Screening Equine Influenza Neutralizing Antibodies Based On B Cell Separation Technology

Equine Influenza is abbreviated as EI,which is an acute outbreak of infectious disease caused by the Orthomyxoviridae influenza virus genus Equine Influenza A virus(EIV).This disease spreads quickly and widely,cause immeasurable economic losses to the global horse industry.The inactivated Equine Influenza vaccine(H3N8 subtype,XJ strain)developed by the Harbin Institute of Veterinary Medicine of the Chinese Academy of Agricultural Sciences can protect the immunized horses against the existing epidemic strains of Equine Influenza virus,and the protective effect is lasting for several months.However,the existing vaccines is not able to produce effective cross-protection due to the characteristics of antigen drift of Influenza virus.Therefore,neutralizing antibody,an important immunological and therapeutic drug,have become a development of Influenza prevention and control strategies.In recent years,through effective antibody screening methods,screening neutralizing antibodies that can effectively neutralize Influenza viruses has become the goal of many researchers.Related research results on Equine Influenza in this field is still unknown.Therefore,this study intends to establish a high-efficiency horse-derived antibody screening platform to screen for neutralizing antibodies from peripheral blood lymphocytes(PBMCs)of horses immunized with Equine Influenza inactivated vaccine.On the one hand,passive immunotherapy can be quickly provided during a pandemic;on the other hand,the recognition of neutralizing epitopes of neutralizing antibodies can also promote the development of new vaccines for Equine Influenza.In recent years,screening of monoclonal antibodies(MAbs)based on single B cell amplification technology has gradually emerged.The heavy and light chains of antibodies are natural combinations,with low side effects,rapid production and good specificity.Therefore,this study chose this method as the main technical solution for building a screening platform for Equine Influenza neutralizing antibodies.First,in order to increase the positive rate of identification of recombinant monoclonal antibodies,it is necessary to obtain IgG⁺phenotype B cells,and there is no commercial horse-derived IgG monoclonal antibody.Therefore,we cooperated with Jinan University to prepare horse-derived IgG monoclonal antibodies using the mouse-derived B cell monoclonal antibody screening platform established by Jinan University.First,we constructed the horse-derived IgG eukaryotic expression plasmid and purified this protein,and immunized the mice by the strategy of DNA prime and protein boost.After that,the mouse spleen cells were separated and sorted into IgG⁺single memory B lymphocytes by flow cytometry.By lysing a single memory B lymphocyte,extracting the total RNA of the single B cell,reverse transcription,single-cell PCR and other methods,the heavy chain and light chain variable region gene fragments of the horse-derived IgG monoclonal antibody were amplified,and than they were connected to the antibody constant region expression vector to construct the horse-derived IgG recombinant antibody expression vector.After co-transfection of HEK293F cells,the cell culture supernatant was collected and the horse-derived IgG recombinant antibody was purified.Subsequently,we screen horse-derived IgG recombinant antibodies with the ability to bind horse-derived IgG protein by the method of ELISA test in vitro.The results showed that we obtained 24 pairs of horse-derived IgG recombinant antibody heavy chain and light chain variable region gene linear expression plasmids by fusion PCR from 870CD4^-/CD8^-/B220⁺/Ig D^-/IgG⁺memory B lymphocyte cells.Among them,21 strains of horse-derived IgG recombinant monoclonal antibodies can bind to horse-derived IgG protein.Among them,the horse-derived IgG recombinant antibody G23 with the strongest binding was selected and used in the horse-derived peripheral memory B lymphocyte antibody gene screening platform after avidin-labeled.Subsequently,we used Equine Influenza inactivated vaccine(XJ strain)to immunize 6 horses,and synchronized the separation of peripheral blood mononuclear cells(PBMC)and serum with the corresponding time points.After the serum tested for HI titer,select horses with HA titer>2¹⁰(HA titer>2⁶can produce immune protection)at the late stage of immunization for flow sorting of memory B cells.We obtained memory B cells with Aq VD^-/CD4^-/CD8^-/IgG⁺/CD21⁺/HA⁺.Using specific primers for the heavy and light chains of the antibody for PCR amplification and the construction of the corresponding recombinant antibody expression vector(this part is finished by the partner).After transfection,Protein A purification,and ELISA binding test,we obtained 10 Equine Influenza HA recombinant antibodies(H16?H24?H38?H58?H74?H81?H87?H100?H125?H129)from 10⁸ equine PBMCs.It is evident that the H81 Equine Influenza HA recombinant antibody has good neutralizing activity by the method of surface plasmon resonance(SPR)and microneutralization experiments.In summary,this study successfully prepared horse-derived IgG recombinant antibodies and established a horse-derived single B cell antibody preparation platform with our partner.We obtained a HA antibody with good neutralizing activity using this platform,which can be used for the next step of research.At the same time,this research also provides an efficient research platform for the screening of neutralizing antibodies for other equine-derived diseases.