Experimental Study On TBK-1 Enhancing Specific Immune Responses To HBcAg Nucleic Acid Vaccine

Hepatitis B Virus (HBV) infection remains a major worldwide infectious disease characterized by degeneration and necrosis of hepatic parenchymal cells. Chronic HBV infection often leads to the development of liver cirrhosis, liver failure, and hepatocellular carcinoma, which is seriously threatening the human health. Viral persistence is thought to be related to the poor HBV-specific immune responses, making the virus hardly removed. In the therapeutic area of hepatitis B, the efficacy of drug therapy is not satisfactory, and new strategies are urgently needed.DNA vaccine, also named nucleic acid vaccine or genetic vaccine, is a novel vaccine which is developed along with gene therapy techniques. It not only plays the role of preventing diseases, but also can cure diseases. In animal models, intramuscular injections of HBV DNA vaccines were proved to induce antigen specific cellular and humoral immune responses. However, DNA vaccines suffer from lower immunogenicity in human species which limits their effectiveness. How to improve the immune effects of HBV DNA vaccines is one of the hot research areas. Recent studies have been focused on strategies to improve the immune effects of DNA vaccines combined with immune adjuvants, that is co-administration of genes or protein which can improve immune effects or other immunocompetent molecules with HBV DNA vaccines. These molecules mainly contain co-stimulatory molecules, chemokines, molecules that induce apoptosis, and ligands for Toll-like receptors (TLRs) or their signaling molecules, etc.Hepatitis B virus core antigen (HBcAg) can be stably expressed in prokaryotes, and retain their inherent immunogenicity. It is a typical T cell-independent and T cell-dependent antigen which can induce strong HBcAg specific humoral and cellular immune response. In recent years, mechanism researches of innate immune responses have been made great progresses. TANK-binding kinase 1 (TBK-1) is a kind of non-canonical IκB kinase, which can phosphorylate interferon regulator factor (IRF3) and IRF7 and increase the production of IFN-β, and is essential for the cell signaling of innate immune responses.In this study, the immunogenicity in the induction of humoral and cellular immune responses by HBV DNA vaccine and the adjuvant effect of TBK-1 were studied in BALB/c mice. The eukaryotic expression plasmid pcTBK-1 was constructed by inserting mouse TBK-1 gene into the vector pcDNA3.1(+). L929 cells transfected with TBK-1 were stimulated with dsDNA in vitro to analyse the role of TBK-1 in the production of IFN-β. Then BALB/c mice were immunized by three intramuscular injections of HBcAg DNA vaccine plasmids alone or in combination with TBK-1 expressing plasmids, humoral and cellular immune responses were measured comparable to the control groups immunized with parent plasmid pcDNA3.1(+) or PBS. As a result, the production of IFN-βwas obviously increased in the TBK-1 transfected group at both mRNA level and protein level (P<0.05). The levels of anti-HBc in groups of mice immunized with DNA vaccine of HBV core gene plus pcTBK-1 were higher than that in group of mice immunized with HBV core DNA vaccine alone (P<0.05). Similarly, the HBcAg specific splenocytes proliferation and cytotoxic activities in the co-immunization group were both significantly higher than those in the HBV core DNA vaccine single immunization group (P<0.05). Besides, the level of IFN-γin the supernatant of splenocytes cultured with HBcAg in vitro was significantly increased (P<0.05) while the level of IL-4 showed no significant difference (P>0.05).In conclusion, TBK-1 can increase the production of IFN-βin L929 cells stimulated with dsDNA. Furthermore, TBK-1 can not only enhance the humoral immune response but also promote the cellular immune response in mice elicited by HBcAg DNA vaccine. Thus, TBK-1 is revealed to be a potent candidate adjuvant for HBV DNA vaccine. Therefore, the results provide the experimental and theoretical basis on the study of therapeutic HBV DNA vaccines, and also open up a new approach for the treatment of chronic hepatitis B.