Study design

This was a phase 1, open-label, dose-escalation, nonrandomized, first-in-human trial to evaluate the safety, tolerability and immunogenicity of mRNA-1215 in healthy adults (ClinicalTrials.gov NCT05398796). Eligible study participants were healthy adults, based on medical history and physical examination, who were 18–60 years of age. Participants were able and willing to complete the informed consent process, provide proof of their identity and were available for clinic follow-up visits for the duration of the trial (52 weeks after the last product administration). The participants were required to have a body mass index of 18–35 kg m⁻² within 56 days before enrollment.

Inclusion criteria

Laboratory-based inclusion criteria within 56 days before enrollment included white blood cell and differential count within the institutional normal range or accompanied by principal investigator (PI) or designee approval; total lymphocyte count ≥800 cells per μl; platelets count of 125,000–500,000 cells per μl; hemoglobin within the institutional normal range or accompanied by PI or designee approval; alanine aminotransferase ≤1.25 × the institutional upper limit of normal (ULN); aspartate aminotransferase ≤1.25 × the institutional ULN; alkaline phosphatase

Exclusion criteria

Study participants were excluded from the study if the following conditions applied: females who were breast-feeding or planning to become pregnant during the study; treatment with systemic immunosuppressive medications for more than 10 days or with cytotoxic medications within the 4 weeks before enrollment or any treatment within the 14 days before enrollment; receipt of blood products within 16 weeks before enrollment; receipt of any vaccine, including COVID-19 vaccines, within 4 weeks before enrollment; receipt of an investigational research agent(s) within 4 weeks before enrollment or planned receipt of investigational products while on the study; current allergy treatment with allergen immunotherapy with antigen injections, unless the participant was on a maintenance schedule; current anti-TB prophylaxis or therapy; a known immediate hypersensitivity to any component of the study product, including polyethylene glycol; a confirmed past NiV infection, previous residence in (for more than 6 months), or planned travel for any length of time during the study to countries where NiV infection is endemic, such as Bangladesh, India or the Philippines. In addition, participants with a history of any of the following medical conditions were excluded: serious reactions to vaccines, including allergic reactions, such as anaphylaxis, urticaria or other allergic reaction requiring medical intervention, to SARS-CoV-2 mRNA vaccines, as determined by the investigator; a history of myocarditis and/or pericarditis, hereditary angioedema, acquired angioedema, or idiopathic forms of angioedema; asthma that is not well controlled; diabetes mellitus (type I or II), with the exception of gestational diabetes; thyroid disease that is not well controlled; idiopathic urticaria within the past year; autoimmune disease or immunodeficiency; hypertension that is not well controlled; bleeding disorder diagnosed by a physician (for example factor deficiency, coagulopathy, or platelet disorder requiring special precautions) or more-than-expected bruising or bleeding difficulties with intramuscular (i.m.) injections or blood draws; malignancy that is active or a history of malignancy that is likely to recur during the period of the study; a seizure disorder other than: (1) febrile seizures; (2) seizures secondary to alcohol withdrawal more than 3 years ago; or (3) seizures that have not required treatment within the last 3 years; asplenia, functional asplenia or any condition resulting in the absence or removal of the spleen; Guillain–Barré syndrome; any medical, psychiatric or social condition, occupational reason or other responsibility that, in the judgment of the investigator, is a contraindication to protocol participation or that impairs a participant’s ability to give informed consent, including but not limited to certain clinical presentations of infectious diseases, drug or alcohol abuse, autoimmune diseases, psychiatric disorders, or heart disease. Because the effects of the vaccine on the fetus are not known, pregnant women were not eligible for the trial. Children were not eligible to participate in this clinical trial because the investigational vaccine had not been previously evaluated in adults.

The study was sponsored by the National Institute of Allergy and Infectious Diseases (NIAID) Division of Microbiology and Infectious Diseases and conducted at the National Institutes of Health (NIH) Clinical Center by the Vaccine Research Center Clinical Trials Program, NIAID, NIH, in Bethesda, MD after approval by the NIH Institutional Review Board. Participants were recruited from the greater Washington, DC area using institutional review board-approved recruitment advertisements and community outreach and engagement. Written informed consent was obtained from all participants before enrollment in the study. Participants were compensated for their time and inconvenience associated with participation in the trial.

Trial vaccine

The investigational mRNA-1215 vaccine is a lipid nanoparticle (LNP) dispersion containing mRNA that encodes for a secreted pre-fusion stabilized F component covalently linked to a G monomer (Pre-F/G of a NiV Malaysian 1999 strain (isolate NV/MY/99/VRI-2794), GenBank AJ564621) with a trimerization domain resulting in secretion of a trimer of heterodimers^21,22. The LNPs are composed of four lipids: one proprietary ionizable lipid, SM-102, and three commercially available lipids including cholesterol, DSPC (dioleoyl-glycero-phosphocholine) and DMG-PEG2000 (di-myristoyl diglyceride-polyethylene glycol). The ratio of mRNA to lipid is constant across all doses used in this trial. mRNA-1215 is formulated in 20 mM Tris buffer, 87 mg ml⁻¹ sucrose and 10.1 mM acetate at a pH of 7.5. The vaccine was co-developed by the VRC, NIAID and Moderna, Inc. and manufactured by Moderna, Inc.

Study procedures

The trial evaluated a dose escalation of the mRNA-1215 vaccine from 25 μg to 50 μg to 100 μg. Each dose was administered intramuscularly (i.m.) in the deltoid muscle by needle and syringe as a two-dose regimen on day 0 and day 28 to 10 participants per dosing group. Enrollment began in the 25-μg dose group. After the protocol safety review team (PSRT) assessed the safety data at 2 weeks after the first vaccination for the first three 25-μg dose recipients and approved dose escalation, the enrollment began for the 50-μg dose group, while sequential enrollment continued in the 25-μg dose group. A similar dose-escalation approval occurred to open the 100-μg dose group for enrollment. After three participants received the 100-μg dose, the PSRT determined that it was safe to continue dosing in the 100-μg group and enrollment into the 50-μg and 100-μg groups alternated in a sequential fashion. After enrollment of the 25-, 50- and 100-μg dose groups was completed, a protocol-specified interim analysis of the cumulative safety and immunogenicity data from 2 weeks after the second vaccine administration was conducted, and a fourth dose, 10 μg, was selected and administered as a two-dose regimen 28 days apart to an additional group of ten participants.

Study participants self-reported sex (male or female), race (American Indian/Alaska Native, Asian, Native Hawaiian or other Pacific Islander, Black or African American, White, Multiracial or unknown/not reported), and ethnic group (Hispanic or Latin American or Not Hispanic or Latin American background) on a demographic form provided to them after enrollment. Subsequently, these data were entered into the study database. Study investigators conducted clinical assessments of participants pre-vaccination and at scheduled visits throughout the study with the last study visit occurring 52 weeks after the second vaccination. Participants were observed for at least 30 min after vaccination, and at the end of the observation period, vital signs were obtained, and the injection site was assessed. A phone evaluation was conducted the day after each vaccination, and a clinic visit was established, if indicated, based on symptoms and signs reported by the participant. Blood for clinical laboratory testing to assess safety was obtained before each vaccination and at various time points after vaccination and included a complete blood count with differential, total bilirubin, aspartate and alanine aminotransferase (AST and ALT), alkaline phosphatase and creatinine. A pregnancy test was obtained before each vaccination for women of reproductive potential and was confirmed to be negative before vaccine administration. Blood was also collected for evaluation of immunogenicity using serum, plasma and peripheral blood mononuclear cells (PBMCs).

Participants recorded solicited local and systemic AEs (reactogenicity signs and symptoms) and concomitant medications on a diary card, daily, for 7 days after each vaccination. Unsolicited AEs were collected for 28 days after each vaccine administration. The severity of AEs was determined using the Toxicity Grading Scale for Healthy Adults and Adolescent Volunteers Enrolled in Preventive Vaccine Clinical Trials (US FDA Administration Guidance-September 2007). Serious AEs (SAEs), AEs leading to withdrawal from the study, new-onset chronic medical conditions and AEs of special interest were collected for assessment of safety after the first vaccination through the last study visit. AEs of special interest included thrombocytopenia, new-onset or worsening neurological diseases (Guillain–Barré syndrome, acute disseminated encephalomyelitis, idiopathic peripheral facial nerve palsy or seizures), anaphylaxis, myocarditis, pericarditis and myopericarditis. Medically attended AEs were collected through 6 months after the second dose.

All secondary and exploratory immunogenicity assessments in this trial were performed using the same cohort of 40 participants: 18 females and 22 males with a mean age of 37 years (range 22–59); therefore, sex and age of the participants were consistent across all assessments.

The clinical trial protocol was amended several times during the course of trial. One amendment, implemented on 20 July 2022, was made in response to a US FDA request to add additional information on product preparation to the protocol (section 7.3.1). Another amendment, implemented on 15 June 2023, updated the group 4 dose (10 μg) and its schedule of administration from a 12-week interval to a 4-week interval and added safety information for groups 1–3 (25, 50 and 100 μg). Additional amendments were related to the change of the study PI and were implemented on 22 December 2022, 20 December 2023 and 15 June 2023.

There were a total of 16 minor protocol deviations that included: ten follow-up visits that occurred out-of-window (ranging from 1–8 days); one missed visit due to participant’s illness; one incomplete sample collection due to side effects experienced by the study participant during an apheresis procedure; two incomplete blood draws due to inability to collect more than 100 ml of the required 120 ml of blood; one incident of a blood collection of 6 ml over the allowable 550 ml per 8-week limit; and one incident of a participant dating the informed consent with the incorrect year. All the protocol deviations were minor and had no impact on the safety of the study participants or integrity of the study results.

Study objectives

The primary objective of the trial was to evaluate the safety and tolerability of the two-dose vaccination regimen at each dose. The secondary objective was to evaluate Pre-F and G antibody responses to the mRNA-1215 vaccine at 2 weeks after the second vaccination at each dose level. Exploratory objectives included evaluation of Pre-F and G antibody responses, neutralizing antibody responses, and antigen-specific T and B cell responses at various time points throughout the study.

Statistical analysis

All participants who received at least one vaccination were included in the analysis of safety and reactogenicity and in the immunogenicity analysis. One participant in the 10-μg dose group, received only the first dose of vaccine, and hence, did not have a visit at 2 weeks after the second dose, the secondary immunogenicity time point. One participant in the 25-μg group missed the visit at 2 weeks after the second dose and also did not have the secondary immunogenicity time point. We conducted a power calculation, based on our primary safety objective, to determine our ability to detect SAEs with group sample sizes of ten. For each group size of ten participants, there was over a 90% chance to observe at least one SAE if the true event rate was at least 0.21, and over a 90% chance to observe no SAE if the true rate was no more than 0.01. To compare immune responses between dose groups, comparisons were made using ANOVA, followed by pairwise two-sample t-tests after log₁₀ transformation of raw titers. Within-group time point comparisons were performed using paired t-tests after log₁₀ transformation of raw titers. Reported GMTs and 95% CIs are asymptotic. Given the small sample size of this trial, group comparisons are reported descriptively. The study was not designed to detect large immunological differences between the dose groups (1.2 × s.d. of the immune response with 80% power to detect between-group differences if the mean difference between groups is at least 1.2 times the standard deviation of the immune response). To maintain statistical power in this smaller study, adjustments for multiple comparisons were not performed. A post hoc analysis of the ELISA immunogenicity analysis was performed by implementing mixed models for repeated measures with effects for week, dose group and interaction between week and dose group using the mmrm package in R⁴⁴. Statistical analyses for the exploratory end points that evaluated NiV(B) neutralization and HeV Pre-F and G ELISA and neutralization assays were conducted in the post hoc manner. No sex-based analyses were performed; because of the small size of our study, such analyses were not preplanned. All data were graphed in Prism (v.10.4.1).

All statistical tests were two-sided. For ELISA binding assays, all titer values, including those below the LLOQ, were detected and used in the analysis. For neutralization assays, titer values that were negative were imputed as the reciprocal of half of the lowest dilution tested, which was 1:50, the LOD. For both ELISA and neutralization assays, measurements were obtained through repeated assessment of the same sample at a single time point, and the mean of the replicates was used for statistical analyses. Analyses were performed using R v.4.3.3, and results were considered statistically significant if P 

Assessment of anti-Pre-F and anti-G IgG binding antibody responses

The methods for the NiV IgG ELISA have been previously described²², and the following modifications were implemented in this study. Immulon 4HBX 384-well ELISA plates (Thermo Scientific) were coated with 40 ng per well of NiV Malaysia 1999 isolate NV/MY/99/VRI-2794 (GenBank AJ564621) stabilized Pre-F or monomeric G protein in 0.2 M BupH carbonate-bicarbonate buffer, pH 9.4 (Thermo Scientific) at 4 °C for 16 h. For HeV assays, 80 ng per well of Pre-F and monomeric G from HeV Australia 1994 (GenBank AF017149) viral isolate was used for plate coating. Each sample was run on two separate 384-well ELISA plates coated with Pre-F or monomeric G protein. After three washes in 1× PBS/0.05% Tween-20 (PBS-T), the plates were blocked with 1× PBS/20%FBS for 1 h at room temperature and then washed again in PBS-T. Serum samples were diluted as fourfold serial dilutions in 1× PBS/1% FBS/0.2% Tween-20 solution, added to the antigen-coated plates, and incubated for 45 min at room temperature. Following PBS-T washes, anti-nonhuman primate IgG-horseradish peroxidase conjugate (Southern Biotech) cross-reacting to human IgG was prepared in 1× PBS/2% milk and added to the plates for a 45-min incubation at room temperature. After final washes with PBS-T, SureBlue 3,5,3′,5′-tetramethylbenzidine (TMB) (SeraCare KPL) was used as the substrate to detect antibody responses. The reactions were stopped with 1 N H₂SO₄ sulfuric acid (Sigma Aldrich) within 15 min. Optical density (OD) measurements were obtained after reading the plates at 450 nm with an Envision plate reader (PerkinElmer). The value of the antibody response was determined by obtaining half of the maximum effective concentration (EC₅₀) titer from the OD450 nm curves for the function of the reciprocal dilution using a 4-PL curve fit in (Prism, v.10.2.2 software). NiV Pre-F and G EC₅₀ titers were normalized using the International Standard and reported as IU ml⁻¹, whereas HeV Pre-F and G EC₅₀ titers were not normalized and reported as such. LLOQ for NiV assays were determined to be 10.15 IU ml⁻¹ for Pre-F and 16.32 IU ml⁻¹ for G, respectively. For assessment of anti-Hendra virus binding antibody responses, the Pre-F and G proteins from HeV Australia (1994 AF017149) were used to coat the plates and assays were run as described above.

Assessment of neutralizing antibody responses

Serum neutralizing activity was measured in a luciferase reporter gene assay using single round infection of HEK293T (ATCC CRL-11268) target cells with pseudo-typed virus particles (pseudovirus). Pseudovirus was produced according to the manufacturer’s protocol via Lipofectamine3000 (Thermo Fisher) transfection of HEK293T cells with DNA plasmids encoding NiV full-length F and truncated G (GΔ34)⁴⁵ proteins in combination with a luciferase reporter plasmid (VRC5601: pHR’ CMV Luc), and a plasmid containing the essential HIV structural genes (VRC5602: pCMV DR8.2) at a plasmid ratio of 6:6:1:1 F:GΔ34:VRC5601:VRC5602. Neutralization was measured by adding 10 μl of serum (nine threefold dilutions ranging from 1:50 to 1:328,050) in complete Dulbecco’s modified Eagle medium (cDMEM), no phenol red, supplemented with 10% heat-inactivated fetal bovine serum (FBS), 0.6 mg ml⁻¹ L-glutamine and 1% penicillin–streptomycin (all from Thermo Fisher) and added to 10 μl of pseudovirus (at a concentration selected to yield relative light units (RLU) of at least ten times above background) in black ViewPlate 384-F tissue culture-treated plates (Perkin Elmer) with a white adhesive bottom seal (Revvity) that were incubated at 37 °C with 5% CO₂, 95% humidity. After 30 min, 20 μl of HEK293T cells (~2,600 cells per well) were added to all wells and incubated at 37 °C with 5% CO₂, 95% humidity. Each experiment plate contained a column of cells only (no serum or virus) as a control for background luciferase activity and a column of virus and cells only (no serum) as a maximal viral entry control. After 72 h, 40 μl Bright-Glo luciferase assay substrate (Promega) was added to each well, mixed by pipetting, and RLU was measured at 570 nm on a SpectraMax L luminometer (Molecular Devices). The 50% inhibitory dilution (ID₅₀), defined as the serum dilution required to achieve 50% reduction in RLU compared to virus control wells after subtraction of background RLUs, was obtained using an asymmetric five parameter fit curve (Prism v.10.0.0). For neutralization curves that did not reach 20% neutralization (background), ID₅₀ is reported as 25 (below the lowest (50-fold) dilution tested), whereas for neutralization curves that reached below 50% but above 20% neutralization, ID₅₀ was reported as 50 (the lowest (50-fold) dilution tested). ID₅₀ values are derived from three NiV Malaysia (GenBank AJ564621) and HeV Redlands (GenBank HM044317) assays or two NiV Bangladesh 2004 (isolate 810398) Hu – MK673564 isolate) assays with two technical replicates per assay. NiV(M) and NiV(B) ID₅₀ titers were normalized using the International Standard and reported as IU ml⁻¹, whereas HeV ID₅₀ titers were not normalized and reported as such. The LOD for neutralization assays was determined to be 17 IU ml⁻¹ for NiV(M), 9 IU ml⁻¹ for NiV(B) and an ID₅₀ titer of 50 for HeV.

Normalization of NiV immunogenicity data to the WHO/NIBSC NiV international standard

All NiV virus ELISA binding and neutralization assays included the WHO International Standard for anti-NiV antibodies as an additional sample (National Institute for Biological Standards and Control (NIBSC) code 22/130) to enable harmonization of the ELISA binding and neutralization data to a common unit, thereby facilitating comparison to different NiV vaccine studies and assays⁴⁶. The International Standard is based on a pool of sera from 36 NiV convalescent individuals: 6 from Malaysia and 30 from Bangladesh. Normalization of ED₅₀ or IC₅₀ titers was achieved by using the equation: EC₅₀ or ID₅₀ of sample × 1,000/EC₅₀ or ID₅₀ International Standard median across assays. Thus, the International Standard had an attributed value of 1,000 IU ml⁻¹ for each graphical representation of data of anti-NiV antibody responses. An International Standard for F glycoprotein binding was not established due to the lack of enough data for anti-Pre-F assays during the International Standard characterization study⁵. However, we decided to use the International Standard for the normalization of anti-NiV Pre-F antibody titers. Due to the lack of an International Standard for HeV, HeV binding and neutralizing titers are expressed as EC₅₀ and ID₅₀ titers, respectively.

Assessment of B cell responses by B cell probe binding

Cryopreserved PBMCs were thawed, washed briefly with phenol-free RPMI/4% heat-inactivated newborn calf serum (R&D Systems), and incubated with an aqua live/dead fixable dead cell stain kit (Thermo Fisher Scientific) for 20 min at room temperature. Cells were stained with the following antibodies (all monoclonal unless indicated) for 20 min at room temperature: IgD FITC (1:160 dilution) (goat polyclonal, Southern Biotech), IgM PerCP-Cy5.5 (1:20 dilution) (clone G20-127, BD Biosciences), IgA Dylight 405 (1:80 dilution) (goat polyclonal, Jackson Immunoresearch), CD20 BV570 (1:40 dilution) (clone 2H7, BioLegend), CD27 BV650 (1:20 dilution) (clone O323, BioLegend), CD14 BV785 (1:80 dilution) (clone M5E2, BioLegend), CD16 BUV496 (1:80 dilution) (clone 3G8, BD Biosciences), CD4 BUV737 (1:320 dilution) (clone SK3, BD Biosciences), CD19 APC (1:80 dilution) (clone J3-119, Beckman), IgG Alexa 700 (1:20 dilution) (clone G18-145, BD Biosciences), CD3 APC-Cy7 (1:40 dilution) (clone SP34-2, BD Biosciences), CD38 PE (1:160 dilution) (clone OKT10, Caprico Biotechnologies), CD21 PE-Cy5 (1:40 dilution) (clone B-ly4, BD Biosciences) and CXCR5 PE-Cy7 (1:40 dilution) (clone MU5UBEE, Thermo Fisher Scientific). Stained cells were then incubated with matched Pre-F trimer and G trimer probe pairs: streptavidin-BUV661 (BD Biosciences) labeled Bangladesh Pre-F probe (1:25 dilution) and streptavidin-BUV395 labeled Malaysia Pre-F probe (1:25 dilution), and streptavidin-BUV661 (BD Biosciences) labeled Bangladesh G probe (1:25 dilution) and streptavidin-BUV395 labeled Malaysia G probe (1:25 dilution) for 30 min at 4 °C (protected from light). The Pre-F probe used in these assays (GenBank AJ627196) is derived from a NiV Malaysia 1999 isolate that differs by only one amino acid, at position T348M, from AJ564621, the isolate sequence used for mRNA-1215. The NiV Malaysian G probe matched the sequence of G in the vaccine. For the Bangladesh probes, both the Pre-F and G amino acid sequences were derived from the 2004 sequence Genebank MK673564. Cells were washed and fixed in 0.5% formaldehyde (Tousimis Research Corp) before data acquisition. All antibodies were titrated on human PBMCs to determine the optimal concentration. Samples were acquired on a BD FACSymphony cytometer and analyzed for the B cell phenotype using FlowJo v.10.10.0 (BD).

Assessment of T cell responses by intracellular cytokine staining

Cryopreserved PBMCs were thawed and incubated overnight at 37 °C/5% CO₂. After incubation, cells were stimulated with Malaysia Pre-F or Malaysia G peptide pools (Malaysia 1999 isolate NV/MY/99/VRI-2794 (GenBank AJ564621); JPT Peptides) at a final concentration of 2 μg ml⁻¹ in the presence of 3 mM monensin for 6 h. The Pre-F and G peptide pools consist of 123 and 108 individual peptides, respectively, as 15mers overlapping by 11 amino acids in 100% dimethylsulfoxide (DMSO). Negative controls received an equal concentration of DMSO to that of peptide pools (final concentration of 0.5%). Intracellular cytokine staining was performed as described previously⁴⁷. The following monoclonal antibodies were used: CD3 APC-Cy7 (1:160 dilution) (clone SP34.2, BD Biosciences), CD4 PE-Cy5.5 (1:80 dilution) (clone SK3, Thermo Fisher), CD8 BV570 (1:40 dilution) (clone RPA-T8, BioLegend), CD45RA PE-Cy5 (1:2,500 dilution) (clone 5H9, BD Biosciences), CCR7 BV650 (1:10 dilution) (clone G043H7, BioLegend), CXCR5 PE (1:10 dilution) (clone MU5UBEE, Thermo Fisher), CXCR3 BV711 (1:20 dilution) (clone 1C6/CXCR3, BD Biosciences), PD-1 BUV737 (1:20 dilution) (clone EH12.1, BD Biosciences), ICOS Pe-Cy7 (1:80 dilution) (clone C398.4A, BioLegend), CD69 ECD (1:40 dilution) (cloneTP1.55.3, Beckman Coulter), IFNγ Ax700 (1:320 dilution) (clone B27, BioLegend), IL-2 BV750 (1:40 dilution) (clone MQ1-17H12, BD Biosciences), IL-4 BB700 (1:20 dilution) (clone MP4-25D2, BD Biosciences), TNF-FITC (1:80 dilution) (clone Mab11, BD Biosciences), IL-13 BV421 (1:20 dilution) (clone JES10-5A2, BD Biosciences), IL-17 BV605 (1:20 dilution) (clone BL168, BioLegend), IL-21 Ax647 (1:10 dilution) (clone 3A3-N2.1, BD Biosciences) and CD154 BV785 (1:20 dilution) (clone 24-31, BioLegend). Aqua live/dead fixable dead cell stain kit (Thermo Fisher Scientific) was used to exclude dead cells. All antibodies were previously titrated to determine the optimal concentration. Samples were acquired on a BD FACSymphony flow cytometer and analyzed using FlowJo v.10.10.0 (TreeStar).

Reporting summary

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