Background: Influenza virus remains a continuous and severe threat to public health worldwide, and its prevention and treatment have always been a major international issue. Because of its ability to evade immune surveillance through rapid antigenic drift and antigenic shift, broad-spectrum vaccines seem increasingly important.

Methods: A mAb named 3C12 from an immortalized hybrid cell was generated via immunizing mice with HA2 protein from A/chicken/Anhui/BRI99/2016 (AH/BRI99/16, H9N2) generated by prokaryotic expression. Then, its broad-spectrum activity was analyzed by WB and IFA. Next, the minimal linear epitope was identified via analyzing the reaction of a series of HA truncations with 3C12. Finally, the protective effects of 3C12 were evaluated in vitro and in vivo infection experiments.

Results: The mAb could react with the viruses of subtypes H1, H2, H5, H8, H9, H12, H13, H16, and HA protein of H18 in group 1, but failed to react with viruses in group 2. The minimal linear epitope targeted by the mAb was 433NAELLVL439 in full length of HA and localized in the C-helix region of HA2 (residue 95-101, HA2 numbering). What´s more, the mAb 3C12 inhibited H1, H2, H5, H8, H9, H12, H13 and H16 virus-replication in vitro and also has shown effectiveness in preventing and treating disease in mice challenged with lethal dose of AH/BRI99/16 (H9N2) virus in vivo. These results suggested that the broadly reactive anti-HA stem mAb 3C12 exhibited prophylactic and therapeutic efficacy.

Conclusions: Here, we have demonstrated that the linear epitope identified in this study could be a novel target for developing broad-spectrum influenza diagnostics or vaccine design, and the HA2-based monoclonal antibody is indeed a promising strategy for broad-spectrum protection against seasonal and pandemic influenza viruses.