Novel HCV core+1 protein, methods for diagnosis of HCV infections, prophylaxis, and for screening of anti-HCV agents

Abstract

The present invention relates to a novel form of core+1 protein of Hepatitis C virus (HCV), designated shorter form core+1 protein. The shorter form core+1 protein of Hepatitis C virus is the product of translation of a coding sequence consisting of all or part of a nucleotide sequence extending from nucleotide 598 to nucleotide 920 within the core+1 ORF of HCV represented on FIG. 3 B. The invention also provides methods for detecting infection by Hepatitis C virus in biological samples, methods of screening compounds which interact with viral propagation in HCV infected cells or screening of compounds impaction on the expression of shorter form core+1 protein and uses of these compounds for the preparation of compositions useful for their anti-viral activities.

1 . A shorter form core+1 protein of Hepatitis C virus (HCV) which is the product of translation of a coding sequence consisting of all or part of a nucleotide sequence extending from nucleotide 598 to nucleotide 920 within the core+1 ORF of HCV represented on  FIG. 3B . 
     
     
         2 . The shorter form core+1 protein according to  claim 1 , which is encoded by a nucleotide sequence having a translation initiation codon (ATG) at position 598 or by a nucleotide sequence having an ATG at position 606 of the HCV core+1 coding sequence. 
     
     
         3 . The shorter form core+1 protein according to  claim 1  or  2 , which is encoded by:
 (i) a nucleotide sequence extending from nucleotide 598 to nucleotide 826 of the sequence represented on  FIG. 3B ; or 
 (ii) a nucleotide sequence extending from nucleotide 598 to nucleotide 897 of the sequence represented on  FIG. 3B ; or 
 (iii) a nucleotide sequence extending from nucleotide 606 to nucleotide 826 of the sequence represented on  FIG. 3B ; or 
 (iv) a nucleotide sequence extending from nucleotide 606 to nucleotide 897 of the sequence represented on  FIG. 3B ; or 
 (v) a nucleotide sequence extending from nucleotide 606 to nucleotide 920 of the sequence represented on  FIG. 3B . 
 
     
     
         4 . A shorter form core+1 protein of HCV obtainable in vivo by expression in transfected cells of the core+1 open reading frame (ORF) which is contained in nucleotide sequence extending from nucleotide at position 342 to nucleotide at position 920 of the nucleotide sequence represented on  FIG. 3B , and which molecular weight is less than 10 kDa. 
     
     
         5 . The shorter form core+1 protein according to  claim 4 , which is the expression product of the core+1 ORF in mammalian cells. 
     
     
         6 . The shorter form core+1 protein according to anyone of  claims 1  to  5 , which is recognized by a serum of patients infected with HCV. 
     
     
         7 . The shorter form core+1 protein according to anyone of  claims 1  to  6  which comprises amino-acid sequence extending from amino-acid residue corresponding to nucleotide 598 to amino-acid residue corresponding to nucleotide 826, or to nucleotide 897, or to nucleotide 920. 
     
     
         8 . The shorter form core+1 protein according to anyone of  claims 1  to  6  which comprises amino-acid sequence extending from amino-acid residue corresponding to nucleotide 606 to amino-acid residue corresponding to nucleotide 826, or to nucleotide 897, or to nucleotide 920. 
     
     
         9 . A peptide contained within the shorter form core+1 protein according to anyone of  claims 1  to  8  which comprises an epitope. 
     
     
         10 . The peptide according to  claim 9 , which comprises the following amino-acid sequence: COOH-T-Y—R-S-S-A-P-L-L-E-A-L-P-G-P—NH 2  or a variant thereof which reacts with antibodies directed against said peptide sequence. 
     
     
         11 . The peptide variant according to  claim 10 , which is derived from the sequence of  FIG. 8 . 
     
     
         12 . A nucleotide sequence consisting in a fragment of the nucleotide sequence extending from nucleotide 342 to nucleotide 920 represented on  FIG. 3B , which fragment is capable of encoding a shorter form core+1 protein of HCV when transfected in mammalian cells under expression conditions. 
     
     
         13 . A nucleotide sequence encoding a shorter form core+1 protein according to anyone of  claim 7  or  8 . 
     
     
         14 . The nucleotide sequence according to  claim 12  or  13  comprising a nucleotide sequence extending from nucleotide 598 or from nucleotide 606 to nucleotide 826 within the core+1 coding sequence represented on  FIG. 36 . 
     
     
         15 . The nucleotide sequence, which is chosen among:
 (i) a nucleotide sequence extending from nucleotide 606 to nucleotide 826 of the sequence represented on  FIG. 3B ;   (ii) a nucleotide sequence extending from nucleotide 606 to nucleotide 897 of the sequence represented on  FIG. 3B ;   (iii) a nucleotide sequence extending from nucleotide 606 to nucleotide 920 of the sequence represented on  FIG. 3B ;   (iv) a nucleotide sequence extending from nucleotide 598 to nucleotide 826 of the sequence represented on  FIG. 3B ;   (v) a nucleotide sequence extending from nucleotide 598 to nucleotide 897 of the sequence represented on  FIG. 3B ;   (vi) a nucleotide sequence extending from nucleotide 598 to nucleotide 920 of the sequence represented on  FIG. 3B ;   (vii) a fragment of sequence (i), (ii), (iii), (iv), (v), or (vi) which is capable of encoding a shorter form core+1 protein according to anyone of  claims 1  to  8  in mammalian cells or an epitope thereof.   
     
     
         16 . A nucleotide sequence comprising a HCV core protein coding sequence, which is derived from the nucleotide sequence represented on  FIG. 3B  as a result of one or several mutations selected among the following:
 in 9 th  and 11 th  codons a mutation which respectively corresponds to a triple substitution of two A to G and of an A to C; or 
 in 9 th , 10 th  and 11 th  codons a mutation which respectively consists of a substitution of one A to G and two A to C; or 
 in 9 th  codon a mutation which consists of a substitution of A to G; or 
 in 10 th  codon a mutation which consists of a substitution of A to C or 
 a substitution of an initiator codon into a terminator codon; or 
 a substitution of the 25 th  codon into a stop codon; or 
 a substitution of the 43 rd  codon into a stop codon; or 
 a substitution of the 79 th  codon into a stop codon; or 
 a substitution of the 87 th  codon into a stop codon; or 
 a substation of the 85 th  codon into a stop codon and/or 
 a substitution of the 87 th  codon into a stop codon. 
 
     
     
         17 . The nucleotide sequence according to any of  claims 12  to  15 , said sequence being a functional variant thereof having at least 70% identity. 
     
     
         18 . A nucleotide sequence hybridizing under stringent conditions to a nucleotide sequence according to anyone of  claims 12  to  17 . 
     
     
         19 . A nucleotide sequence which is a sequence complementary to a nucleotide sequence according to anyone of  claims 12  to  18 . 
     
     
         20 . A nucleotide sequence hybridizing under stringent conditions with at least a complementary sequence of a nucleotide sequence according to anyone of  claims 12  to  17 . 
     
     
         21 . A chimeric gene comprising a promoter operatively linked to a nucleotide sequence according to any of  claims 12  to  15  and  17  to  20 . 
     
     
         22 . The chimeric gene according to  claim 21 , wherein said promoter is selected from the group consisting of lactose promoter system, tryptophan promoter system, tac promoter and CMV promoter 
     
     
         23 . The chimeric gene according to  claim 21 , comprising a CMV/T7 promoter and a chloramphenicol acetyl transferase (CAT) gene in a first cistron and the entire IRES of the HCV core coding sequence and part of the wild type core coding sequence fused to LUC gene in a second cistron. 
     
     
         24 . The chimeric gene according to  claim 23 , wherein LUC gene is fused to the core sequence in a 0, +1 or −1 frames. 
     
     
         25 . A vector comprising a chimeric gene according to anyone of  claims 21  to  24 . 
     
     
         26 . The vector of  claim 24  which, is a plasmid, a cosmid, a phage or a virus. 
     
     
         27 . The vector according to  claim 24  is preferably a plasmid selected from the group consisting of pHPI-1333, pHPI-1335 represented on  FIG. 1 . 
     
     
         28 . Recombinant cells transfected with a nucleotide sequence according to anyone of  claims 1  to  27 . 
     
     
         29 . The recombinant cells according to  claim 28 , which are animal, mammalian or human cells. 
     
     
         30 . The recombinant cells according to  claim 29 , which are BHK-21 or Huh-1 cells. 
     
     
         31 . Purified antibodies which specifically bind to shorter form core+1 protein according to anyone of  claims 1  to  8 , without cross-reacting with core protein and/or core+1 protein. 
     
     
         32 . Purified antibodies which specifically bind to polypeptide fragments common for shorter form core+1 protein according to anyone of  claims 1  to  8 , and core+1 protein and optionally core protein. 
     
     
         33 . Purified antibodies which specifically bind to peptide according to anyone of  claims 9  to  11 . 
     
     
         34 . Purified antibodies according to anyone of  claims 31  to  33 , which are monoclonal antibodies. 
     
     
         35 . A method for producing antibodies, wherein the shorter form core+1 protein according to anyone of  claims 1  to  8 , or fragment thereof is used as antigen. 
     
     
         36 . A purified polypeptide which specifically binds to at least one antibody according to anyone of  claims 31  to  34  or to an antibody produced by the method according to  claim 35 . 
     
     
         37 . An in vitro method for the detection of infection by Hepatitis C virus, in a biological sample, said method comprising determining the presence or absence of the shorter form core+1 protein according to anyone of  claims 1  to  8 . 
     
     
         38 . The method according to  claim 37 , wherein said shorter form core+1 protein is detected with antibodies which are immunologicaly reactive with the shorter form core+1 protein according to anyone of  claims 1  to  8 . 
     
     
         39 . The method according to  claim 37 , wherein said shorter form core+1 protein is detected with antibodies which are immunologicaly reactive with the peptide according to anyone of  claims 9  to  11 . 
     
     
         40 . A method for the in vitro detection of infection by Hepatitis C virus, which comprises detecting antibodies recognizing the shorter form core+1 protein according to anyone of  claims 1  to  8 , in a biological sample. 
     
     
         41 . The method according to anyone of  claims 37  to  40 , wherein the formation of antigen-antibody complex is detected by immunoassay (direct detection) or ELISA (indirect detection). 
     
     
         42 . A method of screening compounds for their capacity to interact with viral propagation in cells infected by HCV, said method comprising:
 a. contacting said cells with a candidate compound;   b. determining interaction between said candidate compound and expression of said shorter form core+1 protein.   
     
     
         43 . The method of screening compounds according to  claim 42  wherein interaction is determined by measuring the expression level of shorter form core+1 protein, prior and after contacting the HCV infected cells with a candidate compound. 
     
     
         44 . The method according to  claim 42  or  43 , wherein said cells infected by HCV are animal, mammalian or human cells. 
     
     
         45 . The method according to  claim 44 , wherein said cells infected by HCV are BHK-21 or Huh-1 cells. 
     
     
         46 . Use of a compound selected according to the method of anyone of  claims 38  to  41  for the preparation of a medicine for the treatment of disorders induced by or associated with infection of HCV.