cDNA construction of vaccine vectors

We inserted codon-optimized Nipah virus glycoprotein gene G (Bangladesh strain, GenBank: AY988601.1) between the N and P genes of the parental BNSP333 rabies vector using BsiWI and NheI restriction sites(16). Codon bias optimization for human codon use was carried out by GenScript Inc. The resulting plasmid was designated BNSP333-coNiV-G (NIPARAB), and the correct sequence of the plasmid was confirmed by sequencing using primers targeting the region between the N and P genes.

Recovery of recombinant vectors

X-tremeGENE 9 transfection reagent (Roche Diagnostics) in Opti-MEM was used to transfect full-length viral cDNA clones along with support plasmids bearing RABV N, P, G, and L genes under the control of a T7 promoter and a plasmid expressing T7 RNA polymerase into BSR cells on 6-well plates as described previously.31 Successful recovery was determined by a rabies focus-forming assay. Briefly, seven days after transfection, supernatant from each transfected well of the 6-well plate was transferred to duplicate wells of a 12-well plate seeded with VERO cells. Forty-eight hours later, cells in the 12-well plate were fixed with 80% acetone and stained with a FITC-conjugated antibody against RABV N (Fujirebio Diagnostics, Inc). Fluorescence microscopy was used to observe the appearance of viral foci, indicative of recovered, infectious recombinant RABV.

Sucrose purification and inactivation of virus particles

NIPARAB was grown large-scale by infecting VERO cells in a 2-stack plate at MOI = 0.001. The supernatant was collected every 4 days for 6 harvests. Harvests were titered using rabies focus-forming assay55 and harvest 4–6 were pooled and concentrated 9× in a stirred 300-ml ultrafiltration cell (Millipore). Concentrated supernatant was then centrifuged for 2 h at 76,755×g through a 20% sucrose cushion using SW32 Ti rotor (Beckman, Inc.) to pellet virus particles. Virion pellets were resuspended in phosphate-buffered saline (PBS), and protein concentrations were determined using a bicinchoninic acid (BCA) assay kit (Pierce). The virus particles were chemically inactivated with β-propiolactone (BPL) at a dilution of 1:2000 overnight at 4 °C. BPL in the virus preparation was inactivated the next day by hydrolysis at 37 °C for 30 min. The absence of infectious particles was verified by inoculating VERO cells in a T25 vessel with 10 μg of BPL-inactivated virus for two passages. Inoculated cells were fixed and stained with FITC-conjugated anti-RABV N mAb and visualized by fluorescence microscopy for the presence of foci of infection.

Immunofluorescence testing of the vaccine

VERO cells were plated onto 12 well plates with 3E5 cells with 15 mm circular diameter coverslips inserted and then incubated overnight at 37 °C. The next day the wells were infected at an MOI of 0.01 in 500 μL of serum-free media (OptiPro) per well with NIPARAB or BNSP333-cover-G mixed by rocking and then stored at 34 °C for 48 h. After 48 h, cells were washed with 1 mL of 1× PBS, then fixed with 500 μL of 2% paraformaldehyde (PFA) diluted in PBS for 15 min at room temperature. PFA was removed by aspiration and cells washed 3× with 1× PBS. 1 mL of blocking solution (4% fetal bovine serum [FBS] in PBS) was added to each well for 1 h at room temperature while on the shaker. Blocking solution was aspirated off, then 500 μL of dual primary stain (1:250 dilution of anti-RABV G human mAb 4C12 at 4 mg/mL (Dr. Scott Dessain, Lankenau Institute for Medical Research, Wynnewood, PA) plus 1:200 dilution of mouse sera) in 2% FBS was added for 1 h while rocking. Cells were washed four times with 1× PBS and then incubated with 500 μL of a 1:250 dilution of both anti-mouse Cy3 (Jackson ImmunoResearch) and anti-human Cy2 (Jackson ImmunoResearch) secondary antibodies containing Cy2 and Cy3 dyes and incubated at room temperature for 45 min. Cells were washed 5 times with 1× PBS and then cells were mounted onto slides with the mounting solution containing DAPI (Abcam) with the coverslips face down onto the slide and stored overnight at room temperature for visualization by confocal microscopy the following day.

Virus characterization

Sucrose-purified virus particles were denatured with urea buffer (125 mM Tris-HCl [pH 6.8], 8 M urea, 4% sodium dodecyl sulfate, 5% β-mercaptoethanol, 0.02% bromophenol blue, Thermo Fisher Scientific) at 95 °C for 5 min. Stained protein gel: 8 μg of particles were resolved by 10% SDS–PAGE and thereafter stained overnight with SYPRO Ruby for total protein analysis. Western blot: 4 μg of particles were resolved on a 10% SDS-PAGE gel and transferred onto a nitrocellulose membrane in Towbin buffer (192 mM glycine, 25 mm Tris, 20% methanol, Thermo Fisher Scientific) for Western blot analysis. The nitrocellulose membrane was then blocked in TBST (100 mM Tris-HCl [pH 7.9], 150 mM NaCl, 0.05% Tween 20, Thermo Fisher Scientific) containing 5% dried milk (MilliporeSigma) at room temperature for 1 h. After blocking, the membrane was incubated overnight with rabbit polyclonal HeV G antisera (Dr. Christopher Broder, Uniformed Services University, Bethesda, MD) at a dilution of 1:1000 in antibody diluent (1% bovine serum albumin (BSA) in PBS with 0.1% Tween-20). After washing, the blot was incubated for 1 h with donkey anti-rabbit IgG conjugated to horseradish peroxidase (HRP) at a 1:20,000 in antibody diluent. Bands were developed with SuperSignal West Dura Chemiluminescent substrate (Pierce). The SDS-PAGE gel and corresponding Western blot in Fig. 4 derived from the same experiment and were processed in parallel.

Pathogenicity and immunogenicity studies

(i) Animal ethics statement

This study was carried out in strict adherence to recommendations described in the Guide for the Care and Use of Laboratory Animals,56 as well as guidelines of the National Institutes of Health, the Office of Animal Welfare, and the United States Department of Agriculture. All animal work was approved by the Institutional Animal Care and Use Committee (IACUC) at Thomas Jefferson University (animal protocols 00990 and 01526). All procedures were carried out under isoflurane anesthesia by trained personnel, under the supervision of veterinary staff. Mice were housed in cages, in groups of 5, under controlled conditions of humidity, temperature, and light (12-h light/12-h dark cycles). Food and water were available ad libitum.

(ii) Immunizations

Three groups of 6-week-old to 8-week-old C56BL/6 mice were immunized intramuscularly with 10 μg of virus particles in a total volume of 100 μL (50 μL per hindlimb). The three groups were as follows: inactivated NIPARAB, live NIPARAB, and inactivated BNSP333. Each group consisted of 5 male and 5 female mice. Mice receiving inactivated vaccine were given two doses, once on day 0 and once on day 28, while mice receiving the live NIPARAB vaccine were only immunized once, on day 0.

(iii) Pathogenicity experiments

Three groups of 6-week-old to 8-week-old C56BL/6 mice were intranasally infected with 10 μL of 5.6 × 105 ffu of live virus (SPBN, BNSP333, or NIPARAB). Each group consisted of 5 male and 5 female mice. The mice were monitored for signs of disease such as ruffled fur, ataxia, and disorientation and weighed until day 40. Mice that lost more than 20% of their original weight were considered to have reached the endpoint and were euthanized.

Production of HA-tagged NiV G

Subconfluent T175 flasks of 293 T cells (human kidney cell line) were transfected with a eukaryotic expression vector (pDisplay) encoding amino acids 71 to 602 of the head and stalk domains of codon-optimized NiV G (Bangladesh strain) fused to an N-terminal hemagglutinin (HA) peptide. Supernatant was collected 48 h after transfection, clarified by centrifugation, and filtered through a 0.45 μm filter before being loaded onto an equilibrated anti-HA agarose column (Pierce) containing a 2.5 ml agarose bed volume. The supernatant was allowed to bind to the column overnight at 4 °C. The next day, the column was washed with 10-bed volumes of TBST (TBS with 0.05% Tween 20) and 2-bed volumes of TBS, and bound HA-coNiV-G was eluted with 5 ml of 250 μg/ml HA peptide in TBS. Fractions were collected and analyzed for the presence of HA-coNiV-G by western blotting with monoclonal anti-HA antibody (Sigma) prepared in 5% BSA-TBST. Peak fractions were pooled and dialyzed against PBS in 10,000 molecular weight cutoff dialysis cassettes (MWCO) (Thermo Scientific) to remove excess HA peptide. After dialysis, the protein was quantified by BCA and frozen in aliquots at −80 °C.

RABV and NiV G responses by ELISA

Sera from immunized mice were collected by retro-orbital eye bleed under isoflurane anesthesia on days 0, 14, and 45, and samples were tested for immunogenicity by indirect ELISA using N-terminus HA-tagged soluble recombinant protein for antibody capture. We tested individual mouse sera, as well as pooled sera for the presence of total IgG specific to NiV G and RABV G. To test for anti-NiV G humoral responses, we produced soluble NiV G (sNiV-G) as described above. sNiV-G was diluted in coating buffer (50 mM Na 2 CO 3 [pH 9.6]) at a concentration of 500 ng/mL and then plated in 96-well ELISA MaxiSorp plates (Nunc) at 100 μL in each well. RABV-G was also resuspended in coating buffer at a concentration of 500 ng/mL and then plated in 96-well ELISA MaxiSorp plates (Nunc) at 100 μl per well. After overnight incubation at 4 °C, plates were washed three times with PBST (0.05% Tween 20 in 1× PBS) and incubated for 1 h at room temperature with blocking buffer (5% dry milk powder in 1× PBST) in a volume of 250 μl per well. The plates were then washed three times with PBST and incubated overnight at 4 °C with 3-fold or 4-fold serial dilutions of sera from immunized mice in PBS containing 0.5% BSA. Plates were washed 3 times the next day, followed by the addition of horseradish peroxidase-conjugated goat anti-mouse-IgG (H + L) secondary antibody (1:10,000) (Jackson ImmunoResearch). After incubation for 2 h at room temperature, plates were washed 3 times with PBST, and 200 μl of o-phenylenediamine dihydrochloride (OPD) substrate (Sigma) was added to each well. The reaction was stopped by the addition of 50 μl of 3 M H 2 SO 4 per well after 15 min. Optical density was determined at 490 nm (OD 490 ).

Fluorescence reduction neutralization assay (FRNA50)

FRNA50 was used to determine the highest serum dilution, which would reduce the infectivity of the virus by 50%. This assay (with minor modifications) was described in Walpita et al., 2017 (ref.14). Briefly, VERO E6 cells were seeded at 40,000 cells/well at 1 day prior to the experiment. Serum samples were heat inactivated for 60 min at 56 °C and serially diluted in serum-free DMEM. After incubation, diluted samples were mixed with 4000 PFU of NiV and incubated for 1 h at 37 °C. Serum-virus mixtures were added to cells’ monolayers and incubated at 37 °C/5% CO 2 for 48 h before fixing with 10% NBF for 24 h. Immunofluorescence assay (IFA) was performed by permeabilizing of cells with 0.25% Triton X-100 in PBS for 5 min, blocking with 3% BSA for 30 min, staining with a NiV G protein-specific rabbit polyclonal antiserum (produced by ThermoFisher Scientific from NiV GP “293 FreeStyle Tet-NiV-sG”, a gift from Dr. Christopher C. Broder, Uniformed Services University) in 3% BSA diluted at 1:2000 at 37 °C for 60 min. In the final step of IFA, goat anti-rabbit IgG (H + L) secondary antibody, Alexa Fluor® 594 conjugate (Life Technologies), and Hoechst 33342 nucleic acid stain (ThermoFisher Scientific) were applied to the cells for 30 min at room temperature (1:2500 dilution each). Cells were washed with 1× PBS between each step, except for “blocking-primary antibody step”. The percentage of cells infected with NiV was detected using High Content Imaging System Operetta (PerkinElmer).

Reporting Summary

Further information on experimental design is available in the Nature Research Reporting Summary linked to this article.