| BackgroundSince the emergence of human cases of H7N9 influenza in early 2013,a total of 1568 confirmed cases of H7N9 influenza infection and 615 deaths have been reported in China,with a morbidity and mortality rate of 39%.Although mandatory vaccination of farmed poultry has been effective in stopping human infections,the H7N9 influenza virus continues to mutate and sporadic cases of human infections have emerged from time to time.There is still no therapeutic drug for H7N9 influenza virus,and neutralizing antibodies have the potential to be an effective treatment for H7N9 influenza virus.Therefore,screening for neutralizing antibodies against H7N9 influenza virus is important for the prevention and treatment of influenza infections.ObjectiveObjective to screen neutralizing antibodies targeting HA protein in H7N9 influenza virus convalescent patients,explore the function and recognition sites of neutralizing antibodies,and find candidate antibodies for H7N9 influenza prevention and treatment,so as to provide reference for the design of H7N9 influenza virus vaccine.MethodIn this study,peripheral blood was collected from recovered patients infected with H7N9 influenza virus,and memory B cells were sorted from peripheral blood mononuclear cells by magnetic bead sorting method for in vitro culture.An enzyme-linked immunosorbent assay was used to detect the binding activity of the antibody supernatant induced by B-cell culture to the HA protein of H7N9 influenza virus.B cells with binding activity were lysed,total cellular RNA was reverse transcribed into c DNA,the variable region genes of the antibody heavy and light chains were amplified by PCR using c DNA as a template,cloned into the corresponding antibody heavy and light chain expression vectors,and the antibody was expressed in 293 F cells.The binding activity and affinity of the antibody to HA protein of H7N9 influenza virus,as well as the neutralization activity and hemagglutination inhibition activity of the antibody against H7N9 influenza virus were detected.The virus escape strain of the antibody was screened in chicken embryos,and the binding ability of the virus escape strain of the antibody to the antibody was detected by immunofluorescence,and the recognition epitope of the antibody was verified by competitive ELISA assay.Results(1)In this study,two monoclonal antibodies 70A3 and 70C3,which are specific antibodies against the HA protein head of H7N9 influenza virus,were successfully isolated,and both of them possess hemagglutination inhibition activity and neutralization activity;(2)the binding activity and neutralization activity of the antibodies were verified,and it was found that antibody 70A3 recognizes the highly conserved amino acid site S158 on the Ca1 epitope of H7N9 influenza virus HA1,while antibody 70C3 recognizes the three medium to highly conserved amino acid sites S132,G133 and S134 on the Ca2 epitope of H7N9 influenza virus HA1;(3)competed with published H7N9 influenza virus-specific neutralizing antibodies and found that antibody 70A3 recognizes the L144 site on the 150 loop of the RBS region of the HA head of H7N9 influenza virus,while antibody 70C3 recognizes three sites W142,D148 and N149 on the 150 loop of the RBS region of the HA head of H7N9 influenza virus.ConclusionsThe antibodies 70A3 and 70C3 screened in this study are specific neutralizing antibodies against the structural domain of the HA head of H7N9 influenza virus;antibodies70A3 and 70C3 recognize immunodominant epitopes in HA proteins,and attention to the changes of these sites can provide a reference for the evolution and mutation of H7N9 influenza virus. |
