A CpG-oupled N-glycosylation Mutant Of HCV E1E2as A Novel Potential Effective Vaccine And Its Associated Neutralizing Monoclonal Antibody Against HCV Infection

Hepatitis C virus (HCV) is a member of the Hepacivirus genus in the Flaviviridae family. HCV has a positive-sense, single stranded RNA genome and enveloped proteins, which infects human hepatocytes and mainly causes chronic infection. HCV infects approximately170million people worldwide and70%cases often develope into chronic hepatitis and even progress into liver fibrosis and hepatocellular carcinoma. HCV contains6-7genotypes. HCV genotypes1,2, and3are distributed in whole world, genotype4is the main type in Egypt, genotype5is widespread in South Africa, and genotype6is found primarily in Southeast Asia. Genotypes lb and2are the major genotypes in China. The virus spreads exclusively by the parenteral route. The World Health Organization (WHO) has estimated that2million individuals are newly infected through unsafe needle injection each year. Nowadays, there is no effective vaccine for HCV. Currently available therapy for HCV is PEGylated IFN-γ combined with Ribavirin. However it is expensive, and not effective for all the infections. The prognosis of this treatment depends on the genotypes of HCV. Thus, the development of an ideal vaccine against hepatitis C virus remains a high priority goal.E1and E2envelop glycoproteins play vital roles for HCV entry and infection. N-linked glycosylations of viral envelope proteins have been implicated in immunicity. The N-glycans on envelope glycoproteins E1and E2regulate antigenicity and immunogenicity. In this study, the effects of the N-linked glycosylation of the HCV El and E2proteins, naturally poor immunogens, on the induction of specific immune responses were examined. We constructed the plasmids containing the genes encoding both wild type and mutant E1E2proteins in which N-linked glycosylation sites were mutated individually or in combination by site-directed mutagenesis. The recombinant DNA plasmids or coupled with CpG were immunized in the Balb/C mouse with immunoadjuvants IL-2via the intramuscular injection (i.m.)(30μg of each plasmid DNA per mouse) by gene gun with an electric square porator. The results showed that both N-glycosylation mutant proteins and E1E2-WT protein were expressed in vivo and vitro. Among all groups, E1E2-M5induced the highest level of E2specific IgG, IgGl and IL-4, while CpG-ElE2-M4increased the highest level of IgG2a and IFN-y. The lactate dehydrogenase (LDH) assay revealed that CpG-E1E2-M4induced the strongest CTL activity and significantly suppressed E1E2-expressing CT26tumor cells growth in mice. These results indicated that CpG-E1E2-M4induced Th1type immune response. Dendritic cells (DCs) from CpG-E1E2-M4immunized mice secreted higher levels of IL-12. Co-cultured with DCs from CpG-E1E2-M4immunized mice enhanced IFN-y production of CD4+T cells, and stimulated productions of perform and Granzyme B of CD8+T cells. These results indicated that CpG-E1E2-M4enhanced DC antigen presentation and induced Th1type immune response.During viral early infection, neutralizing antibody (N-Ab) is important for HCV clearance. E1E2is the primary target for N-Ab production. The anti-serum from immunized mouse was incubated with HCVcc (JFH-1,2a) and then the mixture was added into Huh7.5.1cells. The results showed that CpG-E1E2-M4induced the highest level of N-Ab response and inhibited HCVcc and HCVpp (6genotypes) infection in vitro. The EC50of CpG-E1E2-M4anti serum was estimated as16.8μg/ml, much lower than those of CpG-E1E2-WT, E1E2-WT and E1E2-M4groups. Monoclonal antibodies (mAb) from CpG-E1E2-M4immunized mice were prepared and the neutralization assay showed that1C2mAb could significantly decrease the infections of HCVcc and HCV positive patient serum to Huh7.5.1cells. And the EC50of1C2was determined as10.82μg/ml. Epitopes mapping showed that1C2recognized two regions of E2, F550to G572(P10) and Y489to C508(P7), which are adjacent to the E2-CD81binding region.1C2was demonstrated to inhibit HCV infection by disturbing the binding between E2and CD81.Humans and chimpanzee are the only hosts for HCV infection. Due to lack of small animal model, development of HCV-specific vaccine has been hampered. To determine the effect of1C2anti-viral infection, human HCV receptors CD81, SRB1, OCLN and CLDN-1transgenic mice were prepared. The effect of1C2mAb was examined in the HCV infected humanized mouse model. To summary, the immunogenicity of wild type E1E2,10mutated E1E2proteins were analyzed in BALB/C mice. We found that CpG-E1E2-M4significantly enhanced the E1E2-specific CTL activities and induced neutralization antibody. MAb1C2was prepared from CpG-E1E2-M4immunized mice and was found to neutralize HCV infection effectively. In the humanized transgenic mice,1C2was evaluated and found to prevent HCV infection in vivo effectively. Our data showed that CpG-E1E2-M4vaccine and1C2provided potential applications for the development of DNA vaccine with enhanced immunogenicity and effective therapeutic neutralizing antibody for the treatment of HCV infection.