Use of a novel hepatitis C virus (HCV) major-epitope chimeric polypeptide for diagnosis of HCV infection

The genome of hepatitis C virus (HCV) consists of seven functional regions: the core, El, E2/NS1, NS2, NS3, NS4, and NS5 regions. The U.S. Food and Drug Administration-licensed 2.0G immunoassay for the detection of anti-HCV uses proteins from the core, NS3, and NS4 regions (McHutchinson et al,, Hepatology 15:19-25, 1992), The 3.0G enzyme-linked immunosorbent assay includes the protein from the NS5 region (Uyttendaele et al,, Vox Sang. 66:122-129, 1994), The necessity of detecting antibodies to viral envelope proteins (El and E2) and to different genotype samples has been demonstrated previously (Chien et al,, Lancet 342:933, 1993; Lok et al,, Hepatology 18:497-502, 1993), In this study we have attempted to improve the sensitivity of the anti-HCV assay by developing a single multiple-epitope fusion antigen (MEFA; MEFA-6) which incorporates all of the major immunodominant epitopes from the seven functional regions of the HCV genome. A nucleic acid sequence consisting of proteins from the viral core, EI, E2, NS3, NS4, and NS5 regions and different subtype-specific regions of the NS4 region was constructed, cloned, and expressed in yeast. The epitopes present on this antigen can be detected by epitope-specific monoclonal and polyclonal antibodies. In a competition assay, the MEFA-6 protein competed with 83 to 96% of genotype-specific antibodies from HCV genotype-specific peptides, This recombinant antigen was subsequently used to design an anti-HCV chemiluminescent immunoassay. We designed our assay using a monoclonal anti-human immunoglobulin G antibody bound to the solid phase. Because MEFA-6 is fused with human superoxide dismutase (h-SOD),,ve used an anti-human superoxide dismutase, dimethyl acridinium ester-labeled monoclonal antibody for detection. Our results indicate that MEFA-6 6 exposes all of the major immunogenic epitopes, Its excellent sensitivity and specificity for the detection of clinical seroconversion are demonstrated by this assay.