Construction, Pathogenicity And Immunogenicity Of The Attenuation Of Vaccinia Tian Tan Strain

Widespread use of vaccinia virus as smallpox vaccine has eradicated variolavirus. Recently, vaccinia virus is a live recombinant vector against HIV, rabies andother infectious diseases that have been intensively studied. However, intracranialinoculation test show a high death rate of mice and neurotoxicity. Clinical vaccinationof vaccinia virus may also lead to severe side effects, such as myocarditis,encephalitis, conjunctivitis and systemic hair blain, etc. Therefore, how to build amore safe and efficient vaccinia virus vaccine strain is becoming a hot spot ofresearch.Deletion of non-essential gene in vaccinia virus genome, is one of the effectivemethod to construct attenuated vaccine or vector by homologous recombination andCre-LoxP technology. Genetic recombination, as one of the important geneticengineering technology, includes the homologous recombination, site specificrestructuring and reorganization. Homologous recombination technology, also knownas gene targeting technology, is currently method in gene function research. Thetechnology of DNA restructuring was done by shuttle vector which containsrestructuring arm, promoter and EGFP. Attenuated vaccinia Tian Tan strain isconstructed by deletion of the target gene. Growth dynamics, virus particlesphenotype, intercellular communication ability, virulence, immunogenicity and hostrange features of vaccinia virus are analysised through in vitro and in vivo. Screeningmarker genes are used in the process of construction. However, screening markergenes are foreign genes which do not conform to the requirements of the commercialvaccines of biological safety. Thus, screening marker genes should be deleted in theprocess of research. Cre-loxP site-specific gene recombination system may removeexogenous gene, which has been used widely in the research of recombinant viruses,bacteria and transgenic animal. Cre recombinase can recognize LoxP site and remove the sequence when LoxP sequence located on the same DNA strand.In the present study, we constructed modified VTT genome (vMVTT1, vMVTT2and vMVTT3) by deleting TC7L-TK2L or TA35R genes, which resulted in reducedvirulence. vMVTT1deletes TC7L-TK2L gene. vMVTT2deletes TA35R gene.vMVTT3deletes both TC7L-TK2L and TA35R genes. Here, we examine the mutantvirus which removed TC7L-TK2L (15,262-25,450) including TC7L, TC6L, TC5L,TC4L, TC3L, TC2L, TC1L, TN1L, TN2L, TM1L, TM2L, TK1L, and TK2L or asingle open reading frame TA35R (138,881-139,570) from vaccinia Tian Tan strain.Growth dynamics, virus particles phenotype, intercellular communication ability,virulence, immunogenicity and host range features of vaccinia virus are analysisedthrough in vitro and in vivo. First, the diffusing capacity and cytotoxicity ofvMVTT1、vMVTT2、vMVTT3and VTT in BHK-21、Vero、MDCK、HeLa and PK15cells were detected by crystal violet staining and MTT colorimetric assay. We alsoevaluate the genetic stability of the strains. Second, the body weight changes ofBALB/c mice were analyzed through intranasal inoculation, and the death rates ofBALB/c mice were analyzed by intracranial inoculation. Rabbits were test thevirulence of strains by intradermal inoculation. The immunogenicity of the vMVTT1,vMVTT2and vMVTT3was evaluated in BALB/c mice. IL-2, IL-4, IL-10, IFN-γ andVTT-specific antiboady was tested in peripheral blood. Protection from lethalchallenge was also tested.The results showed that three attenuated strains (vMVTT1, vMVTT2andvMVTT3) deleting TC7L-TK2L or TA35R gene were successful constructed, andthey had well genetic stability. There were no significant differences in morphologyamong vMVTT1, vMVTT2, vMVTT3and VTT, suggesting that deletion of theTC7L-TK2L and TA35R genes did not affect the virus morphogenesis. However, ithas a great influence on the growth dynamics, intercellular communication ability,toxicity and immunogenicity. Replication was decreased in the cells compared toVTT (VTT> vMVTT2> vMVTT1> vMVTT3). Spread of cell-to-cell were changed(VTT> vMVTT2> vMVTT1> vMVTT3). vMVTT3spread in BHK-21cells. ButvMVTT3nearly could not spread in Vero, HeLa, MDCK and PK15cells. The mutantvMVTT3did not cause detectable ulceration when it was inoculated intradermally in rabbits. And skin virulence of vMVTT2was stronger than vMVTT1. Additionally,size of the spots also related to the titer of virus. Inoculation of virus was important tovirulence. The vMVTT3was found to be100-fold less virulent than VTT based onthe body weight loss of mice intranasally infected with either virus, and3200-fold lessvirulent based on the intracranial lethal infectious dose in mice. In addition, wedemonstrated mutants induced immune responses was equal to those induced by VTTimmunization. vMVTT3protect mice effectively from the challenge of a massivedose of VTT, suggesting that removal of TC7L-TK2L and TA35R genes fromvaccinia Tian Tan strain-based vaccines will keep their immunogenicity. In this study,the virulence of vMVTT1, vMVTT2and vMVTT3was decreased. The vMVTT3mutant was shown to be avirulent and immunogenic.Base on VTT, TC7L-TK2L and TA35R genes are deleted without inserting ofEGFP. vMVTT3can be used as a safe viral vector or as platform of vaccines forinfectious diseases and cancer.