Screening, Expression And Characterization Of Hypervariable Region1 Of Hepatitis C Virus

Hypervariable region 1 (HVR1) is a highly heterogeneous sequence located at the N terminus of the second envelope protein (E2) of hepatitis C virus (HCV) and has been proved to be the target of neutralizing antibodies but with isolate specificity, which involved in the escape from host immune recognition. However, some studies revealed that the protective antibodies might be obtained through conserved sites of HVR1 by convenient immunization process. Based on the variability analysis of HVR1 sequences, gene fragments of HVR1 of four HCV strains (three genotype 1b and one genotype 2a) were selected and amplified from pGEMT-E plasmids and sub-cloned into pQE40 vectors respectively to construct recombinant expression plasmids which expressed HVR1 fused downstream to DHFR in Escherichia coli. The reactivity of the four fusion proteins was detected in serum samples of patients with chronic hepatitis C. Among them, SH1b was the most reactive one (66.67%). The results revealed that the intensity and/or quality of the immune response against HCV might be a critical factor determining the response to interferon treatment. Meanwhile, the purified DHFR- HVR1 proteins were used to immunize rabbits and antibodies anti-HVR1 were obtained. Three of the four HVR1 sequences were also expressed as fusion proteins with HBsAg in mammalian cells.To further improve the cross-reactivity of HVR1 sequences, thirty-one HVR1 cDNA fragments were digested by DNase â…  into a pool of random fragments and reassembled by repeated cycles of annealing in the presence of DNA polymerase to their original size. The shuffled HVR1 were then inserted into the gene III phagemid vector pCANTAB-5E and displayed on the surface of the phage. After four rounds of biopanning against anti-HVR1, the reactivity of eight selected clones to this panel of sera were from 46.7%-66.7% to 53.3%-80%. The two highest reactive phages (phage 13,80% and phage 23, 76%) had the similar amino acid sequences with the consensus sequences defined by Puntoriero et al, which might play a particular role in determining the frequency of reactivity. In conclusion, this study has successfully expressed four selected HVR1 sequences in prokaryotic and mammalian system and evaluated their characteristics. Meanwhile, DNA shuffling combined with phage display technology was effectively utilized to identify broadly cross-reactive mimotopes recognized by human polyclonal antibodies. Compared with synthetic peptides widely used in other studies, these techniques, costing much less and covering more variants, had got the broadly reactive sequences. Mimotope 13 and 23 appeared to be immunologically most reactive and could be immunogen candidate. Efforts are now underway to identify their neutralizing antibodies by immunization of animals.