Synthesis Of A Multi-epitope Combination Antigen Of Hepatitis C Virus And Analyses Of Its Immunogenicity

Hepatitis C virus (HCV) is a major causative agent of chronic hepatitis and is associated with liver cirrhosis which may develop into hepatocellular carcinoma. At present there are 1.7-2.0 hundred million infected persons. The prevalence of HCV is about 2% in our country. It is currently believed that more than 50~80% of HCV-infected patiens will become chronic carriers. While there are no efficacious HCV vaccinations, only a minority of HCV-infected patiens benefit from antiviral therapies (IFN and/or ribavirin). The development of a prophylactic, and possibly even more, a therapeutic vaccine is thus highly desirable. The HCV genome is a linear, positive-sense single-stranded RNA molecular of 9,379~9,481nt. It encodes a poly-protein precursor of ~3,000 amino acids. This poly-protein is cleaved by both host and viral proteases to generate several distinct polypeptides, with structural proteins located in the N-terminal portion and nonstrucrural protein in the C-terminal portion. From N- to C-terminus of the poly-protein, the componential proteins are core protein(C), envelope glycoprotein (E1.E2), p7, and non-structural proteins NS2, NS3, NS4A, NS4B, NS5A and NS5B. Although the hypervariability of HCV antigens and the weakness of the immunity to HCV infection in vivo has hindered the preparation of HCV vaccines, the development of HCV vaccine candidates using a synthetic multi-epitope antigen is practicable on the basis of the last study reports and progresses, which E2 hypervariable region 1 (HVR1) has a conserved structure of global and some neutralization epitopes, and some conserved T cell epitopes in the HCV componential proteins can induce eliminating-virus immunity responses.In our studies, a multiple-epitope fragment combination antigen gene (mfc) of HCV was synthesized, which contains nine B-cell HVR1 mimtopes (aa 384~410), two conserved CTL epitopes from C (aa 35~ 44, aa 132~ 140), one conserved CTL epitopes from NS3(aa I073~ 1081), and one conserved Th epitope from NS3(aa 1251~ 1259). Then three styles of HCV vaccine candidates (protein-kind, DNA-kind and virus-like particle) were constructed using MFC. Their immunogenicity was observed in vitro and in vivo. 1. Design, synthesis, expression and immunogenicity of MFC antigen of hepatits C virusOn the basis of our early results and other published data, we selected and identified nine B-cell HVR1 mimtopes in E2, two conserved CTL epitopes in C, one conserved CTL epitope in NS3, one conserved Th epitope in NS3. These thirteen epitopes gene linked together by three amino acids constituted a multiple-epitope fragment combination antigen gene, which was abbreviated to mfc. The mfc gene containing 969bp was synthesized by PCR method, and then cloned into a GST expression vector pGEX-4T-l to generate a fusion protein GST-MFC, whose reactivity frequency with HCV patients' sera was assessed by the ELISA and Western-blot. The experimental data show that the harvest rate of the purified GST-MFC fusion protein in E.coli was 0.29mg / g (wet bacterium), which was able to react with 15 720 anti-HCV sera samples from HCV patients. The reactivity frequency was 75%. The conformation and hydrophilism of MFC protein coded by gene mfc were analyzed by INSIGHT II and DNAMAN software. The software outputs indicated that each conformation of HCV epitope in MFC protein possesed no samebut relative independent conformation. The whole MFC configuration seems untight, but was separated from spherical shape GST subunit in GST-MFC fusion protein.2. Immunological responses of GST-MFC protein in mice and rabbitsAfter sufficient purified GST-MFC fusion protein were harvested, Mice and rabbits were injected with GST-MFC fusion protein emulsified in Freund's complete adjuvant (CFA)or Freund's incomplete adjuvant(IFA) with a routine immunization , which contains 5 tunes multi-point inoculation with 2 weeks interval. In mice experiments the specific antibody with high titer(l : 10s) in sera can be detected by ELISA and western blotting methods in week 8 after the first vaccin...