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Published in final edited form as: Science. 2022 Mar 3;375(6587):1373–1378. doi: 10.1126/science.abm5561

Architecture and antigenicity of the Nipah virus attachment glycoprotein

Zhaoqian Wang 1, Moushimi Amaya 2,3, Amin Addetia 1, Ha V Dang 1, Gabriella Reggiano 1, Lianying Yan 2,3, Andrew C Hickey 2,4, Frank DiMaio 1, Christopher C Broder 2, David Veesler 1,5,*
PMCID: PMC12303162  NIHMSID: NIHMS2096797  PMID: 35239409

Abstract

Nipah virus (NiV) and Hendra virus (HeV) are zoonotic henipaviruses (HNVs) responsible for outbreaks of encephalitis and respiratory illness. HNVs entry into host cells requires the attachment (G) and fusion (F) glycoproteins which are the main targets of antibody responses. To understand viral infection and host immunity, we determined a cryo-electron microscopy structure of the NiV G homotetrameric ectodomain in complex with the nAH1.3 broadly neutralizing antibody Fab fragment. We show that a cocktail of two non-overlapping G-specific antibodies neutralizes NiV and HeV synergistically and limits the emergence of escape mutants. Analysis of polyclonal serum antibody responses elicited by vaccination of macaques with NiV G indicates that the receptor-binding head domain is immunodominant. These results pave the way for implementing multi-pronged therapeutic strategies against these deadly pathogens.

Nipah virus (NiV) and Hendra virus (HeV) are bat-borne zoonotic pathogens of the Henipavirus (HNV) genus causing encephalitis and respiratory symptoms in humans with fatality rates between 50% and 100% (1). Over the past two decades, NiV has spilled over into humans almost annually in Bangladesh (Bangladesh strain, NiV-B) (2) and has also caused outbreaks in India (NiV-B) and the Philippines (Malaysia strain, NiV-M) (3–5). The detection of cross-reactive HNVs antibodies (Abs) in humans and Pteropus bats in Africa underscored that 2 billion people worldwide live in regions threatened by HNV spillovers (6). Moreover, the recent discovery of a new HeV genotype (7, 8) is an urgent reminder of the HNV zoonotic threat. To date, no approved vaccines or therapeutics for use in people exist against HNV infections (9).

HNV entry into host cells requires fusion of the viral membrane and the host plasma membrane through the concerted action of an attachment (G) glycoprotein and a fusion (F) glycoprotein. The NiV and HeV entry receptors at the surface of host cells are the transmembrane protein tyrosine kinases ephrin-B2 or ephrin-B3 (10–13). G and F have been proposed to undergo a cascade of conformational changes to promote membrane fusion upon receptor engagement (14, 15). HNV G and F are the targets of the humoral immune response (16–19) and serum neutralizing antibodies (Abs) are a correlate of protection in animals experimentally infected with NiV or HeV (20–23). The cross-reactive NiV/HeV G-specific m102.4 monoclonal Ab (mAb) has been administered on an emergency compassionate use basis to 15 individuals with high risk exposure to HeV or NiV infection and recently completed a phase I clinical trial in Australia (24).

A soluble HeV G tetrameric ectodomain immunogen (HeV sG) elicits high titers of cross-reactive neutralizing Abs and protects against both NiV and HeV challenge in preclinical studies (20, 25–30). A vaccine using HeV sG is commercially available for use in horses in Australia (Equivac® HeV, Zoetis Inc.), and a formulation suitable for human use has recently entered phase I clinical trials (NCT04199169). Although crystal structures of the isolated NiV and HeV G head domains were previously determined (10, 13), no structural information is available for any HNV G tetramer, hindering both our understanding of immunity directed towards G and the rational design of improved vaccine candidates.

Results

Architecture of the NiV G tetramer

To unveil the 3D organization of the HNV G protein and provide a blueprint for vaccine design, we determined a cryoEM structure of the NiV G ectodomain homotetramer bound to the mouse nAH1.3 neutralizing antibody Fab fragment (31) at 3.5Å overall resolution (Figure S1). We used local refinement to account for the flexibility of the viral membrane proximal region relative to the distal region, yielding reconstructions at 3.5Å and 3.2Å resolution, respectively, allowing us to build a (composite) model of the NiV G ectodomain tetramer (Figure 1A-C, Figure S1 and Table S1). The final model comprises nearly the entire NiV sG ectodomain from residues 96 to 602 along with the nAH1.3 Fab variable domains. NiV G forms a 200Å long and 120Å wide intertwined homotetramer. At the core is an N-terminal 4-helix bundle (stalk) followed by an interlaced β-sandwich (designated neck) that connects to a C-terminal β-propeller head domain on each protomer (Figure 1A-C). The NiV G tetramer is stabilized by inter-protomer disulfide bonds formed in the neck and in the stalk (Figure 1D).

Figure 1. Architecture of the NiV G homotetramer.

Figure 1.

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(A) Linear representation of the NiV G ectodomain (as resolved in the cryoEM map), which contains an N-terminal stalk (residue 96 to 147), a neck domain (residue 148 to 165), a linker region (residue 166 to 177) and a C-terminal Head domain (residue 178 to 602). Green arrows indicate N-linked glycosylation sites. Yellow lines refer to cysteine residues. (B-C) Ribbon diagram of the NiV G ectodomain bound to the broadly neutralizing nAH1.3 Fab fragment in two orthogonal orientations. Each of the four NiV G protomers is colored distinctly and resolved N-linked glycans are rendered as green surfaces. The nAH1.3 heavy and light chains are colored gold and yellow, respectively, and only the variable domains were modeled in density. The linkers connecting the neck to the two proximal head domains are shown as dashed lines due to weaker density in the cryoEM reconstruction. (D) Zoomed-in view of the interlaced β-sandwich neck domain showing the four antiparallel inter-protomer disulfide bonds between residues C158 and C162 and the glycan at position N159 protruding from the two chains shown in the foreground. (E) Superimposition of NiV G protomers based on the stalk highlighting that the same polypeptide chain adopts three distinct folds in the homotetrameric assembly. (F) Schematic representation of the NiV G homotetramer showing that only one out of four head domains orients its receptor-binding site (arrow) towards the host cell membrane (light grey) whereas the other three sites point towards the viral membrane (dark grey). EB2: ephrin-B2.

NiV G residues 96–147 form a pseudo 2-fold symmetric helical bundle, which is presumably extended at its N-terminal end towards the viral membrane in which the tetramer is anchored (residues 70–95 are weakly resolved in the cryoEM map and were not modeled) (Figure 1A-C). The interlaced β-sandwich neck domain is formed by apposition of two 4-stranded mixed β-sheets, packed against each other through hydrophobic contacts, comprising residues 153–163 from each of the four protomers (Figure 1D). The neck is covalently cross-linked through formation of two antiparallel inter-protomer disulfide bonds between residues C158 and C162 of chains A and D and two antiparallel disulfides between residues C158 and C162 of chains B and C. The neck is decorated with four N-linked glycans protruding from residue N159 on each protomer, partially shielding this domain on both sides of the β-sandwich (Figure 1D). Each of the four C-terminal head domains form a 6-bladed β-propeller decorated with five N-linked oligosaccharides at positions N306, N378, N417, N481 and N529, which are all resolved in the cryoEM map (Figure 1A-C). Two head domains are connected to the neck through flexible linkers folding back towards the viral membrane with the β-propellers docked on either side of the stalk (Figure 1B-C). The other two head domains form a head-to-tail dimer positioned distal to the viral membrane and are connected to the neck through linkers that adopt distinct folds (Fig 1B-C). As a result, the NiV G tetramer is assembled from four identical polypeptides adopting three distinct folding patterns (Fig 1E). Interactions between the two distal head domains maintain their approximately antiparallel orientation whereas contacts between each proximal head and the NiV G stalk lead to their pseudo-2-fold symmetrical arrangement. Only one out of four head domains orients its receptor-binding site towards the host cell surface whereas the other three sites point towards the viral membrane (Fig 1F), suggesting that conformational dynamics would allow reorienting the head domains for receptor engagement. Although the cryoEM map does not resolve the topology of the two stalk C146 disulfide bonds unambiguously, previous biochemical data suggest they form across pairs of protomers that are not covalently cross-linked in the neck (25, 32). The overall NiV G architecture described here adopts a unique two heads up and two heads down conformation that is different from structures of any other paramyxovirus attachment glycoproteins. As a comparison, Newcastle disease virus HN adopts a four heads down conformation whereas parainfluenza virus 5 HN exhibits a distinct two heads up and two heads down conformation with the receptor binding sites oriented differently than for NiV G (33, 34) (Figure S2).

nAH1.3-mediated broad neutralization of NiV and HeV

The nAH1.3 mAb was previously shown to potently neutralize NiV-M, NiV-B and HeV in vitro (31). Using a green fluorescent protein (GFP)-encoding, replication-competent, Cedar (henipa)virus (rCedV) chimeras in which the native glycoproteins are substituted with the NiV-B (rCedV-NiV-B-GFP) or the HeV (rCedV-HeV-GFP) F and G glycoproteins (35), we determined nAH1.3 half-maximum inhibitory concentrations (IC50) of 33 ng/mL and 32 ng/mL, respectively (Figure S3A). This neutralization potency is comparable to the human mAb m102.4 for which we determined IC50 values of 17 ng/mL and 58 ng/mL against rCedV-NiV-B-GFP and rCedV-HeV-GFP, respectively (Figure S3B). Our cryoEM structure reveals that one nAH1.3 Fab fragment is bound to each NiV G head domain (Figure 1B-C and Figure 2A). nAH1.3 heavy and light chains interact with an antigenic site located on the side of the β-propeller that is opposite to the ephrin-B2/B3-binding site (Figure 2A). As a result, nAH1.3 does not compete with receptor binding to the NiV G ectodomain (Figure 2B), unlike m102.4 which forms interactions mimicking receptor interactions (18). nAH1.3 buries ~1,000 Å2 of its paratope upon binding through shape complementarity and hydrogen bonding involving complementary determining regions (CDR) H1, H3, L1 and L2, explaining its high affinity binding to NiV G (Figure 2C-D and Figure S3C). nAH1.3 is conformation-dependent and recognizes a discontinuous epitope, spanning residues 172, 182–191, 358, 448–451, 468–478, 515–518 and 570–571, which is in close proximity to the oligosaccharide at position N481 (Figure 1A and Figure 2A). 21 out of 26 epitope residues are strictly conserved across NiV G and HeV G and 3 residues are conservatively substituted from NiV G to HeV G (N187Q, N478Q and I517L). Only 2 residues are non-conservatively substituted (L470D and K571V and these map to the periphery of the epitope (Fig 2E). Our structural data suggest that none of the 5 substitutions would affect nAH1.3 recognition, as confirmed by the similar neutralization potencies determined against rCedV-NiV-B-GFP and rCedV-HeV-GFP (Figure S3A). However, no cross-reactivity was detected with more divergent HNV sG ectodomain tetramers from CedV, Ghana virus (GhV) and Mojiang virus (MojV) (Figure S3D–E). Since mAb nAH1.3 does not appear to affect the overall NiV G conformation (36) nor to bind to the ephrin virus entry receptor binding site, we propose that the mAb inhibits HNV entry into cells by interfering with its F fusion triggering mechanism, as supported by membrane fusion assays (37).

Figure 2. Structural basis for nAH1.3-mediated broad neutralization of NiV and HeV.

Figure 2.

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(A) Superimposition of the NiV G head domain (blue surface) bound to nAH1.3 (heavy and light chains colored gold and yellow, respectively) or to ephrin-B2 (purple, PDB 2VSM) showing that they bind to opposite sides of the β-propeller (13). (B) Biolayer interferometry analysis of binding of the nAH1.3 Fab and ephrin-B2 (EB2) to immobilized NiV G ectodomain showing absence of competition irrespective of their order of addition. Each NiV G loaded anti-penta His biosensor probe was sequentially dipped in a solution containing 25 nM nAH1.3 Fab (red) and then 50 nM EB2 + 25 nM nAH1.3 Fab (red) or 50 nM EB2 and then 25 nM of nAH1.3 Fab + 50 nM EB2 (green). Controls with only nAH1.3 Fab (blue) or EB2 (orange) are shown for comparison. (C) Zoomed-in view of the interface between NiV G and nAH1.3 with selected side chains shown as sticks. (D) Ribbon diagram of a NiV G head domain (blue) with the interacting nAH1.3 heavy and light chains CDRs rendered in gold and yellow. (E) Molecular surface representation of the NiV G head showing the nAH1.3 footprint colored by residue conservation between NiV G and HeV G. Conservative sub, conservative substitution. Semi-conservative sub, semi-conservative substitution. (F) Molecular surface representation of the NiV G head showing the nAH1.3 escape mutations identified here (I520T introducing an N-linked glycosylation site at position N518; N186D comes from HeV G escape mutant) and Q450K and R516K (previously described (31)).

To validate our structural data, we passaged chimeric rCedV-NiV-B-GFP and rCedV-HeV-GFP in the presence of a sub-neutralizing concentration of nAH1.3. Deep sequencing revealed the selection of a clone harboring the I520T mutation (T1559C nucleotide substitution) introducing an N-linked glycosylation site at position N518 of NiV G. This is within the epitope recognized by nAH1.3 and likely abrogates mAb binding and neutralization via steric hindrance (Figure 2F and Figure S4A). For HeV G, we identified the T117A/N186D escape mutations. The latter substitution resides within the epitope and likely disrupts interactions with the nAH1.3 light chain (Figure 2D,F and Figure S4B). Previously identified Q450K and R516K nAH1.3 escape mutations (31), further validate our structure as both residues form extensive interactions with the Fab heavy chain (Figure 2F). Moreover, passaging of rCedV-HeV-GFP in the presence of m102.4 led to the emergence of a single amino acid mutation in HeV G (D582N) (Figure S4C) which is identical to the one previously identified using authentic HeV and NiV (31), validating the use of the chimeras as a surrogate system for pathogenic henipaviruses.

Synergistic neutralization of NiV and HeV by a mAb cocktail

mAb cocktails have had success in preventing or treating infections with RNA viruses (38). For instance, although the individual Regeneron 10933 (casirivimab) and 10987 (imdevimab) mAbs against SARS-CoV-2 are affected by a range of residue substitutions detected in clinical isolates, the two-mAb cocktail proved more resilient to escape mutations (39, 40). Because experimental passaging of NiV and HeV with low concentrations of neutralizing mAbs can favor the emergence of neutralization escape mutants, we tested a cocktail of m102.4 and nAH1.3 which recognize epitopes located on opposite sides of the β-propeller (Figure 3A). Analysis of the negatively stained complex between NiV G and the nAH1.3 and m102.4 Fabs by single particle analysis confirmed formation of a ternary complex (Figure S5A–C), allowing the determination of a 3D reconstruction with three nAH1.3 and three m102.4 Fabs bound to a NiV G tetramer (Figure S5C). We subsequently used biolayer interferometry to confirm that both mAbs can recognize the NiV G ectodomain immobilized at the surface of biosensors irrespective of their order of addition (Fig 3B). We then carried out virus neutralization assays using rCedV-NiV-B-GFP or rCedV-HeV-GFP to examine whether these mAbs possessed synergistic virus-neutralizing activity (35). Using a concentration matrix of each mAb, we found that combining m102.4 and nAH1.3 led to synergistic neutralization of the two rCedV chimeras (Figure 3C-D and Figure S6A–B). Passaging chimeric rCedV-HeV-GFP in the presence of the cocktail of nAH1.3 and m102.4 led to the emergence of the T117A, N186D and T507I triple mutant with T507I mapping to the m102.4 epitope and N186D to the nAH1.3 epitope, as described above (Figure 2D,F and Figure S4D). These findings support the potential use of a cocktail of these two non-competing G glycoprotein-directed neutralizing mAbs as a therapeutic against these two deadly HNVs due to their synergy and the higher barrier for resistance due to the requirement for multiple mutations to escape neutralization.

Figure 3. A HNV G head-directed mAb cocktail with synergistic neutralizing activity.

Figure 3.

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(A) Superimposition of the NiV G head domain (blue surface) bound to nAH1.3 (heavy and light chains colored gold and yellow, respectively) or to the m102.3 Fab (heavy and light chains colored dark and light grey, respectively, PDB 6CMI) showing that they bind to opposite sides of the β-propeller. m102.3 is closely related to the m102.4 mAb. (B) Biolayer interferometry analysis of binding of the nAH1.3 and m102.4 IgGs to the immobilized NiV G ectodomain showing absence of competition irrespective of the order of addition. Each NiV G loaded anti-penta His biosensor probe was sequentially dipped in a solution containing 100 nM nAH1.3 IgG (red) and then 100 nM m102.4 IgG + 100 nM nAH1.3 IgG (red) or 100 nM m102.4 IgG and then 100 nM of nAH1.3 IgG + 100 nM m102.4 IgG (green). Controls with only nAH1.3 IgG (blue) or m102.4 IgG (orange) are shown for comparison. (C-D) Synergy maps for neutralization of replication competent rCedV-NiV-B-GFP (C) and rCedV-HeV-GFP (D) by varying concentrations of the m102.4 and nAH1.3 mAb cocktail analyzed with SynergyFinder (41). ZIP synergy score greater than 10 indicates strong synergistic relationship. The white box indicates the most synergistic region on each plot.

The NiV G head domain is the main target of vaccine-elicited serum neutralizing activity

We next investigated the nature and fine-specificity of polyclonal Ab responses against the NiV G and HeV G glycoprotein using sera from two rhesus macaques that were immunized three times (4 weeks apart) with 200 μg of an alum-adjuvanted equimolar mixture of the purified NiV-B and NiV-M sG tetramers (Figure 4A). The two immunizations elicited potent serum neutralizing activity at day 84 with geometric mean neutralization titers of 1/8,434 and 1/992 against rCedV-NiV-B-GFP and rCedV-HeV-GFP, respectively (Figure 4B). Using competition ELISA with the nAH1.3 (31), m102.4 (42) and HENV-32 (43) mAbs, we showed that vaccine-elicited Abs target at least three distinct antigenic sites at the surface of the NiV G head domain with comparable magnitudes, illustrating the diversity of polyclonal Ab responses elicited by vaccination (Figure 4C-E). To directly visualize the epitopes recognized by serum Abs, we used single particle electron microscopy analysis of negatively stained complexes formed between NiV G and purified polyclonal Fab fragments (obtained from IgG cleavage) after size-exclusion chromatography (44–46). 3D classification of the data led to the identification of at least three distinct classes of head binding Abs and one class of stalk-specific Ab (Figure 4F-J and Figure S5D–F). Overall, Ab responses are virtually exclusively directed to the NiV G head domain, in spite of using the full ectodomain tetramer for immunization, underscoring an apparent focusing of Ab responses on the receptor-binding domain (Figure 4F-J and Figure S5D–F).

Figure 4. The NiV G receptor-binding head domain is immunodominant and accounts for most of the neutralizing activity elicited by vaccination.

Figure 4.

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(A) Study design for vaccination of rhesus macaques, where two rhesus macaques have been immunized three times (4 weeks apart) with 200 μg of an alum-adjuvanted equimolar mixture of the purified NiV-B and NiV-M sG tetramers. Blood was collected on day 42 and day 84 post immunization. (B) Serum neutralizing Ab titers against rCedV-NiV-B-GFP and rCedV-HeV-GFP measured at day 84. The curves for each animal are shown in black and blue. (C-E) Competition ELISAs showing binding of biotinylated nAH1.3 (C), m102.4 (D) and HENV-32 (E) mAbs to the immobilized NiV G ectodomain in the presence of various dilutions of vaccine-elicited rhesus macaque sera (obtained at day 84). The curves for each animal are shown in black and blue whereas binding of the mAb in the absence of sera is shown in green (control). (F-I) Representative EM reconstructions (grey surfaces) of negatively stained complexes formed between purified polyclonal serum Fab fragments and NiV G fitted with atomic models for visualization of antigenic sites targeted. (J) Surface representation of the NiV G tetramer with three head domain-specific Fabs bound (left) and a stalk-directed Fab bound (right) highlighting antigenic sites detected in vaccine elicited polyclonal serum Abs. (K-L) Neutralizing Ab titers against rCedV-NiV-B-GFP (K) and rCedV-HeV-GFP (L) before and after depletion with the NiV G head domain (Head) or the ectodomain tetramer (Ecto).

To understand the quantitative contribution of the different NiV G domains to serum neutralizing activity induced by vaccination, we depleted polyclonal Abs using either the purified isolated NiV G head domain or the tetrameric NiV G ectodomain. Neutralizing Ab titers for each of the two rhesus macaques were reduced 9 and 19-fold against rCedV-NiV-B-GFP (Figure 4K and Figure S6C–F) and 9 and 93-fold against rCedV-HeV-GFP (Figure 4L and Figure S6G–H) following depletion of NiV G head-specific Abs. Serum Ab depletion using the NiV G ectodomain tetramer resulted in a 11 and 13-fold dampening of neutralizing activity against rCedV-NiV-B-GFP (Figure 4K and Figure S6I–L) and a 69 and 2.5-fold reduction against rCedV-HeV-GFP (Figure 4L and Figure S6M–N). Collectively, these data indicate that the NiV G head domain is immunodominant and the target of most serum neutralizing activity elicited by NiV G vaccination in rhesus macaques.

Discussion

Our structure of the NiV G ectodomain tetramer reveals the ultrastructural organization of this key target of the immune system, informs the mechanism of HNV entry into host cells and provides a blueprint for engineering next-generation vaccine candidates with improved stability and immunogenicity, as was successful for respiratory syncytial virus (47, 48) and SARS-CoV-2 (49–55). The discovery that the NiV/HeV G head domain is the target of most neutralizing activity in the serum of rhesus macaques vaccinated with tetrameric NiV G ectodomains motivates the development of vaccines focusing Ab responses on this domain of vulnerability. Presenting the head antigen as an ordered array through multivalent display bears the promise of enhancing immunogenicity, as recently described for SARS-CoV-2 (51, 56, 57), and would allow presentation of a mosaic of HNV head domains with the goal of inducing neutralizing Ab responses with maximal breadth (45, 57–59). Finally, the enhanced expression yield, stability and homogeneity of the NiV G head domain compared to HeV sG (35) would improve scalability of the manufacturing process of such a vaccine.

Supplementary Material

SuppMat

Figs S1 to S6

Table S1

References 60–79

Acknowledgements:

This study was supported by the National Institute of Allergy and Infectious Diseases (DP1AI158186 and HHSN272201700059C to D.V., AI077995 to C.C.B. and U19AI142764 to C.C.B. and D.V.), the National Institute of Health Cellular and Molecular Biology Training Grant (T32GM007270 to A.A.), a Pew Biomedical Scholars Award (D.V.), an Investigators in the Pathogenesis of Infectious Disease Awards from the Burroughs Wellcome Fund (D.V.), the University of Washington Arnold and Mabel Beckman cryoEM center and the National Institute of Health grant S10OD032290 (to D.V.). The authors thank Auro Vaccines, LLC, for providing the rhesus macaque NiV-M and NiV-B sG immune sera, and also acknowledge the Coalition for Epidemic Preparedness Innovations (CEPI) for their support of the Nipah virus vaccine program. Molecular graphics and analyses performed with UCSF ChimeraX, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from National Institutes of Health R01-GM129325 and the Office of Cyber Infrastructure and Computational Biology, National Institute of Allergy and Infectious Diseases. The opinions and assertions expressed herein are those of the author(s) and do not necessarily reflect the official policy or position of the Uniformed Services University or the Department of Defense, or the Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc. D.V is an Investigator of the Howard Hughes Medical Institute.

Footnotes

Competing interests: C.C.B is a United States federal employee and co-inventor on US and foreign patents pertaining to soluble forms of Nipah virus and Hendra virus G glycoproteins and C.C.B. and M.A. are co-inventors on US and foreign patents pertaining to Cedar virus and methods of use and recombinant Cedar virus chimeras, whose assignees are the US as represented by the Henry M. Jackson Foundation for the Advancement of Military Medicine (Bethesda, MD, USA).

Data and Materials Availability.

The cryoEM maps and coordinates have been deposited to the Electron Microscopy Databank and Protein Data Bank with accession numbers listed in Table S1. Materials generated in this study will be made available on request after signing a materials transfer agreement with the University of Washington (Uniform Biological MTA).

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Associated Data

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Supplementary Materials

SuppMat
NIHMS2096797-supplement-SuppMat.docx (2.6MB, docx)

Data Availability Statement

The cryoEM maps and coordinates have been deposited to the Electron Microscopy Databank and Protein Data Bank with accession numbers listed in Table S1. Materials generated in this study will be made available on request after signing a materials transfer agreement with the University of Washington (Uniform Biological MTA).

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