Development of an mRNA vaccine for Nipah virus to prepare for pandemics

HEK293FT and Vero cells (Korean Cell Line Bank, Seoul, Korea) were cultured in DMEM (Cytiva, Washington, USA) and RPMI (Cytiva, Washington, USA) containing 10% fetal bovine serum (FBS, Cytiva, Washington, USA) and 1% antibiotic–antimycotic (Gibco, Massachusetts, USA). Cells were cultured at 37 °C in a 5% CO₂ atmosphere.

NiV-F and NiV-G gene sequences were obtained from the Nipah virus Malaysia strain (NCBI GenBank accession: UMMC1, AY029767.1↗). To stabilize the prefusion conformation of NiV-F, four mutations (I114C, L104C, S191P, and L172F) were introduced. For trimer formation, a GCN4 trimerization domain was fused to the C-terminus of NiV-F, and a foldon (Fd) domain was added to the C-terminus of NiV-G (13). Both antigens were codon-optimized for expression in mice. The designed coding sequences (CDSs) were cloned into the CUK3–1 plasmid vector (provided by The Catholic University of Korea, Gyeonggi, Korea), which contains a T7 promoter, 5′ and 3′ untranslated regions (UTRs), and an approximately 100-nucleotide poly(A) tail. To enable CleanCap^® (TriLink, California, USA) incorporation during in vitro transcription (IVT), the CUK3–1 vector was further modified by site-directed mutagenesis. IVT was performed using the HiScribe^® T7 High Yield RNA Synthesis Kit (New England Biolabs, Massachusetts, USA) at 37 °C for 2 h, and no modified nucleotides were incorporated into the synthesized mRNA. The transcribed mRNA was first purified by lithium chloride (LiCl) precipitation and subsequently subjected to a second purification step using the Min-Immune Gold dsRNA Removal Kit (CellScript, Wisconsin, USA) according to the manufacturer’s protocol.

Double-stranded RNA (dsRNA) contamination in purified mRNA samples was evaluated using a dot blot assay. Briefly, 500 ng of purified mRNA was spotted onto a membrane and allowed to air-dry. The membrane was then blocked with 5% skim milk in TBST for 1h. After blocking, membrane was incubated with J2 anti-dsRNA antibody (Cell signaling Technology, Massachusetts, USA) diluted 1:1000 in 1% skim milk in TBST for 1h at room temperature (RT). HRP-conjugated anti-mouse IgG (SouthernBiotech, Alabama, USA) diluted 1:3000 in 1% skim milk in TBST was used as the secondary antibody and incubated at RT for 1 h. Signals were detected using an Enhanced Chemiluminescence (ECL) substrate (BIOMAX, Gyeonggi, Korea). Poly I:C (Invitrogen, Massachusetts, USA) was used as a positive control, and signal intensity was quantified using the ImageJ software.

NiV-F and NiV-G mRNA-LNP were prepared according to a previously established protocol (15). Briefly, mRNA was prepared in multiples of 120 µg, corresponding to the amount used per formulation. The mRNA was diluted in citrate buffer (pH 4) to generate an mRNA solution at a concentration of 120 µg per 1.5 mL. The lipid mixture was prepared by mixing SM-102 (MedChemExpress, New Jersey, USA), DSPC (Avanti Polar Lipids, Alabama, USA), cholesterol (Avanti Polar Lipids, Alabama, USA), and DEG-PEG2000 (Avanti Polar Lipids, Alabama, USA) in ethanol at a molar ratio of 9.2:2:3.8:1 and dissolved by sonication at 25 °C for 5–10 min. The mRNA solution and lipid mixture were then combined at a volume ratio of 3:1. LNP formulation was performed using the EnCELL Master system (ENPARTICLE, Busan, Korea). The formulated LNPs were transferred to Amicon Ultra-15 centrifugal filter units (Merck Millipore, Massachusetts, USA) and subjected to centrifugation at 3000 g for 20 min to perform buffer exchange into phosphate-buffered saline (PBS). Particle size and polydispersity index (PDI) were subsequently measured by dynamic light scattering (DLS).

HEK293FT cells were seeded into plates to reach 70–80% confluency. NiV-F or NiV-G mRNA was diluted in Opti-MEM (Gibco, Massachusetts, USA), and Lipofectamine 2000 (Thermo Fisher Scientific, Massachusetts, USA) was added at 2.5 times the amount of mRNA. After transfection, cells were incubated for 24 h, the supernatant was removed and proteins were extracted. Protein expression was analyzed by Western blotting. Proteins were separated on 12% SDS–PAGE gels and transferred onto membranes. Membranes were blocked with 5% skim milk in TBST for 1 h. Membranes were then incubated overnight at 4 °C with either NiV-F antibody or NiV-G antibody (Creative Diagnostics, New York, USA) diluted 1:1000 in 5% BSA in TBST. HRP-conjugated anti-mouse IgG (SouthernBiotech, Alabama, USA) diluted 1:10,000 in 5% skim milk in TBST was used as the secondary antibody and incubated at room temperature for 1 h. Signals were detected using an Enhanced Chemiluminescence (ECL) substrate (BIOMAX, Gyeonggi, Korea).

Lentivirus-based Nipah pseudovirus was generated as previously described (14). Briefly, HEK293FT cells were seeded in 10 cm dishes to reach 70–80% confluency on the following day. NiV-F and NiV-G sequences were synthesized and cloned into the pcDNA3.1 expression vector (VectorBuilder, Illinois, USA). A total of 22 µg of plasmid DNA was prepared by mixing pcDNA3.1-NiV-F (2 µg), pcDNA3.1-NiV-G (4 µg), Gag/Pol plasmid (6 µg; pMDLg/pRRE, Addgene, Massachusetts, USA), Rev plasmid (2 µg; pRSV-Rev, Addgene, Massachusetts, USA), and a mammalian gene expression lentiviral vector (8 µg; pLV[Exp]-EGFP/Puro-EF1A>Luciferase, VectorBuilder, Illinois, USA) at a ratio of 1:2:3:1:4. Transfection was performed using Lipofectamine 2000 (Thermo Fisher Scientific, Massachusetts, USA). At 5 h post-transfection, the medium was replaced with 10 mL of DMEM-5% FBS. At 48 h post-transfection, culture supernatants were collected and centrifuged at 450 g for 15 min. The clarified supernatants were collected using a 0.45 µm syringe filter. VSV-based Nipah pseudovirus was generated as previously described (16). HEK293FT cells were seeded in 10 cm dishes to reach 70–80% confluency. Cells were transfected with pcDNA3.1-NiV-F (4 μg) and pcDNA3.1-NiV-G (8 μg) at a 1:2 ratio, for a total of 12 μg of plasmid DNA, using Lipofectamine 2000 (Thermo Fisher Scientific, Massachusetts, USA). At 5 h post-transfection, the medium was replaced with 10 mL of DMEM-5% FBS. At 24 h post-transfection, the culture supernatant was removed, and cells were infected with pseudotyped ΔG-luciferase recombinant VSV (G*ΔG-luciferase-rVSV; ΔG-VSV-Luc; Kerafast, North Carolina, USA) at a multiplicity of infection (MOI) of 0.5 in 4 mL of serum-free DMEM. After incubation for 2 h, the virus inoculum was removed, and cells were washed 3–4 times with PBS to reduce background infectivity. Cells were then incubated for 24 h in DMEM-5% FBS supplemented with anti–VSV-G antibody (Kerafast, North Carolina, USA) diluted 1:1000 to further suppress residual non-specific infectivity. At 24 h post-infection, culture supernatants were collected and centrifuged at 450 g for 15 min. The clarified supernatants were collected using 0.45 µm syringe filter.

Vero cells were seeded into 96-well plates to reach 70–80% confluency on the following day. VSV-based NiV pseudovirus was serially diluted 10-fold from 10¹ to 10¹¹ in serum-free RPMI in a separate plate. The culture medium was removed from the Vero cell–seeded 96-well plates, and 50 µL of each virus dilution was transferred to the corresponding wells. After incubation for 2 h, 50 µL of RPMI-5% FBS was added to each well, and incubated at 37 °C under 5% CO₂ for 24 h. At 24 h post-infection, luminescence was measured using a One-Glo luciferase assay reagent (Promega, Wisconsin, USA) according to the manufacturer’s instructions using a luminometer (Victor Nivo Multimode Plate Reader, BMG Labtech, Ortenberg, Germany). The 50% tissue culture infectious dose (TCID₅₀) was calculated using the Reed–Muench method (17).

6-week-old female BALB/c mice were purchased from Orient Bio (Gyeonggi, Korea) and maintained under specific pathogen-free (SPF) conditions at the Center of Animal Care and Use (CACU, Incheon, Korea). For the mRNA vaccine group, NiV-F mRNA-LNP (10 µg) and NiV-G mRNA-LNP (10 µg) were mixed in PBS to a total volume of 100 µL and administered intramuscularly (IM) into the thigh muscle of the hind limb at weeks 0 and 2, for a total of two immunizations (13). For the plant-based recombinant protein vaccine (provided by the Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea), 10 µg of F+G proteins was mixed with 700 µg of aluminum hydroxide (InvivoGen, California, USA) in PBS at a 3:7 ratio and administered via the same route following the same immunization schedule. Blood was collected at weeks 0, 2, and 4 by retro-orbital bleeding under anesthesia with 1–5% isoflurane (Ifran^®, Hana Pharm, Seoul, Korea) in oxygen and serum was isolated by centrifugation at 1,000 g for 10 min. Animal experiments were conducted with approval from the Center of Animal Care and Use of the Lee Gil Ya Cancer and Diabetes Institute of Gachon University (Incheon, Korea, LCDI-2023-0144).

NiV-F or NiV-G proteins (provided by the Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea) were coated onto 96-well ELISA plates (Greiner Bio-One, Kremsmünster, Austria) and incubated at 37 °C under 5% CO₂ for 24 h. On the following day, plates were blocked and immunized mouse sera were added. For the mRNA vaccine group, mouse sera were diluted 1:500 in 1% BSA in PBS, whereas sera from the recombinant protein vaccine group were diluted 1:2500. Plates were incubated at 37 °C under 5% CO₂ for 3 h. After incubation with primary antibodies, HRP-conjugated anti-mouse IgG (SouthernBiotech, Alabama, USA) diluted 1:2000 in 1% BSA in PBS was added and incubated at 37 °C under 5% CO₂ for 1 h. Following incubation, TMB substrate (GenDEPOT, Texas, USA) was added and incubated for 15 min at room temperature in the dark. Absorbance was measured at 450 nm.

PBNA was performed as previously described (16). Briefly, Vero cells were seeded into 96-well plates to reach 70–80% confluency on the following day. Mouse sera were serially diluted 3-fold starting at 1:20 and ending at 1:43,740 in serum-free RPMI. VSV-based NiV pseudovirus was diluted in serum-free RPMI to a concentration of 200 TCID₅₀ and incubated with diluted sera at 50 µL per well on ice for 1 h. After removal of the culture medium, 100 µL of the virus–serum mixtures were added to the cells and incubated at 37 °C under 5% CO₂ for 24 h. Luciferase activity was measured using a luminometer, and relative luminescence units (RLU) were recorded. The percentage of neutralization was calculated using the following equations.

Neutralization Antibody Titer 50% (NT₅₀) was calculated using GraphPad Prism.

All statistical analyses were performed using GraphPad Prism version 8.0.2. A p value of less than 0.05 was considered statistically significant (*P< 0.05; **P< 0.01; ***P< 0.001; ****P< 0.0001; compared with the control group). All data are presented as mean ± SD.

ChatGPT (OpenAI, GPT-5.2) was used for English language translation.

To generate mRNA vaccines, NiV-F and NiV-G of the Nipah virus Malaysia strain were synthesized as individual antigens. For the NiV-F antigen, four stabilizing mutations were introduced to maintain the prefusion conformation. In addition, trimerization domains were fused to each antigen to promote trimer formation, specifically the GCN4 domain linked to NiV-F and the foldon trimerization domain linked to NiV-G (10, 13). (Figure 1A). To enhance expression efficiency in the mouse model, the coding sequences were codon-optimized for mouse expression, and the optimized CDSs were cloned into the IVT vector CUK3-1 (Figure 1A). IVT of the NiV-F and NiV-G antigens were subsequently performed, and agarose gel electrophoresis was used to verify successful transcription. Electrophoretic analysis showed that the NiV-F mRNA and NiV-G mRNA were approximately 2.1 kb and 2.3 kb in length, respectively, consistent with their expected transcript sizes, suggesting successful IVT of both antigens (Figure 1B).

Following IVT, LiCl precipitation and dsRNA removal were performed sequentially for mRNA purification. No smear-like impurities were observed after purification (Figure 1B). To further assess dsRNA contamination, dot blot analysis was conducted using an anti-dsRNA antibody. Compared with the positive control (poly I:C), little to no signal was detected in either the LiCl-precipitated samples or the dsRNA-depleted NiV-F and NiV-G mRNA samples, indicating effective removal of dsRNA (Figure 1C). Quantitative analysis of the dot blot results using ImageJ further demonstrated that residual dsRNA levels were extremely low relative to poly I:C. These results suggested that the synthesized mRNA-LNP vaccines exhibited high purity and low residual dsRNA levels following purification.

To evaluate antigen expression from the synthesized mRNA, HEK293FT cells were transfected with NiV-F or NiV-G mRNA, followed by Western blot analysis (Figure 2A). Although overall signal intensity was relatively weak, distinct bands were observed at the expected molecular weights of 60.3 kDa for NiV-F and 66.3 kDa for NiV-G. These bands were reproducibly detected at the expected molecular weights across independent experiments, suggesting qualitative expression of both antigens. To characterize the physicochemical properties of LNPs encapsulating NiV-F and NiV-G mRNA, DLS analysis was performed (Figure 2B). DLS analysis showed that the mean particle size of NiV-F mRNA-LNPs was 129.8 nm (PDI 0.225), and NiV-G mRNA-LNPs also exhibited a similar mean particle size of 128 nm (PDI 0.176), indicating relatively uniform particle size distribution characteristics of the formulated LNPs (15).

We attempted to generate NiV pseudovirus using lentivirus and VSV-based system. During lentivirus-based production of NiV pseudovirus (Figure 3A), syncytium formation mediated by NiV-F was observed at 24-48h post-transfection, and GFP expression from the lentiviral backbone was confirmed (Figure 3B). However, when the harvested virus was used for infection, the signal was comparable to background levels, indicating that infectious pseudovirus was not successfully produced (Figure 3D).

In contrast, when NiV pseudovirus was generated using a VSV-based system (Figure 3A), NiV-F–mediated syncytium formation was observed at 24 h post-transfection (Figure 3C). In infectivity assays, the harvested virus showed a signal of approximately 10⁴ RLU, confirming the production of infectious pseudovirus. However, we observed unexpectedly high levels of residual non-specific infectivity in supernatants collected from cells infected with ΔG-VSV-Luc in the absence of NiV envelope protein expression (Bald-VSV) when used without purification. Therefore, purification conditions were established, and when the optimized purification protocol was applied and evaluated in comparison with Bald-VSV, non-specific infectivity was effectively suppressed to background levels. As a result, only infectious NiV pseudovirus was selectively retained, and ultimately, purified NiV pseudovirus was successfully obtained (Figure 3E).

To evaluate the immunogenicity of the co-administered NiV mRNA-LNP vaccines, three experimental groups were established: a group receiving co-administration of NiV-F and NiV-G mRNA-LNPs, a group receiving a plant-based recombinant NiV F+G protein vaccine used in previous studies, and a PBS control group. The co-administration group receiving NiV-F and NiV-G mRNA-LNPs in combination was immunized intramuscularly with a mixture of NiV-F mRNA-LNP (10 μg) and NiV-G mRNA-LNP (10 μg) at a 1:1 ratio and was designated as group G1. The second group, designated G2, was immunized with the plant-based recombinant NiV F+G protein vaccine consisting of 10 μg of protein mixed with 700 μg of alum as an adjuvant. The PBS-treated group was designated as G3. All groups were immunized twice in weeks 0 and 2. Serum samples were collected at week 0 (pre-immunization), week 2 (pre-boost), and week 4. (Figure 4A).

To assess vaccine-induced binding antibody responses, IgG ELISA specific for NiV-F and NiV-G glycoproteins were performed (Figure 4B). Sera collected at week 0 and sera from the PBS control group at all time points exhibited uniformly low absorbance values (O.D.< 0.2) across all experimental groups, indicating the absence of antigen-specific antibodies. During week 2 post-immunization, distinct response patterns were observed depending on the antigen. NiV-F specific IgG levels in both the mRNA-LNP group and the recombinant protein vaccine group remained comparable to those of the PBS group, with no marked increase in antibody responses. In contrast, in the recombinant protein vaccine group, NiV-G specific IgG levels began to increase at 2 weeks post-immunization, with all animals exhibiting antibody responses (O.D. 1.2 ± 0.2), whereas in the mRNA-LNP group, antibody responses were observed only in a subset of animals at 2 weeks post-immunization. At week 4 post-immunization, both the mRNA-LNP group and the recombinant protein vaccine group showed pronounced increases in NiV-F and NiV-G specific antibody responses. Notably, under co-administration conditions, antibody responses against NiV-G appeared to be more strongly induced than those against NiV-F. In the mRNA-LNP group, NiV-G specific IgG levels reached an O.D. value of 2.6 ± 1.1, higher than the NiV-F specific IgG levels under the co-administration condition (O.D. 0.6 ± 0.2). Similarly, in the recombinant protein vaccine group, NiV-G specific IgG levels (O.D. 3.3 ± 0.6) were approximately threefold higher than NiV-F specific IgG levels (O.D. 1.1 ± 0.4). Overall, antigen-specific IgG responses were detected in both the mRNA-LNP vaccine group and the recombinant protein vaccine group. Our findings confirmed that the NiV-F and NiV-G antigens used in this study retained their capacity to elicit antigen-specific immune responses.

To evaluate neutralizing antibody responses induced by co-administered NiV mRNA-LNP vaccines, a pseudovirus-based neutralization assay was performed (Figures 5A, B). Sera collected at week 2 post-immunization showed little to no neutralizing activity across the entire dilution range and exhibited levels comparable to those of the PBS control group. In contrast, sera collected at week 4 post-immunization displayed clear dose-dependent neutralizing activity (Figure 5A). Based on these results, neutralizing antibody titers were quantitatively assessed using NT₅₀ values (Figure 5B). No neutralizing activity was detected in the PBS control group, and NT₅₀ values were therefore denoted as not detected (N.D.). Similarly, sera collected at week 2 post-immunization showed no detectable neutralizing activity in most animals, with only a subset exhibiting very low NT₅₀ values at approximately 1 × 10¹. In contrast, sera collected at week 4 post-immunization showed a significant increase in NT₅₀ values, reaching approximately 10³–10⁴ in all animals, confirming robust induction of neutralizing antibodies. A commercial polyclonal anti-NiV-G antibody used as a positive control exhibited the highest NT₅₀ values (>10⁴) and served as a validation control demonstrating the appropriate performance of the assay.