Immunoprotection Study Of Recombinant Live Virus Vector Vaccine Candidate Strains Of African Swine Fever Virus

African swine fever(ASF)is an acute,haemorrhagic infectious disease caused by the African swine fever virus(ASFV)that spreads mainly among domestic pigs and wild boars and can cause 100%mortality.ASFV,as a double-stranded DNA virus,is the only member of the genus Asfivirus in the family Depending on the strain,the ASFV genome is 170 to 194 kb long and encodes 151 to 167 open reading frames,among which the function of only part of the genes have been elucidated,while the function of about half of the genes remains unknown.After 2007,ASF appeared a new round of international prevalence,with Eastern Europe,Siberia,East Asia,Southeast Asia and the Caribbean becoming ASF epidemic areas one after another.In 2018,ASF was introduced to most of China,causing substantial economic loss.Today,the ASF epidemic is still spreading globally,posing a lasting danger to the pig industry,and seriously affecting world food security.Developing a safe and effective vaccine or treatment has become an urgent priority.Various types of ASFV vaccines are being tried,including inactivated vaccines,subunit vaccines,DNA vaccines,live virus vector vaccines,gene deletion vaccines,etc.Among them,live virus vector vaccines are safe,low-cost,effective and have significant application prospect.In this study,SY18,a genotype II ASFV strain prevalent in China and maintained in our laboratory,was used as the subject for the following preliminary work on live virus vector vaccine:1.Eight ASFV immunoprotection-related genes(B119L,DP96R,I329L,O61R,B438L,E199L,MGF110-5L6L,MGF505-5R)were initially screened for their homology at the amino acid level among different genotypes to investigate their feasibility as target genes for live virus vector vaccines.Homology comparison results showed that these eight genes were highly conserved(100%homology)in the genotype II endemic strains of ASFV and relatively conserved in the other genotypes and could be used as target genes for the construction of live vector viruses in this study.2.We packaged eight recombinant adenovirus strains expressing the target genes based on the safe and efficient replication-deficient human type 5 adenovirus packaging system(CELL BIOLABS,USA)and investigated the in vitro biological properties of each recombinant adenovirus strain.The results show that the recombinant adenovirus can be inherited stably,correctly express the target gene over 20 generations,and has preliminary feasibility as a vaccine candidate.3.Porcine reproductive and respiratory syndrome virus(PRRSV)is another virus that needs to be prevented and controlled in pig farms and it is also an excellent live virus vector.We constructed an infectious cloning plasmid(pcDNA-HH-PRRSV)based on the PRRSV vaccine strain JL-att kept in our laboratory and modified it to construct eight recombinant PRRSV strains that can be stably inherited and correctly express the target gene within 15 generations.4.Before in vivo studies with recombinant live virus vectores,we evaluated a challenge model of the potent ASFV strain SY18 in pigs.The results of different challenge doses and different routes of challenge in pigs showed that intranasal infection with ASFV SY18 at 40-5000 TCID50(50%tissue culture infective dose)and intranasal infection at 40-1000 TCID50 produced the same acute ASF symptoms as after intramuscular challenge(200 TCID50)and led to death or a near-death state within 21 days after challenge.Showing that intraoral and intranasal challenges can be used as alternative attack models for vaccine efficacy evaluation.5.We evaluated the safety and immunoprotective efficacy of the live virus vector expressing the target gene constructed in this study by conducting safety and immunoprotective efficacy tests in pigs.The results showed that all recombinant live vector virus strains constructed in this study were safe for pigs,did not affect pig fattening,and could induce the production of antibodies against the target gene and antibodies against the vector virus,and that the recombinant live virus vector constructed in this study could prolong the process of acute ASF caused by the challenge of a highly virulent ASFV strain after immunizing pigs.Moreover,combining recombinant adenovirus and inactivated ASFV antigens could achieve a 60%protection effect in pigs.And,Increased activation of ASFV-specific T cells(IFNY+)was detected only in pigs immunized with inactivated ASFV antigen,suggesting that some structural proteins of ASFV may be critical in eliciting specific T cell responses.In summary,the eight ASFV immunoprotective genes screened in this study were conserved in the prevalent strains,and recombinant adenovirus and recombinant PRRSV expressing these eight genes are all safe for pigs and could provide partial protection to pigs after immunization,and one of the immunization combinations had a 60%protective effect.Preliminary studies on the mechanism of the effective combination of immune protection have revealed that the structural protein of ASFV is crucial in inducing a specific T-cell response in pigs.The above data and results may provide a partial reference for the subsequent development of a live vector and subunit vaccine for ASFV.