Potent efficacy of a NA-targeting antibody against a broad spectrum of H5N1 influenza viruses

Potent efficacy of a NA-targeting antibody against a broad spectrum of H5N1 influenza viruses | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Potent efficacy of a NA-targeting antibody against a broad spectrum of H5N1 influenza viruses Matteo Samuele Pizzuto, Saya Moriyama, Julia di Iulio, Fabrizia Zatta, and 21 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5999144/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Mar, 2026 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract For nearly 30 years, Goose/Guangdong (Gs/Gd)-derived highly pathogenic avian influenza (HPAI) H5N1 viruses have posed significant risks to economic stability, food security, and public health. Virus evolution has resulted in various clades, including the panzootic subclade 2.3.4.4b, recognized for its pandemic potential. Here we present the potent in vitro activity of FNI9, a pan-influenza NA-inhibiting monoclonal antibody, against a range of pseudoparticles with NA spanning 27 years of Gs/Gd-derived H5N1 virus evolution. FNI9 also shows strong prophylactic protection in mice against lethal challenges with H5N1 from clade 1 and 2.3.4.4b. Cryo-EM and molecular dynamics analysis reveal that FNI9 binds to 7 highly conserved H5N1 NA residues (R118, E119, D151, E228, E278, R293, and R368). In silico evolutionary escape profiling and machine learning indicate low escapability, high fitness costs, and minimal spread likelihood for viral mutations that evade FNI9 binding. These findings support FNI9 broad protection and underscore the NA role in future influenza vaccine design. Biological sciences/Immunology/Infectious diseases/Influenza virus Health sciences/Diseases/Infectious diseases/Influenza virus Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Since 1997, highly pathogenic avian influenza (HPAI) A/Goose/Guangdong (Gs/Gd)-derived H5N1 viruses have continued to spread globally causing huge economic losses in the poultry industry, representing a food security problem in low-income countries, and a constant pandemic threat 1 , 2 . The HA of Gs/Gd H5N1 viruses has evolved extensively via reassortment into 10 phylogenetic clades (clade 0 to 9) and multiple sub-clades 3 . Clade 2 viruses in particular have been under scrutiny due to the alarming numbers of outbreaks in birds 4 and sporadic human infections with a cumulative case fatality rate of more than 50% 5 . Besides respiratory symptoms, common clinical signs reported for HPAI H5N1 viruses in mammals and in birds arResultse neurological (e.g. encephalitis) 6 , as the virus can reach the central nervous system via the olfactory bulb 7 , 8 . Since 2020–2021, HPAI H5N1 clade 2.3.4.4b has spread to an unprecedented global scale in the animal reservoir and is considered panzootic 6 , 9 . Additionally concerning is the evidence of transmission amongst mammals living in close proximity 10 – 12 as well as the detection of mammal-adaptive mutations such as E627K and D701N in polymerase basic protein 2 (PB2) observed in recent isolates 12 , 13 . Furthermore, several cases of human infection by H5N1 clade 2.3.4.4b have been reported in various countries, although so far with no evidence of human-to-human transmission 14 – 16 . Oseltamivir treatment is recommended for outpatients and hospitalized patients who have suspected, probable, or confirmed infection by HPAI viruses. However, reduced sensitivity and emergence of resistance to this NA-inhibitor has been documented during treatment of H5N1 infections 17 , 18 . Despite the multitude of universal influenza vaccine candidates proposed in the past decades, only a handful of them have advanced beyond animal models, with mixed results 19 – 21 . In response to high circulation of HPAI H5N1 virus from clade 1 in 2003, a vaccine derived from A/Vietnam/1203/2004 was manufactured and shown to be safe and immunogenic in humans 22 . However, while the HA antigen and adjuvant in the vaccine formulations were shown to maintain their functional integrity and to generate antibody responses following stockpiling for several years 23 , virus drifting and reassortment might result in the same mismatch problem and ensuing reduced activity experienced by seasonal influenza vaccines 24 . Furthermore, limited immunogenicity and waning of the vaccine-elicited immune response could limit effectiveness in high-risk individuals. Broadly neutralizing monoclonal antibodies (bnAbs) offer an off-the-shelf tool for immediate deployment to protect high risk individuals, and healthcare personnel, thus potentially complementing vaccines in a pandemic setting. Here, we report the potent in vitro and in vivo activity of the pan-influenza NA-targeting mAb FNI9 25 against Gs/Gd-derived H5N1 influenza viruses including the panzootic subclade 2.3.4.4b. Results FNI9 displays superior NAI activity to oseltamivir and peramivir against H5N1 clade 2.3.4.4b. The neuraminidase inhibition activity (NAI) of FNI9 across a panel of pseudoparticles bearing NAs representative of different H5N1 clades ( Fig. 1, S1, S2 and Table S1 ) was compared with NA-targeting antivirals oseltamivir and peramivir in the enzyme-linked lectin assay (ELLA) 26 . Conversely to the MUNANA assay that measures only the NAI activity derived from direct engagement of the enzymatic pocket, ELLA uses a more physiological substrate and can also measure the contribution of steric hindrance exerted by the mAb. Indeed, previous publications have shown how NA-targeting mAbs can inhibit sialidase activity by steric hindrance without directly engaging the enzymatic pocket 27 . FNI9 displayed similar NAI activity compared to oseltamivir and peramivir against the ancestral clade 0 and across clade 1 viruses, consistent with the use of N1 from H5N1 A/Vietnam/1203/2004 (clade 1) for the original isolation of the anti-NA mAb 25 . FNI9 also demonstrated equivalent or superior NAI activity compared to the antivirals against pseudoparticles bearing NAs from Egyptian human-like (HL) H5N1 clade 2.2.1.2, which is a clade of particular concern due to HA mutations that enhance virus affinity to the human-type 2,6-sialic acid (SA) receptors 28 , thereby increasing replication in human cells 29 . Similarly, potent inhibitory activity by FNI9 was observed against NA from H5N1 clade 2.3.2.1c, which is responsible for at least eleven human deaths in Southeast Asia within the last years 14 . Remarkably, FNI9 displayed consistent superior NAI activity compared to oseltamivir and peramivir across pseudoparticles representative of the panzootic subclade 2.3.4.4b, including the H5N1 viruses associated to the recent outbreak in dairy cattle and human infections in the United States 14 , 15 , 30 . Of note, FNI9 retained NAI activity against N1 bearing the S247N oseltamivir-resistant mutation 31 . Overall, these results indicate that FNI9 is highly active across the entire Gs/Gd-derived family of H5N1 viruses, spanning 27 years of virus evolution and including mutants that are resistant to frontline therapy. Structural basis for FNI9 broad activity across H5N1 viruses To elucidate the epitope of FNI9 on N1 and the structural basis for the broad NAI activity, we determined a cryo-EM structure of FNI9 Fab in complex with NA from H5N1 A/Vietnam/1203/2004 (clade 1) at 2.4 Å resolution ( Fig. 2A ). The FNI9–NA complex adopts a C4 symmetry, with each of the four NA protomers in the tetrameric quaternary complex bound to one Fab. FNI9 adopts a mode of binding to N1 similar to the one previously described with N2 25 , inserting its HCDR3 into the enzymatic pocket. R106 in HCDR3 forms a bidentate bridge with E228 and D151 and it is within 5 Å of E278 and E119; D107 in HCDR3 forms a tridentate cruciform interaction with R118, R293, and R368 ( Fig. 2B ). To evaluate potential differences in the interaction between FNI9 and the NA of more contemporary H5N1 viruses, the sequence of the N1 of A/Texas/37/2024 belonging to the panzootic clade 2.3.4.4b was overlayed with the structure obtained from N1 A/Vietnam/1203/2004 ( Fig. 2C ). None of the residues that differ between the two NAs are within 9 Å of the FNI9 paratope ( Fig. S3A ) and therefore are not expected to impact FNI9 binding to NA, in line with the retained activity of the mAb against contemporary H5N1 strains ( Fig. 1 ). To obtain a more detailed understanding of the binding epitope of FNI9, we performed molecular dynamics (MD) simulations of the FNI9–N1/Vietnam/1203/2004 complex ( Fig. S3B, C ). MD revealed additional sites that participate in antibody binding by accounting for molecular motion. Even with a broader definition of epitope residues to include those which appear as contacts during the MD simulation, designated the “dynamic” epitope, none of the epitope residues differ in the N1/Texas/37/2024 strain ( Fig. 2C and Fig. S3B, C ). Since energies derived from charge-charge interactions may be overestimated when utilizing implicit solvent-based energy calculations 32 , the contact energies across the dynamic epitope residues were normalized by the fraction of the time the contacts were present in the MD simulation and the results were designated as J score (higher score = stronger contact). The normalization ranked seventeen major contacts with J score > 0.3 including seven key contacts whose score was greater than 1.5: R118, E119, D151, E228, E278, R293, and R368 ( Fig. 3A ). The seven key contacting residues are highly conserved on the N1 across all the different Gs/Gd H5N1 clades and subclades isolated from avian, human and other mammals since 1997 ( Fig. 3A and Fig. S4 ). To predict the likelihood of emergence of FNI9-resistant H5N1 variants, we assessed the escape potential of mutations in N1 at the major FNI9 contact positions using two different in silico approaches, which were then overlaid with previously published experimental deep mutational scanning (DMS) data 25 . The first approach, called EVEscape 33 , associates a high likelihood of escapability to a mutation that is fit, accessible, and in which the new amino acid is dissimilar from the reference amino acid. The model was validated by the significantly higher EVEscape scoring of known N1 escaping mutations (from either H5N1 or H1N1) 34 in comparison to other amino acid mutations ( Fig. S5 ). The analysis revealed that mutations in the key contacting residues have low escape potential, as none of them display an EVEscape score within the top 5th percentile ( Fig. 3B ). The second in silico approach was developed in-house to further assess whether previously experimentally identified escapes (in a H1N1 background) 25 were likely to spread in the long-term. This novel approach accommodates ranking scores alongside structural and biophysical features, using a machine learning meta-model trained on historical data. Rather than predicting escape directly, the model predicts probability of spread, building on previous work from our group 35 . Spreading probabilities can then be intersected with escape mutations obtained experimentally from DMS 25 analysis. Expanding this analysis to surface exposed sites beyond the epitope, we identify only one FNI9 binding altering mutation (K432P) that has a probability of spread > 1% ( Fig. S6 ). Of note, this mutation requires at least 2 nucleotides change to occur, indicating it would need to pass through an intermediate amino acid. Overall, these data indicate that FNI9 interacts with highly conserved and functionally constrained residues on the N1 protein of H5N1 viruses, thus minimizing the likelihood of emerging viral variants that can evade the anti-NA mAb activity. Prophylactic activity of FNI9 against lethal challenge with H5N1 viruses To dissect the protection against respiratory disease from the neurotropism of highly pathogenic (HP) H5N1 viruses, we first evaluated the prophylactic activity of FNI9 against lethal challenge with a low pathogenic (LP) H5N1 virus (NIBRG-14) in BALB/c mice. NIBRG-14 is a recombinant virus bearing the internal genes from H1N1 PR8 and the HA and NA from A/Vietnam/1194/2004 (clade 1). In addition, the polybasic cleavage site on the HA of the virus was engineered to monobasic with consequent transition from HP to LP phenotype. In this study, FNI9 was administered to the animals as single intravenous injection at four different doses (0.3, 0.9, 3, 9 mg/kg) 24h before lethal intranasal challenge with H5N1 NIBRG-14. The anti-HA stem-directed mAb VIR-2482 derived from Fc engineering of MEDI8852 36 was tested in parallel at two different doses (0.9 and 9 mg/kg) as comparator. The animals were observed for 14 days, and body weight loss and survival were recorded. A single administration of FNI9 at 0.3 mg/kg provided 100% survival in comparison to 12.5% of the vehicle group ( Fig. 4A ). Mice receiving VIR-2482 at 0.9 mg/kg also displayed 100% survival although higher body weight loss was recorded in comparison to those administered with a three times lower dose of FNI9. All the animals treated with higher doses of FNI9 or VIR-2482 were completely protected from morbidity. Of note, body weight loss inversely correlated with the serum mAb concentration measured 2 hours before infection ( Fig. S7 ). Next, we tested the prophylactic activity of FNI9 against a HP H5N1 A/red_fox/Hokkaido/1/2022 virus belonging to clade 2.3.4.4b using different LD50s in two separate studies. In the first study BALB/c mice were administered with FNI9 at 5 and 10 mg/kg 24h before intranasal infection with 2LD50 of HP H5N1 virus ( Fig. 4B ). Five out of eight animals (62.5%) in the vehicle group died in the 14 days following infection, with two succumbed animals displaying limited body weight loss but neurological symptoms such as tremors and loss of balance, suggesting spreading of the infection to the CNS. All the mice treated with FNI9 at 5 mg/kg were protected from morbidity and mortality except for one animal that started to lose body weight at day 11 post-infection and displayed neurological symptoms before dying. Single administration of FNI9 at 10 mg/kg conferred complete protection to all the mice from mortality and morbidity similarly to those receiving VIR-2482 at the same dose. A second prophylactic study with HP H5N1 clade 2.3.4.4b was performed using higher virus challenge (5LD50) ( Fig. 4C ). All the animals in the vehicle group died by day 6 post-infection with different mice presenting modest body weight loss (12–15%) but symptomatology consistent with potential infection of the CNS. Three (3/6) and two (2/6) mice administered with FNI9 at 5 and 10 mg/kg, respectively, had their survival prolonged between 8- or 11-days post-challenge before succumbing to the infection. All the remaining mice in these groups displayed no or minimal body weight loss through 14 days. Animals administered with either FNI9 or VIR-2482 at 20 mg/kg were completely protected from morbidity and mortality during the study. Overall, these data demonstrate that FNI9 provides strong in vivo protection against lethal challenges from H5N1 viruses including the panzootic clade 2.3.4.4b. Discussion Since their emergence in the late 1990s, highly pathogenic Gs/Gd H5N1 viruses have raised global concerns due to their high mortality rate in humans and significant pandemic potential 1 , 2 . Viruses from clade 2 have been particularly concerning, as they show a widespread geographic distribution. This clade has given rise to multiple subclades detected across a diverse array of wild and domestic animals 37 , 38 , and responsible for numerous human infections and fatalities 5 . First isolated in Europe in 2016, H5 viruses from clade 2.3.4.4b re-emerged in 2020 and rapidly spread worldwide becoming panzootic 6 , 9 . Particularly alarming is the detection in U.S. dairy cattle of H5N1 clade 2.3.4.4b viruses with mutations (such as PB2-E627K) that enhance influenza polymerase activity in human cells 39 . Relatedly, highly pathogenic avian influenza (HPAI) H5N1 2.3.4.4b viruses, isolated from the eye of a farm worker infected through dairy cow contact, were lethal in ferrets and transmissible via respiratory droplets leading to animal fatalities 40 . While neuraminidase inhibitors (e.g. oseltamivir) and vaccines can be stockpiled for a pandemic response, long-term pre-exposure prophylaxis may be challenging for the small molecules due to their short half-life 41 , and H5 vaccines could face drawbacks in common with seasonal influenza vaccines, such as mismatch, poor immunogenicity, and immunological imprinting 24 , 42 , 43 . Broadly neutralizing anti-hemagglutinin (HA) stem mAbs have previously been shown to protect animals against influenza A viruses, including HP H5 strains 44 , 45 . However, this class of mAbs display a trade-off between their wide breadth and the modest neutralization potency. In contrast, mAbs engaging the highly conserved NA enzymatic pocket can combine wide breadth of coverage, encompassing in different cases both influenza A and B viruses, and high in vitro and in vivo potency 25 , 46 , 47 . In addition, the NA evolves independently from the HA 48 – 50 and it is poorly represented in current vaccines, suggesting that anti-NA mAbs could expand the protection elicited by active immunoprophylaxis 51 . Here, we report the potent NAI activity of the recently described anti-NA mAb FNI9 against a broad panel of NA-based pseudotypes spanning over 27 years of Gs/Gd-derived H5N1 virus evolution. Remarkably, FNI9 exhibited superior potency than NA antivirals oseltamivir and peramivir against NAs from H5N1 clade 2.3.4.4b viruses. In addition, FNI9 provided potent in vivo protection against highly pathogenic avian influenza H5N1 viruses from the panzootic clade and its prophylactic activity against these viruses is currently under evaluation in non-human primates. Overall, FNI9 demonstrates strong in vitro and in vivo activity against H5N1 viruses including the panzootic clade 2.3.4.4b. The structural analysis presented in this study with N1 from H5N1 revealed that FNI9 interacts with 7 highly conserved key residues on the NA enzymatic pocket (R118, E119, D151, E228, E278, R293, and R368) mimicking the sialic acid receptor and NA-targeting antivirals similar to our previous observations of the mAb in complex with N2 25 . Our in-silico analysis of NA conservation reveals that the key contact sites are functionally constrained, thus anticipating a high barrier for the emergence of FNI9 escape variants. In conclusion, our data support the concept that "off-the-shelf" mAbs such as FNI9 could be developed to complement vaccination and to provide a critical layer of protection to healthcare workers and farmers facing high exposure risks during human and animal outbreaks, and to high-risk populations such as the elderly and immunocompromised. Methods Production of NA-based pseudoparticles NA-only based particles (NA-PVs) were produced transfecting Lenti-X 293T cells (Takara) in 6-well plates (Corning). Cells were co-transfected using X-tremeGENE HP (Roche) with 0.1 µg of NA-expressing plasmid and with 1.6 µg of a complementing viral-genome reporter vector, pNL4-3.Luc+.E-R-. A 1:3 DNA/X-tremeGENE HP ratio was used and the transfection was performed following the manufacturer’s protocol. Transfected cells were incubated for 72 hours, then the supernatant was harvested and stored at -80°C into aliquots. The pseudoparticles used in this study were generated with NAs from A/HongKong/156/1997, A/Vietnam/CL01/2004, A/Vietnam/1203/2004, A/Thailand/NBL1/2006, A/Cambodia/X0207301/2013, A/Indonesia/NIHRD/15023/2015, A/Egypt/N04915/2014, A/Nepal/19FL1997/2019, A/Victoria/149/2024, A/Cambodia/NPH230032/2023, A/Vietnam/KhanhhoaRV1-005/2024, A/CommonBuzzard/GermanyHH/2024AI01435/2024, A/bottlenose dolphin/Florida/UFTt2203/2022, A/Mink/Spain/3691-8_22VIR10586-10/2022, A/Chile/25945/2023, A/Texas/37/2024, A/Ezo red fox/Hokkaido/1/2023, A/Michigan/90/2024, A/California/135/2024, A/California/168/2024, A/Washington/240/2024. Production of recombinant NA protein N1 A/Vietnam/1203/2004 protein (residues 63–449 from GenBank ID: AAT73329.1) containing an N-terminal IgK light chain secretion sequence, a 6xHis-Tag, AviTag, a Tetrabrachion tetramerization domain and a Thrombin protease site was expressed in Expi293F cells at 37°C and 8% CO 2 . Cell culture supernatant was collected four days post transfection and protein was purified using Nickel-NTA agarose (Qiagen) followed by size exclusion chromatography using a Superdex 200 10/300 GL Increase column equilibrated in 50 mM Tris, 150 mM NaCl, 10 mM CaCl2, pH 8.0. Enzyme-linked lectin assay (ELLA) 96-well half area ELISA plates (Corning) were coated overnight at 4°C with 25 µg/ml of Fetuin. Plates were blocked with a 1% w/v solution of Bovine Serum Albumin (BSA; Sigma-Aldrich) in PBS containing Ca 2+ Mg 2+ (Gibco) and incubated for 1 hour at room temperature. In the meantime, mAbs were serially diluted and mixed with a fixed amount of recombinant NA-PVs and the mix was transferred to fetuin-coated plates, previously washed. After an overnight incubation at 37°C, the plates were washed and 1 µg/ml lectin from peanut-agglutinin (PNA, Sigma-Aldrich) horseradish peroxidase labelled (HRP) was added and incubated at room temperature for 1 hour. After further washing, SureBlue Reserve TMB 1-Component (KPL) was added, and plates were read at 450 nm after blocking the reaction with 1% hydrochloric acid solution. Cryo-EM sample preparation N1 NA protein antigen from H5N1 A/Vietnam/1203/2004 was mixed with a 1.2-fold molar excess of FNI9 Fab and incubated on ice for one hour. The complex was diluted to 0.3 mg/mL in Tris-HCl pH 8.0, 150 mM NaCl, 10 mM CaCl 2 buffer. DDM at 0.70xCMC was added to the complex right before vitrification. 3 µL of 0.3 mg/ml of NA-Fab complex with DDM were loaded onto a freshly glow discharged 1.2/1.3 UltrAuFoil grid prior to plunge freezing using a Vitrobot MarkIV (ThermoFisher Scientific) with a blot force of 0 and 3.5 s blot time at 100% humidity and 4°C. Cryo-EM data collection Data were acquired on a 300kV FEI Titan Krios TEM equipped with a Gatan K3 Summit direct detector and Gatan Quantum GIF energy filter, operated in zero-loss mode with a slit width of 20 eV. Automated data collection was carried out using SerialEM at a nominal magnification of 105,000x and pixel size of 0.8189 Å. The dose rate was adjusted to 16 counts/pixel/s with a total dose of 47.7 (e − /A 2 ), and each movie was fractionated in 79 frames of 25ms each. A total of 3060 movies were collected with a defocus range comprised between − 0.8 and − 2 µm. A summary of data collection is provided in Extended Data Table 2. Cryo-EM data processing Dose-weighted movie frame alignment to account for stage drift and beam-induced particle motion was performed in Relion 52 . The contrast transfer function is estimated for each micrograph using Gctf 53 . Individual particles are selected first using Gautomatch (Zhang) and extracted into particle stacks in Relion. Extracted particles were subjected to 2D classification. Initial maps were generated ab initio from selected 2D classes and used for 3D classification of particles from selected 2D classes. The best 3D classes were submitted to 3D refinement with C4 for 4-Fab bound NA) symmetry applied. A soft mask was applied to postprocess the unsharpened map. After refinement, particles were subject to CTF refinement and bayesian polishing, re-refined as above, and postprocessed to produce the final map. Reported resolutions are based on the gold standard FSC = 0.143 criterion. A summary of cryo-EM data statistics is provided in Extended Data Table 2. Cryo-EM model building and analysis The atomic model of N1 NA from Influenza A virus strain A/Vietnam/1203/2004 H5N1 (PDB: 2HU0) and FNI9 Fab from N2 neuraminidase of A/Tanzania/205/2010 H3N2 in complex with 4 FNI9 Fab molecules (PDB: 8G3N) was used as an initial model to fit into cryo-EM maps by UCSF Chimera. Coot was used to manually rebuild amino acid mutations and glycans on NAs and build the Fab 54 . Models were refined using Rosetta and Servalcat 55 , 56 . Model validation and map-model FSC were generated by Validation: model tool in ccpem v1.6.0. Figures were generated using Pymol 57 . Epitopes in all NA complexes were identified by determining NA residues within 5.0 Å of any heteroatom in FNI9 Fabs using MOE (v2020.0901, https://www.chemcomp.com ). MOE contacts for the static structure were generated for the FNI9:N1 complex after having run QuickPrep. Glycan profiling of neuraminidase with peptide mapping LC-MS Peptide mapping with liquid chromatography-mass spectrometry (LC-MS) was used to profile the site-specific glycosylation sites on neuraminidases (H5N1 A/Vietnam/1203/2004). Glycopeptides containing only one specific glycan were achieved by selectively digesting with trypsin, Glu-C, Lys-C, or Asp-N protease, depending on the sequence context. 25 µg of each digest product (peptide with a single glycan) was analysed by LC-MS (Agilent AdvanceBio peptide mapping column and Thermo Q Exactive Plus Orbitrap MS). Peptide mapping data were analyzed on Biopharma Finder 3.2 data analysis software. Technical replicates were performed by injecting 25 µg of digested product three times from the same sample vial into the LC-MS. Molecular dynamics methods The coordinates of FNI9:NA (N1 A/Vietnam/1203/2004) were obtained from cryo-EM (see above) with glycans determined by peptide mapping LC-MS (see above) modelled using GLYCAM ( http://glycam.org ), based on an early iteration of the published structure. The glycosylated FNI9:NA complex was prepared using QuickPrep (MOE v2020.0901, https://www.chemcomp.com ). The FNI9:NA tetrameric complex structure was parameterized as previously described using tleap 58 : ff14SB for protein 59 , GLYCAM_06j-1 for glycans 60 , TIP3P for water 61 , Joung & Cheatham for ions 62 . We generated 4.0 µs of aggregate MD from 5 independent 0.8 µs MD simulations of FNI9:N1 seeded with unique initial velocities and were minimized and equilibrated as previously described 63 . With only experimentally resolved atoms restrained, minimize for 10,000 steps, heat to 300 K in NVT, 100 ps of NPT MD at 300 K. With the same atoms restrained but a 10-fold reduced force constant, 250 ps of NPT MD at 300 K. With only the experimentally resolved backbone atoms, 10,000 steps of minimization, 100 ps of NPT MD at 300K; 100 ps of NPT MD at 300K. Then, the last two stages progressively reduce the restraint force constant by 10-fold for 100 ps NPT MD at 300K. The final restraint force constant was 0.1 kcal/mol Å 2 . The last stage of equilibration was 2.5 ns of unrestrained NPT MD at 300 K. Production MD continued dynamics (NPT, 300 K, velocities) from the last stage of equilibration for an additional 0.8 µs of unrestrained MD sampling. This entire procedure was repeated five times for the 5 independent FNI9:N1 MD simulations. MD trajectory files were stripped of water and ions using cpptraj 64 as previously described 63 . FNI9:N1 interactions were defined using the protein contact analysis in MOE (CCG MOE 2020.09). The analysis classifies contacts as hydrogen bond, metal, ionic, and arene interactions based on participating atom types and their relative geometries; and uses bonded and non-bonded force field energies and implicit solvent solvation energies to compute per-residue interaction energies (within 5 Å). Contacts were calculated using MD frames sampled every 10 ns. QuickPrep was performed for each snapshot prior to evaluation. MOE contact energies reported above are the average energy of each unique interaction pair normalized by the number of frames in which the contact appears. Number of contacts (# Contacts) counted the number of unique residues in FNI9 that formed a contact with an epitope residue in N1 during the MD simulation. Percent energy (% energy) normalizes the MOE contact energies (see above) by summing the energies across the epitope, dividing the energy of each epitope residue by that sum, and multiplying by 100. Contact energy renormalization by the fraction of the time the contacts were present in the MD was computed by multiplying the percent energy scores by the sum of the fraction occupancy divided by the number of contacts (multiply percent energy by 0.75 if there were four contacts and the sum of their fraction occupancy was 3.0). The value obtained was defined as J score . A cutoff of 0.3 was derived by weighing its ability to separate strong and significant contacts (energy, persistence, contact order) from weaker ones. Percent solvent exposure was calculated using MOE by dividing solvent exposure of NA residues within the structure by the solvent exposure that the same amino acid would have in a Gly-X-Gly peptide. In vivo studies BALB/c mice were purchased from Japan SLC. Clade 1 (NIBRG-14) and clade 2.3.3.4b (A/Ezo red fox/Hokkaido/1/2022) viruses were obtained from NIBSC and Dr. Yoshihiro Sakoda at Hokkaido University, respectively. FNI9, VIR-2482, or PBS control were administered intravenously into mice at doses described in figure legends. On the next day, plasma samples were collected to quantify transferred IgG in the mouse plasma, and the mice were challenged intranasally with 2 or 5 mouse lethal dose 50 (LD 50 ) in 50µL. Body weight and CNS disease behavior were monitored daily from day 4 post-infection until day 14, and the body weight loss compared to 2 days before infection was calculated. Mouse with more than 25% of body weight loss was humanely euthanized. Animal procedures were approved by the Animal Ethics Committee of the NIID, and animal experiments were performed at SPF ABSL2 and ABSL3 animal facilities at the NIID. Sequence conservation analysis H5N1 NA protein sequences (up to January 23, 2025) were retrieved from GISAID EpiFlu project (ww.gisaid.org). Protein sequences were aligned using MAFFT 65 [parameters: --mapout --thread 1 --auto --op 4.5 --keeplength –-addfragments] to a modified version of the N1 of A/Vietnam/1203/2004 strain, that introduced 20 amino acids at residues 49–68 to match the nomenclature of the H1N1 Cal09 strain. The logo plots were generated with R package ( https://www.R-project.org/ ) “ggseqlogo” v.0.1 66 . The residue and haplotype conservations were computed with the R package “Biostrings” v.2.58.0 ( https://bioconductor.org/packages/Biostrings ). EVEscape computation The EVEscape scores were computed as described in 33 . Briefly the EVEscape model relies on three components: 1.) EVE fitness, as estimated by a neural network fit on the multiple sequence alignment of the sequence of interest to distantly related sequences, 2.) solvent accessibility and 3.) biochemical dissimilarity. The code from the following repository was used: https://github.com/OATML-Markslab/EVEscape/tree/main . The following non-default parameters were used: theta = 0.01; batch_size = 512; num_training_steps = 20000; num_samples_compute_evol_indices = 20000; computation_mode = all_singles. The multiple sequence alignment used as input was obtained as follows: All N1 protein sequences from H1N1 (up to February 2, 2024) and H5N1 (up to May 8, 2024) were downloaded from GISAID EpiFlu project ( www.gisaid.org ). To retrieve homolog sequences 33 , we downloaded all UniRef100 sequences available as of February 27, 2024 and searched for homologs using the A/Vietnam/1203/2004 strain as input sequence using jackhmmer (HMMER 3.4; http://hmmer.org/ ). We selected homologs with an E value < 0.001. Both sets of sequences were aligned to a modified version of the N1 of A/Vietnam/1203/2004 strain, that introduced 20 amino acids at residues 49–68 to match the nomenclature of the H1N1 Cal09 strain, with MAFFT [parameters: --mapout --thread 1 --auto --op 4.5 --keeplength –-addfragments;]. The protein structure used is the one described in this paper. Spreading Forecast Analysis The machine learning meta-model was trained on historical GISAID flu protein sequences from October 2015 to February 2019 for N1 from H1N1 and N2 from H3N2. Mutations relative to reference strains (A/California/233/2019 [H1N1] and A/Darwin/9/2021 [H3N2]) were measured and prevalence for each mutation was calculated for each month. A mutation was considered highly prevalent if it achieved greater than 10% prevalence for any month within a flu season (October – February in the northern hemisphere), across all years studied. Features for the machine learning meta-model were calculated from ESM-2 65 and ESM-IF 66 , from the same reference sequences and matched PDB structures (6LXK and 7U4G). Likelihood scores for each mutation from each model were used as features in the meta-model. Logistic regression was performed on the features and mutation prevalence targets for the H1N1 and H3N2 mutations together (Python and scikit-learn). The model was evaluated on corresponding data for H5N1. GISAID flu NA protein sequences from October 2021 – February 2023 were collected and mutations were calculated relative to the previously described modified A/Vietnam/1203/2004 N1 strain. Statistical Analysis All statistical tests were performed as described in the indicated figure legends using Prism v9.0. The number of independent experiments performed are indicated in the relevant figure legends. Declarations Competing interests J.d.I., F.Z., K.H, H. E. M., J.M.E., H.V.D., N.C., A.C.,Y.C., A.P., E.V., M.C.M., L.E.R., G.S., D.C, and M.S.P are current or former employees of and may hold shares in Vir Biotechnology Inc. M.S.P., D.C., and G.S. are currently listed as an inventor on multiple patent applications, which disclose the subject matter described in this manuscript. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Acknowledgments We thank Dr. Yoshihiro Sakoda at Hokkaido University for providing A/Ezo red fox/Hokkaido/1/2022 virus. We also thank Eriko Izumiyama, Akira Dosaka, and Ryoko Itami at Research Center for Drug and Vaccine Development, NIID for their technical support as well as Siro Bianchi at VIR Biotechnology for his graphical support. This work was supported by Japan Agency for Medical Research and Development Grant Numbers JP22fk0108141, JP243fa627005, and JP243fa727002 (YT). Author contributions M.S.P., D.C., and Y.T., conceived the research and designed the study. Experiment design: S.M., J.d.I, F.Z., K.H., H.A., H.E.M., J.M.E., Y.A., H.V.D., N.C., E.S., A.C., Y.C., R.K., A.P., E.V., T.O., M.C.M., L.E.R., M.S., G.S., H.H., Y.T., D.C., and M.S.P.; NA-based pseudoparticles production and ELLA testing: F.Z., and M.S.P.; NA antigen design and production: H.V.D., and N.C., Complex formation for cryo-EM: H.V.D., and N.C.; Cryo-EM Data Collection, Processing, and Model Building: J.M.E., and H.V.D.; Glycan analysis and MD analysis: K.H., A.C., and L.E.R.; Bioinformatic epitope conservation and escape prediction: J.d.I., H.E.M., Y.C., M.C.M.; in vivo studies: S.M., H.A., Y.A, E.S., R.K., T.O., M.S., H.H., and Y.T.; Data analysis: S.M., J.d.I, F.Z., K.H., H.A., H.E.M., J.M.E., Y.A., H.V.D., N.C., E.S., A.C., Y.C., R.K., A.P., E.V., T.O., M.C.M., L.E.R., M.S., H.H., Y.T., D.C., and M.S.P.; Supervision: S.M., J.d.I., K.H., N.C., A.P., M.C.M., L.E.R., G.S., Y.T., D.C., and M.S.P.; Y.T., D.C., and M.S.P. wrote the manuscript with input from all the authors. 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Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Nadine","middleName":"","lastName":"Czudnochowski","suffix":""},{"id":418099285,"identity":"c391f517-8eb3-4d0b-9c0b-536cebe990ad","order_by":11,"name":"Eita Sasaki","email":"","orcid":"https://orcid.org/0000-0002-3384-7414","institution":"National Institute of Infectious Diseases","correspondingAuthor":false,"prefix":"","firstName":"Eita","middleName":"","lastName":"Sasaki","suffix":""},{"id":418099286,"identity":"aefb4e82-f587-44f5-817d-b1b619dc2c33","order_by":12,"name":"Alex Chen","email":"","orcid":"","institution":"Vir Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Alex","middleName":"","lastName":"Chen","suffix":""},{"id":418099287,"identity":"3912f308-209c-492f-8f4b-656ee3cea418","order_by":13,"name":"Yi-Pei Chen","email":"","orcid":"","institution":"Vir Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Yi-Pei","middleName":"","lastName":"Chen","suffix":""},{"id":418099288,"identity":"0eebeea3-aeac-49d3-9adc-3b7a862174e1","order_by":14,"name":"Ryutaro Kotaki","email":"","orcid":"https://orcid.org/0000-0001-7965-1671","institution":"Hokkaido University","correspondingAuthor":false,"prefix":"","firstName":"Ryutaro","middleName":"","lastName":"Kotaki","suffix":""},{"id":418099289,"identity":"a13db556-d683-4aa1-952d-578e1d68fc68","order_by":15,"name":"Alessia Peter","email":"","orcid":"","institution":"Humabs BioMed SA, a Subsidiary of Vir Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Alessia","middleName":"","lastName":"Peter","suffix":""},{"id":418099290,"identity":"8f18dd56-aab2-44b9-9e01-6e2aa7f8b431","order_by":16,"name":"Eneida Vetti","email":"","orcid":"","institution":"Humabs BioMed SA, a Subsidiary of Vir Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Eneida","middleName":"","lastName":"Vetti","suffix":""},{"id":418099291,"identity":"a886b285-945d-449f-9145-c62d29935c40","order_by":17,"name":"Taishi Onodera","email":"","orcid":"","institution":"National Institute of Infectious Diseases","correspondingAuthor":false,"prefix":"","firstName":"Taishi","middleName":"","lastName":"Onodera","suffix":""},{"id":418099292,"identity":"827eef9c-1f7f-44a7-8783-2cdbe8046b16","order_by":18,"name":"M. Cyrus Maher","email":"","orcid":"","institution":"VIR Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"M.","middleName":"Cyrus","lastName":"Maher","suffix":""},{"id":418099293,"identity":"fb3eb684-81d9-43fe-a0a6-e13762b73a13","order_by":19,"name":"Laura Rosen","email":"","orcid":"","institution":"VIR Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Laura","middleName":"","lastName":"Rosen","suffix":""},{"id":418099294,"identity":"4fdb7262-d67f-4f8f-b2eb-effc25c4ca36","order_by":20,"name":"Masayuki Shirakura","email":"","orcid":"","institution":"National Institute of Infectious Diseases","correspondingAuthor":false,"prefix":"","firstName":"Masayuki","middleName":"","lastName":"Shirakura","suffix":""},{"id":418099295,"identity":"56e2be80-d511-4d8d-a5e1-54c66b6b2016","order_by":21,"name":"gyorgy snell","email":"","orcid":"","institution":"Vir Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"gyorgy","middleName":"","lastName":"snell","suffix":""},{"id":418099296,"identity":"e9125d3a-9be0-4290-bb8f-5ac1b33b877d","order_by":22,"name":"Hideki Hasegawa","email":"","orcid":"https://orcid.org/0000-0002-6558-2297","institution":"National Institute of Infectious Diseases","correspondingAuthor":false,"prefix":"","firstName":"Hideki","middleName":"","lastName":"Hasegawa","suffix":""},{"id":418099297,"identity":"5976b48b-c0d7-40ac-a050-e7004b1d2190","order_by":23,"name":"Yoshimasa Takahashi","email":"","orcid":"https://orcid.org/0000-0001-6342-4087","institution":"National Institute of Infcetious Diseases","correspondingAuthor":false,"prefix":"","firstName":"Yoshimasa","middleName":"","lastName":"Takahashi","suffix":""},{"id":418099298,"identity":"7ebc97a5-1cee-4767-9522-1da6aed74cd1","order_by":24,"name":"Davide Corti","email":"","orcid":"https://orcid.org/0000-0002-5797-1364","institution":"Vir Biotechnology (Switzerland)","correspondingAuthor":false,"prefix":"","firstName":"Davide","middleName":"","lastName":"Corti","suffix":""}],"badges":[],"createdAt":"2025-02-10 12:25:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5999144/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5999144/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-026-70036-8","type":"published","date":"2026-03-02T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":76778434,"identity":"3a6841de-5e83-43d0-ac4e-359b2721812a","added_by":"auto","created_at":"2025-02-20 15:47:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":260636,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNAI activity of FNI9 mAb against pseudoparticles bearing NAs from Gs/Gd- derived H5N1 influenza viruses. \u003c/strong\u003eScattered phylogeny tree of NAs from Gs/Gd-derived H5N1 viruses isolated between 1997 and 2025. Viruses selected to generate NA-based pseudoviruses are highlighted by large size dots and the corresponding clade/subclades are indicated based on GISAID (https://gisaid.org) definition. Adapted from Nextstrain: https://nextstrain.org/avian-flu/h5n1/na/all-time?c=gisaid_clade\u0026amp;l=scatter. Inhibition of neuraminidase activity (NAI) IC50s against the selected NA-based pseudoparticles bearing N1 from highly pathogenic H5N1 viruses are reported for FNI9, peramivir, and oseltamivir with colors matching the corresponding clades shown in the scattered phylogeny tree.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-5999144/v1/1c72da0426845229437c0623.png"},{"id":76779026,"identity":"08a8f5db-49ce-41a9-99ae-644047077043","added_by":"auto","created_at":"2025-02-20 15:55:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1159931,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStructure of anti-NA mAb targeting the SA-binding site of H5N1 NA.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA \u003c/strong\u003eThe complex formed by FNI9 Fab (VH: light blue; VL: light green) binding to N1/Vietnam/1203/2004 (grey). Calcium ions appear at the center of the NA tetramer and the NA–Fab interface, shown as red spheres. Three glycans decorate each NA protomer, shown in green sticks (Asn88, Asn146, Asn235). \u003cstrong\u003eB\u003c/strong\u003e Network of salt bridges and hydrogen bond interactions between R106 and D107 (sticks; carbon light blue) in the HCDR3 of FNI9 and R118, E119, D151, E228, E278, R293, and R368 in N1 (sticks; carbon light grey). The backbone H atoms of R106 and D107 were added using PyMol. Distance of key contacts below 5 Å are shown as dotted lines. \u003cstrong\u003eC\u003c/strong\u003eResidue positions in N1/Vietnam/1203/2004 NA sequence that differ from N1/Texas/37/2024 clade 2.3.4.4b are highlighted in red in the FNI9:N1 complex structure.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-5999144/v1/e4cc9f55a01913e77de8c86a.png"},{"id":76778432,"identity":"437050ff-94f0-4be0-99b3-cfd03b684064","added_by":"auto","created_at":"2025-02-20 15:47:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":226617,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConservation and escape potential of major FNI9-contacting NA residues. A \u003c/strong\u003eLogo plot of amino acid conservation of the major FNI9-contacting residues on the NA based on GISAID NA sequences from H5N1 IAVs submitted up to January 23, 2025 (upper panel: N=19,998 from avian host; middle panel: N=642 from human host, lower panel: N=3,489 from other mammal hosts). Amino acids are colored based on contact strength, designated J\u003csub\u003escore\u003c/sub\u003e. Red arrows indicate the 7 key contact residues (R118, E119, D151, E228, E278, R293, and R368) in the H5N1 NA. \u003cstrong\u003eB \u003c/strong\u003eLikelihood of escape mutations in the N1 backbone of the H5N1 A/Vietnam/1203/2004, as measured by EVEscape\u003csup\u003e33\u003c/sup\u003e, for the major FNI9-contacting residues. The higher the score (brown), the higher the probability of escape. The color scale is based on the protein wide EVEscape score distribution, where the white color is set at the 5\u003csup\u003eth\u003c/sup\u003e percentile, indicating that mutations colored as brown have a score within the top 5% of escapability for this protein. Amino acids present in the sequence of the NA of H5N1 A/Vietnam/1203/2004 strain are shown in grey. The residue number is shown at the bottom in bold based on N1 numbering.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-5999144/v1/1e4bcc5c2eacaf07e26a82f4.png"},{"id":76778441,"identity":"7b94c653-3b92-4ccb-8067-c4af12e7ae2e","added_by":"auto","created_at":"2025-02-20 15:47:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":420328,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFNI9 protects mice against lethal challenge with H5N1 viruses. A \u003c/strong\u003ePercentage of body weight loss of BALB/c mice prophylactically administered with FNI9 (n = 8/group) or VIR-2482 (n = 5/group) mAbs 24 h before challenge with 5xLD50 of LP H5N1 NIBRG-14. \u003cstrong\u003eB-C\u003c/strong\u003ePercentage of body weight loss of BALB/c mice prophylactically administered with FNI9 (n = 8/group in B; n = 6/group in C) and VIR-2482 (n = 5/group) mAbs 24 h before challenge with 2xLD50 (B) or 5xLD50 (C) of HP H5N1 A/red_fox/Hokkaido/1/2022. The 0% dotted line indicates baseline body weight loss; the −25% dotted line indicates body weight loss % for euthanasia.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-5999144/v1/964f381669a8188cb34a15bf.png"},{"id":106582949,"identity":"63735d9b-fa61-4125-9e63-5c3ee7f980f9","added_by":"auto","created_at":"2026-04-10 07:05:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3507074,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5999144/v1/862dd749-0c74-47f8-a5b0-031a1e8ddc7b.pdf"},{"id":76779024,"identity":"30b76602-6a42-4205-8357-b9574ac6fc23","added_by":"auto","created_at":"2025-02-20 15:55:07","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3158315,"visible":true,"origin":"","legend":"","description":"","filename":"Supp.Data.docx","url":"https://assets-eu.researchsquare.com/files/rs-5999144/v1/21a57ba61895680247fef2d8.docx"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nJ.d.I., F.Z., K.H, H. E. M., J.M.E., H.V.D., N.C., A.C.,Y.C., A.P., E.V., M.C.M., L.E.R., G.S., D.C, and M.S.P are current or former employees of and may hold shares in Vir Biotechnology Inc. M.S.P., D.C., and G.S. are currently listed as an inventor on multiple patent applications, which disclose the subject matter described in this manuscript. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.","formattedTitle":"Potent efficacy of a NA-targeting antibody against a broad spectrum of H5N1 influenza viruses","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSince 1997, highly pathogenic avian influenza (HPAI) A/Goose/Guangdong (Gs/Gd)-derived H5N1 viruses have continued to spread globally causing huge economic losses in the poultry industry, representing a food security problem in low-income countries, and a constant pandemic threat\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The HA of Gs/Gd H5N1 viruses has evolved extensively via reassortment into 10 phylogenetic clades (clade 0 to 9) and multiple sub-clades\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Clade 2 viruses in particular have been under scrutiny due to the alarming numbers of outbreaks in birds\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e and sporadic human infections with a cumulative case fatality rate of more than 50%\u003csup\u003e5\u003c/sup\u003e. Besides respiratory symptoms, common clinical signs reported for HPAI H5N1 viruses in mammals and in birds arResultse neurological (e.g. encephalitis) \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, as the virus can reach the central nervous system via the olfactory bulb\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eSince 2020\u0026ndash;2021, HPAI H5N1 clade 2.3.4.4b has spread to an unprecedented global scale in the animal reservoir and is considered panzootic\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Additionally concerning is the evidence of transmission amongst mammals living in close proximity\u003csup\u003e\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e as well as the detection of mammal-adaptive mutations such as E627K and D701N in polymerase basic protein 2 (PB2) observed in recent isolates\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Furthermore, several cases of human infection by H5N1 clade 2.3.4.4b have been reported in various countries, although so far with no evidence of human-to-human transmission\u003csup\u003e\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eOseltamivir treatment is recommended for outpatients and hospitalized patients who have suspected, probable, or confirmed infection by HPAI viruses. However, reduced sensitivity and emergence of resistance to this NA-inhibitor has been documented during treatment of H5N1 infections\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Despite the multitude of universal influenza vaccine candidates proposed in the past decades, only a handful of them have advanced beyond animal models, with mixed results\u003csup\u003e\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. In response to high circulation of HPAI H5N1 virus from clade 1 in 2003, a vaccine derived from A/Vietnam/1203/2004 was manufactured and shown to be safe and immunogenic in humans\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. However, while the HA antigen and adjuvant in the vaccine formulations were shown to maintain their functional integrity and to generate antibody responses following stockpiling for several years\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, virus drifting and reassortment might result in the same mismatch problem and ensuing reduced activity experienced by seasonal influenza vaccines\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Furthermore, limited immunogenicity and waning of the vaccine-elicited immune response could limit effectiveness in high-risk individuals.\u003c/p\u003e\u003cp\u003eBroadly neutralizing monoclonal antibodies (bnAbs) offer an off-the-shelf tool for immediate deployment to protect high risk individuals, and healthcare personnel, thus potentially complementing vaccines in a pandemic setting. Here, we report the potent in vitro and in vivo activity of the pan-influenza NA-targeting mAb FNI9\u003csup\u003e25\u003c/sup\u003e against Gs/Gd-derived H5N1 influenza viruses including the panzootic subclade 2.3.4.4b.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eFNI9 displays superior NAI activity to oseltamivir and peramivir against H5N1 clade 2.3.4.4b.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe neuraminidase inhibition activity (NAI) of FNI9 across a panel of pseudoparticles bearing NAs representative of different H5N1 clades (\u003cb\u003eFig.\u0026nbsp;1, S1, S2\u003c/b\u003e and \u003cb\u003eTable S1\u003c/b\u003e) was compared with NA-targeting antivirals oseltamivir and peramivir in the enzyme-linked lectin assay (ELLA) \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Conversely to the MUNANA assay that measures only the NAI activity derived from direct engagement of the enzymatic pocket, ELLA uses a more physiological substrate and can also measure the contribution of steric hindrance exerted by the mAb. Indeed, previous publications have shown how NA-targeting mAbs can inhibit sialidase activity by steric hindrance without directly engaging the enzymatic pocket\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eFNI9 displayed similar NAI activity compared to oseltamivir and peramivir against the ancestral clade 0 and across clade 1 viruses, consistent with the use of N1 from H5N1 A/Vietnam/1203/2004 (clade 1) for the original isolation of the anti-NA mAb\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. FNI9 also demonstrated equivalent or superior NAI activity compared to the antivirals against pseudoparticles bearing NAs from Egyptian human-like (HL) H5N1 clade 2.2.1.2, which is a clade of particular concern due to HA mutations that enhance virus affinity to the human-type 2,6-sialic acid (SA) receptors\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, thereby increasing replication in human cells\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Similarly, potent inhibitory activity by FNI9 was observed against NA from H5N1 clade 2.3.2.1c, which is responsible for at least eleven human deaths in Southeast Asia within the last years\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Remarkably, FNI9 displayed consistent superior NAI activity compared to oseltamivir and peramivir across pseudoparticles representative of the panzootic subclade 2.3.4.4b, including the H5N1 viruses associated to the recent outbreak in dairy cattle and human infections in the United States\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Of note, FNI9 retained NAI activity against N1 bearing the S247N oseltamivir-resistant mutation\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Overall, these results indicate that FNI9 is highly active across the entire Gs/Gd-derived family of H5N1 viruses, spanning 27 years of virus evolution and including mutants that are resistant to frontline therapy.\u003c/p\u003e\u003cp\u003e\u003cb\u003eStructural basis for FNI9 broad activity across H5N1 viruses\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo elucidate the epitope of FNI9 on N1 and the structural basis for the broad NAI activity, we determined a cryo-EM structure of FNI9 Fab in complex with NA from H5N1 A/Vietnam/1203/2004 (clade 1) at 2.4 \u0026Aring; resolution (\u003cb\u003eFig.\u0026nbsp;2A\u003c/b\u003e). The FNI9\u0026ndash;NA complex adopts a C4 symmetry, with each of the four NA protomers in the tetrameric quaternary complex bound to one Fab. FNI9 adopts a mode of binding to N1 similar to the one previously described with N2\u003csup\u003e25\u003c/sup\u003e, inserting its HCDR3 into the enzymatic pocket. R106 in HCDR3 forms a bidentate bridge with E228 and D151 and it is within 5 \u0026Aring; of E278 and E119; D107 in HCDR3 forms a tridentate cruciform interaction with R118, R293, and R368 (\u003cb\u003eFig.\u0026nbsp;2B\u003c/b\u003e). To evaluate potential differences in the interaction between FNI9 and the NA of more contemporary H5N1 viruses, the sequence of the N1 of A/Texas/37/2024 belonging to the panzootic clade 2.3.4.4b was overlayed with the structure obtained from N1 A/Vietnam/1203/2004 (\u003cb\u003eFig.\u0026nbsp;2C\u003c/b\u003e). None of the residues that differ between the two NAs are within 9 \u0026Aring; of the FNI9 paratope (\u003cb\u003eFig. S3A\u003c/b\u003e) and therefore are not expected to impact FNI9 binding to NA, in line with the retained activity of the mAb against contemporary H5N1 strains (\u003cb\u003eFig.\u0026nbsp;1\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eTo obtain a more detailed understanding of the binding epitope of FNI9, we performed molecular dynamics (MD) simulations of the FNI9\u0026ndash;N1/Vietnam/1203/2004 complex (\u003cb\u003eFig. S3B, C\u003c/b\u003e). MD revealed additional sites that participate in antibody binding by accounting for molecular motion. Even with a broader definition of epitope residues to include those which appear as contacts during the MD simulation, designated the \u0026ldquo;dynamic\u0026rdquo; epitope, none of the epitope residues differ in the N1/Texas/37/2024 strain (\u003cb\u003eFig.\u0026nbsp;2C\u003c/b\u003e and \u003cb\u003eFig. S3B, C\u003c/b\u003e). Since energies derived from charge-charge interactions may be overestimated when utilizing implicit solvent-based energy calculations\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, the contact energies across the dynamic epitope residues were normalized by the fraction of the time the contacts were present in the MD simulation and the results were designated as J\u003csub\u003escore\u003c/sub\u003e (higher score\u0026thinsp;=\u0026thinsp;stronger contact). The normalization ranked seventeen major contacts with J\u003csub\u003escore\u003c/sub\u003e \u0026gt; 0.3 including seven key contacts whose score was greater than 1.5: R118, E119, D151, E228, E278, R293, and R368 (\u003cb\u003eFig.\u0026nbsp;3A\u003c/b\u003e). The seven key contacting residues are highly conserved on the N1 across all the different Gs/Gd H5N1 clades and subclades isolated from avian, human and other mammals since 1997 (\u003cb\u003eFig.\u0026nbsp;3A\u003c/b\u003e and \u003cb\u003eFig. S4\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eTo predict the likelihood of emergence of FNI9-resistant H5N1 variants, we assessed the escape potential of mutations in N1 at the major FNI9 contact positions using two different \u003cem\u003ein silico\u003c/em\u003e approaches, which were then overlaid with previously published experimental deep mutational scanning (DMS) data\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe first approach, called EVEscape\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, associates a high likelihood of escapability to a mutation that is fit, accessible, and in which the new amino acid is dissimilar from the reference amino acid. The model was validated by the significantly higher EVEscape scoring of known N1 escaping mutations (from either H5N1 or H1N1)\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e in comparison to other amino acid mutations (\u003cb\u003eFig. S5\u003c/b\u003e). The analysis revealed that mutations in the key contacting residues have low escape potential, as none of them display an EVEscape score within the top 5th percentile (\u003cb\u003eFig.\u0026nbsp;3B\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eThe second \u003cem\u003ein silico\u003c/em\u003e approach was developed in-house to further assess whether previously experimentally identified escapes (in a H1N1 background) \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e were likely to spread in the long-term. This novel approach accommodates ranking scores alongside structural and biophysical features, using a machine learning meta-model trained on historical data. Rather than predicting escape directly, the model predicts probability of spread, building on previous work from our group\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Spreading probabilities can then be intersected with escape mutations obtained experimentally from DMS\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e analysis. Expanding this analysis to surface exposed sites beyond the epitope, we identify only one FNI9 binding altering mutation (K432P) that has a probability of spread\u0026thinsp;\u0026gt;\u0026thinsp;1% (\u003cb\u003eFig. S6\u003c/b\u003e). Of note, this mutation requires at least 2 nucleotides change to occur, indicating it would need to pass through an intermediate amino acid.\u003c/p\u003e\u003cp\u003eOverall, these data indicate that FNI9 interacts with highly conserved and functionally constrained residues on the N1 protein of H5N1 viruses, thus minimizing the likelihood of emerging viral variants that can evade the anti-NA mAb activity.\u003c/p\u003e\u003cp\u003e\u003cb\u003eProphylactic activity of FNI9 against lethal challenge with H5N1 viruses\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo dissect the protection against respiratory disease from the neurotropism of highly pathogenic (HP) H5N1 viruses, we first evaluated the prophylactic activity of FNI9 against lethal challenge with a low pathogenic (LP) H5N1 virus (NIBRG-14) in BALB/c mice. NIBRG-14 is a recombinant virus bearing the internal genes from H1N1 PR8 and the HA and NA from A/Vietnam/1194/2004 (clade 1). In addition, the polybasic cleavage site on the HA of the virus was engineered to monobasic with consequent transition from HP to LP phenotype. In this study, FNI9 was administered to the animals as single intravenous injection at four different doses (0.3, 0.9, 3, 9 mg/kg) 24h before lethal intranasal challenge with H5N1 NIBRG-14. The anti-HA stem-directed mAb VIR-2482 derived from Fc engineering of MEDI8852\u003csup\u003e36\u003c/sup\u003e was tested in parallel at two different doses (0.9 and 9 mg/kg) as comparator. The animals were observed for 14 days, and body weight loss and survival were recorded. A single administration of FNI9 at 0.3 mg/kg provided 100% survival in comparison to 12.5% of the vehicle group (\u003cb\u003eFig.\u0026nbsp;4A\u003c/b\u003e). Mice receiving VIR-2482 at 0.9 mg/kg also displayed 100% survival although higher body weight loss was recorded in comparison to those administered with a three times lower dose of FNI9. All the animals treated with higher doses of FNI9 or VIR-2482 were completely protected from morbidity. Of note, body weight loss inversely correlated with the serum mAb concentration measured 2 hours before infection (\u003cb\u003eFig. S7\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eNext, we tested the prophylactic activity of FNI9 against a HP H5N1 A/red_fox/Hokkaido/1/2022 virus belonging to clade 2.3.4.4b using different LD50s in two separate studies. In the first study BALB/c mice were administered with FNI9 at 5 and 10 mg/kg 24h before intranasal infection with 2LD50 of HP H5N1 virus (\u003cb\u003eFig.\u0026nbsp;4B\u003c/b\u003e). Five out of eight animals (62.5%) in the vehicle group died in the 14 days following infection, with two succumbed animals displaying limited body weight loss but neurological symptoms such as tremors and loss of balance, suggesting spreading of the infection to the CNS. All the mice treated with FNI9 at 5 mg/kg were protected from morbidity and mortality except for one animal that started to lose body weight at day 11 post-infection and displayed neurological symptoms before dying. Single administration of FNI9 at 10 mg/kg conferred complete protection to all the mice from mortality and morbidity similarly to those receiving VIR-2482 at the same dose.\u003c/p\u003e\u003cp\u003eA second prophylactic study with HP H5N1 clade 2.3.4.4b was performed using higher virus challenge (5LD50) (\u003cb\u003eFig.\u0026nbsp;4C\u003c/b\u003e). All the animals in the vehicle group died by day 6 post-infection with different mice presenting modest body weight loss (12\u0026ndash;15%) but symptomatology consistent with potential infection of the CNS. Three (3/6) and two (2/6) mice administered with FNI9 at 5 and 10 mg/kg, respectively, had their survival prolonged between 8- or 11-days post-challenge before succumbing to the infection. All the remaining mice in these groups displayed no or minimal body weight loss through 14 days. Animals administered with either FNI9 or VIR-2482 at 20 mg/kg were completely protected from morbidity and mortality during the study.\u003c/p\u003e\u003cp\u003eOverall, these data demonstrate that FNI9 provides strong in vivo protection against lethal challenges from H5N1 viruses including the panzootic clade 2.3.4.4b.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eSince their emergence in the late 1990s, highly pathogenic Gs/Gd H5N1 viruses have raised global concerns due to their high mortality rate in humans and significant pandemic potential\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Viruses from clade 2 have been particularly concerning, as they show a widespread geographic distribution. This clade has given rise to multiple subclades detected across a diverse array of wild and domestic animals\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, and responsible for numerous human infections and fatalities\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. First isolated in Europe in 2016, H5 viruses from clade 2.3.4.4b re-emerged in 2020 and rapidly spread worldwide becoming panzootic\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Particularly alarming is the detection in U.S. dairy cattle of H5N1 clade 2.3.4.4b viruses with mutations (such as PB2-E627K) that enhance influenza polymerase activity in human cells\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Relatedly, highly pathogenic avian influenza (HPAI) H5N1 2.3.4.4b viruses, isolated from the eye of a farm worker infected through dairy cow contact, were lethal in ferrets and transmissible via respiratory droplets leading to animal fatalities\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eWhile neuraminidase inhibitors (e.g. oseltamivir) and vaccines can be stockpiled for a pandemic response, long-term pre-exposure prophylaxis may be challenging for the small molecules due to their short half-life\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, and H5 vaccines could face drawbacks in common with seasonal influenza vaccines, such as mismatch, poor immunogenicity, and immunological imprinting\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eBroadly neutralizing anti-hemagglutinin (HA) stem mAbs have previously been shown to protect animals against influenza A viruses, including HP H5 strains\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. However, this class of mAbs display a trade-off between their wide breadth and the modest neutralization potency. In contrast, mAbs engaging the highly conserved NA enzymatic pocket can combine wide breadth of coverage, encompassing in different cases both influenza A and B viruses, and high in vitro and in vivo potency\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. In addition, the NA evolves independently from the HA\u003csup\u003e\u003cspan additionalcitationids=\"CR49\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e and it is poorly represented in current vaccines, suggesting that anti-NA mAbs could expand the protection elicited by active immunoprophylaxis\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eHere, we report the potent NAI activity of the recently described anti-NA mAb FNI9 against a broad panel of NA-based pseudotypes spanning over 27 years of Gs/Gd-derived H5N1 virus evolution. Remarkably, FNI9 exhibited superior potency than NA antivirals oseltamivir and peramivir against NAs from H5N1 clade 2.3.4.4b viruses. In addition, FNI9 provided potent in vivo protection against highly pathogenic avian influenza H5N1 viruses from the panzootic clade and its prophylactic activity against these viruses is currently under evaluation in non-human primates. Overall, FNI9 demonstrates strong in vitro and in vivo activity against H5N1 viruses including the panzootic clade 2.3.4.4b.\u003c/p\u003e\u003cp\u003eThe structural analysis presented in this study with N1 from H5N1 revealed that FNI9 interacts with 7 highly conserved key residues on the NA enzymatic pocket (R118, E119, D151, E228, E278, R293, and R368) mimicking the sialic acid receptor and NA-targeting antivirals similar to our previous observations of the mAb in complex with N2\u003csup\u003e25\u003c/sup\u003e. Our in-silico analysis of NA conservation reveals that the key contact sites are functionally constrained, thus anticipating a high barrier for the emergence of FNI9 escape variants.\u003c/p\u003e\u003cp\u003eIn conclusion, our data support the concept that \"off-the-shelf\" mAbs such as FNI9 could be developed to complement vaccination and to provide a critical layer of protection to healthcare workers and farmers facing high exposure risks during human and animal outbreaks, and to high-risk populations such as the elderly and immunocompromised.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cb\u003eProduction of NA-based pseudoparticles\u003c/b\u003e\u003c/p\u003e\u003cp\u003eNA-only based particles (NA-PVs) were produced transfecting Lenti-X 293T cells (Takara) in 6-well plates (Corning). Cells were co-transfected using X-tremeGENE HP (Roche) with 0.1 \u0026micro;g of NA-expressing plasmid and with 1.6 \u0026micro;g of a complementing viral-genome reporter vector, pNL4-3.Luc+.E-R-. A 1:3 DNA/X-tremeGENE HP ratio was used and the transfection was performed following the manufacturer\u0026rsquo;s protocol. Transfected cells were incubated for 72 hours, then the supernatant was harvested and stored at -80\u0026deg;C into aliquots. The pseudoparticles used in this study were generated with NAs from A/HongKong/156/1997, A/Vietnam/CL01/2004, A/Vietnam/1203/2004, A/Thailand/NBL1/2006, A/Cambodia/X0207301/2013, A/Indonesia/NIHRD/15023/2015, A/Egypt/N04915/2014, A/Nepal/19FL1997/2019, A/Victoria/149/2024, A/Cambodia/NPH230032/2023, A/Vietnam/KhanhhoaRV1-005/2024, A/CommonBuzzard/GermanyHH/2024AI01435/2024, A/bottlenose dolphin/Florida/UFTt2203/2022, A/Mink/Spain/3691-8_22VIR10586-10/2022, A/Chile/25945/2023, A/Texas/37/2024, A/Ezo red fox/Hokkaido/1/2023, A/Michigan/90/2024, A/California/135/2024, A/California/168/2024, A/Washington/240/2024.\u003c/p\u003e\u003cp\u003e\u003cb\u003eProduction of recombinant NA protein\u003c/b\u003e\u003c/p\u003e\u003cp\u003eN1 A/Vietnam/1203/2004 protein (residues 63\u0026ndash;449 from GenBank ID: AAT73329.1) containing an N-terminal IgK light chain secretion sequence, a 6xHis-Tag, AviTag, a Tetrabrachion tetramerization domain and a Thrombin protease site was expressed in Expi293F cells at 37\u0026deg;C and 8% CO\u003csub\u003e2\u003c/sub\u003e. Cell culture supernatant was collected four days post transfection and protein was purified using Nickel-NTA agarose (Qiagen) followed by size exclusion chromatography using a Superdex 200 10/300 GL Increase column equilibrated in 50 mM Tris, 150 mM NaCl, 10 mM CaCl2, pH 8.0.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEnzyme-linked lectin assay (ELLA)\u003c/b\u003e\u003c/p\u003e\u003cp\u003e96-well half area ELISA plates (Corning) were coated overnight at 4\u0026deg;C with 25 \u0026micro;g/ml of Fetuin. Plates were blocked with a 1% w/v solution of Bovine Serum Albumin (BSA; Sigma-Aldrich) in PBS containing Ca\u003csup\u003e2+\u003c/sup\u003eMg\u003csup\u003e2+\u003c/sup\u003e (Gibco) and incubated for 1 hour at room temperature. In the meantime, mAbs were serially diluted and mixed with a fixed amount of recombinant NA-PVs and the mix was transferred to fetuin-coated plates, previously washed. After an overnight incubation at 37\u0026deg;C, the plates were washed and 1 \u0026micro;g/ml lectin from peanut-agglutinin (PNA, Sigma-Aldrich) horseradish peroxidase labelled (HRP) was added and incubated at room temperature for 1 hour. After further washing, SureBlue Reserve TMB 1-Component (KPL) was added, and plates were read at 450 nm after blocking the reaction with 1% hydrochloric acid solution.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCryo-EM sample preparation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eN1 NA protein antigen from H5N1 A/Vietnam/1203/2004 was mixed with a 1.2-fold molar excess of FNI9 Fab and incubated on ice for one hour. The complex was diluted to 0.3 mg/mL in Tris-HCl pH 8.0, 150 mM NaCl, 10 mM CaCl\u003csub\u003e2\u003c/sub\u003e buffer. DDM at 0.70xCMC was added to the complex right before vitrification. 3 \u0026micro;L of 0.3 mg/ml of NA-Fab complex with DDM were loaded onto a freshly glow discharged 1.2/1.3 UltrAuFoil grid prior to plunge freezing using a Vitrobot MarkIV (ThermoFisher Scientific) with a blot force of 0 and 3.5 s blot time at 100% humidity and 4\u0026deg;C.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCryo-EM data collection\u003c/b\u003e\u003c/p\u003e\u003cp\u003eData were acquired on a 300kV FEI Titan Krios TEM equipped with a Gatan K3 Summit direct detector and Gatan Quantum GIF energy filter, operated in zero-loss mode with a slit width of 20 eV. Automated data collection was carried out using SerialEM at a nominal magnification of 105,000x and pixel size of 0.8189 \u0026Aring;. The dose rate was adjusted to 16 counts/pixel/s with a total dose of 47.7 (e\u003csup\u003e\u0026minus;\u003c/sup\u003e/A\u003csup\u003e2\u003c/sup\u003e), and each movie was fractionated in 79 frames of 25ms each. A total of 3060 movies were collected with a defocus range comprised between \u0026minus;\u0026thinsp;0.8 and \u0026minus;\u0026thinsp;2 \u0026micro;m. A summary of data collection is provided in Extended Data Table\u0026nbsp;2.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCryo-EM data processing\u003c/b\u003e\u003c/p\u003e\u003cp\u003eDose-weighted movie frame alignment to account for stage drift and beam-induced particle motion was performed in Relion\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. The contrast transfer function is estimated for each micrograph using Gctf\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Individual particles are selected first using Gautomatch (Zhang) and extracted into particle stacks in Relion. Extracted particles were subjected to 2D classification. Initial maps were generated \u003cem\u003eab initio\u003c/em\u003e from selected 2D classes and used for 3D classification of particles from selected 2D classes. The best 3D classes were submitted to 3D refinement with C4 for 4-Fab bound NA) symmetry applied. A soft mask was applied to postprocess the unsharpened map. After refinement, particles were subject to CTF refinement and bayesian polishing, re-refined as above, and postprocessed to produce the final map. Reported resolutions are based on the gold standard FSC\u0026thinsp;=\u0026thinsp;0.143 criterion. A summary of cryo-EM data statistics is provided in Extended Data Table\u0026nbsp;2.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCryo-EM model building and analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe atomic model of N1 NA from \u003cem\u003eInfluenza A virus strain A/Vietnam/1203/2004 H5N1\u003c/em\u003e (PDB: 2HU0) and FNI9 Fab from \u003cem\u003eN2 neuraminidase of A/Tanzania/205/2010 H3N2 in complex with 4 FNI9 Fab molecules\u003c/em\u003e (PDB: 8G3N) was used as an initial model to fit into cryo-EM maps by UCSF Chimera. Coot was used to manually rebuild amino acid mutations and glycans on NAs and build the Fab\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. Models were refined using Rosetta and Servalcat\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e,\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. Model validation and map-model FSC were generated by Validation: model tool in ccpem v1.6.0. Figures were generated using Pymol\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. Epitopes in all NA complexes were identified by determining NA residues within 5.0 \u0026Aring; of any heteroatom in FNI9 Fabs using MOE (v2020.0901, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.chemcomp.com\u003c/span\u003e\u003cspan address=\"https://www.chemcomp.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). MOE contacts for the static structure were generated for the FNI9:N1 complex after having run QuickPrep.\u003c/p\u003e\u003cp\u003e\u003cb\u003eGlycan profiling of neuraminidase with peptide mapping LC-MS\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePeptide mapping with liquid chromatography-mass spectrometry (LC-MS) was used to profile the site-specific glycosylation sites on neuraminidases (H5N1 A/Vietnam/1203/2004). Glycopeptides containing only one specific glycan were achieved by selectively digesting with trypsin, Glu-C, Lys-C, or Asp-N protease, depending on the sequence context. 25 \u0026micro;g of each digest product (peptide with a single glycan) was analysed by LC-MS (Agilent AdvanceBio peptide mapping column and Thermo Q Exactive Plus Orbitrap MS). Peptide mapping data were analyzed on Biopharma Finder 3.2 data analysis software. Technical replicates were performed by injecting 25 \u0026micro;g of digested product three times from the same sample vial into the LC-MS.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMolecular dynamics methods\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe coordinates of FNI9:NA (N1 A/Vietnam/1203/2004) were obtained from cryo-EM (see above) with glycans determined by peptide mapping LC-MS (see above) modelled using GLYCAM (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://glycam.org\u003c/span\u003e\u003cspan address=\"http://glycam.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), based on an early iteration of the published structure. The glycosylated FNI9:NA complex was prepared using QuickPrep (MOE v2020.0901, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.chemcomp.com\u003c/span\u003e\u003cspan address=\"https://www.chemcomp.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e ).\u003c/p\u003e\u003cp\u003eThe FNI9:NA tetrameric complex structure was parameterized as previously described using tleap\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e: ff14SB for protein\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e, GLYCAM_06j-1 for glycans\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e, TIP3P for water\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e, Joung \u0026amp; Cheatham for ions\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. We generated 4.0 \u0026micro;s of aggregate MD from 5 independent 0.8 \u0026micro;s MD simulations of FNI9:N1 seeded with unique initial velocities and were minimized and equilibrated as previously described\u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eWith only experimentally resolved atoms restrained, minimize for 10,000 steps, heat to 300 K in NVT, 100 ps of NPT MD at 300 K. With the same atoms restrained but a 10-fold reduced force constant, 250 ps of NPT MD at 300 K. With only the experimentally resolved backbone atoms, 10,000 steps of minimization, 100 ps of NPT MD at 300K; 100 ps of NPT MD at 300K. Then, the last two stages progressively reduce the restraint force constant by 10-fold for 100 ps NPT MD at 300K. The final restraint force constant was 0.1 kcal/mol \u0026Aring;\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The last stage of equilibration was 2.5 ns of unrestrained NPT MD at 300 K. Production MD continued dynamics (NPT, 300 K, velocities) from the last stage of equilibration for an additional 0.8 \u0026micro;s of unrestrained MD sampling. This entire procedure was repeated five times for the 5 independent FNI9:N1 MD simulations.\u003c/p\u003e\u003cp\u003eMD trajectory files were stripped of water and ions using cpptraj\u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e as previously described\u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. FNI9:N1 interactions were defined using the protein contact analysis in MOE (CCG MOE 2020.09). The analysis classifies contacts as hydrogen bond, metal, ionic, and arene interactions based on participating atom types and their relative geometries; and uses bonded and non-bonded force field energies and implicit solvent solvation energies to compute per-residue interaction energies (within 5 \u0026Aring;). Contacts were calculated using MD frames sampled every 10 ns. QuickPrep was performed for each snapshot prior to evaluation. MOE contact energies reported above are the average energy of each unique interaction pair normalized by the number of frames in which the contact appears. Number of contacts (# Contacts) counted the number of unique residues in FNI9 that formed a contact with an epitope residue in N1 during the MD simulation. Percent energy (% energy) normalizes the MOE contact energies (see above) by summing the energies across the epitope, dividing the energy of each epitope residue by that sum, and multiplying by 100. Contact energy renormalization by the fraction of the time the contacts were present in the MD was computed by multiplying the percent energy scores by the sum of the fraction occupancy divided by the number of contacts (multiply percent energy by 0.75 if there were four contacts and the sum of their fraction occupancy was 3.0). The value obtained was defined as J\u003csub\u003escore\u003c/sub\u003e. A cutoff of 0.3 was derived by weighing its ability to separate strong and significant contacts (energy, persistence, contact order) from weaker ones. Percent solvent exposure was calculated using MOE by dividing solvent exposure of NA residues within the structure by the solvent exposure that the same amino acid would have in a Gly-X-Gly peptide.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vivo studies\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBALB/c mice were purchased from Japan SLC. Clade 1 (NIBRG-14) and clade 2.3.3.4b (A/Ezo red fox/Hokkaido/1/2022) viruses were obtained from NIBSC and Dr. Yoshihiro Sakoda at Hokkaido University, respectively. FNI9, VIR-2482, or PBS control were administered intravenously into mice at doses described in figure legends. On the next day, plasma samples were collected to quantify transferred IgG in the mouse plasma, and the mice were challenged intranasally with 2 or 5 mouse lethal dose\u003csub\u003e50\u003c/sub\u003e (LD\u003csub\u003e50\u003c/sub\u003e) in 50\u0026micro;L. Body weight and CNS disease behavior were monitored daily from day 4 post-infection until day 14, and the body weight loss compared to 2 days before infection was calculated. Mouse with more than 25% of body weight loss was humanely euthanized. Animal procedures were approved by the Animal Ethics Committee of the NIID, and animal experiments were performed at SPF ABSL2 and ABSL3 animal facilities at the NIID.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSequence conservation analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eH5N1 NA protein sequences (up to January 23, 2025) were retrieved from GISAID EpiFlu project (ww.gisaid.org). Protein sequences were aligned using MAFFT\u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e [parameters: --mapout --thread 1 --auto --op 4.5 --keeplength \u0026ndash;-addfragments] to a modified version of the N1 of A/Vietnam/1203/2004 strain, that introduced 20 amino acids at residues 49\u0026ndash;68 to match the nomenclature of the H1N1 Cal09 strain. The logo plots were generated with R package (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.R-project.org/\u003c/span\u003e\u003cspan address=\"https://www.R-project.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) \u0026ldquo;ggseqlogo\u0026rdquo; v.0.1\u003csup\u003e66\u003c/sup\u003e. The residue and haplotype conservations were computed with the R package \u0026ldquo;Biostrings\u0026rdquo; v.2.58.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bioconductor.org/packages/Biostrings\u003c/span\u003e\u003cspan address=\"https://bioconductor.org/packages/Biostrings\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eEVEscape computation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe EVEscape scores were computed as described in \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Briefly the EVEscape model relies on three components: 1.) EVE fitness, as estimated by a neural network fit on the multiple sequence alignment of the sequence of interest to distantly related sequences, 2.) solvent accessibility and 3.) biochemical dissimilarity. The code from the following repository was used: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/OATML-Markslab/EVEscape/tree/main\u003c/span\u003e\u003cspan address=\"https://github.com/OATML-Markslab/EVEscape/tree/main\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. The following non-default parameters were used: theta\u0026thinsp;=\u0026thinsp;0.01; batch_size\u0026thinsp;=\u0026thinsp;512; num_training_steps\u0026thinsp;=\u0026thinsp;20000; num_samples_compute_evol_indices\u0026thinsp;=\u0026thinsp;20000; computation_mode\u0026thinsp;=\u0026thinsp;all_singles. The multiple sequence alignment used as input was obtained as follows: All N1 protein sequences from H1N1 (up to February 2, 2024) and H5N1 (up to May 8, 2024) were downloaded from GISAID EpiFlu project (\u003cspan class=\"ExternalRef\"\u003ewww.gisaid.org\u003cspan address=\"http://www.gisaid.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). To retrieve homolog sequences\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, we downloaded all UniRef100 sequences available as of February 27, 2024 and searched for homologs using the A/Vietnam/1203/2004 strain as input sequence using \u003cem\u003ejackhmmer\u003c/em\u003e (HMMER 3.4; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://hmmer.org/\u003c/span\u003e\u003cspan address=\"http://hmmer.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). We selected homologs with an E value\u0026thinsp;\u0026lt;\u0026thinsp;0.001. Both sets of sequences were aligned to a modified version of the N1 of A/Vietnam/1203/2004 strain, that introduced 20 amino acids at residues 49\u0026ndash;68 to match the nomenclature of the H1N1 Cal09 strain, with MAFFT [parameters: --mapout --thread 1 --auto --op 4.5 --keeplength \u0026ndash;-addfragments;]. The protein structure used is the one described in this paper.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSpreading Forecast Analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe machine learning meta-model was trained on historical GISAID flu protein sequences from October 2015 to February 2019 for N1 from H1N1 and N2 from H3N2. Mutations relative to reference strains (A/California/233/2019 [H1N1] and A/Darwin/9/2021 [H3N2]) were measured and prevalence for each mutation was calculated for each month. A mutation was considered highly prevalent if it achieved greater than 10% prevalence for any month within a flu season (October \u0026ndash; February in the northern hemisphere), across all years studied. Features for the machine learning meta-model were calculated from ESM-2\u003csup\u003e65\u003c/sup\u003e and ESM-IF\u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e, from the same reference sequences and matched PDB structures (6LXK and 7U4G). Likelihood scores for each mutation from each model were used as features in the meta-model. Logistic regression was performed on the features and mutation prevalence targets for the H1N1 and H3N2 mutations together (Python and scikit-learn). The model was evaluated on corresponding data for H5N1. GISAID flu NA protein sequences from October 2021 \u0026ndash; February 2023 were collected and mutations were calculated relative to the previously described modified A/Vietnam/1203/2004 N1 strain.\u003c/p\u003e\u003cp\u003e\u003cb\u003eStatistical Analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAll statistical tests were performed as described in the indicated figure legends using Prism v9.0. The number of independent experiments performed are indicated in the relevant figure legends.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eJ.d.I., F.Z., K.H, H. E. M., J.M.E., H.V.D., N.C., A.C.,Y.C., A.P., E.V., M.C.M., L.E.R., G.S., D.C, and M.S.P are current or former employees of and may hold shares in Vir Biotechnology Inc. M.S.P., D.C., and G.S. are currently listed as an inventor on multiple patent applications, which disclose the subject matter described in this manuscript. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e \u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eWe thank Dr. Yoshihiro Sakoda at Hokkaido University for providing A/Ezo red fox/Hokkaido/1/2022 virus. We also thank Eriko Izumiyama, Akira Dosaka, and Ryoko Itami at Research Center for Drug and Vaccine Development, NIID for their technical support as well as Siro Bianchi at VIR Biotechnology for his graphical support. This work was supported by Japan Agency for Medical Research and Development Grant Numbers JP22fk0108141, JP243fa627005, and JP243fa727002 (YT).\u003c/p\u003e\n\u003ch2\u003eAuthor contributions\u003c/h2\u003e\n\u003cp\u003eM.S.P., D.C., and Y.T., conceived the research and designed the study. Experiment design: S.M., J.d.I, F.Z., K.H., H.A., H.E.M., J.M.E., Y.A., H.V.D., N.C., E.S., A.C., Y.C., R.K., A.P., E.V., T.O., M.C.M., L.E.R., M.S., G.S., H.H., Y.T., D.C., and M.S.P.; NA-based pseudoparticles production and ELLA testing: F.Z., and M.S.P.; NA antigen design and production: H.V.D., and N.C., Complex formation for cryo-EM: H.V.D., and N.C.; Cryo-EM Data Collection, Processing, and Model Building: J.M.E., and H.V.D.; Glycan analysis and MD analysis: K.H., A.C., and L.E.R.; Bioinformatic epitope conservation and escape prediction: J.d.I., H.E.M., Y.C., M.C.M.; in vivo studies: S.M., H.A., Y.A, E.S., R.K., T.O., M.S., H.H., and Y.T.; Data analysis: S.M., J.d.I, F.Z., K.H., H.A., H.E.M., J.M.E., Y.A., H.V.D., N.C., E.S., A.C., Y.C., R.K., A.P., E.V., T.O., M.C.M., L.E.R., M.S., H.H., Y.T., D.C., and M.S.P.; Supervision: S.M., J.d.I., K.H., N.C., A.P., M.C.M., L.E.R., G.S., Y.T., D.C., and M.S.P.; Y.T., D.C., and M.S.P. wrote the manuscript with input from all the authors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eYamaji R et al (2099) (2020) Pandemic potential of highly pathogenic avian influenza clade 2.3.4.4 A(H5) viruses. \u003cem\u003eRev Med Virol\u003c/em\u003e 30, e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1002/rmv.2099\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1002/rmv.2099\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorens DM, Park J, Taubenberger JK (2023) Many potential pathways to future pandemic influenza. 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Virus evolution has resulted in various clades, including the panzootic subclade 2.3.4.4b, recognized for its pandemic potential. Here we present the potent in vitro activity of FNI9, a pan-influenza NA-inhibiting monoclonal antibody, against a range of pseudoparticles with NA spanning 27 years of Gs/Gd-derived H5N1 virus evolution. FNI9 also shows strong prophylactic protection in mice against lethal challenges with H5N1 from clade 1 and 2.3.4.4b. Cryo-EM and molecular dynamics analysis reveal that FNI9 binds to 7 highly conserved H5N1 NA residues (R118, E119, D151, E228, E278, R293, and R368). In silico evolutionary escape profiling and machine learning indicate low escapability, high fitness costs, and minimal spread likelihood for viral mutations that evade FNI9 binding. These findings support FNI9 broad protection and underscore the NA role in future influenza vaccine design.\u003c/b\u003e \u003c/p\u003e","manuscriptTitle":"Potent efficacy of a NA-targeting antibody against a broad spectrum of H5N1 influenza viruses","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-20 15:47:02","doi":"10.21203/rs.3.rs-5999144/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"75837496-b84e-453f-ae7b-3573b1a11211","owner":[],"postedDate":"February 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":44578976,"name":"Biological sciences/Immunology/Infectious diseases/Influenza virus"},{"id":44578977,"name":"Health sciences/Diseases/Infectious diseases/Influenza virus"},{"id":44578978,"name":"Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy"}],"tags":[],"updatedAt":"2026-04-10T07:05:44+00:00","versionOfRecord":{"articleIdentity":"rs-5999144","link":"https://doi.org/10.1038/s41467-026-70036-8","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2026-03-02 05:00:00","publishedOnDateReadable":"March 2nd, 2026"},"versionCreatedAt":"2025-02-20 15:47:02","video":"","vorDoi":"10.1038/s41467-026-70036-8","vorDoiUrl":"https://doi.org/10.1038/s41467-026-70036-8","workflowStages":[]},"version":"v1","identity":"rs-5999144","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5999144","identity":"rs-5999144","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}