Study On Specific Immune Responses Induced By Fusion Proteins Of African Swine Fever Virus Antigens

African swine fever(ASF)is an acute and severe animal infectious disease caused by African swine fever virus(ASFV),with a fatality rate of up to 100% in domestic pigs for highly pathogenic strains.The disease was introduced into China in 2018 and has not been effectively controlled,posing a serious threat to our country’s pig industry.A safe and effective vaccine is urgently needed for the prevention and control of the disease.Due to the complex structure of ASFV,traditional inactivated vaccines cannot induce immune protection,and commercial ASF vaccines have not yet been developed.As a safe,effective and controllable vaccine type,subunit vaccines have been widely used and are valuable in the development of ASF vaccines.However,insufficient immunogenicity and protective efficacy of ASF subunit vaccines is a key problem to be solved in research.Bacterial lipoprotein Opr I,as a natural intramolecular adjuvant,can significantly enhance the immune responses induced by subunit vaccines and improve the protective efficacy of vaccines,which may be of great significance for improving the immune efficacy of ASF subunit vaccines.In this study,Opr I and a universal foreign T cell epitope(TT-P2)were introduced into the design of fusion proteins of ASFV antigens,and three fusion proteins of ASFV antigens containing Opr I and TT-P2 were constructed,namely Opr I-p30-modified p54-TT-P2(OPMT),Opr I-p72-△p E248R-TT-P2(OPET),Opr I-△ CD2v-△ p EP153R-TT-P2(OCET)and a Opr I-fused ASFV antigens:Opr I-p30-modified p54(OPM).Nine fusion proteins of ASFV antigens without Opr I were also constructed,namely p30-modified p54(PM),p72-△p E248R(PE),CD2v-△p EP153R(CE),p30,modified p54,p72,△p E248 R,△CD2v and △p EP153 R.After protein expression and purification,the immunostimulatory activity,immunogenicity and immune effects of combined recombinant proteins were evaluated.The results of recombinant protein expression and purification showed that 13 recombinant proteins were effectively expressed in E.coli.All recombinant proteins were obtained by Ni chelation affinity chromatography,which could be specifically recognized by anti-ASFV positive serum in Western-blotting identification,indicating good immunoreactivity.The results of immunostimulatory activity assay showed that the Opr I-ASFV antigen fusion proteins(OPM,OPMT,OPET and OCET)significantly increased the expression of CD40,CD80,CD86 and MHC-II on BMDCs and the secretion of cytokines after incubation with BMDCs,promoting the activation of BMDCs.In the experiment of immunogenicity evaluation,the specific antibody levels,lymphocyte proliferation and activation,and the neutralizing activity against ASFV of immune serum from mice immunized with OPM,OPMT,OPET and OCET were significantly higher than other recombinant proteins.Vaccination of mice with 1:1:2 of OPMT,OPET and OCET induced a more comprehensive specific antibody responses,high level of lymphocytes proliferation and activation after stimulation with inactivated ASFV,and a higher ASFV neutralization ratio of the immune serum.Immunization of the mixed antigens with ISA206 adjuvant(O-Ags-T “cocktail” vaccine)increased antibody levels against p72,p E248 R,CD2v and p EP153 R,but the immune sera exhibited no significant promotion of neutralization rate against ASFV.The experiment of immunization of pigs with O-Ags-T “cocktail” vaccine showed that the vaccine induced high levels of ASFV-specific antibody,and peripheral blood mononuclear cells(PBMCs),spleen lymphocytes,and cells isolated from lymph node showed good proliferation and activation ability after stimulation with inactivated ASFV.The neutralization rate of immune sera(42 dpv)at 1:5dilution against ASFV was about 80%,and the inhibition rate of ASFV infection in vitro by PBMCs(42dpv)from immunized pigs was more than 90%.Taken together,the results of this study show that the O-Ags-T “cocktail” vaccine induces potent anti-ASFV immune responses,which lays a foundation for the vaccine to carry out the challenge experiment,and provides a reference for the development of ASF subunit vaccines.