The animal experiments were approved by the Animal Ethics Committee of the Wuhan Institute of Virology, Chinese Academy of Sciences (approval number: WIVA21202104). All animal experiments involving NiV were executed in the animal biosafety level 4 (ABSL-4) facility at the National Biosafety Laboratory (Wuhan), Chinese Academy of Sciences.
Cells and virus
HEK293T (ATCC: ACS-4500), HEK293 (ATCC: CRL-1573) and Vero E6 (ATCC: CRL-1586) cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) containing 10% Fetal bovine serum (FBS, Gibco), penicillin (100 units/ml) and streptomycin (100 μg/ml) at 37 °C in 5% CO2. 293 Freestyle (293 F) cells were maintained in FreeStyle293 expression medium containing penicillin (100 units/ml) and streptomycin (100 μg/ml) in shaker incubators at 150 rpm, 37 °C, 8% CO2. The NiV Malaysia (AF212302.2) and NiV Bangladesh (AY988601.1) strains used in the challenge studies were obtained from the National Virus Resource Center, Wuhan Institute of Virology, Chinese Academy of Sciences. The virus stock was propagated in Vero E6 cells.
AdC68-Based NiV vaccine production
The full-length G sequences used in this study were obtained by optimizing the NiV G sequences of two different NiV-M and NiV-B strains in the software Geneious using the principle of sequence homology. Specifically, all the sequences of G proteins were downloaded from GenBank and the vaccine consensus sequences were acquired according to the sequence consistency principle. To understand the conservation of the acquired consensus sequence, a phylogenetic tree was constructed by the maximum likelihood method in Mega11. The G sequence was then codon optimized for humans and synthesized by GenScript (Nanjing, China). The codon optimized G gene was cloned into a transgene expression plasmid pShuttle2 between the restriction sites NotI and KpnI. Subsequently, after digestion with I-CeuI and PI-SceI restriction enzymes, the whole G expression cassette containing the CMV promoter, G gene, and BGH polyA tail was inserted into the E1-deleted region of the chimpanzee adenoviral vector pAdC68 to generate the recombinant pAdC68-G. The resultant pAdC68-G was linearized by PacI and transfected into HEK293 cells to rescue AdC68-G, Finally, AdC68-G as well as empty control AdC68 were propagated and purified by cesium chloride density gradient centrifugation, titrated, and stored at −80 °C.
NiV DNA vaccine construction
For the construction of the NiV DNA vaccine (also termed DNA-G), The optimized gene was subcloned into the clinically used vector pVAX1 between the restriction sites KpnI and BamHI with the Kozak sequence incorporated at the 5’ end of the genes. The plasmid was transformed into E. Coli Top 10 cells for plasmid amplification and purified using an endotoxin-free Plasmid Plus Maxi Kit (Qiagen).
Western blot analysis
HEK293T cells cultured in 6-well plates were transfected with 2 μg of DNA-G or pVAX1 for 48 h. HEK293 cells were infected with AdC68-G and AdC68 at doses of 109 VPs per well. After transfection or infection, the culture supernatant was discarded, and the cells were harvested and lysed with 150 μl of RIPA lysis buffer containing a protease inhibitor cocktail. The samples were run on a 10% PAGE gel and transferred to a PVDF membrane (Millipore). After that, skim milk (5%) was added to block the membrane for about 1 h, and a mouse antibody against NiV-G serum (prepared by our laboratory) was used to incubate the membrane with a 1:500 dilutions. After washes, a horseradish peroxidase (HRP)-conjugated secondary anti-mice IgG (Proteintech, Cat No. SA00001-1, diluted 1:2000) was used to bind to the primary antibody. Finally, the membranes were developed with an ECL substrate (Thermo Scientific). The expression of GAPDH (Abcam, Cat No. ab8245, diluted 1:5000) was used as a loading control. Uncropped and unprocessed scans of are unloaded in the Supplementary Fig. 5.
BALB/c mice (6–8 weeks old, female) and Syrian hamsters (5–6 weeks old, female) were purchased from Vital River Laboratories (Beijing, China). BALB/c mice were randomly allocated into 10 groups (Fig. 2b). In batch 1 (groups 1–4), two groups (groups 1 and 3) of mice were vaccinated (one or two doses) with 5 × 1010 VPs AdC68 via the i.n. route and were used as controls. Two groups (groups 2 and 4) of mice were vaccinated (one or two doses) with 5 × 1010 VPs AdC68-G by i.n. route. In batch 2 (groups 5–10), three groups (groups 5, 7 and 9) of mice were vaccinated with 5 × 1010 VPs AdC68 (i.m.) or 50 μg pVAX1 (i.m./EP) at day 0 and AdC68 (i.m.) at day 21 and were used as controls. Three groups (groups 6, 8 and 10) of mice were vaccinated with 5 × 1010 VPs AdC68-G (i.m.) or 50 μg DNA-G (i.m./EP) and AdC68-G (i.m.) at day 21. For DNA vaccine immunization in BALB/c mice, anesthetized mice were injected with 50 μl of solution containing indicated DNA, following instillation, the injection site was subject to electroporation at 100 V constant voltage with 3 pulses at 50 msec/pulse and 1 s intervals between pulses. Electric pulses were delivered by the electric pulse generator (ECM830; BTX, San Diego, CA). At day 10 after each vaccination, mice were sacrificed and splenocytes were harvested for the detection of cellular immune responses. Sera were collected at predetermined time intervals for the detection of humoral immune responses (Fig. 2a). The mice were anesthetized by isoflurane before the needle vaccination and retro-obital bleeding. For all the DNA vaccination, the nosecone connected with vaporizer machine was used to maintain the anesthesia stage. For the euthanasia procedure, the mice were anesthetized by isoflurane followed by the cervical dislocation.
Hamsters were randomly divided into 8 groups. Four groups of animals were homologously vaccinated with 5 × 1010 VPs AdC68-G or AdC68 via an i.m. or i.n. route at a 21-day interval. Two groups of animals were prime vaccinated with 100 μg of DNA-G or pVAX1 and boosted with 5 × 1010 VPs AdC68-G or AdC68 via i.m. route at a 21-day interval. Two groups of animals were immunized thrice with 100 μg of DNA-G or pVAX1 on days 0, 21, and 42, respectively. For DNA vaccine immunization in hamsters, the injection site was electroporated immediately after instillation (100 μl/per animals) with a 75 V constant voltage with 10 pulses at 50 msec/pulse and 100-msec intervals between pulses. The serum samples were collected at the indicated time points and subjected to immunological assays (Fig. 3a). Three weeks after final immunization, all animals were transferred to the ABSL-4 and challenged with 1000 LD50 of NiV Malaysia (8.55 × 103 TCID50) or Bangladesh (22.33 × 103 TCID50) strains in 500 μl DMEM via the i.p. route. Six animals in each group (except for the AdC68-G (i.n.) group that was challenged with NiV-M, n = 3) were euthanized 5 d.p.i. The lungs, brain and spleen were collected and subjected to analyze for virology and histology. The remaining animals (n = 3 per control group, n = 6 per vaccine group) were monitored over a 21-day period. During the monitored period, the clinical scores of the challenged hamsters were recorded daily50. The weight of the animals was recorded over the 21-day period. Hamsters with a weight loss of more than 25% (recorded as dead) or at the end of protocol are euthanized by cervical dislocation under isoflurane anesthesia. All animal studies follow the ARRIVE reporting guidelines51.
NiV G protein expression and purification
The codon-optimized stalk and ectodomain of NiV G (residues Q71-T602) was cloned into the pcDNA3.4 mammalian expression vector using NotI and XbaI to fuse an N-terminal signal peptide and a C-terminal His-Tag. The constructs were expressed by transfection of 293 F cells with PEI transfection reagent (Sigma) according to the manufacturer’s protocol. Transfected cells were incubated in shaker incubators at 150 rpm, 37 °C and 8% CO2. The supernatant was harvested 5 days after transfection and centrifuged at 1000 rpm for 30 min to remove cellular debris. The supernatant was filtered through a 0.45 µm filter before the G protein was purified using Ni Sepharose High Performance histidine-tagged protein purification resin (Cytiva) eluted with 500 mM imidazole in 50 mM Tris and 100 mM NaCl pH 8.0. Pooled fractions were dialyzed overnight into 20 mM Tris and 150 mM NaCl and 10% (v/v) glycerol at pH 8.0.
96-well EIA/RIA plates (Corning) were coated overnight at 4 °C with 20 μg of G protein per plate in coating buffer (Solarbio). All wells were blocked with 100 µl of blocking buffer (5% skim milk in PBS) for 1.5 h at 37 °C. After standard washes and blocks, serum (2x serially diluted, starting at 100x dilution) in 5% skim milk in PBS was incubated at 37 °C for 2 h. Plates were washed four times in PBS with 0.1% Tween 20 (PBST) before addition of HRP-conjugated goat anti-mouse IgG (Abcam, Cat No. ab6789, diluted 1:20,000), IgA (Abcam, Cat No. ab97235, diluted 1:10,000) or HRP-conjugated goat anti- hamster IgG (Abcam, Cat No. ab6892, diluted 1:15,000) diluted in 5% milk in PBS for 1.5 h at 37 °C. Plates were washed as before prior to being developed with 100 µl/well of TMB chromogen solution (Beyotime) for 15 min. Substrate reactions were stopped by the addition of 50 µl/well of stop solution for TMB Substrate (Beyotime) before reading plate absorbance at 450 nm (OD450). The cutoff value was defined as 2.1-fold of OD450 values from the sample of nonvaccinated mice. The reciprocal of the maximum sample dilution with OD450 values equal to or greater than the cutoff value was used to calculate the endpoint binding antibody titers.
An ELISpot assay for the detection of IFN-γ-secreting mouse splenocytes was performed with a mouse IFN-γ ELISpot kit (Mabtech). Feshly harvested splenocytes of 5 × 105 per well incubated with pools of NiV G peptides (18-mers with 10 amino acid overlap). The final concentration of each peptide was 2 μg/ml. The cells were then incubated with 5% CO2 at 37 °C. 24 h later, IFN-γ spot-forming cells were detected by staining membranes with anti-mouse IFN-γ-biotin (1 μg/ml) followed by streptavidin-ALP. Phorbol 12-myristate 13-acetate and 10 μg/ml ionomycin (Dakewe) was added to the positive-control group, whereas the negative-control group received no stimuli. Analysis was performed using the CTL ImmunoSpot Analyzer and ImmunoSpot Software (Cellular Technology). Spot-forming unit (SFU) per million cells was calculated.
Neutralization assay based on NiV pseudotyped lentivirus
NiV pseudovirus bearing the F25 (1-523 aa) and G33 (34-602 aa) proteins of NiV was produced in an Env-defective, luciferase-expressing HIV-1 backbone (pNL4-3. Luc-R-E-). Briefly, the F25 and G33 sequences of NiV were synthesized and subcloned into the pcDNA3.1(+) plasmid (GenScript). A total of 8 × 106 HEK293T cells were seeded into a 75 cm2 flask and co-transfected with 30 μg of pNL4-3. Luc-R-E- and 1.7 μg of pcDNA3.1( + )-F25 and 3.3 μg of pcDNA3.1( + )-G33 using the Lipofectamine 2000 transfection reagent (Thermo Scientific). 48 h later, the supernatants were filtered and stored below −80 °C. In the serum neutralization assay, samples were heat-inactivated at 56 °C for 30 min, and pseudovirus was diluted and mixed with 3-fold serial dilution of samples in 96-well plates. After 1 h incubation at 37 °C, 3 × 104 of HEK293T cells were seeded on a serum pseudovirus-mixture. 48 h later, the activity of luciferase was measured using a Luciferase Assay System (Promega). Neutralization titers were calculated as the serum dilution at which RLU were reduced by 50% compared with RLU in virus control wells.
Neutralization assay based on NiV live virus
Three-fold serial dilutions, starting at a 1:20 dilution, of heat-inactivated (30 min, 56 °C) sera were prepared in DMEM containing 2% FBS. The samples were then added to either 100 TCID50 NiV-M or 50 TCID50 NiV-B, subsequently incubated at 37 °C for 1 h. Following incubation, the virus-serum mixtures were incubated for 1 h with Vero E6 cells, at 37 °C with 5% CO2. The cytopathic effect (CPE) was scored on wells at 5 dpi. The neutralization titer was expressed as the reciprocal of highest dilution of the serum that prevented infection of 50% of quadruplicate inoculations.
Virus titration assay
Tissue sections were weighed and homogenized in 1 ml of DMEM, Then, 100 μl of 10-fold serial dilutions of tissue homogenate was added to Vero E6 cells in a 96-well plate in quadruplicate. After 1 h incubation at 37 °C and 5% CO2, cells were washed with PBS and 100 μl of DMEM containing 2% FBS was added. Cells were incubated at 37 °C and 5% CO2. Cytopathogenic effects were assessed 5 days later, and results are expressed as TCID50 according to the method of Reed and Muench.
After the hamsters were euthanized, lungs, brain and spleen were collected for viral load examination. The tissues were homogenized in 1 ml DMEM in a tissue grinder. 140 μl of the homogenate supernatant was then subjected to RNA isolation using the Qiagen RNeasy Mini kit (Qiagen), and the total RNA was eluted in 50 μl RNase-free water. Real-time quantitative RT-PCR (qRT-PCR) was performed on a CFX96 Real-Time System (Bio Rad) using the HiScript II One Step qRT-PCR Probe Kit (Vazyme) targeting the NiV nucleocapsid (N) gene. Primers and probes used were: forward primer (5’-AACATCAGCAGGAAGGCAAGA-3’), reverse prime (5’-GCCACTCTGTTCTATAGGTTCTTC-3’), probe (5’-FAM-TTGCTGCAGGAGGTGTGCTC-BHQ1-3’) The standard curve was constructed with nine points in a 20 µl reaction system (109–101 copies). Samples < 101 copies were defined as negative.
Histology and immunohistochemistry
Lungs, brain and spleen tissue were fixed in 10% formalin for 7 days with two volume change before being transferred out of the BSL-4, following standard operating procedure approved by the Institutional Biosafety Committee. Then the samples were embedded in paraffin, sequentially sectioned to 4 µm thickness, and stained with haematoxylin and eosin (HE) prior to examination by light microscopy. To obtain a representative result, different sites of the lung tissue and the almost whole spleen were sampled for the histopathology analysis.
Specific anti-NiV immunoreactivity was detected using a rabbit anti-NiV N protein antibody (prepared by our laboratory) at 1:3000 dilutions for overnight at 4 °C. The secondary antibody used was biotinylated goat anti-rabbit IgG (SeraCare, Cat No.5220-0336) at 1:500 dilutions for 50 min. Slides were developed with DAB chromogen for 15 s and counterstained with hematoxylin for 45 s. The image was collected by a Pannoramic MIDI system (3DHISTECH, Budapest, Hungary).
The statistical analysis was performed with GraphPad Prism 8.0 software. Two-tailed unpaired Student’s t tests were conducted to compare differences between two experimental groups. The One-way ANOVA with Tukey’s multiple comparisons tests were applied to compare more than two experimental groups. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n.s., not significant.
Further information on research design is available in the Nature Research Reporting Summary linked to this article.