The Modification Of Vaccinia Virus Based On EEV Egress Related Genes For Potential Application As Vaccine Vector

Vaccinia virus（VV）has been widely used as a smallpox vaccine.With the global eradication of smallpox,VV has been modified as a vaccine vector against infectious diseases including AIDS,influenza,etc.and tumors.To date,several vaccines based on VV have stepped into clinical research stage.However,VV is a DNA virus with a large genome and a large number of genes and remains many unknowns to be further investigated.In order to meet the needs of various applications,vaccinia virus needs to be further modified and optimized.VV contains two distinct infectious forms:intracellular mature virus（IMV）and extracellular enveloped virus（EEV）.Compared with the majority-IMVs,the minority-EEV consists less than 1%of the total progeny virus,but EEV has several unique advantages in that they are early released,capable of early infection,and long-distance transmission.Thus,we hypothesized that the changes on EEV release may have a impact on the characteristics of viral infection and the induced immune responses.The release of EEV involves two key steps:the microtubule-dependent transport of intracellular enveloped virus to the cell membrane periphery;and the egress of EEV to the extracellular space.In this study,we used the Tiantan vaccinia virus（VTT）as a model,modified the wild type VTT by deletion or mutation of VV genes related to the key steps of EEV release and then systematically investigated the replication,EEV release,and antigen expression of VTT recombinants,and finally,to assess and screen for an optimized recombinant VTT vector with enhanced immunogenicity.First,we attempted to alter the microtubule-dependent EEV release by the deletion of related A36R and F12L genes,thereby facilitating the release of virus to the mucosal basolateral.As the F12L gene deletion mutant was difficult to amplify,therefore in the following study,we focused on the comparison of A36R-deleted recombinant virus with the wild-type VTT in their EEV release and replication capability.The results showed that:1)In non-polarized Caco-2 epithelial cells,the EEV release and viral replication of rVTTΔA36R-GFP was decreased when compared to the wild type VTT;2)In polarized Caco-2 cells of trans-well system,the progeny virus released to the luminal and basolateral surface were also decreased.Moreover,in the in vivo experiments,recombinant virus rVTTΔA36R-GFP produced less total progeny virus in the nasal and lung tissues of mice after the intranasal inoculation.The results above proved that:the deletion of A36R gene inhibited viral EEV release,reduced the replication of vaccinia virus in epithelial cells and in respiratory mucosal of mice.Therefore,it is not a potent way to improve the immunogenicity of VTT vectored vaccines.Second,we attempted to improve the EEV release by point mutation on A34R and B5R gene that related to the egress of enveloped virus from cell membrane.The results showed that:compared with wild type VTT,recombinant virus rVTT-B5R_mut only slightly increased EEV release and total progeny virus production,while rVTT-A34R_mutut significantly increased EEV release and total progeny virus production.Based on this result,the HIV-1 env gene was inserted into rVTT-A34R_mut as a model antigen to construct the rVTT-A34R_mut vectored HIV vaccine rVTT-A34R_mut-Env.Compared to the control virus rVTT-Env,rVTT-A34R_mut-Env significantly increased the production of progeny virus and the expression of foreign antigens in vitro and in mice muscle after intramuscular inoculation,and induced a stronger HIV Env specific T cell and antibody responses.In addition,the recombinant vaccine rVTT-A34R_mut-Env not only showed no-increased cytotoxicity,but also showed no-increased virulence in mice,demonstrating its good safety profiles.Thus in this study,we proved that the A34R gene mutated recombinant rVTT-A34R_mut with enhanced EEV release is a promising vaccine vector to improve its immunogenicity.Moreover,we applied the A34R point mutation to VTKgpe,a VTT based vaccine that has stepped into clinical trials,and proved that the A34R point mutation is sufficient to improve the HIV specific responses in mice.Thus,we demonstrated that the A34R point mutation can also be effective in improving the induced immune responses of other vaccinia virus vectored vaccines,and that promotion of the EEV release by A34R point mutation is a promising way to improve the immunogenicity of VTT vectored vaccines.In summary,this paper systematically studied the VTT modification that affecting the EEV release on the influence of recombinant virus replication,foreign antigen expression,and the immunogenicity of VTT vectored vaccines,and screened for a highly efficient and safe VTT vaccine vector rVTT-A34R_mut,and further proved that the modification of the A34R point mutation is also effective in other vaccinia virus vectors.Therefore,this study not only provides an example of how to construct a safe and efficient vaccinia virus vaccine vector,but also provides a new way for VV vector optimization.