# Patent

## Title
Streptococcus antigens

## Abstract

Streptococcus polypeptides and polynucleotides encoding them are disclosed. The 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.

## Background
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







TABLE D, E and F Variants and Epitope of BVH-11-










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







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'Université 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







TABLE 1







List of PCR oligonucleotide primers












SEQ






ID

Nucleotide
Restriction


Primer
NO
Sequence 5′–3′
position
sites














OCRR
25
cagtagatctgtgcct
SEQ ID 1:
BglII


479

atgcactaaac
61–78





SEQ ID 9:





 1–18


OCRR
26
gatctctagactactg
SEQ ID 2:
XbaI


480

ctattccttacgctat
4909–4887




g





SEQ ID 9:





2528–2519


OCRR
27
atcactcgagcattac
SEQ ID 1:
XhoI


497

ctggataatcctgt
1525–1506


OCRR
28
ctgctaagcttatgaa
SEQ ID 1:
HindIII


498

agatttagat
1534–1548


OCRR
29
gatactcgagctgcta
SEQ ID 2:
XhoI


499

ttccttac
4906–4893


HAMJ
30
gaatctcgagttaagc
SEQ ID 1:
XhoI


172

tgctgctaattc
675–661


HAMJ
31
gacgctcgagcgctat
SEQ ID 1:
XhoI


247

gaaatcagataaattc
3117–3096


HAMJ
32
gacgctcgagggcatt
SEQ ID 1:
XhoI


248

acctggataatcctgt
1527–1501




tcatg


HAMJ
33
cagtagatctcttcat
SEQ ID 2:
BgIII


249

catttattgaaaagag
1749–1771




g


HAMJ
34
ttatttcttccatatg
SEQ ID 1:
NdeI


278

gacttgacagaagagc
1414–1437




aaattaag


HAMJ
35
cgccaagcttcgctat
SEQ ID 1:
HindIII


279

gaaatcagataaattc
3117–3096


HAMJ
36
cgccaagcttttccac
SEQ ID 1:
HindIII


280

aatataagtcgattga
2400–2377




tt


HAMJ
37
ttatttcttccatatg
SEQ ID 1:
NdeI


281

gaagtacctatcttgg
2398–2421




aaaaagaa


HAMJ
38
ttatttcttccatatg
SEQ ID 1:
NdeI


300

gtgcctatgcactaaa
62–82




ccagc


HAMJ
39
ataagaatgcggccgc
SEQ ID 1:
NotI


313

ttccacaatataagtc
2400–2377




gattgatt


OCRR
40
cagtagatctgtgctt
SEQ ID 3:
BglII


487

atgaactaggtttgc
58–79


OCRR
41
gatcaagcttgctgct
SEQ ID 4:
HindIII


488

acctttacttactctc
2577–2556


HAMJ
42
ctgagatatccgttat
SEQ ID 3:
EcoRV


171

cgttcaaacc
1060–1075


HAMJ
43
ctgcaagcttttaaag
SEQ ID 3:
HindIII


251

gggaataatacg
1059–1045


HAMJ
44
cagtagatctgcagaa
SEQ ID 3:
BglII


264

gccttcctatctg
682–700


HAMJ
45
tcgccaagcttcgtta
SEQ ID 3:
HindIII


282

tcgttcaaaccattgg
1060–1081




g


HAMJ
46
ataagaatgcggccgc
SEQ ID 3:
NotI


283

cttactctcctttaat
2520–2492




aaagccaatagtt


HAMJ
47
catgccatggacattg
SEQ ID 3:
NcoI


284

atagtctcttgaaaca
856–880




gc


HAMJ
48
cgccaagcttcttact
SEQ ID 3:
HindIII


285

ctcctttaataaagcc
2520–2494




aatag


HAMJ
49
cgacaagcttaacatg
SEQ ID 3:
HindIII


286

gtcgctagcgttacc
2139–2119





SEQ ID 5:





2210–2190


HAMJ
50
cataccatgggccttt
SEQ ID 3:
NcoI


287

atgaggcacctaag
2014–2034


HAMJ
51
cgacaagcttaagtaa
SEQ ID 3:
HindIII


288

atcttcagcctctctc
2376–2353




ag


HAMJ
52
gataccatggctagcg
SEQ ID 3:
NcoI


289

accatgttcaaagaa
2125–2146


HAMJ
53
cgccaagcttatcatc
SEQ ID 3:
HindIII


290

cactaacttgacttta
1533–1508




tcac


HAMJ
54
cataccatggatattc
SEQ ID 3:
NcoI


291

ttgccttcttagctcc
1531–1554




g


HAMJ
55
catgccatggtgctta
SEQ ID 3:
NcoI


301

tgaactaggtttgc
59–79


HAMJ
56
cgccaagctttagcgt
SEQ ID 3:
HindIII


302

taccaaaaccattatc
2128–2107


HAMJ
57
gtattagatctgttcc
SEQ ID 5:
BglII


160

tatgaacttggtcgtc
172–196




acca


HAMJ
58
cgcctctagactactg
SEQ ID 5:
XbaI


186

tataggagccgg
2613–2630


HAMJ
59
catgccatggaaaaca
SEQ ID 5:
NcoI


292

tttcaagccttttacg
925–948




tg


HAMJ
60
cgacaagcttctgtat
SEQ ID 5:
HindIII


293

aggagccggttgactt
2627–2604




tc


HAMJ
61
catgccatggttcgta
SEQ ID 5:
NcoI


294

aaaataaggcagacca
2209–2232




ag


HAMJ
62
catgccatggaagcct
SEQ ID 5:
NcoI


297

attggaatgggaag
793–812


HAMJ
63
catgccatggaagcct
SEQ ID 5:
NcoI


352

attggaatgggaagc
793–813


HAMJ
64
cgccaagcttgtaggt
SEQ ID 5:
HindIII


353

aatttgcgcatttgg
1673–1653


HAMJ
65
cgccaagcttctgtat
SEQ ID 5:
HindIII


354

aggagccggttgac
2627–2608


HAMJ
66
catgccatggatattc
SEQ ID 5:
NcoI


355

ttgccttcttagctcc
1603–1624


HAMJ
67
ttatttcttccatatg
SEQ ID 1:
NdeI


404

catggtgatcatttcc
1186–1207




attaca


HAMJ
68
gatgcatatgaatatg
SEQ ID 1:
NdeI


464

caaccgagtcagttaa
697–720




gc


HAMJ
69
gatgctcgagagcatc
SEQ ID 1:
XhoI


465

aaatccgtatccatc
1338–1318


HAMJ
70
gatgcatatggatcat
SEQ ID 1:
NdeI


466

ttccattacattcca
1192–1212


HAMJ
71
gacaagcttggcatta
SEQ ID 1:
HindIII


467

cctggataatcctg
1527–1507


HAMJ
72
catgccatggaagcct
SEQ ID 5:
NcoI


352

attggaatgggaagc
793–813


HAMJ
73
ataagaatgcggccgc
SEQ ID 1:
NotI


470

cgctatgaaatcagat
3096–3117




aaattc


HAMJ
168
atatgggcccctgtat
SEQ ID 5:
Apa I


471

aggagccggttgactt
2626–2604




tc


HAMJ
169
atatgggcccaatatg
SEQ ID 1:
Apa I


472

caaccgagtcagttaa
720–697




gc


HAMJ
170
atatgggcccaacatg
SEQ ID 3:
Apa I


350

gtcgctagcgttacc
2139–2119


HAMJ
171
tcccgggcccgacttg
SEQ ID 1:
Apa I


351

acagaagagcaaatta
1414–1437




ag


HAMJ
172
catgccatgggacttg
SEQ ID 1:
NcoI


358

acagaagagcaaatta
1415–1437




ag


HAMJ
173
tcccgggccccgctat
SEQ ID 1:
Apa I


359

gaaatcagataaattc
3116–3096


HAMJ
174
atatgggcccgacatt
SEQ ID 3:
Apa I


403

gatagtctcttgaaac
856–880




agc


HAMJ
175
cgccaagcttaacatg
SEQ ID 3:
HindIII


361

gtcgctagcgttacc
2139–2119


HAMJ
176
atatgggccccttact
SEQ ID 3:
Apa I


483

ctcctttaataaagcc
2520–2494




aatag









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 DH5α 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
No 6)
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–


NEW 13


HAMJ283 (NEW 13)


HAMJ284–HAMJ350
NEW17
Chimera*
M- NEW 5 -GP -
pET 21 d (+)


(NEW 5) HAMJ351–


NEW 1


HAMJ279 (NEW 1)


HAMJ358–HAMJ359
NEW20
Chimera*
M- NEW 1 -GP -
pET 21 d (+)


(NEW 1) HAMJ403–


NEW 5


HAMJ361 (NEW 5)


HAMJ292–HAMJ471
NEW26
Chimera*
M- NEW 10 -GP -
pET 21 d (+)


(NEW 10) HAMJ472–


NEW 25


HAMJ470 (NEW 25)


HAMJ355–HAMJ471
NEW27
Chimera*


M- NEW 19 -GP -
pET 21 d (+)


(NEW 19) HAMJ472–


NEW 25


HAMJ470 (NEW 25)


HAMJ292–HAMJ471
NEW28
Chimera*
M- NEW 10 -GP -
pET 21 d (+)


(NEW 10) HAMJ351–


NEW 1


HAMJ279 (NEW 1)


HAMJ284–HAMJ350
NEW29
Chimera*
M- NEW 5 -GP -
pET 21 d (+)


(NEW 5) HAMJ472–


NEW 25


HAMJ470 (NEW 25)


HAMJ284–HAMJ483
NEW30
Chimera*
M- NEW 4 -GP -
pET 21 d (+)


(NEW 4) HAMJ472–


NEW 25


HAMJ470 (NEW 25)


HAMJ284–HAMJ483
NEW31
Chimera*
M- NEW 4 -GP -
pET 21 d (+)


(NEW 4) HAMJ351–


NEW 1


HAMJ279 (NEW 1)


HAMJ355–HAMJ471
NEW32
Chimera*
M- NEW 19 -GP -
pET 21 d (+)


(NEW 19) HAMJ351–


NEW 1


HAMJ279 (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-11M 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 His•Tag-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


Experiment
Immunogen
Alive:Dead
post-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









Gene products tested



















Mabs










BVH-



(IgG
BVH-
BVH-
BVH-
BVH-
NEW
NEW
NEW
NEW
NEW
NEW
3
BVH-


isotype)
3M
3AD
3B
3C
1
2
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























Mabs














BVH-



(IgG
BVH-
BVH-
BVH-
BVH-
NEW
NEW
NEW
NEW
NEW
NEW
New
New
New
New
11-
BVH-


isotype)
11M
11A
11B
11C
5
6
7
8
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/







Protein
Nucleotide
Amino acid

SEQ 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
7G11.7
SEQ ID 1:
SEQ ID 6:
GIQAEQIVIKITDQGYVTSHGDHYHYYNGKVPYDALFSEEL
12




193–316
 64–105
L


 


H11-7G11
7G11.9
SEQ ID 1:
SEQ ID 6:
TAYIVRHGDHFHYIPKSNQIGQPTLPNNSLATPSPSLPI
13




1171–1284
390–428


 


H3-4D3
4D3.4
SEQ ID 1:
SEQ ID 6:
TSNSTLEEVPTVDPVQEKVAKFAESYGMKLENVLFN
14




2565–2670
855–890


 


HN1-8E3
8E3.1
SEQ ID 1:
SEQ ID 6:
MDGTIELRLPSGEVIKKNLSDFIA
15




3004–3120
1016–1039


 


HN1-1G2
1G2.2
SEQ ID 1:
SEQ ID 6:
YGLGLDSVIFNMDGTIELRLPSGEVIKKNLSDFIA
16




3017–3120
1005–1039


 


HN1-10C12
10C12.7
SEQ ID 1:
SEQ ID 6:
PALEEAPAVDPVQEKLEKFTASYGLGLDSVIFNMDGTIELR
17




2936–3120
 983–1039
LPSGEVIKKNLSDFIA


 


HN1-14F6
14F6.3
SEQ ID 1:
SEQ ID 6:
KVEEPKTSEKVEKEKLSETGNSTSNSTLEEVPTVDPVQEK
18




2501–2618
833–872


 


HN1-12D8
B12D8.2
SEQ ID 1:
SEQ ID 6:
MKDLDKKIEEKIAGIMKQYGVKRESIVVNKEKNAIIYPHGD
19




1433–1767
512–589
HHHADPIDEHKPVGIGHSHSNYELFKPEEGVAKKEGN


 


H3V-7F4
7F4.1
SEQ ID 1:
SEQ ID 6:
AIIYPHGDHHHADPIDEHKPVGIGHSHSNYELFKPEEGVAK
20




1633–1785
545–595
KEGNKVYTGE


 


H112-10D7
10D7.5
SEQ ID 5:
SEQ ID 8:
IQVAKLAGKYTTEDGYIFDPRDITSDEGD
21




1685–1765
525–553


 


H112-10G9
10G9.3
SEQ ID 5:
SEQ ID 8:
DHQDSGNTEAKGAEAIYNRVKAAKKVPLDRMPYNLQYTVEV
22




1893–2150
594–679
KNGSLIIPHYDHYHNIKFEWFDEGLYEAPKGYSLEDLLATV






KYYV


 


H112-10A2
10A2.2
SEQ ID 5:
SEQ ID 8:
DHQDSGNTEAKGAEAIYNRVKAAKKVPLDRMPYNLQYTVEV
22




1893–2150
594–679
KNGSLIIPHYDHYHNIKFEWFDEGLYEAPKGYSLEDLLATV






KYYV


 


H11B-11B8
B11B8.1
SEQ ID 5:
SEQ ID 8:
DHQDSGNTEAKGAEAIYNRVKAAKKVPLDRMPYNLQYTVEV
22




1893–2150
594–679
KNGSLIIPHYDHYHNIKFEWFDEGLYEAPKGYSLEDLLATV






KYYV


 


H11B-11B8
11B8.4
SEQ ID 5:
SEQ ID 8:
GLYEAPKGYSLEDLLATVKYYVEHPNERPHSDNGFGNASDH
23




2085–2217
658–698


 


H112-3A4
3A4.1
SEQ ID 5:
SEQ ID 8:
VENSVINAKIADAEALLEKVTDPSIRQNAMETLTGLKSSLL
24




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














Sera

BVH-
S.


Rabbit
Immunogen
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-3AD
Preimmune
<100
NT
NT



(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.
1 h before
0:5
2, 2, 2, 2, 2


meningitidis
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
Alive:
Days to death


preparation
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-11 and/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.










Exper-

Alive:
Days to death


iment
Mab
Dead
post-infection





1
H112 3A4 + H112-10G9
6:0
6X >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
6X >10



None
0:6
2, 2, 2, 2, 2, 2


3
H112-10D7 + H11B-11B8
6:0
6X >10



H112-10G9 + H11B-15G2
3:3
2, 6, 6, 3X >10



None
0:6
2, 2, 2, 2, 2, 2


4
H112-10G9 + H112-10D7
5:0
5X >11



None
0:5
2, 2, 2, 2, 2


5
H112-10G9 + H11-10B8
4:1
8, 4X >14



H112-10G9 + H11B-7E11
5:0
5X >14



None
0:3
1, 2, 2


6
H112-10G9 + H11B-1C9
4:1
4, 4X >14



None
0:3
2, 2, 2


7
H112-10C5 + H11B-13D5
5:0
5X >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



Mabs
Protection
Mab-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 λDE3 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 T7 gene 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 NO6 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 ID
Primer SEQUENCE


set
identification
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










Experi-





ment
Immunogen
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



NEW 56
8:0
8 X >10



NEW 40
7:1
6, >10, >10, >10, >10, >10, >10,





>10


3
Quil A
0:8
3, 3, 4, 4, 4, 4, 4, 4



NEW 25
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.









Fluorescence index












Experiment
Experiment
Experiment



Antisera
1
2
3
Mean ± 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 ID

Nucleotide
Restriction


Primer
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


 


HAMJ354
65
cgccaagcttctgtatagg
SEQ ID 5:
HindIII




agccggttgac
2627–2608


 


HAMJ583
266
ggatcccgggaggtatgat
SEQ ID 5:
SmaI




taaactaccg
2039–2021


 


HAMJ584
267
catgcccgggaacatcaaa
SEQ ID 5:
SmaI




tttgagtggtttgac
2058–2081


 


HAMJ610
268
cttgatcgacatatgttgg
SEQ ID 5:
NdeI




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 the NEW43 variant gene products generated, respectively.







TABLE 19







List of PCR oligonucleotide primer sets used for site-directed


mutagenesis on NEW43 gene












SEQ



Primer
Primer
ID
Primer SEQUENCE


set
identification
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












Poly-

PCR




pep-

primer



tide

set



SEQ

(ref.
Gene used


Polypeptide
ID
Polypeptide
table
for 


designation
NO
identification*
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 NEW 19 and NEW 43 fragments or


variants thereof in experimental pneumonia












Exper-

Alive:




iment
Immunogen
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, 6X >14




NEW 83
6:2
8, 10, 6X >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, 6X >8




  NEW 88D2
5:3
6, 6, 6, 6X >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
8X >8




NEW 84
8:0
8X >8




NEW 85
5:3
5, 7, 7, 5X >8




NEW 86
5:3
5, 6, 6, 5X >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















VP 89
369
M-New56 -GP- New43*



VP 90
370
M-New43 -GP- New56



VP 91
371
M-New52 -GP- New43



VP 92
372
M-New43 -GP- New52



VP 93
373
M-New56 -GP- New60



VP 94
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




Gene used for
fragment on the


Primer
PCR-primer
PCR
chimeric protein


set
identification
amplification
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

1. An isolated 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.

2. An isolated polypeptide which has the amino acid sequence of SEQ ID NO:332.

3. A vaccine composition comprising a polypeptide according to claim 1 and a pharmaceutically acceptable carrier, diluent or adjuvant.

4. A vaccine composition comprising a polypeptide according to claim 2 and a pharmaceutically acceptable carrier, diluent or adjuvant.

5. A method of treating or preventing Streptococcus pneumoniae infection in an animal comprising administering to the animal a therapeutically or prophylactically effective amount of a vaccine composition according to claim 3.

6. A method of treating or preventing Streptococcus pneumoniae infection in an animal comprising administering to the animal a therapeutically or prophylactically effective amount of a vaccine composition according to claim 4.