoded protein to the sequence of E. coli fucose isomerase (Fuel) and to several HSP70 gene family members, also known as DnaK genes. Alignment of SEQ ID NO:3 and those of the E. coli Fuel and HSP70 (Dnak) proteins indicated that the N-terminal portion corresponding to amino acids 1 to 545 (SEQ ID
NO:3 ) of the 74 kDa chimeric protein is highly homologous to E. coli Fuel, while the C-terminal portion corresponding to amino acids 546-714 (SEQ ID NO:3 ) is similar to HSP70 (DnaK) proteins. It is noteworthy that there is an EcoRI restriction site lying in the junction of these two portions of the gene coding for the 74 kDa protein (SEQ ID NO:l, between nucleotides 2404 and 2405) .
Other restriction sites exist between nucleotides 971 and_ 972 (Pst I), nucleotides 1916 and 1917 (Pst I), nucleotides 1978 and 1979 (Xho I), and nucleotides 3164 and 3165 (EcoRV) . From these data we concluded that the 74 kDa protein was a chimeric protein encoded by two pieces of S. pneumoniae chromosomal DNA, a 2.4 kb EcoRI- EcoRI fragment derived from the Fuel homologous gene and a 2.3 kb EcoRI-EcoRI fragment derived from the HSP72 gene. D. Southern Blot Analysis
Southern blotting was performed in order to confirm that the 74 kDa protein is a chimeric protein and to attempt to clone the entire pneumococcal HSP72 gene. Chromosomal S. pneumoniae DNA was digested with Hindlll to completion, separated on a 0.8% agarose gel, and transferred onto two positively charged nylon membranes (Boehringer Mannheim) . The membranes were then blotted with either the 0.8 kb EcoRI-EcoRV probe, derived from the 2.3 kb EcoRI-EcoRI fragment, or the 1 kb Pstl-PstI probe, obtained from the 2.4 kb EcoRI-EcoRI fragment. Both probes had been previously labelled with digoxigenin-dUTP. These two probes hybridized two individual Hindlll fragments of different sizes (FIGS. 10B and IOC) . The 0.8 kb EcoRI-EcoRV probe recognized the 3.2 kb Hindlll fragment and the 1 kb Pstl-PstI probe reacted with the 4 kb Hindlll fragment. This result further indicated that the gene responsible for the expression of the 74 kDa chimeric protein was generated by fusion, in frame, of two pieces of EcoRI fragments, one originated from the fragment containing the 5 ' portion of the S. pneumoniae Fuel homologue, the other derived from the segment carrying the C-169 fragment of the pneumococcal HSP72 gene. The fact that the 0.8 kb EcoRI-EcoRV probe hybridized a single 3.2 kb fragment suggested that there is only a single HSP72 gene copy in S. pneumoniae .
E. Production of Recombinant HSP72
A partial pneumococcal genomic library was generated by ligation of the pool of Hindlll digests of chromosomal DNA, with sizes ranging from 2.8 to 3.7 kb, into plasmid pWSK29/Hindlll. The ligation mixture was used to transform E. coli strain JM 109 and the transformants were screened by hybridization with the 0.8 kb EcoRI-EcoRV probe. One representative plasmid from four positive hybridizing clones was named pJBD291. Restriction analysis of the insert and Western blot of the cell lysate of transformants were employed to verify that the plasmid pJBD291 indeed carries the 3.2 kb Hindlll fragment containing the HSP72 gene expressing the recombinant HSP72 protein (FIG. 10B) . The HSP72 protein expressed by the transformants (pJBD291) migrated on the SDS-PAGE gel at the same position as the native HSP72 protein (FIG. 12) . To sequence the entire HSP72 gene and to overexpress the full-length HSP72 protein, the 3.2 kb Hindlll fragment was isolated from plasmid pJBD291, and subcloned into plasmids pDELTA 1 and pT7-5 to generate pJBDΔ4 and pJBDk51, respectively.
The entire 3.2 kb Hindlll DNA fragment carried on the plasmid pJBDΔ4 and the 2.3 kb EcoRI-EcoRI DNA fragment contained on the plasmid pJBD177 were sequenced. Altogether, the nucleotide sequence comprised 4320 base pairs and revealed two ORFs (SEQ ID NO:4) . The first ORF, starting at nucleotide 682 and ending at nucleotide 2502 (SEQ ID NO:4), was identified as the pneumococcal HSP72 gene, and the second ORF, spanning from nucleotide 3265 to nucleotide 4320 (SEQ ID NO:4) , was located 764 base pairs downstream from the HΞP72 structural gene and was identified as the 5 ' portion of the pneumococcal DnaJ gene. The putative ribosome binding site ( "AGGA") was located 9 base pairs upstream from the start codon of the HSP72 structural gene, while the typical ribosome binding
site ("AGGA") was found 66 base pairs upstream from the - start codon of the DnaJ structural gene. No typical 5' regulatory region was identified in front of these two genes . Restriction sites are located between nucleotides 1 and 2 (Hindlll), nucleotides 1318 and 1319 (EcoRI) , nucleotides 1994 and 1995 (EcoRI) , nucleotides 3343 and 3344 (Hindlll), and nucleotides 4315 and 4316 (EcoRI) . The gene organization of HSP72 (DnaK) and DnaJ in S. pneumoniae is similar to that of E. coli [Saito, H. and Uchida, Mol. Gen. Genet. 164, 1-8 (1978)] as well as several other Gram positive bacteria [Wetzstein, M. et al., J. Bacteriol. 174, 3300-3310 (1992)] . However, the intragenic region of S. pneumoniae is significantly larger and no ORF for the grpE gene was found upstream of the HSP72 (DnaK) structural gene.
The predicted HSP72 protein has 607 amino acids and a calculated molecular mass of 64,755 daltons, as compared to the 72 kDa molecular mass estimated by SDS- PAGE. The predicted HSP72 protein is acidic with an isoelectric point (pi) of 4.35. Automated Edman degradation of the purified native HSP72 protein extracted from S. pneumoniae strain 64 revealed SKIIGIDLGTTN-AVAVLE as the 19 amino acid N-terminal sequence of the protein. The amino-terminal methionine was not detected, presumably due to in si tu processing which is known to occur in many proteins. No amino acid residue was identified on position 13. The 19 amino acid N-terminal sequence obtained from the native HSP72 protein is in full agreement with the 19 amino acid N-terminal sequence deduced from the nucleotide sequence of the recombinant
S. pneumoniae HΞP72 gene (SEQ ID NO: 5) thus confirming the cloning. This N-terminal sequence showed complete identity with the DnaK protein from Lactococcus lactis and 68.4% identity with the DnaK protein from Escherichia Coli . Similarly, the alignment of the predicted amino acid sequence of HSP72 (SEQ ID NO:5) with those from other bacterial HSP70 (DnaK) proteins also revealed high
homology (FIGS. 13A-13D) . For example, HSP72 showed 54% - identity with the E. coli DnaK protein. The highest identity value was obtained from comparison with the Gram positive bacterium Lactococcus lactis , showing 85% identity with HSP72. Like other HSP70 proteins of Gram positive bacteria, HSP72 misses a stretch of 24 amino acids near the amino terminus when compared with DnaK proteins from Gram negative bacteria (FIGS. 13A-13D) .
Although HSP72 shares homology with HSP70 (DnaK) proteins from other organisms, it does possess some unique features. Sequence divergence of the HSP70 (DnaK) proteins is largely localized to two regions (residues 244 to 330 and 510 to 607, SEQ ID N0:5) . 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 determinants specific to S. pneumoniae . Consistent with this hypothesis, monoclonal antibodies directed against the C- 169 fragment of HSP72 (infra) , were not reactive with E. coli and S. aureus , which are known to express DnaK proteins similar to HSP72.
The truncated DnaJ protein of S. pneumoniae (SEQ ID NO: 6) has 352 amino acids, which show a high degree of similarity with the corresponding portions of the L . lactis DnaJ protein (72% identity) and the E. coli DnaJ protein (51% identity) . The predicted truncated DnaJ protein contains high glycine content (15%) . Four Gly-, Cys-rich repeats, each with the Cys-X-X-Cys-X-Gly-X-Gly motif characteristic of DnaJ proteins [P.A. Silver and J.C. Way, Cell, 74, pp. 5-6 (1993)], were identified between amino acids 148 and 212 of the S. pneumoniae DnaJ protein (SEQ ID NO: 6 ) . Three repeated GGFGG sequences (residues 75-79, 81-85, and 90-94) were found near the N- terminus .
F. Reactivity of MAbs Against Recombinant Antigens The four HSP72 specific MAbs (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 λJBD7, thus localizing the epitopes recognized by these MAbs to the C-terminal 169 residues. Surprisingly, the proteins encoded by the pneumoccocal inserts in λJBD17 and pJBDΔl were recognized by only 3 of 4 Mabs . These results suggest that although the C-169 fragments synthesized in E. coli infected with λJBD7 and λJBD17 have the same primary structure, they have distinct conformation. The lack of reactivity of MAb F2-Pn3.2 with some recombinant proteins raised the possibility that this particular MAb recognizes a more complex epitope.
Although complex, F2-Pn3.2 epitopes are still recognizable on Western immunoblots . The complete HSP72rβc protein expressed by E . col i containing the recombinant plasmid pJBDΔ4 was reactive with all four MAbs.
EXAMPLE 4 - Antigenic Specificity and
Reactivity of HSP72-Specific Monoclonal Antibodies
The reactivity of MAbs 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
and then suspended in PBS, pH 7.4. A volume of 5 μl of a suspension containing approximately 109 CFU/ml was applied to a nitrocellulose paper, blocked with PBS containing 3% bovine serum albumin, and then incubated sequentially with MAbs and peroxydase-labeled secondary antibody. Whole cell extracts were prepared for Western blot analysis by boiling bacterial suspensions in sample buffer for 5 minutes.
TABLE 2:LIST OP NON-PNEUMOCOCCAL ISOLATES TESTED BY DOT ENZYME IMMUNOASSAY
Genus species group or type
Streptococcus pyogenes group A
Streptococcus agalactiae group B Enterococcus faecalis group D Streptococcus bovis group D Streptococcus mutans Streptococcus salivarius Streptococcus sanguis I Streptococcus sanguis I Streptococcus sanguis I Streptococcus sanguis II Streptococcus sanguis II Streptococcus sanguis II Streptococcus sanguis II Gemella morbillorum Staphylococcus aureus Bacillus
Escherichia coli
When tested by dot enzyme immunoassay, each MAb reacted with each of the S. pneumoniae strains and none of the non-pneumococcal isolates. These results were unexpected since comparison studies revealed that HSP72 is
very similar to other known bacterial HSP70 (DnaK) proteins, for example those from E. coli and S. aureus .
Immunoblots were then performed to further investigate the immunoreactivities of our MAbs. As shown in Table 3, each MAb exhibited some reactivity. Although the percent identity of the E. coli amino acid sequence and the HSP72 amino acid sequence (SEQ ID NO:5) is 54%, the four HSP72-specific MAbs did not recognize the E. coli HSP70 (DnaK) protein. Similarly, the HSP72-specific MAbs did not react with the C. trachomatis HSP70 (DnaK) protein, which has 56% amino acid identity with the amino acid sequence of HSP72. High amino acid sequence homology is observed between HSP72 and the HSP70 (DnaK) proteins from gram positive bacterial species. However, again, none of the HSP72-specific MAbs reacted with S. aureaus or Bacillus gram positive species, which exhibit 74% and 76% amino acid sequence homology, respectively, with HSP72. From these data it is clear that although HSP70 (DnaK) proteins may be structurally related to HSP72, they are immunologically distinct. Among the non-pneumococcal isolates that reacted with at least one MAb, there is S. pyogenes , Enterococcus faecalis , S. mutans and S. sanguis, which all belong to the Streptococcus or Streptococcus- related En terococcus genus. So far, neither the HSP70 protein, nor the gene structure has been identified in these Streptococcus or En terococcus species. Altogether, these observations indicate that hypervariable amino acid sequences or residues within HSP70 (DnaK) proteins are involved in antigenicity. Interestingly, immunoblotting analysis revealed that there was no significant variation in the molecular mass of the HSP70 (DnaK) proteins among both S. pneumoniae isolates and immunoreactive non- pneumococcal isolates .
TABLE 3: REACTIVITY OP MABS WITH NON-PNEUMOCOCCAL ISOLATES IN
WESTERN IMMUNOBLOTTING
Bacterial Strain MAbs
± indicates a weak signal compared to the reactivity observed with S. pneumoniae antigens b C. trachomatis purified elementary bodies were tested.
EXAMPLE 5 - Purification of HSP72 And Its Use As An Immunogen to Protect Against Lethal S. Pneumoniae Infection
A. Procedures
1. Preparation of Purified
Recombinant HSP72 Protein and Recombinant C-169
High level exclusive expression of the HSP72 gene was achieved by employing the bacteriophage T7 RNA polymerase/T7 promoter system in E. coli . The 3.2 kb Hindlll fragment was cloned in both orientations in front of the T7 promoter Φ10 in the plasmid pT7-5. The resulting plasmid pJBDk51 was then transformed into E. coli strain BL21 (DE3) . Overexpression of the recombinant HSP72 protein (HSP72rβc) was induced by culturing in broth supplemented with antibiotics for a 3- hour period after the addition of IPTG to a final concentration of 1 mM. E. coli expressing high levels of HSP72rβc were concentrated by centrifugation and lysed by mild sonication in 50 mM Tris-Cl (pH 8.0) , 1 mM EDTA and 100 mM NaCl lysis buffer containing 0.2 mg/ml lysozyme. The cell lysates were centrifuged at 12,000 g for 15 minutes and the supernatants were collected. HSP72rβc 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-169rβo) was expressed in the form of insoluble inclusion bodies in E. coli strain JM109 transformed with the plasmid pJBDΔl. Protein inclusion bodies were recovered from pelleted bacterial cells disrupted by sonication as described before. The pellets were washed in lysis buffer containing 1 mg/ml of deoxycholate to remove contaminating materials, and the protein inclusion bodies were then solubilized in urea 6 M. The protein solution was
centrifuged at 100,000 g and the cleared supernatant collected and dialysed against phosphate-buffered saline. After purification, the protein content was determined by the Bio-Rad protein assay (Bio-Rad Laboratories, Mississauga, Ontario, Canada) .
2. Active Immunoprotection Studies
Two groups of 10 female Balb/c mice (Charles River Laboratories) were immunized subcutaneously three times at two-week intervals with 0.1 ml of purified HSP72rβc or C-169rβc antigens absorbed to Alhydrogel adjuvant. Two antigen doses, approximately 1 and 5 μg, were tested. A third group of 10 control mice were immunized identically via the same route with Alhydrogel adjuvant alone. Blood samples were collected from the orbital sinus prior to each immmunization and five to seven days following the third injection. The mice were then challenged with approximately 106 CFU of the type 3 S. pneumoniae strain WU2. Samples of the S. pneumoniae challenge inoculum were plated on chocolate agar plates to determine the CFU and to verify the challenge dose. Deaths were recorded at 6-hour intervals for the first 3-4 days post-infection and then at 24-hour intervals for a period of 14 days. On days 14 or 15, the surviving mice were sacrificed and blood samples tested for the presence of S. pneumoniae organisms. Antibody responses to the recombinant HSP72 antigens are described in Example 7.
3. Passive Immunoprotection Studies
One NZW rabbit (Charles River Laboratories) was immunized subcutaneously at multiple sites with approximately 50 μg of the purified C-169rβc protein adsorbed to Alhydrogel adjuvant. The rabbit was boosted three times at two-week intervals with the same antigen and blood samples collected 7 and 14 days following the
last immunization. The serum samples were pooled and antibodies were purified by precipitation using 40% saturated ammonium sulfate.
Severe-combined immunodeficient SCID mice were injected intraperitoneally with 0.25 ml of the purified rabbit antibodies 1 hour before intravenous challenge with 5000 or 880 CFU of the type 3 S. pneumoniae strain WU2. Control SCID mice received sterile buffer or antibodies purified from nonimmune rabbit sera. Samples of the S. pneumoniae challenge inoculum were plated on chocolate agar plates to determine the CFU and to verify the challenge dose. The SCID mice were chosen because of their high susceptibility to S. pneumoniae infection. Blood samples (20 μl each) obtained 24 hours post- challenge were plated on chocolate agar and tested for the presence of S. pneumoniae organisms. The level of detection was 50 CFU/ml. Deaths were recorded at 24-hour intervals for a period of 5 days. B. Results
The availability of cloned S. pneumoniae DNA inserts encoding the complete or partial (C-169) HSP72 protein and the expression of recombinant proteins in E. coli allowed the obtention of purified proteins useful for the investigation of the vaccinogenic potential of HSP72 protein. Both HSP72rβc and C-169rβc 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 HSP72rβc to elicit a protective immune response. Groups of 10 mice were immunized with full-length HSP72rβo (1 μg or 5 μg dose) and challenged with 4.2 million CFU of S. pneumoniae type 3 strain WU2. Eighty percent (80%) of the mice dosed with 1 μg HSP72rβc survived the challenge, as did 50% of the mice
dosed with 5 μg HSP72. None of the naive mice immunized with Alhydrogel adjuvant alone without antigen survived the challenge (FIG. 16) . No S. pneumoniae organisms were detected in any of the blood samples collected on days 14 or 15 from mice surviving infection. The observation that HSP72rβc elicited protection against type 3 strain WU2 pneumococci indicated that HSP72 derived from DNA extracted from a type 6 strain contains epitopes capable of eliciting protection against a heterologous strain having a different capsular type.
We further examined the immune response to the HSP72 protein by using recombinant protein fragments expressed from E. coli transformed with a chimeric fucT- HSP72 gene. Mice immunized with purified C-169rβc 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-169rβc (1 μg or 5 μg doses) and challenged with 6 million CFU of S. pneumoniae type 3 strain WU2. Sixty percent (60%) of the mice dosed with 1 μg C-169rβc survived the challenge, as did 70% of the mice dosed with 5 μg C-169rβc (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-169rβc antibodies were protected from fatal infection with S. pneumoniae WU2. In contrast, none of the 15 control mice survived. The control mice received antibodies from nonimmune rabbit sera or received sterile buffer alone. In addition, all mice from the control groups had positive S. pneumoniae hemoculture 24 hours post-challenge, while S. pneumoniae
organisms were detected in only 2 out of a total of 10 immunized SCID mice.
TABLE 4: PASSIVE IMMUNIZATION STUDIES SHOWING PROTECTION OP SCID MICE FROM EXPERIMENTAL S. PNEUMONIAE INFECTION BY ANTI-C-169roc RABBIT ANTIBODIES
Experiment Injection No. of Mice No. of Mice
Surviving Testing
Challenge Positive for after 5 days the Presence of
In experiments 1 and 2 (Table 4), mice were challenged with 5000 and 880 CFU of type 3 S. pneumoniae strain WU2, respectively. Results in Table 4 are expressed as the number of mice surviving challenge, or testing positive for the presence of S. pneumoniae , compared to the total number of mice in each group.
Demonstration of the anti-HSP72 specificity of the antibody elicited by immunization with recombinant HSP72 or C-169 proteins came from Western Blot analyses using S. pneumoniae cell lysates as antigens. A single band corresponding to HSP72 was detected by all rabbit and mouse antisera tested. These serologic results suggested that the protection following the immunization with recombinant proteins was due to the production of antibodies reactive with S. pneumoniae HSP72.
EXAMPLE 6 - Heat-Indueible Expression System for High Level Production of the C-151 Terminal Portion of the HSP72 Protein
A. Construction of Plasmid pURV3 Containing the C- 151 terminal coding region of the HSP72 of S. pneumoniae The DNA region coding for 151 amino acids at the carboxyl end of the HSP72 of S. pneumoniae was inserted downstream of the promoter λ PL into the translation vector p629 [H. J. George et al., Bio/Technology 5, pp. 600-603 (1987)] . This vector contains a cassette of the bacteriophage λ CI857 temperature sensitive repressor gene from which the functional PR promoter has been deleted. The inactivation of the cl857 repressor by a temperature increase from the ranges of 30-37°C to 37-42°C results in the induction of the gene under the control of λ PL. The induction of gene expression in E. coli cells by a temperature shift is advantageous for large scale fermentation since it can easily be achieved with modern fermenters. However, it should be understood that while E. coli was the microorganism of choice in the experiments herein described, other host organisms, such as yeast, are intended to be included within the scope of this invention.
A fragment of 477 nucleotides, including the region of 457 bases between 2050 to 2506 in HSP72 gene of S. pneumoniae (see SEQ ID NO 4), was amplified by the polymerase chain reaction (PCR) from the S. pneumoniae type 6 strain 64 genomic DNA using the oligonucleotide primers OCRR26 (5 ' -GGCAGATCTATGAAGGCCAAAGACCTTGGAAC) and OCRR27 (5 ' -CGCGGATCCTTACTTTTCCGTAAACTCTCCGT) . Chromosomal DNA was prepared from a 90 ml culture of exponentionally growing cells of S. pneumoniae in heart infusion broth using the method of Jayarao et al. [J. Clin. Microbiol., 29, pp. 2774-2778 (1991)] . DNA amplification reactions were made using a DNA Thermal Cycler, Perkin Elmer, San Jose, CA. In OCRR26, an ATG start codon is present in frame just upstream of the
coding region for the amino-terminus region of the C-15X The primers OCRR26 and OCRR27 contain, respectively, a Bgill (AGATCT) and a BamHI (GGATCC) recognition site in order to facilitate the cloning of the PCR product into the dephosphorylated restriction sites Bgill and BamHI of p629. The PCR product was purified from agarose gels by the method of phenol freeze [S. A. Benson, Biotechniques 2, pp. 67-68 (1984)] and digested with the restriction enzymes Bgill and BamHI. The Bglll-BamHI fragment of 471 base pairs was then ligated into the Bgill. and BamHI recognition sites dephosphorylated of p629. A partial map of the resulting plasmid pURV3 is shown in FIG. 18. This plasmid was transformed by the method of Simanis [Hanahan, D. In D. M. Glover (ed.), DNA Cloning, pp. 109-135, (1985)] into the E. coli strain XLI Blue MRF' (A (mcrA) 183 A (mcrCB-hsdSMR-mrr) 173 endAl supE44 thi -1 recAl gyrA96 relAl lac [F ' proAB lacI^ZAMlδ Tnl O (Tetr)]c ) which was obtained from Stratagene, La Jolla, CA. The transformants grown at 37°C were screened by colony immunoblot [J. Sambrook et al . (supra) ] using the MAb Fl-Pn3.1 reactive with C-169rec. Plasmid DNA was purified from a selected transformant and the DNA insert was seguenced by PCR using the Taq Dye Deoxy Terminator Cycle Sequencing kit of Applied Biosystems Inc. (ABI) and DNA electrophoresis was performed on automated DNA sequencer 373A (ABI) . The nucleotide sequence of the insert perfectly matched the nucleotide sequence of the C-151 coding region of the HSP72 gene. (See SEQ ID No: 25 and corresponding amino acid sequence at SEQ ID No : 26. ) The plasmid was transformed into the prototrophic E . coli strain W3110 (ATCC 27325) for the production of C-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
grown at 30°C in LB broth containing 100 μg of ampicillin- per ml until the AgQO reached a value of 0.6. The cells were then cultivated at 40°C for 18 hours to induce the production of C-151rec protein. A semi-purified C-151rec protein was prepared using the following procedures. The bacterial cells were harvested by centrifugation and the resulting pellet was washed and resuspended in phosphate- buffered saline. Lysozyme was added and the cells were incubated for 15 min on ice before disruption by pulse sonication. The cell lysates were cleared by centrifugation and the supernatants were collected and subjected to separation using an Amicon's ultrafiltration equipment (stirred cells series 8000, Amicon Canada Ltd. Oakville, Ontario) . The ultrafiltrate not retained by a YM30 membrane was recovered, analysed by SDS-PAGE and stained with Coomassie blue R-250. Protein concentrations were estimated by comparing the staining intensity of the C-151rec protein with those obtained with defined concentrations of soybean trypsin inhibitor.
C . Reactivity of MAbs Against C-151rec A panel of 10 monoclonal antibodies selected for their reactivity with the_S. pneumoniae HSP72 protein were tested for their reactivity to C-151rec by Western blot analysis using YM30-ultrafiltrates prepared as described above. The MAbs included a series of six monoclonal antibodies raised to the HSP72rec protein (F3- Pn3.5 to 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-169rec also recognized the C-151rec fragment. All other MAbs were only reactive with HSP72rec thus indicating that they may be directed against epitopes present in the amino terminal region of the HSP72 protein.
EXAMPLE 7 - Antibody Response of Balb/c Mice and Macaca- Fascicularis (cynomolgus) Monkeys to Recombinant HSP72 Antigens
A. Procedures 1. Immunization of Animals
Groups of 10 female Balb/c mice were immunized subcutaneously with either HSP72 rec or C-169 rec as described in Example 5. In order to assess the antibody response to C-151rec, a group of 6 mice were immunized three times at two-week intervals with 0.5 μg 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. pneumoniae by ELISA using plates coated with S. pneumoniae cell wall extracts.
Female cynomolgus monkeys were immunized intramuscularly at Day 1, 22 and 77 with 0.5 ml containing 150 μg of purified HSP72rθC or C-169rθC antigens absorbed to Alhydrogel adjuvant. Blood samples were collected regularly before and after each immunization and the sera were tested for antibody reactive with S. pneumoniae HSP72 antigen by Western blot analysis .
The specificity of the raised antibodies for_S. pneumoniae HSP72 was confirmed by Western blot analyses to S. pneumoniae cell extracts and purified recombinant antigens .
B. Results The results previously described in Example 5 clearly demonstrate the protective nature of the antibody response elicited following immunization with recombinant HSP72 antigens . Here we monitored the appearance of serum antibody response in mice (FIG. 19, 20 and 21) and in monkeys (FIG. 22) during the immunization schedule. Both species responded strongly to the full-length and truncated recombinant HSP72 proteins used as immunogens
with average titers of 1:64000 after the third injection.- Detailed analysis of individual sera revealed that each animal responded to the immunization in developping antibodies reactive with S. pneumoniae HSP72. In mice immunized with C-169rβc, the two doses tested, i.e. 1 and 5 μg, were similarly efficient with the induction of similar antibody titers (FIG. 20) . A strong boost response was observed after the second injection with C-169rθC with no enhancement in the antibody titers after a third injection. In contrast to this, we observed that the immune response to the HSP72rβc was dose- dependent. Increases in the specific antibody titers were observed after a second and a third injection with either HSP72rec or C-151rec (FIG. 19 and 21) . Study of the immune response of monkeys clearly indicated that the immunogenicity of recombinant HSP72 antigens is not restricted to rodents such as rabbit and mouse. The humoral response following the second injection with either antigen is characterized by a strong increase in HSP72-specific antibody titers that can persist for several weeks without any detectable decrease in their antibody titers (FIG. 22) . In addition, specific serum antibodies were detectable in the sera of each monkey after a single injection of recombinant antigens.
EXAMPLE 8 - B-Cell Epitope Mapping of HSP72 Stress Protein
In Example 3 , it was shown that significant variability in the primary sequence of the HSP70 proteins was mainly localized to two regions corresponding to amino acid residues 244 to 330 and 510 to 607 of the S. pneumoniae HSP72 protein. These variable regions may contain B-cell epitopes responsable for the antigenic heterogeneity reported in Example 4. To investigate this possibility, the reactivity of polyclonal and monoclonal
antibodies to S. pneumoniae HSP72 were tested against fourteen peptides selected to cover most of these regions. A. Procedures
Fourteen peptides of 14 to 30 amino acids residues were synthesized. The peptide sequences and their locations in the protein are summarized in Table 5. Peptides CS870, CS873, CS874, CS875, CS876, CS877, CS878, CS879, CS880 and CS882 were synthesized by Biochem Immunosystem Inc. (Montreal, Canada) using an automated peptide synthesizer. Peptides MAPI, MAP2, MAP3 and MAP4 were synthesized onto a branching lysine core as Multiple Antigenic Peptides (MAP) by the Service de Sequence de Peptides de l'Est du Quebec, Centre de recherche du CHUL (Sainte-Foy, Canada) . Peptides were purified by reverse- phase high-pressure liquid chromatography. Peptides were solubilized in distilled water except for peptides CS874 and CS876 which were solubilized in a small volume of either 6M guanidine-HCl or dimethyl sulfoxide and then adjusted to 1 mg/ml with distilled water. Peptide ELISA were performed by coating synthetic peptides onto Immunolon 4 microtitration plates (Dynatech Laboratories, Inc., Chantilly, VA) at a concentration of 50 μg/ml according to the prodedures described in J. Hamel et al . [supra] . To confirm the reactivity of MAbs with peptides, the ability of fluid-phase peptides to inhibit MAb binding to solid HSP72 was determined. For the inhibition assay, microtitration plates were coated with S. pneumoniae cell wall extracts. Hybridoma culture supernatants containing the HSP72-specific MAbs were incubated overnight at 4°C with several concentrations of peptide. Peptide treated and control supernatants were then tested by ELISA as described above.
Immune sera were from animals immunized three times with recombinant HSP72 antigens. One rabbit was immunized with 37.5 μg of purified HSP72rec according to the immunization protocol described in Example 5. Pool murine sera were from three Balb/c mice immunized with
HSP72rec from Example 5 and monkey pool sera were from groups of two animals immunized with either HSP72rec or C- 169 ',rec •
TABLE 5: SEQUENCES AND LOCATIONS OF SYNTHETIC PEPTIDES CORRESPONDING TO S. PNEUMONIAE HSP72 AMINO ACID RESIDUES
B. Identification and Localization of Linear B-
Cell Epitopes
The results presented in FIG. 23 revealed that most of the immunological reactivity was observed with the
peptides localized within amino acid residues 457 and 607- corresponding to the C-151 fragment of HSP72. Rabbit, mice and monkey sera antibody from animals immunized with either recombinant HSP72rec 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 AEGAQATGNAGDDW (residues 586 to 600) defined as exclusive to S. pneumoniae HSP72 based on the comparison of HSP70 protein sequences available in the data banks. Our data thus revealed that both peptide sequences contain linear B-cell epitopes. In addition, the peptide MAP4 alone was also recognized by the MAb Fl-Pn3.1. This reactivity was confirmed by fluid-phase inhibition assays in which 10 μg/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, MAPI and MAP3. All together these data indicate that the hypervariable C-151 terminal fragment of the HSP72 stimulates B-cell responses and possibly constitutes the immunodominant portion of the HSP72 protein. The lack of reactivity of MAbs F2-Pn3.3 and F2-Pn3.4 with the synthetic peptides suggest that they react with conformational determinants present on the C-terminal region of the HSP72. The existence of protective epitopes in the C-151 region was strongly suggested in Example 5 where mice immunized with purified C-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
CS877 (amino acids 257 to 271) and CS878 (amino acids 268- to 281), peptides CS880 (amino acis 286-299) and peptides CS882 (amino acids 315-333) were identified by hyperimmune sera.
EXAMPLE 9 - HSP70 (DnaK) from Streptococcus pyogenes and Streptococcus agalactiae : Molecular Cloning and DNA Sequencing of the hsp70 Genes; Nucleotide and Protein Sequence Analyses; Antigenic Relatedness to S. pneumoniae; Increased Streptococcus agalactiae HSP70 synthesis"in response to heat.
A. Procedures
1. Bacterial Strains and Plasmid Vector The strains of S. pyogenes (Group A
Streptococcus ) and S. agalactiae (Group B Streptococcus) used in this study were provided by the Laboratoire de la Sante Publique du Quebec (LSPQ) , Sainte-Anne de Bellevue, Quebec, Canada. S. agalactiae type II strain V8 corresponds to the ATCC strain 12973. S. pyogenes strain Bruno corresponds to the ATCC strain 19615. The E. coli strain XLI Blue MRF' was obtained from Stratagene.
Streptococcal strains were grown at 37°C in a 5 % CO2 incubator. The streptococci were streaked on tryptic soy agar plates containing 5 % sheep blood (Les Laboratoires Quelab, Montreal, Canada), liquid cultures were made in heart infusion broth (Difco Laboratories, Detroit, MI) without agitation. The E. coli strain was grown at 37°C in L-broth with agitation at 250 rpm or on L- agar.
The general cloning phagemid pBluescript KS(-) was purchased from Stratagene.
2. Recombinant DNA Techniques Restriction enzymes, T4 DNA ligase, and calf intestinal phosphatase were used as recommended by the suppliers (Pharmacia [Canada] Inc., Baie d'Urfe, Canada; and New England Biolabs Ltd. , Mississauga, Canada) .
Preparation of plasmids by equilibrium centrifugation in - CsCl-ethidium bromide gradients, agarose gel electrophoresis of DNA fragments, Southern hybridization, and colony DNA hybridization were performed as described by J. Sambrook et al. [ supra] . Chromosomal DNA of the streptococcal bacteria was prepared using the procedure of B. M. Jayarao et al. [J. Clin. Microbiol., 29, pp. 2774- 2778 (1991)] adapted for bacterial cultures of 90 ml. Rapid plasmid preparations were made accordingly to D. Ish-Horowicz et al. [Nucl. Acids Res. 9, pp. 2989-2998 (1981)] . Plasmids used for DNA sequencing were purified using plasmid kits from Qiagen Inc. (Chatsworth, CA) . DNA fragments were purified from agarose gels by the method of phenol freeze [S. A. Benson, Biotechniques 2, pp. 67-68 (1984)] . DNA probes were labeled with a32P-dCTP or digoxigenin (DIG) -11-dUTP using the random primer labeling kits of Boehringer Mannheim (Laval, Canada) . Plasmid transformations were carried out by the method of Simanis [Hanahan, D. Jn D. M. Glover (ed. ) , DNA Cloning, pp. 109- 135, (1985)] . The sequencing of genomic DNA inserts in plasmids was done using synthetic oligonucleotides . The sequencing reactions were carried out by the polymerase chain reaction (PCR) using the Taq Dye Deoxy Terminator Cycle Sequencing kit (ABI) and DNA electrophoresis was performed on automated DNA sequencer 373A (ABI) . The assembly of the DNA sequence was performed using the program Sequencher 3.0 from the Gene Codes Corporation (Ann Arbor, MI) . Analysis of the DNA sequences and their predicted polypeptides were performed with the program Gene Works version 2.45 from Intelligenetics, Inc.
(Mountain View, CA) . DNA amplification reactions were made using a DNA Thermal Cycler 480, Perkin Elmer. Oligonucleotides were synthesized by oligonucleotide synthesizer model 394 (ABI) .
3. Molecular Cloning of the Genes hsp70/dnak- of S. agalactiae and S. pyogenes
Chromosomal DNA from S. agalactiae and S. pyogenes was digested to completion with various restriction enzymes with palindromic hexanucleotide recognition sequences. The digests were analysed by Southern hybridization using a labeled PCR-amplified DNA probe corresponding to a 782 base-pairs region starting at base 332 downstream from the ATG initiation codon of the HSP72 gene of S. pneumoniae (see SEQ ID NO 4) . This DNA region was selected because it is relatively well conserved among the hsp70 genes of Gram-positive bacteria that have been characterized. The PCR amplification was done on the genomic DNA of S. pneumoniae using the oligonucleotides OCRR2 (5 ' -AAGCTGTTATCACAGTTCCGG) and OCRR3 (5 '-GATACCAAGTGACAATGGCG) . Hybridizing genomic restriction fragments of sufficient size to code for a 70- kDa polypeptide (>1.8 kb) were partially purified by extraction of genomic fragments of corresponding size from agarose gel. Verification of the presence of the hsp70 gene among the purified genomic restriction fragments was done by Southern hybridization using the labeled 782-bp S. pneumoniae DNA probe.
The purified genomic DNA restriction fragments were cloned into dephosphorylated compatible restriction sites of pBluescript KS(-) and transformed into the E. coli strain XLI Blue MRF' . The colonies were screened by DNA hybridization using the labeled 782-bp S. pneumoniae DNA probe. Extracted plasmids were digested with various restriction enzymes to evaluate the size of the inserts and to verify the presence of the hsp70 gene by Southern hybridization using the labeled 782-bp S. pneumoniae DNA probe. Plasmid pURV5 contains a 4.2-kb Hindlll insert of the genomic DNA of S. agalactiae . Plasmid pURV4 contains a 3.5-kb Hindlll fragment of the genomic DNA of S. pyogenes .
4. Heat Shock and Protein Labeling The stress response of S. agalactiae to an heat shock was assayed by pulse-labeling with [35s]met ion e as described before in Example 1. S. agalactiae bacteria grown overnight in SMAM (Methionine assay Medium supplemented with 1 mg/1 methionine, 1% (v/v) Isovitalex and 1 mg/1 choline chloride) were pelleted by centrifugation and then resuspended in the methionine-free SMAM medium. The bacteria were incubated at 37°C for 1 h and then divided into two fractions of equal volume. The samples were either incubated at 37 or 43°C for 10 minutes and then labeled with 100 μCi/ml [^5S] ethionine for 30 minutes at 37°C. The bacteria were extensively washed with PBS and cell extracts were prepared by treatment with mutanolysine and lysozyme as described for the DNA isolation (M.Jayarao et al. , supra) followed by sonication.
5. Immunological Characterization A series of six monoclonal antibodies raised to the HSP72rec 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. _agalactiae_by Western blot analysis. Cell lysates from S. pyogenes and_S. agalactiae were obtained from treatment with mutanolysine and lysozyme (M.Jayarao et al . , supra) . , sonication and boiling in SDS-PAGE sample buffer. Cell lysates from E. coli transformed with either pURV4 or pURV6 producing truncated S . _pyogenes HSP70 antigens were tested after boiling in SDS-PAGE sample buffer.
B. DNA Sequence Analysis of the hsp70 /dnak Genes of Streptococcus pyogenes, Streptococcus agalactiae and Streptococcus pneumoniae
A region of 2438 bases in the 4.2-kb Hindlll insert of plasmid pURV5 was sequenced. This sequence
contains an open reading frame (ORF) of 1830 nucleotides- coding for a polypeptide of 609 amino acids with a molecular weight of 64907 (see SEQ ID NO: 7) . The ORF has an ATG start codon beginning at position 248 and TAA stop codon ending at position 2077. The ATG start codon is preceeded by the sequence GAGG, starting at position 237, which is complementary to 16S rRNA and serves as a ribosome binding site in E. coli [G. D. Stormo et al. , Nucleic Acids Res. 10, pp. 2971-2996 (1982)] . The ORF and the polypeptide of the HSP70 of S. agalactiae are, respectively, identical at 85 and 95 % to the ORF and polypeptide of the HSP72 of S. pneumoniae .
Preliminary sequence comparisons with the HSP72 of S. pneumoniae showed that the 3.5-kb Hindlll insert in plasmid pURV4 lacks the 3 ' -end coding region of the hsp70 of S. pyogenes . An attempt to clone a 3-kb Sail genomic fragment containing the entire coding region of hsp70 of S. pyogenes yielded plasmid pURV6 containing a 3.1-kb insert lacking the 5 ' -end coding region of the gene. The assembly of the hsp70 gene regions present in plasmids pURV4 and pURV6 gave a 2183 nucleotide region containing an ORF of 1824 bases coding for a polypeptide of 608 amino acids with a molecular weight of 64847 (see SEQ ID NO: 20) . The ATG start codon begins at position 204 and the TAA stop codon extends to position 2030. Similarly to the hsp70 of S. agalactiae, the ATG start codon is preceeded by a putative ribosome binding site sequence GAGG starting at position 193 [G. D. Stormo, supra] . The ORF and the deduced polypeptide of the hsp70 of S. pyogenes are, respectively, identical at 85 and 94 % to the ORF and polypeptide of the HSP72 of S. pneumoniae . The ORF of plasmid pURV4 lacks 125 base pairs coding for 41 amino acids at the carboxyl end of the HSP70 of S. pyogenes ; the ORF thus codes for the 567 amino acids of the amino end of that HSP70 (N-567rec) . The ORF of plasmid pURVδ lacks 114 base pairs coding for 38 amino acids at the amino end of the HSP70 of S. pyogenes ; the ORF thus codes
for the 570 amino acids of the carboxyl end of that HSP7Θ
(C-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 cell extracts of heat-shocked and control S. agalactiae pulse-labeled with [35s] ethionine revealed that the synthesis of a 70 kDa- protein was significantly increased after a thermal stress (FIG. 26, lanes 1 and 2) . Radioimmunoprecipitation analysis revealed that the heat inducible 70kDa-protein was easily detected at 43°C using monoclonal antibody F2- Pn3.4 thus indicating that the protein belongs to the heat shock protein 70 (hsp70/DnaK) family (FIG. 26, lanes 3 and 4) .
D. Antigenic Relatedness of HSP70 Proteins in S. pneumoniae ,_S. pyogenes and S. agalactiae
In this study, a panel of MAbs were used to investigate the antigenic relatedness of S. pyogenes, S. agalactiae and S. pneumoniae HSP70 proteins. Eight of ten MAbs reacted with all three Streptoccocus species thus indicating that some B-cell epitopes are widely distributed among S. pneumoniae , S. pyogenes and S. agalactiae . The MAb 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 HSP70 antigens from either S. pyogenes or S. agalactiae . Comparison of this region among the three Streptococcus species revealed differences in 5 to 8 amino acids located between amino acids 589 and 596. The MAb F2-Pn3.3 which
was also directed against epitopes present in the C-151 _ 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
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
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.
SEQUENCE LISTING
(1) GENERAL INFORMATION
(l) APPLICANT Hamel, Josee
Brodeur, Bernard R Martin, Denis Rio x, Clement
(ll) TITLE OF INVENTION STREPTOCOCCAL HEAT SHOCK PROTEINS MEMBERS OF THE HSP70 FAMILY
(lll) NUMBER OF SEQUENCES 26
(IV) CORRESPONDENCE ADDRESS
(A) ADDRESSEE Goudreau Gage Dubuc & Martmeau Walker
(B) STREET 800 Place Victoria, Suite 3400, Stock
Exchange Tower (C) CITY Montreal
(D) STATE Quebec
(E) COUNTRY CANADA
(F) ZIP H4Z1E9 (v) COMPUTER READABLE FORM
(A) MEDIUM TYPE Floppy disk
(B) COMPUTER IBM PC compatible
(C) OPERATING SYSTEM PC-DOS/MS-DOS
(D) SOFTWARE Patentin Release #1 0, Version #1 25
(vi) CURRENT APPLICATION DATA
(A) APPLICATION NUMBER.
(B) FILING DATE
(C) CLASSIFICATION.
(vii) PRIOR APPLICATION DATA
(A) APPLICATION NUMBER US 08/472,534
(B) FILING DATE 07-JUN-1995 (vii) PRIOR APPLICATION DATA
(A) APPLICATION NUMBER US (PROVIS) 60/001, 805
(B) FILING DATE 04-AUG-1995
(vm) ATTORNEY/AGENT INFORMATION (A) NAME Leclerc/Dubuc/Prince, Alain/Jean/Gaetan
(C) REFERENCE/DOCKET NUMBER BIOVAC2-PCT
(IX) TELECOMMUNICATION INFORMATION (A) TELEPHONE (514) 397-7400 (B) TELEFAX (514) 397-4382
(2) INFORMATION FOR SEQ ID NO 1 (I) SEQUENCE CHARACTERISTICS
(A) LENGTH 3167 base pairs
(B) TYPE nucleic acid
(C) STRANDEDNESS double
(D) TOPOLOGY linear
(ιι) MOLECULE TYPE DNA (genomic)
(ill) HYPOTHETICAL NO (iv) ANTI-SENSE NO
(vi) ORIGINAL SOURCE
(A) ORGANISM Streptococcus pneumoniae (lx) FEATURE
(A) NAME/KEY CDS
(B) LOCATION: 30..755
(ix) FEATURE:
(A) NAME/KEY: CDS (B) LOCATION: 771..2912
(D) OTHER INFORMATION: /product= "FucI/HSP72 (C-169)"
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:1 :
GAACTTCATT TTTAGAAAGG AGTGAGTTT ATG TCT CAA GAT GAA AAA TTA ATT 53
Met Ser Gin Asp Glu Lys Leu lie 1 5 CGT GAA CAG ATT TGT GAT GTT TGT CAT AAG ATG TGG CAA CTT GGT TGG 101 Arg Glu Gin lie Cys Asp Val Cys His Lys Met Trp Gin Leu Gly Trp 10 15 20
GTT GCT GCT AAC GAT GGG AAT GTA TCT GTT CGA TTA GAT GAG GAT ACC 149 Val Ala Ala Asn Asp Gly Asn Val Ser Val Arg Leu Asp Glu Asp Thr 25 30 35 40
ATT CTT GCA ACA CCT ACT GGT ATC AGC AAA AGT TTT ATT ACA CCA GAA 197 lie Leu Ala Thr Pro Thr Gly lie Ser Lys Ser Phe lie Thr Pro Glu 45 50 55
AAG CTG GTG AAG TTA AAT CTT AAA GGA GAG ATT TTA GAA GCA GAA GGT 245 Lys Leu Val Lys Leu Asn Leu Lys Gly Glu lie Leu Glu Ala Glu Gly 60 65 70
GAT TAC TGT CCT TCT AGT GAA ATT AAA ATG CAC ATT CGG TGC TAC GAA 293 Asp Tyr Cys Pro Ser Ser Glu lie Lys Met His lie Arg Cys Tyr Glu 75 80 85 GAA CGT GAG GAT GTT CGT TCA GTT GTT CAC GCG CAT CCA CCG ATT GCA 341 Glu Arg Glu Asp Val Arg Ser Val Val His Ala His Pro Pro lie Ala 90 95 100
ACA GGA TTT GCT CTT GCA CAC ATT CCT TTA GAT ACT TAT TCA CTA ATT 389 Thr Gly Phe Ala Leu Ala His lie Pro Leu Asp Thr Tyr Ser Leu lie 105 110 115 120
GAG AGC GCG ATT GTG GTT GGG GCA ATT CCT ATT ACC CCA TTT GGA GTA 437 Glu Ser Ala He Val Val Gly Ala He Pro He Thr Pro Phe Gly Val 125 130 135
CCG TCT ACA ATG GAA GTG CCA GAA GCA ATT ACA CCT TAT CTG CCC GAT 485 Pro Ser Thr Met Glu Val Pro Glu Ala He Thr Pro Tyr Leu Pro Asp 140 145 150
CAT GAT GTC ATG CTA TTA GAA AAT CAT GGA GCT CTG ACT GTC GGA AGC 533 His Asp Val Met Leu Leu Glu Asn His Gly Ala Leu Thr Val Gly Ser 155 160 165 GAT GTC ATT ACA GCA TAC TAC CGT ATG GAA ACT TTA GAA TTA GTC GCA 581 Asp Val He Thr Ala Tyr Tyr Arg Met Glu Thr Leu Glu Leu Val Ala 170 175 180
AAG ACA ACC TTC CAC GGA AGA ATG TTA CTT TCT ACA AAG GGC ATT GAG 629 Lys Thr Thr Phe His Gly Arg Met Leu Leu Ser Thr Lys Gly He Glu 185 190 195 200
GAG CAA GAA ATT GCT CGT CCG ACT TTA GAA CGT CTA TTC TCA ATG CGA 677 Glu Gin Glu He Ala Arg Pro Thr Leu Glu Arg Leu Phe Ser Met Arg 205 210 215
GAA AAT TAT AAG GTT ACA GGT CGT CAC CCA GGC TAC CGT AAA TAT AAT 725 Glu Asn Tyr Lys Val Thr Gly Arg His Pro Gly Tyr Arg Lys Tyr Asn 220 225 230
GGC GAT GGT AGT ATA AAA GAA ACA AAA AAA TAAGAGGAAA GTATT ATG ATC 776 Gly Asp Gly Ser He Lys Glu Thr Lys Lys Met He
235 240 1
CAA CAT CCA CGT ATT GGG ATT CGT CCG ACT ATT GAT GGT CGT CGT CAA 824 Gin His Pro Arg He Gly He Arg Pro Thr He Asp Gly Arg Arg Gin 5 10 15 GGT GTA CGC GAA TCA CTT GAA GTA CAA ACA ATG AAC ATG GCT AAA AGT 872 Gly Val Arg Glu Ser Leu Glu Val Gin Thr Met Asn Met Ala Lys Ser 20 25 30
GTG GCA GAT TTG ATT TCA AGC ACA TTG AAA TAT CCA GAT GGG GAA CCT 920 Val Ala Asp Leu He Ser Ser Thr Leu Lys Tyr Pro Asp Gly Glu Pro 35 40 45 50
GTG GAA TGT GTG ATT TCT CCA TCT ACC ATT GGT CGT GTT CCA GAG GCT 968 Val Glu Cys Val He Ser Pro Ser Thr He Gly Arg Val Pro Glu Ala 55 60 65
GCA GCT TCC CAT GAG TTG TTT AAA AAA TCA AAT GTT TGC GCA ACA ATT 1016 Ala Ala Ser His Glu Leu Phe Lys Lys Ser Asn Val Cys Ala Thr He 70 75 80
ACA GTT ACA CCA TGC TGG TGT TAT GGT AGT GAA ACT ATG GAT ATG TCT 1064 Thr Val Thr Pro Cys Trp Cys Tyr Gly Ser Glu Thr Met Asp Met Ser 85 90 95 CCA GAT ATT CCT CAT GCT ATT TGG GGA TTT AAT GGG ACA GAA CGC CCA 1112 Pro Asp He Pro His Ala He Trp Gly Phe Asn Gly Thr Glu Arg Pro 100 105 110
GGA GCT GTC TAT CTT GCA GCT GTA CTA GCT TCA CAT ACT CAA AAA GGG 1160 Gly Ala Val Tyr Leu Ala Ala Val Leu Ala Ser His Thr Gin Lys Gly 115 120 125 130
ATT CCA GCC TTT GGG ATT TAT GGT AGA GAT GTT CAG GAA GCT AAT GAT 1208 He Pro Ala Phe Gly He Tyr Gly Arg Asp Val Gin Glu Ala Asn Asp 135 140 145
ACA GCT ATT CCA GAA GAT GTC AAA GAA AAA CTT TTA CGT TAT GCG CGG 1256
Thr Ala He Pro Glu Asp Val Lys Glu Lys Leu Leu Arg Tyr Ala Arg 150 155 160
GCA GTT CTT GCA ACT GGC TTG ATG AGA GAC ACT GCT TAC CTA TCA ATG 1304
Ala Val Leu Ala Thr Gly Leu Met Arg Asp Thr Ala Tyr Leu Ser Met 165 170 175 GGT AGT GTT TCG ATG GGG ATT GGT GGT TCT ATT GTA AAT CCA GAT TTC 1352 Gly Ser Val Ser Met Gly He Gly Gly Ser He Val Asn Pro Asp Phe 180 185 190
TTC CAA GAA TAC TTA GGA ATG CGA AAT GAA TCG GTA GAT ATG ACG GAG 1400 Phe Gin Glu Tyr Leu Gly Met Arg Asn Glu Ser Val Asp Met Thr Glu 195 200 205 210
TTC ACG CGC CGT ATG GAC CGT GGT ATT TAC GAC CCT GAA GAG TTC GAA 1448 Phe Thr Arg Arg Met Asp Arg Gly He Tyr Asp Pro Glu Glu Phe Glu 215 220 225
CGT GCG CTC AAA TGG GTG AAA GAA AAC GTA AAA GAA GGA TTC GAC CAT 1496 Arg Ala Leu Lys Trp Val Lys Glu Asn Val Lys Glu Gly Phe Asp His 230 235 240
AAC CGT GAA GAC CTT GTT TTA AGC CGT GAA GAA AAA GAT AGA CAA TGG 1544 Asn Arg Glu Asp Leu Val Leu Ser Arg Glu Glu Lys Asp Arg Gin Trp 245 250 255
GAA TTT GTT ATT AAG ATG TTC ATG ATT GGA CGT GAC TTA ATG GTT GGT 1592
Glu Phe Val He Lys Met Phe Met He Gly Arg Asp Leu Met Val Gly
260 265 270
AAC CCA AGA CTT GCT GAA CTT GGT TTT GAG GAA GAA GCA GTT GGT CAC 1640
Asn Pro Arg Leu Ala Glu Leu Gly Phe Glu Glu Glu Ala Val Gly His
275 280 285 290 CAT GCT TTA GTA GCT GGT TTC CAA GGT CAA CGT CAG TGG ACA GAC CAT 1688 His Ala Leu Val Ala Gly Phe Gin Gly Gin Arg Gin Trp Thr Asp His 295 300 305
TTT CCA AAT GGG GAC TTT ATG GAA ACT TTC CTC AAT ACT CAG TTT GAC 1736 Phe Pro Asn Gly Asp Phe Met Glu Thr Phe Leu Asn Thr Gin Phe Asp 310 315 320
TGG AAT GGT ATT CGA AAA CCA TTT GTA TTT GCG ACA GAG AAT GAT TCA .- 1784 Trp Asn Gly He Arg Lys Pro Phe Val Phe Ala Thr Glu Asn Asp Ser 325 330 335
CTA AAT GGT GTG TCT ATG CTC TTT AAT TAT CTA TTA ACA AAT ACT CCA 1832 Leu Asn Gly Val Ser Met Leu Phe Asn Tyr Leu Leu Thr Asn Thr Pro 340 345 350
CAA ATC TTT GCT GAT GTG CGT ACT TAT TGG AGT CCA GAG GCT GTT GAA 1880 Gin He Phe Ala Asp Val Arg Thr Tyr Trp Ser Pro Glu Ala Val Glu 355 360 365 370 CGT GTA ACA GGA TAT ACT TTA GAG GGT CGT GCT GCA GCT GGA TTC TTA 1928 Arg Val Thr Gly Tyr Thr Leu Glu Gly Arg Ala Ala Ala Gly Phe Leu 375 380 385
CAT CTA ATC AAC TCT GGA TCT TGT ACA TTG GAT GGT ACA GGT CAA GCT 1976 His Leu He Asn Ser Gly Ser Cys Thr Leu Asp Gly Thr Gly Gin Ala 390 395 400
ACT CGA GAT GGC AAA CCT GTT ATG AAA CCA TTC TGG GAG TTG GAT GAA 2024 Thr Arg Asp Gly Lys Pro Val Met Lys Pro Phe Trp Glu Leu Asp Glu 405 410 415
AGT GAA GTA CAG GCT ATG CTT GAA AAT ACA GAC TTC CCA CCA GCA AAC 2072
Ser Glu Val Gin Ala Met Leu Glu Asn Thr Asp Phe Pro Pro Ala Asn 420 425 430
CGC GAA TAC TTC CGT GGA GGA GGA TTC TCA ACT CGT TTC TTG ACG AAG 2120
Arg Glu Tyr Phe Arg Gly Gly Gly Phe Ser Thr Arg Phe Leu Thr Lys 435 440 445 450 GGG GAT ATG CCA GTA ACA ATG GTA CGT CTC AAT CTT TTA AAA GGG GTT 2168 Gly Asp Met Pro Val Thr Met Val Arg Leu Asn Leu Leu Lys Gly Val 455 460 465
GGT CCA GTG CTA CAA ATT GCA GAA GGT TAC ACA CTT GAA CTT CCT GAA 2216 Gly Pro Val Leu Gin He Ala Glu Gly Tyr Thr Leu Glu Leu Pro Glu 470 475 480
GAT GTT CAC CAT ACT TTA GAT AAT CGT ACA GAT CCA GGA TGG CCA ACT 2264 Asp Val His His Thr Leu Asp Asn Arg Thr Asp Pro Gly Trp Pro Thr 485 490 495
ACT TGG TTT GCT CCA CGT TTG ACA GGA AAA GGT GCT TTC AAG TCT GTC 2312 Thr Trp Phe Ala Pro Arg Leu Thr Gly Lys Gly Ala Phe Lys Ser Val 500 505 510
TAT GAC GTC ATG AAT AAT TGG GGA GCT AAT CAC GGA GCC ATA ACA TAT 2360 Tyr Asp Val Met Asn Asn Trp Gly Ala Asn His Gly Ala He Thr Tyr 515 520 525 530
GGA CAC ATT GGA GCA GAC TTG ATT ACC TTG GCT TCT ATG TTG AGA ATT 2408 Gly His He Gly Ala Asp Leu He Thr Leu Ala Ser Met Leu Arg He 535 540 545
CCT CAA ATC GAA GTA ACA TTT GAC ATC GAC AAG AAC GGT ATC GTG TCT 2456 Pro Gin He Glu Val Thr Phe Asp He Asp Lys Asn Gly He Val Ser 550 555 560 GTT AAG GCC AAA GAC CTT GGA ACT CAA AAA GAA CAA ACT ATT GTC ATC 2504 Val Lys Ala Lys Asp Leu Gly Thr Gin Lys Glu Gin Thr He Val He 565 570 575
CAA TCG AAC TCA GGT TTG ACT GAC GAA GAA ATC GAC CGC ATG ATG AAA 2552 Gin Ser Asn Ser Gly Leu Thr Asp Glu Glu He Asp Arg Met Met Lys 580 585 590
GAT GCA GAA GCA AAC GCT GAA TCC GAT AAG AAA CGT AAA GAA GAA GTA 2600 Asp Ala Glu Ala Asn Ala Glu Ser Asp Lys Lys Arg Lys Glu Glu Val 595 600 605 610
GAC CTT CGT AAT GAA GTG GAC CAA GCA ATC TTT GCG ACT GAA AAG ACA 2648 Asp Leu Arg Asn Glu Val Asp Gin Ala He Phe Ala Thr Glu Lys Thr 615 620 625
ATC AAG GAA ACT GAA GGT AAA GGC TTC GAC GCA GAA CGT GAC GCT GCC 2696 He Lys Glu Thr Glu Gly Lys Gly Phe Asp Ala Glu Arg Asp Ala Ala 630 635 640 CAA GCT GCC CTT GAT GAC CTT AAG AAA GCT CAA GAA GAC AAC AAC TTG 2744 Gin Ala Ala Leu Asp Asp Leu Lys Lys Ala Gin Glu Asp Asn Asn Leu 645 650 655
GAC GAC ATG AAA GCA AAA CTT GAA GCA TTG AAC GAA AAA GCT CAA GGA 2792 Asp Asp Met Lys Ala Lys Leu Glu Ala Leu Asn Glu Lys Ala Gin Gly 660 665 670
CTT GCT GTT AAA CTC TAC GAA CAA GCC GCA GCA GCG CAA CAA GCT CAA 2840 Leu Ala Val Lys Leu Tyr Glu Gin Ala Ala Ala Ala Gin Gin Ala Gin 675 680 685 690
GAA GGA GCA GAA GGC GCA CAA GCA ACA GGA AAC GCA GGC GAT GAC GTC 2888 Glu Gly Ala Glu Gly Ala Gin Ala Thr Gly Asn Ala Gly Asp Asp Val 695 700 705
GTA GAC GGA GAG TTT ACG GAA AAG TAAGATGAGT GTATTGGATG AAGAGTATCT 2942 Val Asp Gly Glu Phe Thr Glu Lys 710 AAAAAATACA CGAAAAGTTT ATAATGATTT TTGTAATCAA GCTGATAACT ATAGAACATC 3002
AAAAGATTTT ATTGATAATA TTCCAATAGA ATATTTAGCT AGATATAGAG AAATTATATT 3062
AGCTGAGCAT GATAGTTGTG TCAAAAATGA TGAAGCGGTA AGGAATTTTG TTACCTCAGT 3122
ATTGTTGTCT GCATTTGTAT CGGCGATGGT ATCAGCTATG ATATC 3167
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 242 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Ser Gin Asp Glu Lys Leu He Arg Glu Gin He Cys Asp Val Cys 1 5 10 15
His Lys Met Trp Gin Leu Gly Trp Val Ala Ala Asn Asp Gly Asn Val 20 25 30 Ser Val Arg Leu Asp Glu Asp Thr He Leu Ala Thr Pro Thr Gly He 35 40 45
Ser Lys Ser Phe He Thr Pro Glu Lys Leu Val Lys Leu Asn Leu Lys 50 55 60
Gly Glu He Leu Glu Ala Glu Gly Asp Tyr Cys Pro Ser Ser Glu He 65 70 75 80
Lys Met His He Arg Cys Tyr Glu Glu Arg Glu Asp Val Arg Ser Val 85 90 95
Val His Ala His Pro Pro He Ala Thr Gly Phe Ala Leu Ala His He 100 105 110 Pro Leu Asp Thr Tyr Ser Leu He Glu Ser Ala He Val Val Gly Ala 115 120 125
He Pro He Thr Pro Phe Gly Val Pro Ser Thr Met Glu Val Pro Glu 130 135 140
Ala He Thr Pro Tyr Leu Pro Asp His Asp Val Met Leu Leu Glu Asn 145 150 155 160
His Gly Ala Leu Thr Val Gly Ser Asp Val He Thr Ala Tyr Tyr Arg 165 170 175
Met Glu Thr Leu Glu Leu Val Ala Lys Thr Thr Phe His Gly Arg Met 180 185 190 Leu Leu Ser Thr Lys Gly He Glu Glu Gin Glu He Ala Arg Pro Thr 195 200 205
Leu Glu Arg Leu Phe Ser Met Arg Glu Asn Tyr Lys Val Thr Gly Arg 210 215 220
His Pro Gly Tyr Arg Lys Tyr Asn Gly Asp Gly Ser He Lys Glu Thr 225 230 235 240
Lys Lys
(2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 714 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
( i) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Met He Gin His Pro Arg He Gly He Arg Pro Thr He Asp Gly Arg 1 5 10 15
Arg Gin Gly Val Arg Glu Ser Leu Glu Val Gin Thr Met Asn Met Ala 20 25 30 Lys Ser Val Ala Asp Leu He Ser Ser Thr Leu Lys Tyr Pro Asp Gly 35 40 45
Glu Pro Val Glu Cys Val He Ser Pro Ser Thr He Gly Arg Val Pro 50 55 60
Glu Ala Ala Ala Ser His Glu Leu Phe Lys Lys Ser Asn Val Cys Ala 65 70 75 80 Thr He Thr Val Thr Pro Cys Trp Cys Tyr Gly Ser Glu Thr Met Asp
85 90 95
Met Ser Pro Asp He Pro His Ala He Trp Gly Phe Asn Gly Thr Glu 100 105 110
Arg Pro Gly Ala Val Tyr Leu Ala Ala Val Leu Ala Ser His Thr Gin 115 120 125
Lys Gly He Pro Ala Phe Gly He Tyr Gly Arg Asp Val Gin Glu Ala 130 135 140
Asn Asp Thr Ala He Pro Glu Asp Val Lys Glu Lys Leu Leu Arg Tyr 145 150 155 160 Ala Arg Ala Val Leu Ala Thr Gly Leu Met Arg Asp Thr Ala Tyr Leu
165 170 175
Ser Met Gly Ser Val Ser Met Gly He Gly Gly Ser He Val Asn Pro 180 185 190
Asp Phe Phe Gin Glu Tyr Leu Gly Met Arg Asn Glu Ser Val Asp Met 195 200 205
Thr Glu Phe Thr Arg Arg Met Asp Arg Gly He Tyr Asp Pro Glu Glu 210 215 220
Phe Glu Arg Ala Leu Lys Trp Val Lys Glu Asn Val Lys Glu Gly Phe 225 230 235 240 Asp His Asn Arg Glu Asp Leu Val Leu Ser Arg Glu Glu Lys Asp Arg
245 250 255
Gin Trp Glu Phe Val He Lys Met Phe Met He Gly Arg Asp Leu Met 260 265 270
Val Gly Asn Pro Arg Leu Ala Glu Leu Gly Phe Glu Glu Glu Ala Val 275 280 285
Gly His His Ala Leu Val Ala Gly Phe Gin Gly Gin Arg Gin Trp Thr 290 295 300
Asp His Phe Pro Asn Gly Asp Phe Met Glu Thr Phe Leu Asn Thr Gin 305 310 315 320 Phe Asp Trp Asn Gly He Arg Lys Pro Phe Val Phe Ala Thr Glu Asn
325 330 335
Asp Ser Leu Asn Gly Val Ser Met Leu Phe Asn Tyr Leu Leu Thr Asn 340 345 350
Thr Pro Gin He Phe Ala Asp Val Arg Thr Tyr Trp Ser Pro Glu Ala 355 360 365
Val Glu Arg Val Thr Gly Tyr Thr Leu Glu Gly Arg Ala Ala Ala Gly 370 375 380
Phe Leu His Leu He Asn Ser Gly Ser Cys Thr Leu Asp Gly Thr Gly 385 390 395 400 Gin Ala Thr Arg Asp Gly Lys Pro Val Met Lys Pro Phe Trp Glu Leu
405 410 415
Asp Glu Ser Glu Val Gin Ala Met Leu Glu Asn Thr Asp Phe Pro Pro 420 425 430
Ala Asn Arg Glu Tyr Phe Arg Gly Gly Gly Phe Ser Thr Arg Phe Leu 435 440 445 Thr Lys Gly Asp Met Pro Val Thr Met Val Arg Leu Asn Leu Leu Lys 450 455 460
Gly Val Gly Pro Val Leu Gin He Ala Glu Gly Tyr Thr Leu Glu Leu 465 470 475 480
Pro Glu Asp Val His His Thr Leu Asp Asn Arg Thr Asp Pro Gly Trp 485 490 495
Pro Thr Thr Trp Phe Ala Pro Arg Leu Thr Gly Lys Gly Ala Phe Lys 500 505 510
Ser Val Tyr Asp Val Met Asn Asn Trp Gly Ala Asn His Gly Ala He 515 520 525 Thr Tyr Gly His He Gly Ala Asp Leu He Thr Leu Ala Ser Met Leu 530 535 540
Arg He Pro Gin He Glu Val Thr Phe Asp He Asp Lys Asn Gly He 545 550 555 560
Val Ser Val Lys Ala Lys Asp Leu Gly Thr Gin Lys Glu Gin Thr He 565 570 575
Val He Gin Ser Asn Ser Gly Leu Thr Asp Glu Glu He Asp Arg Met 580 585 590
Met Lys Asp Ala Glu Ala Asn Ala Glu Ser Asp Lys Lys Arg Lys Glu 595 600 605 Glu Val Asp Leu Arg Asn Glu Val Asp Gin Ala He Phe Ala Thr Glu 610 615 620
Lys Thr He Lys Glu Thr Glu Gly Lys Gly Phe Asp Ala Glu Arg Asp 625 630 635 640
Ala Ala Gin Ala Ala Leu Asp Asp Leu Lys Lys Ala Gin Glu Asp Asn 645 650 655
Asn Leu Asp Asp Met Lys Ala Lys Leu Glu Ala Leu Asn Glu Lys Ala 660 665 670
Gin Gly Leu Ala Val Lys Leu Tyr Glu Gin Ala Ala Ala Ala Gin Gin 675 680 685 Ala Gin Glu Gly Ala Glu Gly Ala Gin Ala Thr Gly Asn Ala Gly Asp 690 695 700
Asp Val Val Asp Gly Glu Phe Thr Glu Lys 705 710
(2) INFORMATION FOR SEQ ID NO:4 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4320 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:
(A) ORGANISM: Streptococcus pneumoniae
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 682..2502 ( D ) OTHER INFORMATION: /product= " "Heat-shock protein 72 " "
( ix) FEATURE :
(A) NAME/KEY: CDS
(B) LOCATION: 3265..4320 (D) OTHER INFORMATION: /product= " "NH2-terminal portion of
DNA J" "
(ix) FEATURE:
(A) NAME/KEY: mat_peptide (B) LOCATION: 682..2502
( i) SEQUENCE DESCRIPTION: SEQ ID NO:4 : AAGCTTGATT CACGCTTTGA AAGAAGAAGG AATTGAAGAA ATCGCAGCAG ATGGCGAATT 60
TGACCATAAC TACCATATGG CCATCCAAAC TCTCCCAGCA GACGATGAAC ACCCAGTAGA 120
TACCATCGCC CAAGTCTTTC AAAAAGGCTA CAAACTCCAT GACCGCATCC TACGCCCAGC 180
AATGGTAGTG GTGTATAACT AAGATACAAA GCCCGTAAAA AGCTCGCAGT AAAAATAGGA 240
GATTGACGAA GTGTTCGATG AACACAAGAA AATCTATCTT TTTTACTCAG AGCTTAGGGC 300 GTGTTCGATT CGGCAATTCT GACGGTAGCT AAAGCAACTC GTCAGAAAAC GGCAGTCGCT 360
ATGGCGTTTG TCTAGCTTCC TTACTAACTC GTCGTCGAAA TAAAATCGAT TTCGACTCTT 420
CGTGTCGCAA TTTACATAAT AGAAAACTTG TCCGAAACGA CAATAAACTA TGAAGAAAGA 480
TAAAATATGT TTGGCTTTGT AATAGTGAGC GAAGCGAACC AAAGACGATA CTCTTCGCTG 540
TGGCGCTATT TGCGCAAATT TTGAGACCTT AGGCTCAAAG TTTAGTCAAA GAGATTGACA 600 AAGTCAAGCT CTGACGGCGT CGCCACTTAA GAAGAGTATC AAAAAGAAAA ATAGAAAATT 660
AACTAACAAG GAGAAAAACA C ATG TCT AAA ATT ATC GGT ATT GAC TTA GGT 711
Met Ser Lys He He Gly He Asp Leu Gly 1 5 10
ACA ACA AAC TCA GCA GTT GCA GTT CTT GAA GGA ACT GAA AGC AAA ATC 759
Thr Thr Asn Ser Ala Val Ala Val Leu Glu Gly Thr Glu Ser Lys He 15 20 25 ATC GCA AAC CCA GAA GGA AAC CGC ACA ACT CCA TCT GTA GTC TCA TTC 807 He Ala Asn Pro Glu Gly Asn Arg Thr Thr Pro Ser Val Val Ser Phe 30 35 40
AAA AAC GGA GAA ATC ATC GTT GGT GAT GCT GCA AAA CGT CAA GCA GTT 855 Lys Asn Gly Glu He He Val Gly Asp Ala Ala Lys Arg Gin Ala Val 45 50 55
ACA AAC CCA GAT ACA GTT ATC TCT ATC AAA TCT AAG ATG GGA ACT TCT 903 Thr Asn Pro Asp Thr Val He Ser He Lys Ser Lys Met Gly Thr Ser 60 65 70
GAA AAA GTT TCT GCA AAT GGA AAA GAA TAC ACT CCA CAA GAA ATC TCA 951 Glu Lys Val Ser Ala Asn Gly Lys Glu Tyr Thr Pro Gin Glu He Ser 75 80 85 90
GCT ATG ATC CTT CAA TAC TTG AAA GGC TAC GCT GAA GAC TAC CTT GGT 999 Ala Met He Leu Gin Tyr Leu Lys Gly Tyr Ala Glu Asp Tyr Leu Gly 95 100 105
GAG AAA GTA ACC AAA GCT GTT ATC ACA GTT CCG GCT TAC TTC AAC GAC 1047 Glu Lys Val Thr Lys Ala Val He Thr Val Pro Ala Tyr Phe Asn Asp 110 115 120
GCT CAA CGT CAA GCA ACA AAA GAC GCT GGT AAA ATT GCT GGT CTT GAA 1095 Ala Gin Arg Gin Ala Thr Lys Asp Ala Gly Lys He Ala Gly Leu Glu 125 130 135 GTA GAA CGT ATT GTT AAC GAA CCA ACT GCA GCA GCT CTT GCT TAT GGT 1143 Val Glu Arg He Val Asn Glu Pro Thr Ala Ala Ala Leu Ala Tyr Gly 140 145 150
TTG GAC AAG ACT GAC AAA GAA GAA AAA ATC TTG GTA TTT GAC CTT GGT 1191 Leu Asp Lys Thr Asp Lys Glu Glu Lys He Leu Val Phe Asp Leu Gly 155 160 165 170
GGT GGT ACA TTC GAC GTC TCT ATC CTT GAA TTG GGT GAC GGT GTC TTC 1239 Gly Gly Thr Phe Asp Val Ser He Leu Glu Leu Gly Asp Gly Val Phe 175 180 185
GAC GTA TTG TCA ACT GCA GGG GAC AAC AAA CTT GGT GGT GAC GAC TTT 1287 Asp Val Leu Ser Thr Ala Gly Asp Asn Lys Leu Gly Gly Asp Asp Phe 190 195 200
GAC CAA AAA ATC ATT GAC CAC TTG GTA GCA GAA TTC AAG AAA GAA AAC 1335 Asp Gin Lys He He Asp His Leu Val Ala Glu Phe Lys Lys Glu Asn 205 210 215 GGT ATC GAC TTG TCT ACT GAC AAG ATG GCA ATG CAA CGT TTG AAA GAT 1383 Gly He Asp Leu Ser Thr Asp Lys Met Ala Met Gin Arg Leu Lys Asp 220 225 230
GCG GCT GAA AAA GCG AAG AAA GAC CTT TCT GGT GTA ACT TCA ACA CAA 1431 Ala Ala Glu Lys Ala Lys Lys Asp Leu Ser Gly Val Thr Ser Thr Gin 235 240 245 250
ATC AGC TTG CCA TTT ATC ACT GCA GGT GAG GCT GGA CCT CTT CAC TTG 1479 He Ser Leu Pro Phe He Thr Ala Gly Glu Ala Gly Pro Leu His Leu 255 260 265
GAA ATG ACT TTA ACT CGT GCG AAA TTT GAT GAT TTG ACT CGT GAC CTT 1527
Glu Met Thr Leu Thr Arg Ala Lys Phe Asp Asp Leu Thr Arg Asp Leu 270 275 280
GTT GAA CGT ACA AAA GTT CCA GTT CGT CAA GCC CTT TCA GAT GCA GGT 1575
Val Glu Arg Thr Lys Val Pro Val Arg Gin Ala Leu Ser Asp Ala Gly 285 290 295 TTG AGC TTG TCA GAA ATC GAC GAA GTT ATC CTT GTT GGT GGT TCA ACT 1623 Leu Ser Leu Ser Glu He Asp Glu Val He Leu Val Gly Gly Ser Thr 300 305 310
CGT ATC CCT GCC GTT GTT GAA GCT GTT AAA GCT GAA ACT GGT AAA GAA 1671 Arg He Pro Ala Val Val Glu Ala Val Lys Ala Glu Thr Gly Lys Glu 315 320 325 330
CCA AAC AAA TCA GTA AAC CCT GAT GAA GTA GTT GCT ATG GGT GCG GCT 1719 Pro Asn Lys Ser Val Asn Pro Asp Glu Val Val Ala Met Gly Ala Ala 335 340 345
ATC CAA GGT GGT GTG ATT ACT GGT GAT GTC AAG GAT GTT GTC CTT CTT 1767 He Gin Gly Gly Val He Thr Gly Asp Val Lys Asp Val Val Leu Leu 350 355 360
GAT GTA ACG CCA TTG TCA CTT GGT ATC GAA ACA ATG GGT GGA GTA TTT 1815 Asp Val Thr Pro Leu Ser Leu Gly He Glu Thr Met Gly Gly Val Phe 365 370 375
ACA AAA CTT ATC GAT CGC AAC ACT ACA ATC CCA ACA TCT AAA TCA CAA 1863
Thr Lys Leu He Asp Arg Asn Thr Thr He Pro Thr Ser Lys Ser Gin 380 385 390
GTC TTC TCA ACA GCA GCA GAC -AAC CAA CCA GCC GTT GAT ATC CAC GTT 1911
Val Phe Ser Thr Ala Ala Asp Asn Gin Pro Ala Val Asp He His Val 395 400 405 410 CTT CAA GGT GAA CGC CCA ATG GCA GCA GAT AAC AAG ACT CTT GGA CGC 1959 Leu Gin Gly Glu Arg Pro Met Ala Ala Asp Asn Lys Thr Leu Gly Arg 415 420 425
TTC CAA TTG ACT GAT ATC CCA GCT GCA CCT CGT GGA ATT CCT CAA ATC 2007 Phe Gin Leu Thr Asp He Pro Ala Ala Pro Arg Gly He Pro Gin He 430 435 440
GAA GTA ACA TTT GAC ATC GAC AAG AAC GGT ATC GTG TCT GTT AAG GCC 2055 Glu Val Thr Phe Asp He Asp Lys Asn Gly He Val Ser Val Lys Ala 445 450 455
AAA GAC CTT GGA ACT CAA AAA GAA CAA ACT ATT GTC ATC CAA TCG AAC 2103
Lys Asp Leu Gly Thr Gin Lys Glu Gin Thr He Val He Gin Ser Asn 460 465 470
TCA GGT TTG ACT GAC GAA GAA ATC GAC CGC ATG ATG AAA GAT GCA GAA 2151
Ser Gly Leu Thr Asp Glu Glu He Asp Arg Met Met Lys Asp Ala Glu 475 480 485 490 GCA AAC GCT GAA TCC GAT AAG AAA CGT AAA GAA GAA GTA GAC CTT CGT 2199 Ala Asn Ala Glu Ser Asp Lys Lys Arg Lys Glu Glu Val Asp Leu Arg 495 500 505
AAT GAA GTG GAC CAA GCA ATC TTT GCG ACT GAA AAG ACA ATC AAG GAA 2247 Asn Glu Val Asp Gin Ala He Phe Ala Thr Glu Lys Thr He Lys Glu 510 515 520
ACT GAA GGT AAA GGC TTC GAC GCA GAA CGT GAC GCT GCC CAA GCT GCC 2295 Thr Glu Gly Lys Gly Phe Asp Ala Glu Arg Asp Ala Ala Gin Ala Ala 525 530 535
CTT GAT GAC CTT AAG AAA GCT CAA GAA GAC AAC AAC TTG GAC GAC ATG 2343 Leu Asp Asp Leu Lys Lys Ala Gin Glu Asp Asn Asn Leu Asp Asp Met 540 545 550
AAA GCA AAA CTT GAA GCA TTG AAC GAA AAA GCT CAA GGA CTT GCT GTT 2391 Lys Ala Lys Leu Glu Ala Leu Asn Glu Lys Ala Gin Gly Leu Ala Val 555 560 565 570 AAA CTC TAC GAA CAA GCC GCA GCA GCG CAA CAA GCT CAA GAA GGA GCA 2439 Lys Leu Tyr Glu Gin Ala Ala Ala Ala Gin Gin Ala Gin Glu Gly Ala 575 580 585
GAA GGC GCA CAA GCA ACA GGA AAC GCA GGC GAT GAC GTC GTA GAC GGA 2487 Glu Gly Ala Gin Ala Thr Gly Asn Ala Gly Asp Asp Val Val Asp Gly 590 595 600
GAG TTT ACG GAA AAG TAAGATGAGT GTATTGGATG AAGAGTATCT AAAAAATACA 2542 Glu Phe Thr Glu Lys 605
CGAAAAGTTT ATAATGATTT TTGTAATCAA GCTGATAACT ATAGAACATC AAAAGATTTT 2602
ATTGATAATA TTCCAATAGA ATATTTAGCT AGATATAGAG AAATTATATT AGCTGAGCAT 2662
GATAGTTGTG TCAAAAATGA TGAAGCGGTA AGGAATTTTG TTACCTCAGT ATTGTTGTCT 2722
GCATTTGTAT CGGCGATGGT ATCAGCTATG ATATCATTAG AAATACAAAC ATATAAATTT 2782 GTAATACCGT TCATAATTGG TATGATTTGG ACAGTAGTTG TATTTCTTAT GATCAATTGG 2842
AATTATATAG GCAAATACTA AGAAGAGACA AAAATATATA AATATTTCTG TACTTATAGG 2902
ATATTTAAAA TCCAAATAAA GTTAATTTAC TTATTTGCAG AGGTTGCAAC CCAGCCTCTG 2962 TTTTTCGATA AAAAGGGACG GAATCTCATT TGTTTGGGTT TTGTCTCATC AATAGAAAGG 3022
AACAAAGAGT GTTCGTAACT GAACACGGGT TTCAGAATTT CTTACTAAAT ATAAAAGAAA 3082
GGAATTGAAC CCGACCTAAA TGGTGGTTCG ATTCAGAACA TCAATAGAAA GGAATAAGGG 3142
TGTTCGTAAC TGAACACGGG CTACGGACTG TGCCAAAAAG ATAGTTTTTT CTAGGACGTA 3202
AGCGTCCGTC GTCAAAACTC CTAGATGGCT GTGTCCGTTT GACGCCCTTT GTATCTTGAA 3262 TT ATG AAC AAT ACT GAA TTT TAT GAT CGT CTG GGG GTA TCC AAA AAC 3309 Met Asn Asn Thr Glu Phe Tyr Asp Arg Leu Gly Val Ser Lys Asn 1 5 10 15
GCT TCG GCA GAC GAA ATC AAA AAG GCT TAT CGT AAG CTT TCC AAA AAA 3357 Ala Ser Ala Asp Glu He Lys Lys Ala Tyr Arg Lys Leu Ser Lys Lys
20 25 30
TAT CAC CCA GAT ATC AAC AAG GAG CCT GGT GCT GAG GAC AAG TAC AAG 3405 Tyr His Pro Asp He Asn Lys Glu Pro Gly Ala Glu Asp Lys Tyr Lys 35 40 45
GAA GTT CAA GAA GCC TAT GAG ACT TTG AGT GAC GAC CAA AAA CGT GCT 3453
Glu Val Gin Glu Ala Tyr Glu Thr Leu Ser Asp Asp Gin Lys Arg Ala
50 55 60
GCC TAT GAC CAG TAT GGT GCT GCA GGC GCC AAT GGT GGT TTT GGT GGA 3501
Ala Tyr Asp Gin Tyr Gly Ala Ala Gly Ala Asn Gly Gly Phe Gly Gly
65 70 75 GCT GGT GGT TTC GGC GGT TTC AAT GGG GCA GGT GGC TTC GGT GGT TTT 3549 Ala Gly Gly Phe Gly Gly Phe Asn Gly Ala Gly Gly Phe Gly Gly Phe 80 85 90 95
GAG GAT ATT TTC TCA AGT TTC TTC GGC GGA GGC GGT TCT TCG CGC AAT 3597 Glu Asp He Phe Ser Ser Phe Phe Gly Gly Gly Gly Ser Ser Arg Asn
100 105 110
CCA AAC GCT CCT CGC CAA GGA GAT GAT CTC CAG TAT CGT GTC AAT TTG 3645 Pro Asn Ala Pro Arg Gin Gly Asp Asp Leu Gin Tyr Arg Val Asn Leu 115 120 125
ACC TTT GAA GAA GCT ATC TTC GGA ACT GAG AAG GAA GTT AAG TAT CAT 3693 Thr Phe Glu Glu Ala He Phe Gly Thr Glu Lys Glu Val Lys Tyr His 130 135 140
CGT GAA GCT GGC TGT CGT ACA TGT AAT GGA TCT GGT GCT AAG CCA GGG 3741
Arg Glu Ala Gly Cys Arg Thr Cys Asn Gly Ser Gly Ala Lys Pro Gly 145 150 155 ACA AGT CCA GTC ACT TGT GGA CGC TGT CAT GGC GCT GGT GTC ATT AAC 3789
Thr Ser Pro Val Thr Cys Gly Arg Cys His Gly Ala Gly Val He Asn 160 165 170 175
GTC GAT ACG CAG ACT CCT CTT GGT ATG ATG CGT CGC CAA GTA ACC TGT 3837 Val Asp Thr Gin Thr Pro Leu Gly Met Met Arg Arg Gin Val Thr Cys
180 185 190
GAT GTC TGT CAC GGT CGA GGA AAA GAA ATC AAA TAT CCA TGT ACA ACC 3885
Asp Val Cys His Gly Arg Gly Lys Glu He Lys Tyr Pro Cys Thr Thr 195 200 205
TGT CAT GGA ACA GGT CAT GAG AAA CAA GCT CAT AGC GTA CAT GTG AAA 3933
Cys His Gly Thr Gly His Glu Lys Gin Ala His Ser Val His Val Lys 210 215 220
ATC CCT GCT GGT GTG GAA ACA GGT CAA CAA ATT CGC CTC GCT GGT CAA 3981 -
He Pro Ala Gly Val Glu Thr Gly Gin Gin He Arg Leu Ala Gly Gin 225 230 235
GGT GAA GCA GGC TTT AAC GGT GGA CCT TAT GGT GAC TTG TAT GTA GTA 4029
Gly Glu Ala Gly Phe Asn Gly Gly Pro Tyr Gly Asp Leu Tyr Val Val 240 245 250 255 GTT TCT GTG GAA GCT AGT GAC AAG TTT GAA CGT GAA GGA ACG ACT ATC 4077 Val Ser Val Glu Ala Ser Asp Lys Phe Glu Arg Glu Gly Thr Thr He 260 265 270
TTC TAC AAT CTC AAC CTC AAC TTT GTC CAA GCG GCT CTT GGT GAT ACA 4125 Phe Tyr Asn Leu Asn Leu Asn Phe Val Gin Ala Ala Leu Gly Asp Thr 275 280 285
GTA GAT ATT CCA ACT GTT CAC GGT GAT GTT GAA TTG GTT ATT CCA GAG 4173 Val Asp He Pro Thr Val His Gly Asp Val Glu Leu Val He Pro Glu 290 295 300
GGA ACT CAG ACT GGT AAG AAA TTC CGC CTA CGT AGT AAG GGG GCA CCG 4221
Gly Thr Gin Thr Gly Lys Lys Phe Arg Leu Arg Ser Lys Gly Ala Pro
305 310 315
AGC CTT CGT GGC GGT GCA GTT GGT GAC CAA TAC GTT ACT GTT AAT GTC 4269
Ser Leu Arg Gly Gly Ala Val Gly Asp Gin Tyr Val Thr Val Asn Val
320 325 330 335 GTA ACA CCG ACA GGC TTG AAC GAC CGC CAA AAA GTA GCC TTG AAA GAA 4317 Val Thr Pro Thr Gly Leu Asn Asp Arg Gin Lys Val Ala Leu Lys Glu 340 345 350
TTC 4320 Phe
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 607 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
( i) SEQUENCE DESCRIPTION: SEQ ID NO: : Met Ser Lys He He Gly He Asp Leu Gly Thr Thr Asn Ser Ala Val 1 5 10 15
Ala Val Leu Glu Gly Thr Glu Ser Lys He He Ala Asn Pro Glu Gly 20 25 30
Asn Arg Thr Thr Pro Ser Val Val Ser Phe Lys Asn Gly Glu He He 35 40 45
Val Gly Asp Ala Ala Lys Arg Gin Ala Val Thr Asn Pro Asp Thr Val 50 55 60
He Ser He Lys Ser Lys Met Gly Thr Ser Glu Lys Val Ser Ala Asn
65 70 75 80 Gly Lys Glu Tyr Thr Pro Gin Glu He Ser Ala Met He Leu Gin Tyr
85 90 95
Leu Lys Gly Tyr Ala Glu Asp Tyr Leu Gly Glu Lys Val Thr Lys Ala 100 105 110
Val He Thr Val Pro Ala Tyr Phe Asn Asp Ala Gin Arg Gin Ala Thr 115 120 125 Lys Asp Ala Gly Lys He Ala Gly Leu Glu Val Glu Arg He Val Asn 130 135 140
Glu Pro Thr Ala Ala Ala Leu Ala Tyr Gly Leu Asp Lys Thr Asp Lys 145 150 155 160
Glu Glu Lys He Leu Val Phe Asp Leu Gly Gly Gly Thr Phe Asp Val 165 170 175
Ser He Leu Glu Leu Gly Asp Gly Val Phe Asp Val Leu Ser Thr Ala 180 185 190
Gly Asp Asn Lys Leu Gly Gly Asp Asp Phe Asp Gin Lys He He Asp 195 200 205 His Leu Val Ala Glu Phe Lys Lys Glu Asn Gly He Asp Leu Ser Thr 210 215 220
Asp Lys Met Ala Met Gin Arg Leu Lys Asp Ala Ala Glu Lys Ala Lys 225 230 235 240
Lys Asp Leu Ser Gly Val Thr Ser Thr Gin He Ser Leu Pro Phe He 245 250 255
Thr Ala Gly Glu Ala Gly Pro Leu His Leu Glu Met Thr Leu Thr Arg 260 265 270
Ala Lys Phe Asp Asp Leu Thr Arg Asp Leu Val Glu Arg Thr Lys Val 275 280 285 Pro Val Arg Gin Ala Leu Ser Asp Ala Gly Leu Ser Leu Ser Glu He 290 295 300
Asp Glu Val He Leu Val Gly Gly Ser Thr Arg He 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 He Gin Gly Gly Val He 340 345 350
Thr Gly Asp Val Lys Asp Val Val Leu Leu Asp Val Thr Pro Leu Ser 355 360 365 Leu Gly He Glu Thr Met Gly Gly Val Phe Thr Lys Leu He Asp Arg 370 375 380
Asn Thr Thr He Pro Thr Ser Lys Ser Gin Val Phe Ser Thr Ala Ala 385 390 395 400
Asp Asn Gin Pro Ala Val Asp He His Val Leu Gin Gly Glu Arg Pro 405 410 415
Met Ala Ala Asp Asn Lys Thr Leu Gly Arg Phe Gin Leu Thr Asp He 420 425 430
Pro Ala Ala Pro Arg Gly He Pro Gin He Glu Val Thr Phe Asp He 435 440 445 Asp Lys Asn Gly He Val Ser Val Lys Ala Lys Asp Leu Gly Thr Gin 450 455 460
Lys Glu Gin Thr He Val He Gin Ser Asn Ser Gly Leu Thr Asp Glu 465 470 475 480
Glu He Asp Arg Met Met Lys Asp Ala Glu Ala Asn Ala Glu Ser Asp 485 490 495 Lys Lys Arg Lys Glu Glu Val Asp Leu Arg Asn Glu Val Asp Gin Ala 500 505 510
He Phe Ala Thr Glu Lys Thr He Lys Glu Thr Glu Gly Lys Gly Phe 515 520 525
Asp Ala Glu Arg Asp Ala Ala Gin Ala Ala Leu Asp Asp Leu Lys Lys 530 535 540
Ala Gin Glu Asp Asn Asn Leu Asp Asp Met Lys Ala Lys Leu Glu Ala 545 550 555 560
Leu Asn Glu Lys Ala Gin Gly Leu Ala Val Lys Leu Tyr Glu Gin Ala 565 570 575 Ala Ala Ala Gin Gin Ala Gin Glu Gly Ala Glu Gly Ala Gin Ala Thr 580 585 590
Gly Asn Ala Gly Asp Asp Val Val Asp Gly Glu Phe Thr Glu Lys 595 600 605
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 352 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6 :
Met Asn Asn Thr Glu Phe Tyr Asp Arg Leu Gly Val Ser Lys Asn Ala 1 5 10 15
Ser Ala Asp Glu He Lys Lys Ala Tyr Arg Lys Leu Ser Lys Lys Tyr 20 25 30
Asp Thr Gin Thr Pro Leu Gly Met Met Arg Arg Gin Val Thr Cys Asp 180 185 190 Val Cys His Gly Arg Gly Lys Glu He Lys Tyr Pro Cys Thr Thr Cys 195 200 205
His Gly Thr Gly His Glu Lys Gin Ala His Ser Val His Val Lys He 210 215 220
Pro Ala Gly Val Glu Thr Gly Gin Gin He Arg Leu Ala Gly Gin Gly 225 230 235 240
Glu Ala Gly Phe Asn Gly Gly Pro Tyr Gly Asp Leu Tyr Val Val Val 245 250 255
Ser Val Glu Ala Ser Asp Lys Phe Glu Arg Glu Gly Thr Thr He Phe 260 265 270 Tyr Asn Leu Asn Leu Asn Phe Val Gin Ala Ala Leu Gly Asp Thr Val 275 280 285
Asp He Pro Thr Val His Gly Asp Val Glu Leu Val He Pro Glu Gly 290 295 300
Thr Gin Thr Gly Lys Lys Phe Arg Leu Arg Ser Lys Gly Ala Pro Ser 305 310 315 320
Leu Arg Gly Gly Ala Val Gly Asp Gin Tyr Val Thr Val Asn Val Val 325 330 335
Thr Pro Thr Gly Leu Asn Asp Arg Gin Lys Val Ala Leu Lys Glu Phe 340 345 350
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids (B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7 :
Thr Ser Thr Gin He Ser Leu Pro Phe He Thr Ala Gly Glu Ala 1 5 10 15
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:8 : Thr Ala Gly Glu Ala Gly Pro Leu His Leu Glu Met Thr Leu Thr 1 5 10 15
(2) INFORMATION FOR SEQ ID NO:9 : (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid (D) 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 10
(2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Asp Asp Leu Thr Arg Asp Leu Val Glu Arg Thr Lys Val Pro Val 1 5 10 15
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
Thr Lys Val Pro Val Arg Gin Ala Leu Ser Asp Ala Gly Leu 1 5 10 (2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Lys Ala Lys Asp Leu Gly Thr Gin Lys Glu Gin Thr He Val He 1 5 10 15
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids (B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
( i ) SEQUENCE DESCRIPTION : SEQ ID NO : 13 :
Leu Thr Asp Glu He Asp Arg Met Met Lys Asp Ala Glu Ala 1 5 10
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 amino acids (B) TYPE: amino acid
(D) 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 15
Val Asp Leu Arg Asn Glu Val Asp 20 (2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
Asn Glu Val Asp Gin Ala He Phe Ala Thr Glu Lys Thr He Lys 1 5 10 15
(2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 amino acids (B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
Glu Lys Thr He Lys Glu Thr Glu Gly Lys Gly Phe Asp Ala Glu Arg 1 5 10 15
Asp Ala Ala Gin Ala Ala Leu Asp Asp Leu Lys Lys 20 25 (2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
Lys Ala Gin Glu Asp Asn Asn Leu Asp Asp Met Lys Ala Lys Leu Glu
1 5 10 15 Ala Leu Asn Glu Lys Ala Gin Gly Leu Ala Val Lys Leu Tyr
20 25 30
(2) INFORMATION FOR SEQ ID NO:18: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 amino acids
(B) TYPE: amino acid (D) 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 15
Val Val Asp Gly Glu Phe Thr Glu Lys 20 25
(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2183 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Streptococcus pyogenes
(ix) FEATURE: (A) NAME/KEY: CDS
(B) LOCATION: 204..2030
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
CAGCGATGGT AGTTGTTTAT AACTAAGGTA AATGAGTTTT CGTTTTTGTC CGTAATGACA 60
GTAAACTAGA TAGCAAGTTA GAAGCTATTT CGCTTGCTGA TTAAACTATA GTGATTGCTT 120 AGAATTGGAA GTAAAATAAT TCGAGTGCTT ACTAAGATAA ATTGAAATAA AAAGTAATAA 180
AGTATAAAAT AAGAGGTATT AAC ATG TCT AAA ATT ATT GGT ATT GAC TTA 230
Met Ser Lys He He Gly He Asp Leu 1 5
GGT ACA ACA AAC TCA GCA GTA GCA GTT CTT GAA GGG ACT GAA TCA AAA 278 Gly Thr Thr Asn Ser Ala Val Ala Val Leu Glu Gly Thr Glu Ser Lys 10 15 20 25 ATC ATT GCT AAC CCA GAA GGC AAT CGT ACA ACT CCT TCA GTA GTA TCA 326 He He Ala Asn Pro Glu Gly Asn Arg Thr Thr Pro Ser Val Val Ser 30 35 40
TTC AAA AAT GGT GAA ATT ATC GTG GGT GAT GCT GCA AAA CGC CAA GCA 374 Phe Lys Asn Gly Glu He He Val Gly Asp Ala Ala Lys Arg Gin Ala 45 50 55
GTG ACA AAC CCA GAA ACA GTA ATC TCT ATT AAA TCT AAA ATG GGA ACT 422
Val Thr Asn Pro Glu Thr Val He Ser He Lys Ser Lys Met Gly Thr 60 65 70
TCT GAA AAA GTT TCT GCA AAT GGT AAA GAA TAT ACT CCT CAA GAA ATT 470
Ser Glu Lys Val Ser Ala Asn Gly Lys Glu Tyr Thr Pro Gin Glu He 75 80 85 TCA GCA ATG ATT CTT CAA TAC CTT AAA GGT TAT GCT GAA GAC TAT CTT 518 Ser Ala Met He Leu Gin Tyr Leu Lys Gly Tyr Ala Glu Asp Tyr Leu 90 95 100 105
GGA GAA AAA GTA GAA AAA GCA GTT ATT ACT GTT CCA GCT TAT TTC AAC 566 Gly Glu Lys Val Glu Lys Ala Val He Thr Val Pro Ala Tyr Phe Asn
110 115 120
GAT GCA CAA CGT CAA GCA ACT AAA GAC GCT GGT AAA ATT GCA GGT CTT 614 Asp Ala Gin Arg Gin Ala Thr Lys Asp Ala Gly Lys He Ala Gly Leu 125 130 135
GAA GTA GAA CGT ATC GTT AAT GAA CCA ACA GCA GCT GCA CTT GCT TAT 662
Glu Val Glu Arg He Val Asn Glu Pro Thr Ala Ala Ala Leu Ala Tyr 140 145 150
GGT ATG GAC AAG ACT GAC AAG GAT GAA AAA ATC TTA GTT TTT GAC CTT 710
Gly Met Asp Lys Thr Asp Lys Asp Glu Lys He Leu Val Phe Asp Leu 155 160 165 GGT GGT GGT ACA TTT GAC GTA TCA ATC CTT GAA TTA GGT GAT GGT GTC 758 Gly Gly Gly Thr Phe Asp Val Ser He Leu Glu Leu Gly Asp Gly Val 170 175 180 185
TTC GAC GTT CTT GCA ACA GCA GGT GAT AAC AAA CTT GGT GGT GAC GAC 806 Phe Asp Val Leu Ala Thr Ala Gly Asp Asn Lys Leu Gly Gly Asp Asp
190 195 200
TTT GAC CAA AAA ATT ATT GAT TTC TTA GTG GCT GAA TTT AAG AAA GAA 854 Phe Asp Gin Lys He He Asp Phe Leu Val Ala Glu Phe Lys Lys Glu 205 210 215
AAT GGT ATT GAC TTA TCA CAA GAT AAG ATG GCA CTT CAA CGC TTG AAA 902 Asn Gly He Asp Leu Ser Gin Asp Lys Met Ala Leu Gin Arg Leu Lys 220 225 230
GAT GCT GCT GAA AAA GCT AAA AAA GAT CTT TCA GGT GTG ACA CAA ACA 950 Asp Ala Ala Glu Lys Ala Lys Lys Asp Leu Ser Gly Val Thr Gin Thr 235 240 245 CAA ATT TCA TTA CCG TTC ATC ACT GCT GGT TCT GCT GGT CCT CTT CAC 998 Gin He Ser Leu Pro Phe He Thr Ala Gly Ser Ala Gly Pro Leu His 250 255 260 265
TTA GAG ATG AGC TTA TCT CGT GCT AAA TTT GAC GAT CTC ACT CGT GAC 1046 Leu Glu Met Ser Leu Ser Arg Ala Lys Phe Asp Asp Leu Thr Arg Asp
270 275 280
CTT GTT GAA CGT ACG AAA ACT CCA GTT CGT CAA GCT CTT TCA GAT GCA 1094 Leu Val Glu Arg Thr Lys Thr Pro Val Arg Gin Ala Leu Ser Asp Ala 285 290 295
GGA TTG TCA TTG TCA GAA ATT GAT GAA GTT ATC CTT GTT GGT GGA TCA 1142 Gly Leu Ser Leu Ser Glu He Asp Glu Val He Leu Val Gly Gly Ser 300 305 310
ACT CGT ATC CCA GCA GTT GTC GAA GCT GTA AAA GCT GAA ACT GGT AAA 1190 Thr Arg He Pro Ala Val Val Glu Ala Val Lys Ala Glu Thr Gly Lys 315 320 325
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 He Gin Gly Gly Val He 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 He 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 He Asp Arg Asn Thr Thr He Pro Thr Ser Lys Ser 380 385 390
CAA GTC TTC 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 He 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 ACT GAT ATC CCA GCT GCA CCT CGT GGA ATC CCA CAA 1526
Arg Phe Gin Leu Thr Asp He Pro Ala Ala Pro Arg Gly He Pro Gin 430 435 440 ATT GAA GTA ACA TTT GAT ATC GAT AAA AAC GGT ATT GTT TCT GTA AAA 1574 He Glu Val Thr Phe Asp He Asp Lys Asn Gly He Val Ser Val Lys 445 450 455
GCT AAA GAC CTT GGT ACG CAA AAG GAA CAA CAC ATC GTT ATC AAA TCA 1622 Ala Lys Asp Leu Gly Thr Gin Lys Glu Gin His He Val He 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 He 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 He Phe Ala Thr Glu Lys Thr He 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 Tyr 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 Asn Asn Asp Asp Val Val 590 595 600
GAT GGC GAA TTT ACA GAA AAG TAATGATTTA GTTATCTAGT AACATTAATA 2057 -
Asp Gly Glu Phe Thr Glu Lys 605
TCCGAATTCA GAGGTTGTAC CAAACCTCTG TTTTTGGCTA AATAAAATGT AAAAATGCTG 2117
ACGTCAAAAT ATTTTAAGAA AGGAATACAA GTTCGATTAT TCGAACACAG GCTAAAGCGT 2177 GTAAAG 2183
(2) INFORMATION FOR SEQ ID NO:20: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 608 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
Met Ser Lys He He Gly He Asp Leu Gly Thr Thr Asn Ser Ala Val 1 5 10 15
Ala Val Leu Glu Gly Thr Glu Ser Lys He He Ala Asn Pro Glu Gly 20 25 30 Asn Arg Thr Thr Pro Ser Val Val Ser Phe Lys Asn Gly Glu He He 35 40 45
Val Gly Asp Ala Ala Lys Arg Gin Ala Val Thr Asn Pro Glu Thr Val 50 55 60
He Ser He Lys Ser Lys Met Gly Thr Ser Glu Lys Val Ser Ala Asn 65 70 75 80
Gly Lys Glu Tyr Thr Pro Gin Glu He Ser Ala Met He Leu Gin Tyr 85 90 95
Leu Lys Gly Tyr Ala Glu Asp Tyr Leu Gly Glu Lys Val Glu Lys Ala 100 105 110 Val He Thr Val Pro Ala Tyr Phe Asn Asp Ala Gin Arg Gin Ala Thr 115 120 125
Lys Asp Ala Gly Lys He Ala Gly Leu Glu Val Glu Arg He 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 He Leu Val Phe Asp Leu Gly Gly Gly Thr Phe Asp Val 165 170 175
Ser He Leu Glu Leu Gly Asp 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 He He Asp 195 200 205
Phe Leu Val Ala Glu Phe Lys Lys Glu Asn Gly He Asp Leu Ser Gin 210 215 220
Asp Lys Met Ala Leu Gin Arg Leu Lys Asp Ala Ala Glu Lys Ala Lys 225 230 235 240
Lys Asp Leu Ser Gly Val Thr Gin Thr Gin He Ser Leu Pro Phe He 245 250 255
Thr Ala Gly Ser Ala Gly Pro 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 He 290 295 300 Asp Glu Val He Leu Val Gly Gly Ser Thr Arg He 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 He Gin Gly Gly Val He 340 345 350
Thr Gly Asp Val Lys Asp Val Val Leu Leu Asp Val Thr Pro Leu Ser 355 360 365
Leu Gly He Glu Thr Met Gly Gly Val Phe Thr Lys Leu He Asp Arg 370 375 380 Asn Thr Thr He Pro Thr Ser Lys Ser Gin Val Phe Ser Thr Ala Ala 385 390 395 400
Asp Asn Gin Pro Ala Val Asp He His Val Leu Gin Gly Glu Arg Pro 405 410 415
Met Ala Ala Asp Asn Lys Thr Leu Gly Arg Phe Gin Leu Thr Asp He 420 425 430
Pro Ala Ala Pro Arg Gly He Pro Gin He Glu Val Thr Phe Asp He 435 440 445
Asp Lys Asn Gly He Val Ser Val Lys Ala Lys Asp Leu Gly Thr Gin 450 455 460 Lys Glu Gin His He Val He Lys Ser Asn Asp Gly Leu Ser Glu Glu 465 470 475 480
Glu He Asp Arg Met Met Lys Asp Ala Glu Ala Asn Ala Glu Ala Asp 485 490 495
Ala Lys Arg Lys Glu Glu Val Asp Leu Lys Asn Glu Val Asp Gin Ala 500 505 510
He Phe Ala Thr Glu Lys Thr He Lys Glu Thr Glu Gly Lys Gly Phe 515 520 525
Asp Thr Glu Arg Asp Ala Ala Gin Ser Ala Leu Asp Glu Leu Lys Ala 530 535 540 Ala Gin Glu Ser Gly Asn Leu Asp Asp Met Lys Ala Lys Leu Glu Ala 545 550 555 560
Leu Asn Glu Lys Ala Gin Ala Leu Ala Val Lys Met Tyr Glu Gin Ala 565 570 575
Ala Ala Ala Gin Gin Ala Ala Gin Gly Ala Glu Gly Ala Gin Ala Asn 580 585 590
Asp Ser Ala Asn Asn Asp Asp Val Val Asp Gly Glu Phe Thr Glu Lys 595 600 605
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2438 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Streptococcus agalactiae
(ix) FEATURE: (A) NAME/KEY: CDS
(B) LOCATION: 248..2077
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
CTTTCAAAAG GGATATAAAT TGCACGAGCG TCTGCTAAGA CCAGCGATGG TAGTTGTCTA 60
TAACTAAGGT AAATGAGTTT TCGTTTTTGT CCGTAATGAC AGTAAACTAG ATAGCAAGTT 120 AGAAGCTATT CAGCTTGCTG ATTAAACTAT AGTGATTGCT TAGAATTGGA AGTAAAATAA 180
TTCGAGTGCT TACTAAGATA AATTGAAATA AAAAGTAATA AAGTATTATA AAATAAGAGG 240
TATTAAC ATG TCT AAA ATT ATT GGT ATT GAC TTA GGT ACA ACA AAC TCA 289 Met Ser Lys He He Gly He Asp Leu Gly Thr Thr Asn Ser
1 5 10
GCA GTA GCA GTT CTT GAA GGG ACT GAA TCA AAA ATC ATT GCT AAC CCA 337 Ala Val Ala Val Leu Glu Gly Thr Glu Ser Lys He He Ala Asn Pro 15 20 25 30
GAA GGC AAT CGT ACA ACT CCT TCA GTA GTA TCA TTC AAA AAT GGT GAA 385
Glu Gly Asn Arg Thr Thr Pro Ser Val Val Ser Phe Lys Asn Gly Glu
35 40 45
ATT ATC GTG GGT GAT GCT GCA AAA CGT CAA GCG GTA ACA AAT CCA GAT 433
He He Val Gly Asp Ala Ala Lys Arg Gin Ala Val Thr Asn Pro Asp
50 55 60 ACT GTT ATC TCT ATC AAA TCA AAG ATG GGA ACT TCT GAA AAA GTT TCT 481 Thr Val He Ser He Lys Ser Lys Met Gly Thr Ser Glu Lys Val Ser 65 70 75
GCA AAT GGT AAA GAA TAT ACT CCT CAA GAA ATT TCA GCA ATG ATT CTT 529 Ala Asn Gly Lys Glu Tyr Thr Pro Gin Glu He Ser Ala Met He Leu 80 85 90
CAA TAC CTT AAA GGT TAT GCT GAA GAC TAT CTT GGA GAA AAA GTA GAA 577 Gin Tyr Leu Lys Gly Tyr Ala Glu Asp Tyr Leu Gly Glu Lys Val Glu 95 100 105 110
AAA GCA GTT ATT ACT GTT CCA GCT TAC TTC AAC GAT GCA CAA CGT CAG 625 Lys Ala Val He Thr Val Pro Ala Tyr Phe Asn Asp Ala Gin Arg Gin 115 120 125
GCA ACT AAA GAC GCT GGT AAA ATT GCA GGT CTT GAA GTA GAA CGT ATC 673 Ala Thr Lys Asp Ala Gly Lys He Ala Gly Leu Glu Val Glu Arg He 130 135 140
GTT AAC GAA CCA ACA GCA GCC GCA CTT GCT TAT GGT ATG GAC AAG ACT 721
Val Asn Glu Pro Thr Ala Ala Ala Leu Ala Tyr Gly Met Asp Lys Thr 145 150 155
GAC AAG GAT GAA AAA ATC TTA GTT TTT GAC CTT GGT GGT GGT ACA TTT 769
Asp Lys Asp Glu Lys He Leu Val Phe Asp Leu Gly Gly Gly Thr Phe 160 165 170 GAC GTA TCA ATC CTT GAA TTA GGT GAT GGT GTC TTC GAC GTT CTT GCA 817 Asp Val Ser He Leu Glu Leu Gly Asp Gly Val Phe Asp Val Leu Ala 175 180 185 190
ACA GCA GGT GAT AAC AAA CTT GGT GGT GAC GAC TTT GAC CAG AAA ATT 865 Thr Ala Gly Asp Asn Lys Leu Gly Gly Asp Asp Phe Asp Gin Lys He
195 200 205
ATT GAT TTC TTG GTA GAA GAA TTC AAG AAA GAA AAT GGT ATT GAT CTT 913 He Asp Phe Leu Val Glu Glu Phe Lys Lys Glu Asn Gly He Asp Leu 210 215 220
TCT CAA GAC AAA ATG GCT CTT CAA CGC TTG AAA GAT GCT GCT GAA AAA 961
Ser Gin Asp Lys Met Ala Leu Gin Arg Leu Lys Asp Ala Ala Glu Lys 225 230 235
GCT AAA AAA GAC CTT TCA GGT GTA ACT CAA ACT CAA ATT TCA TTA CCG 1009
Ala Lys Lys Asp Leu Ser Gly Val Thr Gin Thr Gin He Ser Leu Pro 240 245 250 TTC ATC ACT GCT GGT TCT GCT GGT CCT CTT CAC TTG GAG ATG AGC TTA 1057 Phe He Thr Ala Gly Ser Ala Gly Pro Leu His Leu Glu Met Ser Leu 255 260 265 270
TCA CGT GCT AAA TTT GAC GAT CTC ACT CGT GAC CTT GTT GAA CGT ACG 1105 Ser Arg Ala Lys Phe Asp Asp Leu Thr Arg Asp Leu Val Glu Arg Thr
275 280 285
AAA ACT CCA GTT CGT CAA GCT CTT TCA GAT GCA GGC TTG TCA TTG TCA 1153 Lys Thr Pro Val Arg Gin Ala Leu Ser Asp Ala Gly Leu Ser Leu Ser 290 295 300
GAA ATT GAT GAA GTT ATC CTC GTT GGT GGA TCA ACA CGT ATC CCA GCA 1201
Glu He Asp Glu Val He Leu Val Gly Gly Ser Thr Arg He Pro Ala
305 310 315
GTT GTT GAA GCT GTA AAA GCT GAA ACT GGT AAA GAA CCA AAT AAA TCT 1249
Val Val Glu Ala Val Lys Ala Glu Thr Gly Lys Glu Pro Asn Lys Ser
320 325 330 GTT AAC CCT GAT GAA GTG GTT GCC ATG GGT GCT GCT ATC CAA GGT GGT 1297 Val Asn Pro Asp Glu Val Val Ala Met Gly Ala Ala He Gin Gly Gly 335 340 345 350
GTT ATC ACT GGG GAT GTG AAA GAC GTT GTA CTT CTT GAC GTA ACA CCA 1345 Val He Thr Gly Asp Val Lys Asp Val Val Leu Leu Asp Val Thr Pro
355 360 365
TTG TCA CTT GGT ATT GAA ACA ATG GGT GGT GTC TTC ACT AAA TTG ATC 1393 Leu Ser Leu Gly He Glu Thr Met Gly Gly Val Phe Thr Lys Leu He 370 375 380
GAC CGC AAC ACA ACT ATC CCA ACA TCT AAA TCA CAA GTC TTC TCA ACA 1441 Asp Arg Asn Thr Thr He Pro Thr Ser Lys Ser Gin Val Phe Ser Thr 385 390 395
GCA GCA GAC AAC CAA CCA GCC GTT GAT ATC CAT GTT CTT CAA GGT GAA 1489 Ala Ala Asp Asn Gin Pro Ala Val Asp He His Val Leu Gin Gly Glu 400 405 410
CGC CCA ATG GCA GCA GAT AAC AAA ACA CTC GGT CGC TTC CAA TTG ACT 1537
Arg Pro Met Ala Ala Asp Asn Lys Thr Leu Gly Arg Phe Gin Leu Thr
415 420 425 430
GAT ATC CCA GCT GCA CCT CGT GGA ATC CCA CAA ATT GAA GTA ACA TTT 1585
Asp He Pro Ala Ala Pro Arg Gly He Pro Gin He Glu Val Thr Phe 435 440 445 GAT ATC GAT AAA AAT GGT ATT GTA TCT GTT AAA GCT AAA GAT CTC GGT 1633 Asp He Asp Lys Asn Gly He Val Ser Val Lys Ala Lys Asp Leu Gly 450 455 460
ACT CAA AAA GAA CAA CAC ATT GTT ATC CAA TCT AAT TCA GGA TTA ACT 1681 Thr Gin Lys Glu Gin His He Val He Gin Ser Asn Ser Gly Leu Thr 465 470 475
GAT GAA GAA ATT GAT AAA ATG ATG AAA GAT GCT GAA GCA AAT GCT GAA 1729 Asp Glu Glu He Asp Lys Met Met Lys Asp Ala Glu Ala Asn Ala Glu 480 485 490
GCA GAT GCA AAA CGT AAA GAA GAA GTT GAT CTT AAA AAT GAA GTT GAC 1777
Ala Asp Ala Lys Arg Lys Glu Glu Val Asp Leu Lys Asn Glu Val Asp 495 500 505 510
CAA GCC ATC TTT GCA ACA GAA AAA ACT ATT AAA GAA ACT GAA GGC AAA 1825
Gin Ala He Phe Ala Thr Glu Lys Thr He Lys Glu Thr Glu Gly Lys 515 520 525 GGT TTT GAT ACA GAA CGC GAT GCA GCG CAA TCA GCA CTT GAT GAG TTG 1873 Gly Phe Asp Thr Glu Arg Asp Ala Ala Gin Ser Ala Leu Asp Glu Leu 530 535 540
AAA AAA GCT CAA GAA TCA GGT AAC CTT GAC GAC ATG AAA GCT AAA CTT 1921 Lys Lys Ala Gin Glu Ser Gly Asn Leu Asp Asp Met Lys Ala Lys Leu 545 550 555
GAA GCT CTT AAC GAA AAA GCA CAA GCT CTT GCA GTT AAA CTT TAC GAA 1969 Glu Ala Leu Asn Glu Lys Ala Gin Ala Leu Ala Val Lys Leu Tyr Glu 560 565 570
CAA GCG GCT GCA GCA CAA CAA GCA GCT CAA GGG GCT GAA GGT GCA CAA 2017 Gin Ala Ala Ala Ala Gin Gin Ala Ala Gin Gly Ala Glu Gly Ala Gin 575 580 585 590
TCA GCT GAT TCA TCA AGC AAG GGT GAT GAT GTT GTA GAT GGC GAA TTC 2065 Ser Ala Asp Ser Ser Ser Lys Gly Asp Asp Val Val Asp Gly Glu Phe 595 600 605 ACT GAG AAA TAATTATTAA TATTGTTCAG ATTCATTTGA ATATAAGCAT 2114
Thr Glu Lys
610
GAAAACTATA CTAGCATAGT AAAGTTCTTC GTGATAGGGA TTGCTCAATA ATCTAGATAA 2174
GTTTCAGATT ACATAAGCTA ATTTCGCTAT CACTAAATAA AAACATATTA ATAATAAATA 2234
GGCGGGGCGC CTCGCTCCGT CTGTTTTATT AAGTGTCATA TATATGTTAA CTATTTAGAG 2294 CTGTAACTGG GCAAGAATAA TTGTTAATCT CTTCAAGTGT AGTATATGAA CAAAATATAA 2354
AGGATTAGAT AATGAACAAT ACAGAATTTT ATGATCGTCT TGGCGTTTCA AAAGATGCTT 2414
CTCAGGACGA AATAAAAAAA GCTT 2438
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 609 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
Met Ser Lys He He Gly He Asp Leu Gly Thr Thr Asn Ser Ala Val 1 5 10 15 Ala Val Leu Glu Gly Thr Glu Ser Lys He He Ala Asn Pro Glu Gly 20 25 30
Asn Arg Thr Thr Pro Ser Val Val Ser Phe Lys Asn Gly Glu He He 35 40 45
Val Gly Asp Ala Ala Lys Arg Gin Ala Val Thr Asn Pro Asp Thr Val 50 55 60
He Ser He Lys Ser Lys Met Gly Thr Ser Glu Lys Val Ser Ala Asn 65 70 75 80
Gly Lys Glu Tyr Thr Pro Gin Glu He Ser Ala Met He Leu Gin Tyr 85 90 95 Leu Lys Gly Tyr Ala Glu Asp Tyr Leu Gly Glu Lys Val Glu Lys Ala 100 105 110
Val He Thr Val Pro Ala Tyr Phe Asn Asp Ala Gin Arg Gin Ala Thr 115 120 125
Lys Asp Ala Gly Lys He Ala Gly Leu Glu Val Glu Arg He 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 He Leu Val Phe Asp Leu Gly Gly Gly Thr Phe Asp Val 165 170 175 Ser He Leu Glu Leu Gly Asp 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 He He Asp 195 200 205
Phe Leu Val Glu Glu Phe Lys Lys Glu Asn Gly He Asp Leu Ser Gin 210 215 220
Asp Lys Met Ala Leu Gin Arg Leu Lys Asp Ala Ala Glu Lys Ala Lys 225 230 235 240
Lys Asp Leu Ser Gly Val Thr Gin Thr Gin He Ser Leu Pro Phe He 245 250 255 Thr Ala Gly Ser Ala Gly Pro 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 He 290 295 300
Asp Glu Val He Leu Val Gly Gly Ser Thr Arg He 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 He Gin Gly Gly Val He 340 345 350
Thr Gly Asp Val Lys Asp Val Val Leu Leu Asp Val Thr Pro Leu Ser 355 360 365 Leu Gly He Glu Thr Met Gly Gly Val Phe Thr Lys Leu He Asp Arg 370 375 380
Asn Thr Thr He Pro Thr Ser Lys Ser Gin Val Phe Ser Thr Ala Ala 385 390 395 400
Asp Asn Gin Pro Ala Val Asp He His Val Leu Gin Gly Glu Arg Pro 405 410 415
Met Ala Ala Asp Asn Lys Thr Leu Gly Arg Phe Gin Leu Thr Asp He 420 425 430
Pro Ala Ala Pro Arg Gly He Pro Gin He Glu Val Thr Phe Asp He 435 440 445 Asp Lys Asn Gly He Val Ser Val Lys Ala Lys Asp Leu Gly Thr Gin 450 455 460
Lys Glu Gin His He Val He Gin Ser Asn Ser Gly Leu Thr Asp Glu 465 470 475 480
Glu He Asp Lys Met Met Lys Asp Ala Glu Ala Asn Ala Glu Ala Asp 485 490 495
Ala Lys Arg Lys Glu Glu Val Asp Leu Lys Asn Glu Val Asp Gin Ala 500 505 510
He Phe Ala Thr Glu Lys Thr He Lys Glu Thr Glu Gly Lys Gly Phe 515 520 525 Asp Thr Glu Arg Asp Ala Ala Gin Ser Ala Leu Asp Glu Leu Lys Lys 530 535 540
Ala Gin Glu Ser Gly Asn Leu Asp Asp Met Lys Ala Lys Leu Glu Ala 545 550 555 560
Leu Asn Glu Lys Ala Gin Ala Leu Ala Val Lys Leu Tyr Glu Gin Ala 565 570 575
Ala Ala Ala Gin Gin Ala Ala Gin Gly Ala Glu Gly Ala Gin Ser Ala 580 585 590
Asp Ser Ser Ser Lys Gly Asp Asp Val Val Asp Gly Glu Phe Thr Glu 595 600 605 Lys
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
Arg He Pro Ala Val Val Glu Ala Val Lys Ala Glu Thr Gly Lys Glu 1 5 10 15 Pro Asn Lys
(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
( ii ) MOLECULE TYPE : peptide
(xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 24 : Gin Thr He Val He Gin Ser Asn Ser Gly Leu Thr Asp Glu Glu
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:25: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 460 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Streptococcus pneumoniae (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..456
(D) OTHER INFORMATION: /product= "C-terminal 151-reεidue fragment (C-151) of HΞP72"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
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 He Val He 1 5 10 15
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 He Asp Arg Met Met Lys 20 25 30
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 35 40 45
GAC CTT CGT AAT GAA GTG GAC CAA GCA ATC TTT GCG ACT GAA AAG ACA 192
Asp Leu Arg Asn Glu Val Asp Gin Ala He Phe Ala Thr Glu Lys Thr 50 55 60 ATC AAG GAA ACT GAA GGT AAA GGC TTC GAC GCA GAA CGT GAC GCT GCC 240 He Lys Glu Thr Glu Gly Lys Gly Phe Asp Ala Glu Arg Asp Ala Ala 65 70 75 80
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
85 90 95
GAC GAC ATG AAA GCA AAA CTT GAA GCA TTG AAC GAA AAA GCT CAA GGA 336 Asp Asp Met Lys Ala Lys Leu Glu Ala Leu Asn Glu Lys Ala Gin Gly 100 105 110
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 Gin 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 Asn 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
(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 152 amino acids
(B) TYPE: amino acid (D) 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 He Val He 1 5 10 15
Gin Ser Asn Ser Gly Leu Thr Asp Glu Glu He Asp Arg Met Met Lys 20 25 30
Asp Ala Glu Ala Asn Ala Glu Ser Asp Lys Lys Arg Lys Glu Glu Val 35 40 45
Asp Leu Arg Asn Glu Val Asp Gin Ala He Phe Ala Thr Glu Lys Thr 50 55 60
He Lys Glu Thr Glu Gly Lys Gly Phe Asp Ala Glu Arg Asp Ala Ala 65 70 75 80 Gin Ala Ala Leu Asp Asp Leu Lys Lys Ala Gin Glu Asp Asn Asn Leu
85 90 95
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
1. A polypeptide selected from the group consisting of:
(a) the HSP72 polypeptide having the amino acid sequence of SEQ ID NO:5;
(b) the HSP70(DnaK) polypeptide having the amino acid sequence of SEQ ID NO: 20;
(c) the HSP70 (DnaK) polypeptide having the amino acid sequence of SEQ ID NO:22;
(d) polypeptides that are immunologically reactive with antibodies generated by infection of a mammalian host with Streptococcus pneumoniae cells, which antibodies are immunologically reactive with the
polypeptide of paragraph (a), (b), or (c);
(e) polypeptides that are capable of eliciting antibodies that are immunologically reactive with the polypeptide of paragraph (a), (b), or (c);
(f) polypeptides that are immunologically reactive with antibodies elicited by immunization with the polypeptide of paragraph (a), (b), or (c); and
(g) fragments of any of the foregoing polypeptides, either alone or in combination with other polypeptides to form a fusion protein.
2. The polypeptide of claim 1, wherein the polypeptides of paragraph (d) are selected from the group consisting of polypeptides of the genera Streptococcus and Enterococcus .
3. The polypeptide of claim 1, wherein the polypeptides of paragraph (d) are selected from the group consisting of polypeptides of the species Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus pyogenes, Streptococcus mutans, Streptococcus sanguis, and Enterococcus faecalis .
4. The polypeptide of claim 1, wherein the polypeptides of paragraph (d) are selected from the group consisting of polypeptides of the species Streptococcus pneumoniae, Streptococcus agalactiae, and Streptococcus pyogenes .
5. The polypeptide of claim 1, wherein the fragments of paragraph (g) are selected from the group consisting of amino acids 439-607 of SEQ ID NO:5 (C-169), amino acids 457-607 of SEQ ID NO:5 (C-151), amino acids 527-541 of SEQ ID NO:5, and amino acids 586-600 of SEQ ID NO:5.
6. A polypeptide having the amino acid sequence of SEQ ID NO : 5 analogues , homologues and
derivatives thereof .
7. A polypeptide having the amino acid sequence of SEQ ID NO:20, analogues, homologues and derivatives thereof.
8. A polypeptide having the amino acid sequence of SEQ ID NO: 22, analogues, homologues and derivatives thereof.
9. A polypeptide having the amino acid sequence of SEQ ID NO:26, analogues, homologues and derivatives thereof.
10. A polypeptide having the amino acid sequence of SEQ ID NO:7, analogues, homologues and derivatives thereof.
11. A polypeptide having the amino acid sequence of SEQ ID NO:8, analogues, homologues and derivatives thereof.
12. A polypeptide having the amino acid sequence of SEQ ID NO:9, analogues, homologues and derivatives thereof.
13. A polypeptide having the amino acid sequence of SEQ ID NO:10, analogues, homologues and derivatives thereof.
14. A polypeptide having the amino acid sequence of SEQ ID NO:11, analogues, homologues and derivatives thereof.
15. A polypeptide having the amino acid sequence of SEQ ID NO:12, analogues, homologues and derivatives thereof.
16. A polypeptide having the amino acid sequence of SEQ ID NO:13, analogues, homologues and derivatives thereof.
17. A polypeptide having the amino acid sequence of SEQ ID NO:14, analogues, homologues and derivatives thereof.
18. A polypeptide having the amino acid
sequence of SEQ ID NO: 15, analogues, homologues and derivatives thereof.
19. A polypeptide having the amino acid
sequence of SEQ ID NO: 16 analogues, homologues and
derivatives thereof.
20. A polypeptide having the amino acid
sequence of SEQ ID NO: 17, analogues, homologues and derivatives thereof.
21. A polypeptide having the amino acid
sequence of SEQ ID NO: 18, analogues, homologues and derivatives thereof.
22. The polypeptide of any one of claims 1 to 21 and 100-101, wherein said polypeptide elicits an immune reaction that is specific to Streptococcal strains.
23. A polypeptide selected from the group consisting of:
(a) the HSP72 polypeptide having the amino acid sequence of SEQ ID NO :5;
(b) polypeptides that are immunologically reactive with antibodies generated by infection of a mammalian host with Streptococcus pneumoniae cells, which antibodies are immunologically reactive with the HSP72 polypeptide of paragraph (a);
(c) polypeptides that are capable of eliciting antibodies that are immunologically reactive with the HSP72 polypeptide of paragraph (a) ; (d) polypeptides that are immunologically reactive with antibodies elicited by immunization with the HSP72 polypeptide of paragraph (a); and
(e) fragments of any of the foregoing polypeptides, either alone or in combination with other polypeptides to form a fusion protein.
24. The polypeptide of claim 23, wherein the polypeptides of paragraph (b) are selected from the group consisting of polypeptides of the genera Streptococcus and Enterococcus .
25. The polypeptide of claim 23, wherein the polypeptides of paragraph (b) are selected from the group consisting of polypeptides of the species Streptococcus pyogenes, Streptococcus mutans, Streptococcus sanguis, and Enterococcus faecalis .
26. The polypeptide of claim 23, wherein the fragments of paragraph (e) are selected from the group consisting of amino acids 439-607 of SEQ ID NO:5 (C-169); amino acids 527-541 of SEQ ID NO:5, and amino acids 586- 600 of SEQ ID NO: 5.
27. The polypeptide of claim 23, wherein the fusion protein of paragraph (e) is the Fucose Isomerase- HSP72 (C-169) protein having the amino acid sequence of SEQ ID NO:3.
28. A DNA sequence selected from the group consisting of:
(a) the HSP72 DNA sequence of SEQ ID NO:4;
(b) the HSP70 (DnaK) DNA sequence of SEQ ID NO:19; (c) the HSP70 (DnaK) DNA sequence of SEQ ID NO: 21;
(d) DNA sequences encoding polypeptides that are immunologically reactive with antibodies
generated by infection of a mammalian host with
Streptococcus pneumoniae cells, which antibodies are immunologically reactive with the HSP72 polypeptide (SEQ ID NO:5);
(e) DNA sequences encoding polypeptides that are capable of eliciting antibodies that are
immunologically reactive with the HSP72 polypeptide (SEQ ID NO :5);
(f) DNA sequences encoding polypeptides that are immunologically reactive with antibodies elicited by immunization with the HSP72 polypeptide (SEQ ID NO:5);
(g) DNA sequences that are degenerate to any of the foregoing DNA sequences; and
(h) fragments of any of the foregoing DNA sequences, either alone or in combination with other DNA sequences to form a fusion DNA sequence.
29. The DNA sequence of claim 28, wherein the DNA sequences of paragraph (d) are selected from the group consisting of DNA sequences of the genera Streptococcus and Enterococcus .
30. The DNA sequence of claim 28, wherein the DNA sequences of paragraph (d) are selected from the group consisting of DNA sequences of the species Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus pyogenes, Streptococcus mutans, Streptococcus sanguis, and Enterococcus faecalis .
31. The DNA sequence of claim 28, wherein the DNA sequences of paragraph (d) are selected from the group consisting of DNA sequences of the species Streptococcus pneumoniae, Streptococcus agalactiae, and Streptococcus pyogenes .
32. A DNA sequence of the formula of SEQ ID
NO: 4 from nucleotide 682 to nucleotide 2502, or
derivatives thereof, coding for HSP72.
33. A DNA sequence of the formula of SEQ ID NO: 4 from nucleotide 1996 to nucleotide 2502, or
derivatives thereof, coding for the C-169 fragment of
HSP72.
34. A DNA sequence of the formula of SEQ ID NO: 4 from nucleotide 2050 to nucleotide 2502, or
derivatives thereof, coding for the C-151 fragment of HSP72.
35. A DNA sequence of the formula of SEQ ID NO: 4 from nucleotide 2260 to nucleotide 2304, or
derivatives thereof.
36. A DNA sequence of the formula of SEQ ID NO: 4 from nucleotide 2437 to nucleotide 2481, or
derivatives thereof.
37. A DNA sequence of the formula of SEQ ID NO: 19 from nucleotide 204 to nucleotide 2027, or
derivatives thereof, coding for HSP70 of Streptococcus agalactiae .
38. A DNA sequence of the formula of SEQ ID NO: 21 from nucleotide 248 to nucleotide 2074, or derivatives thereof, coding for HSP70 of Streptococcus pyogenes .
39. A DNA sequence of the formula of SEQ ID NO: 25 from nucleotide 4 to nucleotide 456, or derivatives thereof, coding for the C-terminal 151-residue fragment (C-151) of HSP72.
40. A DNA sequence coding for a polypeptide according to any one of claims 1-21 and 100-101.
41. A DNA sequence selected from the group consisting of:
(a) the HSP72 DNA sequence of SEQ ID NO:4; (b) DNA sequences encoding polypeptides that are immunologically reactive with antibodies
generated by infection of a mammalian host with
Streptococcus pneumoniae cells, which antibodies are immunologically reactive with the HSP72 polypeptide (SEQ ID NO:5);
(c) DNA sequences encoding polypeptides that are capable of eliciting antibodies that are
immunologically reactive with the HSP72 polypeptide (SEQ ID NO :5);
(d) DNA sequences encoding polypeptides that are immunologically reactive with antibodies elicited by immunization with the HSP72 polypeptide (SEQ ID NO:5) ;
(e) DNA sequences that are degenerate to any of the foregoing DNA sequences; and
(f) fragments of any of the foregoing DNA sequences, either alone or in combination with other DNA sequences to form a fusion DNA sequence.
42. The DNA sequence of claim 41, wherein the DNA sequences of paragraph (b) are selected from the group consisting of DNA sequences of the genera Streptococcus and Enterococcus .
43. The DNA sequence of claim 41, wherein the DNA sequences of paragraph (b) are selected from the group consisting of DNA sequences of the species Streptococcus pyogenes, Streptococcus mutans, Streptococcus sanguis, and Enterococcus faecalis .
44. The DNA sequence of claim 41, wherein the fragments of paragraph (f) are selected from the group consisting of nucleotide 1996-2502 (amino acids 439-607) of SEQ ID N0:4 (C-169); nucleotide 2260-2304 (amino acids 527-541) of SEQ ID NO:4; and nucleotide 2437-2481 (amino acids 586-600) of SEQ ID NO:4.
45. The DNA sequence of claim 41, wherein the fusion DNA sequence of paragraph (f) is the Fucose
Isomerase-HSP72 (C-169) DNA sequence of SEQ ID NO:1
(nucleotides 771-2912).
46. An expression vector including at least one DNA sequence according to claim 41 operably linked to a promoter.
47. A recombinant DNA molecule comprising a DNA sequence according to any one of claims 28 to 40, and one or more expression control sequence operably linked to the DNA sequence.
48. The recombinant DNA molecule of claim 47, wherein said expression control sequence is an inducible expression vector.
49. The recombinant molecule of claim 48, wherein said expression vector comprises the λ PL
promoter.
50. A recombinant molecule according to claim
47 consisting of a plasmid selected from the group
consisting of: pURV3 , pURV4, pURV5 , pURV6, pJBD291, pJBDΔ4, pJBDk51, pJBD177, pJBD171, pJBD177, pJBD179, pJBDΔ1, pJBDf51, and pJBDf62.
51. A unicellular host transformed with an expression vector of claim 46.
52. A unicellular host transformed with a recombinant DNA molecule of claim 47.
53. A unicellular host according to claim 52, wherein said host is selected from the group consisting of: E.coli strains XLI Blue MRF', W3110, JM109, Y1090 and BL2KDE3).
54. A method for producing a polypeptide or fragment thereof comprising the steps of culturing the unicellular host of claim 51 and isolating said
polypeptide or fragment.
55. An antibody or fragment thereof that specifically binds to a polypeptide of claim 23.
56. An antibody or fragment thereof that specifically binds to the epitope recognized by monoclonal antibody F1-Pn3.1.
57. The antibody or fragment of claim 55, which is a monoclonal antibody.
58. The monoclonal antibody or fragment of claim 57, which is of murine origin.
59. The monoclonal antibody or fragment of claim 58, which is of IgG type.
60. The monoclonal antibody of claim 59, which is selected from the group consisting of F1-Pn3.1, F2- Pn3.2, F2-Pn3.3, and F2-Pn3.4.
61. The monoclonal antibody F1-Pn3.1.
62. A method for isolating the antibody of claim 55 comprising:
(a) introducing a preparation of the polypeptide of claim 23 into a mammal; and
(b) isolating serum from the mammal containing said antibody.
63. A method for isolating the monoclonal antibody of claim 57 comprising:
(a) introducing a preparation of the polypeptide of claim 23 to antibody producing cells of a mammal;
(b) fusing the antibody producing cells with myeloma cells to form hybridoma cells, and
(c) isolating said monoclonal antibody from the hybridoma cells.
64. A pharmaceutical composition comprising a polypeptide of claim 23.
65. The pharmaceutical composition of claim 64, which is a vaccine.
66. The pharmaceutical composition of claim 64, further comprising one or more pharmaceutically acceptable excipients.
67. A method for preventing infection of a patient by Streptococcus pneumoniae or related bacteria comprising the administration of a pharmaceutically effective amount of the vaccine of claim 65.
68. A pharmaceutical composition comprising one or more antibodies or fragments thereof according to claim 55.
69. The pharmaceutical composition of claim 68, which is a vaccine.
70. The pharmaceutical composition of claim 69, further comprising a pharmaceutically acceptable
excipient.
71. The pharmaceutical composition of claim 69, wherein the antibody is selected from the group consisting of F1-Pn3.1, F2-Pn3.2, F2-Pn3.3, and F2-Pn3.4.
72. The pharmaceutical composition of claim 69, wherein the antibody is F1-Pn3.1.
73. A method for treating a patient infected with or suspected of being infected with Streptococcus pneumoniae or related bacteria comprising the administration of a pharmaceutically effective amount of the vaccine of claim 69.
74. A method for the detection of Streptococcus pneumoniae or related bacteria in a biological sample comprising:
(a) isolating the biological sample from a patient;
(b) incubating the antibody or fragment of claim 55 with the biological sample to form a mixture; and
(c) detecting specifically bound antibody or fragment in the mixture which indicates the presence of Streptococcus pneumoniae or related bacteria.
75. The method of claim 74, wherein the
antibody is selected from the group consisting of F1- Pn3.1, F2-Pn3.2, F2-Pn3.3, and F2-Pn3.4.
76. The method of claim 74, wherein the
antibody is F1-Pn3.1.
77. A method for the detection of antibodies specific to Streptococcus pneumoniae or related bacteria in a biological sample comprising:
(a) isolating the biological sample from a patient;
(b) incubating a polypeptide of claim 23 with the biological sample to form a mixture; and
(c) detecting specifically bound
polypeptide in the mixture, which indicates the presence of antibodies specific to Streptococcus pneumoniae or related bacteria.
78. A method for the detection of Streptococcus pneumoniae or related bacteria in a biological sample comprising:
(a) isolating the biological sample from a patient;
(b) incubating a DNA probe having the DNA sequence of claim 41 with the biological sample to form a mixture; and
(c) detecting specifically bound DNA probe in the mixture which indicates the presence of
Streptococcus pneumoniae and related bacteria.
79. The method of claim 78, wherein the DNA probe is an oligomer having a sequence complementary to at least about 6 contiguous nucleotides of a DNA sequence of claim 41.
80. The method of claim 79, which further comprises:
(a) providing a set of oligomers which are primers for a polymerase chain reaction method and which flank the target region; and
(b) amplifying the target region via the polymerase chain reaction method.
81. The use of a pharmaceutically effective amount of the polypeptide of claim 23 for the prevention of Streptococcus pneumoniae or related bacterial
infections in humans.
82. The use of a pharmaceutically effective amount of an antibody specific to HSP72 for the prevention of Streptococcus pneumoniae or related bacterial
infections in humans.
83. A method for producing a polypeptide or fragment thereof comprising the steps of culturing the unicellular host of claim 52 or 53 and isolating said polypeptide or fragment.
84. A polypeptide in substantially pure form as obtained by the method of claim 83.
85. An antibody or fragment thereof that specifically binds to a polypeptide of claim 1 or 22.
86. A method for isolating the antibody of claim 86 comprising:
(a) introducing a preparation of the polypeptide of claim 1 or 22 into a mammal; and
(b) isolating serum from the mammal containing said antibody.
87. A pharmaceutical composition comprising a polypeptide of claim 1 or 22.
88. The pharmaceutical composition of claim 87, which is a vaccine.
89. The pharmaceutical composition of claim 87, further comprising one or more pharmaceutically acceptable excipients.
90. A method for preventing infection of a patient by Streptococcus pneumoniae, Streptococcus
pyogenes or Streptococcus agalactiae comprising the administration of a pharmaceutically effective amount of the vaccine of claim 88.
91. An antibody or fragment thereof that specifically binds to a polypeptide of claim 1 or 22.
92. A method for the detection of Streptococcus pneumoniae, Streptococcus pyogenes ox Streptococcus agalactiae in a biological sample comprising:
(a) isolating the biological sample from a patient;
(b) incubating the antibody or fragment of claim 91 with the biological sample to form a mixture; and
(c) detecting specifically bound antibody or fragment in the mixture which indicates the presence of Streptococcus pneumoniae, Streptococcus pyogenes or
Streptococcus agalactiae.
93. A method for the detection of antibodies specific to Streptococcus pneumoniae, Streptococcus pyogenes or Streptococcus agalactiae in a biological sample comprising:
(a) isolating the biological sample from a patient;
(b) incubating a polypeptide of claim 1 or 22 with the biological sample to form a mixture; and
(c) detecting specifically bound
polypeptide in the mixture, which indicates the presence of antibodies specific to Streptococcus pneumoniae.
Streptococcus pyogenes or Streptococcus agalactiae .
94. A method for the detection of Streptococcus pneumoniae. Streptococcus pyogenes or Streptococcus agalactiae in a biological sample comprising:
(a) isolating the biological sample from a patient; (b) incubating a DNA probe having the DNA sequence of claim 28 with the biological sample to form a mixture; and
(c) detecting specifically bound DNA probe in the mixture which indicates the presence of
Streptococcus pneumoniae, Streptococcus pyogenes or
Streptococcus agalactiae .
95. The method of claim 94, wherein the DNA probe is an oligomer having a sequence complementary to at least about 6 contiguous nucleotides of a DNA sequence of claim 28.
96. The method of claim 95, which further comprises:
(a) providing a set of oligomers which are primers for a polymerase chain reaction method and which flank the target region; and
(b) amplifying the target region via the polymerase chain reaction method.
97. The use of a pharmaceutically effective amount of the polypeptide of claim 1 or 22 for the
prevention of Streptococcus pneumoniae, Streptococcus pyogenes or Streptococcus agalactiae infection in humans.
98. The use of a pharmaceutically effective amount of an antibody specific to HSP72 for the prevention of Streptococcus pneumoniae, Streptococcus pyogenes or Streptococcus agalactiae infection in humans.
99. The use of a pharmaceutically effective amount of a polypeptide according to any one of claims 2 to 21 for the prevention of Streptococcal infections in humans.
100. A polypeptide having the amino acid sequence of SEQ ID NO:23, analogues, homologues, or derivatives thereof.
101. A polypeptide having the amino acid sequence of SEQ ID NO: 24, analogues, homologues or derivatives thereof.
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US08/472,534 US5919620A (en) | 1995-06-07 | 1995-06-07 | Heat shock protein HSP72 of Streptococcus pneumoniae |
US180595P | 1995-08-04 | 1995-08-04 | |
US1805P | 1995-08-04 | ||
PCT/CA1996/000322 WO1996040928A1 (en) | 1995-06-07 | 1996-05-17 | Streptococcal heat shock proteins members of the hsp70 family |
US472534 | 1999-12-27 |
Publications (1)
Publication Number | Publication Date |
---|---|
EP0832238A1 true EP0832238A1 (en) | 1998-04-01 |
Family
ID=26669494
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP96914821A Withdrawn EP0832238A1 (en) | 1995-06-07 | 1996-05-17 | Streptococcal heat shock proteins members of the hsp70 family |
Country Status (18)
Country | Link |
---|---|
EP (1) | EP0832238A1 (en) |
JP (1) | JPH11507214A (en) |
KR (1) | KR19990022742A (en) |
CN (1) | CN1192241A (en) |
AP (1) | AP9701163A0 (en) |
AR (1) | AR003124A1 (en) |
AU (1) | AU700080B2 (en) |
BR (1) | BR9609399A (en) |
CA (1) | CA2224015A1 (en) |
CZ (1) | CZ394297A3 (en) |
EA (1) | EA199800046A1 (en) |
HU (1) | HUP0600442A3 (en) |
IL (1) | IL118329A0 (en) |
NO (1) | NO975752L (en) |
PL (1) | PL323781A1 (en) |
SK (1) | SK168497A3 (en) |
TR (1) | TR199701537T1 (en) |
WO (1) | WO1996040928A1 (en) |
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US6245335B1 (en) | 1996-05-01 | 2001-06-12 | The Rockefeller University | Choline binding proteins for anti-pneumococcal vaccines |
ATE323721T1 (en) * | 1996-05-01 | 2006-05-15 | Univ Rockefeller | CHOLINE-BINDING PROTEIN USED AS AN PNEUMOCOCCAL VACCINE |
EP1042480A1 (en) * | 1997-12-31 | 2000-10-11 | Stressgen Biotechnologies Corporation | Streptococcal heat shock proteins of the hsp60 family |
US6497880B1 (en) | 1998-12-08 | 2002-12-24 | Stressgen Biotechnologies Corporation | Heat shock genes and proteins from Neisseria meningitidis, Candida glabrata and Aspergillus fumigatus |
TR200200633T2 (en) * | 1998-12-23 | 2002-06-21 | Shire Biochem Inc. | New streptococcus antigens |
US7128918B1 (en) | 1998-12-23 | 2006-10-31 | Id Biomedical Corporation | Streptococcus antigens |
WO2000056360A2 (en) | 1999-03-19 | 2000-09-28 | Smithkline Beecham Biologicals S.A. | Vaccine against antigens from bacteriae |
US7015309B1 (en) | 1999-06-23 | 2006-03-21 | The Wistar Institute Of Anatomy And Biology | Pyrrhocoricin-derived peptides, and methods of use thereof |
GB9918319D0 (en) | 1999-08-03 | 1999-10-06 | Smithkline Beecham Biolog | Vaccine composition |
AU2005204321B2 (en) * | 1999-08-19 | 2008-07-10 | Immunobiology Limited | Vaccines from Infectious Agents |
GB9919734D0 (en) * | 1999-08-19 | 1999-10-20 | Colaco Camilo | Vaccines from infectious agents |
AU2795801A (en) * | 2000-01-21 | 2001-07-31 | Creighton University | Biocidal molecules, macromolecular targets and methods of production and use |
US6833134B2 (en) | 2000-06-12 | 2004-12-21 | University Of Saskacthewan | Immunization of dairy cattle with GapC protein against Streptococcus infection |
DK1294771T3 (en) | 2000-06-12 | 2008-12-15 | Univ Saskatchewan | Streptococcus chimeric GapC protein and its use for vaccination and diagnosis |
US6866855B2 (en) | 2000-06-12 | 2005-03-15 | University Of Saskatchewan | Immunization of dairy cattle with GapC protein against Streptococcus infection |
GB0021757D0 (en) * | 2000-09-04 | 2000-10-18 | Colaco Camilo | Vaccine against microbial pathogens |
GB0022742D0 (en) | 2000-09-15 | 2000-11-01 | Smithkline Beecham Biolog | Vaccine |
EP1961427A3 (en) | 2002-08-02 | 2009-11-04 | GlaxoSmithKline Biologicals S.A. | Neisserial vaccine compositions comprising a combination of antigens |
US7927858B2 (en) | 2002-11-01 | 2011-04-19 | Glaxosmithkline Biologicals, S.A. | Drying process |
DK1601770T3 (en) * | 2003-03-04 | 2009-11-02 | Intercell Ag | ANTIGENES OF THE STREPTOCOCCUS PYOGENES |
EP2311989A1 (en) * | 2003-04-15 | 2011-04-20 | Intercell AG | S. pneumoniae antigens |
ES2346314T3 (en) | 2003-10-02 | 2010-10-14 | Glaxosmithkline Biolog Sa | ANTIGENS OF B. PERTUSSIS AND USE OF THE SAME IN VACCINATION. |
GB0505996D0 (en) | 2005-03-23 | 2005-04-27 | Glaxosmithkline Biolog Sa | Fermentation process |
TWI457133B (en) | 2005-12-13 | 2014-10-21 | Glaxosmithkline Biolog Sa | Novel composition |
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CN102869378A (en) | 2010-03-10 | 2013-01-09 | 葛兰素史密丝克莱恩生物有限公司 | Immunogenic composition |
GB201015132D0 (en) | 2010-09-10 | 2010-10-27 | Univ Bristol | Vaccine composition |
CN103146734B (en) * | 2013-03-12 | 2014-10-01 | 中国人民解放军军事医学科学院军事兽医研究所 | Anti-burn and scald infection multiple organ failure Pseudomonas aeruginosa toxin vaccine |
CN104001164A (en) * | 2014-04-23 | 2014-08-27 | 杭州师范大学 | Aeromonas hydrophila heat shock protein subunit vaccine and preparation method thereof |
CN114805567B (en) * | 2022-06-27 | 2022-09-16 | 和元生物技术(上海)股份有限公司 | Monoclonal antibody, method and application of marker protein HSPA1A for recognizing exosomes |
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ZA921025B (en) * | 1991-02-15 | 1992-11-25 | Uab Research Foundation | Structural gene of pneumococcal protein |
IT1262896B (en) * | 1992-03-06 | 1996-07-22 | CONJUGATE COMPOUNDS FORMED FROM HEAT SHOCK PROTEIN (HSP) AND OLIGO-POLY-SACCHARIDES, THEIR USE FOR THE PRODUCTION OF VACCINES. |
-
1996
- 1996-05-17 CN CN96195891A patent/CN1192241A/en active Pending
- 1996-05-17 EP EP96914821A patent/EP0832238A1/en not_active Withdrawn
- 1996-05-17 AU AU56828/96A patent/AU700080B2/en not_active Ceased
- 1996-05-17 JP JP9500026A patent/JPH11507214A/en active Pending
- 1996-05-17 PL PL96323781A patent/PL323781A1/en unknown
- 1996-05-17 BR BR9609399-4A patent/BR9609399A/en not_active Application Discontinuation
- 1996-05-17 SK SK1684-97A patent/SK168497A3/en unknown
- 1996-05-17 KR KR1019970709184A patent/KR19990022742A/en not_active Application Discontinuation
- 1996-05-17 WO PCT/CA1996/000322 patent/WO1996040928A1/en not_active Application Discontinuation
- 1996-05-17 HU HU0600442A patent/HUP0600442A3/en unknown
- 1996-05-17 CZ CZ973942A patent/CZ394297A3/en unknown
- 1996-05-17 AP APAP/P/1997/001163A patent/AP9701163A0/en unknown
- 1996-05-17 EA EA199800046A patent/EA199800046A1/en unknown
- 1996-05-17 CA CA002224015A patent/CA2224015A1/en not_active Abandoned
- 1996-05-17 TR TR97/01537T patent/TR199701537T1/en unknown
- 1996-05-20 AR ARP960102631A patent/AR003124A1/en unknown
- 1996-05-20 IL IL11832996A patent/IL118329A0/en unknown
-
1997
- 1997-12-05 NO NO975752A patent/NO975752L/en unknown
Non-Patent Citations (1)
Title |
---|
See references of WO9640928A1 * |
Also Published As
Publication number | Publication date |
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NO975752L (en) | 1998-02-06 |
CA2224015A1 (en) | 1996-12-19 |
IL118329A0 (en) | 1996-09-12 |
AU700080B2 (en) | 1998-12-17 |
CZ394297A3 (en) | 1998-04-15 |
HUP0600442A3 (en) | 2007-03-28 |
BR9609399A (en) | 2001-08-28 |
HUP0600442A2 (en) | 2006-08-28 |
AU5682896A (en) | 1996-12-30 |
AP9701163A0 (en) | 1998-01-31 |
JPH11507214A (en) | 1999-06-29 |
KR19990022742A (en) | 1999-03-25 |
CN1192241A (en) | 1998-09-02 |
SK168497A3 (en) | 1998-07-08 |
TR199701537T1 (en) | 1998-03-21 |
NO975752D0 (en) | 1997-12-05 |
EA199800046A1 (en) | 1998-06-25 |
WO1996040928A1 (en) | 1996-12-19 |
PL323781A1 (en) | 1998-04-27 |
AR003124A1 (en) | 1998-07-08 |
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