Use of a Novel Hepatitis C Virus (HCV) Major-Epitope Chimeric Polypeptide for Diagnosis of HCV Infection

Abstract

The genome of hepatitis C virus (HCV) consists of seven functional regions: the core, E1, 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 (E1 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, E1, 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), we used an anti-human superoxide dismutase, dimethyl acridinium ester-labeled monoclonal antibody for detection. Our results indicate that MEFA-6 exposes all of the major immunogenic epitopes. Its excellent sensitivity and specificity for the detection of clinical seroconversion are demonstrated by this assay.

High levels of subgenomic HCV plasma RNA in immunosilent infections

“…The serological test used throughout was the HCV 3.0 enzyme-immunoassay (EIA) (Ortho Clinical Diagnostics, Raritan, New Jersey) which includes recombinant proteins representing linear epitopes from the Core, NS3, NS4 and NS5 regions (Chien et al, 1999;Lin et al, 2005) and is used to screen approximately 80% of the blood supply in the United States (US) for antibodies to HCV. Three unlicensed EIAs containing antigenic proteins not present in HCV 3.0 EIA 1) MEFA 7.1-NS3/4a, 2) F and Core, and 3) E1/E2 proteins were also used for a subset of samples (Tobler et al, 2007).…”

“…NS3/4a is a conformational protein retaining protease and helicase enzymatic activities. E1/E2 proteins are derived from Chinese hamster ovary cells (Chien et al, 1999;Lin et al, 2005).…”

Genotyping of hepatitis B and C virus Russian isolates for reference serum panel construction

“…Human serum samples are tested for anti‐hepatitis C virus antibodies using enzyme‐linked immunosorbent assay based on several HCV antigens—Core, NS3, NS4, and NS5—as individual recombinant proteins or, for example, in the form of single multiple epitope fusion antigen [Chien et al, ]. In spite of the proven efficacy for blood screening and for the diagnosis of HCV infection in symptomatic patients, it is important to continue monitoring assays for their ability to detect samples with low titer anti‐HCV antibodies of all genotypes.…”