EP0832238A1 - Streptokokken-hitzeschock-proteine, mitglieder der hsp70-familie - Google Patents

Streptokokken-hitzeschock-proteine, mitglieder der hsp70-familie

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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
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European Patent Office
Prior art keywords
ala
polypeptide
seq
glu
gly
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EP96914821A
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English (en)
French (fr)
Inventor
Josée Hamel
Bernard Brodeur
Denis Martin
Clément Rioux
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Biochem Vaccines Inc
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Biochem Vaccines Inc
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Priority claimed from US08/472,534 external-priority patent/US5919620A/en
Application filed by Biochem Vaccines Inc filed Critical Biochem Vaccines Inc
Publication of EP0832238A1 publication Critical patent/EP0832238A1/de
Withdrawn legal-status Critical Current

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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)
    • 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
    • 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
    • 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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EP96914821A 1995-06-07 1996-05-17 Streptokokken-hitzeschock-proteine, mitglieder der hsp70-familie Withdrawn EP0832238A1 (de)

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US472534 1995-06-07
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

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US6245335B1 (en) 1996-05-01 2001-06-12 The Rockefeller University Choline binding proteins for anti-pneumococcal vaccines
JP2000511411A (ja) * 1996-05-01 2000-09-05 ザ ロックフェラー ユニヴァーシティ 抗―肺炎球菌ワクチン用のコリン結合タンパク質
AU1866099A (en) * 1997-12-31 1999-07-26 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
US7128918B1 (en) 1998-12-23 2006-10-31 Id Biomedical Corporation Streptococcus antigens
CN1191362C (zh) * 1998-12-23 2005-03-02 夏尔生化公司 新颖的链球菌抗原
EP1880735A3 (de) 1999-03-19 2008-03-12 GlaxoSmithKline Biologicals S.A. Impfstoff
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
DE60136356D1 (de) 2000-06-12 2008-12-11 Univ Saskatchewan Chimäres GapC Protein aus Streptococcus und dessen Verwendung zur Impfung und Diagnostik
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
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MXPA05001265A (es) 2002-08-02 2005-04-28 Glaxosmithkline Biolog Sa Composiciones de vacuna de neisserial que comprenden una combinacion de antigenos.
LT1556477T (lt) 2002-11-01 2017-10-25 Glaxosmithkline Biologicals S.A. Džiovinimo būdas
ES2330334T3 (es) * 2003-03-04 2009-12-09 Intercell Ag Antigenos de streptococcus pyogenes.
EP2311989A1 (de) * 2003-04-15 2011-04-20 Intercell AG S. pneumoniae Antigene
CA2539715C (en) 2003-10-02 2015-02-24 Glaxosmithkline Biologicals S.A. Pertussis antigens and use thereof in vaccination
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KR20080096775A (ko) 2006-01-17 2008-11-03 아르네 포르스그렌 표면 노출된 헤모필루스 인플루엔자 단백질 （단백질 Ｅ ―ｐＥ）
CN103002910A (zh) 2010-03-10 2013-03-27 葛兰素史密丝克莱恩生物有限公司 疫苗组合物
GB201015132D0 (en) 2010-09-10 2010-10-27 Univ Bristol Vaccine composition
CN103146734B (zh) * 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 抗烧烫伤感染多器官衰竭绿脓杆菌毒素疫苗
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NO975752L (no) 1998-02-06

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