AU2005209689B2

AU2005209689B2 - Novel streptococcus antigens

Info

Publication number: AU2005209689B2
Application number: AU2005209689A
Authority: AU
Inventors: Bernard R Brodeur; Nathalie Charland; Josee Hamel; Denis Martin; Isabelle Pineau; Clement Rioux
Original assignee: ID Biomedical Corp of Quebec
Current assignee: ID Biomedical Corp of Quebec
Priority date: 1998-12-23
Filing date: 2005-09-13
Publication date: 2008-07-17
Anticipated expiration: 2019-12-20
Also published as: AU2005209689A1; AU2008229967A1; AU2008229967B2

Description

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Applicant: ID Biomedical Corporation Invention Title: NOVEL STREPTOCOCCUS ANTIGENS The following statement is a full description of this invention, including the best method of performing it known to me/us: r 1 NOVEL STREPTOCOCCUS ANTIGENS

(N

SThe entire disclosure in the complete specification of our Australian Patent Application No. 17649/00 is by this cross-reference incorporated into the present specification.

00 \D FIELD OF THE INVENTION (1 10 The present invention is related to antigens, more particularly protein antigens of Streptococcus C, pneumoniaepathogen which are useful as vaccine components for therapy and/or prophylaxis.

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.

H:\rochb\Keep\P58259.doc 13/09/05 2 0 Effective pneumococcal vaccines could have a major impact on the morbidity and mortality associated with S. pneumoniae disease. Such vaccines would also potentially be useful to prevent otitis media in infants and young children.

Efforts to develop a pneumococcal vaccine have generally concentrated on generating immune responses to the 00 pneumococcal capsular polysaccharide. More than C pneumococcal capsular serotypes have been identified on the C- 10 basis of antigenic differences. The currently available pneumococcal vaccine, comprising 23 capsular polysaccharides Sthat most frequently cause disease, has significant shortcomings related primarily to the poor immunogenicity of some capsular polysaccharides, the diversity of the serotypes and the differences in the distribution of serotypes over time, geographic areas and age groups. In particular, the failure of existing vaccines and capsular conjugate vaccines currently in development to protect young children against all serotypes spurres evaluation of other S. pneumoniae components. Although immunogenicity of capsular polysaccharides can be improved, serotype specificity will still represent a major limitation of polysaccharide-based vaccines. The use of an antigenically conserved immunogenic pneumococcal protein antigen, either by itself or in combination with additional components, offers the possibility of a protein-based pneumococcal vaccine.

PCT Publication number W098/18930 published May 7 1998 entitled "Streptococcus Pneumoniae antigens and vaccines" describes certain polypeptides which are claimed to be antigenic. However, no biological activity of these polypeptides is reported.

Therefore there remains an unmet need for Streptococcus antigens that may be used as vaccine components for the prophylaxis and/or therapy of Streptococcus infection.

H:\rochb\Keep\P58259.doc 13/09/05 2a SUMMARY OF THE INVENTION

(N

SAccording to one aspect, the present invention provides an isolated polynucleotide encoding a polypeptide having at 5 least 70% identity to a second polypeptide comprising a sequence chosen from SEQ ID NOs: 2, 4, 6, 8, 10, 14, 16, 00 Io o H:\rochb\Keep\P58259.doc 13/09/05 to 75, 77 to 79, 81, 83 or fragments, analogs or derivatives thereof.

C In other aspects, there are provided vectors comprising polynucleotides of the invention operably linked to an expression control region, as well as host cells Stransfected with said vectors and methods of producing polypeptides comprising culturing said host cells under conditions suitable for expression.

C\ 00 oO ND In yet another aspect, there are provided novel S polypeptides encoded by polynucleotides of the invention.

Cq 15 BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is the DNA sequence of BVH-3 gene; SEQ ID NO: 1.

Figure 2 is the amino acid sequence of BVH-3 protein; SEQ ID NO: 2.

Figure 3 is the DNA sequence of BVH-11 gene; SEQ ID NO: 3.

Figure 4 is the amino acid sequence of BVH-11 protein; SEQ ID NO: 4.

Figure 5 is the DNA sequence of BVH-28 gene; SEQ ID NO: Figure 6 is the amino acid sequence of BVH-28 protein; SEQ ID NO: 6.

Figure 7 is the DNA sequence of BVH-3A gene which corresponds to the 5' terminal end of BVH-3; SEQ ID NO: 7.

Figure 8 is the amino acid sequence of BVH-3A protein; SEQ ID NO: 8.

C( Figure 9 is the DNA sequence of BVH-3B gene which corresponds to the 3' terminal end of BVH-3; SEQ ID NO: 9.

Figure 10 is the amino acid sequence of BVH-3B protein; SEQ ID NOS 00 1 Figure 11 depicts the comparison of the predicted amino acid sequences of the BVH-3 open reading frames from WU2, V RX1, JNR.7/87, SP64, P4241 and A66 S. pneumoniae strains 0 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 12 depicts the comparison of the predicted amino acid sequences of the BVH-11 open reading frames from WU2, Rxl, JNR.7/87, SP64, P4241, A66 and SP63 S. pneumoniae strains 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 13 depicts the comparison of the predicted amino acid sequences of the BVH-11 proteins from various S.

pneumoniae strains. The degrees of identity and similarity were determined by using the program Clustal W from MacVector sequence analysis software (version Figure 14 is a DNA sequence containing the complete BVH-3 gene (open reading frame "ORF" at nucleotides 1777 to 4896); SEQ ID NO: 11.

Figure 15 is a DNA sequence containing the complete BVH-11 gene (ORF at nucleotides 45 to 2567); SEQ ID NO: 12.

Figure 16 is a DNA sequence containing the complete BVH-11- 2 gene (ORF at nucleotides 114 to 2630); SEQ ID NO: 13.

Figure 17 is the amino acid sequence of BVH-11-2 protein; SEQ ID NO: 14.

0 Figure 18 is the DNA sequence of SP63 BVH-3 gene; SEQ ID Figure SEQ ID Figure ID NO: 19 is the NO: 16.

20 is the 21 is the NO: 56.

amino acid sequence of SP63 BVH-3 protein; amino acid sequence of BVH-3M protein;

SEQ

Figure SEQ ID amino acid sequence of BVH-3AD protein; Figure SEQ ID Figure ID NO: 22 is the NO: 57.

23 is the 58.

amino acid sequence of L-BVH-3-AD protein; amino acid sequence of NEW12 protein;

SEQ

Figure 24 is the ID NO: 59.

amino acid sequence of BVH-3C protein;

SEQ

Figure 25 is the amino acid sequence SEQ ID NO: of BVH-11M protein; Figure 26 is the amino acid sequence of BVH-11A protein; SEQ ID NO: 61.

Figure 27 is the amino acid sequence of BVH-11B (also called Newl3) protein; SEQ ID NO: 62.

Figure 28 is the amino acid sequence of BVH-11C protein; SEQ ID NO: 63.

Figure 29 is ID NO: 64.

the amino acid sequence of NEW1 protein;

SEQ

C 00

ND

o1 Cq 15 Figure ID NO: Figure ID NO: 30 is 31 is 66.

32 is 67.

33 is 68.

the amino acid sequence of NEW2 protein;

SEQ

the amino acid sequence of NEW3 protein;

SEQ

the amino acid sequence of NEW4 protein;

SEQ

the amino acid sequence of NEWS protein;

SEQ

Figure ID NO: Figure ID NO: Figure ID NO: Figure ID NO: 34 is 69.

35 is the amino acid sequence of NEW6 protein;

SEQ

the amino acid sequence of NEW7 protein;

SEQ

Figure 36 is ID NO: 71.

the amino acid sequence of NEWS protein;

SEQ

Figure 37 is the amino acid sequence of NEW9 protein;

SEQ

ID NO: 72.

Figure 38 is the amino acid sequence of BVH-11-2M protein; SEQ ID NO: 73.

Figure 39 is ID NO: 74.

Figure 40 is ID NO: the amino acid sequence of NEW10 protein; SEQ the amino acid sequence of NEW11 protein; SEQ Figure 41 is the DNA sequence of NEW12 gene; SEQ ID NO: 76.

Figure 42 is ID NO: 77.

Figure 43 is ID NO: 78.

the amino acid sequence of NEW14 protein; SEQ the amino acid sequence of NEW15 protein; SEQ Figure 44 is ID NO: 79.

the amino acid sequence of NEW16 protein; SEQ Figure 45 is NO: the DNA sequence of GBS BVH-71 gene; SEQ ID Figure 46 is the amino acid sequence of GBS BVH-71 protein; SEQ ID NO: 81.

Figure 47 is the DNA sequence of GAS BVH-71 gene; SEQ ID NO:82.

Figure 48 is the amino acid sequence of GAS BVH-71 protein; SEQ ID NO:83.

DETAILED DESCRIPTION OF THE INVENTION 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 SEQ ID NOs: 2, 4, 6, 8, 10, 14, 16, to 75, 77 to 79, 81, 83 or fragments, analogs or derivatives thereof.

S According to one aspect, the present invention provides an isolated polynucleotide encoding a polypeptide having at Sleast 95% identity to a second polypeptide comprising a Ssequence chosen from SEQ ID NOs: 2, 4, 6, 8, 10, 14, 16, to 75, 77 to 79, 81, 83 or fragments, analogs or derivatives thereof.

oO IND According to one aspect, the present invention provides an S isolated polynucleotide encoding a polypeptide having at (N least 70% identity to a second polypeptide comprising a sequence chosen from SEQ ID NOs: 2, 4, 8, 10, 14, 16, 55 to C 15 75, 77 to 79, 81, 83 or fragments, analogs 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 SEQ ID NOs: 2, 4, 10, 14, 16, 55 to 77 to 79, 81, 83 or fragments, analogs 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 SEQ ID NOs: 2, 4, 8, 10, 14, 16, 55 to 77 to 79 or fragments, analogs 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 SEQ ID NOs: 2, 8, 10, 16, 55, 56, 57, 58, 59, 64, 65, 66, 78 or fragments, analogs or derivatives thereof.

According to one aspect, the present invention provides an 0 isolated polynucleotide encoding a polypeptide having at 0 least 70% identity to a second polypeptide comprising a CL 5 sequence chosen from SEQ ID NOs: 2, 8, 10, 16, 55, 56, 57, 59, 64, 65, 66, 78 or fragments, analogs or derivatives cthereof.

According to one aspect, the present invention provides an 0 10 isolated polynucleotide encoding a polypeptide having at 00 ID least 70% identity to a second polypeptide comprising a S sequence chosen from SEQ ID NOs: 4, 14, 58, 60, 61, 62, 63, V) 67, 68, 69, 70, 71, 72, 73, 74, 75, 77, 79 or fragments, analogs or derivatives thereof.

Cq 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 SEQ ID NOs: 4, 14, 60, 61, 62, 63, 67, 68, 69, 70, 71, 72, 73, 74, 75, 77, 79 or fragments, analogs 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 SEQ ID NOs: 2, 4, 10, 14, 16, 55 to 77 to 79 or fragments, analogs 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 sequence chosen from SEQ ID NOs: 10, 55 to 75, 77, 78, 79 or fragments, analogs 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 sequence chosen from SEQ ID NOs: 55 to 75, 77, 78, 79 or Sfragments, analogs or derivatives thereof.

(N According to one aspect, the present invention provides an Sisolated polynucleotide encoding a polypeptide having at least 70% identity to a second polypeptide comprising a sequence chosen from SEQ ID NOs: 2, 4, 6, 8, 10 or S10 fragments, analogs or derivatives thereof.

00 aO i According to one aspect, the present invention provides an Sisolated polynucleotide encoding a polypeptide having at C( 15 least 70% identity to a second polypeptide comprising a sequence chosen from SEQ ID NOs: 2, 4, 10, 14, 16 or fragments, analogs 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 SEQ ID NOs: 2, 4, 14, 16 or fragments, analogs 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 sequence SEQ ID NO: 2 or fragments, analogs 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 sequence SEQ ID NO: 4 or fragments, analogs or derivatives thereof.

According to one aspect, the present invention provides an isolated polynucleotide encoding a polypeptide having at 3 least 70% identity to a second polypeptide comprising sequence SEQ ID NO: 10 or fragments, analogs or derivatives thereof.

SAccording to one aspect, the present invention provides an isolated polynucleotide encoding a polypeptide having at least 70% identity to a second polypeptide comprising 10 sequence SEQ ID NO: 14 or fragments, analogs or derivatives ND thereof.

4 According to one aspect, the present invention provides an Sisolated polynucleotide encoding a polypeptide having at Cl 15 least 70% identity to a second polypeptide comprising sequence SEQ ID NO: 16 or fragments, analogs 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 sequence SEQ ID NO: 58 or fragments, analogs 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 sequence SEQ ID NO: 60 or fragments, analogs 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 sequence SEQ ID NO: 62 or fragments, analogs or derivatives thereof.

According to one aspect, the present invention provides an isolated polynucleotide encoding a polypeptide having at 0 least 70% identity to a second polypeptide comprising C) sequence SEQ ID NO: 64 or fragments, analogs or derivatives C 5 thereof.

Mn According to one aspect, the present invention provides an isolated polynucleotide encoding a polypeptide having at least 70% identity to a second polypeptide comprising 00 10 sequence SEQ ID NO: 67 or fragments, analogs or derivatives IND thereof.

V) According to one aspect, the present invention provides an isolated polynucleotide encoding a polypeptide having at N 15 least 70% identity to a second polypeptide comprising sequence SEQ ID NO: 68 or fragments, analogs 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 sequence SEQ ID NO: 69 or fragments, analogs 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 sequence SEQ ID NO: 72 or fragments, analogs 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 sequence SEQ ID NO: 74 or fragments, analogs or derivatives thereof.

According to one aspect, the present invention provides an isolated polynucleotide encoding a polypeptide having at 0 least 70% identity to a second polypeptide comprising C-i sequence SEQ ID NO: 77 or fragments, analogs or derivatives 0 5 thereof.

C According to one aspect, the present invention relates to polypeptides characterized by the amino acid sequence chosen from SEQ ID NOs: 2, 4, 6, 8, 10 or fragments, 00 10 analogs or derivatives thereof.

\O

O According to one aspect, the present invention relates to Vt polypeptides characterized by the amino acid sequence chosen from SEQ ID NOs: 2, 4, 6, 8, 10, 14, 16, 55 to 77 to 79, 81, 83 or fragments, analogs or derivatives thereof.

According to one aspect, the present invention relates to polypeptides characterized by the amino acid sequence chosen from SEQ ID NOs: 2, 4, 8, 10, 14, 16, 55 to 75, 77 to 79, 81, 83 or fragments, analogs or derivatives thereof.

According to one aspect, the present invention relates to polypeptides characterized by the amino acid sequence chosen from SEQ ID NOs: 2, 4 10, 14, 16, 55 to 75, 77 to 79, 81, 83 or fragments, analogs or derivatives thereof.

According to one aspect, the present invention relates to polypeptides characterized by the amino acid sequence chosen from SEQ ID NOs: 2, 4, 8, 10, 14, 16, 55 to 75, 77 to 79 or fragments, analogs or derivatives thereof.

According to one aspect, the present invention relates to polypeptides characterized by the amino acid sequence chosen from SEQ ID NOs: 2, 4, 10, 14, 16, 55 to 75, 77 to 79 or fragments, analogs or derivatives thereof.

According to one aspect, the present invention relates to polypeptides characterized by the amino acid sequence C chosen from SEQ ID NOs: 2, 4, 10, 14, 16 or fragments, analogs or derivatives thereof.

cu C According to one aspect, the present invention relates to polypeptides characterized by the amino acid sequence comprising sequence SEQ ID NO: 2 or fragments, analogs or 0C 10 derivatives thereof.

00 \O O According to one aspect, the present invention relates to C polypeptides characterized by the amino acid sequence Scomprising sequence SEQ ID NO: 4 or fragments, analogs or Ci 15 derivatives thereof.

According to one aspect, the present invention relates to polypeptides characterized by the amino acid sequence comprising sequence SEQ ID NO: 10 or fragments, analogs or derivatives thereof.

According to one aspect, the present invention relates to polypeptides characterized by the amino acid sequence comprising sequence SEQ ID NO: 14 or fragments, analogs or derivatives thereof.

According to one aspect, .the present invention relates to polypeptides characterized by the amino acid sequence comprising sequence SEQ ID NO: 16 or fragments, analogs or derivatives thereof.

According to one aspect, the present invention relates to polypeptides characterized by the amino acid sequence chosen from SEQ ID NOs: 10, 55 to 75, 77, 78, 79 or fragments, analogs or derivatives thereof.

According to one aspect, the present invention relates to polypeptides characterized by the amino acid sequence S chosen from SEQ ID NO: 10, 58, 60, 62, 64, 67, 68, 69, 72, 74, 77 or fragments, analogs or derivatives thereof.

d, SAccording to one aspect, the present invention relates to polypeptides characterized by the amino acid sequence chosen from SEQ ID NO: 10, 58, 60, 62, 64, 67, 68, 69, 72, 74, 77 or fragments, analogs or derivatives thereof.

00 ND According to one aspect, the present invention relates to C polypeptides characterized by the amino acid sequence V' chosen from SEQ ID NO: 10, 58, 60, 62, 64, 67, 68, 69, 72, 74, 77 or fragments, analogs or derivatives thereof.

Cq According to one aspect, the present invention relates to polypeptides characterized by the amino acid sequence chosen from SEQ ID NO: 10, 62, 64, 67, 68, 74, 77 or fragments, analogs or derivatives thereof.

According to one aspect, the present invention relates to polypeptides characterized by the amino acid sequence comprising sequence SEQ ID NO: 58 or fragments, analogs or derivatives thereof.

According to one aspect, the present invention relates to polypeptides characterized by the amino acid sequence comprising sequence SEQ ID NO: 62 or fragments, analogs or derivatives thereof.

According to one aspect, the present invention relates to polypeptides characterized by the amino acid sequence comprising sequence SEQ ID NO: 64 or fragments, analogs or derivatives thereof.

According to one aspect, the present invention relates to polypeptides characterized by the amino acid sequence comprising sequence SEQ ID NO: 67 or fragments, analogs or n derivatives thereof.

According to one aspect, the present invention relates to Spolypeptides characterized by the amino acid sequence Scomprising sequence SEQ ID NO: 68 or fragments, analogs or derivatives thereof.

0C 10 According to one aspect, the present invention relates to 00 \0 polypeptides characterized by the amino acid sequence CO comprising sequence SEQ ID NO: 74 or fragments, analogs or derivatives thereof.

C 15 According to one aspect, the present invention relates to polypeptides characterized by the amino acid sequence comprising sequence SEQ ID NO: 77 or fragments, analogs or derivatives thereof.

In a further embodiment, the present invention also relates to chimeric polypeptides which comprise one or more polypeptides or fragments, analogs or derivatives thereof as described in the present application.

In a further embodiment, the present invention also relates to chimeric polypeptides which comprise one or more polypeptides or fragments, analogs or derivatives thereof as defined in the figures of the present application.

In a further embodiment, the present application also relates to chimeric polypeptides which comprise two or more polypeptides chosen from SEQ ID NOs: 2, 4, 6, 8, 10, 14, 16, 55 to 75, 77 to 79, 81, 83 or fragments, analogs or derivatives thereof ;provided that the polypeptides or fragments, analogs or derivatives thereof are linked as to form a chimeric polypeptide.

In a further embodiment, the chimeric polypeptide will comprise two or more polypeptides chosen from SEQ ID NOs :10, 58, 60, 62, 64, 67, 68, 69, 72, 74, 77 or fragments, analogs or derivatives thereof; provided that the polypeptides or fragments, analogs or derivatives cthereof are linked as to form a chimeric polypeptide.

In a further embodiment, the chimeric polypeptide will 00 10 comprise two or more polypeptides chosen from SEQ ID

\O

IN NOs :10, 58, 60, 62, 64, 67, 68, 74, 77 or fragments, eC analogs or derivatives thereof; provided that the 3 polypeptides or fragments, analogs or derivatives thereof 0 are linked as to form a chimeric polypeptide.

In a further embodiment, the chimeric polypeptide will comprise two or more polypeptides chosen from SEQ ID NOs :10, 62, 64, 67, 68, 74, 77 or fragments, analogs or derivatives thereof; provided that the polypeptides or fragments, analogs or derivatives thereof are linked as to form a chimeric polypeptide.

In a further embodiment, the chimeric polypeptide will comprise between 2 and 5 polypeptides.

In a further embodiment, the chimeric polypeptide will comprise between 2 and 4 polypeptides.

In a further embodiment, the chimeric polypeptide will comprise between 2 and 3 polypeptides.

In a further embodiment, the chimeric polypeptide will comprise 2 polypeptides.

00

IN

oO Cq 15 In a further embodiment, there is provided a chimeric polypeptide of formula

(I)

Wherein; m is 0 or 1, n is 0 or 1, A is chosen from SEQ ID NOs: 2, 4, 6, 8, 10, 14, 16, 55 to 77 to 79, 81, 83 or fragments, analogs or derivatives thereof; B is chosen from SEQ ID NOs: 2, 4, 6, 8, 10, 14, 16, 55 to 75, 77 to 79, 81, 83 or fragments, analogs or derivatives thereof; C is chosen from SEQ ID NOs: 2, 4, 6, 8, 10, 14, 16, 55 to 75, 77 to 79, 81, 83 or fragments, analogs or derivatives thereof; and D is chosen from SEQ ID NOs: 2, 4, 6, 8, 10, 14, 16, 55 to 77 to 79, 81, 83 or fragments, analogs or derivatives thereof.

In a further embodiment, A is chosen from SEQ ID NOs :10, 58, 60, 62, 64, 67, 68, 69, 72, 74, 77 or fragments, analogs or derivatives thereof; B is chosen from SEQ ID NOs :10, 58, 60, 62, 64, 67, 68, 69, 72, 74, 77, or fragments, analogs or derivatives thereof; C is chosen from SEQ ID NOs :10, 58, 60, 62, 64, 67, 68, 69, 72, 74, 77 or fragments, analogs or derivatives thereof; and D is chosen from SEQ ID NOs :10, 58, 60, 62, 64, 67, 68, 69, 72, 74, 77 or fragments, analogs or derivatives thereof.

In a further embodiment, A is chosen from SEQ ID NOs :10, 74, 77 or fragments, analogs or B is chosen from SEQ ID NOs :10, 74, 77, or fragments, analogs or C is chosen from SEQ ID NOs :10, 74, 77 or fragments, analogs or D is chosen from SEQ ID NOs :10, 74, 77 or fragments, analogs or 58, 60, 62, derivatives 58, 60, 62, derivatives 58, 60, 62, derivatives 58, 60, 62, derivatives 64, 67, 68, thereof; 64, 67, 68, thereof; 64, 67, 68, thereof; and 64, 67, 68, thereof.

"j Oc oo sD Ca Vo oq 5 In one embodiment, chimeric polypeptides of the present invention comprise those wherein the following embodiments are present, either independently or in combination.

In a further embodiment, A is SEQ ID NOs :10, 58, 62, 64, 67, 68, 74, 77 or fragments, analogs or derivatives thereof.

In a further embodiment, A is SEQ analogs or derivatives thereof.

In a further embodiment, A is SEQ analobs or derivatives thereof.

ID NO :10 or fragments, ID NO :58 or fragments, ID NO :62 or fragments, ID NO :64 or fragments, ID NO :67 or fragments, ID NO :68 or fragments, ID NO :74 or fragments, In a further embodiment, A is SEQ ID NO :77 or fragments, analogs or derivatives thereof.

In a further embodiment, B is SEQ ID NOs :10, 58, 62, 64, 67, 68, 74, 77 or fragments, analogs or derivatives thereof.

In a further embodiment, B is SEQ analogs or derivatives thereof.

ID NO :10 or fragments, ID NO :58 or fragments, ID NO :64 or fragments, ID NO :64 or fragments, ID NO :67 or fragments, ID NO :68 or fragments, ID NO :74 or fragments, ID NO 77 or fragments, In a further embodiment, C is SEQ ID NOs :10, 58, 62, 64, 67, 68, 74, 77 or fragments, analogs or derivatives thereof.

In a further embodiment, C is SEQ ID NO :10 or fragments, analogs or derivatives thereof.

In a further embodiment, C is SEQ ID NO :58 or fragments, analogs or derivatives thereof.

In a further embodiment, C is SEQ ID NO 62 or fragments analogs or derivatives thereof.

In a further embodiment, C is SEQ ID NO :64 or fragments, analogs or derivatives thereof.

In a further embodiment, C is SEQ ID NO 67 or fragments analogs or derivatives thereof.

I

In a further embodiment, C is SEQ ID NO 68 or fragments, analogs or derivatives thereof.

In a further embodiment, C is SEQ ID NO analogs or derivatives thereof.

In a further embodiment, D is SEQ ID NO 67, 68, 74, 77 or fragments, analogs or thereof.

In a further embodiment, D is SEQ ID NO 74 or fragments, 77 or fragments, :10, 58, 62, 64, derivatives :10 or fracments.

analogs or derivatives thereof.

In a further embodiment, D is SEQ analogs or derivatives thereof.

ID NO :58 or fragments, ID NO :62 or fragments, ID NO :64 or fragments, ID NO :67 or fragments, ID NO :68 or fragments, ID NO :74 or fragments, ID NO :77 or fragments, In a further embodiment, m is 0.

In a further embodiment, n is 0.

In a 'further embodiment, m and n are 0.

In a further embodiment, m and n are 0, A is SEQ ID NO:64 or fragments, analogs or derivatives thereof, B is SEQ ID 22 NO:62 or fragments, analogs or derivatives thereof.

SIn a further embodiment, m and n are 0, A is SEQ ID NO:62 Sor fragments, analogs or derivatives thereof, B is SEQ ID J NO:64 or fragments, analogs or derivatives thereof.

In accordance with the present invention, all nucleotides encoding polypeptides and chimeric polypeptides are within 00 the scope of the present invention.

\O

10 In a further embodiment, the polypeptides or chimeric polypeptides in accordance with the present invention are Santigenic.

In a further embodiment, the polypeptides or chimeric polypeptides in accordance with the present invention can elicit an immune response in an individual.

In a further embodiment, the present invention also relates to polypeptides which are able to raise antibodies having binding specificity to the polypeptides or chimeric polypeptides of the present invention as defined above.

An antibody that "has binding specificity" is an antibody that recognizes and binds the selected polypeptide but which does not substantially recognize and bind other molecules in a sample, a biological sample, which naturally includes the selected peptide. Specific binding can be measured using an ELISA assay in which the selected polypeptide is used as an antigen.

In the claims which follow and in the preceding description of the invention, except where the 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 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.

H:\rochb\Keep\P58259.doc 13/09/05 22a Unless otherwise defined, all technical and scientific Sterms 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 00

IO

o oq H:\rochb\Keep\P58259.doc 13/09/05 case of conflict, the present specification, including definitions, will control. In addition, the materials, y methods, and examples are illustrative only and not Sintended to be limiting.

cu As used herein, "fragments", "derivatives" or "analogs" of M 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 C 10 amino acid residue (preferably conserved) and which may be 00 DO natural or unnatural. In one embodiment, derivatives and S analogs of polypeptides of the invention will have about 70% identity with those sequences illustrated in the Sfigures or fragments thereof. That is, 70% of the residues C'q 15 are the same. In a further embodiment, polypeptides will have greater than 75% homology. In a further embodiment, polypeptides will have greater than 80% homology. In a further embodiment, polypeptides will have greater than homology. In a further embodiment, polypeptides will have greater than 90% homology. In a further embodiment, polypeptides will have greater than 95% homology. In a further embodiment, polypeptides will have greater than 99% homology. In a further embodiment, derivatives and analogs of polypeptides of the invention will have fewer than about 20 amino acid residue substitutions, modifications or deletions and more preferably less than 10. Preferred substitutions are those known in the art as conserved i.e.

the substituted residues share physical or chemical properties such as hydrophobicity, size, charge or functional groups.

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) 3 to increase half-life; leader or secretory amino acid sequences for ease of purification; prepro- and pro- Q 5 sequences; and (poly)saccharides.

9 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 10 mimic the different epitopes of the different streptococcus IO strains.

i Moreover, the polypeptides of the present invention can be O modified by terminal -NH, acylation (eg. by acetylation, or Cl 15 thioglycolic acid amidation, terminal carbosy amidation, e.g. with ammonia or methylamine) to provide stability, increased hydrophobicity for linking or binding to a support or other molecule.

Also contemplated are hetero and homo polypeptide multimers of the polypeptide fragments, analogues and derivatives.

These polymeric forms include, for example, one or more polypeptides that have been cross-linked with cross-linkers such as avidin/biotin, gluteraldehyde or dimethylsuperimidate. Such polymeric forms also include polypeptides containing two or more tandem or inverted contiguous sequences, produced from multicistronic mRNAs generated by recombinant DNA technology.

Preferably, a fragment, analog 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 synthetic multimers), polypeptides may be utilized 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 Sof a linking group of at least two, typically at least CD four, and not more than 16, but usually not more than about 14 carbon atoms.

cu SIn a particular embodiment, polypeptide fragments, analogs and derivatives of the invention do not contain a methionine (Met) starting residue. Preferably, OC 10 polypeptides will not incorporate a leader or secretory 00 ND sequence (signal sequence). The signal portion of a S polypeptide of the invention may be determined according to C established molecular biological techniques. In general, Sthe polypeptide of interest may be isolated from a C- 15 streptococcus culture and subsequently sequenced to determine the initial residue of the mature protein and therefore the sequence of the mature polypeptide.

According to another aspect, there are provided vaccine compositions comprising one or more streptococcus polypeptides of the invention in admixture with a pharmaceutically acceptable carrier diluent or adjuvant.

Suitable adjuvants include oils i.e. Freund's complete or incomplete adjuvant; salts i.e. AlK(SO,) 2 AlNa(SO,),, A1NH,(SO,),, silica, kaolin, carbon polynucleotides i.e.

poly IC and poly AU. Preferred adjuvants include QuilA and Alhydrogel. Vaccines of the invention may be administered parenterally by injection, rapid infusion, nasopharyngeal absorption, dermoabsorption, or bucal or oral.

Pharmaceutically acceptable carriers also include tetanus toxoid.

I

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 Sone embodiment, vaccine compositions of the present 5 invention are used for the treatment or prophylaxis of Smeningitis, otitis media, bacteremia or pneumonia. In one c embodiment, vaccine compositions of the invention are used for the treatment or prophylaxis of streptococcus infection and/or diseases and symptoms mediated by streptococcus (C 10 infection, in particular S.pneumoniae, group A 00 NO streptococcus (pyogenes), group B streptococcus (GBS or Sagalactiae), dysgalactiae, uberis, nocardia as well as Staphylococcus aureus. In a further embodiment, the Sstreptococcus infection is S.pneumoniae.

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

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

Vaccine compositions are preferably in unit dosage form of about 0.001 to 100 pg/kg (antigen/body weight) and more preferably 0.01 to 10 g/kg and most preferably 0.1 to 1 gg/kg 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 characterized by the amino'acid sequence chosen from SEQ ID NOs: 2, 4, 6, 8, 14, 16, 55 to 75, 77 to 79, 81, 83 or fragments, analogs or derivatives thereof.

In one embodiment, polynucleotides are those illustrated in SEQ ID Nos: 1, 3, 5, 7, 9, 11, 12, 13, 15, 76, 80, 82 V which may include the open reading frames (ORF), encoding Spolypeptides of the invention. It will be appreciated that the polynucleotide sequences illustrated in the figures may be altered with degenerate codons yet still encode the Spolypeptides of the invention. Accordingly the present invention further provides polynucleotides which hybridize to the polynucleotide sequences herein above described (or 10 the complement sequences thereof) having 50% identity between sequences. In one embodiment, at least 70% identity ID between sequences. In one embodiment, at least 75% identity S between sequences. In one embodiment, at least 80% identity S between sequences. In one embodiment, at least 85% identity between sequences. In one embodiment, at least 85% identity CN 15 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.

In a further embodiment, polynucleotides are those illustrated in SEQ ID NOs 1, 3, 7, 9, 11, 12, 13, 15, 76, 82 encoding polypeptides of the invention.

In a further embodiment, polynucleotides are those illustrated in SEQ ID NOs 1, 3, 9, 11, 12, 13, 15, 76, 82 which may include the open reading frames (ORF), encoding polypeptides of the invention.

In a further embodiment, polynucleotides are those illustrated in SEQ ID NOs 1, 3, 9, 11, 12, 13, 15, 76 which'may include the open reading frames (ORF), encoding polypeptides of the invention.

In a further embodiment, polynucleotides are those illustrated in SEQ ID NOs 1, 3, 7, 9, 11, 12, 13, 15, 76 which may include the open reading frames (ORF), encoding 0 polypeptides of the invention.

Q^ 5 In a further embodiment, polynucleotides are those illustrated in SEQ ID NOs 1, 7, 9, 11, 15, 76 which may CM include the open reading frames (ORF), encoding polypeptides of the invention.

00 10 In a .further embodiment, polynucleotides are those I illustrated in SEQ ID NOs 1, 9, 11, 15, 76 which may include the open reading frames (ORF), encoding n polypeptides of the invention.

In a further embodiment, polynucleotides are those illustrated in SEQ ID NOs 1, 7, 9, 11 which may include the open reading frames (ORF), encoding polypeptides of the invention.

In a further embodiment, polynucleotides are those illustrated in SEQ ID NO 1, encoding polypeptides of the invention.

In a further embodiment, polynucleotides are those illustrated in SEQ ID NO encoding polypeptides of the invention.

In a further embodiment, polynucleotides are those illustrated in SEQ ID NO :11, encoding polypeptides of the invention.

In a further embodiment, polynucleotides are those illustrated in SEQ ID NO :15, encoding polypeptides of the invention.

In a further embodiment, polynucleotides are those illustrated in SEQ ID NOs 3, 12, 13, 76, encoding r<f polypeptides of the invention.

In a further embodiment, polynucleotides are those 00 10 illustrated in SEQ ID NO encoding polypeptides of the

\O

Nh invention.

*V In a further embodiment, polynucleotides are those illustrated in SEQ ID NO :12, encoding polypeptides of the invention.

In a further embodiment, polynucleotides are those illustrated in SEQ ID NO :13, encoding polypeptides of the invention.

In a further embodiment, polynucleotides are those illustrated in SEQ ID NO :76, encoding polypeptides of the invention.

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

The present invention also includes polynucleotides complementary to the polynucleotides described in the present application.

In a further aspect, polynucleotides encoding polypeptides of the invention, or fragments, analogs or derivatives thereof, may be used in a DNA immunization method. That is, they can be incorporated into a vector which is replicable and expressible upon injection thereby producing the antigenic polypeptide in vivo. For example Spolynucleotides may be incorporated into a plasmid vector 0 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 Cs 10 techniques by expressing a polynucleotide encoding said 00 00 polypeptide in a host cell and recovering the expressed g polypeptide product. Alternatively, the polypeptides can C be produced according to established synthetic chemical 0 techniques i.e. solution phase or solid phase synthesis of oligopeptides which are ligated to produce the full polypeptide (block ligation).

General methods for obtention and evaluation of polynucleotides and polypeptides are described in the following references: Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd ed, Cold Spring Harbor, 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, 3rd Edition, 1993, 380 pages; Current Protocols in Immunology, Edited by Coligan J.E. et al., John Wiley Sons Inc., New York which are herein incorporated by reference.

For recombinant production, host cells are transfected with vectors which encode the polypeptide, and then cultured in a nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the genes.

Suitable vectors are those that are viable and replicable in the chosen host and include chromosomal, non-chromosomal and synthetic DNA sequences e.g. bacterial plasmids, phage SDNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA. The polypeptide sequence may be incorporated in the vector at the Sappropriate site using restriction enzymes such that it is operably linked to an expression control region comprising a promoter, ribosome binding site (consensus region or 10 Shine-Dalgarno sequence), and optionally an operator 00 0D (control element). One can select individual components of S the expression control region that are appropriate for a given host and vector according to established molecular g biology principles (Sambrook et al, Molecular Cloning: A C 15 Laboratory Manual, 2nd 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 P, promoter.

Vectors will preferably incorporate an origin of replication as well as selection markers i.e. ampicilin resistance gene. Suitable bacterial vectors include pET, pQE60, pQE-9, pbs, pD10 phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a, pNH18A, pNH46A, ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 and eukaryotic vectors pBlueBacIII, pWLNEO, pSV2CAT, pOG44, pXT1, pSG, pSVK3, pBPV, pMSG and pSVL. Host cells may be bacterial i.e. E.coli, Bacillus subtilis, Streptomyces; fungal i.e.

Aspergillus nicer, Aspercillus nidulins; yeast i.e.

Saccharomyces or eukaryotic i.e. CHO, COS.

Upon expression of the polypeptide in culture, cells are typically harvested by centrifugation then disrupted by physical or chemical means (if the expressed polypeptide is not secreted into the media) and the resulting crude .extract retained to isolate the polypeptide of interest.

Purification of the polypeptide from culture media or lysate may be achieved by established techniques depending C, on the properties of the polypeptide i.e. using ammonium L 5 sulfate or ethanol precipitation acid extraction, anion or cation exchange chromatography, phosphocellulose Cc, chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography and lectin chromatography.

Final purification may be achieved using HPLC.

00 IO The polypeptide may be expressed with or without a leader Sor 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 C 15 herein by reference) or be chemically removed subsequent to purifying the expressed polypeptide.

According to a further aspect, the streptococcus polypeptides of the invention may be used in a diagnostic test for streptococcus infection, in particular S.

pneumoniae infection. Several diagnostic methods are possible, for example detecting streptococcus organism in a biological sample, the following procedure may be followed: a) obtaining a biological sample from a patient; b) incubating an antibody or fragment thereof reactive with a streptococcus polypeptide of the invention with the biological sample to form a mixture; and c) detecting specifically bound antibody or bound fragment in the mixture which indicates the presence of streptococcus.

Alternatively, a method for the detection of antibody specific to a streptococcus antigen in a biological sample containing or suspected of containing said antibody may be performed as follows: a) obtaining a biological sample from a patient; b) incubating one or more streptococcus polypeptides of the invention or fragments thereof with the biological sample to form a mixture; and c) detecting specifically bound antigen or bound fragment in the mixture which indicates the presence of antibody specific to streptococcus.

One of skill in the art will recognize that this diagnostic test may take several forms, including an immunological 0 10 test such as an enzyme-linked immunosorbent assay (ELISA), 00 \O a radioimmunoassay or a latex agglutination assay, essentially to determine whether antibodies specific for Cl the protein are present in an organism.

C 15 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.pneumoniaenucleic acids in a sample, for example using a polymerase chain reaction, as a method of diagnosing streptococcus infections. The probe may be synthesized using conventional techniques and may be immobilized on a solid phase, or may be labelled with a detectable label. A preferred DNA probe for this application is an oligomer having a sequence complementary to at least about 6 contiguous nucleotides of the streptococcus pneumoniae polypeptides of the invention.

Another diagnostic method for the detection of streptococcus in a patient comprises: C a) labelling an antibody reactive with a polypeptide of the invention or fragment thereof with a detectable label; 10 b) administering the labelled antibody or labelled 00 IND fragment to the patient; and c) detecting specifically bound labelled antibody or labelled fragment in the patient which indicates the Spresence of streptococcus.

C A further aspect of the invention is the use of the streptococcus polypeptides of the invention as immunogens for the production of specific antibodies for the diagnosis and in particular the treatment of streptococcus infection.

Suitable antibodies may be determined using appropriate screening methods, for example by measuring the ability of a particular antibody to passively protect against streptococcus infection in a test model. One example of an animal model is the mouse model described in the examples herein. The antibody may be a whole antibody or an antigen-binding fragment thereof and may belong to any immunoglobulin class. The antibody or fragment may be of animal origin, specifically of mammalian origin and more specifically of murine, rat or human origin. It may be a natural antibody or a fragment thereof, or if desired, a recombinant antibody or antibody fragment. The term recombinant antibody or antibody fragment means antibody or antibody fragment which was produced using molecular biology techniques. The antibody or antibody fragments may be polyclonal, or preferably monoclonal. It may be specific for a number of epitopes associated with the streptococcus pneumoniae polypeptides but is preferably specific for one.

Without limiting its scope, the present invention also relates to new antigens designated BVH-3, BVH-ll, BVH-1l-2, BVH-28 and BVH-71. The present invention also relates to truncated polypeptides comprising fragments of the new antigens designated BVH-3, BVH-1l, BVH-11-2, BVH-28 and BVH-71. The present invention also relates to chimeric polypeptides comprising fragments of the new antigens designated BVH-3, BVH-11, BVH-l1-2, BVH-28 and BVH-71. The following is a reference table summarizing the relation between the antigens of the present invention: Family Nucleotide SEQ ID Polypeptide SEQ ID NO NO BVH -3 BVH-3 1, 11 2 BVH-3A 7 8 BVH-3B 9 BVH-3 SF63 15 16 BVH- 3M BVH- 3AD 56 L -BVH- 3AD 57 Newl2 76 58 BVH-3 C Newl 64 New2 New3 BVH- 11 BVH-11 3, 12 4 BVH-11-2 13 14 BVH-11M BVH-11A 61 BVH-11B also 62 ref drred to as NEW13 BVH- 110 63 New4 67 68 Family Nucleotide SEQ ID Polypeptide SEQ ID NO NO New6 69 New7 New8 71 New9 72 BVH-11-2M 73 NewlO 74 Newll Newl2 76 58 Newl4 77 Newl6 79 BVH-28 BVH-28 5 6 BVH-71 GBS 80 81 GAS 82 83 EXAMPLE 1 This example illustrates the cloning of S. pneumoniae genes.

The coding region of S. pneumoniae gene BVH-3 (SEQ ID NO: 1) and the coding region of S. pneumoniae gene BVH-28 (SEQ ID NO: 5) were amplified by PCR (DNA Thermal Cycler GeneAmp PCR system 2400 Perkin Elmer, San Jose, CA) from genomic DNA of serogroup 6 S. pneumoniae strain SP64 using the oligos that contained base extensions for the addition of restriction sites BglII (AGATCT) and XbaI (TCTAGA). PCR products were purified from agarose gel using a QIAquick gel extraction kit from QIAgen (Chatsworth, CA), digested BglII- XbaI (Pharmacia Canada Inc, Baie d'Urf6, Canada), extracted with phenol chloroform and precipitated with ethanol. The Superlinker vector pSL301 (Invitrogen, San Diego, CA) was digested with BglII and XbaI and purified from agarose gel using a QIAquick gel extraction kit from QIAgen (Chatsworth, CA). The BglII-XbaI genomic DNA fragments were ligated to the BglII-XbaI pSL301 vector. The ligated products were transformed into E. coli strain DH5a [f80 lacZ DM15 endAl 0 recAl hsdR17 (rK-mK) supE44 thi-11l gyrA96 relAl D(lacZYA- CI argF)U169] (Gibco BRL, Gaithersburg, MD) according to the method of Simanis (Hanahan, D. DNA Cloning, 1985, D.M.

Glover pp. 109-135). Recombinant pSL301 plasmids (rpSL301) containing either BVH-3 or BVH-28 gene were purified using a QIAgen kit (Chatsworth, CA) and DNA inserts OO were confirmed by nucleotide sequence analysis (Taq Dye 0O Deoxy Terminator Cycle Sequencing kit, ABI, Foster City, CA). Recombinant rpSL301 (rpSL301) were digested with the restriction enzymes BglII (AGATCT) and XhoI (CTCGAG). DNA S fragments BglII-XhoI were purified using the QIAquick gel extraction kit from QIAgen (Chatsworth, CA). pET-32c(+) expression vector (Novagen, Madison, WI) containing the thioredoxin-His-Tag sequence was digested with BamHI (GGATCC) and XhoI and gel extracted using the QIAquick gel extraction kit from QIAgen (Chatsworth, CA). The BglII-XhoI DNA fragments were ligated to the BamHI-XhoI pET-32c(+) vector to create the coding sequence for thioredoxin- His-Tag-BVH-3 or thioredoxin-His-Tag-BVH-28 fusion protein.

The ligated products were transformed into E. coli strain [f80 lacZ DM15 endAl recAl hsdRl7 (rK-mK) supE44 thi-llgyrA96 relAl D(1acZYA-argF)U169] (Gibco BRL, Gaithersburg, MD) according to the method of Simanis (Hanahan, D. DNA Cloning, 1985, D.M. Glover pp. 109-135). Recombinant pET-32c(+) plasmids were purified using a QIAgen kit (Chatsworth, CA) and the nucleotide sequences at the fusion sites of thioredoxin-His-Tag and DNA insert were verified by DNA sequencing (Taq Dye Deoxy Terminator Cycle Sequencing kit, ABI, Foster City, CA).

EXAMPLE 2 C- This example illustrates the cloning of S. pneumoniae e 5 protein genes in CMV plasmid pCMV-GH.

The DNA coding region of a S. pneumoniae protein was inserted in phase downstream of a human growth hormone (hGH) gene which was under the transcriptional control of the 00 ~O 10 cytomegalavirus (CMV) promotor in the plasmid vector pCMV-GH (Tang et al., Nature, 1992, 356 :152). The CMV promotor is V non functional plasmid in E. coli cells but active upon Sadministration of the plasmid in eukaryotic cells. The vector also incorporated the ampicillin resistance gene.

The coding region of BVH-3 gene (SEQ ID NO: 1) and BVH-28 gene (SEQ ID NO: 5) were obtained from rpSL301 (see example 1) using restriction enzymes BglII (AGATCT) and XbaI (TCTAGA). The digested products were purified from agarose gel using the QIAquick gel extraction kit from QIAgen (Chatsworth, CA). The pCMV-GH vector (Laboratory of Dr.

Stephen A. Johnston, Department of Biochemistry, The University of Texas, Dallas, Texas) containing the human growth hormone to create fusion proteins was digested with BglII and XbaI and purified from agarose gel using the QIAquick gel extraction kit from QIAgen (Chatsworth,

CA).

The BglII-XbaI DNA fragments were ligated to the BglII-XbaI pCMV-GH vector to create the hGH-BVH-3 or hGH-BVH-28 fusion protein under the control of the CMV promoter. The ligated products were transformed into E. coli strain DH5a[f80 lacZ endAl recAl hsdR17 (rK-mK') supE44 thi-11l gyrA96 relAl D(lacZYA-argF)U169] (Gibco BRL, Gaithersburg, MD) according to the method of Simanis (Hanahan, D. DNA Cloning, 1985, D.M. Glover pp. 109-135). The recombinant pCMV O plasmids were purified using a QIAgen kit (QIAgen, C< Chatsworth, CA).

dj The coding region of BVH-11 gene (SEQ ID NO: 3) was amplified by PCR (DNA Thermal Cycler GeneAmp PCR system 2400 Perkin Elmer, San Jose, CA) from genomic DNA of serogroup 6 SO S. pneumoniae strain SP64 using the oligos that contained 00 base extensions for the addition of restriction sites BglII (AGATCT) and HindIII (AAGCTT). The PCR product was purified 3 from agarose gel using a QIAquick gel extraction kit from QIAgen (Chatsworth, CA), digested with restriction enzymes (Pharmacia Canada Inc, Baie d'Urfe, Canada), extracted with phenol chloroform and precipitated with ethanol. The pCMV-GH vector (Laboratory of Dr. Stephen A. Johnston, Department of Biochemistry, The University of Texas, Dallas, Texas) was digested with BglII and HindIII and purified from agarose gel using the QIAquick gel extraction kit from QIAgen (Chatsworth, CA). The BglII-HindIII DNA fragment was ligated to the BglII-HindIII pCMV-GH vector to create the hGH-BVH-11 fusion protein under the control of the CMV promoter. The ligated products were transformed into E. coli strain DH5a[f80 lacZ DM15 endAl recAl hsdR17 (rK-mK+) supE44 thi-ll- gyrA96 relAl D(lacZYA-argF)U169] (Gibco BRL, Gaithersburg, MD) according to the method of Simanis (Hanahan, D. DNA Cloning, 1985, D.M. Glover pp. 109- 135). The recombinant pCMV plasmid was purified using a QIAgen kit (Chatsworth, CA) and the nucleotide sequence of the DNA insert was verified by DNA sequencing.

EXAMPLE 3 9 This example illustrates the use of DNA to elicit an immune response to S. pneumoniae antigens.

e( 1 A group of 8 female BALB/c mice (Charles River, St-Constant, Qu6bec, Canada) were immunized by intramuscular injection of pl three times at two- or three-week intervals with 100 OO ~g of recombinant pCMV-GH encoding the BVH-3, BVH-11 or the \0O OC 10 BVH-28 gene in presence of 50 pg of granulocyte-macrophage (c colony-stimulating factor (GM-CSF)- expressing plasmid pCMV- 0 GH-GM-CSF (Laboratory of Dr. Stephen A. Johnston, Department of Biochemistry, The University of Texas, Dallas, Texas).

As control, a group of mice were injected with 100 jg of pCMV-GH in presence of 50 Ag of pCMV-GH-GM-CSF. Blood samples were collected from the orbital prior to each immunization and seven days following the third injection and serum antibody responses were determined by ELISA using thioredoxin-His.Tag-S. pneumoniae fusion protein as coating antigen. DNA immunization with recombinant plasmid pCMV-GH encoding the BVH-3, BVH-11 or the BVH-28 S. pneumoniae protein induced antibody reactive against the respective recombinant protein. The reciprocal antibody titers, defined as the highest serum dilution at which the absorbance values were 0.1 above the background values, were above 4x10 3 EXAMPLE 4 This example illustrates the production and purification of recombinant S. pneumoniae proteins.

The recombinant pET plasmids containing the BVH-3, BVH-11 or 0 the BVH-28 gene corresponding to the SEQ ID NO: 1 SEQ ID C- NO: 3 or the SEQ ID NO: 5 respectively were transformed by i 5 electroporation (Gene Pulser II apparatus, BIO-RAD Labs, r Mississauga, Canada) into E. coli strain AD494 (DE3) (Daraleu7697 DlacX74 DphoA PvuII phoR DmalF3 F'[lac'(lacI q pro] trxB::Kan) (Novagen, Madison, WI). In this strain of E.

coli, the T7 promotor controlling expression of the fusion 00 10 protein is specifically recognized by the T7 RNA polymerase (present on the 1DE3 prophage) whose gene is under the S control of the lac promotor which is inducible by isopropyl- 0 -d-thio-galactopyranoside (IPTG). The transformant AD494(DE3)/rpET was grown at 37 0 C with agitation at 250 rpm in LB broth (peptone 10g/L, yeast extract 5g/L, NaC1 containing 100pg of ampicillin (Sigma-Aldrich Canada Ltd., Oakville, Canada) per ml until the A 600 reached a value of 0.6. In order to induce the production of the thioredoxin- His-Tag-BVH-3, thioredoxin-His*Tag-BVH-11 or thioredoxin- His-Tag-BVH-28 fusion protein, the cells were incubated for 2 additional hours in the presence of IPTG at a final concentration of 1 mM. Induced cells from a 100 ml culture were pelleted by centrifugation and frozen at -70 0

C.

The purification of the fusion proteins from the soluble cytoplasmic fraction of IPTG-induced AD494(DE3)/rpET was done by affinity chromatography based on the properties of the His-Tag sequence (6 consecutive histidine residues) to bind to divalent cations (Ni 2 immobilized on the His-Bind metal chelation resin. Briefly, the pelleted cells obtained from a 100mL culture induced with IPTG were resuspended in phosphate-buffered (PBS):500mM NaCI pH7.1, sonicated and spun at 20,000 X g for 20 min to remove debris. The g supernatant was filtered (0.22pm pore size membrane) and (C deposited on a HiTrap® imL chelating pre-packed ready-to-use q 5 column (Pharmacia Biotech, Bale d'Urf6, Canada). The thioredoxin-HisTag-S. pneumoniae fusion protein was eluted with 1M imidazole-500mM NaCl-PBS pH7.1. The removal of the salt and imidazole from the sample was done by dialysis O .against PBS at 4 0 C. The quantities of fusion protein C' 10 obtained from the soluble fraction of E. coli was estimated by MicroBCA (Pierce, Rockford, Illinois).

EXAMPLE This example illustrates the protection of mice against fatal pneumococcal infection by immunization.

Groups of 8 female BALB/c mice (Charles River) were immunized subcutaneously three times at three-week intervals with either 25 ig of affinity purified thioredoxin-His-Tag- BVH-3 fusion protein in presence of 15 xg of QuilA adjuvant (Cedarlane Laboratories Ltd, Hornby, Canada) or, as control, with QuilA adjuvant alone in PBS. Blood samples were collected from the orbital sinus on day 1, 22 and 43 prior to each immunization and seven days (day 50) following the third injection. One week later the mice were challenged with approximately 106 CFU of the type 3 S. pneumoniae strain WU2. Samples of the S. pneumoniae challenge inoculum were plated on chocolate agar plates to determine the CFU and to verify the challenge dose. Deaths were recorded for a period of 14 days and on day 14 post-challenge, the surviving mice were sacrificied and blood samples tested for 0 the presence of S. pneumoniae organisms. The survival data c-i are shown in table 1.

Cu Prechallenge sera were analyzed for the presence of antibodies reactive with S. pneumoniae by standard immunoassays. Elisa and immunoblot analyses indicated that C immunization with recombinant S. pneumoniae protein produced O 10 in E. coli elicited antibodies reactive with both, 0 recombinant and native pneumococcal protein.

ci S Table 1. Protection mediated by recombinant BVH-3 protein Immunogen No. of mice alive no. of mice Median day of dead death 14 days post-challenge BVH-3 8 0 >14 none 0 8 1 All mice immunized with BVH-3 recombinant protein survived to infection while none of the control mice given adjuvant alone survived. There was a significant difference in survival between the two groups of mice (P<0.0001, log rank test for nonparametric analysis of survival curves; P=0.0002, Fisher's exact test). All hemocultures from surviving mice were negative at day 14 post-challenge.

EXAMPLE 6 This example describes the cloning of BVH-3 and BVH-11 genes from a variety of S_ pneumoniae strains and the molecular conservation of these genes.

Molecular analysis of chromosomal DNA from various S.

pneumoniae isolates with DNA probes spanning different Sregions of BVH-3 or BVH-11 revealed the presence of one BVH-3 gene copy and two BVH-11 gene copies. The two BVH-11 gene copies are not identical and the genes were C 10 arbitrarily designated BVH-11 (SEQ ID NO:12; ORF at 00 \D nucleotides 45 to 2567) and BVH-11-2 (SEQ ID NO:13; ORF at Q nucleotides 114 to 2630).

The first amino acids of the BVH-3 and BVH-11 coding C( 15 regions have the characteristics of leader sequences also known as signal peptides. The consensus signal peptidase cleavage site L-X-X-C of lipoprotein modification/processing sites was present in the sequences.

Mature BVH-3, BVH-11 and BVH-11-2 proteins from S.

?0 pneumoniae SP64 have 1019, 821 and 819 amino acids, respectively. The regions of S. pneumoniae genes coding for mature BVH-3, termed BVH-3M, (nucleotides 1837 4896; SEQ. ID. NO: 11), BVH-11M (nucleotides 102-2567; SEQ. ID.

NO: 12) and BVH-11-2M (nucleotides 171-2630; SEQ. ID. NO: 13), were amplified by PCR(DNA Thermal Cycler GeneAmp PCR system 2400 Perkin Elmer, San Jose, CA) from genomic DNA of 6 or 7 S. pneumoniae strains. Serogroup 6 pneumoniae SP64 and serogroup 9 SP63 clinical isolates were provided by the laboratoire de la sante publique du Qu6bec, Sainte- Anne-de-Bellevue; serotype 4 strain JNR.7/87 was provided by Andrew Camilli, Tufts University School of Medicine, Boston; Rxl strain, a nonencapsulated derivative of the type 2 strain D39 and the type 3 strains A66 and WU2 were provided by David E. Briles from University of Alabama, Birmingham and the type 3 clinical isolate P4241 was provided by the centre de recherche en infectiologie du centre hospitalier de l'universit6 Laval, Sainte-Foy. The sets of oligonucleotide primers OCRR479-OCRR480; HAMJ160- OCRR488 and HAMJ160-HAMJ186, that contained base extensions 0 for the addition of restriction sites were used for the amplification of BVH-3, BVH-11 and BVH-11-2 gene, Srespectively, with the exception of BVH-11 gene from SP64 Sstrain which was amplified using the set of primers consisting of HAMJ487 and OCRR488. Primer sequences are listed below (Table PCR products were purified from C* 10 agarose gel using a QIAquick gel extraction kit from QIAgen 00 IO (Chatsworth, CA) and digested BglII-XbaI or BglII-HindIII O (Pharmacia Canada Inc, Baie d'Urf4, Canada). Digestions were cleaned using a QIAquick PCR purification kit from QIAgen (Chatsworth, CA). The PCR products were ligated to C- 15 the BglII-XbaI or BglII-HindIII pSL301 vector. The ligated products were transformed into E. coli strain DH5cx [080 lacZ AM15 endAl recAl hsdR17 supE44 thi-lX gyrA96 relAl A(lacZYA-argF)U169] (Gibco BRL, Gaithersburg,

MD)

according to the method of Simanis (Hanahan, D. DNA Cloning, 1985, D.M. Glover pp. 109-135). Recombinant pSL301 plasmids (rpSL301) containing BVH-3, BVH-11 or BVH11-2 were purified using a QIAgen kit (Chatsworth,

CA)

and DNA inserts were sequenced (Taq Dye Deoxy Terminator Cycle Sequencing kit, ABI, Foster City, CA). The figures 11 and 12 depict the consensus sequence established from the BVH-3, and BVH-11 deduced amino acid sequences, respectively. Comparison of BVH-3 protein sequences revealed 99 to 100% identity of sequences for all strains with the exception that BVH-3 from serogroup 9 SP63 strain (SEQ. ID. NO: 15 and SEQ. ID. NO: 16) misses a stretch of 177 amino acids corresponding to residues 244 to 420 on BVH-3'protein sequence of S. pneumoniae SP64. Analysis of sequences of additional serogroup 9 strains revealed BVH-3 molecule having the same deletion in 3 out of 4 strains thus suggesting that the 3 strains are members of a S.

pneumoniae serogroup 9 clone.

O Comparison of 13 BVH-11 nucleotide sequences obtained from 7 S. pneumoniae strains, revealed that the nucleotide sequences are very similar. Computer analysis (MacVector, SClustal W 1.4) using multiple alignment of the predicted BVH-11 protein sequences revealed that these sequences were identical and 82 homologous on a length of 834 amino C 10 acids. Pairwise alignment revealed 80 to 100% identity 00 ID (Figure 13). The sequences showed great similarity in S overall organization. Variability in the primary sequence 'q of these proteins is almost restricted to the last 125 amino acids in the C-terminal portion of the proteins. This C 15 region constitutes a domain. Close examination of this domain revealed two groups of sequences. The first 9 sequences from the figure 13 belong to one group while the last 4 sequences belong to another group. A 39% identity value is obtained when the domain sequences of the 13 proteins are compared (MacVector, Clustal W The identity value increased to more than 92% when sequences belonging to a same group are compared.

EXAMPLE 7 This example illustrates the homology of portions of BVH-3 and BVH-11 genes.

Molecular analysis with DNA probes derived from BVH-3 and BVH-11 genes indicated that BVH-3 and BVH-11 were related.

In dot blot hybridization studies, DNA probe consisting of either, BVH-3 or BVH-11, gene sequence hybridized to both, BVH-3 and BVH-11 genes thus indicating that BVH-3 and BVH- 11 genes shared homologous sequences. Comparison of sequences revealed that the ORFs and the proteins were 43 and 33% identical, respectively. Closer examination revealed that the region corresponding to amino acids 1 to S225 in BVH-3 and 1 to 228 in BVH-11 were 73 and Sidentical at the DNA and protein level, respectively. In contrast, the 3' regions corresponding to amino acids 226 Sto 1039 from BVH-3 and amino acids 229-840 from BVH-11 were Sonly 34 and 22% identical at the DNA and protein level, respectively. Thus the 5' termini of BVH-3 and BVH-11 genes appear to contain highly conserved sequences while 10 the remaining parts of the genes are highly divergent.

00 k0 These results suggest that BVH-3 and BVH-11 might share Ssimilar functions mediated by sequences present in the conserved region whereas BVH-3- and BVH-11-specific 0 functions might be mediated by sequences in the divergent 15 region.

EXAMPLE 8 This example describes the cloning of truncated BVH-3, BVH- 11 and BVH-11-2 genes by polymerase chain reaction (PCR) and the expression of truncated BVH-3 and BVH-11 molecules.

Gene fragments were amplified by PCR using pairs of oligonucleotide engineered to amplify fragments spanning the BVH-3 (SEQ ID NO: 1 and SEQ ID NO: 11), BVH-11 (SEQ ID NO: 3 and SEQ ID NO: 12) or BVH-11-2 (SEQ ID NO: 13) gene from S pneumoniae strain SP64. Each of the primers had a restriction endonuclease site at the 5' end, thereby allowing directional in-frame cloning of the amplified product into the digested plasmid vector (Tables 2 and 3).

PCR-amplified products were digested with restriction endonucleases and ligated to either linearized plasmid pSL301 (see example pCMV-GH (see example 2) or pET (Novagen, Madison, WI) expression vector digested likewise or digested with enzymes that produce compatible cohesive ends. Recombinant pSL301 and recombinant pCMV-GH plasmids were digested with restriction enzymes for the in-frame cloning in pET expression vector. Clones were first stabilized in E. coli DH5a before introduction into E. coli e 5 BL21(XDE3) or AD494 (ADE3) for expression of truncated BVH- S3 or BVH-11 molecules. Each of the resultant plasmid constructs was confirmed by nucleotide sequence analysis.

The recombinant proteins were expressed as N-terminal fusions with the thioredoxin and His-tag or as C-terminal 00 10 fusions with an His-tag. The expressed recombinant h proteins were purified from supernatant fractions obtained C-i from centrifugation of sonicated IPTG-induced E. coli cultures using a His-Bind metal chelation resin (QIAgen, 0 Chatsworth, CA). The gene products generated are listed in the table 3. The gene products corresponding to the Nterminal region including the signal sequence are designated as Lipidated-proteins or lipoproteins (Lproteins). The gene products corresponding to the Nterminal region lacking the signal sequence are identified as protein without signal sequence (w/o ss).

Table 2. List of PCR oligonucleotide primers Primer SEQ. Sequence 5' 3' Nucleotide Restric- ID. position tion sites OCRR 479 17 cagtagatctgtgcctatgcactaaac SEQ ID 1:61- Bgll 78 OCRR 480 18 gatctctagactactgctattccttacgctatg SEQ XbaI ID 11:4909- 4887 OCRR 497 19 atcactcgagcattacctggataatcctgt SEQ XhoI ID 1:1525- _1506 OCRR 498 20 ctgctaagcttatgaaagatttagat SEQ HindMI ID 1:1534- 1548 OCRR 499 21 gatactcgagctgctattccttac SEQ XhoI ID 11 :4906- 4893 HAMJ 172 22 gaatctcgagttaagctgctgctaattc SEQ ID 1: XhoI 675-661 HAMJ 247 23 gacgctcgagcgctatgaaatcagatattc SEQ ID XhoI 1:3117-3096 I{AMJ 248 24 gacgctcgagggcattacctggataatcctgttcatg SEQ ID XhoI 1 1:1527-1501 1-AMJ 249 25 cagtagatctcttcatcatttattgaaaagagg SEQ lID I1I BglIII 177 1 HAMJ 278 26 ttatttcttccatatggacttgacagaagagcaaattaag SEQ ID ENdel 1 1:1414-1437 HAMJ 279 27 cgccaagcttcgctatgaaatcagataaattc- SEQ ID HinduHI 1:3117-3096 HAMJ 280 28 cgccaagcttttccacaatataagtcgattgatt SEQ ID HindlIl :2400-2377 HAMJ 281 29 ttatttcttccatatggaagtacctatcttggaaaaagaa SEQ ID Ndel :2398-2421 l-AMJ 300 30 ttatttcttccatatggtgcctatgcactaaaccagc SEQ ID 1 :62- NdeI 82 HAMI 313 31 ataagaatgcggccgcttccacaatataagt-cgattgatt SEQ ID Nodi 1 :2400-2377 OCRR 487 32 cagtagatctgtgcttatgaactaggtttgc SEQ ID 3 :58- BglI OCRR 488 33 gatcaagcttgctgctacctttacttactctc SEQ ID HinduI :2577-2556 HAMJ 171 34 ctgagatatccgttatcgttcaaacc SEQ ID EcoRV 3 :1060-1075 HAMJ 251 35 ctgcaagcttttaaaggggaataatacg SEQ ID HindIII :1059-1045 HAMJ 264 36 cagtagatctgcagaagccttcctatctg SEQ ID 3 :682- Bglll HAMJ 282 37 tcgccaagcttcgttatcgttcaaaccattggg SEQ ID HindIll :1060-1081 HAMJ 283 38 ataagaatgcggccgccttactctcctttaata-aagccaat SEQ ID NdeI 3 :2520-2492 J-AMJ 284 39 catgccatggacattgatagtctcttgaaacagc SEQ ID 3 :856- NcoI 880 HAMJ 285 40 cgccaagcttcttactctcctttaataaagccaatag SEQ ID HindUl :2520-2494 HAMvJ 286 41 cgacaagcttaacatggtcgctagcgtcc SEQ ID Hindul :2139-21 19 HAMJ 287 42 cataccatgggcctttatgaggcacctaag SEQ ID NcoI 3 :2014-2034 HIAMJ 288 43 1 cgacaagcttaagtaaatcttcagcctctctcag SEQ ID H-indM :2376-2353 HAMJ 289 44 gataccatggctagcgaccatgttcaaagaa SEQ ID NcoI :2125-2146 HAMJ 290 45 cgccaagcttatcatccactaacttgactttatcac SEQ ID HindII 3 3:1533-1508 HAMJ 291 46 cataccatggatattcttgccttcttagctccg SEQ lID NcoI :1531-1554 HAMJ 301 47 catgccatggtgcttatgaactaggtttgc SEQ ID 3 :59- NcoI HAMJ 302 48 cgccaagctttagcgttaccaaaaccattatc SEQ ID HindIII :2128-2107 I-AMJ. 160 49 gtattagatctgttcctatgaacttggtcgtcacca SEQ ID 13: Bglll _____172-196 HAMJ 186 50 cgcctctagactactgtataggagccgg SEQ ID 13: XbaI HAMJ 292 51 catgccatggaaaacatttcaagccttttacgtg SEQ ID 11: NcoI HAMJ 293 52 cgacaagcttctgtataggagccggttgactttc SEQ ID 11: H-induI HAMJ 294 53 catgccatggttcgtaaaaataaggcagaccaag SEQ ID I1I NcoI I-AMJ 297 54 catgccatggaagcctattggaatgggaag SEQ ID 11: NcoI 1622-642 2005209689 13 Sep 2005 Table 3. Lists of truncated BVH-3 and BVH-l1 gene Products generated from s. pneumoniae SP64 PCR-priner sets OCIRR47 9-OCRR48 OCRR4 79 -0CRR49 IIAMJ248-HAMJ24 OCRR498-OCRR49 OCRR479-HAM~J172 OCRR487-OCRR48 HAMJ251-OCRR487 HAMAJ171-0CRR488 HAMAJ264-OCRR488 HAMvJ278-HAMyJ27 HAM4J278-HAM~J280 HAMJ 281. -HAMJ 279 HAMJ284-HAM4J285 HAMJ284 -HAMJ286 HAMJ287-Hi MJ-288 HAMJ285-HAM4J289 HAMJ284-HAMJ290 ProtLein designation EVHl-3M BVH-3AD L-BVH-3

AD

BVH-3B BVH -3 C BVHq 11M BVHW-11A BVH-11B BVH*-l1C

NEW.

NEW2 NEW3 NEW4 NEW5 NEW 6-

NEW?

NEW8:: Identification (encoded amino acids) I3VH-3 w/o ss (21-1039) BVH-3 N'end w/o ss (21-509) BVH-3 N'end (1-509) BVH-3 C'end (512-1039) BVH-3 N'end w/o ss (21-225) BVH--11 w/o ss (20-840) BVH-11 N'end w/o ss (20:-3-53) BVH-11 C'end (354-840) BVH-11 C'end (228-840) BVH-3 C'end (472-10-39) BVH-3 C'end (472-800) BVH-3 &'end (800-1039) BVH-11 C'end (286-840) EVH-11 internal (286-713)- BVH-11 internal (672-792)- ByE -h 1internal (709-840) BVH-11 internal (286-511) SEQ. Cloning ID.NO. vector 55 pSL3OI 5 6 pSL3OI 61 pET-32 62 pE-'T-32 c(± 63 -pET-32 64 pET-2 65 pET-21b(±) 66 pET-21b(+) 67 pET-21d(+) 6-8 pET-2ld(+) 69 pET-21 70 pET-2 Id(±) 71 pET-21Id(+i) 2005209689 13 Sep 2005 HAMJ286-HAMJ291 NEW9 BVH-11 internal (511-713) 72 pET-21d(+) HAMJ160-HAMJ186 BTH-11-2M BVH-11-2 w/o ss (20-838) 73 pSL3OI HAM292-HAMJ293 NEW16 BVH-11-2 C'end (271-838) 74 pET-21d(+) 14AM.T9Q -uOA tr".t 0 0 A I 4.uuu- -Jn ~0S4 .j V4J~VV.L.

u'en (699-838) 4- I I lurLu -lzLtj z 0 j .BvH.-LBt HAMJ286-HAMJ297 HAMJ300-HAMJ313 NEW14 BVH-11 Cend (354-840) BVH-11-2 internal (227-699) 62 77 INEWI 5I

I

pET-21 pET-21 pET-2 pET-21b(+) pET-2 ld(+) BV'H-J N'end w/o ss (21-800) rr~iiin? rNEW15~ I 7 7 1 tJ -uJ-1. -fAi3d02 NEW16 BVH-11 N'end w/o ss (20-709) 8 9 J I EXAMPLE 9 This example describes the isolation of monoclonal antibodies (Mabs) and the use of Mabs to characterize

BVH-

3, BVH-11 and BVH-11-2 protein epitopes.

Female BALB/c mice (Charles River) were immunized subcutaneously with BVH-3, BVH-11 or BVH-11-2 gene products from S_ pneumoniae strain SP64 in presence of 15 pg of C* 10 QuilA adjuvant (Cedarlane Laboratories Ltd, Hornby, 00 k0D Canada). One set of mice (fusion experiment 1) were S immunized on day 1 and 14 with 25 gg of affinity purified l^ thioredoxin-HiseTag-BVH-3M fusion protein. A second group S of mice (fusion experiment 2) were immunized three times at

C

N 15 three-week intervals with 25 4g of affinity purified thioredoxin-His*Tag-BVH-11M. A third group of mice (fusion experiment 3) were immunized on day 1 and day 15 with 25 4g of affinity purified thioredoxin-HiseTag-BVH-ll-2M fusion protein. A fourth group of mice (fusion experiment 4) were immunized on day 1 with 25 gg of affinity purified thioredoxin-HisBVH-11lB fusion protein and boosted by intravenous injection on day 16 and on day 37 with recombinant BVH-11B in PBS. Three to four days before fusion, mice were injected intravenously with 25 pg of the respective antigen suspended in PBS alone. Hybridomas were produced by fusion of spleen cells with nonsecreting myeloma cells as previously described by J. Hamel et al.

Med. Microbiol., 23, pp163-170 (1987)]. Culture supernatants of hybridomas were initially screened by enzyme-linked-immunoassay according to the procedure described by Hamel et al. (Supra) using plates coated with preparations of purified recombinant proteins or suspensions of heat-killed neumoniae cells. Positive hybridomas selected on the basis of ELISA reactivity with a variety of antigens were then cloned by limiting dilutions, expanded and frozen.

Hybridomas were tested by ELISA or Western irmunoblotting against BVH-3 and BVH-11 gene products in order to characterize the epitopes recognized by the Mabs. BV}H-3 and BVH-11 shared common epitopes with 6 Mabs (H3-l-F9, H3- 1-D4, H3-1-H12, H11-l--E7, H1l-1-HlO andHll-l.l-G1l) showing reactivities with both proteins (Table BVH-11 and BVH-11-2 molecules from S. loneumoniae SP64 shared common epitopes not present on BVH-3 with Mabs (3A1, 13C11, 1OHlO, 1D8, 10G9, 10A2, 3E8, 10D7, 2H7 and 6H7) reactive with both, BVH-11 and BVH-1l-2, recombinant proteins (Table 00 IND 1 Table 4. Reactivity of BVH-3-immunoreactive Mabs with a panel of BVH-3 and BVH-11 gene products a. Immunoreactivity with MAbs BVH-3M BVH-3A BVH-3B BVH-3C NEW2 N\EW3 BXTH-11M- 21-1039 21-509 512-1039 21-225 472-800 800-1039 20-840 H3-1-F9 H3-1-D4 H3-1-H12 H3-2--G2 H3-3-A1 H3-4-D3 H11-1-E7 Hl-- H11- 1.1-G1l Table 5. Reactivity of Mabs raised against BVH--11-2 protein from S. pneumoniae strain SP64 with a panel of BVH- 11 gene products mabs* b. Immiunoreactivity with c.BVH-ll products dBT-l2pout BVH-11-2 products

I

20-840 286-511 511-713 BVH-11B 354-840 BVH-11-2 20-838 271-838 NEW11 NEW14 699-838 227-6 t I

II

3AI 13C1 1D8 10OG9 10OA2 3E8 10D7 2117 6H17 3A4 14H16 7G2 13HI0- 7E8 I 7116 I Ia Mabs listed in this table were not reactive -with recombinant BVH-3 molecule The results obtained from the i'munoreactivity studies of the Mabs (Table 4 and Table 5) are in agreement with the protein sequences derived from the respective gene sequences. Indeed the Mabs cross-reactive with BVHi-3 and .0 BVH-11 molecules recognized BVH-3C protein corresponding to the conserved region, and BVH-l1 and BVTH-11-2 specific Mabs were reactive with epitopes located on variable parts of these molecules. BVH-3 and ByE-11, and BVH-11 and BVH-11-2 can be distinguished by their reactivity with Mabs.

EXAMPLE This example illustrates the simultaneous expression of BVH-3 and BVH-11 gene products by S. pneumoniae.

A standard Western blot technique was used to investigate whether BVH-3 and BVH-11 genes were expressed in S.

pneumoniae. S. pneumoniae strain SP64 and SP63 were grown Sovernight at 37 0 C in 5% CO, on chocolate agar plates, bacteria were suspended in PBS and heat-killed at 56 0 C for min. For the preparation of antigens, suspensions of S.

O\ 10 pneumoniae were treated with sample buffer containing SDS IND and 2-mercaptoethanol for 5 min at 100 0 C. Pneumococcal S protein antigens were resolved by SDS-PAGE electrophoresis S according to the method of Laemmli [Nature, 227, pp. 680- 685 (1970)]. After SDS-PAGE, the proteins were transferred C~ 15 electrophoretically from the gel to nitrocellulose paper by the method of Towbin [Proc. Natl. Acad. Sci. USA, 76, pp.

4350-4354 (1979)] and probed with mouse antiserum or monoclonal antibodies. The detection of antigens reactive with the antibodies was performed by indirect enzymeimmunoassay using conjugated-anti-mouse immunoglobulins and a colour substrate. When antiserum raised to recombinant BVH-3 was tested against S. pneumoniae SP64 antigens, two reactive bands having apparent molecular masses of 127 kDa and 99 kDa were detected. Bands having the same apparent molecular masses were also detected when Mabs H3-1-F9, H3- 1-D4, H3-1-H12, H11-1-E7, H11-1-H10 andHll-l.l-G11 were used individually as immunological probes. In contrast, Mabs specific for the BVH-3 molecule detected the 127 kDa band only and Mabs specific for BVH-11 detected the 99 kDa band only thus confirming the identity of the 127 and 99 kDa bands as BVH-3 and BVH-11, respectively. These studies provide evidence that BVH-3 and BVH-11 proteins are simultaneously present on S. pneumoniae. Moreover, the results are consistent with our previous observations that BVH-3 and BVH-11 possess epitopes that are common to both proteins and epitopes that are exclusive to either protein.

In S. pneumoniae SP64, mature BVH-3, BVH-11 and BVH-11-2 are proteins of 1019, 821 and 819 amino acids with predicted molecular mass of 112.5 kDa, 92.4 kDa, and 91.7 kDa, respectively. Although there is a discrepancy between the molecular mass predicted from the sequence and the Smolecular mass calculated on SDS-PAGE, BVH-3 can be distinguished from BVH-11 by its higher molecular mass.

Moreover, BVH-3 molecules from S. pneumoniae strain SP63 10 have an apparent molecular mass of 112 kDa in SDS-PAGE 00 NO compared to 127 kDa for BVH-3 of SP64 strain. This data is S consistent with the deletion of a stretch of 177 amino acid residues in BVH-3 of S. pneumoniae strain SP63.

C- EXAMPLE 11 This example describes the protection conferred in experimental infection of mice vaccinated with recombinant BVH-3 or BVH-11 gene products.

Groups of 7 or 8 female BALB/c mice (Charles River) were immunized subcutaneously three times at three-week intervals with either affinity purified thioredoxin- His*Tag-BVH-3M fusion protein, affinity purified thioredoxin-His*Tag-BVH-11M fusion protein or, as control, with QuilA adjuvant alone in PBS. Twelve to 14 days following the third immunization, the mice were challenged intravenously with S. pneumoniae WU2 strain or intranasally with P4241 strain. Samples of the S. pneumoniae challenge inoculum were plated on chocolate agar plates to determine the CFU and to verify the challenge dose. The challenge dose was approximately 106 CFU. Deaths were recorded for a period of 14 days and on day 14 post-challenge, the surviving mice were sacrificed and blood samples tested for 1 the presence of S. pneumoniae organisms. The survival data are shown in Tables 6 and 7.

Table 6. Protection mediated by recombinant BVH-3M and BVH-11M proteins in experimental infection with virulent S.

pneumoniae WU2 Experiment Immunogen Alive dead" Median days alive 1 BVH-3M 8 0 >14 none 0 8 1 2 BVH-11M 8 0 >14 none 0 8 1 The number of mice alive the number of mice dead on day 14 post-challenge.

Table 7. Protection mediated by recombinant BVH-3M and BVH-11M proteins in experimental pneumonia with virulent S.

pneumoniae P4241 Experiment Immunogen Alive dead" Median day alive 1 BVH-3M 6 1 >14 none 1 7 2 BVH-3M 8 0 >14 BVH-11M 8 0 >14 none 0 8 4

C-

i e nLumler ULo mice alive 14 post-challenge.

tne number of mice dead on day All mice immunized with recombinant BVH-3M or BVH-11M protein survived to infection with WU2 while none of the control mice given adjuvant alone survived. All except one mice immunized with recombinant BVH-3M or BVH-11M protein survived to infection with P4241 while only one control mice given adjuvant alone survived. All hemocultures from surviving mice were negative at day 14 post-challenge.

These results clearly indicate that both, BVH-3M and BVH- 11M, elicit protective anti-pneumococcal immune responses in mice. The fact that these proteins are highly conserved among S. pneumoniae isolates emphasize the potential of BVH-3 and BVH-11 as universal vaccine candidates. Indeed, the BVH-3 and BVH-11 proteins from serogroup 6 S.

pneumoniae strain SP64 elicited protection against pneumococcal infections with strains of different capsular serotypes.

00 S Ideally, a vaccine that could protect against pneumococcal disease, could protect against meningitis, otitis media, S bacteremia and pneumonia. BVH-3 and BVH-11 were protective C( 15 against lethal systemic- and pneumonia-infection models thus suggesting that, in humans, BVH-3- and BVHll-proteinbased vaccines could reduce the incidence of a wide spectrum of disease caused by virtually all S. pneumoniae independently of the capsular serotype.

Data from Tables 6 and 7 clearly demonstrate that BVH-3 and BVH-11 were, both, protection-eliciting molecules of S.

pneumoniae. It was not known, however, whether protection can be mediated by specific sequences that were not shared on BVH-3 and BVH-11 molecules. Groups of female BALB/c mice (Charles River) were immunized subcutaneously three times at three-week intervals with either affinity purified thioredoxin-His*Tag- BVH-3AD, -BVH-3B or -BVH-3C fusion protein in presence of 15 ig of QuilA adjuvant (Cedarlane Laboratories Ltd, Hornby, Canada). Control mice were immunized with QuilA adjuvant alone in PBS or affinity purified thioredoxin-HiseTag or thioredoxin-His*Tag-fusion protein (His-Thio) in presence of QuilA.

To determine the protective ability of a set of truncated proteins, termed NEW4, NEW5, NEW6, NEW7, NEW8, NEW9, NEW11, NEW14 and BVH-11B, groups of female BALB/c mice (Charles River) were immunized subcutaneously two times at three-week intervals with 25 4g of either affinity purified His*Tag-fusion protein in presence of 15 gg of QuilA p 5 adjuvant. Ten to 14 days following the last immunization, iy the mice were challenged with virulent S. pneumoniae. Our mf results indicate that, BVH-3B, a truncated BVH-3 molecule consisting of amino acids 512-1039, elicited protection against the mouse-virulent strains WU2 and P4241.

00 10 Similarly, BVH-11B, NEW4 and NEW5 molecules, three D truncated BVH-11 molecules consisting of amino acids 354- 0 840, amino acids 286-840 and amino acids 286-713, i3 respectively, elicited protection against experiment O intravenous challenge with WU2 and intranasal challenge with P4241. Moreover, vaccination with NEW10 and NEW14, consisting of amino acids 272-838 and amino acids 227-699 from BVH-11-2 molecule also resulted in protection against death with the pneumococcal strains. These results indicate that the region comprising 428 amino acids extending from amino acids 286-713 and amino acids 272-699 on S_ pneumoniae SP64 BVH-11 and BVH-11-2 protein sequences, respectively, contains protective epitopes.

This region is highly conserved with a global 91% identity and 94% homology among thirteen BVH-11 protein sequences.

Table 8. Evaluation of protection elicited by vaccination of mice with BVH-3 and BVH-11 gene products Challenge with WU2 Challenge with P4241 Experiment Immunogen Alive: deada Median day Alive: dead Median day alive alive i b None 0 8 1.5 1 :7 NEW4 8 :0 >14 8 :0 >14 NEWS 8 0 >14 8 0 >14 NEW7 0 :8 2 0 :8 BVH-11M 8 0 >14 8 0 >14 2b None 0 8 1 0 8 4 8 :0 >14 8 0 >14 NEW8 0 8 1.5 0 :8 NEW9 3 5 3.5 2 :6 7 BVH-11M 8 0 >14 8 0 >14 3 b None 0 8 1 0 8 4 NEW6 0 8 1 4 :4 10.5 8 0 >14 8 0 >14 NEW11 0 8 1.5 1 7 6 BVH-11M 8 0 >14 8 0 >14 4 b None 0 8 2 0 :8 4 BVH-11B 7 1 >14 8 0 >14 NEW14 8 0 >14 8 0 >14 0 I A His-Tnio BVH-3AD BVH-3B 2 >14 6 His-Thio 0 8 1 BVH-3C 0 8 1 SThe number of mice alive 14 post-challenge.

b The WU2 challenge dose was Mice living longer than 14 the number of mice dead on day 10 5

CPU.

days were assigned a survival time of 14 days for the determination of median values.

EXAMPLE 12 Cl| This example described the cloning and expression of a chimeric gene encoding for a chimeric polypeptide corresponding to the carboxy-terminal region of BVH-3 in fusion at the C' end to the carboxy-terminal region of BVH- 11 and the additive protection observed after vaccination with a chimeric polypeptide.

00 kD 0 It is clear from the studies described above that BVH-3 and SBVH-1l are serologically distinct molecules simultaneously f present on S. pneumoniae. The results of immunological 0studies of mice indicate that both proteins are good vaccine candidates. These proteins have the potential to provide protection against all pneumococci, regardless of serotype. Even though the two proteins share epitopes and sequences, they have different characteristics and may serve different biological functions. Thus, immunization against the two proteins may provide a higher level of protection than that imparted by each individually. To examine this, several avenues where full-length or truncated BVH-3 and BVH-ll are administered in combination or in conjugation can be explored. Here we describe the genetic engineering of a BVH-3-BVH-11 fusion gene and protein, termed NEW12 (SEQ ID NO:76 and SEQ ID NO:58, respectively), and the potential use of NEW12 protein as a vaccine.

BVH-3 and BVH-1l gene fragments corresponding to the 3'end of the genes were amplified by PCR using pairs of oligopucleotides engineered to amplify fragments spanning nucleotides 1414 to 3117(SEQ ID NO: 1) and nucleotides 1060 to 2520 (SEQ ID NO: 3) from S. pneumoniae strain SP64 BVH-3 and BVH-ll genes, respectively. The primers used, HAMJ278 and HAMJ279; HAMJ282 and HAMJ283 had a restriction endonuclease site at the 5' end, thereby allowing directional in-frame cloning of the amplified product into 0 the digested pET21b(+) plasmid vector (Table PCR- Samplified products were digested with restriction C 5 endonucleases and ligated to linearized plasmid pET21b(+) vector digested likewise. The resultant plasmid constructs were confirmed by nucleotide sequence analysis. The recombinant pET21b(+) plasmid containing the NdeI-HindIII BVH-3 PCR product was linearized by digestion with the C 10 restriction enzymes HindIII and NotI for the in-frame N cloning of the HindIII-NotI DNA fragment obtained from the 0 recombinant pET21(+) vector containing the BVH-11 gene Vm fragment. Clones were first stabilized in E. coli before introduction into E. coli BL21(XDE3) for expression of a chimeric pneumococcal protein molecule. The recombinant chimeric polypeptide, termed NEW 12, was expressed as C-terminal fusion with an His-tag. The expressed recombinant NEW 12 protein was purified from supernatant fractions obtained from centrifugation of sonicated IPTG-induced E. coli cultures using a His-Bind metal chelation resin (QIAgen, Chatsworth,

CA).

According to the same procedure described above, it is possible to construct other chimeric polypeptides, as a result of a simultaneous expression of New 1 and New 4, New 1 and New 5, New 1 and New 10, or New. 1 and New 14. The construction can be with New 1 upstream or downstream of New 4, New 5, New 10, BVH-11B or New 14. It is also possible to construct other chimeric polypeptides as a result of a simultaneous expression of more than two fragments of either genes of BVH-3, BVH-11 or BVH-11-2.

Groups of 8 female BALB/c mice (Charles River) were immunized subcutaneously two times at three-week intervals with 25 gg of either affinity purified HiseTag-fusion NEW1, BVH-11B or NEW12 protein in presence of 15 4g of QuilA adjuvant. Ten to 14 days following the last immunization, S the mice were challenged with virulent S. pneumoniae. As C demonstrated before, NEW1 and BVH-11B molecules comprising CL 5 amino acids 472 to 1039 from BVH-3 protein and amino acids 354-840 from BVH-11 protein, respectively, correspond to c portions of the proteins capable of eliciting a protective immune response. To determine if a chimeric polypeptide would significantly improve the protection compared with 00 10 those seen for the individual counterparts, the challenge ND dose was adjusted in a manner that protection was not 0 expected with NEW1 and BVH-11B molecules. Interestingly, ln the chimeric NEW12 protein, elicited protection against the S mouse-virulent strains WU2 and P4241. Seven out of 8 mice C 15 immunized with NEW12 were still alive 10 days after the challenge while 28 out of 32 mice immunized with NEW1, BVH- 11B, BVH-3M or adjuvant alone were dead by five days postchallenge. Thus, vaccination of mice with NEW12 provided the highest degree of protection against WU2 challenge.

These results indicate that immunization with a chimeric polypeptide and possibly a combination of BVH-3 and BVH-11 gene products can provide additional protection to that obtained by administration of BVH-3 or BVH-11 antigens alone.

Table 9. Evaluation of protection elicited by vaccination of mice with the chimeric NEW12 molecule Challenge with WU2 Challenge with P4241 Immunogen Alive deada Median day Alive dead Median day alive alive None 0 8 1 0 8 NEW1' 2 6 2 1 7 8 BVH-11B 1 7 3.5 8 0 >14 NEW12 6 2 >14 7 1 >14 BVH-3M 1 7 3 8 1 >14 EXAMPLE 13 This example illustrates the identification of additional BVH-3 and BVH-11 related sequences in Streptococcus species other than S. pneumoniae.

0 10 It was previously shown that BVH-3, BVH-11 and BVH-11-2 are C a family of related proteins sharing common sequences.

Cl Homology searches were performed with the nucleotide Q sequence from the conserved region of these genes and C compared with GenBank and.EMBL sequences using FASTA. The most significant homology was observed with a 2.469-kb gene coding for a calculated 92-kDa protein (SEQ ID NO: 81) of unknown function in S. agalactiae also called group B streptococcus or GBS. The gene was designated BVH-71. A protein demonstrating 99.2% identity and 99.5% similarity with that of GBS was also identified in S. pvoenes also called group A streptococcus or GAS (SEQ ID NO: 83). The region of the BVH-71 sequences (SEQ ID NO: 80 and SEQ ID NO: 82), spanning nucleotides 1 to 717, demonstrated 58 and identity with the conserved regions of BVH-3 (nucleotides 1 to 675) and BVH-11 (nucleotides 1 to 684) genes respectively. The first 239 amino acids of the translated sequences of the GBS and GAS BVH-71 open reading frames are 51 and 54% identical to the first 225 and 228 amino acids of BVH-3 and BVH-11, respectively. In addition to structural similarities, streptococcal BVH-3, BVH-11 and BVH-71 proteins also share antigenic epitopes. A 97-kDa band was revealed on Western blots of GAS or GBS whole cells, using Mab H11-1.1-G11 reactive with the BVH-3 and BVH-11 conserved regions. Similarly, GAS and GBS recombinant BVH-71 proteins were detected in Western immunoblot analysis.

These results indicate that BVH-71, BVH-3 and BVH-11 C proteins might share similar functions. Our results also suggest that BVH-71 proteins can be used as protein vaccine components of anti-streptococcus. In a further embodiment M BVH-71 proteins can be used as protein vaccine components of anti-GAS or anti-GBS vaccines.

00 O\ IND oD oD

(N

Claims

1. An isolated polynucleotide encoding comprising: an amino acid the amino acid sequence 64, 65, or 66; an amino acid the amino acid sequence 55, 64, 65, or 66; an amino acid the amino acid sequence 64, 65, or 66; or sequence at least set forth in SEQ sequence at least set forth in SEQ I t polypeptide 80% identical to :D NO:2, 10, 16, 90% identical to D NO:2, 10, 16, sequence at least 95% identical to set forth in SEQ ID NO:2, 10, 16, I the amino acid sequence set fort NO:2, 10, 16, 55, 64, 65, or 66, wherein the encoded polypeptide elici streptococcal immune response when adminis individual.

2. The isolated polynucleotide according wherein the isolated polynucleotide compri a nucleotide sequence at least 8 the nucleotide sequence set forth in SEQ I h in SEQ ID ts an tered anti- to an to claim 1, ses: 0% identical to D NO:1, 9, or 3% identical to D NO:1, 9, or the the 15; a nucleotide sequence at least 9 nucleotide sequence set forth in SEQ I a nucleotide sequence at least 9 identical to nucleotide sequence set forth in SEQ I NO:l, 9, or or the nucleotide sequence set fort NO:l, 9, or h in SEQ ID iplementary to

3. the An isolated polynucleotide that is coi polynucleotide of claim 1 or claim 2. N\M<elboume\a -\%P.let 000-42919P42B27.AU.1\Spl s

42827.AU.1 SpOccficsllon 200-7-2.doc COMS ID No: ARCS-196815 Received by IP Australia: Time 14:41 Date 2008-07-02 02/07 2008 14:39 FAX 61 3 92438333 GRIFFITH HACK IPAUSTRALIA 008 68

4. The polynucleotide of any one of clai wherein said polynucleotide is DNA or polynucleotide is RNA.

5. The polynucleotide according to claim wherein the encoded polypeptide elicits an specifically binds to a polypeptide consis amino acid sequence set forth in SEQ ID NO

6. A vector comprising the polynucleotid claim 2, wherein said polynucleotide is op an expression control region. ms 1 to 3, said 1 or claim 2, antibody that ting of the 16, or e of claim 1 or -rably linked to A host cell transfected with the vectbr of claim 6. 8. A process for producing a polypeptide polynucleotide of claim 1 or claim 2, said comprising culturing the host cell of clai conditions suitable for expression of said 9. An isolated polypeptide comprising: encoded by the process n 7 under polypeptide. an amino acid sequence at least 80% identical to the amino acid sequence 64, 65, or 66; an amino acid the amino acid sequence 64, 65, or 66; an amino acid the amino acid sequence 55, 64, 65, or 66; or set forth in SEQ ID NO:2, 10, 16, sequence at least 10% identical to set forth in SEQ ID NO 2, 10, 16, sequence at least set forth in SEQ I] the amino acid sequence set fortl NO:2, 10, 16, 55, 64, 65, or 66, wherein the isolated polypeptide elicj streptococcal immune response when administ individual. identical to NO:2, 10, 16, Sin SEQ ID ts an anti- ered to an !tlMlBtOtnmcasescPatnL%2000429wwe2aZ.AuIlspa1P42a27AU.1 Speciicaron2005-7-2_de COMS ID No: ARCS-196815 Received by IP Australia: Time 14:41 Date 2008-07-02 02/07 2008 14:39 FAX 61 3 92438333 GRIFFITH HACK 69 The isolated polypeptide of claim 9, isolated polypeptide is fused to a sacchar 11. The isolated polypeptide of claim 9, anti-streptococcal immune response is an a Streptococcus pneumoniae immune response. IPAUSTRALIA wherein the ide. herein the nti- 1009 12. A chimeric polypeptide comprising two or more polypeptides, wherein the amino acid sequence of each of the two or more polypeptides comprises an amino acid sequence chosen from SEQ ID NO:2, 10, 16, 5, 64, 65, and 66, wherein the two or more polypeptides are linked to form a chimeric polypeptide, and wherein the chimeric polypeptide elicits an anti-streptococcal immune response when administered to an individual. 13. A chimeric polypeptide comprising two polypeptides, wherein the amino acid seque: the two or more polypeptides comprises an sequence chosen from SEQ ID NO: 10 and 64, or more polypeptides are linked to form a polypeptide, and wherein the chimeric poly an anti-streptococcal immune response when an individual. 14. A chimeric polypeptide of formula (I) A D (I) wherein; m is 0 or 1, n is 0 or 1, A is chosen from SEQ ID NO:2, 10, 16, or more ice of each of amino acid wherein the two -himeric )eptide elicits administered to 55, 64, 65, and 66; 66; B is chosen from SEQ ID NO:2, 10, 16, 55, 64, 65, and C is 66; and chosen from SEQ ID NO:2, 10, 16, 55, 64, 65, and r(:WMelbovna\L Ca\PyontW&20W-814299BW4207 I Spo~iW42527.AU.1 Speeiicn 4-MMo7.2,DC COMS ID No: ARCS-196815 Received by IP Australia: Time 14:41 Date 2008-07-02 02/07 2008 14:39 FAX 61 3 92438333 GRIFFITH HACK IPAUSTRALIA 010 70 D is chosen from SEQ ID NO:2, 10, 16, 66, wherein the chimeric polypeptide elic streptococcal immune response when adminis s individual. A chimeric polypeptide of formula (I) A (B)m D (1) wherein; m is 0 or 1, n is 0 or 1, A is chosen from SEQ ID NO: 10 and 64 B is chosen from SEQ ID NO: 10 and 64 C is chosen from SEQ ID NO: 10 and 64 D is chosen from SEQ ID NO: 10 and 64 wherein the chimeric polypeptide elic streptococcal immune response when adminisi individual. 55, 64, 65, and its an anti- tered to an i and its an anti- :ered to an 16. The chimeric polypeptide of any one o: wherein the chimeric polypeptide is fu saccharide. 17. The isolated polypeptide according to claims 9 to 11 or the chimeric polypeptide any one of claims 12 to 16, wherein the isJ polypeptide or the chimeric polypeptide ell antibody that specifically binds to a polyI consisting of the amino acid sequence set f NO:2, 16, or 18. A vaccine composition comprising (a) polypeptide according to any one of claims or the chimeric polypeptide according to a 12 to 17, and a pharmaceutically accept diluent or adjuvant. claims 12 to sed to a any one of according to )lated cits an eptide orth in SEQ ID he isolated 9 to 11 and 17 y one of claims able carrier, N;:Molbomrns\aCeaPalan4200-429gggP42e27AU.iSpeda2B27AU.1 Spciicaion 20085-2doc COMS ID No: ARCS-196815 Received by IP Australia: Time 14:41 Date 2008-07-02 02/07 2008 14:40 FAX 61 3 92438333 GRIFFITH HACK 4IPAUSTRLIA ol 71 19. A method for therapeutic or prophylac meningitis, otitis media, bacteremia, or p infection in an individual susceptible to otitis media, bacteremia, or pneumonia inf s comprising administering to said individua or prophylactic amount of the isolated pol according to any one of claims 9 to 11 and chimeric polypeptide according to any one 17, and/or the composition according to cl A method for therapeutic or prophylac streptococcal infection in an individual s streptococcal infection, comprising admini individual a therapeutic or prophylactic ai s1 isolated polypeptide according to any one 11 and 17, the chimeric polypeptide accord of claims 12 to 17, and/or the composition claim 18. tic treatment of neumonia meningitis, ection, 1 a therapeutic peptide 17, the of claims 12 to aim 18. tic treatment of isceptible to 3tering to said nount of the Df claims 9 to ing to any one according to 21. Use of the isolated polypeptide accor3ing to any of claims 9 to 11 and 17, the chimeric pol peptide according to any one of claims 12 to 17, ahd/or the composition according to claim 18, in the therapeutic prophylactic treatment of meningitis, otitis media, bacteremia, or pneumonia infection. one or 22. Use of the isolated polypeptide accor of claims 9 to 11 and 17, the chimeric pol according to any one of claims 12 to 17, a] composition according to claim 18, in the I prophylactic treatment of streptococcal ini 23. Use of the isolated polypeptide accorc of claims 9 to 11 and 17, the chimeric pol] according to any one of claims 12 to 17, ai composition according to claim 18, in the I medicament for the therapeutic or prophylac ling to any one rpeptide id/or the ;herapeut'ic or .ection. ling to any one peptide Id/or the reparation of a tic treatment ctKhaounmkeaPslen%42ma42sA1P428r27.Au.1psIP4252rAu1 Spedfioao 2067-.doc COMS ID No: ARCS-196815 Received by IP Australia: Time 14:41 Date 2008-07-02 02/07 200<8 14:40 FAX 61 3 92438333 GRIFFITH HACK 72 of meningitis, otitis media, bacteremia, c infection in an individual susceptible to otitis media, bacteremia, or pneumonia inf 24. Use of the isolated polypeptide accor of claims 9 to 11 and 17, the chimeric pol according to any one of claims 12 to 17, a composition according to claim 18, in the medicament for the therapeutic or prophyla of streptococcal infection. The method according to claim 19 or c use according to any one of claims 21 to 2 individual is a mammal. is 26. The method according to any one of cl or the use according to any one of claii wherein said individual is a human. IPAUSTRALIA @1012 r pneumonia meningitis, ection. ding to any one ypeptide ad/or the preparation of a 2tic treatment laim 20 or 4, wherein the said aims 19, ns 21 to 20 or 27. The method according to any one of cl ims 20, 25, or 26 or the use according to any one of claims 22 or 24 to 26, wherein said streptococcal infection i a S. pneumoniae, group A Streptococcus pyognes), group B Streptococcus (GBS or S. agalactiae), S. dysgalactiae, or S. uberis infection, 28. The method according to any one of cl 27, or a use according to any one of claim: 27, wherein said streptococcal infection ii pneumoniae infection. 29. A polynucleotide according to claim 1, according to claim 9, a chimeric polypeptic any one of claims 12 to 15, a vaccine compc according to claim 18, a method according t claim 20, or a use according to any one of aims 20 or 25 to s 22 or 24 to an S. a polypeptide Ie according to sition o: claim 19 or claims 21 to NeI.elabcerCraesBpMP2no.42sp42827,Au 1lSpeO '4227AU.1 Spedrrkn 2008-?4a.c COMS ID No: ARCS-196815 Received by IP Australia: Time 14:41 Date 2008-07-02 02/07 2008 14:40 FAX1 61 3 92438333GRFIhHC-4PASRLA[]3 GRIFFITH HACK 4,IPAUSTRALIA IN13 73 24, subutantially as herein described witT aniy of the examples or figures. reference to N:V~bouXC&9ePt-nM0W-ZNMPC287AU.%Sp-AP4927AU-ped~r.C162WO--2.doc COMS ID No: ARCS-196815 Received by IP Australia: Time 14:41 Date 2008-07-02