Isolation, Functional Study And Unique Structural Discovery Of Full Human Neutralizing Antibodies Against Group2 Influenza Viruses

Influenza is a severe disease caused by influenza viruses and has been a major threat to human health,causing serious economic damage.About 5%-15% of the world's population is infected with seasonal influenza viruses every year,of which 250,000-500,000 people die.Because of the limitations of small molecule drugs and seasonal vaccines,it is urgent to seek new treatment and prevention methods.The paratopes of influenza antibodies are mainly classified into three types.Type I is represented by FI6v3,CR6261,F10,CR9114 and 39.29,which recognize the fusion subdomain of HA.CR6261,F10 only neutralize Group 1 flu viruses while FI6v3,39.29 and CR9114 neutralize both Group1 and Group2 viruses.The epitope of Type II is closer to the membrane-binding site of HA,including the ? sheet before Helix A sequence and the fusion peptide on HA2,which can only neutralize Group 2 influenza virus,represented by CR8020.In addition,MEDI8852 and 3E1 possessed the epitope of both type I and type II.MEDI8852 can broadly neutralize all Group viruses,while 3E1 can only neutralize Group1 viruses.However,we found that MEDI8852 could not bind to certain H3N2 HA,which suggested that more broad-spectrum and better neutralization of influenza antibodies against H3N2 is still the main research direction.In addition,we are unable to clearly distinguish the similarities and differences of the recognition patterns between Group1 and Group2 through the above research.Does Group2 viruses have specific epitopes different from Group1 viruses? The discovery of new epitopes contributes a lot to the development of universal vaccines.To further clarify the above problems,we initiated this project to develop a Group2 influenza antibody and find unique epitopes.In this study,we successfully isolated a Group2 biased neutralizing antibody,12 mAb,from a volunteer vaccinated with trivalent seasonal influenza vaccine.12 mAb could bind all subtypes of Group2(H3/H4/H7/H10/H14/H15).12 mAb could protect mice against H3N2.Mechanism study showed that 12 mAb could inhibit the low pH induced conformational change of HA,thus inhibiting viral invasion into host cells.In order to understand the detailed interaction mechanism between antigen and antibody,we analyzed the 12 mAb Fab-H3N2 HA complex by cryo-electron microscopy.The results showed that 12 mAb Fab target the HA stem region of H3N2,including the C-terminus of Helix A and the N-terminus of loop region between Helix A and Helix B which has never been reported before.12 mAb bound to H3N2 HA through both the heavy and light chain to form a triangular scaffold.We believed that this binding mode is more stable which indicates that 12 mAb has a high affinity for H3N2 HA.In the following study,we found that the binding and neutralizing activity of 12 mAb to H3N2 subtypes prevalent in different years was better than MEDI8852,especially for H3N2 influenza viruses prevalent in recent years,which was consistent with the cryo-EM data.This provided a new and better candidate drug for H3N2 viruses.Cryo-EM showed that the binding epitope of 12 mAb to H3N2 HA includes the Nterminus of loop region between Helix A and Helix B which is a linearized sequence on HA.In order to further prove the existence of this linear epitope,we carried out ELISA and Western Blot experiments.The results showed that 12 mAb could bind to linearized H3N2 HA protein,but not for MEDI8852 and CR8020.Next,we mapped the linear epitope of 12 mAb,which located on 58-75 amino acid of H3N2 HA2.To further explore whether the epitope of 12 mAb to H1N1 HA is different from or the same as H3N2 HA,we also performed structural analysis of the 12 mAb FabH1N1 HA complex.12 mAb Fab mainly targeted at Helix A of H1N1 HA,which was similar to the epitopes of antibodies such as FI6v3 and CR6261,but significantly different from the recognition epitope of H3N2 HA by 12 mAb Fab.There have been no reports of influenza antibodies that have different epitopes between different HA subtypes,which represents a new class of antibody-antigen interaction patterns.In brief,we identified a Group2 specific epitope that was different from Group1.The discovery of new epitopes shed light for the design of influenza vaccines,and suggested that the design of broad-spectrum influenza vaccines should consider not only the conserved sequences of HA in all subtypes,but also the differences in HA of different subtypes.