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WO1996040928A1 - Proteines streptococciques de choc thermique membres de la famille 70 des proteines de choc thermique - Google Patents

Proteines streptococciques de choc thermique membres de la famille 70 des proteines de choc thermique Download PDF

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Publication number
WO1996040928A1
WO1996040928A1 PCT/CA1996/000322 CA9600322W WO9640928A1 WO 1996040928 A1 WO1996040928 A1 WO 1996040928A1 CA 9600322 W CA9600322 W CA 9600322W WO 9640928 A1 WO9640928 A1 WO 9640928A1
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WIPO (PCT)
Prior art keywords
polypeptide
seq
hsp72
streptococcus
dna
Prior art date
Application number
PCT/CA1996/000322
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English (en)
Inventor
Josée Hamel
Bernard Brodeur
Denis Martin
Clément Rioux
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Biochem Vaccines Inc.
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Filing date
Publication date
Priority claimed from US08/472,534 external-priority patent/US5919620A/en
Priority to AU56828/96A priority Critical patent/AU700080B2/en
Priority to HU0600442A priority patent/HUP0600442A3/hu
Priority to EP96914821A priority patent/EP0832238A1/fr
Priority to BR9609399-4A priority patent/BR9609399A/pt
Priority to JP9500026A priority patent/JPH11507214A/ja
Application filed by Biochem Vaccines Inc. filed Critical Biochem Vaccines Inc.
Priority to PL96323781A priority patent/PL323781A1/xx
Priority to EA199800046A priority patent/EA199800046A1/ru
Priority to SK1684-97A priority patent/SK168497A3/sk
Priority to APAP/P/1997/001163A priority patent/AP9701163A0/en
Publication of WO1996040928A1 publication Critical patent/WO1996040928A1/fr
Priority to NO975752A priority patent/NO975752L/no

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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
  • 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.
  • pneumococcal capsular serotypes are identified on the basis of antigenic differences. Antibodies are the mechanism of protection and the importance of anticapsular antibodies in host defenses against S. pneumoniae is well established [R. Austrian, Am. J . Med., 67, pp. 547-549 (1979)]. Nevertheless, the currently available
  • pneumococcal vaccine comprising 23 capsular
  • polysaccharides that most frequently caused disease has significant shortcomings such as the poor immunogenicity of capsular polysaccharides, the diversity of the
  • pneumoniae proteins have been studied. This might result from the lack of protein-specific antibodies which renders difficult the study of the role of protein antigens in protection and pathogenicity. It is believed that the pneumococcal protein antigens are not very immunogenic and that most antibody responses are to the ph ⁇ sphocholine and the capsular polysaccharides [L.S. McDaniel et al., J. Exp . Med., 160, pp. 386-397 (1984); R.M. Krause, Adv.
  • pneumoniae proteins are poor immunogens [McDaniel et al., supra].
  • Streptococcus agalactiae also called Group B
  • GBS blood infection
  • meningitis meningitis
  • GBS is also a frequent cause of newborn pneumonia. Approximately 8,000 babies in the United States get GBS disease each year; 5%-15% of these babies die. Babies that survive, particularly those who have meningitis, may have long-term problems, such as hearing or vision loss or learning disabilities. In pregnant women, GBS can cause urinary tract infections, womb infections (amnionitis,
  • GBS infections in both newborns and adults are usually treated with
  • antibiotics e.g., penicillin or ampicillin
  • 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
  • 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
  • TSS shock and multiple organ system failure.
  • TSS have a 30 to 70% fatality rate in spite of aggressive treatment involving the removing of the focus of bacterial infection and antibiotic therapy.
  • the incidence of TSS is 10 to 20 cases per 100,000. No vaccine against GAS is presently available.
  • 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 W.J. Welch, Ann. Rev. Cell. Biol., 9, pp. 601-634 (1993); D. Ang et al., J. Biol. Chem., 266, pp. 24233-24236 (1991)].
  • HSPs are likely involved in protecting cells from the deleterious effects of stress.
  • HSPs are major antigens of many pathogens.
  • Members of the hsp60 family also called GroEL-related proteins for their similarity to the E. coli GroEL protein, are major antigens of a variety of bacterial pathogens including Mycobacterium leprae and Mycobacterium tuberculosis [D. Young et al., Proc. Natl. Acad. Sci. USA, 85, pp. 4267-4270 (1988)], Legionella pneumophila [B.B. Plikaytis et al., J. Clin. Microbiol., 25, pp. 2080-2084 (1987)], Borrelia burgdorferi [B.J. Lucas et al., J. Immunol., 146, pp. 2776-2782 (1991)], and
  • 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
  • the present invention addresses the problems referred to above by providing novel heat shock proteins from S. pneumoniae, S. pyogenes and S. agalactiae, and immunologically related polypeptides. Also provided are DNA sequences that code for the foregoing polypeptides, vectors containing the polypeptides, unicellular hosts transformed with those vectors, and a process for making substantially pure, recombinant polypeptides. Also provided are antibodies specific to the foregoing
  • polypeptides The polypeptides, DNA sequences and
  • antibodies of this invention provide the basis for novel methods and pharmaceutical compositions for the detection, prevention and treatment of disease.
  • this invention provides a novel vaccine based on fragments of these polypeptides that are specific to streptococcal strains.
  • the novel heat shock protein is the
  • HSP72 approximately 72 kDa heat shock protein of Streptococcus pneumoniae
  • HSP70 approximately 70 kDa heat shock protein of Streptococcus pyogenes
  • HSP70 approximately 70 kDa heat shock protein of Streptococcus agalactiae
  • HSP70/72 include the C-terminal portion of the HSP70/72 polypeptides. More particularly, it includes the C_terminal 169-residue fragment ("C-169") (residues 439-607, SEQ ID NO:5), the C-terminal 151-residue fragment (“C-151”) (residues 457-607, SEQ ID No:5), and smaller fragments consisting of peptide epitopes within the C-169 region. Particularly preferred fragments within the C-169 region of HSP72 include the peptide sequences
  • GFDAERDAAQAALDD (residues 527-541 of SEQ ID NO:5) and AEGAQATGNAGDDVV (residues 586-600 of SEQ ID NO:5), which are exclusive to HSP72 of Streptococcus pneumoniae . Even more preferred are fragments that elicit an immune
  • Such fragments may be selected from the following peptides: CS870, CS873, CS874, CS875, CS876, CS877, CS878, CS879, CS880, CS882, MAP1, MAP2 , MAP3 and MAP4 (see TABLE 5, supra).
  • Preferred antibodies of this invention are the F1-Pn3.1, F2-Pn3.2, F2-Pn3.3 and F2-Pn3.4 monoclonal antibodies (“MAbs”), which are specific to HSP72.
  • MAbs monoclonal antibodies
  • More preferred antibodies are the F2-Pn3.2 and F2-Pn3.4 monoclonal anibodies that are specific to both HSP 70 and HSP72. Even more preferred are the F1-Pn3.1 antibodies that are specific for Streptococcus pneumoniae.
  • the preferred polypeptides and antibodies of this invention provide the basis for novel methods and pharmaceutical compositions for the detection, prevention and treatment of pneumococcal diseases.
  • 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
  • HSP80, HSP72 and HSP62 are shown by arrows at the right-hand side of each panel.
  • FIG. 2 is a graphical depiction of a comparison of the electrophoretic profiles of [ 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
  • S. pneumoniae are shown in lanes 1 and 2, respectively.
  • the position of HSPs is indicated by the arrows at the left of the fluorogram.
  • FIG. 5 depicts a photograph, which shows the S. pneumoniae antigens detected by Western blot analysis.
  • Whole cell extracts were probed with sera from 15 mice (lanes 1-15) immunized with heat-killed S. pneumoniae bacteria.
  • Lane 16 shows the HSP72 protein detected by MAb F1-Pn3.1.
  • 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 F1-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 F1-Pn3.1 (lane 5,6) and then analyzed by SDS-PAGE and fluorography.
  • 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 F1-Pn3.1.
  • Lane 1 contains heat-shocked cell lysates (45°C).
  • Lane 2 contains non heat-shocked cell lysates (37°C).
  • Panel B depicts a fluorogram of the immunoblot shown in panel A.
  • FIG. 8 depicts a Western Blot, which shows subcellular localization of S. pneumoniae HSP72.
  • Sample containing 15 ⁇ 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 F1-Pn3.1.
  • FIG. 9 is a photograph of an immunoblot showing the reactivity of recombinant fusion proteins containing the C-169 region of S. pneumoniae HSP72 with MAb F1-Pn3.1.
  • Lane 1 contains whole cell extracts from S. pneumoniae strain 64 probed with HSP72-specific MAb F1-Pn3.1.
  • Lanes 2 and 3 contain phage lysates from E. coli infected with ⁇ JBD17 cultured in the presence (+) or absence (-) of IPTG and probed with HSP72-specific MAb F1-Pn3.1.
  • Lanes 4 and 5 contain phage lysates from E. coli infected with ⁇ JBD7 cultured in the presence (+) or absence (-) of IPTG and probed with HSP72-specific MAb F1-Pn3.1.
  • Molecular mass markers are shown to the left. The positions of the 74kDa- and 160 kDa-reactive proteins are shown on the left and on the right, respectively.
  • FIG. 10 is a schematic representation of the restriction map of the HSP72 (DnaK) and Fuc loci and inserts of recombinant clones. The relationships between DNA fragments are shown with respect to each other.
  • FIGS. 10A and IOC illustrate the restriction map of the HSP72(DnaK) and Fuc loci, respectively.
  • FIG 10B illustrates the restriction map of the HSP72(DnaK) and Fuc loci, respectively.
  • 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 F1-Pn3.1.
  • Molecular mass markers in kilodaltons are shown to the left. The arrow at the left-hand side of each panel marks the 74 kDa protein marker.
  • FIG. 12 depicts the detection of native and recombinant HSP72 antigens by Western blot analysis.
  • FIGS. 13A-13D depict a comparison of the
  • HSP72 SPNEU predicted amino acid sequence of the S. pneumoniae HSP72 open reading frame 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,
  • 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 E. coli (pJBDk51)
  • Lane 3 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
  • FIG. 16 is a graphical depiction of the survival curve of Balb/c mice protected from S. pneumoniae
  • FIG. 17 is a graphical depiction of the survival curve of Balb/c mice protected from S. pneuiTioniae
  • 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 ,
  • T1 transcription terminator T1 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.
  • FIG. 19 illustrates the
  • FIG. 20 illustrates the distribution of anti-S.
  • FIG. 21 illustrates the distribution of anti-S.
  • pneumoniae titers in sera from Balb/c mice immunized with C-151 rec 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
  • HSP72 antigens 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
  • Streptococcus pyogenes sga-orf
  • agalactiae indicates the substitutions and insertions of nucleotides specific to each species.
  • FIG. 25 depicts the consensus sequence established from the protein sequences of the Hsp70 of Streptococcus pneumoniae (spn-prot), Streptococcus pyogenes (sga-prot) and Streptococcus agalactiae (sgb-prot) and indicates the substitutions and insertions of amino acids specific to each species.
  • FIG. 26 depicts a fluorogram, which shows the effect of heat shock on S. agalactiae protein synthesis and the S. agalactiae protein antigen immunoprecipitated by MAb F2-Pn3.4.
  • Cell lysates from [ 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
  • 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
  • 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.
  • 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 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,
  • the "fragments" of this invention will have at least one immunogenic epitope.
  • An "immunogenic epitope” is an epitope that is instrumental in eliciting an immune response.
  • the preferred fragments of this invention will elicit an immune response sufficient to prevent or lessen the severity of infection, e.g., S. pneumoniae infection.
  • Preferred fragments of HSP72 include the C-terminal region of the polypeptides.
  • More preferred fragment include the C-terminal 169-residue fragment ("C-169”) (SEQ ID NO: 5, residues 439-607), the C-terminal 151-residue ("C-151”) (SEQ ID No: 5, residues 457-607) and smaller fragments consisting of peptide epitopes within the C-169 region.
  • Particularly preferred fragments within the C-169 region of HSP72 include the peptide sequences GFDAERDAAQAALDD (residues 527-541 of SEQ ID NO: 5) and AEGAQATGNAGDDVV
  • 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). Even more preferred are fragments that elicit a specific immune reaction against Streptococcal strains . Such fragments may be selected from the
  • CS870 CS873, CS874, CS875, CS876, CS877, CS878, CS879, CS880, CS882, MAP1, MAP2 , MAP3 and MAP4 (see TABLE 5, supra), or homologues thereof.
  • polypeptides that are immunologically related to HSP70/72.
  • immunologically related polypeptides are characterized by one or more of the following properties:
  • HSP72 SEQ ID NO: 5
  • HSP70 SEQ ID NO:20 and SEQ ID NO:22
  • analogues By definition, analogues, homologues and
  • HSP70/72 derivatives of HSP70/72 are immunologically related polypeptides. Moreover, all immunologically related polypeptides contain at least one HSP70/72 antigen.
  • HSP70/72 antigens may be found in HSP70/72 itself, or in immunologically related polypeptides.
  • polypeptides that are immunologically related to HSP72.
  • immunologically related polypeptides are characterized by one or more of the following properties:
  • analogues By definition, analogues, homologues and
  • polypeptides Moreover, all immunologically related polypeptides contain at least one HSP72 antigen.
  • 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 .
  • polypeptides of this invention include, for example, one or more polypeptides that have been crosslinked with crosslinkers such as avidin/biotin, glutaraldehyde or dimethylsuberimidate.
  • polymeric forms also include polypeptides containing two or more tandem or inverted contiguous protein sequences, produced from multicistronic mRNAs generated by recombinant DNA technology.
  • This invention provides substantially pure HSP72 and immunologically related polypeptides.
  • 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
  • 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
  • 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.
  • separation techniques such as ion exchange and gel chromatography and electrophoresis, or by the use of recombinant DNA techniques.
  • 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 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
  • 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
  • Useful expression vectors for bacterial hosts include bacterial plasmids, such as those from E. coli , including pBluescript, pGEX2T, pUC vectors, col E1, pCR1, pBR322, pMB9 and their
  • phage DNAs e.g., the numerous derivatives of phage lambda, e.g. ⁇ gt10 and ⁇ gt11, 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. Examples of useful expression control sequences include, for example, the early and late
  • promoters of SV40 or adenovirus the lac system, the trp system, the TAC or TRC system, the T3 and T7 promoters the major operator and promoter regions of phage lambda, the control regions of fd coat protein, the promoter for 3- phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters of the yeast alpha-mating system and other constitutive and indueible promoter sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.
  • the T7 RNA polymerase promoter ⁇ 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 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.
  • the host in selecting a vector, the host must be considered because the vector must replicate in it.
  • the vector's copy number, the ability to control that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered.
  • an expression control sequence a variety of factors should also be considered. These include, for example, the relative strength of the sequence, its controllability, and its compatibility with the DNA sequences of this invention, particularly as regards potential secondary structures.
  • Unicellular hosts should be selected by consideration of their compatibility with the chosen vector, the toxicity of the product coded for by the DNA sequences of this invention, their
  • 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
  • polypeptides of this invention may be generated by any of several chemical techniques.
  • they may be prepared using the solid-phase synthetic technique originally described by R. B. Merrifield, "Solid Phase Peptide Synthesis. I. The
  • compositions and methods of this invention comprise polypeptides having enhanced
  • 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
  • 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
  • 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.
  • substitutions which are less conservative may also result in desired derivatives, e.g., by causing changes in charge, conformation and other biological properties .
  • Such substitutions would include for example, substitution of a hydrophilic residue for a hydrophobic residue, substitution of a cysteine or proline for another residue, substitution of a residue having a small side chain for a residue having a bulky side chain or substitution of a residue having a net positive charge for a residue having a net negative charge.
  • polypeptides may also be prepared with the objective of increasing stability or rendering the
  • fusion proteins comprising other S. pneumoniae or non- S. pneumoniae sequences. It is preferred that- the fusion proteins comprising the polypeptides of this invention be produced at the DNA level, e.g., by constructing a nucleic acid molecule encoding the fusion, transforming host cells with the molecule, inducing the cells to express the fusion protein, and recovering the fusion protein from the cell culture. Alternatively, the fusion proteins may be produced after gene expression according to known methods.
  • An example of a fusion protein according to this invention is the FucI/HSP72 (C-169) protein of Example 3, infra.
  • 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.
  • 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.
  • 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
  • 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
  • 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 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.
  • 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.
  • polypeptides of this invention may be used to produce other monoclonal antibodies which could be screened for their ability to confer protection against S. pneumoniae , S. pyogenes, S.
  • 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
  • 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
  • polypeptides, DNA sequences and antibodies of this invention are useful in prophylactic, therapeutic and diagnostic compositions for preventing, treating and diagnosing disease.
  • any suitable host may be any suitable host.
  • polypeptide is selected from the group consisting of
  • 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
  • polypeptide is used, it will be administered with a pharmaceutically acceptable adjuvant, such as complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptides) or ISCOM (immunostimulating complexes).
  • a pharmaceutically acceptable adjuvant such as complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptides) or ISCOM (immunostimulating 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
  • 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
  • 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.
  • 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.
  • polypeptides, DNA sequences and antibodies of this invention may also form the basis for diagnostic methods and kits for the detection of pathogenic organisms.
  • diagnostic methods are possible.
  • this invention provides a method for the
  • Antibodies for use in this method include monoclonal antibodies F1-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:
  • ELISA enzyme-linked immunosorbent assay
  • radioimmunoassay or a latex agglutination assay.
  • the diagnostic agents may be included in a kit which may also comprise instructions for use and other appropriate reagents, preferably a means for detecting when the polypeptide or antibody is bound.
  • 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 11 C , 15 O, 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. For instance, monoclonal antibody molecules produced by a hybridoma may be metabolically labeled by incorporation of radioisotope-containing amino acids in the culture medium, or polypeptides may be conjugated or coupled to a detection means through
  • 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
  • the probe-based detection method of this invention comprises the steps of:
  • sequence of this invention with the biological sample to form a mixture; and (c) detecting specifically bound DNA probe in the mixture which indicates the presence of Streptococcus pneumoniae or related bacteria.
  • the DNA probes of this invention may also be used for detecting circulating nucleic acids in a sample, for example using a polymerase chain reaction, as a method of diagnosing Streptococcus pneumoniae or related
  • 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).
  • 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
  • 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
  • Example 5 describes processes for obtaining substantially pure HSP72, and the use of HSP72 or antibodies against it to protect against
  • 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 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% CO 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.
  • 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 Male Balb/c mice (Charles River Laboratories, St-Constant, Quebec, Canada) were immunized with
  • 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
  • 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.
  • 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,
  • 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).
  • FIG. 1 The most prominent induced protein was about 72 kDa (HSP72), whereas the other two were approximately 80 kDa (HSP80) and 62 kDa (HSP62). Increased protein synthesis was already apparent after 10 minutes of labeling (FIG. 1, lanes 1, 2 and 7, 8) and became more significant when the labeling period was prolonged to 30 minutes (FIG. 1, lanes 3, 4 and 9, 10) and 60 minutes (FIG. 1, lanes 5, 6). The effect of elevated temperature on the protein
  • FIG. 2 Radioimmunoprecipitation analysis revealed, however, that HSP72 was undetectable at 37°C (supra; and FIGS. 3, 4 and 6) thus indicating that peak 9 from FIG. 2 corresponds to protein component(s) comigrating with
  • FIGS. 3 and 4 relate to detergent soluble protein preparations.
  • FIG. 3 relates to detergent soluble protein preparations.
  • FIG. 4 relates to heat-killed bacterial preparation. Although many bands were detected by most antisera, HSP72 was a major precipitation product. The specificity of antibodies for HSP72 was demonstrated by the detection of proteins among heat-shocked products only (FIG. 3, lanes 4, 6, 8 and 10; FIG. 4, lanes 4, 6 and 8 ) . Interestingly, all immunized mice consistently recognized HSP72. The antibodies reactive with the HSP72 were not specific to the strain used during the immunization since strong reactivities were observed with heterologous
  • HSP72 S. pneumoniae HSP72. It should be noted that in addition to HSP72, one sera precipitated comigrating product labeled at both 37°C and 45°C (FIG. 4, lane 4). This 72 kDa-product probably corresponds to component from peak 9 in FIG. 2 and was not detected in immunoblots. HSP62 is another immune target which was precipitated by some but not all immune sera (FIG. 3, lane 6 and, FIG. 4, lanes 4 and 6). None of the sera tested reacted with HSP80. No proteins were precipitated when preimmune sera taken from the mice used in this study were tested for the presence of antibodies reactive with the labeled products.
  • 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.
  • Pneumococci were separated into subcellular fractions according to the technique described by Pearce et al. [Mol. Microbiol., 9, pp. 1037-1050 (1993)].
  • S. pneumoniae strain 64 (type 6) was grown in Todd Hewitt broth supplemented with 0.5% (w/v) yeast extract for 6 hours at 37°C and isolated by centrifugation. Cell pellets were resuspended in 25 mM Tris-HCl pH 8.0, 1 mM EDTA, 1 mM phenylmethylsulphonylfluoride (PMSF) and sonicated for 4 minutes with 15 second bursts. Cellular debris were removed by centrifugation. The bacterial membranes and cytoplasmic contents were separated by centrifugation at 98,000 g for 4 hours. The cytoplasmic (supernatant) and the membrane (pellet) fractions were adjusted to 1 mg protein per ml and subjected to SDS-PAGE and immunoblot analyses. B. Identification and Characterization
  • hybridomas with anti- S. pneumoniae reactivity in immunoblot four were found to recognize epitopes present on a protein band with an apparent molecular mass of 72 kDa.
  • the four hybridomas were designated F1-Pn3.1 (from fusionn experiment 1) and F2-Pn3.2, F2-Pn3.3 and F2-Pn3.4 (from fusion experiment 2). Isotype analysis revealed that hybridoma F1-Pn3.1
  • FIG. 6 (lanes 5 and 6) demonstrates the results obtained for MAb F1-Pn3.1. The same results were obtained with MAbs F2-Pn3.2, F2-Pn3.3 and F2-Pn3.4
  • FIG. 7, panel A shows the results obtained for MAb F1-Pn3.1.
  • MAbs F2-Pn3.2, F2-Pn3.3 and F2-Pn3.4 were obtained with MAbs F2-Pn3.2, F2-Pn3.3 and F2-Pn3.4. Accordingly, the heat shock stress did not significantly increase the reactivity of anti-HSP72 monoclonal antibodies.
  • the fluorograph of the immunoblots clearly showed that the heat shock response had occurred (FIG. 7, panel B).
  • S. pneumoniae HSP72 increases in response to heat shock, but that the absolute amounts of HSP72 do not increase after heat shock.
  • 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
  • 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 ⁇ gt11 arms, packaged, and the resulting phage mixtures used to infect E. coli Y1090.
  • Immunoscreening of plaques expressing recombinant HSP72 antigens was performed using HSP72- specific monoclonal antibody F1-Pn3.1, supra. Plaque clones expressing peptides recognized by MAb F1-Pn3.1 were isolated and purified. Liquid lysates were prepared and DNA was purified from a Promega LambdaSorb phage adsorbent according to the manufacturer's directions followed by conventional DNA purification procedures.
  • 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
  • DNA fragments sequenced in this example were first cloned into plasmid pDELTA 1 (GIBCO BRL Life Technologies, Burlington, Ontario). A series of nested deletions were generated from both strands by in vivo deletion mediated by Tn 1000 transposon transposition (Deletion Factory System, GIBCO BRL) following the
  • 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
  • Pneumococcal HSP72 was purified by
  • PVDF polyvinylidene difluoride
  • PVDF membrane was stained with Coomassie Blue, the HSP72 band excised and then analyzed in an automated protein sequencer (ABI), according to standard procedures.
  • ABSI automated protein sequencer
  • the ⁇ gt11 S. pneumoniae genomic DNA library was screened with the HSP72-specific MAb F1-Pn3.1. Seventeen (17) immunoreactive clones were isolated and purified from a total of 1500 phages tested. To confirm the specificity of the proteins expressed by the recombinant phages,
  • 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 F1-Pn3.1.
  • the region responsible for expression of the 74 kDa chimeric protein was found to localize on the 3.2 kb EcoRI-EcoRV fragment, which
  • 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 NO: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 NO:1, nucleotides 18-21 and 760-763). No obvious -10 and -35 promoter sequences were detected.
  • ORF27 spans nucleotides 30-755 (SEQ ID NO:1) and encodes a protein of 242 amino acids with a calculated molecular weight of 27,066 daltons .
  • the deduced amino acid sequence of this protein is SEQ ID NO:2.
  • This gene orf27 is designated this gene orf27, and compared it to other known sequences. No homologous gene or protein was found.
  • the large ORF (nucleotides 771-2912, SEQ ID NO:1) specifies a protein of 714 amino acids with a predicted molecular mass of 79,238 daltons.
  • the deduced amino acid sequence of this protein is SEQ ID NO:3.
  • This ORF was compared with other known sequences to determine its relationship to other amino acid sequences. This analysis revealed a high degree of similarity of the encoded protein to the
  • E. coli fucose isomerase (Fuel) and to several HSP70 gene family members, also known as DnaK genes.
  • 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.
  • 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.
  • FIG. 10B The HSP72 protein expressed by the transformants (pJBD291) migrated on the SDS-PAGE gel at the same position as the native HSP72 protein (FIG. 12).
  • 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 entire 3.2 kb Hindlll DNA fragment carried on the plasmid pJBD ⁇ 4 and the 2.3 kb EcoRI-EcoRI DNA fragment contained on the plasmid pJBD177 were sequenced. Altogether, the nucleotide sequence comprised 4320 base pairs and revealed two ORFs (SEQ ID NO : 4).
  • the putative ribosome binding site (“AGGA”) was located 9 base pairs upstream from the start codon of the HSP72 structural gene, while the typical ribosome binding site (“AGGA”) was found 66 base pairs upstream from the start codon of the DnaJ structural gene. No typical 5' regulatory region was identified in front of these two genes. Restriction sites are located between nucleotides 1 and 2 (Hindlll), nucleotides 1318 and 1319 (EcoRI), nucleotides 1994 and 1995 (EcoRI), nucleotides 3343 and 3344 (Hindlll), and nucleotides 4315 and 4316 (EcoRI).
  • S. pneumoniae is similar to that of E. coli [Saito, H. and Uchida, Mol. Gen. Genet. 164, 1-8 (1978)] as well as several other Gram positive bacteria [Wetzstein, M.
  • 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.
  • 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%
  • HSP72 Like other HSP70 proteins of Gram positive bacteria, HSP72 misses a stretch of 24 amino acids near the amino terminus when compared with DnaK proteins from Gram negative bacteria (FIGS. 13A-13D).
  • HSP72 shares homology with HSP70 (DnaK) proteins from other organisms, it does possess some unique features. Sequence divergence of the HSP70 (DnaK) proteins is largely localized to two regions (residues 244 to 330 and 510 to 607, SEQ ID NO:5). More specifically, the peptide sequences GFDAERDAAQAALDD (residues 527 to 541, SEQ ID NO:5) and AEGAQATGNAGDDVV (residues 586 to 600, SEQ ID NO:5) are exclusive to HSP72. The fact that the C-terminal portion of HSP72 is highly variable
  • 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 .
  • Pn3.2, F2-Pn3.3 and F2-Pn3.4, supra were tested for their reactivity against proteins expressed by E. coli infected or transformed with recombinant phages and plasmids containing HSP72 sequences .
  • the four individual MAbs reacted with the lacZ-HSP72 fusion protein expressed by the clone ⁇ JBD7, thus localizing the epitopes recognized by these MAbs to the C-terminal 169 residues.
  • capsular serotypes types 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 14, 15, 19, 20, and 22
  • 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.
  • proteins for example those from E. coli and S. aureus .
  • HSP72 HSP70 (DnaK) proteins from gram positive bacterial species.
  • HSP70 (DnaK) proteins may be structurally related to HSP72, they are immunologically distinct.
  • S. pyogenes Enterococcus faecalis , S.
  • 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.
  • HSP72 rec recombinant HSP72 protein
  • HSP72 rec was purified by immunoaffinity using monoclonal antibody F1- Pn3.1 immobilized on sepharose 4B beads (Pharmacia). The purity of eluates was assessed on SDS-PAGE.
  • C-169 rec The recombinant C-169 protein (C-169 rec ) was expressed in the form of insoluble inclusion bodies in E. coli strain JM109 transformed with the plasmid pJBD ⁇ 1. Protein inclusion bodies were recovered from pelleted bacterial cells disrupted by sonication as described before. The pellets were washed in lysis buffer
  • 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 rec or C-169 rec antigens absorbed to Alhydrogel
  • mice 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.
  • NZW rabbit (Charles River Laboratories) was immunized subcutaneously at multiple sites with
  • 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.
  • S. pneumoniae challenge inoculum were plated on chocolate agar plates to determine the CFU and to verify the
  • SCID mice were chosen because of their high susceptibility to S. pneumoniae infection.
  • HSP72 rec and C-169 rec proteins were obtained in a relatively pure state with no contaminants detected on Coomassie Blue-stained SDS polyacrylamide gels (FIGS. 14 and 15, respectively) .
  • HSP72 rec To evaluate the vaccinogenic potential of HSP72, we first examined the ability of HSP72 rec to elicit a protective immune response. Groups of 10 mice were immunized with full-length HSP72 rec (1 ⁇ 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 rec 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. pneumoniae organisms were detected in any of the blood samples collected on days 14 or 15 from mice surviving infection. The observation that HSP72 rec elicited protection against type 3 strain WU2 pneumococci indicated that HSP72 derived from DNA
  • extracted from a type 6 strain contains epitopes capable of eliciting protection against a heterologous strain having a different capsular type.
  • mice were immunized with C-169 rec (1 ⁇ g or 5 ⁇ g doses) and challenged with 6 million CFU of S. pneumoniae type 3 strain WU2. Sixty percent (60%) of the mice dosed with 1 ⁇ g C-169 rec survived the challenge, as did 70% of the mice dosed with 5 ⁇ g C-169 rec (FIG. 17). In contrast, all of the naive mice were dead by 2 days post-challenge. Therefore, the C-terminal portion of S. pneumoniae HSP72, which includes the region of maximum divergence among DnaK proteins, is a target for the protective immune response.
  • mice transferred with rabbit anti-C-169 rec antibodies were protected from fatal infection with S. pneumoniae WU2. In contrast, none of the 15 control mice survived. The control mice received antibodies from nonimmune rabbit sera or received sterile buffer alone. In addition, all mice from the control groups had positive S. pneumoniae hemoculture 24 hours post-challenge, while S. pneumoniae organisms were detected in only 2 out of a total of 10 immunized SCID mice.
  • mice were challenged with 5000 and 880 CFU of type 3 S. pneumoniae strain WU2 , respectively. Results in Table 4 are
  • 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.
  • This vector contains a cassette of the bacteriophage ⁇ CI857
  • PCR polymerase chain reaction
  • 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.
  • amplification reactions were made using a DNA Thermal Cycler, Perkin Elmer, San Jose, CA.
  • OCRR26 an ATG start codon is present in frame just upstream of the coding region for the amino-terminus region of the C-15X
  • the primers OCRR26 and OCRR27 contain, respectively, a Bglll (AGATCT) and a BamHI (GGATCC) recognition site in order to facilitate the cloning of the PCR product into the dephosphorylated restriction sites Bglll and BamHI of p629.
  • the PCR product was purified from agarose gels by the method of phenol freeze [S. A. Benson, Biotechniques 2, pp.
  • 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
  • the recombinant C-151 rec was synthesized with a methionine residue at its amino end in E. coli strain W3110 harboring the plasmid pURV3.
  • E. coli cells were grown at 30°C in LB broth containing 100 ⁇ g of ampicillin- per ml until the A 600 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
  • a panel of 10 monoclonal antibodies selected for their reactivity with the_S. pneumoniae HSP72 protein were tested for their reactivity to C-151 rec by Western blot analysis using YM30-ultrafiltrates prepared as described above.
  • the MAbs included a series of six monoclonal antibodies raised to the HSP72 rec protein (F3- Pn3.5 to F3-Pn3.10) and monoclonal antibodies F1-Pn3.1, F2-Pn3.2, F2-Pn3.3, F2-Pn3.4.
  • the three MAbs F1-Pn3.1, F2- Pn3.3 and F2-Pn3.4 that were reactive with C-169 rec also recognized the C-151 rec fragment.
  • mice Groups of 10 female Balb/c mice were immunized subcutaneously with either HSP72 rec or C-169 rec as described in Example 5.
  • HSP72 rec HSP72 rec
  • C-169 rec C-169 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
  • 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.
  • Example 5 The results previously described in Example 5 clearly demonstrate the protective nature of the antibody response elicited following immunization with recombinant HSP72 antigens .
  • mice FIG. 19, 20 and 21
  • monkeys FIG. 22
  • HSP72 proteins used as immunogens with average titers of 1:64000 after the third injection.- Detailed analysis of individual sera revealed that each animal responded to the immunization in developping antibodies reactive with S. pneumoniae HSP72.
  • mice immunized with C-169 rec the two doses tested, i.e. 1 and 5 ⁇ g, were similarly efficient with the induction of similar antibody titers (FIG. 20).
  • a strong boost response was observed after the second injection with C-169 rec with no enhancement in the antibody titers after a third injection.
  • the immune response to the HSP72 rec was dose- dependent.
  • Increases in the specific antibody titers were observed after a second and a third injection with either HSP72 rec or C-151 rec (FIG. 19 and 21).
  • 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.
  • variable regions may contain B-cell epitopes that are responsible for the antigenic heterogeneity reported in Example 4.
  • the reactivity of polyclonal and monoclonal antibodies to S. pneumoniae HSP72 were tested against fourteen peptides selected to cover most of these regions.
  • Peptides MAP1, MAP2 , MAP3 and MAP4 were synthesized onto a branching lysine core as Multiple Antigenic Peptides (MAP) by the Service de Sequence de Peptides de l'Est du Quebec, Centre de noted du CHUL (Sainte-Foy, Canada). Peptides were purified by reverse- phase high-pressure liquid chromatography. Peptides were solubilized in distilled water except for peptides CS874 and CS876 which were solubilized in a small volume of either 6M guanidine-HCl or dimethyl sulfoxide and then adjusted to 1 mg/ml with distilled water.
  • Peptide ELISA were performed by coating synthetic peptides onto Immunolon 4 microtitration plates (Dynatech Laboratories, Inc., Chantilly, VA) at a concentration of 50 ⁇ g/ml according to the prodedures described in J. Hamel et al. [supra].
  • microtitration plates were coated with S. pneumoniae cell wall extracts. Hybridoma culture supernatants containing the HSP72-specific MAbs were incubated overnight at 4°C with several concentrations of peptide. Peptide treated and control supernatants were then tested by ELISA as described above.
  • Immune sera were from animals immunized three times with recombinant HSP72 antigens.
  • One rabbit was immunized with 37.5 ⁇ 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 .
  • TABLE 5 SEQUENCES AND LOCATIONS OF SYNTHETIC PEPTIDES CORRESPONDING TO S. PNEUMONIAE HSP72
  • AEGAQATGNAGDDVV (residues 586 to 600) defined as exclusive to S. pneumoniae HSP72 based on the comparison of HSP70 protein sequences available in the data banks.
  • Our data thus revealed that both peptide sequences contain linear B-cell epitopes.
  • the peptide MAP4 alone was also recognized by the MAb F1-Pn3.1. This reactivity was confirmed by fluid-phase inhibition assays in which 10 ⁇ g/ml of MAP4 caused complete inhibition of F1-Pn3.1 binding to HSP72.
  • Polyclonal antisera from animals immunized with the complete HSP72 recombinant protein also recognized B-cell epitopes localized on peptides CS875, MAPI and MAP3. All together these data indicate that the hypervariable C-151 terminal fragment of the HSP72
  • 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.
  • 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.
  • Plasmids used for DNA sequencing were purified using plasmid kits from Qiagen Inc. (Chatsworth, CA). DNA fragments were purified from agarose gels by the method of phenol freeze [S. A. Benson, Biotechniques 2, pp. 67-68 (1984)]. DNA probes were labeled with a 32 P-dCTP or digoxigenin (DIG)-11-dUTP using the random primer labeling kits of Boehringer Mannheim (Laval, Canada).
  • Plasmid transformations were carried out by the method of Simanis [Hanahan, D. In D. M. Glover (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.
  • DNA amplification reactions were made using a DNA Thermal Cycler 480, Perkin Elmer.
  • Oligonucleotides were synthesized by oligonucleotide synthesizer model 394 (ABI). 3. Molecular Cloning of the Genes hsp70/dnak- of S. agalactiae and S. pyogenes
  • the PCR amplification was done on the genomic DNA of S. pneumoniae using the oligonucleotides OCRR2 (5'-AAGCTGTTATCACAGTTCCGG) and OCRR3 (5'-GATACCAAGTGACAATGGCG).
  • Hybridizing genomic restriction fragments of sufficient size to code for a 70- kDa polypeptide 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.
  • 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 [ 35 S]methionine for 30 minutes at 37°C. The bacteria were extensively washed with PBS and cell extracts were prepared by treatment with mutanolysine and lysozyme as described for the DNA
  • lysozyme M.Jayarao et al., supra., sonication and boiling in SDS-PAGE sample buffer.
  • Cell lysates from E. coli transformed with either pURV4 or pURV6 producing truncated S._pyogenes HSP70 antigens were tested after boiling in SDS-PAGE sample buffer.
  • 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
  • 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].
  • the ORF and the deduced polypeptide of the hsp70 of S. pyogenes are, respectively, identical at 85 and 94 % to the ORF and polypeptide of the HSP72 of S. pneumoniae .
  • the ORF of plasmid pURV4 lacks 125 base pairs coding for 41 amino acids at the carboxyl end of the HSP70 of S.
  • 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 ⁇
  • HSP70/DnaK of S. pyogenes, S. agalactiae, and S. pneumoniae gave percentages of identity of 82 and 93 %, respectively.
  • [ 35 S]methionine revealed that the synthesis of a 70 kDa- protein was significantly increased after a thermal stress (FIG. 26, lanes 1 and 2). Radioimmunoprecipitation analysis revealed that the heat inducible 70kDa-protein was easily detected at 43°C using monoclonal antibody F2- Pn3.4 thus indicating that the protein belongs to the heat shock protein 70 (hsp70/DnaK) family (FIG. 26, lanes 3 and 4).
  • S. pneumoniae distributed among S. pneumoniae , S. pyogenes and S.
  • antigens from either S. pyogenes or S. agalactiae are antigens from either S. pyogenes or S. agalactiae .
  • 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
  • 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.
  • boosters will be administered at about 1 and 6 months after the initial injection.
  • mucosal immunity 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 are particulate carriers or recombinant live vectors.
  • 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 agalactiae or related bacteria.
  • the dosage forms and regimens for such passive immunization would be similar to those of other passive immunotherapies .
  • An antibody according to this invention is exemplified by a hybridoma producing MAb F1-Pn3.1

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Abstract

L'invention concerne des protéines de choc thermique du Streptococcus pneumoniae, du Streptococcus pyogenes et du Streptococcus agalactiae de masse moléculaire apparente comprise entre 70 et 72 kDa, les polypeptides qui leur sont apparentés du point de vue immunologique, les séquences de nucléotides et d'acides aminés dérivés de la famille 72 des protéines de choc thermique de S. pneumoniae (SEQ ID no. 4, SEQ ID No. 5), les séquences de nucléotides et d'acides aminés dérivés de la famille 70 des protéines de choc thermique de S. pyogenes (SEQ ID No. 19, SEQ ID No. 20), les séquences de nucléotides et d'acides aminés dérivés de la famille 70 des protéines de choc thermique de S. agalactiae (SEQ ID No. 21, SEQ, ID No 22), les anticorps qui se lient aux protéines de choc thermique, ainsi que des procédés de recombinaison d'ADN utiles pour produire ces protéines de choc thermique et les polypeptides qui leur sont apparentés du point de vue immunologique. Ces polypeptides, séquences d'ADN et anticorps constituent de nouveaux moyens de diagnostic, de prévention et/ou de traitement de maladies causées par des streptocoques.
PCT/CA1996/000322 1995-06-07 1996-05-17 Proteines streptococciques de choc thermique membres de la famille 70 des proteines de choc thermique WO1996040928A1 (fr)

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APAP/P/1997/001163A AP9701163A0 (en) 1995-06-07 1996-05-17 Streptococcal heat shock proteins members of the HSP70 family.
HU0600442A HUP0600442A3 (en) 1995-06-07 1996-05-17 Streptococcal heat shock proteins members of the hsp70 family
EP96914821A EP0832238A1 (fr) 1995-06-07 1996-05-17 Proteines streptococciques de choc thermique membres de la famille 70 des proteines de choc thermique
BR9609399-4A BR9609399A (pt) 1995-06-07 1996-05-17 Elementos de proteìnas de choque térmico estreptocócicos da famìlia hsp70
JP9500026A JPH11507214A (ja) 1995-06-07 1996-05-17 Ｈｓｐ７０ファミリーに属する連鎖球菌の熱ショック蛋白質メンバー
AU56828/96A AU700080B2 (en) 1995-06-07 1996-05-17 Streptococcal heat shock proteins members of the HSP70 family
PL96323781A PL323781A1 (en) 1995-06-07 1996-05-17 Streptococco thermal shock proteins belonging to a family hsp70
EA199800046A EA199800046A1 (ru) 1995-06-07 1996-05-17 Полипептид, последовательность днк, вакцинная композиция (варианты), антитело или его фрагмент, вакцина, применения указанных полипептида, последовательности днк и антитела или его фрагмента
SK1684-97A SK168497A3 (en) 1995-06-07 1996-05-17 Streptococcal heat shock proteins members of the hsp70 family
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WO2001013944A3 (fr) * 1999-08-19 2001-09-20 Immunobiology Ltd Vaccins contre des agents infectieux
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US6497880B1 (en) 1998-12-08 2002-12-24 Stressgen Biotechnologies Corporation Heat shock genes and proteins from Neisseria meningitidis, Candida glabrata and Aspergillus fumigatus
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US7015309B1 (en) 1999-06-23 2006-03-21 The Wistar Institute Of Anatomy And Biology Pyrrhocoricin-derived peptides, and methods of use thereof
EP1797894A2 (fr) 1999-08-03 2007-06-20 GlaxoSmithKline Biologicals S.A. Composition vaccinale
WO2007084053A1 (fr) 2006-01-17 2007-07-26 Arne Forsgren NOUVELLE PROTÉINE DE HAEMOPHILUS INFLUENZAE À SURFACE EXPOSÉE (PROTÉINE; pE)
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US7635482B2 (en) 1998-12-23 2009-12-22 Id Biomedical Corporation Streptococcus antigens
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EP2314313A1 (fr) 2000-09-15 2011-04-27 GlaxoSmithKline Biologicals S.A. Vaccin contre streptococcus pneumoniae
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DE20321890U1 (de) 2002-08-02 2012-03-12 Glaxosmithkline Biologicals S.A. Impfstoffzusammensetzung
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AU5682896A (en) 1996-12-30
CA2224015A1 (fr) 1996-12-19
HUP0600442A3 (en) 2007-03-28
NO975752D0 (no) 1997-12-05
EP0832238A1 (fr) 1998-04-01
NO975752L (no) 1998-02-06
CZ394297A3 (cs) 1998-04-15
AU700080B2 (en) 1998-12-17
IL118329A0 (en) 1996-09-12
PL323781A1 (en) 1998-04-27
AR003124A1 (es) 1998-07-08
CN1192241A (zh) 1998-09-02
TR199701537T1 (xx) 1998-03-21
EA199800046A1 (ru) 1998-06-25
SK168497A3 (en) 1998-07-08
BR9609399A (pt) 2001-08-28
AP9701163A0 (en) 1998-01-31
JPH11507214A (ja) 1999-06-29
KR19990022742A (ko) 1999-03-25
HUP0600442A2 (en) 2006-08-28

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