Fc-dependent protective efficacy of non-inhibitory antibodies targeting influenza A virus neuraminidase is limited by epitope availability

Antibodies targeting hemagglutinin and neuraminidase (NA) are key components of the adaptive immune response against influenza A virus (IAV). However, antigenic drift allows the virus to escape inhibition by such antibodies. In this study, we aimed to isolate antibodies with cross-subtype reactivity against human H1N1 and H3N2 IAVs from transgenic mice bearing genes encoding the human immunoglobulin variable regions. We immunized these mice with recombinant N1 and N2 NA proteins, presenting them either as unconjugated soluble proteins or conjugated to self-assembling protein nanoparticles. This approach yielded a panel of NA-specific monoclonal antibodies (mAbs) with various levels of intra-and inter-subtype reactivity for N1 and N2 NA. Three of these mAbs, which collectively recognize two distinct epitopes, were cross-reactive against N1 and N2 NAs in ELISA, but did not inhibit NA enzymatic activity. Two of these mAbs, 21H8 and 45D9, were selected for further characterization. These recognized different epitopes and induced Fc-mediated effector functions to varying extents. Prophylactic administration of 21H8, but not 45D9, protected mice against challenge with H1N1 IAV, while neither mAb protected against a H3N2 challenge. The observed protective efficacy correlated with the mAbs capacity, or lack thereof, to bind membrane-associated full-length NA. The introduction of Fc silencing mutations in mAb 21H8 resulted in an inability to activate NK cells or mediate phagocytosis in vitro and significantly reduced protection in vivo, indicating that the protective efficacy of mAb 21H8 is Fc-dependent. However, mAb 21H8 expressed with reduced core fucosylation of its Fc N-glycan, which specifically enhanced NK cell activation in vitro, failed to improve protection against H1N1 challenge in vivo. Future work is needed to decipher in more detail the mechanism of Fc-mediated protection against influenza via NA-specific antibodies and to identify the optimal strategies for their enhancement.