Development Of Novel Recombinant Swinepox Live Vaccine Against TGEV And PEDV And Bio-safety Evaluation Of The Swine Pox Virus Vector

Transmissible gastroenteritis virus(TGEV)and Porcine epidemic diarrhea virus(PEDV)were the major pathogen of swine viral diarrhea.Since the outbreak of the global epidemic diarrhea from 2010,commercial vaccines are available,but they are not significantly effective on farm animals against the complex and changeable infection situation in pig farms.The incidence of viral diarrhea of infection was 100%,and the mortality rate was very high within the age of 10 days.The study constructed recombinant Swinepox virus which expressed TGEV and PEDV neutralizing antigens and evaluation of swinepox virus vector attenuation optimization and biosafety.1.Efficacy and Immunogenicity of Recombinant swinepox virus expressing the A site of the TGEV S proteinTo explore the possibility of developing a vaccine against transmissibleastroenteritis virus(TGEV)infections,a recombinant swinepox virus(rSPV-SA)expressing a TGEV protective antigen has been constructed.Immune responses and protection efficacy of the vaccination vector were assessed in both mice and pig models.An indirect ELISA assay approach suggested that when mice were vaccinated with rSPV-SA,synthesis of IgG against TGEV was enhanced dramatically.The cytokine assays were employed and the results indicated that both the Thl-type and Th2-type cytokine levels increased dramatically after vaccination with rSPV-SA in mice models.Results from the passive immunity protection test of new born piglets demonstrate that the recombinant live-vector vaccine,SPV-SA,could 100%protect piglets from the SPV infection,and there was no significant clinical symptom in the SPV-SA treatment group during this experiment.The data suggest that the novel recombinant swinepox virus is a potential vaccine against TGEV infection.2.A recombinant swinepox virus vaccine provides protective immunity against challenge with novel porcine epidemic diarrhea virus strainPorcine Epidemic Diarrhea virus(PEDV)causes significant economic losses to the swine industry.Commercial vaccines are available,but are not effective on farms against the outbreak in Chinaof a novel PEDV strain,SQ2014.A recombinant swinepox virus vaccine(rSPV-Sa)expressing the truncated S protein from PEDV strain SQ2014 was constructed,and its immunogenicity and efficacy were tested and characterized.Vaccination robustly increased the IgG titer specific for PEDV(SQ2014)and induced neutralizing antibodies with a mean titer of 1:24 at 42 days post-inoculation.The rSPV-Sa group serum IgA levels were significantly higher than those killed vaccine groups.The efficacy of the rSPV-Sa vaccine was tested in passive transfer models using homologous(SQ2014)and heterologous(CV777)challenges.In the homologous challenge,rSPV-Sa provided complete protection.None of the challenged animals showed clinical symptoms of PEDV infection and there was no mortality.The rSPV-Sa vaccine did not show protective efficacy in the heterologous challenge(100%mortality in challenged animals).Differences in the amino acid sequences of the two viruses were identified in the neutralizing epitopes and might have contributed to the divergent clinical results.The data suggest that rSPV-Sa is a potential vaccine against new strain of PEDV infection.3.Construction and immunogenic analysis of recombinant swinepox co-expressing truncated S gene of PEDV and TGEVTo develop a vaccine against porcine epidemic diarrhea virus and transmissible gastroenteritis virus co-infection,the genes of truncated S of TGEV and PEDV were inserted into the swinepox virus(SPV)genome.The recombinant swinepox virus rSPV-TP was verified by PCR and immunofluorescence assays.The growth rate of rSPV-TP was lower than wtSPV.To evaluate the immunogenicity of rSPV-TP,twenty five Balb/c mice were immunized with rSPV-TP,inactived-TGEV,inactived-PEDV,wtSPV and PBS.The results showed that TGEV and PEDV S-specific antibody of the rSPV-TP immunized group increased significantly compared to the wtSPV treated group after vaccination and increased continuously.The neutralizing antibody titers could be up to 1:600 of TGEV or 1:30 of PEDV.The levels of IFN-yand IL-4 in the rSPV-TP inoculated group were significantly higher than the other groups.Accordingly,the data suggest that the novel recombinant swinepox virus is a potential vaccine against TGEV and PEDV infection.4.Evaluation of swine poxvirus vector attenuation optimization and biosafetyThe Swine pox virus(SPV),a member of the pox virus family,has several advantages to become a potential clinical vector vaccine.Nevertheless,the SPV vector vaccine is still not commercialized nor broadly applied in term of clinical use.In this study,in order to acquire safer and more efficient SPV vector vaccine,three mutant strains of SPV,?003,?010,and ?TK,were successfully constructed.The virus replication experiment showed these three mutant strains of SPV had lower replication rate,comparing to the wild type SPV.Especially,the titer of ?003 and ATK dropped significantly.The animal experiments on mice and rabbit model demonstrated the three mutant strains and wtSPV cannot induce any symptoms of dermatitis.No fatality nor any clinical symptom was observed during the peritoneal challenge assay of these three mutant strains and WT SPV in mice model.And all the mutant strains and wtSPV was indicated to be not infectious in 60 hours after the vaccination.Furthermore,39 one month old piglets were applied to evaluate the safety.The results showed there was no symptom of dermatitis in the ?003 and ?TK vaccinated group,and mild symptoms in AO 10 vaccinated groups when the titer was high,while the apparent symptoms were observed at the vaccinated region.No virus load was detected,and there was no significant difference in terms of the cellular immunity level and humoral immunity level in all vaccinated groups.To sum up,this study suggests two mutant strains of SPV,?003 and ?TK,can become promising candidates as an attenuated virus vector vaccine in veterinary medicine.