Hcv ns3 recombinant antigens and mutants thereof for improved antibody detection

Abstract

The present disclosure relates to polypeptides, including fusions thereof, nucleic acids, vectors, host cells, immunodiagnostic reagents, kits, and immunoassays for use detecting the presence of HCV antibodies. More specifically, the present invention describes specific NS3 antigens that can be used for the detection of anti-HCV antibodies.

1 . A recombinant HCV NS3 antigen comprising a NS3 helicase sequence that comprises each of domains I, II and III of said helicase, wherein said antigen has increased immunoreactivity against HCV antibodies from test sample as compared to C33 antigen, wherein said recombinant HCV NS3 antigen comprises one or more of the characteristics selected from the group consisting of:
 diminished ATP-binding activity as compared to the ATP-binding activity of wild-type NS3 helicase   diminished ATPase activity as compared to wild-type NS3 as compared to the ATP-binding activity of wild-type NS3 helicase, and   increased redox stability as compared to the redox stability of wild-type NS3 helicase.   
     
     
         2 . The recombinant HCV NS3 antigen of  claim 1 , wherein said antigen further comprises addition of at least one cysteine residue in the C-terminus end of said NS3 helicase. 
     
     
         3 . The recombinant HCV NS3 antigen of  claim 2 , wherein said antigen comprises addition of two cysteine residues in the C-terminus end of said NS3 helicase. 
     
     
         4 . The recombinant HCV NS3 antigen of  claim 1 , wherein said wild-type HCV NS3 comprises a sequence of SEQ ID NO: 87 and wherein said antigen comprises at least one mutation as compared to the sequence of SEQ ID NO:87. 
     
     
         5 . The recombinant HCV NS3 antigen of  claim 4 , wherein said mutation comprises a mutation of one or more of the cysteine residues of said SEQ ID NO:87 to any other amino acid. 
     
     
         6 . The recombinant HCV NS3 antigen of  claim 4 , wherein said mutation comprises a mutation of said one or more cysteine residues to corresponding serine residues. 
     
     
         7 . The recombinant HCV NS3 antigen of  claim 6 , wherein said mutation comprises one or more of the mutations of the cysteine residues from domain III of HCV NS3 helicase. 
     
     
         8 . The recombinant HCV NS3 antigen of  claim 5 , wherein said cysteine residue mutation comprises a mutation of one or more of the cysteine residues selected from the group consisting C292, C368, C374, C499, and C525 of SEQ ID NO:87. 
     
     
         9 . The recombinant HCV NS3 antigen of  claim 5 , wherein said antigen is an HCV NS3 mutant in which at least two of said cysteine residues are replaced by corresponding serine residues. 
     
     
         10 . The recombinant HCV NS3 antigen of  claim 5 , wherein said antigen further comprises addition of at least one cysteine residue at the C-terminus end of said NS3 helicase. 
     
     
         11 . The recombinant HCV NS3 antigen of  claim 4 , wherein said mutation that diminishes ATP binding or diminishes ATPase activity is a replacement of one or more of the amino acid residues selected from the group consisting of K210, S211, T212, Y241, D290, E291, H293, T419, Q460, R464, R467 and W501 of SEQ ID NO:87 with any other amino acid residue. 
     
     
         12 . The recombinant HCV NS3 antigen of  claim 11 , wherein said mutation is selected from the group consisting of K210N, S211A, T212E, Y241S, D290N, E291Q, H293A, T419G, Q460H, R464A, R467K and W501A as compared to SEQ ID NO:87. 
     
     
         13 . The recombinant HCV NS3 antigen of  claim 11 , wherein said antigen further comprises a mutation of one or more of the cysteine residues of said SEQ ID NO:87 to any other amino acid. 
     
     
         14 . The recombinant HCV NS3 antigen of  claim 13 , wherein said mutation of one or more of the cysteine residues of said SEQ ID NO:87 comprises a mutation of one or more of the cysteine residues selected from the group consisting C292, C368, C374, C499, and C525 of SEQ ID NO:87. 
     
     
         15 . The recombinant HCV NS3 antigen of  claim 11 , wherein said antigen further comprises addition of at least one cysteine residue at the C-terminus end of said NS3 helicase. 
     
     
         16 . The recombinant HCV NS3 antigen of  claim 14 , wherein said antigen further comprises addition of at least one cysteine residue at the C-terminus end of said NS3 helicase. 
     
     
         17 . The recombinant HCV NS3 antigen of  claim 15 , wherein said addition of a cysteine residue at the C-terminus end of said NS3 helicase comprises addition of a sequence selected from the group consisting of GGCSGGA, DECHSTD, and SKKKCDE to the C-terminus end of said NS3 helicase. 
     
     
         18 . The recombinant HCV NS3 antigen of  claim 16 , wherein said addition of a cysteine residue at the C-terminus end of said NS3 helicase comprises addition of a sequence selected from the group consisting of GGCSGGA, DECHSTD, and SKKKCDE to the C-terminus end of said NS3 helicase. 
     
     
         19 . The recombinant HCV NS3 antigen of  claim 15 , wherein said addition of at least one cysteine residue at the C-terminus end of said NS3 helicase comprises addition of a sequence selected from the group consisting of GSGSGHHHHHHHHGGCSGGARSGC; GSGSGHHHHHHHHDECHSTDRSGC; and GSGCGHHHHHHHHGGCSGGA. 
     
     
         20 . The recombinant HCV NS3 antigen of  claim 16 , wherein said addition of at least one cysteine residue at the C-terminus end of said NS3 helicase comprises addition of a sequence selected from the group consisting of GSGSGHHHHHHHHGGCSGGARSGC; GSGSGHHHHHHHHDECHSTDRSGC; and GSGCGHHHHHHHHGGCSGGA. 
     
     
         21 . The recombinant HCV NS3 antigen of any one of  claim 18 ,  19  or  20 , wherein said C-terminus sequence is modified by conjugation to a signal generating moiety. 
     
     
         22 . The recombinant HCV NS3 antigen of  claim 15 , wherein said antigen further comprises a histidine tag. 
     
     
         23 . The recombinant  claim 16 , wherein said antigen further comprises a histidine tag. 
     
     
         24 . The recombinant HCV NS3 antigen of  claim 22 , wherein said histidine tag is located between the C-terminus of SEQ ID NO:87 and the N-terminus of said added sequence. 
     
     
         25 . The recombinant HCV NS3 antigen of  claim 23 , wherein said histidine tag is located between the C-terminus of SEQ ID NO:87 and the N-terminus of said added sequence. 
     
     
         26 . The recombinant HCV NS3 antigen of any of  claims 1 - 25  wherein said antigen is biotinylated. 
     
     
         27 . The recombinant HCV NS3 antigen of  claim 26  wherein said biotinylation is at the N-terminus of said antigen. 
     
     
         28 . The recombinant HCV NS3 antigen of  claim 26  wherein said biotinylation is at the C-terminus of said antigen. 
     
     
         29 . The recombinant HCV NS3 antigen of  claim 26  wherein said biotinylation is site-specific biotinylation. 
     
     
         30 . An isolated nucleic acid encoding a recombinant HCV antigen of any of  claims 1 - 29 . 
     
     
         31 . An expression vector comprising an isolated nucleic acid of  claim 30 . 
     
     
         32 . A host cell transformed or transfected with an expression vector of  claim 31 . 
     
     
         33 . The host cell of  claim 32 , wherein said host cell is an  E. coli  cell. 
     
     
         34 . An immunodiagnostic reagent comprising the recombinant HCV antigen of any of  claims 1 - 29 . 
     
     
         35 . The immunodiagnostic reagent of  claim 34 , further comprising a solid support. 
     
     
         36 . The immunodiagnostic reagent of  claim 34  wherein said solid support is a microparticle and said recombinant antigen is bound to said microparticle. 
     
     
         37 . The immunodiagnostic reagent of  claim 34  wherein said recombinant antigen is detectably labeled with a fluorescent label. 
     
     
         38 . A kit comprising an immunodiagnostic reagent of  claim 34  and further comprising an additional isolated HCV antigen comprising an epitope that is immunoreactive with an anti-HCV antibody. 
     
     
         39 . The kit of  claim 38  wherein said additional HCV antigen is an HCV core antigen. 
     
     
         40 . The kit of  claim 38  wherein said recombinant HCV NS3 antigen and said additional HCV antigen are co-coated on the same solid phase. 
     
     
         41 . The kit of  claim 38  wherein said recombinant HCV NS3 antigen and said core antigen are coated on the separate solid phases. 
     
     
         42 . The kit of  claim 41  further comprising antibodies for detection of human antibodies. 
     
     
         43 . The kit of  claim 41  further comprising anti-HCV antibodies, optionally comprising a detectable label. 
     
     
         44 . An immunoassay method of determining the presence of anti-HCV antibodies in a test sample, comprising contacting said test sample with an immunodiagnostic agent of  claim 34  under conditions to allow a complex to from between said recombinant HCV NS3 antigen and said anti-HCV antibodies in said test sample, and detecting the presence of said complex, wherein presence of said complex is indicative of anti-HCV antibodies in said test sample. 
     
     
         45 . The immunoassay method of  claim 44  wherein said detection of said complex formation is detected by determining binding of labeled anti-human antibodies to said complex. 
     
     
         46 . The immunoassay method of  claim 45  wherein said labeled anti-human antibodies are labeled with a fluorescent label. 
     
     
         47 . The immunoassay method of  claim 45  wherein said labeled anti-human antibodies are labeled with acridinium. 
     
     
         48 . The immunoassay method of  claim 44 , wherein the recombinant HCV NS3 antigen is coated on microparticles. 
     
     
         49 . The immunoassay method of  claim 44  wherein said method further comprises assaying said test sample to determine the presence of antibodies against HCV core antigen. 
     
     
         50 . The immunoassay method of  claim 49  wherein said recombinant HCV NS3 antigen and said HCV core antigen are co-coated on the same microparticle. 
     
     
         51 . The immunoassay method of  claim 49  wherein said recombinant HCV NS3 antigen and said HCV core antigen are coated on the separate microparticles. 
     
     
         52 . The immunoassay method of  claim 49 , wherein the test sample was obtained from a patient and the method further comprises diagnosing, prognosticating, or assessing the efficacy of a therapeutic/prophylactic treatment of the patient, wherein, if the method further comprises assessing the efficacy of a therapeutic/prophylactic treatment of the patient, the method optionally further comprises modifying the therapeutic/prophylactic treatment of the patient as needed to improve efficacy. 
     
     
         53 . The immunoassay method of  claim 49 , wherein the method is adapted for use in an automated system or a semi-automated system. 
     
     
         54 . A recombinant HCV NS3 antigen comprising a NS3 helicase sequence that comprises each of domains I, II and III of said helicase, wherein said antigen has increased immunoreactivity against HCV antibodies from serum as compared to C33 antigen, wherein said recombinant HCV NS3 antigen comprises increased redox stability as compared to the redox stability of wild-type NS3 helicase. 
     
     
         55 . A recombinant HCV NS3 antigen comprising a NS3 helicase sequence that comprises each of domains I and II of said helicase, wherein said antigen has increased immunoreactivity against HCV antibodies from serum as compared to C33 antigen, and wherein said recombinant HCV NS3 antigen comprises increased redox stability as compared to the redox stability of wild-type NS3 helicase.