Antigenic polypeptides

Abstract

The invention relates to a method for the identification of antigenic polypeptides expressed by pathogenic microbes; vaccines comprising said polypeptides; recombinant methods to manufacutre said polypeptides; and therapeutic antibodies directed to said polypeptides.

1 . An isolated nucleic acid molecule comprising a DNA sequence selected from the group consisting of: 
 (i) the DNA sequence as represented in SEQ ID NO's 1-13;    (ii) DNA sequences which hybridise to the sequence presented in the SEQ ID No's 1-13 identified in (i) above and which encode a polypeptide expressed by a pathogenic organism; and    (iii) DNA sequences which are degenerate as a result of the genetic code to the DNA sequences defined in (i) and (ii).    
     
     
         2 . An isolated nucleic acid molecule according to  claim 1  which is genomic DNA.  
     
     
         3 . An isolated nucleic acid molecule according to  claim 1  or  2  which anneals under stringent hybridisation conditions to the sequences presented in SEQ ID NO's 1-13.  
     
     
         4 . A vector comprising a nucleic acid molecule according to any of claims  1 - 3 .  
     
     
         5 . A vector according to  claim 4  wherein the vector is adapted for recombinant expression of the polypeptide encoded by the nucleic acid.  
     
     
         6 . A vector according to  claim 4  or  5  wherein said vector is an expression vector adapted for prokaryotic gene expression.  
     
     
         7 . A vector according to  claim 4  or  5  wherein said vector is an expression vector adapted for eukaryotic gene expression.  
     
     
         8 . A vector according to any of  claims 4  to  7  wherein the adaptation of the vector includes the provision of promoter sequences.  
     
     
         9 . A vector according to  claim 8  wherein the promoter sequences provide for cell specific, inducible or constitutive expression.  
     
     
         10 . A method to identify antigenic polypeptides comprising: 
 (i) providing a nucleic acid library encoding genes or partial gene sequences of a pathogenic organism;    (ii) transforming/transfecting said library into a host cell;    (iii) contacting the polypeptides expressed by the genes/partial gene sequences with autologous antisera derived from an animal infected with, or has been infected with, said pathogenic organism; and    (iv) purifying the nucleic acid encoding the polypeptide or partial polypeptide binding to said autologous antisera.    
     
     
         11 . A method according to  claim 10  wherein said library comprises genomic DNA of a pathogenic organism.  
     
     
         12 . A method according to  claim 10  or  claim 11  wherein said pathogenic organism is bacterial.  
     
     
         13 . A method according to any of  claims 10  to  12  wherein said bacterial organism is selected from the following:  Staphylococcus aureus; Staphylococcus epidermidis; Enterococcus faecalis; Mycobacterium tuberculsis;  Streptococcus group B;  Streptoccocus pneumoniae; Helicobacter pylori; Neisseria gonorrhea;  Streptococcus group A;  Borrelia burgdorferi; Coccidiodes immitis; Histoplasma sapsulatum; Neisseria meningitidis  type B;  Shigella flexneri; Escherichia coli; Haemophilus influenzae    
     
     
         14 . A method according to any of  claim 13  wherein said pathogenic organism is  Staphylococcus aureus.    
     
     
         15 . A method according to any of  claim 13  wherein said pathogenic organism is  Staphylococcus epidermidis.    
     
     
         16 . A method according to any of  claims 10  to  15  wherein said nucleic acid library is a lambda library.  
     
     
         17 . A polypeptide identified by the method according to any of  claims 10  to  16 .  
     
     
         18 . A polypeptide according to  claim 17  which is selected from the group consisting of SEQ ID NO's: 14-19.  
     
     
         19 . A method for the production of the polypeptides according to any of claims  17  or  18  comprising: 
 (i) providing a cell transformed/transfected with a vector according to any of  claims 4  to  9  and with cell culture conditions; and  
 (ii) purifying said polypeptide from said cell, or its growth environment.  
 
     
     
         20 . A method according to  claim 19  wherein said vector encodes, and thus said recombinant polypeptide is provided with, a secretion signal to facilitate purification of said polypeptide.  
     
     
         21 . A cell transformed or transfected with the vector according to any of  claims 4  to  9 .  
     
     
         22 . A cell according to  claim 21  which is a prokaryotic cell.  
     
     
         23 . A cell according to  claim 21  which is a eukaryotic cell selected from the group consisting of: fungal cell, insect cell, amphibian cell; mammalian cell; plant cell.  
     
     
         24 . A vaccine comprising at least one polypeptide according to claims  16  or  17 .  
     
     
         25 . A vaccine according to  claim 24  which further comprises a carrier and/or adjuvant.  
     
     
         26 . A method to immunise an animal against a pathogenic microbe comprising administering to the animal at least one polypeptide, or part thereof, according to any previous claim or the vaccine of any previous claim.  
     
     
         27 . A method according to  claim 26  wherein the animal is human.  
     
     
         28 . A method according to  claim 26  or  27  wherein the vaccine, or antigenic polypeptide, is delivered by direct injection either intravenously, intramuscularly or subcutaneously.  
     
     
         29 . A method according to  claim 25  or  26  wherein the vaccine or antigenic polypeptide is taken orally.  
     
     
         30 . A method according to any of  claims 26  to  29  wherein the vaccine is against the bacterial genus Staphylococcus spp.  
     
     
         31 . A method according to  claim 30  wherein the vaccine is against the bacterial species  Staphylococcus aureus.    
     
     
         32 . A method according to  claim 30  wherein the vaccine is against the bacterial species  Staphylococcus epidermidis.    
     
     
         33 . An antibody, or at least an effective part thereof, which binds at least with a selective part of the polypeptide according to  claim 16  or  17 .  
     
     
         34 . An antibody according to  claim 33  which is a monoclonal antibody.  
     
     
         35 . An antibody according to  claim 33  or  34  wherein said effective part comprises FAb fragments.  
     
     
         36 . An antibody according to any of  claims 33  to  35  which is a chimeric antibody.  
     
     
         37 . An antibody according to any of  claims 33  to  35  which is a humanised antibody.  
     
     
         38 . An antibody according to any of  claims 33  to  37  wherein said antibody is provided with a marker, label or tag.  
     
     
         39 . An antibody according to  claim 38  wherein said antibody is provided with a marker selected from a group consisting of: a radioactive label, a fluorescent label; an epitope tag.  
     
     
         40 . An antibody according to any of  claims 34  to  39  which is produced as a fusion polypeptide.  
     
     
         41 . A vector which is adapted for the expression of the antibodies according to any of claims  34 - 40 .  
     
     
         42 . A cell which has been transformed or transfected with the vector according to  claim 41 .  
     
     
         43 . A method for the production of the antibody according to any of claims  34  or  40  comprising: 
 i) providing a cell transformed or transfected with the vector according to  claim 41  and with cell culture conditions; and  
 ii) purifying said antibody from said cell, or its growth environment.  
 
     
     
         44 . A hybridoma cell line which produces an antibody according to  claim 34 .  
     
     
         45 . Use of the antibodies according to any of  claims 33  to  40  for the manufacture of a medicament for the treatment of  Staphylococcus aureus -associated septicaemia, food-poisoning or skin disorders.  
     
     
         46 . Use of the antibodies according to any of  claims 33  to  40  for the manufacture of a medicament for the treatment of  Staphylococcus epidermidis -associated septicaemia, peritonitis or endocarditis  
     
     
         47 . A method for preparing a hybridoma cell-line producing monoclonal antibodies according to  claim 34 , comprising the steps of: 
 i) immunising an immunocompetent mammal with an immunogen comprising at least one polypeptide having the amino acid sequence as set forward in SEQ ID No: 14-19, or fragments thereof;    ii) fusing lymphocytes of the immunised immunocompetent mammal with myeloma cells to form hybridoma cells;    iii) screening monoclonal antibodies produced by the hybridoma cells of step (ii) for binding activity to the amino acid sequences of (i);    iv) culturing the hybridoma cells to proliferate and/or to secrete said monoclonal antibody; and    v) recovering the monoclonal antibody from the culture supernatant.    
     
     
         48 . A method according to  claim 47 , wherein said immunocompetent mammal is a mouse  
     
     
         49 . A method according to  claim 47 , wherein said immunocompetent mammal is a rat