Cloning, Expression And Immunogenicity Of A Synthetic Multi-epitope Antigen Gene Pcx3 Of Hepatitis C Virus

HCV is a positive RNA virus, which genome is approximately 9.6 kb and encodes a polyprotein of 3 010 to 3 030 aa residues. From N- to C-terminus of the polyprotein, the component proteins are core protein (C), envelope glycoproteins (E1, E2), p7, and non-structural proteins NS2, NS3, NS4A, NS4B, NS5A and NS5B. The high rate of mutation makes the virus easy to escape from the host defenses, which is one of the major obstacle in the vaccine development. Recent study indicates that the effective vaccine must elicit the high-level, multi-specific(cross-reactivity among genetically diverse isolates) immune response.A multi-epitope antigen gene pcx was assembled by five highly conserved B-and/or T-cell epitopes, which were selected from C (1-16aa, 132-141aa), E1 (126-135aa), NS3 (139-147aa) and NS5B (768-775aa) protein, as well as a T-cell epitope of tetanus toxin (TT), which was added to enhance T-cell immunity. Previous studies indicated that the antigen PCX fused with β-galactosidase showed promising immunogenicity against HCV.The pcx3 gene was constructed by triple tandem of pcx gene as designed and was further cloned into pET-28b(+) or pCDNA3.1(+) expression vector respectively. PCX3 protein was expressed in E. coli with molecular weight about 32kDa and purified up to 98% by Ni²⁺-NTA-agarose.BCA kit was used to quantify the purified proteins.The specific antigenicity of the PCX3 protein expressed in E.coli or SMMC 7721 cell was verified by Western blot with human anti-HCV antibody positive sera, which showed that B-cell epitopes in PCX3 protein were able to be recognized by human anti-HCV antibody. The result of ELISA turned out that PCX3 protein reacted with 83.8% out of the 62 positive samples, with no reactivity to 15 samples of negative control, which determined that PCX3 protein was broadly recognized by anti-HCVantibodies from different people of different regions. Meanwhile, PCX protein reacted with 70% of the same 62 positive samples, which meaned the antigenicity of PCX3 protein was stronger than PCX protein.BALB/c mice were immunized by PCX3 or PCX antigen mixed with Freund's complete adjuvant. It was observed that high titer antibodies(1×10⁶) were detectable and kept about one month in BALB/c mice after immunization by PCX3. The comparison of four dose groups of PCX3 showed that the humoral response was optimal in 50μg dose group. In BALB/c mice, the titer of antibodies induced by PCX was 1 × 10⁴, which meaned that the immunity of PCX3 protein was stronger than PCX as predicted. 6 epitopes designed for the multi-epitope antigen showed different reactivity to anti-PCX3 antibodies. The core protein epitope P1 (MTSTNPKPQRKTKRNTN), E1 protein epitope P5 (RMAWDMMMW) and NS3 protein epitope P6 (TGDFDSVID) had strong activity, which meaned that all of them played important role in humoral response against HCV.CTL response was detected in immunized mice by PCX3 antigen. It is noteworthy that the spleen lymphocytes from the immunized mice specifically killed the target SP2/O myeloma cells incubated with PCX3 protein and epitope peptides. In addition, peripheral CD3⁺CD4⁺ T cells increased significantly detected at 6 weeks after immunization. Meanwhile, the evaluation of antigen toxicity showed that PCX3 protein was safe for mice. These results preliminarily indicate that PCX3 antigen is safe and effective in stimulating the peripheral CD3⁺CD4⁺ Th cells proliferation and generating CTL responses that recognize the epitopes of HCV.After incubation with anti-PCX3 antibodies overnight, the high copies of HCV RNA (genotype 2a) were detected in the pellet of mixture. At the same time, HCV RNA was undetectable in pellet of mixture contained negative control antibody. The result indicates that anti-PCX3 antibodies are able to bind HCV particles in vitro.The inhibition of HCV infection by anti-PCX3 antibodies was further studied in SCID/Alb-uPA transgenic mice with chimeric human liver. In this kind of transgenic mice, human hepatocytes were transplanted and formed proliferative nodules throughout the mouse liver that were able to be infected with HCV by inoculation ofmice with human positive serum. This system is capable of supporting long-term stable infection with HCV and provides greatest utility in evaluation of antibodies neutralizing activities, by passive transfer of antibodies from immunized mice. Two weeks after inoculation of HCV positive serum from patient(1.5×10⁵ RNA copies per mouse) mixed with anti-PCX3 antibody or mouse normal sera, 3 mice were negative and 2 mice were infected with relatively low titer of HCV viroload in the study group. Meanwhile, 7 mice in the control group were all carried significantly higher titer of HCV viroload. It seems that the anti-sera have protection from HCV infection. Unfortunately, this experiment was only tested in genotype la and the data was assayed at one time point, a further study needs to be continued. Overall, these two results provide evidence to prove that the specific antibody against PCX3 should be indicator for protective immune response against HCV.Western blot of anti-PCX3 antisera reaction with core protein and NS3 protein further indicates that the antisera consist of anti-core and anti-NS3 antibody. The preparation of monoclonal antibody against PCX3 antigen by hybridoma showed that two strains of monoclonal antibodies against core protein or NS3 protein respectively and one strain against the epitope of El protein P5(RMAWDMMMW) were obtained. These mAbs would be used for the detail study about the neutralization. After that, cDNA sequences were cloned from variable region of antibody against core protein to construct single chain variable fragment, which will contribute to the application of antibody in therapy.These findings demonstrate that this designed multiple-epitope antigen appears to be a potent HCV vaccine candidate, which is able to induce both potent cell-mediated and humoral immune response against HCV, including the virus capture(genotype 2a) in vitro and more important, inhibition of HCV infection(genotype la) in transgenic mice with chimeric human liver by anti-PCX3 antibody.