The Analysis Of Porcine Antibody Repertoire Following PEDV Infection And Vaccination

Porcine epidemic diarrhea virus is one of the most common diarrhea-causing pathogens in newborn piglets.It is transmitted mainly through the fecal-oral route and primarily infects small intestinal epithelia in vivo.It can cause infectious disease characterized by clinical symptoms including watery diarrhea,vomiting,dehydration,weight loss,high morbidity and mortality in piglets,which causes serious damage to the pork industry.To date,there is no effective drugs available for the therapy of PEDV infection,and vaccination is still the optimal strategy to control PEDV infection.And the major protective mechanism of vaccination is to induce effective humoral immunity,especially the gut local mucosal antibody response against PEDV spike protein which is the main determinant of viral cellular tropism and the main target recognized by neutralizing antibodies.So,to explore deeply and understand antibody response is of importance to effectively prevent PEDV infection.Potent humoral immunity fundamentally depends on the generation of a substantially diverse antibody repertoire,which has gradually become a key factor to evaluate antibody response.However,there are few studies to date about porcine antibody repertoire upon pathogens stimulation except for pre-immune antibody repertoire in fetuses and newborn piglets by traditional cloning and Sanger sequencing.And the information about the total antibody repertoire and the antigen-specific antibody repertoire elicited by PEDV remains unclear.Additionally,with the development of high-throughput sequencing,it is more practical to have a global understanding of such a vast antibody repertoire in healthy hosts or following vaccination or infection.Therefore,the purpose of the study is to characterize PEDV S-specific antibody repertoire and the overall immunoglobulin variable gene usage in pigs following PEDV vaccination or infection by single B cell RT-PCR and high-throughput sequencing.At first,the single lymphocyte suspensions from two Landrace pigs'mesenteric lymph nodes were used to sort PEDV S-specific B cells to clone and express PEDV S-specific antibodies by single cell RT-PCR,and then identified their neutralizing activity and analyzed the variable gene sequence information.Finally,fifty-eight Ig HV genes from 360 single PEDV S-specific Ig G~+cells of pig#242and eighty-five Ig HV genes from 320 single PEDV S-specific Ig G~+cells of pig#243 were successfully cloned.However,there is no highly neutralizing antibody to be cloned and expressed in vitro.The statistical analysis results of PEDV S-specific variable gene usage show that antibody variable genes of PEDV S-specific B cells preferentially adopted Ig HV1-4 for heavy chain,Ig KV1-11 for kappa chain and Ig LV3-3 for lambda chain,up to 37.93%?96.77%and 32.56%,respectively.What is more,the diversity and frequency of variable genes are similar to the total antibody repertoire following PEDV vaccination.Additionally,Next-generation sequencing technology was used to investigate the total antibody repertoire in PBL and MLN following PEDV vaccination,and found that all members of 13 functional porcine Ig HV genes,4 different Ig KV functional families and 5 porcine Ig LV functional families could be expressed in MLN and PBL.But,Ig HV1S2,Ig KV1-11,and Ig LV3-4 are the most prevalent gene segments for heavy,kappa,and lambda chains,respectively,in PBL and MLN.There is no significant difference in the diversity and gene usage of the total antibody repertoire between MLN and PBL.These data indicate that selection of specific B cell clone and the expansion of selected B cell clone occurred,which can be explained by the Clonal Selection Theory.Furthermore,to explore the effect of PEDV acute infection on antibody response in piglets,two 5-day old Landrace SPF piglets were infected with PEDV-JMS.Histopathological analysis of MLN in#3and#4 piglets were performed and antibody repertoire in MLN,spleen and peripheral blood following PEDV infection were analyzed by high-throughput sequencing.Hematoxylin-eosin staining results showed that more nuclei have become pyknotic and have undergone fragmentation and dissolution compared with control piglets.At the same time,the results of one-step TUNEL apoptosis detection further demonstrated that numerous cells were induced apoptosis in MLN after PEDV infection,especially in#4 piglet following PEDV infection 2 days to be dead.The analysis results of antibody repertoire in different immune tissues in piglets following PEDV infection displayed that 13 functional porcine Ig HV genes,two different Ig KV families Ig KV1 and Ig KV2,two porcine Ig LV families Ig LV3and Ig LV8 could be expressed in MLN,spleen and PBL.The more prevalent gene segments for heavy,kappa,and lambda chains are Ig HV1-4 and Ig HV1S2,Ig KV1-11 and Ig KV2-10,Ig LV3-5,respectively.The diversity of antibody repertoire in these immune tissues has no significant difference.However,there are some difference in the frequency of lambda chain gene usage between MLN and the other two tissues.Antibody lambda chain variable gene in MLN is more inclined to use Ig LV3-5,reached 64.04%in#4 MLN.In addition,the analysis results of antibody repertoire in PBL before and after PEDV infection demonstrated that all members of 13 functional porcine Ig HV genes,two different Ig KV families Ig KV1 and Ig KV2,two porcine Ig LV families Ig LV3 and Ig LV8 can be expressed in PBL before and after PEDV infection.So,PEDV infection does not cause the difference in the diversity of antibody repertoire.However,the frequency and preference of gene usage in PBL following PEDV infection have been changed.The frequency of Ig HV1-6 is noticeably decreased in PBL after PEDV infection,up to 39.10%compared with only 5.10%in PBL before PEDV infection.In pre-infection kappa chain variable genes,Ig KV2-10 is the most prevalent gene accounted for 40.30%of the total Ig KV genes.However,Ig KV1-11 is the most prevalent Ig KV gene after PEDV infection,which accounted for54.31%.Moreover,Ig LV3-5 significantly dominated in the total pre-infection Ig KV genes,up to72.34%compared with 23.81%in PBL after PEDV infection.In summary,the study represents a comprehensive analysis to characterize the Ig V-gene segment usage in the overall and PEDV spike-specific antibody repertoire in PBL and MLN following PEDV vaccination as well as the overall antibody repertoire upon PEDV infection or not.The results supplemented and improved the information of porcine antibody repertoire,play a role in the development of veterinary immunology.