Design of Novel Conformational and Genotype-Specific Antigens for Improving Sensitivity of Immunoassays for Hepatitis C Virus-Specific Antibodies

Abstract

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.Hepatitis C virus (HCV) is the major etiologic agent for blood transfusion-associated and community-acquired non-A, non-B viral hepatitis (1,9,19). HCV currently affects approximately 3% of the world's population, and 70% of those individuals develop chronic HCV infection, which often progresses to liver cirrhosis and hepatocellular carcinomas (3,19,23). The incidence of posttransfusion HCV has steadily declined since the implementation of routine screening for HCV antibodies and HCV nucleic acid amplification testing among blood donors (21). Despite the proven utility of these assays for blood screening and for the diagnosis of HCV infection in symptomatic patients, important challenges to the improvement of immunoassay performance remain. Such challenges include detecting antibody earlier, improving the detection of HCV samples from immunosuppressed patients, and increasing assay sensitivity to detect antibodies to the different HCV genotype-specific epitopes.HCV is an enveloped virus with a single-stranded positivesense RNA genome of approximately 9.5 kb that encodes about 3,010 amino acids (10, 24). The HCV polyprotein is processed by host and viral proteases into several mature proteins: core protein (C), envelope glycoproteins (E1 and E2), and six nonstructural proteins (NS2, NS3, NS4a, NS4b, NS5a, NS5b) (14,17). NS3 is a 630-amino-acid protein with three enzymatic activities: the N-terminal 180 amino ...

Sensitivity of a rapid immuno-chromatographic test for Hepatitis C antibodies detection

“…The Immunoflow test uses antigens derived from the same regions as those used in third-generation assays, including NS5. However, the improvement of anti-HCV detection by third-generation assays has been shown to be more likely due to the use of conformational core and NS3 antigens rather than the inclusion of the NS5 antigen (Lin et al, 2005). Thus, suboptimal design of HCV antigens may account for the lack of sensitivity observed for the rapid test.…”

Antibodies to a novel antigen in acute hepatitis C virus infections

“…The first EIA contained two antigenic proteins: a conformational antigen that retains both protease and helicase enzymatic activities (NS3/4a), and a single multiple‐epitope fusion protein (MEFA 7·1). MEFA 7·1 represents an enhanced antigen incorporating known major epitopes of the HCV polyprotein [15]. Expression of MEFA 7·1 in yeast produces proteins from the HCV Core, E1, E2, NS3, NS4, and NS5 regions with genotypes 1–3 specific epitopes for subtype‐specific regions of the core, E2 and NS4 proteins.…”

“…We therefore tested three research EIAs, containing E1/E2, F/Core and multiple‐epitope fusion antigen (MEFA) 7·1‐NS3/4a proteins which are not present in commercially available assays. The rationale behind using these three research EIAs was the concept of delayed antibody detection by HCV 3·0 EIA due to either absence of the antigen (F protein [12] and E1/E2 [13,14] antigens) or new and uniquely configured antigens (MEFA 7·1 and NS3/4a [15]) with heightened sensitivity to non‐genotype 1 specimens. To investigate the presence of previously undetected anti‐HCV antibodies, we evaluated 42 ‘NAT yield’ specimens, defined as HCV RNA positive but HCV antibody negative using EIA 3·0.…”

Hepatitis C virus polyprotein vaccine formulations capable of inducing broad antibody and cellular immune responses

“…The termini of the polyprotein-encoding DNA were modified to be compatible with restriction sites in the pBS24.1 yeast shuttle vector, which contains 2m sequences for autonomous replication in yeast, the a-factor terminator to ensure transcription termination, the yeast genes leu2-d and URA3 for selection, the Col E1 origin of replication and the b-lactamase gene required for plasmid replication in bacteria (Lin et al, 2005). Thereafter, the hybrid ADH2/GAPDH promoter was cloned 59 to the polyprotein-coding sequence to generate pd.DNS3NS5.PJ.…”

“…The plasmid pCMV-KMDNS3-5 was generated by ligating a PCR fragment that encodes a Kozak initiator methionine followed by aa 1242 of HCV-1a to a 39 StuI site. This product was ligated to a StuI-XbaI fragment from pT7-HCV along with pCMV-KM2 vector (Murphy et al, 1998).The termini of the polyprotein-encoding DNA were modified to be compatible with restriction sites in the pBS24.1 yeast shuttle vector, which contains 2m sequences for autonomous replication in yeast, the a-factor terminator to ensure transcription termination, the yeast genes leu2-d and URA3 for selection, the Col E1 origin of replication and the b-lactamase gene required for plasmid replication in bacteria (Lin et al, 2005). Thereafter, the hybrid ADH2/GAPDH promoter was cloned 59 to the polyprotein-coding sequence to generate pd.DNS3NS5.PJ.…”