Research Of Human IFN-α Eukaryotic Expression Plasmid Induced Anti-HBV Effects And Mechanisms

ObjectAt present, there are about four hundred million Chronic Hepatitis B (CHB) patients around the world. It is estimated that one million people died from CHB related hepatocellular carcinoma (HCC) and liver cirrhosis, of which China accounts for thirty percent. HBV has also been classified as one of the most important human carcinogens by World Health Organization (WHO). HBV infection threatens people’s health and life, and brings heavy burden to the state and society.Interferon alpha (IFN-a), as the main approach of antiviral treatment, plays a crucial role in the practice of the clinical treatment of CHB. IFN-a has the obvious advantages in the treatment of HBV, especially for the patients characterized by young, low viral load and low level of alamine aminotransferase (ALT).However, due to the small molecular weight and short half-life of ordinary IFN-a, people need frequent injection, then reducing the compliance of patients. Pegylation improves the half-life of IFN-a, but lowers its biological activity. No matter what the type of IFN-a is, flu-like symptoms such as headache, muscle aches, chills, fever, occur with each injection. Therefore, improving the therapeutic efficacy of IFN-a plays a vital role in the treatment of HBV.With the consecutive development of molecular biology technology and molecular pharmacology, gene therapy based on the genetic and cell engineering, brings new hope to conquer human diseases. Gene therapy has a lot of special advantages compared with the traditional drug therapy, including sustained drug delivery, targeted drug delivery, etc. In light of these findings, an attempt to investigate the effects of human IFN-a on HBV replication in vitro by transfecting a eukaryotic expression plasmid (pSecTagB-IFN-a) was carried out.MethodsFirst of all, HBV-transfected HepG2.2.15cell line and its parental cell HepG2were transfected with pSecTagB-IFN-a or empty plasmid using LipofectamineTM 2000reagent. The expressions of IFN-a after transfecting pSecTagB-IFN-a in HepG2and HepG2.2.15cells were determined by RT-PCR and ELISA. Then, the effects of transfecting pSecTagB-IFN-a on HBV mRNA, DNA and antigens were tested by qRT-PCR and ELISA. Then, RT-PCR, qRT-PCR and western blot were employed to research antiviral mechanism by assessing the influence of pSecTagB-IFN-a transfection on IFN-a-induced signal pathway. Furthermore, through the analysis of qRT-PCR and ELISA, the suppressive effects of endogenously expressed IFN-a and the combination with lamivudine on HBV were also examined. In addition, by means of flow cytometry and RT-PCR, the levels of MHC I and Fas were detected after transfection of pSecTagB-IFN-a.Results(1) Human IFN-α (hIFN-α) was efficiently expressed in hepatocytes transfected with recombinant pSecTagB-IFN-α vector. The recombinant eukaryotic expression vector (pSecTagB-IFN-α) could stably express and secrete high levels of IFN-a in the supernatants of HepG2.2.15and HepG2cells.(2) pSecTagB-IFN-a inhibited HBV replication in HepG2.2.15cells. Endogenous expression of IFN-a could suppress HBV characterized by a decline of HBs and HBc mRNA, a reduction of HBV DNA load and a decrease of HBsAg and HBeAg.(3) pSecTagB-IFN-a activated JAK-STAT signal pathway and promoted the induction of IFN-stimulated genes. The expressions of STAT-1and p-STAT-1were up-regulated after transfection of pSecTagB-IFN-a, meanwhile, the levels of typical IFN-a-induced antiviral effectors including ISG15, OAS-1, IFIT-1and IFIT-3were increased in HCC cells.(4) Double-stranded RNA (dsRNA) sensing receptors were up-regulated by IFN-a over-expression. Delivering pSecTagB-IFN-a into HepG2.2.15and HepG2cells brought about enhancive levels of RIG-I, MDA5, LGP2, PKR and TLR3.(5) Endogenously expressed IFN-a could act more efficiently in inhibiting HBV than exogenous IFN-α. Transfecting pSecTagB-IFN-a can be more effective than equivalent dose of exogenous IFN-a in suppressing HBV, including the decrease of HBx, HBs and HBc mRNA, the reduction of HBV DNA load, and the low contents of HBsAg and HBeAg.(6) Lamivudine combined with pSecTagB-IFN-α displayed enhanced anti-HBV effect than with exogenously supplemented IFN-a.The combination of endogenous expression of IFN-α and lamivudine can be more efficacious in inhibiting HBV in contrast with the combination of exogenously supplemented IFN-a and lamivudine, characterized by the decrease of HBx, HBs and HBc mRNA, the loss of HBV DNA load and the reduction of HBsAg and HBeAg.(7) pSecTagB-IFN-a up-regulated MHC I and Fas. The levels of MHC I and Fas were increased after transfecting pSecTagB-IFN-α into HepG2and HepG2.2.15cells.ConclusionThe experimental results showed that endogenously expressed IFN-a can more effectively and persistently inhibit HBV replication in HBV infected cells. These observations might provide a promising way to design new antiviral genetic engineering drugs based on IFN-a and probably bring some enlightenment about the gene therapy combined with the traditional drug.