The Study Of Immune Response And Its Mechanism Induced By HCV HVR1 Novel T Cell Epitope

There has no effective treatment and prophylaxis way for chronic HCV infection until now. HCV therapeutics vaccine, which can induce cellular immune response, can re-establish immune response for HCV in patients with chronic HCV infection and utilize self immune response to eliminate virus, so therapeutics vaccine may reduce virus relapse and become one of the important treatments in future. The research was established on the study of HCV strains (II/1b, III/2a) envelope protein E2 hypervariable regions 1 (HVR1) sequence variation, and a series of HCV HVR1 mimic epitopes were synthesized according to the conservation site. In our research, BALB/c mouse was used as the object to investigate the immune response and its molecule mechanism induced by a synthesized peptide (7P) which contains seven amino acids. The research was divided to three sections. (1) The study of immune response induced by 7P on mouse.the assay which included compute homologization analogue build, MTS prolifetation assay, flow cytometry, ELISpot and ELISA was used to study immune response mode induced by 7P on mouse. The results showed that 7P was a linear construction. Splenocyte prolifetation in the mouse immunized by 7P was increased. The value of OD₄₉₀ in experiment group was higher than that of control group after the splenocyte cultured 3 days in vitro (0.99 ±0.16 vs 0.53 ± 0.07). The ratio of CD45⁺CD3⁺, CD3⁺CD8⁺ cell increased (36%; 34.1%), and the level of IFN-γ , IL-2 and IL-10 secreted by splenocyte in experimental group were also higher than those of control group (164.3±111.7SFC/10⁷vs 27.9±17.1SFC/10⁷; 285.3± 157.6SFC/10⁷ vs 103 ± 66SFC/10⁷; 271.7 ±173.7SFC/10⁷ vs 70 ± 30.4SFC/10⁷). However, no specific antibody to 7P was detected. These results indicated that 7P was a T cell epitope andmight induce cellular immune response.(2) The study of cytotoxicity of splenocyte of the mouse immunized by 7PCell sorting technology was used to separate CD4⁺ or CD8⁺ cell and ELISpot was used to detect the level of cytokines secreted by subgroup cells. The results showed that IFN-γ was secreted mainly by CD8⁺ cell and IL-2, IL-10 were secreted mainly by CD4⁺ cell. Used P815 cell as target cell, cytotoxicity of the splenocyte of the mouse immunized by 7P was tested. The results showed that the splenocyte had the specific cytotoxicity on target cell. The rate of cytotoxicity was 42% and 14% in experimental group and control group respectively when the ratio of effector cell and target cell was 100. However, when the MHC I or II monoclonal antibody were added respectively before the effector cell was added to target cell, the cytotocixity of the splenocyte of the mouse immunized by 7P could be blocked by the MHC I monoclonal antibody. The rate of cytotoxicity was 14.1% and 54.8% respectively after blocked by MHC I and MHC II monoclonal antibody. These indicated that the cytotoxicity was restricted by MHC I antigen.(3) The study of the mechanism of immune response induced by 7PRT-PCT and Western blot were used to detect the mRNA level of cytokine and the pathway of MAPK singal transduction in mouse immunized by 7P. The results showed that the mRNA level of IFN-γ , IL-2 and IL-10 were increased in experimental group. The level of phosphorylation MEK1/2, JNK and C-Jun in CD 19^- cell in experimental group were higher than that of control group. But they didn't increase in CD19⁺ cell. These results indicated that 7P was a novel T cell epitope and induced cellular immune response and active the MAPK pathway. MAPK pathway was one of the important pathways in the immune response induced by 7P.