Lentiviral Vector-encoding Ubiquitinated HBcAg Transduced Murine Bone Marrow-derived Dendritic Cells To Stimulate Cytotoxic T Lymphocytes Response In Vitro

Chronic hepatitis B virus (HBV) infection is still a severe human health problem, and there are about 350 million chronic HBV carriers worldwide. Chronic HBV infection is associated with serious complications as a result of long-term sequelae such as liver cirrhosis or hepatocellular carcinoma. It has been reported that specific cellular immunity against HBV is a key factor for the control of HBV infection. Specific cytotoxic T lymphocytes (CTLs) generated by efficient antigen-stimulation may provide a possibility for virus elimination in chronic HBV patients.The ubiquitin-proteasome system (UPS) is a highly selective ATP-dependent proteolytic system in all eukaryotic cells and plays a key role in antigen presentation. UPS is composed of ubiquitin (Ub), ubiquitin-activating enzyme (E1), ubiquitin -conjugating enzyme (E2), ubiquitin-ligasing enzyme (E3), 26S proteasome and deubiquitinating- enzyme (DUB). Ubiquitinated protein is recognized by proteasome complex and progressively degradated to a number of small peptides,which could combine with major histocompatibility complex (MHC) class I and was identified by specific CD8+ T lymphocytes to induce specific CTL immune response.The aim of this study was to investigate whether ubiquitinated hepatitis B virus core antigen (Ub-HBcAg) transduced DC by lentiviral vector could induce a HBcAg-specific CTL response in vitro. This experiment was composed of three parts: (1) Construction and expression of a lentiviral vector encoding ubiquitinated HBcAg gene; (2) Effect of LV-Ub-HBcAg on inducing murine bone marrow-derived dendritic cells maturation and promoting T lymphocytes proliferation in vitro; (3) CTLs induced by DCs transduced with LV-Ub-HBcAg in vitro.PartⅠConstruction and expression of a lentiviral vector encoding ubiquitinated HBcAg geneObjective: To construct a lentiviral vector encoding ubiquitinated HBcAg gene and investigate its expression in 293T cells.Methods: Ubiquitin-fused HBcAg gene was amplified from the plasmid pcDNA3.1(-)-Ub-HBcAg using PCR and inserted into the lentiviral vector pWPXLd to construct the recombinant lentiviral expression vector pWPXLd-Ub-HBcAg. The recombinant lentivirus LV-Ub-HBcAg generated by 293T cells co-transfected with pWPXLd-Ub-HBcAg and the packaging plasmid psPAX2 and envelope plasmid PMD2.G. The recombinant lentiviral LV-Ub-HBcAg carrying Ub-HBcAg gene expressed in 293T cell was detected by western blot.Results: The fusion gene Ub-HBcAg was inserted into the right direction of plasmid pWPXLd. LV-Ub-HBcAg could be expressed in 293T cells. Conclusion: The recombinant lentiviral LV-Ub-HBcAg has been successfully constructed, which is capable of delivering the target gene into 293T cells for its stable expression.PartⅡEffect of LV-Ub-HBcAg on inducing murine bone marrow-derived dendritic cells maturation and promoting T lymphocytes proliferation in vitro Objective: To observe the effects of LV-Ub-HBcAg on inducing murine bone marrow-derived DCs maturation and promoting T 1ymphocyte proliferation in vitro.Methods: Bone marrow derived DCs isolated from Balb/c mice were cultured with recombinant granulocyte-macrophage colony stimulating factor (rGM-CSF) and recombinant interleutin-4 (rIL-4) for 5 days. LV-Ub-HBcAg, LV-HBcAg, LV or LPS were added to induce DC maturation. The DC phenotypes were analyzed by flow cytometry. The level of IL-12 in the supernatant was detected by enzyme linked immunosorbent assay (ELISA). The proliferation of T lymphocytes was performed by using cell counting kit-8 (CCK-8). All data were analyzed using t test.Results: On day 8, 56.1% of GFP-expressing DCs were detected after infection with LV-Ub-HBcAg. DC surface molecules, such as CD80, CD86 and MHC-II were upregulated by LV-Ub-HBcAg. IL-12 level induced by LV-Ub-HBcAg was (128.08±15.09) pg/ml, which was significantly higher than that induced by LV-HBcAg (P<0.01). The proliferation of T lymphocytes induced by LV-Ub-HBcAg was much higher than that in LV-HBcAg group.Conclusions: DCs were efficiently infected by LVs. LV-Ub-HBcAg could up-regulate the expressions of costimulatory molecules on surface of DC, and enhance the ability of DC to stimulate T lymphocytes proliferation and IL-12 production.PartⅢCTLs induced by DCs transduced with LV-Ub-HBcAg in vitroObjective: To investigate the effect of DCs transduced with LV-Ub-HBcAg on inducing specific cytotoxic T lymphocyte in vitro. Methods: DCs were cultured and induced to maturate by different LVs, and co-cultured with allogeneic T cells to detect the secretion level of IL-2, IL-4, IL-10 and IFN-γin the supernatants of T cells by ELISA. Intracellular cytokine of proliferative T cells was analyzed by flow cytometry.Results: Sensitized dendritic cells by LV-Ub-HBcAg could promote cytokine secretion effectively, the levels of IL-2 (436.5±44.6 pg/ml) and IFN-γ(144.6±15.6 pg/ml) induced by LV-Ub-HBcAg were higher than that induced by LV-HBcAg (367.8±59.4 pg/ml and 102.1±13.3 pg/ml respectively). The amount of CTLs induced by LV-Ub-HBcAg were more than other groups by the analysis of intracellular cytokine of proliferative T cells.Conclusions: Sensitised DCs by LV-Ub-HBcAg can stimulate cytokine secretion and promote cytotoxic T lymphocytes generation.