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 Loop01 and the loop connecting β-strands 2 and 3 is termed Loop23. The twelve 01 and 23 loops in each monomer point up and surround and contribute to the enzyme active site. b) Antigenic surface formation. 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 twelve loops and the C-terminal domain in one monomer are coloured to show the top surface surrounding the active site. The remaining four Beta strands and the Loops 12 and 34 we refer 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. 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 22.

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, 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 (https://www.crick.ac.uk/sites/default/files/2022-10/Crick%20report%20Sep2022%20for%20SH2023_to%20post.pdf). Loops 01 and 23 that form the top antigenic surface are highlighted. Loops annotation is based on Varghese et al. (see Supplementary Table 2 and supplementary Figure 4 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 mS N1 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.

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 supplementary figure 2 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 2nd column. N1/19 protein didn’t express in ExpiCHO cells in two instances. Size-exclusion chromatography graphs are shown in the 3rd column. Elution volume of 10-14 mL indicates tetrameric form of the protein. MUNANA and ELLA activity of the NA proteins are in 4th and 5th columns and the nanoDSF thermal melting temperature is in 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 Supplementary Fig. 6a) ‘+++’ 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 (see Supplementary Fig. 5b). mAb Z2C2 is specific for mSN1 and N1/09, hence did not bind N1/19 or its hybrid form.

The fold of the scaffold neuraminidase is strictly conserved in the loops-grafted hybrid proteins.

a, X-ray crystal structures of the protomers of H5N1/21 (mSN1) (pale brown), N1/09Loops-mS hybrid (N1/09 hybrid) (pale blue), and N1/19Loops-mS (N1/19 hybrid) (pale violet) are shown in cartoon representation. In the mSN1 protein, six L01 and six L23 loops are labelled and coloured in magenta and cyan blue, respectively. The C-terminal domain (CTD) is shown in pale green, and the calcium ion is shown as green sphere. Eight highly conserved residues in the catalytic site (R118, D151, R152, R225, E277, R293, R368, and Y402) surrounding oseltamivir are shown as yellow sticks. Grafted L01 and L23 residues in the N1/09 and N1/19 hybrid proteins are shown as yellow-green sticks. Residues that are the same in N1/09 and N1/19 hybrids are labelled in blue, and those that differ in violet. b, N1/09 and N1/19 hybrid structures in putty tube representation with colour and radius reflecting local RMS deviation values (generally <0.5 Å) throughout the structures, with modestly higher values in the inherently flexible B1L23 and B6L23 loops. c, Structures in surface representation displaying the active site cavities. The msN1 structures revealed heterogeneity in the active site conformation. Within a tetramer, one monomer showed a wider cavity (i) and another showed a narrower cavity (ii) due to different trajectories of the B1L23 loop (150-loop). The Y344N in N1/09 and N1/19 hybrids, compared to msN1, caused a minor, local widening of the rim of the cavity.

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 AddaVaxTM (squalene-based oil-in-water nano-emulsion). Intramuscular immunisations were done twice at the interval of three weeks and sera were harvested three 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 two 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 Supplementary Fig. 7. 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.

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) 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 2nd column. Size-exclusion chromatography graphs are in the 3rd 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 ELLA activity of the NA proteins are in 4th column 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 (see Supplementary figure 3b).) ‘+++’ 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 and showed binding to PR8Loops-mS but not the PR8 and mSLoops-PR8 (see Supplementary Fig. 6b). Similarly, NmAb-20 is a mSN1 Loops + scaffold dependent mAb. mSN1 Loops specificity is shown by NmAb-02 and NmAb-13.

NA hybrid proteins elicited loop-specific NA inhibiting antibodies and provided loop-specific protection in vivo against virus challenge.

a,b,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 AddaVaxTM (squalene-based oil-in-water nano-emulsion). Intramuscular immunisations were done twice at the interval of three weeks and sera were harvested three 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,e,f,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 two 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.