Development And Application Of Novel DNA Vaccine Candidates Against HCV Based On Enhancement Of Immune Response In Mice

Hepatitis C virus (HCV) is the major etiological agent of transfusion-associated and community-acquired hepatitis worldwide. Approximately 70% of patients develop chronic hepatitis, of which 20 to 30% progress onto liver cirrhosis, and all cases of infection carry an increased risk of hepatocellular carcinoma. Presently, the only available therapies are alpha interferon (IFN-a) alone or in combination with ribavirin. Such treatments are expensive, show low-response rates, and carry the risk of significant side effects. Consequently, the development of a effective vaccine against hepatitis C infection remains a high priority goal.Efficient vaccination against hepatitis C infection requires potent and sustained T cell-mediated immune responses. DNA immunization is one of the most evaluated approaches to obtain an effective vaccine against HCV infection. Several vaccine candidates have been evaluated in pre-clinic, but only a few have reached the clinical evaluation. Two DNA vaccine candidates against HCV have already reached clinical evaluation, and are well tolerated and immunogenic in HCV-chronically infected individuals.DNA vaccination has emerged as an effective and safe strategy for inducing protective T cell immunity in preclinical models. However, experiments in nonhuman primates and human trials suggested that DNA vaccines are not nearly as immunogenic in these species as they are in rodents. In the last few years a group of technical refinements in DNA vaccines has allowed to increase their immunogenicity,include:1) involving gene delivery systems as electroporation to enhance DNA uptake and therefore expression; 2) targeting Dentritic cells(DCs) or other APCs via encoding fusion antigen through genetically fusing the Ag to molecules binding receptors at the cell surface of APCs;3)enhancing the immune respose by codelivery DNA vaccine with immunostimulatory or molecular adjuvants.To develop novel HCV DNA vaccine candidates and explore the strategy enhancing the immune response during DNA vaccination,In this study, several plasmids were constructed to express coding antigen of HCV E1/E2 protein derived from JFH1/HeBei isolates, NS3, NS3 and Core fusion protein, NS3 fused with an antibody to DEC205(an endocytic receptor and expressed at high levels on DCs), and replicating(pSCK) or non-replicating(pVRC) backbone of DNA vaccine vectors was applied.The expression in vitro of novel DNA vaccine candidates were identified with Western Blotting (WB) after transient transfection. These plasmids immunized Balb/C mice via direct injection intramuscularly (i.m.) or intrademally(i.d.), or injection combined with electroporation. The humoral and cellular immune response after vaccination was measured by ELISA or Elispot assay,so as to provide a basis to optimize their immunogenicity.The major findings are as follows:1.To character immune responces between the protein and DNA vaccine in mouse and to optimize the immune protocol and evaluate the immunogenicy of DNA vaccine with electroporation. Construct a novel DNA vaccine expressing fusion protein coding partial NS3 and Core and then confirmed its expression in vitro by WB. The DNA vaccine was immunized mice twice by injection (i.m./i.d.),,and the technique of electroporation was used to facilitate DNA delivery in vivo. And the C44P protein was immunized as control. The results demonstrated that DNA vaccines induced stronger cellular responces in mice than protein vaccination. And Elispot assay showed that IFN-γinduced by injection(i.m.) combined with 2 needle array electrode were stronger than that of without electrical pulses. Injection (i.d.) combined with caliper electrodes induced more IFN-γsecreted than that combined with 2 needle array electrode. Subclass IgG1 was the predominant antibody detected in each DNA vaccination group.2. To compare the immunogenicity of DNA vaccines based on replicative backbone vector (pSCK) to which based on common non-replicative backbone vector (pVRC),several DNA vaccine candidates were constructed expressing E1E2 derived from Hebei(1b) or JFH1(2a)isolate, named as pVRC-JFH1-E1E2, pVRC-HeBei-E1E2, pSCK-HeBei-E1E2. These DNA vaccines were used to immunize mice, and detect the specific cellular immune response by IFN-γELISPOT assay with spleen cells stimulated by synthetic peptides derived from amino acid sequence of HeBei isolate. The results showed that pSCK-HeBei-E1E2(derived from a replicating backbone vector) induce a significant increase in secreting anti-E1E2 specific IFN-γthan that of pVRC-HeBei-E1E2(derived from a non-replicating backbone vector), and pVRC-HeBei-E1E2 induced stronger cellular immune responses than pVRC-JFH1-E1E2. The results also indicated that immunization by intradermal injection with electroporation using caliper electrodes was mostly effective to improve the immunogenicity of DNA vaccines. And the total specific IgG titers induced were lower than 1:50 by ELISA with converse peptides as antigen in this study.3. To evaluvate the effectiveness of a noval adjuvant to DNA vaccines by targeting the encoded protein to dendritic cells, we constructed a novel HCV DNA vaccine coding fusion protein, in which engineered NS3 protein into the heavy chain of anti-DEC-205 and nemed as pVRC-DEC-NS3, then transfect it into 293 cells to confirmed the expression in vitro. Immunize Balb/C mice by twice intradermal injection with electroporation using caliper electrodes, compared with the codelivery of pVRC-DEC or/and pVRC-NS3 only with the same immune protocol. The results indicated that the humoral immune responces as well as cellular immune responces induced by DNA vaccine pVRC-DEC-NS3 was significant increased compared to DNA vaccines expressing NS3 with or without the codelivery of the plasmid encoded anti-DEC-205 molecule.In summary, all the novel HCV DNA vaccines constructed in this study,with either structural proteins (Core, E1/E2) or non-structural protein NS3 as the target antigen, could induce specific cellular immune response.Vaccination applying with electroporation can improve the immunogenicity of DNA vaccines. And plasmids immunized by intradermal injection with electroporation using caliper electrodes could induce most significant increased in CMI, compared with other immune protocol. DNA vaccine based on replicative backbone vector could induce more IFN-Y secreting than which based on a non-replicative vector. And DNA vaccine coding fusion protein, in which engineered target protein into the heavy chain of anti-DEC-205, would improve the immunogenicity of DNA vaccinaton. These data pave a way for the development and application of novel DNA vaccines against HCV infection and other persistant infection.,...