Construction And Immunological Study Of The Attenuated Vaccinia Tiantan Strain With Multiple Genes Deleted

Vaccinia virus(VACV) has a large genome, wide range of hosts and exhibits a strict replication in cytoplasm. Thus, it is widely used in the treatment of infectious diseases, cardiovascular diseases, immune deficiency diseases and tumors. Although the conventional vaccinia virus, like vaccinia virus TianTan strain, as a vaccine has played a key role in the eradication of smallpox process, but induce different side effects, such as fatigue, fever, rash, encephalitis and ocular vaccinia infection, which are severer especially in patients with immunossuppression, chronic skin disease and heart disease.The live genetically engineered vector vaccines using vaccinia virus as the vectors have been developed. Although the related research on vaccinia virus as expressed vectors has made remarkable achievements, there are still some problems including the complexity of vector construction procedures, the insertion of exogenous selection markers and the difficulty in the screening of recombinant virus. Therefore, to further improve the efficiency of vector vaccines, reduce the side effects of vaccinia virus and simplify the preparation procedures have become a hot topic in the research on vaccinia virus vector.In this study, we used homologous recombination technique and Cre-loxP recombination system to knock out gene segments including TC7L-TK2L(TC7L, TC6 L, TC5 L, TC4 L, TC3 L, TC2 L, TC1 L, TN1 L, TN2 L, TM1 L, TM2 L, TK1 L and TK2L), TE3 L, TA35 L, TB13 R and TA66 R on VTT, ultimately to achieve the result of attenuating virulence and narrowing host range. Then, the virulence of recombinant vaccinia virus was analyzed by a series of in vivo and in vitro experiments. Firstly, cell proliferation ability and cytotoxicity of recombinant vaccinia virus were analyzed by crystal violet staining and MTT testing experiment. Secondly, the virulence level of recombinant vaccinia virus was analyzed by observeing weight change and survival rate. Finally, the skin damage of recombinant vaccinia virus was analyzed by observeing pock size in the back of rabbits.Further, the immunogenicity of recombinant vaccinia virus was analyzed by immunization of mice. T lymphocyte proliferation assays, cytokine detection experiments, specific antibody and neutralizing antibody detection experiments were used to verify the immunogenicity of recombinant vaccinia virus.The results show that the recombinant vaccinia virus MVTTEAB, which is deleted TC7 L ~ TK2 L, TE3 L, TA35 R, TB13 R and TA66 R genes is successfully constructed and has a excellent genetic stability. The deletion of five gene fragments does not affect the morphogenesis and normal replication of recombinant vaccinia virus. In vitro experiment, the cell proliferation capacity and cytotoxicity of recombinant vaccinia virus are significantly weakened. In vivo experiment indicates that the recombinant vaccinia virus has a high safety and immunogenicity.The above results show that the deletion of TC7 L ~ TK2 L, TE3 L, TA35 R, TB13 R and TA66 R genes has reduced the virulence and host range of recombinant vaccinia virus MVTTEAB without affecting the immunogenicity. The viral vector lays a foundation for recombinant vector vaccine research.