Broad neuraminidase antibodies confer protection against seasonal and avian influenza viruses

Abstract

Neuraminidase (NA) is a critical target for universal influenza vaccines and therapeutic antibodies, yet its antigenic landscape remains incompletely understood. Here we identify two broadly cross-protective monoclonal antibodies, CAV-F6 and CAV-F34, from influenza-infected individuals. These antibodies inhibit NA enzymatic activity across multiple subtypes and confer protection against seasonal influenza in female mouse models. Importantly, the two antibodies also neutralize emerging avian strains, including recent bovine H5N1 and H7N9 strains, both with pandemic potential. Structural studies reveal that both antibodies target conserved regions of the NA active site via HCDR3, blocking sialic acid interaction. Furthermore, we observe distinct occupancy for the two antibodies on N2 tetramer, which is likely due to differences in binding affinity. Our findings provide molecular insights into NA-targeted immunity and offer a foundation for developing broadly protective influenza vaccines and therapeutics.

Fig. 1. Identification and characterization of broadly cross-reactive NA-targeting antibodies.

A Schematic representation of the antibodies screening process. B Binding heatmap of mAbs to recombination NAs representative of IAV and IBV strains by ELISA. Colored boxes represent the value of the optical density at 405 nm (OD405). C Phylogenetic tree of IAV and IBV NAs constructed using maximum likelihood analysis of amino acid sequences. Pentagrams indicate antibody-binding subtypes (OD405 over twice the negative control value; red: CAV-F6; yellow: CAV-F34). D Immunoglobulin variable heavy-chain and light-chain gene usage, HCDR3 and LCDR3 sequences of NA mAbs. E Half maximal inhibitory concentration (IC50) values of CAV-F34 and CAV-F6 against NAs via ELLA (orange) and MUNANA (red) assay. F–I Inhibitory activity of CAV-F34 and CAV-F6 against drug-resistant NA mutants in MUNANA assays. Data represents one of three independent experiments, shown as mean ± SD of three technical replicates.

Fig. 2. In vivo protection of CAV-F34 and CAV-F6 against seasonal influenza viruses.

A, B Experimental design for pre-exposure prophylaxis (A) and post-exposure therapy (B). BALB/c mice (6-8 weeks old, female) were intraperitoneally administered 10 mg/kg mAbs either 24 h before challenge (prophylaxis) or 24 h (IBV)/48 h (IAV) post-challenge (therapy). Virus dose was 5 × LD₅₀. Survival was monitored for 14 days. C, D Protection against A/California/04/2009(H1N1): Prophylaxis (C) and therapy (D) with CAV-F34 (green) and CAV-F6 (blue). Yellow: PBS control. Left panels: mean percentage change in body weight relative to baseline. Right panels: survival curves. Five animals were used per group (n = 5), and error bars represent SD. E, F Protection against A/Guizhou/54/1989 (H3N2): Prophylaxis (E) and therapy (F). G, H Protection against B/Colorado/06/2017 (Victoria): Prophylaxis (G) and therapy (H). I, J Protection against MA-B/Florida/4/2006 (Yamagata): Prophylaxis (I) and therapy (J). For (E–J) Data presentation identical to (C–D).

Fig. 3. In vivo protection of CAV-F34 and CAV-F6 against avian influenza viruses.

A, B Experimental design for pre-exposure prophylaxis (A) and post-exposure therapy (B). BALB/c mice (6-8 weeks old, female) were intraperitoneally administered 10 mg/kg (H7N9) or 20 mg/kg (H5N1) mAbs either 24 h before challenge (prophylaxis) or 24 h post-challenge (therapy). Survival was monitored for 14 days. C, D Protection against A/Anhui/1/2013 (H7N9) NIBGR-268: Prophylaxis (C) and therapy (D) with CAV-F34 (green) and CAV-F6 (blue). Yellow: PBS control. Left panels: mean percentage change in body weight relative to baseline. Right panels: survival curves. Five animals were used per group (n = 5), and error bars represent SD. E, F Protection against A/dairy cattle/Texas/24-008749-003-original/2024 (H5N1): Prophylaxis (E) and therapy (F). For (E–F) Data presentation identical to (C–D).

Fig. 4. Cryo-EM structures of the NA-antibody complexes.

A Overall structures of the complexes of NA tetramers and Fabs. Calcium ions (red spheres) and glycans (green) are shown. B Overall structures of Fab-NA protomers. Both CAV-F34 and CAV-F6 Fab utilize HCDR3 to approach into the enzymatic site. C Binding epitopes on the NAs. Footprints of CAV-F34 and CAV-F6 Fabs on the NAs are represented by red surface. The interacted CDRs and FR loops are shown. For all the panels, A/California/04-CIP100_RGCM_1868/2009 (H1N1) NA tetramer, A/Switzerland/9715293/2013 (H3N2) NA tetramer, A/Red knot/Delaware Bay/310/2016 (H10N4) NA tetramer, and A/dairy cow/Minnesota/24_016288-003/2024 (H5N1) NA tetramer are colored by light purple, cyan, cornflower blue and dark violet, respectively. CAV-F34 and CAV-F6 are colored as orange and pink, respectively. Abbreviations: variable domain of heavy chain (VH); variable domain of kappa chain (VK); complementarity-determining region (CDR); framework region (FR). D–H Detailed interactions between the NA enzymatic site and HCDRs. Salt bridges and hydrogen bonds are depicted by solid lines. I–M Detailed interactions between sialic acid (SA) and the NA enzymatic site of A/Tanzania/205/2010 (H3N2) (PDB ID: 4GZQ) (I), A/Tanzania/205/2010 (H3N2) NA and oseltamivir (OSE) (PDB ID: 4GZP) (J), A/Tanzania/205/2010 (H3N2) NA and FNI9 (PDB ID: 4G3N) (K), A/California/04/2009 (H1N1) NA and 1G01 (PDB ID: 6Q23) (L) and A/Kansas/14/2017 (H3N2) NA and 4N2C402 (PDB ID: 8ZR4) (M). Salt bridges and hydrogen bonds are depicted by solid lines, and other interactions within 4 Å are represented by dashed lines. For all the panels, A/Tanzania/205/2010 N2 NA, A/California/04/2009 (H1N1) NA and A/Kansas/14/2017 (H3N2) NA are colored by dark blue, light purple and salmon, respectively. SA, OSE, FNI9, 1G01 and 4N2C402 are colored as yellow, lime, dark green, grey and brown, respectively. N, Sequence logo plot illustrating amino acid conservation of a portion of NA sequences from H1N1 and H5N1 IAVs. O, Sequence logo plot illustrating amino acid conservation of a portion of NA sequences from human seasonal H3N2 IAVs.

Fig. 5. CAV-F6 R54S mutation reduces binding affinity and consequentially diminishes inhibition potency.

A, B Two-dimensional classification and three-dimensional models of the A/Switzerland/9715293/2013 (H3N2) in complex with CAV-F6 (A) and CAV-F34 (B). C Comparison of key amino acid sites between CAV-F34 and CAV-F6 binding to the A/Switzerland/9715293/2013 (H3N2) NA tetramer. Hydrogen bonds and salt bridge interactions formed between NA and CAV-F6 are marked with blue triangles, while those with CAV-F34 are marked with orange inverted triangles. Amino acid residues that form plenty of contacts with NA are marked with black circles. D The IC₅₀ value of enzyme inhibitory ability of CAV-F6 and its single point mutant including R54S, D102N and W105L against A/Switzerland/9715293/2013 (H3N2) NA protein via ELLA and MUNANA assay. E–G Binding affinity of CAV-F34, CAV-F6 and CAV-F6 R54S with A/Switzerland/9715293/2013 (H3N2) NA via BLI assay. H, I Detailed interactions between the wild-type CAV-F6-R54 residue (H) and mutated CAV-F6-R54S residue (I) with the A/Switzerland/9715293/2013 (H3N2) NA protomer. Salt bridges and hydrogen bonds are depicted by solid lines. J Superimposition of the CAV-F6-R54S fab and the unbounded NA protomer of the A/Switzerland/9715293/2013 (H3N2) NA/CAV-F6-R54S complex displayed an obvious steric hindrance. K Superimposition of the CAV-F6-R54S fab of the A/Switzerland/9715293/2013 (H3N2) NA/CAV-F6-R54S complex and the protomer of apo A/Switzerland/9715293/2013 (H3N2) NA tetramer also displayed a similar steric hindrance. L Fold change in IC50 values for CAV-F6-R54S and CAV-F6 in NAI of A/Switzerland/9715293/2013 (H3N2) NA with different mutations. M–Q Inhibitory activity of CAV-F6-R54S and CAV-F6 against N2 NA carrying K199R, K199A, K199D, K199E mutations in MUNANA assay. Data represents one of three independent experiments, shown as mean ± SD of three technical replicates.