Generation And Immunogenicity Analysis Of An Attenuated Vaccinia Virus Tiantan Strain Deleting TE3L And TE4L

Since vaccinia virus was used in expression of exogenous gene in1982, it hadbeen generally applicated in the study of the expression of exogenous gene and liverecombinant viral vaccines. In the whole development of vaccinia virus, it hadexperience four generations vaccines―the wild-type vaccinia virus, the new typevaccinia virus developed through tissue culture, an attenuated vaccinia virusdeveloped through sequential passage and gene defective virus ovtained by geneticengineering. However, our goal of study still is raising the safety of virus andremaining or improving immunogenicity.VACV causes relatively mild symptoms in humans infected naturally. However,it has been reported that VACV results in several serious side effects, includingencephalitis and progressive poxvirus infections, as well as some less severe reactions.But, because vaccinia virus has a lot of merits, for example the long history ofapplication in human, allowing the insertion of considerable exogenous genes, theeffective expression of proteins and immunogenicity. Thus, it is highly important todevelop a safe and effective VACV vector for the study of live viral vaccines for theprevention or therapy of infectious diseases.In the present study, we analyzed the genome of vaccinia virus Tian Tan (VTT)and constructed a TE3L and TE4L deleted strain (TE3/4L-VTT) by homologousrecombination. Based on VTT, the recombinant arms were designed and the TE3L andTE4L were replaced with enhanced green fluorescent protein (EGFP). And then theEGFP gene was deleted from the genome of TE3/4L-VTT-EGFP+through theCre/LoxP recombination system. The mutant virus TE3/4L-VTT was obtained byplaque screening. Then to determine whether the deletion of TE3L and TE4L impactsthe morphopoiesis of virus or not, we detected the bionomics such as viral morphousand replication. In addition, in this study, the virulence of TE3/4L-VTT as the viral vector wasanalyzed in vivo and vitro. First, the diffusing capacity and cytotoxicity ofTE3/4L-VTT and VTT in Vero, HeLa, MDCK, PK15and BHK-21cells weredetected by crystal violet staining and MTT colorimetric assay. Then, the virulenceof TE3/4L-VTT and VTT in vivo was evaluated through different routes of infectionin BALB/c mice and rabbits as animal models. The body weight changes of BALB/cmice were analyzed post-infection through intranasal inoculation, and the death ratesof BALB/c mice were analyzed post-infection by intranasal inoculation. To test theseverity of pathology of TE3/4L-VTT to the skin, the skin of rabbit was infected byintradermal injection.The immunogenicity of the mutant was evaluated in BALB/c mice as animalmodels immunized through intramuscular injection (IM). The immune response stateof mice after immunized was analyzed through lymphocyte proliferative analysis,cellular surface antigen of splenocytes through flow cytometry, the number of T cellssecreting IFN-γ, and IL-2and IL-4and VTT-specific antiboady in peripheral bloodmeasuring by ELISA.The results were following. An attenuated vaccinia virus TianTan strain deletingTE3L and TE4L was constructed successfully using shuttle vector, green fluorescentprotein and Cre-LoxP system, and it had well genetic stability. There were nosignificant differences in morphology between TE3/4L-VTT and VTT throughnegative staining and ultrathin section, suggesting that deletion of the TE3L and TE4Lgene did not affect the particle formation of VACV. The replicate ability ofTE3/4L-VTT and VTT was detected in Vero and BHK-21cells, the results suggestedthat the deletion of TE3L and TE4L did not affect the replication of TE3/4L-VTT.However, the crystal violet staining and MTT analysis revealed that the toxicity ofTE3/4L-VTT was reduced compared to those of VTT at the same infection time andcells. The results of the body weight changes of BALB/c mice infected withTE3/4L-VTT showed that the mutant did not affect the weight gain significantly. Bycontrast, BALB/c mice infected with VTT lost weight markedly post-inoculation.Additionally, after infection through intranasal inoculationl, there was no death in theTE3/4L-VTT-infected group within14days or even the following month, but the mice infected with VTT were dead post-infection. And there were no visible scars at thesite of infection of mice infected with TE3/4L-VTT, and the skin snapped back. Therabbits infected intradermally with VTT displayed relatively severe lesions at theinjection site, and the ulcerations required longer times to heal. These results wereshowed that the defection of TE3L and TE4L did not affect the morphogenesis andreplication of VTT, but the virulence of the mutant was attenuated compared with thewild type VTT.Immunogenicity is the significance aspect to evaluate a candidate vaccine or anantigen. In BALB/c mice models, the immunogenicity of a VTT mutant deletingTE3L and TE4L was analyzed through measuring VTT-specific antiboady, cytokinesin peripheral blood, cellular surface antigen of splenocytes and the number of T cellssecreting IFN-γ, and so on. We found that the expression of this immunogen wassufficient to facilitate the development of substantial number of antigen-specific Tcells secreting IFN-γ, and the number of CD4+and CD8+T cells was significantlyimproved. And VTT-specific antibody, IL-2and IL-4were induced to produce aftermmunization with the mutant virus TE3/4L-VTT.60days after the boostimmunization with the mutant, the results of ELISPOT assay had shown that themutant virus could induce to generate immune remember cells. Hence, TE3/4L-VTTdeleting TE3L and TE4L could induce robust immune responses.In conclusion, the defection of TE3L and TE4L attenuated the virulence of VTT,but the mutant TE3/4L-VTT remained the immunogenicity of VTT. The vacciniavirus mutant would provide a new material for the study of recombinant vacciniavirus vector vaccines or vaccine strain.