Oropouche virus mRNA vaccines provide full protection against related virus strains in mice

To evaluate antigenic differences critical for vaccine design, we compared the envelope glycoprotein complexes of the well-characterized prototype strain OROV BeAn19991 (Brazil, 1960) and OROV AM0059, a contemporary clinical isolate associated with the 2024 resurgence in Brazil that reflects the antigenic diversity of currently circulating OROV. Gn and Gc form a surface heterodimer; across orthobunyaviruses, Gc is the principal target of neutralizing antibodies and a key mediator of entry and fusion (22–24), whereas Gn mainly supports virion assembly and shields Gc epitopes. Accordingly, structural and sequence comparisons focused on Gc as the most relevant antigen for neutralization-based immunity.

We compared Gc from the prototype BeAn19991 strain and the 2024 Brazilian isolate AM0059. Monomeric ectodomain structures were predicted with AlphaFold3 (25). Both models had high confidence (pTM = 0.86; Fig. S1A) and aligned well to Schmallenberg virus Gc (PDB: 6H3S [22]) (Fig. S1B). Sequence comparison revealed 14 amino acid substitutions—seven in the core domain and seven in the head/stalk region. Of the seven head/stalk substitutions, five are predicted to be surface-exposed (Fig. 1A). These substitutions did not produce global conformational changes or alter electrostatic surface potentials in a characteristic manner (Fig. S1B and C), suggesting that strain-specific modifications are more likely to modulate local dynamics or stability than to remodel the overall fold.

Although Gc is the dominant neutralizing antigen, we also engineered full-length GnGc constructs to test whether Gn affects antigen expression, folding, or immunogenicity in vivo. Based on the sequence analysis, we generated four human-codon-optimized mRNA-LNP constructs encoding either BeAn19991 or AM0059 glycoproteins: BeAn19991 GnGc, BeAn19991 Gc, AM0059 GnGc, and AM0059 Gc (Fig. 1B). Each transcript included an N-terminal signal peptide and a poly(A) tail; the NSm region, a non-structural membrane protein encoded within the M segment and not required for virion assembly or immunogenicity, was omitted (26).

HEK293T cells were transfected with plasmid DNA, and western blot results showed that only the human codon-optimized plasmids produced readily detectable levels of glycoprotein expression, highlighting the importance of codon optimization for efficient antigen production (Fig. S2A). We further tested using in vitro-transcribed mRNA (Fig. 1C) and LNP-formulated mRNA (Fig. 1D), and western blots with OROV Gc-specific antibodies demonstrated robust expression across all formats. To verify Gn expression, an additional GnGc construct bearing an N-terminal V5 tag was probed with anti-V5 antibody, confirming efficient co-expression of Gn and Gc at the expected sizes (Fig. S2B and C). Collectively, these data validate the vaccine design and demonstrate that both glycoproteins are produced as intended.

To characterize the antibody responses induced by the codon-optimized OROV mRNA-LNP vaccines, we immunized BALB/c mice intramuscularly with LNP-formulated mRNAs encoding either the BeAn19991 or AM0059 glycoprotein, expressed as full-length GnGc or Gc-only constructs. Mice received a prime-boost regimen at weeks 0 and 3, and sera were collected at weeks 0, 2, 5, and 9 (Fig. 2A).

Humoral responses were first assessed by ELISA using purified BeAn19991 virion lysate to quantify prototype strain-specific antibodies. All vaccinated groups developed significantly higher total anti-BeAn19991 IgG titers than the phosphate-buffered saline (PBS) group (Fig. 2B). IgG levels were markedly increased after the booster immunization, indicating a strong immune response. Although all mRNA-LNP formulations induced comparable antibody titers, no significant differences were observed between the GnGc- and Gc-only constructs, suggesting that both vaccine designs elicited similarly robust systemic IgG response against the BeAn19991.

Neutralizing capacity was next examined using a VSV-based pseudovirus displaying the BeAn19991 glycoprotein. All vaccine constructs elicited strong pseudovirus-neutralizing activity, as shown by high 50% inhibitory concentration (IC₅₀) titers measured at weeks 5 and 9 following the booster immunization (Fig. 2C). Notably, the AM0059 Gc construct induced the highest neutralizing titers against the BeAn19991 pseudovirus, indicating that antibodies generated from Brazilian isolate AM0059 antigen effectively recognized and neutralized the prototype strain. In contrast, sera from PBS-treated mice showed no inhibition of pseudovirus entry, confirming the specificity of vaccine-induced responses.

To further confirm these findings, we evaluated neutralization using the authentic rOROV^BeAn19991 strain in a focus reduction microneutralization test (FRµNT). Consistent with the pseudovirus results, sera from all vaccinated groups showed significantly higher neutralization activity than the PBS control (Fig. 2D and E), with corresponding infection data provided in Fig. S3A and B. Among the vaccine formulations, sera from the AM0059 Gc group exhibited the highest neutralization activity, significantly exceeding the other vaccine groups, whereas PBS sera showed no detectable inhibition.

Together, these results demonstrate that the human codon-optimized OROV mRNA-LNP vaccines elicit robust antibody responses directed against the prototype rOROV^BeAn19991 strain, characterized by high systemic IgG levels and strong neutralizing activity in both pseudovirus and authentic-virus assays.

Having established that both prototype- and outbreak-based mRNA vaccines elicit strong antibody responses against the prototype BeAn19991 strain, we next evaluated the breadth of these responses against a more recently isolated OROV strain. Because the Brazilian AM0059 isolate was unavailable for downstream assays, we used the closely related Cuba isolate OROV²⁴⁰⁰²³, obtained from BEI Resources (NIAID, NIH). This choice is epidemiologically relevant, as over 100 imported OROV cases linked to Cuba have been reported in the United States, underscoring the importance of assessing vaccine responses against this circulating lineage (6). Pairwise comparison of the OROV^AM0059 and OROV²⁴⁰⁰²³ Gc glycoproteins revealed 99.79% sequence identity and 99.89% amino acid similarity, differing by only two residues (one conservative), indicating that OROV²⁴⁰⁰²³ serves as an appropriate surrogate for evaluating recognition of contemporary OROV variants (Fig. 3A). Sera from mice immunized with either BeAn19991- or AM0059-based constructs were analyzed for binding and neutralizing activity against OROV²⁴⁰⁰²³ antigens. The immunization regimen was identical to that used in the prototype strain study (Fig. 2A).

ELISA using purified OROV²⁴⁰⁰²³ virion lysate revealed a significant increase in total IgG titers across all vaccinated groups following prime-boost immunization (Fig. 3B). Antibody levels were comparable among the constructs, indicating that both BeAn19991- and AM0059-based vaccines effectively elicited binding antibodies against the outbreak antigen.

Functional neutralization was first evaluated using a VSV-based pseudovirus displaying the AM0059 glycoprotein. As expected, sera from AM0059-vaccinated mice exhibited strong, dose-dependent inhibition of pseudovirus entry at weeks 5 and 9, consistent with an antigen-matched response (Fig. 3C). In contrast, sera from BeAn19991-vaccinated mice showed minimal inhibition, suggesting weaker neutralization against the outbreak antigen.

To further validate these observations using authentic virus, we next performed FRµNT assays with the OROV²⁴⁰⁰²³ strain. Consistent with the pseudovirus results, sera from AM0059-vaccinated mice displayed significantly higher neutralizing titers than sera from BeAn19991-vaccinated mice (Fig. 3D and E), with corresponding infection data provided in Fig. S3C and D. The PBS control group showed no viral entry inhibition.

Collectively, these data confirm that AM0059 mRNA-LNP vaccination induces potent neutralizing antibodies that recognize both pseudotyped and authentic OROV²⁴⁰⁰²³ viruses. The reduced neutralization observed in BeAn19991-immunized sera occurred despite comparable binding titers. This finding suggests that even subtle amino acid changes in Gc can alter neutralization sensitivity and impact vaccine performance.

To characterize the immune responses elicited by OROV mRNA-LNP vaccines beyond neutralizing antibody production, we assessed antigen-specific B cell, cytokine, and T cell responses in vaccinated mice at weeks 5 and 10 post-immunization.

First, BeAn19991 Gc-specific B cells were quantified in week 10 splenocytes by flow cytometry using a Cy3-conjugated recombinant BeAn19991 Gc probe (Fig. S4A). Vaccination increased the frequency of Gc⁺ B cells relative to PBS controls (Fig. 4A), with the AM0059 Gc group showing the highest proportion of antigen-specific B cells.

Next, we stimulated splenocytes with BeAn19991 Gc overlapping peptide pools and analyzed cytokine secretion to investigate vaccine-induced immune responses (Fig. 4B through G). Vaccinated mice exhibited elevated levels of Th1-associated cytokines, such as IL-2, IFN-γ, and TNF-α, and a moderate increase in Th2-associated cytokines, such as IL-4, IL-5, and IL-10, indicating a Th1-skewed immune response. Among the groups, AM0059-based vaccines appeared to induce higher overall cytokine concentrations, consistent with their stronger B cell activation profiles.

Finally, antigen-specific T cell responses were measured ex vivo after restimulation with the same peptide pools. Both CD4⁺ and CD8⁺ T cell activation increased in vaccinated mice compared with PBS controls (Fig. 4H through K). AM0059 Gc vaccination elicited the most pronounced activation of both subsets at weeks 5 and 10, including upon heterologous restimulation with BeAn19991 antigen, supporting enhanced cross-reactive cellular immunity.

Overall, these results show that OROV mRNA-LNP vaccines induce robust adaptive immunity characterized by OROV-specific B cells, a Th1-skewed cytokine profile, and strong CD4⁺/CD8⁺ T cell responses. The AM0059 Gc vaccine generated the most potent and broadly reactive cellular responses, consistent with its superior neutralizing activity.

To determine vaccine-mediated protection against OROV infection, the in vivo efficacy of the OROV mRNA-LNP vaccines was assessed in a lethal challenge model. Because GnGc and Gc-only formulations elicited comparable immunogenicity, subsequent studies focused on Gc constructs from the BeAn19991 and AM0059 strains.

A129 mice received intramuscular prime-boost at weeks 0 and 3 with LNP-encapsulated mRNA encoding either BeAn19991 Gc or AM0059 Gc. To evaluate antibody responses before viral challenge, sera were collected at weeks 2 and 5 for serological analysis (Fig. 5A). ELISA showed a marked increase in total anti-BeAn19991 IgG titers in both vaccinated groups. In contrast, PBS controls remained seronegative (Fig. 5B). Consistent with previous immunogenicity results, both vaccines induced potent neutralizing activity against the prototype OROV strain in VSV-based pseudovirus and authentic-virus FRµNT assays (Fig. 5C) with corresponding infection data provided in Fig. S3E. No significant difference was observed between the BeAn19991 Gc and AM0059 Gc formulations, indicating that both antigens elicited comparable pre-challenge neutralizing responses against the prototype virus.

At week 5, the mice were challenged subcutaneously via hock injection with a lethal dose (1 × 10⁴ plaque-forming units [PFU]) of authentic BeAn19991 virus, and clinical outcomes were monitored for 10 days. Both vaccinated groups maintained stable body weight, whereas PBS-treated mice exhibited rapid weight loss beginning on day 3 post-infection and succumbed by day 5 (Fig. 5E). Notably, all mice vaccinated with either BeAn19991 Gc or AM0059 Gc survived the challenge, whereas 100% of control mice died within 5 days (Fig. 5F).

Taken together, these data demonstrate that OROV mRNA-LNP vaccination provides complete protection against lethal rOROV^BeAn19991 infections. The equivalent protection achieved by BeAn19991 Gc and AM0059 Gc constructs indicates that the Gc antigen alone is sufficient to elicit robust protective immunity against the prototype strain.

To determine whether the OROV mRNA-LNP vaccines protect against newly circulating strains, we evaluated their efficacy against the recent outbreak strain (Cuba isolate OROV²⁴⁰⁰²³) (Fig. 6A).

Prior to the viral challenge, both mRNA-LNP vaccine formulations elicited strong antibody responses, consistent with earlier immunogenicity findings. By week 5, sera from vaccinated mice exhibited high anti-OROV²⁴⁰⁰²³ IgG titers (Fig. 6B) and robust neutralizing activity against both pseudotyped VSV^AM0059-GP and authentic OROV²⁴⁰⁰²³ viruses, with the AM0059 Gc group showing the stronger response (Fig. 6C and D, corresponding infection data provided in Fig. S3F). These results confirmed that both vaccines remained immunogenic prior to the challenge, with the AM0059 Gc vaccine inducing stronger neutralization against the outbreak strain.

Following a lethal challenge with the authentic Cuba isolate OROV²⁴⁰⁰²³ virus, mice were monitored for 10 days for body weight and survival (Fig. 6E and F). The AM0059 Gc vaccine conferred complete protection, with 100% survival and stable body weights throughout the observation period. In contrast, BeAn19991-Gc-vaccinated mice exhibited partial protection (75% survival) accompanied by moderate weight loss, indicating reduced efficacy against the contemporary isolate. All PBS control mice succumbed to infection between 4 and 6 days post-challenge.

Together, these results demonstrate that both OROV mRNA vaccines are protective, but the AM0059-Gc formulation provides enhanced efficacy against the Cuba isolate OROV²⁴⁰⁰²³. This suggests incorporating contemporary sequence variants to improve cross-strain protection as OROV continues to evolve.

HEK293T (human embryonic kidney), Vero E6 (African green monkey kidney), and BHK-21 (baby hamster kidney fibroblast) cell lines were obtained from the American Type Culture Collection (ATCC). Cells were maintained in Dulbecco’s modified Eagle medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (Gibco) at 37°C in a humidified incubator containing 5% CO₂.

The recombinant prototype strain (rOROV^BeAn19991) was kindly provided by Dr. Natasha Tilston-Lunel (Indiana University School of Medicine). The outbreak-associated strain OROV²⁴⁰⁰²³ was obtained through BEI Resources (NIAID, NIH: OROV, 240023, NR-59930).

Working stocks of rOROV^BeAn19991 and OROV²⁴⁰⁰²³ were propagated in Vero E6 cells at a multiplicity of infection of 0.01. Infected cells were incubated at 37°C with 5% CO₂ until the development of visible cytopathic effect. Virus-containing supernatant was harvested, clarified by centrifugation at 2,500 rpm for 10 min to remove cellular debris, aliquoted, and stored at −80°C until use.

Virus titers were determined by a plaque assay on BHK-21 cells seeded at a density of 3 × 10⁵ cells/mL in a 12-well plate. Cell monolayers were infected with 10-fold serial dilutions of the virus and incubated for 3 days at 37°C under an overlay of DMEM containing 1% methylcellulose (Sigma-Aldrich). Following incubation, cells were fixed with 4% paraformaldehyde and stained with 1% crystal violet to visualize plaques. Plaques were enumerated to calculate viral titers expressed as PFU per milliliter (PFU/mL).

Codon-optimized glycoprotein sequences encoding either the full-length GnGc polyprotein or the Gc full-length from the prototype strain BeAn19991 (GenBank accession no. KP052851.1↗) and the 2024 outbreak strain AM0059 (GenBank accession no. PP992528.1↗) were synthesized by GenScript (Piscataway, NJ, USA). The native viral signal peptide was replaced with the Lucia luciferase signal peptide (InvivoGen) to promote efficient secretion of the recombinant glycoproteins.

Each optimized coding sequence was cloned into a custom pZMV backbone containing a bacteriophage T7 promoter and 5′ and 3′ untranslated regions designed to support efficient in vitro transcription and translation (21). The resulting plasmids encoded either BeAn19991 GnGc, BeAn19991 Gc, AM0059 GnGc, or AM0059 Gc constructs, which were subsequently used for mRNA production and vaccine formulation.

In vitro transcription of OROV mRNA constructs was performed using the HiScribe T7 High Yield RNA Synthesis Kit (New England Biolabs, USA) with the incorporation of a CleanCap AG Cap1 analog (TriLink Biotechnologies, USA) for co-transcriptional capping. To enhance mRNA stability and minimize activation of innate immune sensors, uridine triphosphate (UTP) was completely substituted with N¹-methyl-pseudouridine triphosphate (TriLink).

Polyadenylation was carried out enzymatically at the 3′ end using Escherichia coli poly(A) polymerase (New England Biolabs). The resulting capped and polyadenylated mRNAs, encoding BeAn19991 or AM0059 GnGc and Gc sequences, were purified using RNA Clean & Concentrator spin columns (Zymo Research, USA) according to the manufacturer’s protocol. The integrity and polyadenylation of mRNAs were confirmed by electrophoresis on a 1% agarose gel prepared in MOPS buffer.

HEK293T cells were transfected with 1 μg of plasmid DNA encoding either the GnGc (with or without V5-tagged) or Gc glycoproteins from the OROV BeAn19991 or AM0059 strains using PolyJet transfection reagent (SignaGen Laboratories, USA) according to the manufacturer’s instructions. For mRNA transfection, 2.5 μg of in vitro-transcribed OROV GnGc or Gc mRNA was introduced into HEK293T cells using the TransIT-mRNA Transfection Kit (Mirus Bio, USA).

Whole-cell lysates were collected 48 h post-transfection for DNA-transfected cells and 24 h post-transfection for mRNA-transfected cells. Total protein concentrations were quantified using the Bradford protein assay (Bio-Rad, USA) prior to subsequent analysis.

mRNAs encoding either the GnGc or Gc glycoproteins from the OROV BeAn19991 and AM0059 strains were encapsulated in LNPs using GenVoy-ILM lipid (Cytiva, USA) at a mRNA-to-lipid ratio of 3:1 (vol/vol). Formulations were prepared on a NanoAssemblr Ignite+ microfluidic system (Precision NanoSystems, Canada) according to the manufacturer’s protocol. The resulting mRNA-LNPs were diluted in PBS, concentrated using Amicon Ultra centrifugal filters (Millipore, USA), and stored at 4°C until use.

mRNA concentration and encapsulation efficiency were quantified using the Quant-iT RiboGreen RNA assay (Thermo Fisher Scientific, USA). For cell transfection, 2.5 μg of mRNA-LNPs supplemented with human apolipoprotein E (Millipore Sigma, USA) in complete DMEM was added to HEK293T cells. Whole-cell lysates were collected 24 h post-transfection, and total protein concentrations were determined using the Bradford protein assay (Bio-Rad, USA).

Whole-cell lysates were mixed with 6× Laemmli SDS sample buffer (bioWORLD, USA), heated at 95°C for 5 min, and subjected to SDS-polyacrylamide gel electrophoresis for protein separation. Proteins were transferred onto polyvinylidene fluoride membranes using the Trans-Blot Turbo Transfer System (Bio-Rad, USA) under semi-dry conditions (25 V, 60 min).

Membranes were blocked for 1 h at room temperature in 5% (wt/vol) non-fat milk prepared in PBS containing 0.1% (vol/vol) Tween-20 (PBST), followed by overnight incubation at 4°C with a polyclonal mouse anti-OROV ascites antibody (VR-1228 AF; ATCC, USA) or anti-V5 antibody (Invitrogen, USA). After washing with PBST, membranes were incubated for 1 h at room temperature with horseradish peroxidase (HRP)-conjugated anti-mouse IgG secondary antibody (Cell Signaling Technology, USA).

For loading controls, membranes were probed with anti-GAPDH (Santa Cruz Biotechnology, USA) or anti-β-actin (Cell Signaling Technology, USA), followed by incubation with the same HRP-conjugated secondary antibody. Protein bands were visualized using the SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, USA) and imaged with a ChemiDoc MP Imaging System (Bio-Rad, USA).

Amino acid sequences of the OROV Gc glycoproteins from strains AM0059 (GenBank: PP992528.1↗) and 240023 (GenBank: PQ417949.1↗) were aligned using Geneious Prime (version 2024.0.1; Biomatters, Auckland, New Zealand). Alignments were generated with the Geneious Alignment algorithm configured for global alignment with free end gaps and the BLOSUM62 substitution matrix. Geneious automatically calculated sequence identity (exact residue matches) and similarity (conservative substitutions based on BLOSUM62). The final pairwise alignment was exported for figure visualization, with mismatches highlighted.

Female BALB/c mice (6–8 weeks old; BALB/cJ, Strain# 000651; RRID: IMSR_JAX:000651) were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). Female interferon-α/β receptor knockout mice (4–8 weeks old; B6.129S2-Ifnar1^tm1Agt/Mmjax; RRID: MMRC_032045-JAX) were obtained from the Mutant Mouse Resource and Research Center at The Jackson Laboratory, an NIH-funded strain repository. This strain was originally contributed by Dr. Michael Aguet (Swiss Institute for Experimental Cancer Research) (53).

mRNA-LNP immunizations were performed under conventional housing conditions, while subsequent OROV challenge experiments were conducted in an animal biosafety level 2 facility.

BALB/c mice were used for immunogenicity studies because they retain a fully functional type I interferon system and are widely used for profiling B cell, cytokine, and T cell responses. Hence, in this study, female BALB/c mice were immunized intramuscularly with either PBS or 3 μg of mRNA-LNPs encoding the GnGc or Gc glycoproteins from the OROV BeAn19991 or AM0059 strains. Immunizations were administered at weeks 0 and 3. Blood samples were collected at the indicated time points via retro-orbital bleeding, and sera were separated by centrifugation and heat-inactivated at 56°C for 30 min. Mice were euthanized at weeks 5 and 10, and spleens were harvested for downstream immunological analyses.

A129 mice (IFNAR1^−/−) were used exclusively for viral challenge studies because immunocompetent mouse strains do not develop clinical OROV disease. A129 mice are an established model for OROV pathogenesis and uniformly develop lethal infection, enabling rigorous assessment of vaccine-mediated protection. In this study, female A129 mice were immunized intramuscularly with either PBS or 3 μg of mRNA-LNPs encoding the Gc glycoproteins from the OROV BeAn19991 or AM0059 strains at weeks 0 and 3. At week 5, mice were challenged subcutaneously via hock injection with a lethal dose (1 × 10⁴ PFU) of rOROV^BeAn19991 or OROV²⁴⁰⁰²³. Following challenge, animals were monitored daily for body weight, clinical signs of disease, and survival for up to 10 days post-infection.

Immulon 4 HBX 96-well plates (Thermo Fisher Scientific) were coated overnight at 4°C with purified rOROV^BeAn19991 or OROV²⁴⁰⁰²³ virion lysate (10 µg/mL) diluted in PBS. Plates were blocked with 1% Block ACE (Bio-Rad) for 1 h at room temperature and washed three times with PBS containing 0.05% Tween-20 (PBST). Heat-inactivated mouse sera were serially diluted in PBS and incubated on the plates overnight at 4°C. After washing, bound antibodies were detected using HRP-conjugated anti-mouse IgG (Thermo Fisher Scientific). Color development was achieved using 3,3′,5,5′-tetramethylbenzidine substrate (BioLegend), and the reaction was stopped with 2 N sulfuric acid. Absorbance was measured at 450 nm using a microplate reader (BioTek). Endpoint titers were determined as the highest serum dilution yielding an absorbance value at least threefold above the background.

Recombinant vesicular stomatitis virus pseudoviruses expressing OROV GnGc from either the BeAn19991 or AM0059 strain, together with a luciferase reporter gene (VSV^BeAn19991-GP and VSV^AM0059-GP, respectively), were generated as previously described (21, 54, 55). Pseudoviruses were harvested 48 h post-infection, clarified by passage through 0.45 μm filters, and stored in aliquots at −80°C until use.

For neutralization assays, serial dilutions of heat-inactivated mouse sera were prepared in DMEM and incubated with pseudoviruses for 1 h at 37°C. The serum-virus mixtures were then added to HEK293T cells and incubated for 24 h under standard culture conditions. Cells were lysed, and luciferase activity was quantified as relative light units (RLU) using the Luciferase Assay System (Promega).

Percent neutralization was calculated by normalizing RLU values to control wells, where virus-only wells (no serum) represented 0% neutralization and uninfected wells (media only) represented 100%. The IC₅₀ values were determined by nonlinear regression (log[serum dilution] vs normalized response, variable slope) using GraphPad Prism v.10.

Neutralizing antibody titers were quantified by FRµNT. Briefly, Vero E6 cells were seeded in 96-well plates (2 × 10⁴ cells/well) and incubated overnight at 37°C to form a confluent monolayer. Mouse sera were heat-inactivated at 56°C for 30 min and serially diluted 10-fold in infection medium (DMEM supplemented with 2% FBS). Each dilution was mixed 1:1 (vol/vol) with OROV (200 PFU per well) and incubated at 37°C for 1 h to allow antibody-virus complex formation.

Following incubation, 50 µL of the serum-virus mixture was added to the Vero E6 monolayers and incubated for 1 h at 37°C with gentle rocking every 15 min. Subsequently, 100 µL of overlay medium (DMEM containing 1.5% carboxymethylcellulose and 2% FBS) was added to each well, and plates were incubated for 48 h at 37°C. Following infection, cells were fixed with ice-cold 80% acetone for 20 min at RT and air-dried.

For immunostaining, fixed monolayers were incubated with a primary mouse anti-OROV Gc antibody (Jung laboratory-generated, 1:1,000 dilution in PBS with 3% BSA) for 1 h at room temperature, followed by an HRP-conjugated anti-mouse IgG secondary antibody (1:2,000 dilution, 1 h at room temperature). Foci were visualized using the Vector VIP Substrate Kit, Peroxidase (HRP) (Vector Laboratories), according to the manufacturer’s protocol. The reaction was stopped by rinsing with distilled water, and plates were air-dried. Foci were enumerated using an ELISpot reader (CTL ImmunoSpot) with automated spot detection.

Splenocytes (1 × 10⁶ cells) collected from immunized BALB/c mice at week 10 after initial immunization were seeded into 96-well V-bottom plate (Corning). Unstained cells were included as a negative control. Cells were incubated with TruStain FcX PLUS (BioLegend) and LIVE/DEAD Fixable Blue Dead Cell Stain (Invitrogen) in FACS buffer (2% heat-inactivated FBS in PBS) at 4°C for 15 min. After two washes with FACS buffer at 4°C for 5 min, cells were incubated with BeAn19991 Gc-Cy3 antigens along with Brilliant Stain Buffer Plus (BD Biosciences) and True-Stain Monocyte Blocker (BioLegend) in FACS buffer at 4°C for 60 min. Following wash with FACS buffer, cells were stained with OROV Gc-specific B cell immunophenotyping antibody cocktails (Table 1) in FACS buffer containing Brilliant Stain Buffer Plus and True-Stain Monocyte Blocker at 4°C for 30 min. After staining and washing, cells were fixed with 4% paraformaldehyde (PFA) at room temperature for 15 min. Prior to acquisition on the SONY ID7000 Spectral Cell Analyzer (Sony Biotechnology), cells were kept in the dark at 4°C in FACS buffer containing Tandem Stabilizer (BioLegend). The results were analyzed using FlowJo v.10.9.0 (BD Biosciences), and the gating strategy is illustrated in Fig. S4A.

A modified AIM assay was employed to assess OROV-specific CD4⁺ and CD8⁺ T cell responses based on previously validated protocols (56–62). Splenocytes were isolated from BALB/c mice immunized with PBS or mRNA-LNPs encoding either GnGc or Gc from the BeAn19991 or AM0059 strains at weeks 5 and 10 after initial immunization. A total of 2 × 10⁶ splenocytes per well were seeded in 96-well V-bottom plates (Corning). A CD40-blocking antibody (0.5 µg/mL) was added and incubated for 15 min to enhance specificity. Cells were then stimulated with 5 µg/mL of an overlapping 15-mer peptide pool (11-aa overlap) derived from BeAn19991 Gc (GenScript) and incubated for 20 h at 37°C. Wells containing unstimulated cells served as negative controls.

Following stimulation, cells were washed twice with FACS buffer (PBS supplemented with 2% heat-inactivated FBS and 0.5 mM EDTA), blocked with anti-CD16/CD32 (TruStain FcX PLUS; BioLegend) for 15 min, and stained for 30 min with a surface antibody cocktail in the presence of True-Stain Monocyte Blocker (BioLegend) and Brilliant Stain Buffer Plus (BD Biosciences). Cells were fixed in 4% PFA for 10 min at room temperature, washed, and resuspended in 100 µL FACS buffer containing a tandem-dye stabilizer (BioLegend). The samples were stored at 4°C in the dark until flow cytometry was performed using a SONY ID7000 Spectral Cell Analyzer (Sony Biotechnology). Data were analyzed using FlowJo software (v.10.9.0, Treestar Inc.), and the gating strategy is illustrated in Fig. S4B. A complete list of antibodies and reagents used is provided in Table 2.

Splenocytes (3 × 10⁵ cells) from immunized BALB/c mice at weeks 5 and 10 following primary immunization were seeded into a 96-well V-bottom plate (Corning). Cells were stimulated ex vivo with 5 μg/mL of an overlapping peptide pool derived from the BeAn19991 Gc sequence, identical to that used in the AIM assay. After incubation for 24 h at 37°C, the plates were centrifuged at 400 × g for 7 min at 4°C, and the supernatants were transferred to 96-well clear flat-bottom Ultra-Low Attachment plates (Corning) for cytokine analysis. Cytokine levels in culture supernatants were quantified using the Olink Target 48 Mouse Cytokine panel on the Signature Q100 platform.

All data analyses were performed using GraphPad Prism version 10 (GraphPad Software Inc.). To compare OROV-specific IgG titers and virus neutralization levels against the BeAn19991 and AM0059 strains, statistical differences were assessed using one-way ANOVA followed by unpaired t-tests. The results were presented as geometric means and 95% confidence intervals. Student’s t-test was used to evaluate the differences in CD4⁺ and CD8⁺ T cell responses measured by the AIM assay. Graphs display mean values with error bars representing the SEM. Mouse survival data were analyzed using the log-rank (Mantel-Cox) test. Statistical significance was defined as follows: P ≤ 0.05 (*), P ≤ 0.01 (**), P ≤ 0.001 (***), and P ≤ 0.0001 (****), with “ns” for non-significant findings.