Effect of Cytotoxic T Lymphocytes Induced by Recombinant Adeno-Associated Virus on Different Hepatitis B Virus Genes

Background: Hepatitis B virus (HBV) infection is a significant global problem. The main therapies for chronic hepatitis B are nucleos(t)ide analogues (NAs) and (pegylated) interferon (IFN). However, the prognosis of chronic hepatitis B is still not so optimistic since the low serological conversion rate and the high recurrence rate after drug withdrawal. Objectives: This aim was to evaluate the killing activity of cytotoxic T lymphocytes (CTLs) induced by dendritic cells transduced recombinant adenovirus associated virus (rAAV) with different hepatitis B virus gene fragment (HBV-S, C, E, X). Methods: Peripheral blood mononuclear cells (PBMCs) were extracted from chronic hepatitis B patients, and the adhered cells were harvested to cultivate with recombinant adeno-associated virus with different hepatitis B virus (HBV) antigen gene fragment (rAAV-HBV-S, C, E, X) for 7 days by adding GM-CSF, IL-4 and TNF-alpha to generate mature dendritic cells (DCs). The DCs' state were observed and their differentiation antigen molecules (CD) were detected by flow cytometry (FACS) to evaluate their maturation and function. DCs were co-cultured with prepared T lymphocytes to induce cytotoxic T lymphocytes (CTLs). The activity of CTLs induced by different DCs were compared by detection of lymphocyte CD molecules and cytokine levels. HepaG2.2.15 cells were target cells and HepaG2 cells as control. The specific killing activity of CTLs were compared by cell killing assay using 3-(4, 5-dimethylthiazol-2yl)- 5(3-carboxymethoxyphenyl)-2-(4-sulfopheny)-2H-tetrazolium (MTS). Results: The expression of phenotype CD14, CD80, CD83, CD86 from DCs transduced with rAAV-HBV-S, C, E, X and the secretion of cytokine IL-10, IL-12 were compared, respectively. CD80 in rAAV-HBV-S was the highest (P < 0.05), and CD83 in rAAV-HBV-X was the highest (P< 0.05), but other results were not significantly different (P > 0.05). The response of cytotoxic T-lymphocytes induced by different adeno-associated virus (AAV) vector delivery into dendritic cells had significant differences. In comparing the four antigens in terms of CD4+ CD25+ T cells, it was found that rAAV HBV-E group was the highest, and rAAV HBV-X group was the lowest (P < 0.05); CD8+ CD69 + T cells in rAAV HBV-X group was the highest, and rAAV HBV-S group was the lowest (P < 0.05); IFN-gamma levels secreted by CTLs in rAAV HBV-X group was the highest, and rAAV HBV-S group was the lowest (P < 0.05). The specific cell killing activity of CTLs induced by DCs transduced by rAAV-HBV-X to the HepaG2.2.15 cells was the highest, but the four groups were no significant difference, respectively (P > 0.05). Conclusions: These data suggest that rAAV-HBV-S, C, E, Xdelivery into DC may be all useful for immunotherapeutic strategies against hepatitis B virus infection and that the HBV X antigen gene may be the most useful.