Purified hepatitis C virus envelope proteins for diagnostic and therapeutic use

Abstract

The present invention relates to a method for purifying recombinant HCV single or specific oligomeric envelope proteins selected from the group consisting of E1 and/or E1/E2 characterized in that upon lysing the transformed host cells to isolate the recombinantly expressed protein a disulphide bond cleavage or reduction step is carried out with a disulphide bond cleavage agent. The present invention also relates to a composition isolated by such a method. The present invention also relates to the diagnostic and therapeutic application of these compositions. Furthermore, the invention relates to the use of HCV E1 protein and peptides for prognosing and monitoring the clinical effectiveness and/or clinical outcome of HCV treatment.

1 . An isolated E1 specific monoclonal antibody, said antibody specifically binding to at least one region within a domain spanning amino acids 192-383 of the hepatitis C virus polyprotein.  
     
     
         2 . The monoclonal antibody according to  claim 1  which has been isolated from a mammal immunized with a composition comprising purified recombinant HCV single or specific oligomeric recombinant E1 envelope proteins.  
     
     
         3 . The monoclonal antibody according to  claim 2  wherein said recombinant HCV E1 envelope proteins are produced by a recombinant mammalian cell.  
     
     
         4 . The monoclonal antibody according to  claim 3  wherein said mammalian cell is infected with recombinant vaccinia virus carrying DNA for expressing said HCV E1 envelope proteins.  
     
     
         5 . The monoclonal antibody according to  claim 2  wherein said recombinant HCV E1 envelope proteins are produced by a recombinant yeast cell.  
     
     
         6 . The isolated antibody of  claim 2  wherein said E1 protein is at least 90% pure.  
     
     
         7 . The isolated antibody of  claim 2  wherein said E1 protein is at least 95% pure.  
     
     
         8 . The isolated protein of  claim 2  wherein said E1 protein is at least 97% pure.  
     
     
         9 . The isolated antibody of  claim 2  wherein said E1 protein is at least 97% pure.  
     
     
         10 . The isolated antibody of  claim 2  wherein said E1 protein is at least 99% pure.  
     
     
         11 . Kit for determining the presence of HCV antigens present in a biological sample, comprising: 
 at least one E1 specific monoclonal antibody according to  claim 1  or  2 , 
 a buffer or components necessary for producing the buffer enabling binding reaction between these antibodies and the HCV antigens present in said biological sample, and  
 a means for detecting the immune complexes formed in the preceding binding reaction.  
   
     
     
         12 . The kit according to  claim 11  wherein said E1 specific monoclonal antibody is in an immobilized form on a solid substrate.  
     
     
         13 . A method for in vitro diagnosis of HCV antigen present in a biological sample, comprising at least the following steps: 
 (i) contacting said biological sample with an E1 specific monoclonal antibody according to  claim 1  or  2  under appropriate conditions which allow the formation of an immune complex,    (ii) removing unbound components,    (iii) incubating the immune complexes formed with heterologous antibodies, with said heterologous antibodies being conjugated to a detectable label under appropriate conditions,    (iv) detecting the presence of said immune complexes visually or mechanically.    
     
     
         14 . The method according to  claim 13  wherein said E1 specific monoclonal antibody is in an immobilized form on a solid substrate.  
     
     
         15 . An isolated E1 specific monoclonal antibody which specifically binds to a region in the E1 domain spanning an amino acid segment selected from the group consisting of the segments spanning E1 amino acids 181 to 200, 193 to 212, 205 to 224, 208 to 227, 217 to 236, 229 to 248, 241 to 260, 253 to 272, 265 to 284, 289 to 308, 301 to 320, 313 to 332, 325 to 344, 337 to 356, 349 to 368, and 373 to 392.  
     
     
         16 . The isolated E1 specific monoclonal antibody according to  claim 14  which specifically binds to a region in the E1 domain spanning an amino acid segment selected from the group consisting of SEQ ID NOs: 56-71.