The Experimental Study Of Specific CTLs Induced By Ub-HBcAg-CTP To Inhibit HBV Replication

Objective: To observe whether the fusion protein of Ubiquitin- modified the epitope HBc Ag could enhance HBV-specific immune responses in HBV transgenic mice and antiviral ability in these animals?Methods: 1. HBV transgenic mice were randomly divided into 6 groups, with 7 mice in each group. Mice were immunized at the tail base one time at 1-week intervals with Ub-HBc Ag-CTP(50ug), HBc Ag-CTP(50ug), Ub-HBc Ag(50ug), IFN-a(20000IU), HBc Ag(50ug) and PBS respectively. Mice were sacrificed, and splenocytes were collected at 7 days after the last immunization. The level of cytokines(IL-2 and IFN-?) was performed by enzyme- linked immunosorbent assay(ELISA). Furthermore, we also detected the level of specific IFN-? secreted by T lymphocytes using enzyme- linked immunospot Assay(ELISPOT). The killing effect of T lymphocytes was detected by using non-radioactive cytotoxicity assay and the intracellular cytokine of induced T cells was analyzed by flow cytometry(FCM). 2. Mice were randomly divided into 6 groups, with 7 mice in each group. Mice were immunized at the tail base one time at 1-week intervals with 50 ug Ub-HBc Ag-CTP, 50 ug HBc Ag-CTP, 50 ug Ub-HBc Ag, 20000 IU IFN-a, 50 ug HBc Ag and PBS respectively. Serum samples were collected at day 7 after the second and the third immunization respectively. Mice were sacrificed, and livers were collected at day 7 after the third immunization. Serum HBs Ag and HBV DNA levels were respectively determined by micro-particle enzyme immunoassay and real-time fluorescent PCR assay. The expression of HBs Ag and HBc Ag in hepatic tissue was detected by immunohistochemistry.Results: 1. These studies showed that synthesized Ub-HBc Ag-CTP not only significantly increased productio ns of interleukin(IL)-2 and interferon(IFN)-?, compared with HBc Ag-CTP, IFN-?, HBc Ag, Ub-HBc Ag and PBS, but also induced the highest percentage of CD8+/IFN-?+ T cells and the cytotoxic T lymphocyte(C TL) responses in HBV transgenic mice. 2. Additionally, at 1 week after the last treatment to HBV transgenic mice, the enhancement of specific CTL activity provoked by the fusion protein Ub-HBc Ag-CTP reduced serum hepatitis B surface antigen(HBs Ag) and HBV DNA serum levels. The hepatocytes in the liver tissue of Ub-HBc Ag-CTP fusion protein group appeared swelling and there were a lot of lymphocytes infiltration and hyperemia. By contrast, these histological changes were not apparent in the mice from other groups. Furthermore, the fusion protein Ub-HBc Ag-CTP also decreased HBs Ag and HBc Ag expressions in livers. To further confirm the specific C TL activity in liver tissue, the results of levels of ALT and AST was higher than others group, which demonstrated that there were significant differences in the immunize d transgenic mice groups.Conclusion: Ub-HBc Ag-CTP fusion protein could enhance the percentages of CTL, trigger functional T cell responses, induce robust HBV-specific C TL activity and inhibit Hepatitis B virus replication in vivo, suggesting the therapeutic effects.