Structure-guided loop grafting improves expression and stability of influenza neuraminidase for vaccine development
- Chinese Academy of Medical Science Oxford Institute, Nuffield Department of Medicine, University of Oxford, United Kingdom
- MRC Human Immunology Unit, MRC Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, United Kingdom
- Division of Structural Biology, Centre for Human Genetics, University of Oxford, United Kingdom
- Department of Pharmacology, University of Cambridge, United Kingdom
- Graduate Institute of Immunology and Department of Paediatrics, National Taiwan University Hospital, College of Medicine, National Taiwan University, Taiwan
- Genomics Research Center, Academia Sinica, Taiwan
Figures
Figure 1 with 3 supplements
Surface antigenic loops transfer method to design NA hybrid protein.
(a) NA anatomy. Each monomer of the tetrameric NA head is composed of a six-bladed propeller-like structure. Each blade unit consists of a β-sheet composed of four β-strands connected by loops arranged in a W-shape. The loop preceding β-strand 1 is termed loop 01 and the loop connecting β-strands 2 and 3 is termed loop 23. The twelve 01 and 23 loops in each monomer point up and surround and contribute to the enzyme active site. (b) Antigenic surface formation. (i) The key antigenic surface on the top of each NA monomer is formed by the twelve 01 and 23 loops from the six β-sheet units and the C-terminal domain. The 12 loops and the C-terminal domain in 1 monomer are coloured to show the top surface surrounding the active site. The remaining four β-strands and the loops 12 and 34 are referred to as the ‘scaffold’. Oseltamivir is shown in red in the sialic acid receptor-binding site. A calcium ion at its binding site is shown as a green circle. (ii) Twelve L01 and L23 loops are coloured cyan blue and antibody epitopes as purple (L01 and L23 loops represent 90% of antibody epitopes – refer to Figure 1—figure supplement 2 for epitope mapping on the aligned sequences). Similar to b(i), oseltamivir and C-terminal domain are shown for orientation. (c) Hybrid NA design. Loops 01 and 23, and the C-terminal domain and the scaffold are shown separately. Oseltamivir and the binding residues are included for reference. The surface antigenic loops 01 and 23 of an NA of interest were transferred on to the scaffold of a high-expressing NA candidate to improve the protein yield and stability. Figures were generated with PDB 4B7J using UCSF ChimeraX (Meng et al., 2023).
Figure 1—figure supplement 1
Graphical abstract.
Figure 1—figure supplement 2
Viral neuraminidases selected for resistance to various monoclonal antibodies and sera with the positions of substitutions shown from seven published studies N1 numbering with H5N1 A/mute swan/England/053054/2021 (mSN1 as reference).
The antibody epitopes from each study are shown in a different coloured blocks with the sequence of the original NA used in these studies. Twelve L01 and L23 loops and C-terminal domain (CTD) are highlighted. The figure shows 45 examples of mAb selection experiments. Fourteen amino acid positions were selected repeatedly (13 in loops 01 and 23, 1 at 309 on the underside of the head at B4L34) by different antibodies so that in total 31 amino acid residue positions have been identified as sites for selection of resistant viruses. Among these 31 sites, 25 are in L01 and L23 or CTD as defined, 3 are immediately adjacent to L01 and L23 and could be considered as part of the top loops in future designs, and 3 sites are in underside positions: at 88 between stem and head, 285 on B4L12 and 309 on B4L34. The mSN1 sequence and the loop assignments are based on Varghese et al., 1983 and Varghese and Colman, 1991 as reference. This shows that ~90% of sites for selection by monoclonal and serum antibodies of resistant viruses reside within the L01, L23, and CTD. Figure was generated using Geneious Prime (Colman et al., 1983, Webster et al., 1987, Saito et al., 1994, Wan et al., 2013, Chen et al., 2018, Kirkpatrick Roubidoux et al., 2022, Strohmeier et al., 2022).
Figure 1—figure supplement 3
Sequence alignment of various neuraminidases showing the contact residues for various mAbs, as defined by cryo-EM or crystal structures.
mAb-binding epitopes from each study are shown in different coloured blocks with the sequence of the original NA used in these studies. The msN1 sequence is included as a reference for sequence alignment, and twelve L12 and L23 loops and the C-terminal domain are highlighted. mAbs NDS.1 and NDS.3 are described as antibodies towards underside loops. However, they form some interactions with L01 and L23 as well. This shows that the majority of key antigenic sites are located within the surface Loops 01 and 23, with some on the underside of the NA head, confirming the data from selections of resistant viruses by NA-specific MAbs. Figure was generated using Geneious Prime. References: FNI9 (Momont et al., 2023), NC41 (Colman et al., 1987; Tulip et al., 1992; Air et al., 1990), NC10 (Tulip et al., 1994; Malby et al., 1994), Z2B3 (Jiang et al., 2020), CD6 (Wan et al., 2015), 1G01 (Stadlbauer et al., 2019; Lederhofer et al., 2024), and NDS.1 and NDS.3 (Lederhofer et al., 2024).
Figure 2 with 2 supplements
NA protein sequence alignment highlighting surface loops and active site.
(a) Protein sequence alignment of the scaffold of N1 from H5N1 A/mute swan/England/053054/2021 (mS), and loop donors H1N1 A/California/7/2009 (N1/09) and A/Wisconsin/588/2019 (N1/19) neuraminidases. The N2 sequence H2N2 A/Tokyo/3/1967 from Varghese et al., 1983; Varghese and Colman, 1991, that was used to define the loops (PDB 1NN2) is also included. mSN1 is used as a reference sequence and identical residues are shown as dots. The sequence conservation is shown by green bars. The numbering here is based on N1 numbering as used in the annual Crick Reports. Loops 01 and 23 that form the top antigenic surface are highlighted. Loops annotation is based on Varghese et al. (Supplementary file 2 and Figure 2—figure supplement 1 for detailed information). Residues that form the catalytic site (8 residues) and conserved framework residues for the catalytic site (11 residues) of the enzymatic cavity are annotated with red and orange bars respectively. These residues are highly conserved between NA subtypes and the majority are part of the surface loops - 7/8 catalytic residues and 8/11 catalytic site framework residues. Two catalytic site framework residues are at the edge of loop B2L01. The figure was generated using Geneious Prime. (b) Number of amino acid differences for the N1/09 and N1/19 loop donors and the mSN1 loop recipient are shown. Twelve dissimilar residues within Loops 01 and 23 of N1/09 were transferred to mSN1 scaffold to form N1/09 hybrid. Similarly, 16 dissimilar residues within Loops 01 and 23 of N1/19 were transferred to mS N1 scaffold to form N1/19 hybrid.
Figure 2—figure supplement 1
Top loops 01 and 23 of neuraminidase, as defined by the first NA structure publications.
(a–c) Loops annotations in Varghese et al., 1983 and Varghese and Colman, 1991 (Colman et al., 1983) on PDB 1NN2 N2 NA sequence. Figures were downloaded and adapted from https://pdbj.org/ (b, c). (d, e) Loops were visualised and coloured on PDB 1NN2 structure using Chimera X1.4.
Figure 2—figure supplement 2
Comparison with the optimised N1 sequence by Ellis et al.
Ten residues mutated in N1/09 (construct N1-CA09-sNAp-155 with VASP tetramerisation domain) for favouring closed conformation by Ellis et al. are highlighted. All 10 residues are unaltered in our msN1 and N1/09 hybrid proteins. Figures were created using Geneious Prime.
Figure 3 with 3 supplements
Characteristics of NA hybrid proteins including epitope specificity.
(a) Characteristics of NA proteins. NA proteins were expressed in a transient mammalian ExpiCHO expression system. Gene constructs including affinity purification tags, SpyTag and an artificial tetramerisation domain tetrabrachion at N-terminus were cloned in the pcDNA3.1- vector. Proteins were purified using the 6His tag and Nickel-sepharose HisTrap purification columns. See Figure 3—figure supplement 1 for constructs design and SDS–PAGE of purified proteins. N1/09 hybrid means residues of loops 01 and 23 of N1/2009 grafted to mSN1 (H5N1/2021) scaffold. N1/19 hybrid means loops 01 and 23 of N1/2019 grafted to mSN1 (H5N1/2021) scaffold. The expression yield of NA proteins and their hybrid forms is included in the second column. N1/19 protein expression was undetectable to low in ExpiCHO cells in two instances. Size-exclusion chromatography graphs are shown in the third column. Elution volume of 10–14 ml indicates tetrameric form of the protein. MUNANA and enzyme-linked lectin assay (ELLA) activity of the NA proteins are in fourth and fifth columns with EC50 (effective concentration 50%) and AUC (area under curve) values, and the nanoDSF thermal melting temperature is in the final column. The sharp narrow peak and the higher melting temperature indicate higher protein stability. (b) Epitope specificity has been transferred with loops. Human monoclonal antibodies, previously published and some new, were titrated for ELISA binding of NA proteins. Areas under the curve were ranked after normalisation with one of the strongest binding mAb (see Figure 3—figure supplement 3) ‘+++’ denotes >70% binding, ‘++’ 40–70% binding, ‘+’ 10–40%, and ‘-’ <10% as a non-binder. Loops cross-reactive mAbs AG7C, AF9C, Z2B3, and 1G01, all defined by crystal structures, show full binding to all NA proteins. Seven mAbs (NmAb) do not bind mSN1 but bind to N1/09, N1/19 and their hybrid forms. NmAb-03 is specific to N1/19 surface loops. mAb CD6 is a scaffold-dependent mAb that shows binding to N1/19 hybrid protein but does not bind the N1/19 protein on the cell surface. mAb Z2C2 is specific for mSN1 and N1/09, hence did not bind N1/19 or its hybrid form.
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Figure 3—source data 1
- https://cdn.elifesciences.org/articles/105317/elife-105317-fig3-data1-v1.xlsx
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Figure 3—source data 2
mAbs binding data related to Figures 3b and 6c.
- https://cdn.elifesciences.org/articles/105317/elife-105317-fig3-data2-v1.xlsx
Figure 3—figure supplement 1
Neuraminidase expression constructs design and protein characterisation.
(a) Gene constructs for NA proteins. All the constructs are the same except for N1/09 which uses the Ig Kappa signal sequence, contains extra residues 69–81 in the NA head and has no Strep tag II. (b) NA and hybrid proteins were expressed as tetramers. Purified proteins on SDS–PAGE with Coomassie staining, in reducing conditions and cross-linked before loading on the gel. 2 μg of NA protein was incubated with 6 mM BS3 cross-linking reagent for 30 min and the reaction was stopped by adding 1 M Tris-HCl pH 8.0. Proteins were separated on reducing 4–12% Bis-Tris SDS–PAGE and 1x MES SDS buffer. (c) Inhibition of enzyme activity of NA hybrid proteins by small molecule inhibitors and mAbs. Oseltamivir and zanamivir inhibit the function of mSN1, N1/09, and N1/19 hybrid proteins, suggesting the active site is intact and functional. mAb 1G01 (Stadlbauer et al., 2019) inhibited mSN1 and N1/09 hybrid, suggesting the cross-reactive epitopes recognised by 1G01 have been preserved. 1G01 fails to inhibit N1/19 due to substitution at N222K (see Figure 2 alignment), and this has been confirmed by the loss of inhibition of NA activity of N1/19 hybrid. (d) Coupling of NA to mi3 virus-like particles (VLPs) using SpyCatcher technology. SDS–PAGE showing the NA + mi3, NA, and mi3. Two μg mi3 with SpyCatcher003 tag covalently linked to 5 μg NA (molar ratio 1:0.4). These NA-VLPs were used for mouse immunisations.
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Figure 3—figure supplement 1—source data 1
PDF file containing reducing SDS–PAGE for Figure 3—figure supplement 1, indicating the relevant lanes.
- https://cdn.elifesciences.org/articles/105317/elife-105317-fig3-figsupp1-data1-v1.zip
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Figure 3—figure supplement 1—source data 2
PDF file containing BS3-cross-linked reducing SDS–PAGE for Figure 3—figure supplement 1, indicating the relevant lanes.
- https://cdn.elifesciences.org/articles/105317/elife-105317-fig3-figsupp1-data2-v1.zip
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Figure 3—figure supplement 1—source data 3
Original files for SDS–PAGE displayed in Figure 3—figure supplement 1.
- https://cdn.elifesciences.org/articles/105317/elife-105317-fig3-figsupp1-data3-v1.zip
Figure 3—figure supplement 2
Epitope and binding pattern of mAb CD6.
(a) Binding by mAb CD6 is predominantly scaffold dependent and occurs across two protomers. Epitope recognised by mAb CD6 on H1N1/09 (PDB 4QNP). mAb CD6 20 forms interactions with two monomers, indicated with olive green and light green. msN1 sequence is used as a reference showing Loops 01 and 23. Interactions with monomer A (olive green) are all on the NA scaffold, whereas interactions with monomer B (light green) are on the top surface Loops and scaffold. CD6 binds to N1/09 and mSN1 but not to N1/19. N1/19 escape from mAb CD6 is likely due to one or more of the following substitutions within the epitope - L269M (B4L01), N270K (B4L01), V389K (B5S4), and N449D (B6L34). However, the N1/19 hybrid that shares two of these substitutions L269M (B4L01) and N270K (B4L01) retained binding, which suggests that loss of binding to N1/19 was due to V389K (B5S4) and or N449D (B6L34). (b) Recognition of N1/09 NA protein expressed as tetramers with VASP or tetrabrachion tetramerisation domains. Note that CD6 did not bind to N1/09-VASP and but did bind to N1/09-TB relatively weakly compared to mAbs AG7C 60 and 1G01 13 that (unlike CD6) bind within a single monomer.
Figure 3—figure supplement 3
Binding titration of mAbs against recombinant soluble proteins or NA expressed on the surface of virus infected cells.
(a) Twenty-five anti-N1 mAbs were titrated for binding against mSN1, N1/09, and N1/19 and their loop transferred hybrid variants. (b) Binding titration of 18 mAbs that bind either PR8 N1 or mSN1 on wild-type sequence proteins and their loop-grafted hybrid proteins. Area under curve (AUC) was calculated and normalised against one of the strongest binders to rank the order.
Figure 4
The structure of the influenza virus neuraminidase is strictly conserved in the loop-grafted hybrid proteins.
(a) X-ray crystal structures. (Top) The influenza virus neuraminidase is shown in the native tetrameric form, an assembly observed in all reported crystal structures. A schematic representation of the stalk and a membrane is shown. (Bottom) Structures of each crystallized construct viewed from above the β-propeller fold (cartoon representation). (Left) Crystal structure of a protomer of the mSN1 neuraminidase. The β-strands of the H5N1/21 protomer (mSN1, left) are coloured pale brown, with the B6L01 and B6L23 loops coloured magenta and cyan blue, respectively. The C-terminal domain (CTD) is shown in pale green, and the calcium ion is depicted as a green sphere. Eight highly conserved residues in the catalytic site (R118, D151, R152, R225, E277, R293, R368, and Y402, N1 numbering based on msN1) are shown as yellow sticks. (Middle) Crystal structure of a protomer of the N1/09 loops-mS hybrid (pale blue). (Right) Crystal structure of a protomer of the N1/19 loops-mS hybrid (pale violet). B6L01 and B6L23 residues grafted into the N1/09 and N1/19 hybrid proteins are shown as yellow–green sticks. Residues identical in the N1/09 and N1/19 hybrids are labelled in blue; residues differing between these hybrids are labelled in violet. (b) Mapping structural differences between N1/09 and N1/19 hybrid structures. Local root-mean square (RMS) deviations between equivalent Cα pairs are mapped following the overlay of N1/09 (left) and N1/19 (right) onto the crystal structure of msN1. The b-propeller of msN1 is shown in a putty tube representation, with colour and radius reflecting the local RMS deviation values between equivalent Cα pairs. RMS deviations are generally below 0.5 Å, with modestly higher values in the inherently flexible B1L23 and B6L23 loops. (c) Active site cavities. The msN1 surface is coloured brown, except for the C-terminal loop region (green). The N1/09 and N1/19 surfaces are in blue and violet, respectively. In mSN1, the rim of the cavity containing the active site is traced with a blue dashed line. The position of the active site is denoted by a sialic acid molecule (grey–blue sticks), taken from a superposed, related structure (PDB ID: 2BAT). The msN1 structure revealed differences among the protomers of the tetramer at the active site entrance. Within the same tetramer, protomers with a relatively narrow cavity (i) combine with protomers showing a wider entrance (ii). This difference is dictated by the trajectory of the B1L23 loop (150-loop; dark blue with yellow side chains). The variation in trajectory between (i) and (ii) is most pronounced at Val149 (red surface). In contrast, the N1/09 and N1/19 hybrids show no noticeable differences between the protomer cavities, all of which closely resemble the wider msN1(ii) conformation, apart from minor, local widening of the rim induced by the substitution of Y344 with an asparagine residue (shown as light-green sticks).
Figure 5 with 1 supplement
NA hybrid proteins are immunogenic and provide in vivo protection against virus challenge.
(a) Immunogenicity of NA hybrid proteins. BALB/c mice (n = 6/group) were immunised with 0.5 μg NA coupled to the mi3 virus-like particles (NA-VLP) adjuvanted with 1:1 vol/vol AddaVax (squalene-based oil-in-water nano-emulsion). Intramuscular immunisations were done twice at the interval of 3 weeks and sera were harvested 3 weeks post booster dose to assess the antibody response. Neuraminidase activity inhibition (NAI) IC50 titres measured using fetuin-based enzyme-linked lectin assay (ELLA) are shown as a separate dot for each mouse. mAb AG7C was used as a positive control. Pooled sera to unconjugated mi3 VLP was negative control and showed no inhibition at 1:40 dilution (not included here). Geometric mean with 95% confidence interval is shown. (b) Protection against virus challenge. DBA/2 mice (n = 6/group) were immunised as above and were challenged with intranasally administered 200 LD50 of H1N1/2009 (X-179A) virus. Weights were monitored for 2 weeks. Loss of ≥20% initial weight was considered an endpoint. mAb AG7C (10 mg/kg prophylaxis) was used as a positive control. Empty VLP pooled sera was a negative control and mice reached the endpoint within day 5–7 post virus infection. The ELLA NA inhibition graphs of these pooled sera are shown in Figure 5—figure supplement 1. Figures were made using GraphPad Prism v10. Kruskal–Wallis test was used for statistical analysis. ns: non-significant (p-value >0.05), ** means p-value <0.005. Kaplan–Meier survival analysis was done with logrank Mantel–Cox test for comparison.
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Figure 5—source data 1
Mouse sera titration data on enzyme-linked lectin assay (ELLA).
- https://cdn.elifesciences.org/articles/105317/elife-105317-fig5-data1-v1.xlsx
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Figure 5—source data 2
Weight curves of mice following X-179A virus challenge.
- https://cdn.elifesciences.org/articles/105317/elife-105317-fig5-data2-v1.xlsx
Figure 5—figure supplement 1
Inhibition of NA activity by mouse antisera measured using ELLA.
(a–c) Pooled sera from immunised mice (n = 6) in Figure 5a (BALB/c mice) and Figure 5b (DBA/2 mice) were titrated in enzyme-linked lectin assay (ELLA). Naive sera and sera from mice immunised with empty VLP were used as negative controls. mAb AG7C was used as a positive assay control. H1N1/2009 virus = X-179A (A/California/07/2009); H1N1/2021 = A/Sydney/5/2021 (differs from N1/19 only by V453M in the C-terminal domain), mSN1 = NA from H5N1 A/mute swan/England/053054/2021. (d) Binding of sera antibodies to H1N1/2009 infected cells, for samples related to Figure 5b and (a) is compared. Similar to the ELLA titres, the binding titres for msN1 are lower, indicating that the protective response must be from the ELLA inhibiting antibodies. AUC: area under curve of the titration curve.
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Figure 5—figure supplement 1—source data 1
Mouse sera titration (pooled sera) on enzyme-linked lectin assay (ELLA).
- https://cdn.elifesciences.org/articles/105317/elife-105317-fig5-figsupp1-data1-v1.xlsx
Figure 6 with 1 supplement
Loop grafting between two distant N1 NAs: H5N1 A/mute swan/England/053054/2021 (mS) and H1N1 A/PR/8/1934 (PR8).
(a) Number of amino acid differences between mSN1 and PR8 N1 and their loop-grafted hybrids are shown. Eighteen dissimilar residues (5%) within loops 01 and 23 were grafted to make the hybrid proteins. (b) Characteristics of proteins. NA proteins were expressed in a transient mammalian ExpiCHO expression system. The expression yield of NA proteins and their hybrid forms is included in the second column. Size-exclusion chromatography graphs are in the third column. Elution volume of 10–14 ml indicates tetrameric form of the protein and 14–15 ml indicates trimeric or dimeric nature of the protein. MUNANA and enzyme-linked lectin assay (ELLA) activity of the NA proteins are in fourth columns and the nanoDSF thermal melting temperature is in the final column. The sharp narrow peak and the higher melting temperature indicate the higher protein stability. (c) Epitope specificity has been transferred with loops with a few exceptions. Antibodies, previously published and some new, were titrated for ELISA binding of NA proteins. Area under curve was ranked after normalisation with one of the strongest binding mAb (refer to Figure 3—figure supplement 3b for binding titration data). ‘+++’ denotes >70% binding, ‘++’ 40–70% binding, ‘+’ 10–40%, and ‘-’ <10% as a non-binder. Loops cross-reactive mAbs recognised both mSN1 and PR8N1 and their hybrid proteins. PR8N1 loops specificity is shown by NmAb-03. mAb CD6 is a major scaffold-dependent mAb (see Figure 3—figure supplement 2) and showed binding to PR8Loops-mS but not the PR8 and mSLoops-PR8. Similarly, NmAb-20 is a mSN1 Loops + scaffold-dependent mAb. mSN1 loops specificity is shown by NmAb-02 and NmAb-13.
Figure 6—figure supplement 1
Loop transfer between two distant N1 NAs: H5N1 A/mute swan/England/053054/2021 (mS) and H1N1 A/PR/8/1934 (PR8).
Twelve loops 01 and 23 in an NA head of a monomer are highlighted within the sequence alignment of mSN1, PR8 N1, and loops-exchanged NA hybrid proteins. There is a residue difference at position 454 in the C-terminal domain (CTD) between mSN1 and PR8N1. CTD appears at the monomeric interface and can form a part of antigenic surface. However, this residue appears not to be solvent exposed and hence was not considered for transfer. Figure was created using Geneious Prime. Refer to Figure 6.
Figure 7
NA hybrid proteins elicited loop-specific NA inhibiting antibodies and provided loop-specific protection in vivo against virus challenge.
(a–c) Immunogenicity of NA hybrid proteins. BALB/c mice (n = 6/group) were immunised with 0.5 μg NA coupled on to the mi3 virus-like particles (NA-VLP) adjuvanted with 1:1 vol/vol AddaVax (squalene-based oil-in-water nano-emulsion). Intramuscular immunisations were done twice at the interval of 3 weeks and sera were harvested 3 weeks post booster dose to assess the antibody response. Neuraminidase activity inhibition (NAI) IC50 titres measured using fetuin-based enzyme-linked lectin assay (ELLA) are shown as a separate dot for each mouse. mAb AG7C was used as a positive control. Empty VLP pool sera were negative controls and showed no inhibition at 1:40 dilution (not included here). Geometric mean with 95% confidence interval is shown. (d–g) Protection against virus challenge. BALB/c mice (n = 6/group) were immunised as above. Pooled sera antibodies were assessed in ELLA assay before virus challenge (e) and IC50 values shown are sera reciprocal dilution. Mice were challenged with intranasally administered 1000 LD50 of PR8 virus (Cambridge strain, 104 TCID50). Weights were monitored for 2 weeks. Loss of ≥20% initial weight was considered an endpoint. mAb AG7C (10 mg/kg prophylaxis) was used as a positive control. Empty VLP pool sera were negative controls and mice reached the endpoint by day 6 post virus infection. Importantly, immunogens with PR8 Loops protected 100% mice from virus challenge, and mS loops did not. Mean and standard deviations are shown. Figures were made using GraphPad Prism v10. Kruskal–Wallis test was used for statistical analysis. ns: non-significant (p-value >0.05), *** denotes p-value <0.0005. Kaplan–Meier survival analysis was used with logrank Mantel–Cox test for comparison.
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Figure 7—source data 1
Mouse sera titration data on enzyme-linked lectin assay (ELLA).
- https://cdn.elifesciences.org/articles/105317/elife-105317-fig7-data1-v1.xlsx
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Figure 7—source data 2
Mouse sera titration (pooled sera) on enzyme-linked lectin assay (ELLA).
- https://cdn.elifesciences.org/articles/105317/elife-105317-fig7-data2-v1.xlsx
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Figure 7—source data 3
Weight curves of mice following PR8 virus challenge.
- https://cdn.elifesciences.org/articles/105317/elife-105317-fig7-data3-v1.xlsx
Appendix 1—figure 1
Power analysis for animal experiments.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | 1G01 (anti-NA human monoclonal) | Chen et al., 2018 | PDB: 6Q23 | 10 µg/ml |
| Antibody | NmAbs, 24-1C (anti-NA human monoclonals) | In-house (Kuan-Ying Huang) | 20 µg/ml | |
| Antibody | Goat-anti-mouse immunoglobulins HRP (polyclonal) | Dako | P0447 | ELISA (1:800) |
| Antibody | Rabbit-anti-human IgG HRP (polyclonal) | Dako | P0214 | ELISA (1:1600) |
| Antibody | AG7C (anti-NA human monoclonal) | Rijal et al., 2020 | 20 µg/ml | |
| Antibody | AF9C (anti-NA human monoclonal) | Rijal et al., 2020 | 20 µg/ml | |
| Antibody | CD6 (anti-NA chimeric-human monoclonal) | Wan et al., 2015; this paper | PDB: 4QNP | 20 µg/ml |
| Antibody | Z2B3 (anti-NA human monoclonal) | Rijal et al., 2020; Jiang et al., 2020 | PDB: 6LXI | 20 µg/ml |
| Cell line (Canis lupus familiaris) | MDCK-SIAT1 | ECACC | RRID:CVCL_Z936 | |
| Cell line (Cricetulus griseus) | ExpiCHO (Chinese hamster ovary) | Thermo Fisher | A29127; RRID:CVCL_5J31 | |
| Chemical compound, drug | N-tosyl-l-phenylalanine chloromethyl ketone (TPCK)-trypsin | Sigma-Aldrich | T1426 | 0.75–1 µg/ml |
| Chemical compound, drug | KPL SureBlue (TMB substrate) | SeraCare | 5120-0077 | |
| Chemical compound, drug | Pierce BS-3 | Thermo Fisher | A39266 | 6 mM |
| Chemical compound, drug | MUNANA [0-(4-methylumbelliferyl)541-a-D-N-acetylneuraminic acid] | Sigma-Aldrich | 69587 | 100 µM |
| Chemical compound, drug | AddaVax | InvivoGen | vac-adx-10 | 1:1 vol/vol |
| Chemical compound, drug | Oseltamivir | Sigma-Aldrich | TA9H9A9A73CA | 500 nM |
| Chemical compound, drug | Zanamivir | Sigma-Aldrich | SML0492 | 500 nM |
| Commercial assay or kit | QIAGEN Plasmid Maxi Kit | QIAGEN | 12162 | |
| Commercial assay or kit | HisTrap HP His tag protein purification columns (5 ml) | Cytiva | 17524801 | |
| Commercial assay or kit | Zeba Spin Desalting Columns, 7K MWCO | Thermo Fisher Scientific | 89889, 89891 | |
| Commercial assay or kit | Expifectamine CHO transfection kit | Thermo Fisher | A29129 | |
| Peptide, recombinant protein | Fetuin | Sigma-Aldrich | F3385 | 25 µg/ml |
| Peptide, recombinant protein | PNA–HRP (peanut agglutinin conjugated to horseradish peroxidase) | Sigma-Aldrich | L7759 | |
| Peptide, recombinant protein | SpyCatcher003-mi3 virus-like particles | Ingenza Ltd | Lot No. 108-23-001-007 | |
| Recombinant DNA reagent | pcDNA3.1- (plasmids) expressing NA | This paper | Cloned between NotI and EcoRI sites | |
| Strain, strain background (Escherichia coli) | DH5α Competent E. coli (High Efficiency) | NEB | C2987; RRID:CVCL_R748 | Electrocompetent cells |
| Strain, strain background (Influenza A) | A/PR/8/1934 (Cambridge) | WHO Influenza Centre (Crick, London) | ||
| Strain, strain background (Influenza A) | X-179A | NIBSC | 14/116 | |
| Strain, strain background (Influenza A) | A/Sydney/5/2021 | WHO Influenza Centre (Crick, London) | ||
| Strain, strain background (Mus musculus; female) | BALB/cOlaHsd | Envigo (inotiv) | Order code: 162 | |
| Strain, strain background (Mus musculus; female) | DBA/2OlaHsd | Envigo (inotiv) | Order code: 870 | |
| Commercial assay or kit | Morpheus II crystallisation screen | Molecular Dimensions | MD1-91 | 96-Reagent screen |
| Commercial assay or kit | Poly gamma glutamic acid crystallisation screen | Molecular Dimensions | MD1-51 | 96-Reagent screen |
| Commercial assay or kit | Pentaerytritol crystallisation screen | Jena Bioscience | CS-210L | 96-Reagent screen |
| Chemical compound, drug | Glycerol | Sigma-Aldrich | G7757 | |
| Software | Xia2 programme suite | Winter et al., 2013 | X-ray data processing software | |
| Software | autoProc/StarAniso | Vonrhein et al., 2018 | X-ray data processing software | |
| Software | Phaser | McCoy et al., 2007 | X-ray data processing software | |
| Software | Coot | Emsley et al., 2010 | Model building and refinement software | |
| Software | Phenix | Liebschner et al., 2019 | Model building and refinement software | |
| Software | ChimeraX | Meng et al., 2023 | Software for structure visualization and analysis |
Additional files
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Supplementary file 1
Recombinant NA expression in various cell expression systems.
- https://cdn.elifesciences.org/articles/105317/elife-105317-supp1-v1.docx
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Supplementary file 2
List of loop annotations in Varghese et al., 1983 used as a reference for creating NA hybrids in this paper.
Loops in mSN1, N1/09, N1/19, and PR8N1 are listed. Text in orange colour where N1 differs from the original N2 numbering system. Residues in the loops that differ from mSN1 sequence are marked in red (‘aa’ denotes amino acids).
- https://cdn.elifesciences.org/articles/105317/elife-105317-supp2-v1.docx
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Supplementary file 3
Crystallographic data collection and refinement statistics.
- https://cdn.elifesciences.org/articles/105317/elife-105317-supp3-v1.docx
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Supplementary file 4
Amino acid sequences of NA gene constructs.
- https://cdn.elifesciences.org/articles/105317/elife-105317-supp4-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/105317/elife-105317-mdarchecklist1-v1.docx
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Structure-guided loop grafting improves expression and stability of influenza neuraminidase for vaccine development
eLife 14:RP105317.
https://doi.org/10.7554/eLife.105317.3
