Development Of Genetically Engineered Monoclonal Antibody From Antigen-Specific Single B Cells Of Cattle Against Foot-and-Mouth Disease Virus Serotype O

Foot-and-mouth disease virus(FMDV)is the pathogen of foot-and-mouth disease affecting pigs,cattle and sheep.Type O is the most prevalent among seven serotypes of FMDV in the world.The epidemic situation of FMDV type O is even more complicated in China with four main lineages within three topotypes introduced and prevalence in recent years,which further increased the difficulty to control this disease.The antigenic variation is obvious among different lineages of FMDV type O indicated by the variable antigenic matching and cross-protection against each other.So,the antigenic variation is one important issue for the development of disease control and prevention products.Single B cell antibody technology is a new method for screening neutralizing mAbs binding to different antigenic sites,which will provide abundant tools for studying the antigenic variation.In this study,peripheral blood mononuclear cells(PBMCs)were isolated from cattle immunized with three strains of FMDV type O to screen single B cells.Single antigen-specific B cell was sorted from the PBMCs by flow cytometry using biotinylated FMDV type O 146 S antigen as a bait.The gene sequences coding for IgG variable regions were amplified from the isolated single B cell by single cell PCR technique.The coding sequences were inserted into the pcDNA3.4 vector containing the constant region genes of the bovine IgG to express the whole antibody molecular.The constructed vectors were transfected into CHO-S suspension cultured cells for antibody expression.The reactivity of the expressed antibodies was verified by VNT,IFA,ELISA,and Western-blot.Finally,69 strains of full cattle mAbs(IgG subtypes)were obtained.The results of ELISA,IFA,VNT suggested that 55 strains of mAbs were FMDV type O-specific.Among the 55 strains of mAbs,28 strains of mAbs have virus neutralizing activity,and 13 strains of them are intra-type broadly neutralizing antibodies(bnAbs),which can neutralize 4 lineage strains of FMDV type O.Polyclonal serum blocking ELISA suggested that the antigenic sites recognized by these intra-type bnAbs were all immunodominant.Of the 55 strains of FMDV-specific mAbs,only 4 mAbs(E32,E53,F104 and B66)can recognize denatured FMDV capsid proteins,of which 3 strains of non-neutralizing mAbs(E32,E53 and F104)bind to the capsid protein VP2,and the other one neutralizing mAb(B66)binds to the C-terminus of VP1 protein.The germline genes of 55 strains of mAbs were analyzed by searching IMGT database.The results showed that germline IGHV 1-7*02 gene fragment were used in VH gene of all mAbs containing ultralong HCDR3.Comparing with non-neutralizing mAbs,cattle germline IGHV(D)J gene segments including 4 IGHV(1-7*02;1-10*01/2;1-20*01;1-21*01/1-33*01),3 IGHD(6-2*01;7-3*01;5-2*01)and 1 sole IGHJ(IGHJ 2-4*01)were preferentially utilized in these FMDV-neutralizing mAbs.In summary,this study for the first time to establish a technical platform for rapid screening and preparation of full cattle-derived antibodies that can neutralize FMDV serotype O.Total 28 neutralizing mAbs were prepared,which made a good basis for exploring the antigenic variation of FMDV type O.