HK1154514A - Compositions and methods of use of orf 554 from beta hemolytic streptococcal strains - Google Patents

Description

Compositions and methods of use of ORF554 from beta hemolytic streptococcus strains

Background

The present invention relates generally to the fields of bacteriology, infectious disease, and immunology. More particularly, the present invention relates to polynucleotides, polypeptides and immunogenic compositions comprising beta hemolytic streptococcus (heterologous streptococcus) polypeptides.

Beta Hemolytic Streptococcus (BHS) species are important pathogens responsible for a wide range of human diseases from superficial infections to more severe diseases. They include species from serology A, B, C and group G. Group A Streptococcus bacteria (GAS; Streptococcus pyogenes) can be responsible for most cases of disease and can lead to non-invasive diseases such as pharyngitis, scarlet fever, impetigo, cellulitis or erysipelas, but certain strains can lead to more severe invasive infections such as toxic shock syndrome, necrotizing fasciitis and septicaemia. In addition, complications of surface infection can lead to immune-mediated sequelae. Lancefield's group B streptococci (GBS; Streptococcus agalactiae) are the leading cause of neonatal sepsis in newborns and can cause pneumonia in elderly patients. Streptococci group C and G were initially recognized as animal pathogens, but have been shown to have strong potential for human disease in recent years. The disease presents itself generally similarly to group a streptococci, but has not been shown to cause immune-mediated sequelae. Group C and G streptococci are often present in patients with potential health problems, are of importance for elderly patients and are dispersed among several streptococcal species.

SUMMARY

In one aspect, the invention provides novel polypeptides encoded by group C or group G streptococcus open reading frame number 554(ORF 554). In one embodiment, the present invention provides a polypeptide comprising SEQ ID NO: 11, which is a consensus sequence derived from several novel different ORF554 sequences as follows: streptococcus dysgalactiae subsp. 2. SEQ ID NO: 8. SEQ ID NO: 10. SEQ ID NO: 26 and SEQ ID NO: 28; streptococci constellata subsp. 4 and SEQ ID NO: 32, a first step of removing the first layer; streptococcus pharyngis (Streptococcus anginosus), i.e., SEQ ID NO: 6 and SEQ ID NO: 30, of a nitrogen-containing gas; or a fragment thereof. In certain embodiments, the isolated polypeptide comprises or consists of an amino acid sequence that is set forth in SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. SEQ ID NO: 8. SEQ ID NO: 10. SEQ ID NO: 26. SEQ ID NO: 28. SEQ ID NO: 30 and/or SEQ ID NO: shown in 32. In certain embodiments, the isolated polypeptide has Peptidyl Prolyl Isomerase (PPI) activity.

In another aspect, the invention provides an isolated polypeptide comprising or consisting of an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. SEQ ID NO: 8. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 26. SEQ ID NO: 28. SEQ ID NO: 30 and/or seq id NO: 32 is at least 90%, 95% or 99% identical. In certain embodiments, the isolated polypeptide has peptidyl-prolyl isomerase activity.

In another aspect, the invention provides an isolated polynucleotide encoding a streptococcus ORF554 polypeptide or fragment thereof. In one embodiment, the present invention provides an isolated polynucleotide encoding a polypeptide comprising SEQ ID NO: 11 or a fragment thereof. In certain embodiments, the isolated polynucleotide encodes a polypeptide comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. SEQ ID NO: 8. SEQ ID NO: 10. SEQ ID NO: 26. SEQ ID NO: 28. SEQ ID NO: 30 and SEQ ID NO: 32, shown in any one or more of figures 32. In certain embodiments, the isolated polynucleotide comprises or consists of a nucleotide sequence that is set forth in SEQ ID NO: 1. SEQ ID NO: 3. SEQ ID NO: 5. SEQ ID NO: 7. SEQ ID NO: 9. SEQ ID NO: 25. SEQ ID NO: 27. SEQ ID NO: 29 and SEQ ID NO: 31, shown in any one or more of figures 31. In certain embodiments, the polynucleotide is operably linked to a regulatory element.

In another aspect, the present invention provides an isolated polynucleotide encoding a polypeptide (or fragment thereof) comprising or consisting of an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. SEQ ID NO: 8. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 26. SEQ ID NO: 28. SEQ ID NO: 30 and SEQ ID NO: 32 is at least 90%, 95%, or 99% identical. In certain embodiments, the isolated polypeptide has peptidyl-prolyl isomerase activity. In other embodiments, the isolated polynucleotide comprises or consists of a nucleotide sequence that is identical to or a fragment of SEQ ID NO: 1. SEQ ID NO: 3. SEQ ID NO: 5. SEQ ID NO: 7. SEQ ID NO: 9. SEQ ID NO: 25. SEQ ID NO: 27. SEQ ID NO: 29 and SEQ ID NO: 31 is at least 90%, 95%, or 99% identical. In certain embodiments, the polynucleotide is operably linked to a regulatory element. In certain embodiments, the regulatory element comprises an inducible promoter and/or a constitutive promoter.

In another aspect, the present invention provides a polynucleotide vector comprising a nucleotide sequence encoding a polypeptide comprising or consisting of an amino acid sequence that hybridizes with SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. SEQ ID NO: 8. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 26. SEQ ID NO: 28. SEQ ID NO: 30 and SEQ ID NO: 32 is at least 90%, 95%, or 99% identical, or 100% identical. In certain embodiments, the isolated polypeptide has peptidyl-prolyl isomerase activity. The vector may be, for example, a plasmid vector, a viral vector, or the like. In other embodiments, the polynucleotide vector comprises a nucleotide sequence (or fragment thereof) that hybridizes to SEQ ID NO: 1. SEQ ID NO: 3. SEQ ID NO: 5. SEQ ID NO: 7. SEQ ID NO: 9. SEQ ID NO: 25. SEQ ID NO: 27. SEQ ID NO: 29 and SEQ ID NO: 31 are at least 90%, 95%, or 99% identical, or 100% identical. In certain embodiments, the polynucleotide vector is an expression vector that enables the production of a recombinant streptococcal PPI protein.

In another aspect, the invention provides an ex vivo cell comprising or consisting of an isolated polynucleotide comprising or consisting of a nucleotide sequence encoding a group C or group G streptococcus PPI encoded by ORF554, or a fragment thereof. In certain embodiments, the group C or group G streptococcal PPI is encoded by ORF554 comprising or consisting of an amino acid sequence that is substantially identical to SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. SEQ ID NO: 8. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 26. SEQ ID NO: 28. SEQ ID NO: 30 and SEQ ID NO: 32 is at least 90%, 95%, or 99% identical, or 100% identical. In certain embodiments, the isolated polynucleotide comprises or consists of a nucleotide sequence that is identical to or a fragment of seq id NO: 1. SEQ ID NO: 3. SEQ ID NO: 5. SEQ ID NO: 7. SEQ ID NO: 9. SEQ ID NO: 25. SEQ ID NO: 27. SEQ ID NO: 29 and SEQ ID NO: 31 are at least 90%, 95%, or 99% identical, or 100% identical. In certain embodiments, the cell produces a group C or group G streptococcus PPI, or fragment thereof. In certain embodiments, the host cell comprises a polynucleotide vector comprising an isolated polynucleotide comprising a regulatory sequence operably linked to the isolated polynucleotide. In certain embodiments, the host cell comprises a polynucleotide vector comprising a regulatory element, which may be a constitutive or inducible promoter. In certain embodiments, the host cell comprises a polynucleotide vector that is a plasmid, a viral vector, or an expression vector. In certain embodiments, the host cell is selected from a bacterium, a mammalian cell, an insect cell, or a yeast cell.

In another aspect, the present invention provides immunogenic compositions useful in eliciting an immune response against a group C or group G streptococcus PPI polypeptide in a subject. In certain embodiments, the immunogenic composition comprises or consists of a polypeptide or fragment thereof comprising or consisting of an amino acid sequence that is identical to the amino acid sequence of SEQ id no: 2. SEQ ID NO: 4. SEQ ID NO: 6. SEQ ID NO: 8. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 26. SEQ ID NO: 28. SEQ ID NO: 30 and SEQ ID NO: 32 is at least 90%, 95%, or 99% identical, or 100% identical. In other embodiments, the immunogenic composition comprises or consists of an isolated polynucleotide or fragment thereof comprising or consisting of a nucleotide sequence that is identical to the nucleotide sequence set forth in SEQ ID NO: 1. SEQ ID NO: 3. SEQ ID NO: 5. SEQ ID NO: 7. SEQ ID NO: 9. SEQ ID NO: 25. SEQ ID NO: 27. SEQ ID NO: 29 and SEQ ID NO: 31 are at least 90%, 95%, or 99% identical, or 100% identical. In certain embodiments, the polynucleotide further comprises a regulatory element, such as an inducible or constitutive promoter that regulates expression of the isolated polynucleotide in the subject.

In another aspect, the invention provides immunological reagents comprising or consisting of an epitope binding region that binds to a streptococcal PPI encoded by group C or group G streptococcal ORF 554. In certain embodiments, the immunological reagent comprises or consists of an antibody that specifically binds to at least one streptococcal PPI polypeptide encoded by ORF 554. In certain embodiments, the antibody binds to a polypeptide comprising or consisting of an amino acid sequence that hybridizes to SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. SEQ ID NO: 8. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 26. SEQ ID NO: 28. SEQ ID NO: 30 and SEQ ID NO: 32 is at least 90%, 95%, or 99% identical, or 100% identical. The antibody may be a monoclonal antibody or a polyclonal antibody. In certain embodiments, the antibody is a chimeric antibody, including, for example, a humanized antibody.

In another aspect, the present invention provides a method for inducing an immune response against beta hemolytic streptococcus or beta hemolytic streptococcus infection in a subject comprising administering to the subject an immunogenic composition comprising a group C or group G streptococcus PPI protein or fragment thereof. In certain embodiments, the PPI protein comprises or consists of an amino acid sequence that is identical to SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. SEQ ID NO: 8. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 26. SEQ ID NO: 28. SEQ ID NO: 30 and SEQ ID NO: 32 is at least 90%, 95%, or 99% identical, or 100% identical.

In another aspect, the invention provides a method for inducing an immune response against beta hemolytic streptococcus or beta hemolytic streptococcus infection in a subject comprising administering to the subject an immunogenic composition comprising a polynucleotide encoding a group C or group G streptococcus PPI protein or fragment thereof. In certain embodiments, the PPI protein comprises or consists of an amino acid sequence that is identical to SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. SEQ ID NO: 8. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 26. SEQ ID NO: 28. SEQ ID NO: 30 and SEQ ID NO: 32 is at least 90%, 95%, or 99% identical, or 100% identical. In other embodiments, the polynucleotide comprises or consists of a nucleotide sequence that is identical to the nucleotide sequence set forth in SEQ ID NO: 1. SEQ ID NO: 3. SEQ ID NO: 5. SEQ ID NO: 7. SEQ ID NO: 9. SEQ ID NO: 25. SEQ ID NO: 27. SEQ ID NO: 29 and SEQ ID NO: 31 are at least 90%, 95%, or 99% identical, or 100% identical. In certain embodiments, the polynucleotide further comprises a regulatory element, such as an inducible or constitutive promoter that regulates expression of the isolated polynucleotide in the subject.

In another aspect, the present invention provides a use of an isolated group C or group G streptococcus PPI polypeptide, or fragment thereof, in the manufacture of a medicament for prophylactically treating a beta hemolytic streptococcus infection in a subject. In certain embodiments, the subject is a human. In certain embodiments, the PPI polypeptide comprises or consists of an amino acid sequence that differs from the amino acid sequence of seq id NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. SEQ ID NO: 8. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 26. SEQ ID NO: 28. SEQ ID NO: 30 and SEQ ID NO: 32 is at least 90%, 95%, or 99% identical, or 100% identical.

In another aspect, the present invention provides the use of an isolated polynucleotide encoding an isolated group C or group G streptococcus PPI polypeptide, or a fragment thereof, in the manufacture of a medicament for the prophylactic treatment of a beta hemolytic streptococcal infection in a subject. In certain embodiments, the subject is a human patient. In certain embodiments, the PPI polypeptide comprises or consists of an amino acid sequence that is identical to SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. SEQ ID NO: 8. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 26. SEQ ID NO: 28. SEQ ID NO: 30 and SEQ ID NO: 32 is at least 90%, 95%, or 99% identical, or 100% identical. In other embodiments, the polynucleotide comprises or consists of a nucleotide sequence that is identical to the nucleotide sequence set forth in SEQ id no: 1. SEQ ID NO: 3. SEQ ID NO: 5. SEQ ID NO: 7. SEQ ID NO: 9. SEQ ID NO: 25. SEQ ID NO: 27. SEQ ID NO: 29 and SEQ ID NO: 31 are at least 90%, 95%, or 99% identical, or 100% identical. In certain embodiments, the polynucleotide further comprises a regulatory element, such as an inducible or constitutive promoter that enables expression of the isolated polynucleotide in the subject.

In another aspect, the invention provides the use of an immunological reagent comprising a region that specifically binds to a group C or group G streptococcus PPI polypeptide in the manufacture of a medicament for the prophylactic treatment of a beta hemolytic streptococcus infection in a subject. In certain embodiments, the immunological agent comprises or consists of an antibody. In certain embodiments, the subject is a human patient. In certain embodiments, the PPI polypeptide comprises or consists of an amino acid sequence that is identical to SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. SEQ ID NO: 8. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 26. SEQ ID NO: 28. SEQ ID NO: 30 and SEQ ID NO: 32 is at least 90%, 95%, or 99% identical, or 100% identical. In certain embodiments, the antibody may be a monoclonal antibody or a polyclonal antibody. In certain embodiments, the antibody may be a chimeric antibody, such as a humanized antibody or a polyclonal antibody.

In another aspect, the present invention provides a method for treating beta hemolytic streptococcus in a subject comprising administering a therapeutically effective amount of an antibody or fragment thereof that specifically binds to an epitope of a group C or group G streptococcus PPI polypeptide in a pharmaceutically effective excipient. In certain embodiments, the subject is a human. In certain embodiments, the PPI polypeptide comprises or consists of an amino acid sequence that is identical to SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. SEQ ID NO: 8. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 26. SEQ ID NO: 28. SEQ ID NO: 30 and SEQ ID NO: 32 is at least 90%, 95%, or 99% identical, or 100% identical. In certain embodiments, the antibody may be a monoclonal antibody or a polyclonal antibody. In certain embodiments, the antibody may be a chimeric antibody, such as a humanized antibody or a polyclonal antibody.

In still other aspects, the invention provides kits comprising: (a) an isolated group C or group G Streptococcus PPI polypeptide having an amino acid sequence identical to SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. SEQ ID NO: 8. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 26. SEQ ID NO: 28. SEQ ID NO: 30 and SEQ ID NO: 32, or at least 90%, 95%, or 99%, or 100% equivalent amino acid sequence; (b) an isolated polynucleotide encoding a group C or group G streptococcus PPI polypeptide (supra); and/or (C) an antibody that specifically binds to an epitope of a group C or group G streptococcus PPI polypeptide (supra).

Brief description of the sequence listing

SEQ ID NO: 1 is the nucleotide sequence of ORF554 in Streptococcus dysgalactiae subspecies equisimilis.

SEQ ID NO: 2 is the amino acid sequence encoded by ORF554 in streptococcus dysgalactiae subsp.

SEQ ID NO: 3 is the nucleotide sequence of ORF554 in constellated streptococcus constellations.

SEQ ID NO: 4 is an amino acid sequence encoded by ORF554 in constellated streptococcus constellations.

SEQ ID NO: 5 is the nucleotide sequence of ORF554 in Streptococcus aryngitis.

SEQ ID NO: 6 is the amino acid sequence encoded by ORF554 in Streptococcus aryngitis.

SEQ ID NO: 7 is the nucleotide sequence of ORF554 in Streptococcus sp (Streptococcus sp.) strain N04A 27.

SEQ ID NO: 8 is an amino acid sequence encoded by ORF554 in Streptococcus sp strain N04A 27.

SEQ ID NO: 9 is the nucleotide sequence of ORF554 in Streptococcus spp strain N04 AFT.

SEQ ID NO: 10 is an amino acid sequence encoded by ORF554 in streptococcus sp strain N04 AFT.

SEQ ID NO: 11 is SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. SEQ ID NO: 8 and SEQ ID NO: 10.

SEQ ID NO: 12 is the nucleotide sequence of ORF554 of streptococcus agalactiae GBS 0827.

SEQ ID NO: 13 is the amino acid sequence encoded by ORF554 of streptococcus agalactiae GBS 0827.

SEQ ID NO: 14 is the nucleotide sequence of ORF554 of streptococcus pyogenes SPY _ 1390.

SEQ ID NO: 15 is the amino acid sequence encoded by ORF554 of streptococcus pyogenes SPY _ 1390.

SEQ ID NO: 16 is the nucleotide sequence of ORF554 of streptococcus zooepidemicus (s.zopedimicus).

SEQ ID NO: 17 is an amino acid sequence encoded by ORF554 of streptococcus zooepidemicus.

SEQ ID NO: 18 is the nucleotide sequence of primer D554F 1.

SEQ ID NO: 19 is the amino acid sequence of primer D554F 2.

SEQ ID NO: 20 is the nucleotide sequence of primer D554F 5.

SEQ ID NO: 21 is the amino acid sequence of primer D554R 1.

SEQ ID NO: 22 is the nucleotide sequence of primer D554R 4.

SEQ ID NO: 23 is the nucleotide sequence of primer ATCC 12394554F.

SEQ ID NO: 24 is the amino acid sequence of primer ATCC 12394554R.

SEQ ID NO: 25 is the nucleotide sequence of ORF554 in Streptococcus dysgalactiae subspecies equisimilis.

SEQ ID NO: 26 is the amino acid sequence encoded by ORF554 in Streptococcus dysgalactiae subspecies equisimilis.

SEQ ID NO: 27 is the nucleotide sequence of ORF554 in Streptococcus dysgalactiae subspecies equisimilis.

SEQ ID NO: 28 is the amino acid sequence encoded by ORF554 in streptococcus dysgalactiae subsp.

SEQ ID NO: 29 is the nucleotide sequence of ORF554 in Streptococcus aryngitis.

SEQ ID NO: 30 is the amino acid sequence encoded by ORF554 in streptococcus valoris.

SEQ ID NO: 31 is the nucleotide sequence of ORF554 in constellated streptococcus constellations.

SEQ ID NO: 32 is an amino acid sequence encoded by ORF554 in constellated streptococcus constellations.

Detailed description of the invention

The present invention describes polypeptides and polynucleotides from group C or group G Streptococcus species that correspond to Streptococcus pyogenes open reading frame 554(ORF 554). The DNA and amino acid sequence for ORF554 is provided in published International patent application No. WO 02/083859. These ORF554 polypeptides are similar to the streptococcus pyogenes peptidyl-prolyl isomerase (PPI) protein. These polynucleotides and polypeptides may be used in immunogenic compositions to induce an immune response against beta hemolytic streptococcus or beta hemolytic streptococcus infection in a subject.

The terms "polynucleotide", "nucleic acid" and "nucleic acid fragment" are used interchangeably herein. These terms encompass nucleotides linked by phosphodiester linkages. A "polynucleotide" may be a polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), which is single-or double-stranded, optionally containing synthetic, non-natural, or altered nucleotide bases. Polynucleotides in the form of a polymer of DNA may comprise one or more segments of cDNA, genomic DNA, synthetic DNA, or mixtures thereof. Nucleotide bases are indicated herein by single letter code: adenine (A), guanine (G), thymine (T), cytosine (C), inosine (I), and uracil (U).

A "protein" or "polypeptide" is a chain of amino acids arranged in a polynucleotide that encodes a polypeptide in a specific order determined by the coding sequence.

The term "isolated" means altered from a natural state by "artificial". If a composition or substance exists in nature, it must have been altered or removed from its original environment, or both, in order for it to be considered "isolated". For example, a polynucleotide or polypeptide naturally occurring in a living animal is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated," as the term is employed herein. An isolated polynucleotide or an isolated polypeptide may be purified from a host cell in which they naturally occur. Conventional nucleic acid purification methods known to those skilled in the art to which the present invention relates can be used to obtain isolated polynucleotides.

Group C or group G streptococcal ORF554 polynucleotides

Group C or group G streptococcus ORF554 polynucleotides described herein can be obtained using standard cloning and screening techniques. These polynucleotides may be obtained, for example, from genomic DNA, cDNA libraries derived from mRNA, genomic DNA libraries, or may be synthesized using well-known and commercially available techniques, for example, from cDNA libraries by PCR or via RT-PCR (reverse transcription polymerase chain reaction).

The term "recombinant" means a polynucleotide prepared, for example, by the artificial combination of 2 or more other separate polynucleotide segments, e.g., by chemical synthesis or by processing the isolated polynucleotide using genetic engineering techniques. "recombinant DNA construct" includes any of the isolated polynucleotides of the present invention operably linked to at least one regulatory element.

In one aspect, the invention provides an isolated polynucleotide encoding a group C or group G streptococcal Prolyl Peptidyl Isomerase (PPI) protein, or a fragment thereof.

In one embodiment, the isolated polynucleotide encodes a polypeptide comprising SEQ ID NO: 11. SEQ ID NO: 11 is a consensus sequence obtained after alignment of a conceptually translated polypeptide sequence derived from group C or group G species streptococcus dysgalactiae subsp. ORF554 consisting of SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. SEQ ID NO: 8 and SEQ ID NO: and 10, respectively.

In one embodiment, the present invention provides an isolated polynucleotide encoding a polypeptide comprising seq id NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. SEQ ID NO: 8. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 26. SEQ ID NO: 28. SEQ ID NO: 30 or SEQ ID NO: 32, or a fragment thereof. Exemplary nucleotide sequences are set forth in SEQ ID NO: 1. SEQ ID NO: 3. SEQ ID NO: 5. SEQ ID NO: 7. SEQ ID NO: 9. SEQ ID NO: 25. SEQ ID NO: 27. SEQ ID NO: 29 and SEQ ID NO: shown in fig. 31. In another embodiment, the present invention provides a method of producing a polypeptide of SEQ ID NO: 1. SEQ ID NO: 3. SEQ ID NO: 5. SEQ ID NO: 7. SEQ ID NO: 9. SEQ ID NO: 25. SEQ ID NO: 27. SEQ ID NO: 29 and SEQ ID NO: 31, or a variant thereof. These polynucleotides encode the same ORF554 polypeptide.

Orthologs and allelic variants of group C or group G Streptococcus ORF554 polynucleotides can be readily identified using methods well known in the art. Allelic variants and orthologs of the ORF554 polynucleotide may include nucleotide sequences that are generally identical to SEQ id no: 1. SEQ ID NO: 3. SEQ ID NO: 5. SEQ ID NO: 7. SEQ ID NO: 9. SEQ ID NO: 25. SEQ ID NO: 27. SEQ ID NO: 29 and SEQ ID NO: 31 or a fragment thereof is at least about 90-95% or more identical. Allelic variants and orthologs of the ORF554 polynucleotide may encode a polypeptide comprising an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. SEQ ID NO: 8. SEQ ID NO: 10. SEQ ID NO: 26. SEQ ID NO: 28. SEQ ID NO: 30 and SEQ ID NO: 32 is at least 90% identical to the amino acid sequence set forth in any one or more of claims 32. Such polynucleotides can be readily identified as capable of hybridizing under stringent conditions to any one or more of the following polynucleotides, or fragments thereof, having the sequence set forth in SEQ ID NO: 1. SEQ ID NO: 3. SEQ ID NO: 5. SEQ ID NO: 7. SEQ ID NO: 9. SEQ ID NO: 25. SEQ ID NO: 27. SEQ ID NO: 29 and/or SEQ id no: 31, or a nucleotide sequence set forth in seq id no.

Furthermore, allelic variants and orthologs of the ORF554 polynucleotide may include only fragments of the coding region of group C or group G streptococcal ORF554 polynucleotides, such as SEQ id no: 1. SEQ ID NO: 3. SEQ ID NO: 5. SEQ ID NO: 7. SEQ ID NO: 9. SEQ ID NO: 25. SEQ ID NO: 27. SEQ ID NO: 29 and/or SEQ ID NO: 31, or a fragment of these nucleotide sequences. In particular embodiments, such fragments encode immunogenic fragments.

Those skilled in the art are well aware of the many levels of sequence identity that are useful in identifying related polynucleotide and polypeptide sequences. Sequence alignment and identityPercent sexual calculation may use LASERGENE^TMMEGALIGN of bioinformatics computing suite (DNASTAR Inc., Madison, Wis.)^TMAnd (6) executing the program. Multiple alignments of sequences can be performed using the Clustal alignment method (Higgins and Sharp, Gene, 73 (1): 237-44, 1988) with default parameters such as GAP PENALTY ═ 10 and GAP LENGTH PENALTY ═ 10. Default parameters for pairwise alignments using the Clustal method may be, for example, KTUPLE 1, GAPPENALTY ═ 3, WINDOW ═ 5, and DIAGONALS SAVED ═ 5.

The ORF554 polynucleotides of the invention can be used to produce recombinant polypeptides for inclusion in immunogenic compositions and other uses. For the production of recombinant polypeptides, the polynucleotide may include a coding sequence for the mature polypeptide, itself, or operably linked to other coding sequences for the mature polypeptide, such as those encoding leader or secretory sequences, protein pre-, pro-, or prepro-sequences, or other fusion peptide portions. For example, a marker sequence that facilitates purification of the fusion polypeptide can be linked to the coding sequence (see Gentz et al, Proc. Natl. Acad. Sci. USA, 86: 821-824, 1989). The polynucleotide may also comprise non-coding 5 'and 3' sequences, such as transcribed, untranslated sequences, splicing, and polyadenylation signals.

In particular embodiments, the polynucleotide sequence information provided herein allows for the preparation of relatively short DNA (or RNA) oligonucleotide sequences that have the ability to specifically hybridize to the nucleotide sequence of a selected polynucleotide disclosed herein. The term "oligonucleotide" as used herein is defined as a molecule comprising 2 or more deoxyribonucleotides or ribonucleotides, typically more than three (3), and generally more than ten (10) and up to one hundred (100) or more (although preferably 20-30). The exact size will depend on many factors, which in turn depend on the ultimate function or use of the oligonucleotide. Thus, in certain embodiments, nucleic acid probes of suitable length are prepared based on a selected nucleotide sequence, such as SEQ ID NO: 1. SEQ ID NO: 3. SEQ ID NO: 5. SEQ ID NO: 7. SEQ ID NO: 9. SEQ ID NO: 25. SEQ ID NO: 27. SEQ ID NO: 29 and SEQ ID NO: 31, or a sequence shown in seq id no. The ability of such nucleic acid probes to specifically hybridize to polynucleotides encoding group C or group G streptococcal PPI polypeptides renders them particularly useful in various embodiments. In certain embodiments, the probes may be used in various assays for detecting the presence of complementary sequences in a given sample.

In certain embodiments, oligonucleotides may be used as primers. These primers may be generated in any manner, including chemical synthesis, DNA replication, reverse transcription, or a combination thereof. The sequences of such primers are designed using the polynucleotides described herein for detecting, amplifying, or mutating a defined segment of the ORF554 polynucleotide encoding a group C or group G streptococcal PPI polypeptide using Polymerase Chain Reaction (PCR) techniques.

In particular embodiments, it is advantageous to employ a polynucleotide described herein in combination with a suitable label for detecting hybrid formation. A wide variety of suitable labels are known in the art, including radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of giving a detectable signal.

And SEQ ID NO: 1. SEQ ID NO: 3. SEQ ID NO: 5. SEQ ID NO: 7. SEQ ID NO: 9. SEQ ID NO: 12. SEQ ID NO: 14. SEQ ID NO: 16 and SEQ ID NO: 18 up to SEQ ID NO: 24. SEQ ID NO: 25. SEQ ID NO: 27. SEQ ID NO: 29 and SEQ ID NO: 31 or a fragment thereof may be used as hybridization probes for cDNA and genomic DNA, or as primers for nucleic acid amplification (PCR) reactions, to isolate full-length cDNA and genomic clones encoding the polypeptides described herein, and to isolate cDNA and genomic clones of other genes, including genes encoding homologs and orthologs from species other than streptococcus dysgalactiae, that are identical or substantially identical to the nucleotide sequences contained in one of the cDNA and genomic DNA fragments of SEQ ID NO: 1. SEQ ID NO: 3. SEQ ID NO: 5. SEQ ID NO: 7. SEQ ID NO: 9. SEQ ID NO: 25. SEQ ID NO: 27. SEQ ID NO: 29 and SEQ ID NO: 31 or a fragment thereof has high sequence similarity. Typically, these polynucleotide sequences are at least about 90% identical to that of the reference polynucleotide sequence to at least about 99% identical. The probe or primer will typically comprise at least 15 nucleotides, at least 30 nucleotides or at least 50 nucleotides.

There are several methods available and well known to those skilled in the art to obtain full-length cDNA or extended short cDNA, such as those based on the Rapid Amplification of CDNA Ends (RACE) method. See Frohman et al, Proc. Natl. Acad. Sci. USA 85, 8998-. For example by MARATHON^TMRecent modifications of this technology exemplified by the technology (Clontech Laboratories Inc.) have significantly simplified the study on longer cdnas. In MARATHON^TMIn the art, cDNA has been prepared from mRNA extracted from selected tissues and "adaptor" sequences ligated on each end. Nucleic acid amplification (PCR) is then performed using a combination of gene-specific and adaptor-specific oligonucleotide primers to amplify the "missing" 5' end of the cDNA. The PCR reaction is then repeated using "nested" primers, i.e., primers designed to anneal within the amplification product (typically adaptor-specific primers that anneal 3 'farther in the adaptor sequence, and gene-specific primers that anneal 5' farther in the known gene sequence). The products of this reaction can then be analyzed by DNA sequencing and a full-length cDNA constructed either by directly ligating the products to the existing cDNA to obtain the full sequence, or by performing a separate full-length PCR using the new sequence information for designing the 5' primers.

Group C or group G streptococcal PPI polypeptides

In one aspect, the invention provides isolated group C or group G streptococcal Peptidyl Prolyl Isomerase (PPI) polypeptides (encoded by ORF 554) that are particularly useful as immunogens and in immunogenic compositions. The PPI polypeptide may be a recombinant polypeptide.

In one embodiment, the present invention provides a polypeptide comprising SEQ ID NO: 11. SEQ ID NO: 11 is a consensus sequence obtained after aligning the polynucleotide sequence obtained for streptococcus dysgalactiae subspecies mariae ORF554 and the amino acid sequence shown in SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. SEQ ID NO: 8 and SEQ ID NO: shown in fig. 10.

In one embodiment, the polypeptide comprises a sequence identical to SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. SEQ ID NO: 8. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 26. SEQ ID NO: 28. SEQ ID NO: 30 and SEQ ID NO: 32, or at least 90%, 95%, or 99%, or 100% equivalent amino acid sequence; functional and non-functional naturally occurring variants or biological equivalents of said polypeptides; a recombinantly produced variant or biological equivalent of said polypeptide; orthologues and/or allelic variants of the polypeptides; and fragments of said polypeptides.

Biological equivalents or variants of group C or group G streptococcus PPI polypeptides include functional and non-functional polypeptides. Functional biological equivalents or variants include naturally occurring amino acid sequence variants of a polypeptide that maintain the ability to elicit an immunological or antigenic response in a subject. The functional variant typically comprises SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. SEQ ID NO: 8. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 26. SEQ ID NO: 28. SEQ ID NO: 30 and SEQ ID NO: 32, conservative substitutions of one or more amino acids of any one or more of; or a substitution, deletion or insertion of a non-critical residue in a non-critical region of the polypeptide.

In certain embodiments, modifications and alterations in the structure of the PPI polypeptide may be made and still obtain a PPI polypeptide having the same antigenicity as an unaltered group C or group G streptococcus PPI polypeptide. For example, a particular amino acid can be substituted for other amino acids in the sequence without a measurable loss of antigenicity. Because this is the interactive capacity and nature of the polypeptide that defines the biological functional activity of the polypeptide, specific amino acid sequence substitutions can be made in the polypeptide sequence and still obtain a polypeptide with similar properties.

In making such modifications and alterations, the hydropathic index of amino acids may be considered. The importance of the hydrophilic amino acid index in conferring interactive biological functions on a polypeptide is generally understood in the art (Kyte and Doolittle, J Mol Biol 157: page 105-132, 1982). It is known in the art that certain amino acids may be substituted for other amino acids having similar hydropathic indices or scores and still result in polypeptides having similar biological activities. Each amino acid has been assigned a hydropathic index based on its hydrophobicity and charge characteristics. These indices are: isoleucine (+ 4.5); valine (+ 4.2); leucine (+ 3.8); phenylalanine (+ 2.8); cysteine/cystine (+ 2.5); methionine (+ 1.9); alanine (+ 1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

It is generally recognized in the art that the relative hydropathic character of amino acid residues determines the secondary and tertiary structure of the resulting polypeptide, which in turn defines the interaction of the polypeptide with other molecules, e.g., enzymes, substrates, receptors, antibodies, antigens, and the like. It is known in the art that an amino acid may be substituted by another amino acid having a similar hydropathic index and still obtain a functionally equivalent polypeptide. In certain embodiments, the modified or altered PPI polypeptide comprises one or more substituted amino acids having a hydropathic index within +/-2 of each original amino acid. In other embodiments, the hydropathic index of each substituted amino acid is within +/-1 of its original amino acid. In still other embodiments, the hydropathic index of each substituted amino acid is within +/-0.5 of its original amino acid.

Substitutions of like amino acids can also be made on the basis of hydrophilicity, particularly when the biologically functionally equivalent polypeptide or peptide so prepared is intended for use in immunological embodiments. U.S. Pat. No. 4,554,101 teaches that the greatest local average hydrophilicity of a polypeptide as controlled by the hydrophilicity of its adjacent amino acids correlates with its immunogenicity and antigenicity.

As the term is used herein, a "variant" is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, respectively, but retains some essential properties. A general variant of a polynucleotide differs from a reference polynucleotide in nucleotide sequence. Changes in the nucleotide sequence of a variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence as described below. A general variant of a polypeptide differs in amino acid sequence from a reference polypeptide. In general, the differences are limited such that the sequences of the reference and variant polypeptides are generally similar and, in many regions, identical. The variant polypeptide and its reference polypeptide may differ in amino acid sequence by one or more substitutions, additions and deletions in any combination. The substituted or inserted amino acid residue may or may not be that encoded by the genetic code. Variants of a polynucleotide or polypeptide may be naturally occurring, e.g., allelic variants, or may be variants that are not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be prepared by mutagenesis techniques or by direct synthesis.

Recombination system

For recombinant production of polypeptides, host cells can be genetically engineered to incorporate expression systems comprising the ORF554 polynucleotides of the invention. Polynucleotides can be introduced into host cells, for example, by methods described IN many standard laboratory manuals, e.g., Davis et al, BASICMOLECULAR BIOLOGY (1986) and Sambrook et al, MOLECULAR CLONING: a LABORATORY MANUAL, 2 nd Ed., Cold Spring Harbor LABORATORY Press, Cold Spring Harbor, N.Y. (1989.) these methods include, for example, calcium phosphate transfection, DEAE-dextran mediated transfection, transduction (transfection), microinjection, ultrasound, cationic lipid mediated transfection, electroporation, transduction, scrape loading, ballistic introduction (ballistic introduction) and infection.

Representative examples of suitable host cells include bacterial cells (e.g., streptococcus, staphylococcus, escherichia, streptomyces, and bacillus subtilis cells), yeast cells (e.g., Pichia and Saccharomyces), mammalian cells (e.g., CHO cells), and insect cells (e.g., Sf9 and Sf 21).

Recombinantly produced polypeptides can be recovered and purified from recombinant cell cultures by well-known methods including high performance liquid chromatography, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography.

Any one or more of a number of vector systems may be used to express and produce group C or group G streptococcus PPI polypeptides in heterologous cell systems. Such vector systems include, inter alia, chromosomal, episomal and virus-derived systems, such as vectors derived from: bacterial plasmids, attenuated bacteria such as Salmonella (Salmonella) (U.S. patent No. 4,837,151), bacteriophages, transposons, yeast episomes, insertion elements, yeast chromosomal elements, viruses such as vaccinia and other poxviruses, sindbis, adenovirus, baculovirus, papova, such as SV40, fowlpox virus, pseudorabies virus and retrovirus, alphaviruses such as venezuelan equine encephalitis virus (U.S. patent No. 5,643,576), non-segmented negative-strand RNA viruses such as vesicular stomatitis virus (U.S. patent No. 6,168,943), and vectors derived from combinations thereof, such as vectors derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. The expression system should include control regions that regulate as well as cause expression, such as promoters and other regulatory elements (e.g., polyadenylation signals). In general, any system or vector suitable for maintaining, propagating or expressing a polynucleotide to produce a polypeptide in a host may be used. Suitable nucleotide sequences can be inserted into the expression system by any of the well known and conventional techniques, such as those set forth in Sambrook et al, Molecularclong, A Lamatory Manual (supra).

In one embodiment, the invention provides an expression vector comprising a group C or group G streptococcus ORF554 polynucleotide encoding a group C or group G streptococcus PPI polypeptide. The expression vector comprises an ORF554 polynucleotide encoding a polypeptide comprising or consisting of an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. SEQ ID NO: 8. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 26. SEQ ID NO: 28. SEQ ID NO: 30 and SEQ ID NO: 32 is at least 90%, 95%, or 99% identical, or 100% identical. Alternatively, the expression vector comprises or consists of a polynucleotide comprising or consisting of a nucleotide sequence that is identical to the nucleotide sequence set forth in SEQ ID NO: 1. SEQ ID NO: 3. SEQ ID NO: 5. SEQ ID NO: 7. SEQ ID NO: 9. SEQ ID NO: 25. SEQ ID NO: 27. SEQ ID NO: 29 and SEQ ID NO: 31 are at least 90%, 95%, or 99% identical, or 100% identical. In other embodiments, the expression vectors of the invention comprise a polynucleotide operably linked to an enhancer-promoter. In yet other embodiments, the expression vector comprises a polynucleotide operably linked to a prokaryotic promoter. Alternatively, the expression vector comprises a polynucleotide operably linked to an enhancer-promoter, which is a eukaryotic promoter. The expression vector further includes a polyadenylation signal located 3' to the carboxy-terminal amino acid and within the transcription unit encoding the polypeptide.

As used herein, the term "regulatory element" refers to a genetic element that controls some aspect of the expression of a nucleic acid sequence. For example, a promoter is a regulatory element that facilitates the initiation of transcription of an operably linked coding region. Other regulatory elements are enhancers, silencers, splicing signals, polyadenylation signals, termination signals, RNA export elements, internal ribosome entry sites, etc.

As used herein, a promoter is a region of a DNA molecule that is generally about 100 nucleotide pairs before (upstream of) the point at which transcription begins (i.e., the transcription start site). That region typically contains several types of DNA sequence elements, which are located in similar relative positions in different genes. As used herein, the term "promoter" includes that which is known in the art as an upstream promoter region, a promoter region or a promoter that ubiquitously emits eukaryotic RNA polymerase II transcription units.

Another type of discontinuous transcriptional regulatory sequence element is an enhancer. Enhancers provide specificity with respect to the time, location, and expression level of a particular coding region (e.g., gene). The primary function of an enhancer is to increase the level of transcription of a coding sequence in a cell that contains one or more transcription factors that bind to that enhancer. Unlike promoters, enhancers can function at variable distances from the transcription start site, provided that a promoter is present.

As used herein, the phrase "enhancer-promoter" means a combined unit comprising enhancer and promoter elements. Enhancer-promoters are operably linked to a coding sequence that encodes at least one gene product. As used herein, the phrase "operably linked" means that an enhancer-promoter is linked to a coding sequence in such a way that transcription of that coding sequence is controlled and regulated by that enhancer-promoter. Methods for operably linking an enhancer-promoter to a coding sequence are well known in the art. As is also well known in the art, the precise orientation and location of a coding sequence with respect to which transcription is controlled depends, inter alia, on the specific nature of the enhancer-promoter. Thus, a TATA box minimal promoter is typically positioned about 25 to about 30 base pairs upstream of the transcription start site, and an upstream promoter element is typically positioned about 100 to about 200 base pairs upstream of the transcription start site. In contrast, an enhancer may be located downstream of the initiation site and may be at a substantial distance from that site.

The enhancer-promoter used in the vector constructs described herein may be any enhancer-promoter that drives expression in the cell to be transfected. By using enhancer-promoters with well-known properties, the level and pattern of gene product expression can be optimized. For example, commonly used promoters may be derived from polyoma, adenovirus 2, cytomegalovirus and simian virus 40. For other suitable expression systems for prokaryotic and eukaryotic cells, see Sambrook et al, "Molecular Cloning: chapters 16 and 17 of A Laboratory Manual "2 nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

In another embodiment, the expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements for expressing the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al, Genes Dev, 1: pp.268-277, 1987), lymphoid-specific promoters (Calame and Eaton, Adv Immunol, 43: pp.235-275, 1988), promoters of T-Cell receptors (Winto and Baltimore, EMBO J, 8: pp.729-733, 1989) and promoters of immunoglobulins (Banerji et al, Cell, 33: pp.729-740, 1983), (Queen and Baltimore, Cell, 33: pp.741-748, 1983), neuron-specific promoters (e.g., neurofilament promoter; Byrne and Ruddle, PNAS, 86: pp.5473-5477, 1989), pancreas-specific promoters (Eund et al, Science, pp.230: 912-916, 1985) and mammary gland-specific promoters (e.g., EP 874, EP 316, 166). In another embodiment, the regulatory elements include developmentally regulated promoters, such as the murine hox promoter (Kessel and Gruss, Science, 249: 374) -379, 1990) and the alpha-fetoprotein promoter (Campes and Tilghman, Genes Dev, 3: 537-546, 1989).

Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation, infection, or transfection techniques. As used herein, the terms "transformation" and "transfection" refer to various art-recognized techniques for introducing exogenous nucleic acid into a host cell, including, for example, calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, sonication, and electroporation methods. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al ("Molecular Cloning: Alabortory Manual" 2 nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) and other Laboratory manuals.

Production of polypeptides in prokaryotes is most commonly performed in E.coli, using vectors containing constitutive or inducible promoters directing the expression of fusion or non-fusion proteins. Constitutive promoters include, for example, lambda PL, spc ribosomes, and beta-lactamases. Inducible promoters include, for example, arabinose, lac, tac, and maltose binding protein. Fusion vectors add a number of amino acids to the protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve 3 purposes: increasing expression of the recombinant protein; increasing the solubility of the recombinant protein; and to aid in the purification of recombinant proteins by acting as a ligand in affinity purification. Typically, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable isolation of the recombinant protein from the fusion moiety after purification of the fusion protein. Such enzymes and their cognate recognition sequences include factor Xa, thrombin and enterokinase. The invention also provides vectors (e.g., expression vectors, sequencing vectors, cloning vectors) comprising at least one polynucleotide of the invention, host cells genetically engineered with the vectors of the invention, and production of the polypeptides of the invention by recombinant techniques. Cell-free translation systems are also used to produce such proteins, using RNA derived from the DNA constructs of the invention.

Group C or group G streptococcal PPI antibodies

The polypeptides of the invention include polypeptides comprising SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. SEQ ID NO: 8. SEQ ID NO: 10 or SEQ ID NO: 11, fragments and analogs thereof, or cells expressing such sequences, fragments and analogs, can also be used as immunogens to generate antibodies that specifically bind to the polypeptides of the present invention. In one aspect, the invention provides (a) an antibody that specifically binds to a group C or group G streptococcal PPI polypeptide, (b) use of such an antibody to detect the presence of or measure the amount or concentration of a streptococcal PPI polypeptide in a cell, cell or tissue extract or biological fluid, and (C) use of such an antibody to treat a beta hemolytic streptococcal infection in a subject.

Antibodies of the invention include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, and anti-idiotypic antibodies. Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen. Monoclonal antibodies are substantially homogeneous populations of antibodies that bind to a specific antigen. In general, antibodies can be displayed, for example, using conventional hybridoma technology (Kohler and Milstein (1975) Nature, 256: 495-499), recombinant DNA methods (U.S. Pat. No. 4,816,567), or phage display using antibody libraries (Clackson et al (1991) Nature, 352: 624-628; Marks et al (1991) J.mol.biol., 222: 581-597). For additional antibody production techniques, see Antibodies: a Laboratory Manual, eds Harlow and Lane, Cold Spring Harbor Laboratory, 1988. The invention is not limited to any particular source, method of production, or other particular characteristics of the antibody.

Other suitable methods of generating or isolating antibodies that specifically bind to epitopes of group C or group G streptococcus PPI polypeptides may be used. In certain embodiments, the recombinant antibody is selected from a peptide or protein display library, such as phage, ribosome, oligonucleotide, RNA and cDNA display libraries (EP368,684; PCT/GB 91/01134; PCT/GB 92/01755; PCT/GB 92/002240; PCT/GB 92/00883; PCT/GB 93/00605; PCT/GB 94/01422; PCT/GB 94/02662; PCT/GB 97/010124; WO 90/14443; WO 90/14424; WO 90/14430; PCT/US 94/1234; WO 92/18619; WO 96/07754; EP614,989; WO 614,989/16027; WO 614,989/06630; WO 614,989/369; U.S. Pat. No. 4,692,692,692,3672/614,989; WO 614,989/3683; EP614,989; EP 363672; EP614,989/3638072; PCT/614,989; WO 363672; PCT/36363672; see, EP 36363672; PCT/614,989; WO 363672; see, EP 3636363672; see, EP 363672; PCT/614,989; see, U.S. Pat. Nos. 3; PCT/614,989; and WO 614,989; see. ) In yet other embodiments, the recombinant antibody is produced in a transgenic animal capable of producing a repertoire of human antibodies (Nguyen et al, microbiol.immunol.41: 901-907, 1997; sandhu et al, crit.rev.biotechnol.16: 95-118, 1996; and Eren et al, Immunol.93: 154-161, 1998) other techniques for producing recombinant antibodies include, for example, (a) single cell antibody production techniques such as the selective lymphocyte antibody method ("SLAM") (U.S. Pat. No. 5,627,052), (b) gel microdroplets and flow cytometry methods (Powell et al, biotechnol.8: 333-337, 1990), and (c) B cell selection (Steenbakkers et al, mol. biol. reports 19: 125-134, 1994). These same methods can also be used to improve the affinity and/or avidity of anti-group C or group G streptococcal PPI antibodies for their specific binding targets.

Intact antibodies are immunoglobulins (Ig) and they are generally tetramerically glycosylated proteins consisting of 2 light chains (25 kDa each) and 2 heavy chains (50 kDa each). The light chains are classified into 2 isotypes (λ and κ), and the heavy chains are classified into 5 isotypes (A, D, E, G and M). Certain heavy chain isotypes are further divided into isotype subclasses, e.g., IgG₁、IgG₂、IgG₃And IgG₄。

The domains and three-dimensional structures of different antibodies are known in the art (Harlow and Lane, supra). Briefly, the light chain is composed of a constant domain (C)_L) And an N-terminal variable domain (V)_L) And (4) forming. The heavy chain consists of 3 or 4 constant domains (C)_H) Hinge region and N-terminal variable domain (V)_H) And (4) forming. And V_HAdjacent domains of C_HIs designated as C_H1。V_HAnd V_LThe domains comprise 4 conserved sequence regions (FR1, FR2, FR3 and FR4), called Framework (FR) regions, which form a scaffold for 3 hypervariable sequence regions, called Complementarity Determining Regions (CDRs). The CDRs (CDR1, CDR2, and CDR3) comprise the majority of antibody amino acids that specifically recognize and bind antigen. The heavy chain CDRs are indicated as H1, H2, and H3, while the light chain CDRs are indicated as L1, L2, and L3.

Fab fragments (antigen binding fragments) are covalently linked by V through disulfide bonds between constant regions_H-C_H1And V_L-C_LDomain composition. F_vFragments are smaller and are composed of non-covalently linked V_HAnd V_LDomain composition. To overcome the tendency of non-covalent domain separation, single-stranded F can be constructed_vSegment (scF)_v)。scF_vComprising an elastic polypeptide which renders (a) V_HC terminal and V of_LIs linked at the N-terminus of (a) or (b) V_LC terminal and V of_HThe N-terminal of (1) is ligated. 15-mer (Gly)₄Ser)₃Peptides may be used as linkers, but other linkers are also known in the art.

Antibody diversity is generated by using multiple germline genes encoding variable regions and various somatic events. Somatic events include recombination of variable gene segments and diverse (D) and joining (J) gene segments to produce complete V_HRecombination of regions and variable, and variable and linked gene segments to produce complete V_LAnd (4) a zone. CDR3(H3) is the largest source of molecular diversity within an antibody sequence. H3 may be as short as 2 amino acid residues or greater than 26, for example. The smallest antigen-binding fragment is Fv, consisting of V_HAnd V_LDomain composition.

The anti-group C or group G streptococcus PPI antibodies of the present invention may optionally include an antibody constant region or portion thereof. For example, V_LThe domain may be attached at its C-terminus to a light chain constant domain such as ck or C λ. Similarly, V_HThe domains or portions thereof may be attached to all or part of heavy chains such as IgA, IgD, IgE, IgG and IgM, as well as any isotype subclass. Antibody isotypes such as IgG₁、IgG₂、IgG₃Or IgG₄From CH₂And CH₃Domain determination. Isoforms can switch by changing these domains without affecting antigen binding. Constant regions are known in the art (see, e.g., Kabat et al, Sequences of Proteins of Immunological Interest, No.91-3242, National Institutes of Health issues, Bethesda, Md., 1991.)

The term "antibody" is also intended to include intact molecules as well as fragments such as Fab, which are capable of binding antigen. Fab fragments lack the Fc fragment of intact antibody, clear more rapidly from circulation, and are likely to have less non-specific tissue binding than intact antibody (Wahl et al, 1983, J.Nucl. Med.24: 316-. It is understood that Fab and other fragments of antibodies useful in the present invention can be used to detect and quantify group C or group G streptococcal PPI polypeptides, according to the methods used for the intact antibody molecule.

Chimeric antibodies are molecules, different parts of which are derived from different animal species, e.g. having variable regions (V) derived from murine monoclonal antibodies_H、V_L) And human immunoglobulin constant region (CH)₁-CH₂-CH₃、C_L) Those of (a). Chimeric antibodies and methods for their production are known in the art (Cabilly et al, 1984, Proc. Natl. Acad. Sci. USA 81: 3273. 3277; Morrison et al, 1984, Proc. Natl. Acad. Sci. USA 81: 6851. 6855; Boulianne et al, 1984, Nature 312: 643. 646; Cabilly et al, European patent application 125023 (published 1984.11.14), Taniguchi et al, European patent application 171496 (published 1985.2.19), Morrison et al, European patent application 173494 (published 1986.3.5), Neuberger et al, PCT application WO 86/01533 (published 1986.13; Kudo et al, European patent application 184187 (published 1986.11.494; Morrison et al, 1986.17344; published 1986.16: 3444; European patent application WO 3.16: 3444; European patent application WO 2/10652; published 1986: 3451: 1986; published 1986: 3444; European patent application 3435; published 1986: 3435; European patent application 3435; published by European patent No. 3435; European patent application 3435; European patent application No. 1986: 1986, 1987, proc.natl.acad.sci.usa 84: 214-218; better et al, 1988, Science 240: 1041-1043). These references are incorporated herein by reference.

Antibodies are used in various ways, for example to confirm protein expression or to confirm where a protein is expressed. Labeled antibodies (e.g., fluorescent labels for FACS) can be incubated with intact bacteria, and the presence of the label on the surface of the bacteria confirms, for example, the localization of the protein.

Immunogenic compositions

The present invention provides immunogenic compositions comprising one or more of group C or group G streptococcus PPI polypeptides. In certain embodiments, an immunogenic composition includes one or more PPI polypeptides comprising a sequence identical to SEQ id no: 2. 4,6, 8 and 10, at least 90%, 95%, 99% or 100% identical.

In other embodiments, the immunogenic compositions of the invention comprise a polynucleotide encoding a group C or group G streptococcus PPI polypeptide, and one or more physiologically acceptable carriers. In certain embodiments, the immunogenic composition comprises a polypeptide having an amino acid sequence that is identical to SEQ ID NO: 1.3, 5,7 and/or 9, at least 90%, 95%, 99% or 100% identical in nucleotide sequence.

As used herein, the term "immunogenic composition" refers to any type of biological agent in an administrable manner that is capable of stimulating an immune response in a subject vaccinated with the immunogenic composition. The immune response may include induction of antibodies and/or induction of a T cell response. The term "protection" when used in reference to an immunogenic composition refers herein to an improvement (partial or complete) in any of the symptoms associated with the disease or condition in question. Thus, protection of a subject from infection by streptococcus species such as streptococcus dysgalactiae (including dysgalactiae and equisimian) by the presented immunogenic composition generally results in the elimination of one or more of bacterial growth and/or clinical symptoms associated with streptococcal infection, including arthritis, endocarditis, meningitis, uveitis, bronchopneumonia, meningitis, permanent hearing loss, and septic shock.

The methods disclosed herein can include inducing an immune response against one or more pathogens including streptococcus species (e.g., streptococcus dysgalactiae subspecies equisimilis, streptococcus dysgalactiae subspecies pyogenes, streptococcus agalactiae, streptococcus pharyngolaris, streptococcus constellatus, streptococcus equisimilis (s.equisimilis), and streptococcus intermedius (s.intermedia)), e.g., the methods can include inducing polyclonal antibody production against one or more streptococcal pathogens, such as streptococcus dysgalactiae subspecies equisimilis. In certain embodiments, the method comprises administering to the subject a composition comprising an isolated group C or group G streptococcus ORF554 polynucleotide or PPI polypeptide.

Various tests are used to assess the in vitro immunogenicity of the polypeptides of the invention. For example, an in vitro opsonin assay is performed by incubating a mixture of cells of a Streptococcus species, a heat-inactivated serum comprising specific antibodies to the polypeptide in question, and an exogenous complement source together. Opsonophagocytosis is performed during the incubation of freshly isolated polymorphonuclear cells (PMN's) and antibody/complement/streptococcus species cell mixtures. Bacterial cells coated with antibodies and complement are killed after opsonophagocytosis. Colony forming units (cfu) of surviving bacteria that escape opsonophagocytosis were determined by the plating assay mixture. Titers are reported as the reciprocal of the highest dilution giving > 50% bacterial kill, as determined by comparison to assay controls. Samples that showed less than 50% killing at the lowest serum dilution tested (1: 8) were reported to have an opsonophagocytosis antibody (OPA) titer of 4. The above method is a modification of the Gray's method (Gray, Conjugate vaccine, pp.694-697, 1990).

A test serum control comprising test serum plus bacterial cells and heat inactivated complement was included for each individual serum. This control is used to assess whether the presence of antibiotics or other serum components is capable of directly killing the bacterial strain (i.e., in the absence of complement or PMN's). Human serum with known opsonin titers was used as a positive human serum control. The opsonin antibody titer for each unknown serum was calculated as the reciprocal of the initial dilution of serum that gave a 50% cfu reduction compared to the control without serum.

Whole cell ELISA assays can also be used to assess in vitro immunogenicity and surface exposure of polypeptide antigens, wherein a bacterial strain of interest is coated on a plate, e.g., a 96-well plate, and test sera from the immunized animal are reacted with the bacterial cells. If any antibody specific for the test polypeptide antigen reacts with a surface-exposed epitope of the polypeptide antigen, it can be detected by standard methods known to those skilled in the art.

Any polypeptide exhibiting the desired in vitro activity can then be tested in an in vivo animal challenge model. In certain embodiments, the immunogenic compositions are used for immunization of animals (e.g., mice) by immunization methods and routes known to those of skill in the art (e.g., intranasal, parenteral, intramuscular, oral, rectal, vaginal, transdermal, intraperitoneal, intravenous, subcutaneous, etc.). Following immunization of an animal with a particular Streptococcus dysgalactiae immunogenic composition, the animal is challenged with Streptococcus dysgalactiae or other Streptococcus species and resistance to infection with Streptococcus dysgalactiae or other Streptococcus species is determined.

Group C or group G streptococcal PPI/ORF 554 polypeptides and polynucleotides are incorporated into immunogenic compositions suitable for administration to a subject. Such compositions generally include a nucleic acid molecule or protein, along with a pharmaceutically acceptable carrier. As used herein, the phrase "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, excipients, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, such media can be used in the compositions of the invention. Supplementary active compounds may also be incorporated into the compositions.

Any immunogenic composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral (e.g., intramuscular, intravenous, intradermal, subcutaneous, intraperitoneal), transmucosal (e.g., oral, rectal, intranasal, vaginal, respiratory), and transdermal (topical). Solutions or suspensions for parenteral, intradermal, or subcutaneous application may include the following components: sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates, and agents for adjusting tonicity such as sodium chloride or dextrose. The pH can be adjusted with an acid or base such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL^TM(BASF, Parsippany, n.j.) or Phosphate Buffered Saline (PBS). In all cases, the composition must be sterile and should flow to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof. Suitable fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, p-hydroxybenzoic acid, chlorobutanol, phenol, ascorbic acid, and the like. In many cases, isotonic agents are included in the compositions, for example, sugars, polyalcohols such as mannitol, sorbitol, and/or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a group C or group G streptococcal PPI polypeptide, ORF554 polynucleotide or an antibody thereto) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, followed by filtered sterilization, if necessary. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Oral compositions typically include an inert diluent or an edible carrier. They may be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compounds may be incorporated with excipients and used in the form of tablets, dragees or capsules. Oral compositions may also be prepared for use as mouthwashes using a fluid carrier in which the compound is applied orally and emits sirtuin and is expectorated or swallowed. Pharmaceutically compatible binding agents and/or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, lozenges, and the like may comprise any of the following ingredients, or compounds of similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch or lactose, disintegrants such as alginic acid, Primogel or corn starch; lubricants such as magnesium stearate or Sterotes; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressurized container or dispenser or nebulizer, the dispenser containing a suitable propellant, e.g., a gas such as carbon dioxide. Systemic administration may also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be penetrated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated as ointments, salves, gels, or creams as generally known in the art.

The compounds may also be prepared for rectal delivery in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas.

In one embodiment, the active compound is prepared with a carrier that protects the compound from rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.

Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used as carriers. Methods for preparing such formulations will be apparent to those skilled in the art. Materials are also commercially available from Alza corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies against viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example as described in U.S. Pat. No. 4,522,811, incorporated herein by reference.

It is advantageous to formulate oral or parenteral compositions in unit dosage form for ease of administration and uniformity of dosage. As used herein, unit dosage form refers to physically discrete units suitable as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the unit dosage form of the invention are indicated by and directly depend on the following: the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in dosage regimens such as the active compound used to treat an individual.

A combination immunogenic composition is provided by combining one or more of the polypeptides of the invention with one or more known streptococcal polysaccharides or polysaccharide-protein conjugates.

The protein component of the polysaccharide-protein conjugate is referred to as the "carrier protein". The term "carrier protein" as a group includes those proteins that are non-toxic, non-reactive and available in sufficient quantity and purity. The carrier protein may be amenable to standard conjugation procedures. For example, CRM₁₉₇Can be used as a carrier protein. CRM₁₉₇(Wyeth, Sanford, N.C.) is a non-toxic variant of diphtheria toxin (toxoid) isolated from Corynebacterium diphtheriae (Corynebacterium diphtheria) strain C7 (. beta.197), which has been grown in a medium based on casamino acids and yeast extract. CRM₁₉₇Purification was performed by ultrafiltration, ammonium sulfate precipitation and ion exchange chromatography. Other diphtheria toxoids are also suitable for use as carrier proteins.

Other suitable carrier proteins include inactivated bacterial toxins such as tetanus toxoid, pertussis toxoid, cholera toxoid (as described in e.g. WO/2004/083251), escherichia coli LT, escherichia coli ST, and exotoxin a from Pseudomonas aeruginosa (Pseudomonas aeruginosa). Bacterial outer membrane proteins such as outer membrane complex c (ompc), porins, transferrin binding proteins, pulmonary lysis, pneumococcal surface protein a (pspa), pneumococcal adhesin protein (PsaA), or haemophilus influenzae (haemophilus fluuenzae) protein D may also be used. Purified protein derivatives of other proteins such as ovalbumin, Keyhole Limpet Hemocyanin (KLH), Bovine Serum Albumin (BSA) or tuberculin (PPD) may also be used as carrier proteins.

Immunogenic compositions comprising the polynucleotides are delivered to a recipient by a variety of vectors and expression systems. Such systems include, inter alia, chromosomal, episomal and virus-derived systems, such as vectors derived from: bacterial plasmids, attenuated bacteria such as salmonella (U.S. patent No. 4,837,151), bacteriophages, transposons, yeast episomes, insertion elements, yeast chromosomal elements, viruses such as vaccinia and other poxviruses, adenoviruses, baculoviruses, papova viruses such as SV40, fowlpox viruses, pseudorabies viruses and retroviruses, alphaviruses such as venezuelan equine encephalitis virus (U.S. patent No. 5,643,576), sindbis virus and semliki forest virus, non-segmented negative strand RNA viruses such as vesicular stomatitis virus (U.S. patent No. 6,168,943), and vectors derived from combinations thereof, such as vectors derived from plasmid and phage genetic elements, such as cosmids and phagemids. The expression system should include control regions that regulate as well as cause expression, such as promoters and other regulatory elements (e.g., polyadenylation signals). In general, any system or vector suitable for maintaining, propagating or expressing a polynucleotide to produce a polypeptide in a host may be used. Suitable nucleotide sequences may be inserted into the expression system by any of a variety of well-known and conventional techniques, such as Sambrook et al, "Molecular Cloning: a Laboratory Manual "2 nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

The immunogenic compositions of the invention are generally administered parenterally in unit dosage formulations containing standard, well known non-toxic physiologically acceptable carriers, adjuvants and vehicles as needed.

A pharmaceutically acceptable vehicle is understood to mean a compound or a combination of compounds destined to come within a pharmaceutical or immunogenic composition that does not cause side effects and makes it possible, for example, to facilitate the administration of the active compound, to increase its lifetime in vivo and/or its efficacy, to increase its solubility in solution or alternatively to enhance its preservation. These pharmaceutically acceptable vehicles are well known and will be modified by those skilled in the art according to the nature and mode of administration of the active compound selected.

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions are formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1, 3-butanediol.

Among the acceptable vehicles and solvents that may be employed are water, ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

The carrier includes a neutral saline solution buffered with phosphate, lactate, Tris, etc. When a viral vector is administered, the vector is sufficiently purified so that it is substantially free of undesirable contaminants, such as defective interfering adenovirus particles or endotoxins and other pyrogens, so that it does not cause any adverse reactions in the individual receiving the vector construct. In certain embodiments, the method of purifying a carrier involves the use of a buoyant density gradient, such as cesium chloride gradient centrifugation.

The carrier may also be a liposome. Methods for using liposomes as delivery vehicles are well known in the art (see, e.g., review by schwendenner RA, adv. exp. med. biol. 620: 117-.

The immunogenic compositions of the invention also include polynucleotide sequences of the invention operably linked to regulatory sequences that control gene expression. The polynucleotide sequence of interest is engineered into an expression vector, such as a plasmid, under the control of regulatory elements that promote expression of the DNA, i.e., promoter and/or enhancer elements. In certain embodiments, the human cytomegalovirus immediate early promoter/enhancer is used (U.S. Pat. No. 5,168,062). The promoter may be cell-specific and allow for substantial transcription of the polynucleotide only in predetermined cells.

The polynucleotides of the invention are introduced directly into the host as "naked" DNA (U.S. Pat. No. 5,580,859) or are formulated in a composition with facilitators, such as bupivacaine and other local anesthetics (U.S. Pat. No. 5,593,972) and cationic polyamines (U.S. Pat. No. 6,127,170.)

In this polynucleotide immunization procedure, the polypeptides of the invention in vivo in transient basis expression; the genetic material is not inserted or integrated into the chromosome of the host. This operation is distinguished from gene therapy, in which the purpose is to insert or integrate genetic material of interest into the chromosome. Assays are used to confirm that polynucleotides administered by immunization do not cause a transformation phenotype in a host (e.g., U.S. Pat. No. 6,168,918).

In particular embodiments, an immunogenic composition as described herein further comprises one or more adjuvants. Adjuvants are substances that enhance the immune response when administered in conjunction with an immunogen or antigen. Many cytokines or lymphokines have been shown to have immunomodulatory activity, and are therefore useful as adjuvants, including, but not limited to, interleukins 1-alpha, 1-beta, 2, 4,5, 6,7, 8, 10, 12 (see, e.g., U.S. Pat. No. 5,723,127), 13, 14, 15, 16, 17, and 18 (and mutated forms thereof); interferon- α, β and γ; granulocyte-macrophage colony stimulating factor (GM-CSF) (see, e.g., U.S. patent No. 5,078,996 and ATCC accession No. 39900); macrophage colony stimulating factor (M-CSF); granulocyte colony stimulating factor (G-CSF); and tumor necrosis factors alpha and beta. Still other adjuvants useful with the immunogenic compositions described herein include chemokines, including but not limited to MCP-1, MIP-1 α, MIP-1 β, and RANTES; adhesion molecules such as selectins, e.g., L-selectin, P-selectin and E-selectin; mucin-like molecules, such as CD34, GlyCAM-1 and MadCAM-1; integrin family members such as LFA-1, VLA-1, Mac-1 and p 150.95; immunoglobulin superfamily members such as PECAM, ICAMs such as ICAM-1, ICAM-2 and ICAM-3, CD2 and LFA-3; co-stimulatory molecules such as CD40 and CD 40L; the growth factors comprise vascular growth factor, nerve growth factor, fibroblast growth factor, epidermal growth factor, B7.2, PDGF, BL-1 and vascular endothelial growth factor; receptor molecules including Fas, TNF receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, and DR 6; and Caspase (ICE).

Suitable adjuvants for enhancing an immune response further include, but are not limited to, MPL^TM(3-O-deacetylated monophosphoryl lipid A, Corixa, Hamilton, MT), which is described in U.S. Pat. No. 4,912,094. Also suitable for use as adjuvants are synthetic lipid a analogs or aminoalkylglucosamine phosphate compounds (AGPs), or derivatives or analogs thereof, available from Corixa (Hamilton, MT) and described in U.S. patent No. 6,113,918. One such AGP is 2- [ (R) -3-tetradecanoyloxytetradecanoylamino [ ] -AGP]Ethyl 2-deoxy-4-O-phosphono-3-O- [ (R) -3-tetradecanoyloxy-tetradecanoyl]-2- [ (R) -3-tetradecanoyloxytetradecanoyl-amino]-b-D-glucopyranoside, also known as 529 (previously known as RC 529). This 529 adjuvant is formulated as an Aqueous Form (AF) or a Stable Emulsion (SE).

Still other adjuvants include muramyl peptides such as N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-orthopyroyl-L-alanine-2- (1 '-2' dipalmitoyl-sn-glycero-3-hydroxyphosphonoxy) -ethylamine (MTP-PE); oil-in-water emulsions, such as MF59 (U.S. patent No. 6,299,884) formulated as submicron particles using a microfluidizer, such as a Model 110Y microfluidizer (Microfluidics, Newton, MA) (containing 5% squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing various amounts of MTP-PE), and SAF (containing 10% squalene, 0.4% Tween 80, 5% pluronic-block polymer L121, and thr-MDP, microfluidized into submicron emulsions or vortexed to produce larger particle size emulsions); incomplete Freund's Adjuvant (IFA); aluminum salts (aluminum) such as aluminum hydroxide, aluminum phosphate, aluminum sulfate; amphigen; alfvudine; l121/squalene; d-lactide-polylactide/glucoside; pluronic polyols; killed Bordetella (Bordetella); saponins, such as Stimulon described in U.S. Pat. No. 5,057,540^TMQS-21 (antibiotics, Framingham, MA.), ISCOMATRIX (CSL Limited, Parkville, Australia) described in U.S. Pat. No. 5,254,339, and Immune Stimulating Complexes (ISCOMS); mycobacterium tuberculosis (Mycobacterium tuberculosis); bacterial lipopolysaccharides; synthetic polynucleotidesFor example, oligonucleotides comprising CpG motifs (e.g., U.S. Pat. No. 6,207,646); IC-31(Intercell AG, Vienna, Austria) described in European patent Nos. 1,296,713 and 1,326,634; pertussis Toxin (PT) or a mutant thereof, cholera toxin or a mutant thereof (e.g., U.S. patent nos. 7,285,281, 7,332,174, 7,361,355, and 7,384,640); or E.coli heat-Labile Toxin (LT) or mutants thereof, particularly LT-K63, LT-R72 (e.g., U.S. Pat. Nos. 6,149,919, 7,115,730 and 7,291,588).

Therapeutic antibodies and antigen binding polypeptides

The present invention is directed, inter alia, to the treatment of streptococcal infections by administering to a subject a therapeutic immunological agent, such as a humanized monoclonal antibody that recognizes a specific epitope within streptococcal PPI, under conditions that produce a favorable therapeutic response in the subject. "immunological reagents" include, for example, antibodies, humanized antibodies, antibody fragments, peptides comprising antigen binding elements or CDRs, and the like. "favorable therapeutic response" includes, for example, induction of phagocytosis or opsonization by beta hemolytic streptococci. The invention also relates to the use of the disclosed immunological reagent in the manufacture of a medicament for the treatment or prevention of beta hemolytic streptococcal infection.

In one aspect, the invention provides a method of preventing or treating a disease associated with beta hemolytic streptococcal infection in a patient. Certain methods of the invention entail administering to a patient an effective dose of an antibody that specifically binds to a streptococcal PPI epitope. Such methods are particularly useful for preventing or treating beta hemolytic streptococcal disease in a subject. "subject" includes any vertebrate animal, such as companion animals, farm animals, mammals, and human patients. Exemplary methods comprise administering an effective dose of an antibody or antigen-binding peptide that binds streptococcal PPI. Certain embodiments include administering an effective dose of an antibody or other peptide comprising an antigen recognition site or CDR that specifically binds to an epitope within a streptococcal PPI, such as an epitope comprising SEQ ID NO: 2. 4,6, 8, 10 and 11.

In another aspect, the invention features administering an antibody or other antigen binding peptide that binds to streptococcal PPI in a subject and induces a clearance response against beta hemolytic streptococci. For example, such a clearing response may be achieved through Fc receptor-mediated phagocytosis.

The therapeutic immunological reagents of the invention are generally substantially purified and free of undesirable contaminants. This means that the immunological reagents are generally at least about 50% w/w (weight/weight) pure and substantially free of interfering proteins and contaminants. In certain embodiments, the immunological reagent is at least about 80% w/w pure. In other embodiments, the immunological reagent is at least about 90 or about 95% w/w pure. However, using conventional protein purification techniques, homogeneous peptides of at least 99% w/w purity can be obtained.

The method can be used on asymptomatic subjects and subjects currently exhibiting symptoms of disease. The antibodies used in such methods can be human, humanized, chimeric or non-human antibodies, or fragments thereof (e.g., antigen-binding fragments, peptides or CDRs comprising epitope-binding regions), and can be monoclonal or polyclonal, as described herein.

In another aspect, the invention features administering the antibody with a pharmaceutical carrier as a pharmaceutical composition. Alternatively, the antibody may be administered to the subject by administering a polynucleotide encoding at least one antibody chain. The polynucleotide is expressed to produce antibody chains in the patient. Optionally, the polynucleotide encodes the heavy and light chains of the antibody. The polynucleotide is expressed to produce the heavy and light chains in the patient. In an exemplary embodiment, the patient is monitored for the level of administered antibody in the blood of the patient.

Treatment-compliant subjects include individuals at risk of disease but who do not show symptoms, as well as patients who currently show symptoms. Thus, the presented immunogenic compositions and therapeutic antibodies can be administered prophylactically to the general population. In asymptomatic subjects, treatment may begin at any age. Treatment can be monitored by measuring antibody levels over time. If the immune response or antibody level is decreased, a booster dose is indicated.

In prophylactic applications, the immunogenic composition or medicament is administered to a subject susceptible to or otherwise at risk of β hemolytic streptococcal infection in an amount sufficient to eliminate or reduce the risk, lessen the severity, or delay the onset of disease, including biochemical, histological, and/or behavioral symptoms of the disease associated with the infection, its complications, and intermediate pathological phenotypes present during disease progression. In therapeutic applications, the composition or medicament is administered to a patient suspected of, or having, such a disease, in an amount sufficient to cure or at least partially arrest the symptoms (biochemical, histological, and/or behavioral) of the disease, including its complications and intermediate pathological phenotypes in the development of the disease.

An amount sufficient to effect a therapeutic or prophylactic treatment is defined as a therapeutically or prophylactically effective dose. In prophylactic and therapeutic regimens, immunological reagents are typically administered in several doses until a sufficient immune response has been achieved. The term "immune response" or "immunological response" includes the development of a humoral (antibody-mediated) and/or cellular (mediated by antigen-specific T cells or their secretory products) response to an antigen in a recipient subject. Such responses may be active, i.e. induced by administration of an immunogen (supra), or passive, i.e. induced by administration of an immunoglobulin or antibody or pre-treated T cells. Typically, the immune response is monitored and if the immune response begins to diminish, repeated doses are administered.

The effective dosage of the compositions of the invention for treating beta hemolytic streptococcal infection varies depending on many different factors, including the mode of administration, the target site, the physiological condition of the patient, whether the patient is a human or other animal, other drugs administered, and whether the treatment is prophylactic or therapeutic. Typically, the subject is a human, but non-human mammals including transgenic animals can also be treated. The therapeutic dose may need to be escalated to optimize safety and efficacy.

For passive immunization with an antibody, the dosage range is about 0.0001-100mg/kg, and more typically 0.01-5mg/kg (e.g., 0.02mg/kg, 0.1mg/kg, 0.15mg/kg, 0.2mg/kg, 0.25mg/kg, 0.5mg/kg, 0.75mg/kg, 1mg/kg, 2mg/kg, etc.) of the host weight. For example, the dose may be about 1mg/kg body weight or about 10mg/kg body weight or in the range of 1-10 mg/kg. Dosages intermediate to the above ranges are also intended to be within the scope of the present invention. The subject may administer such doses daily, every other day, weekly, monthly, every 2 months, every 3 months, or according to any other schedule determined by empirical analysis. Exemplary treatments entail administering multiple doses over an extended period of, for example, at least 6 months. Additional exemplary treatment regimens must be administered once every 2 weeks, or once a month or once every 3-6 months. Exemplary dosage schedules include 1-10mg/kg or 15mg/kg on consecutive days, 30mg/kg on alternate days or 60mg/kg weekly. In certain methods, 2 or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dose administered for each antibody is included in the ranges indicated.

Antibodies are typically administered in multiple occasions. The interval between individual doses may be weekly, monthly or yearly. The intervals may also be irregular, as indicated by measuring blood levels of antibodies against streptococcal PPI in the patient. In some methods, the dose is adjusted to achieve a plasma antibody concentration of 1-1000. mu.g/ml, and in some methods, 25-300. mu.g/ml. Alternatively, the antibody may be administered as a sustained release formulation, in which case less frequent administration is required. The dose and frequency vary depending on the half-life of the antibody in the patient. In general, humanized antibodies exhibit the longest half-life, followed by chimeric and non-human antibodies.

The dosage and frequency of administration may vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, compositions comprising the presented antibodies or mixtures thereof are administered to a patient who is not in a disease state to enhance the patient's resistance. Such amounts are defined as "prophylactically effective doses". In this use, the precise amount again depends on the health and systemic immunity of the patient, but generally ranges from 0.1 to 25 mg/dose, especially from 0.5 to 2.5 mg/dose. Relatively low doses are administered at relatively infrequent intervals over a long period of time.

In therapeutic applications, relatively high doses (e.g., about 1-200mg antibody per dose, with doses of 5-25mg more commonly used) at relatively short intervals are sometimes required until disease progression is reduced or terminated, and preferably until the patient exhibits partial or complete improvement in disease symptoms. Thereafter, the patient may be administered a prophylactic regimen.

The dosage range for the nucleic acid encoding the antibody is about 10ng-1g, 100ng-100mg, 1 μ g-10mg, or 30-300 μ g DNA/patient. The dose for infectious viral vectors varies from 10 to 100 or more virions per dose.

Therapeutic immunological agents may be administered by parenteral, topical, intravenous, oral, subcutaneous, intraarterial, intracranial, intraperitoneal, intranasal, or intramuscular methods for prophylactic and/or therapeutic treatment. The most common route of administration of immunological reagents is intravenous infusion or subcutaneous administration, although other routes are equally effective. The next most common route is intramuscular injection. This type of injection is most commonly performed in the arm or leg muscles. In certain methods, the immunological agent is injected directly into a particular tissue in which deposits have accumulated, e.g., intracranially. Intramuscular injection or intravenous infusion is preferred for antibody administration. In certain methods, the antibody is administered as a sustained release composition or device, such as a microinfusor device (e.g., medipod)^TMA device; see Meehan et al, Journal of Controlled Release, 46: 107-119, 1997.)

As described above, an immune response against beta hemolytic streptococcal infection can be developed in vivo (or ex vivo) by administering nucleic acids encoding antibodies and component chains thereof for passive immunization. Such nucleic acids may be DNA or RNA. Nucleic acid segments encoding immunological agents are typically linked to regulatory elements, such as promoters and enhancers, that allow for expression of the DNA segment in the intended target cells of the patient. For expression in blood cells, promoter and enhancer elements from light or heavy chain immunoglobulin genes or CMV major immediate early promoter and enhancer are suitable to direct expression, as desired for inducing an immune response. The regulatory elements and coding sequences to be ligated are usually cloned into a vector. For the administration of diabodies, the 2 strands may be cloned into the same or separate vectors.

Many viral vector systems are available, including retroviral systems (see, e.g., Lawrie and Tumin, Cur. Opin. Genet. Defelop.3: 102109 (1993)); adenoviral vectors (see, e.g., Bett et al, J.Virol.67: 5911 (1993)); adeno-associated viral vectors (see, e.g., Zhou et al, J.Exp.Med.179: 1867(1994)), viral vectors from the pox family including vaccinia and fowlpox virus, viral vectors from the alphavirus genus such as those derived from sindbis and Semliki forest viruses (see, e.g., Dubensky et al, J.Virol.70: 508(1996)), Venezuela equine encephalitis virus (see Johnston et al, U.S. Pat. No. 5,643,576) and rhabdoviruses such as vesicular stomatitis virus (see Rose, U.S. Pat. No. 6,168,943) and papilloma virus (Ohe et al, Human Gene Therapy 6: 325 (1995); Woo et al, WO 94/12629 and Xiao & Brandsma, Nucleic acids.Res.24, 26302622 (1996)).

DNA encoding an antibody or antibody fragment including CDRs, or a vector comprising the same, can be packaged into liposomes. Suitable lipids and related analogs are described by Eppstein et al, U.S. patent No. 5,208,036, Felgner et al, U.S. patent No. 5,264,618, Rose, U.S. patent No. 5,279,833, and Epand et al, U.S. patent No. 5,283,185. Carriers and DNA encoding immunogens can also be adsorbed or bound to particulate carriers, examples of which include polymethylmethacrylate polymers and polylactide and poly (lactide-co-glycolide), see, e.g., McGee et al, j.micro encap.14 (2): 197-210(1997).

The polynucleotide vector or naked polynucleotide (e.g., DNA) may be administeredIn vivo delivery to an individual patient, typically by systemic administration (e.g., intravenous, intraperitoneal, nasal, gastric, intradermal, intramuscular, subcutaneous, or intracranial infusion) or topical application (see, e.g., Anderson et al, U.S. patent No. 5,399,346). The term "naked polynucleotide" refers to a polynucleotide that is not administered in conjunction with a transfection facilitating agent. Naked polynucleotides are sometimes cloned in plasmid vectors. Plasmid vectors can further include transfection facilitating agents such as bupivacaine (Weiner et al, U.S. Pat. No. 5,593,972). DNA can also be administered using a gene gun. See Xiao for Xiao&Brandsma, supra. DNA encoding the antibody (or fragments including the CDRs) is precipitated onto the surface of the micro-metallic beads. Microprojectiles bombardment (microprojectiles) is accelerated with a shock wave or expanding helium gas and penetrates the tissue to a depth of several cell layers. For example, ACCEL manufactured by Agricetus, Inc. Middleton Wis^TMGene Delivery Device, a DNA gun, is suitable for use in the practice of the present invention. Alternatively, naked DNA can be passed through the skin into the bloodstream simply by spotting the DNA on the skin with chemical or mechanical stimulation (see Howell et al, WO 95/05853).

In another embodiment, the vector encoding the immunological agent may be delivered ex vivo to cells, such as cells explanted from an individual patient (e.g., lymphocytes, bone marrow aspirate, tissue biopsy) or universal donor hematopoietic stem cells, followed by re-implantation of the cells into the patient, typically after selecting for cells that have incorporated the vector.

The immunological reagents of the invention may optionally be administered in combination with other reagents which are at least partially effective in the treatment of beta hemolytic streptococcal disease. The immunological reagents of the invention may also be administered in combination with other reagents that enhance the entry of the therapeutic immunological reagent into the target cell or tissue, such as liposomes and the like. Co-administration of such agents can reduce the dose of therapeutic immunological agent (e.g., therapeutic antibody or antibody chain) needed to achieve a desired effect.

The immunological reagents of the invention are typically administered as a pharmaceutical composition comprising the active therapeutic agent, i.e., together with various other pharmaceutically acceptable components. See Remington's Pharmaceutical Science (15 th edition, Mack Publishing Company, Easton, Pa. (1980)). The preferred form depends on the intended mode of administration and therapeutic application. Depending on the desired formulation, the composition may also include a pharmaceutically acceptable, non-toxic carrier or diluent, which is defined as a vehicle commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate buffered saline, ringer's solution, dextrose solution and hank's solution. In addition, the pharmaceutical composition or formulation may also include other carriers, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizers, and the like.

The pharmaceutical composition may also include large, slowly metabolized macromolecules such as proteins, polysaccharides such as chitosan, polylactic acid, polyglycolic acid, and copolymers (e.g., latex functionalized sepharose)^TMAgarose, cellulose, etc.), polymeric amino acids, amino acid copolymers, and lipid aggregates (e.g., oil droplets or liposomes). In addition, these carriers can act as immunostimulants (i.e., adjuvants).

For parenteral administration, the immunological reagents of the invention may be administered as an injectable dose of a solution or suspension of the substance in a pharmaceutically acceptable diluent with a pharmaceutical carrier, which may be a sterile liquid such as water oil, saline, glycerol or ethanol. In addition, auxiliary substances such as wetting or emulsifying agents, surfactants, pH buffering substances and the like may be present in the composition. Other components of the pharmaceutical compositions are those of petroleum, animal, vegetable or synthetic origin, for example peanut oil, soybean oil and mineral oil. Generally, glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions. The antibody may be administered in the form of a depot injection or implant preparation, which may be formulated in such a way as to allow sustained release of the active ingredient. An exemplary composition includes a monoclonal antibody at 5mg/mL formulated in an aqueous buffer consisting of 50mM L-histidine, 150mM NaCl, adjusted to pH6.0 with HCl.

Generally, the compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution or suspension in a liquid vehicle prior to injection can also be prepared. The preparation may also be emulsified or encapsulated in liposomes or microparticles, such as polylactide, polyglycolide or copolymers, for enhanced adjuvant effect, as described above (see Langer, Science 249: 1527(1990) and Hanes, Advanced Drug Delivery Reviews 28: 97 (1997)). The immunological reagents of the invention may be administered in the form of depot injections or implant preparations, which may be administered in such a way as to allow sustained or pulsatile release of the active ingredient.

Additional formulations suitable for other modes of administration include oral, intranasal and pulmonary formulations, suppositories and transdermal applications. For suppositories, binders and carriers include, for example, polyethylene glycol or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1% to 2%. Oral formulations include excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained-release preparations or powders and contain 10% to 95% of active ingredient, preferably 25% to 70%.

Alternatively, transdermal delivery can be achieved using skin patches or using transferosomes (Paul et al, Eur. J. Immunol.25: 3521 (1995); Cevc et al, biochem. Biophys. acta 1368: 20115 (1998)).

The invention also provides a method of monitoring treatment in a patient suffering from or susceptible to a beta hemolytic streptococcal infection, i.e. for monitoring the course of treatment administered to the patient. The method can be used to monitor therapeutic treatment of asymptomatic patients and prophylactic treatment of asymptomatic patients. In particular, the method is useful for monitoring passive immunity (e.g., measuring the level of antibody administered).

Certain methods entail determining a baseline value for the antibody level or profile in the patient, e.g., prior to administration of a dose of the immunological agent, and comparing it to a value for the post-treatment profile or level. A significant increase in the value of the level or spectrum (i.e., greater than the general margin of experimental error in repeated measurements of the same sample, expressed as 1 standard deviation from the mean of such measurements) indicates a positive treatment result (i.e., administration of the immunological agent has achieved the desired response). If the value for the immune response does not change or decrease significantly, a negative treatment result is indicated. If the treatment is passive immunotherapy, then the antibody levels are expected to decrease over time with a characteristic half-life.

The tissue sample for analysis is typically blood, plasma, serum, mucus fluid or cerebrospinal fluid from the patient. The sample is analyzed, for example, for antibody levels or titers against streptococcal PPI. ELISA methods for detecting antibodies specific for streptococcal PPI are described in the examples section. In certain methods, the level or titer of the administered antibody is determined using a clearance assay, such as in an in vitro phagocytosis assay (see, e.g., Jansen et al, Clin. Diagn. Lab. Immunol., 8 (2): 245-

Antibody profiles following passive immunization generally show an immediate peak in antibody concentration followed by an exponential decay. If there were no further doses, the attenuation approached pre-treatment levels over a period of days to months, depending on the half-life of the administered antibody.

In certain methods, a baseline measurement of antibody to streptococcal PPI in the patient is taken prior to administration, followed shortly thereafter by a second measurement to determine peak antibody levels, and one or more further measurements are taken at intervals to monitor the decay in antibody levels. Administration of further doses of antibody is used when the antibody level has dropped to the baseline or a predetermined percentage (e.g., 50%, 25%, or 10%) of the peaks below the baseline. In certain methods, the peak or subsequently measured level less than background is compared to a previously determined reference level to constitute a prophylactic or therapeutic treatment regimen that is advantageous in other patients. If the measured antibody level is significantly less than the reference level (e.g., less than the mean of the reference value minus 1 standard deviation in the patient population benefiting from treatment), then administration of an additional dose of antibody is indicated.

Additional methods include any art-recognized exemplary symptoms that are routinely relied upon by researchers or physicians to diagnose or monitor streptococcal infection or related disease as the course of treatment is monitored. For example, cellulitis, erysipelas, pustular herpes, necrotizing fasciitis, sore throat, red larynx (retchroat), chills, fever, headache, nausea, vomiting, increased heartbeat, malaise, enlarged tonsils, enlarged lymph nodes, and/or rashes may be monitored.

The specification is best understood from the teachings of the references cited within the specification, all of which are incorporated herein by reference in their entirety. The embodiments within the specification provide illustrations of embodiments of the invention and should not be construed as limiting the scope of the invention. An artisan will recognize that many other embodiments are encompassed by the invention, and that the specification and examples are intended to be exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

Example 1: cloning of Streptococcus dysgalactiae ORF554

Twelve (12) genomic sequences for group A and multiple genomic sequences for group B Streptococcus species are publicly available. The DNA and protein sequences of Open Reading Frame (ORF) number 554(ORF 554) have been identified in many of these Streptococcus genomes; see, for example, published international patent application No. WO 02/083859. However, there is limited sequence information about group C or group G genomes. Disclosed herein is the sequence of this ORF554 in other group G and group C streptococcus strains.

Known Streptococcus sequences comprising ORF554 are aligned in AlignX (vector NTI) and the regions of homology are used for degenerate primer construction (see Wessner, Science 286 (5554): 1495-. Oligonucleotide primer nucleotide sequences for isolating, amplifying and identifying novel group C or group G streptococcus ORF554 polynucleotides are set forth in SEQ ID NO: 18-26.

Initial PCR studies were performed using genomic DNA preparations prepared from Streptococcus C isolate ATCC12394 (S.lactis subsp.agalactiae). Partial gene sequences were obtained for the 5-initial and 3-initial ends of ORF 554. Forward and reverse primers (SEQ ID NO: 23 and SEQ ID NO: 24, respectively) were then designed based on these sequences and subsequently used to generate by PCR approximately 700-900bp ORF554 sequences from different G and C strains.

The genomic or protein sequence of ORF554 from these strains was not defined prior to the present disclosure. The nucleotide sequence obtained for ORF554 from isolate ATCC12394 is set forth in SEQ ID NO: 1 and its amino acid sequence is depicted in SEQ ID NO: 2.

ORF554 is found in five (5) isolates: ATCC12394, ATCC35666, and ATCC27823 obtained from ATCC, and 2 lansfield group G isolates, N04a27 and N04AFT obtained as part of preclinical screening [ Study No. 6122K1-9000, Cross Sectional sequential Study, Australia, f. Their respective nucleotide sequences are set forth in SEQ ID NOs: 1.3, 5,7, 9, 25, 27, 29 and 31. Their respective amino acid sequences are set forth in SEQ ID NOs: 2. 4,6, 8, 10, 26, 28, 30 and 32. Preparation of the peptide set forth in SEQ ID NO: 2. 4,6, 8 and 10, and as SEQ ID NO: and 11, presenting.

Alignment and Clust of protein and nucleic acid sequences between novel group C or group G Streptococcus PPI and ORF554 sequences and known group A and group B Streptococcus PPI and ORF554 sequencesalW analysis (Chenna et al,Nuc.Acids Res.，313497-3500(2003)) provides the percentages summarized in Table 1.

Table 1: percent identity

A: amino acid identity

	GAS	GBS	SEQ ID NO：10
				GAS	100	56	76
GBS		100	57
				SEQ ID NO：10			100

B: nucleotide identity

	GAS	GBS	SEQ ID NO：9
				GAS	100	52	76
GBS		100	53
				SEQ ID NO：9			100

Example 2: antibodies to PPI epitopes of group C/G staphylococci

Binding of antibodies to bacteria, a process known as opsonization, can result in the uptake and killing of bacteria by phagocytic cells. Such antibodies, whether derived from a number of human or animal sources, or human or murine or chimeric monoclonal sources, and used alone or in combination, may be used in a prophylactic or therapeutic setting where BHS may be present in the bloodstream, such as neonatal sepsis or sepsis after surgery or abscess leakage.

Antibodies are raised in mice against recombinant group C or group G staphylococcal peptidyl prolyl isomerase polypeptides encoded by ORF 554. In screening these anti-beta hemolytic streptococcal antisera and monoclonal antibodies against multiple Beta Hemolytic Streptococcal (BHS) strains, it should be noted that certain antisera and antibodies were cross-reactive against many BHS strains, including streptococcus pyogenes (group a streptococci), streptococcus agalactiae (group B streptococci), and group C and group G streptococci (this includes streptococcus species streptococcus pharyngolaryngitis, constellar streptococcus, streptococcus intermedius, streptococcus dysgalactiae subspecies equi and dysgalactiae subspecies dysgalactiae) (table 2.) screening for antibodies was performed by Fluorescence Activated Cell Sorting (FACS). Briefly, heat killed streptococci were incubated with mouse anti-group C and group G streptococcal PPI antibodies for 45 minutes on ice followed by 2 washes. The streptococci were then incubated with goat anti-mouse Alexa-488 antibodies (Molecular Probes, Eugene, OR) for 30 minutes on ice followed by 2 washes. Thus the cross-reactivity of the treated cells when run on the FACS machine (see, e.g., DeMaster et al, infection. Immun., 70 (1): 350-359, 2002.) also means that group C or group G ORF554 or the polypeptide encoded thereby can be used in immunogenic compositions to induce an immune response that is effectively protected against infection by group A or group B streptococci as well as by group C or group G streptococci.

Table 2 describes the cross-reactivity of antisera and antibodies against PPIs from group C or group G streptococci encoded by ORF 554. According to table 2, the symbol "+" means that the antibody reacts with the antigen at least 3-fold over background; the symbol "+/-" means that the antibody reacts between 2-fold and 3-fold over background with antigen; and the symbol "-" means that the detection of antibody signal is at or below background.

Table 2: antibody cross-reactivity

Bacterial strains	Species (II)	Reactivity towards alpha PPI
			GAR 1165	Streptococcus pyogenes	+
GAR 1199	Streptococcus pyogenes	+
			GAR 1251	Streptococcus pyogenes	+
GAR 1278	Streptococcus pyogenes	+
			GAR 1362	Streptococcus pyogenes	+
GAR 1439	Streptococcus pyogenes	+
			GAR 1530	Streptococcus pyogenes	+
GAR 1566	Streptococcus pyogenes	+
			GAR 1672	Streptococcus pyogenes	+
GAR 1839	Streptococcus pyogenes	+
			GAR 1923	Streptococcus pyogenes	+
GAR 2107	Streptococcus pyogenes	+
			GAR 2330	Streptococcus pyogenes	+
GAR 2646	Streptococcus pyogenes	+
			GAR 2650	Streptococcus pyogenes	+
GAR 2869	Streptococcus pyogenes	+

GAR 3104	Streptococcus pyogenes	+
			GAR 3549	Streptococcus pyogenes	+
GAR 3784	Streptococcus pyogenes	+
			GAR 4029	Streptococcus pyogenes	+
GAR 4030	Streptococcus pyogenes	+
			GAR 4230	Streptococcus pyogenes	+
GAR 4773	Streptococcus pyogenes	+
			GAR 4983	Streptococcus pyogenes	+
GAR 4987	Streptococcus pyogenes	+
			GAR 5861	Streptococcus pyogenes	+
GAR 5991	Streptococcus pyogenes	+
			GAR 6084	Streptococcus pyogenes	+
GAR 7055	Streptococcus pyogenes	+
			GS20	Streptococcus pyogenes	+
GS21	Streptococcus pyogenes	+
			GS22	Streptococcus pyogenes	+
GS23	Streptococcus pyogenes	+
			GS24	Streptococcus pyogenes	+
GS25	Streptococcus pyogenes	+

GS26	Streptococcus pyogenes	+
			GS27	Streptococcus pyogenes	+
GS28	Streptococcus pyogenes	+
			GS29	Streptococcus pyogenes	+
GS30	Streptococcus pyogenes	+
			GS31	Streptococcus pyogenes	+/-
GS32	Streptococcus pyogenes	+
			GS33	Streptococcus pyogenes	+
GS34	Streptococcus pyogenes	+
			GS35	Streptococcus pyogenes	+
GS36	Streptococcus pyogenes	+/-
			GS37	Streptococcus pyogenes	+
GS38	Streptococcus pyogenes	+
			GS39	Streptococcus pyogenes	+
GS40	Streptococcus pyogenes	+
			GS41	Streptococcus pyogenes	+
GS42	Streptococcus pyogenes	+
			GS43	Streptococcus pyogenes	+
GS44	Streptococcus pyogenes	+

GS45	Streptococcus pyogenes	+
			GS46	Streptococcus pyogenes	+
GS47	Streptococcus pyogenes	+
			GS 48	Streptococcus pyogenes	+
GS 49	Streptococcus pyogenes	+
			GS 50	Streptococcus pyogenes	+
GS 51	Streptococcus pyogenes	+
			GS 52	Streptococcus pyogenes	+
GS 53	Streptococcus pyogenes	+
			GS 54	Streptococcus pyogenes	+/-
GS 55	Streptococcus pyogenes	+
			GS 56	Streptococcus pyogenes	+
GS 57	Streptococcus pyogenes	+
			GS 58	Streptococcus pyogenes	+
GS 59	Streptococcus pyogenes	+
			GS 60	Streptococcus pyogenes	+
GS 61	Streptococcus pyogenes	+
			GS 62	Streptococcus pyogenes	+
GS 63	Streptococcus pyogenes	+
			GS 64	Streptococcus pyogenes	+
GS 65	Streptococcus pyogenes	+
			GS 66	Streptococcus pyogenes	+
GAR 1	Streptococcus agalactiae	+
			GAR 1012	Streptococcus agalactiae	+/-
GAR 1023	Streptococcus agalactiae	-
			GAR 1049	Streptococcus agalactiae	-
GAR 10895	Streptococcus agalactiae	-
			GAR 1192	Streptococcus agalactiae	+/-
GAR 127	Streptococcus agalactiae	-
			GAR 12790	Streptococcus agalactiae	-
GAR 1305	Streptococcus agalactiae	-
			GAR 131	Streptococcus agalactiae	-

GAR 1355	Streptococcus agalactiae	-
			GAR 1446	Streptococcus agalactiae	-
GAR 1494	Streptococcus agalactiae	-
			GAR 154	Streptococcus agalactiae	+
GAR 176	Streptococcus agalactiae	-
			GAR 18	Streptococcus agalactiae	+

GAR 1844	Streptococcus agalactiae	-
			GAR 1931	Streptococcus agalactiae	-
GAR 2369	Streptococcus agalactiae	-
			GAR 252	Streptococcus agalactiae	-
GAR 2533	Streptococcus agalactiae	-
			GAR 2682	Streptococcus agalactiae	+
GAR 2717	Streptococcus agalactiae	-
			GAR 2723	Streptococcus agalactiae	-
GAR 2724	Streptococcus agalactiae	-
			GAR 2842	Streptococcus agalactiae	+/-
GAR 287	Streptococcus agalactiae	-
			GAR 3003	Streptococcus agalactiae	-
GAR 3751	Streptococcus agalactiae	-
			GAR 381	Streptococcus agalactiae	-
GAR 3830	Streptococcus agalactiae	-
			GAR 4131	Streptococcus agalactiae	+/-
GAR 4293	Streptococcus agalactiae	-
			GAR 4398	Streptococcus agalactiae	-
GAR 462	Streptococcus agalactiae	-
			GAR 4837	Streptococcus agalactiae	+/-
GAR 54	Streptococcus agalactiae	-
			GAR 562	Streptococcus agalactiae	+
GAR 6016	Streptococcus agalactiae	+
			GAR 614	Streptococcus agalactiae	+
GAR 63	Streptococcus agalactiae	+
			GAR 6332	Streptococcus agalactiae	+/-
GAR 6387	Streptococcus agalactiae	+
			GAR 6505	Streptococcus agalactiae	+/-
GAR 67	Streptococcus agalactiae	-
			GAR 864	Streptococcus agalactiae	+/-
GAR 967	Streptococcus agalactiae	-
			GS19	GGS	+/-
GS27	GGS	+/-
			ATCC 33397	Streptococcus aryngitis	+/-
ATCC 33397	Streptococcus aryngitis	-
			GAR 10823	Streptococcus aryngitis	+/-
GAR 1272	Streptococcus aryngitis	-
			GAR 1370	Streptococcus aryngitis	-

GAR 1425	Streptococcus aryngitis	+/-
			GAR 1592	Streptococcus aryngitis	-
GAR 1595	Streptococcus aryngitis	-
			GAR 2044	Streptococcus aryngitis	-
GAR 2523	Streptococcus aryngitis	-
			GAR 2565	Streptococcus aryngitis	-

GAR 2697	Streptococcus aryngitis	+/-
			GAR 2822	Streptococcus aryngitis	+/-
GAR 3091	Streptococcus aryngitis	-
			GAR 3560	Streptococcus aryngitis	+
GAR 3576	Streptococcus aryngitis	-
			GAR 3858	Streptococcus aryngitis	+/-
GAR 3938	Streptococcus aryngitis	-
			GAR 4133	Streptococcus aryngitis	-
GAR 4158	Streptococcus aryngitis	+
			GAR 4234	Streptococcus aryngitis	-
GAR 4426	Streptococcus aryngitis	+/-
			GAR 4680	Streptococcus aryngitis	+/-
GAR 4834	Streptococcus aryngitis	-
			GAR 4896	Streptococcus aryngitis	+
GAR 5093	Streptococcus aryngitis	-
			GAR 5094	Streptococcus aryngitis	+/-
GAR 5675	Streptococcus aryngitis	-
			GAR 5776	Streptococcus aryngitis	+
GAR 5831	Streptococcus aryngitis	-
			GAR 6187	Streptococcus aryngitis	+/-
GAR 6590	Streptococcus aryngitis	+/-
			GAR 7000	Streptococcus aryngitis	+/-
GAR 7023	Streptococcus aryngitis	+/-
			GAR 7190	Streptococcus aryngitis	-
GAR 7214	Streptococcus aryngitis	+
			GAR 7468	Streptococcus aryngitis	-
GAR 7818	Streptococcus aryngitis	+
			GAR 8620	Streptococcus aryngitis	+
GAR 8693	Streptococcus aryngitis	+/-
			GAR 8722	Streptococcus aryngitis	-
GAR 8736	Streptococcus aryngitis	-
			GAR 8954	Streptococcus aryngitis	+/-

ATCC 27823	Constellation streptococcus	+/-
			GAR 1235	Constellation streptococcus	-
GAR 1384	Constellation streptococcus	+
			GAR 1811	Constellation streptococcus	+
GAR 2421	Constellation streptococcus	+/-
			GAR 3145	Constellation streptococcus	-
GAR 3355	Constellation streptococcus	-
			GAR 4048	Constellation streptococcus	+/-
GAR 4083	Constellation streptococcus	+/-
			GAR 4861	Constellation streptococcus	+
GAR 4870	Constellation streptococcus	+/-
			GAR 5757	Constellation streptococcus	-
GAR 6129	Constellation streptococcus	+
			GAR 6147	Constellation streptococcus	-
GAR 6258	Constellation streptococcus	+/-
			GAR 7224	Constellation streptococcus	+
GAR 7369	Constellation streptococcus	+
			ATCC 12394	Streptococcus dysgalactiae	+
ATCC 12394	Streptococcus dysgalactiae	+

ATCC 40378	Streptococcus dysgalactiae	-
			ATCC 40378	Streptococcus dysgalactiae	-
GAR 3868	Streptococcus dysgalactiae	+/-
			GAR 4272	Streptococcus dysgalactiae	+
ATCC 35666	Streptococcus dysgalactiae subsp	+
			BAA-338	Streptococcus dysgalactiae subsp	-
GAR 3015	Streptococcus equisimilis	+
			ATCC 27335	Streptococcus intermedius	+
ATCC 27335	Streptococcus intermedius	+
			GAR 2407	Streptococcus intermedius	+/-
GS28	unk	+
			GS67	GGS/GCS	+
GS68	GGS/GCS	+/-
			GS69	GGS/GCS	+/-
GS70	GGS/GCS	+/-
			GS71	GGS/GCS	+
GS72	GGS/GCS	+
			GS73	GGS/GCS	+/-
GS74	GGS/GCS	-

GS75	GGS/GCS	+/-
			GS77	GGS/GCS	+
GS78	GGS/GCS	+/-
			GS79	GGS/GCS	+/-
GS80	GGS/GCS	-
			GS81	GGS/GCS	-
GS82	GGS/GCS	+/-
			GS83	GGS/GCS	+
GS84	GGS/GCS	-
			GS85	GGS/GCS	+/-
GS86	GGS/GCS	+/-
			GS88	GGS/GCS	+
GS89	GGS/GCS	+/-
			GS90	GGS/GCS	-
GS91	GGS/GCS	+/-
			GS92	GGS/GCS	+
GS93	GGS/GCS	+
			GS94	GGS/GCS	+

Claims (19)

1. An isolated polypeptide comprising an amino acid sequence that is substantially identical to SEQ id no: 2. SEQ ID NO: 4. SEQ ID NO: 6. SEQ ID NO: 8. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 26. SEQ ID NO: 28. SEQ ID NO: 30 and SEQ ID NO: 32 are at least 90% identical.

2. The isolated polypeptide of claim 1, wherein the polypeptide comprises SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. SEQ ID NO: 8. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 26. SEQ ID NO: 28. SEQ ID NO: 30 and SEQ ID NO: 32, or a pharmaceutically acceptable salt thereof.

3. An isolated polynucleotide encoding any one of the isolated polypeptides of claim 1 or claim 2.

4. The isolated polynucleotide of claim 3, wherein the polynucleotide comprises a nucleotide sequence identical to SEQ id no: 1. SEQ ID NO: 3. SEQ ID NO: 5. SEQ ID NO: 7. SEQ ID NO: 9. SEQ ID NO: 25. SEQ ID NO: 27. SEQ ID NO: 29 and SEQ ID NO: 31, or a nucleotide sequence at least 90% identical to any one or more of 31.

5. The isolated polynucleotide of claim 3 or claim 4, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 1. SEQ ID NO: 3. SEQ ID NO: 5. SEQ ID NO: 7. SEQ ID NO: 9. SEQ ID NO: 25. SEQ ID NO: 27. SEQ ID NO: 29 and SEQ ID NO: 31, or a nucleotide sequence of any one of.

6. The isolated polynucleotide of any one of claims 3-5, wherein the polynucleotide is operably linked to a regulatory element.

7. A polynucleotide vector comprising the isolated polynucleotide of any one of claims 3-6.

8. The polynucleotide vector of claim 7, wherein the polynucleotide vector is any one or more of a plasmid, a viral vector and an expression vector.

9. A cell comprising the isolated polynucleotide of any one of claims 3-6, or the polynucleotide vector of any one of claims 7 and 8, wherein the cell is ex vivo.

10. The cell of claim 9, wherein the cell is selected from the group consisting of a bacterium, a yeast cell, a mammalian cell, and an insect cell.

11. An immunogenic composition comprising the isolated polypeptide of claim 1 or claim 2.

12. An immunogenic composition comprising the isolated polynucleotide of any one of claims 3-6, or the polynucleotide vector of any one of claims 7 and 8.

13. A method for inducing an immune response against a beta hemolytic streptococcus bacterium or a beta hemolytic streptococcus infection in a patient comprising administering to the patient the immunogenic composition of claim 11 or claim 12.

14. The method of claim 13, wherein the beta hemolytic streptococcal bacterium or beta hemolytic streptococcal infection is from group a, group B, group C, or group G.

15. Use of an isolated polypeptide according to claim 1 or claim 2 in the manufacture of a medicament useful in the prophylactic treatment of a β -hemolytic streptococcal infection in a patient.

16. Use of the isolated polynucleotide of any one of claims 3-6 or the polynucleotide vector of any one of claims 7 and 8 in the manufacture of a medicament useful in the prophylactic treatment of a beta hemolytic streptococcal infection in a patient.

17. A kit comprising the isolated polypeptide of claim 1 or claim 2.

18. A kit comprising the isolated polynucleotide of any one of claims 3-6 or the polynucleotide vector of any one of claims 7 and 8.

19. A method of producing an isolated polypeptide comprising transforming, transfecting or infecting a cell with a plasmid comprising an isolated polynucleotide encoding the isolated polypeptide of claim 1 or claim 2 and culturing the cell under conditions that allow expression of the polypeptide by the cell and purifying the polypeptide from the cell.