HCV multiple epitope fusion antigens with modified proteolytic cleavage sites and uses thereof
View Patent ↗Modified HCV multiple epitope fusion antigens (MEFAs) are described. The proteins include modified sequences such that proteolytic cleavage of the MEFAs by HCV NS3 protease is inhibited. HCV immunoassays including the modified MEFAs are also described.
1. A modified hepatitis C virus (HCV) multiple epitope fusion antigen (MEFA), wherein said MEFA comprises at least one epitope from the HCV helicase domain that comprises an HCV NS3 proteolytic cleavage site, and at least one epitope from an NS4 region that comprises an HCV NS3 proteolytic cleavage site, wherein the HCV NS3 proteolytic cleavage sites present in said helicase domain epitope and said NS4 epitope are mutated, such that proteolytic cleavage of the modified MEFA by NS3 is inhibited relative to proteolytic cleavage of a corresponding MEFA lacking the mutations, and further wherein said modified MEFA reacts specifically with anti-HCV antibodies present in a biological sample from an HCV-infected individual, wherein the MEFA comprises a sequence of amino acids with at least 90% sequence identity to the contiguous sequence of amino acids depicted in SEQ ID NO:4, with the proviso that the amino acids at positions 466, 467, 493, 494, 695, 696, 721, 722, 770, 771, 819 and 820 of SEQ ID NO:4 are maintained.
2. The modified MEFA of claim 1 , wherein said MEFA comprises the contiguous sequence of amino acids depicted in SEQ ID NO:4.
3. The modified MEFA of claim 1 , wherein said MEFA consists of the contiguous sequence of amino acids depicted in SEQ ID NO:4.
4. An immunoassay solid support comprising the modified MEFA of claim 1 .
5. The immunoassay solid support of claim 4 , further comprising at least one HCV NS3/4a conformational epitope, wherein said NS3/4a conformational epitope reacts specifically with anti-HCV antibodies present in a biological sample from an HCV-infected individual.
6. The immunoassay solid support of claim 5 , wherein said NS3/4a conformational epitope comprises the contiguous sequence of amino acids depicted in SEQ ID NO:6.
7. The immunoassay solid support of claim 5 , wherein said NS3/4a conformational epitope consists of the contiguous sequence of amino acids depicted in SEQ ID NO:6.
8. A method of detecting hepatitis C virus (HCV) infection in a biological sample, said method comprising:
(a) providing an immunoassay solid support according to claim 4 ;
(b) combining a biological sample with said solid support under conditions which allow HCV antibodies, when present in the biological sample, to bind to said MEFA and said NS3/4a conformational epitope if present, to form a first immune complex;
(c) adding to the solid support from step (b) under complex forming conditions a detectably labeled antibody, wherein said labeled antibody is reactive with said immune complex;
(d) detecting second immune complexes formed between the detectably labeled antibody and the first immune complex, if any, as an indication of HCV infection in the biological sample.
9. An immunodiagnostic test kit comprising the immunoassay solid support of claim 4 , and instructions for conducting the immunodiagnostic test.
10. A method of producing an immunoassay solid support, comprising:
(a) providing a solid support; and
(b) binding to the solid support at least one modified MEFA according to claim 1 .
11. The method of claim 10 , further comprising binding to the solid support at a discrete position an HCV NS3/4a conformational epitope, wherein the conformational epitope reacts specifically with anti-HCV antibodies present in a biological sample from an HCV-infected individual.
12. The method of claim 11 , wherein said NS3/4a conformational epitope comprises the contiguous sequence of amino acids depicted in SEQ ID NO:6.
13. The method of claim 11 , wherein said NS3/4a conformational epitope consists of the contiguous sequence of amino acids depicted in SEQ ID NO:6.
14. A polynucleotide comprising a coding sequence for the modified MEFA of claim 1 .
15. A recombinant vector comprising:
(a) a polynucleotide according to claim 14 ;
(b) and control elements operably linked to said polynucleotide whereby the coding sequence can be transcribed and translated in a host cell.
16. A host cell transformed with the recombinant vector of claim 15 .
17. A method of producing a recombinant MEFA comprising:
(a) providing a population of host cells according to claim 16 ; and
(b) culturing said population of cells under conditions whereby the multiple epitope fusion antigen encoded by the coding sequence present in said recombinant vector is expressed.