Design of Novel Conformational and Genotype-Specific Antigens for Improving Sensitivity of Immunoassays for Hepatitis C Virus-Specific Antibodies
2005
The current commercially licensed enzyme-linked immunosorbent assays (ELISAs) for hepatitis C virus (HCV) mainly use recombinant proteins containing linear epitopes. There is evidence, however, that conformational epitopes of HCV are more immunoreactive. Thus, we have designed an HCV antibody assay that employs a conformational protein, NS3NS4a PI (with functional protease and helicase activities), and a linear fusion protein, multiple-epitope fusion antigen 7.1 (MEFA 7.1) or MEFA 7.2. We have shown that NS3NS4a PI detects early-seroconversion conformation-sensitive antibodies better than c33c antigen. The correct conformation of NS3NS4a PI also cross-reacts with different genotype samples better than the c33c antigen. MEFA 7.1 and MEFA 7.2 incorporate all the major immunodominant and genotype-specific epitopes of HCV core, E1, E2 hypervariable region 1 (HVR1), E2 HVR1-plus-HVR2 consensus, NS3, NS4, and NS5. Since MEFA 7.1 is degraded by the active NS3NS4a PI protease, we designed a second MEFA 7.2 construct in which the six protease cleavage sites found in MEFA 7.1 were eliminated by amino acid mutation. We demonstrate here that MEFA 7.2 remains intact in the presence of NS3NS4a PI and preserves the epitopes present in MEFA 7.1. Compared to currently licensed assays, an ELISA incorporating a combination of the two antigens NS3NS4a PI and MEFA 7.1 or 7.2 demonstrates better serotype sensitivity and detects seroconversion earlier in many commercially available panels. We believe that an assay using NS3NS4a PI and MEFA 7.1 or 7.2 may have the potential to replace current HCV immunoassays for better sensitivity.
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