HCV DNA Vaccine And Anti-HCV Strategy By Targeting RNA G-quadruplex

Hepatitis C virus (HCV) is a major cause of liver cirrhosis and hepatocacinoma. The current standard of care (peginterferon-a/ribavirin), and recently developed direct antiviral agents (DAA) are not efficient enough to cure all chronically infected patients. Moreover, their enormous costs limit their uses. Additionally, since most HCV infection is asymptomatic, medical care is often not in time. Therefore, the development of vaccine against HCV for prevention and treatment is urgently needed, though it is extremely challenging.HCV is an enveloped virus with positive sense single-stranded RNA genome. Viral ribonucleocapsid is composed of genome RNA and CORE protein, while viral envelope glycoproteins E1 and E2 form dimers as the surface components of HCV virion. E2 plays an important role in virus entry and also is the primary target for the binding of neutralizing antibodies. In this thesis, on one hand, we conducted comprehensive studies on HCV DNA vaccines based on E2 protein; on the other hand, we identified a G quadruplex within core gene and carried out research of anti-HCV drugs specifically targeting the G quadruplex.HCV E2 exhibits a naturally poor immunogenicity, which is caused, at least partially, by the heavy N-glycosylations. Here, we further investigated the roles of viral N-glycans on E2 immunogenicity and meanwhile screened E2 mutant(s) with improved immunogenicity by N-glycan deletion engineering. Six plasmids were thus constructed, encoding soluble E2 (sE2,384aa-661aa) protein and the mutants (sE2-N1, N2, N4, N6, N11) in which five neutralization-related N-linked glycosylation sites of the sE2 protein were mutated separately. Immunogenicity of these sE2 mutants were analyzed in BALB/c mice using DNA prime-protein boost immunization strategy. We demonstrated that specific de-glycosylation could enhance the naturally poor immunogenicity of sE2 without influence on the expression and secretion of the sE2 protein. We found that the point mutation at N-glycosylation position 423 (N423D) (named as sE2N2 for the corresponding mutant) induced the highest levels of IFN-y, GrB, perforin and cytotoxic T lymphocyte (CTL) response compared to the wild type sE2 and all other mutants. Additionally, sE2Nl (N417D) also displayed prominent effects as an overall improved immunogen. Furthermore, sera from sE2N1 or sE2N2 (N423D) vaccination could block the infection of HCV cell culture (HCVcc, genotype lb) to human Huh7.5.1 hepatocytes, suggesting that both sE2N1 and sE2N2 can elicit effective HCV neutralization antibodies. Further, we selected a monoclonal antibody (mAb) 1C1 from sE2N2-immunized mice.1C1 inhibited viral infection to both Huh7.5.1 cells and more importantly, also restricted HCV infection to ICRTg4R+ mice which support HCV complete life cycle. Collectively, sE2N2 can induce both of robust cellular and humoral immune response.On the other hand, we employed a vaccination strategy through targeting the extracellular domain of HCV envelope glycoprotein E2 (sE2) to B cells via a CD19 single-chain variable fragment mini-Ab (scFV). We found that the chimeric protein, denoted as sE2-scFV, could enhance the arrest of sE2 by B cells both in vivo and in vitro. Then, the immunogenicity of sE2-scFV was also evaluated in BALB/c mice. The results suggest that sE2-scFV boosts the capacity of E2 to induce both cellular and humoral immune responses. Moreover, sE2N2-scFV DNA vaccine seems to be better than sE2-scFV DNA vaccine in provoking IFN-? production.Therefore, the sE2N2 mutant offers a promising efficient strategy for the development of an effective B and T-cell-based HCV vaccine, and this study highlights the role of glycosylation of viral envelope protein in viral immunogenicity and immune evasion.Except for evelope glycoprotein E1E2, another structure protein of HCV is CORE. We chose HCV core as a target of antiviral drug research, because CORE plays essential roles in both of virion assembly and RNA replication. Moreover, our bioinformatics analysisdemonstrated a guanine-rich consensus sequence with high conservation in HCV core gene, which potentially may be able to form G quadruplex. G quadruplex structure ubiquitously exists in cellular genome and RNAs, contributing to the regulation of gene expression; however, little is known in G quadruplex of viral RNA.By a collection of biochemical methods, two representative sequences, derived from genomes of HCV subtype la and lb, were shown to possess a high potential to fold into unimolecular G4 RNA structures. Next, two G4 DNA ligands were found to bind to HCV G4 RNAs with high affinities. Our further investigation provides the first finding and direct evidence that G4 ligands can target HCV core gene and function as efficient agents to inhibit viral replication, through the action of G4 stabilization. Thus, the stabilization of G4 RNA in HCV genome would represent a promising and new strategy for the design and development of novel anti-HCV drugs.In summary, we have conducted comprehensive studies on HCV vaccine and modulation of viral RNA synthesis targeting viral structural proteins and RNA. These studies will benefit the understanding of HCV-host interactions and the development of anti-HCV therapies.