Hcv e1 comprising specific disulfide bridges

Abstract

The invention relates to recombinantly or synthetically produced HCV E1 envelope proteins or parts thereof comprising disulfides between specific cysteine residues. The invention further relates to viral-like particles and compositions comprising said HCV E1 envelope proteins or parts thereof as well as to methods using said HCV E1 envelope proteins or parts thereof, and to kits comprising said HCV E1 envelope proteins or parts thereof.

1 . A recombinant or synthetic HCV E1 envelope protein or a part thereof comprising at least one of the following disulfides:
 a disulfide between the pair of cysteines at amino acid positions 207 and 226,   a disulfide between the pair of cysteines at amino acid positions 229 and 238, or   a disulfide between the pair of cysteines at amino acid positions 272 and 281,   
       wherein said amino acid positions are relative to the HCV polyprotein which is starting with the methionine of the Core protein at amino acid position 1 of said HCV polyprotein. 
     
     
         2 . The HCV E1 envelope protein or a part thereof according to  claim 1  further comprising at least one of the cysteines at amino acid positions 304 or 306 wherein said at least one cysteine is carrying a free thiol group or a thiol group which is blocked. 
     
     
         3 . The HCV E1 envelope protein or a part thereof according to  claim 2  wherein said blocked thiol group is reversibly or irreversibly blocked. 
     
     
         4 . The HCV E1 envelope protein or a part thereof according to  claim 2  wherein said at least one cysteine at amino acid position 304 or 306 is mutated to a non-cysteine amino acid or is deleted. 
     
     
         5 . A viral-like particle comprising the HCV E1 envelope protein or part thereof according to any one of  claims 1  to  4 . 
     
     
         6 . A composition comprising the HCV E1 envelope protein or part thereof according to any one of  claims 1  to  4  or the viral-like particle according to  claim 5  and at least one of a pharmaceutically acceptable carrier, adjuvant or vehicle. 
     
     
         7 . An HCV vaccine comprising the HCV E1 envelope protein or part thereof according to any one of  claims 1  to  4  or the viral-like particle according to  claim 5  and at least one of a pharmaceutically acceptable carrier, adjuvant or vehicle. 
     
     
         8 . The HCV vaccine according to  claim 7  which is a prophylactic or therapeutic HCV vaccine. 
     
     
         9 . An antibody selectively binding to an HCV E1 envelope protein or a part thereof comprising at least one of the following disulfides:
 a disulfide between the pair of cysteines at amino acid positions 207 and 226,   a disulfide between the pair of cysteines at amino acid positions 229 and 238, or   a disulfide between the pair of cysteines at amino acid positions 272 and 281,   
       wherein said amino acid positions are relative to the HCV polyprotein which is starting with the methionine of the Core protein at amino acid position 1 of said HCV polyprotein; or selectively binding to a viral-like particle comprising any of said HCV E1 envelope proteins or parts thereof, 
       an antibody capable of competing with said antibody for selectively binding said HCV E1 envelope protein, 
       or a fragment of any of said antibodies. 
     
     
         10 . The antibody according to  claim 9  that is isolated from a mammal immunized with the HCV E1 envelope protein or part thereof according to any one of  claims 1  to  4  or with the viral-like particle according to  claim 5 . 
     
     
         11 . The antibody according to  claim 9  or  10  which is a monoclonal antibody. 
     
     
         12 . The antibody according to any of  claims 9  to  11  which is secreted by the hybridoma cell line of DSM deposit with accession number ACC 2470. 
     
     
         13 . The hybridoma cell line of DSM deposit with accession number ACC 2470. 
     
     
         14 . A method for determining the presence of HCV antibodies in a sample, said method comprising the steps of:
 (i) contacting said sample with the HCV E1 envelope protein or part thereof according to any one of  claims 1  to  4  or the viral-like particle according to  claim 5  under conditions allowing the formation of an immunological antigen-antibody complex;   (ii) determining the immunological complex formed in (i);   (iii) inferring from (ii) the presence of HCV antibodies in said sample.   
     
     
         15 . A method for determining HCV E1 antigens in a sample, said method comprising the steps of:
 (i) contacting said sample with the antibody according to any one of  claims 9  to  12  under conditions allowing the formation of an immunological antigen-antibody complex;   (ii) determining the immunological complex formed in (i);   (iii) inferring from (ii) the presence of HCV E1 antigens in said sample.   
     
     
         16 . The method according to  claim 14  or  15  wherein in step (i) the HCV E1 envelope protein or part thereof according to any one of  claims 1  to  4 , the viral-like particle according to  claim 5  or the antibody according to any one of  claims 9  to  12  is added as competitor; and wherein said HCV E1 envelope protein or part thereof, viral-like particle or antibody used in said step (i) or said HCV E1 envelope protein or part thereof, viral-like particle or antibody added as competitor are labeled. 
     
     
         17 . A method for screening compounds capable of modulating the binding between an HCV E1 envelope protein and an E1 ligand, said method comprising:
 (i) contacting said E1 ligand with an HCV E1 envelope protein or part thereof according to any one of  claims 1  to  4  or with the viral-like particle according to  claim 5  under conditions allowing the formation of an HCV E1-E1 ligand complex;   (ii) adding a compound suspected of modulating the binding between an HCV E1 envelope protein and an E1 ligand to the HCV E1-E1 ligand complex formed in (i);   (iii) determining the change in amount of HCV E1-E1 ligand complex formed in (i) and (ii);   (iv) inferring from (iii) whether the compound added in (ii) is a modulator of binding between an HCV E1 envelope protein and an E1 ligand.   
     
     
         18 . A method for screening compounds capable of modulating the binding between an HCV E1 envelope protein and an E1 ligand, said method comprising:
 (i) contacting, under conditions allowing the formation of an HCV E1-E1 ligand complex, said E1 ligand with an HCV E1 envelope protein or part thereof according to any one of  claims 1  to  4  or with the viral-like particle according to  claim 5  in the presence and absence, respectively, of a compound suspected of modulating the binding between an HCV E1 envelope protein and an E1 ligand;   (ii) determining the amount of HCV E1-E1 ligand complex formed in (i) in the presence and absence of said compound;   (iii) inferring from (ii) whether said compound is a modulator of binding between an HCV E1 envelope protein and an E1 ligand.   
     
     
         19 . A method according to  claim 17  or  18  wherein in step (i) the HCV E1 envelope protein or part thereof according to any one of  claims 1  to  4  or the viral-like particle according to  claim 5  is added as competitor; and wherein said HCV E1 envelope protein or part thereof or viral-like particle used in said step (i) or said HCV E1 envelope protein or part thereof or viral-like particle added as competitor are labeled. 
     
     
         20 . A diagnostic kit for determining the presence of HCV antibodies in a sample, said kit comprising at least one of an HCV E1 envelope protein or part thereof according to any one of  claims 1  to  4 , the viral-like particle according to  claim 5  and the antibody according to any of  claims 9  to  12 . 
     
     
         21 . A diagnostic kit for determining the presence of HCV E1 envelope protein antigens in a sample comprising at least one of an HCV E1 envelope protein or part thereof according to any one of  claims 1  to  4 , the viral-like particle according to  claim 5  and the antibody according to any of  claims 9  to  12 . 
     
     
         22 . A method of producing the recombinant HCV E1 envelope protein or part thereof according to any one of  claims 1  to  4 , said method comprising the steps of
 (i) expressing of an HCV E1 envelope protein or part thereof in a eukaryotic host;   (ii) isolating from the pool of HCV E1 envelope protein or part thereof expressed in (i) the non-aggregated monomeric fraction of HCV E1 envelope protein or part thereof according to any one of  claims 1  to  4 .   
     
     
         23 . A method of producing the synthetic HCV E1 envelope protein or part thereof according to any one of  claims 1  to  4  wherein said method comprises:
 (i) chemical synthesis of an HCV E1 envelope protein or part thereof;   (ii) introducing during or after the chemical synthesis of said HCV E1 envelope protein or part thereof at least one disulfide according to  claim 1 .   
     
     
         24 . Use of a HCV E1 envelope protein or part thereof according to any of  claims 1  to  4 , the viral-like particle according to  claim 5  and/or the antibody according to any of  claims 9  to  12  for the preparation of a medicament or a vaccine or for the preparation of an immunoassay or immunoassay kit. 
     
     
         25 . Use of a HCV E1 envelope protein or part thereof according to any of  claims 1  to  4  or the viral-like particle according to  claim 5  as a carrier of another protein or of a non-proteinaceous molecule. 
     
     
         26 . An isolated protein comprising the HCV E1 envelope protein or part thereof according to any of  claims 1  to  4 . 
     
     
         27 . The isolated protein according to  claim 26  further comprising at least one of:
 an N-terminal flanking amino acid or amino acid sequence of an HCV protein or part thereof not naturally contiguous with said HCV E1 envelope protein or part thereof;   a C-terminal flanking amino acid or amino acid sequence of an HCV protein or part thereof not naturally contiguous with said HCV E1 envelope protein or part thereof;   an N-terminal flanking non-HCV amino acid or amino acid sequence;   a C-terminal flanking non-HCV amino acid or amino acid sequence.   
     
     
         28 . The isolated protein according to  claim 26  comprising the HCV E1 envelope protein or part thereof as carrier protein.