Immune Efficacy Of MVA Expressing HBsAg In Mice

Hepatitis B virus infection can cause both acute and chronic disease. According to report from WHO (World Health Organization), an estimated 240 million people are chronically infected with hepatitis B. Approximately 780000 persons die each year. Hepatitis B virus infection is a major global health problem. Chronic hepatitis B can be treated by drugs including IFN, PEG-IFN-a and Nucleic acid analogs. Drugs can slow the progression of cirrhosis, reduce incidence of liver cancer and improve long term survival. However, hepatitis B virus cannot be completely eliminated in patients. The main limitations of drugs are drug resistance, side effects and poor compliance of patients. During chronic HBV infection, persistent high HBV DNA copies and HBeAg lead to T cells exhausted and immune responses impaired. Thus using therapeutic vaccine could induce specific cellular immune responses, active immune inhibitory mechanisms and achieve eradication of HBV.Viral vector vaccines offer an interesting approach to chronic hepatitis B treatment. The modified vaccinia Ankara (MVA) has been an ideal vector for recombinant viral vaccines due to its large capability of foreign genes, defective replication and safety. In present study, the HBsAg gene was cloned into a plasmid pSC11. The recombinant plasmid pSCll-S was transfected into BHK-21 cells infected with MVA. Homologous recombination occurs between MVA genome and homologous sequences within the recombinant plasmid pSC11-S. The recombinant virus was selected by several rounds of blue/clear plaques. The gene of interest was identified by PCR. The recombinant virus with HBsAg gene (MVA-S) was generated. The expressing HBsAg was detected by Western Blotting.BALB/c mice were primed and boosted with MVA-S and DNA vaccines using different immune strategy. Mouse splenocytes were stimulated by specific synthetic peptides to secret IFN-y. The level of specific cellular immune response was measured by ELISpot assay. Analyses of ELISpot data from different groups may allow evaluating efficacy of different immunization strategies.In first phase, BALB/c mice were immunized by DNA vaccines (pVR-S, pcDNA-S) and viral vaccine (MVA-S), respectively. The results of single immunization strategy showed that both MVA-S and two DNA vaccines can induce significant specific cellular immune responses. However, there was no significant difference in the SFCs among three groups (F= 0.61, P= 0.5599).In second phase, heterologous consecutive immunization strategy was evaluated. BALB/c mice of experimental group were immunized by DNA prime/MVA-S boost strategy, while mice of control groups were boosted by MVA after DNA vaccine to be primed. The ELISpot results showed that SFCs of experimental groups and control groups were significantly different (P<0.05). The prime/boost regiment could induce higher cellular immune responses than single immunization.The prime/boost regimens were used in the different doses of MVA-S, including low dose group, median dose group and high dose group. The results of ELISpot assay were significantly different among three dose groups (F=12.67, P=0.0036).The high doses of MVA-S could induce strongest immune responses among the three different dose groups.To investigate effects of DNA vaccine vectors, BALB/c mice were primed with two DNA vaccines (pVR-S, pcDNA-S) followed by same MVA-S boost. The ELSpot assay showed there was no significant difference between different two DNA vaccines using prime-boost immunization strategy. The DNA vaccine vectors did not influence boost effects.