Ethics statement

Animal experiments were approved by the Animal Ethics Committee of Wuhan University (WDSKY0202201) and the Wuhan Institute of Virology, Chinese Academy of Sciences (WIVAF21202301). The authentic virus infections were performed in the animal biosafety level 4 (ABSL-4) facility at the National Biosafety Laboratory (Wuhan), Chinese Academy of Sciences.

Viruses and cells

The NiV Malaysia (AF212302.2) and NiV Bangladesh (AY988601.1) strains used in the authentic virus neutralization assay and the challenge studies were obtained from the National Virus Resource Center, Wuhan Institute of Virology, Chinese Academy of Sciences. All authentic viruses were propagated in Vero E6 cells. Vero E6 or 293 T cells were maintained in DMEM (Monad Biotech) with 8% or 10% heat-inactivated FBS (ExCell Bio) at 37 °C with 5% CO2.

Cloning, protein expression, and purification

The genes encoding G (NP_112027.1) and F (NP_112026.1) of NiV-M, G (AAY43916.1) and F (AAY43915.1) of NiV-B, G (NP_047112.2) and F (NP_047111.2) of HeV, G (YP_009094095.1) of MojV, G (UUV47206.1) of LayV, Helicobacter pylori ferritin (WP_000949190.1) and bullfrog ferritin fragment (ESQVRQQF) were codon optimized and synthesized by Tsingke Biotechnology Co. Genes encoding the full-length G and F of NiV-M, NiV-B, and HeV were cloned into a mammalian expression vector respectively for pseudovirus generation. The gene encoding Helicobacter pylori-bullfrog hybrid ferritin was constructed as previously described38 and cloned into a mammalian expression vector with an N-terminal 6×His-tag without signal-peptide for protein production. The gene encoding NiV G-ferritin was constructed by fusing the C-terminal of the NiV G head domain with the N-terminal extension of ferritin. Genes encoding NiV G-ferritin, NiV sG (residues:176–602), NiV-B G head domain (residues:176–602), HeV G head domain (residues:176–605), LayV G head domain (residues:176–625) and MojV G head domain (residues:176–625) were individually cloned into a mammalian expression vector with an N-terminal 6×His-tag and signal peptide for protein production. All plasmids were verified by DNA sequencing.

For protein purification, the above expression vectors were transiently transfected into Expi293F cells with Polyethylenimine (PEI, Polysciences), and the supernatants of cultured cells (except the cells transfected ferritin) were harvested 108 h after transfection. The supernatants were filtered through a 0.45 μm filter, and the recombinant proteins were purified by Ni-Charged Resin (GenScript). For ferritin purification, the transfected Expi293F cells were harvested 108 h post-transfection, resuspended in the lysis buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.25% NP-40, and 100 μg/mL PMSF) and broken with a Dounce grinder. Then, the cell lysate was centrifuged at 12,000 × g for 40 min to remove cell debris. The supernatant containing ferritin protein was filtered through a 0.45 μm filter and then purified by Ni-TED resin (GenScript). The proteins above were further purified by size-exclusion chromatography (SEC) using Superose 6 Increase 10/300 GL column (Cytiva) or Superdex 200 Increase 10/300 GL column (Cytiva).

Negative stain electron microscopy

Purified nanoparticles were serially diluted to 800/400/200/100/50 nM, and 10 μL samples were then adsorbed onto a 300-mesh copper grid (Beijing Daji Keyi Technology Co., Ltd, D11023) for 1 min. Then, the excess solution was blotted away with filter paper. The grids were stained with 2% uranyl acetate for 30 s, and the excess solution was blotted away. Images were collected using a transmission electron microscope (JEOL, JEM-1400plus) or a Talos L120C (Thermo Scientific) electron microscope.

Dynamic light scattering (DLS)

Proteins were filtered by a 0.22 μm filter and diluted to approximately 0.5 mg/mL using a buffer containing 20 mM Tris-HCl pH 8.0 and 150 mM NaCl. Then, the samples were loaded into a microcuvette to measure the hydrodynamic diameters and polydispersity using the Zetasizer Nano ZSP instrument (Malvern Panalytical). Each sample was performed triplicate measurements, and each parallel repeat lasted 60 s at 25 °C. The hydrodynamic diameters and polydispersity index of the samples were analyzed using instrument software (Malvern Panalytical).

Differential scanning fluorimetry (DSF)

The thermostability of target proteins was determined using a PSA-16 instrument (BEST Science & Technology, Beijing, China). The sample was diluted to 0.5 mg/mL, and 20 μL of diluted sample was loaded into a quartz glass tube (Cat#LG-002, BEST Science & Technology, Beijing, China). The intensity of the intrinsic protein fluorescence at 330 and 350 nm was measured using a linear temperature scan from 23 to 97 °C at a heating rate of 1 °C/min. The thermal transition midpoint (Tm) was calculated based on the slope of the F350/F330 curve. Each sample was measured four times.

Size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS)

A 100 μL volume of each sample (0.5 mg/mL for NiV G-ferritin and 2 mg/mL for NiV sG) was injected onto the WTC-030S5 column (Wyatt Technology, Santa Barbara, CA, USA) with an isocratic run at 0.5 mL/min for 40 min. 20 mM Tris-HCl pH 8.0 and 150 mM NaCl were used as the mobile phase. A MALS detector (DAWN®) and a refractive index (RI) detector (Wyatt Technology, Santa Barbara, CA, USA) were connected in series to the UV detector on the SEC system. Bovine Serum Albumin (BSA, Thermo Scientific) was used to normalize the static light scattering detector. The light scattering, differential refractive index (dRI) measurements, and molecular weights were analyzed using ASTRA software (6.1.2.84) (Wyatt Technology, Santa Barbara, CA, USA).

Enzyme-linked immunosorbent assay (ELISA)

The antigenicity of NiV G-ferritin was measured by ELISA. Briefly, 96-well ELISA plates (Corning) were coated overnight at 4 °C with 3 μg/mL of NiV sG or NiV G-ferritin in coating buffer (0.1 M carbonate, pH 9.6). After four times washes with PBS-T (PBS with 0.05% Tween 20), all wells were blocked with 50 μL of blocking buffer (1% BSA in PBS-T) for 2 h at 37 °C. After blocking, NiV G mAbs HENV-32, HENV-26, nAH1.3, and anti-Rabies virus mAb RVC20 (a negative control) were serially diluted from 5 μg/mL and then added to the plates and incubated at 37 °C for 2 h. Plates were washed four times in PBS-T before adding HRP conjugated goat anti-human IgG (1∶20000 dilution, ABclonal, AS002) for 1 h at 37 °C. Plates were washed and colored with 50 μL/well of one component TMB chromogen solution (NCM Biotech) for 10–30 min at 37 °C. Substrate reactions were stopped by adding 50 μL/well of 1 M HCl, and the absorbance was measured at 450 nm (OD450). For the serum binding titer detection, we performed a similar procedure, and G head proteins of NiV-M, NiV-B, HeV, LayV, and MojV were coated on the ELISA plates, and HRP conjugated goat anti-mouse IgG (1∶8000 dilution, ABclonal, AS003) was used.

Animal experiments-BALB/c mice

BALB/c mice (6–8 weeks old, female) were purchased from Vital River Laboratories (Beijing, China). For the vaccine immunogenicity assessment experiment, BALB/c mice were randomly allocated into six groups, with each group containing six mice. Mice were vaccinated with 1 μg or 10 μg of NiV sG or corresponding amounts of NiV G-ferritin containing an equimolar ratio of NiV sG. Control group mice were immunized with PBS or equimolar (compared to 10 μg NiV sG) ferritin. All the mice were vaccinated three times via the intramuscular (i.m.) route with the AddaVax adjuvant (InvivoGen) and injection interval of three weeks. Sera were collected at different times (Days 7, 14, 21, 42, and 61 after the first immunization) to detect humoral immune responses. On Day 61, mice were sacrificed by the cervical dislocation after being anesthetized by 2% isoflurane, and splenocytes and lymph node cells were harvested for cellular immune response analysis and antigen-specific B cell sorting.

For the temperature stability assay, BALB/c mice were randomly allocated into nine groups with each group containing five mice. NiV G-ferritin vaccine was prepared by mixing NiV G-ferritin with Imject Alum adjuvant (Thermo Scientific). Then, the prepared NiV G-ferritin-alum vaccine was stored at 4 °C, 27 °C, and 37 °C for 7 or 14 days, respectively. The sample of NiV G-ferritin-alum stored at 27 °C for 1 h served as a positive control, and mice immunized with PBS or equimolar ferritin were negative controls. All the mice were vaccinated three times via the i.m. route with an interval of three weeks. Sera were collected at predetermined times to detect humoral immune responses.

Animal experiments-Syrian hamsters

Syrian hamsters (six weeks old, female) were purchased from Vital River Laboratories (Beijing, China) and randomly divided into three groups, with each group containing 12 animals. 2 μg of NiV sG, NiV G-ferritin (containing equivalent 2 μg of NiV G antigen), or equimolar ferritin was adjuvanted with 50% (volume ratio) AddaVax adjuvant (InvivoGen). Then, these immunogens were intramuscularly administrated to hamsters three times at a three-week interval. Serum samples were collected and subjected to immunological assays at the indicated time points. Three weeks after the final immunization, all animals were transferred to the ABSL-4, and each hamster was challenged with 1000 LD50 NiV-M via the intraperitoneal injection route. Before vaccination, retro-orbital bleeding and virus inoculation, hamsters were anesthetized with 5% isoflurane. Half of the animals in each group were euthanized by cervical dislocation under isoflurane anesthesia five days after being challenged with NiV-M, and the organs of the lung, brain, and spleen were harvested for viral load detection through qRT-PCR. The other half of the animals in each group were monitored for 21 days after the challenge, and the survival rate was calculated at the end of the monitoring time. The weight change was monitored daily for 14 days.

In the two-dose immunization regimen, hamsters were randomly divided into eight groups, with each group containing three animals. The hamsters were vaccinated with 2 μg, 6 μg, or 20 μg NiV sG, or equimolar NiV G-ferritin mixed with AddaVax adjuvant (1:1 volume ratio) twice with a three-week interval, respectively. Control group hamsters were immunized with PBS or equimolar (compared to 20 μg NiV sG) ferritin. Three weeks after the second immunization, all animals were transferred to the ABSL-4, and each was challenged with 1000 LD50 NiV-M via the i.p. route. The hamsters were monitored for weight change and survival rate for 21 days after the NiV-M challenge.

During the experiments, all hamsters were acclimated to the Specific Pathogen Free (SPF) barrier environment for at least one week prior to the experiment. They were euthanized by cervical dislocation under isoflurane anesthesia if they experienced more than 25% weight loss after the challenge or at the end of the experiment. Animal immunization and challenge experiments do not allow for blinding. The individual who performed the tissue sample analysis did not know the identity of the samples.

Pseudovirus neutralization assay

NiV-M, NiV-B, or HeV full-length F and G glycoproteins were pseudotyped using VSVΔG-eGFP, a reporter virus in which the VSV-G glycoprotein has been replaced with an eGFP reporter gene. To increase the NiV-B pseudovirus titer, we introduced S207L and G252D point mutations on the NiV-B F protein. For the pseudovirus production, 293 T cells were transiently transfected to overexpress F and G glycoproteins. 24 h after transfection, the cells were infected with VSVΔG-eGFP for 4–6 h and then washed with PBS and incubated with 1 μg/mL of anti-VSV G monoclonal antibody-I157. 24 h post infection, supernatants were collected and centrifuged at 1500 × g for 5 min to remove the cell debris and aliquoted appropriately before storage at −80 °C to avoid multiple freeze thaws.

For the pseudovirus neutralization assay, serum samples were heat-inactivated at 56 °C for 30 min before experiments. Pseudovirus was diluted and mixed with serial dilutions of sera/mAbs in 96-well plates and incubated for 1 h at 37 °C. The virus-serum/mAb mixtures were added to Vero E6 cells seeded at a density of 1.5 × 104 cells/well in the 96-well plates the day before. 24 h later, the cells were fixed with 4% paraformaldehyde, and then the green fluorescent dots (GFP signal) were counted and analyzed using CTL ImmunoSpot Analyzer (S6 Ultra M2, Cellular Technology) and ImmunoSpot Software (ImmunoSpot 7.0.34.0, Cellular Technology). Half-maximal inhibitory concentrations (NT50) values were calculated from neutralization curves using a nonlinear regression model and plotted with GraphPad Prism (v.8.0).

Authentic virus neutralization assay

The virus stock was propagated in Vero E6 cells. The serum samples of mice and hamsters (sera were heat-inactivated for 30 min at 56 °C) were serially diluted starting from 1:40 dilution in DMEM containing 2% FBS and incubated with either 100 TCID50 NiV-M or 50 TCID50 NiV-B for 1 h at 37 °C. Virus and serum mixtures were added to Vero E6 cells at 37 °C with 5% CO2 for 1 h, and the cytopathic effect in each well was calculated five days after infection. Four replicates were set for each serum dilution. NT50 values were calculated using IBM SPSS Statistics 27.

qRT-PCR

Five days after the challenge, the lungs, brains, and spleens of hamsters were collected after euthanasia to measure the viral RNA copies in the tissues. Each hamster tissue was homogenized in 1 mL DMEM in a tissue grinder, and the samples were inactivated before removal from the BSL-4 laboratory. The tissue homogenate supernatant was taken to RNA extraction using a Qiagen RNeasy Mini kit. RNA was analyzed using primers/probes targeting the NiV nucleocapsid (N) gene for quantitative real-time PCR (qRT-PCR), and the primers and probes were used as follows: forward primer (5’-AACATCAGCAGGAAGGCAAGA-3’), reverse prime (5’-GCCACTCTGTTCTATAGGTTCTTC-3’), probe (5’FAM-TTGCTGCAGGAGGTGTGCTC-BHQ1-3’). NiV RNA was detected using the CFX96 Real-Time system (Bio-Rad) in HiScript II One Step qRT-PCR Probe Kit (Vazyme). The standard curve was constructed with 9 points in a 20 μL reaction system (1 × 109 to 1 × 101 copies). Samples with fewer than ten copies were defined as negative.

Measurement of IFN-γ and IL-4 in spleen cell supernatant by ELISA

The production of IFN-γ and IL-4 in the cell culture supernatant was measured using IFN-γ/IL-4 Mouse Uncoated ELISA Kit (Invitrogen, 88-7314-88/88-7044-86). Briefly, the isolated splenocytes were diluted to 5 × 106 cells/mL with RPMI 1640, and then NiV sG was added to the cells at a final concentration of 5 μg/mL. Then, the mixtures were divided into 96-well plates. The cells stimulated with Cell Stimulation Cocktail (500×, eBioscience™, Invitrogen) were used as a positive control, and the cells incubated with 5 μg/mL BSA were used as a negative control. The plates were incubated at 37 °C with 5% CO2 for 72 h, and then IFN-γ and IL-4 in the culture supernatants were determined using IFN-γ/IL-4 Mouse Uncoated ELISA Kit according to the manufacturer’s protocol.

Intracellular cytokine staining assay

The isolated splenocytes were diluted to 5 × 106 cells/mL with RPMI 1640, and then 5 μg/mL NiV sG was added to the cells for the stimulation. The mixtures were divided into 6-well plates, and the cells stimulated with Cell Stimulation Cocktail (500×, eBioscience™, Invitrogen) were served as a positive control; the cells added with 5 μg/mL BSA were used as a negative control, with PBS-treated cells as blank control. The plates were incubated for 2 h in a cell incubator at 37 °C with 5% CO2, and then Monensin Solution (1000×, eBioscience™, Invitrogen) was added to cells, and cells were cultured for another 4 h. After fixing with 4% paraformaldehyde and washing with PBS, the cells were stained with Ms CD4-BV510 (1∶100 dilution, BD Pharmingen™, 563106), IFN-γ-PE (1∶80 dilution, Invitrogen, 12-7311-82), IL4-APC (1∶80 dilution, Invitrogen, 17-7041-82) or Ms CD8a-BV510 (1∶100 dilution BD Pharmingen™, 563068), IFN-γ-PE, IL4-APC. The percentage of IFN-γ+/IL4+ cells was measured using CytoFLEX Flow Cytometer (Beckman). The data were processed using FlowJo software.

Isolation of NiV G-specific B cells and amplification of antibody variable region sequence

After 21 days of the third immunization, mice immunized with the NiV G nanoparticle were sacrificed, and splenocytes and lymph node cells were harvested for antigen-specific B cell sorting. Briefly, the NiV sG were randomly biotinylated with EZ-Link NHS-PEG4-Biotin (Thermo Scientific), and the isolated cells were stained with 0.5 μg/mL of biotinylated NiV sG for 30 min at 4 °C. After washing with PBS (1 mM EDTA, 2% FBS), the cells were stained with Fixable Viability Stain 780 (1∶1000 dilution, FVS780, BD Biosciences, 565388), Ms CD3e-BV510 (1∶100 dilution, BD Biosciences, 563024), Ms CD4-BV510 (1∶100 dilution, BD Biosciences, 563106), Ms CD8a-BV510 (1∶100 dilution, BD Biosciences, 563068), Ms CD19-PE-Cy7 (1∶100 dilution, BD Biosciences, 552854), Ms IgD-PerCP-Cy5.5 (1∶100 dilution, BD Biosciences, 564273), CD138-FITC-A (1∶100 dilution, BD Biosciences, 564511), CD95-PE-A (1∶100 dilution, BD Biosciences, 554258) and Streptavidin-APC (1∶1000 dilution, BD Biosciences, 554067) for 30 min at 4 °C. After washing, the cells were loaded on FACS Aria III (BD Biosciences). The target cells (780−, CD3/4/8−, CD19+, IgD−, CD95+, and NiV G+) were sorted into 96-well PCR plates (Bio-Rad), with each well containing 7 μL of catch buffer (10 mM Tris-HCl, 1U/μL Rnain), and stored at −80 °C. NiV G-specific B cell genes were isolated following a previously published protocol58.

Expression and purification of monoclonal antibodies

The paired heavy-chain and light-chain plasmids of antibodies at a molar ratio of 1:1.2 were transiently co-transfected into Expi293F cells with Polyethylenimine (PEI, Polysciences). The supernatants of cultured cells were harvested six days post-transfection for purification. Then, mAbs were purified by protein A Resin (smart-lifesciences) and eluted with 0.1 M glycine pH 2.7. The elution containing the mAbs was exchanged into PBS buffer using a 30 kDa ultrafiltration tube (Millipore).

Biolayer interferometry (BLI) assay

The competition between NiV G mAbs was detected by BLI using an Octet-Red96 device (Pall ForteBio). Briefly, 10 μg/mL of the first mAb was loaded onto Octet ProA Biosensors (Sartorius) for 10 min. After a 10 s washing in running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% Tween-20, and 1% BSA), the biosensor tips were dipped into the running buffer containing 500 nM NiV sG for 120 s. Then, the biosensors were dipped into a buffer containing 10 μg/mL of the second mAb or the same first mAb as a control for 120 s. The biosensors were regenerated with 10 mM glycine (pH 2.0). The running buffer was used to define the background.

For antigenic stability detection of the NiV G-ferritin, 10 μg/mL of NiV mAbs HENV-26 or nAH1.3 were loaded on the protein A biosensors for 10 min. Then, the biosensors were dipped into buffer containing the analytes (NiV G-ferritin, NiV G-ferritin-AddaVax, or NiV G-ferritin-alum) which have been stored at 4 °C, 27 °C, 37 °C or 80 °C for different times, and the maximum binding signals (Rmax) was recorded and normalized by loading signals of HENV-26 or nAH1.3. The relative binding percentage of each sample was calculated by dividing the Rmax of the tested sample to the Rmax of corresponding samples stored at −80 °C, and the maximum binding signal of samples stored at −80 °C was defined as 100%.

Competition ELISA of the sera from immunized mice with NiV G mAbs

96-well ELISA plates (Corning) were coated with 3 μg/mL of NiV sG proteins overnight at 4 °C in coating buffer, and then plates were blocked with 50 μL of blocking buffer for 2 h at 37 °C. Sera, collected on Day 61 after the final vaccination, were diluted 100-fold in blocking buffer, added to the wells, and incubated at 37 °C for 1.5 h. Wells with blocking buffer (without serum) served as controls, with their OD450 values defined as 100%. NiV G monoclonal antibodies from different groups were diluted to 0.05 μg/mL and added to the wells for a 25-minute incubation at 37 °C. Plates were washed four times in PBS-T, followed by adding 50 μL HRP goat anti-Human IgG (ABclonal, AS002) for 1 h at 37 °C. Plates were washed, colored, stopped, and read as described above in antigenicity detection ELISA. In another competition ELISA, after blocking, 10 μg/mL of NiV G mAbs from different groups or a negative control mAb T3D9 were added to the plates. Then the sera from NiV sG or NiV G-ferritin vaccinated mice (20000-fold dilution) were added for binding, and the HRP goat anti-mouse IgG (Thermo Scientific, #31430) was used as the detection antibody.