The invention relates to subunit immunogenic or vaccine compositions which may comprise at least one polypeptide of Streptococcus equi and methods for preparing and/or formulating such compositions. The invention also relates to the use of such subunit compositions, such as a method for eliciting an immunogenic response or a protective immune response, which may comprise administering the composition to a mammal susceptible to streptococcal infection.

Description of the Invention

The present invention relates to the identification of polypeptides of Streptococcus equi able, when administered to a mammal, to elicit an immunogenic or immune response, and to the identification of polynucleotides encoding these polypeptides. These polypeptides are useful for the production of subunit immunogenic compositions or subunit vaccines. These polynucleotides are useful for the production of DNA immunogenic compositions, DNA vaccines, recombinant viral vector immunogenic compositions or recombinant viral vector vaccines.

The invention concerns compositions, uses and methods against infections caused by bacteria of the Streptococcal family, notably caused by Streptococcus equi, e.g. strangles disease in equine, camelid, canine and human, and against infections caused by Streptococcus zooepidemicus in equine, camelid, canine and human.

The invention concerns polypeptides, polynucleotides and genes obtained or derived from Streptococcus equi. The present invention may relate also to polypeptides, polynucleotides and genes obtained or derived from other streptococci, notably from Streptococcus zooepidemicus, Streptococcus suis, Streptococcus uberis, Streptococcus dysgalactiae, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes.

A particular aspect of the invention is a polypeptide selected from the amino acid sequences SEQ ID NOS: 18, 20, 22, 24, 26, 48, 50 and 52.

The whole genome of Streptococcus equi is available in the Sanger database (http://www.sanger.ac.uk/Projects/S_equi/).

The polypeptides are identified as SEQ ID NOS: 18, 20, 22, 24, 26, 48, 50 and 52 for Se50, Se1459, Se595, Se528, Se358, Se1631, Se1681 and S1a, respectively.

A particular aspect of the invention is a polynucleotide selected from the nucleotide sequences SEQ ID NOS: 17, 19, 21, 23 and 25.

The polynucleotides are identified as SEQ ID NOS: 17, 19, 21, 23 and 25 for Se50, Se1459, Se595, Se528 and Se358, respectively.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA technology, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press; DNA Cloning, Vols. I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Animal Cell Culture (R. K. Freshney ed. 1986); Immobilized Cells and Enzymes (IRL press, 1986); Perbal, B., A Practical Guide to Molecular Cloning (1984); the series, Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); and Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell eds., 1986, Blackwell Scientific Publications).

Before describing the present invention in detail, it is to be understood that this invention is not limited to particular DNA, polypeptide sequences or process parameters as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.

The terms "immunogenic" protein or polypeptide as used herein also refers to an amino acid sequence which elicits an immunological response as described above. An "immunogenic" protein or polypeptide, as used herein, includes the full-length sequence of the protein, analogs thereof, or immunogenic fragments thereof. By "immunogenic fragment" is meant a fragment of a protein which includes one or more epitopes and thus elicits the immunological response described above. Such fragments can be identified using any number of epitope mapping techniques, well known in the art. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, N.J. For example, linear epitopes may be determined by e.g., concurrently synthesizing large numbers of peptides on solid supports, the peptides corresponding to portions of the protein molecule, and reacting the peptides with antibodies while the peptides are still attached to the supports. Such techniques are known in the art and described in, e.g., U.S. Pat. No. 4,708,871; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81:3998-4002; Geysen et al. (1986) Molec. Immunol. 23:709-715, all incorporated herein by reference in their entireties. Similarly, conformational epitopes are readily identified by determining spatial conformation of amino acids such as by, e.g., x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols, supra.

Synthetic antigens are also included within the definition, for example, polyepitopes, flanking epitopes, and other recombinant antigens. See, e.g., Bergmann et al. (1993) Eur. J. Immunol. 23:2777-2781; Bergmann et al. (1996) J. Immunol. 157:3242-3249; Suhrbier, A. (1997) Immunol. and Cell Biol. 75:402-408; Gardner et al. (1998) 12th World AIDS Conference, Geneva, Switzerland, Jun. 28-Jul. 3, 1998. Immunogenic fragments, for purposes of the present invention, will usually include at least about 3 amino acids, preferably at least about 5 amino acids, more preferably at least about 10-15 amino acids, and most preferably 25 or more amino acids, of the molecule. There is no critical upper limit to the length of the fragment, which could comprise nearly the full-length of the protein sequence, or even a fusion protein comprising at least one epitope of the protein.

Accordingly, a minimum structure of a polynucleotide expressing an epitope is that it comprises or consists essentially of or consists of nucleotides to encode an epitope or antigenic determinant of the protein or polyprotein of interest. A polynucleotide encoding a fragment of the total protein or polyprotein, more advantageously, comprises or consists essentially of or consists of a minimum of 21 nucleotides, advantageously at least 42 nucleotides, and preferably at least 57, 87 or 150 consecutive or contiguous nucleotides of the sequence encoding the total protein or polyprotein. Epitope determination procedures, such as, generating overlapping peptide libraries (Hemmer B. et al., Immunology Today, 1998, 19 (4), 163-168), Pepscan (Geysen et al., (1984) Proc. Nat. Acad. Sci. USA, 81, 3998-4002; Geysen et al., (1985) Proc. Nat. Acad. Sci. USA, 82, 178-182; Van der Zee R. et al., (1989) Eur. J. Immunol., 19, 43-47; Geysen H. M., (1990) Southeast Asian J. Trop. Med. Public Health, 21, 523-533; Multipin.RTM.. Peptide Synthesis Kits de Chiron) and algorithms (De Groot A. et al., (1999) Nature Biotechnology, 17, 533-561), and in PCT Application Serial No. PCT/US2004/022605 all of which are incorporated herein by reference in their entireties, can be used in the practice of the invention, without undue experimentation. Other documents cited and incorporated herein may also be consulted for methods for determining epitopes of an immunogen or antigen and thus nucleic acid molecules that encode such epitopes.

In one embodiment, the fragments of polypeptide are selected from the sequences SEQ ID NOS: 32, 34, 36, 38, 40, 54, 56 and 58, which are respectively encoded by the nucleotide sequences SEQ ID NOS: 31, 33, 35, 37, 39, 53, 55 and 57. The fragments of polypeptides SEQ ID NOS: 32, 34, 36, 38, 40, 54, 56 and 58 are part of Se50, Se1459, Se595, Se528, Se358, Se1631, Se1681 and Sla, respectively.

The polynucleotides encoding polypeptides according to the present invention, analogs thereof or fragments thereof are inserted into a vector, linked to regulatory elements such as promoter, ribosome binding region and terminator, and start codon and stop codon.

The polypeptides, analogs thereof and fragments thereof can be produced by in vitro expression in host cells. The in vitro expression vectors are expression vectors used for the in vitro expression of proteins in an appropriate cell system. This can be produced in prokaryotic host cells, i.e. in Escherichia coli (Mahona F et al., Biochimie 1994, 46(1): 9-14; Watt M A et al., Cell Stress Chaperones 1997, 2(3): 180-90; Frey J Res. Microbiol. 1992, 143(3): 263-9) or in Lactobacillus (Seegers J F, Trends Biotechnol. 2002, 20(12): 508-515; Pouwels P H et al, Methods Enzymol. 2001, 336: 369-389), or in eukaryotic host cells, i.e. in yeast (Gerngross T U, Nat. Biotechnol. 2004, 22(11): 1409-1414; Malissard M et al., Glycoconj J. 1999, 16(2): 125-139), in insect cells (Oker-Blom C et al., Brief Funct Genomic Proteomic. 2003, 2(3), 244-253), mammal cells or avian cells.

For Escherichia coli, different strains can be used, notably BL21 (DE3) strain (Novagen or Invitrogen; see Hedayati M A et al., Protein Expr Purif, 2005, 43(2): 133-139) Origami2(DE3) strain (Novagen or Invitrogen), Y1089 (Galan J E et al., Infect Immun, 1987, 55(12): 3181-3187). Vectors are advantageously plasmids, i.e. pGEX plasmids (GE Healthcare), pET plasmids (Novagen or Invitrogen) (Jiang X Y et al., J Biochem Mol. Biol. 2006, 39(1): 22-25; Hedayati M A et al., Protein Expr Purif, 2005, 43(2): 133-139; Jedrzejas M J et al., Protein Expr Purif, 1998, 13(1): 83-89). Vectors can also be a virus, notably a bacteriophage, i.e. lambda-gt11 phage (Galan J E et al., Infect Immun, 1987, 55(12): 3181-3187). Another approach is to use fused genes for the production of chimeric proteins between a Streptococcus protein and a Escherichia coli protein, notably lipoprotein (Cullen P A et al., Plasmid, 2003, 49(1): 18-29), or with GST (Zhao G et al., Protein Expr Purif, 1999, 16(2): 331-339). The Streptococcal insert can be linked to promoter, i.e. bacteriophage T7 promoter (Yamamoto M et al., FEMS Microbiol Lett, 1995, 132(3): 209-213), signal peptide sequence, i.e. Borrelia burgdorferi outer surface protein A signal peptide (De B K et al., Vaccine, 2000, 18(17): 1811-1821), an Escherichia coli major outer membrane lipoprotein signal sequence (Cullen P A et al., Plasmid, 2003, 49(1): 18-29).

For Lactobacillus, different strains can be used, notably Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus casei (Seegers J F, Trends Biotechnol., 2002, 20(12): 508-515). Promoters and regulatory genes involved in production of sakacin P are suitable for establishing inducible high-level gene expression in Lactobacillus (Mathiesen G et al., Lett Appl Microbiol., 2004, 39(2): 137-143), or lactose operon promoter (Oliveira M L et al., FEMS Microbiol Lett., 2003, 227(1): 25-31). Another approach is to use fused genes for the production of chimeric proteins between a Lactobacillus protein and a Streptococcus protein (Hung J et al., FEMS Microbiol Lett., 2002, 211(1): 71-75).

For in vitro expression in insect cells, vectors are advantageously viruses, i.e. baculoviruses (see, e.g., U.S. Pat. No. 4,745,051; Vialard J. et al., J. Virol., 1990 64 (1), 37-50; Verne A., Virology, 1988, 167, 56-71; Oker-Blom C et al., Brief Funct Genomic Proteomic. 2003, 2(3), 244-253), e.g. Autographa californica Nuclear Polyhedrosis Virus AcNPV, and insect cells are Sf9 Spodoptera frugiperda cells (ATCC CRL 1711; see also U.S. Pat. Nos. 6,228,846, 6,103,526). Protein production can take place by the transfection of mammalian cells by plasmids, by replication or expression without productive replication of viral vectors on mammal cells or avian cells. Mammalian cells which can be used are advantageously hamster cells (e.g. CHO or BHK-21) or monkey cells (e.g. COS or VERO) or bovine cells (e.g. MDBK), i.e. culture of EHV-1 vectors in MDBK cells (Ibrahim el S M et al., Microbiol. Immunol., 2004, 48(11): 831-842) or in Vero cells (U.S. Pat. No. 4,110,433), or culture of VEEV replicons in BHK cells (Lee J S et al., Infect. Immun., 2001, 69(9): 5709-5715).

It is understood to one of skill in the art that conditions for culturing in vitro a host cell varies according to the particular gene and that routine experimentation is necessary at times to determine the optimal conditions for culturing a protein depending on the host cell. A "host cell" denotes a prokaryotic or eukaryotic cell that has been genetically altered, or is capable of being genetically altered by administration of an exogenous polynucleotide, such as a recombinant plasmid or vector. When referring to genetically altered cells, the term refers both to the originally altered cell and to the progeny thereof.

The in vitro expression vectors can be introduced into a suitable host cell for replication and amplification. The vectors containing the polynucleotides of interest can be introduced into the host cell by any of a number of appropriate means, including direct uptake, endocytosis, transfection, f-mating, electroporation, transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; microprojectile bombardment; lipofection; and infection (where the vector is infectious, for instance, a retroviral vector). The choice of introducing vectors or polynucleotides will often depend on features of the host cell.

The expressed proteins can be harvested in or from the culture supernatant after, or not after secretion (if there is no secretion a cell lysis typically occurs or is performed), optionally concentrated by concentration methods such as ultrafiltration and/or purified by purification means, such as affinity, ion exchange or gel filtration-type chromatography methods, notably by affinity chromatography, i.e. using Ni NTA Superflow (Qiagen) or Ni Sepharose fastflow (Amersham), or by gel-filtration, i.e. using Sephacryl.RTM. or Superdex.RTM. (Amersham GE Healthcare).

The polypeptides and fragments thereof can also be obtained by extraction, notably acid extraction or mutanolysin extraction (Boschwitz J S et al., Cornell Vet., 1991, 81(1): 25-36), and purification, notably immunoprecipitation (Erickson E D et al., Can J Comp Med., 1975, 39(2): 110-115), from crude culture of streptococcal bacteria, notably of Streptococcus equi.

The polypeptides and fragments thereof can also be synthesised chemically (Luo Y et al., Vaccine 1999, 17(7-8): 821-31).

By "subunit vaccine composition" is meant a composition containing at least one immunogenic polypeptide or a polynucleotide able to express at least one, but not all, antigen derived from or homologous to an antigen from a pathogen of interest. Such a composition is substantially free of intact pathogen cells or particles, or the lysate of such cells or particles. Thus, a "subunit vaccine composition" is prepared from at least partially purified (preferably substantially purified) immunogenic polypeptides from the pathogen, or recombinant analogs thereof. A subunit vaccine composition can comprise the subunit antigen or antigens of interest substantially free of other antigens or polypeptides from the pathogen.

An object of the invention is a subunit immunogenic composition or subunit vaccine comprising at least one polypeptide, analog thereof or fragment thereof according to the invention, and a pharmaceutically acceptable excipient, diluent or vehicle, and optionally an adjuvant and/or a stabilizer.

The subunit immunogenic or vaccine composition can comprise at least one polypeptide selected from the group consisting of sequences SEQ ID NOS: 18, 20, 22, 24, 26, 48, 50 and 52, or analogs thereof, or fragments thereof. The subunit immunogenic or vaccine composition can advantageously comprise two or three or four or five polypeptides selected from the group consisting of sequences SEQ ID NOS: 18, 20, 22, 24, 26, 48, 50 and 52, or analogs thereof, or fragments thereof.

The subunit immunogenic or vaccine composition can comprise at least one polypeptide selected from the group consisting of sequences SEQ ID NOS: 32, 34, 36, 38, 40, 54, 56 and 58, or analogs thereof.

The subunit immunogenic or vaccine composition can advantageously comprise two or three or four or five polypeptides selected from the group consisting of sequences SEQ ID NOS: 32, 34, 36, 38, 40, 54, 56 and 58, or analogs thereof.

Another object of the invention is a recombinant immunogenic composition or vaccine comprising at least one recombinant in vivo expression vector, and a pharmaceutically acceptable excipient, diluent or vehicle, and optionally an adjuvant and/or a stabilizer. The recombinant in vivo expression vectors are vectors comprising polynucleotides or their fragments inserted hereinto and able to express in vivo in the targeted mammal the polypeptide encoded by this polynucleotide or fragment thereof. The vectors can be a polynucleotide vectors or plasmids (EP-A2-1001025; Chaudhuri P Res. Vet. Sci. 2001, 70(3), 255-6) for DNA immunogenic compositions and DNA vaccines, or can be viruses (e.g. herpesvirus such as equine herpesvirus type 1 (Trapp S et al., J. Virol. 2005, 79(9): 5445-5454), equine herpesvirus type 2, equine herpesvirus type 4; poxvirus virus such as vaccinia virus or avipox virus, like fowlpox (U.S. Pat. No. 5,174,993 U.S. Pat. No. 5,505,941 and U.S. Pat. No. 5,766,599) or canarypox (U.S. Pat. No. 5,756,103); adenovirus such as human adenovirus (Chroboczek J et al., Virol. 1992, 186: 280-285); encephalitis virus such as venezuelean equine encephalitis virus (Pushko P et al. Virol. 1997, 239: 389-401; Davis N L et al. J. Virol. 2000, 74: 371-378)) for recombinant viral vector immunogenic compositions and recombinant viral vector vaccines. In a further embodiment, the polynucleotides or their fragments may be inserted into recombinant in vivo expression bacterial vectors, notably into Salmonella, to produce live recombinant bacterial immunogenic compositions or vaccines.

The term plasmid covers any DNA transcription unit comprising a polynucleotide according to the invention and the elements necessary for its in vivo expression in a cell or cells of the desired host or target; and, in this regard, it is noted that a supercoiled or non-supercoiled, circular plasmid, as well as a linear form, are intended to be within the scope of the invention. In a specific, non-limiting example, the pVR1020 or 1012 plasmid (VICAL Inc.; Luke C. et al., Journal of Infectious Diseases, 1997, 175, 91-97; Hartikka J. et al., Human Gene Therapy, 1996, 7, 1205-1217) can be utilized as a vector for the insertion of a polynucleotide sequence. The pVR1020 plasmid is derived from pVR1012 and contains the human tPA signal sequence. Each plasmid comprises or contains or consists essentially of, in addition to the polynucleotide or analog thereof or fragment thereof, operably linked to a promoter or under the control of a promoter or dependent upon a promoter. In general, it is advantageous to employ a strong promoter functional in eukaryotic cells. The preferred strong promoter is the immediate early cytomegalovirus promoter (CMV-IE) of human or murine origin, or optionally having another origin such as the rat or guinea pig. The CMV-IE promoter can comprise the actual promoter part, which may or may not be associated with the enhancer part. Reference can be made to EP-A-260 148, EP-A-323 597, U.S. Pat. Nos. 5,168,062, 5,385,839, and 4,968,615, as well as to PCT Application No WO-A-87/03905. The CMV-IE promoter is advantageously a human CMV-IE (Boshart M. et al., Cell, 1985, 41, 521-530) or murine CMV-IE. In more general terms, the promoter has either a viral or a cellular origin. A strong viral promoter other than CMV-IE that may be usefully employed in the practice of the invention is the early/late promoter of the SV40 virus or the LTR promoter of the Rous sarcoma virus. A strong cellular promoter that may be usefully employed in the practice of the invention is the promoter of a gene of the cytoskeleton, such as e.g. the desmin promoter (Kwissa M. et al., Vaccine, 2000, 18, 2337-2344), or the actin promoter (Miyazaki J. et al., Gene, 1989, 79, 269-277). Functional sub fragments of these promoters, i.e., portions of these promoters that maintain an adequate promoting activity, are included within the present invention, e.g. truncated CMV-IE promoters according to PCT Application No. WO-A-98/00166 or U.S. Pat. No. 6,156,567 can be used in the practice of the invention. A promoter in the practice of the invention consequently includes derivatives and sub fragments of a full-length promoter that maintain an adequate promoting activity and hence function as a promoter, preferably promoting activity substantially similar to that of the actual or full-length promoter from which the derivative or sub fragment is derived, e.g., akin to the activity of the truncated CMV-IE promoters of U.S. Pat. No. 6,156,567 to the activity of full-length CMV-IE promoters. Thus, a CMV-IE promoter in the practice of the invention can comprise or consist essentially of or consist of the promoter portion of the full-length promoter and/or the enhancer portion of the full-length promoter, as well as derivatives and sub fragments. Advantageously, the plasmids comprise or consist essentially of other expression control elements. It is particularly advantageous to incorporate stabilizing sequence(s), e.g., intron sequence(s), preferably the first intron of the hCMV-IE (PCT Application No. WO-A-89/01036), the intron II of the rabbit .beta.-globin gene (van Ooyen et al., Science, 1979, 206, 337-344). As to the polyadenylation signal (polyA) for the plasmids and viral vectors other than poxviruses, use can more be made of the poly(A) signal of the bovine growth hormone (bGH) gene (see U.S. Pat. No. 5,122,458), or the poly(A) signal of the rabbit .beta.-globin gene or the poly(A) signal of the SV40 virus.

For recombinant vectors based on equine herpesvirus, in particular attenuated strains (i.e. EHV-1 KyA strains (Zhang Y et al., Virology, 2000, 268(2): 482-492)) or attenuated by mutation or deletion of genes involve in pathogenicity (Kirisawa R et al., Vet. Microbiol., 2003, 95(3): 159-174) or by serial culture passages (U.S. Pat. No. 4,110,433) can be used. For EHV-1, the insertion can be made in the intergenic region between ORF 62 and ORF 63 (Ibrahim el S M et al., Microbiol. Immunol., 2004, 48(11): 831-842; Csellner H et al., Arch. Virol., 1998, 143(11): 2215-2231), or in the genes, eventually after a partial or complete deletion of the genes, i.e. in thymidine kinase (TK) gene, in genes 1 or 71 (Kirisawa R et al., Vet. Microbiol., 2003, 95(3): 159-174), in glycoprotein I gene and glycoprotein E gene (Matsumura T. et al., Virology, 1998, 242(1): 68-79), in IR6 protein gene (Osterrieder N et al., Virology, 1996, 217(2): 442-451), in gene 15 (EP-B1-0,668,355). For EHV-4, the insertion can be made in glycoprotein I gene and glycoprotein E gene (Damiani A M et al., Virus Res., 2000, 67(2): 189-202), in US2 or TK or glycoprotein E gene (U.S. Pat. Nos. 5,741,696; 5,731,188), eventually after a partial or complete deletion of the genes. In one embodiment the polynucleotide to be expressed is inserted under the control of a promoter functional in eukaryotic cells, advantageously a CMV-IE promoter (murine or human). A poly(A) sequence and terminator sequence can be inserted downstream the polynucleotide to be expressed, e.g. bovine growth hormone or a rabbit .beta.-globin gene polyadenylation signal.

For recombinant vector based on poxvirus vector, a vaccinia virus or an attenuated vaccinia virus, (for instance, MVA, a modified Ankara strain obtained after more than 570 passages of the Ankara vaccine strain on chicken embryo fibroblasts; see Stickl & Hochstein-Mintzel, Munch. Med. Wschr., 1971, 113, 1149-1153; Sutter et al., Proc. Natl. Acad. Sci. U.S.A., 1992, 89, 10847-10851; available as ATCC VR-1508; or NYVAC, see U.S. Pat. No. 5,494,807, for instance, Examples 1 to 6 and et seq of U.S. Pat. No. 5,494,807 which discuss the construction of NYVAC, as well as variations of NYVAC with additional ORFs deleted from the Copenhagen strain vaccinia virus genome, as well as the insertion of heterologous coding nucleic acid molecules into sites of this recombinant, and also, the use of matched promoters; see also WO-A-96/40241), an avipox virus or an attenuated avipox virus (e.g., canarypox, fowlpox, dovepox, pigeonpox, quailpox, ALVAC or TROVAC; see, e.g., U.S. Pat. Nos. 5,505,941, 5,494,807) can be used. Attenuated canarypox viruses are described in U.S. Pat. No. 5,756,103 (ALVAC) and WO-A-01/05934. Reference is also made to U.S. Pat. No. 5,766,599 which pertains to the attenuated fowlpox strain TROVAC. Reference is made to the canarypox available from the ATCC under access number VR-111. Numerous fowlpox virus vaccination strains are also available, e.g. the DIFTOSEC CT strain marketed by MERIAL and the NOBILIS VARIOLE vaccine marketed by INTERVET. For information on the method to generate recombinants thereof and how to administer recombinants thereof, the skilled artisan can refer documents cited herein and to WO-A-90/12882, e.g., as to vaccinia virus mention is made of U.S. Pat. Nos. 4,769,330, 4,722,848, 4,603,112, 5,110,587, 5,494,807, and 5,762,938 inter alia; as to fowlpox, mention is made of U.S. Pat. Nos. 5,174,993, 5,505,941 and 5,766,599 inter alia; as to canarypox mention is made of U.S. Pat. No. 5,756,103 inter alia. When the expression vector is a vaccinia virus, insertion site or sites for the polynucleotide or polynucleotides to be expressed are advantageously at the thymidine kinase (TK) gene or insertion site, the hemagglutinin (HA) gene or insertion site, the region encoding the inclusion body of the A type (ATI); see also documents cited herein, especially those pertaining to vaccinia virus. In the case of canarypox, advantageously the insertion site or sites are ORF(s) C3, C5 and/or C6; see also documents cited herein, especially those pertaining to canarypox virus. In the case of fowlpox, advantageously the insertion site or sites are ORFs F7 and/or F8; see also documents cited herein, especially those pertaining to fowlpox virus. The insertion site or sites for MVA virus area advantageously as in various publications, including Carroll M. W. et al., Vaccine, 1997, 15 (4), 387-394; Stittelaar K. J. et al., J. Virol., 2000, 74 (9), 4236-4243; Sutter G. et al., 1994, Vaccine, 12 (11), 1032-1040; and, in this regard it is also noted that the complete MVA genome is described in Antoine G., Virology, 1998, 244, 365-396, which enables the skilled artisan to use other insertion sites or other promoters. Advantageously, the polynucleotide to be expressed is inserted under the control of a specific poxvirus promoter, e.g., the vaccinia promoter 7.5 kDa (Cochran et al., J. Virology, 1985, 54, 30-35), the vaccinia promoter I3L (Riviere et al., J. Virology, 1992, 66, 3424-3434), the vaccinia promoter HA (Shida, Virology, 1986, 150, 451-457), the cowpox promoter ATI (Funahashi et al., J. Gen. Virol., 1988, 69, 35-47), the vaccinia promoter H6 (Taylor J. et al., Vaccine, 1988, 6, 504-508; Guo P. et al. J. Virol., 1989, 63, 4189-4198; Perkus M. et al., J. Virol., 1989, 63, 3829-3836), inter alia.

For recombinant vector based on adenovirus vector, a human adenovirus (HAV), advantageously, a human adenovirus serotype 5 (Ad5) vector, an E1-deleted and/or disrupted adenovirus, an E3-deleted and/or disrupted adenovirus or an E1- and E3-deleted and/or disrupted adenovirus can be used. Optionally, E4 may be deleted and/or disrupted from any of the adenoviruses described above. For example, the human Ad5 vectors described in Yarosh et al. and Lutze-Wallace et al. can be used (see, e.g., Yarosh et al., Vaccine. 1996 September; 14(13):1257-64 and Lutze-Wallace et al., Biologicals. 1995 December; 23(4):271-7). In one embodiment the viral vector is a human adenovirus, in particular a serotype 5 adenovirus, rendered incompetent for replication by a deletion in the E1 region of the viral genome. The deleted adenovirus is propagated in E1-expressing 293 cells or PER cells, in particular PER.C6 (F. Falloux et al Human Gene Therapy 1998, 9, 1909-1917). The human adenovirus can be deleted in the E3 region eventually in combination with a deletion in the E1 region (see, e.g. J. Shriver et al. Nature, 2002, 415, 331-335, F. Graham et al Methods in Molecular Biology Vol 0.7: Gene Transfer and Expression Protocols Edited by E. Murray, The Human Press Inc, 1991, p 109-128; Y. Ilan et al Proc. Natl. Acad. Sci. 1997, 94, 2587-2592; S. Tripathy et al Proc. Natl. Acad. Sci. 1994, 91, 11557-11561; B. Tapnell Adv. Drug Deliv. Rev. 1993, 12, 185-199; X. Danthinne et al Gene Therapy 2000, 7, 1707-1714; K. Berkner Bio Techniques 1988, 6, 616-629; K. Berkner et al Nucl. Acid Res. 1983, 11, 6003-6020; C. Chavier et al J. Virol. 1996, 70, 4805-4810). The insertion sites can be the E1 and/or E3 loci eventually after a partial or complete deletion of the E1 and/or E3 regions. Advantageously, when the expression vector is an adenovirus, the polynucleotide to be expressed is inserted under the control of a promoter functional in eukaryotic cells, such as a strong promoter, preferably a cytomegalovirus immediate-early gene promoter (CMV-IE promoter). The CMV-IE promoter is advantageously of murine or human origin. The promoter of the elongation factor 1.alpha. can also be used. A muscle specific promoter can also be used (X. Li et al Nat. Biotechnol. 1999, 17, 241-245). Strong promoters are also discussed herein in relation to plasmid vectors. A poly(A) sequence and terminator sequence can be inserted downstream of the polynucleotide to be expressed, e.g. a bovine growth hormone gene or a rabbit .beta.-globin gene polyadenylation signal.

For recombinant vector based on Encephalitis virus, a venezuelean equine encephalitis virus (VEEV) can be used (Nelson E L et al., Breast Cancer Res. Treat., 2003, 82(3): 169-183), in particular as a replicon, that is to say as a self-replicating RNA containing all of the VEEV non-structural genes and a multiple-cloning site in place of the VEEV structural genes (Lee J S et al., Infect. Immun., 2003, 71(3): 1491-1496; Velders M P et al., Cancer Res., 2001, 61(21): 7861-7867). The polynucleotides to be expressed are inserted into this multiple-cloning site, optionally linked to the nucleotide sequence encoding a secretory sequence or a tissue plasminogen activator secretory sequence. The polynucleotides to be expressed can also be inserted downstream of the subgenomic 26S promoter in place of the viral VEEV structural genes (Lee J S et al., Infect. Immun., 2001, 69(9): 5709-5715; Pushko P et al., Vaccine, 2000, 19(1): 142-153).

For recombinant vectors based on bacteria, Salmonella, notably Salmonella typhimurium (Yang X L et al., Biomed. Environ. Sci., 2005, 18(6): 411-418; Dunstan S J et al., FEMS Immunol. Med. Microbiol., 2003, 37(2-3): 111-119), Salmonella typhi (Santiago-Machuca A E et al., Plasmid., 2002, 47(2): 108-119) can be used. The polynucleotides to be expressed can be inserted into the flagellin gene of Salmonella (Chauhan N et al., Mol Cell Biochem., 2005, 276(1-2): 1-6), or into the aroC gene (Santiago-Machuca A E et al., Plasmid., 2002, 47(2): 108-119). The polynucleotides to be expressed can also be inserted under the control of the anaerobically inducible nirB promoter (Santiago-Machuca A E et al., Plasmid., 2002, 47(2): 108-119).

The recombinant immunogenic or vaccine composition can contain at least one recombinant expression vector comprising at least one polynucleotide encoding a polypeptide selected from the group consisting of sequences SEQ ID NOS: 18, 20, 22, 24, 26, 48, 50 and 52, or analogs thereof or fragments thereof. The recombinant immunogenic or vaccine composition can contain at least one recombinant expression vector comprising at least one polynucleotide encoding a polypeptide selected from the group consisting of sequences SEQ ID NOS: 32, 34, 36, 38, 40, 54, 56 and 58, or analogs thereof. The recombinant immunogenic or vaccine composition can advantageously contain at least one recombinant expression vector comprising at least one polynucleotide encoding two or three or four or five polypeptides selected from the group consisting of sequences SEQ ID NOS: 32, 34, 36, 38, 40, 54, 56 and 58, or analogs thereof.

The vectors of the invention may further comprise at least one heterologous polynucleotide. This is useful for reproducing or replicating heterologous polynucleotides and/or for expression of heterologous polynucleotides, either in vivo or in vitro. Such vectors are also useful for preparing multivalent immunogenic or vaccine compositions, notably multivalent DNA immunogenic compositions or vaccines and multivalent recombinant viral vector immunogenic compositions or vaccines. The heterologous nucleic acid sequence advantageously codes for an immunogen, antigen or epitope from a pathogenic viral, parasitic or bacterial agent, such bacterial agent is different from the streptococcal bacteria at the origin of the polynucleotides encoding the polypeptides according to the invention. This heterologous sequence may encode an immunogen, antigen or epitope from western equine encephalitis virus (WEEV), eastern equine encephalitis virus (EEEV), venezuelean equine encephalitis virus (VEEV), equine influenza virus, equine herpesvirus type 1 (EHV-1), equine herpesvirus type 2 (EHV-2), equine herpesvirus type 4 (EHV-4), Equine Artheritis virus (EAV), West Nile virus (WNV), tetanus, rhodococcus. In a particular embodiment, the heterologous sequence may encode an immunogen, antigen or epitope from equine influenza virus and from EHV-1 and/or EHV-4. An immunogen or antigen is a protein or polypeptide able to induce an immune response against the pathogenic agent or a secreted antigen of the pathogenic agent, and contains one or more epitopes; an epitope is a peptide or polypeptide which is able to induce an immune response against the pathogenic agent or a secreted antigen of the pathogenic agent.

Optionally, the subunit immunogenic composition or vaccine of the invention can be combined with one or more immunogens, antigens or epitopes selected from other pathogenic micro-organisms or viruses to form a multivalent subunit immunogenic composition or vaccine. For the equine, such a multivalent subunit immunogenic composition or vaccine may comprises at least one polypeptide according to the present invention and at least one immunogen, antigen or epitope from WEEV, EEEV, VEEV, equine influenza virus, EHV-1, EHV-4, EAV, WNV, tetanus, rhodococcus. In a particular embodiment, such a multivalent subunit immunogenic composition or vaccine may comprises at least one polypeptide according to the present invention and at least one immunogen, antigen or epitope from equine influenza virus and from EHV-1 and/or EHV-4.

The pharmaceutically or veterinary acceptable excipient, diluent or vehicle may be water or saline, buffer but it may, for example, also comprise Streptococcus culture medium.

The pharmaceutically or veterinarily acceptable carriers or vehicles or excipients are well known to the one skilled in the art. For example, a pharmaceutically or veterinarily acceptable carrier or vehicle or excipient can be a 0.9% NaCl (e.g., saline) solution or a phosphate buffer. Other pharmaceutically or veterinarily acceptable carrier or vehicle or excipients that can be used for methods of this invention include, but are not limited to, poly-(L-glutamate) or polyvinylpyrrolidone. The pharmaceutically or veterinarily acceptable carrier or vehicle or excipients may be any compound or combination of compounds facilitating the administration of the vector (or protein expressed from an inventive vector in vitro); advantageously, the carrier, vehicle or excipient may facilitate transfection and/or improve preservation of the vector (or protein). Doses and dose volumes are herein discussed in the general description and can also be determined by the skilled artisan from this disclosure read in conjunction with the knowledge in the art, without any undue experimentation.

The cationic lipids containing a quaternary ammonium salt which are advantageously but not exclusively suitable for plasmids, are advantageously those having the following formula -- see Original Patent.

Among these cationic lipids, preference is given to DMRIE (N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propane ammonium; WO96/34109), advantageously associated with a neutral lipid, advantageously DOPE (dioleoyl-phosphatidyl-ethanol amine; Behr J. P., 1994, Bioconjugate Chemistry, 5, 382-389), to form DMRIE-DOPE.

Advantageously, the plasmid mixture with the adjuvant is formed extemporaneously and advantageously contemporaneously with administration of the preparation or shortly before administration of the preparation; for instance, shortly before or prior to administration, the plasmid-adjuvant mixture is formed, advantageously so as to give enough time prior to administration for the mixture to form a complex, e.g. between about 10 and about 60 minutes prior to administration, such as approximately 30 minutes prior to administration.

When DOPE is present, the DMRIE:DOPE molar ratio is advantageously about 95:about 5 to about 5:about 95, more advantageously about 1:about 1, e.g., 1:1.

The DMRIE or DMRIE-DOPE adjuvant:plasmid weight ratio can be between about 50:about 1 and about 1:about 10, such as about 10:about 1 and about 1:about 5, and advantageously about 1:about 1 and about 1:about 2, e.g., 1:1 and 1:2.

The immunogenic compositions and vaccines according to the invention may comprise or consist essentially of one or more adjuvants. Suitable adjuvants for use in the practice of the present invention are (1) polymers of acrylic or methacrylic acid, maleic anhydride and alkenyl derivative polymers, (2) immunostimulating sequences (ISS), such as oligodeoxyribonucleotide sequences having one or more non-methylated CpG units (Klinman et al., Proc. Natl. Acad. Sci., USA, 1996, 93, 2879-2883; WO98/16247), (3) an oil in water emulsion, such as the SPT emulsion described on p 147 of "Vaccine Design, The Subunit and Adjuvant Approach" published by M. Powell, M. Newman, Plenum Press 1995, and the emulsion MF59 described on p 183 of the same work, (4) cation lipids containing a quaternary ammonium salt, e.g., DDA (5) cytokines, (6) aluminum hydroxide or aluminum phosphate, (7) saponin or (8) other adjuvants discussed in any document cited and incorporated by reference into the instant application, or (9) any combinations or mixtures thereof.

The oil in water emulsion (3), which is especially appropriate for viral vectors, can be based on: light liquid paraffin oil (European pharmacopoeia type), isoprenoid oil such as squalane, squalene, oil resulting from the oligomerization of alkenes, e.g. isobutene or decene, esters of acids or alcohols having a straight-chain alkyl group, such as vegetable oils, ethyl oleate, propylene glycol, di(caprylate/caprate), glycerol tri(caprylate/caprate) and propylene glycol dioleate, or esters of branched, fatty alcohols or acids, especially isostearic acid esters.

The oil is used in combination with emulsifiers to form an emulsion. The emulsifiers may be nonionic surfactants, such as: esters of on the one hand sorbitan, mannide (e.g. anhydromannitol oleate), glycerol, polyglycerol or propylene glycol and on the other hand oleic, isostearic, ricinoleic or hydroxystearic acids, said esters being optionally ethoxylated, or polyoxypropylene-polyoxyethylene copolymer blocks, such as Pluronic, e.g., L121. Among the type (1) adjuvant polymers, preference is given to polymers of crosslinked acrylic or methacrylic acid, especially crosslinked by polyalkenyl ethers of sugars or polyalcohols. These compounds are known under the name carbomer (Pharmeuropa, vol. 8, no. 2, June 1996). One skilled in the art can also refer to U.S. Pat. No. 2,909,462, which provides such acrylic polymers crosslinked by a polyhydroxyl compound having at least three hydroxyl groups, preferably no more than eight such groups, the hydrogen atoms of at least three hydroxyl groups being replaced by unsaturated, aliphatic radicals having at least two carbon atoms. The preferred radicals are those containing 2 to 4 carbon atoms, e.g. vinyls, allyls and other ethylenically unsaturated groups. The unsaturated radicals can also contain other substituents, such as methyl. Products sold under the name Carbopol (BF Goodrich, Ohio, USA) are especially suitable. They are crosslinked by allyl saccharose or by allyl pentaerythritol. Among them, reference is made to Carbopol 974P, 934P and 971P. As to the maleic anhydride-alkenyl derivative copolymers, preference is given to EMA (Monsanto), which are straight-chain or crosslinked ethylene-maleic anhydride copolymers and they are, for example, crosslinked by divinyl ether. Reference is also made to J. Fields et al., Nature 186: 778-780, Jun. 4, 1960.

With regard to structure, the acrylic or methacrylic acid polymers and EMA are preferably formed by basic units having the following formula -- see Original Patent.

These polymers are soluble in water or physiological salt solution (20 g/l NaCl) and the pH can be adjusted to 7.3 to 7.4, e.g., by soda (NaOH), to provide the adjuvant solution in which the expression vector(s) can be incorporated. The polymer concentration in the final vaccine composition can range between 0.01 and 1.5% w/v, advantageously 0.05 to 1% w/v and preferably 0.1 to 0.4% w/v.

The cytokine or cytokines (5) can be in protein form in the immunogenic or vaccine composition, or can be co-expressed in the host with the immunogen or immunogens or epitope(s) thereof. Preference is given to the co-expression of the cytokine or cytokines, either by the same vector as that expressing the immunogen or immunogens or epitope(s) thereof, or by a separate vector therefor.

The invention comprehends preparing such combination compositions; for instance by admixing the active components, advantageously together and with an adjuvant, carrier, cytokine, and/or diluent.

Cytokines that may be used in the present invention include, but are not limited to, granulocyte colony stimulating factor (G-CSF), granulocyte/macrophage colony stimulating factor (GM-CSF), interferon .alpha. (IFN .alpha.), interferon .beta. (IFN .beta.), interferon .gamma., (IFN .gamma.), interleukin-1.alpha. (IL-1 .alpha.), interleukin-1 .beta. (IL-1 .beta.), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-11 (IL-11), interleukin-12 (IL-12), tumor necrosis factor .alpha. (TNF .alpha.), tumor necrosis factor .beta. (TNF .beta.), and transforming growth factor .beta. (TGF .beta.). It is understood that cytokines can be co-administered and/or sequentially administered with the immunogenic or vaccine composition of the present invention. Thus, for instance, the vaccine of the instant invention can also contain an exogenous nucleic acid molecule that expresses in vivo a suitable cytokine, e.g., a cytokine matched to this host to be vaccinated or in which an immunological response is to be elicited (for instance, a canine cytokine for preparations to be administered to dogs).

Advantageously, the pharmaceutical and/or therapeutic compositions and/or formulations according to the invention comprise or consist essentially of or consist of an effective quantity to elicit a therapeutic response of one or more expression vectors and/or polypeptides as discussed herein; and, an effective quantity can be determined from this disclosure, including the documents incorporated herein, and the knowledge in the art, without undue experimentation.

The recombinant viral vector immunogenic compositions and the recombinant viral vector vaccines according to the invention may be freeze-dried advantageously with a stabiliser. Freeze-drying can be done according to well-known standard freeze-drying procedures. The pharmaceutically or veterinary acceptable stabilisers may be carbohydrates (e.g. sorbitol, mannitol, lactose, sucrose, glucose, dextran, trehalose), sodium glutamate (Tsvetkov T et al., Cryobiology 1983, 20(3): 318-23; Israeli E et al., Cryobiology 1993, 30(5): 519-23), proteins such as peptone, albumin, lactalbumin or casein, protein containing agents such as skimmed milk (Mills C K et al., Cryobiology 1988, 25(2): 148-52; Wolff E et al., Cryobiology 1990, 27(5): 569-75), and buffers (e.g. phosphate buffer, alkaline metal phosphate buffer). An adjuvant may be used to make soluble the freeze-dried preparations.

Further the present invention concerns the use of at least one polypeptide having an amino acid sequence as shown in SEQ ID NOS: 18, 20, 22, 24, 26, 48, 50 and 52, or analogs thereof or fragments thereof, for the treatment and/or vaccination of mammals against streptococcal infection, notably of equine, canine and human species. In one embodiment, these fragments are selected from the group consisting of sequences SEQ ID NOS: 32, 34, 36, 38, 40, 54, 56 and 58, or analogs thereof. A particular embodiment concerns the use of at least one polypeptide selected from the group consisting of sequences SEQ ID NOS: 18, 20, 22, 24, 26, 32, 34, 36, 38, 40, 48, 50, 52, 54, 56 and 58, or analogs thereof, for the treatment and/or vaccination of equines against strangles disease. A preferred embodiment concerns the use of at least one polypeptide selected from the group consisting of sequences SEQ ID NOS: 32, 34, 36, 38, 40, 54, 56 and 58, or analogs thereof, for the treatment and/or vaccination of equines against strangles disease.

A further embodiment concerns the use of at least one polypeptide having an amino acid sequence as shown in SEQ ID NOS: 18, 20, 22, 24, 26, 48, 50 and 52, or analogs thereof or fragments thereof, for the preparation of a subunit vaccine protecting equine against Streptococcus equi infection. This further embodiment concerns preferrably the use of at least one polypeptide having an amino acid sequence as shown in SEQ ID NOS: 32, 34, 36, 38, 40, 54, 56 and 58, or analogs thereof, for the preparation of a subunit vaccine protecting equine against Streptococcus equi infection.

Another further embodiment concerns the use of at least one recombinant vector and of at least one polynucleotide inserted therein coding for a polypeptide having an amino acid sequence as shown in SEQ ID NOS: 18, 20, 22, 24, 26, 48, 50 and 52, or an analog thereof or a fragment thereof, and said vector is able to express in vivo this polypeptide in a mammal susceptible to streptococcal infection, for the preparation of a recombinant vaccine protecting equine against Streptococcus equi infection. This further embodiment concerns preferrably the use of at least one recombinant vector and of at least one polynucleotide inserted therein coding for a polypeptide having an amino acid sequence as shown in SEQ ID NOS: 32, 34, 36, 38, 40, 54, 56 and 58, or an analog thereof, and said vector is able to express in vivo this polypeptide in a mammal susceptible to streptococcal infection, for the preparation of a recombinant vaccine protecting equine against Streptococcus equi infection.

An additional embodiment concerns the use of at least one recombinant vector and at least one polynucleotide inserted therein, wherein said polynucleotide has a nucleotide sequence as shown in SEQ ID NOS: 17, 19, 21, 23, 25, 31, 33, 35, 37, 39, 47, 49, 51, 53, 55 and 57, or an analog thereof, and said vector is able to express in vivo the polypeptide encoded by said polynucleotide in a mammal susceptible to streptococcal infection, for the preparation of a recombinant vaccine protecting equine against Streptococcus equi infection.

In particular, combinations of polypeptides to be used for the treatment and/or vaccination of equine against strangles disease are combination of at least one polypeptide selected from a first group of polypeptides consisting of SEQ ID NOS: 18, 20, 22, 24, 26, 32, 34, 36, 38, 40, 48, 50, 52, 54, 56 and 58, or analogs thereof or combinations thereof, and at least one another Streptococcus equi immunogen, which is not present in this first group, notably at least one immunogen selected from a second group of polypeptides consisting of FNZ protein, EAG protein, SFS protein, SEC protein, SFSC1 fragment, FNZN fragment, SEC2.16 fragment, SEC1.18 fragment, SclC1 fragment (WO-A-2004/032957) and SEQ ID NOS: 28, 30 or analogs thereof or fragments thereof, or combinations thereof. In preferred combinations of polypeptides, this first group consists of SEQ ID NOS: 32, 34, 36, 38, 40, 54, 56 and 58, or analogs thereof or combinations thereof. Further the present invention also concerns the use of these combinations of peptides for the preparation of subunit vaccines protecting equines against Streptococcus equi infection. In these embodiments, the use of polypeptides may be replaced by the use of recombinant expression vectors according to the present invention comprising at least one polynucleotide encoding said polypeptides. In particular, at least one recombinant expression vector comprising at least one polynucleotide encoding at least one polypeptide selected from a first group of polypeptides consisting of SEQ ID NOS: 18, 20, 22, 24, 26, 32, 34, 36, 38, 40, 48, 50, 52, 54, 56 and 58, or analogs thereof, and at least one polynucleotide encoding at least one another Streptococcus equi immunogen, which is not present in this first group, notably at least one immunogen selected from a second group of polypeptides consisting of FNZ protein, EAG protein, SFS protein, SEC protein, SFSC1 fragment, FNZN fragment, SEC2.16 fragment, SEC1.18 fragment, SclC1 fragment (WO-A-2004/032957) and SEQ ID NOS: 28, 30 or analogs thereof or fragments thereof, or combinations thereof, are used for the treatment and/or vaccination of equines against strangles disease. In a preferred embodiment, this first group consists of SEQ ID NOS: 32, 34, 36, 38, 40, 54, 56 and 58, or analogs thereof or combinations thereof. Further the present invention also concerns the use of these recombinant expression vectors for the preparation of recombinant vaccines protecting equines against Streptococcus equi infection.

In a particular embodiment, the combinations of polypeptides to be used for the treatment and/or vaccination of equine against strangles disease are combination of polypeptides SEQ ID NOS: 52, 48, 22 and 26, or analogs thereof, or fragments thereof. In a more preferred embodiment, the combinations of polypeptides to be used for the treatment and/or vaccination of equine against strangles disease are combination of polypeptides SEQ ID NOS: 58, 54, 36 and 40, or analogs thereof.

In a particular embodiment, the combinations of polypeptides to be used for the treatment and/or vaccination of equine against strangles disease are combination of polypeptides SEQ ID NOS: 52, 48, 22, 26 and 50, or analogs thereof, or fragments thereof. In a more preferred embodiment, the combinations of polypeptides to be used for the treatment and/or vaccination of equine against strangles disease are combination of polypeptides SEQ ID NOS: 58, 54, 36, 40 and 56, or analogs thereof.

In a particular embodiment, the combinations of polypeptides to be used for the treatment and/or vaccination of equine against strangles disease are combination of polypeptides SEQ ID NOS: 52, 48, 22, 26 and 20, or analogs thereof, or fragments thereof. In a more preferred embodiment, the combinations of polypeptides to be used for the treatment and/or vaccination of equine against strangles disease are combination of polypeptides SEQ ID NOS: 58, 54, 36, 40 and 34, or analogs thereof.

In a particular embodiment, the combinations of polypeptides to be used for the treatment and/or vaccination of equine against strangles disease are combination of polypeptides SEQ ID NOS: 52, 48, 22, 26 and 18, or analogs thereof, or fragments thereof. In a more preferred embodiment, the combinations of polypeptides to be used for the treatment and/or vaccination of equine against strangles disease are combination of polypeptides SEQ ID NOS: 58, 54, 36, 40 and 32, or analogs thereof.

Further the present invention relates to methods to immunise against or to prevent streptococcal infection in mammals, preferably in equine, canine and in human species. According to these methods, (1) a subunit immunogenic composition or vaccine of the present invention, or (2) a recombinant immunogenic composition or vaccine of the present invention, or their combinations, are administered. Of course, embodiments of the invention may be employed with other vaccines or immunogenic compositions that are not of the invention, e.g., in prime-boost processes, such as where a vaccine or immunogenic composition of the invention is administered first and a different vaccine or immunogenic composition is administered thereafter, or vice versa. Particular prime-boost processes may be that a subunit vaccine or immunogenic composition of the invention is administered first and a recombinant vaccine or immunogenic composition of the invention is administered thereafter, or vice versa.

The administration may be notably made by intramuscular (IM), intradermal (ID), subcutaneous (SC) or transdermal injection or via intranasal, intratracheal, oral administration, or through the ears or the lip. The immunogenic composition or the vaccine according to the invention is administered by syringe, a syringe with a microneedle (i.e. BD.TM. Intradermal Delivery System of Becton, Dickinson and Company, Franklin Lakes, N.J., USA), needleless apparatus (like for example Pigjet, Avijet, Dermojet or Biojector (Bioject, Oreg., USA), see U.S. Pat. No. 2006/0034867) or a spray. The administration is preferrably made by IM injection with a syringe, or by transdermal injection with a needleless apparatus or with a syringe with a microneedle (i.e. BD.TM. Intradermal Delivery System), or by intranasal or oral administration with a spray, i.e. a liquid nebulisation of a vaccine of the invention, or by oral or nasal administration of a micronized powder of a freeze-dried vaccine according to the invention.

The quantity of immunogenic compositions or vaccines can be determined and optimised by the skilled person, without undue experimentation from this disclosure and the knowledge in the art. Generally an animal (including a human) may be administered approximately 10.sup.4-10.sup.9 CFUs, advantageously approximately 10.sup.5-10.sup.8 CFUs and more advantageously approximately 10.sup.6-10.sup.8 CFUs in a single dosage unit of recombinant viral immunogenic compositions or vaccines of the present invention; approximately 10 ng-1 mg, advantageously approximately 100 ng-500 .mu.g and more advantageously approximately 1 .mu.g-250 .mu.g per plasmid type in a single dosage unit of recombinant DNA immunogenic compositions or vaccines of the present invention; approximately 5 .mu.g-1 mg, advantageously approximately 50 .mu.g-500 .mu.g and more advantageously approximately 100 .mu.g-200 .mu.g in a single dosage unit of subunit immunogenic compositions or vaccines of the present invention.

In the case of therapeutic and/or pharmaceutical compositions based on a plasmid vector, a dose can comprise, consist essentially of or consist of, in general terms, about in 1 .mu.g to about 2000 .mu.g, advantageously about 50 .mu.g to about 1000 .mu.g and more advantageously from about 100 .mu.g to about 800 .mu.g of plasmid expressing the antigen, epitope, immunogen, peptide or polypeptide of interest. When the therapeutic and/or pharmaceutical compositions based on a plasmid vector is administered with electroporation the dose of plasmid is generally between about 0.1 .mu.g and 1 mg, advantageously between about 1 .mu.g and 100 .mu.g, advantageously between about 2 .mu.g and 50 .mu.g. The dose volumes can be between about 0.1 and about 2 ml, advantageously between about 0.2 and about 1 ml. These doses and dose volumes are suitable for the treatment mammalian target species.

The volume of one single dosage unit by syringe can be between about 0.2 ml and about 5.0 ml and advantageously between about 0.5 ml and about 2.0 ml and more advantageously about 1.0 ml. The volume of one single dosage unit by needleless apparatus can be between about 0.1 ml and about 1.0 ml and advantageously between about 0.2 ml and about 0.5 ml. The volume of one single dosage unit by liquid spray can be between about 2.0 ml and about 10.0 ml and advantageously about 5.0 ml (for powder spray, the quantities administered are corresponding to the equivalent volumes).

In a particular method, foals, that is to say horses of 2 to 6 months old and preferably 3 to 4 months old, are vaccinated with subunit vaccines of the present invention, adjuvanted with CTB and/or CTA and/or Carbopol.RTM., via the intranasal or oral route.

In another particular method, foals are vaccinated with recombinant viral vaccines of the present invention, adjuvanted or not with Carbopol.RTM., via the oral route with a spray and liquid nebulisation. Preferrably, the viral vectors of these recombinant viral vaccines are EHV-4 or EHV-2, or EHV-1.

In another particular method, foals are vaccinated with recombinant viral vaccines of the present invention, via the lips with a syringe. Preferrably, the viral vectors of these recombinant viral vaccines are EHV-4 or EHV-2 or EHV-1.

Preferably for the vaccination of horses and mares, two administrations of subunit vaccines of the present invention, adjuvanted with Carbopol.RTM. and/or CpG, and or emulsion are made by IM injection with a syringe. Boost administrations may be injected every 6 months or annually. For pregnant mares, a boost administration may be injected 2 to 4 weeks before the expected foaling date.

Polypeptides of the invention and fragments thereof may also be used in therapy.

The polypeptides and fragments may also be used as reagents in antibody-antigen reactions. Accordingly, another aspect of the invention is thus a diagnostic method and/or kit for detecting infection by the streptococcal bacterium. Kits, e.g. ELISA, can include at least one polypeptide or fragment according to the invention (e.g., at least one polypeptide identified by sequence herein or a fragment thereof as herein discussed).

Antibodies against the herein polypeptides or fragments (e.g., polypeptides identified by sequence herein or fragments thereof as herein discussed) can be used as a diagnostic reagent or in passive immunization or vaccination or in therapy. The amounts of antibody administered in passive immunization can be the same as or analogous to amounts used in the art, such that from the knowledge in the art, the skilled artisan can practice passive immunization without undue experimentation.

Proteins encoded by the novel viruses of the present invention, or their fragments, can be used to produce antibodies, both polyclonal and monoclonal. If polyclonal antibodies are desired, a selected mammal, (e.g., mouse, rabbit, goat, horse, etc.) is immunized with an antigen of the present invention, or its fragment, or a mutated antigen. Serum from the immunized animal is collected and treated according to known procedures. See, e.g., Jurgens et al. (1985) J. Chrom. 348:363-370. If serum containing polyclonal antibodies is used, the polyclonal antibodies can be purified by immunoaffinity chromatography, using known procedures.

Another aspect of the invention is an antibody preparation comprising an antibody specific to a polypeptide or a fragment according to the invention and methods of diagnosis using the same. With respect to an antibody specific to a polypeptide, it is meant that the antibody binds preferentially to the polypeptide, e.g., the antibody binds to the polypeptide and not to other polypeptides or has a specificity to the polypeptide that is acceptably particular to the polypeptide such that the antibody can be used to isolate the polypeptide from a sample or detect its presence in a sample with no more than 5% false positives, using techniques known in the art or discussed in documents cited herein, including Sambrook, infra.

Antibodies can be polyclonal or monoclonal.

If polyclonal antibodies are desired, a selected animal (e.g. mouse, rabbit, goat, horse, etc.) is immunized with a polypeptide or a fragment. Serum from the immunized animal is collected and treated according to known procedures and possibly purified. See, e.g. Jurgens et al. J. Chrom., 1985, 348: 363-370.

Monoclonal antibodies to the proteins and to the fragments thereof, can also be readily produced by one skilled in the art. The general methodology for making monoclonal antibodies by using hybridoma technology is well known. Immortal antibody-producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. See, e.g., M. Schreier et al., Hybridoma Techniques (1980); Hammerling et al., Monoclonal Antibodies and T-cell Hybridomas (1981); Kennett et al., Monoclonal Antibodies (1980); see also U.S. Pat. Nos. 4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,452,570; 4,466,917; 4,472,500, 4,491,632; and 4,493,890. Panels of monoclonal antibodies produced against the desired protein, or fragment thereof, can be screened for various properties; i.e., for isotype, epitope, affinity, etc. Monoclonal antibodies are useful in purification, using immunoaffinity techniques, of the individual antigens which they are directed against. Both polyclonal and monoclonal antibodies can also be used for passive immunization or can be combined with subunit vaccine preparations to enhance the immune response. Polyclonal and monoclonal antibodies are also useful for diagnostic purposes.

One embodiment of the invention is a method of eliciting an immune response against the antigen, epitope, immunogen, peptide or polypeptide of interest in an animal, comprising administering a formulation for delivery and expression of a recombinant vaccine in an effective amount for eliciting an immune response. Still another embodiment of the invention is a method of inducing an immunological or protective response in an animal, comprising administering to the animal an effective amount of a formulation for delivery and expression of an antigen, epitope, immunogen, peptide or polypeptide of interest wherein the formulation comprises a recombinant vaccine and a pharmaceutically or veterinarily acceptable carrier, vehicle or excipient.

The invention relates to a method to elicit, induce or stimulate the immune response of an animal, advantageously a mammal or a vertebrate.

Another embodiment of the invention is a kit for performing a method of inducing an immunological or protective response against an antigen, epitope, immunogen, peptide or polypeptide of interest in an animal comprising a recombinant vaccine and instructions for performing the method of delivery in an effective amount for eliciting an immune response in the animal.
 

Claim 1 of 4 Claims

1. An immunogenic composition comprising at least one polypeptide comprising the amino acid sequence of SEQ ID NO: 22.
 

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