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Oct 17, 2024

A human monoclonal antibody targeting the monomeric N6 neuraminidase confers protection against avian H5N6 influenza virus infection | Nature Communications

Nature Communications volume 15, Article number: 8871 (2024) Cite this article 3 Altmetric Metrics details The influenza neuraminidase (NA) is a potential target for the development of a

Nature Communications volume 15, Article number: 8871 (2024) Cite this article

3 Altmetric

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The influenza neuraminidase (NA) is a potential target for the development of a next-generation influenza vaccine, but its antigenicity is not well understood. Here, we isolate an anti-N6 human monoclonal antibody, named 18_14D, from an H5N6 avian influenza virus (AIV) infected patient. The antibody weakly inhibits enzymatic activity but confers protection in female mice, mainly via ADCC function. The cryo-EM structure shows that 18_14D binds to a unique epitope on the lateral surface of the N6 tetramer, preventing the formation of tightly closed NA tetramers. These findings contribute to the molecular understanding of protective immune responses to NA of AIVs in humans and open an avenue for the rational design of NA-based vaccines.

Influenza viruses are continuing to evolve and cause seasonal flu and occasional worldwide pandemics, which have posed a great challenge to public health1,2,3,4. Influenza viruses have two major surface glycoproteins, hemagglutinin (HA), and neuraminidase (NA). HA mediates the binding of viruses to the cellular sialic acid receptors and fusion of the virion membrane with the intracellular endosome membranes5. NA removes decoy receptors from mucins and cleaves off the sialic acid, allowing the release of progeny viruses from the infected cells6,7.

Human protective antibodies elicited by vaccination or infection can bind to both the HA and NA proteins8,9. In the past years, the NA has been historically understudied compared to the surface protein counterpart HA. However, increasing studies highlighted the importance of NA-targeting antibodies and their implications for therapy and design of universal flu vaccines, as they have shown that NA-targeted immunity can confer protective efficacy against influenza virus challenge in animal models10,11,12,13. NA-targeting monoclonal antibodies (mAbs) are shown to reduce the viral loads and symptoms in infected mice and even correlate with the protection in humans14,15,16, and antigenic drifts also occurred on NA protein16,17,18,19,20.

Most of the NA-targeting antibodies directly inhibit enzymatic activity of the NA via binding to enzyme active site21,22,23,24,25, or indirectly inhibit enzymatic activity of the NA through steric hindrance24,26,27,28. Otherwise, there are some other NA antibodies with no inhibitory activity to the NA, could also contribute to protection through antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP)16.

In addition to seasonal H1N1, H3N2 influenza A virus and influenza B virus, the recent rise in the frequency of avian influenza viruses (AIVs) infection has also raised serious public health concerns4,5,29. Among them, H5N6 AIV has obtained global attention. Severe disease occurred in 93.8% of human H5N6 infection cases, and the case fatality rate was >50%30. However, the role of NA in human immune response to H5N6 AIV infection has not been well investigated.

Here, we isolated an N6-specific antibody, 18_14D, from an H5N6-infected human case in Shenzhen, China, in 2015. Antibody 18_14D can bind to N6 and weakly inhibit enzymatic activity of the H5N6 virus at a high concentration. Moreover, 18_14D could protect mice from lethal virus infection via ADCC function. Further cryo-electron microscopy (cryo-EM) structure revealed that 18_14D recognize a distinctive epitope on the lateral surface of NA, and the antibody binding would induce conformational change to generate steric conflict at the interface of NA protomer in the tetramer under closed state, acting as a “bayonet” to unclench the N6 tetramer and keep the NA tetramer in an open state. These findings will contribute to the molecular understanding of protective immune response to the NA of AIVs in human, and provide a new direction for the rational design of NA-based vaccines.

Peripheral blood mononuclear cell (PBMC) samples were collected at 18 days post illness onset (d.p.o) from a hospitalized H5N6 AIV infected patient in Shenzhen, China31. IgG+ memory B cells were selected and cultured. On the 13th day of the culture, the positive rate of IgG+ B cells was 34.3%, and the positive rate of H5N6-positive B cells was 10.1%. A total of 9 cell wells were successfully amplified with heavy chain and light chain genes of antibodies and the antibody genes were analyzed (Supplementary Table. 1). The putative antibodies from the H5- or N6-positive cell wells were expressed by 293 T cells, and ELISA was used to measure their binding activities for H5 HA or N6 NA (Supplementary Table. 2). Finally, only one antibody named as 18_14D was identified to bind the N6 protein (Supplementary Fig. 1). 18_14D was encoded by the genes IGHV1-69/IGLV2-14. IGHV1-69 was one of the most polymorphic loci within the human IGHV gene cluster, and IGHV1-69 germline gene was frequently found in the broadly neutralizing antibodies to the stem domain of influenza A hemagglutinin32. Remarkably, the VH gene of 18_14D was entirely germline without any somatic mutation (Supplementary Fig. 2).

The Bio-Layer Interferometry (BLI) assay showed that 18_14D Fab could bind both the N6_DQ (the head domain of NA from an H5N6 virus, A/Yunan/DQ001/2016) tetramer and monomer, with affinities of 26 nM and 18 nM, respectively (Fig. 1a,b). To assess the direct inhibition ability on virus reproduction for 18_14D, virus inhibition assay (Egress) and enzyme-linked lectin assay (ELLA) were conducted. 18_14D could not inhibit the replication of the H5N6 virus and showed no NA inhibition activity in the ELLA even at 100 µg/mL (Fig. 1c,d). ELLA was also conducted using H5N6 virus, and the results showed that 18_14D exhibited about 70% inhibition activity at a high concentration of 1 mg/mL, which was weak compared with other anti-NA antibodies (Supplementary Fig. 3). As several previously reported anti-NA antibodies conferred in vivo protection due to ADCC function16, the capacity of 18_14D to activate ADCC-signaling pathways was also assessed using an ADCC reporter assay. We found that 18_14D was capable of inducing ADCC reporter activity in both human and mouse cells (Fig. 1e,f).

The binding kinetics between 18_14D Fab and N6 monomer (a) or tetramer (b) were measured using BLI. (c) Inhibition of N6 enzymatic activity by 18_14D measured in ELLA. Fetuin was used as substrate. Data represents one of three independent experiments, shown as mean ± SEM of three technical replicates. 1G01 was used as a positive control. (d) Egress inhibition of H5N6 virus by 18_14D and 1G01. Data represents one of three independent experiments. 1G01 was used as a positive control. (e,f) ADCC activity of the 18_14D was measured. An anti-HA antibody FI6 was used as a positive control, and an anti-Ebola GP antibody 13C6 was used as a negative control. Data represents one of three independent experiments, shown as mean ± SEM (standard deviation) of two technical replicates. Both of human (e) and mouse (f) Jurkat cells were examined.

The prophylactic and therapeutic efficacy against H5N6 AIV in vivo was further investigated in a murine model. 18_14D IgG with doses of 20, 5, 1 mg/kg were used in a prophylactic treatment setting before challenge with 3 LD50 (50% lethal dose) of A/Yunan/DQ001/2016 (H5N6, DQ001) virus. It shows that 18_14D conferred full protection (6 out of 6 mice survived) at a dose of 20 mg/kg, and 5 or 1 mg/kg of 18_14D was also protective (5 out of 6 mice survived per group) (Fig. 2a,b). In addition, 18_14D was administered at 48 hours post infection to test the therapeutic efficacy, and 18_14D provided partial protection against the H5N6 challenge, with 1 out of 6 mice, 3 out of 6 mice survived in the 5 mg/kg and 20 mg/kg groups, respectively, and all the mice experienced obvious weight loss (Fig. 2c,d).

6–8-week-old female BALB/c mice were used for prophylaxis and therapeutic study. The mean percentage of body weight change and survival curves post-infection are shown. Six animals were used per group (n = 6), and error bars indicate SEM. The humane endpoint, which was defined as a weight loss of 25% from initial weight on day 0, is shown as a dotted line. For prophylaxis study, 1, 5 or 20 mg/kg of wild type 18_14D (a–d) and 20 mg/kg 18_14D_LALA (e, f) were injected intraperitoneally 3 h before 3 LD50 of A/Yunan/DQ001/2016 (H5N6) virus challenge. For therapeutic study, 5 or 20 mg/kg of 18_14D were injected intraperitoneally 48 h after 3 LD50 of A/Yunan/DQ001/2016 (H5N6) virus challenge.

To examine the importance of Fc effector function, we generated an 18_14D LALA variant to eliminate its Fc effector functions. The mice prophylactically administered with 20 mg/kg of 18_14D LALA variant had significantly more weight loss than those with wild type 18_14D, and only 3 mice survived (Fig. 2e,f). Taken together, we concluded that 18_14D protected mice from lethal infection with H5N6 AIV mainly via ADCC function.

To elucidate the binding epitope of 18_14D on NA, we performed single particle cryo-electron microscopy (cryo-EM) to determine the three-dimensional structure of 18_14D Fab in complex with the N6_DQ. Firstly, N6_DQ tetramer was incubated with 18_14D Fab to generate the protein complex. Under negative staining EM, N6_DQ tetramer displayed different conformations including both closed and open states. The binding of 18_14D Fab made the N6_DQ tetramer into a fully open state with inhomogeneous performance, which could not be used to solve the structure (Supplementary Fig. 4). We then prepared a ternary complex (N6_DQ/18_14D/1G01), using N6_DQ monomer, 18_14D Fab, and the previously reported 1G01 Fab, a broadly protective human monoclonal antibody that targets the active site of influenza virus neuraminidase. Initially, we got a final EM map of 3.2 Å (Supplementary Fig. 5). In the map, we noticed the EM densities of the CH1 and CL of two Fabs were much weak and flexible, which would affect the alignment during 3D refinement. To improve the resolution and obtain better EM densities on the interfaces between NA and antibodies, we performed signal subtraction to eliminate the influence of CH1 and CL of two Fabs and focused on the NA, VH and VL of two Fabs for further refinement, and we got a final EM density map of 3.0 Å (Supplementary Fig. 5, Supplementary Table. 3).

The overall structure showed that 18_14D recognized a unique epitope far away from the enzyme active site of N6_DQ. Two antibodies bind to the N6_DQ in an almost vertical orientation. 1G01 binds to the enzyme active site, while 18_14D binds to the lateral surface of the neuraminidase head (Fig. 3a). Further analysis showed that there were 20 residues in the N6 involved in the interaction with 18_14D, in which 19 residues (N330, R365, I367, K369, E376, L378, P391, T392, S393, Y394, Q395, L396, N400, R454, L455, G456, S457, W458, W460) and 2 (Q401, S457) residues interacted with the antibody heavy and light chains, respectively (Fig. 3b,c and Supplementary Table. 4). The heavy chain of 18_14D (including HCDR1, HCDR2 and HCDR3) contributed to most of the interactions, while the light chain (including LCDR1 and LCDR2) contributed much less to the interaction, only with 8 atom-to-atom contacts (Fig. 3b,c and Supplementary Table. 4). Residues G27, T28, Y32 in HCDR1 and R102, Y103, F104, D105 in HCDR3 form hydrogen-bond interactions with N6_DQ (Figure 3d). We also found that the main chain of HCDR3 (Y394, L396) formed 3 hydrogen-bond interactions with N6_DQ, while the side chain was away from the binding interface, which was relatively rare in the antigen-antibody interaction (Supplementary Fig. 6).

a Overall structures of N6_DQ/18_14D/1G01, N6_DQ was displayed in a surface representation. The 18_14D heavy chain (orange), light chain (yellow), 1G01 heavy chain (blue), light chain (light blue) are shown in cartoon representation. Two antibodies bind N6_DQ in an almost vertical orientation. b Surface representations of paratope 18_14D, CDR1 loop of heavy chain (HCDR1) is colored in forest green; CDR2 loop of heavy chain (HCDR2), purple; CDR3 loop of heavy chain (HCDR3), orange; framework region 3 (FR3), magenta, CDR1 loop of light chain (LCDR1), blue; CDR2 loop of light chain (LCDR2), yellow. c The epitope of 18_14D is shown in surface representation. Footprint of 18_14D on N6_DQ is colored according to the component that mediates the contact. Otherwise, S457 interacting with LCDR1 and HCDR3 is colored in cyan, N330 interacting with HCDR1 and FR3 is colored in salmon, R365 interacting with HCDR1 and HCDR2 is colored in teal, I367 and N400R interacting with HCDR1 and HCDR3 are colored in green, T392 and S393 interacting with HCDR2 and HCDR3 are colored in pink, Y394 interacting with HCDR1, HCDR2 and HCDR3 is colored in medium purple. Footprints for the N6_DQ and 18_14D binding sites is defined by residue contacts within 4.5 Å. d The detailed polar interactions between N6_DQ and 18_14D. N6_DQ is colored in gray, CDR loop is labeled with the same colors as in (b). The residues involved in the interaction are shown in stick representation and labeled in saturated color. Red spheres represent the enzyme active site. Single-letter abbreviations for the amino acid residues are as follows: D, Asp; E, Glu; F, Phe; G, Gly; I, Ile; K, Lys; L, Leu; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; W, Trp; and Y, Tyr.

Negative staining EM indicated that N6_DQ_tetramer in N6_DQ_tetramer/18_14D complex showed a more “open” form, compared with N6_DQ_tetramer alone or N6_DQ_tetramer/1G01 complex (Supplementary Fig. 4). Therefore, we deduced that 18_14D could unclench the closed tetramerization state of N6. Structural analysis indicated the binding footprint of 18_14D was partially hidden in the NA tetramer, according to the previously reported N6 tetramer structure [Protein Data Bank (PDB) ID: 2CML]33. The 454–458 loop of NA was at the interface of the NA tetramer (Fig. 4a), and it forms intensive interactions with 18_14D.

a The epitope of 18_14D is mapped onto N6 (PDB: 2CML) (orange). b Superimposition of the N6_DQ/18_14D/1G01 complex structure to an English duck N6 tetramer (PDB: 2CML, colored in gray) and N6_H3 (PDB: 4QN6, colored in light sea green). The N6_DQ, heavy chain and light chain of 18_14D are colored in purple, green and orange respectively. The side chain of residues involved in the interaction are shown in sticks. Single-letter abbreviations for the amino acid residues are as follows: D, Asp; G, Gly; I, Ile; L, Leu; N, Asn; P, Pro; R, Arg; S, Ser; W, Trp; and Y, Tyr.

By superimposing N6_DQ/18_14D/1G01 complex structure to the previously reported two N6 tetramer structures (PDB: 2CML and 4QN6), we found that the 454–458 loop of N6_DQ showed a significant structural displacement compared with the apo N6 protomers in the two tetramer structures (PDB: 2CML and 4QN6) (Fig. 4b). The displacement of 454–458 loop would generate the structural clash between S457 in N6_DQ and N201 in the adjacent N6 protomer 2 (PDB: 2CML and 4QN6) and then unclench the N6 tetramer interaction (Fig. 4b).

Sequence alignment of different N6 proteins showed that the most of binding epitope residues of 18_14D were highly conserved except polymorphism on residues 392, 393, 394 and 396 (Supplementary Table. 5). We expressed an N6 protein from A/Chicken/Nanchang/7-010/2000 (H3N6, named N6_H3)34, and sequence alignment showed that there are 3 different amino acids on the epitope: I392, H394 and V396 (Supplementary Fig. 7). BLI results showed that 18_14D Fab did not bind to N6_H3. Therefore, we deduced that residues 392, 394 and 396 were critical for the binding. Mutagenesis experiment showed that Y394H or L396V single substitution significantly reduced the binding affinity of 18_14D, with KD values of 937 and 345 nM, respectively. T392I single substitution also slightly affect the binding ability. Combination of I392T, H394Y and V396L triple-substitution significantly increase the binding activity of N6_H3 to 18_14D antibody, with KD value of 185 nM (Fig. 5a). Structural analysis indicated that residues T392, Y394 and L396 interact with HCDR1, HCDR2 and HCDR3 of 18_14D (Fig. 5b). Y394H substitution would destroy the hydrophobic interaction between 18_14D residues and Y394, while T394I and L396V would also alter the interactions (Fig. 5c).

a Binding affinities of 18_14D and 1G01 Fabs to different N6 proteins. N.D. represents not detected. b The interactions between 18_14D and residues T392, Y394 and L396 of N6_DQ. 18_14D and N6_DQ are shown in cartoon representation. HCDR1, HCDR2 and HCDR3 of 18_14D are colored in light green, plum and wheat, respectively. N6_DQ is colored in light blue. c Paratope of 18_14D is shown in surface representation, N6_DQ is colored in light blue, and N6_H3 (PDB: 4QN6) is colored in light green. The residues 392, 394 and 396 involved in the interaction are shown in stick representation. Single-letter abbreviations for the amino acid residues are as follows: F, Phe; H, His; I, Ile; L, Leu; Q, Gln; R, Arg; T, Thr; V, Val; W, Trp; and Y, Tyr.

According to the sequence analysis, except for clade 2.3.4.4b H5N6 viruses, >97% of H5N6 viruses contained Y394 and L396 for N6, indicating that the binding epitope of 18_14D is highly conserved. However, only about 20% of clade 2.3.4.4b H5N6 viruses carried Y394 and L396, which indicated that 18_14D antibody would show weak or no binding to most of clade 2.3.4.4b H5N6 viruses. For other HxN6 subtypes AIV, such as H3N6, H4N6 and H13N6, most of them contain H394 (Supplementary Table. 5), while residue 396 showed more polymorphism, indicating that 18_14D may also show weak or no binding to these viruses. Other NA subtypes including N2, N3, N7 and N9 from group 2 and N5 from group 1 were also used to test the binding to 18_14D, and no binding was detected (Supplementary Fig. 8). Overall, the 18_14D is an H5N6-specific antibody.

Previous studies have showed the antibody epitopes on NA can be mainly divided into four groups. (1) The enzyme active site. Generally, antibodies targeting enzyme active site or the edge of enzyme active site show strong enzyme inhibition activity and broad-spectrum neutralizing ability, such as FNI9, 1G01 and DA03E1721,22,25 (Fig. 6a). (2) The underside of NA, such as 1F04, 3A10, 3H03 and NDS.326,27,28 (Fig. 6b). (3) The region across adjacent NA protomers in the lateral surface, such as NA-22 and CD624,35 (Fig. 6c). (4) Another region in the lateral surface (not across the promoter-promoter interface), such as NA-6324 (Fig. 6d). The mAbs targeting the lateral surface or the underside of NA head can show good in vivo protection by both interfering NA cleavage of fetuin substrate and Fc effector functions.

The NA tetramer is displayed in a surface representation, and the antibodies are displayed in a cartoon representation. Red spheres represent the enzyme active site. a Anti-NA antibodies targeting the enzyme active site, such as 1G01 (PDB: 6Q23, medium purple) and NA-73 (PDB: 6PZY, salmon) (b) Anti-NA antibodies targeting the underside of NA, such as 3A10 (PDB: 8EZ3, purple), 1F04 (PDB: 8EZ7, orange), 3H03 (PDB: 8E6J, green) and NDS.3 (PDB: 8GAV, sky blue). c Anti-NA antibodies targeting the region across adjacent NA protomers in the lateral surface, such as CD6 (PDB: 4QNP, blue) and NA-22 (PDB: 6PZW, hot pink). d Anti-NA antibodies targeting the other region in the lateral surface (not across the promoter-promoter interface), such as NA-63 (PDB: 6U02, cyan). e Anti-NA antibodies targeting the interface of the N6 tetramer, such as 18_14D (yellow).

Unlike the binding epitopes mentioned above, 18_14D recognizes a distinctive epitope on the lateral surface of NA head (Fig. 6e). 18_14D binds to the interface of the N6 tetramer, however, 18_14D only interacts with one NA protomer, which is different from NA-22 and CD624,35. Moreover, like a “bayonet”, 18_14D could unclench the N6 tetramer by inducing the conformational change of NA to generate steric clash between the NA promoters. We propose that the binding epitope of 18_14D would be a fifth group for classification of antibody epitopes on NA.

Both anti-NA and anti-HA antibodies could confer protection against influenza virus in humans and animals. Recent studies of NA immunity showed that NA could be a promising target for broad-spectrum protection against influenza virus, and the antigenicity profiles of seasonal human influenza A virus N1, N2 and influenza B virus NAs have been widely investigated13,22,23,25,26,27. However, the role of N6 protein in highly pathogenic H5N6 AIV infection has not been well illustrated. A previous study has investigated murine mAbs to the N6 and found that the antibodies have broad reactivity across the American and Eurasian N6 lineages, but relatively little binding to the H5N6 NA36. Here, we described the isolation of a human anti-N6 mAbs induced by H5N6 AIV infection and characterized its protective function and binding epitopes.

In accordance with most of mAbs targeting the lateral surface of NA, 18_14D confers protection against H5N6 lethal challenge in a murine model. Our results also showed that 18_14D could activate ADCC-signaling pathways, which help to suppress virus replication in vivo. Of note, the VH gene of 18_14D was entirely germline without any somatic mutation, and lack of affinity maturation might be responsible for the relatively low affinity. However, germline mAbs typically exhibit excellent drugability properties, especially lower immunogenicity. Therefore, 18_14D mAb could have great potential for prophylaxis and therapeutics of H5N6 infection in humans. Although 18_14D did not broadly interact with different NA subtypes, the binding epitope of 18_14D was still worthy of our attention due to its effectiveness and novelty. Antibodies targeting a similar region on other NAs may also exist, which deserves to be studied in the future.

Previous studies have shown that tetrameric NA rather than monomeric NA confer protection in mouse model12,13,37. Ellis and Lederhofer et al. showed that NA could adopt different tetrameric conformations, including both “open” state and “closed” state, and tried to stabilize NA tetramer by introducing mutations38. Considering the unique epitope of 18_14D, we speculated 18_14D recognizes the “open” state of NA tetramer, and an “open” N6 tetramer is required for eliciting the protective antibodies like 18_14D. Overall, our results suggest that stabilized recombinant NA tetramers would not be the only road to Rome, and we could consider the strategy of multimeric NA monomers for the vaccine design.

In conclusion, our results have elucidated a distinctive epitope on the NA, and help to further illuminate the dark side of NA in influenza immunity. These findings contribute to the molecular understanding of protective immune response to the NA of AIVs in human and promote the design of next-generation flu vaccine.

All procedures in this study involving H5N6 AIV infection was performed in the biosafety level 3 (BSL-3) facility and animal biosafety level 3 (ABSL-3) facility. All the procedures in the murine study were reviewed and approved by the Laboratory Animal Welfare and Ethics Committee in Shenzhen Third People’s Hospital, China. Written informed consent was obtained from the donor following the approval from the Research Ethics Committee of Shenzhen Third People’s Hospital, China.

6–8-week-old female BALB/c mice were purchased from Vital River and housed in a specific pathogen-free (SPF) animal facility on a 12-hour light/dark cycle under ambient conditions with free access to food and water.

HEK293T cells and MDCK cells were cultured at 37°C with 5% CO2 in Dulbecco’s Modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS). HEK293F cells were cultured at 37°C with 5% CO2 in SMM 293-TII Expression Medium (Sino Biological, M293TII). ADCC bioeffector cell FcγRIIIa cells (Promega, G7102) were cultured at 37°C with 5% CO2 in RMPI 1640 with 10% FBS.

H5N6 virus strain A/Yunan/DQ001/2016 (H5N6, GISAID ID: EPI_ISL_200837, DQ001) was isolated by Shenzhen Key Laboratory of Pathogen and Immunity, Shenzhen Third People’s Hospital.

The screening method of human antibody was carried out according to the method reported previously39. Memory B cells secreting IgG subtype antibodies from convalescent PBMC of H5N6 infected patients in Shenzhen Third People’s Hospital (collected on day 18 after illness onset) were sorted out by BD Aria III by gating of CD19+IgM–IgA–IgD– memory B cells and cultured three cells per well in 96-well cell culture plates (3T3-msCD40L-IL21 as feeder cells) (Antibodies purchased from BD Biosciences). ELISA was used to detect the secretion of IgG antibody and H5 or N6 specific antibody in the culture supernatant. The positive cell wells were screened, and the heavy chain and light chain coding genes of B cells in positive cell wells were amplified by primers specific to the heavy and light chains of human antibodies and sequenced using both the first-generation and high-throughput sequencing technologies. Due to being plated at a density of 3 cells per well, each well can obtain more than one heavy chain and one light chain gene sequences, and we will explore the different combinations of heavy chain and light chain genes in the same well.

The heavy chain and light chain of antibodies (18_14D, 1G01, MEDI8852, 13C6, HENV-32 and mutant antibody) were cloned into pCAGGS plasmids, and plasmids were transfected into HEK293T or HEK293F cells at a ratio of 1:2 to express antibody proteins. After 5 days, the supernatants were collected, and soluble protein were purified using a Protein A column (Cytiva). The proteins were further purified via gel filtration chromatography with a Superdex 200 column (Cytiva). 18_14D and 1G01 Fabs were generated by papain digestion and further purified by Protein A column (Cytiva) as described previously40,41,42. Briefly, antibodies were concentrated to ~20 mg/mL and digested using papain (Pierece) protease with an antibody to papain ratio of 160:1 (wt/wt) at 37 °C for 6 h. The digestion mixture was loaded into a Protein A column to separate the Fab fragment with the Fc region and undigested antibody. Fab fragments were collected, and purified via gel filtration using a Superdex 200 column (Cytiva).

All genes used in this study were synthesized by Azenda and performed codon optimization. For ELISA assay during human antibody isolation, the sequence encoding the ectodomain of HA from A/duck/Guangdong/04.23 DGQTXC213-O/2015 (H5N6, GISAID ID: EPI_ISL_199291, H5_DG) or A/Shenzhen/TH002/2016 (H5N6, GISAID ID: EPI_ISL_207051, H5_TH002) was cloned into the baculovirus transfer vector pFastBac1 (Invitrogen) as previously described43,44, in-frame with an N-terminal gp67 signal peptide for secretion, a C-terminal thrombin cleavage site, a trimerization foldon sequence and a His6-tag at the extreme C terminus for purification. The NA head domain, which contained residues 83-470 (N6 numbering) of DQ001 (N6_DQ), A/duck/Guangdong/04.23 DGQTXC213-O/2015 (H5N6, N6_DG) or A/Chicken/Nanchang/7-010/2000 (H3N6, N3_H6) was cloned into the baculovirus transfer vector pFastBac1 (Invitrogen) as described previously34, with a GP67 signal peptide, a His6-tag, a tetramerization sequence, and a thrombin cleavage site at the N terminus. Transfection and virus amplification were performed according to the Bac-to-Bac baculovirus expression system manual (Invitrogen). HA or NA proteins were produced by infecting suspension cultures of Hi5 cells (Invitrogen) for 48 h. Soluble proteins were collected from the cell supernatant by metal affinity chromatography using a 5 mL HisTrap HP column (Cytiva), and then initially purified by ion-exchange chromatography (IEX) using a RESOURCE Q column (Cytiva), and further purified by gel filtration chromatography using a Superdex 200 Increase 10/300 GL (Cytiva).

For NA binding affinity assay, ELLA and structural analysis, the NA head domain of A/Yunnan/DQ001/2016 (H5N6, N6_DQ, residues 83–470), N6_DQ T392I (residues 83–470), N6_DQ Y394H (residues 83–470), N6_DQ L396V (residues 83–470), N6_ H3 I392T/H394Y/V396L (N6_H3 MUT, residues 83–470), A/New York/3147/2009 (H3N2, N2, residues 80–466), A/swine/Missouri/2124514/2006 (H2N3, N3, residues 83–469), A/mallard duck/ALB/60/1976(H12N5, N5, residues 80–473), A/mallard/ALB/196/1996 (H10N7, N7, residues 81–490) and A/pigeon/Shanghai/S1423/2013(H7N9, N9, residues 78–465) was fused to an N-terminal signal peptide (signal peptide from low-affinity nerve growth factor receptor, LNGFR), His6-tag, a human vasodilator-stimulated phosphoprotein (VASP) tetramerization domain, and a thrombin cleavage site, and constructed into pCAGGS vector and expressed in HEK293F cells. After 5 days, the supernatants were collected, and soluble proteins were purified using a 5 mL HisTrap HP column (Cytiva). The proteins were further purified via gel filtration chromatography with a Superdex 200 Increase 10/300 GL. To generate N6_DQ monomer, thrombin enzyme (Sigma-Aldrich, 5 U/mg) was added into the protein and incubated overnight at 4 °C, The monomer was then acquired via gel filtration chromatography with a Superdex 200 Increase 10/300 GL (Cytiva).

Microtiter plates (Sangon Biotech) were coated with 100 ng of each recombinant proteins overnight at 4 °C. The plates were then washed twice with PBS containing 0.1% v/v Tween-20 (PBST) and blocked with blocking solution (PBS containing 5% w/v bovine serum albumin) overnight at 4 °C. On the day of the experiment, the plates were then washed once with PBST. The serially diluted sera or monoclonal antibodies were added to the wells and incubated at 37 °C for 60 min. Then the plates were washed five times using PBST and added 100 µL horseradish peroxidase (HRP)-conjugated goat anti-human or mouse IgG antibody solution (Sangon Biotech) to each plate, respectively, and incubated at 37 °C for 60 min. Wash each plate with PBST five times, and tetramethylbenzidine (TMB) substrate (Sangon Biotech) was added to each plate at room temperature in the dark and reacted for 15 min. After the reaction, the plate was stopped with a 2 M HCL solution. The absorbance was measured at 450 nm using a microplate reader (Thermo Scientific).

The antibody binding affinity and the competitive binding of monoclonal antibodies were measured by biolayer interferometry (BLI) using the Octet RED96 system (FortéBio). All experiments were performed at 25 °C, and the biosensors were pre-equilibrated in BLI buffer (20 mM Tris, pH 7.4 and 150 mM NaCl and 0.005% (v/v) Tween-20) for 10 min. For the antibody binding affinity assay, Biotinylated NAs were loaded onto SA biosensors for 160 s and baseline was measured in BLI buffer. Following baseline measurements, sensors were loaded with 18_14D or 1G01 Fab with different concentrations and association and dissociation was measured. The data were analyzed using the Octet-Red96 software.

ELLA experiments were performed described45. Briefly, each well of a 96-well microtiter plate (Corning) was coated with 100 μL fetuin (Sigma) at 25 μg/mL in coating buffer at 4 °C overnight. The fetuin was discarded and washed three times with PBS with 0.05% Tween-20 (PBST). Then 100 μL blocking solution (5% skimmed milk) was added to the plate and incubated for 1 h. After washing the plate three times, 50 μL antibodies or sera at the indicated concentration in PBS were mixed with an equal volume of N6_DQ tetramer (0.014 mg/mL) or H5N6 virus in plate for 2 h at 37 °C. Then, the mixture was added to the fetuin-coated plate and incubated for 2 h at 37 °C. The plate was then washed four times with PBST. Subsequently, 100 μL of HRP-conjugated peanut agglutinin lectin (PNA-HRPO, Sigma–Aldrich) in PBS was added and incubated for 1 h at room temperature in the dark. The plate was washed four times and incubated TMB (Sangon Biotech) for 15 min in the dark. Then 2 M HCl was added to the plate. Absorbance was read at 450 nm by microplate reader (Thermo Scientific). Data points were analyzed using GraphPad Prism 8 and the 50% inhibition concentration (IC50) was defined as the concentration at which 50% of the NA activity was inhibited compared to the negative control.

MDCK cells were seeded in a 96-well, flat-bottom cell culture plate (Thermo Fisher) and cultured for 24 h. 100 TCID50 virus was diluted using DMEM and was incubated with two-fold diluted antibodies at 37 °C for 1 h. MDCK cells were washed twice with PBS and then incubated with the antibody-virus mixture. After 48 h incubation, the presence of virus was detected by hemagglutination assay. The results were analyzed using Prism software.

100 μL/well MDCK cell (2 × 105) in DMEM media were seeded into white, flat bottom, 96-well cell culture plates (Corning) and incubated overnight at 37 °C. Cells were recounted and infected with DQ001 virus at multiplicity of infection (MOI) of 3, and then were incubated at 37 °C. After 16 h, the medium was replaced with ADCC bioeffector cell FcγRIIIa cell (Promega) and 10-fold serial dilutions of mAb in RPMI 1640 media (starting at 100 μg/mL). After 6 h’s incubation, Bio-Glo Luciferase (Promega) was added to each well and incubated for 15 min in the dark at room temperature. The luciferase was measured by a microtiter plate reader (BMG CLARIOstar), and the data was processed by Microsoft Excel and GraphPad Prism 8.

In the prophylactic study against DQ001 virus, groups of six BALB/c mice (Vital River) at 6–8 week age received doses of 1, 5 or 20 mg/kg of intraperitoneal (i.p.) 18_14D antibody or 5, 20 mg/kg of LALA mutant antibody respectively. An equivalent volume of PBS was i.p. administered as a control. After 3 h of treatment, mice were anaesthetized with isoflurane and then intranasally inoculated with 50 μL PBS containing a 3 LD50 of DQ001 virus. For the therapeutic setting, the mice were given 5 or 20 mg/kg mAb 48 h after infection with DQ001 virus. Body weights and survival rates of all mice were monitored and recorded for 14 days. Mice that displayed 25% or more weight loss were subsequently euthanized.

Proteins were diluted to a final concentration between 0.01–0.02 mg/mL using 20 mM Tris pH 8.0, 150 mM NaCl. Samples were adsorbed to glow-discharged carbon-coated copper grids. The grids were either washed with double distilled water two times, and stained with stained directly with 1% uranyl acetate. Images were recorded at 100 k magnification in 1.02 Å/pixel. Data were collected with a JEM-1400 instrument (JEDL, Japan) equipped with an Orius SC1000A CCD camera using SerialEM79.

To prepare the cryo-EM sample of NA in complex with Fabs, the Fab regions of 18_14D and 1G01 were added at 2 times molar excess to the N6_DQ monomer and incubated on ice for 20 min. About 3 μL solution was loaded onto a glow-discharged Au grid coated with graphene, which was blotted for 3.0 seconds with a humidity of 100% at 4 °C and then plunged frozen with an FEI Vitrobot Mark IV. Cryo-EM images were automatically collected using Serial-EM software (http://bio3d.colorado.edu/SerialEM/) using beam-image shift imaging scheme. Stacks were recorded with a K2-subunit detector using the super-resolution counting mode at a pixel size of 0.82 Å. The exposure was performed with a dose rate of 10 e−1pixel−1s−1 and an accumulative dose of 60 e−1 Å2 for each micrograph, which fractionated into 32 frames. The defocus range of this dataset was roughly −1.0 to −2.5 μm.

For the dataset, 3,437 raw movies were aligned and corrected using MotionCor246 and the contrast transfer function (CTF) values of each micrograph were determined using CTFFind4. About 1,000,000 particles were picked out from 1000 micrographs using bolb picker, which were subjected to two-dimensional (2D) classification to generate good particles with different views for Topaz training47. All subsequent classification and reconstruction procedures were performed using cryoSPARC v3.148. A total of ~780,000 particles were selected from all micrographs using the Topaz model. After a round of 2D classification, about 720,000 particles were used for further heterogeneous refinement. After three rounds of heterogeneous refinement, 116,206 particles remained for non-uniform refinement and yielded a reconstruction map of 3.4 Å. To polish the resolution, we performed dose weighting using MotionCor2 by discarding the first two and last fourteen frames in each stack to generate a reduced dataset with total dose of 30 e−1 Å2 which generated a better density map of 3.2 Å. In the density map, we noticed that distal regions of CH1 and CL of two antibodies were much flexible and they had no interactions with the NA which would affect the alignment during reconstruction. Thus, we performed particle signal subtraction to focus on the NA and scFvs of two antibodies for another round of local refinement which yielded a final map with 3.0 Å resolution. The local resolution maps were evaluated by ResMap49.

The structure of NA and 1G01 complex (PDB: 6Q23) was rigidly docked into the EM density maps using CHIMERA50. The structure of 18_14D was predicted using AlphaFold2 and then fitted into the EM density maps. The side chains were manually adjusted to improve the local fit using COOT51. The coordinates were refined against corresponding maps in real space using PHENIX52, in which the secondary structure restraints and Ramachandran restraints were applied. The stereochemical quality of each model was assessed using MolProbity. Structural figures were prepared by Pymol (https://pymol.org/) and CHIMERAX53.

All statistical tests were performed as described in the indicated figure legends using GraphPad Prism software. The number of independent experiments performed is indicated in the relevant figure legends. No samples were excluded from the analysis.

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

The atomic coordinates data generated in this study have been deposited in the Protein Data Bank (PDB) database under accession code 8Z4E. The corresponding electron microscopy maps have been deposited in the Electron Microscopy Data Bank (EMDB) with the accession codes EMD-39762. Other structures for analysis, including 2CML, 4QN6, 6Q23, 6PZY, 8EZ3, 8EZ7, 8E6J, 8GAV, 4QNP, 6PZW, 6U02, were obtained from the PDB. Source data are provided as a Source Data file. Source data are provided with this paper.

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We thank all the staff members at the Center for Biological Imaging (CBI), Institute of Biophysics (IBP), Chinese Academy of Sciences (CAS) for assistance with data collection. We would like to thank Wenjuan Zhang for her help on negatively stained sampling and imaging at the Cryo-electron Microscopy Platform, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences (IGDB). We also thank Zheng Fan, Qian Wang from the Institute of Microbiology, Chinese Academy of Sciences for help in BLI and ELLA assay. This study was supported by National Natural Science Foundation of China (NSFC) (32192452 to Y.S.), the National Key Research and Development Program of China (2023YFC2307800 to M.W.), National Natural Science Foundation of China (NSFC) (81902058 to C.S., and 32100119 to Q.P.), Shenzhen High‐level Hospital Construction Fund (23250G1001 to Y.Y.) and a grant from Sanofi Pasteur company.

These authors contributed equally: Min Wang, Yuan Gao, Chenguang Shen.

CAS Key Laboratory of Pathogen Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences (CAS), Beijing, China

Min Wang, Yuan Gao, Qi Peng, Jinlong Cheng, George Fu Gao & Yi Shi

Shenzhen Key Laboratory of Pathogen and Immunity, Shenzhen Third People’s Hospital, Shenzhen, China

Min Wang, Chenguang Shen, Yang Yang & Yi Shi

Faculty of Health Sciences, University of Macau, Macau, China

Yuan Gao & Han-Ming Shen

BSL-3 Laboratory (Guangdong), Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health; Southern Medical University, Guangzhou, China

Chenguang Shen

Medical School, University of Chinese Academy of Sciences, Beijing, China

Wei Yang, George Fu Gao & Yi Shi

Beijing Life Science Academy, Beijing, China

George Fu Gao & Yi Shi

Health Science Center, Ningbo University, Ningbo, China

Yi Shi

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Y.S., G.F.G. and Y.Y. conceived the study. M.W., Y.G., C.S., W.Y., Q.P., and J.C performed the experiments. Y.S., M.W., C.S., and Y.Y. conducted the analysis. M.W., Y.G., C.S., H.S., Y.Y., and Y.S. wrote the manuscript. All authors participated in the discussion and manuscript editing. Y.S. supervised all the work.

Correspondence to Yang Yang, George Fu Gao or Yi Shi.

A patent describing potential treatments for influenza using 18_14D was in application, and the authors listed on the application are Y.S., G.F.G., M.W., Y.G., Q.P., Y.Y., C.S. The remaining authors declare no competing interests.

Nature Communications thanks the anonymous reviewers for their contribution to the peer review of this work. A peer review file is available.

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Wang, M., Gao, Y., Shen, C. et al. A human monoclonal antibody targeting the monomeric N6 neuraminidase confers protection against avian H5N6 influenza virus infection. Nat Commun 15, 8871 (2024). https://doi.org/10.1038/s41467-024-53301-6

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Received: 17 May 2024

Accepted: 09 October 2024

Published: 15 October 2024

DOI: https://doi.org/10.1038/s41467-024-53301-6

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