Development Of Neutralizing Antibody Against H1N1 Influenza Virus By Culturing Human Memory B Cells In Vitro

BackgroundIn the global health and world economic development process,influenza virus infection still gives a series of threats to our life.The annual outbreak will res?lt in approximately 3-5 million patients admitted to hospital.Antiviral drugs are the main treatment at this stage.But because of its existence of drug tolerance,short treatment window and other issues,the urgent need for other means to more effective treatment of influenza virus infection.Antibody therapy is a hotspot in recent years.Through effective antibody screening,screening for antibodies that can effectively neutralize influenza viruses has become the goal of many scientists.ObjectiveAn antibody with neutralizing H1N1 influenza virus was screened from the volunteers'blood of the immunized influenza vaccine by using the memory B cell culture experiment platform.The antibody was screened by in vitro binding activity,neutralizing activity.It is potential for the treatment of influenza patientsMethodsBased on the memory B cell culture platform,PBMCs were isolated from the blood of volunteers who was recently vaccine.CD19~+CD27~+memory B cells were isolated and the cells were culturing in the medium containing IL-2,IL-21 and 293T-mCD40L trophoblast cells for 13 days.The positive cells were screened by ELISA.The antibody gene was obtained by cleavage,reverse transcription and single cell PCR on the positive cell lines,and constructed and expressed on the IgG antibody expression vector.The expression of the antibody was detected by ELISA,Western-Blot experiment,SPR assay,HAI experiment and so on.ResultsWe successfully established a platform for memory B cell culture and screened 16antibodies with binding activity.Among them,7 strains had neutralizing activity(monoclonal antibodies Hb-B3,B5,C6,E7,A8,A9,C9 had strong neutralizing activity).Moreover,Hb-B5 has broad-spectrum binding activity to Group 1 and 2 HA(H1,H3,H5,H7,H10).Hb-A9 can neutralize group1(H1N1,H5N1,H5N6)and group2(H7N9)influenza virus.ConclusionsMonoclonal antibodies Hb-B3,B5,C6,E7,A8,A9 and C9 have strong in vitro viral neutralizing activity.On the one hand,Hb3-B5 has activity to bind different groups of HA protein and it can be developed to be a diagnostic reagent.On the other hand,Hb-A9 has activity to neutralize Group 1 and 2 influenza virus.So this antibody can be used as potential antibodies for the next step experiments,and applied to clinical treatment.