Preliminary Study on Hepatitis B Theraputic MVA Vector Vaccine
|School||PLA Postgraduate Medical School|
|Keywords||hepatitis B therapeutic vaccine heterologous vector|
DNA vaccine,with a predominant advantage in inducing specific cellular immunity in vivo,DNA vaccine has been widely determined as a potential immunotherapy method for treating CHB.How to enhance the efficiency should be particularly focused on DNA vaccine discovery.One possible way is to adopt suitable immunoadjuvants as well as to optimize immunization strategies.The immunization strategy of the heterologous vector(prime/boost)is achieved by the co-expression of the same protective antigen with different vectors,i.e.prime immunization by a DNA vaccine(prime)and boost immunization by a viral vector (boost).As an ideal prime immunization vaccine,the DNA vaccine has the advantages of high purity,continuous expression of antigens with low dose, repeatable immunization and inducement of memory CTL with high titer.Similarly, recombinant virus can not only highly express the target antigen,simulate the inducement process of virus infection on body immunity,and induce various cytokines,but also be beneficial to the interaction of high-affinity antibodies with T cell receptors,and the generation of Th1 reaction and CTL reaction.Interleukin-18(IL-18),also called interferon-gamma inducing factor(IGIF), can not only promote T cells and NK cells to generate cytokines,such as IFN-γ,IL-2 and GM-CSF,but also strengthen the expression on Fas ligand in Th1 cells and NK cells in order to mediate cytotoxicity and promote the development of Th1 cells.In this paper,the genes expressing PreS2-S、C、PreC/C and two mutanted PreC/C of HBV adr subtype were inserted into a shuttle vector pSC11,which is homologous with modified vaccinia virus Ankara(MVA),to construct and screen recombinant plasmid.The recombinant plasmid was then transfected into MVA virus. Recombinant MVA viruses respectively carrying the genes were screened after subculture of nine generations.Besides,monoclonal virus without wild virus was obtained during the subculture process.Indirect immunofluorescent analysis was used to identify the recombinant virus.The results showed that the recombinant virus could be recognized by the specific antibody against the antigen expressed by the recombinant virus.It was thus proved that the recombinant virus could express target antigen correctly and had good antigenicity.The DNA vaccine,the recombinant virus and mouse IL-18(mIL-18)were prepared on large scale.C57BL/6 mice were immunized by different immunization processes,including single DNA vaccine immunization,single rMVA immunization, DNA prime/rMVA boost and DNA prime/rMVA boost + mIL-18.The effects of humoral immunity and cellular immunity were evaluated by ELISA and ELISPOT. The results showed that there was no statistic difference among the vaccine groups capable of expressing two antigens,in the respect of antibody generation time and titer.The ELISPOT detection of IFN-γshowed that all immunization groups could induce cellular immunity against the specific antigen.The effect of the single rMVA immunization group was better than that of the single DNA vaccine immunization group,while weaker than that of the DNA prime/rMVA boost group and the DNA prime/rMVA boost + mIL-18 group.There was no significant difference between the DNA prime/rMVA boost group and the DNA prime/rMVA boost + mIL-18 group.The recombinant MVA virus induced specific cellular and humoral immune response toward HBV antigen.With the strategy of DNA prime/rMVA boost, stronger cellular immune were induced than DNA vaccine or rMVA alone,which provides basic evidence for the application of gene therapy for HBV.