Preparation Of Cross-reactive Monoclonal Antibodies Of Bovine Origin Against Foot-and-mouth Disease Virus Type O And A And Preliminary Identification Of The Recognized Epitopes

Foot-and-mouth disease(FMD)is a highly contagious animal disease caused by foot-and-mouth disease virus,which mainly affects cloven-hoofed animals,such as cattle,sheep and pigs,and has a significant impact on international trade related to livestock and their products.There are seven serotypes of foot-and-mouth disease virus(O,A,C,Asia 1,SAT 1 ~ 3).Each serotype contains multiple topotypes with geographical characteristics,and each topotype can be divided into multiple virus lineages.The prevalence of FMDV type O in China is rather complex with at least 4 lineages among the3 topotypes of the viruses circulated,which is currently the most difficult FMDV serotype to control.FMDV type A is the serotype with the greatest variation in antigen structure.Although cross-protection are absent among different serotypes of FMDV,there are historical data indicate that the cross-protection of other serotype FMDV can be produced by O?A and Asia 1 FMDV sequentially vaccination.This indicated that conserved antigenic sites exist among different serotypes of FMDV.Whether these antigenic sites can induce broadly neutralizing antibody(bn Ab)responses needs further study.In this study,three lineages of FMDV type O virus strains and one type A strain were used to sequentially vaccinate cattle.Antigenic specific single B cells were isolated from the collected peripheral blood by flow cytometry using Fluo Probes 647 H and Pacific Blue labeled 146 S particles of FMDV type O and type A as bait antigens.The complete antibody expression plasmid was constructed and transfected into 293 F suspension cells for expression of monoclonal antibodies(mAbs).The biological activity of antibodies was identified by indirect immunofluorescent assay(IFA),enzyme-linked immunosorbent assay(ELISA),western blot(WB)and virus neutralization test(VNT).In this study,a total of 56 bovine IgG mAbs were expressed.After identification by ELISA and IFA,forty-two mAbs were FMDV-specific,of which 14 mAbs were FMDV type O and type A cross-reactive antibodies and the remaining 28 mAbs are FMDV monospecific antibodies.Seven of 42 mAbs bind the linear epitopes of type O and A antigens indicated by WB,the rest were determined to recognize conformational epitopes.Twenty-one of 56 mAbs show virus neutralizing activity,twenty of them can neutralize A/GDMM/CHA/2013,four of 20 mAbs can neutralize A/WH/CHA/09 contemporary;Three of 21 mAbs can neutralize type O epidemic strains,but only one mAb(R50)shows characteristics of bnAb,which can neutralize different lineages of FMDV type O and type A.Through the IMGT database,the IGHV(D)J germline gene of R50 mAb was mapped and found that this bnAb contained an ultra-long HCDR3 and was confirmed to use germline IGHV 1-7*02 and IGLV 1-47*01/1-52*01 gene fragments in the VH and VL sequences,respectively.In summary,using different fluorescence to label FMDV type O and type A 146 S particles,15 FMDV type O and A cross-reactive antibodies were obtained by a single B cell antibody technique including one bnAb against different lineages of FMDV type O and type A.This study lays a good foundation for analysis of conserved antigen structure among different FMDV serotypes.