Rapid Isolation Of Human Neutralizing Antibodies Against H7N9 Avian Influenza Virus And Antiviral Mechanisms Research

H7N9 avian influenza virus?H7N9 AIV?is a influenza A virus that can be transmitted to humans by infected poultries.Human infection with H7N9 avian influenza is an acute respiratory infectious disease,which usually causes severe pneumonia and acute respiratory distress syndrome?ARDS?with a case fatality rate of 40%.There is still a lack of effective drugs and vaccines.Currently,the emergence of highly pathogenic H7N9 avian influenza virus has posed a serious threat to poultry farming,animal husbandry and human health,emphasizing that new and effective therapeutic approaches in both the human and animal health sectors are crucial.To solve the problem,human monoclonal antibodies?HmAbs?are being developed as alternatives to prevent and treat influenza viruses due to their significant effects on neutralizing avian influenza viruses and promoting killing of infected cells.In the present study,we have mastered single memory B cell PCR technology and established the microneutralization assay.Two neutralizing human antibody clones named 3L11 and 5J13 have been rapidly isolated from the switched memory B cells of a patient infected with H7N9 in two weeks,and their antiviral mechanisms have been investigated.The antibody clone 3L11 was encoded by a rare lineage carrying VH1-8*01/D2-15*01/JH5*02 and VL2-13*01/JL3*02 gene segments that had undergone somatic mutations of 8.87%and 5.55%,respectively,and conferred high affinity binding to H7N9 hemagglutinins?HAs?,with KD values of 5.32×10^-9M.We have established the CHO cell lines stably expressing the antibody 3L11.The yield and purity were 250.12mg/L and 99%,respectively.3L11 could broadly neutralize H7N9 viruses,including A/Anhui/1/2013?AH1?,A/Shanghai/2/2013?SH2?,A/Shenzhen/Th004/2017?SZ4?,A/Shanghai/1/2013?SH1?.It promoted killing of infected cells by antibody-dependent cell-mediated cytotoxicity?ADCC?.Epitope mapping by mass spectroscopy?MS?indicated that 3L11 bound to the peptide149–175 of HAs that contained the 150-loop of the receptor-binding site?RBS?.Additionally,the 3L11 escape strains had G151R?Gly151?Arg151?and S152P?Ser152?Pro152?mutations within a conserved antigenic site A near the RBS that were not observed in field strains.Importantly,3L11 fully protected mice against a lethal H7N9 virus challenge,in both pre-and postexposure administration regimens.The antibody clone 5J13 was encoded by VH3-30*03/D2-2*01/JH6*02 and V?4-1*01/J?4*01 gene segments that had undergone somatic mutations of 15.25%and 4.42%,respectively,and conferred high affinity binding to H7N9 hemagglutinins?HAs?,with KD values of 1.95×10^-10M.We also established the CHO cell lines stably expressing the antibody 5J13.The yield and purity were 84.48mg/L and 99%,respectively.5J13 could neutralize H7N9 viruses by inhibiting hemagglutination and HA-mediated membrane fusion activity in vitro,and in vivo 5J13 fully protected mice against a lethal H7N9 virus challenge,in both pre-and postexposure administration regimens.Importantly,we found that 5J13 targeted a unique epitope named antigenic site J which was located opposite to RBS on the head of H7N9 HA by epitope mappings using hydrogen deuterium exchange mass spectrometry?HDX-MS?,sequencing of escape mutants and competition binding analysis.The E89K(Glu⁸⁹?Lys⁸⁹)mutation within antigenic site J resulted in loss of binding for HA to the 5J13 antibody.Altogether,this work demonstrates the feasibility of rapid isolation of neutralizing H7N9 antibodies from infected patients,expands the potential antibody repertoire against influenza A virus and provides two potential prophylactic and therapeutic agents against H7N9 viruses.In addition,we have defined the protective mechanisms of two human antibodies and discovered a novel epitope that could help the potential development of influenza vaccines.