WO2004048575A2

WO2004048575A2 - Streptococcus pneumoniae surface polypeptides

Info

Publication number: WO2004048575A2
Application number: PCT/CA2003/001835
Authority: WO
Inventors: Josée Hamel; Nathalie Charland; Denis Martin; Bernard R. Brodeur; Normand Blais; Isabelle Pineau; Steve LABBÉ
Original assignee: Id Biomedical Corporation
Priority date: 2002-11-26
Filing date: 2003-11-26
Publication date: 2004-06-10
Also published as: AU2003286056A1; AU2003286056A8; WO2004048575A3

Abstract

Streptococcus polypeptides and polynucleotides encoding them, antigens, epitopes, antibodies directed to these epitopes, which may be used to prevent, diagnose and/or treat Streptococcus pneumoniae infections in mammals, such as humans are disclosed.

Description

STREPTOCOCCUS PNEUMONIAE POLYPEPTIDES

FIELD OF THE INVENTION The present invention is related to Streptococcus pneumoniae polypeptides and their corresponding DNA fragments, antigens, epitopes, antibodies directed to these epitopes, which may be used to prevent, diagnose and/or treat Streptococcus pneumoniae infections in mammals, such as humans .

BACKGROUND OF THE INVENTION

S . pneumoniae is an important agent of disease in man especially among infants, the elderly and immunocompromised persons. It is a bacterium frequently isolated from patients with invasive diseases such as bacteraemia, septicaemia, pneumonia, meningitis with high morbidity and mortality throughout the world. Even with appropriate antibiotic therapy, pneumococcal infections still result in many deaths. Although the advent of antimicrobial drugs has reduced the overall mortality from pneumococcal disease, the presence of resistant pneumococcal organisms has become a major problem in the world today. Effective pneumococcal vaccines could have a major impact on the morbidity and mortality associated with S_-_ pneumoniae disease. Such vaccines would also potentially be useful to prevent otitis media in infants and young children.

Efforts to develop a pneumococcal vaccine have generally concentrated on generating immune responses to the pneumococcal capsular polysaccharide . More than 80 pneumococcal capsular serotypes have been identified on the basis on antigenic differences. The currently available polysaccharide vaccine comprising 23 capsular polysaccharides that most frequently caused disease has significant shortcomings related primarily to the poor immunogenicity of many of the polysaccharides in infants, the diversity of the serotypes and the differences in the distribution of serotypes over time, geographic areas and age groups . Although immunogenicity of capsular polysaccharides can be improved by conjugation of the polysaccharides to a protein carrier, the cost, the serotype specificity and serotype substitution following vaccination provide a strong rationale for developing vaccines capable of overcoming these limitations .

Several polynucleotide and polypeptide sequences from Streptococcus pneumoniae have been disclosed in WO 98/18930, WO 98/18931, WO 00/06738, US 6,348,328.

Phagocytosis and the subsequent killing of the ingested S . pneumoniae compose an immune mechanism that could clear the bacteria from infected individuals. Bacterial surface antigens appear to be important targets for opsonic and protective antibody. The use of a group-common immunogenic and protective pneumococcal surface protein antigen, either by itself or in combination with additional components, offers the possibility of a protein-based pneumococcal vaccine. This is the basis of our invention. Several studies including structural protein analysis, hydrophobicity/hydrophilicity protein profile characterization, gene amplification, cloning and expression, gene and protein conservation studies, protein purification, and immunization and infection of animals were used to identify genes encoding for novel conserved pneumococcal surface proteins that are associated with protective immunity.

There remains an unmet need for polypeptides that may be used for the prophylaxis, diagnosis and/or therapy of Streptococcus pneumoniae infection.

SUMMARY 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 No : 2, 4, 6, 18 or fragments or analogs thereof . According to one aspect, the present invention relates to polypeptides comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof.

In other aspects, there are provided polypeptides encoded by polynucleotides of the invention, pharmaceutical compositions, vectors comprising polynucleotides of the invention operably linked to an expression control region, as well as host cells transfected with said vectors and processes for producing polypeptides comprising culturing said host cells under conditions suitable for expression.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 represents the DNA sequence of SHB-SP-101 gene from SP64 strain; SEQ ID NO: 1

Figure 2 represents the amino acid sequence of SHB-SP-101 polypeptide from SP64 strain; SEQ ID NO: 2

Figure 3 represents the DNA sequence of SHB-SP-102 gene from

SP64 strain; SEQ ID NO: 3

Figure 4 represents the amino acid sequence of SHB-SP-102 polypeptide from SP64 strain; SEQ ID NO: 4

Figure 5 represents the DNA sequence of SHB-SP-103 gene from

SP64 strain; SEQ ID NO: 5

Figure 6 represents the amino acid sequence of SHB-SP-103 polypeptide from SP64 strain; SEQ ID NO: 6

Figure 7 represents the comparison of the amino acid sequences of SHB-SP-101 from ∑ - pneumoniae strains SP64, TIGR4 and R6 by using the program Align X from Vector NTI® sequence analysis software (version 7.0). Underneath the alignment, there is a consensus line where * and . characters indicate identical and similar amino acid residues, respectively. Figure 8 depicts the comparison of the amino acid sequences of SHB-SP-102 from S__ pneumoniae strains SP64, TIGR4 and R6 by using the program Align X from Vector NTI® sequence analysis software (version 7.0). Underneath the alignment, there is a consensus line where * and . characters indicate identical and similar amino acid residues, respectively.

Figure 9 depicts the comparison of the amino acid sequences of SHB-SP-103 from S_^ pneumoniae strains SP64, TIGR4 and R6 by using the program Align X from Vector NTI® sequence analysis software (version 7.0). Underneath the alignment, there is a consensus line where * and . characters indicate identical and similar amino acid residues, respectively.

Figure 10 represents the DNA sequence of SHB-SP-104 polypeptide; SEQ ID NO: 17.

Figure 11 epresents the amino acid sequence of SHB-SP-104 polypeptide; SEQ ID NO: 18.

DETAILED DESCRIPTION OF THE INVENTION

According to one aspect, the present invention provides an isolated polynucleotide comprising a polynucleotide chosen from:

(a) a polynucleotide encoding a polypeptide having at least 70% identity to a second polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof; (b) a polynucleotide encoding a polypeptide having at least 80% identity to a second polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof; (c) a polynucleotide encoding a polypeptide having at least 95% identity to a second polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof; (d) a polynucleotide encoding a polypeptide comprising a sequence chosen from SEQ ID No : 2 , 4, 6, 18 or fragments or analogs thereof;

(e) a polynucleotide encoding a polypeptide capable of raising antibodies having binding specificity for a polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof;

(f) a polynucleotide encoding an epitope bearing portion of a polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof;

(g) a polynucleotide comprising a sequence chosen from SEQ ID No : 1, 3, 5, 17 or fragments or analogs thereof;

(h) a polynucleotide that is complementary to a polynucleotide in (a) , (b) , (c), (d) , (e), (f) or (g) .

(a) a polynucleotide encoding a polypeptide having at least 70% identity to a second polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6 or 18;

(b) a polynucleotide encoding a polypeptide having at least 80% identity to a second polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6 or 18; (c) a polynucleotide encoding a polypeptide having at least 95% identity to a second polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6 or 18; (d) a polynucleotide encoding a polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6 or 18; (e) a polynucleotide encoding a polypeptide capable of raising antibodies having binding specificity for a polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6 or 18;

(f) a polynucleotide encoding an epitope bearing portion of a polypeptide comprising a sequence chosen from SEQ ID No :

2, 4, 6 or 18;

(g) a polynucleotide comprising a sequence chosen from SEQ ID No : 1, 3, 5 or 17; (h) a polynucleotide that is complementary to a polynucleotide in (a) , (b) , (c) , (d) , (e) , (f) or (g) .

According to one aspect, the present invention provides an isolated polypeptide comprising a polypeptide chosen from:

(a) a polypeptide having at least 70% identity to a second polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof;

(b) a polypeptide having at least 80% identity to a second polypeptide comprising a sequence chosen from SEQ ID No :

2, 4, 6, 18 or fragments or analogs thereof;

(c) a polypeptide having at least 95% identity to a second polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof; (d) a polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof;

(e) a polypeptide capable of raising antibodies having binding specificity for a polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof;

(f) an epitope bearing portion of a polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof;

(g) the polypeptide of (a), (b) , (c) , (d) , (e) or (f) wherein the N-terminal Met residue is deleted;

(h) the polypeptide of (a), (b) , (c) , (d) , (e) , (f) or (g) wherein the secretory amino acid sequence is deleted.

(a) a polypeptide having at least 70% identity to a second polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6 or 18;

2, 4, 6 or 18; (c) a polypeptide having at least 95% identity to a second polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6 or 18;

(d) a polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6 or 18;

(e) a polypeptide capable of raising antibodies having binding specificity for a polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6 or 18;

(f) an epitope bearing portion of a polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6 or 18;

(g) the polypeptide of (a), (b) , (c), (d) , (e) or (f) wherein the N-terminal Met residue is deleted;

(h) the polypeptide of (a), (b) , (c), (d) , (e) , (f) or (g) wherein the secretory amino acid sequence is deleted.

The present invention provides purified and isolated polynucleotides, which encode polypeptides which may be used to prevent, diagnose and/or treat Streptococcus pneumoniae infection.

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 No : 2, 4, 6, 18 or fragments or analogs thereof.

According to one aspect, the present invention provides an isolated polynucleotide encoding a polypeptide having at least 80% identity to a second polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof .

According to one aspect, the present invention provides an isolated polynucleotide encoding a polypeptide having at least 90% identity to a second polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof . According to one aspect, the present invention provides an isolated polynucleotide encoding a polypeptide having at least 95% identity to a second polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs 5 thereof.

According to one aspect, the present invention provides an isolated polynucleotide encoding a polypeptide having at least 98% identity to a second polypeptide comprising a sequence 10 chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof .

According to one aspect, the present invention provides an isolated polynucleotide encoding a polypeptide having at least 1599% identity to a second polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof .

According to one aspect, the present invention provides an 20 isolated polynucleotide encoding a polypeptide having at least 70% identity to a second polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6 or 18.

According to one aspect, the present invention provides an 25 isolated polynucleotide encoding a polypeptide having at least

80% identity to a second polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6 or 18.

According to one aspect, the present invention provides an isolated polynucleotide encoding a polypeptide having at least 3090% identity to a second polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6 or 18.

According to one aspect, the present invention provides an isolated polynucleotide encoding a polypeptide having at least 3595% identity to a second polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6 or 18. According to one aspect, the present invention provides an isolated polynucleotide encoding a polypeptide having at least 98% identity to a second polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6 or 18.

According to one aspect, the present invention provides an isolated polynucleotide encoding a polypeptide having at least 99% identity to a second polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6 or 18.

According to one aspect, the present invention relates to polypeptides comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof.

According to one aspect, the present invention relates to polypeptides comprising a sequence chosen from SEQ ID No : 2, 4, 6 or 18.

According to one aspect, the present invention relates to polypeptides characterized by the amino acid sequence comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof .

According to one aspect, the present invention relates to polypeptides characterized by the amino acid sequence comprising a sequence chosen from SEQ ID No : 2, 4, 6 or 18.

According to one aspect, the present invention provides a polynucleotide encoding an epitope bearing portion of a polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof.

According to one aspect, the present invention provides a polynucleotide encoding an epitope bearing portion of a polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6 or 18. According to one aspect, the present invention relates to epitope bearing portions of a polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof .

According to one aspect, the present invention relates to epitope bearing portions of a polypeptide comprising a sequence chosen from SEQ ID No : 2 , 4, 6 or 18.

(a) a polynucleotide encoding a polypeptide having at least 70% identity to a second polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof;

(b) a polynucleotide encoding a polypeptide having at least 80% identity to a second polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof;

(c) a polynucleotide encoding a polypeptide having at least 95% identity to a second polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof; (d) a polynucleotide encoding a polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof;

(f) a polynucleotide encoding an epitope bearing portion of a polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof; (g) a polynucleotide comprising a sequence chosen from SEQ ID No : 1, 3, 5, 17 or fragments or analogs thereof; (h) a polynucleotide that is complementary to a polynucleotide in (a), (b) , (c), (d) , (e), (f) or (g) . According to one aspect, the present invention provides an isolated polynucleotide comprising a polynucleotide chosen from: (a) a polynucleotide encoding a polypeptide having at least

70% identity to a second polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6 or 18;

(b) a polynucleotide encoding a polypeptide having at least 80% identity to a second polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6 or 18;

(c) a polynucleotide encoding a polypeptide having at least 95% identity to a second polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6 or 18;

(d) a polynucleotide encoding a polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6 or 18;

(e) a polynucleotide encoding a polypeptide capable of raising antibodies having binding specificity for a polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6 or 18; (f) a polynucleotide encoding an epitope bearing portion of a polypeptide comprising a sequence chosen from SEQ ID No :

2, 4, 6 or 18; (g) a polynucleotide comprising a sequence chosen from SEQ ID

No : 1, 3, 5 or 17; (h) a polynucleotide that is complementary to a polynucleotide in (a), (b) , (c), (d) , (e) , (f) or (g) .

According to one aspect, the present invention provides an isolated polypeptide comprising a polypeptide chosen from: (a) a polypeptide having at least 70% identity to a second polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof;

(b) a polypeptide having at least 80% identity to a second polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof;

(e) a polypeptide capable of raising antibodies having binding specificity for a polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof ;

(f) an epitope bearing portion of a polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof; (g) the polypeptide of (a), (b) , (c) , (d) , (e) or (f) wherein the N-terminal Met residue is deleted; (h) the polypeptide of (a), (b) , (c) , (d) , (e) , (f) or (g) wherein the secretory amino acid sequence is deleted.

According to one aspect, the present invention provides an isolated polypeptide comprising a polypeptide chosen from: (a) a polypeptide having at least 70% identity to a second polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6 or 18; (b) a polypeptide having at least 80% identity to a second polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6 or 18;

(c) a polypeptide having at least 95% identity to a second polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6 or 18;

(d) a polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6 or 18;

(g) the polypeptide of (a), (b) , (c) , (d) , (e) or (f) wherein the N-terminal Met residue is deleted; (h) the polypeptide of (a), (b) , (c) , (d) , (e) , (f) or (g) wherein the secretory amino acid sequence is deleted. Those skilled in the art will appreciate that the invention includes DNA molecules, i.e. polynucleotides and their complementary sequences that encode analogs such as mutants, variants, homologues and derivatives of such polypeptides, as described herein in the present patent application. The invention also includes RNA molecules corresponding to the DNA molecules of the invention. In addition to the DNA and RNA molecules, the invention includes the corresponding polypeptides and monospecific antibodies that specifically bind to such polypeptides .

In a further embodiment, the polypeptides in accordance with the present invention are antigenic.

In a further embodiment, the polypeptides in accordance with the present invention are immunogenic.

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

In a further embodiment, the present invention also relates to polypeptides which are able to raise antibodies having binding specificity to the 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, e.g., 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 accordance with the present invention, "protection" in the biological studies is defined by a significant increase in the survival curve, rate or period. Statistical analysis using the Log rank test to compare survival curves, and Fisher exact test to compare survival rates and numbers of days to death, respectively, might be useful to calculate P values and determine whether the difference between the two groups is statistically significant, p values of 0.05 are regarded as not significant . In an additional aspect of the invention there are provided antigenic/immunogenic fragments of the polypeptides of the invention, or of analogs thereof.

The fragments of the present invention should include one or more such epitopic regions or be sufficiently similar to such regions to retain their antigenic/immunogenic properties. Thus, for fragments according to the present invention the degree of identity is perhaps irrelevant, since they may be 100% identical to a particular part of a polypeptide or analog thereof as described herein. The present invention further provides fragments having at least 10 contiguous amino acid residues from the polypeptide sequences of the present invention. In one embodiment, at least 15 contiguous amino acid residues. In one embodiment, at least 20 contiguous amino acid residues. In one embodiment, at least 25 contiguous amino acid residues. In one embodiment, at least 30 contiguous amino acid residues. In one embodiment, at least 35 contiguous amino acid residues. In one embodiment, at least 40 contiguous amino acid residues.

The skilled person will appreciate that analogs of the polypeptides of the invention will also find use in the context of the present invention, i.e. as antigenic/immunogenic material. Thus, for instance proteins or polypeptides which include one or more additions, deletions, substitutions or the like are encompassed by the present invention.

As used herein, "fragments", "analogs" or "derivatives" of the polypeptides of the invention include those polypeptides in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably conserved) and which may be natural or unnatural. In one embodiment, derivatives and analogs of polypeptides of the invention will have about 80% identity with those sequences illustrated in the figures or fragments thereof. That is, 80% of the residues are the same. In a further embodiment, polypeptides will have greater than 80% identity. In a further embodiment, polypeptides will have greater than 85% identity. In a further embodiment, polypeptides will have greater than 90% identity. In a further embodiment, polypeptides will have greater than 95% identity. In a further embodiment, polypeptides will have greater than 99% identity. In a further embodiment, 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.

These substitutions are those having a minimal influence on the secondary structure and hydropathic nature of the polypeptide.

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. These include substitutions such as those described by Dayhoff, M. in Atlas of Protein Sequence and

Structure 5, 1978 and by Argos, P. in EMBO J. 8, 779-785, 1989.

For example, amino acids, either natural or unnatural, belonging to one of the following groups represent conservative changes : ala, pro, gly, gin, asn, ser, thr, val; cys, ser, tyr, thr; val, ile, leu, met, ala, phe; lys, arg, orn, his; and phe, tyr, trp, his. The preferred substitutions also include substitutions of D- enantiomers for the corresponding L-amino acids.

In an alternative approach, the analogs or derivatives could be fusion polypeptides, incorporating moieties which render purification easier, for example by effectively tagging the desired polypeptide. It may be necessary to remove the "tag" or it may be the case that the fusion polypeptide itself retains sufficient antigenicity to be useful . The percentage of homology is defined as the sum of the percentage of identity plus the percentage of similarity or conservation of amino acid type. In one embodiment, analogs of polypeptides of the invention will have about 70% homology with those sequences illustrated in the figures or fragments thereof. In a further embodiment, polypeptides will have greater than 80% homology. In a further embodiment, polypeptides will have greater than 85% homology. In a further embodiment, polypeptides will have greater than 90% homology. In a further embodiment, polypeptides will have greater than 95% homology. In a further embodiment, polypeptides will have greater than 99% homology. In a further embodiment, 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.

One can use a program such as the CLUSTAL program to compare amino acid sequences. This program compares amino acid sequences and finds the optimal alignment by inserting spaces in either sequence as appropriate. It is possible to calculate amino acid identity or homology for an optimal alignment. A program like BLASTx will align the longest stretch of similar sequences and assign a value to the fit. It is thus possible to obtain a comparison where several regions of similarity are found, each having a different score. Both types of identity analysis are contemplated in the present invention.

It is well known that it is possible to screen an antigenic polypeptide to identify epitopic regions, i.e. those regions which are responsible for the polypeptide' s antigenicity or immunogenicity. Methods for carrying out such screening are well known in the art. Thus, the fragments of the present invention should include one or more such epitopic regions or be sufficiently similar to such regions to retain their antigenic/immunogenic properties. Thus, what is important for analogs, derivatives and fragments is that they possess at least a degree of the antigenicity/ immunogenicity of the protein or polypeptide from which they are derived.

Also included are polypeptides which have fused thereto other compounds which alter the polypeptides biological or pharmacological properties i.e. polyethylene glycol (PEG) to increase half-life; leader or secretory amino acid sequences for ease of purification; prepro- and pro- sequences; and (poly) saccharides .

Furthermore, in those situations where amino acid regions are found to be polymorphic, it may be desirable to vary one or more particular amino acids to more effectively mimic the different epitopes of the different Streptococcus pneumoniae strains .

Moreover, the polypeptides of the present invention can be modified by terminal -NH₂ acylation (eg. by acetylation, or thioglycolic acid amidation, terminal carboxy amidation, e.g. with ammonia or methylamine) to provide stability, increased hydrophobicity for linking or binding to a support or other molecule .

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

In a further embodiment, the present invention also relates to chimeric polypeptides which comprise one or more polypeptides or fragments or analogs thereof as defined in the figures of the present application. In a further embodiment, the present invention also relates to chimeric polypeptides comprising two or more polypeptides comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof; provided that the polypeptides are linked as to formed a chimeric polypeptide.

In a further embodiment, the present invention also relates to chimeric polypeptides comprising two or more polypeptides comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 provided that the polypeptides are linked as to formed a chimeric polypeptide.

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 (i.e. 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 polypeptides may be a single bond or may be composed of a linking group of at least two, typically at least four, and not more than 16, but usually not more than about 14 carbon atoms.

In a particular embodiment, polypeptide fragments and analogs of the invention do not contain a starting residue, such as methionine (Met) or valine (Val) . Preferably, polypeptides will not incorporate a leader or secretory sequence (signal sequence) . The signal portion of a polypeptide of the invention may be determined according to established molecular biological techniques. In general, the polypeptide of interest may be isolated from a Streptococcus pneumoniae culture and subsequently sequenced to determine the initial residue of the mature protein and therefore the sequence of the mature polypeptide. It is understood that polypeptides can be produced and/or used without their start codon (methionine or valine) and/or without their leader peptide to favor production and purification of recombinant polypeptides. It is known that cloning genes without sequences encoding leader peptides will restrict the polypeptides to the cytoplasm of E. coli and will facilitate their recovery (Glick, B.R. and Pasternak, J.J. (1998) Manipulation of gene expression in prokaryotes . In "Molecular biotechnology: Principles and applications of recombinant DNA", 2nd edition, ASM Press, Washington DC, p.109-143).

According to another aspect of the invention, there are also provided (i) a composition of matter containing a polypeptide of the invention, together with a carrier, diluent or adjuvant; (ii) a pharmaceutical composition comprising a polypeptide of the invention and a pharmaceutically acceptable carrier, diluent or adjuvant; (iii) a vaccine comprising a polypeptide of the invention and a pharmaceutically acceptable carrier, diluent or adjuvant; (iv) a method for inducing an immune response against Streptococcus pneumoniae , in a host, by administering to the host, an immunogenically effective amount of a polypeptide of the invention to elicit an immune response, e.g., a protective immune response to Streptococcus pneumoniae ; and particularly, (v) a method for preventing and/or treating a Streptococcus pneumoniae infection, by administering a prophylactic or therapeutic amount of a polypeptide of the invention to a host in need.

According to another aspect of the invention, there are also provided (i) a composition of matter containing a polynucleotide of the invention, together with a carrier, diluent or adjuvant; (ii) a pharmaceutical composition comprising a polynucleotide of the invention and a carrier, diluent or adjuvant; (iii) a method for inducing an immune response against Streptococcus pneumoniae , in a host, by administering to the host, an immunogenically effective amount of a polynucleotide of the invention to elicit an immune response, e.g., a protective immune response to Streptococcus pneumoniae; and particularly, (iv) a method for preventing and/or treating a Streptococcus pneumoniae infection, by administering a prophylactic or therapeutic amount of a polynucleotide of the invention to a host in need. Before immunization, the polypeptides of the invention can also be coupled or conjugated to carrier proteins such as tetanus toxin, diphtheria toxin, hepatitis B virus surface antigen, poliomyelitis virus VP1 antigen or any other viral or bacterial toxin or antigen or any suitable proteins to stimulate the development of a stronger immune response. This coupling or conjugation can be done chemically or genetically. A more detailed description of peptide-carrier conjugation is available in Van Regenmortel, M.H.V., Briand J.P., Muller S., Plaue S., «Synthetic Polypeptides as antigens» in Laboratory Techniques in Biochemistry and Molecular Biology, Vol.19 (ed. ) Burdou, R.H. & Van Knippenberg P.H. (1988), Elsevier New York.

According to another aspect, there are provided pharmaceutical compositions comprising one or more Streptococcus pneumoniae polypeptides of the invention in a mixture with a pharmaceutically acceptable adjuvant. Suitable adjuvants include (1) oil-in-water emulsion formulations such as MF59™, SAF™, Ribi™ ; (2) Freund's complete or incomplete adjuvant; (3) salts i.e. A1K(S0₄)₂, AlNa(S0₄)₂, AlNH₄(S0₄)₂, Al(0H)₃, AlP0₄, silica, kaolin; (4) saponin derivatives such as Stimulon™ or particles generated therefrom such as ISCOMs (immunostimulating complexes); (5) cytokines such as interleukins, interferons, macrophage colony stimulating factor (M-CSF) , tumor necrosis factor (TNF) ; (6) other substances such as carbon polynucleotides i.e. poly IC and poly AU, detoxified cholera toxin (CTB)and E.coli heat labile toxin for induction of mucosal immunity; (7) liposomes. A more detailed description of adjuvant is available in a review by M.Z.I Khan et al . in Pharmaceutical Research, vol. 11, No. 1 (1994) pp2-ll, and also in another review by Gupta et al . , in Vaccine, Vol. 13, No. 14, ppl263-1276 (1995) and in WO 99/24578. Preferred adjuvants include QuilA™, QS21™, Alhydrogel™ and Adjuphos™. Pharmaceutical compositions of the invention may be administered parenterally by injection, rapid infusion, nasopharyngeal absorption, dermoabsorption, or buccal or oral.

The term "pharmaceutical composition" is also meant to include antibodies. In accordance with the present invention, there is also provided the use of one or more antibodies having binding specificity for the polypeptides of the present invention for the treatment or prophylaxis of Streptococcus pneumoniae infection and/or diseases and symptoms mediated by Streptococcus pneumoniae infection.

Pharmaceutical compositions of the invention are used for the prophylactic or therapeutic treatment of streptococcal infection and/or diseases and symptoms mediated by streptococcal infection as described in Manual of Clinical Microbiology, P.R. Murray (Ed, in chief),E.J. Baron, M.A. Pfaller, F.C. Tenover and R.H. Yolken. ASM Press, Washington, D.C. seventh edition, 1999, 1773p. In one embodiment, pharmaceutical compositions of the present invention are used for the prophylactic or therapeutic treatment of otitis media, sinusitis, pneumonia, meningitis, bacteremia, septicaemia. In one embodiment, pharmaceutical compositions of the invention are used for the prophylactic or therapeutic treatment of streptococcal infection and/or diseases and symptoms mediated by streptococcal infection, in particular S.pneumoniae, group A streptococcus ( Streptococcus pyoqenes ) , group B streptococcus (GBS or Streptococcus aqalactiae) , Streptococcus dvsgalactiae. Streptococcus uberis , Streptococcus nocardia as well as Staphylococcus aureus . In a further embodiment, the streptococcus infection is S.pneumoniae.

In a further embodiment, the invention provides a method for prophylactic or therapeutic treatment of Streptococcus pneumoniae infection in a host susceptible to Streptococcus pneumoniae infection comprising administering to said host a prophylactic or therapeutic amount of a composition of the invention. As used in the present application, the term "host" includes mammals. In a further embodiment, the mammal is human. In a further embodiment, the human is a neonate, infant or child. In a further embodiment, the human is an adult.

In a particular embodiment, pharmaceutical compositions are administered to those hosts at risk of Streptococcus pneumoniae infection such as neonates, infants, children, elderly, immunocompromised hosts, hosts with an underlying disease that affect the innate mucosal immune system, such as chronic obstructive pulmonary disease and cystic fibrosis.

In a particular embodiment, pharmaceutical compositions of the invention are administered to adults.

Pharmaceutical compositions are preferably in unit dosage form of about 0.001 to 100 μg/kg (antigen/body weight) and more preferably 0.01 to 10 μg/kg and most preferably 0.1 to 1 μg/kg 1 to 3 times with an interval of about 1 to 6 week intervals between immunizations.

Pharmaceutical compositions are preferably in unit dosage form of about 0.1 μg to 10 mg and more preferably lμg to 1 mg and most preferably 10 to 100 μg 1 to 3 times with an interval of about 1 to 6 week intervals between immunizations.

According to another aspect, there are provided polynucleotides encoding polypeptides characterized by the amino acid sequence comprising SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof .

In one embodiment, polynucleotides are those illustrated in SEQ ID No: 1, 3, 5, 17 which may include the open reading frames (ORF) , encoding the polypeptides of the invention.

It will be appreciated that the polynucleotide sequences illustrated in the figures may be altered with degenerate codons yet still encode the polypeptides of the invention. Accordingly the present invention further provides polynucleotides which hybridize to the polynucleotide sequences herein above described (or the complement sequences thereof) having 70% identity between sequences. In one embodiment, at least 80% identity between sequences. In one embodiment, at least 85% identity between sequences. In one embodiment, at least 90% identity between sequences. In a further embodiment, polynucleotides are hybridizable under stringent conditions i.e. having at least 95% identity. In a further embodiment, more than 97% identity. In a further embodiment, more than 98% identity. In a further embodiment, more than 99% identity.

Suitable stringent conditions for hybridation can be readily determined by one of skilled in the art (see for example Sambrook et al . , (1989) Molecular cloning : A Laboratory Manual, 2^nd ed, Cold Spring Harbor, N.Y. ; Current Protocols in Molecular Biology, (1999) Edited by Ausubel F.M. et al . , John Wiley 6- Sons, Inc., N.Y. ) .

In a further embodiment, the present invention provides polynucleotides that hybridize under stringent conditions to either

(a) a DNA sequence encoding a polypeptide or (b) the complement of a DNA sequence encoding a polypeptide; wherein said polypeptide comprises a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof.

(a) a DNA sequence encoding a polypeptide or

(b) the complement of a DNA sequence encoding a polypeptide; wherein said polypeptide comprises a sequence chosen from SEQ ID NO: 2, 4, 6 or 18. In a further embodiment, the present invention provides polynucleotides that hybridize under stringent conditions to either

(a) a DNA sequence encoding a polypeptide or (b) the complement of a DNA sequence encoding a polypeptide; wherein said polypeptide comprises at least 10 contiguous amino acid residues from a polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof.

(a) a DNA sequence encoding a polypeptide or (b) the complement of a DNA sequence encoding a polypeptide; wherein said polypeptide comprises at least 10 contiguous amino acid residues from a polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6 or 18.

In a further embodiment, polynucleotides are those illustrated in SEQ ID NO: 1, 3, 5 or 17 or fragments or analogs thereof encoding polypeptides of the invention.

In a further embodiment, polynucleotides are those illustrated in SEQ ID NO: 1, 3, 5 or 17 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.

According to another aspect, there is provided a process for producing polypeptides of the invention by recombinant techniques by expressing a polynucleotide encoding said polypeptide in a host cell and recovering the expressed polypeptide product. Alternatively, the polypeptides can be produced according to established synthetic chemical techniques i.e. solution phase or solid phase synthesis of oligopeptides which are ligated to produce the full polypeptide (block ligation) .

General methods for obtention and evaluation of polynucleotides and polypeptides are described in the following references: Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd ed, Cold Spring Harbor, N.Y., 1989; Current Protocols in Molecular Biology, Edited by Ausubel F.M. et al., John Wiley and Sons, Inc. New York; PCR Cloning Protocols, from Molecular Cloning to Genetic Engineering, Edited by White B.A., Humana Press, Totowa, New Jersey, 1997, 490 pages; Protein Purification, Principles and Practices, Scopes R.K., Springer-Verlag, New York, 3rd Edition, 1993, 380 pages; Current Protocols in Immunology, Edited by Coligan J.E. et al . , John Wiley & Sons Inc . , New York .

The present invention provides host cells transfected with vectors comprising the polynucleotides of the invention.

For recombinant production, host cells are transfected with vectors which encode the polypeptides of the invention, and then cultured in a nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the genes. Suitable vectors are those that are viable and replicable in the chosen host and include chromosomal, non- chromosomal and synthetic DNA sequences e.g. bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA. The polypeptide sequence may be incorporated in the vector at the appropriate site using restriction enzymes such that it is operably linked to an expression control region comprising a promoter, ribosome binding site (consensus region or Shine- Dalgarno sequence) , and optionally an operator (control element) . One can select individual components of the expression control region that are appropriate for a given host and vector according to established molecular biology principles (Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd ed, Cold Spring Harbor, N.Y., 1989; Current Protocols in Molecular Biology, Edited by Ausubel F.M. et al . , John Wiley and Sons, Inc. New York) . Suitable promoters include but are not limited to LTR or SV40 promoter, E.coli lac, tac or trp promoters and the phage lambda P_L promoter. Vectors will preferably incorporate an origin of replication as well as selection markers i.e. ampicilin resistance gene. Suitable bacterial vectors include pET, pQE70, pQE60, pQE-9, pDlO phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNHlβa, PNH18A, pNH46A, ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 and eukaryotic vectors pBlueBacIII, pWLNEO, pSV2CAT, pOG44, pXTl , pSG, pSVK3, pBPV, pMSG and pSVL. Host cells may be bacterial i.e. E.coli, Bacillus subtilis, Streptomyces ; fungal i.e. Asperqillus niqer, Asperqillus nidulins; yeast i.e. Saccharomyces or eukaryotic i.e. CHO, COS.

The present invention provides a process for producing a polypeptide comprising culturing a host cell of the invention under conditions suitable for expression of said polypeptide.

Upon expression of the polypeptide in culture, cells are typically harvested by centrifugation then disrupted by physical or chemical means (if the expressed polypeptide is not secreted into the media) and the resulting crude extract retained to isolate the polypeptide of interest. Purification of the polypeptide from culture media or lysate may be achieved by established techniques depending on the properties of the polypeptide i.e. using ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography and lectin chromatography. Final purification may be achieved using HPLC. The polypeptides may be expressed with or without a leader or secretion sequence. In the former case the leader may be removed using post-translational processing (see US 4,431,739; US 4,425,437; and US 4,338,397) or be chemically removed subsequent to purifying the expressed polypeptide.

According to a further aspect, the Streptococcus pneumoniae polypeptides of the invention may be used in a diagnostic test for Streptococcus pneumoniae infection.

Several diagnostic methods are possible, for example detecting Streptococcus pneumoniae organism in a biological sample, or for diagnostic of a Streptococcus pneumoniae infection in an host susceptible to Streptococcus pneumoniae infection, the following procedure may be followed: a) obtaining a biological sample from a host; b) incubating an antibody or fragment thereof reactive with a Streptococcus pneumoniae 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 pneumoniae.

Alternatively, a method for the detection of antibody specific to a Streptococcus pneumoniae antigen in a biological sample containing or suspected of containing said antibody may be performed as follows: a) obtaining a biological sample from a host; b) incubating one or more Streptococcus pneumoniae 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 pneumoniae .

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

The DNA sequences encoding polypeptides of the invention may also be used to design DNA probes for use in detecting the presence of Streptococcus pneumoniae in a biological sample suspected of containing such bacteria. The detection method of this invention comprises : a) obtaining the biological sample from a host; 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 pneumoniae bacteria.

The DNA probes of this invention may also be used for detecting circulating Streptococcus pneumoniae i.e. Streptococcus pneumoniae nucleic acids in a sample, for example using a polymerase chain reaction, as a method of diagnosing Streptococcus pneumoniae 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. In a further embodiment, the preferred DNA probe will be an oligomer having a sequence complementary to at least about 15 contiguous nucleotides of the Streptococcus pneumoniae polypeptides of the invention. In a further embodiment, the preferred DNA probe will be an oligomer having a sequence complementary to at least about 30 contiguous nucleotides of the Streptococcus pneumoniae polypeptides of the invention. In a further embodiment, the preferred DNA probe will be an oligomer having a sequence complementary to at least about 50 contiguous nucleotides of the Streptococcus pneumoniae polypeptides of the invention.

Another diagnostic method for the detection of Streptococcus 5 pneumoniae in a host comprises : a) labelling an antibody reactive with a polypeptide of the invention or fragment thereof with a detectable label ; b) administering the labelled antibody or labelled 10 fragment to the host; and c) detecting specifically bound labelled antibody or labelled fragment in the host which indicates the presence of Streptococcus pneumoniae .

15 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

20 vivo. For example polynucleotides may be incorporated into a plasmid vector under the control of the CMV promoter which is functional in eukaryotic cells. Preferably the vector is injected intramuscularly.

25 The use of a polynucleotide of the invention in genetic immunization will preferably employ a suitable delivery method or system such as direct injection of plasmid DNA into muscles [Wolf et al. H M G (1992) 1: 363, Turnes et al., Vaccine (1999), 17 : 2089, Le et al . , Vaccine (2000) 18 : 1893, Alves

30 et al., Vaccine (2001) 19 : 788], injection of plasmid DNA with or without adjuvants [Ulmer et al . , Vaccine (1999) 18: 18, MacLaughlin et al . , J. Control Release (1998) 56: 259, Hartikka et al., Gene Ther. (2000) 7: 1171-82, Benvenisty and Reshef, PNAS USA (1986) 83:9551, Singh et al . , PNAS USA (2000) 97:

35 811] , targeting cells by delivery of DNA complexed with specific carriers [Wa et al . , J Biol Chem (1989) 264: 16985, Chaplin et al . , Infect. Immun. (1999) 67: 6434], injection of plasmid complexed or encapsulated in various forms of liposomes [Ishii et al . , AIDS Research and Human Retroviruses (1997) 13: 142, Perrie et al . , Vaccine (2001) 19: 3301], administration of DNA with different methods of bombardment [Tang et al . , Nature (1992) 356: 152, Eisenbraun et al . , DNA Cell Biol (1993) 12: 5791, Chen et al . , Vaccine (2001) 19: 2908], and administration of DNA with lived vectors [Tubulekas et al . , Gene (1997) 190: 191, Pushko et al., Virology (1997) 239: 389, Spreng et al . FEMS (2000) 27: 299, Dietrich et al., Vaccine (2001) 19: 2506]

0 A further aspect of the invention is the use of the Streptococcus pneumoniae polypeptides of the invention as immunogens for the production of specific antibodies for the diagnosis and in particular the treatment of Streptococcus pneumoniae infection. Suitable antibodies may be determined 5 using appropriate screening methods, for example by measuring the ability of a particular antibody to passively protect against Streptococcus pneumoniae 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 0 an antigen-binding fragment thereof and may belong to any immunoglobulm 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 5 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 0 number of epitopes associated with the Streptococcus pneumoniae polypeptides but is preferably specific for one.

According to one aspect, the present invention provides the use of an antibody for treatment and/or prophylaxis of 5 Streptococcus pneumoniae infections.

A further aspect of the invention is the use of the antibodies directed to the polypeptides of the invention for passive immunization. One could use the antibodies described in the present application.

A further aspect of the invention is a method for immunization, whereby an antibody raised by a polypeptide of the invention is administered to a host in an amount sufficient to provide a passive immunization.

In a further embodiment, the invention provides the use of a pharmaceutical composition of the invention in the manufacture of a medicament for the prophylactic or therapeutic treatment of Streptococcus pneumoniae infection.

In a further embodiment, the invention provides a kit comprising a polypeptide of the invention for detection or diagnosis of Streptococcus pneumoniae infection.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

EXAMPLE 1

This example describes the amplification, cloning and expression of S_j_ pneumoniae gene fragments SHB-SP-101, SHB-SP- 102 and SHB-SP-103.

Gene fragments were amplified from genomic DNA isolated from S . pneumoniae serogroup 6 strain SP64 by PCR using pairs of oligonucleotide engineered to amplify the SHB-SP-101 (SEQ ID NO: 1), SHB-SP-102 (SEQ ID NO: 3) and SHB-SP-103 (SEQ ID NO: 5) DNA sequences. 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 (see Table 1) . PCR-amplified products were digested with restriction endonucleases and ligated to linearized plasmid pET19b (Novagen, Madison, WI ) expression vector. pET plasmids were digested with restriction enzymes for the in-frame cloning. Clones were first stabilized in E_;_ coli DH5α before introduction into E__ coli BL21(λDE3) or BL21 STAR (λDE3) for expression of SHB-SP-101, SHB-SP-102 and SHB-SP-103 molecules . Each of the resultant plasmid constructs was confirmed by nucleotide sequence analysis. The recombinant polypeptides were expressed as N-terminal fusions with His-tag. The expressed recombinant polypeptides were purified from supernatant fractions obtained from centrifugation of sonicated IPTG-induced E_;_ coli cultures using a His-Bind metal chelation resin (QIAgen, Chatsworth, CA) . The gene products generated are listed in the Table 2.

Table 2. List of SHB-SP-101, SHB-SP-102 and SHB-SP-103 gene products generated from S__ pneumoniae SP64

^a The polypeptide sequences also contain a methionine starting residue.

EXAMPLE 2 This example describes the immunization of animals with recombinant gene products to elicit a protective immune response against pneumococcal disease.

Groups of 8 female BALB/c mice (Charles River, St-Constant, Canada) were immunized subcutaneously three times at three-week intervals with either 20 μg of SHB-SP-101, SHB-SP-102 or SHB- SP-103 polypeptides in presence of 10 μg 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. Five to seven days later, the mice were challenged with approximately 10⁵ CFU of the type 3 £___ pneumoniae strain 5 P4241. 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 sacrificed and blood samples tested for the presence of S___ 10 pneumoniae organisms . The survival data are shown in Table 3.

Prechallenge sera were analyzed for the presence of antibodies reactive with S__ pneumoniae by standard immunoassays . Elisa analyses indicated that immunization with recombinant SHB-SP- 15101, SHB-SP-102 and SHB-SP-103 polypeptides produced in E__ coli elicited antibodies reactive with both, recombinant and native pneumococcal polypeptide .

20 Table 3. Protection mediated by recombinant SHB-SP-101, SHB- SP-102 and SHB-SP-103 in experimental pneumococcal pneumonia.

For statistical analysis, P values were calculated using the logrank test to compare the survival curves for immunized mice to controls. 5

All control mice injected with adjuvant alone succumbed to infection while 6, 7 and 3 mice immunized with SHB-SP-101, SHB- SP-102 and SHB-SP-103, respectively, survived. The observed difference in survival between immunized and control animals 0 indicates that SHB-SP-101, SHB-SP-102 and SHB-SP-103 may be useful antigens to prevent pneumococcal disease. EXAMPLE 3

This example describes the immunization of animals with a combination of recombinant gene products to elicit a protective immune response against pneumococcal disease.

Groups of 8 female BALB/c mice (Charles River) were immunized as described above in Example 1 with either 20 μg of SHB-SP- 101, SHB-SP-102 or SHB-SP-103 polypeptides alone or in combination with a chimeric protective polypeptide SHB-SP-104 (SEQ ID No: 18) prior to intranasal challenge with virulent S . pneumoniae P4241. The survival data are shown in Table 4.

Table 4. Protection mediated by recombinant SHB-SP-101 or SHB-SP-102 alone or in combination to SHB-SP-104 in experimental pneumococcal pneumonia.

For statistical analysis, P values were calculated using the logrank test to compare the survival curves for immunized mice to controls.

All control mice injected with adjuvant alone succumbed to infection while 3 to 8 mice immunized with SHB-SP-101 or SHB- SP-102 alone or in combination with a protective antigen survived to the challenge. The highest protection level as defined by the survival rate and survival period was achieved when animals were vaccinated with combinations of SHB-SP-101 or SHB-SP-102 with SHB-SP-104 thus indicating that mixtures of polypeptides could afford superior protection by directing immune responses towards various protective targets on S___ 5 pneumoniae cells .

EXAMPLE 4

This example illustrates the molecular conservation of 10 pneumococcal gene fragments .

Comparison of DNA sequences of SHB-SP-101 (SEQ ID NO: 1), SHB- SP-102 (SEQ ID NO: 3) and SHB-SP-103 (SEQ ID NO : 5) to the complete genome of S_;_ pneumoniae type 4 strain TIGR4 (Science

15 2001. 293: 498-506) and nonencapsulated S_-- pneumoniae R6 (J. Bacteriol.2001. 183: 5709-5717) revealed that the sequences from various _____ pneumoniae isolates share very high level of homology. Computer analysis (Align X program, Vector NTI® Suite, version 7.0, Bethesda, MD) using multiple alignment of

20 the predicted protein sequences from these three distinct S . pneumoniae strains revealed that the SHB-SP-101, SHB-SP-102 and SHB-SP-103 sequences were 96.1, 98.2 and 97.7 identical, respectively, while the sequences showed 99.9% similarity (Figures 7, 8 and 9). These data suggest that vaccination with 5 SHB-SP-101, SHB-SP-102 or SHB-SP-103 polypeptides could provide protection against all pneumococcal strains independently of the capsular serotype. In agreement with this, data shown in Examples 2 and 3 clearly established that recombinant gene products generated from serogroup 6 strain SP64 prevented

30 lethal experimental infection caused by type 3 P4241 S ■ pneumoniae.

Claims

What is claimed is :

1. An isolated polynucleotide comprising a polynucleotide chosen from: (a) a polynucleotide encoding a polypeptide having at least 70% identity to a second polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof;

(b) a polynucleotide encoding a polypeptide. having at least 80% identity to a second polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof;

(c) a polynucleotide encoding a polypeptide having at least 95% identity to a second polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof;

(d) a polynucleotide encoding a polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof; (e) a polynucleotide encoding a polypeptide capable of raising antibodies having binding specificity for a polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof;

2, 4, 6, 18 or fragments or analogs thereof;

(h) a polynucleotide that is complementary to a polynucleotide in (a), (b) , (c) , (d) , (e) , (f) or (g) .

2. An isolated polynucleotide comprising a polynucleotide chosen from:

(a) a polynucleotide encoding a polypeptide having at least 70% identity to a second polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6 or 18; (b) a polynucleotide encoding a polypeptide having at least 80% identity to a second polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6 or 18;

(e) a polynucleotide encoding a polypeptide capable of raising antibodies having binding specificity for a polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6 or 18;

(f) a polynucleotide encoding an epitope bearing portion of a polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6 or 18;

(g) a polynucleotide comprising a sequence chosen from SEQ ID No : 1, 3, 5, 17;

(h) a polynucleotide that is complementary to a polynucleotide in (a) , (b) , (c) , (d) , (e) , (f) or (g) .

3. The polynucleotide of claim 1, wherein said polynucleotide is DNA.

4. The polynucleotide of claim 2, wherein said polynucleotide is DNA.

5. The polynucleotide of claim 1, wherein said polynucleotide is RNA.

6. The polynucleotide of claim 2, wherein said polynucleotide is RNA.

7. The polynucleotide of claim 1 that hybridizes under stringent conditions to either (a) a DNA sequence encoding a polypeptide or

(b) the complement of a DNA sequence encoding a polypeptide; wherein said polypeptide comprises SEQ ID No : 2, 4, 6 or 18 or fragments or analogs thereof.

8. The polynucleotide of claim 2 that hybridizes under stringent conditions to either

(a) a DNA sequence encoding a polypeptide or

(b) the complement of a DNA sequence encoding a polypeptide; wherein said polypeptide comprises SEQ ID No : 2, 4, 6 or 18.

9. The polynucleotide of claim 1 that hybridizes under stringent conditions to either

(a) a DNA sequence encoding a polypeptide or

(b) the complement of a DNA sequence encoding a polypeptide; wherein said polypeptide comprises at least 10 contiguous amino acid residues from a polypeptide comprising SEQ ID No : 2, 4, 6 or 18 or fragments or analogs thereof.

10. The polynucleotide of claim 2 that hybridizes under stringent conditions to either

(a) a DNA sequence encoding a polypeptide or

(b) the complement of a DNA sequence encoding a polypeptide; wherein said polypeptide comprises at least 10 contiguous amino acid residues from a polypeptide SEQ ID No : 2, 4, 6 or 18.

11. A vector comprising the polynucleotide of claim 1, wherein said DNA is operably linked to an expression control region.

12. A vector comprising the polynucleotide of claim 2, wherein said DNA is operably linked to an expression control region.

13. A host cell transfected with the vector of claim 12.

14. A host cell transfected with the vector of claim 13.

15. A process for producing a polypeptide comprising culturing a host cell according to claim 14 under conditions suitable for expression of said polypeptide.

16. A process for producing a polypeptide comprising culturing a host cell according to claim 15 under condition suitable for expression of said polypeptide.

17. An isolated polypeptide comprising a polypeptide chosen from:

(a) a polypeptide having at least 70% identity to a second polypeptide having an amino acid sequence SEQ ID No : 2 ,

4, 6, 18 or fragments or analogs thereof;

(b) a polypeptide having at least 80% identity to a second polypeptide having an amino acid sequence comprising SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof; (c) a polypeptide having at least 95% identity to a second polypeptide having an amino acid sequence comprising SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof; (d) a polypeptide comprising SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof; (e) a polypeptide capable of raising antibodies having binding specificity for a polypeptide comprising SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof; (f) an epitope bearing portion of a polypeptide comprising SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof; (g) the polypeptide of (a), (b) , (c) , (d) , (e) or (f) wherein the N-terminal Met residue is deleted; (h) the polypeptide of (a), (b) , (c) , (d) , (e) , (f) or (g) wherein the secretory amino acid sequence is deleted.

18. An isolated polypeptide comprising a polypeptide chosen from:

(a) a polypeptide having at least 70% identity to a second polypeptide having an amino acid sequence comprising SEQ ID No : 2, 4, 6 or 18;

(b) a polypeptide having at least 80% identity to a second polypeptide having an amino acid sequence comprising SEQ ID No : 2, 4, 6 or 18;

(c) a polypeptide having at least 95% identity to a second polypeptide having an amino acid sequence comprising SEQ

ID No : 2, 4, 6 or 18;

(d) a polypeptide comprising SEQ ID No : 2, 4, 6 or 18;

(e) a polypeptide capable of raising antibodies having binding specificity for a polypeptide comprising SEQ ID No : 2, 4, 6 or 18;

(f) an epitope bearing portion of a polypeptide comprising SEQ ID No : 2, 4, 6 or 18;

(g) the polypeptide of (a), (b) , (c) , (d) , (e) or (f) wherein the N-terminal Met residue is deleted; (h) the polypeptide of (a), (b) , (c) , (d) , (e) , (f) or (g) wherein the secretory amino acid sequence is deleted.

19. A chimeric polypeptide comprising two or more polypeptides comprising SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof; provided that the polypeptides are linked as to formed a chimeric polypeptide .

20. A chimeric polypeptide comprising two or more polypeptides comprising SEQ ID No : 2, 4, 6 or 18 provided that the polypeptides are linked as to formed a chimeric polypeptide.

21. A pharmaceutical composition comprising a polypeptide according to any one of claims 17 to 20 and a pharmaceutically acceptable carrier, diluent or adjuvant.

22. A method for prophylactic or therapeutic treatment of S. pneumoniae infection in a host susceptible to S . pneumoniae infection comprising administering to said host a prophylactic or therapeutic amount of a composition according to claim 21.

23. A method according to claim 22 wherein the host is a neonate, an infant or a child.

24. A method according to claim 22 wherein the host is an immunocompromised host.

25. A method according to claim 22 wherein the host is an adult .

26. A method for therapeutic or prophylactic treatment of otitis media, sinusitis, pneumonia, meningitis, bacteremia, or septicaemia comprising administering to said host a therapeutic or prophylactic amount of a composition according to claim 21.

27. A method for diagnostic of S . pneumoniae infection in an host susceptible to S. pneumoniae infection comprising

(a) obtaining a biological sample from a host;

(b) incubating an antibody or fragment thereof reactive with a polypeptide according to any one of claims 17 to 20 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 S . pneumoniae .

28. A method for the detection of antibody specific to a S . pneumoniae antigen in a biological sample containing or suspected of containing said antibody comprising

(a) obtaining a biological sample from a host; (b) incubating one or more polypeptides according to any one of claims 17 to 20 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 S . pneumoniae .

29. Use of the pharmaceutical composition according to claim 21 in the manufacture of a medicament for the prophylactic or therapeutic treatment of S . pneumoniae infection.

30. A Kit comprising a polypeptide according to any one of claims 17 to 20 for detection or diagnosis of S. pneumoniae infection.