The Experimental Study Of A Novel DC-Targeting Lentivector Which Could Elicit HBV-specific CTL Response By Autophagy

Objective: To construct a DC-targeting lentiviral vector encoding ubiquitinated HBcAg and LIGHT gene(LVDC-UbHBcAg-LIGHT)using the third generation lentivirus packaging system;To evaluate the DC-targeting specificity of the lentiviral vector and investigate whether the additional expression of LIGHT in DCs can enhance the capacity of lentiviral vector to induce HBV-specific CTL response both ex vivo and in vivo;To compare the immune effect between the DC-targeting lentiviral vector and its non-targeting form and explore the anti-viral efficacy of the lentiviral vector in HBV transgenic mice;To further assess the molecular mechanisms underlying LVDC-UbHBcAg-LIGHT mediated CTL activation.Methods: 1.The expression plasmid pLOV-UBC-Ub-HBcAg-EGFP-P2A-Tnfsf14-3FLAG was constructed by inserting the gene EGFP-P2A-Tnfsf14-3FLAG into BamHI/XbaI sites of the pLOV-UBC-UB-HBcAg-EGFP-3FLAG which was kept in our lab.The SVG gene was engineered with the alterations to blind its canonical binding receptor heparin while leave its intact ability to interact with DC-SIGN.We cloned the mutant gene into BamHI sites of pHCMV-VSV-G to replace the VSVG gene and achieved the DC-targeting envelope plasmid.The lentiviral vector LVDC-UbHBcAg-LIGHT was generated by co-transfecting 293 T cells with pLOV-UBC-Ub-HBcAg-EGFP-P2A-Tnfsf14-3FLAG and the packaging plasmids pLP1,pLP2 and the envelope plasmid pLP/SVG.Then,LVDC-UbHBcAg-LIGHT was purified and identified by Western blot.2.293 T cell models with gradient protein levels of DC-SIGN were established and transduced with the DC-targeting lentiviral vector and its non-targeting form respectively.The relationship between DC-SIGN expression in 293 T cells and transduction efficiencies of the lentiviral vectors was analyzed by flow cytometry;The capacity of both the two lentivectors to transduce DCs within ex vivo cultured murine bone marrow cells and bone marrow-derived DCs(BMDCs)was examined with or without the presence of a blocking DC-SIGN antibody;Furthermore,specificity of LVDC-UbHBcAg-LIGHT for DCs in vivo was also confirmed through live animal imaging studies.3.The expression of DC activation markers and IL-12 p levels in the medium after different lentivectors transduction were detected;The capacity of LVDC-UbHBcAg-LIGHT-loaded DCs to induce HBV-specific CTLs and the expression levels of proteins related to autophagy,apoptosis and cell cycle during T cells activation were investigated.Moreover,the role of autophagy in T cell activation was explored by blocking autophagy using 3-MA or ATG5 siRNA.4.The anti-viral efficacy of LVDC-UbHBcAg-LIGHT in HBV transgenic mice was evaluated by the activities of HBV-specific CTL,serum HBsAg,HBV DNA levels and the expression of HBsAg and HBcAg in liver tissues of HBV transgenic mice.In addition,molecular mechanism underlying the activation of CD8+ T cells was explored by analyzing the expression and co-localization of autophagy,cell apoptosis and cell cycle associated proteins.Results: 1.The gene EGFP-P2A-Tnfsf14-3FLAG was inserted into the expression plasmid pLOV-UBC-UB-HBcAg-EGFP-3FLAG in the right direction.After lentiviral transduction of 293 T cells,the expression of the HBcAg and LIGHT was confirmed by western blot.2.The transduction efficiency of LVDC-UbHBcAg-LIGHT increased with the protein levels of DC-SIGN in 293 T cells.In comparison,LV-UbHBcAg-LIGHT could transduce all the 293 T cell lines with similar transduction efficiency;LV-UbHBcAg-LIGHT could transduce mixed bone marrow cells more efficiently,however,the percentage of DCs within the transduced cells was relatively low;By contrast,almost all the transduced cells were DCs in the LVDC-UbHBcAg-LIGHT group.Additionally,a neutralizing DC-SIGN antibody obviously decreased the transduction efficiency of LVDC-UbHBcAg-LIGHT for DCs,but not the efficiency LV-UbHBcAg-LIGHT;Live animal imaging studies showed that following subcutaneous administration,no luminescence signal was detected at any site one month after the injection in the targeting group.In contrast,mice in the non-targeting group had obvious luminescence signals at the injection site and other sites of the body.3.LVDC-UbHBcAg-LIGHT could significantly elevate the expression of DC activation markers when compared with LVDC-UbHBcAg,however,there was no difference between LV-UbHBcAg-LIGHT and LVDC-UbHBcAg-LIGHT.LVDC-UbHBcAg-LIGHT-loaded DCs were highly effective in stimulating T cell proliferation,promoting the secretion of cytokine IFN-?,IL-2,IL-6,and TNF-?,increasing the percentages of IFN-?+CD8+ and TNF-?+CD8+ T cells,and enhancing the activity of specific cytotoxic T cells,compared with LVDC-Ub-HBcAg-loaded DCs.However,there was no difference between LV-UbHBcAg-LIGHT-loaded DCs and LVDC-UbHBcAg-LIGHT-loaded DCs.Furthermore,autophagy was induced in the activated T cells as evidenced by western blot,transmission electron microscopy and confocal microscopy analyses.We also observed obviously increased percentages of CD8+ T cells entering into S-phase,decreased percentages of apoptotic CD8+ T cells and decreased expression of proteins associated with apoptosis and cell-cycle in the activated CD8+ T cells.More importantly,autophagy blocking impaired degradation of the proteins associated with apoptosis and cell-cycle leading to a significant increase in apoptosis and obvious inhibition of CD8+ T cells entry into S-phase,correspondingly attenuated all the LVDC-UbHBcAg-LIGHT-loaded DC-induced T cell responses.4.Immunization with LVDC-UbHBcAg-LIGHT elicited the most potent T cell responses in HBV transgenic mice evidenced by a wider mix of cytokines produced by the HBV specific CD8+ and CD4+ T cells,increased percentages of IFN-?,TNF-? and GZMB secreting CD8+ T cells as well as IFN-? secreting CD4+ T cells,and improved HBcAg-specific CTL activity.Additonally,vaccination with LVDC-UbHBcAg-LIGHT efficiently reduced serum HBsAg,HBV DNA levels,the expression of HBsAg and HBcAg in liver tissues of HBV transgenic mice and increased serum AST and ALT levels.Autophagy was also induced in the CD8+ T cells of mice immunized by LVDC-UbHBcAg-LIGHT and the induced autophagy noticeably promoted the proliferation of T cells and decreased the percentage of apoptotic CD8+ T cells by selectively degrading ubiquitinated apoptosis and cell cycle-associated protein aggregates.Futhermore,we confirmed the interaction between autophagosomes and ubiquitinated aggregates by confocal microscopy and immunoprecipitation analysis.Conclusion: The engineered lentivirus vector LVDC-UbHBcAg-LIGHT could specifically transduce DCs both in vivo and ex vivo;LIGHT expression in DCs strongly enhanced the capacity of lentiviral particles to activate T cells and generated more significant antiviral effects in HBV transgenic mice;Moreover,the results showed that selective degradation of proteins associated with apoptosis and cell cycle through the autophagy-lysosomal pathway,which was induced after TCR stimulation,could promote the survival,proliferation and killing activities of CTLs.