WO2004007725A9

WO2004007725A9 - Polypeptide of streptococcus pyogenes

Info

Publication number: WO2004007725A9
Application number: PCT/CA2003/001067
Authority: WO
Inventors: Stephane Rioux; Denis Martin; Josee Hamel; Bernard R Brodeur
Original assignee: Shire Biochem Inc; Stephane Rioux; Denis Martin; Josee Hamel; Bernard R Brodeur
Priority date: 2002-07-15
Filing date: 2003-07-15
Publication date: 2004-06-03
Also published as: AU2003246495A1; WO2004007725A1

Abstract

The present invention relates to a polypeptide of S. pyogenes which may be useful to prevent, diagnose and/or treat S. pyogenes infections.

Description

POLYPEPTIDE OF STREPTOCOCCUS PYOGENES

FIELD OF THE INVENTION

The present invention is related to SHB-GAS-101 polypeptide of S. pyogenes (Group A Streptococcus) and corresponding DNA fragments, which may be used to prevent, diagnose and/or treat £>__;_ pyogenes infections .

BACKGROUND OF THE INVENTION

Streptococci are gram (+) bacteria which are differentiated by group specific carbohydrate antigens A through 0 which are found at the cell surface. £!__;_ pyogenes isolates are further distinguished by type- specific M protein antigens . M proteins are important virulence factors which are highly variable both in molecular weights and in sequences. Indeed, more than 100-M protein types have been identified on the basis of antigenic differences.

S_^ pyogenes is responsible for many diverse infection types, including pharyngitis, erysipelas and impetigo, scarlet fever, and invasive diseases such as bacteremia and necrotizing fasciitis. A resurgence of invasive disease in recent years has been documented in many countries, including those in North America and Europe. Although the organism is sensitive to antibiotics, the high attack rate and rapid onset of sepsis results in high morbidity and mortality.

To develop a vaccine that will protect individuals from S _ pyogenes infection, efforts have focused on virulence factors such as the type- specific M proteins. However, the amino-terminal portion of proteins was found to induce cross-reactive antibodies which reacted with human myocardium, tropomyosin, myosin, and vimentin, which might be implicated in autoimmune diseases . Others have used recombinant techniques to produce complex hybrid proteins containing amino- terminal peptides of M proteins from different serotypes . However, a safe vaccine containing all S^ pyogenes serotypes will be highly complex to produce and standardize. The sequencing project at the Sanger Institute of the genome of S . pyogenes strain Manfredo, serotype M5, is available at http://www.sanger.ac.uk. Smoot at al . have described a genome sequence analysis of serotype Ml8 of Group A Streptococcus in PNAS, 2002, vol.99 No.7, 4668-4673. Telford et al . have filed a PCT application WO 02/34771 describing nucleic acids and proteins from Streptococcus groups A and B .

In addition to the serotype-specific antigens, other S^ pyogenes proteins have generated interest as potential vaccine candidates . The C5a peptidase, which is expressed by at least S^ pyogenes 40 serotypes, was shown to be immunogenic in mice, but its capacity to reduce the level of nasopharyngeal colonization was limited. Other investigators have also focused on the streptococcal pyrogenic exotoxins which appear to play an important role in pathogenesis of infection. Immunization with these proteins prevented the deadly symptoms of toxic shock, but did not prevent colonization.

Therefore there remains an unmet need for S^ pyogenes antigens that may be used as vaccine components for the prophylaxis and/or therapy of S^ pyogenes 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 SEQ ID No : 2, 4, 6, 8 or fragments or analogs thereof .

According to one aspect, the present invention relates to polypeptides comprising SEQ ID No : 2, 4, 6, 8 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-GAS-101 gene from serotype Ml S^ pyogenes strain ATCC700294; SEQ ID NO: 1. The underlined portion of the sequence represents the region coding for the leader peptide.

Figure 2 represents the amino acid sequence SHB-GAS-101 polypeptide from serotype Ml S__;_ pyogenes strain ATCC700294; SEQ ID NO: 2. The underlined sequence represents the 26 amino acid residues leader peptide .

Figure 3 represents the DNA sequence of SHB-GAS-101-1 gene from serotype Ml EL pyogenes strain ATCC700294; SEQ ID NO: 3.

Figure 4 represents the amino acid sequence SHB-GAS-101-1 polypeptide from serotype Ml S^ pyogenes strain ATCC700294; SEQ ID NO: 4.

Figure 5 represents the DNA sequence of SHB-GAS-101-2 gene from serotype Ml S^_ pyogenes strain ATCC700294; SEQ ID NO: 5.

Figure 6 represents the amino acid sequence SHB-GAS-101-2 polypeptide from serotype Ml £ pyogenes strain ATCC700294; SEQ ID NO: 6.

Figure 7 represents the DNA sequence of SHB-GAS-101-3 gene from serotype Ml S^ pyogenes strain ATCC700294; SEQ ID NO: 7.

Figure 8 represents the amino acid sequence SHB-GAS-101-3 polypeptide from serotype Ml S^ pyogenes strain ATCC700294; SEQ ID NO: 8.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides purified and isolated polynucleotides, which encode S . pyogenes polypeptides which may be used to prevent, diagnose and/or treat S . pyogenes 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 SEQ ID No : 2 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 SEQ ID No : 2 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 SEQ ID No : 2 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 SEQ ID No : 2 or fragments or analogs 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 SEQ ID No : 2 or fragments or analogs 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 SEQ ID No : 2.

According to one aspect, the present invention provides an isolated polynucleotide encoding a polypeptide having at least 80% identity to a second polypeptide comprising SEQ ID No : 2.

According to one aspect, the present invention provides an isolated polynucleotide encoding a polypeptide having at least 90% identity to a second polypeptide comprising SEQ ID No : 2.

According to one aspect, the present invention provides an isolated polynucleotide encoding a polypeptide having at least 95% identity to a second polypeptide comprising SEQ ID No : 2.

According to one aspect, the present invention provides an isolated polynucleotide encoding a polypeptide having at least 98% identity to a second polypeptide comprising SEQ ID No : 2. According to one aspect, the present invention relates to polypeptides comprising SEQ ID No : 2 or fragments or analogs thereof .

According to one aspect, the present invention relates to polypeptides comprising SEQ ID No : 2.

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

According to one aspect, the present invention provides a polynucleotide encoding an epitope bearing portion of a polypeptide comprising SEQ ID No : 2.

According to one aspect, the present invention relates to epitope bearing portions of a polypeptide comprising SEQ ID No : 2 or fragments or analogs thereof .

According to one aspect, the present invention relates to epitope bearing portions of a polypeptide comprising SEQ ID No : 2.

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 SEQ ID No : 2 or fragments or analogs thereof;

(b) a polynucleotide encoding a polypeptide having at least 80% identity to a second polypeptide comprising SEQ ID No : 2 or fragments or analogs thereof;

(c) a polynucleotide encoding a polypeptide having at least 95% identity to a second polypeptide comprising SEQ ID No : 2 or fragments or analogs thereof;

(d) a polynucleotide encoding a polypeptide comprising SEQ ID No : 2 or fragments or analogs thereof;

(e) a polynucleotide encoding a polypeptide capable of raising antibodies having binding specificity for a polypeptide comprising SEQ ID No : 2 or fragments or analogs thereof;

(f) a polynucleotide encoding an epitope bearing portion of a polypeptide comprising SEQ ID No : 2 or fragments or analogs thereof; (g) a polynucleotide comprising SEQ ID No : 1 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 SEQ ID No : 2;

(b) a polynucleotide encoding a polypeptide having at least 80% identity to a second polypeptide comprising SEQ ID No : 2;

(c) a polynucleotide encoding a polypeptide having at least 95% identity to a second polypeptide comprising SEQ ID No : 2;

(d) a polynucleotide encoding a polypeptide comprising SEQ ID No : 2;

(e) a polynucleotide encoding a polypeptide capable of raising antibodies having binding specificity for a polypeptide comprising SEQ ID No : 2;

(f) a polynucleotide encoding an epitope bearing portion of a polypeptide comprising SEQ ID No : 2;

(g) a polynucleotide comprising SEQ ID No : 1; (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 SEQ ID No : 2 or fragments or analogs thereof;

(b) a polypeptide having at least 80% identity to a second polypeptide comprising SEQ ID No : 2 or fragments or analogs thereof;

(c) a polypeptide having at least 95% identity to a second polypeptide comprising SEQ ID No : 2 or fragments or analogs thereof; (d) a polypeptide comprising SEQ ID No : 2 or fragments or analogs thereof;

(e) a polypeptide capable of raising antibodies having binding specificity for a polypeptide comprising SEQ ID No : 2 or fragments or analogs thereof; (f) an epitope bearing portion of a polypeptide comprising SEQ ID No : 2 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) or (f) wherein the secretory amino acid sequence is deleted.

(a) a polypeptide having at least 70% identity to a second polypeptide comprising SEQ ID No : 2;

(b) a polypeptide having at least 80% identity to a second polypeptide comprising SEQ ID No : 2 ;

(c) a polypeptide having at least 95% identity to a second polypeptide comprising SEQ ID No : 2; (d) a polypeptide comprising SEQ ID No : 2;

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

(f) an epitope bearing portion of a polypeptide comprising SEQ ID No : 2; (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) or (f) wherein the secretory amino acid sequence is deleted.

According to one aspect, the present invention provides an isolated polynucleotide encoding a polypeptide having at least 70% identity to a second polypeptide comprising SEQ ID No : 4, 6, 8 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 SEQ ID No : 4, 6, 8 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 SEQ ID No : 4, 6, 8 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 SEQ ID No : 4, 6, 8 or fragments or analogs 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 SEQ ID No : 4, 6, 8 or fragments or analogs 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 SEQ ID No : 4, 6, 8.

According to one aspect, the present invention provides an isolated polynucleotide encoding a polypeptide having at least 80% identity to a second polypeptide comprising SEQ ID No : 4, 6, 8.

According to one aspect, the present invention provides an isolated polynucleotide encoding a polypeptide having at least 90% identity to a second polypeptide comprising SEQ ID No : 4, 6, 8.

According to one aspect, the present invention provides an isolated polynucleotide encoding a polypeptide having at least 95% identity to a second polypeptide comprising SEQ ID No : 4, 6, 8.

According to one aspect, the present invention provides an isolated polynucleotide encoding a polypeptide having at least 98% identity to a second polypeptide comprising SEQ ID No : 4, 6, 8.

According to one aspect, the present invention provides a polynucleotide encoding an epitope bearing portion of a polypeptide comprising SEQ ID No : 4, 6, 8 or fragments or analogs thereof.

According to one aspect, the present invention provides a polynucleotide encoding an epitope bearing portion of a polypeptide comprising SEQ ID No : 4, 6, 8. According to one aspect, the present invention relates to epitope bearing portions of a polypeptide comprising SEQ ID No : 4, 6, 8 or fragments or analogs thereof .

According to one aspect, the present invention relates to epitope bearing portions of a polypeptide comprising SEQ ID No : 4, 6, 8.

(a) a polynucleotide encoding a polypeptide having at least 70% identity to a second polypeptide comprising SEQ ID No : 4, 6, 8 or fragments or analogs thereof;

(b) a polynucleotide encoding a polypeptide having at least 80% identity to a second polypeptide comprising SEQ ID No : 4, 6, 8 or fragments or analogs thereof;

(c) a polynucleotide encoding a polypeptide having at least 95% identity to a second polypeptide comprising SEQ ID No : 4, 6, 8 or fragments or analogs thereof;

(d) a polynucleotide encoding a polypeptide comprising SEQ ID No : 4, 6, 8 or fragments or analogs thereof;

(e) a polynucleotide encoding a polypeptide capable of raising antibodies having binding specificity for a polypeptide comprising SEQ ID No : 4, 6, 8 or fragments or analogs thereof;

(f) a polynucleotide encoding an epitope bearing portion of a polypeptide comprising SEQ ID No : 4, 6, 8 or fragments or analogs thereof ; (g) a polynucleotide comprising SEQ ID No : 3, 5, 7 or fragments or analogs thereof;

(a) a polynucleotide encoding a polypeptide having at least 70% identity to a second polypeptide comprising SΞQ ID No : 4, 6, 8;

(b) a polynucleotide encoding a polypeptide having at least 80% identity to a second polypeptide comprising SEQ ID No : 4, 6, 8; (c) a polynucleotide encoding a polypeptide having at least 95% identity to a second polypeptide comprising SEQ ID No : 4, 6, 8;

(d) a polynucleotide encoding a polypeptide comprising SEQ ID No : 4, 6, 8; (e) a polynucleotide encoding a polypeptide capable of raising antibodies having binding specificity for a polypeptide comprising SEQ ID No : 4, 6, 8 ;

(f) a polynucleotide encoding an epitope bearing portion of a polypeptide comprising SEQ ID No : 4, 6, 8; (g) a polynucleotide comprising SEQ ID No : 3, 5, 7;

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 SEQ ID No : 4, 6, 8 or fragments or analogs thereof;

(b) a polypeptide having at least 80% identity to a second polypeptide comprising SEQ ID No : 4, 6, 8 or fragments or analogs thereof;

(c) a polypeptide having at least 95% identity to a second polypeptide comprising SEQ ID No : 4, 6, 8 or fragments or analogs thereof ;

(d) a polypeptide comprising SEQ ID No : 4, 6, 8 or fragments or analogs thereof;

(e) a polypeptide capable of raising antibodies having binding specificity for a polypeptide comprising SEQ ID No : 4, 6, 8 or fragments or analogs thereof;

(f) an epitope bearing portion of a polypeptide comprising SEQ ID No : 4, 6, 8 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) or (f) 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 SEQ ID No : 4, 6, 8 ; (b) a polypeptide having at least 80% identity to a second polypeptide comprising SEQ ID No : 4, 6, 8;

(c) a polypeptide having at least 95% identity to a second polypeptide comprising SEQ ID No : 4, 6, 8;

(d) a polypeptide comprising SEQ ID No : 4, 6, 8; (e) a polypeptide capable of raising antibodies having binding specificity for a polypeptide comprising SEQ ID No : 4, 6, 8;

(f) an epitope bearing portion of a polypeptide comprising SEQ ID No : 4, 6, 8;

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 accordance with the present invention, all polynucleotides encoding polypeptides of the present invention are within the scope of the present invention.

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. 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 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% identity with those sequences illustrated in the figures or fragments thereof. That is, 70% of the residues are the same. In a further embodiment, polypeptides will have greater than 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.

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.

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 protein or 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 .

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 S . pyogenes 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 dimethyl- superimidate . 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 SEQ ID No : 2, 4, 6, 8 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 SEQ ID No : 2, 4, 6, 8 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 S__;_ pyogenes 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) .

In another embodiment, the polypeptides of the invention may be lacking an N-terminal leader peptide, and/or a transmembrane domain and/or a C-terminal anchor domain.

The present invention further provides a fragment of the polypeptide comprising substantially all of the extra cellular domain of a polypeptide which has at least 70% identify, preferably 80% identity, more preferably 95% identity, to a second polypeptide comprising a sequence chosen from SEQ ID NOs : 2, 4, 6, 8 or fragments or analogs thereof, over the entire length of said sequence.

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 carrier, diluent or adjuvant; (iii) a vaccine comprising a polypeptide of the invention and a carrier, diluent or adjuvant; (iv) a method for inducing an immune response against S . pyogenes , 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 S . pyogenes ; and particularly, (v) a method for preventing and/or treating a S . pyogenes 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 S . pyogenes , 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 S . pyogenes ; and particularly, (iv) a method for preventing and/or treating a S . pyogenes 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 S . pyogenes 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, A1NH₄(S0₄)₂, Al(OH)₃, A1P0₄, 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. A more detailed description of adjuvants 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 infection and/or diseases and symptoms mediated by streptococcus infection.

Pharmaceutical compositions of the invention are used for the prophylaxis of S . pyogenes infection and/or diseases and symptoms mediated by S . pyogenes 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 treatment or prophylaxis of many diverse infection types, including pharyngitis, erysipelas and impetigo, scarlet fever, and invasive diseases such as bacteremia and necrotizing fasciitis.

In one embodiment, pharmaceutical compositions of the invention are used for the treatment or prophylaxis of Streptococcus infection and/or diseases and symptoms mediated by Streptococcus infection, in particular group A Streptococcus (Streptococcus pyogenes) , group B Streptococcus (GBS or S .agalactiae) , S .pneumoniae, S . dysgalactiae, S .uberis, S .nocardia as well as Staphylococcus aureus . In one embodiment, pharmaceutical compositions of the invention are used for the treatment or prophylaxis of S . pyogenes infection and/or diseases and symptoms mediated by S . pyogenes infection. In a further embodiment, the S . pyogenes infection is nontypeable S . pyogenes .

In a further embodiment, the invention provides a method for prophylaxis or treatment of streptococcus infection in a host susceptible to streptococcus infection comprising administering to said host a prophylactic or therapeutic amount of a composition of the invention.

In a further embodiment, the invention provides a method for prophylaxis or treatment of S . pyogenes infection in a host susceptible to S . pyogenes 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 particular embodiment, pharmaceutical compositions are administered to those hosts at risk of S . pyogenes infection such as neonates, infants, children, elderly and immunocompromised hosts.

In a particular embodiment, pharmaceutical compositions are administered to those hosts at risk of S . pyogenes infection such as 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, 8 or fragments or analogs thereof.

In one embodiment, polynucleotides are those illustrated in SEQ ID No: 1 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, polynucleotides are hybridizable under stringent conditions.

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

"Suitable stringent conditions", as used herein, means, for example, incubating a blot overnight ( e . g. , at least 12 hours) with a long polynucleotide probe in a hybridization solution containing, e . g. , about 5X SSC, 0.5% SDS, 100 μg/ml denatured salmon sperm DNA and 50% formamide, at 42 °C. Blots can be washed at high stringency conditions that allow, e.g., for less than 5% bp mismatch { e . g. , wash twice in 0.1X SSC and 0.1% SDS for 30 min at 65°C), thereby, selecting sequences having, e . g. , 95% or greater sequence identity.

Other non-limiting examples of suitable stringent conditions include a final wash at 65 °C in aqueous buffer containing 30 mM NaCl and 0.5% SDS . Another example of suitable stringent conditions is hybridization in 7% SDS, 0.5 M NaP0₄, pH 7, 1 mM EDTA at 50°C, e . g. , overnight, followed by one or more washes with a 1% SDS solution at 42 °C. Whereas high stringency washes can allow for less than 5% mismatch, reduced or low stringency conditions can permit up to 20% nucleotide mismatch. Hybridization at low stringency can be accomplished as above, but using lower formamide conditions, lower temperatures and/or lower salt concentrations, as well as longer periods of incubation time .

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 SEQ ID No : 2, 4, 6, 8 or fragments or analogs thereof .

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 SEQ ID NO: 2, 4, 6, 8. 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 SEQ ID No : 2, 4, 6, 8 or fragments or analogs thereof.

(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, 8.

In a further embodiment, polynucleotides are those illustrated in SEQ ID NO: 1, 3, 5, 7 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, 7 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 polynucleotides may be incorporated into a plasmid vector under the control of the CMV promoter which is functional in eukaryotic cells . Preferably the vector is injected intramuscularly. According to another aspect, there is provided a process for producing polypeptides of the invention by recombinant techniques by expressing a polynucleotide encoding said polypeptide in a host cell and recovering the expressed polypeptide product .

Alternatively, the polypeptides can be produced according to established synthetic chemical techniques i.e. solution phase or solid phase synthesis of oligopeptides which are ligated to produce the full polypeptide (block ligation) .

General methods for obtention and evaluation of polynucleotides and polypeptides are described in the following references: Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd ed, Cold Spring Harbor, N.Y., 1989; Current Protocols in Molecular Biology, Edited by Ausubel F.M. et al . , John Wiley and Sons, Inc. New York; PCR Cloning Protocols, from Molecular Cloning to Genetic Engineering, Edited by White B.A., Humana Press, Totowa, 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 a process for producing a polypeptide comprising culturing a host cell of the invention under conditions suitable for expression of said polypeptide .

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, pDIO phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a, 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. Aspergillus niger, Aspergillus nidulins; yeast i.e. Saccharomyces or eukaryotic i.e. CHO, COS.

Upon expression of the polypeptide in culture, cells are typically harvested by centrifugation then disrupted by physical or chemical means (if the expressed polypeptide is not secreted into the media) and the resulting crude extract retained to isolate the polypeptide of interest. Purification of the polypeptide from culture media or lysate may be achieved by established techniques depending on the properties of the polypeptide i.e. using ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography and lectin chromatography. Final purification may be achieved using HPLC.

The 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 S . pyogenes polypeptides of the invention may be used in a diagnostic test for Streptococcus infection, in particular S . pyogenes infection.

Several diagnostic methods are possible, for example detecting S__;_ pyogenes organism in a biological sample, the following procedure may be followed: a) obtaining a biological sample from a host; b) incubating an antibody or fragment thereof reactive with a S . pyogenes 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 S . pyogenes .

Alternatively, a method for the detection of antibody specific to a S . pyogenes 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 S . pyogenes 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 S . pyogenes .

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

The DNA sequences encoding polypeptides of the invention may also be used to design DNA probes for use in detecting the presence of S^ pyogenes 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 S . pyogenes bacteria.

The DNA probes of this invention may also be used for detecting circulating S . pyogenes i.e. S . pyogenes nucleic acids in a sample, for example using a polymerase chain reaction, as a method of diagnosing S . pyogenes 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 S . pyogenes 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 S . pyogenes 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 S . pyogenes 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 S . pyogenes polypeptides of the invention.

Another diagnostic method for the detection of S . pyogenes 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 fragment to the host; and c) detecting specifically bound labelled antibody or labelled fragment in the host which indicates the presence of S . pyogenes . A further aspect of the invention is the use of the S . pyogenes polypeptides of the invention as immunogens for the production of specific antibodies for the diagnosis and in particular the treatment of S . pyogenes infection. Suitable antibodies may be determined using appropriate screening methods, for example by measuring the ability of a particular antibody to passively protect against S . pyogenes 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 S . pyogenes 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 S . pyogenes 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.

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 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: 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: 791; 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].

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 S^ pyogenes infection.

In a further embodiment, the invention provides a kit comprising a polypeptide of the invention for detection or diagnosis of S . pyogenes 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 illustrates the cloning and molecular characteristics of SHB-GAS-101 gene and corresponding polypeptide

The coding region of £X_ pyogenes SHB-GAS-101 (SEQ ID NO: 1) gene was amplified by PCR (Robocycler Gradient 96 Temperature cycler, Stratagene, La Jolla, CA) from genomic DNA of serotype Ml S^ pyogenes strain ATCC700294 using the following oligonucleotide primers that contained base extensions for the addition of restriction sites Ndel (CATATG) and Xhol (CTCGAG) : DMAR1174 and DMAR1175, which are presented in Table 1. PCR products were purified from agarose gel using a QIAquick gel extraction kit from QIAgen following the manufacturer's instructions (Chatsworth, CA) , and digested with Vdel and Xhol

(Amersham Pharmacia Biotech Inc, Baie d'Urfe, Canada). The pET-19b(+) vector (Novagen, Madison, WI) was digested with JVdel and Xhol and purified from agarose gel using a QIAquick gel extraction kit from QIAgen (Chatsworth, CA) . The Ndel-Xhol PCR products were ligated to the Ndel-Xhol pET-19b(+) expression vector. The ligated products were transformed into EL coli strain DH5c- [φ80dlacZΔM15 Δ (2acZYA-a-r9rF)U169 ertdAl recAl iιsdR17 (r_κ-m_κ+) deoR thi-1 supE44 λ^"gyrA96 relAl] (Gibco BRL, Gaithersburg, MD) according to the method of Simanis (Hanahan, D. DNA Cloning, 1985, D.M. Glover (ed) , pp. 109-135). Recombinant pET-19b(+) plasmid (rpET19b(+)) containing SHB-GAS-101 gene was purified using a QIAgen plasmid kit (Chatsworth, CA) and DNA insert was sequenced (Taq Dye Deoxy Terminator Cycle Sequencing kit, ABI, Foster City, CA) .

Table 1. Oligonucleotide primers used for PCR amplifications of S . pyogenes SHB-GAS-101 gene and truncated genes .

It was determined that the 1626-bp including a stop codon (TAA) open reading frame (ORF) of SHB-GAS-101 encodes a 541 amino-acid-residues polypeptide with a predicted pl of 6.70 and a predicted molecular mass of 59,796.0 Da. Analysis of the predicted amino acid residues sequence (SEQ ID NO: 2) using the Spscan software (Wisconsin Sequence Analysis Package; Genetics Computer Group) suggested the existence of a 26 amino acid residues signal peptide (MKKRKLLAVTLLSTILLNSAVPLWA) . To confirm the presence by PCR amplification of SHB-GAS-101 (SEQ ID NO :1) gene, the following 3 serologically distinct S^_ pyogenes strains were used: the serotype Ml S^ pyogenes strain ATCC700294 and the serotype M3 S^ pyogenes strain ATCC12384 were obtained from the American Type Culture Collection (Rockville, MD) , and the serotype M6 S . pyogenes SPY67 clinical isolate was provided by the Centre de recherche en infectiologie du Centre hospitalier de 1'Universite Laval, Sainte-Foy, Canada. The E__;_ coli strain XLl-Blue MRF' was used in these experiments as negative control . Chromosomal DNA was isolated from each strain as previously described (Jayarao BM et al . 1991. J. Clin. Microbiol. 29:2774-2778) . SHB-GAS-101 (SEQ ID NO :1) gene was amplified by PCR (Robocycler Gradient 96 Temperature cycler) from the genomic DNA purified from the 3 S__;_ pyogenes strains, and the control E. coli strain using the oligonucleotide primers DMAR1174 and DMAR1175 (Table 1) . PCR was performed with 30 cycles of 45 sec at 95°C, 45 sec at 50°C and 1 min at 72°C and a final elongation period of 7 min at 72°C. The PCR products were size fractionated in 1% agarose gels and were visualized by ethidium bromide staining. The results of these PCR amplifications are presented in Table 2. The analysis of the amplification products revealed that SHB-GAS-101 (SEQ ID NO :1) gene was present in the genome of all of the 3 S^ pyogenes strains tested. No such product was detected when the control E^ coli DNA was submitted to identical PCR amplifications with these oligonucleotide primers. The PCR data as well as the sequence comparison presented in the previous paragraphs clearly demonstrated that the SHB-GAS-101 gene is highly conserved among streptococcal strains .

Table 2. Identification of S^ pyogenes SHB-GAS-101 gene by PCR amplification in the genome of three serologically distinct S . pyogenes strains

EXAMPLE 2

This example illustrates the cloning of S^ pyogenes SHB-GAS-101 gene in CMV plasmid pCMV-GH.

The DNA coding region of E pyogenes polypeptide was inserted in phase downstream of a human growth hormone (hGH) gene which was under the transcriptional control of the cytomegalovirus (CMV) promotor in the plasmid vector pCMV-GH (Tang et al . , Nature, 1992, 356 :152). The CMV promotor is a non-functional plasmid in E_-_ coli cells but active upon administration of the plasmid in eukaryotic cells . The vector also incorporated the ampicillin resistance gene.

The coding regions of SHB-GAS-101 (SEQ ID NO: 1) gene was amplified by PCR (Robocycler Gradient 96 Temperature cycler) from genomic DNA of serotype Ml S^ pyogenes strain ATCC700294 using oligonucleotide primers DMAR1174a and DMAR1175a that contained base extensions for the addition of restriction sites BairiΑI (GGATCC) and Sail (GTCGAC) which are described in Table 1. The PCR products were purified from agarose gel using a QIAquick gel extraction kit from QIAgen (Chatsworth, CA) , and digested with restriction enzymes (A ersham Pharmacia Biotech Inc) . The pCMV-GH vector (Laboratory of Dr. Stephen A. Johnston, Department of Biochemistry, The University of Texas, Dallas, Texas) was digested with BamHI and Sail and purified from agarose gel using the QIAquick gel extraction kit from QIAgen (Chatsworth, CA) . The SamHI-Sall DNA fragment was ligated to the BamHI-Sall-pCMV-GH vector to create the hGH-SHB-GAS-101 fusion polypeptide under the control of the CMV promoter. The ligated product was transformed into E^ coli strain DH5α [φ80dlacZΔM15 Δ(2acZYA-argF)U169 endAl recAl lxsdR17 (r_κ-m_κ+) deoR thi-1 supΞ44 λ^"gryrA96 relAl] (Gibco BRL) according to the method of Simanis (Hanahan, D. DNA Cloning, 1985, D.M. Glover (ed) , pp. 109- 135) . The recombinant pCMV plasmid was purified using a QIAgen plasmid kit (Chatsworth, CA) and the nucleotide sequence of the DNA insert was verified by DNA sequencing. EXAMPLE 3

This example illustrates the use of DNA to elicit an immune response to EL pyogenes SHB-GAS-101 polypeptide antigen.

Groups of 8 female BALB/c mice (Charles River, St-Constant, Quebec, Canada) were immunized by intramuscular injection of 100 μl three times at two- or three-week intervals with 50 μg of recombinant pCMV- GH encoding SHB-GAS-101 (SEQ ID NO: 1) gene in presence of 50 μg of granulocyte-macrophage colony-stimulating factor (GM-CSF) -expressing plasmid pCMV-GH-GM-CSF (Laboratory of Dr. Stephen A. Johnston, Department of Biochemistry, The University of Texas, Dallas, Texas) . As control, groups of mice were injected with 50 μg of pCMV-GH in presence of 50 μg of pCMV-GH-GM-CSF. Blood samples were collected from the orbital sinus prior to each immunization and seven days following the third injection and serum antibody responses were determined by ELISA using the SHB-GAS-101 His-tagged labeled S. pyogenes recombinant polypeptide as coating antigen. The production and purification of this SHB-GAS-101 His-tagged labeled £L_ pyogenes recombinant polypeptide is presented in Example 4.

EXAMPLE 4

This example illustrates the production and purification of S . pyogenes SHB-GAS-101 recombinant polypeptide.

The recombinant pET-19 (+) plasmid with SHB-GAS-101 (SEQ ID NO: 1) gene was used to transform by electroporation (Gene Pulser II apparatus, BIO-RAD Labs, Mississauga, Canada) EL coli strain BLR (DE3) (F^" ompT hsdS_B {r_B ^"m _B ^") gal dcm A (srl-recA) 306 : :Tnl0 (Tc^R) (DE3)) (Novagen) . In this strain of EL_ coli, the T7 promotor controlling expression of the recombinant polypeptide is specifically recognized by the T7 RNA polymerase (present on the λDE3 prophage) whose gene is under the control of the lac promotor, which is inducible by isopropyl-β-d-thio- galactopyranoside (IPTG) . The transformants BLR (DE3)/rpET19 (+) were grown at 37°C with agitation at 250 rpm in LB broth (peptone lOg/L, yeast extract 5g/L, NaCl lOg/L) containing 100 μg of carbenicillin (Sigma-Aldrich Canada Ltd., Oakville, Canada) per ml until the A_60o reached a value of 0.6. In order to induce the production of SHB-GAS- 101 His-tagged S^ pyogenes recombinant polypeptide, the cells were incubated for 120 minutes in the presence of IPTG at a final concentration of 1 mM. Induced cells from a 400 ml culture were pelleted by centrifugation and frozen at -70°C.

The purification of the SHB-GAS-101 His-tagged recombinant polypeptide from the soluble fraction of IPTG-induced BLR (DE3) /rpET19b (+) was done by affinity chromatography based on the properties of the His»Tag sequence (10 consecutive histidine residues) to bind to divalent cations (Ni²⁺) immobilized on the His»Bind metal chelation resin. Briefly, the pelleted cells obtained from a 400 mL culture induced with IPTG was resuspended in lysis buffer (20 mM Tris, 500 mM NaCl, 10 mM imidazole, pH 7.9), sonicated and centrifuged at 12,000 X g for 20 min to remove debris. The supernatant was incubated with Ni-NTA agarose resin (QIAgen) for 45 min at 4°C. The SHB-GAS-101 His-tagged S . pyogenes recombinant polypeptide was eluted from the resin with a solution of 250 mM imidazole-500mM NaCl-20 mM Tris, pH 7.9. The removal of the salt and imidazole from the samples was done by dialysis against PBS buffer overnight at 4°C. The amount of recombinant polypeptide was estimated by MicroBCA (Pierce, Rockford, 111.) .

EXAMPLE 5

This example describes the cloning of truncated SHB-GAS-101 gene products by polymerase chain reaction (PCR) and the expression of truncated SHB-GAS-101 molecules.

Gene fragments were amplified by PCR (DNA Thermal Cycler GeneAmp PCR system 2400 Perkin Elmer) from genomic DNA of serotype Ml S . pyogenes strain ATCC700294 using oligonucleotide primers presented in Table 1. The methods used for cloning the truncated SHB-GAS-101 gene products into an expression vector and sequencing are similar to the methods described in Example 1. The recombinant SHB-GAS-101-1 and SHB-GAS- 101-3 proteins were purified from supernatant fractions obtained after centrifugation of sonicated IPTG-induced E. coli cultures using a His- Bind metal chelation resin (QIAgen) as described in Example 4. The His-tagged recombinant SHB-GAS-101-2 protein was purified from non- soluble fraction of IPTG-induced BL21 (DE3) /rpET19b (+) . Briefly, the pelleted cells obtained from a 400 mL culture induced with IPTG was resuspended in lysis buffer (20 mM Tris, 500 mM NaCl, 10 mM imidazole, pH 7.9) containing 8M urea, sonicated and centrifuged at 12,000 X g for 20 min to remove debris. The supernatant was incubated with Ni- NTA agarose resin (Qiagen) for 45 min at 4°C. The His-tagged recombinant SHB-GAS-101-2 protein was eluted from the resin with a solution containing 8M urea and 250 mM imidazole-500mM NaCl-20 mM Tris, pH 7.9. The removal of the salt and imidazole from the samples was done by dialysis against lOmM Tris, 0.9% NaCl and 2 M urea, pH 7.9 overnight at 4°C. The amount of recombinant protein was estimated by MicroBCA (Pierce, Rockford, Illinois) . The gene products generated are listed in the Table 3.

Table 3. List of truncated SHB-GAS-101 gene products generated from serotype Ml S . pyogenes strain ATCC700294.

EXAMPLE 6

This example illustrates the reactivity of the recombinant SHB-GAS-101 and truncated proteins with human sera.

As shown in Table 4, purified recombinant SHB-GAS-101 and truncated proteins were recognized in immunoblots by the antibodies present in a normal human serum. This is an important result since it clearly indicates that human which are normally in contact with EL pyogenes do develop antibodies that are specific to those proteins . These particular human antibodies might be implicated in the protection against £L_ pyogenes infection.

Table 4. Reactivity in immunoblots of antibodies present in human serum with SHB-GAS-101 and truncated proteins.

Recombinant SHB-GAS-101 and truncated proteins produced and purified as described in Examples 4 and 5 were used to perform the immunoblots , ²Molecular weight of the recombinant proteins was estimated after SDS- PAGE.

³Serum collected from healthy human volunteer was diluted 1/500 to perform the immunoblots.

EXAMPLE 7

This example illustrates the accessibility to antibodies of the S . pyogenes SHB-GAS-101 polypeptide at the surface of intact streptococcal cells.

Bacteria were grown in Todd Hewitt (TH) broth (Difco Laboratories, Detroit, MI) with 0.5% Yeast extract (Difco Laboratories) and 1% peptone extract (Merck, Darmstadt, Germany) at 37°C in a 8% C0₂ atmosphere to give an OD_60on of 0.600 (~10⁹ CFU/ml) . Dilutions of anti- SHB-GAS-101 or control sera were then added and allowed to bind to the cells, which were incubated for 2 h at 4°C. Samples were washed 2 times in blocking buffer [phosphate-buffered saline (PBS) containing 2% bovine serum albumin (BSA)], and then 0.5 ml of goat fluorescein (FITC) -conjugated anti-mouse IgG + IgM diluted in blocking buffer was added. After an additional incubation of 60 min at room temperature, samples were washed 2 times in blocking buffer and fixed with 0.25 % formaldehyde in PBS buffer for 18-24 h at 4°C. Cells were kept in the dark at 4°C until analyzed by flow cytometry (Epics^® XL; Beckman Coulter, Inc.) . Ten thousands intact £L_ pyogenes cells were analyzed per sample .

EXAMPLE 8

This example illustrates the protection against fatal SX_ pyogenes infection induced by passive immunization of mice with rabbit hyper- immune sera.

New Zealand rabbits (Charles River) were injected subcutaneously at multiple sites with 50 μg or 100 μg of the purified recombinant SHB- GAS-101 or truncated SHB-GAS-101 proteins that were produced and purified as described in Examples 4 and 5 and adsorbed to Alhydrogel adjuvant (Superfos Biosector a/s) . Rabbits were immunized three times at three-week intervals with the recombinant SHB-GAS-101 and truncated proteins . Blood samples were collected three weeks after the third injection. The antibodies present in the serum were purified by precipitation using 40% saturated ammonium sulfate. Groups of 10 female Balb/c mice (Charles River) were injected intravenously with 500 μl of purified serum collected from rabbits immunized with the recombinant SHB-GAS-101 and truncated proteins, or rabbits immunized with an unrelated control recombinant polypeptide. Eighteen hours later the mice were challenged with approximately 2x10^s CFU of the type 3 £L_ pyogenes strain ATCC12384. Samples of the £L_ pyogenes challenge inoculum were plated on blood agar plates to determine the CFU and to verify the challenge dose. Deaths were recorded for a period of 5 days .

EXAMPLE 9

This example illustrates the protection of mice against fatal S . pyogenes infection induced by immunization with purified recombinant SHB-GAS-101 (full-length), SHB-GAS-101-1 and SHB-GAS-101-3 proteins.

Groups of female Balb/c mice (Charles River) were immunized subcutaneously three times at two-week intervals with 20 μg of affinity purified recombinant SHB-GAS-101, SHB-GAS-101-1 or SHB-GAS- 101-3 proteins 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, 14 and 28 prior to each immunization and 14 days (day 42) following the third injection. One week later the mice were challenged with approximately 4x10^s CFU of the type 3 EL_ pyogenes strain ATCC12384. Samples of the EL pyogenes challenge inoculum were plated on blood agar plates to determine the CFU and to verify the challenge dose. Deaths were recorded for a period of 7 days.

As presented in Table 5, 58% of the mice immunized with recombinant SHB-GAS-101 protein were protected against a lethal challenge with heterologous GAS strain. Interestingly, mice immunized with recombinant SHB-GAS-101-1 protein, representing the N-terminal region, were also protected against a lethal challenge with heterologous GAS strain. On the contrary, immunization of mice with recombinant SHB- GAS-101-3 protein (aa 186 to 413) or with adjuvant only did not confer such protection (Table 5) .

Table 5. Evaluation of the protective potential of SHB-GAS-101 and truncated proteins .

^^■Number of survivors was evaluated for 7 days after challenge. The mice were immunized subcutaneously three times with 20 μg of purified recombinant proteins or adjuvant only. After immunization, the mice were challenged with a lethal dose of a GAS strain.

Claims

CLAIMS :

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 SEQ ID NO: 2, 4, 6, 8 or fragments or analogs thereof;

(b) a polynucleotide encoding a polypeptide having at least 80% identity to a second polypeptide comprising SEQ ID NO: 2, 4, 6, 8 or fragments or analogs thereof;

(c) a polynucleotide encoding a polypeptide having at least 95% identity to a second polypeptide comprising SEQ ID NO: 2, 4, 6, 8 or fragments or analogs thereof;

(d) a polynucleotide encoding a polypeptide comprising SEQ ID NO: 2, 4, 6, 8 or fragments or analogs thereof;

(e) a polynucleotide encoding a polypeptide capable of raising antibodies having binding specificity for a polypeptide comprising SEQ ID NO: 2, 4, 6, 8 or fragments or analogs thereof;

(f) a polynucleotide encoding an epitope bearing portion of a polypeptide comprising SEQ ID NO: 2, 4, 6, 8 or fragments or analogs thereof;

(g) a polynucleotide comprising SEQ ID NO: 1, 3, 5, 7 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 SEQ ID NO: 2, 4, 6, 8; (b) a polynucleotide encoding a polypeptide having at least 80% identity to a second polypeptide comprising SEQ ID NO: 2, 4, 6, 8;

(c) a polynucleotide encoding a polypeptide having at least 95% identity to a second polypeptide comprising SEQ ID NO: 2, 4, 6, 8;

(d) a polynucleotide encoding a polypeptide comprising SEQ ID NO: 2, 4, 6, 8;

(e) a polynucleotide encoding a polypeptide capable of raising antibodies having binding specificity for a polypeptide comprising SEQ ID NO: 2, 4, 6, 8;

(f) a polynucleotide encoding an epitope bearing portion of a polypeptide comprising SEQ ID NO: 2, 4, 6, 8;

(g) a polynucleotide comprising SEQ ID NO: 1, 3, 5, 7;

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. An isolated polynucleotide that hybridizes under stringent conditions to either

wherein said polypeptide comprises SEQ ID NO: 2, 4, 6, 8 or fragments or analogs thereof .

8. 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;

9. 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, 8.

10. 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, 8 or fragments or analogs thereof.

11. 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 5 amino acid residues from a polypeptide SEQ ID NO: 2, 4, 6, 8.

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

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

10 14. A host cell transfected with the vector of claim 12.

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

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

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

18. An isolated polypeptide comprising a polypeptide chosen from:

20 (a) a polypeptide having at least 70% identity to a second polypeptide having an amino acid sequence SEQ ID NO: 2, 4, 6, 8 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,

25 6, 8 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, 8 or fragments or analogs thereof; (d) a polypeptide comprising SEQ ID NO: 2, 4, 6, 8 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, 8 or fragments or analogs thereof;

(f) an epitope bearing portion of a polypeptide comprising SEQ ID NO: 2, 4, 6, 8 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) , or (f) wherein the secretory amino acid sequence is deleted.

19. 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, 8;

(b) a polypeptide having at least 80% identity to a second polypeptide having an amino acid sequence comprising SEQ ID NO: 2, 4, 6, 8;

(c) a polypeptide having at least 95% identity to a second polypeptide having an amino acid sequence comprising SEQ ID NO: 2, 4,. 6, 8;

(d) a polypeptide comprising SEQ ID NO: 2, 4, 6, 8;

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

8;

(f) an epitope bearing portion of a polypeptide comprising SEQ ID NO: 2, 4, 6, 8; (g) the polypeptide of (a) , (b) , (c) , (d) , (e) or (f) wherein the N-terminal Met residue is deleted;

520. A chimeric polypeptide comprising two or more polypeptides comprising SEQ ID NO: 2, 4, 6, 8 or fragments or analogs thereof; provided that the polypeptides are linked as to formed a chimeric polypeptide.

21. A chimeric polypeptide comprising two or more polypeptides 10 comprising SEQ ID NO: 2, 4, 6, 8 provided that the polypeptides are linked as to formed a chimeric polypeptide.

22. A pharmaceutical composition comprising a polypeptide according to any one of claims 18 to 21 and a pharmaceutically acceptable carrier, diluent or adjuvant.

1523. A method for prophylactic or therapeutic treatment of S . pyogenes infection in a host susceptible to S . pyogenes infection comprising administering to said host a prophylactic or therapeutic amount of a composition according to claim 22.

24. A method according to claim 23 wherein the host is a 20 neonate, an infant or a child.

25. A method according to claim 23 wherein the host is an immunocompromised host.

26. A method according to claim 23 wherein the host is an adult .

2527. A method for therapeutic or prophylactic treatment of infections, including pharyngitis, erysipelas, impetigo, scarlet fever, and invasive diseases such as bacteremia and necrotizing fasciitis comprising administering to said host a therapeutic or prophylactic amount of a composition according to claim 22.

28. A method for diagnostic of S . pyogenes infection in an host susceptible to S . pyogenes 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 18 to 21 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 . pyogenes .

29. A method for the detection of antibody specific to a S . pyogenes 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 18 to 21 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 . pyogenes .

30. Use of the pharmaceutical composition according to claim 22 in the manufacture of a medicament for the prophylactic or therapeutic treatment of S . pyogenes infection.

31. Kit comprising a polypeptide according to any one of claims 18 to 21 for detection or diagnosis of S . pyogenes infection.