The impact of HCV genotypes on seroreactivity against HCV antigens immobilized on the RIBA™ 3.0 SIA

Abstract

To the Editor: Hepatitis C virus (HCV) is known for its genetic heterogeneity and has been classified into six major genotypes based on phylogenetic analysis (1). The diverse genetic heterogeneity also translates into variations of the encoded viral proteins, which may impact on virologic behavior, host-virus interaction, and the viral epitopes recognized by the host B and T cells. The current serologic assay for diagnosing HCV infection is based on the detection of host antibody against relatively conserved HCV viral polypeptides. Recent studies have shown differences in seroreactivity between HCV genotypes based on the commercial assays, and suggested that a large proportion of epitopes of the type la or lb recombinant proteins used in the current assays are genotype specific (2). The antigens used for Ortho HCV version 3.0 ELISA and RIBA TM HCV 3.0 Strip Immunobtot assay (SIA) were not modified to improve the detection of various genotypes. However, the sensitivity of these assays for the detection of seroreactivities in patients infected with different HCV genotypes has not been extensively investigated. To determine the impact of HCV genotypes on seroreactivity in patients infected with different HCV genotypes, 108 patients infected with different HCV genotypes were studied. The HCV genotypes in these patients were determined by both restriction fragment length polymorphism and a line probe assay (InnoLiPA-HCV, Innogenetics, Ghent, Belgium) (3). Seroreactivities to HCV antigens from structural and non-structural regions were evaluated with RIBA TM HCV 3.0 SIA according to manufacturer's instructions. RIBA TM HCV 3.0 SIA strip contains four HCV antigen lanes coated with 5 different capture HCV-encoded recombinant antigens/peptides: lane 1 with cl00(p) and 5-1-1(p); lane 2 with c33c; lane 3 c22(p); and lane 4 with NS5; in addition to a fifth lane with a recombinant human superoxide dismutase (hSOD) as a control for anti-SOD reactivities. Seroreactivity seen on developed strip was graded from - (minus) to 4+ according to product insert. Table 1 shows the results. Most patients had either strong reactivity or no reactivity to a particular antigen, and hence the results are given as 0 (no reactivity), 1 to 3+, and 4+. Two patients infected with HCV type 4 were non-reactive to all four antigens, and they both had severe renal insufficiency (serum creatinine 6.2 and 8.4 mg/dl), suggesting that host factors may play a role in the non-reactivity in these two patients. Sequencing of the HCV peptide for HCV core(p) region (a.a. 10-53) by reverse transcription-polymerase chain reaction and dideoxy chain termination sequencing showed no specific amino acid substitutions at this region to account for the loss of seroreactivity, further supporting the hypothesis that the non-reactivity was related to the host factors in these two patients. In general, antibody reactivity to c33c and c22(p) was strong and found to be present in 104/108 (96.3%) of the chronic HCV patients tested, demonstrating that the sequence selected for c33c and c22(p) in the RIBA TM HCV 3.0 SIA was conserved for its B-cell reactivity amongst the various genotypes, and offering the ability to detect the presence of these antibodies in the specimens. Antibody reactivities towards cl00(p)/5-1-1(p) occurred in >90% of genotypes 1, 2, 3, 5, and 6 specimens, but in only 12/20 (60.0%) of the type 4 specimens. The prevalence of antibody reactivity towards NS5 ranged from 75% to 95% in the various genotype specimens. With the current set of data, non-reactivity to c-100(p) and 5-1l(p) in patients with established HCV infection suggested genotypes 2, 3 or 4 infection. As all but two of the 108 patients genotyped as type 1 to type 6 were detected by RIBA TM HCV 3.0 SIA, the current RIBA HCV 3.0 SIA is satisfactory for confirming the diagnosis of HCV infection in most HCV genotypes.