AU2007207883A1 - Streptococcus antigens - Google Patents

Description

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION Standard Patent Applicant: ID Biomedical Corporation Invention Title: STREPTOCOCCUS ANTIGENS The following statement is a full description of this invention, including the best method for performing it known to us: la STREPTOCOCCUS ANTIGENS SFIELD 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 00 5 which may be useful for prophylaxis, diagnostic or treatment of streptococcal infection.

The entire disclosure in the complete specification of our Australian Patent Application SNo. 2001270381 is by this cross-reference incorporated into the present specification.

BACKGROUND OF THE INVENTION All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

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 2 5 morbidity and mortality associated with S. pneumoniae disease. Such vaccines would also potentially be useful to prevent otitis media in infants and young children.

N \Mcl ine\Cases\Palncn\48OO-48999\P48 171 AU\Spcis\P48171 AU Spccification 2007-5-8 doc 15108/07 lb O Efforts to develop a pneumococcal vaccine have generally concentrated on generating immune responses to the pneumococcal capsular polysaccharide. More than Z pneumococcal capsular serotypes have been identified on the basis of antigenic N differences. The currently available pneumococcal vaccine, comprising 23 capsular polysaccharides that most frequently caused disease, has significant shortcomings Cc related primarily to the poor immunogenicity of some capsular polysaccharides, the 00 OO diversity of the serotypes and the differences in the distribution of serotypes over time, Sgeographic areas and age 0 Os N Melhourc\Cases\Pscrn\48000.48999\P48171 AU\Spc~is\P48 171 AU Spcification 2007.5.8 do 15/08/07 1 groups. In particular, the failure of existing vaccines and 0 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 C(f pneumococcal protein antigen, either by itself or in 00 00 combination with additional components, offers the possibility of a protein-based pneumococcal vaccine.

0 PCT WO 98/18930 published May 7, 1998 entitled "Streptococcus C(N 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 polynucleotide encoding a polypeptide having at least identity to a second polypeptide chosen from: table A, B, D, E or H; O a polynucleotide encoding a polypeptide having at least C 95% identity to a second polypeptide chosen from: table A, B, D, E or H; 5 a polynucleotide encoding a polypeptide having an amino IN sequence chosen from table A, B, D, E or H; or fragments, analogs or derivatives thereof; a polynucleotide encoding a polypeptide chosen from: 00 table A, B, D, E or H; 00 0. 10 a polynucleotide encoding a polypeptide capable of e generating antibodies having binding specificity for a polypeptide having a sequence chosen from: table A, B, D, SE or H; a polynucleotide encoding an epitope bearing portion of a polypeptide chosen from table A, B, D, E or H; and a polynycleotide complementary to a polynucleotide in or 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 Figure 1 is the DNA sequence of SP64 BVH-3 gene; SEQ ID NO: 1 Figure 2 is a DNA sequence containing the complete SP64 BVH-3 gene at nucleotides 1777 to 4896; SEQ ID NO: 2 Figure 3 is the DNA sequence of SP64 BVH-11 gene; SEQ ID NO: 3 Figure 4 is a DNA sequence containing the complete SP64 BVH-11 gene at nucleotides 45 to 2567; SEQ ID NO: 4 O Figure 5 is a DNA sequence containing the complete SP64 BVH- C 11-2 gene at nucleotides 114 to 2630; SEQ ID NO: 5 Figure 6 is the amino acid sequence of SP64 BVH-3 polypeptide; \D SEQ ID NO: 6 Figure 7 is the amino acid sequence of SP64 BVH-11 00 polypeptide; SEQ ID NO: 7 00 00 SFigure 8 is the amino acid sequence of SP64 BVH-11-2 polypeptide; SEQ ID NO: 8 Figure 9 is the DNA sequence of SP63 BVH-3 gene; SEQ ID NO:9 Figure 10 is the amino acid sequence of SP63 BVH-3 polypeptide; SEQ ID NO: Figure 11 is the amino acid sequence of 4D4.9 polypeptide;

SEQ

ID NO: 11 Figure 12 is the amino acid sequence of 7G11.7 polypeptide; SEQ ID NO: 12 Figure 13 is the amino acid sequence of 7G11.9 polypeptide; SEQ ID NO: 13 Figure 14 is the amino acid sequence of 4D3.4 polypeptide;

SEQ

ID NO: 14 Figure 15 is the amino acid sequence of 8E3.1 polypeptide;

SEQ

ID NO: Figure 16 is the amino acid sequence of 1G2.2 polypeptide;

SEQ

ID NO: 16 Figure 17 is the amino acid sequence of 10C12.7 polypeptide; 0 SEQ ID NO: 17 SFigure 18 is the amino acid sequence of 14F6.3 polypeptide; SEQ ID NO: 18 Figure 19 is the amino acid sequence of B12D8.2 polypeptide; SEQ ID NO: 19 00 00 10 Figure 20 is the amino acid sequence of 7F4.1 polypeptide; SEQ q ID NO: Figure 21 is the amino acid sequence of 10D7.5 polypeptide; SEQ ID NO: 21 Figure 22 is the amino acid sequence of 10G9.3 polypeptide, 10A2.2 polypeptide and B11B8.1 polypeptide; SEQ ID NO: 22 Figure 23 is the amino acid sequence of 11B8.4 polypeptide; SEQ ID NO: 23 Figure 24 is the amino acid sequence of Mab H11B-11B8 target epitope; SEQ ID 163 Figure 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.

Figure 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.

Figure 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 0 DNA fragments are shown with respect to each other.

(N

W Figure 28 is a schematic representation of the BVH-3 protein and the location of internal and surface epitopes recognized \O by certain monoclonal antibodies.

Figure 29 is a schematic representation of the BVH-11-2 00 protein and the location of protective surface epitopes 00 0. 10 recognized by certain monoclonal antibodies.

Figure 30 is a map of plasmid pURV22.HIS. KanR, kanamycinresistance coding region; ci857, bacteriophage X cI857 temperature-sensitive repressor gene; lambda pL, bacteriophage X transcription promotor; His-tag, 6-histidine coding region; terminator, T1 transcription terminator; ori, colEl origin of replication.

Figure 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 Underneath the alignment, there is a consensus line where and characters indicate identical and similar amino acid residues, respectively.

Figure 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 Underneath the alignment, there is a consensus line where and characters indicate identical and similar amino acid residues, respectively.

Figure 33 is the DNA sequence of the NEW43 gene (SEQ ID No 257).

7 Figure 34 is the deduced amino acid sequence of NEW43 polypeptide (SEQ ID NO S) 258).

0O DETAILED DESCRIPTION OF THE INVENTION c In the claims which follow and in the description of the invention, except where the 00 5 context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive

C',

sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

C

It was determined that portions of the BVH-3 and BVH-1 I 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 polypcptides 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 AUUJ -\Ii171 Ali S 21W- 'I 11107117 7a and BVH-lI I in which certain portions were deleted and/or modified and chimeras of to these polypeptides have 00 00 N, \MtCIN Va e (NX IS'.L'AIS 171 AItJ\S pcc P I. 17 1 A L! .SWC I Ilo 2IM17-5S 911)S/0)7 biological properties and generate a specific immune response.

N- According to one .aspect, the Present invention provides an isolated PolYnucleotide encoding a polypeptide having aL least 70* identity to a second polypeptide comprising a sequence as IND disclosed in the present application, the tables and figures.

In accordance with one aspect of the present invention, there is provided an isolated polyflucleotide comprising a 00 00 10 polynucleotide chosen from: c-i(a) a polynucleotide encoding a polypeptide having at least identity to a second polypeptide chosen from: table c-i B, E or H, a polynucleotide encoding a polypeptide having at least identity to a second polypeptide chosen from: table BBor H; a polynucleotide encoding a polypeptide having an amino sequence chosen from table B, E or H or fragments, analogs or derivatives thereof; a polynucleotide encoding a polypeptide chosen from: table B, E or H; 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;

a polynucleotide encoding an epitope bearing portion of a polypeptide chosen from table B, E or R; and a polynucJleotide complementary to a polyflucl.eotide in or According to one aspect, the present invention provides an igolated polynucleotide encoding a polypeptide having at least identity to a second polypeptide comprising a sequence chosen from table A, B, D, E, G or H or fragments, analogues or derivatives thereof.

O According to one aspect, the present invention provides an CI isolated polynucleotide encoding a polypeptide having at least Z 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 00 polypeptides characterised by the amino acid sequence chosen 10 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 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.

(111 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 IND thereof.

According to one aspect, the present invention provides an isolated polynucleotide encoding a polypeptide having at least 00 00 10 7O* identity to a second polypeptide comprising a sequence chosen from table R, 9 ox 31.

*According to one aspect, the present invention provides an C1 isolated polynucleotide encoding a polypeptide having at least 95* identity to a second polypeptide comprising a sequence chosen from B, E or Hi.

According to one aspect, the present invention rtelategs to polypeptides characterised by the amino acid sequence chosen from table B, E or H.

in accordance with the present the present invention, all polynucjleotides encoding polypept ides 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 ant igenic.

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 imimune response in an individual.

SIn a further embodiment, the present invention also relates to C polypeptides which are able to raise antibodies having binding specificity to the polypeptides or chimeric polypeptides of 5 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 00 portion.

00 010 C- In a further embodiment, the fragments of the polypeptides of the present invention will comprise one or more epitope O 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 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 C-i 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 00 survival curve, rate or period. Statistical analysis using the 00 00 10 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 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 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 Preferred substitutions are those known in the art as O conserved i.e. the substituted residues share physical or C- chemical properties such as hydrophobicity, size, charge or O functional groups.

IND 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, 00 i.e. as antigenic/immunogenic material. Thus, for instance 00 proteins or polypeptides which include one or more additions, C 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 0 antigenic/immunogenic fragments of the proteins or Cq 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.

00 Thus, for fragments according to the present invention the 00 10 degree of identity is perhaps irrelevant, since they may be pq 100% identical to a particular part of a protein or polypeptide, analogue or derivative as described herein. The 0 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 -NH 2 acylation by acetylation, or O thioglycolic acid amidation, terminal carboxy amidation, e.g.

CI with ammonia or methylamine) to provide stability, increased hydrophobicity for linking or binding to a support or other molecule.

\O

Also contemplated are hetero and homo polypeptide multimers of the polypeptide fragments, analogues and derivatives. These 00 polymeric forms include, for example, one or more polypeptides 00 10 that have been cross-linked with cross-linkers such as C- avidin/biotin, gluteraldehyde or dimethylsuperimidate. Such Spolymeric forms also include polypeptides containing two or O 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 0 protein and therefore the sequence of the mature polypeptide.

&Ji According to another aspect, there are provided vaccine S 5 compositions comprising one or more streptococcus polypeptides \O 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; 00 salts i.e. AlK(S0 4 2 AlNa(SO 4 2 A1NH 4 (0 4 2 silica, kaolin, 0 10 carbon polynucleotides i.e. poly IC and poly AU. Preferred Sadjuvants include QuilA and Alhydrogel. Vaccines of the invention may be administered parenterally by injection, rapid Sinfusion, 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 0 streptococcus (GBS or agalactiae), dysgalactiae, uberis, CA nocardia as well as Staphylococcus aureus. In a further embodiment, the streptococcus infection is S.pneumoniae.

ND In a particular embodiment, vaccines are administered to those individuals at risk of streptococcus infection such as infants, elderly and immunocompromised individuals.

00 00 10 As used in the present application, the term individuals" C, include mammals. In a further embodiment, the mammal is human.

0 Vaccine compositions are preferably in unit dosage 'form of about 0.001 to 100 pg/kg (antigen/body weight) and more preferably 0.01 to 10 [g/kg and most preferably 0.1 to 1 pg/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 jig to 10 mg and more preferably lpg to 1 mg.and most preferably 10 to 100 pg 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.

C-N In one embodiment, polynucleotides are those illustrated in o table B, E or H which encodes polypeptides of the invention.

\O 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.

00 Accordingly the present invention further provides 00 00 10 polynucleotides which hybridise to the polynucleotide C, sequences herein above described (or the complement sequences thereof) having 50% identity between sequences. In one O embodiment, at least 70% identity between sequences. In one embodiment, at least 75% identity between sequences. In one embodiment, at least 80% identity between sequences. In one embodiment, at least 85% identity between sequences. In one embodiment, at least 90% identity between sequences. In a further embodiment, polynucleotides are hybridizable under stringent conditions i.e. having at least 95% identity. In a further embodiment, more than 97% identity.

Suitable stringent conditions for hybridation can be readily determined by one of skilled in the art (see for example Sambrook et al., (1989) Molecular cloning A Laboratory Manual, 2 nd ed, Cold Spring Harbor, Current Protocols in Molecular Biology, (1999) Edited by Ausubel F.M. et al., John Wiley Sons, Inc., In a further embodiment, the present invention provides polynucleotides that hybridise under stringent conditions to either a DNA sequence encoding a polypeptide or 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.

C In a further embodiment, the present invention provides o polynucleotides that hybridise under stringent conditions to 5 e.ither _O a DNA sequence encoding a polypeptide or the complement of a DNA sequence encoding a polypeptide; 00 wherein said polypeptide comprising a sequence chosen from 00 0 table B, E or H or fragments or analogues thereof.

In a further embodiment, the present invention provides O polynucleotides that hybridise under stringent conditions to either a DNA sequence encoding a polypeptide or 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 a DNA sequence encoding a polypeptide or 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.

SAs will be readily appreciated by one skilled in the art, Spolynucleotides include both DNA and RNA.

The present invention also includes polynucleotides complementary to the polynucleotides described in the present NO application.

In a further aspect, polynucleotides encoding polypeptides of 00 the invention, or fragments, analogues or derivatives thereof, 00 10 may be used in a DNA immunization method. That is, they can Cg be incorporated into a vector which is replicable and expressible upon injection thereby producing the antigenic Spolypeptide 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, 2 nd ed, Cold Spring Harbor, 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 Humana Press, Totowa, New Jersey, 1997, 490 pages; Protein Purification, Principles and Practices, Scopes Springer-Verlag, New York, 3 rd Edition, 1993, 380 O pages; Current Protocols in Immunology, Edited by Coligan J.E.

Cl et al., John Wiley Sons Inc., New York which are herein Sincorporated by reference.

ND 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 00 promoters, selecting transformants or amplifying the genes.

00 S 10 Suitable vectors are those that are viable and replicable in «s the chosen host and include chromosomal, non-chromosomal and synthetic DNA sequences e.g. bacterial plasmids, phage DNA, 0 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, 2 nd ed, Cold Spring Harbor, 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, 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 O i.e. Saccharomyces or eukaryotic i.e. CHO, COS.

SUpon expression of the polypeptide in culture, cells are typically harvested by centrifugation then disrupted by 0 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 00 of the polypeptide from culture media or lysate may be 00 S 10 achieved by established techniques depending on the properties C4 of the polypeptide i.e. using ammonium sulfate or ethanol precipitation ,acid extraction, anion or cation exchange O 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 US 4,431,739; US 4,425,437; and US 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.

SAlternatively, a method for the detection of antibody specific CI to a streptococcus antigen in a biological sample containing Sor suspected of containing said antibody may be performed as follows: NO 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 00 form a mixture; and 00 S 10 c)detecting specifically bound antigen or bound fragment in C- 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 C< detectable label. A preferred DNA probe for this application O is an oligomer having a sequence complementary to at least about 6 contiguous nucleotides of the streptococcus pneumoniae ND polypeptides of the invention.

Another diagnostic method for the detection of streptococcus 00 in a patient comprises: 00 0- 10 a)labelling an antibody reactive with a polypeptide of the Sinvention 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 O pneumoniae polypeptides but is preferably specific for one.

SA further aopect of the invention is the use of the antibodies 5 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 0O by measuring the ability of a particular antibody to passively OO 10 protect against streptococcus infection in a test model. One Sexample of an animal model is the mouse model described in the examples herein. The antibody may be a whole antibody or an Santigen-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 0 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 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 BVHr-3 NEW1-mutl** 235 1NEW35A 236 NEW42 349 NEW49 350 NEWSO 351 N"EW51 352 l'1E52 353 NIEW5335 56 NEWS6 NEP"6-MUt2**'5 flEW5G-mut3* NRWS7 360 NEW63 361 N~EW64 362 NEW69363 NEWGG 364 1T276 365 ME105 NEW106 367 L14 a r.LU I 36B silent mutation, the polypeptide Is the same as Newi or New 56 TABE C titoeof BV-3 TABLE D, E and F Variants and Epitope of BVH-11- TABLE D- New19 aa 497-838 of Seq.ID 8 New24 aa 227-838 of Seq.ID 8 TABLE Ea .2r.1~ j';flr./ 4*P New 43 258 NEWGO 293 NEW61 294 NEW62 295 NEWBO 296 NEW81. 297 NEW82 298 NEW83 299 NEW84 300 301 NEW88DI 302 b7EW88D2 303 NEW88 304 TABLE F- epitopes of BVH-12l 10D7.5 21 2.0G9.3 22 11B8.1 22 10A2.2 22 i1b8.4 123 3A4.1 24 TABLE G and H Chimeras- TABLE G 1NEW32 M*-N1-*P-M (384 00) OPTIONAL AMINO ACID 00 TABLE H P.~i c itl.3 t. ti dC~F k and BVE~- VP 89 _VP 90 37 _VP 9137 VP. 9237 VP 9337 VP 9432 VP 10833 VP 10934 VP 11035 =P111 3436 VP12 337 VP113 345 VP1162 341 VP120 34 VP121 348 EXAMPLE 1 This example describes the bacterial atrains, plasrnids,

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 Sante CI Publique du Quebec, Sainte-Anne-de-Bellevue; Rxl strain, a Snonencapsulated derivative of the type 2 strain D39 and the 5 type 3 strain WU2 were provided by David E. Briles from _O 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'Universit6 Laval, 00 Sainte-Foy. E. coli strains DH5a (Gibco BRL, Gaithesburg, MD); AD494 (XDE3) (Novagen, Madison, WI) and BL21 (XDE3) (Novagen) as well as plasmid superlinker pSL301 vector S(Invitrogen, San Diego, CA); pCMV-GH vector (gift from Dr.

C Stephen A. Johnston, Department for Biochemistry, University of Texas, Dallas, Texas); pET32 and pET21 (Novagen) and pURV22.HIS expression vectors (Figure 30) were used in this study. The pURV22.HIS vector contains a cassette of the bacteriophage X cI857 temperature-sensitive repressor gene from which the functional PR promoter has been deleted. The inactivation of the c1857 repressor by a temperature increase from the range of 30-37 0 C to 37-42 0 C results in the induction of the gene under the control of promoter .PL. The PCR primers used for the generation of the recombinant plasmids had a restriction endonuclease site at the 5'end, thereby allowing directional cloning of the amplified product into the digested plasmid vector. The PCR oligonucleotide primers used are listed in the following Table 1. The location of the gene sequences coding for BVH-3, BVH-11 and BVH-11-2 gene products is summarized in the Figure 25, Figure 26 and Figure 27, respectively.

Table 1. List of PCR oligonucleotide primers Primer SEQ Sequence 5' 3' Nucleotide Restriction ID position sites NO OCRR 25 cagtagatctgtgcctatgcact SEQ ID 1: BglII 479 aaac 61-78 SEQ ID 9: 1-18 OCRR 26 gatctctagactactgctattcc SEQ ID 2: XbaI 480 ttacgctatg 4909-4887 SEQ ID 9: 2528-2519' OCRR 27 atcactcgagcattacctggata SEQ ID 1: XhoI 497 atcctgt 1525-1506 OCRR 28 ctgctaagcttatgaaagattta SEQ ID 1; HindIll.

498 gat 1534-1548 OCRR 29 gatactcgagctgctattcctta SEQ ID 2: XhoI- 499 C 4906-4893 HAI4J 30 gaatctcgagttaagctgctgct SEQ ID 1: XhoI 172 aattc 675-661 HAMJ 31 gacgctcgagcgctatgaaatca SEQ ID 1: XhoI 247 gataaattc 3117-3096 HAMJ 32 gacgctcgagggcattacctgga SEQ ID 1: XhoI 248 taatcctgttcatg 1527-1501 HANJ. 33 cagtagatctcttcatcatttat SEQ ID 2: BglII 249 tgaaaagagg 1749-1771 HANJ 34 ttatttcttccatatggacttga SEQ ID 1: NdeI 278 cagaagagcaaattaag 1414-1437 HANJ 35 cgccaagcttcgctatgaaatca SEQ ID 1: HindIII 279 gataaattc 3117-3096 HAMJ 36 cgccaagcttttccacaatataa SEQ ID 1: HindlII 280 gtcgattgatt 2400-2377 HANJ 37 ttatttcttccatatggaagtac SEQ ID 1: NdeI 281 ctatcttggaaaaagaa 2398-2421 HAMJ 38 ttatttcttccatatggtgccta SEQ ID 1: NdeI 300 tgcactaaaccagc 62-82 ;0 00 Primer SEQ Sequence 5' 3' Nucleotide Restriction ID position sites

___NO

HAMJ 39 ataagaatgcggccgcttccaca SEQ ID 1: NotI 313 atataagtcgattgatt 2400-2377 OCRR 40 cagtagatctgtgcttatgaact SEQ ID 3: BgIII 487 aggtttgc 58-79 OCRR 41 gatcaagcttgctgctaccttta SEQ ID 4: HindlIl 488 cttactctc 2577-2556 HAMJ 42 ctgagatatccgttatcgttcaa SEQ ID 3: EcoRV 171 acc 1060-1075 HAMJ 43 ctgcaagcttttaaaggggaata SEQ ID 3:7 HindIll 251 atacg 1059-1045 }{AIVJ 44 cagtagatctgcagaagccttcc SEQ ID 3: BgI 264 tatctg 682-700 1{AMJ 45 tcgccaagcttcgttatcgttca SEQ ID 3: HindIll 282 aaccattggg 1060-1081 HANJ 46 ataagaatgcggccgccttactc SEQ ID 3: NotI 283 tcctttaataaagccaatagtt 2520-2492 HANJ 47 catgccatggacattgatagtct SEQ ID 3: NcoI 284 cttgaaacagc 856-880 HAkMJ 48 cgccaagcttcttactctccttt SEQ ID 3: HindIll 285 aataaagccaatag 2520-2494 HANJ 49 cg3acaagcttaacatggtcgcta SEQ ID 3: HindIIl 286 gcgttacc 2139-2119 SEQ ID 2210-2190 IIAMJ 50 cataccatg(ggcctttatgaggc SEQ ID 3: NcoI 287 acctaag 2014-2034 HANJ 51 cgacaagcttaagtaaatcttca SEQ ID 3: HindIll 288 gcctctctcag 2376-2353 HANJ 52 gataccatggctagcgaccatgt SEQ ID 3: NcoI 289 tcaaagaa 2125-2146 HAMJ 53 cgccaagcttatcatccactaac SEQ ID 3: HindIll 290 ttgactttatcac 1533-1508 00 00 Primer SEQ Sequence 5' 3' Nucleotide Restriction ID position sites NO HIAMJ 54 cataccatggatattcttgcctt SEQ ID 3: *NcoI 291 cttagCtCCg 1531-1554 HAMJ 55 catgccatggtgcttatgaacta SEQ ID 3: NcoI 301 ggtttgc 59-79 HAMJ 56 cgccaagctttagcgttaccaaa SEQ ID-3: HindlIl 302 accattatc 2128-2107 HANJ 57 gtattagatctgttcctatgaac SEQ ID 5: BgIII 160 ttggtcgtcacca .172-196 HAMJ 58 cgcctctagactactgtatagga SEQ ID XbaI 186 gccgg 2613-2630 HAMVJ 59 catgccatggaaaacatttcaag SEQ ID 5: NCoI 292 ccttttacgtg 925-948 HAMvJ 60 cgacaagcttctgtataggagcc SEQ ID 5: HindlIl 293 Iggttgactttc 2627-2604 HAMJ 61 catgccatggttcgtaaaaataa SEQ ID 5: NcoI 294 ggcagaccaag 2 209-2232 HAMJ 62 catgccatggaagcctattggaa SEQ ID 5: NcoI 297 tgggaag 793-812 HAMJ 63 catgccatggaagcctattggaa SEQ ID 5: NcoI 352 tgggaagc 793-813 HAMJ 64 cgccaagcttgtaggtaatttgc SEQ ID 5: HindIII 353 gcatttgg 1673-1653 HAMJ 65 cgccaagcttctgtataggagcc SEQ ID 5: HindIIl 354 ggttgac .2627-2608 RAMJ 66 cat9ccatggatattcttgcctt SEQ ID 5: Ncol 355 ctitagCtCC 1603-1624 HAMJ 67 ttatttcttccatatgcatggtg SEQ ID 1: NdeI 404 atcatttccattaca 1186-1207 HAMJ 68 gatgcatatgaatatgcaaccga SEQ ID 1: NdeI 464 gtcagttaagc 697-720 HI J 69 gatgctcgagagcatcaaatccg SEQ ID 1: XhoI 465 tatccatc 1338-1318 Primer SEQ Sequence 5' 3' Nucleotide Restriction ID position sites NO HAMJ 70 gatgcatatggatcatttccatt SEQ ID 1: NdcI 466 acattcca 1192-1212 H.AMJ 71 gacaagcttggcattacctggat SEQ ID 1: HindlII 467 aatcctg 1527-1507 HAMJ 72 catgccatggaagcctattggaa SEQ ID 5: NcoI 352 tgggaagc 793-813 HAJ 73 ataagaatgcggccgccgctatg SEQ ID 1: NotI 470 aaatcagataaattc 3096-3117 HAMJ 168 atatgggcccctgtataggagcc SEQ ID 5: Apa I 471 ggttgactttc 2G26-2604 HAMJ 169 atatgggcccaatatgcaaccga SEQ ID 1: Apa I 472 gtcagttaagc 720-697 HAMJ 170 atatgggcccaacatggtcgcta SEQ ID 3: Apa I 350 ___gcgttacc 2139-2119 HAMJ 171 tcccgggcccgacttgacagaag SEQ ID 1: Apa I 351. agcaaattaag 1414-1437 HAMJ 172 catgccatgggacttgacagaag SEQ ID 1: NcoI 358 agcaaattaag 1415-1437 H-AIAJ 173 tcccgggccccgctatgaaatca SEQ ID 1: Apa I 359 gataaattc 3116-3096 HAMJ 174 atatgggcccgacattgatagtc SEQ ID 3: Apa I 403 tcttgaaacagc 856-880 HAMJ 175 cgccaagcttaacatggtcgcta SEQ ID 3: Hind III 361 ___gcgttacc 2139-2119 HAMJ 176 atatgggccccttactctccttt SEQ ID 3: .Apa I 483~~ aataaagccaatag 2520-2494 M1-becular biology tech-niques were perforrrei accrdxing to standard rrthxds. See for exanple, Samrorok, Fish, E Mniatis, "1 becular clcning. A laboratory rrn-na1" Vol.1-2-3 (secad eition) (bid Sprirxg Ha:ir Laboratory Press, 1989, New York, which is herein incorprated by reference. PR arrpified proaicts were digested with restriction er-Aaiceases and] 1igated to either linearized plasmid pSL3o1, FJCM'V-CI-, p~ or pUV2.1-ES expression vec=o digested likewise or digested with enzyffes that procice cmrpatible cabesive endis. Re~rrbiraant PSL301 and re~rbinant pQvIV-CE plasnids were digested with restrictin enzyrres for the in-frarre clanirg in pEr expression vector. &eni pET vectors were used, clones were first stabilized in S. coil.

DHScz before introduction into E. coli BL2l(.XD83) or AD494 RD23) for expression of full-length or truncated BVH-3, BVH- 11 or BVII-11-2 molecules. Each of *the resultant plasniid ;Z constructs was confirmed by nucleotide sequence analysis. The recombinant proteins were expressed as N-terminal 'fusions with the thioredoxin and HiB-tag (pET32 expression aystem); as Cterminal fusions with an His-tag (pET21 expression system); or as N-terminal fusions with an His-tag (ptTRV22.HIS expression 00 00 system). The expressed recombinant proteins were purified from supernatant fractions obtained after centrifugation of sonicated IPTG- (pET systems) or heat- (ptJRV22.HIS) induced

R.

Scoli using a His-Bind metal chelation resin (Ql[Ageri, Chatsworth, CA). The gene products' generated from S.

pneumonjae SP64 are listed in the following Table 2. The gene fragment encoding BVH-3-Sp63 protein (amino acid residues 21 to 840 on SFQ ID NO: 10) was generated from S. pneumoniae SP63 using the PCR-primer sets OCRR479-OCRR480 and the cloning vector pSL3O1. The recombinant pSL30l-BVH-3SpE3 was digested for the in-frame cloning in pET32 vector for the expression of the BVH4-3-SpG3 molelcule.

Table 2. Lists of truncated BVH-3, BVH-l1, BVH-11-2 and Chimeric gene products generated from S. pneurnoniae SP64 PCR-primer 3ets Protein Identification Encoded amino Cloning designation acids (SEQ IOD vector No:6, 7 or 8) OCRR479-OCRR480 BVII-3M BVH-3 w/o ss 2 1-1039 pSL301 OCRR479-OCR4R497 BVH-3AD BVI.3 NWend w/o ss 2 1-509 pSL3O1 5857 HAM248--AM1249 L-BVH--3AD BVH-3 M'end 1-509 pJET-21I(+) OCRR498-0CRR499 BVH-3B IIVH-3 C'crid 512-1039 pSL301 0CRR479-HAMI72 B9VH-3C DVH-3 Mend w/o ss 21-225 pET-32 0CRR487-OCRR48 I3VH-1 IM D-I I w/o ss 20-840 pCMV-QH HIAMJ251-0CRR487 BYN-IIA BVH-I I N'eid w/o 20-353 pET-32 HAMJ171-ocRR,4gj BVHWIIB BVH-l I C'end HAM1264-OCRR488 BVH-1IIC BVH-1 I Vead- H1AMJ278-HAMJ279 NEW] BVII-3 C'end HM~J 278-HAMvU 279 11AM1278-1IAMJ2so _NEW2 l4A1-1.

354-M4 228-840 472-1039 JA~~fl~ HIAM] 278-HAM) 280 HAMJ)28l-HAMT279 11AM1284-HAMJ2SS 11AMJ284-11AMJ286_ HAM1287-HAMJr288 NEW4 REW6 00 00 HAM]2S5-HAMJ289 HAM]284-ILAMfl90 H1AMJ296-HAMJ291I HIAMII6O-HAMJI 86 HAM1292-HAM]293 kHAM3293-HAM1294 HAMJ282-HAM12 83 HAMJ286-HAMJ297 BVH- I 1-2M BVH-3 C'end BVH- I I Cend BVI- I I internal BVH-1 I intenmal BVH-11 C-eud BVH.i I Inmternal BVH-1I I internal JBVH-I 1-2 w/o so 800-1039 286-840 586--713 672-792 709-840 286-511 72 511-713 pET-32 pET-32 PET-21b(+) pET-2lb(-f) PET-2lb(+) VET-21d(+) pET-2 Id(+) pET-2lId(+) pET-Zi d(-s) pET-Zld(+) PET-21Id+ NEWIO 1BVH- 1-2 C'end j}271-838 j pET-2 Id(+) NEWI I tBVH-1 1-2 C'end f 699-838 NEW 1 NEW 14 BVH-1 I C'e-d BVH-1 1-2 internal 354-340 pET-2l pET-2lb(+) 2 3_'n 2-69 R-IPET-2 I HIAMJ301I-HAM)302 tNEW1 6 BVJ-I I N 'end w/o 120-709 1 pET-21d(.) I I H 1AIJ352-HAM1353 HAMJ354-HAMvJ355 HAMJ404-HAMJ279 HAMJ464JJ.AMJW465 NEW18 NEW] 9 NEW21 WEW22 BVH-1 1-2 itra1227-520 E2d) ~BVH-1 11-2 Cend BVH-3 C'end flVIR- internal [497-838 396-1039 pET71b(+) I3-4 IE-Ia+ HAMJ466-HAMJ467 1NEW21 BVH-3 ntenal- 39-509 1DT2Ib+ HAM1352-HA1293 NEW-24 HAMJ464-H1AMJ470 NEW-25 HAM127 8-HAMJ279 NEW 12 (NEW 1) l*AMJ282- LHAMJ283 (NW 13) HAMJ284-HAMJ350 NEW 17 (NEW 5) HAMJ351- HAMJ279 (NW 1) IIAM]3S 8-HAMJ359 NEW20 (NEW 1) HAMJ4O3.

H-AM3 61 (NW5) JBVi- 11-2 C'end 22f 3 [BvH-3 CVend_ Chimera' IChimera 4 Chimeraa 233-1039 NEW 13 M- NEW 5 -GP -NEW I M- NEW I G NEW pET-21 pET.2 Ib(-4) pET 21 b(+ pET 21 d(+ pET 21 d(4 ;0 00 HAMJ361 I RAM292-RAMb~471 NEW2 Chimera* M- NEW 10 -GP pET 21 d (NEW 10) HAMJ472- 6 -NEW H{AMJ470 (NEW 25) HAN1J355-HAMAJ71 NEW2 Chimera* M- NEW 19-G pET 21 d (NEW 19) iAMJ472- 7 -NEW HA.MJ470 (NEW HAMJ292-HAM~J471 NEW2 Chimiera* M- NEW 10 -GP pET 21 (NEW 10) HAMJ351 9 -NEW I -HAI4J279 (NEW 1) HAM1284-HAM1350 NEW2 Chimiera* M. NEW 5 _GP pET 21 (NEW 5) HAM1472- 9 NEW H{AMJ470 (NEW HAMJ284-1JAMJ483 NEW3 Chimiera* M-NW4GP-pT2d(+ (NEW 4) HAMJ472- 0 NEW HAMJ470 (NW25) HIAM34284-HAMJ1483 NEW3 Chimera* M- NEW 4 -GP IpLET 21 d H+ (NEW 4) HAMvJ351- 1 NEW I HAMJ279 (N3W 1) (NEW 19) HAM1351- H.AMJ279 (NEW 1) NkW 3 2 Chimera* M- NEW 19 -GP pET 21 d(+ -NEW 1 11 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 lysinie, L is leucine, G is glycine and P is praline.

Monoclonal antibody (Mab)-secreting hybridomas were obtained by fusions of spleen cells from immunized mice and nonsecreting, HGPRT-deficient mouae myeloma SP2/o cells by the methods of Fazekas De St-Groth and Seheidegger CJ Immunol Methods 35 :1-21, 1980) with modifications Hamel et J Med Microbiol 23 :163-170, 1987). Female aALB/c mice (Charles River, St-Constant, Quebec, Canada) were immunized with either BVH-3M (thioredoxin-His9Tag-BVH-3M fusion protein/ pET32 system), BVH-11M (thioredoxin-His.Tag-BVH-11M fusion protein/ O pET32 system), BVH-11-2M (thioredoxin-His*Tag-BVH-11-2M fusion protein/ pET32 system), BVH-11B (thioredoxin-His*Tag-BVH-11B Sfusion protein/ pET32 system), BVH-3M (His*Tag-BVH-3 fusion D 5 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-, Sand HN1-, respectively. Culture supernatants of hybridomas 00 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 heatc 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,

AL).

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 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 0 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 0O the generation of a chimeric gene. The recombinant chimeric 00 10 polypeptides listed in Table 2 were as C-terminal fusion with 0 an His-tag. The expressed recombinant proteins were purified .s from supernatant fractions obtained from centrifugation of sonicated IPTG-induced E. coli cultures using a His-Bind metal C chelation resin (QIAgen, Chatsworth, CA).

Groups of 8 female BALB/c mice (Charles River) were immunized subcutaneously two times at three-week intervals with 25 yg of either affinity purified His*Tag-fusion protein identifed in presence of 15-20 Ag 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 Experiment Immunogen Alive :Days to death post- Dead infection 1 none 0 8 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, >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, 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.

2007207883 16 Aug 2007 Table 3. EILISA reactivity of BVI-I-3-reactive Mabs with a panel of eleven BVH-3 gene products and the BVH-11M molecule Gene products tested Mabs (IgG BVH- BVH- BVH- BVH- NEW NEW NEW NEW NEW NEW BXTH- BVHisotype) 3M 3AD 3B 3C 1 2 3 21 .22 23 3 11M Sp 63 H3-4F9 H-3-4D4 I H3-9H12 H3-7G2 H3-1OA1 H3-4D3 H11-6E7 NT NT NT H11-1OH1Q NT NT NT (2a)

I

Hll-7G11 NT NT NT (2b) H3V-4F3 H3V-2F2 H3V-7F4 H3V-7H3 2007207883 16 Aug 2007 Gene products Mabs (IgG BVH- BVH- BVH- BVH- NEW NEW NEW NEW NEW NEW BVH- BVHisotype) 3M 3AD 32B 3C 1 2 3 21 22 23 3 1iM Sp 63 H3V-13B8 (1) H3V-9C2()+ +1 H3V-9C6 H3V-16A7 (1) H3V-15A10 +1+ H3V-,6B3 NT NT NT (1/2) HN1-5113 NT (2b) HN1-8E3 NT (2a) HN1-14F6 NT (2a)I HN1-2G2 NT I+ 1 2007207883 16 Aug 2007 HN1-10 +VH NT BV NE NE NEW E E (2a) H1-l-10C12 NT (1) HN1-1E(1G+ 2 NT NT not tested 1- very low reactivity but higher than background, possible non-specific Mab binding Table 4. ELISA reactivity of BVH-11 and/or BVH-ll-2-reactive Mabs with a panel of fourteen

BVH-

11 and BVH-11-2 gene products and the BVil-3M molecule 2007207883 16 Aug 2007 Gene products tes ted Mabs BVH- BVH- BVH- BVH- NEW NEW NEW NEW NEW NEW NEW NEW NEW NEW BH-BH (IgG 11M 11A 11B 1iC 5 6 7 8 9 10 11 14 18 19 2-M 3M i so type) H3-4F9 (1) H3-4D4 H3-9H12 (1) H11-6E7 (1) Hit- 1OH10 (2a) H11-7G11 (2b) H11-lB12 (1) Hh1-7B19 2007207883 16 Aug 2007 Gene products tested Mabs BVH- BXTH- BVH- BVH- NEW NEW NEW NEW NEW NEW NEW NEW NEW NEW BVH- H (i gGt p li A 11B 11C 5 6 7 8 9 10 11 14 18 19 2-M! 3M (2a) H11-3H5 (1) Nl12-31B+ NT (1) H112 NT

(I)

(1) (2a) 16 Aug 2007 2007207883 2007207883 16 Aug 2007 Gene products tested Mabs BVH- BVH- BV-I- BVH- NEW NEW NEW NEW NEW NEW NEW NEW NEW NEW BVH- H (IgG 11M 11A 11B 11C 5 6 7 8 9 10 11 14 18 19 2-M 3M iso type) 10C5 (1) H112- NT +4 14H6 (1) H112-7G2 NT H1112- N'T 131110 (2a) H112-7E8 NT (2b) H112-7H6 NT 1- (1) H11B-

I

H11B- 15G2 (1) H11B- 2007207883 16 Aug 2007 G ~ene products tested Mabs BVH- BVH- EVil- BVil- NEW NEW NEW NEW NEW NEW NEW NEW NEW NEW EVil- B (IgG 11M IIA 11B 11C 5 6 7 8 9 10 11 14 18 19 2-M 3M isotype) 13D5 (2) Ni E 11B8 H1l- 7E11 (1) H11B-1C9+ (1) H1B-5E3 (2) H11B-6E8+ (1)

NT

1- very low reactivity but higher than background, possible non-specific Mab binding a strong signal was detected when tested by Western itmunoblotting SThe deduced locations of the epitopes are summarized in Figure O 28 and Figure 29. As can be seen from the data in Table 3, BVH-3-reactive Mabs can be divided into two groups BVH-3AkD 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 00 00 10 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- Sand BVH-ll-cross-reactive Mabs were also reactive with BVH-11A 0N 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 17 7-amino acid fragment comprised between amino acids 244 and 420 on BVH-3 molecule of S.

pneumoniae SP64 (Figure 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 M recognize DVH-3 molecules can be divided into three groups according to their reactivities with BVH-11A and D 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.

00 00 10 EXAMPLE 3 This example describes the construction of BVH-3 and BVH-11-2 Sgene 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, CA) and digested with the restriction enzymes BglII and Xbal 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 Sconformation-dependent epitopes consisting of different protein domains. Sequencing of DNA inserts from positive 0 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 00 vector can be purified and used as immunogens as described OO 10 below in Example 6.

r The peptide sequences obtained from the screening of BVH-3 and SBVH-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).

2007207883 16 Aug 2007 Table S. Peptide sequences obtained from the screening of BVH-3 and BVH-1l-2 gene libraries with Mabs Mab Clone/ Nucleotide Amino acid Amino acid sequence

SEQ

Protein position position ID NO designat ion H3 -4D4 4D4.9 SEQ ID 1: SEQ ID 6: DQGYVTSIIGDIY1YYGVPDAFSEELLMKDPNYQLKDA 11 226-509 76-169 DIVNEVKGGYII1VDGKYYVYAHANVTGEINRQK QEHVKDNEKVNsF 1 Hil- 7G11.7 SEQ ID 1: SEQ ID 6: G;IQAEQIVIKITDQGYVTSHGDHYHYYNGKVPYDAIJFSEEL :12 7G11 193-316 64-105

L

1111- 7G11.9 SEQ ID 1:SEQ ID 6: TAYIVRHGDHFHYIPKSNQIGQPTLPNSLTPSPSLPI -13 7G11 1171-1284 390-428 H3-4D3 4D3.4 SEQ ID 1: SEQ ID 6: TSSLEPVPQKAKASGKEVF 14 2565-2670 855-890 HL 1- 8E23.l SEQ ID 1: SEQ ID- 6: MDGTIELRLPSGEVIKKJJSDFIA. HNi- 1G2.2 SEQ ID 1: SEQ ID 6: YGLGLDSVIFNMDGTIELRLPSGEV;IKKNLSDFIA 16 1C22936-3120 983-1039

LPSGEVIKKULSDFIA

-9M- 14F6.3 SEQ ID 1: SEQ ID. 6: KVE EVKKSTNTNTEVTDVE 18 14F62501-2618 833-872 2007207883 16 Aug 2007 Mab Clone/ Nucleot Protein positlo designat ion 11N1- B12D8.2 SEQ 12D8 1:1433- 1767 H3V- 7F4.1 SEQ ID 7F4 1633-17f 11112- 10D7.5 SEQ ID 10D7 1685-176 11112- 10G39.3 SEQ ID 10G9 1893-215 1 ide n Amino acidAino acid sequence position

SEQ

ID

NO

I I IDJEEQ ID 6:1 MKDLDKKIEEKIAGIMKQYGKESIVNKEKAI IYPHGD4 HHHADP IDEHKPVGIGHSHSNYELFPEEGVKKEG 5: 5: 5: 0l bhU ID) 6:1 545-59 Al PGHHAPDHPGIHHNELKEGA

KEGNKVYTGE

I

I

SEQ ID 525 -553 SEQ ID 594-67 9 8: IQVAKLAGKYTTEDGYIFDPRDITSDEGD 4 0.

KNGSLI IPHDHYHNIKFEWFDEGLYEPKGYSLEDLtATV

KYYV

H112 1 0A2 1 0A2 .2 SEQ ID 5: 1893 -2150 SEQ ID 8:- 594-679

DHQDSGNTE-AKGAEAIYNRVKAKKVPLDMPYNLQYTE

KNGSLI IPHYDHYRNIKEWFDEGLYE-APKGYSLEDLAT 4 H1113- 21128.1 S EQ ID 5: SEQ ID 8: DHQDSGNTEAKGAEAIYNRVK AKPLDRMPYNLQYTVEV 22 1128 1893-2150 594-679

KNSIPYHHIFWDGYAKYLDLT

KYYV

H11B- 11B8.4 SEQ ID 5: SEQ ID 8: GLYEAPKGYSLEDLLATVYYVEHPNERPHSDNGFGMpA5DH 23 11B8 2085-2217 658-698 2007207883 16 Aug 2007 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.

\O

NZW rabbits (Charles River Laboratories, St-Constant, Qu6bec, Canada) were immunized subcutaneously at multiple sites with 00 50 zg or 100 yg of the purified BVH-3M, L-BVH-3AD, NEW1, 00 10 NEW13, or L-BVH-11 recombinant protein in presence of 80 yg 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 lt, post 2 nd or post 3 rd injection. The rabbit immune response to immunization was evaluated by ELISA using recombinant BVH-3M (BVH-3M-His*Tag fusion protein/ pET21 system) or BVH-11M (BVH-11M-His.Tag fusion protein/ pET21 system) proteins or suspensions of heat-killedS. pneumoniae Rx-1 cells as coating antigens. ELISA titer was defined as the reciprocal of the highest sera dilution at which absorbance

A

410 value was 0.1 above the background value.

Antibodies reactive with BVH-3 and/or BVH-11 epitopes were elicited following immunization in all animals as shown in the following Table 6. Antibody reactive with recombinant or pneumococcal antigens was not present in the preimmune sera.

The immune response to immunization was detectable in the sera of each rabbit after a single injection of recombinant antigen. The antibody response following the second injection with either antigen tested was characterized by a strong increase in antibody titer. 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 54 data support the potential use of BVH-3, BVH-11 and/or BVH-11a 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 pneumococca. disease, respectively.

Table 6. Rabbit Antibody response to immunization with BVH-3 and BVH-11 gene products Rabbit Immunogt BVJ{-3M 0gg) BVH -3M (10011Lg) ELISA Titer antigen en Sera BVH-3M

TIFI--

sample 11M Pre immune NT

NT

Post-i 8 NT

NT

Post- 2 c -NT

NT

Pot3 512,000

NT

Preimmune NT

NT

post I NT

NT

post 2n NT

NT

post 3r 10

NT

Preimmune <100

NT

Jpost 1s 16,000

NT

post 2 512,000

NT

post 3r 2x10

NT

=Preimmune <100

NT

post 1" 16,000

NT

post 2 512,000

NT

post 3 r 10

NT

Preimmune NT <100

I

post 1, NT 16,000 post 2" NT 64,000 post 3r NT 256,00 3 0 Preimmun INT<10

N

with coating

S.

pneumioniae 200 1,600 2,000 40,000 200 1,600 40,000 80,000

NT

NT

32, 000

-ITT

24, 000

T

#112 L-BVH-3A] 'Ug) #113 New 1 jg) #114 New 13 0 jig) NT not tested EXAMPLE 00 This example describes the protection of animals against fatal 00 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,

MA)

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 CPU 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 CPU and verify the challenge dose. The CBA/N mice were chosen because of their high susceptibility to S. pneumoniae infection. The LDs 5 of WU2 injected intravenously to CBA/N g mice is estimated to be <10 CFU. Deaths were recorded at 24-h C intervals for a period of at least 7 days.

5 The protection data obtained from mice injected with rabbit NO 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 Scontrol antibody or PBS bufer. The observation that antibody 00 10 raised to the BVH-3-M molecule passively protected even when Sadministered after the challenge demonstrated the ability of Santi-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 Alive Days to death preparation Days to administration Dead 1 post-infection 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 before0 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 folowing Table 8, all immunized mie strainved ^wng lafle 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.

SS. pneumoniae hemocultures on day 11 post-challenge were J 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 Sagainst. BVH-11/BVH-11-2.

00 00 10 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, Antibody from 0 5 3, 3, 3, 6, 6 preimmune sera H112-10G9 4 0 >11, >11, >11, >11 H112-10G9+H1B- 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 7 sets of 2 Mabs conferred complete protection against experimental infection. These data indicated that antibody Cl targetting BVH-11 and/or BVH-11-2 epitopes gave efficient Sprotection. The Mabs H112-3A4, H112-10G9, H112-10D7, H112- 5 10A2, H112-3E8, H112-10C5, H11B-11B8, H11B-15G2, H11B-1C9, D 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 00 protection-conferring epitopes on BVH-11-2 molecules are 10 summarized in Table 10 and Figure 29 Protective Mabs H112- 3A4, H112-10G9, H112-10D7, H112-10A2, H112-3E8, H112-10C5, g H11B-11B8, H11B-15G2, H11B-1C9, H11B-7E11, H11B-13D5 and H11- C 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-ll 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-ll-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 (Figure 32).

Altogether the data set forth in Table 9 and Table 10 suggest that the protection eliciting BVH-11- and BVH-ll-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-11-2ci specific Mabs can protect mice from lethal experimental pneumococcal infection.

Experim Nab Alive Days to death ent Dead post-infection 1 H112 3A4 H112-lOGS9 6 :0 6 X H112-3A4 H112-105D7 5 :1 4, 5X 00 Noe0 6 2,2,,2,26 002 H112-l0 A2 H112-10D7 5 :1 3, 5X H1l2-3E8 H112-10G9 6 :0 6 X 'oe0 :6 2, 2, 2, 2, 2, 2 3 H112-10D7 HllB-1lB8 6 :0 6 X H112-I0G9 HllB-15_G2 3 3 2, 6, 6, 3 X None 0 6 2, 2, 2, 2, 2, 2 4H112-10G9 H112-10D7 5 0 5 X >11 None ,2 H112-10G9 H11l-10B8 4 1 8, 4 X >14 H112-lOG9 H11lB-7Ell- 5 :0 5 X >14 6H112-10G9 Hh1B-1C 4 1 4, 4 X >14 7 H112-10CS H11B13DS 5 5:0 5 X >14 CBA/N mice were injected intraperitoneally with a total of 0.2 ml of ascites fluid 4 hours before intravenous challenge with Pneumoniae WU2.

Table 10. Deduced locations of protection- conferring epitopes on BVH-11-2 molecules.

Mabs Protection Gene products carrying Mabepi tope 11112-3A4 NEW 19 and NEW 11 H112-2.0G9 NEW 19 11112-10D7 NEW 14 and NEW E112-lOA2-7+ NEW 19 H112-3E8 NEW 19 H11B-ITB-8 NEW 19 H11B-15G2 NEW 18 H11B-7E.1 NEW 14 and NEW NEW 18 H11B-1C9 NEW 14 and NEW H1.12-A. NEW 18 and NEW 8 11112-101110 NEW 18 and NEW 8 21112-2117 BVH-l1-2M H112-6H BVH-11-2M H11- 71-1 -BVH-11A and BVH-3C -H11-E7 -BVH-11A and BVH-3C H112-7-CS +NEW 19, NEW11 and 3A4.1 H112-3DS +NEW 19 H112-7G2NEW 18 H1127EBBVH -11- 2M H3-4F9BVH-11A and BVH-3C Altogether the data presented in this example substantiate the potential use of antibodies raised to BVH-3, BV11 or BVH1-11 2 molecules as therapeutic means to prevent, diagnose or treat pneumoniae diseases.

EXAMPLE 6 This example describes the localization of surface-exposed peptide domains using Mabs described in Example 1.

SS. pneumoniae type 3 strain WU2 was grown in Todd Hewitt (TH) 5 broth (Difco Laboratories, Detroit MI) enriched with Yeast extract (Difco Laboratories) at 37*C in a 8% CO 2 atmosphere to give an OD 600 of '0.260 (-108 CFU/ml). The bacterial suspension was then aliquoted in 1 ml samples and 00 the S. pneumoniae cells were pelletted by centrifugation and 0 10 resuspended in hybridoma culture supernatants. The bacterial suspensions were then incubated for 2 h at 40C. Samples were Swashed twice in blocking buffer [PBS containing 2% bovine serum albumin 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 Ml 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 cellsurface 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 C- 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 5 or NEW 23 BVH-3 gene products were detected at the surface of ND 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 00 portions of the sequence corresponding to the 177-amino acids 00 10 absent from the BVH-3 molecule of S. pneumoniae SP63 appears not to be accessible to antibodies.

SResults 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 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 Mab FI Bindin Gene products carrying Fluoresce g Mab-epitope nce 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 Mab Fl Fluoresce ac e H3-4D 33.2 3.5 H-3V-4F3 24.4 2.9 H3V-2F2 15.6 2.4 H3V-7F4 58.7 5.6 H3V-7H3 68.8 6.9 H3V-J.3B8 75.07.

H3V-9C2 66.4 6.2 H3V-9C6 2.91.

H3V-16A7 16.61.

H3V- 58.75.

15A1O HN1-.5H3 43.4 5. 3 HN1-BE3 57.4 6.6 HN1-J.4p6 57.8 6.7 HN-22 54.8 6.3 HN1i-2D8 14.3 3.0 HN-1-14B2 11.5 2.7 HiN 1 -1 G2 5-9. 9 7.0 HN1-7J 813.2 9.0 H11-GE71 3.1 1.2.

H11-7B 2.4 1.16 H11-10B8 81.1 9.

T

H11-1A2 84.4 10- J-11-3H5 84.0 9.8 11112- 49.3 5.9 13Cl11 H112- 0.4 Bindin Gene products carrying g Mab-epitope NEW 3 NEW I NEW 2

NEW

NEW 22 NEW22 NEW23 NEW 22 andNW2 6- NEW 2 NEW 3 NEW 3 NEW-3CadVI-1 BVH-3C and BVH-11A BVH-3C and NEW 2 BVH-1J.A BVH-13-A NEW 18 and NEW 8 NEW 18 and NEW 8 NEW 18 and NEW 8 NEW 18 and NEW 8 BVH-11A and NEW 18 4 Mab H112-1D H112- 1 0G9 H112 1 0A2 H112-3E 11112- 1 0D7 11112-2117 H112 -6H7 11112- 3A4 H1112- 1 H112- 14H6 H112 1 4H6 H112-7G2 H112 131110 H112 -7E8 H11B-1I H11B- 1 5G2 H1B 13 H11B- 11B F1 Fluoresce nce 0.41.

Bindin Gene products carrying 9 Mab-epitope 75.5 62.5 64.5 0.7 0.3 8 6.3 9.6 7.7 1.0 87.92 BVH-11A and NEW 18 NEW 19 NEW 19 NEW 19 NEW 14 BVH-11A BVH-11A NEW 11 NEW 11 AND 3A4.1 89.6 ii NEW 11 0.8 1.4 NEW 11 4.7 0.5 0.4 0.2 3.1 1.0 2.0 1.0 1.1- [NEW 18 NEW 18 BVH- 11 2M BVH 1 1- 2M NEW 1 60.2 NEW 18 and NEW 8 7S.7 8. 3 7+ NEW 19 78.4 8. 37+ NEW 19 32.3 [NW4 NEW 14 oCO 00 00 EXAMPLE 7 5 This example describes the immunization of animals 'with Speptide epitopes of BVH-3 and BVH-11-2.

The recombinant pSCREEN-T vector (Novagen, Madison,

WI)

containing DNA fragment (nucleotides 2421 to 2626 on SEQ ID NO 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 (XDE3) pLysS [BL21 ompT hsdSB (rB-mB-) gal dcm lacYI pLysS (Cmr)] (Novagen). In this strain, the expression of the fusion protein is controlled by the T7 promoter which is recognized by the T7 RNA polymerase (present on the XDE3 prophage, itself under the control of the lac promoter inducible by isopropyl- P-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 0 C with 250 RPM agitation in LB broth (peptone 10g/l, yeast extract 5g/l, NaC1 supplemented with 50mM glucose, 100yg/ml carbenicillin and 3 4pg/ml chloramphenicol, until the absorbance at 600nm reached a value of 0,7. The overexpression of T7gene 10 protein- His*Tag-3A4.1 fusion protein was then induced by the addition of IPTG to a final concentration of 1mM and further C incubation at 25 0 C with 250 RPM agitation for 3 hours. Induced O cells from a 800-ml culture were pelleted by centrifugation and frozen at -70 0 C. The fusion protein was purified from the Ssoluble cell fraction by affinity chromatography based on the binding of a six histidine residues sequence (His-Tag) to divalent cations (Ni 2 immobilized on a metal chelation Ni- NTA resin (Qiagen, Mississauga, Canada). Briefly, the Spelleted cells were thawed and resuspended in Tris buffered 00 sucrose solution (50mM Tris, 25%(w/v) sucrose) and frozen at 70 0 C for 15 minutes. Cells were incubated 15 minutes on ice in the presence of 2mg/ml lysozyme before disruption by 0 sonication. The lysate was centrifuged at 12000 RPM for minutes and Nickel charged Ni-NTA resin (QIAgen) was added to the supernatant for an overnight incubation at 4 0 C, with 100 RPM agitation. After washing the resin with a buffer consisting of 20mM Tris, 500mM NaC1, 20mM imidazole pH 7,9, the fusion 3A4.1 protein was eluted with the same buffer supplemented with 250mM imidazole. The removal of the salt and imidazole was done by dialysis against PBS at 4 0 C. The protein concentration was determined with BCA protein assay reagent kit (Perce, Rockford,IL) and adjusted to 760 pg/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 T7genel0 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 CPU 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 P- 14 post-challenge, the surviving mice are sacrificed and blood 0 samples tested for the presence of S. pneumoniae organisms.

C The 3A4.1 protein or other tested protein is said protective Swhen the number of mice surviving the infection or the median number of days to death is significantly greater in the 3A4.1- ND immunized group compared to the control mock-immunized group.

pg EXAMPLE 8 00 00 r 10 This example illustrates the improvement of the antibody 9 response to pneumococci using BVH-3 fragments and variants Sthereof.

The combined results obtained from studies of Mab reactivity with truncated gene products, epitope-expressing colonies and live intact pneumococci presented in examples 2, 3 and 6, allowed to delineate between surface-exposed and internal epitopes. The epitopes detected by Mabs that efficiently bound to pneumococci cells mapped to a region comprised between amino acid residues 223 to 1039 of BVH-3 described in SEQ ID NO 6. The existence of protective epitopes in the BVH- 3-carboxyl half was confirmed by demonstrating that mice immunized with NEW1 molecule were protected from fatal infection with P4241 strain.

Gene sequence comparison revealed that in some strains, the region of BVH-3 encoding for amino acids 244 to 420 as described in SEQ ID N06 is absent thus suggesting the lack of utility of this sequence in vaccine to prevent disease caused by such strains (SEQ ID NO: 9 versus SEQ ID NO: 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 Q PCR using pairs of oligonucleotides engineered for the amplification of the appropriate gene fragment. Each of the Sprimers had a restriction endonuclease site at the thereby.allowing directional in-frame cloning of the amplified N product into the digested plasmid vector. PCR-amplified products were digested with restriction endonucleases and Mp ligated to linearized plasmid pET21 (Novagen) expression 00 00 vector digested likewise. Oligonucleotide primers HAMJ489 (ccgaattccatatgcaaattgggcaaccgactc; NdeI) and HAMJ279 q (cgccaagcttcgctatgaaatcagataaattc; HindIII) were used for the 0 NEW 40 construction. Clones were first stabilized in E. coli C- DH5a before introduction into E. coli BL21 (1DE3) 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 Cterminus 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.

2007207883 16 Aug 2007 Table 12. List of PCR oligonucleotide primer sets used for site-directed mutagenesis gene truncates Primer Primer SEQ Primer SEQUENCE set identification ID N~o 51 3' 9 H{AMJ5 13 177 GAATCAGGTTTTGTCATGAGTTCCGGACACATCATTATTTC HAM4J514 178 GAAATAATGATTGTGGTCTCCGGCTGAACCTGATTC H-IAMJ515 179 GTAGGTCGGCCAACATTTTCAAG 16 180 CCTTGAAAATATGATTCGAT-TA 11 HAMJ517 181 ATGAGTTCGGAGACTCCTTCTTATTTCTTAGAGGACTTG 18 182 CAAGTCCTTCTTGAAGAATAGATTGGAGTCTCCGGAACTCAT 14 CHAN5 1 183 GCATTTTCTTGGTACTAG 2 184 GCATGATGGTGATCTCCAGACGTAATTCGC 17 CHIANS 3 185 CCGTCTGAGATGGCCATCATGCGATCCG 4 186 CGGATCTGCATGATGGCATCTCAGACG 19 CHAN4 7 187 CCGCAGGGAGATAAGCGTATGCAGATCCATTG ____CHAN4 8 188 CATCGGATCTGCATGACGCTTATCTCCCTGCGG Cij5 189 CCGTCTGGAGATGGACTC.TGCGATCCG7ATTG CHAN56 190 r '~rATTCTATCACCAAG 22 CHAN57 191 C::LCGTCGGAGATGACTTCTGCA.GATCCGATTGATG 8 192 CATCAATCGGATCTGCAGGTGCATCTCACG 23 HAMJ523 1.93 CCCTGGTGCACTCGTC 24 194 CGGATCTGCATGATGGCATCTCATGCG 24 HAMJ526 195 GTAGGCCGGCCATATTTTCAAG 27 196 CCTTGAAAATATGGCCGGCCTA HAMJ5 28 197 ATATAGAACCATTATCTAGAGCT HIAMJ52 9 198 CAGCTCTAGATAATGGTTCTATA 2 F9 HMI'J569 199 TACCTCATTATGACTCTTACTC

TATCAATTGAGTGTTTG

70 200 CAACCCATTAGTGGAGGCTAGGT HAMJ5 71 201 TACCTTCTTATGACCATTACAATCAATTTAGTGTTTG -AMJ5 72 202 -AACCCATTAGTGGATGCTAAGT 351 HAMJ5 73 1203 IACGATTACTCTCAAA

ATCAACT

74 12 04L GATGTTATGGTAATGGTCTGTA~TGAT"TAACTACCGTT on BVH-3 2007207883 16 Aug 2007 Prme rimer SEQ Primer SEQUENCE sne lidentification ID No 5' 3' 32 HAMJ575 205 CGTGTATAACCAAGATTACTAACA HAMJ5 76 206 TTTGATGTTATGGTAGAGTCCTTATGGTATATTACTACCG 33 HAMJ5 77 207 ACGGTAGTTTAATCATACCTGCCATTACCATCATATTG 78 208 CAAATTTGATGTTATGGTATGGTCAGTATGATTACTACCGTT 34 HAMJ5 79 209 AACGGTAGTTTAATCATACCTTACCATCTAATTTGAGTGG 80 210 CCACTCAATTTGATGTTATGGTAGGTATGATAACTACCGTT HAMJ5 81 211 ACCGGTAGTTTAATCATACCTAACATAAATTTGAGTGGTTTAC 82 212 GTCAAACCACTCATTTGATGTTAGTATGATTACTACCGTT 37 HAMJ5 36 213 CCTATGTACTCCACATATAACCCTAGCCCTGG 37 214 CCAGTGGCTATGGGTTATATGTGGAGTTACATAGG 39 HAMJ550 215 CGTGAAAGTATTGTCGTAATAAAAAATGCG 51 216 CGCATTTTTTTCTTTATTTACGAATACTTCG HAMJ5 86 217 CATGAAGAAGATGGTTACGGTTTCGATGCTA3ACCGTATTATCGCTG 87 218 CTTCAGCGATAATACGGTTAGCATCGACCGTAACCTCTTCTTCTG -1 HAMvJ5 88 219 GAATCAGGTTTTGTCATGAGTGACCAATCATTATTTCTTC 89 220 GAAGAAATAATGATTGTGGTCACTCATGACAAAACCTGATTC 42 HAMJ5 90 221 GAGTATAGTTTAGGATATTTTCA H{AMJ591 222 CTTGAAGAAATAATGATTACTCATGAAAAACCTGATTCATCTTC 43 HAMJ5 92 223 GAAATGATCAGGTTTTGTCATGAGTTATCTTAGAGGC 93 224 GTCTTGAAAACCTAAACTATACT 44 HNJ-594 225 IAAAATGCGATTATTTATCCGCACCATCATGCAGATCCGATTG 95 226 CATGACGAGTGGCGTATACCTT HAMJG00 227 IAAAATGCGATTATTTATCCGGCAGATCCGATTGATGCTAC 1{AMJ6 01 228 GTTTTCTATGACGCGTATACCTT 46 HAMJ604 229 GAGTACTTACCGAAGACGTTGCT HAMJ6 05 230 -CATGACAAAACCTGATTCGTCTTCGGCGATTATACTAGCTC 47 HAMJ6 06 231 CGCAGCATCGTTTAGGCCGGCC HAMJ6 07 232 GGAGTCTCCGTGACTCATTACAAGCCGATTCGTCTTCGGCG -8 HAMJ6 08 233 CACCTACGATTCGCAGCAGATAA HIAMJ6 09 1234 ICTTTCATTTCTTTGGCGTTGCCGGTTCCTGTTCATAGTG 2007207883 16 Aug 2007 Tabl e 13. Lists of truncated variant BVI{-3 gene products generated frorm S. rneumoniae SP64 Poen Cn/ Pratein !dentiticat-lon* VCR primer~ not Gone used far designation Pratein eftal12 ugnsi 33Q ID NO NEW1- 25 NW39E1 Mutl* 256 NEWI 550-SGDGTS-5S55 41,02 i ?1EW42 34 rT.

NEW4 9 NEWS 0 NEW 51 NEWS 2 NEW53 NEWS 4 NEW 55

NEWSE

NEWS 6- NEW56 Inut3** 'WEWS 7 NEW6 3 NBW6 4 NEW6 S 350 351 353 .353 3554 356 357- 358 az-Ev-VYS-6 1 44-SGDGTS-149 NEW40 55-SGDHNH-60 NM1W40 55-SGDSNH-60 NTEW4 Q 55-S--GIDH-60 144-SG()llI{FH-14g.

NEW40 SS-SGDSNH-60 144-SGDGH- FNEW40 55-HGDHN-so6 244-SGDHLWH-3149 NEW40 55-HGDI*7H-ESo 144-SGDGHH-149 NEW1 550-HGDGM1-5,55L NEW40 55-HGUDSNH-60 144 -SGDHR-1495 NEWS6 16, -11,14, 17, 20, 22 10 14 10, 17 14 17 23 24 40 NEW4 6 NEW4 0 NEW4 9 NEW4 NEWS i NEW4 0 NEWK3 NEW1 NEW53 NEW56 359 NE I5 56 4 -647,413 NEW56 360 362 3i6 3 NEW40 5-5-HGDHKS-60 144-SGDHHH-1459 tBW4O S5-J-IDsNH-60 144-HGDMlHI-149 NEW40 bb-EiGDUS-60 144-HGDEMU-149 NE3W40 b5-HGDS9KH- 60 144-HGDGHw-149- 25 24 25 NE-W53 RE4 0 NEW4 0 2007207883 16 Aug 2007 Protein Gen./ ProteJin Tdenti Lcation* IRpie e ee ue o designation Protein (C rimer ibe-t One gused fo NEW6 3N EW D 55-IGDKNS-60 144-HGDGHR-149 23 NEW64 NEW76 365 NZW4 5-HGD~iNfS-60 144-SGDGHH-149 17NE6 NEW105 366 NEW 0O 5- -60 41,42,43 NEW'4 0 NEW106 367 New4O 144- -149 44,45 0E17 368 INEW40 55- -60 144- -149------W1O The underl~ined amino acid residues represent the odfctn in potein sqene Nuceoids/ain aidresidues are deleted in NEW10S, NEW106 and NEw31O7 constructs.

S* ilent mutation, i.e. the polypeptide is the same as Newl.

SGroups 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 M obtained for NEW40- and NEW56-immunized groups with 7 and 8 00 00 10 survivors out of 8 animals, respectively. Similarly, Scomprising amino acid residues 233 to 1039 protected 7 out of [p 8 animals from lethal infection.

Table 14. Protection mediated by BVH-3 fragments or variants thereof in experimental pneumonia Expe Immunogen Alive Days to death post-infection rime Dead nt 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, 34, 4, 4, 4, 4, 4 NEW 52 4 4 8, 9, >10, >10, >10, NEW56 8 0 8 X NEW 40 7 :1 6, >10, >10, >10, >10, >10, >10, 3 QuilA 0 8 3, 3, 4, 4, 4, 4, 4 4 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 Table 15. Binding of mouse sera antibodies at the surface of S. pneumoniae type 3 strain WU2 as measured by flow cytometry.

Antisera Fluorescence index Experiment Experiment Experiment Mean SE 1 2 3 BVH-3M 9.2 11.4 14.5 11.7 NEW1 11.5 10.1 nd* 10.8 0.7 14.3 12.9 nd 13.6 0.7 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 Cl amplified by PCR using pairs of oligonucleotides engineered to amplify fragments originating from SEQ ID NO :5 spanning 5 nucleotides 1662 to 1742, 1806 to 2153, 2193 to 2414 and 2484 ND to 2627 from S. pneumoniae strain Sp64 BVH-11-2 gene.

c The primers used, HAMJ490-491, HAMJ492-HAMJ493, HAMJ494- 00 HAMJ495, HAMJ496-HAMJ354 had a restriction endonuclease site at the 5' end, thereby allowing directional in-frame cloning C- of -the amplified product into the digested pET21b(+) plasmid Svector (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, CA) and ligated into pGEM-T plasmid vector without any prior restriction endonuclease digestion. The resultant plasmid constructs were confirmed by nucleotide sequence analysis. The recombinant plasmids containing each of the four were digested with restriction endonucleases corresponding with the 5' end of each primer pair used for the PCR-amplification. The fragments were purified from agarose gel like described earlier and were all ligated to linearized plasmid pET21b digested with the restriction enzymes NdeI and HindIII for the in-frame cloning of the four different regions of the BVH11-2 gene. Clones were first stabilized in E.coli DH5 before introduction into E.coli BL21 (XDE3) for expression of a chimeric pneumococcal protein molecule.

The resulting NEW43 gene sequence (SEQ ID No 257) is described in Figure 33.

The deduced amino acid sequence of NEW43 protein (SEQ ID No 258) is described in Figure 34.

O

ND

00 0 O0 Table 16. List of PCR oligonucleotide primers used to construct the NEW43 VP43S and NEWBE6 Primer SEQ Sequence 5' 3' Nucleotide Restrictio ;ZID NO position n sites IND HAMJ490 259 ccgaattccatatgcaaat SE ID NdeI tacctacactgatgatg 5 :1662- 1683 HAMJ491. 260 ggactagtatcaaagatat SEQ ID SpeI aaccgtcttc 5 :1742- 00 HAMJ492 261 ggactagttggattaaaaa SEQ ID SpeI agatagtttgtctg 5 :1806c-I 1830 HAMJ4.93 262 ttcccgcggttcgacatag SEQ ID SaclI tacttgacagtcg 5 :2153cagI 2131 HAMJ494 q263 ttcccgcggaacgctagtg SEQ ID SaclI accatgttcg 5 :2193- 2212 HAMJ495 264 cggggtaccaggaatttca SEQ ID KpnI gcctcatctgtg 5 :2414- 2393 HAMJ49G 265 cccggtacccctagtatta SEQ ID KpnI gacaaaatgctatggag 5 :2484- 2510 HAMJ 65 cgccaagcttctgtatagg SEQ ID H-indIII 354 agccggttgac 5 :2627- 2608 I-IAMJ 266 ggatcccgggaggtatgat SEQ I DSia 583 taaactaccg 5 :2039- 2021 HAMJ 267 cagcggaaca SEQ ID SinaI 584 titgagtggtttgac 5 :2058- 2081 HAMJ 268 cttgatcgacatatgttgg SEQ ID NdeI 610 caggcaagtacacaacag 5 :1701- 1 1722 79 Table 17. List of truncated BVH-1 11-2 gene fragments generated from S. pneumoniae SP64 for the construction of NEW43 PCR-prer sets Gene fragrment Corresponding Cloning vector designation amino acid residues on SEQ MD NO: 8 HAMJ490-HAMJ491 NEW43a 517-543 pGEM-T FIAMJ492-HAMJ493 NEW43b 565-680 pSL3OI HAMJ494-HAMJ495 NEW43c 694-767 pSL3OI HAMJ496-HAMJ354 NEW43d 791-838 pSL3OI Table 18. Properties of NEW86 and VP43S genes generated from NEW43 gene PCIR-primer sets Gene/Protein Identification designation HAMJ610-HAMJ354 VP43S NEW43 Cend corresponding to residues 272) (SEQ ID NO:374) HAMJ490-HAMJ583 NEW86 NEW43 109-__PG_- 114 (SEQ ID NO:375) FIAMJ584-FIAMJ354 NEW43-derived molecules designated VP43S and NEW86 were generated from gene amplification and cloning experiments uIsing PCR primers described in Tables 16 and 18 and pET2 I expression plasmid vector. Variants from NEW43 were generated by Mu.tagenesis using the Quickchange Site-Directed Mutagenesis kit from Stratagene and the oligonuILcleotides designed to incorporate the appropriate Mutation. The presence of' N \XMCI IN J I 979\P-( 71 AUkS[-.\P(9171 A U SNCI~c.,t..m 21W- 5- 8 I~x 9/15/( 79a histidine tag residues on the C-terminus of the recombinant molecules simplified the puiiaio I:of the proteins by nickel chromatography. The following tables 19 and ;Z describe the sequences of the piriers used for the mnutagenesis experiments and the NEW43 variant gene products generated, respectively.

00 00 N \M 1I Ic,1w\SINW).JS99\PAIKJ7 1I I: \S I ?4 1I At, Sr'cjI, 2(H)7. X J, 9/f)Sil7 Table 19. List of PCR oligonucleotide primer sets used for site-directed mutagenesis on N'EW43 gene Prim IPrimer set 2 3 2 6_ 2 7 31 34 identi f ication HAMJZ497 HAMJ 498 HAM~J4 99 0 0 HAMJ501 HAMJ502 HAMJ530 HAMJS 3 1 HAMJ532 HAMJ533 69 70 HAMJ571 HAMJ572 HAMJ5 7 HAMJ574 75 HAMJ576 HAMJ57 HAMJ578 HAMJ5 79 HAMJ580 HAMJ58.

H{AMJ582 SEQ Primer

SEQUENCE

In 31

NO

269 RA:GATTACTCTCTAGCATCAACT 270 GAGTTGATGCTAAGTTATACACT 271 FLrAACTTAOCCTCAAC~iTT]rT 272 CATAATTAGTTGAAATAAGAGAGT 273 TA 'r:.ETATTATCACTAATGGGTT 274 CAAACCACTCTGATGTTAGAGTAGAGTCATAAGAGGTA 275 AACTC

GCCTCCTAACATTAT

276 CATAATGTTAGTACATAATAGAGT 277 TA CTAGCATCCACTAATGGCTT 278 CAACCCATTAGTGGATGCTAGGT 279* TA UXVTATTATCA AATGqGT 280 CAAACCACTCATGATGTTAGAGTAMGAGTCATATGAGT 282 AACATAATGTTAGGATGCTAAGT 284 GAGTTGATGCTAAGTTATACACT 285 CG GTACTCTAAGATTACTAACA 286 TTTGATGTTATGGTAGAGTCCTATGAGGTATGATAACTACCG 2 87 IILU'rU1TATAACU-CTACTAACATT 28.8 CAATGTTAGTAGTCGTTATACACT 2 89 AA TG ATAACTCAACTAATGGG 290 CCCCATTAGTTGAGTTATACACT 291 AC L.'A.'AACACT; 7 AT(TTA 292 GTCAAACCACTCTTATGTTAGGTATGATTAACACCGTT Table 20. List of NEW43 variant gene products generated from S. pneumonjae SP64 Polypeptide Polypep Polypeptide PCR Gene used designation tide pie o SEQ ID identification* sre forgnei ;ZNO (ref.

table 22) 293 NEW43 109-SYDHYH.114 I. NEW43 N EWGI 294 NEW43 109-HYDSYHl-114 26 NEW43 00 NEW62 295 NEW43 109-HYDHYS-114 27 NEW43 00NEWBO 296 NEW43 109-SYDSYH-.114 2 NEW81 297 NEW43 l09-SYDSyS-114 3 NEIW8O NEW82 298 N~EW43 109-HYDSYS..114 29 NEW61 NEW83 299 NEW43 l09-SYDHYS-114 30 NEW84 300 NEW43 l0-K~j~J4 31 301 NEW43 109-HKD)yj114 32 NEW61 NEWSBD. 302 NEW43 109-__DHYH-114- 33' NEW43 NEW8D 303 NEW43 109- Y-1 34 NEW88Dl NEW88 304 NEW43 1 09- -114 35 NEW88D2 The underl ined amino acid residues represent t *he modification in protein sequence. Nucleotides/amino acid residues are deleted in NEW88Dl, NEW88D2 and NEW8B 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.

pneumOni~ae P4241 strain. Data from Table 21 clearly indicate that NEW 19, NEW43 and variants thereof provided protection against experimental pneumonia.

Table 21. Protection mediated by NEW19 and NEW43 fragments or variants thereof in experimental pneumonia

I

Exper Immunogen iment 1 Quil A NEW 19 NEW 43 2 Quil A NEW 43 NEW 80 NEW 83 3 Quil A NEW 43 NEW 88D1 NEW 88D2 NEW 88 3 Quil A NEW 60 NEW 84 NEW 85 NEW 86 Alive Dead 0 :8 7 :1 8: 0 0 :8 7 :1 6 :2 6 :2 0:8 7 :1 5 :3 5 :3 7 :1 0 :8 8:0 8 :0 5 :3 5 :3 Median day alive 4, 4, 4, 4, 4, 4, 4, 5, 7X >14 8X >14 4, 4, 4, 4, 4, 5, 5, 8, 7X >14 5, 6, 6 X >14 8, 10, 6 X >14 4, 4, 4, 4, 5, 5, 5, 5, 7X >8 5, 6, 6, 6 X >8 6, 6, 6, 6 X >8 6, 7X >8 4, 4, 4, 5, 5, 5, 5, 6 8 X >8 8 X >8 5, 7, 7, 5 X >8 5, 6, 6, 5 X >8 EXAMPLE 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 N amplified by PCR using pairs of oligonucleotides engineered so ;Z 5 that the primers had a restriction endonuclease site at the end, thereby allowing directional in-frame cloning of the amnplifiled product into digested plasmid vectors (Table '23 and Table 24). PCR-amplified products were digested with restriction endonucleases and ligated to linearized plasmid 00 00 10 pSL30l vector. The resultant plasrnid construct were confirmed by nucleotide sequence analysis. The recombinant pSL3O1 plasmids containing a PCR product were redigested with the same endonuclease restriction enzyme for the obtention of the CI DNA inserts. The resulting insert DNA fragments were purified and inserts corresponding to a given chimeric gene were ligated into pURV22-N'del vector for the generation of a chimeric gene. The expressed recombinant proteins wer-e purified from supernatant fractions obtained from centrifugation of Boflicated 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 IE DN dniiai designationSQ DOjtiato VP 0 359M-Nev5E -GP- New43* VP 90 1370 M-NeW43 -P VP 91 1371. M-New52 -GP- NeW43 VP 92 372 M-New43 -GP- New52 VP 93 373 M-NewSE -GP- New6O VP 94 332 IM-New6O -GP- New56 VP 108 333 M-New56 -GP- New88 VPl09 334 M-New88 -GP- NewS6 Polypeptide E DN d n i ca o designationSQIDNIenicai VP 110 33S M-New6O -GP- VP ill 336 M-NewGO -GP- New1O7 IND VP112 337 M -New 88T -GP- -NewlO VP113 338 M-New88 -GP- NewlO7 VP114 339 M-NeW80-GP- NewlOS 00 00 P 134 e 8 -G N w 7 VP116 340 M-New83 NewlO7 ciVP116 341 M-New83 -GP- VP119 342 M-New83S -GP- NewlO7--' 343 M-New43S- VP1220 344 M-New43S- GP-New1O7 V122 345 M-New8OS- VP123 3 47 M-ew88S- G6P-New105 VP 12 4 38M-New88S- GP-New1o7 Enode amio aids 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.

;ZPrimer PCR-primer Gene used for Corresponding position amplifcation the chimricprti a_ mo ecule 49 HAMJ49O-nHz447l Variant New43 fl-terminal s0 HAMJ5 6 4 -{AJ5 56 Variant.New43 C-terminal 00 00 51 H{MJ489-HAMJ359 Variant NeW4O N-terminal 5HJ559-HAMJ557 Variant New4O C-terminal 53HMJ610-HA?1471 IVariant New436 N-terminal Table 24. List of PCR oligonucleotide primers designed for the generation of the chimeric genes encoding the polypeptides listed in Table 22.

Primer S EQ Seqence 5' 3' ID Pestriction NO a.i te HMJ-49O 2i59 ccatcaagaatct NdeI HAMJ471 168 atatgggcccctgtataggagccgg Apal ttgactttc HAM~J564 327 attgccaattacacact Apal 9atgatgagattcagg 1.55 SG 5- 328 ataagaatgcggccgcctactgtat NotI aggagccggttgactttc 11AMJ489 329 ccgaattccatatgcaaattyggca fidel accgactc HMAJ3 59 173 tcccgggccccgetatgaaatca a Apal taaattc EAMJ 5 59) 330 jatatgggccccaaattgggcaaccg Apa!I AN34165 cgccaagcttctgtataggagccgg Hindlill HAMJ610 268 cttgatcgacatatgttggcaggca NdeI agtacacaacag HAJ557 331 ataagaatgcggccgcttaegctat NotI gaaatcagataaattc RMJ2 9 3 5 cgccaagcttcgctatgaaatcaga H-indLIll 11AMJ79 taaattc

Claims (18)

1. An isolated p olynucleotide comprising a polynucleotide chosen from: a polynucleotide encoding a polypeptide having at least 701 identity to a second Volypeptide chosen from: table B, E or H; a polynucleotide encoding a polypeptide having at least 9 5 9 E identity to a second polypeptide chosen from: table B, E or X; a polynucleotide encoding a polypeptide having an amino sequence chosen from table B, E or H or fragments, analogs or derivatives thereof; a polynucleotide encoding a polypeptide chosen from: table 8, E or H; a polynucleotide encoding a polypeptide capable of generating antibodies having binding specificity for a polypeptide having a sequence chosen from: table B, S or H; a polynucleotide encoding an epitope bearing portion of a polypeptide chosen from table B, E or H; and a polynucleotide complementary to a Polynucleotide in or

2. The isolated polynucleotide polynucleotide is

3. The isolated polynucleotide polynucleotide isa

4. The isolated polynucleotide polynucleotidle is The isolated polynucleotide polynucleotide is

6. The isolated polynucleotide polynucleotide is of claim 1. wherein said of claim I wherein said of claim 1 wherein said of claim I wherein said of claim 1 wherein said

7. The isolated polynucleotide of claim 1 wherein polynucleotide is

8. The isolated polynucleotide of claim 1 wherein polynucleotide is

9. The isolated polynucleotide of claim 7 wherein polynucleotide is chosen from table B. The isolated polynucleotide of claim 9 wherein epitope bearing portion is chosen from table C.

11. The isolated polynucleotide of claim 7 wherein polynucleotide is chosen from table E.

12. The isolated polynucleotide of claim 11 wherein epitope bearing portion is chosen from table F. said said said said said said

13. The polynucleotide of anyone of claims 1 said polynucleotide is DNA.

14. The polynucleotide of anyone of claims 1 said polynucleotide is RNA. A vector comprising the polynucleotide wherein said DNA is operably linked to control region. to 12, wherein to 12, wherein of claim 13, an expression

16. A host cell transfected with the vector of claim

17. A process for producing a polypeptide comprising culturing a host cell according to claim 16 under conditions suitable for expression of said polypeptide.

18. An isolated polypeptide comprising a member chosen from: a polypeptide having at least 7DW identity to a second r- polypeptide having an amino acid sequence chosen from: table B, E or H; a polypeptide having at least 95%s identity to a second ;Z polypeptide having an amino acid sequence chosen from; table 5, E or H; a polypeptide having an amino acid sequence chosen from table B, E or H; a polypeptide having amino acid sequence chosen from: 00 table 13, E or H or fragments, analogs or derivatives thereof; a polypeptide capable of generating antibodies having binding specificity for a second polypeptide having a sequence chosen from table R, E or HFl an epitope bearing portion of a polypeptide having an, amino acid sequence chosen from: table S, E or H; the polypeptide of or (f) wherein wherein the N-terminal Met residue is deletedl; or the polypeptide of or (f) wherein the secretory amino acid sequence is deleted. 1.9. The polypeptide of claim 18 wherein said polypeptide is (f) The polypeptide of claim 1.9 wherein said polypeptide is chosen from table B.

21. The polypeptide of claim 20 wherein said epitope bearing portion is chosen from table C.

22. The polypeptide of claim 19 wherein said polypeptide is chosen from table E.

273. The polypeptide of claim 22 wherein said epitope bearing portion is chosen from table F. 24. A chimeric polypeptide comprising two or more O polypeptides chosen from table B, E or H thereof; cI provided that the polypeptides are linked as to form a Schimeric polypeptide. O 25. A vaccine composition comprising a polypeptide according to any. one of claims 18 to 24 and a pharmaceutically Sacceptable carrier, diluent or adjuvant. 00 00 26. A method for therapeutic or prophylactic treatment of 4 meningitis, otitis media, bacteremia or pneumonia infection Sin an individual susceptible to meningitis, otitis media, q bacteremia or pneumonia infection comprising administering to said individual a therapeutic or prophylactic amount of a composition according to claim 27. A method for therapeutic or prophylactic treatment of streptococcal bacterial infection in an individual susceptible to streptococcal infection comprising administering to said individual a therapeutic or prophylactic amount of a composition according to claim 28. A method according to claim 26, wherein said individual is a mammal. 29. A method according to claim 27, wherein said individual is a mammal A method according to claim 26, wherein said individual is a human. 31. A method according to claim 27, wherein said individual is a human 32. A method according to claim 27, wherein said bacterial infection is S.pneumoniae, group A streptococcus 89 p- (pyogenes), group B streptococcus (GBS or agalactiae), D dysgalactiae, uberis, nocardia or Staphylococcus aureus. 33. A method according to claim 27, wherein said bacterial infection is S.pneumoniae. \O 34. Use of a vaccine composition according to claim 25 for the prophylactic or therapeutic treatment of Streptococcal 00 00 infection in an animal susceptible to or infected with streptococcal infection comprising administering to said animal a prophylactic or therapeutic amount of the Scomposition. 0D rsl