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      • CpG coupled N-glycosylation mutant of HCV E1-E2 as a potential therapeutic DNA vaccine with enhanced cellular and neutralizing immune responses

        Yushan Ren,Miao Lin,Yuanqin Min,Xiao-Lian Zhang 한국당과학회 2012 한국당과학회 학술대회 Vol.2012 No.1

        N-linked glycosylation of viral proteins have been implicated in shielding antigen epitopes and blocking neutralizing antibodies production and contribute to the evasion of HCV from the host immune response. In this study, the effects of N-linked glycosylation of Hepatitis C virus (HCV) envelope E1 and E2 proteins, the naturally poor immunogens, on the induction of specific immune response were examined. We constructed plasmids containing the genes encoding both wild type E1E2 proteins and mutated E1E2 proteins in which N-linked glycosylation sites are mutated individually or in combination by site-directed mutagenesis or coupled with CpG, a TLR9 ligand. The immunoreactivity of wild type E1E2 and mutated E1E2 proteins or coupled with immune activator CpG was analyzed in BALB/C mice using a DNA-based vaccination approach. We found that E1E2 specific N-glycosylated mutant induced much higher IgG titer than wild type (WT) E1E2. E1E2 mutant vaccinated mice developed a mixture of IgG1 and IgG2a, but CpG-E1E2 mutant and E1-E2 wild type dominantly developed IgG2a isotype. And CpG-E1E2 mutant significantly stimulated Th1-type response and specific CD8+T cells cytotoxic T lymphocytes (CTL) activities. More important we found that CpG-E1E2 mutant significantly induced much higher neutalizing antibody than E1-E2-WT and had the highest level of neutralizing antibodies among all groups. The monoclonal clonal antibody (McAb) were prepared from CpG-E1E2 mutant immunized mice and the main neutralizing McAb 1C2 had significantly neutralizing Ab activity with blocking HCVcc infection in Huh7.5.1 cells in vitro. Epitope mapping of binding of a neutralizing McAb 1C2 was also analyzed. Our data indicate that the presence of glycans at the surface of HCV envelope proteins could help explain how HCV evades the humoral immune response and why most HCV infections lead to chronicity. CpG have the ability to augment both neutralizing antibody and cellular responses of E1E2 mutant. The CpG-E1E2 mutant holds potential applicant for the development of therapeutic vaccine with enhanced cellular and neutralizing immune responses.

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