| Hepatitis C virus (HCV) is a major causative agent of acute and chronic hepatitis and mainly transmitted by blood transfusion. It is estimated that there are 170 million HCV infected individuals worldwide and so far, interferon a combined with Ribavirin treatment is a commonly accepted therapeutic strategy. However, only about 40% of treated patients develop long-term responses. 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 nonstructural protein in the C-terminal portion. From N- to C-terminus of the poly-protein, the component proteins are core protein(C), envelope glycoprotein (E1,E2), p7, and non-structural proteins NS2, NS3, NS4A, NS4B, NS5A and NS5B. Evidences have showed that the hypervariable region 1(HVR1) of the HCV E2 envelope protein contains a principal neutralizing epitope. Antibodies against HVR1 play an important role in blocking viral attachment and recovery, however, it is only in an isolate-specific manner because of the extreme variability of HVR1, which make vaccine development particularly difficult.We have previously synthesized a multiple-epitope fragment combination antigen gene (mfc) of HCV, which contains nine B-cell HVR1 mimtopes (responded to 384~410aa), two conserved CTL epitopes from C (responded to 35~44aa, 132~ 140aa), one conserved CTL epitopes from NS3(responded to1073~1081aa), and one conserved Th epitope from NS3(responded to1251~1259aa), and found it can elicit HCV-specific immune responses by the immunogenicity analysis. In this study, we have completed three works. Firstly, to enhance the immunogenicity of the mfc, we fused HBsAg gene to the N-terminal of the mfc and constructed an HCV DNA vaccine candidate (pVAX1-HBs-MFC) using HBsAg as an adjuvant. BALB/c mice were intramuscularly injected with the pVAXl-HBs-MFC, and the effects of HBsAg gene on the immunity of HCV multiple-epitope DNA vector was evaluated in vitro and in vivo. Secondly, we selected five HVR1 mimtopes (named hcvme) from mfc gene and cloned it into a prokaryotic expression vector pGEX-4T-l to generate a fusion protein GST-HCVME, then its immunogenicity was analysed. Thirdly, wedetected the proliferation of the HCV patients' peripheral blood lymphocyte stimulated by GST-MFC, which was the product of the mfc gene expressed in colibacillus.1. Construction of an HCV DNA vaccine candidate using HBsAg gene as adjuvant and estimation of its immunogenicity in miceAn HCV DNA vector (pVAXl-HBs-MFC) was constructed by fusing HBsAg gene to the N-terminal of an HCV multiple-epitope antigen gene. The pVAXl-HBs-MFC was transfected into HEK 293T cells and its expression was measured by ELISA and Western blot analyses. BALB/c mice were intramuscularly immunized with the pVAXl-HBs-MFC, and an ELISA approach was applied to determine the specific antibody titers and subtypes in the mouse serum. The cross-reactivity of the antibodies was also checked with 2 synthesized HCV hypervariable region 1 (HVR1) peptides. The IFN-y production and cell proliferation of the mouse spleen cells were evaluated by ELISA and MTS (3-(4,5-dimethylthiazol -2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) assays, respectively. Results: The expression of pVAXl-HBs-MFC was detectable in the transfected HEK 293T cells. The serum antibody response was effectively elicited in BALB/c mice injected with pVAXl-HBs-MFC. The highest titer of antibody against HCV (MFC) was 1:1280, and the ratio of IgG2a/IgGl was 1.50+0.12 at the fifth week after first immunization. Moreover, the collected mouse serum antibody had the ability to cross-react with the 2 synthesized HCV HVR1 peptides. The stimulation index of the mouse splenocytes to MFC was 1.79±0.07, and the IFN-y level was 287±6pg/ml at week 21 after first immunization. The highest titer of the antibody in control BALB/c mice immunized with pVAXl-MFC was 1:320, and the ratio of IgG2a/IgGl was 1.33±0.11 at week 5 post-immunization. Furthermore, the stimulation index of the mouse splenocytes cells to MFC was 1.52+0.06, and the IFN-y level was 225+9.3pg/ml at week 21 post-immunization. These data indicate that the HBsAg gene can enhance the effects of an HCV multiple-epitope DNA construct on its humoral and cellular immune responses. This HBsAg enhanced HCV multiple-epitope DNA vector may be of potential use in the development of HCV vaccines.2. Expression and purification of the multiple-mimotope antigen of hepatitis C virus and analysis of its immunogenicityFive HVR1 mimtopes (named hcvme) oimfc gene were selected and cloned into a prokaryotic expression vector pGEX-4T-l to generate a fusion protein GST-HCVME. The reactivity frequency of the GST-HCVME with the sero of HCV patients was measured by Western-blot and ELISA. BALB/c mice were injected with GST-HCVME emulsified in Freund's complete adjuvant (CFA) or Freund's incomplete adjuvant(IFA) with a routine immunization , which contain 3 times multi-point inoculation with 2 weeks interval, and the immune responses induced by GST-HCVME were detected. Results: The GST-HCVME protein was able to react with 25 samples of 35 HCV patients' sero. The serum antibody response was effectively elicited in BALB/c mice injected with GST-HCVME. The highest titer of antibody against HCV (GST-HCVME) was about l:104 at the eighth week after first immunization. Moreover, the collected mouse serum antibody had the ability to cross-react with the 2 synthesized HCV HVR1 peptides. The stimulation index of the mouse splenocytes to GST-HCVME was 2.23±0.18, and the IFN-y level was 1326.5±227.1 pg/ml at week 20 after first immunization. These results showed that GST-HCVME can efficiently induce immune responses in mice and antibodies elicited by GST-HCVME have the ability to neutralize different HCV strains. HCVME protein can be regarded as a prophylactic-therapeutics protein-kind HCV vaccine candidate.3. Proliferation of the HCV patients' peripheral blood lymphocyte stimulated by GST-MFCTen peripheral blood samples of HCV-infected patients and two peripheral blood samples of healthy people were colleted and the lymphocytes were separated aseptically. Using 96-well flat-bottomed microculture plated, lymphocytes at a density of 1X105 cells per well were stimulated with the GST-MFC or GST at a final concentration of 25 H g/ml. After incubation for 72 h at 37°C in a humidified 5% CO2 incubator, assay for cell proliferation was performed. The data were expressed as stimulating index (SI= mean counts per minute of antigen-stimulated cultures per mean counts per minute of medium-control cultures). Results: Eight lymphocyte samples of ten patients were found MFC-specific cell proliferation (average SI=1.42), which showed a significant difference with the group stimulated with GST. These results mean that the T-cell epitopes contained in mfc are relatively conserved, and can be efficiently presented in HCV-infected patients. |
