Generation And Immunogenic Comparison Of African Swine Fever Gene-expressing Recombinant Viral Vectors

Currently,African swine fever(ASF)can be controlled only by killing infected pigs and biosafety measures,and thus the effective vaccine is urgently needed.Since the failure of traditional vaccine development,new ASFV vaccine development is focused on gene deletion and recombinant vector vaccines.For the recombinant virus vectored vaccine development,however,the suitable protective antigens,viral vectors and immunization strategy remain to be defined.In this study,we selected ASFV genes with immune protective potentials and generated different viral vectors for expression of ASFV fusion genes.The immunogenicity of the viral vectors were compared by immunization of pigs and detection of the antigen-specific antibody and cytokine responses.1.Generation and identification of recombinant pseudorabies virus vectorsBy using PRV DNA as the template,the homologous arms of TK gene were amplified by high fidelity PCR.PRV transfer vector was constructed by cloning the homologous arms into a eukaryotic expression vector.Twelve ASFV genes(EP402R、CP204L、E183L、EP153R、B646L、D117L、F317L、EP364R、A151R、B438L、K205R and A104R)were divided into 4 fusion genes,optimized to pig codon usage,and cloned into PRV transfer vector.The four rPRVs were generated by co-transfection with the transfer vectors and PRV DNA.After plague selection,four rPRV vectors,namely rPRV-F1,rPRV-F2,rPRV-F3 and rPRV-F4,were obtained with viral titers of higher than 109 TCID50/ml.Both immunofluorescent and Western blotting analyses showed that the three rPRV vectors could express the ASFV fusion genes,and thud could be used for pig immunization and challenge experiment.2.Generation and identification of surface display baculovirus vectorsThe GED coding sequence of vesicular stomatitis virus(VSV)G protein was synthesized and the pseudotyped baculovirus vector was constructed by coning the synthetic GED sequence into pFastBac Dual vector.The CTD coding sequence of baculovirus gp64 protein was synthesized and the surface display baculovirus vector was constructed by cloning the CTD sequence into the GED-containing vector.Sixteen ASFV genes(EP402R、CP204L、E183L、EP153R、B646L、D117L、F317L、EP364R、E248R、E199L、P12、B119L、DP148R、EP152R、4 copies of DBD and I177L)were divided into six fusion genes and optimized to pig codon usage.After cloning into the surface display baculovirus vector,6 Bac vectors,namely rBac-F1,rBac-F2,rBac-F3,rBac-F4,rBac-F5 and rBac-F6,were generated.After PEG precipitation and sucrose gradient centrifugation,the six Bac vectors were concentrated to 2 × 108 TCID50/ml.Both immunofluorescent and Western blotting analyses showed that all Bac vectors could express ASFV fusion genes and therefore could be used for pig immunization experiment.3.Preparation of highly immunogenic ASFV CD2v antigenBased on the secondary structure analysis,the intracellular domain of ASFV CD2v protein with high antigenic index was synthesized and cloned into elastin-like polypeptide(ELP)gene-containing vector.After transformation into BL21(DE3)E.coli and induction with IPTG,the ELP-CD2v fusion protein was efficiently expressed as a soluble protein and purified 76.3%purity by inverse transition cycling(ITC)under optimized conditions.After cleavage with TEV protease and an additional ITC,the tag-free CD2v protein was purified to 91.7%purity and could be recognized by ASFV antibody.By testing 30 known anti-ASFV serum samples,the recombinant CD2v antigen ELISA had an identical antibody detection performance with the validated multi-antigen ELISA.These data suggest the prepared CD2v antigen could be used to detect the antigen-specific antibody.4.Primary study on immunization strategy with rAd and rPRV vectorsA total of 12 piglets were obtained from an ASFV-free pig farm.After 5-day observation,some piglets were positive for ASFV p54 antibody.Four rAd and four rPRV vectors were assigned into rAd/rAd,rPRV/rPRV,rAd/rPRV and rPRV/rAd combinations.Three piglets in each group were intramuscularly injected with each of four vaccine formulations at the same dose,and boosted on day 21 after primary immunization.ASFV p54-specific antibody levels in immunized pigs increased at different degrees,which were positively related to that of preimmunization.After stimulation with ASFV p54 or CD2v antigen,the antigen recall IFN-y expression in the peripheral blood mononuclear cells(PBMC)from all immunized pigs were significantly increased,which was not influenced by pre-existing antibody.Among the four immunization strategies tested,the priming with rPRV and boosting with rAd strategy stimulated slightly higher antigen-specific antibody and IFN-y responses than other three strategies,which warrants further investigation.5.Comparison of mucosal immune responses in different viral vector-immunized pigsA total of 11 piglets were intranasally immunized with rAd-F1,rPRV-F1 or rBac-F1(n=3)at the same dose and boosted with the same viral vector at day 21 after immunization.On day 7 post immunization,all immunized pigs were positive for ASFV p30,p54 and CD2v IgG antibodies,serum IgA antibodies and nasal S-IgA antibodies,which were significantly boosted by secondary immunization.Among three viral vectors tested,rBac-F1 immunization stimulated the highest antibody responses than other two viral vectors.On day 35 post immunization,the antigen-specific S-IgA antibodies were detected in the tracheal washes and lung lavages from all immunized pigs with the highest S-IgA antibody level in rBac-Fl-immunized pigs.After stimulation with the three ASFV antigens,the expression levels of antigen recall Th1(IL-2 and IL-4)and Th2(IL-10 and IFN-y)cytokines in PBMC from all immunized pigs were significantly increased with the highest expression level in rBac-F1 immunization group.The expression levels of S-IgA-related cytokines in PAM from all immunized pigs were also significantly increased with the highest expression level in rBac-F1 immunization group.The increased S-IgA-related cytokine expression was correlated with IL-1β,IL-4,IL-12and IFN-α,but not with IL-10.Among the three viral vectors tested,surface display Bac vector showed the best performance for pig mucosal immunization.