Antibodies to a novel antigen in acute hepatitis C virus infections.

BACKGROUND: Conformational viral proteins potentially play an important role in the immunobiology of acute hepatitis C virus (HCV) infection and may enable earlier antibody detection. MATERIALS AND METHODS: HCV RNA was detected using nucleic acid testing. Early antibody production was evaluated using three enzyme immunoassays (EIAs) containing antigenic proteins not present in licensed EIAs. Respectively, these contained: (1) multiple-epitope fusion antigen (MEFA) 7.1-NS3/4a, (2) F and Core, and (3) E1/E2 proteins. NS3/4a is a conformational antigen retaining protease and helicase enzymatic activities. MEFA 7.1 contains the linear epitopes used in licenced EIAs, including the latest EIA-3.0, in combination with genotype 1-3 specific epitopes. Forty-two RNA positive, EIA-3.0 negative samples, including two persistently serosilent cases, were used to evaluate these research EIAs. As controls, 54 EIA-3.0 negative/RNA negative and three HCV RNA+/antibody positive specimens were included. RESULTS: Only the MEFA 7.1-NS3/4a EIA was positive in seven (17%) of the 42 HCV RNA + specimens, in all three EIA-3.0 positive controls but in none of 54 EIA-3.0 negative/HCV RNA negative controls. Notably, six of the seven (86%) specimens had evidence of active hepatitis (ALT > 210 IU/l). The two serosilent cases were research EIA negative. CONCLUSION: A novel EIA with conformational and linear epitopes detected HCV antibodies in 17% of viraemic specimens missed by the standard reference EIA-3.0. Our research EIA appears to detect HCV antibodies closer to the initiation of acute hepatitis. Given that the average RNA-positive, antibody-negative window period is 56.4 days, this 17% yield would translate into a 10-day earlier detection of antibodies.

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, 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.