Generation of an Attenuated Tiantan Vaccinia Virus Strain by Deletion of Multiple Genes

An attenuated vaccinia virus-MVTT(EAB)-was constructed by deletion of non-essential gene segments related to the immunomodulatory and virulence functions of the vaccinia virus Tiantan strain (VVTT). The shuttle plasmids pTC-EGFP, pTE-EGFP, pTA35-EGFP, pTB-EGFP, and pTA66-EGFP were constructed and combined with the early and late strong promoter pE/L and EGFP as an exogenous selectable marker. Then, through the homologous recombination technology and Cre/loxP system, the following gene fragments were gradually knocked out one by one: TC7L-TK2L, TE3L, TA35R, TB13R, and TA66R. Ultimately, the five-segment-deleted attenuated strain MVTT(EAB) was obtained. Knockout of these segments and genetic stability of MVTT(EAB) were confirmed, and it was also shown that knockout of these segments did not affect the replication ability of the virus. Further, a series of in vivo and in vitro experiments demonstrated that the virulence of MVTT(EAB) was attenuated significantly, but at same time, high immunogenicity was maintained. These results indicate that MVTT(EAB) has potential for clinical use as a safe viral vector or vaccine with good attenuation and immunogenicity.