Abstract:
Rhodococcus equi  ( R.equi ) has been determined to have a major adhesion factor encoded by a rpl pathogenicity island which enables host colonisation, wherein the rpl pathogenicity islandis absent from non-pathogenic  Rhodococcus  species. Further, the proteins (Rpl) encoded by the rpl pathogenicity island have been determined to be major immunodominant antigens. There is provided a novel diagnostic marker and vaccine candidate for  R. equi  in horses and other susceptible species.

Description:
FIELD OF THE INVENTION 
       [0001]    The present invention relates to polypeptides encoded by  Rhodococcus  ( Corynebacterium )  equi  ( R. equi ), compositions including such polypeptides (Rpl) and antibodies to such polypeptides, which can be useful in the treatment of animals, specifically horses and foals, to minimise infection of animals, by  R. equi . The invention further relates to methods of detection of  R. equi  using polypeptides (Rpl), antibodies with binding specificity to said polypeptides or nucleic acids or the like with binding specificity to nucleic acids encoding such polypeptides using, for example, PCR. 
       BACKGROUND TO THE INVENTION 
       [0002]      Rhodococcus equi  is a Gram-positive, facultative intracellular coccobacillus classified in the order of Acitnomycetales. It is primarily a soil organism. It has been recognised as a positive agent of a debilitating and potentially fatal bronchopneumonia affecting foals worldwide.  R. equi  is considered to be one of the most significant pathogens in the equine breeding industry. 
         [0003]    The successful early diagnosis and treatment of Rhodococcus equi in foals and management of the foals environment to reduce the risk of contracting the disease are, arguably, among the most challenging experiences currently facing equine stud farms. Presently the treatment of  R. equi  disease is by the prolonged administration of a combination of antimicrobials, macrolides, i.e. erythromycin, azithromycin or clarithromycin, and rifampicin. However, as this therapy risks antibiotic resistance and adverse drug reactions in the foal and the dam, improved means of therapy and prophylactic treatment are required. 
         [0004]      R. equi  can also affect non-equine species. In pigs  R. equi  is associated with granulomatous lymphadenitis of cervical lymphatic tissue and in man  R. equi  can cause cavitary pneumonia, predominantly in immunocompromised individuals especially those with acquired immune deficiency syndrome (AIDS). As a consequence of the AIDS pandemic,  R. equi  pneumonia has become a disease of increasing significance in human medicine.  R. equi  infections have also been described in cattle, sheep, goats, lama, cats and dogs, but disease in these species is rare with lesions confined to lymph node abscessation or wound infection. 
         [0005]    Infection by  R. equi  relies on the ability of  R. equi  to colonise the airways and replicate inside macrophages which is dependent on its capacity to interfere with endosomal maturation following phagocytosis and to prevent acidification of the vacuole in which it resides. Eventually, intracellular proliferation of the pathogen leads to the necrotic death of the marcophages accompanied by massive damage to lung tissue characterised by cavitation and granuloma formation. 
         [0006]    Studies of the virulent strains of  R. equi  have determined that such strains possess an extra chromosomal DNA element known as a plasmid, which is associated with virulence. Plasmids isolated from regular strains infecting foals have been proposed to include a region that represents a pathogenicity island, which is a DNA fragment containing genes required for virulence. The pathogenicity island identified contains a family of nine virulence associated protein (Vap) chains (VapA-VapC-Vap-I, pseudo-VapE). Killed/inactivated  R. equi  organisms do not illicit protective immunity, and there is no consistent evidence that protein or DNA vaccines, based on the highly immunogenic VapA surface antigen, are efficacious in producing protection against a Rhodococcal pneumonia in foals. In view of the lack of an efficacious vaccine,  R. equi  infection is a major cause of mortality in young foals and the heavy economic losses incurred due to  R. equi  has a major economic impact in countries where thoroughbred racing and breeding is important (USA, Australia, Ireland, Argentina, UK, France, Spain, Germany, Austria, Japan etc.). There is a need for treatment regimes and a vaccine to be developed which can be used to control  R. equi  on farms, in particular stud farms. 
       SUMMARY OF THE INVENTION 
       [0007]    The inventors have determined a novel diagnostic marker and vaccine candidate for  Rhodococcus equi  in horses and other susceptible species and treatment means. Specifically, the inventors have identified a rpl pathogenicity island that differs from the yap pathogenicity island and the inventors have determined the rpl pathogenicity island, in particular RplB, encodes a major adhesion factor of  R. equi  which enables host colonisation. The proteins (Rpl) encoded by the rpl pathogenicity island are considered to be major immunodominant antigens. The inventors have further determined that the rpl pathogenicity island is absent from non-pathogenic  Rhodococcus  species. These findings allow the use of probes to proteins or nucleic acid of the rpl pathogenicity island and antibodies with binding specificity to the proteins encoded by the rpl pathogenicity island in methods of detection of  R. equi . Further, it enables the use of nucleic acids encoding proteins or proteins of the rpl pathogenicity island as immune system modulators, in particular to provoke a protective immune response to subsequent antigen challenge in an animal. 
         [0008]    Accordingly, a first aspect of the invention provides at least one immunogenic  R. equi  polypeptide having an amino acid sequence, encoded by a polynucleotide sequence comprising a polynucleotide sequence of a gene selected from a gene as listed at table one, or a fragment, derivative or variant of such a polypeptide. 
         [0000]    
       
         
               
               
               
               
               
             
           
               
                 TABLE ONE 
               
               
                   
               
               
                   
                   
                 Proposed 
                   
                   
               
               
                   
                   
                 function of 
                   
                 SEQ 
               
               
                 rpl 
                   
                 encoded 
                 Position in  R. equi   
                 ID 
               
               
                 locus 
                 Identifier 
                 protein 
                 103S 
                 NO 
               
               
                   
               
             
             
               
                 rplA 
                 REQ_18350 
                 Prepilin 
                 Position 1938280-1939068 
                 1 
               
               
                   
                   
                 peptidase 
                 (complement) in 
               
               
                   
                   
                   
                 103S genome 
               
               
                 rplB 
                 REQ_18360 
                 Pilin subunit 
                 Position 1939395-1939601 
                 2 
               
               
                   
                   
                   
                 in 103S 
               
               
                   
                   
                   
                 genome 
               
               
                 rplC 
                 REQ_18370 
                 Minor pilin 
                 Position 
                 3 
               
               
                   
                   
                 protein 
                 1939683.-1940084 
               
               
                   
                   
                   
                 in 103S 
               
               
                   
                   
                   
                 genome 
               
               
                 rplD 
                 REQ_18380 
                 Putative 
                 Position 1940093-1941037 
                 4 
               
               
                   
                   
                 lipoprotein 
                 1940084 
               
               
                   
                   
                   
                 in 103S genome 
               
               
                 rplE 
                 REQ_18390 
                 Pilus assembly 
                 Position 1941047-1941784 
                 5 
               
               
                   
                   
                 protein 
                 in 103S 
               
               
                   
                   
                   
                 genome 
               
               
                 rplF 
                 REQ_18400 
                 Pilus assembly 
                 Position 1941781-1942980 
                 6 
               
               
                   
                   
                 ATPase 
                 in 103S 
               
               
                   
                   
                   
                 genome 
               
               
                 rplG 
                 REQ_18410 
                 Secretion 
                 Position 1942977-1944374 
                 7 
               
               
                   
                   
                 apparatus 
                 in 103S 
               
               
                   
                   
                 ATPsae 
                 genome 
               
               
                 rplH 
                 REQ_18420 
                 Secretion 
                 Position 1944371-1946239 
                 8 
               
               
                   
                   
                 apparatus 
                 in 103S 
               
               
                   
                   
                 integral 
                 genome 
               
               
                   
                   
                 membrane 
               
               
                   
                   
                 protein 
               
               
                 rplI 
                 REQ_18430 
                 Secretion 
                 Position 1946262-1947152 
                 9 
               
               
                   
                   
                 apparatus 
                 in 103S 
               
               
                   
                   
                 integral 
                 genome 
               
               
                   
                   
                 membrane 
               
               
                   
                   
                 protein 
               
               
                   
               
             
          
         
       
     
         [0009]    In embodiments of the invention, the polypeptide or derivative or variant or fragment thereof can be encoded by a polynucleotide sequence comprising a polynucleotide sequence of a gene as listed in Table 2 
         [0000]    
       
         
               
               
               
               
               
             
           
               
                 TABLE TWO 
               
               
                   
               
               
                   
                   
                 Proposed 
                   
                   
               
               
                   
                   
                 function of 
                   
                 SEQ 
               
               
                 rpl 
                   
                 encoded 
                 Position in  R. equi   
                 ID 
               
               
                 locus 
                 Identifier 
                 protein 
                 103S 
                 NO 
               
               
                   
               
             
             
               
                 rplA 
                 REQ_18350 
                 Prepilin 
                 Position 1938280-1939068 
                 1 
               
               
                   
                   
                 peptidase 
                 (complement) in 
               
               
                   
                   
                   
                 103S genome 
               
               
                 rplB 
                 REQ_18360 
                 Pilin subunit 
                 Position 1939395-1939601 
                 2 
               
               
                   
                   
                   
                 in 103S 
               
               
                   
                   
                   
                 genome 
               
               
                 rplC 
                 REQ_18370 
                 Minor pilin 
                 Position 
                 3 
               
               
                   
                   
                 protein 
                 1939683.-1940084 
               
               
                   
                   
                   
                 in 103S 
               
               
                   
                   
                   
                 genome 
               
               
                 rplD 
                 REQ_18380 
                 Putative 
                 Position 1940093-1941037 
                 4 
               
               
                   
                   
                 lipoprotein 
                 1940084 
               
               
                   
                   
                   
                 in 103S genome 
               
               
                 rplE 
                 REQ_18390 
                 Pilus assembly 
                 Position 1941047-1941784 
                 5 
               
               
                   
                   
                 protein 
                 in 103S 
               
               
                   
                   
                   
                 genome 
               
               
                 rplH 
                 REQ_18420 
                 Secretion 
                 Position 1944371-1946239 
                 8 
               
               
                   
                   
                 apparatus 
                 in 103S 
               
               
                   
                   
                 integral 
                 genome 
               
               
                   
                   
                 membrane 
               
               
                   
                   
                 protein 
               
               
                 rplI 
                 REQ_18430 
                 Secretion 
                 Position 1946262-1947152 
                 9 
               
               
                   
                   
                 apparatus 
                 in 103S 
               
               
                   
                   
                 integral 
                 genome 
               
               
                   
                   
                 membrane 
               
               
                   
                   
                 protein 
               
               
                   
               
             
          
         
       
     
         [0010]    In particular embodiments the polypeptide or a derivative or variant or fragment thereof can be encoded by a polynucleotide sequence comprising a polynucleotide sequence of a gene selected from rplB (SEQ ID NO 2), rplC (SEQ ID NO 3), or rplD (SEQ ID NO 4). In an alternative embodiment, the polypeptide or a derivative can be encoded by a polynucleotide sequence comprising a polynucleotide sequence of a gene selected from rplB (SEQ ID NO 2), rplA (SEQ ID NO 1) or rplE (SEQ ID NO 5). 
         [0011]    In embodiments of the invention, the polypeptide or a derivative or fragment thereof is encoded by a polynucleotide sequence comprising a polynucleotide sequence of a gene selected from the list of genes of Table 1, more preferably selected from the list of genes of Table 2. 
         [0012]    In embodiments of the invention, the polypeptide or a derivative or fragment or variant thereof is encoded by a polynucleotide sequence consisting essentially of or consisting of a polynucleotide sequence of a gene selected from the list of genes of Table 1, more preferably selected from the list of genes of Table 2. 
         [0013]    In embodiments, the polypeptide is encoded by a polynucleotide sequence comprising the polynucleotide sequence of a gene encoding Rpl pilin ATGAACCTCTTCTTCGCGAACCTGTACCTCATGGGCTTAGACGTCAA GGACCGTCTGACCCGTGACGACCGCGGCGCCACTGCGGTCGAGTAC GGACTGATGGTCGCCGGCATCGCGATGGTGATCATTGTTGCGGTTTT CGCCTTCGGCGATAAGATTACCGACCTCTTCGATGGCTTCAACTTCG ACGATCCCGGCGGCGAGTAG (SEQ ID NO 2). 
         [0014]    In embodiments, the polypeptide is encoded by a polynucleotide sequence consisting essentially of or consisting of the polynucleotide sequence of a gene encoding Rpl pilin ATGAACCTCTTCTTCGCGAACCTGTACCTCATGGGCTTAGACGTCAA GGACCGTCTGACCCGTGACGACCGCGGCGCCACTGCGGTCGAGTAC GGACTGATGGTCGCCGGCATCGCGATGGTGATCATTGTTGCGGTTTT CGCCTTCGGCGATAAGATTACCGACCTCTTCGATGGCTTCAACTTCG ACGATCCCGGCGGCGAGTAG (SEQ ID NO 2). 
         [0015]    In embodiments, the polypeptide is encoded by a polynucleotide sequence comprising a fragment of the polynucleotide sequence of a gene encoding Rpl pilin ATGAACCTCTTCTTCGCGAACCTGTACCTCATGGGCTTAGACGTCAA GGACCGTCTGACCCGTGACGACCGCGGCGCCACTGCGGTCGAGTAC GGACTGATGGTCGCCGGCATCGCGATGGTGATCATTGTTGCGGTTTT CGCCTTCGGCGATAAGATTACCGACCTCTTCGATGGCTTCAACTTCG ACGATCCCGGCGGCGAGTAG (SEQ ID NO 2) 
         [0016]    wherein the polypeptide encoded by the fragment is a biologically active immunogenic fragment of a polypeptide encoded by the polynucleotide sequence comprising the polynucleotide sequence of the gene encoding Rpl pilin ATGAACCTCTTCTTCGCGAACCTGTACCTCATGGGCTTAGACGTCAA GGACCGTCTGACCCGTGACGACCGCGGCGCCACTGCGGTCGAGTAC GGACTGATGGTCGCCGGCATCGCGATGGTGATCATTGTTGCGGTTTT CGCCTTCGGCGATAAGATTACCGACCTCTTCGATGGCTTCAACTTCG ACGATCCCGGCGGCGAGTAG (SEQ ID NO 2). 
         [0017]    In embodiments, a derivative or fragment or variant can be an immunogenic derivative or fragment or variant that can provide an immune response in which antibodies with binding specificity to at least one of SEQ ID NO 1, 2, 3, 4, 5, 6, 7, 8, and 9 are generated for example antibodies cross-reactive to the biologically active immunogenic fragment and at least one of SEQ ID NO 1, 2, 3, 4, 5, 6, 7, 8 and 9. 
         [0018]    In particular embodiments such fragments, derivatives or variants can functionally provide a pilus in  R. equi . Such derivatives, fragments or variants can be biologically active derivatives, fragments or variants. 
         [0019]    In embodiments the Rpl pilin polypeptide (RplB) can comprise an amino acid sequence MNLFFANLYLMGLDVKDRLTRDDRGATAVEYGLMVAGIAMVIIVAVFAFG DKITDLFDGFNFDDPGGE (SEQ ID NO 10). 
         [0020]    In embodiments, a polypeptide of the invention can consist of an amino acid sequence MNLFFANLYLMGLDVKDRLTRDDRGATAVEYGLMVAGIAMVIIVAVFAFG DKITDLFDGFNFDDPGGE (SEQ ID NO 10). 
         [0021]    In embodiments a polypeptide of the invention can comprise DKITDLFDGFNFDDPGGE (SEQ ID NO 11) or can be a variant thereof wherein such variant has at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 amino acids different to that of SEQ ID NO 11. Substituted amino acids may suitably be conservative or non conservative amino acids. Alternatively, the variant may include insertions or deletions. Suitably, in embodiments a variant can demonstrate analogous biological function as a RplB pilin subunit or SEQ ID NO 11. In embodiments, a conserved variant may be provided by amino acid sequences comprising DKITDLFDGFNFDDPGGE (SEQ ID NO 11) wherein amino acids as shown are replaced by amino acids which are structurally conservative. For example, an aliphatic amino acid (alanine, serine, valine, leucine, isoleucine or the like) can be substituted with another suitable aliphatic amino acid, a hydrophobic amino acid (tyrosine, phenylalanine, tryptophan) can be substituted by another hydrophobic amino acid or a charged amino acid can be substituted by another charged amino acid. In such conserved variants, additional amino acids may be substituted. 
         [0022]    In embodiments a polypeptide of the invention can comprise the amino acid sequence DKITDLFDGFNFDDPGGE (SEQ ID NO 11). In embodiments a polypeptide of the invention consists of, or consists essentially of the amino acid sequence DKITDLFDGFNFDDPGGE (SEQ ID NO 11). 
         [0023]    A polypeptide of the invention may be provided using recombinant means or may be a synthetic polypeptide or may be extracted from  R. equi  bacteria,  R. equi  culture supernatant or from biological material infected with  R. equi . In embodiments an isolated immunogenic polypeptide of the invention is expressed at the bacterial cell surface of a  R. equi , or is secreted from  R. equi.    
         [0024]    In embodiments, a polypeptide of the invention, or a fragment, derivative or variant thereof comprises an amino acid sequence of at least one polypeptide selected from the group consisting of the list provided by Table 3 or as set out in the sequences of  FIG. 9 . 
         [0000]    
       
         
               
               
               
               
             
           
               
                 TABLE THREE 
               
               
                   
               
               
                 Rpl protein 
                 Identifier 
                 Proposed function 
                 SEQ ID NO 
               
               
                   
               
             
             
               
                 RplA 
                 REQ_18350 
                 Prepilin peptidase 
                 12 
               
               
                   
                 product 
               
               
                 RplB 
                 REQ_18360 
                 Pilin subunit 
                 13 
               
               
                   
                 product 
               
               
                 RplC 
                 REQ_18370 
                 Minor pilin protein 
                 14 
               
               
                   
                 product 
               
               
                 RplD 
                 REQ_18380 
                 Putative lipoprotein 
                 15 
               
               
                   
                 product 
               
               
                 RplE 
                 REQ_18390 
                 Pilus assembly 
                 16 
               
               
                   
                 product 
                 protein 
               
               
                 RplF 
                 REQ_18400 
                 Pilus assembly 
                 17 
               
               
                   
                 product 
                 ATPase 
               
               
                 RplG 
                 REQ_18410 
                 Secretion apparatus 
                 18 
               
               
                   
                 product 
                 ATPsae 
               
               
                 RplH 
                 REQ_18420 
                 Secretion apparatus 
                 19 
               
               
                   
                 product 
                 integral membrane 
               
               
                   
                   
                 protein 
               
               
                 RplI 
                 REQ_18430 
                 Secretion apparatus 
                 20 
               
               
                   
                 product 
                 integral membrane 
               
               
                   
                   
                 protein 
               
               
                   
               
             
          
         
       
     
         [0025]    All of the polypeptides shown in Table 3 are encoded in the rpl locus and are part of the  R. equi  Rpl pilus biogenesis machinery. 
         [0026]    In embodiments a polypeptide of the invention can be encoded by an  R. equi.  strain isolated from horses. In embodiments the polypeptide can be isolated from horses and can be from a virulent strain of  R. equi . In embodiments, polypeptides of the invention can be made synthetically or recombinantly using techniques which are widely available in the art. 
         [0027]    The polypeptide of the invention may be optionally linked to an immunogenic carrier. Said immunogenic carrier may be a heterologous polypeptide, lipid, liposome, or another acceptable carrier molecule. Suitably, a polypeptide of the invention may be linked to the immunogenic carrier by chemical coupling or a polypeptide of the invention may be expressed as a fusion protein with the immunogenic carrier. A polypeptide of the invention, and/or a biologically active and/or immunogenic fragment, or derivative, or variant thereof, can be provided in an immunogenic composition, for example to raise antisera or monoclonal antibodies for passive immunisation, or as a vaccine. Alternatively, a polypeptide of the invention, fragment, derivative or variant thereof may be useful in an assay to detect antibodies specific for the polypeptide, including diagnostic assays. As set out herein, in embodiments, a derivative of a polypeptide of the invention can be a composite of specific polypeptide sequences of the invention, for example composites of SEQ ID NO 10, SEQ ID NO 11 and a polypeptide as set out in Table 3 or fragments thereof, or nucleotide sequences for example as set out at Table 1 or Table 2 disclosed herein. In embodiments, the nucleic acid sequences can be used to form concatemers and may be used to provide polypeptide sequences, for example relevant epitopes may be put in tandem or provided in multiples of 3, 4, 5, 6, or greater than 10, greater than 20 or more. Further, in embodiments a derivative can include a scrambled or chimeric polypeptide containing combinations of different relevant Rpl polypeptides. In such embodiments the combinations of relevant Rpl polypeptides can be provided in multiples of 2, 3, 4, 5, 6, or greater than 10, greater than 20 or more. 
         [0028]    It is important to note that even with knowledge of the genome of  R. equi  strain 103S, it would not be apparent that  R. equi  produced pili appendages or that the nine-gene locus encompassing nucleotide positions 1,938,280 to 1,947,152 (locus tags REQ18350-430) encoded a pilus biogenesis apparatus responsible for the production of  R. equi  pili involved in virulence and host colonisation. Pili are widespread among bacteria and can serve many functions unrelated to virulence. For example pili can facilitate attachment of bacteria to environmental surfaces such as soil particles, biofilm formation, be mediators of bacterial motility or enable adhesion to other bacteria. As will be appreciated, depending on pili function, in some instances, pili may not provide an immunogenic determinant suitable for vaccine development or be able to act as a diagnostic marker. 
         [0029]    Using visualisation by electron microscopy and genetic molecular analysis, the inventors demonstrated for the first that  R. equi  produces pili appendages or fimibriae, identified that the rpl locus  R. equi  encodes the pilus biogenesis apparatus, and further determined that proteins of  R. equi  pili are major virulence factors involved in host colonisation and that they are major immunodominant antigens. The latter determination would not have been suggested from sequence data alone. 
         [0030]    According to a second aspect of the present invention there is provided an isolated or recombinant nucleic acid encoding a polypeptide associated with pilus formation in  R. equi . In embodiments of the invention there is provided an isolated or recombinant nucleic acid comprising a polynucleotide sequence comprising or consisting of a sequence as set forth in any one of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, and SEQ ID NO 9 or a variant or derivative or fragment thereof, for example as illustrated in the sequences of  FIG. 10 . 
         [0031]    Due to the known degeneracy of the genetic code, a polynucleotide sequence which differs from those indicated by any one of SEQ ID 1, 2, 3, 4, 5, 6, 7, 8 or 9 can encode an active immunogenic derivative, variant or fragment and/or a biologically active derivative, variant or fragment of a polypeptide of the invention. In embodiments, a polynucleotide sequence which encodes such a derivative, fragment or variant sequence or an immunogenic biologically active derivative or fragment can result from silent mutations (e.g., occurring during PCR amplification), or nucleotide substitutions, deletions or insertions or the like or can be the product of deliberate mutagenesis of a native sequence. Variant polypeptides may be encoded by variant polynucleotide sequences having sequence homology (identity) of greater than at least 85%, 86%, 87%, 88%, 89%, preferably at least 90%, 91%, 92%, 93%, 94%, and more preferably 95%, 96%, 97%, 98%, 99% but less than 100% contiguous nucleotide sequence homology to any one of SEQ ID NO 1, 2, 3, 4, 5, 6, 7, 8, or 9 or fragments thereof. A variant polypeptide may be encoded by a polynucleotide sequence including nucleotide bases not present in the corresponding wild type nucleic acid molecule and/or internal deletions relative to the corresponding wild type nucleic acid molecule, such as SEQ ID NOs 1, 2, 3, 4, 5, 6, 7, or 8. Polynucleotide sequences encoding fragments of a polypeptide of the invention may be greater than 30 nucleotides in length, greater than 50 nucleotides in length, greater than 100 nucleotides in length, or greater than 150 nucleotides in length. The invention also provides isolated nucleic acids useful in the production of polypeptides. Suitably said biologically active immunogenic derivative, fragment or variant can elicit an immune response wherein the antibodies generated to said derivative, fragment or variant have a binding specificity to any one of SEQ ID NO 1, 2, 3, 4, 5, 6, 7, 8 or 9. In embodiments, there can be provided a polynucleotide sequence comprising or consisting of a sequence as set out in any one of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, and SEQ ID NO 9. 
         [0032]    In further embodiments there is provided an isolated or recombinant nucleic acid comprising a polynucleotide sequence comprising or consisting of a sequence as set forth in any one of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 8, and SEQ ID NO 9. In additional embodiments, there is provided an isolated or recombinant nucleic acid comprising a polynucleotide sequence comprising or consisting of a sequence as set forth in any one of SEQ ID NO 2, SEQ ID NO 3, and SEQ ID NO 4. In specific embodiments there is provided an isolated or recombinant nucleic acid comprising a polynucleotide sequence comprising or consisting of a sequence as set forth in SEQ ID NO 2. 
         [0033]    Polypeptides of the invention or a biologically active immunogenic fragment, derivative, or variant thereof may be prepared as a pharmaceutical preparation or composition. Such preparations will comprise the polypeptide or a biologically active immunogenic fragment, derivative, or variant thereof and a suitable carrier, diluent or excipient. These preparations may be administered by a variety of routes, for example, oral, buccal, topical, intramuscular, intravenous, subcutaneous, intranasal or the like. 
         [0034]    In a third aspect of the present invention, there is provided a composition comprising a polypeptide or antibody according to the invention, or a biologically active immunogenic fragment, derivative, or variant thereof, together with a pharmaceutically acceptable carrier. A carrier and/or excipient useful in a composition of the present invention will generally not inhibit to any significant degree a relevant biological activity of the polypeptide or antibody of the invention. Alternatively, or in addition, the carrier or excipient can comprise a compound that enhances uptake and/or delivery and/or efficacy of the polypeptide and/or antibody as described herein. Alternatively, or in addition, the carrier or excipient can comprise a compound that enhances the activity of a polypeptide and/or antibody as described herein and/or reduces inhibition of said polypeptide or antibody by degradative enzymes in the site of administration and/or on route to the site of action and/or at the site of action. For example, the carrier or excipient may comprise a protease inhibitor and/or a DNase inhibitor and/or an RNase inhibitor to thereby enhance the stability of a polypeptide and/or antibody as described herein above or nucleic acid encoding same. 
         [0035]    As will be apparent to the person skilled in the art based on the foregoing description, the methods of the present invention further comprise providing, producing or obtaining a composition comprising a polypeptide and/or an antibody or nucleic acid encoding said polypeptide. Suitable methods for producing such compositions will be apparent to the skilled artisan based on the disclosure herein. A polypeptide can also be delivered with other relevant antigens in a polyvalent protein vaccine. 
         [0036]    In certain further aspects, the present invention provides an antibody which has binding specificity to at least one of the polypeptides of the invention or a fragment, derivative, or variant thereof, or an antigen binding fragment of said antibody. Accordingly, in a fourth aspect of the invention there is provided an antibody which specifically binds to a polypeptide of the invention or an epitope, fragment, derivative or variant thereof. Antibodies of the present invention may confer protection against infection with  R. equi . Additionally or alternatively, an antibody can specifically bind to a polypeptide of the invention or can bind to an epitope of the pili provided on  R. equi  or an  R. equi  antigen of the pili and whilst not conveying protection against infection with  R. equi , may be a useful in an immunoassay for the detection of polypeptides of the invention or for diagnosis of  R. equi  infection. 
         [0037]    In certain embodiments, the antibody can be a polyclonal antibody. Alternatively, the antibody can be a monoclonal antibody, a chimeric antibody, or a synthesized or a synthetic antibody. Methods for producing a polyclonal and monoclonal antibodies are well known in the art and an antibody provided against a polypeptide of the pili is described herein. 
         [0038]    In certain further aspects, the present invention further extends to a method of producing an antibody which specifically binds to at least one polypeptide of the present invention, or a biologically active and/or immunogenic fragment, derivative or variant thereof, said method comprising:
       (i) immunising a host with a polypeptide or a fragment, derivative, or variant thereof as described herein according to any embodiment, and   (ii) recovering antibodies generated by the host against said polypeptide or a fragment, derivative, or variant thereof.       
 
         [0041]    The present invention also provides a method for producing an antibody that binds to an antibody which specifically binds to at least one polypeptide of the present invention or a fragment, derivative, or variant thereof (i.e., a method for producing an anti-idiotypic antibody), said method comprising:
       (i) immunising a host with an antibody that binds to a polypeptide of the invention or a fragment, derivative, or variant thereof or an antigen binding fragment of said antibody,   (ii) identifying antibodies generated by the host against an antigen binding site of said antibody; and   (iii) recovering the antibodies identified at (ii).       
 
         [0045]    The present invention also provides an anti-idiotypic antibody that selectively binds to an antibody that binds to a polypeptide or a fragment, derivative, or variant thereof as described herein or an antigen binding fragment of said antibody. 
         [0046]    In a fifth aspect of the present invention there is provided a composition comprising an antibody of the invention together with a pharmaceutical carrier. 
         [0047]    The invention also provides vectors comprising nucleic acids of the invention and cells comprising such vectors. 
         [0048]    In the sixth aspect of the invention there is provided a construct comprising a nucleic acid molecule which encodes a polypeptide of the invention, for example an isolated nucleic acid, or a fragment, derivative, or variant thereof operably linked to a promoter which is functional to allow transcription of the nucleic acid sequence and the expression of an  R. equi  polypeptide of the invention. 
         [0049]    The present invention also provides a process for producing a polypeptide or a fragment, derivative, or variant thereof as described herein according to any embodiment, said method comprising culturing a cell comprising a nucleic acid encoding a polypeptide or a fragment, derivative, or variant thereof of the present invention operably linked to a promoter under conditions suitable for expression of the polypeptide or a fragment, derivative, or variant thereof. A suitable nucleic acid may comprise a polynucleotide sequence or fragment thereof of a gene selected from Table 1, or more preferably Table 2. In one example, the method additionally comprises recovering the polypeptide from the cell culture, e.g., from the medium in which the cell is cultured. 
         [0050]    In embodiments the present invention provides a method of producing a polypeptide or a fragment, derivative, or variant thereof of the invention, said method comprising the steps of:
       (i) culturing a host cell comprising a nucleic acid encoding a polypeptide of the present invention or a vector encoding the same, and   (ii) recovering the polypeptide of the present invention from the host cell or culture medium.       
 
         [0053]    In embodiments, the construct comprises an isolated nucleic acid which encodes a polypeptide of the invention or a fragment, derivative, or variant thereof operably linked to a promoter which is functional in a host cell to allow transcription of the nucleic acid sequence and the expression of a  R. equi  polypeptide of the invention. 
         [0054]    In alternative embodiments, the construct comprises an isolated nucleic acid which encodes a polypeptide of the invention or a fragment, derivative, or variant thereof operably linked to a promoter which is functional in a heterologous host system, for example an attenuated vaccinal strain, including, but not limited to, a microbial system, a virus, a parasite, an attenuated pathogen or normal or immuno-stimulating microbiota. Suitably, the heterologous host system construct may be delivered as a live vaccine alone or in combination with other relevant protective antigens in a polyvalent vaccine. 
         [0055]    In embodiments, the construct can comprise a nucleic acid comprising a polynucleotide sequence of a gene selected from at least one gene identified by Table 1, more preferably a gene selected from Table 2, operably linked to a promoter. 
         [0056]    In embodiments, the construct can comprise a nucleic acid sequence comprising a polynucleotide sequence of SEQ ID NO 1, 2, 3, 4, 5, 6, 7, 8 or 9, more preferably a polynucleotide sequence which can encode SEQ ID NO 10 or 11. 
         [0057]    In a seventh aspect of the invention there is provided a construct of the sixth aspect of the present invention in combination with a pharmaceutical carrier. 
         [0058]    In an eighth aspect of the present invention there is provided a composition capable of treating or preventing a disease caused by  R. equi , comprising one or more surface-associated (a polypeptide naturally associated to the surface structures or on the outer surface of  R. equi .) or secreted polypeptides of  R. equi  wherein said polypeptides form pili of  R. equi.  In embodiments the composition can be a vaccine capable of preventing a disease caused by  R. equi , which results in the production of antibodies against a polypeptide of  R. equi  which can form the pili of  R. equi  and wherein the polypeptide is reactive against antibodies or immune cells recovered from animals repeatedly infected with  R. equi.    
         [0059]    In embodiments, the polypeptide of  R. equi  which can form the pili of  R. equi , wherein the polypeptide is reactive against antibodies and/or immune cells recovered from animals repeatedly infected with  R. equi  comprises the amino acid sequence encoded by a polynucleotide sequence of a gene selected from Table 1, or more preferably Table 2 or is an immunogenic fragment or variant or derivative of such a polypeptide. 
         [0060]    In embodiments of the invention, the subject for which the vaccine can be administered is a foal and immunisation results in an immune response which inhibits or prevents  R. equi  infection and results in the production of antibodies employed as an immunogen. 
         [0061]    In embodiments the subject to which the vaccine is administered can be a horse and immunisation results in an immune response which inhibits or prevents  R. equi ., or in the production of antibodies to the polypeptide employed as an immunogen. 
         [0062]    While the invention is particularly directed to polypeptide suitable as antigen in a vaccine for use in horses or foals, it will be clearly understood that it is applicable to any other animal which is susceptible to infection with  R. equi , including humans, pigs, cattle, sheep, goats, lama, cats or animals which have a similar biology and would be understood to share a high degree of genomic similarity to horses. It will also be appreciated that the diagnostic, therapeutic and prophylactic aspects of the invention are also applicable to subjects which have been exposed to an animal infected with  R. equi , or an environmental source contaminated with  R. equi  such as faeces, soil, or the like. 
         [0063]    According to a ninth aspect of the present invention there is provided a method of treating or preventing a disease or condition caused by  R. equi  comprising the step of administering a polypeptide of the invention or a fragment, derivative, or variant, an antibody, a nucleic acid, composition and/or a vaccine of the invention to subjects suffering from, or suspected to be suffering from, or at risk of a condition mediated by  R. equi.    
         [0064]    There is provided the use of a polypeptide of the invention or a biologically active and/or immunogenic fragment, derivative, or variant, an antibody, a nucleic acid, composition and/or a vaccine of the invention in the preparation of a medicament for the treatment of a condition mediated by  R. equi . In embodiments the treatment may be prophylactic treatment to prevent or inhibit infection. 
         [0065]    There is provided a polypeptide of the invention or a fragment, derivative, or variant, an antibody, a nucleic acid, composition and/or a vaccine of the invention for use in the treatment of a condition mediated by  R. equi . In embodiments the treatment may be prophylactic treatment to prevent or inhibit infection. 
         [0066]    According to a tenth aspect of the present invention there is provided a method of detecting  R. equi  comprising the step of detecting a polypeptide of the invention or a fragment, derivative, or variant, or an antibody of the invention in a sample, or a polynucleotide of the invention which can encode a polypeptide of the invention or fragment thereof. In embodiments, a sample may be a soil sample. 
         [0067]    In embodiments, there is provided a method of diagnosing a disease or condition caused by  R. equi  comprising the step of detecting a polypeptide of the invention or a fragment, derivative, or variant, or an antibody of the invention in a biological sample from a subject suffering from, suspected to be suffering from, or at risk of such a condition, or a polynucleotide of the invention which can encode a polypeptide of the invention or fragment thereof. 
         [0068]    Detection of a polypeptide or an antibody of the invention may be achieved by a variety of methods, including but not limited immunoassay methods such as radioimmuno assay, enzyme linked immunoabsorbent assays (ELISA), chemiluminescence assays, immunohistochemistry, immunoblotting, for example Western blotting, immunofluorescence and mass spectrometry. An example of use of an antibody to detect a polypeptide of a  R. equi  pili (RplB) is provided in the Examples herein. 
         [0069]    Suitably, detection of antibodies with binding specificity to a polypeptide encoded by any one of SEQ ID NO 1, 2, 3, 4, 5, 6, 7, 8, or 9 may be used as a test for R. equi in horses. In embodiments, PCR testing for nucleic acids encoding a polypeptide of the pili, for example as encoded by any one of SEQ ID NO 1, 2, 3, 4, 5, 6, 7, 8, or 9 may be used as a test for  R. equi,  particularly where a quantitative detection is preferred. Based on the nucleic acid sequence data provided herein, suitable primers or probes for use in the detection of nucleic acid sequences which can encode polypeptides of the pili of  R. equi  could be provided as would be understood in the art. As will be understood, suitably, in embodiments, said probes or primers can hybridise to the nucleic acid sequences encoding peptides associated with pilus formation, preferably any one of SEQ ID NO 1, 2, 3, 4, 5, 6, 7, 8, or 9, under stringent conditions. Hybridisation refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA. Stringent hybridisation occurs when a nucleic acid binds the target nucleic acid with minimal background. Typically, to achieve stringent hybridisation, temperatures of around 1° C. to about 20° C., more preferably 5° C. to about 20° C. below the Tm (melting temperature at which half the molecules dissociate from their partner) are used. However, it is further defined by ionic strength and pH of the solution. An example of highly stringent wash conditions is 0.15 M NaCl at 72° C. for about 15 minutes. An example of a stringent wash condition is a 0.2×SSC wash at 65° C. for 15 minutes (see, Sambrook and Russell, infra, for a description of SSC buffer). Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal. An example of a medium stringency wash for a duplex of, for example, more than 100 nucleotides, is 1×SSC at 45° C. for 15 minutes. An example of a low stringency wash for a duplex of for example more than 100 nucleotides, is 4-6×SSC at 40° C. for 15 minutes. For short probes (for example about 10 to 50 nucleotides), stringent conditions typically involve salt concentrations of less than about 1.5 M, more preferably about 0.01 to 1.0 M, Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is typically at least about 30° C. and at least about 60° C. for long probes (for example, &gt;50 nucleotides). Detection of a polynucleotide of the invention may be by any suitable means, for example using PCR, a microarray or the like as would be known in the art. 
         [0070]    In an eleventh aspect of the present invention there is provide a kit to detect  R. equi  wherein the kit comprises a polypeptide or antibody of the invention or a nucleic acid probe. In embodiments a kit can comprise a polypeptide or antibody of the invention. 
         [0071]    In embodiments, the kit is for use in the method of diagnosing a disease or condition caused by  R. equi  wherein the kit comprises a polypeptide or antibody of the invention or a nucleic acid probe. In embodiments a kit can comprise a polypeptide or antibody of the invention. 
         [0072]    In embodiments, the kit can include a solid support, for example a test strip, plastic bead or the like to which polypeptide or antibody of the invention can be coated. The kit may include a detection antibody capable of binding to a polypeptide or antibody of the invention which comprises a detectable label or binding site for a detectable label. Suitably a labelling molecule can include an enzyme, fluorescent label or radiolabel. Binding sites for detectable labels include avidin, biotin, streptavidin and the like. 
         [0073]    Additionally, the kit can include instructions for using the kit to practise the present invention. The instructions should be in writing in a tangible form or stored in an electronically retrievable form. A further aspect of the present invention provides a method of screening immunogenic  R. equi  polypeptides of the invention or a fragment, derivative, or variant thereof to determine if a test agent can bind to said polypeptide comprising the steps: providing a candidate immunogenic  R. equi  polypeptide of the invention or a fragment, derivative, or variant thereof, providing a test agent to the candidate immunogenic  R. equi  polypeptide and determining whether said test agent can bind to said candidate immunogenic  R. equi  polypeptide. 
         [0074]    A test agent which can bind to a  R. equi  polypeptide of the invention may inhibit the activity of said polypeptide, minimise its secretion or inhibit its ability to form functional pili. In embodiments, such a test agent may be a useful therapeutic. 
         [0075]    The present invention also provides the use of a polypeptide or a fragment, derivative, or variant thereof or an antibody as described herein in medicine. 
         [0076]    In a twelfth aspect, the present invention provides the use of a polypeptide of the invention or a fragment, derivative, or variant thereof, an antibody, composition and/or vaccine of the invention in the treatment or prevention of a disease or condition caused by  R. equi.    
         [0077]    In one embodiment of the invention, a method of treatment comprises the steps: 
         [0078]    (i) identifying a subject suffering from a disorder associated with or  R. equi  or at risk of developing  R. equi;    
         [0079]    (ii) administering a polypeptide, or composition as described herein to said subject. 
         [0080]    In another embodiment, the invention provides a method of treatment comprising administering or recommending a polypeptide, or a fragment, derivative, or variant thereof or an antibody or composition as described herein to a subject previously identified as having  R. equi  infection or suffering from a condition associated with  R. equi  infection. The invention may also provide a method of treatment of a subject in need thereof, said method comprising:
       (i) identifying a subject suffering from a disorder associated with or  R. equi  or at risk of developing  R. equi;      (ii) obtaining a polypeptide or a fragment, derivative, or variant thereof as described herein according to any embodiment,   (iii) formulating the polypeptide or antibody with a suitable carrier and/or excipient to form a composition wherein said composition is in an amount sufficient to reduce or prevent or inhibit  R. equi  infection or suffering from a condition associated with  R. equi  infection and   (iv) administering said composition to said subject.       
 
         [0085]    In a further embodiment there is provided a method of treatment of a subject in need thereof, said method comprising:
       (i) identifying a subject suffering from a disorder associated with or  R. equi . or at risk of developing  R. equi.;      (ii) obtaining a polypeptide or a fragment, derivative, or variant thereof or an antibody as described herein according to any embodiment,   (iii) formulating the polypeptide or antibody with a suitable carrier and/or excipient to form a composition wherein said composition is in an amount sufficient to reduce or prevent or inhibit  R. equi  infection or suffering from a condition associated with  R. equi  infection and   (iv) recommending administration of a composition at (iii).       
 
         [0090]    In embodiments a polypeptide on the invention can be provided to a subject to generate a protective immune response in the subject. In particular embodiments the polypeptide may act as a vaccine. 
         [0091]    Sequences identified in the patent application include: 
         [0000]    
       
         
               
             
           
               
                 SEQ ID NO 1 
               
               
                 rpIA REQ_18350&gt; - 3104103:3104891 
               
               
                 GTGATCGTCGCAGCGGGCGTCGGCGCCGCACTCCTGGGTATCCTCG 
               
               
                   
               
               
                 CCGGGGCGTTCGCGAACAGTGCGATCGACCGCGTGCGCCTGGAGA 
               
               
                   
               
               
                 CCGCGTGCGCCGAGCCGAAGTCGACCCCCACCGGCTCAACCCCGC 
               
               
                   
               
               
                 CGCCCCCCTCCCCTGCGTCCGCGGTAGCCACCCGGATCGCGATGAT 
               
               
                   
               
               
                 CGACACCATCACGCGACGACACGACATCAGTGCCCGCCGCGTGCTC 
               
               
                   
               
               
                 GTCGAACTCGCAACGGCCCTCCTGTTCGTCGGGATCACTCTCCGTCT 
               
               
                   
               
               
                 CGCCGCTCTCGGTCTTCTCCCGGCAACACCGGCCTATCTCTTGCAAA 
               
               
                   
               
               
                 CGGCTGCCGAACTTCCTCGTCGTACCGTCGTACCCGATCGTATTCGC 
               
               
                   
               
               
                 CTGCCTTTCAGTGGGTTCCGTCGTGCCGTTCTGTTCGGGGTCTACTT 
               
               
                   
               
               
                 CGTACTAGCCCTGATCTATCCGGCCGGCATGGGGTTCGGCGACGTC 
               
               
                   
               
               
                 AAACTTGCCGGCGTCATCGGCGCCGTCCTCGCCTACCTGTCGTACG 
               
               
                   
               
               
                 GCACATTGCTCGTCGGGGCGTTTCTCGCGTTCCTGGTGGCCGCACT 
               
               
                   
               
               
                 CGTCGGCCTGATCATCCTGGTCACCCGTCGCGGTCGGATCGGGACC 
               
               
                   
               
               
                 ACGATTCCCTTCGGGCCGTACATGATTGCGGCGGCCATCGTTGCGAT 
               
               
                   
               
               
                 CCTGGCGGCCGATCCGCTGGCGCGCGCGTATCTGGACTGGGCCGC 
               
               
                   
               
               
                 CGCGGCCTGA 
               
               
                   
               
               
                 SEQ ID NO 2 
               
               
                 rpIB REQ_18360&gt; - 1939395:1939601 
               
               
                 ATGAACCTCTTCTTCGCGAACCTGTACCTCATGGGCTTAGACGTCAA 
               
               
                   
               
               
                 GGACCGTCTGACCCGTGACGACCGCGGCGCCACTGCGGTCGAGTAC 
               
               
                   
               
               
                 GGACTGATGGTCGCCGGCATCGCGATGGTGATCATTGTTGCGGTTTT 
               
               
                   
               
               
                 CGCCTTCGGCGATAAGATTACCGACCTCTTCGATGGCTTCAACTTCG 
               
               
                   
               
               
                 ACGATCCCGGCGGCGAGTAG 
               
               
                   
               
               
                 SEQ ID NO 3 
               
               
                 rpIC REQ_18370&gt; - 1939683:1940084 
               
               
                 ATGAAGCGCCTCACTTCCGATTCAGGGGTCGCCGCAGTCGAATTCGC 
               
               
                   
               
               
                 TCTCGTCGTTCCGATCCTGATCACACTGGTCCTCGGCATCGTGGAGT 
               
               
                   
               
               
                 TCGGTCGGGGTTACAACGTCCAGAACGCGGTCAGCGCTGCTGCCCG 
               
               
                   
               
               
                 CGAGGGTGCACGGACGATGGCGATCAAGAAGGATCCGGCGGCGGC 
               
               
                   
               
               
                 GCGTGCTGCCGTGAAGGGCGCGGGTGTGTTCAGTCCGGCGATCACC 
               
               
                   
               
               
                 GATGCGGAGATCTGCATCAGCACTTCGGGAACGCAGGGCTGTTCGG 
               
               
                   
               
               
                 CAACGTCGTGTCCGAGCGGAAGTACCGTGACGCTCACGGTCAGCTA 
               
               
                   
               
               
                 TCCACTCGAGTACATGACGGGACTCTTTCCCGGTAAGCCGACGCTCA 
               
               
                   
               
               
                 CCGGCACGGGGGTCATGCGATGCGGTGGGTGA 
               
               
                   
               
               
                 SEQ ID NO 4 
               
               
                 rpID REQ_18380&gt; - 1940093:1941037 
               
               
                 ATGTCGAATGACGAGCGCGGGGTCGTCGCCGTGCTCGTTGCGATCC 
               
               
                   
               
               
                 TCATGGTCGTGCTCCTGGGATGTGCTGCGATCTCGGTCGACATCGGT 
               
               
                   
               
               
                 GCGAACTATGTCGTCAAACGTCAGTTGCAGAACGGGGCCGATGCGG 
               
               
                   
               
               
                 CTGCGCTCGCCGTAGCTCAGGAATCCAGTTGCAAGGCAGGATCTTCC 
               
               
                   
               
               
                 GCCTCATCCGTGTCGAGCCTTGTCCAGGCGAACGTCAACAGCTCGTC 
               
               
                   
               
               
                 GGCTGCAAGTGCGGCGGTGATCGACGGTGTGAAGCGGAAGGTGAC 
               
               
                   
               
               
                 GGTCACTGCGTCGGCGGTGGGTGACGACGGCCTCGCCGGCCGGAG 
               
               
                   
               
               
                 GAACGTGTTCGCTCCGGTCCTCGGAGTCGACCGCAGCGAGATCTCG 
               
               
                   
               
               
                 GCGTCTGCGACTGCAAGCTGCGTGTTTCCCCTCGGGGGGACCGCGG 
               
               
                   
               
               
                 AACTCCCGCTCACGTTCCACAAGTGCCATTTCGACGAATCCCGCAGT 
               
               
                   
               
               
                 CTGGACGTGAAGATCCTCGTCGCCTACAACGTGACGGCGCCGCGCT 
               
               
                   
               
               
                 GCAATGGAACCTCGGGAAATGCGGCACCGGGCAATTTCGGCTGGCT 
               
               
                   
               
               
                 GCAGGGGGCGAACGGTCGATGCCCGGCGAAGATCGACGCCGCCGT 
               
               
                   
               
               
                 CTACGCAACACCGGGCGACACCGGTAACAACATTCCGGGGCCGTGC 
               
               
                   
               
               
                 AAGGACACCATCAAGCAGTTTCAGAATGCCGTCGTGCGGGTCCCGAT 
               
               
                   
               
               
                 CTACGACGTCGCAGGTGGAACCGGAAGCGGTGGATGGTTTCACGTC 
               
               
                   
               
               
                 GTCGGTTTGGCTGCCTTCAAGATTCAGGGCTACCGGCTGAGCGGCA 
               
               
                   
               
               
                 ACCCGGAGTTCAACTGGAACAACGATGTTCACGGGGCGCTGAGTTG 
               
               
                   
               
               
                 CACCGGCAGCTGTCGCGGCATCATCGGCACCTTCGTGAAGATTGTCA 
               
               
                   
               
               
                 GCCTCGATTCGGATCTGACGCCGGGAGGGATCGATTTCGGCGTGAG 
               
               
                   
               
               
                 TACGATCAGCTTGCTCGATTAG 
               
               
                   
               
               
                 SEQ ID NO 5 
               
               
                 rpIE REQ_18390&gt; - 1941047:1941784 
               
               
                 TTGAGAACCCGAATCATTGCTGCGATCTGTGCGATCGTTCTCGCGGT 
               
               
                   
               
               
                 CGCGGGAACCCTCGCCCTGATCTCGTATGTACGCGGGGCCGATGCC 
               
               
                   
               
               
                 CGCGCCCTGGCGGGTACACGCACCGTCGATGTGCTCGTCGCCGATC 
               
               
                   
               
               
                 AGACGATTCCGAAGAACACTCCCGCTGATTCGCTCGTGGGAATGGTT 
               
               
                   
               
               
                 GTGGTCAAGAAACTTCCGGAAATGGCGGTGCTACCCGATCGGGTGA 
               
               
                   
               
               
                 CCAGTCTCGACCAACTGTCCGGCAAGGTCGCGCTGACCGACCTCCT 
               
               
                   
               
               
                 GCCTGGCGAACAACTGGTCTCGGCGCGATTCGTCGACCCGGCGACC 
               
               
                   
               
               
                 GCCCGAAGTCAGGACCAGGGAGGAATCCCCGAGGGGATGCAGGAG 
               
               
                   
               
               
                 GTGACGGTTCTTCTCGAGCCGCAACGCGCACTGGGAGGCCACATCG 
               
               
                   
               
               
                 CGTCGGGCGATACCGTCGGCGTCTTCATGTCCTTCTCGCCGCCCGT 
               
               
                   
               
               
                 CAAGAACTACGAAACACATCTGAGATTGCAGAAAGTGCGAGTCACGC 
               
               
                   
               
               
                 GGGTCCAGGGAACGTTCTCCAACGCCGACGAAGGGGATTCGGCCAC 
               
               
                   
               
               
                 GGTCGACTCGTCGCCGAGCCCTGCTCCCACCGAGGCCTTTCTCGTC 
               
               
                   
               
               
                 TCGCTGGCGGTCGACGTGCCGATGGCGGAGCGCGTCGTTTTCGCCG 
               
               
                   
               
               
                 CGGAGCACGGGACCATCTGGCTTTCCAATGAGCCGCCGAGTTCGAA 
               
               
                   
               
               
                 CGAGGCCGGGGCATCCGTGGTCTCCCCGGAAGGAGTGTTCCGATGA 
               
               
                   
               
               
                 SEQ ID NO 6 
               
               
                 rpIF REQ_18400&gt; - 1941781:1942980 
               
               
                 ATGAGCCGCATCGTCCTGCTGACCGATCGCGACGATTTCGCCCGCC 
               
               
                   
               
               
                 GCGTGTACCACGCCGCGGACGGCAACCTTCTGGTGTTGCCGGCGCA 
               
               
                   
               
               
                 GCCGGTTCCCCGGGGGCCGGCGCAGTTGGTCGGGCTCGGCGTGAC 
               
               
                   
               
               
                 CGTGCAACCAGAAGTTCTCGTTCTCGGTCCGGACGTGCCGGAAGTG 
               
               
                   
               
               
                 GAGGGCCTCTCCCTCGCCGGCCGGATCGATCATTCGACGCCCGGCA 
               
               
                   
               
               
                 CCACGGTGGTTCTGGCCAGTGATGCGGGCACCGACGTGTGGTTGCG 
               
               
                   
               
               
                 GGCGATGCGCGCCGGCGTGCGGGACGTGATGTCGCCGGAGGCGGA 
               
               
                   
               
               
                 GATCGCGGACGTTCGTGCGGTACTCGATCGAGCGGGCCAGGCCGCA 
               
               
                   
               
               
                 CTGGCGCGACGTCAGGGGGCGAGTGCACCGGCGGAGCAGCATGCG 
               
               
                   
               
               
                 GTTCAAGGGAAGGTCATCGTGGTCGCGTCGCCGAAAGGCGGAACCG 
               
               
                   
               
               
                 GAAAGACCACCGTTGCGACGAATCTTGCAGTAGGACTCGCGGCGGC 
               
               
                   
               
               
                 AGCGCCTCACTCGACGGTGTTGGTGGACCTCGACGTGCAGTTCGGG 
               
               
                   
               
               
                 GACGTTGCCAGTGCTCTCCAGTTGGTTCCGGAACATTGCCTGACCGA 
               
               
                   
               
               
                 CGCCGTCGCGGGCCCGGCCAGCCAGGACATGATCGTCCTCAAGACC 
               
               
                   
               
               
                 GTCCTGACACCCCATTCCACAGGACTGCATGCGCTGTGTGGGTCGG 
               
               
                   
               
               
                 ACTCGCCCGCGGCGGGCGACAGCATCACCGGCGAGCAGGTGAGCA 
               
               
                   
               
               
                 CTCTGCTGACGCAGTTGGCGGCCGAATTCCGGTACGTGGTCGTCGA 
               
               
                   
               
               
                 CACCGCGCCCGGTTTGCTCGAACACACCCTGGCGGCGCTCGACCTT 
               
               
                   
               
               
                 GCTACCGACGTCGTGTTGGTGTCGGGTATGGACGTGCCCAGCGTCC 
               
               
                   
               
               
                 GCGGGATGCACAAGGAACTGCAATTGCTGACGGAGCTGAATCTGGG 
               
               
                   
               
               
                 TCCGGTCGTGCGGCATGTCGTGCTCAACTTTGCGGATCGACGCGAG 
               
               
                   
               
               
                 GGGCTGACGGTCCAGGACATCCAGAACACCATCGGGGTCCCCGCCG 
               
               
                   
               
               
                 ATATCGTGATCAAGCGCTCGAAAGCCGTTGCCCTCTCGACGAACCGG 
               
               
                   
               
               
                 GGGGTTCCACTGCTTCAGAACCCGGGTCGGGATCGCACTGCGAAAG 
               
               
                   
               
               
                 AGTTGTGGCGACTCGTCGGCCGTATCGATCCGGCTCCCGATACCGC 
               
               
                   
               
               
                 CAAGGGTGGACGCGCGCGGCATCGGGCAGCCGAGGCGGTGGGTGC 
               
               
                   
               
               
                 GAAATGA 
               
               
                   
               
               
                 SEQ ID NO 7 
               
               
                 rpIG REQ_18410&gt; - 1942977:1944374 
               
               
                 ATGAGACTGTCCCAACGGCTCGAGGCCGTGCGCGGAGCCGCACCC 
               
               
                   
               
               
                 GTCGAAGCCGCCGCACCGATCCCGCCGGGGAAGCAGGGGAAGGCG 
               
               
                   
               
               
                 AAAACGTCCCTCCCTCCGGCCGACGCTCTCGCCGAACTGAAGGACC 
               
               
                   
               
               
                 GTGCGAGTGCGGCCCTGTACACCCGGATCGGCACCCGCTTCAACGA 
               
               
                   
               
               
                 CTCCTCGTTGAGCGAGGAGCAACTGCATCTCCTGGTCCGTGAGGAA 
               
               
                   
               
               
                 CTGGCCGAAATCGTGGAGCAGGAGACGACGCCACTCACCTTCGACG 
               
               
                   
               
               
                 AACGGCAGCGCCTGCTCCGTGAGGTTGCCGACGAGGTACTGGGGCA 
               
               
                   
               
               
                 CGGACCGCTCCAGCGGCTACTGGAGGACCCGTCGGTCACCGAGATC 
               
               
                   
               
               
                 ATGGTCAACAGCCACGACATGGTCTACGTCGAGCGGGACGGCACCC 
               
               
                   
               
               
                 TCGTCCGCAGCTCCGCGCGATTCGCGGACGAGGCGCACCTGCGTCG 
               
               
                   
               
               
                 CGTGATCGAACGCATCGTTTCCGCCGTCGGTCGACGGATCGACGAA 
               
               
                   
               
               
                 TCGTCCCCGCTCGTGGATGCACGCTTGGCGGATGGCTCCCGTGTCA 
               
               
                   
               
               
                 ACGCGGTGATCCCACCGCTCGCATTCAACGGCTCCTCGCTCACCATT 
               
               
                   
               
               
                 CGAAAGTTCTCGAAAGATCCGTTCCAGGTCGACGATCTCATCGCCTT 
               
               
                   
               
               
                 CGGCACTCTCTCGCACGAGATGGCCGAACTGCTCGACGCGTGTGTG 
               
               
                   
               
               
                 CAGGCGCGACTGAACGTCATCGTCTCGGGCGGCACGGGCACGGGG 
               
               
                   
               
               
                 AAGACGACGCTGCTCAACGTGCTCTCGTCGTTCATTCCGGAAGGGGA 
               
               
                   
               
               
                 GCGGATCGTCACCATCGAGGACGCCGTGGAACTGCAACTTCAGCAG 
               
               
                   
               
               
                 GACCACGTCGTACGGTTGGAGAGCCGACCGCCGAACATCGAGGGCA 
               
               
                   
               
               
                 AGGGTGCCGTCACCATCCGCGACCTGGTGCGGAACTCGCTGCGTAT 
               
               
                   
               
               
                 GCGTCCCGACCGCATCGTGGTGGGGGAGTGTCGCGGAGGCGAGAG 
               
               
                   
               
               
                 TCTCGACATGCTGCAAGCGATGAACACCGGTCACGACGGGTCGCTG 
               
               
                   
               
               
                 TCGACGGTGCATGCGAATTCGCCCCGTGACGCCATCGCGCGCTTGG 
               
               
                   
               
               
                 AGACGCTCGTGTTGATGGCCGGCATGGACCTGCCGTTGCGGGCGAT 
               
               
                   
               
               
                 CCGGGAGCAGATTGCTTCGGCGGTCGACGTGATCGTGCAGCTCACT 
               
               
                   
               
               
                 CGACTACGTGACGGCACTCGGCGAGTGACCCACGTGACCGAGGTCC 
               
               
                   
               
               
                 AGGGCATGGAGGGTGAGATCGTCACCCTGCAGGATGCCTTCCTGTT 
               
               
                   
               
               
                 CGACTACAGCGCCGGCGTCGACGCGCGCGGGCGATTCCTCGGCAG 
               
               
                   
               
               
                 ACCGCAGCCGACCGGAGTGCGGCCGCGGTTCACCGACAGATTCCGA 
               
               
                   
               
               
                 GATCTCGGTATTGCTTTGTCGCCGAGTGTTTTCGGGGTGGGAGAACC 
               
               
                   
               
               
                 CTCCCGGGGGCGGGTATGA 
               
               
                   
               
               
                 SEQ ID NO 8 
               
               
                 rpIH REQ_18420&gt; - 1944371:1946239 
               
               
                 ATGAGCCGGTGCGTGGTGGCCGTCGTGCTCGCCCTCGGTGCGGGT 
               
               
                   
               
               
                 GTTCTGGGAATTCCCGCCGTAGCCGCGGCGGCCGAGGAGGCTGTCC 
               
               
                   
               
               
                 AGGTCTCGGCGGTCGACACGACCCGGTTTCCCGACATCGAGGTGTC 
               
               
                   
               
               
                 CATCCTCGCGCCGCCCGGTATCGAAGGGCAGGCGATCGATCCGGGA 
               
               
                   
               
               
                 ACGTTCGCGCTCACCGAGGGCGGCGTGCCGCGAGAGATCGAGGTC 
               
               
                   
               
               
                 AGGCAGCAGCCGGGTTCCGAGCAGGACATCGTGCTCGCAATCGACG 
               
               
                   
               
               
                 TGTCCGGGGGCATGTCGGGTCCGGCGCTGGACGACGTGAAGCGCG 
               
               
                   
               
               
                 CCGCATCGGATTTCGTGCGGCAGGCGCCGGCCGGCGCCCACATCG 
               
               
                   
               
               
                 GAATCGTCGCGATCTCGTCGACGCCACAGGTGCTCTCGGAACTGAC 
               
               
                   
               
               
                 GACGGACTCCGAGGACCTGCTCCGCAGGATCGACGGACTGAAGGCG 
               
               
                   
               
               
                 GGCGGCAACAGCGCGATCGCAGATTCGGTGGTGACCGCCGCCGAG 
               
               
                   
               
               
                 ATGCTCGAGCGCGGCGAAGCGGCCAACAACATCCTGCTTCTGTTGA 
               
               
                   
               
               
                 CGGACGGCGCCGACACGTCGAGTGCACACTCGATGTCGGAACTCCC 
               
               
                   
               
               
                 GTCCGTCCTGAGTCGGTCGCGCGCGTCGCTGTACGCCGTGCAGATG 
               
               
                   
               
               
                 TCGACACCCGAGACGAACTCTGCTCTCCTGCAGCAGGTTGCGCGGG 
               
               
                   
               
               
                 AGTCGCGCGGTCAGTACGCGTCTGCGGGTGATACGGCGGCGCTGG 
               
               
                   
               
               
                 GTGCGATCTACCAGTCGGCCGCTCGCGCGCTCGGAAACCTGTACGT 
               
               
                   
               
               
                 CGTCCGATACCGATCGGAAGCGAATGGCGATACCCAGGTGGTGGCG 
               
               
                   
               
               
                 AGCGTGCGCAGCGGCGCAGCCGGCCGAGTGAGCGATCCGTTCCCG 
               
               
                   
               
               
                 GTGACATTGCCCGGTGTGGTGCCGACGCCGAGCGTCGTCGCCGGG 
               
               
                   
               
               
                 ACCGTCGACGGTTTCTTCACGTCTTCGACGGGGCTGGTGATCGGGC 
               
               
                   
               
               
                 TCCTAGCGTGCTACTCGGCGCTTGCGGGAGGCGTGCTGGCGGTCGC 
               
               
                   
               
               
                 CGGTAGAGCGCCCGCGAGGATTTCGGCAGCACGTCGTGGGCGGCA 
               
               
                   
               
               
                 GGACGGACGGGACTCGATGCTGTCCCGATTCGCGGAACGGCTGGTG 
               
               
                   
               
               
                 CAGTGGATCGATCAGAACCTGAGGAGACGCGGACGCATCGCTGCCC 
               
               
                   
               
               
                 GCACCCAGGCGCTACAGGAGGCGGGGCTGAAGCTTCGTCCAGGTGA 
               
               
                   
               
               
                 CTTCATCGCCCTGGTCGGTGCTGCGGCGATCACCGCTGCGGCGATC 
               
               
                   
               
               
                 GGTCTCCTGGCTTCGGGCATCGTGGCGGCGCTCTTGCTCGCGGCGA 
               
               
                   
               
               
                 TCACAGTGGGATTGTCGAGAATCTATCTCCGTGTGATGGCCGGTAGG 
               
               
                   
               
               
                 CGTCGGGCCGCGTTCGCTGATCAGCTCGACGATTCCCTGCAGCTGC 
               
               
                   
               
               
                 TGGCCAGCAATCTCCGAGCCGGGCACAGCATGCTCCGAGCGCTCGA 
               
               
                   
               
               
                 TTCCCTTTCCCGAGAGGCGGAGGTGCCGACTTCGGAGGAGTTCGCT 
               
               
                   
               
               
                 CGGATCGTCAACGAGACTCGGGTGGGACGTGATCTCAACGAGTCTC 
               
               
                   
               
               
                 TCGACGACGTGGCCCGGCGGATGCGAAGTGACGATTTCAACTGGAT 
               
               
                   
               
               
                 AGCTCAGGCAATCGCCATCAACCGTGAGGTCGGAGGCGACCTCGCG 
               
               
                   
               
               
                 GAAGTCCTCGACCAGGTGGGCAACACCATTCGAGAGCGAAATCAGAT 
               
               
                   
               
               
                 TCGACGGCAGGTGAAAGCCCTTGCTGCCGAGGGGAAACTGTCCGCC 
               
               
                   
               
               
                 TACGTGCTGATGGCGCTGCCCTTCGGTCTCACCGCATTTCTGCTCGT 
               
               
                   
               
               
                 CTCGAATCCGGACTACCTGTCGAAGTTGACGGGTAGCGCCATCGGC 
               
               
                   
               
               
                 TACGTGATGATCGCGGTGGGGCTCGTCATGCTGACCGTCGGTGGGC 
               
               
                   
               
               
                 TGTGGATGAACAAGGTTGTCTCGGTCAAGTTCTAG 
               
               
                   
               
               
                 SEQ ID NO 9 
               
               
                 rpII REQ_18430&gt; - 1946262:1947152 
               
               
                 GTGATTCCACCGCTGGTGCTCATGGCGGCGCTGTCCGTCGGCGGGG 
               
               
                   
               
               
                 CGTTGGGTGTTCTGGTGTGGTTGACGGTCGGCGCCCGAGATCCGGA 
               
               
                   
               
               
                 ACGCGGACCCGCCCTTCGGAACCTGCAGTCGCAGCTGGCGTTGCCG 
               
               
                   
               
               
                 ATTCCGGAGTCGGGAGGCGCGCCACCGCTTTCGCTCGGCCGATTCG 
               
               
                   
               
               
                 TGAAGCTGCTGTCGCCGCCCGGGACGATGGCCCGCTTGGAACGACT 
               
               
                   
               
               
                 GCACATCCTTGCCGGTCGTCCAGCGGCGTGGGTTCCGGAACGGGCC 
               
               
                   
               
               
                 GCGATGGCGAAGATCGTTCTCGCCGCGGCCGCCGCCCTGCTCGGC 
               
               
                   
               
               
                 CTTCTCGCGGTGGGTGCGTCGCCTGGCGTCGGCCGGGTGCTGTTCG 
               
               
                   
               
               
                 CTGCGGCCGCCGTCGCGCTGGCGTATTTCGTCCCGGAACTTCTCCT 
               
               
                   
               
               
                 GCAGAGCAGGGGGCAGGAGCGCCAAGCCGCGATCGAACTGGCGCT 
               
               
                   
               
               
                 TGCCGACACCCTCGACCAGATGACGATCGCAGTCGAGGCGGGCCTG 
               
               
                   
               
               
                 GGGTTCGAAGCCGCCATGCAGCGGGCCGCGAAGAACGGAAAGGGG 
               
               
                   
               
               
                 CCGCTGGCCGAGGAATTCATCCGGACATTGCAGGACATACAGATGG 
               
               
                   
               
               
                 GGCAGTCGAGGCGAATCGCGTACCTGGATCTTGCCGCCAGAACGAA 
               
               
                   
               
               
                 AGCACCCAACTTGCGGAGGTTCCTTCGGGCCGTCATCCAAGCCGAC 
               
               
                   
               
               
                 GAGTACGGCGTGGCCATCGCCGAGGTCCTGCGGACCCAGGCCTCG 
               
               
                   
               
               
                 GAGATGCGTCTGAAACGCCGTCAGAGTGCTGAGGAGAAGGCGATGA 
               
               
                   
               
               
                 AGGTTCCGGTGAAGGTGCTGTTTCCGTTGATGACCTGCATCCTGCCG 
               
               
                   
               
               
                 ACCATCTTCATCGTGATCCTGGGTCCGGCGGTGATCAACATGATGGA 
               
               
                   
               
               
                 GGTCTTGGGCGGTATGTAA 
               
               
                   
               
               
                 SEQ ID NO 12 
               
               
                 RpIA: 
               
               
                 VIVAAGVGAALLGILAGAFANSAIDRVRLETACAEPKSTPTGSTPPPPSP 
               
               
                   
               
               
                 ASAVATRIAMIDTITRRHDISARRVLVELATALLFVGITLRLAALGLLPA 
               
               
                   
               
               
                 TPAYLWFAAVGIALAVIDIDCKRLPNFLVVPSYPIVFACLSVGSVVTGDW 
               
               
                   
               
               
                 SALLRAAIGAAVLFGVYFVLALIYPAGMGFGDVKLAGVIGAVLAYLSYGT 
               
               
                   
               
               
                 LLVGAFLAFLVAALVGLIILVTRRGRIGTTIPFGPYMIAAAIVAILAADP 
               
               
                   
               
               
                 LARAYLDWAAAA 
               
               
                   
               
               
                 SEQ ID NO 13 
               
               
                 RpIB: 
               
               
                 MNLFFANLYLMGLDVKDRLTRDDRGATAVEYGLMVAGIAMVIIVAVFAFG 
               
               
                   
               
               
                 DKITDLFDGFNFDDPGGE 
               
               
                   
               
               
                 SEQ ID NO 14 
               
               
                 RpIC: 
               
               
                 MKRLTSDSGVAAVEFALVVPILITLVLGIVEFGRGYNVQNAVSAAAREGA 
               
               
                   
               
               
                 RTMAIKKDPAAARAAVKGAGVFSPAITDAEICISTSGTQGCSATSCPSGS 
               
               
                   
               
               
                 TVTLTVSYPLEYMTGLFPGKPTLTGTGVMRCGG 
               
               
                   
               
               
                 SEQ ID NO 15 
               
               
                 RpID: 
               
               
                 MSNDERGVVAVLVAILMVVLLGCAAISVDIGANYVVKRQLQNGADAAALA 
               
               
                   
               
               
                 VAQESSCKAGSSASSVSSLVQANVNSSSAASAAVIDGVKRKVTVTASAV 
               
               
                   
               
               
                 GDDGLAGRRNVFAPVLGVDRSEISASATASCVFPLGGTAELPLTFHKCH 
               
               
                   
               
               
                 FDESRSLDVKILVAYNVTAPRCNGTSGNAAPGNFGWLQGANGRCPAKI 
               
               
                   
               
               
                 DAAVYATPGDTGNNIPGPCKDTIKQFQNAVVRVPIYDVAGGTGSGGWF 
               
               
                   
               
               
                 HVVGLAAFKIQGYRLSGNPEFNWNNDVHGALSCTGSCRGIIGTFVKIVSL 
               
               
                   
               
               
                 DSDLTPGGIDFGVSTISLLD 
               
               
                   
               
               
                 SEQ ID NO 16 
               
               
                 RpIE: 
               
               
                 LRTRIIAAICAIVLAVAGTLALISYVRGADARALAGTRTVDVLVADQTIP 
               
               
                   
               
               
                 KNTPADSLVGMVVVKKLPEMAVLPDRVTSLDQLSGKVALTDLLPGEQLVS 
               
               
                   
               
               
                 ARFVDPATARSQDQGGIPEGMQEVTVLLEPQRALGGHIASGDTVGVFMSF 
               
               
                   
               
               
                 SPPVKNYETHLRLQKVRVTRVQGTFSNADEGDSATVDSSPSPAPTEAFL 
               
               
                   
               
               
                 VSLAVDVPMAERVVFAAEHGTIWLSNEPPSSNEAGASVVSP 
               
               
                   
               
               
                 EGVFR 
               
               
                   
               
               
                 SEQ ID NO 17 
               
               
                 RpIF: 
               
               
                 MSRIVLLTDRDDFARRVYHAADGNLLVLPAQPVPRGPAQLVGLGVTVQP 
               
               
                   
               
               
                 EVLVLGPDVPEVEGLSLAGRIDHSTPGTTVVLASDAGTDVWLRAMRAGV 
               
               
                   
               
               
                 RDVMSPEAEIADVRAVLDRAGQAALARRQGASAPAEQHAVQGKVIVVA 
               
               
                   
               
               
                 SPKGGTGKTTVATNLAVGLAAAAPHSTVLVDLDVQFGDVASALQLVPEH 
               
               
                   
               
               
                 CLTDAVAGPASQDMIVLKTVLTPHSTGLHALCGSDSPAAGDSITGEQVST 
               
               
                   
               
               
                 LLTQLAAEFRYVVVDTAPGLLEHTLAALDLATDVVLVSGMDVPSVRGMH 
               
               
                   
               
               
                 KELQLLTELNLGPVVRHVVLNFADRREGLTVQDIQNTIGVPADIVIKRSK 
               
               
                   
               
               
                 AVALSTNRGVPLLQNPGRDRTAKELWRLVGRIDPAPDTAKGGRARHRAA 
               
               
                   
               
               
                 EAVGAK 
               
               
                   
               
               
                 SEQ ID NO 18 
               
               
                 RpIG: 
               
               
                 MRLSQRLEAVRGAAPVEAAAPIPPGKQGKAKTSLPPADALAELKDRASA 
               
               
                   
               
               
                 ALYTRIGTRFNDSSLSEEQLHLLVREELAEIVEQETTPLTFDERQRLLRE 
               
               
                   
               
               
                 VADEVLGHGPLQRLLEDPSVTEIMVNSHDMVYVERDGTLVRSSARFADEA 
               
               
                   
               
               
                 HLRRVIERIVSAVGRRIDESSPLVDARLADGSRVNAVIPPLAFNGSSLTI 
               
               
                   
               
               
                 RKFSKDPFQVDDLIAFGTLSHEMAELLDACVQARLNVIVSGGTGTGKTTL 
               
               
                   
               
               
                 LNVLSSFIPEGERIVTIEDAVELQLQQDHVVRLESRPPNIEGKGAVTIRD 
               
               
                   
               
               
                 LVRNSLRMRPDRIVVGECRGGESLDMLQAMNTGHDGSLSTVHANSPRDAI 
               
               
                   
               
               
                 ARLETLVLMAGMDLPLRAIREQIASAVDVIVQLTRLRDGTRRVTHVTEVQ 
               
               
                   
               
               
                 GMEGEIVTLQDAFLFDYSAGVDARGRFLGRPQPTGVRPRFTDRFRDLGI 
               
               
                   
               
               
                 ALSPSVFGVGEPSRGRV 
               
               
                   
               
               
                 SEQ ID NO 19 
               
               
                 RpIH: 
               
               
                 MSRCVVAVVLALGAGVLGIPAVAAAAEEAVQVSAVDTTRFPDIEVSILAP 
               
               
                   
               
               
                 PGIEGQAIDPGTFALTEGGVPREIEVRQQPGSEQDIVLAIDVSGGMSGPA 
               
               
                   
               
               
                 LDDVKRAASDFVRQAPAGAHIGIVAISSTPQVLSELTTDSEDLLRRIDGL 
               
               
                   
               
               
                 KAGGNSAIADSVVTAAEMLERGEAANNILLLLTDGADTSSAHSMSELPSV 
               
               
                   
               
               
                 LSRSRASLYAVQMSTPETNSALLQQVARESRGQYASAGDTAALGAIYQSA 
               
               
                   
               
               
                 ARALGNLYVVRYRSEANGDTQVVASVRSGAAGRVSDPFPVTLPGVVPT 
               
               
                   
               
               
                 PSVVAGTVDGFFTSSTGLVIGLLACYSALAGGVLAVAGRAPARISAARRG 
               
               
                   
               
               
                 RQDGRDSMLSRFAERLVQWIDQNLRRRGRIAARTQALQEAGLKLRPGD 
               
               
                   
               
               
                 FIALVGAAAITAAAIGLLASGIVAALLLAAITVGLSRIYLRVMAGRRRAA 
               
               
                   
               
               
                 FADQLDDSLQLLASNLRAGHSMLRALDSLSREAEVPTSEEFARIVNETRV 
               
               
                   
               
               
                 GRDLNESLDDVARRMRSDDFNWIAQAIAINREVGGDLAEVLDQVGNTIRE 
               
               
                   
               
               
                 RNQIRRQVKALAAEGKLSAYVLMALPFGLTAFLLVSNPDYLSKLTGSAIG 
               
               
                   
               
               
                 YVMIAVGLVMLTVGGLWMNKVVSVKF 
               
               
                   
               
               
                 SEQ ID NO 20 
               
               
                 RpII: 
               
               
                 VIPPLVLMAALSVGGALGVLVWLTVGARDPERGPALRNLQSQLALPIPES 
               
               
                   
               
               
                 GGAPPLSLGRFVKLLSPPGTMARLERLHILAGRPAAWVPERAAMAKIVLA 
               
               
                   
               
               
                 AAAALLGLLAVGASPGVGRVLFAAAAVALAYFVPELLLQSRGQERQAAIE 
               
               
                   
               
               
                 LALADTLDQMTIAVEAGLGFEAAMQRAAKNGKGPLAEEFIRTLQDIQMG 
               
               
                   
               
               
                 QSRRIAYLDLAARTKAPNLRRFLRAVIQADEYGVAIAEVLRTQASEMRLK 
               
               
                   
               
               
                 RRQSAEEKAMKVPVKVLFPLMTCILPTIFIVILGPAVINMMEVLGGM 
               
             
          
         
       
     
         [0092]    Preferred features and embodiments of each aspect of the invention are as for each of the other aspects mutatis mutandis unless context demands otherwise. 
         [0093]    Each document, reference, patent application or patent cited in this text is expressly incorporated herein in their entirety by reference, which means it should be read and considered by the reader as part of this text. That the document, reference, patent application or patent cited in the text is not repeated in this text is merely for reasons of conciseness. Reference to cited material or information contained in the text should not be understood as a concession that the material or information was part of the common general knowledge or was known in any country. 
         [0094]    Throughout the specification, unless the context demands otherwise, the terms ‘comprise’ or ‘include’, or variations such as ‘comprises’ or ‘comprising’, ‘includes’ or ‘including’ will be understood to imply the includes of a stated integer or group of integers, but not the exclusion of any other integer or group of integers. 
         [0095]    By “consisting essentially of” it is meant that a nucleic acid does not include additional, substituted or deleted nucleotide(s) to a polynucleotide sequence of the invention described herein or a polypeptide does not include additional, substituted, or deleted amino acids which significantly alter the character of a sequence of the invention such that it is not immunogenic and biologically active. 
         [0096]    As used herein, the singular forms “a”, “an”, and “the” include the corresponding plural reference unless the context clearly dictates otherwise. 
         [0097]    Where a range of values is expressed, it will be understood that this range encompasses the upper and lower limits of the range and all values in between these limits. 
         [0098]    The terms “polypeptide”, “protein” and “peptide” are herein used interchangeably. 
         [0099]    The term “isolated” refers to materials, such as nucleic acid molecules, which are substantially free or otherwise removed from components that normally accompany or interact with the materials in a naturally occurring environment. An isolated nucleic acid typically contains less than about 50%, preferably less than about 75%, and most preferably less than about 90% of the components with which it was originally associated. Polypeptides, antibodies and nucleic acids of the invention as disclosed herein can be isolated. 
         [0100]    The terms “polynucleotide”, “polynucleotide sequence”, and “nucleic acid sequence” are used interchangeably herein. A “polynucleotide” as used herein refers to purine- and pyrimidine-containing polymers of any length, either polyribonucleotides or polydeoxyribonucleotides, which can be single or double stranded, such as, for example, DNA-DNA, DNA-RNA and RNA-RNA. A polynucleotide may optionally contain synthetic, non-natural or altered nucleotide bases. A polynucleotide in the form of a polymer of DNA may be comprised of one or more strands of cDNA, genomic DNA, synthetic DNA, or mixtures thereof. 
         [0101]    A “derivative” of a polypeptide as used herein will be understood to include polypeptides which have been subject to chemical modifications, including esterification, amidation, reduction, methylation, fusion to another peptide and the like. The polypeptide derivatives may be modified such that the modifications increase the stability and/or immunogenicity and/or bioavailability of the polypeptide derivative in comparison to the unmodified polypeptide. Covalent derivatives of the peptides or polypeptides of the invention can be prepared by linking the chemical moieties to functional groups on the amino acid side chains or at the N-terminus or C-terminus of the antigenic polypeptide. Conjugation of a polypeptide to another peptide may further be achieved by genetic means through the use of recombinant DNA techniques that are well know in the art, such as those set forth in the teachings of Sambrook et al. Molecular Cloning: A Laboratory Manual, 2 ed. Vol. 1, pp. 1.101-104, Cold Spring Harbor Laboratory Press, (1989) and F.M. Ausubel et al. Current Protocols in Molecular Biology, Eds. J.Wiley Press (2006), the relevant portions of which are incorporated herein by reference. 
         [0102]    A “variant” polypeptide of the invention can be a polypeptide which has an amino acid sequence which differs from the polypeptide encoded by SEQ ID NO 1, 2, 3, 4, 5, 6, 7, 8 or 9 due to the presence of one or more deletions, insertions, or substitutions of amino acid residues. In embodiments, a variant has at least 85%, 86%, 87%, 88%, 89%, preferably at least 90%, 91%, 92%, 93%, 94%, and more preferably 95%, 96%, 97%, 98%, 99% but less than 100% contiguous amino acid sequence identity to the corresponding polypeptide encoded by the nucleotide sequence as disclosed herein. Percentage identity may be determined using, for example computer programs as would be known by one skilled in the art. 
         [0103]    Variants can include polypeptides in which individual amino acids of the polypeptide of the invention are substituted by other amino acids which are closely related as understood in the art, for example, substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as arginine for lysine, glutamic for aspartic acid or glutamine for asparagine. 
         [0104]    In embodiments, a fragment of a polypeptide of the present invention can consist of a truncated version of a polypeptide of the invention which has been truncated by 1, 2, 3, 4 or more than 5, more than 10, or more than 20 amino acids. An antigenic fragment may be generated using for example C-terminal deletion of any one of the polynucleotide sequences of the genes as listed in Table 1 or Table 2 and said C-terminal deletion constructs may then be inserted into a suitable prokaryotic or eukaryotic expression plasmid. The antigenic activity of the expression products derived from the constructs may then be tested by assessing reactivity with antisera from naturally and/or experimentally infected horse or foals using immunoblotting methods. Alternatively a series of synthetic polypeptide fragments with greater than 85%, greater than 90%, greater than 95%, or 100% sequence identity to portions of any one the polypeptides encoded by a polynucleotide sequence of a gene of Table 1 or more preferably Table 2 can be generated. These peptides may then be reacted with antisera from naturally or experimentally infected horses using an ELISA method to determine which peptide fragments are antigenic. Alternatively, synthetic peptides may be used to immunise, for example, mice, rabbits, or horses and the antisera produced can be assessed for reactivity with  R. equi  using indirect immunofluorescence assays. In this way immunogenic fragments may be identified and  R. equi -specific antisera may be produced. These two latter approaches described are particularly advantageous for small peptides that contain linear, continuous epitopes. 
         [0105]    “Operably linked” means that a nucleic acid molecule is placed in functional relationship with another nucleic acid molecule. Generally an operably linked promoter will be linked such that it is contiguous with and in the same reading phase as the gene to be expressed. 
         [0106]    Generally the terms “treating”, “treatment” and the like are used to mean affecting a subject tissue or cell to obtain a desired pharmacological and/or physiological effect. As used herein, the term “treatment” and associated terms such as “treat” and “treating” means the reduction of the progression, severity and/or duration of infection or for the amelioration of at least one of the symptoms thereof by  R. equi  or may be prophylactic (preventative treatment). The term ‘treatment’ therefore refers to any regimen that can benefit a subject. References herein to “therapeutic” and “prophylactic” treatments are to be considered in their broadest context. The term “therapeutic” does not necessarily imply that a subject is treated until total recovery. Similarly, “prophylactic” does not necessarily mean that the subject will not eventually contract a disease condition. 
         [0107]    As used herein, the term “subject” refers to an animal, preferably a mammal and in particular a horse. 
     
    
     
       FIGURES 
         [0108]    Embodiments of the present invention will now be described by way of example only with reference to the accompanying figures in which: 
           [0109]      FIG. 1  illustrates the  R. equi  pilus locus (rpl). (A) The 9 Kb rpl horizontally acquired (HGT) island (REQ18350-430) is absent from nonpathogenic  Rhodococcus  spp. (e.g.  R. jostii  RHA1 and  R. erythropolis  PR4). rpl genes have were detected in all  R. equi  clinical isolates (≈300 isolates tested). rpl gene products which are considered to be encoded are: A, prepilin peptidase; B, pilin subunit; C, TadE minor pilin; D, putative lipoprotein; E, CpaB pilus assembly protein; F, CpaE pilus assembly protein; GHI, Tad transport machinery. (B) Electron micrograph of  R. equi  103S pili (indicated by arrowheads). Bar=0.5 μm. (C)  R. equi  pili visualized by immunofluorescence microscopy (×1,000 magnification). Reproduced from Letek et al. 2010, PLoS Genet. 6: e1001145). 
           [0110]      FIG. 2  illustrates a demonstration by targeted mutant construction and genetic re-complementation analysis that the rpl locus encodes the  R. equi  pilus. Negative staining transmission electron micrographs of wild-type  R. equi  103S (WT) (panel A), isogenic rplB deletion mutant of 103S (ΔrplB, apiliated) (panel B), rplB-complemented mutant (piliated) (panel C), and mock-complemented mutant with an empty vector (no rplB gene). Bar=0.5 μm (panel D). 
           [0111]      FIG. 3  illustrates the effect of rplB gene deletion and complementation on  R. equi  adhesion to (A) macrophages (J774A.1 cell line) and (B) epithelial cells (HeLa cell line), two key target cell types in the pathogenesis of airborne lung infection. Data expressed as percentage of the control (WT); mean of at least three independent duplicate experiments±SEM. 
           [0112]      FIG. 4  illustrates the adhesion phenotype to (A) epithelial cells (HeLa cell line) and (B) macrophages (J774A.1 cell line) with additional rpl knock-out mutants (rplA and rplE). 
           [0113]      FIG. 5  illustrates Rpl pili are essential for  R. equi  lung colonization in mice as demonstrated using a novel in vivo lung infection model in mice developed by the inventors. It is based on a competitive virulence assay in which each mouse receives an intranasal inoculum containing 50% of wild-type (WT)  R. equi  bacteria and 50% of mutant (ΔrplB)  R. equi  bacteria. t=0 means 60 min after delivery of the intranasal inoculum. 
           [0114]      FIG. 6  illustrates production in rabbits of a specific antibody against the putative  R. equi  pilin subunit (RplB). (A) Amino acid sequence of putative RplB prepilin and of the C-terminal peptide used to raise a rabbit polyclonal antibody (boxed). Arrowhead indicates putative cleavage site of the prepilin. (B) Immunodetection of the RplB pilin by SDS-PAGE western blot analysis of whole cell extracts of wild-type  R. equi  (WT), an isogenic in-frame deletion rplB mutant (ΔrplB), the rplB-complemented mutant (ΔrplB+rplB), and a mock-complement mutant (ΔrplB+vector), using the anti-RplB peptide antibody (diluted 1:1,000; secondary antibody, alkaline phosphatase-conjugated mouse anti-rabbit monoclonal antibody, 1:10,000 diluted; reaction revealed with NBT/BCIP substrate. The anti-Rpl antibody specifically detects the Rpl pilin subunit in WT and re-completed rpl mutant, not in the apiliated rpl mutant and mock-complemented mutant. (C) Detection of Rpl pili production in  R. equi  by immunofluorescence using the anti-RplB peptide antibody and the same bacteria as in (B) (630× magnification, Leica AF6000 microscope). 
           [0115]      FIG. 7  illustrates Inhibition of  R. equi  attachment to (A) macrophages and (B) epithelial cells by an anti-RplB antibody. Prior to the adhesion assay, the antibody raised against the RplB (pilin subunit) peptide (see  FIG. 6A ) was incubated for 60 min at 37° C. (40 μl/ml of a suspension in cell culture medium of exponentially grown R. equi bacteria at a density calculated for a multiplicity of infection of 15:1). As a control, the  R. equi  bacterial cell suspension was pre-incubated with an irrelevant antiserum (anti-Listeria monocytogenes rabbit polyclonal antibody). 
           [0116]      FIG. 8  illustrates RplB pilin antigens are recognized in vivo and elicit a strong antibody response in naturally infected foals. Representative example of the reactivity against the Rpl pilin of horse sera from bacteriologically confirmed cases of foal pneumonia, as determined by SDS-PAGE western blot analysis with whole cell extracts of wild-type  R. equi  (WT) and the isogenic ΔrplB mutant. All convalescent sera tested to date gave a strong reaction against the RplB pilin antigen whereas normal (non-case) sera did not. The Rpl pili dissociate into 18 kDa polypeptides that probably correspond to SDS-resistant homo-tetramers (predicted molecular mass of RplB pilin, 4.95 kDa) that remain non-covalently bound by strong monomer-monomer interactions via the N-terminal hydrophobic region of the pilin subunit. (A) indicates RplB is the first antigen detected in a curde  R. equi  protein preparation by the antibodies present in case sera. 
           [0117]      FIG. 9  illustrates variability of RplB amino acid sequence in  R. equi  strains and of other Rpl proteins. 
           [0118]      FIG. 10  illustrates the nucleotide sequences encoding Rpl proteins of other strains of  R. equi.    
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0119]    As indicated above, the inventors have identified polypeptides which play an important role in virulence of  R equi  and have used this knowledge to identify polypeptides which can be used to mediate an immune response in infected subjects, particularly horses, and in particular foals. Whilst the amino acid sequences of the polypeptides determined for the identified strain are noted, as will be understood, biologically active immunogenic fragments, derivatives or variants of such a polypeptide can also be used. As discussed variant polypeptides can comprise amino acid percent identity with the amino acid sequences disclosed herein. Alternatively, polypeptides of the invention may be encoded by variant nucleic acid sequences which have nucleotide percent identity with the polynucleotide sequences disclosed herein. 
         [0120]    The percent identity of two or more sequences may be determined by visual inspection and mathematical calculation. Alternatively, the percent identity of two nucleic acid sequences can be determined by comparing sequence information using the GAP computer program, version 6.0 described by Devereux et al. (Nucl. Acids Res. 12:387, 1984) and available from the University of Wisconsin Genetics Computer Group (UWGCG). The preferred default parameters for the GAP program include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res. 14:6745, 1986, as described by Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358, 1979; (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps. Other programs used by one skilled in the art of sequence comparison may also be used. 
         [0121]    Polypeptides of the invention may be prepared by any of a number of conventional techniques. A nucleic acid encoding a peptide or a biologically active immunogenic fragment, derivative, or variant thereof, may be subcloned into an expression vector for production of the polypeptide or fragment. The DNA sequence advantageously is fused to a sequence encoding a suitable leader or signal peptide and/or a promoter operable in a cell into which the nucleic acid is to be introduced. Alternatively, the desired fragment may be chemically synthesized using known techniques. DNA fragments also may be produced by restriction endonuclease digestion of a full length cloned DNA sequence, and isolated by electrophoresis on agarose gels. If necessary, oligonucleotides that reconstruct the 5′ or 3′ terminus to a desired point may be ligated to a DNA fragment generated by restriction enzyme digestion. Such oligonucleotides may additionally contain a restriction endonuclease cleavage site upstream of the desired coding sequence, and position an initiation codon (ATG) at the N-terminus of the coding sequence. 
         [0122]    Polymerase chain reaction (PCR) procedure also may be employed to isolate and amplify a DNA sequence encoding a desired polypeptide fragment. Oligonucleotides that define the desired termini of the DNA fragment are employed as 5′ and 3′ primers. The oligonucleotides may additionally contain recognition sites for restriction endonucleases, to facilitate insertion of the amplified DNA fragment into an expression vector. PCR techniques are described in Saiki et al., Science 239:487 (1988); Recombinant DNA Methodology, Wu et al., eds., Academic Press, Inc., San Diego (1989), pp. 189-196; and PCR Protocols: A Guide to Methods and Applications, Innis et al., eds., Academic Press, Inc. (1990). 
         [0123]    The invention encompasses polypeptides and biologically active immunogenic fragments, derivatives, or variants thereof in various forms, including those that are naturally occurring or produced through various techniques such as procedures involving recombinant DNA technology. For example, nucleotides encoding polypeptides of the invention can be derived from SEQ ID NO 1, 2, 3, 4, 5, 6, 7, 8, or 9 by in vitro mutagenesis, which includes site-directed mutagenesis, random mutagenesis, and in vitro nucleic acid synthesis. Such forms include, but are not limited to, derivatives, variants, and oligomers, as well as fusion proteins or fragments thereof. 
       Polypeptide Derivatives 
       [0124]    Embodiments of a derivative of a polypeptide of the invention can comprise one or more non-naturally occurring amino acids or amino acid analogs, including non-genetically encoded L-amino acids, synthetic L-amino acids or D-enantiomers of an amino acid. Suitably, embodiments of a derivative can comprise one or more residues selected from the group consisting of: hydroxyproline, β-alanine, 2,3-diaminopropionic acid, α-aminoisobutyric acid, N-methylglycine (sarcosine), ornithine, citrulline, t-butylalanine, t-butylglycine, N-methylisoleucine, phenylglycine, cyclohexylalanine, norleucine, naphthylalanine, pyridylananine 3-benzothienyl alanine 4-chlorophenylalanine, 2-fluorophenylalanine, 3-fluorophenylalanine, 4-fluorophenylalanine, penicillamine, 1,2,3,4-tetrahydrotic isoquinoline-3-carboxylic acid β-2-thienylalanine, methionine sulfoxide, homoarginine, N-acetyl lysine, 2,4-diamino butyric acid, p-aminophenylalanine, N-methylvaline, homocysteine, homoserine, ε-amino hexanoic acid, δ-amino valeric acid, 2,3-diaminobutyric acid and mixtures thereof. Other amino acid residues that are useful for making the polypeptides and polypeptide derivatives described herein can be found, e.g., in Fasman, 1989, CRC Practical Handbook of Biochemistry and Molecular Biology, CRC Press, Inc., and the references cited therein. 
         [0125]    In embodiments, derivatives of polypeptides of the invention can also comprise an isostere of a polypeptide. The term “isostere” as used herein is intended to include a chemical structure that can be substituted for a second chemical structure because the steric conformation of the first structure fits a binding site specific for the second structure. The term specifically includes peptide back-bone modifications (i.e., amide bond mimetics) known to those skilled in the art. Such modifications include modifications of the amide nitrogen, the α-carbon, amide carbonyl, complete replacement of the amide bond, extensions, deletions or backbone crosslinks. Several peptide backbone modifications are known, including ψ[CH2S], ψ[CH2NH], ψ[CSNH2], ψ[NHCO], ψ[COCH2], and ψ[(E) or (Z) CH═CH]. In the nomenclature used above, ψ indicates the absence of an amide bond. The structure that replaces the amide group is specified within the brackets. Other modifications include, for example, an N-alkyl (or aryl) substitution (ψ[CONR]), or backbone crosslinking to construct lactams and other cyclic structures. In another example, a polypeptide derivative may be a retro-peptide analog. A retro-peptide analog comprises a reversed amino acid sequence of a polypeptide described herein. For example, a retro-peptide analog of a polypeptide comprises a reversed amino acid sequence of a sequence set forth in any one of SEQ ID NO 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. Retro-inverso polypeptides may be complete or partial. Complete retro-inverso peptides are those in which a complete sequence of a polypeptide described herein is reversed and the chirality of each amino acid in a sequence is inverted, other than glycine, because glycine does not have a chiral analog. Partial retro-inverso polypeptides are those in which only some of the peptide bonds are reversed and the chirality of only those amino acid residues in the reversed portion is inverted. For example, one or two or three or four or five or more than 10, more than 20, more than 30, more than 40 or more than 50 amino acid residues are D-amino acids. Suitably a polypeptide of and for use in the present invention may be further modified using at least one of C and/or N-terminal capping, and/or cysteine residue capping. Suitably, a polypeptide of and for use in the present invention may be capped at the N terminal residue with an acetyl group. Suitably, a polypeptide of and for use in the present invention may be capped at the C terminal with an amide group. Suitably, thiol groups of cysteines of polypeptides of the invention may be capped with acetamido methyl groups. In embodiments, the term derivative can include scrambled polypeptides comprising immunodominant epitopes of the rpl encoded pilus for example fragments of SEQ ID NOs 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In embodiments derivatives can be encoded by rpl genes or fragments thereof which encode immunodominant epitopes of Rpl pilus provided in tandem, or as longer repeat stretches, for example concatemerized, to increase the immunogenicity of the encoded polypeptides. In embodiments, combinations of polypeptides of the invention (and corresponding nucleic acid sequences) can be fused in a single polypeptide. 
       Polypeptide Synthesis 
       [0126]    A polypeptide or a biologically active immunogenic fragment, derivative, or variant thereof may be synthesized using any suitable chemical method known to the person skilled in the art. For example, synthetic peptides can be prepared using known techniques of solid phase, liquid phase, or peptide condensation, or any combination thereof, and can include natural and/or unnatural amino acids. Amino acids used for peptide synthesis may be standard Boc (Nα-amino protected Nα-t-butyloxycarbonyl) amino acid resin with the deprotecting, neutralization, coupling and wash protocols of the original solid phase procedure of Merrifield, J. Am. Chem. Soc., 85:2149-2154, 1963, or the base-labile Na-amino protected 9-fluorenylmethoxycarbonyl (Fmoc) amino acids described by Carpino and Han, J. Org. Chem., 37:3403-3409, 1972. Both Fmoc and Boc Nα-amino protected amino acids can be obtained from various commercial sources, such as, for example, Fluka, Bachem, Advanced Chemtech, Sigma, Cambridge Research Biochemical, Bachem, or Peninsula Labs. 
         [0127]    Generally, chemical synthesis methods comprise the sequential addition of one or more amino acids to a growing peptide chain. Normally, either the amino or carboxyl group of the first amino acid is protected by a suitable protecting group. The protected or derivatized amino acid can then be either attached to an inert solid support or utilized in solution by adding the next amino acid in the sequence having the complementary (amino or carboxyl) group suitably protected, under conditions that allow for the formation of an amide linkage. The protecting group is then removed from the newly added amino acid residue and the next amino acid (suitably protected) is then added, and so forth. After the desired amino acids have been linked in the proper sequence, any remaining protecting groups (and any solid support, if solid phase synthesis techniques are used) are removed sequentially or concurrently, to render the final polypeptide. By simple modification of this general procedure, it is possible to add more than one amino acid at a time to a growing chain, for example, by coupling (under conditions which do notracemize chiral centers) a protected tripeptide with a properly protected dipeptide to form, after deprotection, a pentapeptide. See, e.g., J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis (Pierce Chemical Co., Rockford, Ill. 1984) and G. Barany and R. B. Merrifield, The Peptides: Analysis, Synthesis, Biology, editors E. Gross and J. Meienhofer, Vol. 2, (Academic Press, New York, 1980), pp. 3-254, for solid phase peptide synthesis techniques; and M. Bodansky, Principles of Peptide Synthesis, (Springer-Verlag, Berlin 1984)and E. Gross and J. Meienhofer, Eds., The Peptides: Analysis. Synthesis. Biology, Vol. 1, for classical solution synthesis. Typical protecting groups include t-butyloxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc) benzyloxycarbonyl (Cbz); p-toluenesulfonyl (Tx); 2,4-dinitrophenyl; benzyl (Bzl); biphenylisopropyloxycarboxy-carbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl, o-bromobenzyloxycarbonyl, cyclohexyl, isopropyl, acetyl, o-nitrophenylsulfonyl and the like. 
         [0128]    Typical solid supports are cross-linked polymeric supports. These can include divinylbenzene cross-linked-styrene-based polymers, for example, divinylbenzene-hydroxymethylstyrene copolymers, divinylbenzene-chloromethylstyrene copolymers and divinylbenzene-benzhydrylaminopolystyrene copolymers. 
         [0129]    A peptide or a biologically active immunogenic fragment, derivative, or variant thereof as described herein according to any embodiment can also be chemically prepared by other methods such as by the method of simultaneous multiple peptide synthesis. See, e. g., Houghten Proc. Natl. Acad. Sci. USA 82: 5131-5135, 1985 or U.S. Pat. No. 4,631,211. 
       Recombinant Polypeptide Production 
       [0130]    Alternatively, or in addition, a peptide or a biologically active immunogenic fragment, derivative, or variant thereof can be produced as a recombinant protein. To facilitate the production of a recombinant polypeptide, nucleic acid encoding the same is preferably isolated or synthesized. Typically the nucleic acid encoding the recombinant protein is/are isolated using a known method, such as, for example, amplification (e.g., using PCR or splice overlap extension) or isolated from nucleic acid from  R. equi  using one or more restriction enzymes or isolated from a library of nucleic acids. 
         [0131]    Methods for such isolation will be apparent to the ordinary skilled artisan and/or described in Ausubel et al (In: Current Protocols in Molecular Biology. Wiley Interscience, ISBN 047 150338, 1987), Sambrook et al (In: Molecular Cloning: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Third Edition 2001). 
         [0132]    For expressing protein by recombinant means, a protein-encoding nucleic acid is placed in operable connection with a promoter or other regulatory sequence capable of regulating expression in a cell-free system or cellular system. For example, nucleic acid comprising a sequence that encodes a polypeptide of the pili of  R. equi  is placed in operable connection with a suitable promoter and maintained in a suitable cell for a time and under conditions sufficient for expression to occur. 
         [0133]    A number of other gene construct systems for expressing a nucleic acid of a gene selected from Table 1 or Table 2 in bacterial cells are well-known in the art and are described for example, in Ausubel et al (In: Current Protocols in Molecular Biology. Wiley Interscience, ISBN 047 150338, 1987), and Sambrook et al (In: Molecular Cloning: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Third Edition 2001). 
         [0134]    A wide range of additional host/vector systems suitable for expressing a polypeptide of the present invention are available publicly, and described, for example, in Sambrook et al (In: Molecular cloning, A laboratory manual, second edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989). 
         [0135]    Following expression of a polypeptide, isolation and purification of the polypeptide may be accomplished by any suitable technique, as would be known in the art. 
       Compositions 
       [0136]    A polypeptide or a biologically active immunogenic fragment, derivative, or variant thereof may be administered alone, but will preferably be administered as a pharmaceutical composition, which will generally comprise a suitable pharmaceutically acceptable excipient, diluent or carrier selected depending on the intended route of administration. Examples of suitable pharmaceutical carriers include; water, glycerol and ethanol. 
         [0137]    The term “carrier or excipient” as used herein, refers to a carrier or excipient that is conventionally used in the art to facilitate the storage, administration, and/or the biological activity of an active compound. A carrier may also reduce any undesirable side effects of the active compound. A suitable carrier is, for example, stable, e.g., incapable of reacting with other ingredients in the formulation. In one example, the carrier does not produce significant local or systemic adverse effect in recipients at the dosages and concentrations employed for treatment. Such carriers and excipients are generally known in the art. Suitable carriers for this invention include those conventionally used, e.g., water, saline, aqueous dextrose, and glycols are preferred liquid carriers, particularly (when isotonic) for solutions. Suitable pharmaceutical carriers and excipients include starch, cellulose, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, glycerol, propylene glycol, water, ethanol, and the like. 
         [0138]    Pharmaceutical composition adapted for oral administration may be presented as discrete units such as capsules, soft gels, or tablets; powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; edible foams or whips; or oil-in-water liquid emulsions or water-in-oil liquid emulsions. 
         [0139]    Pharmaceutical compositions provided as formulations adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions which contain a polypeptide or a biologically active immunogenic fragment, derivative, or variant thereof or a antibody of the invention and optionally, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets. 
       Administration 
       [0140]    As will be appreciated by a person of skill in the art, selecting an administration regimen for a therapeutic composition or vaccine of the invention depends on several factors, including the serum or tissue turnover rate of a polypeptide of the invention or an antibody invention, the level of symptoms, the immunogenicity of the polypeptide, and the accessibility of the target cells in the biological matrix. Preferably, an administration regimen maximizes the amount of therapeutic compound delivered to the subject consistent with an acceptable level of side effects. Accordingly, the amount of polypeptide, antibody or composition delivered depends in part on the polypeptide, antibody or composition and the severity of the condition being treated. 
         [0141]    A polypeptide or antibody can be provided, for example, by continuous infusion, or by doses at intervals of, e.g., one day, one week, or 1-7 times per week. A preferred dose protocol is one involving the maximal dose or dose frequency that avoids significant undesirable side effects. A total weekly dose depends on the type and activity of the compound being used. Determination of the appropriate dose is made by a veterinarian or clinician, for example using parameters or factors known or suspected in the art to affect treatment or predicted to affect treatment. 
       EXAMPLES 
     Example 1   
       [0142]    Using electron microscopy and other microscopical techniques we demonstrated that  R. equi  produces long, thick and apparently rigid pili appendages, typically between two and four per bacteria cell ( FIG. 1  panels BC). 
       Example 2 
     Genome Sequencing 
       [0143]    Genome sequencing of the complete genome sequence of  R. equi  strain 103S was determined in an international collaborative venture. The genome has just over 5 million base pairs and encodes 4598 genes of average length value 1009 pairs of nucleotides. 
       Example 3  
       [0144]    Demonstration that the rpl ( R. equi  pili) locus (nucleotide positions 1,938,280 to 1,947,152, locus tags REQ18350-430) encodes the  R. equi  pilus by targeted mutant construction and genetic re-complementation analysis. 
         [0145]    An in-frame deletion mutant was constructed in the rplB gene putatively encoding the Rpl pilin subunit (RplB). Homologous recombination methodology previously devised (Navas et al. 2001, Identification and mutagenesis by allelic exchange of choE, encoding a cholesterol oxidase from the intracellular pathogen  Rhodococcus equi.  J. Bacteriol. 183: 4796-4805), and a novel suicide vector, pSelAct, for positive selection of double recombinants on 5-fluorocytosine (5-FC) (van der Geize et al. 2008, A novel method to generate unmarked gene deletions in the intracellular pathogen  Rhodococcus equi  using 5-fluorocytosine conditional lethality. Nucleic Acids Res. 36: el 51) was used in this approach. The ΔrplB mutant was complemented by stably inserting the rplB gene plus corresponding promoter region into the  R. equi  chromosome using the integrative vector pSET152 (Hong and Hondalus 2008, Site-specific integration of  Streptomyces  PhiC31 integrase-based vectors in the chromosome of  Rhodococcus equi . FEMS Microbiol. Lett. 287: 63-68). As shown in  FIG. 2 , the inactivation of the rplB gene results in loss of pili formation by  R. equi . Pili formation is restored upon reintroduction of the rplB gene in the ΔrplB mutant but not by complementation with an empty vector, demonstrating that rplB is a gene directly responsible for the piliated phenotype. 
       Example 4 
       [0146]    Demonstration that the  R. equi  pili mediate attachment to mammalian cells. 
         [0147]    The ΔrplB mutant was tested in adhesion assays using monolayers of two cell types relevant to  R. equi  infection: epithelial cells to which the pathogen have to adhere to during the initial stages of lung colonization, and macrophages, which are used as host cells for bacterial intracellular replication. The rplB mutant was severely impaired in attachment to both eukaryotic cell types, and its complementation with the rplB gene but not an empty vector restored wild-type cytoadhesiveness ( FIG. 3 ). 
         [0148]    Two additional mutants were constructed in rplA and rplE ( FIG. 1A ) and they also caused a significant reduction of  R. equi  cytoadhesiveness ( FIG. 4 ), indicating that other genes from the rpl locus are involved in pilus-mediated attachment to eukaryotic cells (not shown). 
       Example 5  
       [0149]    Demonstration that the  R. equi  pili are essential for lung colonization in vivo in a mouse model of  R. equi  infection. 
         [0150]    A novel in vivo model of competitive  R. equi  lung infection in mice was developed and used to test the virulence of the rplB mutant in comparison to rplB-proficient (wild-type) bacteria.  R. equi  wild-type and an isogenic rplB knock-out mutant in equal amounts were inoculated intranasally to Balb/c mice. At specific time points after infection, the bacterial population was determined in lungs and tracheas to assess airway colonisation. The spleens were also analysed to determine the capacity of the bacteria to overwhelm local defences and spread deeper into host tissues.  FIG. 4  shows that the mutant, initially accounting for 50% of the inoculum, was only detectable—in much less proportion—during the two first time points sampled (0 and 24 hour post inoculation), indicating that apiliated bacteria are immediately cleared from the lungs and thus substantially less virulent. In the first time point, only a very small fraction of the bacteria that translocated to the spleen were mutants. These data indicate that a wild-type capacity to attach to host cells via the Rpl pili is essential for host colonisation by  R. equi.    
       Example 6  
       [0151]    Demonstration that the RplB (putative pilin subunit) protein is antigenic in vivo in rabbits. 
         [0152]    The synthetic RplB peptide indicated in  FIG. 6A  was used to hyperimmunize rabbits. The antiserum specifically detected the RplB pilin subunit in whole cell extracts of  R. equi  ( FIG. 6B ) and the production of Rpl pili in  R. equi  by immunofluorescence ( FIG. 6C ), indicating that it is immunogenic in vivo in rabbits. 
       Example 7  
       [0153]    Demonstration that RplB elicits neutralizing antibodies that inhibit  R. equi  attachment. 
         [0154]    The rabbit hyperimmune anti-RplB antiserum was used in attachment-inhibition assays in HeLa epithelial cells and J774A.1 macrophages.  FIG. 7  shows that the RplB antiserum, but not an irrelevant antiserum, inhibited  R. equi  cytoadhesion. Given the key role of the Rpl pili in lung colonization by  R. equi  ( FIG. 4 ), these data indicate that RplB is a vaccine target to prevent lung infection by the pathogen. 
         [0155]    This is evidence that indicates that the pilin subunit RplB is recognised by the immune system in vivo and the animal body mounts a specific immune response with production of specific antibodies to the  R. equi  pilin subunit RplB. As the polyclonal antiserum containing anti-RplB antibodies inhibits attachment of  R. equi  to monolayers of HeLa epithelial cells or J774 macrophages if added to the infection assays, which effect is not seen if the Rpl antiserum is not added, or if an unrelated control antiserum raised against other bacteria (e.g.  Listeria ) is used, this indicates a protective function of the antibodies through inhibition of bacterial attachment to host cells, the first phase of host colonisation during infection. 
       Example 8 
       [0156]    Demonstration that the RplB putative pilin subunit is an immunodominant antigen in naturally infected foals. 
         [0157]    Using SDS-PAGE western immunoblotting and whole-cell extracts from wild-type and rplB (apiliated) deletion mutant bacteria, it was shown that the sera from natural cases of  R. equi  infection in foals contain antibodies to the RplB putative pilin subunit ( FIG. 8 ). The RplB protein is the first detected in the crude  R. equi  protein preparation by the antibodies present in the case sera. Thus, the RplB pilin subunit is recognized in vivo by the foal&#39;s immune system during  R. equi  infection and is an immunodominant antigen. Normal, non-case sera did not react against the RplB protein, indicating that this antigen provides a suitable maker for the early detection and diagnosis of  R. equi  infection in foals. 
         [0158]    Although the invention has been particularly shown and described with reference to particular examples, it will be understood by those skilled in the art that various changes in the form and details may be made therein without departing from the scope of the present invention.