The Experimental Study Of Specific Immune Response Induced By Recombinant Lentivirus Encoding Ubiquitinated Hepatitis B Core Antigen To Inhibit HBV Replication

Objective:To construct a lentiviral vector encoding ubiquitinated HBcAg gene(Ub-HBcAg).To obtain the high titer of recombinant lentivirus particles with the stable Ub-HBcAg gene expression using the four plasmid packaging system.To investigate the effect of HBV specific humoral and cellular immune responses induced by the recombinant lentivirus LV-Ub-HBcAg in BALB/c mice.To compare the immune effect between direct injection of LV-Ub-HBcAg and injection of in vitro-transduced DCs.To investigate the effect of HBV specific humoral and cellular immune responses induced by recombinant lentivirus LV-Ub-HBcAg on inhibiting HBV replication in HBV transgenic mice.Methods:1.The plasmid pcDNA3.1(-)-Ub-HBcAg was used as a template to amplify the Ub-HBcAg fused gene using PCR,then the recombinant Ub-HBcAg lentiviral expression vector plasmid was constructed by inserting the fused gene into the lentiviral vector plasmid pLOV.UBC.EGFP.3FLAG.The recombinant lentivirus particles LV-Ub-HBcAg were generated by co-transfecting 293T cells with pLOV.UBC.Ub-HBcAg.EGFP.3FLAG and the packaging plasmids pLP1,pLP2 and the envelope plasmid pLP/VSVG.Then,LV-Ub-HBcAg was purified,and was identified by Western blot.2.BALB/c mice were randomly divided into eight groups.Mice were injected subcutaneously in the hind footpads with 5×10~5LV-Ub-HBcAg-transduced DCs(LV/DC,LV-HBcAg/DC,or LV-Ub-HBcAg/DC)or directly with 2×10~7 IU lentivirus particals(LV,LV-HBcAg,or LV-Ub-HBcAg)twice at an interval of 2 weeks.The mice injected with PBS or untransduced DCs served as the controls.ELISA was performed to detect the levels of HBcAb in the sera and the levels of cytokines IFN-?,IL-2,IL-4 and IL-10 secreted by T lymphocytes.The intracellular cytokine of T lymphocytes was analyzed by flow cytometry and IFN-?-secreting specific T lymphocytes were determined using enzyme-linked immunospot assay(ELISPOT).The proliferation of T lymphocytes was detected using CCK-8.Specific CTL activity was analyzed by a LDH release assay.3.HBV transgenic mice were randomly divided into six groups.Mice were injected subcutaneously in the hind footpads with LV-Ub-HBcAg,LV-HBcAg,HBcAg,IFN-?,LV and PBS twice at an interval of 2 weeks.The levels of HBcAb in the sera and the levels of cytokines IFN-?,IL-2,TNF-?,IL-4 and IL-10 secreted by T lymphocytes were detected by ELISA.The levels of HBsAg in the sera were detected by MEIA.The levels of HBV DNA were detected by real-time fluorescent PCR assay kits.The levels of ALT and AST were measured by biochemical analysis.The intracellular cytokine of T lymphocytes was analyzed by flow cytometry and IFN-?-secrecting specific T lymphocytes were determined by ELISPOT.Specific CTL activity was analyzed by a LDH release assay.The histology of the hepatic tissues was observed by HE staining and the protein expressions of HBsAg and HBcAg were detected by immunohistochemistry.The expressions of T-bet and GATA-3 were detected by real-time PCR and western blot.Results:1.We inserted the correct sequence of the fusion gene Ub-HBcAg into the expression plasmid pLOV.UBC.EGFP.3FLAG in the right direction.After lentiviral transfection of 293T cells,the expression of the target protein was detected in 293T cells using western blot.2.BALB/c mice were immunized with LV-Ub-HBcAg and LV-Ub-HBcAg/DC.The levels of anti-HBcAg IgG antibodies were higher in mice.The titer of IgG2?antibody was higher in the LV-Ub-HBcAg and LV-Ub-HBcAg/DC groups than that in other groups.The levels of IgG1 antibodies changed little among all the groups.Both groups stimulated the proliferation of T lymphocytes and specific CTL activities.The levels of IL-2 and IFN-?(Th1-like cytokines)were significantly enhanced.The specific CTL responses detected by flow cytometry and ELISPOT in the two groups were higher than the other groups.However,there was no substantial difference in the immune effect between the two groups.3.HBV transgenic mice were immunized with LV-Ub-HBcAg.LV-Ub-HBcAg induced IgG2?-dominant HBcAb responses,enhanced cytokines(IFN-?,IL-2 and TNF-?)secretion,increased the numbers of HBcAg-specific IFN-?-secreting CD8~+and CD4~+T cells in the spleen,and boosted HBcAg-specific CTL activity.The numbers of lymphocytes in the liver tissues from LV-Ub-HBcAg mice group were more than from the other groups.LV-Ub-HBcAg could decrease HBV DNA and HBsAg level in serum as well as HBsAg and HBcAg expression in liver tissue.LV-Ub-HBcAg increased the levels of AST and ALT.LV-Ub-HBcAg could upregulate the expression of T-bet and downregulate the expression of GATA-3.Conclusion:The recombinant lentivirus carrying ubiquitinated HBcAg gene has been constructed,which can transfect 293T cells resulting in stable target gene expression.Recombinant lentivirus encoding Ub-HBcAg either to modify DCs in vitro or to transduce DCs in vivo after direct injection efficiently induced IgG2?-dominant HBcAb responses,stimulated the secretion of Th1-like cytokines,promoted T lymphocyte proliferation and enhanced the activity of specific CTL in BALB/c mice.Lentiviral vectors encoding Ub-HBcAg induced IgG2?-dominant HBcAb responses and stimulated the secretion of Th1-like cytokines in HBV transgenic mice.LV-Ub-HBcAg could induce Th1 immune responses,CTL activity and therapeutic effects in HBV transgenic mice.The upregulation of T-bet and downregulation of GATA-3 may participate in the induction of Th1 differentiation.