Screening And Evaluation Of Humoral Immunity-Associated Antigens And Epitopes Of African Swine Fever Virus

African swine fever(ASF)is an highly contagious animal infectious disease which is caused by African swine fever virus(ASFV)infected pigs with acute,severe and hemorrhagic symptoms.ASF does great harm to the world pig industry because of its short course,strong infectivity,wide range of infection,high morbidity and mortality.Hence,it is of great significance to study and develop a safe and effective ASFV vaccine for the Prevention and control of ASF.However,there is no safe and effective commercial ASFV vaccine available so far.Humoral immunity,one of essential component of adaptive immunity,which is particularly crucial to exert immune protection against ASFV vaccine.Though the ASFV antigens found in previous studies can make the body produce specific antibodies,it cannot provide complete immune protection and heterologous protection for pigs,which is due to our incomplete understanding of the main immunogen of ASFV and unclear positioning of the protective antigen.In this study,we studied the key antigens involved in humoral immunity during ASFV infection and their role and molecular mechanism in stimulating the immune system,through high-throughput phage screening,preparation of monoclonal antibodies and synthetic peptides and other technical methods.We carried out a series of work including the epitope screening and verification of ASFV humoral immunity-related antigens and the preliminary design and application of epitope peptide recombinant proteins,in response to the unclear information on the key antigens and epitopes of ASFV humoral immunity.(1)We analyzed the antigen types which is corresponding to the specific antibodies produced in ASF-positive serum by phage peptide library screening and high-throughput sequencing of amplified fragments.we obtained a total of 166 ASFV antigens,including 34 structural proteins,55 non-structural proteins,and 77 unknown functional proteins.A total of 5,540 antigen epitopes were identified.It was shown that the epitopes belonged to linear epitopes by displaying some of the screened antigenic epitopes on the protein structure.Western blot validation was performed on some of the screened ASFV antigens,which showed good reactivity with ASF-positive serum.(2)In order to obtain more epitope information,we obtained 19 strains of monoclonal antibodies that can specifically recognize ASFV d UTPase through monoclonal antibody preparation technology,targeting the key ASFV antigen p E165R(ASFV d UTPase)screened in the experiment 1.Through the analysis of the main antigenic regions,we determined that the main antigens of ASFV d UTPase were located at 100-120 aa,120-140 aa and 140-165 aa regions respectively.And we obtained 8F9 m Ab,a strain of monoclonal antibody,that can not only significantly inhibit the catalytic activity of ASFV d UTPase but also has no cross-reaction with porcine d UTPase.It was identified that 8F9 m Ab antigen recognition epitope region is located at 149–RGEGRFGSTG–158 aa which is 100% conserved in all ASFV strains.We elucidated the key amino acid sites(Glu151,Arg153)of 8F9 which is the key amino acid site of 8F9 specifically binding to ASFV d UTPase.(3)The BCE,CTL,MHC and other immune epitopes of antigens,the ASFV partial humoral immune-related antigen obtained from the previous screening,were identified through immunoinformatics methods.And key antigenic epitopes were identified by combining epitope information form high-throughput screening and immunoinformatics analysis.Antigenic epitopes were verified by Dot blot reactogenicity,stimulated cells in vitro and conjugated bovine serum albumin(BSA)for immune evaluation in mice.A total of 43 epitopes that specifically reacted with ASF-positive sera were screened out of the 45 synthesized epitopes which is key epitopes identified in the previous step.In the peptide conjugate carrier immunological assay,p72-EP-1 could enhance the proportion of CD4+ cells significantly in the mouse spleen lymphocytes,while p37-EP inhibit it.p37-EP,p72-EP-2,and p15-EP could enhance the proportion of CD8+cells in the mouse spleen significantly.Immunization that induced by p72-EP-1,p72-EP-2,p15-EP,and p37-EP was biased towards Th1-type cell immunity,while that induced by p72(423-641 aa),p150(64-190 aa),p150-EP,p15,p37,and EP153R-EP was biased towards Th2-type humoral immunity in mouse.The gene transcription level of IL-4,IL-6,IL-12,IFN-α6,IFN-β and IFN-γ was significantly increased in the p37-EP and EP153R-EP immune group.Transcription level of IFN-α6 was significantly enhanced by p72-EP-1.In addition,p72-EP-2,p150-EP,p37-EP,EP153R-EP increase gene transcription level of IL-4 significantly,p37-EP elevates that’s of IL-6,p37-EP and p150-EP increase that’s of IL-12,p72、p72-EP-2、p37-EP and EP153R-EP increase that’s of IFN-α,p37-EP increase that’s of IFN-β,while p150-EP and p37-EP increase that’s of IFN-γ.(4)Using Rosetta protein design software,we designed the ASFV recombinant epitope peptide(REP-23)which based on the ASFV epitope information which through screening verification.And structural prediction showed that REP monomer structure is similar to a "clover".And gel filtration chromatography shows that REP-23 could form a trimeric structure.In addition,the results show that REP-23 had no significant cytotoxicity,and the monoclonal antibody corresponding to epitope and ASF positive serum had good reactogenicity.Moreover,REP-23 could enhance the transcription levels of multiple cytokines by incubating with porcine-derived PBMCs in vitro.To sum up,in this study we obtained antigens and epitope information of ASFV during its humoral immune process.Afterwards,we analyzed and evaluated the immunological response characteristics of some ASFV antigens and their epitopes.Finally,based on the obtained antigen and its epitope information,we tried to develop ASFV recombinant epitope peptide protein,and initially evaluated the recombinant epitope peptide protein.This study not only provides key antigen information for the development of ASFV subunit vaccines,but also provides new ideas for the development of new epitope vaccines.