Preliminary Study On Broad-spectrum Monoclonal Antibodies Targeting Influenza A Virus

Influenza A virus(IAV),a highly contagious respiratory pathogen,poses a major threat to global public health due to the lack of effective control methods.Hemagglutinin(HA)and Neuraminidase(NA)are important glycoproteins located on the surface of the capsule of influenza A virus.According to the different antigens of HA and NA,there are 18 subtypes of IVA HA and 11 subtypes of NA.HA is mainly responsible for binding with host cell sialic acid residues and achieving the fusion of virus and cell membrane,and is the main target of viral neutralization antibodies.NA has sialase activity,which is responsible for hydrolyzing sialic acid residues at the end of cell surface to promote the release of new viruses,and is also an important target antigen for influenza therapy.In this study,phage surface display technology was used to screen broad-spectrum antibodies against the surface proteins HA and NA(N1subtype)of influenza A virus,and functional evaluation was conducted.The study mainly includes the following two aspects:1.Functional evaluation of broad-spectrum antibodies targeting the HA protein of influenza A virusBased on the previous work of our research group,a single-chain antibody fragment(sc Fv)FHA3with multi-subtype HA protein binding activity was obtained by phage library screening technology.According to the sc Fv sequence information,the light and heavy chain variable region of the antibody was amplified by PCR and connected to the expression vector to construct the recombinant plasmid pFRT-Ig G1κ-FHA3.The antibody protein FHA3 was prepared by Expi CHO cells and affinity purification.FHA3 was identified and bound to HA proteins of influenza A viruses(H1N1,H2N2,H3N2,H5N1,H7N9 and H9N2)in a concentration-dependent manner by ELISA.Antibody neutralization experiments showed that FHA3 had good neutralization activity in vitro,and could effectively block the invasion of target cells by H1N1,H5N1 and H7N9 pseudoviruses at low concentrations.Next,we developed a visual model of infection in mice based on H5N1 pseudovirus and used this model to evaluate the protective activity of FHA3 in vivo.In the prophylactic protection experiment,FHA3 showed neutralizing activity in vivo,which could reduce the infection of pseudovirus in mice.In conclusion,FHA3 has good neutralizing activity in vivo and in vitro.2.Screening and identification of antibodies targeting NA protein of influenza A virus N1 subtypeIn this part,we designed an antibody library for N1 subtype NA protein by using the computer molecular simulation technology and the variable region of antibody sequence retrieved as the template.Using Phage antibody library technology,we obtained a single-chain antibody fragment FNA1 with binding activity by using influenza A virus H1N1 NA as the target antigen through "adsorption-elution-infection" cycle screening.The heavy chain and light chain variable region sequences of antibody were synthesized and connected to the antibody mammalian cell expression vector pFRT-Ig G1κ to construct the recombinant expression plasmid.The expression plasmid was transiently transfected into Expi CHO cells.The supernatant of cell culture was collected and the antibody Protein FNA1 from the supernatant was purified by Protein A affinity purification technique.The binding of FNA1 to the NA subtype protein was detected by ELISA,WB and flow cytometry,and it was found that FNA1 specifically recognized the N1 subtype(H1N1,H5N1)NA antigen.ELLA and NA-XTD experiments showed that FNA1 might bind proximal to the active site of NA enzyme to inhibit its sialase activity through steric hindrance.The pseudovirus infection experiment further confirmed that FNA1 could effectively block the release of newly packaged H5N1 pseudovirus particles from the cell surface and inhibit the spread of the pseudovirus in vitro at a low concentration(5 μg/m L).Next,35 sites on H1N1 NA protein were selected for site-specific mutation,and the binding of FNA1 to each NA mutant protein was detected.The results showed that FNA1 lost its binding to 4 mutant proteins mutant15(W219A),mutant21(K254A),mutant31(W358A)and mutant33(S388A)among the 35 mutant proteins.It is suggested that W219,K254,W358 and S388 are the key epitopes for FNA1 to recognize H1N1 NA protein.Thus,we obtained a monoclonal antibody against N1 subtype neuraminidase,and verified the in vitro binding activity and NA inhibitory activity of the antibody.