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US20060177465A1 - Streptococcus antigens - Google Patents

Streptococcus antigens Download PDF

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US20060177465A1
US20060177465A1 US11/338,847 US33884706A US2006177465A1 US 20060177465 A1 US20060177465 A1 US 20060177465A1 US 33884706 A US33884706 A US 33884706A US 2006177465 A1 US2006177465 A1 US 2006177465A1
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bvh
seq
new
polypeptide
polypeptides
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US11/338,847
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Josee Hamel
Catherine Ouellet
Nathalie Charland
Denis Martin
Bernard Brodeur
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ID Biomedical Corp of Washington
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ID Biomedical Corp of Washington
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    • 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
    • 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

Definitions

  • the present invention is related to antigens, epitopes and antibodies directed to these epitopes, more particularly polypeptide antigens of streptococcus pneumoniae pathogen which may be useful for prophylaxis, diagnostic or treatment of streptococcal infection.
  • S. pneumoniae is an important agent of disease in man especially among infants, the elderly and immunocompromised persons. It is a bacterium frequently isolated from patients with invasive diseases such as bacteraemia/septicaemia, pneumonia, meningitis with high morbidity and mortality throughout the world. Even with appropriate antibiotic therapy, pneumococcal infections still result in many deaths. Although the advent of antimicrobial drugs has reduced the overall mortality from pneumococcal disease, 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 otitis media in infants and young children.
  • pneumococcal capsular polysaccharide More than 80 pneumococcal capsular serotypes have been identified on the basis of antigenic differences.
  • the currently available pneumococcal vaccine comprising 23 capsular polysaccharides that most frequently caused disease, has significant shortcomings related primarily to the poor immunogenicity of some capsular polysaccharides, the diversity of the serotypes and the differences in the distribution of serotypes over time, geographic areas and age groups. In particular, the failure of existing vaccines and capsular conjugate vaccines currently in development to protect young children against all serotypes spurres evaluation of other S. pneumoniae components.
  • PCT WO 00/39299 describes polypeptides and polynucleotides encoding these polypeptides.
  • PCT WO 00/39299 demonstrates that polypeptides designated as BVH-3 and BVH-11 provide protection against fatal experimental infection with pneumococci.
  • Streptococcus antigens that may be used as components for the prophylaxis, diagnostic and/or therapy of Streptococcus infection.
  • An isolated polynucleotide comprising a polynucleotide chosen from;
  • FIG. 1 is the DNA sequence of SP64 BVH-3 gene; SEQ ID NO: 1.
  • FIG. 2 is a DNA sequence containing the complete SP64 BVH-3 gene at nucleotides 1777 to 4896; SEQ ID NO: 2.
  • FIG. 3 is the DNA sequence of SP64 BVH-11 gene; SEQ ID NO: 3.
  • FIG. 4 is a DNA sequence containing the complete SP64 BVH-11 gene at nucleotides 45 to 2567; SEQ ID NO: 4.
  • FIG. 5 is a DNA sequence containing the complete SP64 BVH-11-2 gene at nucleotides 114 to 2630; SEQ ID NO: 5.
  • FIG. 6 is the amino acid sequence of SP64 BVH-3 polypeptide; SEQ ID NO: 6.
  • FIG. 7 is the amino acid sequence of SP64 BVH-11 polypeptide; SEQ ID NO: 7.
  • FIG. 8 is the amino acid sequence of SP64 BVH-11-2 polypeptide; SEQ ID NO: 8.
  • FIG. 9 is the DNA sequence of SP63 BVH-3 gene; SEQ ID NO:9.
  • FIG. 10 is the amino acid sequence of SP63 BVH-3 polypeptide; SEQ ID NO: 10.
  • FIG. 11 is the amino acid sequence of 4D4.9 polypeptide; SEQ ID NO: 11.
  • FIG. 12 is the amino acid sequence of 7G11.7 polypeptide; SEQ ID NO: 12.
  • FIG. 13 is the amino acid sequence of 7G11.9 polypeptide; SEQ ID NO: 13.
  • FIG. 14 is the amino acid sequence of 4D3.4 polypeptide; SEQ ID NO: 14.
  • FIG. 15 is the amino acid sequence of 8E3.1 polypeptide; SEQ ID NO: 15.
  • FIG. 16 is the amino acid sequence of 1G2.2 polypeptide; SEQ ID NO: 16.
  • FIG. 17 is the amino acid sequence of 10C12.7 polypeptide; SEQ ID NO: 17.
  • FIG. 18 is the amino acid sequence of 14F6.3 polypeptide; SEQ ID NO: 18.
  • FIG. 19 is the amino acid sequence of B12D8.2 polypeptide; SEQ ID NO: 19.
  • FIG. 20 is the amino acid sequence of 7F4.1 polypeptide; SEQ ID NO: 20.
  • FIG. 21 is the amino acid sequence of 10D7.5 polypeptide; SEQ ID NO: 21.
  • FIG. 22 is the amino acid sequence of 10G9.3 polypeptide, 10A2.2 polypeptide and B11B8.1 polypeptide; SEQ ID NO: 22.
  • FIG. 23 is the amino acid sequence of 11B8.4 polypeptide; SEQ ID NO: 23.
  • FIG. 24 is the amino acid sequence of Mab H11B-11B8 target epitope; SEQ ID 163.
  • FIG. 25 is a schematic representation of the BVH-3 gene as well as location of gene sequences coding for the full length and truncated polypeptides. The relationships between DNA fragments are shown with respect to each other.
  • FIG. 26 is a schematic representation of the BVH-11 gene as well as location of gene sequences coding for the full length and truncated polypeptides. The relationships between DNA fragments are shown with respect to each other.
  • FIG. 27 is a schematic representation of the BVH-11-2 gene as well as location of gene sequences coding for the full length and truncated polypeptides. The relationships between DNA fragments are shown with respect to each other.
  • FIG. 28 is a schematic representation of the BVH-3 protein and the location of internal and surface epitopes recognized by certain monoclonal antibodies.
  • FIG. 29 is a schematic representation of the BVH-11-2 protein and the location of protective surface epitopes recognized by certain monoclonal antibodies.
  • FIG. 30 is a map of plasmid pURV22.HIS. Kan R , kanamycin-resistance coding region; cI857, bacteriophage ⁇ cI857 temperature-sensitive repressor gene; lambda pL, bacteriophage ⁇ transcription promotor; His-tag, 6-histidine coding region; terminator, T1 transcription terminator; ori, colE1 origin of replication.
  • FIG. 31 depicts the comparison of the amino acid sequences of BVH-3M (sp64) and BVH-3 (Sp63) proteins by using the program Clustal W from MacVector sequence analysis software (version 6.5.3). Underneath the alignment, there is a consensus line where * and . characters indicate identical and similar amino acid residues, respectively.
  • FIG. 32 depicts the comparison of the amino acid sequences of BVH-3, BVH-11 and BVH-11-2 proteins by using the program Clustal W from MacVector sequence analysis software (version 6.5.3). Underneath the alignment, there is a consensus line where * and . characters indicate identical and similar amino acid residues, respectively.
  • FIG. 33 is the DNA sequence of the NEW43 gene (SEQ ID No 257).
  • FIG. 34 is the deduced amino acid sequence of NEW43 polypeptide (SEQ ID No 258).
  • portions of the BVH-3 and BVH-11 polypeptides were internal. Other portions were not present in important strains such as encapsulated s. pneumonia causing disease strains. It would be advantageous to have a polypeptide that comprises a portion that is not internal. When large portions of a polypeptide are internal, these portions are not exposed on the bacteria. However, these portions can be very immunogenic in a recombinant polypeptide and will not confer protection against infections. It would also be advantageous to have a polypeptide that comprises a portion that is present in most strains.
  • the present invention is concerned with polypeptides in which undesired portions have been deleted and/or modified in order to obtain a specific immune response.
  • polypeptides or polynucleotides encoding such polypeptides comprising protective domains.
  • the polypeptides when the undesired portion of the polypeptides are deleted or modified, the polypeptides have desired biological properties. This is surprising in view of the fact that some of these portions were described as being epitope bearing portion in the patent application PCT WO 98/18930. In other publications such as PCT WO 00/37105, portions identified as histidine triad and coil coiled regions were said to be of importance. The present inventors have found that variants of the polypeptide BVH-3 and BVH-11 in which certain portions were deleted and/or modified and chimeras of these polypeptides have biological properties and generate a specific immune response.
  • the present invention provides an isolated polynucleotide encoding a polypeptide having at least 70% identity to a second polypeptide comprising a sequence as disclosed in the present application, the tables and figures.
  • an isolated polynucleotide comprising a polynucleotide chosen from;
  • the present invention provides an isolated polynucleotide encoding a polypeptide having at least 70% identity to a second polypeptide comprising a sequence chosen from table A, B, D, E, G or H or fragments, analogues or derivatives thereof.
  • the present invention provides an isolated polynucleotide encoding a polypeptide having at least 95% identity to a second polypeptide comprising a sequence chosen from table A, B, D, E, G or H or fragments, analogues or derivatives thereof.
  • the present invention relates to polypeptides characterised by the amino acid sequence chosen from table A, B, D, E, G or H or fragments, analogues or derivatives thereof.
  • the present invention provides an isolated polynucleotide encoding a polypeptide having at least 70% identity to a second polypeptide comprising a sequence chosen from table A, B, D, E, G or H.
  • the present invention provides an isolated polynucleotide encoding a polypeptide having at least 95% identity to a second polypeptide comprising a sequence chosen from table A, B, D, E, G or H.
  • the present invention relates to polypeptides characterised by the amino acid sequence chosen from table A, B, D, E, G or H.
  • the present invention provides an isolated polynucleotide encoding a polypeptide having at least 70% identity to a second polypeptide comprising a sequence chosen from table B, E or H or fragments, analogues or derivatives thereof.
  • the present invention provides an isolated polynucleotide encoding a polypeptide having at least 95% identity to a second polypeptide comprising a sequence chosen from B, E or H or fragments, analogues or derivatives thereof.
  • the present invention relates to polypeptides characterised by the amino acid sequence chosen from table B, E or H or fragments, analogues or derivatives thereof.
  • the present invention provides an isolated polynucleotide encoding a polypeptide having at least 70% identity to a second polypeptide comprising a sequence chosen from table B, E or H.
  • the present invention provides an isolated polynucleotide encoding a polypeptide having at least 95% identity to a second polypeptide comprising a sequence chosen from B, E or H.
  • the present invention relates to polypeptides characterised by the amino acid sequence chosen from table B, E or H.
  • nucleotides encoding polypeptides and chimeric polypeptides are within the scope of the present invention.
  • polypeptides or chimeric polypeptides in accordance with the present invention are antigenic.
  • polypeptides or chimeric polypeptides in accordance with the present invention are immunogenic.
  • polypeptides or chimeric polypeptides in accordance with the present invention can elicit an immune response in an individual.
  • the present invention also relates to polypeptides which are able to raise antibodies having binding specificity to the polypeptides or chimeric polypeptides of the present invention as defined above.
  • polypeptides of table A (BVH-3) or table D (BVH-11) comprise at least one epitope bearing portion.
  • the fragments of the polypeptides of the present invention will comprise one or more epitope bearing portion identified in Table C and F.
  • the fragment will comprises at least 15 contiguous amino acid of the polypeptide of table C and F.
  • the fragment will comprises at least 20 contiguous amino acid of the polypeptide of table C and F.
  • the epitope bearing portion of the polypeptide of table A(BVH-3) comprises at least one polypeptide listed in Table C.
  • the epitope bearing portion of the polypeptide of table B(BVH-11) comprises at least one polypeptide listed in Table F.
  • An antibody that “has binding specificity” is an antibody that recognises and binds the selected polypeptide but which does not substantially recognise and bind other molecules in a sample, such as a biological sample. Specific binding can be measured using an ELISA assay in which the selected polypeptide is used as an antigen.
  • “protection” in the biological studies is defined by a significant increase in the survival curve, rate or period.
  • Statistical analysis using the Log rank test to compare survival curves, and Fisher exact test to compare survival rates and numbers of days to death, respectively, might be useful to calculate P values and determine whether the difference between the two groups is statistically significant. P values of 0.05 are regarded as not significant.
  • fragments”, “derivatives” or “analogues” of the polypeptides of the invention include those polypeptides in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably conserved) and which may be natural or unnatural.
  • derivatives and analogues of polypeptides of the invention will have about 70% identity with those sequences illustrated in the figures or fragments thereof. That is, 70% of the residues are the same.
  • polypeptides will have greater than 75% homology.
  • polypeptides will have greater than 80% homology.
  • polypeptides will have greater than 85% homology.
  • polypeptides will have greater than 90% homology. In a further embodiment, polypeptides will have greater than 95% homology. In a further embodiment, polypeptides will have greater than 99% homology. In a further embodiment, derivatives and analogues of polypeptides of the invention will have less than about 20 amino acid residue substitutions, modifications or deletions and more preferably less than 10. Preferred substitutions are those known in the art as conserved i.e. the substituted residues share physical or chemical properties such as hydrophobicity, size, charge or functional groups.
  • proteins or polypeptides of the invention will also find use in the context of the present invention, i.e. as antigenic/immunogenic material.
  • proteins or polypeptides which include one or more additions, deletions, substitutions or the like are encompassed by the present invention.
  • This program compares amino acid sequences and finds the optimal alignment by inserting spaces in either sequence as appropriate. It is possible to calculate amino acid identity or similarity (identity plus conservation of amino acid type) for an optimal alignment.
  • a program like BLASTX will align the longest stretch of similar sequences and assign a value to the fit. It is thus possible to obtain a comparison where several regions of similarity are found, each having a different score. Both types of identity analysis are contemplated in the present invention.
  • the analogues or derivatives could be fusion proteins, incorporating moieties which render purification easier, for example by effectively tagging the desired protein or polypeptide, It may be necessary to remove the “tag” or it may be the case that the fusion protein itself retains sufficient antigenicity to be useful.
  • antigenic/immunogenic fragments of the proteins or polypeptides of the invention or of analogues or derivatives thereof.
  • the fragments of the present invention should include one or more such epitopic regions or be sufficiently similar to such regions to retain their antigenic/immunogenic properties.
  • the degree of identity is perhaps irrelevant, since they may be 100% identical to a particular part of a protein or polypeptide, analogue or derivative as described herein.
  • the key issue, once again, is that the fragment retains the antigenic/immunogenic properties.
  • analogues, derivatives and fragments possess at least a degree of the antigenicity/immunogenic of the protein or polypeptide from which they are derived.
  • polypeptides of the invention include both polypeptides and chimeric polypeptides.
  • polypeptides which have fused thereto other compounds which alter the polypeptides biological or pharmacological properties i.e. polyethylene glycol (PEG) to increase half-life; leader or secretory amino acid sequences for ease of purification; prepro- and pro- sequences; and (poly)saccharides.
  • PEG polyethylene glycol
  • amino acid regions are found to be polymorphic, it may be desirable to vary one or more particular amino acids to more effectively mimic the different epitopes of the different streptococcus strains.
  • polypeptides of the present invention can be modified by terminal —NH 2 acylation (e.g. by acetylation, or thioglycolic acid amidation, terminal carboxy amidation, e.g. with ammonia or methylamine) to provide stability, increased hydrophobicity for linking or binding to a support or other molecule.
  • terminal —NH 2 acylation e.g. by acetylation, or thioglycolic acid amidation, terminal carboxy amidation, e.g. with ammonia or methylamine
  • hetero and homo polypeptide multimers of the polypeptide fragments, analogues and derivatives include, for example, one or more polypeptides that have been cross-linked with cross-linkers such as avidin/biotin, gluteraldehyde or dimethylsuperimidate.
  • polymeric forms also include polypeptides containing two or more tandem or inverted contiguous sequences, produced from multicistronic mRNAs generated by recombinant DNA technology.
  • a fragment, analogue or derivative of a polypeptide of the invention will comprise at least one antigenic region i.e. at least one epitope.
  • polypeptides may be utilised having bishaloacetyl groups, nitroarylhalides, or the like, where the reagents being specific for thio groups. Therefore, the link between two mercapto groups of the different peptides may be a single bond or may be composed of a linking group of at least two, typically at least four, and not more than 16, but usually not more than about 14 carbon atoms.
  • polypeptide fragments, analogues and derivatives of the invention do not contain a methionine (Met) starting residue.
  • polypeptides will not incorporate a leader or secretory sequence (signal sequence) .
  • the signal portion of a polypeptide of the invention may be determined according to established molecular biological techniques.
  • the polypeptide of interest may be isolated from a streptococcus culture and subsequently sequenced to determine the initial residue of the mature protein and therefore the sequence of the mature polypeptide.
  • vaccine compositions comprising one or more streptococcus polypeptides of the invention in admixture with a pharmaceutically acceptable carrier diluent or adjuvant.
  • suitable adjuvants include oils i.e. Freund's complete or incomplete adjuvant; salts i.e. AlK(SO 4 ) 2 , AlNa(SO 4 ) 2 , AlNH 4 (SO 4 ) 2 , silica, kaolin, carbon polynucleotides i.e. poly IC and poly AU.
  • Preferred adjuvants include QuilA and Alhydrogel.
  • Vaccines of the invention may be administered parenterally by injection, rapid infusion, nasopharyngeal absorption, dermoabsorption, or bucal or oral.
  • Pharmaceutically acceptable carriers also include tetanus toxoid.
  • vaccine is also meant to include antibodies.
  • one or more antibodies having binding specificity for the polypeptides of the present invention for the treatment or prophylaxis of streptococcus infection and/or diseases and symptoms mediated by streptococcus infection.
  • Vaccine compositions of the invention are used for the treatment or prophylaxis of streptococcus infection and/or diseases and symptoms mediated by streptococcus infection as described in P. R. Murray (Ed, in chief), E. J. Baron, M. A. Pfaller, F. C. Tenover and R. H. Yolken. Manual of Clinical Microbiology, ASM Press, Washington, D.C. sixth edition, 1995, 1482p which are herein incorporated by reference.
  • vaccine compositions of the present invention are used for the treatment or prophylaxis of meningitis, otitis media, bacteremia or pneumonia.
  • vaccine compositions of the invention are used for the treatment or prophylaxis of streptococcus infection and/or diseases and symptoms mediated by streptococcus infection, in particular S. pneumoniae, group A streptococcus ( pyogenes ), group B streptococcus (GBS or agalactiae ), dysgalactiae, uberis, nocardia as well as Staphylococcus aureus.
  • streptococcus infection is S. pneumoniae.
  • vaccines are administered to those individuals at risk of streptococcus infection such as infants, elderly and immunocompromised individuals.
  • mammals include mammals.
  • the mammal is human.
  • Vaccine compositions are preferably in unit dosage form of about 0.001 to 100 ⁇ g/kg (antigen/body weight) and more preferably 0.01 to 10 ⁇ g/kg and most preferably 0.1 to 1 ⁇ g/kg 1 to 3 times with an interval of about 1 to 6 week intervals between immunizations.
  • Vaccine compositions are preferably in unit dosage form of about 0.1 ⁇ g to 10 mg and more preferably 1 ⁇ g to 1 mg and most preferably 10 to 100 ⁇ g 1 to 3 times with an interval of about 1 to 6 week intervals between immunizations.
  • polypeptides characterised by the amino acid sequence chosen from table A, B, D, E, G or H or fragments, analogues or derivatives thereof.
  • polypeptides characterised by the amino acid sequence chosen from table B, E or H or fragments, analogues or derivatives thereof.
  • polynucleotides are those illustrated in table A, B, D, E, G or H which encodes polypeptides of the invention.
  • polynucleotides are those illustrated in table B, E or H which encodes polypeptides of the invention.
  • polynucleotide sequences illustrated in the figures may be altered with degenerate codons yet still encode the polypeptides of the invention. Accordingly the present invention further provides polynucleotides which hybridise to the polynucleotide sequences herein above described (or the complement sequences thereof) having 50% identity between sequences. In one embodiment, at least 70% identity between sequences. In one embodiment, at least 75% identity between sequences. In one embodiment, at least 80% identity between sequences. In one embodiment, at least 85% identity between sequences. In one embodiment, at least 90% identity between sequences. In a further embodiment, polynucleotides are hybridizable under stringent conditions i.e. having at least 95% identity. In a further embodiment, more than 97% identity.
  • Suitable stringent conditions for hybridation can be readily determined by one of skilled in the art (see for example Sambrook et al., (1989) Molecular cloning: A Laboratory Manual, 2 nd ed, Cold Spring Harbor, N.Y.; Current Protocols in Molecular Biology, (1999) Edited by Ausubel F. M. et al., John Wiley & Sons, Inc., N.Y.).
  • the present invention provides polynucleotides that hybridise under stringent conditions to either
  • the present invention provides polynucleotides that hybridise under stringent conditions to either
  • the present invention provides polynucleotides that hybridise under stringent conditions to either
  • the present invention provides polynucleotides that hybridise under stringent conditions to either
  • polynucleotides are those encoding polypeptides of the invention illustrated in table A, B, D, E, G or H.
  • polynucleotides include both DNA and RNA.
  • the present invention also includes polynucleotides complementary to the polynucleotides described in the present application.
  • polynucleotides encoding polypeptides of the invention, or fragments, analogues or derivatives thereof may be used in a DNA immunization method. That is, they can be incorporated into a vector which is replicable and expressible upon injection thereby producing the antigenic polypeptide in vivo.
  • polynucleotides may be incorporated into a plasmid vector under the control of the CMV promoter which is functional in eukaryotic cells.
  • the vector is injected intramuscularly.
  • polypeptides of the invention by recombinant techniques by expressing a polynucleotide encoding said polypeptide in a host cell and recovering the expressed polypeptide product.
  • the polypeptides can be produced according to established synthetic chemical techniques i.e. solution phase or solid phase synthesis of oligopeptides which are ligated to produce the full polypeptide (block ligation).
  • host cells are transfected with vectors which encode the polypeptide, and then cultured in a nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the genes.
  • Suitable vectors are those that are viable and replicable in the chosen host and include chromosomal, non-chromosomal and synthetic DNA sequences e.g. bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA.
  • the polypeptide sequence may be incorporated in the vector at the appropriate site using restriction enzymes such that it is operably linked to an expression control region comprising a promoter, ribosome binding site (consensus region or Shine-Dalgarno sequence), and optionally an operator (control element).
  • an expression control region comprising a promoter, ribosome binding site (consensus region or Shine-Dalgarno sequence), and optionally an operator (control element).
  • Suitable promoters include but are not limited to LTR or SV40 promoter, E. coli lac, tac or trp promoters and the phage lambda P L promoter.
  • Vectors will preferably incorporate an origin of replication as well as selection markers i.e. ampicilin resistance gene.
  • Suitable bacterial vectors include pET, pQE70, pQE60, pQE-9, pbs, pD10 phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a, pNH18A, pNH46A, ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 and eukaryotic vectors pBlueBacIII, pWLNEO, pSV2CAT, pOG44, pXT1, pSG, pSVK3, pBPV, pMSG and pSVL.
  • Host cells may be bacterial i.e. E. coli, Bacillus subtilis, Streptomyces; fungal i.e. Aspergillus niger, Aspergillus nidulins; yeast i.e. Saccharomyces or eukaryotic i.e. CHO, COS.
  • polypeptide Upon expression of the polypeptide in culture, cells are typically harvested by centrifugation then disrupted by physical or chemical means (if the expressed polypeptide is not secreted into the media) and the resulting crude extract retained to isolate the polypeptide of interest. Purification of the polypeptide from culture media or lysate may be achieved by established techniques depending on the properties of the polypeptide i.e. using ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography and lectin chromatography. Final purification may be achieved using HPLC.
  • the polypeptide may be expressed with or without a leader or secretion sequence.
  • the leader may be removed using post-translational processing (see U.S. Pat. No. 4,431,739; U.S. Pat. No. 4,425,437; and U.S. Pat. No. 4,338,397 incorporated herein by reference) or be chemically removed subsequent to purifying the expressed polypeptide.
  • the streptococcus polypeptides of the invention may be used in a diagnostic test for streptococcus infection, in particular S. pneumoniae infection.
  • a diagnostic test for streptococcus infection in particular S. pneumoniae infection.
  • Several diagnostic methods are possible, for example detecting streptococcus organism in a biological sample, the following procedure may be followed:
  • a method for the detection of antibody specific to a streptococcus antigen in a biological sample containing or suspected of containing said antibody may be performed as follows:
  • this diagnostic test may take several forms, including an immunological test such as an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay or a latex agglutination assay, essentially to determine whether antibodies specific for the polypeptide are present in an organism.
  • an immunological test such as an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay or a latex agglutination assay, essentially to determine whether antibodies specific for the polypeptide are present in an organism.
  • the DNA sequences encoding polypeptides of the invention may also be used to design DNA probes for use in detecting the presence of streptococcus in a biological sample suspected of containing such bacteria.
  • the detection method of this invention comprises:
  • the DNA probes of this invention may also be used for detecting circulating streptococcus i.e. S. pneumoniae nucleic acids in a sample, for example using a polymerase chain reaction, as a method of diagnosing streptococcus infections.
  • the probe may be synthesized using conventional techniques and may be immobilized on a solid phase, or may be labelled 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 the streptococcus pneumoniae polypeptides of the invention.
  • Another diagnostic method for the detection of streptococcus in a patient comprises:
  • a further aspect of the invention is the use of the streptococcus polypeptides of the invention as immunogens for the production of specific antibodies for the diagnosis and in particular the treatment of streptococcus infection.
  • Suitable antibodies may be determined using appropriate screening methods, for example by measuring the ability of a particular antibody to passively protect against streptococcus infection in a test model.
  • an animal model is the mouse model described in the examples herein.
  • the antibody may be a whole antibody or an antigen-binding fragment thereof and may belong to any immunoglobulin class.
  • the antibody or fragment may be of animal origin, specifically of mammalian origin and more specifically of murine, rat or human origin.
  • the term recombinant antibody or antibody fragment means antibody or antibody fragment which was produced using molecular biology techniques.
  • the antibody or antibody fragments may be polyclonal, or preferably monoclonal. It may be specific for a number of epitopes associated with the streptococcus pneumoniae polypeptides but is preferably specific for one.
  • a further aspect of the invention is the use of the antibodies directed to the streptococcus polypeptides of the invention for passive immunization.
  • One example of an animal model is the mouse model described in the examples herein.
  • the antibody may be a whole antibody or an antigen-binding fragment thereof and may belong to any immunoglobulin class.
  • the antibody or fragment may be of animal origin, specifically of mammalian origin and more specifically of murine, rat or human origin. It may be a natural antibody or a fragment thereof, or if desired, a recombinant antibody or antibody fragment.
  • the term recombinant antibody or antibody fragment means antibody or antibody fragment which was produced using molecular biology techniques.
  • the antibody or antibody fragments may be polyclonal, or preferably monoclonal. It may be specific for a number of epitopes associated with the streptococcus pneumoniae polypeptides but is preferably specific for one.
  • This example describes the bacterial strains, plasmids, PCR primers, recombinant proteins and hybridoma antibodies used herein.
  • S. pneumoniae SP64 (serogroup 6) and SP63 (serogroup 9) clinical isolates were provided by the Laboratoire de la Santé Publique du Québec, Sainte-Anne-de-Bellevue; Rx1 strain, a nonencapsulated derivative of the type 2 strain D39 and the type 3 strain WU2 were provided by David E. Briles from University of Alabama, Birmingham and the type 3 clinical isolate P4241 was provided by the Centre de mecanic en Infectiologie du Centre Hospitalier de l'Universite Laval, Sainte-Foy. E.
  • coli strains DH5 ⁇ Gabco BRL, Gaithesburg, Md.
  • AD494 ⁇ DE3 (Novagen, Madison, Wis.) and BL21 ( ⁇ DE3) (Novagen) as well as plasmid superlinker pSL301 vector (Invitrogen, San Diego, Calif.); PCMV-GH vector (gift from Dr. Stephen A. Johnston, Department for Biochemistry, University of Texas, Dallas, Tex.); pET32 and pET21 (Novagen) and pURV22.HIS expression vectors ( FIG. 30 ) were used in this study.
  • the pURV22.HIS vector contains a cassette of the bacteriophage ⁇ cI857 temperature-sensitive repressor gene from which the functional P R promoter has been deleted.
  • the inactivation of the cI857 repressor by a temperature increase from the range of 30-37° C. to 37-42° C. results in the induction of the gene under the control of promoter ⁇ PL.
  • the PCR primers used for the generation of the recombinant plasmids had a restriction endonuclease site at the 5′end, thereby allowing directional cloning of the amplified product into the digested plasmid vector.
  • the PCR oligonucleotide primers used are listed in the following Table 1.
  • PCR-amplified products were digested with restriction endonucleases and ligated to either linearized plasmid pSL301, pCMV-GH, pET or pURV22.HIS expression vector digested likewise or digested with enzymes that produce compatible cohesive ends.
  • Recombinant psL301 and recombinant pCMV-GH plasmids were digested with restriction enzymes for the in-frame cloning in pET expression vector.
  • pET vectors were used, clones were first stabilized in E. coli DH5a before introduction into E. coli BL21( ⁇ DE3) or AD494 ( ⁇ DE3) for expression of full-length or truncated BVH-3, BVH-11 or BVH-11-2 molecules.
  • Each of the resultant plasmid constructs was confirmed by nucleotide sequence analysis.
  • the recombinant proteins were expressed as N-terminal fusions with the thioredoxin and His-tag (pET32 expression system); as C-terminal fusions with an His-tag (pET21 expression system); or as N-terminal fusions with an His-tag (pURV22.HIS expression system).
  • the expressed recombinant proteins were purified from supernatant fractions obtained after centrifugation of sonicated IPTG- (pET systems) or heat- (pURV22.HIS) induced E. coli using a His-Bind metal chelation resin (QIAgen, Chatsworth, Calif.).
  • the gene products generated from S. pneumoniae SP64 are listed in the following Table 2.
  • the gene fragment encoding BVH-3-Sp63 protein (amino acid residues 21 to 840 on SEQ ID NO: 10) was generated from S. pneumoniae SP63 using the PCR-primer sets OCRR479-OCRR480 and the cloning vector pSL301.
  • the recombinant pSL301-BVH-3Sp63 was digested for the in-frame cloning in pET32 vector for the expression of the BVH-3-Sp63 molecule.
  • Monoclonal antibody (Mab)-secreting hybridomas were obtained by fusions of spleen cells from immunized mice and non-secreting, HGPRT-deficient mouse myeloma SP2/0 cells by the methods of Fazekas De St-Groth and Scheidegger (J Immunol Methods 35: 1-21, 1980) with modifications (J. Hamel et al. J Med Microbiol 23: 163-170, 1987).
  • mice Female BALB/c mice (Charles River, St-Constant, Quebec, Canada) were immunized with either BVH-3M (thioredoxin-His.Tag-BVH-3M fusion protein/ pET32 system), BVH-11M (thioredoxin-His.Tag-BVH-llM fusion protein/pET32 system), BVH-11-2M (thioredoxin-His.Tag-BVH-11-2M fusion protein/pET32 system), BVH-11B (thioredoxin-His.Tag-BVH-11B fusion protein/pET32 system), BVH-3M (His.Tag-BVH-3 fusion protein/pURV22.HIS system) or NEW1 (NEW1-His.Tag fusion protein/pET21 system) gene products from S.
  • BVH-3M thioredoxin-His.Tag-BVH-3M fusion protein/ pET32 system
  • BVH-11M thioredoxin-His
  • BVH-3 and BVH-11 gene fragments corresponding to the 3′end of the genes were amplified by PCR using pairs of oligonucleotides engineered to amplify gene fragments to be included in the chimeric genes.
  • the primers used had a restriction endonuclease site at the 5′ end, thereby allowing directional in-frame cloning of the amplified product into digested plasmid vectors (Table 1 and Table 2).
  • PCR-amplified products were digested with restriction endonucleases and ligated to linearized plasmid pET21 or pSL301 vector. The resultant plasmid constructs were confirmed by nucleotide sequence analysis.
  • the recombinant pET21 plasmids containing a PCR product were linearized by digestion with restriction enzymes for the in-frame cloning of a second DNA fragment and the generation of a chimeric gene encoding for a chimeric pneumococcal protein molecule.
  • Recombinant pSL301 plasmids containing a PCR product were digested with restriction enzymes for the obtention of the DNA inserts.
  • the resulting insert DNA fragments were purified and inserts corresponding to a given chimeric gene were ligated into pET21 vector for the generation of a chimeric gene.
  • the recombinant chimeric polypeptides listed in Table 2 were as C-terminal fusion with an His-tag.
  • the expressed recombinant proteins were purified from supernatant fractions obtained from centrifugation of sonicated IPTG-induced E. coli cultures using a His-Bind metal chelation resin (QIAgen, Chatsworth, Calif.).
  • mice Groups of 8 female BALB/c mice (Charles River) were immunized subcutaneously two times at three-week intervals with 25 ⁇ g of either affinity purified HisTag-fusion protein identifed in presence of 15-20 ⁇ g of QuilA adjuvant. Ten to 14 days following the last immunization, the mice were challenged challenged intravenously with 10E5-10E6 CFU of S. pneumoniae type 3 strain WU2. The polypeptides and fragments are capable of eliciting a protective immune response.
  • This example describes the identification of peptide domains carrying target epitopes using Mabs and recombinant truncated proteins described in example 1.
  • Hybridomas were tested by ELISA against truncated BVH-3, BVH-11 or BVH-11-2 gene products in order to characterize the epitopes recognized by the Mabs.
  • the truncated gene products were generated from S. pneumoniae SP64 strain except for BVH-3-Sp63 which was generated from S. pneumoniae SP63 strain.
  • the reactivity of each antibody was examined with full-length BVH-3, BVH-11 or BVH-11-2 recombinant proteins.
  • the Mab reactivity was evaluated by Western immunoblotting after separation of the gene product by SDS-PAGE and transfer on nitrocellulose paper. The reactivities observed is set forth in the following Table 3 and Table 4.
  • BVH-3-reactive Mabs can be divided into two groups: BVH-3A- and BVH-3B-reactive Mabs with the exception of Mabs H11-7G11 and H3V-15A10 which reacted with both, BVH-3A and BVH-3B molecules.
  • the BVH-3A-reactive Mabs can be subdivided in two subgroups of antibodies depending of their reactivity or lack of reactivity with BVH-3C recombinant protein. Mab reactive with BVH-3C protein recognized epitopes shared by both, BVH-3 and BVH-11 proteins.
  • BVH-3- and BVH-11-cross-reactive Mabs were also reactive with BVH-11A and BVH-11-2M recombinant proteins.
  • BVH-3B-reactive Mabs can be subdivided into three subgroups according to their reactivity with NEW1, NEW2 and NEW3 recombinant proteins. Some Mabs were only reactive with the NEW1 protein while other Mabs were reactive with either, NEW1 and NEW2 or NEW1 and NEW3 recombinant proteins.
  • Mabs H11-7G11 and H3V-15A10 react with epitopes in more than one position on BVH-3.
  • H11-7G11 epitope might comprised HXXHXH sequence. This sequence is repeated, respectively, 6 and 5 times in BVH-3 and BVH-11/BVH-11-2 protein sequences.
  • the lack of reactivity of Mab H11-7G11 with NEW 10 molecule suggests that the epitope includes the HGDHXH sequence.
  • Multiple-position mapping of H3V-15A10 epitope on BVH-3 is suggested by the reactivity of the Mab with two BVH-3 fragments that do not overlap.
  • Mabs H3-7G2, H3V-9C6 and H3V-16A7 were not reactive with BVH-3 Sp63 thus allowing the location of their corresponding epitopes on a 177-amino acid fragment comprised between amino acids 244 and 420 on BVH-3 molecule of S. pneumoniae SP64 ( FIG. 31 ).
  • the Mabs that are reactive with BVH-11- and/or BVH-11-2 and that do not recognize BVH-3 molecules can be divided into three groups according to their reactivities with BVH-11A and NEW10 recombinant proteins. Some Mabs reacted exclusively with either BVH-11A or NEW10 protein while other Mabs were reactive with both, BVH-11A and NEW10 recombinant proteins.
  • This example describes the construction of BVH-3 and BVH-11-2 gene libraries for the mapping of epitopes.
  • BVH-3 and BVH-11-2 gene libraries were constructed using recombinant PCMV-GH and PSL301 plasmid DNA containing respectively, BVH-3 gene sequence spanning nucleotides 1837 to 4909 (SEQ ID NO: 2) or BVH-11-2 gene sequence spanning nucleotides 172 to 2630 (SEQ ID NO: 5) and the Novatope® library construction and screening system (Novagen).
  • the recombinant plasmids containing BVH-3 or BVH-11-2 gene fragment were purified using QIAgen kit (Chatsworth, Calif.) and digested with the restriction enzymes BglII and XbaI respectively.
  • the resulting BglII-XbaI DNA fragments were purified using the QIAquick gel extraction kit from QIAgen and digested with Dnase I for the generation of randomly cleaved DNA.
  • DNA fragments of 50 to 200 bp were purified, treated with T4 DNA polymerase to blunt the target DNA ends and add a single 3′dA residue, and ligated into pSCREEN-T-Vector (Novagen) following the procedures suggested by the manufacturer (Novatope® System, Novagen).
  • the gene libraries of E. coli clones, each of which expressing a small peptide derived from BVH-3 or BVH-11-2 genes were screened by standard colony lift methods using Mabs as immunoprobes.
  • Mabs H112-10G9, H112-10A2 and H11B-11B8 were reactive against the same peptide sequence (amino acid residues 594 to 679 on BVH-11-2 protein sequence), clones corresponding to the sequence spanning from amino acid residues 658 to 698 were only picked up by Mab H11B-11B8 thus revealing the location of H11B-11B8 epitope between amino acid residues 658 to 679 (SEQ ID NO: 163).
  • Mabs H112-10G9, H112-10A2, and H11B-11B8 are directed against 3 distinct non overlapping epitopes located closely on the peptide sequence corresponding to amino acid residues 594 to 679 (SEQ ID NO: 22).
  • This example describes the immunization of animals with recombinant proteins for the generation of antibody reactive with BVH-3, BVH-11 and/or BVH-11-2.
  • NZW rabbits (Charles River Laboratories, St-Constant, Québec, Canada) were immunized subcutaneously at multiple sites with 50 ⁇ g or 100 ⁇ g of the purified BVH-3M, L-BVH-3AD, NEW1, NEW13, or L-BVH-11 recombinant protein in presence of 80 ⁇ g of QuilA adjuvant (Cedarlane Laboratoratories Ltd, Hornby, Canada).
  • the rabbits were boosted two times at three-week intervals with the same antigen and blood samples were collected before each immunization and 6 to 28 days following the last immunization.
  • the sera samples were designated preimmune, post 1 st , post 2 nd or post 3 rd injection.
  • the rabbit immune response to immunization was evaluated by ELISA using recombinant BVH-3M (BVH-3M-His.Tag fusion protein/pET21 system) or BVH-11M (BVH-11M-His.Tag fusion protein/pET21 system) proteins or suspensions of heat-killed S. pneumoniae Rx-1 cells as coating antigens.
  • ELISA titer was defined as the reciprocal of the highest sera dilution at which absorbance A 410 value was 0.1 above the background value.
  • Antibodies reactive with BVH-3 and/or BVH-11 epitopes were elicited following immunization in all animals as shown in the following Table 6.
  • Antibody reactive with recombinant or pneumococcal antigens was not present in the preimmune sera.
  • the immune response to immunization was detectable in the sera of each rabbit after a single injection of recombinant antigen.
  • the antibody response following the second injection with either antigen tested was characterized by a strong increase in antibody titer.
  • good titers of antibody reactive with S. pneumoniae cells, with an average titer of 52,000 after the third immunization were obtained, thus establishing that native pneumococcal epitopes are expressed on the recombinant E. coli gene products.
  • This example describes the protection of animals against fatal experimental pneumococcal infection by administration of antibody raised to BVH-3, BVH-11 or BVH-11-2 gene products.
  • High-titer Mab preparations were obtained from ascites fluid of mice inoculated intraperitoneally with Mab-secreting hybridoma cells according to the method described by Brodeur et al (J Immunol Methods 71 :265-272, 1984).
  • Sera samples were collected from rabbits immunized with BVH-3M as described in Example 4.
  • the rabbit sera collected after the third immunization and ascites fluid were used for the purification of antibodies by precipitation using 45 to 50% saturated ammonium sulfate.
  • the antibody preparations were dissolved and dialyzed against phosphate-buffered saline (PBS).
  • CBA/N mice National Cancer Institute, Frederick, Mass. mice (National Cancer Institute, Frederick, Mass.) were injected intraperitoneally with either 0.1 ml of purified rabbit antibodies or 0.2 ml of ascites fluid before intravenous challenge with approximately 200 CFU of the type 3 S. pneumoniae strain WU2.
  • Control mice received sterile PBS or antibodies purified from preimmune rabbit sera or sera from rabbits immunized with an unrelated N. meningitidis recombinant protein antigen.
  • One group of mice was challenged with S. pneumoniae before the administration of anti-BVH-3 antibody. Samples of the S. pneumoniae challenge inoculum were plated on chocolate agar plates to determine the number of CFU and verify the challenge dose.
  • the CBA/N mice were chosen because of their high susceptibility to S. pneumoniae infection.
  • the LD 50 of WU2 injected intravenously to CBA/N mice is estimated to be ⁇ 10 CFU. Deaths were recorded at 24-h intervals for a period of at least 7 days.
  • mice injected with rabbit anti-BVH-3 antibody are set forth in the following Table 7.
  • TABLE 7 Protective effects of rabbit antibody to BVH-3-M gene in CBA/N mice challenged i.v. with WU2 pneumococci Antibody Time of antibody Days to death preparation administration Alive:Dead post-infection Anti-BVH3M 1 h before 5:0 >14, >14, >14, infection >14, >14 Anti-N.
  • meningitidis 1 h before 0:5 2, 2, 2, 2, 2 infection Anti-BVH-3M 0.5 h post- 4:1 2, >14, >14, infection >14, >14 None (PBS) 1 h before 0:5 1, 2, 2, 2, 2 infection
  • CBA/N mice were infected with 1000 CFU of WU2 S. pneumoniae before or after intraperitoneal administration of 0.1 ml of rabbit antibody.
  • mice 0.1 ml of rabbit antibody prepared from preimmune and immune sera were administered intraperitoneally to CBA/N mice four hours before intranasal challenge with 280 CFU of S. pneumoniae P4241 type 3 strain.
  • all immunized mice survived the challenge while none of 9 mice receiving preimmune sera antibody or buffer alone were alive on day 6 post-infection.
  • S. pneumoniae hemocultures on day 11 post-challenge were negative for all surviving mice.
  • 100% protection was observed in mice receiving monoclonal antibodies H112-10G9 or a mixture of H112-10G9 and H11B-7E11 which are directed against BVH-11/BVH-11-2.
  • the protection data obtained for mice injected with ascites fluid are set forth in the following Table 9.
  • Administration of a volume of 0.2 ml of ascites fluid of 0.2 ml of some sets of ascites fluid prevented death from experimental infection.
  • H112-3A4+H112-10G9 and H112-10G2+H112-10D7 sets of 2 Mabs conferred complete protection against experimental infection.
  • the Mabs H112-3A4, H112-10G9, H112-10D7, H112-10A2, H112-3E8, H112-10C5, H11B-11B8, H11B-15G2, H11B-1C9, H11B-7E11, H11B-13D5 and H11-10B8 were present in at least one protective pair of Mabs and were said to be protective and reactive against protective epitopes.
  • the locations of protection-conferring epitopes on BVH-11-2 molecules are summarized in Table 10 and FIG. 29 .
  • Protective Mabs H112-3A4, H112-10G9, H112-10D7, H112-10A2, H112-3E8, H112-10C5, H11B-11B8, H11B-15G2, H11B-1C9, H11B-7E11, H11B-13D5 and H11-10B8 were all reactive with New 10 protein corresponding to amino acid residues 271 to 838 on the BVH-11-2 molecule.
  • Six out of these 12 Mabs were directed against epitopes present in the NEW 19 protein and 3 protective Mabs recognized NEW 14.
  • Mab H112-3A4 and H112-10C5 reacted with distinct epitopes exclusive to BVH-11-2 located at the carboxyl end comprised between amino acid residues 769 and 837.
  • Mabs H11-7G11, H11-6E7 and H3-4F9 reactive with epitopes shared by pneumococcal BVH-3, BVH-11 and BVH-11-2 molecules did not succeed to protect even if given in combination with protective H112-10G9 or H112-11B8 Mab.
  • These Mabs recognized epitopes located at the amino end of the BVH-3, BVH-11 and BVH-11-2 molecules comprising, respectively, the first 225, 228 and 226 amino acid residues.
  • the comparison of the BVH-3, BVH-11 and BVH-11-2 protein sequences revealed that a large number of amino acids were conserved in the amino end portion comprising these 225-228 residues with a global 72.8% identity ( FIG. 32 ).
  • This example describes the localization of surface-exposed peptide domains using Mabs described in Example 1.
  • S. pneumoniae type 3 strain WU2 was grown in Todd Hewitt (TH) broth (Difco Laboratories, Detroit Mich.) enriched with 0.5% Yeast extract (Difco Laboratories) at 37° C. in a 8% CO 2 atmosphere to give an OD 600 of 0.260 ( ⁇ 10 8 CFU/ml).
  • the bacterial suspension was then aliquoted in 1 ml samples and the S. pneumoniae cells were pelletted by centrifugation and resuspended in hybridoma culture supernatants. The bacterial suspensions were then incubated for 2 h at 4° C.
  • % Fluorescence and Fluorescence index (FI) values.
  • the % Fluorescence is the number of fluorescein-labelled S. pneumoniae cells divided by 100 and the FI value is the median fluorescence value of pneumococci treated with Mab supernatant divided by the fluorescence value of pneumococci treated with the conjugate alone or with a control unrelated Mab.
  • a FI value of 1 indicated that the Mab has not been detected at the surface of the bacteria whereas a FI value higher than 2 was considered positive when at least 10% of the pneumococcal cells were labelled and indicated that the Mab was reactive with cell-surface exposed epitopes.
  • Table 11 summarized the data obtained with the Mabs tested by flow cytometry.
  • This example describes the immunization of animals with peptide epitopes of BVH-3 and BVH-11-2.
  • the recombinant PSCREEN-T vector (Novagen, Madison, Wis.) containing DNA fragment (nucleotides 2421 to 2626 on SEQ ID NO: 5) encoding the Mab 3A4-epitope (SEQ ID NO: 24) was transformed by electroporation (Gene Pulser II apparatus, BIO-RAD Labs, Mississauga, Canada) into E. coli Tuner ( ⁇ DE3) pLysS [BL21 (F′ ompT hsdSB (rB ⁇ mB ⁇ ) gal dcm lacYI pLysS (Cm r )] (Novagen).
  • the expression of the fusion protein is controlled by the T7 promoter which is recognized by the T7 RNA polymerase (present on the XDE3 prophage, itself under the control of the lac promoter inducible by isopropyl- ⁇ -D-thiogalactopyranoside (IPTG).
  • IPTG isopropyl- ⁇ -D-thiogalactopyranoside
  • the pLysS plasmid reduces the basal fusion protein expression level by coding for a T7 lysozyme, which is a natural inhibitor of the T7 RNA polymerase.
  • the transformants were grown at 37° C. with 250 RPM agitation in LB broth (peptone 10 g/l, yeast extract 5 g/l, NaCl 5 g/l) supplemented with 50 mM glucose, 100 ⁇ g/ml carbenicillin and 34 ⁇ g/ml chloramphenicol, until the absorbance at 600 nm reached a value of 0,7.
  • the overexpression of T7gene 10 protein-His.Tag-3A4.1 fusion protein was then induced by the addition of IPTG to a final concentration of 1 mM and further incubation at 25° C. with 250 RPM agitation for 3 hours. Induced cells from a 800-ml culture were pelleted by centrifugation and frozen at ⁇ 70° C.
  • the fusion protein was purified from the soluble cell fraction by affinity chromatography based on the binding of a six histidine residues sequence (His-Tag) to divalent cations (Ni 2+ ) immobilized on a metal chelation Ni-NTA resin (Qiagen, Mississauga, Canada). Briefly, the pelleted cells were thawed and resuspended in Tris buffered sucrose solution (50 mM Tris, 25% (w/v) sucrose) and frozen at ⁇ 70° C. for 15 minutes. Cells were incubated 15 minutes on ice in the presence of 2 mg/ml lysozyme before disruption by sonication.
  • Tris buffered sucrose solution 50 mM Tris, 25% (w/v) sucrose
  • the lysate was centrifuged at 12000 RPM for 30 minutes and Nickel charged Ni-NTA resin (QIAgen) was added to the supernatant for an overnight incubation at 4° C., with 100 RPM agitation. After washing the resin with a buffer consisting of 20 mM Tris, 500 mM NaCl, 20 mM imidazole pH 7,9, the fusion 3A4.1 protein was eluted with the same buffer supplemented with 250 mM imidazole. The removal of the salt and imidazole was done by dialysis against PBS at 4° C. The protein concentration was determined with BCA protein assay reagent kit (Perce, Rockford, Ill.) and adjusted to 760 ⁇ g/ml.
  • BCA protein assay reagent kit Perce, Rockford, Ill.
  • mice Female CBA/N mice (National Cancer Institute) are immunized subcutaneously three times at three-week intervals with affinity purified T7gene10 protein-His.Tag-3A4.1 fusion protein or, as control, with QuilA adjuvant alone in PBS. Twelve to fourteen days following the third immunization, the mice are challenged intravenously with S. pneumoniae WU2 strain or intranasally with P4241 strain. Samples of the S. pneumoniae challenge inoculum are plated on chocolate agar plates to determine the number of CFU and to verify the challenge dose. The challenge dose are approximalety 300 CFU.
  • the 3A4.1 protein or other tested protein is said protective when the number of mice surviving the infection or the median number of days to death is significantly greater in the 3A4.1-immunized group compared to the control mock-immunized group.
  • This example illustrates the improvement of the antibody response to pneumococci using BVH-3 fragments and variants thereof.
  • BVH-3 Gene sequence comparison revealed that in some strains, the region of BVH-3 encoding for amino acids 244 to 420 as described in SEQ ID N06 is absent thus suggesting the lack of utility of this sequence in vaccine to prevent disease caused by such strains (SEQ ID NO: 9 versus SEQ ID NO: 1). Further BVH-3 fragments or variants thereof were designed in the purpose to develop a universal highly effective vaccine that would target the immune response to ubiquitous surface-exposed protective epitopes. BVH-3 gene fragments designated NEW1 (encoding amino acid residues 472 to 1039 from SEQ ID NO: 6) and NEW40 (encoding amino acid residues 408 to 1039 from SEQ ID NO: 6) were amplified from the S.
  • PCR-amplified products were digested with restriction endonucleases and ligated to linearized plasmid pET21 (Novagen) expression vector digested likewise.
  • Oligonucleotide primers HAMJ489 ccgaattccatatgcaaattgggcaaccgactc; NdeI
  • HAMJ279 cgccaagcttcgctatgaaatcagataaattc; HindIII
  • Clones were first stabilized in E. coli DH5 ⁇ before introduction into E. coli BL21 ( ⁇ DE3) for expression of the truncated gene products.
  • Variants from NEW1 and NEW40 were generated by mutagenesis using the Quickchange Site-Directed Mutagenesis kit from Stratagene and the oligonucleotides designed to incorporate the appropriate mutation.
  • mice Male BALB/c mice (Charles River) immunized as described earlier in example 1 were used for protection experiments against intranasal challenge with virulent S. pneumoniae P4241 strain. The mice were observed for 10 to 14 days post-infection. Data from Table 15 clearly indicate that the NEW35A molecule was equivalent to the parental NEW1 in term of protection. Interestingly, high survival rates where obtained for NEW40- and NEW56-immunized groups with 7 and 8 survivors out of 8 animals, respectively. Similarly, NEW25 comprising amino acid residues 233 to 1039 protected 7 out of 8 animals from lethal infection.
  • Cytometry results are expressed as fluorescence index value where the fluorescence index is the median fluorescence value of pneumococci treated with test sera divided by the background fluorescence value of pneumococci treated with the fluorescein conjugate alone.
  • fluorescence index is the median fluorescence value of pneumococci treated with test sera divided by the background fluorescence value of pneumococci treated with the fluorescein conjugate alone.
  • all sera were used at a dilution of 1:50 and the sera from mice immunized with BVH-3C fragment or QuilA adjuvant alone gave a value similar to the background value.
  • This example describes the cloning and expression of a chimeric deletant BVH-11-2 gene encoding for a chimeric polypeptide corresponding to BVH-11-2 conserved protective surface-exposed epitopes present in most if not all S. pneumoniae strains.
  • BVH-11-2 gene fragments corresponding to 4 gene regions were amplified by PCR using pairs of oligonucleotides engineered to amplify fragments originating from SEQ ID NO :5 spanning nucleotides 1662 to 1742, 1806 to 2153, 2193 to 2414 and 2484 to 2627 from S. pneumoniae strain Sp64 BVH-11-2 gene.
  • HAMJ490-491, HAMJ492-HAMJ493, HAMJ494-HAMJ495, HAMJ496-HAMJ354 had a restriction endonuclease site at the 5′ end, thereby allowing directional in-frame cloning of the amplified product into the digested pET21b(+) plasmid vector (Table 16).
  • PCR-amplified products were digested with restriction endonucleases and ligated to linearized plasmid pSL301 vector digested likewise except for the PCR-amplified fragment obtained with the primer pair HAMJ490-HAMJ491.
  • the HAMJ490-HAMJ491 PCR-amplified product was purified from agarose gel using a QIAquick gel extraction kit from QIAgen (Chatsworth, Calif.) and ligated into PGEM-T plasmid vector without any prior restriction endonuclease digestion.
  • the resultant plasmid constructs were confirmed by nucleotide sequence analysis.
  • the recombinant plasmids containing each of the four were digested with restriction endonucleases corresponding with the 5′ end of each primer pair used for the PCR-amplification.
  • the fragments were purified from agarose gel like described earlier and were all ligated to linearized plasmid pET21b (+) digested with the restriction enzymes NdeI and HindIII for the in-frame cloning of the four different regions of the BVH11-2 gene. Clones were first stabilized in E. coli DH5 ⁇ before introduction into E. coli BL21 ( ⁇ DE3) for expression of a chimeric pneumococcal protein molecule.
  • the resulting NEW43 gene sequence (SEQ ID No 257) is described in FIG. 33 .
  • NEW43-derived molecules designated VP43S and NEW86 were generated from gene amplification and cloning experiments using PCR primers described in Tables 16 and 18 and pET21 expression plasmid vector. Variants from NEW43 were generated by mutagenesis using the Quickchange Site-Directed Mutagenesis kit from Stratagene and the oligonucleotides designed to incorporate the appropriate mutation. The presence of 6 histidine tag residues on the C-terminus of the recombinant molecules simplified the purification of the proteins by nickel chromatography. The following tables 19 and 20 describe the sequences of the primers used for the mutagenesis experiments and the NEW43 variant gene products generated, respectively.
  • mice Male BALB/c mice (Charles River) immunized as described earlier in example 1 were used for protection experiments against intranasal challenge with virulent S. pneumoniae P4241 strain. Data from Table 21 clearly indicate that NEW 19, NEW43 and variants thereof provided protection against experimental pneumonia.
  • This example describes the cloning and expression of chimeric genes encoding for a chimeric protein corresponding to the carboxy-terminal region of BVH-3 or variants thereof in fusion, at either the carboxyl end or the amino end, to NEW43 or variants thereof.
  • the chimeric genes comprising a BVH-3 truncate variant gene and a NEW43 or NEW43 variant gene have been designed following the procedure described in example 1.
  • the polypeptides encoded by these chimeric genes are listed in the table 22.
  • gene fragments to be included in a chimeric gene were amplified by PCR using pairs of oligonucleotides engineered so that the primers had a restriction endonuclease site at the 5′ end, thereby allowing directional in-frame cloning of the amplified product into digested plasmid vectors (Table 23 and Table 24).
  • PCR-amplified products were digested with restriction endonucleases and ligated to linearized plasmid pSL301 vector. The resultant plasmid construct were confirmed by nucleotide sequence analysis.
  • the recombinant pSL301 plasmids containing a PCR product were redigested with the same endonuclease restriction enzyme for the obtention of the DNA inserts.
  • the resulting insert DNA fragments were purified and inserts corresponding to a given chimeric gene were ligated into pURV22-NdeI vector for the generation of a chimeric gene.
  • the expressed recombinant proteins were purified from supernatant fractions obtained from centrifugation of sonicated heat-induced E. coli cultures using multiple chromatographic purification steps.

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Abstract

Streptococcus polypeptides and polynucleotides encoding them are disclosed. Said polypeptides may be useful vaccine components for the prophylaxis or therapy of streptococcus infection in animals. Also disclosed are recombinant methods of producing the protein antigens as well as diagnostic assays for detecting streptococcus bacterial infection.

Description

  • This application claims the benefit of U.S. provisional application 60/212,683 filed Jun. 20, 2000 which is herein incorporated by reference.
    • FIELD OF THE INVENTION
  • The present invention is related to antigens, epitopes and antibodies directed to these epitopes, more particularly polypeptide antigens of streptococcus pneumoniae pathogen which may be useful for prophylaxis, diagnostic or treatment of streptococcal infection.
    • BACKGROUND OF THE INVENTION
  • S. pneumoniae is an important agent of disease in man especially among infants, the elderly and immunocompromised persons. It is a bacterium frequently isolated from patients with invasive diseases such as bacteraemia/septicaemia, pneumonia, meningitis with high morbidity and mortality throughout the world. Even with appropriate antibiotic therapy, pneumococcal infections still result in many deaths. Although the advent of antimicrobial drugs has reduced the overall mortality from pneumococcal disease, 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 otitis media in infants and young children.
  • Efforts to develop a pneumococcal vaccine have generally concentrated on generating immune responses to the pneumococcal capsular polysaccharide. More than 80 pneumococcal capsular serotypes have been identified on the basis of antigenic differences. The currently available pneumococcal vaccine, comprising 23 capsular polysaccharides that most frequently caused disease, has significant shortcomings related primarily to the poor immunogenicity of some capsular polysaccharides, the diversity of the serotypes and the differences in the distribution of serotypes over time, geographic areas and age groups. In particular, the failure of existing vaccines and capsular conjugate vaccines currently in development to protect young children against all serotypes spurres evaluation of other S. pneumoniae components. Although immunogenicity of capsular polysaccharides can be improved, serotype specificity will still represent a major limitation of polysaccharide-based vaccines. The use of a antigenically conserved immunogenic pneumococcal protein antigen, either by itself or in combination with additional components, offers the possibility of a protein-based pneumococcal vaccine.
  • PCT WO 98/18930 published May 7, 1998 entitled “Streptococcus Pneumoniae antigens and vaccines” describes certain polypeptides which are claimed to be antigenic. However, no biological activity of these polypeptides is reported. Similarly, no sequence conservation is reported, which is a necessary species common vaccine candidate.
  • PCT WO 00/39299 describes polypeptides and polynucleotides encoding these polypeptides. PCT WO 00/39299 demonstrates that polypeptides designated as BVH-3 and BVH-11 provide protection against fatal experimental infection with pneumococci.
  • Therefore there remains an unmet need for Streptococcus antigens that may be used as components for the prophylaxis, diagnostic and/or therapy of Streptococcus infection.
    • SUMMARY OF THE INVENTION
  • An isolated polynucleotide comprising a polynucleotide chosen from;
  • (a) a polynucleotide encoding a polypeptide having at least 70% identity to a second polypeptide chosen from: table A, B, D, E or H;
  • (b) a polynucleotide encoding a polypeptide having at least 95% identity to a second polypeptide chosen from: table A, B, D, E or H;
  • (c) a polynucleotide encoding a polypeptide having an amino sequence chosen from table A, B, D, E or H; or fragments, analogs or derivatives thereof;
  • (d) a polynucleotide encoding a polypeptide chosen from: table A, B, D, E or H;
  • (e) a polynucleotide encoding a polypeptide capable of generating antibodies having binding specificity for a polypeptide having a sequence chosen from: table A, B, D, E or H;
  • (f) a polynucleotide encoding an epitope bearing portion of a polypeptide chosen from table A, B, D, E or H; and
  • (g) a polynycleotide complementary to a polynucleotide in (a), (b), (c), (d), (e) or (f).
  • In other aspects, there are provided novel polypeptides encoded by polynucleotides of the invention, pharmaceutical or vaccine composition, vectors comprising polynucleotides of the invention operably linked to an expression control region, as well as host cells transfected with said vectors and methods of producing polypeptides comprising culturing said host cells under conditions suitable for expression.
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 is the DNA sequence of SP64 BVH-3 gene; SEQ ID NO: 1.
  • FIG. 2 is a DNA sequence containing the complete SP64 BVH-3 gene at nucleotides 1777 to 4896; SEQ ID NO: 2.
  • FIG. 3 is the DNA sequence of SP64 BVH-11 gene; SEQ ID NO: 3.
  • FIG. 4 is a DNA sequence containing the complete SP64 BVH-11 gene at nucleotides 45 to 2567; SEQ ID NO: 4.
  • FIG. 5 is a DNA sequence containing the complete SP64 BVH-11-2 gene at nucleotides 114 to 2630; SEQ ID NO: 5.
  • FIG. 6 is the amino acid sequence of SP64 BVH-3 polypeptide; SEQ ID NO: 6.
  • FIG. 7 is the amino acid sequence of SP64 BVH-11 polypeptide; SEQ ID NO: 7.
  • FIG. 8 is the amino acid sequence of SP64 BVH-11-2 polypeptide; SEQ ID NO: 8.
  • FIG. 9 is the DNA sequence of SP63 BVH-3 gene; SEQ ID NO:9.
  • FIG. 10 is the amino acid sequence of SP63 BVH-3 polypeptide; SEQ ID NO: 10.
  • FIG. 11 is the amino acid sequence of 4D4.9 polypeptide; SEQ ID NO: 11.
  • FIG. 12 is the amino acid sequence of 7G11.7 polypeptide; SEQ ID NO: 12.
  • FIG. 13 is the amino acid sequence of 7G11.9 polypeptide; SEQ ID NO: 13.
  • FIG. 14 is the amino acid sequence of 4D3.4 polypeptide; SEQ ID NO: 14.
  • FIG. 15 is the amino acid sequence of 8E3.1 polypeptide; SEQ ID NO: 15.
  • FIG. 16 is the amino acid sequence of 1G2.2 polypeptide; SEQ ID NO: 16.
  • FIG. 17 is the amino acid sequence of 10C12.7 polypeptide; SEQ ID NO: 17.
  • FIG. 18 is the amino acid sequence of 14F6.3 polypeptide; SEQ ID NO: 18.
  • FIG. 19 is the amino acid sequence of B12D8.2 polypeptide; SEQ ID NO: 19.
  • FIG. 20 is the amino acid sequence of 7F4.1 polypeptide; SEQ ID NO: 20.
  • FIG. 21 is the amino acid sequence of 10D7.5 polypeptide; SEQ ID NO: 21.
  • FIG. 22 is the amino acid sequence of 10G9.3 polypeptide, 10A2.2 polypeptide and B11B8.1 polypeptide; SEQ ID NO: 22.
  • FIG. 23 is the amino acid sequence of 11B8.4 polypeptide; SEQ ID NO: 23.
  • FIG. 24 is the amino acid sequence of Mab H11B-11B8 target epitope; SEQ ID 163.
  • FIG. 25 is a schematic representation of the BVH-3 gene as well as location of gene sequences coding for the full length and truncated polypeptides. The relationships between DNA fragments are shown with respect to each other.
  • FIG. 26 is a schematic representation of the BVH-11 gene as well as location of gene sequences coding for the full length and truncated polypeptides. The relationships between DNA fragments are shown with respect to each other.
  • FIG. 27 is a schematic representation of the BVH-11-2 gene as well as location of gene sequences coding for the full length and truncated polypeptides. The relationships between DNA fragments are shown with respect to each other.
  • FIG. 28 is a schematic representation of the BVH-3 protein and the location of internal and surface epitopes recognized by certain monoclonal antibodies.
  • FIG. 29 is a schematic representation of the BVH-11-2 protein and the location of protective surface epitopes recognized by certain monoclonal antibodies.
  • FIG. 30 is a map of plasmid pURV22.HIS. KanR, kanamycin-resistance coding region; cI857, bacteriophage λ cI857 temperature-sensitive repressor gene; lambda pL, bacteriophage λ transcription promotor; His-tag, 6-histidine coding region; terminator, T1 transcription terminator; ori, colE1 origin of replication.
  • FIG. 31 depicts the comparison of the amino acid sequences of BVH-3M (sp64) and BVH-3 (Sp63) proteins by using the program Clustal W from MacVector sequence analysis software (version 6.5.3). Underneath the alignment, there is a consensus line where * and . characters indicate identical and similar amino acid residues, respectively.
  • FIG. 32 depicts the comparison of the amino acid sequences of BVH-3, BVH-11 and BVH-11-2 proteins by using the program Clustal W from MacVector sequence analysis software (version 6.5.3). Underneath the alignment, there is a consensus line where * and . characters indicate identical and similar amino acid residues, respectively.
  • FIG. 33 is the DNA sequence of the NEW43 gene (SEQ ID No 257).
  • FIG. 34 is the deduced amino acid sequence of NEW43 polypeptide (SEQ ID No 258).
    • DETAILED DESCRIPTION OF THE INVENTION
  • It was determined that portions of the BVH-3 and BVH-11 polypeptides were internal. Other portions were not present in important strains such as encapsulated s. pneumonia causing disease strains. It would be advantageous to have a polypeptide that comprises a portion that is not internal. When large portions of a polypeptide are internal, these portions are not exposed on the bacteria. However, these portions can be very immunogenic in a recombinant polypeptide and will not confer protection against infections. It would also be advantageous to have a polypeptide that comprises a portion that is present in most strains.
  • The present invention is concerned with polypeptides in which undesired portions have been deleted and/or modified in order to obtain a specific immune response.
  • In accordance with the present invention, there are also provided polypeptides or polynucleotides encoding such polypeptides comprising protective domains.
  • Surprisingly, when the undesired portion of the polypeptides are deleted or modified, the polypeptides have desired biological properties. This is surprising in view of the fact that some of these portions were described as being epitope bearing portion in the patent application PCT WO 98/18930. In other publications such as PCT WO 00/37105, portions identified as histidine triad and coil coiled regions were said to be of importance. The present inventors have found that variants of the polypeptide BVH-3 and BVH-11 in which certain portions were deleted and/or modified and chimeras of these polypeptides have biological properties and generate a specific immune response.
  • According to one aspect, the present invention provides an isolated polynucleotide encoding a polypeptide having at least 70% identity to a second polypeptide comprising a sequence as disclosed in the present application, the tables and figures.
  • In accordance with one aspect of the present invention, there is provided an isolated polynucleotide comprising a polynucleotide chosen from;
  • (a) a polynucleotide encoding a polypeptide having at least 70% identity to a second polypeptide chosen from: table B, E or H;
  • (b) a polynucleotide encoding a polypeptide having at least 95% identity to a second polypeptide chosen from: table B, E or H;
  • (c) a polynucleotide encoding a polypeptide having an amino sequence chosen from table B, E or H or fragments, analogs or derivatives thereof;
  • (d) a polynucleotide encoding a polypeptide chosen from: table B, E or H;
  • (e) a polynucleotide encoding a polypeptide capable of generating antibodies having binding specificity for a polypeptide having a sequence chosen from: table B, E or H,
  • (f) a polynucleotide encoding an epitope bearing portion of a polypeptide chosen from table B, E or H; and
  • (g) a polynycleotide complementary to a polynucleotide in (a), (b), (c), (d), (e) or (f).
  • According to one aspect, the present invention provides an isolated polynucleotide encoding a polypeptide having at least 70% identity to a second polypeptide comprising a sequence chosen from table A, B, D, E, G or H or fragments, analogues or derivatives thereof.
  • According to one aspect, the present invention provides an isolated polynucleotide encoding a polypeptide having at least 95% identity to a second polypeptide comprising a sequence chosen from table A, B, D, E, G or H or fragments, analogues or derivatives thereof.
  • According to one aspect, the present invention relates to polypeptides characterised by the amino acid sequence chosen from table A, B, D, E, G or H or fragments, analogues or derivatives thereof.
  • According to one aspect, the present invention provides an isolated polynucleotide encoding a polypeptide having at least 70% identity to a second polypeptide comprising a sequence chosen from table A, B, D, E, G or H.
  • According to one aspect, the present invention provides an isolated polynucleotide encoding a polypeptide having at least 95% identity to a second polypeptide comprising a sequence chosen from table A, B, D, E, G or H.
  • According to one aspect, the present invention relates to polypeptides characterised by the amino acid sequence chosen from table A, B, D, E, G or H.
  • According to one aspect, the present invention provides an isolated polynucleotide encoding a polypeptide having at least 70% identity to a second polypeptide comprising a sequence chosen from table B, E or H or fragments, analogues or derivatives thereof.
  • According to one aspect, the present invention provides an isolated polynucleotide encoding a polypeptide having at least 95% identity to a second polypeptide comprising a sequence chosen from B, E or H or fragments, analogues or derivatives thereof.
  • According to one aspect, the present invention relates to polypeptides characterised by the amino acid sequence chosen from table B, E or H or fragments, analogues or derivatives thereof.
  • According to one aspect, the present invention provides an isolated polynucleotide encoding a polypeptide having at least 70% identity to a second polypeptide comprising a sequence chosen from table B, E or H.
  • According to one aspect, the present invention provides an isolated polynucleotide encoding a polypeptide having at least 95% identity to a second polypeptide comprising a sequence chosen from B, E or H.
  • According to one aspect, the present invention relates to polypeptides characterised by the amino acid sequence chosen from table B, E or H.
  • In accordance with the present invention, all nucleotides encoding polypeptides and chimeric polypeptides are within the scope of the present invention.
  • In a further embodiment, the polypeptides or chimeric polypeptides in accordance with the present invention are antigenic.
  • In a further embodiment, the polypeptides or chimeric polypeptides in accordance with the present invention are immunogenic.
  • In a further embodiment, the polypeptides or chimeric polypeptides in accordance with the present invention can elicit an immune response in an individual.
  • In a further embodiment, the present invention also relates to polypeptides which are able to raise antibodies having binding specificity to the polypeptides or chimeric polypeptides of the present invention as defined above.
  • In one embodiment, the polypeptides of table A (BVH-3) or table D (BVH-11) comprise at least one epitope bearing portion.
  • In a further embodiment, the fragments of the polypeptides of the present invention will comprise one or more epitope bearing portion identified in Table C and F. The fragment will comprises at least 15 contiguous amino acid of the polypeptide of table C and F. The fragment will comprises at least 20 contiguous amino acid of the polypeptide of table C and F.
  • In a further embodiment, the epitope bearing portion of the polypeptide of table A(BVH-3) comprises at least one polypeptide listed in Table C.
  • In a further embodiment, the epitope bearing portion of the polypeptide of table B(BVH-11) comprises at least one polypeptide listed in Table F.
  • An antibody that “has binding specificity” is an antibody that recognises and binds the selected polypeptide but which does not substantially recognise and bind other molecules in a sample, such as a biological sample. Specific binding can be measured using an ELISA assay in which the selected polypeptide is used as an antigen.
  • Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
  • In accordance with the present invention, “protection” in the biological studies is defined by a significant increase in the survival curve, rate or period. Statistical analysis using the Log rank test to compare survival curves, and Fisher exact test to compare survival rates and numbers of days to death, respectively, might be useful to calculate P values and determine whether the difference between the two groups is statistically significant. P values of 0.05 are regarded as not significant.
  • As used herein, “fragments”, “derivatives” or “analogues” of the polypeptides of the invention include those polypeptides in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably conserved) and which may be natural or unnatural. In one embodiment, derivatives and analogues of polypeptides of the invention will have about 70% identity with those sequences illustrated in the figures or fragments thereof. That is, 70% of the residues are the same. In a further embodiment, polypeptides will have greater than 75% homology. In a further embodiment, polypeptides will have greater than 80% homology. In a further embodiment, polypeptides will have greater than 85% homology. In a further embodiment, polypeptides will have greater than 90% homology. In a further embodiment, polypeptides will have greater than 95% homology. In a further embodiment, polypeptides will have greater than 99% homology. In a further embodiment, derivatives and analogues of polypeptides of the invention will have less than about 20 amino acid residue substitutions, modifications or deletions and more preferably less than 10. Preferred substitutions are those known in the art as conserved i.e. the substituted residues share physical or chemical properties such as hydrophobicity, size, charge or functional groups.
  • The skilled person will appreciate that analogues or derivatives of the proteins or polypeptides of the invention will also find use in the context of the present invention, i.e. as antigenic/immunogenic material. Thus, for instance proteins or polypeptides which include one or more additions, deletions, substitutions or the like are encompassed by the present invention. In addition, it may be possible to replace one amino acid with another of similar “type”. For instance replacing one hydrophobic amino acid with another hydrophilic amino acid.
  • One can use a program such as the CLUSTAL program to compare amino acid sequences. This program compares amino acid sequences and finds the optimal alignment by inserting spaces in either sequence as appropriate. It is possible to calculate amino acid identity or similarity (identity plus conservation of amino acid type) for an optimal alignment. A program like BLASTX will align the longest stretch of similar sequences and assign a value to the fit. It is thus possible to obtain a comparison where several regions of similarity are found, each having a different score. Both types of identity analysis are contemplated in the present invention.
  • In an alternative approach, the analogues or derivatives could be fusion proteins, incorporating moieties which render purification easier, for example by effectively tagging the desired protein or polypeptide, It may be necessary to remove the “tag” or it may be the case that the fusion protein itself retains sufficient antigenicity to be useful.
  • In an additional aspect of the invention there are provided antigenic/immunogenic fragments of the proteins or polypeptides of the invention, or of analogues or derivatives thereof.
  • The fragments of the present invention should include one or more such epitopic regions or be sufficiently similar to such regions to retain their antigenic/immunogenic properties. Thus, for fragments according to the present invention the degree of identity is perhaps irrelevant, since they may be 100% identical to a particular part of a protein or polypeptide, analogue or derivative as described herein. The key issue, once again, is that the fragment retains the antigenic/immunogenic properties.
  • Thus, what is important for analogues, derivatives and fragments is that they possess at least a degree of the antigenicity/immunogenic of the protein or polypeptide from which they are derived.
  • In accordance with the present invention, polypeptides of the invention include both polypeptides and chimeric polypeptides.
  • Also included are polypeptides which have fused thereto other compounds which alter the polypeptides biological or pharmacological properties i.e. polyethylene glycol (PEG) to increase half-life; leader or secretory amino acid sequences for ease of purification; prepro- and pro- sequences; and (poly)saccharides.
  • Furthermore, in those situations where amino acid regions are found to be polymorphic, it may be desirable to vary one or more particular amino acids to more effectively mimic the different epitopes of the different streptococcus strains.
  • Moreover, the polypeptides of the present invention can be modified by terminal —NH2 acylation (e.g. by acetylation, or thioglycolic acid amidation, terminal carboxy amidation, e.g. with ammonia or methylamine) to provide stability, increased hydrophobicity for linking or binding to a support or other molecule.
  • Also contemplated are hetero and homo polypeptide multimers of the polypeptide fragments, analogues and derivatives. These polymeric forms include, for example, one or more polypeptides that have been cross-linked with cross-linkers such as avidin/biotin, gluteraldehyde or dimethylsuperimidate. Such polymeric forms also include polypeptides containing two or more tandem or inverted contiguous sequences, produced from multicistronic mRNAs generated by recombinant DNA technology.
  • Preferably, a fragment, analogue or derivative of a polypeptide of the invention will comprise at least one antigenic region i.e. at least one epitope.
  • In order to achieve the formation of antigenic polymers (i.e. synthetic multimers), polypeptides may be utilised having bishaloacetyl groups, nitroarylhalides, or the like, where the reagents being specific for thio groups. Therefore, the link between two mercapto groups of the different peptides may be a single bond or may be composed of a linking group of at least two, typically at least four, and not more than 16, but usually not more than about 14 carbon atoms.
  • In a particular embodiment, polypeptide fragments, analogues and derivatives of the invention do not contain a methionine (Met) starting residue. Preferably, polypeptides will not incorporate a leader or secretory sequence (signal sequence) . The signal portion of a polypeptide of the invention may be determined according to established molecular biological techniques. In general, the polypeptide of interest may be isolated from a streptococcus culture and subsequently sequenced to determine the initial residue of the mature protein and therefore the sequence of the mature polypeptide.
  • According to another aspect, there are provided vaccine compositions comprising one or more streptococcus polypeptides of the invention in admixture with a pharmaceutically acceptable carrier diluent or adjuvant. Suitable adjuvants include oils i.e. Freund's complete or incomplete adjuvant; salts i.e. AlK(SO4)2, AlNa(SO4)2, AlNH4(SO4)2, silica, kaolin, carbon polynucleotides i.e. poly IC and poly AU. Preferred adjuvants include QuilA and Alhydrogel. Vaccines of the invention may be administered parenterally by injection, rapid infusion, nasopharyngeal absorption, dermoabsorption, or bucal or oral. Pharmaceutically acceptable carriers also include tetanus toxoid.
  • The term vaccine is also meant to include antibodies. In accordance with the present invention, there is also provided the use of one or more antibodies having binding specificity for the polypeptides of the present invention for the treatment or prophylaxis of streptococcus infection and/or diseases and symptoms mediated by streptococcus infection.
  • Vaccine compositions of the invention are used for the treatment or prophylaxis of streptococcus infection and/or diseases and symptoms mediated by streptococcus infection as described in P. R. Murray (Ed, in chief), E. J. Baron, M. A. Pfaller, F. C. Tenover and R. H. Yolken. Manual of Clinical Microbiology, ASM Press, Washington, D.C. sixth edition, 1995, 1482p which are herein incorporated by reference. In one embodiment, vaccine compositions of the present invention are used for the treatment or prophylaxis of meningitis, otitis media, bacteremia or pneumonia. In one embodiment, vaccine compositions of the invention are used for the treatment or prophylaxis of streptococcus infection and/or diseases and symptoms mediated by streptococcus infection, in particular S. pneumoniae, group A streptococcus (pyogenes), group B streptococcus (GBS or agalactiae), dysgalactiae, uberis, nocardia as well as Staphylococcus aureus. In a further embodiment, the streptococcus infection is S. pneumoniae.
  • In a particular embodiment, vaccines are administered to those individuals at risk of streptococcus infection such as infants, elderly and immunocompromised individuals.
  • As used in the present application, the term “individuals” include mammals. In a further embodiment, the mammal is human.
  • Vaccine compositions are preferably in unit dosage form of about 0.001 to 100 μg/kg (antigen/body weight) and more preferably 0.01 to 10 μg/kg and most preferably 0.1 to 1 μg/kg 1 to 3 times with an interval of about 1 to 6 week intervals between immunizations.
  • Vaccine compositions are preferably in unit dosage form of about 0.1 μg to 10 mg and more preferably 1 μg to 1 mg and most preferably 10 to 100 μg 1 to 3 times with an interval of about 1 to 6 week intervals between immunizations.
  • According to another aspect, there are provided polynucleotides encoding polypeptides characterised by the amino acid sequence chosen from table A, B, D, E, G or H or fragments, analogues or derivatives thereof.
  • According to another aspect, there are provided polynucleotides encoding polypeptides characterised by the amino acid sequence chosen from table B, E or H or fragments, analogues or derivatives thereof.
  • In one embodiment, polynucleotides are those illustrated in table A, B, D, E, G or H which encodes polypeptides of the invention.
  • In one embodiment, polynucleotides are those illustrated in table B, E or H which encodes polypeptides of the invention.
  • It will be appreciated that the polynucleotide sequences illustrated in the figures may be altered with degenerate codons yet still encode the polypeptides of the invention. Accordingly the present invention further provides polynucleotides which hybridise to the polynucleotide sequences herein above described (or the complement sequences thereof) having 50% identity between sequences. In one embodiment, at least 70% identity between sequences. In one embodiment, at least 75% identity between sequences. In one embodiment, at least 80% identity between sequences. In one embodiment, at least 85% identity between sequences. In one embodiment, at least 90% identity between sequences. In a further embodiment, polynucleotides are hybridizable under stringent conditions i.e. having at least 95% identity. In a further embodiment, more than 97% identity.
  • Suitable stringent conditions for hybridation can be readily determined by one of skilled in the art (see for example Sambrook et al., (1989) Molecular cloning: A Laboratory Manual, 2nd ed, Cold Spring Harbor, N.Y.; Current Protocols in Molecular Biology, (1999) Edited by Ausubel F. M. et al., John Wiley & Sons, Inc., N.Y.).
  • In a further embodiment, the present invention provides polynucleotides that hybridise under stringent conditions to either
      • (a) a DNA sequence encoding a polypeptide or
      • (b) the complement of a DNA sequence encoding a polypeptide;
        wherein said polypeptide comprising a sequence chosen from table A, B, D, E, G or H or fragments or analogues thereof.
  • In a further embodiment, the present invention provides polynucleotides that hybridise under stringent conditions to either
      • (c) a DNA sequence encoding a polypeptide or
      • (d) the complement of a DNA sequence encoding a polypeptide;
        wherein said polypeptide comprising a sequence chosen from table B, E or H or fragments or analogues thereof.
  • In a further embodiment, the present invention provides polynucleotides that hybridise under stringent conditions to either
      • (a) a DNA sequence encoding a polypeptide or
      • (b) the complement of a DNA sequence encoding a polypeptide;
        wherein said polypeptide comprises at least 10 contiguous amino acid residues from a polypeptide comprising a sequence chosen from table A, B, D, E, G or H or fragments or analogues thereof.
  • In a further embodiment, the present invention provides polynucleotides that hybridise under stringent conditions to either
      • (c) a DNA sequence encoding a polypeptide or
      • (d) the complement of a DNA sequence encoding a polypeptide;
        wherein said polypeptide comprises at least 10 contiguous amino acid residues from a polypeptide comprising a sequence chosen from table B, E or H or fragments or analogues thereof.
  • In a further embodiment, polynucleotides are those encoding polypeptides of the invention illustrated in table A, B, D, E, G or H.
  • As will be readily appreciated by one skilled in the art, polynucleotides include both DNA and RNA.
  • The present invention also includes polynucleotides complementary to the polynucleotides described in the present application.
  • In a further aspect, polynucleotides encoding polypeptides of the invention, or fragments, analogues or derivatives thereof, may be used in a DNA immunization method. That is, they can be incorporated into a vector which is replicable and expressible upon injection thereby producing the antigenic polypeptide in vivo. For example polynucleotides may be incorporated into a plasmid vector under the control of the CMV promoter which is functional in eukaryotic cells. Preferably the vector is injected intramuscularly.
  • According to another aspect, there is provided a process for producing polypeptides of the invention by recombinant techniques by expressing a polynucleotide encoding said polypeptide in a host cell and recovering the expressed polypeptide product. Alternatively, the polypeptides can be produced according to established synthetic chemical techniques i.e. solution phase or solid phase synthesis of oligopeptides which are ligated to produce the full polypeptide (block ligation).
  • General methods for obtention and evaluation of polynucleotides and polypeptides are described in the following references: Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd ed, Cold Spring Harbor, N.Y., 1989; Current Protocols in Molecular Biology, Edited by Ausubel F. M. et al., John Wiley and Sons, Inc. New York; PCR Cloning Protocols, from Molecular Cloning to Genetic Engineering, Edited by White B. A., Humana Press, Totowa, N.J., 1997, 490 pages; Protein Purification, Principles and Practices, Scopes R. K., Springer-Verlag, New York, 3rd Edition, 1993, 380 pages; Current Protocols in Immunology, Edited by Coligan J. E. et al., John Wiley & Sons Inc., New York which are herein incorporated by reference.
  • For recombinant production, host cells are transfected with vectors which encode the polypeptide, and then cultured in a nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the genes. Suitable vectors are those that are viable and replicable in the chosen host and include chromosomal, non-chromosomal and synthetic DNA sequences e.g. bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA. The polypeptide sequence may be incorporated in the vector at the appropriate site using restriction enzymes such that it is operably linked to an expression control region comprising a promoter, ribosome binding site (consensus region or Shine-Dalgarno sequence), and optionally an operator (control element). One can select individual components of the expression control region that are appropriate for a given host and vector according to established molecular biology principles (Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd ed, Cold Spring Harbor, N.Y., 1989; Current Protocols in Molecular Biology, Edited by Ausubel F. M. et al., John Wiley and Sons, Inc. New York incorporated herein by reference). Suitable promoters include but are not limited to LTR or SV40 promoter, E. coli lac, tac or trp promoters and the phage lambda PL promoter. Vectors will preferably incorporate an origin of replication as well as selection markers i.e. ampicilin resistance gene. Suitable bacterial vectors include pET, pQE70, pQE60, pQE-9, pbs, pD10 phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a, pNH18A, pNH46A, ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 and eukaryotic vectors pBlueBacIII, pWLNEO, pSV2CAT, pOG44, pXT1, pSG, pSVK3, pBPV, pMSG and pSVL. Host cells may be bacterial i.e. E. coli, Bacillus subtilis, Streptomyces; fungal i.e. Aspergillus niger, Aspergillus nidulins; yeast i.e. Saccharomyces or eukaryotic i.e. CHO, COS.
  • Upon expression of the polypeptide in culture, cells are typically harvested by centrifugation then disrupted by physical or chemical means (if the expressed polypeptide is not secreted into the media) and the resulting crude extract retained to isolate the polypeptide of interest. Purification of the polypeptide from culture media or lysate may be achieved by established techniques depending on the properties of the polypeptide i.e. using ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography and lectin chromatography. Final purification may be achieved using HPLC.
  • The polypeptide may be expressed with or without a leader or secretion sequence. In the former case the leader may be removed using post-translational processing (see U.S. Pat. No. 4,431,739; U.S. Pat. No. 4,425,437; and U.S. Pat. No. 4,338,397 incorporated herein by reference) or be chemically removed subsequent to purifying the expressed polypeptide.
  • According to a further aspect, the streptococcus polypeptides of the invention may be used in a diagnostic test for streptococcus infection, in particular S. pneumoniae infection. Several diagnostic methods are possible, for example detecting streptococcus organism in a biological sample, the following procedure may be followed:
  • a) obtaining a biological sample from a patient;
  • b) incubating an antibody or fragment thereof reactive with a streptococcus polypeptide of the invention with the biological sample to form a mixture; and
  • c) detecting specifically bound antibody or bound fragment in the mixture which indicates the presence of streptococcus.
  • Alternatively, a method for the detection of antibody specific to a streptococcus antigen in a biological sample containing or suspected of containing said antibody may be performed as follows:
  • a) obtaining a biological sample from a patient;
  • b) incubating one or more streptococcus polypeptides of the invention or fragments thereof with the biological sample to form a mixture; and
  • c) detecting specifically bound antigen or bound fragment in the mixture which indicates the presence of antibody specific to streptococcus.
  • One of skill in the art will recognize that this diagnostic test may take several forms, including an immunological test such as an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay or a latex agglutination assay, essentially to determine whether antibodies specific for the polypeptide are present in an organism.
  • The DNA sequences encoding polypeptides of the invention may also be used to design DNA probes for use in detecting the presence of streptococcus in a biological sample suspected of containing such bacteria. The detection method of this invention comprises:
  • a) obtaining the biological sample from a patient;
  • b) incubating one or more DNA probes having a DNA sequence encoding a polypeptide of the invention or fragments thereof with the biological sample to form a mixture; and
  • c) detecting specifically bound DNA probe in the mixture which indicates the presence of streptococcus bacteria.
  • The DNA probes of this invention may also be used for detecting circulating streptococcus i.e. S. pneumoniae nucleic acids in a sample, for example using a polymerase chain reaction, as a method of diagnosing streptococcus infections. The probe may be synthesized using conventional techniques and may be immobilized on a solid phase, or may be labelled 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 the streptococcus pneumoniae polypeptides of the invention.
  • Another diagnostic method for the detection of streptococcus in a patient comprises:
  • a) labelling an antibody reactive with a polypeptide of the invention or fragment thereof with a detectable label;
  • b) administering the labelled antibody or labelled fragment to the patient; and
  • c) detecting specifically bound labelled antibody or labelled fragment in the patient which indicates the presence of streptococcus.
  • A further aspect of the invention is the use of the streptococcus polypeptides of the invention as immunogens for the production of specific antibodies for the diagnosis and in particular the treatment of streptococcus infection. Suitable antibodies may be determined using appropriate screening methods, for example by measuring the ability of a particular antibody to passively protect against streptococcus infection in a test model. One example of an animal model is the mouse model described in the examples herein. The antibody may be a whole antibody or an antigen-binding fragment thereof and may belong to any immunoglobulin class. The antibody or fragment may be of animal origin, specifically of mammalian origin and more specifically of murine, rat or human origin. It may be a natural antibody or a fragment thereof, or if desired, a recombinant antibody or antibody fragment. The term recombinant antibody or antibody fragment means antibody or antibody fragment which was produced using molecular biology techniques. The antibody or antibody fragments may be polyclonal, or preferably monoclonal. It may be specific for a number of epitopes associated with the streptococcus pneumoniae polypeptides but is preferably specific for one.
  • A further aspect of the invention is the use of the antibodies directed to the streptococcus polypeptides of the invention for passive immunization. One could use the antibodies described in the present application. Suitable antibodies may be determined using appropriate screening methods, for example by measuring the ability of a particular antibody to passively protect against streptococcus infection in a test model. One example of an animal model is the mouse model described in the examples herein. The antibody may be a whole antibody or an antigen-binding fragment thereof and may belong to any immunoglobulin class. The antibody or fragment may be of animal origin, specifically of mammalian origin and more specifically of murine, rat or human origin. It may be a natural antibody or a fragment thereof, or if desired, a recombinant antibody or antibody fragment. The term recombinant antibody or antibody fragment means antibody or antibody fragment which was produced using molecular biology techniques. The antibody or antibody fragments may be polyclonal, or preferably monoclonal. It may be specific for a number of epitopes associated with the streptococcus pneumoniae polypeptides but is preferably specific for one.
  • The following are reference tables summarizing the sequences disclosed in the present application:
  • TABLE A, B and C Variants and Epitope of BVH-3
    TABLE A
    Family Polypeptide SEQ ID NO
    BVH-3
    New 21 aa 396-1039 of SEQ ID. 6
    New 25 aa 233-1039 of SEQ ID. 6
    New 40 aa 408-1039 of SEQ ID. 6
  • TABLE B
    Family Polypeptide SEQ ID NO
    BVH-3
    NEW1-mut1** 255
    NEW35A 256
    NEW42 349
    NEW49 350
    NEW50 351
    NEW51 352
    NEW52 353
    NEW53 354
    NEW54 355
    NEW55 356
    NEW56 357
    NEW56-mut2** 358
    NEW56-mut3** 359
    NEW57 360
    NEW63 361
    NEW64 362
    NEW65 363
    NEW66 364
    NEW76 365
    NEW105 366
    NEW106 367
    NEW107 368

    **silent mutation, i.e. the polypeptide is the same as New1 or New 56
  • TABLE C
    Epitopes of BVH-3
    7G11.7 12
    7G11.9 13
    B12D8.2 19
    7F4.1 20
    14F6.3 18
    4D3.4 14
    10C12.7 17
    8E3.1 15
    1G2.2 16
  • TABLE D, E and F Variants and Epitope of BVH-11
    TABLE D
    Family Polypeptide SEQ ID NO
    BVH-11
    New19 aa 497-838 of Seq. ID 8
    New24 aa 227-838 of Seq. ID 8
  • TABLE E
    Family Polypeptide SEQ ID NO
    BVH-11
    New 43 258
    NEW60 293
    NEW61 294
    NEW62 295
    NEW80 296
    NEW81 297
    NEW82 298
    NEW83 299
    NEW84 300
    NEW85 301
    NEW88D1 302
    NEW88D2 303
    NEW88 304
  • TABLE F
    epitopes of BVH-11
    10D7.5 21
    10G9.3 22
    B11B8.1 22
    10A2.2 22
    11b8.4 23
    3A4.1 24
  • TABLE G and H Chimeras
    TABLE G
    Family Polypeptide SEQ ID NO
    Chimeras with BVH-11
    and BVH-3
    New17 M*-NEW5-G*P*-NEW1 (376)
    New20 M*-NEW1-G*P*-NEW5 (377)
    New26 M*-NEW10-G*P*-NEW25 (378)
    New27 M*-NEW19-G*P*-NEW25 (379)
    New28 M*-NEW10-G*P*-NEW1 (380)
    New29 M*-NEW5-G*P*-NEW25 (381)
    New30 M*-NEW4-G*P*-NEW25 (382)
    New31 M*-NEW4-G*P*-NEW1 (383)
    NEW32 M*-NE19-G*P*-NEW1 (384)

    *OPTIONAL AMINO ACID
  • TABLE H
    Family Polypeptide SEQ ID NO.
    Chimeras with BVH-
    11 and BVH-3
    VP 89 369
    VP 90 370
    VP 91 371
    VP 92 372
    VP 93 373
    VP 94 332
    VP 108 333
    VP 109 334
    VP 110 335
    VP 111 336
    VP 112 337
    VP 113 338
    VP 114 339
    VP 115 340
    VP 116 341
    VP 117 342
    VP 119 343
    VP 120 344
    VP 121 345
    VP 122 346
    VP 123 347
    VP 124 348
    • EXAMPLE 1
  • This example describes the bacterial strains, plasmids, PCR primers, recombinant proteins and hybridoma antibodies used herein.
  • S. pneumoniae SP64 (serogroup 6) and SP63 (serogroup 9) clinical isolates were provided by the Laboratoire de la Santé Publique du Québec, Sainte-Anne-de-Bellevue; Rx1 strain, a nonencapsulated derivative of the type 2 strain D39 and the type 3 strain WU2 were provided by David E. Briles from University of Alabama, Birmingham and the type 3 clinical isolate P4241 was provided by the Centre de Recherche en Infectiologie du Centre Hospitalier de l'Universite Laval, Sainte-Foy. E. coli strains DH5α (Gibco BRL, Gaithesburg, Md.); AD494 (λDE3) (Novagen, Madison, Wis.) and BL21 (λDE3) (Novagen) as well as plasmid superlinker pSL301 vector (Invitrogen, San Diego, Calif.); PCMV-GH vector (gift from Dr. Stephen A. Johnston, Department for Biochemistry, University of Texas, Dallas, Tex.); pET32 and pET21 (Novagen) and pURV22.HIS expression vectors (FIG. 30) were used in this study. The pURV22.HIS vector contains a cassette of the bacteriophage λ cI857 temperature-sensitive repressor gene from which the functional PR promoter has been deleted. The inactivation of the cI857 repressor by a temperature increase from the range of 30-37° C. to 37-42° C. results in the induction of the gene under the control of promoter λPL. The PCR primers used for the generation of the recombinant plasmids had a restriction endonuclease site at the 5′end, thereby allowing directional cloning of the amplified product into the digested plasmid vector. The PCR oligonucleotide primers used are listed in the following Table 1. The location of the gene sequences coding for BVH-3, BVH-11 and BVH-11-2 gene products is summarized in the FIG. 25, FIG. 26 and FIG. 27, respectively.
    TABLE 1
    List of PCR oligonucleotide primers
    SEQ
    ID Nucleotide Restriction
    Primer NO Sequence 5′ - 3′ position sites
    OCRR 25 cagtagatctgtgcctatgcact SEQ ID 1: BglII
    479 aaac 61-78
    SEQ ID 9:
    1-18
    OCRR 26 gatctctagactactgctattcc SEQ ID 2: XbaI
    480 ttacgctatg 4909-4887
    SEQ ID 9:
    2528-2519
    OCRR 27 atcactcgagcattacctggata SEQ ID 1: XhoI
    497 atcctgt 1525-1506
    OCRR 28 ctgctaagcttatgaaagattta SEQ ID 1: HindIII
    498 gat 1534-1548
    OCRR 29 gatactcgagctgctattccttac SEQ ID 2: XhoI
    499 4906-4893
    HAMJ 30 gaatctcgagttaagctgctgct SEQ ID 1: XhoI
    172 aattc 675-661
    HAMJ 31 gacgctcgagcgctatgaaatca SEQ ID 1: XhoI
    247 gataaattc 3117-3096
    HAMJ 32 gacgctcgagggcattacctgga SEQ ID 1: XhoI
    248 taatcctgttcatg 1527-1501
    HAMJ 33 cagtagatctcttcatcatttat SEQ ID 2: BglII
    249 tgaaaagagg 1749-1771
    HAMJ 34 ttatttcttccatatggacttga SEQ ID 1: NdeI
    278 cagaagagcaaattaag 1414-1437
    HAMJ 35 cgccaagcttcgctatgaaatca SEQ ID 1: HindIII
    279 gataaattc 3117-3096
    HAMJ 36 cgccaagcttttccacaatataa SEQ ID 1: HindIII
    280 gtcgattgatt 2400-2377
    HAMJ 37 ttatttcttccatatggaagtac SEQ ID 1: NdeI
    281 ctatcttggaaaaagaa 2398-2421
    HAMJ 38 ttatttcttccatatggtgccta SEQ ID 1: NdeI
    300 tgcactaaaccagc 62-82
    HAMJ 39 ataagaatgcggccgcttccaca SEQ ID 1: NotI
    313 atataagtcgattgatt 2400-2377
    OCRR 40 cagtagatctgtgcttatgaact SEQ ID 3: BglII
    487 aggtttgc 58-79
    OCRR 41 gatcaagcttgctgctaccttta SEQ ID 4: HindII
    488 cttactctc 2577-2556
    HAMJ 42 ctgagatatccgttatcgttcaa SEQ ID 3: EcoRV
    171 acc 1060-1075
    HAMJ 43 ctgcaagcttttaaaggggaata SEQ ID 3: HindIII
    251 atacg 1059-1045
    HAMJ 44 cagtagatctgcagaagccttcc SEQ ID 3: BglII
    264 tatctg 682-700
    HAMJ 45 tcgccaagcttcgttatcgttca SEQ ID 3: HindIII
    282 aaccattggg 1060-1081
    HAMJ 46 ataagaatgcggccgccttactc SEQ ID 3: NotI
    283 tcctttaataaagccaatagtt 2520-2492
    HAMJ 47 catgccatggacattgatagtct SEQ ID 3: NcoI
    284 cttgaaacagc 856-880
    HAMJ 48 cgccaagcttcttactctccttt SEQ ID 3: HindIII
    285 aataaagccaatag 2520-2494
    HAMJ 49 cgacaagcttaacatggtcgcta SEQ ID 3: HindIII
    286 gcgttacc 2139-2119
    SEQ ID 5:
    2210-2190
    HAMJ 50 cataccatgggcctttatgaggc SEQ ID 3: NcoI
    287 acctaag 2014-2034
    HAMJ 51 cgacaagcttaagtaaatcttca SEQ ID 3: HindIII
    288 gcctctctcag 2376-2353
    HAMJ 52 gataccatggctagcgaccatgt SEQ ID 3: NcoI
    289 tcaaagaa 2125-2146
    HAMJ 53 cgccaagcttatcatccactaac SEQ ID 3: HindIII
    290 ttgactttatcac 1533-1508
    HAMJ 54 cataccatggatattcttgcctt SEQ ID 3: NcoI
    291 cttagctccg 1531-1554
    HAMJ 55 catgccatggtgcttatgaacta SEQ ID 3: NcoI
    301 ggtttgc 59-79
    HAMJ 56 cgccaagctttagcgttaccaaa SEQ ID 3: HindIII
    302 accattatc 2128-2107
    HAMJ 57 gtattagatctgttcctatgaac SEQ ID 5: BglII
    160 ttggtcgtcacca 172-196
    HAMJ 58 cgcctctagactactgtatagga SEQ ID 5: XbaI
    186 gccgg 2613-2630
    HAMJ 59 catgccatggaaaacatttcaag SEQ ID 5: NcoI
    292 ccttttacgtg 925-948
    HAMJ 60 cgacaagcttctgtataggagcc SEQ ID 5: HindIII
    293 ggttgactttc 2627-2604
    HAMJ 61 catgccatggttcgtaaaaataa SEQ ID 5: NcoI
    294 ggcagaccaag 2209-2232
    HAMJ 62 catgccatggaagcctattggaa SEQ ID 5: NcoI
    297 tgggaag 793-812
    HAMJ 63 catgccatggaagcctattggaa SEQ ID 5: NcoI
    352 tgggaagc 793-813
    HAMJ 64 cgccaagcttgtaggtaatttgc SEQ ID 5: HindIII
    353 gcatttgg 1673-1653
    HAMJ 65 cgccaagcttctgtataggagcc SEQ ID 5: HindIII
    354 ggttgac 2627-2608
    HAMJ 66 catgccatggatattcttgcctt SEQ ID 5: NcoI
    355 cttagctcc 1603-1624
    HAMJ 67 ttatttcttccatatgcatggtg SEQ ID 1: NdeI
    404 atcatttccattaca 1186-1207
    HAMJ 68 gatgcatatgaatatgcaaccga SEQ ID 1: NdeI
    464 gtcagttaagc 697-720
    HAMJ 69 gatgctcgagagcatcaaatccg SEQ ID 1: XhoI
    465 tatccatc 1338-1318
    HAMJ 70 gatgcatatggatcatttccatt SEQ ID 1: NdeI
    466 acattcca 1192-1212
    HAMJ 71 gacaagcttggcattacctggat SEQ ID 1: HindIII
    467 aatcctg 1527-1507
    HAMJ 72 catgccatggaagcctattggaa SEQ ID 5: NcoI
    352 tgggaagc 793-813
    HAMJ 73 ataagaatgcggccgccgctatg SEQ ID 1: NotI
    470 aaatcagataaattc 3096-3117
    HAMJ 168 atatgggcccctgtataggagcc SEQ ID 5: Apa I
    471 ggttgactttc 2626-2604
    HAMJ 169 atatgggcccaatatgcaaccga SEQ ID 1: Apa I
    472 gtcagttaagc 720-697
    HAMJ 170 atatgggcccaacatggtcgcta SEQ ID 3: Apa I
    350 gcgttacc 2139-2119
    HAMJ 171 tcccgggcccgacttgacagaag SEQ ID 1: Apa I
    351 agcaaattaag 1414-1437
    HAMJ 172 catgccatgggacttgacagaag SEQ ID 1: NcoI
    358 agcaaattaag 1415-1437
    HAMJ 173 tcccgggccccgctatgaaatca SEQ ID 1: Apa I
    359 gataaattc 3116-3096
    HAMJ 174 atatgggcccgacattgatagtc SEQ ID 3: Apa I
    403 tcttgaaacagc 856-880
    HAMJ 175 cgccaagcttaacatggtcgcta SEQ ID 3: Hind III
    361 gcgttacc 2139-2119
    HAMJ 176 atatgggccccttactctccttt SEQ ID 3: Apa I
    483 aataaagccaatag 2520-2494
  • Molecular biology techniques were performed according to standard methods. See for example, Sambrook, J., Fritsch, E. F., Maniatis, T., “Molecular cloning. A laboratory manual” Vol.1-2-3 (second edition) Cold Spring Harbour Laboratory Press, 1989, New York, which is herein incorporated by reference. PCR-amplified products were digested with restriction endonucleases and ligated to either linearized plasmid pSL301, pCMV-GH, pET or pURV22.HIS expression vector digested likewise or digested with enzymes that produce compatible cohesive ends. Recombinant psL301 and recombinant pCMV-GH plasmids were digested with restriction enzymes for the in-frame cloning in pET expression vector. When pET vectors were used, clones were first stabilized in E. coli DH5a before introduction into E. coli BL21(λDE3) or AD494 (λDE3) for expression of full-length or truncated BVH-3, BVH-11 or BVH-11-2 molecules. Each of the resultant plasmid constructs was confirmed by nucleotide sequence analysis. The recombinant proteins were expressed as N-terminal fusions with the thioredoxin and His-tag (pET32 expression system); as C-terminal fusions with an His-tag (pET21 expression system); or as N-terminal fusions with an His-tag (pURV22.HIS expression system). The expressed recombinant proteins were purified from supernatant fractions obtained after centrifugation of sonicated IPTG- (pET systems) or heat- (pURV22.HIS) induced E. coli using a His-Bind metal chelation resin (QIAgen, Chatsworth, Calif.). The gene products generated from S. pneumoniae SP64 are listed in the following Table 2. The gene fragment encoding BVH-3-Sp63 protein (amino acid residues 21 to 840 on SEQ ID NO: 10) was generated from S. pneumoniae SP63 using the PCR-primer sets OCRR479-OCRR480 and the cloning vector pSL301. The recombinant pSL301-BVH-3Sp63 was digested for the in-frame cloning in pET32 vector for the expression of the BVH-3-Sp63 molecule.
    TABLE 2
    Lists of truncated BVH-3, BVH-11, BVH-11-2 and
    Chimeric gene products generated from S. pneumoniae SP64
    Encoded amino
    Protein acids (SEQ ID Cloning
    PCR-primer sets designation Identification No6) vector
    OCRR479-OCRR480 BVH-3M BVH-3 w/o ss  21-1039 pSL301
    OCRR479-OCRR497 BVH-3AD BVH-3 N′ end w/o ss  21-509 pSL301
    HAMJ248-HAMJ249 L-BVH-3AD BVH-3 N′ end  1-509 pET-21(+)
    OCRR498-OCRR499 BVH-3B BVH-3 C′ end 512-1039 pSL301
    OCRR479-HAMJ172 BVH-3C BVH-3 N′ end w/o ss  21-225 pET-32 c(+)
    OCRR487-OCRR488 BVH-11M BVH-11 w/o ss  20-840 pCMV-GH
    HAMJ251-OCRR487 BVH-11A BVH-11 N′ end w/o ss  20-353 pET-32 c(+)
    HAMJ171-OCRR488 BVH-11B BVH-11 C′ end 354-840 pET-32 a(+)
    HAMJ264-OCRR488 BVH-11C BVH-11 C′ end 228-840 pET-32 a(+)
    HAMJ278-HAMJ279 NEW1 BVH-3 C′ end 472-1039 pET-21b(+)
    HAMJ278-HAMJ280 NEW2 BVH-3 C′ end 472-800 pET-21b(+)
    HAMJ281-HAMJ279 NEW3 BVH-3 C′ end 800-1039 pET-21b(+)
    HAMJ284-HAMJ285 NEW4 BVH-11 C′ end 286-840 pET-21d(+)
    HAMJ284-HAMJ286 NEW5 BVH-11 internal 286-713 pET-21d(+)
    HAMJ287-HAMJ288 NEW6 BVH-11 internal 672-792 pET-21d(+)
    HAMJ285-HAMJ289 NEW7 BVH-11 C′ end 709-840 pET-21d(+)
    HAMJ284-HAMJ290 NEW8 BVH-11 internal 286-511 pET-21d(+)
    HAMJ286-HAMJ291 NEW9 BVH-11 internal 511-713 pET-21d(+)
    HAMJ160-HAMJ186 BVH-11-2M BVH-11-2 w/o ss  20-838 pSL301
    HAMJ292-HAMJ293 NEW10 BVH-11-2 C′ end 271-838 pET-21d(+)
    HAMJ293-HAMJ294 NEW11 BVH-11-2 C′ end 699-838 pET-21d(+)
    HAMJ282-HAMJ283 NEW13 BVH-11 C′ end 354-840 pET-21b(+)
    HAMJ286-HAMJ297 NEW14 BVH-11-2 internal 227-699 pET-21d(+)
    HAMJ300-HAMJ313 NEW15 BVH-3 N′ end w/o ss  21-800 pET-21b(+)
    HAMJ301-HAMJ302 NEW16 BVH-11 N′ end w/o ss  20-709 pET-21d(+)
    HAMJ352-HAMJ353 NEW18 BVH-11-2 internal 227-520 pET21d(+)
    HAMJ354-HAMJ355 NEW19 BVH-11-2 C′ end 497-838 pET21d(+)
    HAMJ404-HAMJ279 NEW21 BVH-3 C′ end 396-1039 pET21b(+)
    HAMJ464-HAMJ465 NEW22 BVH-3 internal 233-446 pET-21a(+)
    HAMJ466-HAMJ467 NEW23 BVH-3 internal 398-509 pET-21b(+)
    HAMJ352-HAMJ293 NEW24 BVH-11-2 C′ end 227-838 pET-21d(+)
    HAMJ464-HAMJ470 NEW25 BVH-3 C′ end 233-1039 pET-21b(+)
    HAMJ278-HAMJ279 NEW12 Chimera* M-NEW 1-KL- pET 21 b (+)
    (NEW 1) HAMJ282-HAMJ283 NEW 13
    (NEW 13)
    HAMJ284-HAMJ350 NEW17 Chimera* M-NEW 5-GP- pET 21 d (+)
    (NEW 5) HAMJ351-HAMJ279 NEW 1
    (NEW 1)
    HAMJ358-HAMJ359 NEW20 Chimera* M-NEW 1-GP- pET 21 d (+)
    (NEW 1) HAMJ403-HAMJ361 NEW 5
    (NEW 5)
    HAMJ292-HAMJ471 NEW26 Chimera* M-NEW 10-GP- pET 21 d (+)
    (NEW 10) HAMJ472-HAMJ470 NEW 25
    (NEW 25)
    HAMJ355-HAMJ471 NEW27 Chimera* M-NEW 19-GP- pET 21 d (+)
    (NEW 19) HAMJ472-HAMJ470 NEW 25
    (NEW 25)
    HAMJ292-HAMJ471 NEW28 Chimera* M-NEW 10-GP- pET 21 d (+)
    (NEW 10) HAMJ351-HAMJ279 NEW 1
    (NEW 1)
    HAMJ284-HAMJ350 NEW29 Chimera* M-NEW 5-GP- pET 21 d (+)
    (NEW 5) HAMJ472-HAMJ470 NEW 25
    (NEW 25)
    HAMJ284-HAMJ483 NEW30 Chimera* M-NEW 4-GP- pET 21 d (+)
    (NEW 4) HAMJ472-HAMJ470 NEW 25
    (NEW 25)
    HAMJ284-HAMJ483 NEW31 Chimera* M-NEW 4-GP- pET 21 d (+)
    (NEW 4) HAMJ351-HAMJ279 NEW 1
    (NEW 1)
    HAMJ355-HAMJ471 NEW32 Chimera* M-NEW 19-GP- pET 21 d (+)
    (NEW 19) HAMJ351-HAMJ279 NEW 1
    (NEW 1)

    w/o ss: without signal sequence. Analysis of the BVH-3, BVH-11 and BVH-11-2 protein sequences suggested the presence of putative hydrophobic leader sequences.

    *encoded amino acids for the chimeras are expressed as the gene product, additional non essential amino acids residue were added M is methionine, K is lysine, L is leucine, G is glycine and P is proline.
  • Monoclonal antibody (Mab)-secreting hybridomas were obtained by fusions of spleen cells from immunized mice and non-secreting, HGPRT-deficient mouse myeloma SP2/0 cells by the methods of Fazekas De St-Groth and Scheidegger (J Immunol Methods 35: 1-21, 1980) with modifications (J. Hamel et al. J Med Microbiol 23: 163-170, 1987). Female BALB/c mice (Charles River, St-Constant, Quebec, Canada) were immunized with either BVH-3M (thioredoxin-His.Tag-BVH-3M fusion protein/ pET32 system), BVH-11M (thioredoxin-His.Tag-BVH-llM fusion protein/pET32 system), BVH-11-2M (thioredoxin-His.Tag-BVH-11-2M fusion protein/pET32 system), BVH-11B (thioredoxin-His.Tag-BVH-11B fusion protein/pET32 system), BVH-3M (His.Tag-BVH-3 fusion protein/pURV22.HIS system) or NEW1 (NEW1-His.Tag fusion protein/pET21 system) gene products from S. pneumoniae strain SP64 to generate the Mab series H3-, H11-, H112-, H11B-, H3V-, and HN1-, respectively. Culture supernatants of hybridomas were initially screened by enzyme-linked-immunoassay (ELISA) according to the procedure described by Hamel et al. (Supra) using plates coated with preparations of purified recombinant BVH-3, BVH-11 and/or BVH-11-2 proteins or suspensions of heat-killed S. pneumoniae cells. The Mab-secreting hybridomas selected for further characterization are listed in Table 3 and Table 4 from the following Example 2. The class and subclass of Mab immunoglobulins were determined by ELISA using commercially available reagents (Southern Biotechnology Associates, Birmingham, Ala.).
  • Furthermore, the cloning and expression of chimeric gene(s) encoding for chimeric polypeptides and the protection observed after vaccination with these chimeric polypeptides are described.
  • BVH-3 and BVH-11 gene fragments corresponding to the 3′end of the genes were amplified by PCR using pairs of oligonucleotides engineered to amplify gene fragments to be included in the chimeric genes. The primers used had a restriction endonuclease site at the 5′ end, thereby allowing directional in-frame cloning of the amplified product into digested plasmid vectors (Table 1 and Table 2). PCR-amplified products were digested with restriction endonucleases and ligated to linearized plasmid pET21 or pSL301 vector. The resultant plasmid constructs were confirmed by nucleotide sequence analysis. The recombinant pET21 plasmids containing a PCR product were linearized by digestion with restriction enzymes for the in-frame cloning of a second DNA fragment and the generation of a chimeric gene encoding for a chimeric pneumococcal protein molecule. Recombinant pSL301 plasmids containing a PCR product were digested with restriction enzymes for the obtention of the DNA inserts. The resulting insert DNA fragments were purified and inserts corresponding to a given chimeric gene were ligated into pET21 vector for the generation of a chimeric gene. The recombinant chimeric polypeptides listed in Table 2 were as C-terminal fusion with an His-tag. The expressed recombinant proteins were purified from supernatant fractions obtained from centrifugation of sonicated IPTG-induced E. coli cultures using a His-Bind metal chelation resin (QIAgen, Chatsworth, Calif.).
  • Groups of 8 female BALB/c mice (Charles River) were immunized subcutaneously two times at three-week intervals with 25 μg of either affinity purified HisTag-fusion protein identifed in presence of 15-20 μg of QuilA adjuvant. Ten to 14 days following the last immunization, the mice were challenged challenged intravenously with 10E5-10E6 CFU of S. pneumoniae type 3 strain WU2. The polypeptides and fragments are capable of eliciting a protective immune response.
    TABLE 2A
    Days to death post-
    Experiment Immunogen Alive:Dead infection
    1 none 0:8 1, 1, 1, 1, 1, 1, 1, 1
    NEW 1 2:6 1, 2, 2, 2, 2, 2,
    >14, >14
    NEW 13 1:7 1, 1, 3, 3, 4, 5, 5,
    >14
    NEW 12 6:2 3, 11, 6X >14
    BVH-3M 1:7 3, 3, 3, 3, 3, 3, 3,
    >14
    2 none 0:8 1, 1, 1, 1, 1, 1, 1, 1
    NEW 17 7:1 4, 7 X >14
    NEW 12 3:5 3, 3, 3, 4, 5, >14,
    >14, >14
    3 none 0:8 2, 2, 2, 2, 2, 2, 2, 2
    NEW 18 1:7 2, 2, 2, 2, 3, 3, 3, 3
    NEW 19 8:0 8 X >14
    NEW 10 8:0 8 X >14
    BVH-11-2 8:0 8 X >14
    • EXAMPLE 2
  • This example describes the identification of peptide domains carrying target epitopes using Mabs and recombinant truncated proteins described in example 1.
  • Hybridomas were tested by ELISA against truncated BVH-3, BVH-11 or BVH-11-2 gene products in order to characterize the epitopes recognized by the Mabs. The truncated gene products were generated from S. pneumoniae SP64 strain except for BVH-3-Sp63 which was generated from S. pneumoniae SP63 strain. As a positive control, the reactivity of each antibody was examined with full-length BVH-3, BVH-11 or BVH-11-2 recombinant proteins. In some cases, the Mab reactivity was evaluated by Western immunoblotting after separation of the gene product by SDS-PAGE and transfer on nitrocellulose paper. The reactivities observed is set forth in the following Table 3 and Table 4.
    TABLE 3
    ELISA reactivity of BVH-3-reactive Mabs with a panel of eleven BVH-3 gene
    products and the BVH-11M molecule
    Mabs Gene products tested
    (IgG BVH- BVH- BVH- BVH- NEW NEW NEW BVH-3 BVH-
    isotype) 3M 3AD 3B 3C NEW 1 NEW 2 NEW 3 21 22 23 Sp63 11M
    H3-4F9 (1) + + − + − − − − − − + +
    H3-4D4 (1) + + − + − − − − − − + +
    H3-9H12 (1) + + − + − − − − − − + +
    H3-7G2 (1) + + − − − − − − + − − −
    H3-10A1 (1) + + − − − − − + − + + −
    H3-4D3 (1) + − + − + − + + − − + −
    H11-6E7 (1) + + − + − − − NT NT NT + +
    H11-10H10 + + − + − − − NT NT NT + +
    (2a)
    H11-7G11 + + + + + + − NT NT NT + +
    (2b)
    H3V-4F3 (1) + − + − + − − + − − + −
    H3V-2F2 (1) + − + − + + − + − − + −
    H3V-7F4 (1) + − + − + + − + − − + −
    H3V-7H3 (1) + − + − + − + + − − + −
    H3V-13B8 + − + − + − + + − − + −
    (1)
    H3V-9C2 (1) + + − +/− − − − − + − +/− +/−
    H3V-9C6 (1) + + − − − − − − + − − −
    H3V-16A7 + + − − − − − + − + − −
    (1)
    H3V-15A10 + + + +/− + + − + + + + +/−
    (1)
    H3V-6B3 + + NT NT + + − + + − NT −
    (1/2)
    HN1-5H3 + − + NT + − − + − − + −
    (2b)
    HN1-8E3 + − + NT + − − + − − + −
    (2a)
    HN1-14F6 + − + NT + − − + − − + −
    (2a)
    HN1-2G2 (1) + − + NT + + − + − − + −
    HN1-12D8 + − + NT + + − + − − + −
    (2a)
    HN1-14B2 + − + NT + + − + − − + −
    (2a)
    HN1-1G2 + − + NT + − + + − − + −
    (2a)
    HN1-10C12 + − + NT + − + + − − + −
    (1)
    HN1-3E5 (1) + + − − + + − + − + + −

    NT: not tested

    +/−: very low reactivity but higher than background, possible non-specific Mab binding
  • TABLE 4
    ELISA reactivity of BVH-11 and/or BVH-11-2-reactive Mabs with a panel of
    fourteen BVH-11 and BVH-11-2 gene products and the BVH-3M molecule
    Gene products tested
    Mabs BVH-
    (IgG BVH- BVH- BVH- BVH- NEW NEW NEW NEW NEW NEW 11- BVH-
    isotype) 11M 11A 11B 11C 5 NEW 6 NEW 7 NEW 8 NEW 9 10 11 14 18 19 2-M 3M
    H3-4F9 + + − − − − − − − − − − − − + +
    (1)
    H3-4D4 + + − − − − − − − − − − − − + +
    (1)
    H3-9H12 + + − − − − − − − − − − − − + +
    (1)
    H11-6E7 + + − − − − − − − − − − − − + +
    (1)
    H11- + + − − − − − − − − − − − − + +
    10H10
    (2a)
    H11-7G11 + + − − − − − − − − − − − − + +
    (2b)
    H11-1B12 + + − − − − − − − − − − − − + −
    (1)
    H11-7B9 + + − − − − − − − − − − − − + −
    (2a)
    H11-3H5 + − + + + − − −* − + − + + − + −
    (1)
    H11-10B8 + − + + + − − −* − + − + + − + −
    (1)
    H11-1A2 + − + + + − − −* − + − + + − + −
    (1)
    H112-3A1 + − + NT + − − + − + − + + − + −
    (1)
    H112- + +/− + NT + − − + − + − + + − + −
    13C11
    (1)
    H112- + + − NT + − − + − + − + + − + −
    10H10
    (1)
    H112-1D8 + + − NT + − − + − + − + + − + −
    (2a)
    H112- + − + NT + − − − + + − + − + + −
    10G9
    (2b)
    H112- + − + NT + − − +/− + + − + − + + −
    10A2 (1)
    H112-3E8 + − + NT + − − +/− − + − + − + + −
    (2a)
    H112- + − + NT + − − − − + − + − − + −
    10D7
    (2a)
    H112-2H7 + + − NT − − − − − − − − − − + −
    (2a)
    H112-6H7 + + − NT − − − − − − − − − − + −
    (1)
    H112-3A4 − − − NT − − − − − + + − − + + −
    (2a)
    H112- − − − NT − − − − − + + − − + + −
    10C5 (1)
    H112- − − − NT − − − − − + + − − + + −
    14H6 (1)
    H112-7G2 − − − NT − − − − − + − + + − + −
    (1)
    H112- − − − NT − − − − − − − + + − + −
    13H10
    (2a)
    H112-7E8 +/− − − NT − − − − − − − − +/− − + −
    (2b)
    H112-7H6 +/− − − NT − − − − − +/− − − − − + −
    (1)
    H11B- + − + + + − − + − + − + + − + −
    5F10 (1)
    H11B- + − + + + − − + − + − + + − + −
    15G2 (1)
    H11B- + − + + + − − − + + − + − + + −
    13D5 (2)
    H11B- + − + + + − − − + + − + − + + −
    11B8 (1)
    H11B- + − + + + − − − − + − + − − + −
    7E11 (1)
    H11B-1C9 + − + + + − − − − + − + − − + −
    (1)
    H11B-5E3 + − + + − − + − − − − − − − − −
    (2)
    H11B-6E8 + − + + − − + − − − − − − − − −
    (1)

    NT: not tested

    +/−: very low reactivity but higher than background, possible non-specific Mab binding

    *a strong signal was detected when tested by Western immunoblotting
  • The deduced locations of the epitopes are summarized in FIG. 28 and FIG. 29. As can be seen from the data in Table 3, BVH-3-reactive Mabs can be divided into two groups: BVH-3A- and BVH-3B-reactive Mabs with the exception of Mabs H11-7G11 and H3V-15A10 which reacted with both, BVH-3A and BVH-3B molecules. The BVH-3A-reactive Mabs can be subdivided in two subgroups of antibodies depending of their reactivity or lack of reactivity with BVH-3C recombinant protein. Mab reactive with BVH-3C protein recognized epitopes shared by both, BVH-3 and BVH-11 proteins. As can be seen in Table 4, these BVH-3- and BVH-11-cross-reactive Mabs were also reactive with BVH-11A and BVH-11-2M recombinant proteins. BVH-3B-reactive Mabs can be subdivided into three subgroups according to their reactivity with NEW1, NEW2 and NEW3 recombinant proteins. Some Mabs were only reactive with the NEW1 protein while other Mabs were reactive with either, NEW1 and NEW2 or NEW1 and NEW3 recombinant proteins. Mabs H11-7G11 and H3V-15A10 react with epitopes in more than one position on BVH-3. The reactivity of H11-7G11 with BVH-3AD, BVH-3B, BVH-3C, BVH-11A and BVH-11-2M molecules suggests that H11-7G11 epitope might comprised HXXHXH sequence. This sequence is repeated, respectively, 6 and 5 times in BVH-3 and BVH-11/BVH-11-2 protein sequences. The lack of reactivity of Mab H11-7G11 with NEW 10 molecule suggests that the epitope includes the HGDHXH sequence. Multiple-position mapping of H3V-15A10 epitope on BVH-3 is suggested by the reactivity of the Mab with two BVH-3 fragments that do not overlap.
  • Interestingly, Mabs H3-7G2, H3V-9C6 and H3V-16A7 were not reactive with BVH-3 Sp63 thus allowing the location of their corresponding epitopes on a 177-amino acid fragment comprised between amino acids 244 and 420 on BVH-3 molecule of S. pneumoniae SP64 (FIG. 31).
  • As can be seen from the data in Table 4, the Mabs that are reactive with BVH-11- and/or BVH-11-2 and that do not recognize BVH-3 molecules can be divided into three groups according to their reactivities with BVH-11A and NEW10 recombinant proteins. Some Mabs reacted exclusively with either BVH-11A or NEW10 protein while other Mabs were reactive with both, BVH-11A and NEW10 recombinant proteins.
    • EXAMPLE 3
  • This example describes the construction of BVH-3 and BVH-11-2 gene libraries for the mapping of epitopes.
  • BVH-3 and BVH-11-2 gene libraries were constructed using recombinant PCMV-GH and PSL301 plasmid DNA containing respectively, BVH-3 gene sequence spanning nucleotides 1837 to 4909 (SEQ ID NO: 2) or BVH-11-2 gene sequence spanning nucleotides 172 to 2630 (SEQ ID NO: 5) and the Novatope® library construction and screening system (Novagen). The recombinant plasmids containing BVH-3 or BVH-11-2 gene fragment were purified using QIAgen kit (Chatsworth, Calif.) and digested with the restriction enzymes BglII and XbaI respectively. The resulting BglII-XbaI DNA fragments were purified using the QIAquick gel extraction kit from QIAgen and digested with Dnase I for the generation of randomly cleaved DNA. DNA fragments of 50 to 200 bp were purified, treated with T4 DNA polymerase to blunt the target DNA ends and add a single 3′dA residue, and ligated into pSCREEN-T-Vector (Novagen) following the procedures suggested by the manufacturer (Novatope® System, Novagen). The gene libraries of E. coli clones, each of which expressing a small peptide derived from BVH-3 or BVH-11-2 genes were screened by standard colony lift methods using Mabs as immunoprobes. The colony screening was not successful with Mabs producing very high backgrounds on colony lifts. Moreover, in some cases, Mabs failed to detect epitope-expressing-colonies. The lack of reactivity can possibly be explained by the small amount of recombinant proteins produced or the recognition of conformation-dependent epitopes consisting of different protein domains. Sequencing of DNA inserts from positive clones determined the location of the segment that encodes the target epitope. The data are presented in Table 5. The peptides encoded by DNA inserts into the recombinant PSCREEN-T vector can be purified and used as immunogens as described below in Example 6.
  • The peptide sequences obtained from the screening of BVH-3 and BVH-11-2 gene libraries with the Mabs are in agreement with the Mab ELISA reactivities against the truncated gene products. As expected, the amino acid sequences obtained from H11-7G11 contained the sequence HGDHXH. These findings provide additional evidence for the location of epitopes recognized with the Mabs. Interestingly, although the Mabs H112-10G9, H112-10A2 and H11B-11B8 were reactive against the same peptide sequence (amino acid residues 594 to 679 on BVH-11-2 protein sequence), clones corresponding to the sequence spanning from amino acid residues 658 to 698 were only picked up by Mab H11B-11B8 thus revealing the location of H11B-11B8 epitope between amino acid residues 658 to 679 (SEQ ID NO: 163). Mabs H112-10G9, H112-10A2, and H11B-11B8 are directed against 3 distinct non overlapping epitopes located closely on the peptide sequence corresponding to amino acid residues 594 to 679 (SEQ ID NO: 22).
    TABLE 5
    Peptide sequences obtained from the screening of BVH-3 and BVH-11-2 gene
    libraries with Mabs
    Clone/ SEQ
    Protein Nucleotide Amino acid ID
    Mab designation position position Amino acid sequence NO
    H3-4D4 4D4.9 SEQ ID 1: SEQ ID 6: DQGYVTSHGDHYHYYNGKVPYDALFSEELLMKDPNYQLKDA 11
    226-509 76-169 DIVNEVKGGYIIKVDGKYYVYLKDAAHADNVRTKDEINRQK
    QEHVKDNEKVNS
    H11- 7G11.7 SEQ ID 1: SEQ ID 6: GIQAEQIVIKITDQGYVTSHGDHYHYYNGKVPYDALFSEELL 12
    7G11 193-316 64-105
    H11- 7G11.9 SEQ ID 1: SEQ ID 6: TAYIVRHCDHFHYIPKSNQIGQPTLPNNSLATPSPSLPI 13
    7G11 1171-1284 390-428
    H3-4D3 4D3.4 SEQ ID 1: SEQ ID 6: TSNSTLEEVPTVDPVQEKVAKFAESYGMKLENVLFN 14
    2565-2670 855-890
    HN1- 8E3.1 SEQ ID 1: SEQ ID 6: MDGTIELRLPSGEVIKKNLSDFIA 15
    8E3 3004-3120 1016-1039
    HN1- 1G2.2 SEQ ID 1: SEQ ID 6: YGLGLDSVIFNMDGTIELRLPSGEVIKKNLSDFIA 16
    1G2 3017-3120 1005-1039
    HN1- 10C12.7 SEQ ID 1: SEQ ID 6: PALEEAPAVDPVQEKLEKFTASYGLGLDSVIFNMDGTIELR 17
    10C12 2936-3120 983-1039 LPSGEVIKKNLSDFIA
    HN1- 14F6.3 SEQ ID 1: SEQ ID 6: KVEEPKTSEKVEKEKLSETGNSTSNSTLEEVPTVDPVQEK 18
    14F6 2501-2618 833-872
    HN1- B12D8.2 SEQ ID 1: SEQ ID 6: MKDLDKKIEEKIAGIMKQYGVKRESIVVNKEKNAIIYPHGD 19
    12D8 1433-1767 512-589 HHHADPIDEHKPVGIGHSHSNYELFKPEEGVAKKEGN
    H3V- 7F4.1 SEQ ID 1: SEQ ID 6: AIIYPHGDHHHADPIDEHKPVGIGHSHSNYELFKPEEGVAK 20
    7F4 1633-1785 545-595 KEGNKVYTGE
    H112- 10D7.5 SEQ ID 5: SEQ ID 8: IQVAKLAGKYTTEDGYIFDPRDITSDEGD 21
    10D7 1685-1765 525-553
    H112- 10G9.3 SEQ ID 5: SEQ ID 8: DHQDSGNTEAKGAEAIYNRVKAAKKVPLDRMPYNLQYTVEV 22
    10G9 1893-2150 594-679 KNGSLIIPHYDHYHNIKFEWFDEGLYEAPKGYSLEDLLATV
    KYYV
    H112- 10A2.2 SEQ ID 5: SEQ ID 8: DHQDSGNTEAKGAEAIYNRVKAAKKVPLDRMPYNLQYTVEV 22
    10A2 1893 -2150 594-679 KNGSLIIPHYDHYHNIKFEWFDEGLYEAPKGYSLEDLLATV
    KYYV
    H11B- B11B8.1 SEQ ID 5: SEQ ID 8: DHQDSGNTEAKGAEATYNRVKAAKKVPLDRMPYNLQYTVEV 22
    11B8 1893-2150 594-679 KNGSLIIPHYDHYHNIKFEWFDEGLYEAPKGYSLEDLLATV
    KYYV
    H11B- 11B8.4 SEQ ID 5: SEQ ID 8: GLYEAPKGYSLEDLLATVKYYVEHPNERPHSDNGFGNASDH 23
    11B8 2085-2217 658-698
    H112- 3A4.1 SEQ ID 5: SEQ ID 8: VENSVINAKIADAEALLEKVTDPSIRQNANETLTGLKSSLL 24
    3A4 2421-2626 769-837 LGTKDNNTISAEVDSLLALLKESQPAPI
    • EXAMPLE 4
  • This example describes the immunization of animals with recombinant proteins for the generation of antibody reactive with BVH-3, BVH-11 and/or BVH-11-2.
  • NZW rabbits (Charles River Laboratories, St-Constant, Québec, Canada) were immunized subcutaneously at multiple sites with 50 μg or 100 μg of the purified BVH-3M, L-BVH-3AD, NEW1, NEW13, or L-BVH-11 recombinant protein in presence of 80 μg of QuilA adjuvant (Cedarlane Laboratoratories Ltd, Hornby, Canada). The rabbits were boosted two times at three-week intervals with the same antigen and blood samples were collected before each immunization and 6 to 28 days following the last immunization. The sera samples were designated preimmune, post 1st, post 2nd or post 3rd injection. The rabbit immune response to immunization was evaluated by ELISA using recombinant BVH-3M (BVH-3M-His.Tag fusion protein/pET21 system) or BVH-11M (BVH-11M-His.Tag fusion protein/pET21 system) proteins or suspensions of heat-killed S. pneumoniae Rx-1 cells as coating antigens. ELISA titer was defined as the reciprocal of the highest sera dilution at which absorbance A410 value was 0.1 above the background value. Antibodies reactive with BVH-3 and/or BVH-11 epitopes were elicited following immunization in all animals as shown in the following Table 6. Antibody reactive with recombinant or pneumococcal antigens was not present in the preimmune sera. The immune response to immunization was detectable in the sera of each rabbit after a single injection of recombinant antigen. The antibody response following the second injection with either antigen tested was characterized by a strong increase in antibody titer. Interestingly, good titers of antibody reactive with S. pneumoniae cells, with an average titer of 52,000 after the third immunization, were obtained, thus establishing that native pneumococcal epitopes are expressed on the recombinant E. coli gene products. These data support the potential use of BVH-3, BVH-11 and/or BVH-11-2 gene products and the antibody raised to BVH-3, BVH-11 and/or BVH-11-2 gene products as vaccines for the prevention and the treatment of pneumococcal disease, respectively.
    TABLE 6
    Rabbit Antibody response to immunization with BVH-3
    and BVH-11 gene products
    ELISA Titer with coating
    antigen
    Immu- Sera BVH- S.
    Rabbit nogen sample BVH-3M 11M pneumoniae
    #
    15 BVH-3M Preimmune NT NT 200
    (50 μg) Post-1st NT NT 1,600
    Post-2nd NT NT 20,000
    Post-3rd 512,000 NT 40,000
    #16 BVH-3M Preimmune NT NT 200
    (100 μg) post 1st NT NT 1,600
    post 2nd NT NT 40,000
    post 3rd    106 NT 80,000
    #112 L-BVH- Preimmune   <100 NT NT
    3AD
    (50 μg) post 1st  16,000 NT NT
    post
    2nd 512,000 NT NT
    post
    3rd 2 × 106 NT 32,000
    #113 New 1 Preimmune   <100 NT NT
    (50 μg) post 1st  16,000 NT NT
    post
    2nd 512,000 NT NT
    post
    3rd    106 NT 64,000
    #114 New 13 Preimmune NT   <100 NT
    (50 μg) post 1st NT  16,000 NT
    post
    2nd NT  64,000 NT
    post
    3rd NT 256,000 32,000
    #116 L-BVH-11 Preimmune NT   <100 NT
    (50 μg) post 1st NT  64,000 NT
    post
    2nd NT    106 NT
    post
    3rd NT 2 × 106 64,000

    NT: not tested
    • EXAMPLE 5
  • This example describes the protection of animals against fatal experimental pneumococcal infection by administration of antibody raised to BVH-3, BVH-11 or BVH-11-2 gene products.
  • High-titer Mab preparations were obtained from ascites fluid of mice inoculated intraperitoneally with Mab-secreting hybridoma cells according to the method described by Brodeur et al (J Immunol Methods 71 :265-272, 1984). Sera samples were collected from rabbits immunized with BVH-3M as described in Example 4. The rabbit sera collected after the third immunization and ascites fluid were used for the purification of antibodies by precipitation using 45 to 50% saturated ammonium sulfate. The antibody preparations were dissolved and dialyzed against phosphate-buffered saline (PBS).
  • CBA/N (xid) mice (National Cancer Institute, Frederick, Mass.) were injected intraperitoneally with either 0.1 ml of purified rabbit antibodies or 0.2 ml of ascites fluid before intravenous challenge with approximately 200 CFU of the type 3 S. pneumoniae strain WU2. Control mice received sterile PBS or antibodies purified from preimmune rabbit sera or sera from rabbits immunized with an unrelated N. meningitidis recombinant protein antigen. One group of mice was challenged with S. pneumoniae before the administration of anti-BVH-3 antibody. Samples of the S. pneumoniae challenge inoculum were plated on chocolate agar plates to determine the number of CFU and verify the challenge dose. The CBA/N mice were chosen because of their high susceptibility to S. pneumoniae infection. The LD50 of WU2 injected intravenously to CBA/N mice is estimated to be ≦10 CFU. Deaths were recorded at 24-h intervals for a period of at least 7 days.
  • The protection data obtained from mice injected with rabbit anti-BVH-3 antibody are set forth in the following Table 7. Nine out of 10 mice receiving the anti-BVH-3 antibody survived the challenge in contrast to none of 10 mice injected with control antibody or PBS buffer. The observation that antibody raised to the BVH-3-M molecule passively protected even when administered after the challenge demonstrated the ability of anti-BVH-3 antibody to prevent death even from an already established infection.
    TABLE 7
    Protective effects of rabbit antibody to BVH-3-M gene
    in CBA/N mice challenged i.v. with WU2 pneumococci
    Antibody Time of antibody Days to death
    preparation administration Alive:Dead post-infection
    Anti-BVH3M 1 h before 5:0 >14, >14, >14,
    infection >14, >14
    Anti-N. meningitidis 1 h before 0:5 2, 2, 2, 2, 2
    infection
    Anti-BVH-3M 0.5 h post- 4:1 2, >14, >14,
    infection >14, >14
    None (PBS) 1 h before 0:5 1, 2, 2, 2, 2
    infection
  • CBA/N mice were infected with 1000 CFU of WU2 S. pneumoniae before or after intraperitoneal administration of 0.1 ml of rabbit antibody.
  • In an other experiment, 0.1 ml of rabbit antibody prepared from preimmune and immune sera were administered intraperitoneally to CBA/N mice four hours before intranasal challenge with 280 CFU of S. pneumoniae P4241 type 3 strain. As seen in the following Table 8, all immunized mice survived the challenge while none of 9 mice receiving preimmune sera antibody or buffer alone were alive on day 6 post-infection. S. pneumoniae hemocultures on day 11 post-challenge were negative for all surviving mice. Furthermore, 100% protection was observed in mice receiving monoclonal antibodies H112-10G9 or a mixture of H112-10G9 and H11B-7E11 which are directed against BVH-11/BVH-11-2.
    TABLE 8
    Protective effects of passive transfer of rabbit
    antibody to BVH-3-M gene product or anti-BVH-11/BVH-11-2
    specific Mabs in CBA/N mice challenged i.n. with P4241
    pneumococci
    Antibody Days to death
    preparation Alive:Dead post-infection
    Anti-BVH-3M 5:0 >11, >11, >11, >11,
    >11
    Antibody from 0:5 3, 3, 3, 6, 6
    preimmune sera
    H112-10G9 4:0 >11, >11, >11, >11
    H112-10G9 + H11B- 5:0 >11, >11, >11, >11,
    7E11 >11
    None (PBS) 0:4 3, 3, 3, 3
  • Altogether, the results from Table 7 and Table 8 clearly establish that immunization of animals with a BVH-3 gene product such as BVH-3M elicited protective antibodies capable of preventing experimental bacteremia and pneumonia infections.
  • The protection data obtained for mice injected with ascites fluid are set forth in the following Table 9. Administration of a volume of 0.2 ml of ascites fluid of 0.2 ml of some sets of ascites fluid prevented death from experimental infection. For example, H112-3A4+H112-10G9 and H112-10G2+H112-10D7 sets of 2 Mabs conferred complete protection against experimental infection. These data indicated that antibody targetting BVH-11and/or BVH-11-2 epitopes gave efficient protection. The Mabs H112-3A4, H112-10G9, H112-10D7, H112-10A2, H112-3E8, H112-10C5, H11B-11B8, H11B-15G2, H11B-1C9, H11B-7E11, H11B-13D5 and H11-10B8 were present in at least one protective pair of Mabs and were said to be protective and reactive against protective epitopes. The locations of protection-conferring epitopes on BVH-11-2 molecules are summarized in Table 10 and FIG. 29. Protective Mabs H112-3A4, H112-10G9, H112-10D7, H112-10A2, H112-3E8, H112-10C5, H11B-11B8, H11B-15G2, H11B-1C9, H11B-7E11, H11B-13D5 and H11-10B8 were all reactive with New 10 protein corresponding to amino acid residues 271 to 838 on the BVH-11-2 molecule. Six out of these 12 Mabs were directed against epitopes present in the NEW 19 protein and 3 protective Mabs recognized NEW 14. Interestingly, Mab H112-3A4 and H112-10C5 reacted with distinct epitopes exclusive to BVH-11-2 located at the carboxyl end comprised between amino acid residues 769 and 837. Also, Mabs H11-7G11, H11-6E7 and H3-4F9 reactive with epitopes shared by pneumococcal BVH-3, BVH-11 and BVH-11-2 molecules did not succeed to protect even if given in combination with protective H112-10G9 or H112-11B8 Mab. These Mabs recognized epitopes located at the amino end of the BVH-3, BVH-11 and BVH-11-2 molecules comprising, respectively, the first 225, 228 and 226 amino acid residues. The comparison of the BVH-3, BVH-11 and BVH-11-2 protein sequences revealed that a large number of amino acids were conserved in the amino end portion comprising these 225-228 residues with a global 72.8% identity (FIG. 32).
  • Altogether the data set forth in Table 9 and Table 10 suggest that the protection eliciting BVH-11- and BVH-11-2-epitopes is comprised in the carboxy terminal product containing amino acids 229 to 840 and 227 to 838, on BVH-11 and BVH-11-2 proteins, respectively.
    TABLE 9
    Passive immunization with BVH-11- and/or BVH-11-2-
    specific Mabs can protect mice from lethal experimental
    pneumococcal infection.
    Ex- Days to death
    periment Mab Alive:Dead post-infection
    1 H112 3A4 + H112-10G9 6:0 6 X >10
    H112-3A4 + H112-10D7 5:1 4, 5X >10
    None 0:6 2, 2, 2, 2, 2, 6
    2 H112-10 A2 + H112-10D7 5:1 3, 5X >10
    H112-3E8 + H112-10G9 6:0 6 X >10
    None 0:6 2, 2, 2, 2, 2, 2
    3 H112-10D7 + H11B-11B8 6:0 6 X >10
    H112-10G9 + H11B-15G2 3:3 2, 6, 6, 3 X >10
    None 0:6 2, 2, 2, 2, 2, 2
    4 H112-10G9 + H112-10D7 5:0 5 X >11
    None 0:5 2, 2, 2, 2, 2
    5 H112-10G9 + H11-10B8 4:1 8, 4 X >14
    H112-10G9 + H11B-7E11 5:0 5 X >14
    None 0:3 1, 2, 2
    6 H112-10G9 + H11B-1C9 4:1 4, 4 X >14
    None 0:3 2, 2, 2
    7 H112-10C5 + H11B-13D5 5:0 5 X >14
    None 3:3 2, 2, 2

    CBA/N mice were injected intraperitoneally with a total of 0.2 ml of ascites fluid 4 hours before intravenous challenge with S. pneumoniae WU2.
  • TABLE 10
    Deduced locations of protection-conferring
    epitopes on BVH-11-2 molecules.
    Gene products carrying Mab-
    Mabs Protection epitope
    H112-3A4 + NEW 19 and NEW 11
    H112-10G9 + NEW 19
    H112-10D7 + NEW 14 and NEW 10
    H112-10A2 + NEW 19
    H112-3E8 + NEW 19
    H11B-11B8 + NEW 19
    H11B-15G2 + NEW 18
    H11B-7E11 + NEW 14 and NEW 10
    H11-10B8 + NEW 18
    H11B-1C9 + NEW 14 and NEW 10
    H112-3A1 − NEW 18 and NEW 8
    H112-10H10 − NEW 18 and NEW 8
    H112-2H7 − BVH-11-2M
    H112-6H7 − BVH-11-2M
    H11-7G11 − BVH-11A and BVH-3C
    H11-6E7 − BVH-11A and BVH-3C
    H112-10C5 + NEW 19, NEW11 and 3A4.1
    H11B-13D5 + NEW 19
    H112-7G2 − NEW 18
    H112-7E8 − BVH-11-2M
    H3-4F9 − BVH-11A and BVH-3C
  • Altogether the data presented in this example substantiate the potential use of antibodies raised to BVH-3, BVH-11 or BVH-11-2 molecules as therapeutic means to prevent, diagnose or treat S. pneumoniae diseases.
    • EXAMPLE 6
  • This example describes the localization of surface-exposed peptide domains using Mabs described in Example 1.
  • S. pneumoniae type 3 strain WU2 was grown in Todd Hewitt (TH) broth (Difco Laboratories, Detroit Mich.) enriched with 0.5% Yeast extract (Difco Laboratories) at 37° C. in a 8% CO2 atmosphere to give an OD600 of 0.260 (˜108 CFU/ml). The bacterial suspension was then aliquoted in 1 ml samples and the S. pneumoniae cells were pelletted by centrifugation and resuspended in hybridoma culture supernatants. The bacterial suspensions were then incubated for 2 h at 4° C. Samples were washed twice in blocking buffer [PBS containing 2% bovine serum albumin (BSA)], and then 1 ml of goat fluorescein (FITC)-conjugated anti-mouse IgG+IgM diluted in blocking buffer was added. After an additional incubation of 60 min at room temperature, samples were washed twice in blocking buffer and fixed with 0.25% formaldehyde in PBS buffer for 18-24 h at 4° C. Cells were washed once in PBS buffer and resuspended in 500 μl of PBS buffer. Cells were kept in the dark at 4° C. until analyzed by flow cytometry (Epics® XL; Beckman Coulter, Inc.). Ten thousands (10,000) cells were analyzed per sample and the results were expressed as % Fluorescence and Fluorescence index (FI) values. The % Fluorescence is the number of fluorescein-labelled S. pneumoniae cells divided by 100 and the FI value is the median fluorescence value of pneumococci treated with Mab supernatant divided by the fluorescence value of pneumococci treated with the conjugate alone or with a control unrelated Mab. A FI value of 1 indicated that the Mab has not been detected at the surface of the bacteria whereas a FI value higher than 2 was considered positive when at least 10% of the pneumococcal cells were labelled and indicated that the Mab was reactive with cell-surface exposed epitopes. The following Table 11 summarized the data obtained with the Mabs tested by flow cytometry.
  • Flow cytometric analysis revealed that the Mabs reactive with BVH-3C and/or BVH-11A molecules did not bind to the cell surface. In contrast, with the exception of Mabs H3V-9C6 and H3V-16A7, the Mabs reactive with NEW 1, NEW 2, NEW 3, NEW 22 or NEW 23 BVH-3 gene products were detected at the surface of pneumococci. These data indicated that the first 225 amino acid residues located at the amino end of BVH-3 are internal. The lack of binding of Mabs H3V-9C6 and H3V-16A7 suggest some portions of the sequence corresponding to the 177-amino acids absent from the BVH-3 molecule of S. pneumoniae SP63 appears not to be accessible to antibodies.
  • Results from BVH-11- and/or BVH-11-2-reactive Mabs revealed that there is a good correlation between surface-exposure and protection. All Mabs reactive with internal epitopes as determined by the flow cytometry assay were not protective whereas all the protective Mabs described in Example 5 gave a positive signal in flow cytometry. Although an FI value of 9.0 and a % Fluorescence of 81.2 were obtained with Mab H11-7G11, this Mab was not shown to protect. Additional assays can be used to further evaluate whether this Mab and its corresponding epitope might participate in anti-infectious immunity.
    TABLE 11
    Results from the binding of Mabs at the surface of
    S. pneumoniae by flow cytometry analysis
    % Gene products carrying
    Mab Fluorescence FI Binding Mab-epitope
    H3-4F9 3.4 1.2 − BVH-3C and BVH-11A
    H3-4D4 3.4 1.2 − BVH-3C and BVH-11A
    H3-9H12 2.5 1.1 − BVH-3C and BVH-11A
    H3-7G2 66.2 6.3 + NEW 22
    H3-10A1 58.8 5.6 + NEW 23
    H3-4D3 33.2 3.5 + NEW 3
    H3V-4F3 24.4 2.9 + NEW 1
    H3V-2F2 15.6 2.4 + NEW 2
    H3V-7F4 58.7 5.6 + NEW 2
    H3V-7H3 68.8 6.9 + NEW 3
    H3V-13B8 75.0 7.7 + NEW 3
    H3V-9C2 66.4 6.2 + NEW 22
    H3V-9C6 2.9 1.0 − NEW 22
    H3V-16A7 6.6 1.7 − NEW 23
    H3V- 58.7 5.7 + NEW 22 and NEW 23
    15A10
    HN1-5H3 43.4 5.3 + NEW 1
    HN1-8E3 57.4 6.6 + NEW 1
    HN1-14F6 57.8 6.7 + NEW 1
    HN1-2G2 54.8 6.3 + NEW 2
    HN1-12D8 14.3 3.0 + NEW 2
    HN1-14B2 11.5 2.7 + NEW 2
    HN1-1G2 59.9 7.0 + NEW 3
    HN1- 13.6 2.8 + NEW 3
    10C12
    H11-6E7 4.9 1.2 − BVH-3C and BVH-11A
    H11- 6.5 1.6 − BVH-3C and BVH-11A
    10H10
    H11-7G11 81.2 9.0 + BVH-3C and NEW 2
    H11-1B12 3.1 1.2 − BVH-11A
    H11-7B9 2.4 1.1 − BVH-11A
    H11-10B8 81.1 9.1 + NEW 18 and NEW 8
    H11-1A2 84.4 10 + NEW 18 and NEW 8
    H11-3H5 84.0 9.8 + NEW 18 and NEW 8
    H112- 49.3 5.9 + NEW 18 and NEW 8
    13C11
    H112- 0.4 1.0 − BVH-11A and NEW 18
    10H10
    H112-1D8 0.4 1.0 − BVH-11A and NEW 18
    H112- 78.9 10.4 + NEW 19
    10G9
    H112- 75.5 9.6 + NEW 19
    10A2
    H112-3E8 62.5 7.5 + NEW 19
    H112- 64.5 7.7 + NEW 14
    10D7
    H112-2H7 0.7 1.1 − BVH-11A
    H112-6H7 0.3 1.0 − BVH-11A
    H112-3A4 70.1 8.9 + NEW 11
    H112- 86.3 9.2 + NEW 11 AND 3A4.1
    10C5
    H112- 89.6 11 + NEW 11
    14H6
    H112- 0.8 1.4 − NEW 11
    14H6
    H112-7G2 4.7 2.0 − NEW 18
    H112- 0.5 1.0 − NEW 18
    13H10
    H112-7E8 0.4 1.0 − BVH-11-2M
    H112-7H6 0.2 1.0 − BVH-11-2M
    H11B- 3.1 1.1 − NEW 18
    5F10
    H11B- 60.2 5.7 + NEW 18 and NEW 8
    15G2
    H11B- 75.7 8.3 + NEW 19
    13D5
    H11B- 78.4 8.3 + NEW 19
    11B8
    H11B- 32.3 3.5 + NEW 14
    7E11
    H11B-1C9 57.3 5.5 + NEW 14
    H11B-5E3 1.8 1.0 − NEW 7
    H11B-6E8 2.4 1.0 − NEW 7
    • EXAMPLE 7
  • This example describes the immunization of animals with peptide epitopes of BVH-3 and BVH-11-2.
  • The recombinant PSCREEN-T vector (Novagen, Madison, Wis.) containing DNA fragment (nucleotides 2421 to 2626 on SEQ ID NO: 5) encoding the Mab 3A4-epitope (SEQ ID NO: 24) was transformed by electroporation (Gene Pulser II apparatus, BIO-RAD Labs, Mississauga, Canada) into E. coli Tuner (λDE3) pLysS [BL21 (F′ ompT hsdSB (rB−mB−) gal dcm lacYI pLysS (Cmr)] (Novagen). In this strain, the expression of the fusion protein is controlled by the T7 promoter which is recognized by the T7 RNA polymerase (present on the XDE3 prophage, itself under the control of the lac promoter inducible by isopropyl-β-D-thiogalactopyranoside (IPTG). The pLysS plasmid reduces the basal fusion protein expression level by coding for a T7 lysozyme, which is a natural inhibitor of the T7 RNA polymerase.
  • The transformants were grown at 37° C. with 250 RPM agitation in LB broth (peptone 10 g/l, yeast extract 5 g/l, NaCl 5 g/l) supplemented with 50 mM glucose, 100 μg/ml carbenicillin and 34 μg/ml chloramphenicol, until the absorbance at 600 nm reached a value of 0,7. The overexpression of T7gene 10 protein-His.Tag-3A4.1 fusion protein was then induced by the addition of IPTG to a final concentration of 1 mM and further incubation at 25° C. with 250 RPM agitation for 3 hours. Induced cells from a 800-ml culture were pelleted by centrifugation and frozen at −70° C. The fusion protein was purified from the soluble cell fraction by affinity chromatography based on the binding of a six histidine residues sequence (His-Tag) to divalent cations (Ni2+) immobilized on a metal chelation Ni-NTA resin (Qiagen, Mississauga, Canada). Briefly, the pelleted cells were thawed and resuspended in Tris buffered sucrose solution (50 mM Tris, 25% (w/v) sucrose) and frozen at −70° C. for 15 minutes. Cells were incubated 15 minutes on ice in the presence of 2 mg/ml lysozyme before disruption by sonication. The lysate was centrifuged at 12000 RPM for 30 minutes and Nickel charged Ni-NTA resin (QIAgen) was added to the supernatant for an overnight incubation at 4° C., with 100 RPM agitation. After washing the resin with a buffer consisting of 20 mM Tris, 500 mM NaCl, 20 mM imidazole pH 7,9, the fusion 3A4.1 protein was eluted with the same buffer supplemented with 250 mM imidazole. The removal of the salt and imidazole was done by dialysis against PBS at 4° C. The protein concentration was determined with BCA protein assay reagent kit (Perce, Rockford, Ill.) and adjusted to 760 μg/ml.
  • To evaluate whether immunization with an epitope peptide sequence could confer protection against disease, groups of 6 female CBA/N (xid) mice (National Cancer Institute) are immunized subcutaneously three times at three-week intervals with affinity purified T7gene10 protein-His.Tag-3A4.1 fusion protein or, as control, with QuilA adjuvant alone in PBS. Twelve to fourteen days following the third immunization, the mice are challenged intravenously with S. pneumoniae WU2 strain or intranasally with P4241 strain. Samples of the S. pneumoniae challenge inoculum are plated on chocolate agar plates to determine the number of CFU and to verify the challenge dose. The challenge dose are approximalety 300 CFU. Deaths are recorded daily for a period of 14 days and on day 14 post-challenge, the surviving mice are sacrificed and blood samples tested for the presence of S. pneumoniae organisms. The 3A4.1 protein or other tested protein is said protective when the number of mice surviving the infection or the median number of days to death is significantly greater in the 3A4.1-immunized group compared to the control mock-immunized group.
    • EXAMPLE 8
  • This example illustrates the improvement of the antibody response to pneumococci using BVH-3 fragments and variants thereof.
  • The combined results obtained from studies of Mab reactivity with truncated gene products, epitope-expressing colonies and live intact pneumococci presented in examples 2, 3 and 6, allowed to delineate between surface-exposed and internal epitopes. The epitopes detected by Mabs that efficiently bound to pneumococci cells mapped to a region comprised between amino acid residues 223 to 1039 of BVH-3 described in SEQ ID NO 6. The existence of protective epitopes in the BVH-3-carboxyl half was confirmed by demonstrating that mice immunized with NEW1 molecule were protected from fatal infection with P4241 strain.
  • Gene sequence comparison revealed that in some strains, the region of BVH-3 encoding for amino acids 244 to 420 as described in SEQ ID N06 is absent thus suggesting the lack of utility of this sequence in vaccine to prevent disease caused by such strains (SEQ ID NO: 9 versus SEQ ID NO: 1). Further BVH-3 fragments or variants thereof were designed in the purpose to develop a universal highly effective vaccine that would target the immune response to ubiquitous surface-exposed protective epitopes. BVH-3 gene fragments designated NEW1 (encoding amino acid residues 472 to 1039 from SEQ ID NO: 6) and NEW40 (encoding amino acid residues 408 to 1039 from SEQ ID NO: 6) were amplified from the S. pneumoniae strain SP64 by PCR using pairs of oligonucleotides engineered for the amplification of the appropriate gene fragment. Each of the primers had a restriction endonuclease site at the 5′end, thereby allowing directional in-frame cloning of the amplified product into the digested plasmid vector. PCR-amplified products were digested with restriction endonucleases and ligated to linearized plasmid pET21 (Novagen) expression vector digested likewise. Oligonucleotide primers HAMJ489 (ccgaattccatatgcaaattgggcaaccgactc; NdeI) and HAMJ279 (cgccaagcttcgctatgaaatcagataaattc; HindIII) were used for the NEW 40 construction. Clones were first stabilized in E. coli DH5α before introduction into E. coli BL21 (λDE3) for expression of the truncated gene products. Variants from NEW1 and NEW40 were generated by mutagenesis using the Quickchange Site-Directed Mutagenesis kit from Stratagene and the oligonucleotides designed to incorporate the appropriate mutation. The presence of 6 histidine tag residues on the C-terminus of the recombinant molecules simplified the purification of the proteins by nickel chromatography. The following tables 12 and 13 describe the sequences of the primers used for the mutagenesis experiments and the variant gene products generated, respectively. Mutagenesis experiments using primer sets 39, 40, 46, 47 or 48 resulted in silent changes and were performed in the purpose of improving the expression of the desired gene or gene fragment since it was observed that during the course of expression, BVH-3 gene and fragments of, shorter secondary translation initiation products were coexpressed.
    TABLE 12
    List of PCR oligonucleotide primer sets used for site-directed mutagenesis on
    BVH-3 gene truncates
    Primer Primer SEQ Primer SEQUENCE
    set identification ID No 5′ - - - > 3′
    9 HAMJ513 177 GAATCAGGTTTTGTCATGAGTTCCGGAGACCACAATCATTATTTC
    HAMJ514
    178 GAAATAATGATTGTGGTCTCCGGAACTCATGACAAAACCTGATTC
    10 HAMJ515 179 GTCATGAGTTCCGGAGACTCCAATCATTATTTCTTCAAGAAGG
    HAMJ516
    180 CCTTCTTGAAGAAATAATGATTGGAGTCTCCGGAACTCATGAC
    11 HAMJ517 181 ATGAGTTCGGAGACTCCAATTCTTATTTCTTCAAGAAGGACTTG
    HAMJ518 182 CAAGTCCTTCTTGAAGAAATAAGAATTGGAGTCTCCGGAACTCAT
    14 CHAN51 183 GCGATTATTTATCCGTCTGGAGATCACCATCATGC
    CHAN52 184 GCATGATGGTGATCTCCAGACGGATAAATAATCGC
    17 CHAN53 185 CCGTCTGGAGATGGCCATCATGCAGATCCG
    CHAN54 186 CGGATCTGCATGATGGCCATCTCCAGACGG
    19 CHAN47 187 CCGCAGGGAGATAAGCGTCATGCAGATCCGATTG
    CHAN48 188 CAATCGGATCTGCATGACGCTTATCTCCCTGCGG
    20 CHAN55 189 CCGTCTGGAGATGGCACTCATGCAGATCCGATTG
    CHAN56 190 CAATCGGATCTGCATGAGTGCCATCTCCAGACGG
    22 CHAN57 191 CCGTCTGGAGATGGCACTTCTGCAGATCCGATTGATG
    CHAN58 192 CATCAATCGGATCTGCAGAAGTGCCATCTCCAGACGG
    23 HAMJ523 193 CCGCATGGAGATGGCCATCATGCAGATCCG
    HAMJ524
    194 CGGATCTGCATGATGGCCATCTCCATGCGG
    24 HAMJ526 195 GTCATGAGTCACGGAGACTCCAATCATTATTTCTTCAAGAAGG
    HAMJ527 196 CCTTCTTGAAGAAATAATGATTGGAGTCTCCGTGACTCATGAC
    25 HAMJ528 197 ATGAGTCACGGAGACCACAATTCTTATTTCTTCAAGAAGGACTTG
    HAMJ529 198 CAAGTCCTTCTTGAAGAAATAAGAATTGTGGTCTCCGTGACTCAT
    29 HAMJ569 199 TACCTCATTATGACTCTTACTCTAACATCAAATTTGAGTGGTTTG
    HAMJ570
    200 CAAACCACTCAAATTTGATGTTAGAGTAAGAGTCATAATGAGGTA
    30 HAMJ571 201 TACCTTCTTATGACCATTACTCTAACATCAAATTTGAGTGGTTTG
    HAMJ572 202 AAACCACTCAAATTTGATGTTAGAGTAATGGTCATAAGAAGGTA
    31 HAMJ573 203 AACGGTAGTTTAATCATACCTTCTAAAGACCATTACCATAACATC
    HAMJ574 204 GATGTTATGGTAATGGTCTTTAGAAGGTATGATTAAACTACCGTT
    32 HAMJ575 205 CGGTAGTTTAATCATACCTCATAAGGACTCTTACCATAACATCAAA
    HAMJ576 206 TTTGATGTTATGGTAAGAGTCCTTATGAGGTATGATTAAACTACCG
    33 HAMJ577 207 AACGGTAGTTTAATCATACCTGACCATTACCATAACATCAAATTTG
    HAMJ578 208 CAAATTTGATGTTATGGTAATGGTCAGGTATGATTAAACTACCGTT
    34 HAMJ579 209 AACGGTAGTTTAATCATACCTTACCATAACATCAAATTTGAGTGG
    HAMJ580 210 CCACTCAAATTTGATGTTATGGTAAGGTATGATTAAACTACCGTT
    35 HAMJ581 211 ACCGGTAGTTTAATCATACCTAACATCAAATTTGAGTGGTTTGAC
    HAMJ582 212 GTCAAACCACTCAAATTTGATGTTAGGTATGATTAAACTACCGTT
    37 HAMJ536 213 CCTATGTAACTCCACATATAACCCATAGCCACTGG
    HAMJ537 214 CCAGTGGCTATGGGTTATATGTGGAGTTACATAGG
    39 HAMJ550 215 CGTGAAAGTATTGTCGTAAATAAAGAAAAAAATGCG
    HAMJ551 216 CGCATTTTTTTCTTTATTTACGACAATACTTTCACG
    40 HAMJ586 217 CATGAAGAAGATGGTTACGGTTTCGATGCTAACCGTATTATCGCTGAAG
    HAMJ587 218 CTTCAGCGATAATACGGTTAGCATCGAAACCGTAACCATCTTCTTCTG
    41 HAMJ588 219 GAATCAGGTTTTGTCATGAGTGACCACAATCATTATTTCTTC
    HAMJ589 220 GAAGAAATAATGATTGTGGTCACTCATGACAAAACCTGATTC
    42 HAMJ590 221 GAAGATGAATCAGGTTTTGTCATGAGTAATCATTATTTCTTCAAG
    HAMJ591 222 CTTGAAGAAATAATGATTACTCATGACAAAACCTGATTCATCTTC
    43 HAMJ592 223 GAAGATGAATCAGGTTTTGTCATGAGTTATTTCTTCAAGAAGGAC
    HAMJ593
    224 GTCCTTCTTGAAGAAATAACTCATGACAAAACCTGATTCATCTTC
    44 HAMJ594 225 AAAATGCGATTATTTATCCGCACCATCATGCAGATCCGATTG
    HAMJ595 226 CAATCGGATCTGCATGATGGTGCGGATAAATAATCGCATTTT
    45 HAMJ600 227 AAAATGCGATTATTTATCCGGCAGATCCGATTGATGAACATAAAC
    HAMJ601 228 GTTTATGTTCATCAATCGGATCTGCCGGATAAATAATCGCATTTT
    46 HAMJ604 229 GATGCTAACCGTATAATCGCCGAAGACGAATCAGGTTTTGTCATG
    HAMJ605 230 CATGACAAAACCTGATTCGTCTTCGGCGATTATACGGTTAGCATC
    47 HAMJ606 231 CGCCGAAGACGAATCCGGCTTTGTAATGAGTCACGGAGACTCC
    HAMJ607 232 GGAGTCTCCGTGACTCATTACAAAGCCGGATTCGTCTTCGGCG
    48 HAMJ608 233 CATCTCATGAACAGGATTATCCCGGCAACGCCAAAGAAATGAAAG
    HAMJ609 234 CTTTCATTTCTTTGGCGTTGCCGGGATAATCCTGTTCATGAGATG
  • TABLE 13
    Lists of truncated variant BVH-3 gene products generated
    from S. pneumoniae SP64
    Gene/
    Protein Protein PCR primer set Gene used for
    designation SEQ ID NO Protein Identification* (ref. table 12) mutagenesis
    NEW1- 255 NEW1 39 NEW1
    mut1**
    NEW35A 256 NEW1 550-SGDGTS-555 14, 17, 20, 22 NEW1
    NEW42 349 NEW40 55-SGDSNS-60 144-SGDGTS-149 9, 10, 11, 14, NEW40
    17, 20, 22
    NEW49 350 NEW40 55-SGDHNH-60 9 NEW40
    NEW50 351 NEW40 55-SGDSNH-60 10 NEW49
    NEW51 352 NEW40 55-SGDHNH-60 144-SGDHHH-149 14 NEW49
    NEW52 353 NEW40 55-SGDSNH-60 144-SGDGHH-149 10, 17 NEW51
    NEW53 354 NEW40 55-HGDHNH-60 144-SGDHHH-149 14 NEW40
    NEW54 355 NEW40 55-SGDHNH-60 144-SGDGHH-149 17 NEW53
    NEW55 356 NEW1 550-HGDGHH-555 23 NEW1
    NEW56 357 NEW40 55-HGDSNH-60 144-SGDHHH-149 24 NEW53
    NEW56- 358 NEW56 40 NEW56
    mut2**
    NEW56- 359 NEW56 46, 47, 48 NEW56
    mut3**
    NEW57 360 NEW40 55-HGDHNS-60 144-SGDHHH-149 25 NEW53
    NEW63 361 NEW40 55-HGDSNH-60 144-HGDHHH-149 24 NEW40
    NEW64 362 NEW40 55-HGDHNS-60 144-HGDHHH-149 25 NEW40
    NEW65 363 NEW40 55-HGDSNH-60 144-HGDGHH-149 23 NEW63
    NEW66 364 NEW40 55-HGDHNS-60 144-HGDGHH-149 23 NEW64
    NEW76 365 NEW40 55-HGDHNS-60 144-SGDGHH-149 17 NEW64
    NEW105 366 NEW40 55-   -60 41, 42, 43 NEW40
    NEW106 367 New40 144-   -149 44, 45 NEW40
    NEW107 368 NEW40 55-   -60 144-   -149 44, 45 NEW105

    * The underlined amino acid residues represent the modification in protein sequence. Nucleotides/amino acid residues are deleted in NEW105, NEW106 and NEW107 constructs.

    ** silent mutation, i.e. the polypeptide is the same as New1.
  • Groups of 7 or 8 female BALB/c mice (Charles River) immunized as described earlier in example 1 were used for protection experiments against intranasal challenge with virulent S. pneumoniae P4241 strain. The mice were observed for 10 to 14 days post-infection. Data from Table 15 clearly indicate that the NEW35A molecule was equivalent to the parental NEW1 in term of protection. Interestingly, high survival rates where obtained for NEW40- and NEW56-immunized groups with 7 and 8 survivors out of 8 animals, respectively. Similarly, NEW25 comprising amino acid residues 233 to 1039 protected 7 out of 8 animals from lethal infection.
    TABLE 14
    Protection mediated by BVH-3 fragments or variants
    thereof in experimental pneumonia
    Ex-
    peri- Immu-
    ment nogen Alive:Dead Days to death post-infection
    1 Quil A 0:8 4, 4, 4, 4, 4, 4, 4, 4
    NEW 1 5:3 5, 7, 7, >14, >14, >14, >14, >14
    NEW 35A 5:2 9, 10, >14, >14, >14, >14, >14
    NEW 40 7:1 13, >14, >14, >14, >14, >14, >14, >14
    BVH-3M 4:4 7, 8, 10, 12, >14, >14, >14, >14
    2 Quil A 0:8 3, 3, 4, 4, 4, 4, 4, 4
    NEW 52 4:4 7, 7, 8, 9, >10, >10, >10, >10
    NEW56 8:0 8 X >10
    NEW 40 7:1 6, >10, >10, >10, >10, >10, >10, >10
    3 QuilA 0:8 3, 3, 4, 4, 4, 4, 4, 4
    NEW25 7:1 6, >13, >13, >13, >13, >13, >13, >13
  • Additionally, flow cytometry analyses of the binding capacity of the sera antibodies from the vaccinated animals revealed that NEW40 and NEW56 antibodies labelled live intact pneumococci more efficiently than antibodies raised to BVH-3M (Table 15).
    TABLE 15
    Binding of mouse sera antibodies at the surface of S. pneumoniae
    type
    3 strain WU2 as measured by flow cytometry.
    Fluorescene index
    Mean ±
    Antisera Experiment 1 Experiment 2 Experiment 3 SE
    BVH-3M 9.2 11.4 14.5 11.7 ± 1.5
    NEW1 11.5 10.1  nd* 10.8 ± 0.7
    NEW35A 14.3 12.9 nd 13.6 ± 0.7
    NEW40 20.4 19.1 20.2 19.9 ± 0.4
    NEW56 nd 16.7 20.2 18.5 ± 1.8
    NEW52 nd 16.6 19.3 18.0 ± 1.4
    Adjuvant 1.9 1.6 1.2  1.6 ± 0.2
    alone

    *nd: not done
  • Cytometry results are expressed as fluorescence index value where the fluorescence index is the median fluorescence value of pneumococci treated with test sera divided by the background fluorescence value of pneumococci treated with the fluorescein conjugate alone. In these flow cytometric assays, all sera were used at a dilution of 1:50 and the sera from mice immunized with BVH-3C fragment or QuilA adjuvant alone gave a value similar to the background value.
  • Altogether the protection and pneumococci antibody binding data indicate that vaccination using NEW1 or NEW40 molecules and variants thereof, directs the immune response to conserved protective surface-exposed epitopes.
    • EXAMPLE 9
  • This example describes the cloning and expression of a chimeric deletant BVH-11-2 gene encoding for a chimeric polypeptide corresponding to BVH-11-2 conserved protective surface-exposed epitopes present in most if not all S. pneumoniae strains.
  • BVH-11-2 gene fragments corresponding to 4 gene regions, were amplified by PCR using pairs of oligonucleotides engineered to amplify fragments originating from SEQ ID NO :5 spanning nucleotides 1662 to 1742, 1806 to 2153, 2193 to 2414 and 2484 to 2627 from S. pneumoniae strain Sp64 BVH-11-2 gene.
  • The primers used, HAMJ490-491, HAMJ492-HAMJ493, HAMJ494-HAMJ495, HAMJ496-HAMJ354 had a restriction endonuclease site at the 5′ end, thereby allowing directional in-frame cloning of the amplified product into the digested pET21b(+) plasmid vector (Table 16). PCR-amplified products were digested with restriction endonucleases and ligated to linearized plasmid pSL301 vector digested likewise except for the PCR-amplified fragment obtained with the primer pair HAMJ490-HAMJ491. The HAMJ490-HAMJ491 PCR-amplified product was purified from agarose gel using a QIAquick gel extraction kit from QIAgen (Chatsworth, Calif.) and ligated into PGEM-T plasmid vector without any prior restriction endonuclease digestion. The resultant plasmid constructs were confirmed by nucleotide sequence analysis. The recombinant plasmids containing each of the four were digested with restriction endonucleases corresponding with the 5′ end of each primer pair used for the PCR-amplification. The fragments were purified from agarose gel like described earlier and were all ligated to linearized plasmid pET21b (+) digested with the restriction enzymes NdeI and HindIII for the in-frame cloning of the four different regions of the BVH11-2 gene. Clones were first stabilized in E. coli DH5α before introduction into E. coli BL21 (λDE3) for expression of a chimeric pneumococcal protein molecule.
  • The resulting NEW43 gene sequence (SEQ ID No 257) is described in FIG. 33.
  • The deduced amino acid sequence of NEW43 protein (SEQ ID No 258). is described in FIG. 34.
    TABLE 16
    List of PCR oligonucleotide primers used to construct
    the NEW43, VP43S and NEW86
    SEQ Nucleotide Restriction
    Primer ID NO Sequence 5′ - 3′ position sites
    HAMJ490 259 ccgaattccatatgcaaat SEQ ID 5: NdeI
    tacctacactgatgatg 1662-1683
    HAMJ491 260 ggactagtatcaaagatat SEQ ID 5: SpeI
    aaccgtcttc 1742-1722
    HAMJ492 261 ggactagttggattaaaaa SEQ ID 5: SpeI
    agatagtttgtctg 1806-1830
    HAMJ493 262 ttcccgcggttcgacatag SEQ ID 5: SacII
    tacttgacagtcg 2153-2131
    HAMJ494 263 ttcccgcggaacgctagtg SEQ ID 5: SacII
    accatgttcg 2193-2212
    HAMJ495 264 cggggtaccaggaatttca SEQ ID 5: KpnI
    gcctcatctgtg 2414-2393
    HAMJ496 265 cccggtacccctagtatta SEQ ID 5: KpnI
    gacaaaatgctatggag 2484-2510
    HAMJ 65 cgccaagcttctgtatagg SEQ ID 5: HindIII
    354 agccggttgac 2627-2608
    HAMJ 266 ggatcccgggaggtatgat SEQ ID 5: SmaI
    583 taaactaccg 2039-2021
    HAMJ 267 catgcccgggaacatcaaa SEQ ID 5: SmaI
    584 tttgagtggtttgac 2058-2081
    HAMJ 268 cttgatcgacatatgttgg SEQ ID 5: NdeI
    610 caggcaagtacacaacag 1701-1722
  • TABLE 17
    List of truncated BVH-11-2 gene fragments generated
    from S. pneumoniae SP64 for the construction of NEW43
    Corresponding
    amino acid
    Gene residues
    fragment on SEQ ID Cloning
    PCR-primer sets designation NO: 8 vector
    HAMJ490-HAMJ491 NEW43a 517-543 pGEM-T
    HAMJ492-HAMJ493 NEW43b 565-680 pSL301
    HAMJ494-HAMJ495 NEW43c 694-767 pSL301
    HAMJ496-HAMJ354 NEW43d 791-838 pSL301
  • TABLE 18
    Properties of NEW86 and VP43S genes
    generated from NEW43 gene
    Gene/
    Protein
    PCR-primer sets designation Identification
    HAMJ610-HAMJ354 VP43S NEW43 C′ end corresponding to
    residues 15-272) (SEQ ID NO:
    374)
    HAMJ490-HAMJ583 NEW86 NEW43 109-_PG_-114
    HAMJ584-HAMJ354 (SEQ ID NO: 375)
  • NEW43-derived molecules designated VP43S and NEW86 were generated from gene amplification and cloning experiments using PCR primers described in Tables 16 and 18 and pET21 expression plasmid vector. Variants from NEW43 were generated by mutagenesis using the Quickchange Site-Directed Mutagenesis kit from Stratagene and the oligonucleotides designed to incorporate the appropriate mutation. The presence of 6 histidine tag residues on the C-terminus of the recombinant molecules simplified the purification of the proteins by nickel chromatography. The following tables 19 and 20 describe the sequences of the primers used for the mutagenesis experiments and the NEW43 variant gene products generated, respectively.
    TABLE 19
    List of PCR oligonucleotide primer sets used for site-
    directed mutagenesis on NEW43 gene
    Primer Primer SEQ Primer SEQUENCE
    set identification ID NO 5′ - - - 3′
    1 HAMJ497 269 AACGGTAGTTTAATCATACCTTCTTATGACCATTACCATAACATC
    HAMJ498 270 GATGTTATGGTAATGGTCATAAGAAGGTATGATTAAACTACCGTT
    2 HAMJ499 271 AATCATACCTTCTTATGACTCTTACCATAACATCAAATTTGAGTG
    HAMJ500 272 CACTCAAATTTGATGTTATGGTAAGAGTCATAAGAAGGTATGATT
    3 HAMJ501 273 TACCTTCTTATGACTCTTACTCTAACATCAAATTTGAGTGGTTTG
    HAMJ502 274 CAAACCACTCAAATTTGATGTTAGAGTAAGAGTCATAAGAAGGTA
    26 HAMJ530 275 AATCATACCTCATTATGACTCTTACCATAACATCAAATTTGAGTG
    HAMJ531 276 CACTCAAATTTGATGTTATGGTAAGAGTCATAATGAGGTATGATT
    27 HAMJ532 277 TACCTCATTATGACCATTACTCTAACATCAAATTTGAGTGGTTTG
    HAMJ533 278 CAAACCACTCAAATTTGATGTTAGAGTAATGGTCATAATGAGGTA
    29 HAMJ569 279 TACCTCATTATGACTCTTACTCTAACATCAAATTTGAGTGGTTTG
    HAMJ570
    280 CAAACCACTCAAATTTGATGTTAGAGTAAGAGTCATAATGAGGTA
    30 HAMJ571 281 TACCTTCTTATGACCATTACTCTAACATCAAATTTGAGTGGTTTG
    HAMJ572 282 AAACCACTCAAATTTGATGTTAGAGTAATGGTCATAAGAAGGTA
    31 HAMJ573 283 AACGGTAGTTTAATCATACCTTCTAAAGACCATTACCATAACATC
    HAMJ574
    284 GATGTTATGGTAATGGTCTTTAGAAGGTATGATTAAACTACCGTT
    32 HAMJ575 285 CGGTAGTTTAATCATACCTCATAAGGACTCTTACCATAACATCAAA
    HAMJ576 286 TTTGATGTTATGGTAAGAGTCCTTATGAGGTATGATTAAACTACCG
    33 HAMJ577 287 AACGGTAGTTTAATCATACCTGACCATTACCATAACATCAAATTTG
    HAMJ578 288 CAAATTTGATGTTATGGTAATGGTCAGGTATGATTAAACTACCGTT
    34 HAMJ579 289 AACGGTAGTTTAATCATACCTTACCATAACATCAAATTTGAGTGG
    HAMJ580 290 CCACTCAAATTTGATGTTATGGTAAGGTATGATTAAACTACCGTT
    35 HAMJ581 291 ACCGGTAGTTTAATCATACCTAACATCAAATTTGAGTGGTTTGAC
    HAMJ582 292 GTCAAACCACTCAAATTTGATGTTAGGTATGATTAAACTACCGTT
  • TABLE 20
    List of NEW43 variant gene products
    generated from S. pneumoniae SP64
    Polypep- PCR primer Gene used
    Polypeptide tide Polypeptide set (ref. for
    designation SEQ ID NO identification* table 22) mutagenesis
    NEW60 293 NEW43 109-SYDHYH-114 1 NEW43
    NEW61 294 NEW43 109-HYDSYH-114 26 NEW43
    NEW62 295 NEW43 109-HYDHYS-114 27 NEW43
    NEW80 296 NEW43 109-SYDSYH-114 2 NEW60
    NEW81 297 NEW43 109-SYDSYS-114 3 NEW80
    NEW82
    298 NEW43 109-HYDSYS-114 29 NEW61
    NEW83
    299 NEW43 109-SYDHYS-114 30 NEW60
    NEW84
    300 NEW43 109-SKDHYH-114 31 NEW60
    NEW85
    301 NEW43 109-HKDSYH-114 32 NEW61
    NEW88D1 302 NEW43 109-  DHYH-114 33 NEW43
    NEW88D2
    303 NEW43 109-    YH-114 34 NEW88D1
    NEW88 304 NEW43 109-      -114 35 NEW88D2

    *The underlined amino acid residues represent the modification in protein sequence. Nucleotides/amino acid residues are deleted in NEW88D1, NEW88D2 and NEW88 constructs.
  • Groups of 7 or 8 female BALB/c mice (Charles River) immunized as described earlier in example 1 were used for protection experiments against intranasal challenge with virulent S. pneumoniae P4241 strain. Data from Table 21 clearly indicate that NEW 19, NEW43 and variants thereof provided protection against experimental pneumonia.
    TABLE 21
    Protection mediated by NEW19 and NEW43 fragments or
    variants thereof in experimental pneumonia
    Experiment Immunogen Alive:Dead Median day alive
    1 Quil A 0:8 4, 4, 4, 4, 4, 4, 4, 5
    NEW 19 7:1 5, 7X >14
    NEW 43 8:0 8X >14
    2 Quil A 0:8 4, 4, 4, 4, 4, 5, 5, 5
    NEW 43 7:1 8, 7X >14
    NEW 80 6:2 5, 6, 6 X >14
    NEW 83 6:2 8, 10, 6 X >14
    3 Quil A 0:8 4, 4, 4, 4, 5, 5, 5, 5
    NEW 43 7:1 5, 7X >8
    NEW 88D1 5:3 5, 6, 6, 6 X >8
    NEW 88D2 5:3 6, 6, 6, 6 X >8
    NEW 88 7:1 6, 7X >8
    3 Quil A 0:8 4, 4, 4, 5, 5, 5, 5, 6
    NEW 60 8:0 8 X >8
    NEW 84 8:0 8 X >8
    NEW 85 5:3 5, 7, 7, 5 X >8
    NEW 86 5:3 5, 6, 6, 5 X >8
    • EXAMPLE 10
  • This example describes the cloning and expression of chimeric genes encoding for a chimeric protein corresponding to the carboxy-terminal region of BVH-3 or variants thereof in fusion, at either the carboxyl end or the amino end, to NEW43 or variants thereof. The chimeric genes comprising a BVH-3 truncate variant gene and a NEW43 or NEW43 variant gene have been designed following the procedure described in example 1. The polypeptides encoded by these chimeric genes are listed in the table 22. Briefly, gene fragments to be included in a chimeric gene were amplified by PCR using pairs of oligonucleotides engineered so that the primers had a restriction endonuclease site at the 5′ end, thereby allowing directional in-frame cloning of the amplified product into digested plasmid vectors (Table 23 and Table 24). PCR-amplified products were digested with restriction endonucleases and ligated to linearized plasmid pSL301 vector. The resultant plasmid construct were confirmed by nucleotide sequence analysis. The recombinant pSL301 plasmids containing a PCR product were redigested with the same endonuclease restriction enzyme for the obtention of the DNA inserts. The resulting insert DNA fragments were purified and inserts corresponding to a given chimeric gene were ligated into pURV22-NdeI vector for the generation of a chimeric gene. The expressed recombinant proteins were purified from supernatant fractions obtained from centrifugation of sonicated heat-induced E. coli cultures using multiple chromatographic purification steps.
    TABLE 22
    List of polypeptides encoded by chimeric genes
    comprising a BVH-3 truncate variant gene and
    a NEW43 or NEW43 variant gene
    Polypeptide
    designation SEQ ID NO Identification
    VP89 369 M-New56-GP-New43*
    VP90 370 M-New43-GP-New56
    VP91 371 M-New52-GP-New43
    VP92 372 M-New43-GP-New52
    VP93 373 M-New56-GP-New60
    VP94 332 M-New60-GP-New56
    VP108 333 M-New56-GP-New88
    VP109 334 M-New88-GP-New56
    VP110 335 M-New60-GP-New105
    VP111 336 M-New60-GP-New107
    VP112 337 M-New88-GP-New105
    VP113 338 M-New88-GP-New107
    VP114 339 M-New80-GP-New105
    VP115 340 M-New80-GP-New107
    VP116 341 M-New83-GP-New105
    VP117 342 M-New83-GP-New107
    VP119 343 M-New43S-GP-New105
    VP120 344 M-New43S-GP-New107
    VP121 345 M-New80S-GP-New105
    VP122 346 M-New80S-GP-New107
    VP123 347 M-New88S-GP-New105
    VP124 348 M-New88S-GP-New107

    *Encoded amino acids for the chimeras are expressed as the gene product, additional amino acid residues were added. M is methionine, G is glycine and P is proline.
  • TABLE 23
    List of PCR oligonucleotide primer pairs designed
    for the generation of the chimeric genes encoding
    the polypeptides listed in Table 22.
    Corresponding
    position of the
    Gene used for gene fragment on
    Primer PCR-primer PCR the chimeric
    set identification amplification protein molecule
    49 HAMJ490-HAMJ471 Variant New43 N-terminal
    50 HAMJ564-HAMJ556 Variant New43 C-terminal
    51 HAMJ489-HAMJ359 Variant New40 N-terminal
    52 HAMJ559-HAMJ557 Variant New40 C-terminal
    53 HAMJ610-HAMJ471 Variant New43S N-terminal
  • TABLE 24
    List of PCR oligonucleotide primers designed for
    the generation of the chimeric genes encoding the
    polypeptides listed in Table 22.
    SEQ
    ID Restriction
    Primer NO Sequence 5′ - 3′ site
    HAMJ490 259 ccgaattccatatgcaaattaccta NdeI
    cactgatgatg
    HAMJ471 168 atatgggcccctgtataggagccgg ApaI
    ttgactttc
    HAMJ564 327 atatgggccccaaattacctacact ApaI
    gatgatgagattcagg
    HAMJ556 328 ataagaatgcggccgcctactgtat NotI
    aggagccggttgactttc
    HAMJ489 329 ccgaattccatatgcaaattgggca NdeI
    accgactc
    HAMJ359 173 tcccgggccccgctatgaaatcaga ApaI
    taaattc
    HAMJ559 330 atatgggccccaaattgggcaaccg ApaI
    actc
    HAMJ354 65 cgccaagcttctgtataggagccgg HindIII
    ttgac
    HAMJ610 268 cttgatcgacatatgttggcaggca NdeI
    agtacacaacag
    HAMJ557 331 ataagaatgcggccgcttacgctat NotI
    gaaatcagataaattc
    HAMJ279 35 cgccaagcttcgctatgaaatcaga HindIII
    taaattc

Claims (7)

1-34. (canceled)
35. A polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO. 332 or a polynucleotide that hybridizes under stringent conditions to the complement of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO. 332, wherein the polypeptide elicits an immune response when administered to an individual.
36. A polynucleotide encoding a polypeptide which has the amino acid sequence of SEQ ID NO. 332.
37. A polynucleotide that hybridizes under stringent conditions to the complement of polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO. 332.
38. A polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO. 332 or a polypeptide having at least 85% sequence similarity to SEQ ID NO. 332, wherein the polypeptide elicits an immune response when administered to an individual.
39. A vector comprising the polynucleotide of claim 32.
40. A method of immunizing an individual against streptococcus infection comprising administering the vector of claim 39 to the individual, wherein the polypeptide is expressed and elicits an immune response.
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