Develpoment Of RAAV-based African Swine Virus Vector Vaccine Candidate And Preliminary Assessment

African swine fever(ASF)is a hemorrhagic swine infectious disease with high fatality rate caused by African swine fever virus(ASFV).ASFV emerged in china in August,2018,it has brought huge economic losses to the pig industry across country.Currently there remains to be no commercial vaccine or effective treatment method for the prevention or treatment of ASF disease.Natural attenuated strains and recombinant live attenuated vaccines(Live Attenuated Vaccine,LAV)with the ASFV virulence gene knocked out have shown good protective effect,however it has the risk of increasing the virulence and causeing continuous infection;ASF subunit vaccine,DNA vaccine and Viral vector vaccines are highly safe and have vaccine protection effects on certain extent,thus having greater research potential and advantages.Therefore,we chosed recombinant adeno-associated virus(r AAV)that can continuously and stably express ASF antigen protein in the body as a viral vector vaccine candidate of ASF.The main research contents are described as follows:1.Construction of ASFV single antigen r AAV vector vaccine candidateHEK293 cells were co-transfected with r AAV’s three-plasmid system,and r AAV virus packaging was performed to obtain antigen gene expressing recombinant adeno-associated virus including r AAV-B646L-HA,EP153R-HA,r AAV-B119L-HA,r AAV-O61R-HA,r AAV-CP204L-HA and r AAV-E183L-HA.2.ASFV single antigen r AAV vector vaccine candidate immunization and immunogenicity evaluationThe mice in the immunized group were intramuscularly injected with virus mixture(total dose was 1.2x10~7CFU,with the single virus 2x10~6CFU,respectively)on day 0 and day 21;the control group mice are inoculated with1.2x10~7CFU of r AAV-GFP virus.At 14,21,35 and 50 days post immunization,blood was collected from the tail vein,then serum was separated for ELISA to determine the antigen-specific antibodies.The result of the ELISA showed B646L,CP204L and E183L-encoded antigen-specific antibodies can be detected in all immunized mice;cellular immunity estimation by q RT-PCR and FACs showed that compared with the control mice,the immunized mice:(1)splenic lymphocytes was significantly increased(P<0.01),(2)cytokines expression including IFN-α,IL-6 and IFN-γincreased significantly(P<0.05),(3)CD4~+T cells in splenic lymphocytes increased extremely significantly(P<0.01),CD8~+T cells showed an increasing trend,but was statiscally insignificant(P>0.05).3.Construction of ASFV multi-antigen r AAV vector vaccine candidater AAV virus packaging was performed to obtain antigen gene expressing recombinant adeno-associated virus r AAV-ASFV-1(B646L,EP153R,B119L,O61R),r AAV-ASFV-2(EP402R,E183L)and r AAV-ASFV-3(A141R,A151R,B438L,B602L,K205R).4.ASFV multi-antigen r AAV vector vaccine immunization and immunogenicity evaluationThe mice in the immunized group were injected intramuscularly with the virus mixture on day 0 and day 21(total dose was 6x10~6CFU,with the single virus2x10~6CFU,respectively));mice in the control group were inoculated with 6x10~6CFU of r AAV-GFP virus.At 14,21,35,and 56 days after immunization,blood was collected from the tail vein,serum was separated and ELISA was performed to determine antigen-specific antibodies.The specific antigen encoded by B646L,EP153R,B119L,O61R,E183L,A151R and B602L can be detected in all mice of immunization group;cellular immunity estimation by q RT-PCR and FACs showed that compared with the control mice,the immunized mice(1)splenic lymphocytes was significantly increased(P<0.05),(2)the expression of cytokines including IFN-α,The expression levels of IL-6 and IFN-γshowed an upward trend,but was statiscally insignificant(P>0.05).(3)CD4~+T cells in splenic lymphocytes increased extremely significantly(P<0.01),CD8~+T cells showed an increasing trend,but was statiscally insignificant(P>0.05).In this study,we successfully constructed single-antigen and multi-antigen r AAV vector vaccines candidate which could express 13 genes of ASFV B646L,EP153R,B119L,O61R,EP402R,E183L.The production of antigen-specific antibody was detected in the serum of mice immunized with the two vector vaccines,and the number of CD4~+T cells in spleen lymphocytes was increased.The expression of IFN-α,IL-6 and IFN-γcytokines in spleen cells of mice immunized with single antigen r AAV vector vaccine increased.