Immunity and protection from an RSV mRNA vaccine against two virus subgroups in mice

HEp-2 cells were obtained from ATCC (CCL-23) and maintained in complete Eagle’s minimum essential medium (EMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and penicillin/streptomycin. Sub-confluent HEp-2 monolayers were inoculated with RSV strains (mKate2 A2, B1, or mKate2 BAF-BAG) and rocked at room temperature (RT) for 1 h to facilitate viral adsorption. Following this, cultures were incubated until peak viral titers were achieved. Virus stocks were then harvested and titrated as previously described.20

Pathogen-free 6- to 8-week-old female BALB/c (H-2^d) mice were purchased from the Jackson Laboratory (Bar Harbor, Maine, USA). All animal procedures and husbandry were conducted in accordance with the guidelines of the Emory University Institutional Animal Care and Use Committee (IACUC), under approved protocol number PROTO201800280.

Plasmid templates for RSV-F mRNAs were prepared using the EndoFree Plasmid Maxi Kit (QIAGEN) according to the manufacturer’s instructions. Approximately 100 μg of supercoiled plasmid DNA was linearized with BspQI (NEB) at 50 °C for 2 h. In vitro transcription was carried out in a 400 μl reaction containing 20 μg linearized DNA, 5 mM each of ATP, CTP, GTP, and m1UTP (N1-methylpseudo UTP), 4 mM SmartCap SC101, 1× T7 RNA polymerase buffer, 16.3 KU T7 RNA polymerase, 320 U RNase inhibitor, and 0.8 U YPP. The reactions were incubated at 42 °C for 4 h followed by DNase I digest (160 U, 37 °C, 1 hr), and mRNA was purified using Amicon Ultra centrifugal filters (30 K, Merck Millipore) by centrifugation (6,000 × g, 16 °C, 10 min) with repeated washes until the A260/230 ratio was ≥2.0. The RNA was recovered by inverted centrifugation (6,000 × g, 16 °C, 2 min). Integrity was assessed by agarose gel electrophoresis following heat denaturation (75 °C, 4 min and 4 °C, 5 min).

STL1244 cells were prepared as described in prior studies.21^,22 Briefly, LNPs were formulated by mixing lipid components and an aqueous mRNA solution using a microfluidic device (Nanoassembly Ignite +, Precision Nanosystems, Canada). A novel ionizable lipid, STP1244 (chemically synthesized by ST Pharm), was mixed with DOPE (helper lipid; Merck, Darmstadt, Germany), cholesterol (structural lipid; Sigma-Aldrich), and C16 PEG2000-Ceramide (PEG-lipid; Avanti Polar Lipids, Alabaster, Alabama, USA) at a defined molar ratio of STP1244: DOPE: cholesterol: C16 PEG2000-Ceramide = 36.5:15:52:1.5 (mol %). The lipid components dissolved in ethanol and mRNA dissolved in 100 mM sodium acetate buffer were mixed at a 1:1 volume ration and formulated using a microfluidic device at a total flow rate of 12 mL/min. The resulting LNPs were diafiltrated with 1 × PBS and concentrated in Tris buffer containing sucrose using ultrafiltration (Amicon Ultra-15 Centrifugal Filter Unit, Sigma-Aldrich). Concentrated LNPs were stored at −20 °C until further use. To determine mRNA concentration and encapsulation efficiency, a RiboGreen assay (Thermo Fisher Scientific, Waltham, Massachusetts, USA) was performed using a spectrofluorometer (FP-8350, Jasco, Tokyo, Japan). The size distribution and zeta potential of the well-dispersed LNPs in 1 × PBS were measured by a dynamic light scattering (DLS) using Zetasizer Ultra instrument (Malvern, UK) at a 173° scattering angle at RT.

DNA template sequence for mRNA in vitro transcription (IVT) consisted of T7 promoter, 5′ untranslated region (UTR), and an open reading frame of RSV prefusion form modified from the RSV fusion glycoprotein (GenBank protein ID: AAB59858.1↗ for genotype A2 and AAB82429.1↗ for B1), 3′ UTR and 120 bases of poly adenine (polyA). The 5′ UTR and 3′ UTR are from the human globin alpha gene. The DNA fragment was synthesized and subcloned into a pUC57-Kan vector by GenScript (Piscataway, New Jersey, USA). The plasmid vector was linearized using the restriction enzyme BspQI (New England Biolabs) to obtain RSV fusion protein. N1-Methylpseudouridine (m1Ψ) was purchased from BOC Sciences (New York, USA). IVT condition was performed according to the manufacture’s recommendation (TranscriptAid T7 High Yield Transcription Kit, ThermoFisher Scientific): ATP/CTP/GTP/m1ψTP: (5 mM each), SmartCap (SC101, 4 mM, ST Pharm, Seoul, ROK), Linear template DNA (1 μg of plasmid), and T7 RNA polymerase enzyme mix (2 μl) was used for the experiment. IVT was carried out in a 20 μl reaction mixture incubated at 37 °C for 2 h. The template DNA was removed using 2 units of DNase I (ThermoFisher Scientific) and treated at 37 °C for 15 min followed by column purification (Monarch RNA Cleanup Kit, New England Biolabs).

One microgram of mRNAs was transfected into 293FT (Thermo Fisher Scientific) cells in a 12 well plate using Lipofectamine MesseangerMax (ThermoFisher Scientific) at a ratio of 1:2 according to the manufacturer’s protocol.

Cell pellets were lysed with NP-40 lysis buffer (150 mM sodium chloride/1% NP-40/50 mM Tris, pH 8.0) after 24 h of transfection. The cell lysates were harvested after centrifugation and loaded onto the SDS-PAGE gel (Bio-Rad) and transferred onto a 0.45 μm PVDF membrane (Bio-Rad). The PVDF membranes were blocked with 1% bovine serum albumin (Sigma-Aldrich, Missouri, USA) in tris-buffered saline buffer (TBS, Bio-Rad) containing 0.1% Tween 20 (Bio-Rad) for 1 h at RT. The membranes were washed 3 times with 1 × TBST and incubated for 1 h with rabbit anti-RSV fusion glycoprotein antibody (10049-R302, Sino Biological at 1:1,000 in TBST) and anti-GAPDH (sc-47724, Santa Cruz at 1:1,000 in TBST) at room temperature. The membranes were then washed three times with 1 × TBST and incubated with horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch at 1:5,000 in TBST) for 1 h at RT. The membranes were washed three times with 1 × TBST and SuperSignal West Pico Plus Chemiluminescent Substrate (Thermo Fisher Scientific) was used for protein detection.

Groups of mice (n = 20 per group) were vaccinated intramuscularly (i.m.) with 2, 10, or 20 μg of VER-027, administered twice at 3-wk intervals. Two control groups were included: a naïve group and an LNP-only control group. Serum samples were collected from all groups at 2, 5, and 7 wk via sub-mandibular bleeding. Mice were anesthetized via intramuscular injection of a ketamine-xylazine solution and infected intranasally (i.n.) with either 3 × 10⁵ PFU mkate2 RSV A2 strain or 2 × 10⁶ PFU RSV B1 strain in 100 μl of plain EMEM. For direct inoculation, mice were held upright with fully extended necks and readily inhaled a 100 μl volume of undiluted stock virus dispensed onto their nostrils using a micropipette. Mice were monitored for pain, distress or suffering. The mice were then euthanized on day 4 post infection (p.i.) and the lungs, spleen, and blood were collected for downstream virological and immunological analyses.

Mice were euthanized on day 4 p.i. by intraperitoneal (i.p.) injection of Fatal Plus, and lungs were harvested for downstream analysis. A mini-BeadBeater-96 (Biospec Products, Bartlesville, Oklahoma, USA) was used to homogenize the lungs as described.23 Lung homogenates were serially diluted in serum-free EMEM and inoculated onto sub-confluent HEp-2 cells in 24-well plates. After a 1 h adsorption period at RT on a rocking platform to ensure uniform viral attachment, the inoculum was removed, and the cells were overlaid with minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS), penicillin/streptomycin, and 0.75% methylcellulose. The cultures were incubated at 37 °C in a 5% CO₂ for 6 d. Following incubation, the overlay medium was carefully removed, and the cell monolayers were fixed in cold methanol at −80 °C for a minimum of 60 min. Viral plaques were then detected via immunostaining with RSV-specific primary antibodies and subsequent colorimetric visualization, as previously described.24–26

Total RNAs were extracted from lung using the Qiagen RNeasy Mini kit (Qiagen, Valencia, California, USA) according to the manufacturer’s instructions and stored at −80 °C. Quantitative real-time PCR was performed by using qScriptTM XLT One-Step RT-qPCR ToughMix (Quantibio LLC.), and a Roche 480 II lightcycler. The thermal cycling conditions were 45 °C for 10 min and 95 °C for 1 min, followed by 45 cycles of amplification at 95 °C for 10 s and 60 °C for 30 s for denaturing and annealing, respectively. Primer 3 software (https://bioinfo.ut.ee/primer3-0.4.0↗) was used to design primers and probe. The primers and probes for RSV B1 Fusion (F) gene (forward, 5′-CCTTCTTTCCACAGGCTGAC-3′; reverse, 5′-TCCCACGATTTTTGTTGGAT-3′, probe, 5′ Fam- CCAAGTGAA/ZEN/GTCAGCCTTTG -BHQ-1 3′) were synthesized by Integrated DNA technologies (IDT) (Coralville, Iowa, USA). All samples were run in triplicate including a nontemplate control (sterile, PCR-grade water) and positive control comprised of an extended standard of the amplicon region. Viral copy numbers were quantified and compared to those of the standard controls.

RSV prefusion (F)-specific IgG antibody titers in serum samples were evaluated using ELISA. Due to institutional animal care guidelines limiting blood collection to approximately 250 µl per 25 g mouse every 2–3 wk, serum volumes obtained at weeks 2, 5, and 7 post-prime were insufficient to perform both IgG ELISA and neutralization assays on individual samples. Therefore, sera were pooled within each experimental group to enable completion of all planned immunogenicity analyses. Briefly, ELISA plate was coated overnight at RT with an RSV prefusion glycoprotein F0 (Acrobiosystems LLC) (2 µg/ml). The next day, the plates were washed with PBS-Tween (PBST) (0.05% Tween 20 in PBS) using the BioTech ELx50 plate washer and blocked with 1% BSA at RT for 2 h. Following blocking and PBST washing, pooled serially diluted mouse serum samples were added and incubated at 4 °C overnight. Then, goat anti-mouse horseradish peroxidase (HRP) diluted 1:3,000 in blocking solution was applied and incubated at RT for 1 h. The plates were again washed with PBST, and 1-Step Ultra TMB ELISA substrate (ThermoFisher LLC.) was applied to catalyze a colorimetric reaction. The reaction was stopped using the SeraCare KPL TMB stop solution. The plates were read at 450 nm using a BioTek Cytation 3. The background absorbance was subtracted from the sample absorbance readings, and the data were plotted on a graph.

HEp-2 cells were seeded at 3.0 × 10⁴ cells per well in 96-well plates and cultured overnight. Mouse serum samples were heat-inactivated at 56 °C for 30 min and serially diluted in 1× PBS without Calcium and Magnesium. mKate2 A2 (RSV A strain) or mKate2 BAF-BAG (RSV B strain) was diluted in in serum-free EMEM to a final concentration of 100 FFU per well and incubated with the serially diluted mouse sera. This virus-serum mixture was rocked at RT for 1 h to allow antibody-RSV interaction. Following incubation HEp-2 cells were infected with the mixture and spinoculated at 3,000 rpm for 30 min. Subsequently, 150 µl of complete EMEM/10% FBS/penicillin G/streptomycin/0.75% methylcellulose was added to each well, and the plates were incubated at 37 °C for 36 to 48 h. FFUs were quantified using the BioTek Cytation 7, which detects the far-red fluorescent mKate2 reporter (561 nm). All samples were evaluated in duplicate, and data were normalized using negative controls (mock-infected HEp-2 cells) and positive controls (RSV without serum).

Teramer staining and intracellular cytokine staining (ICS) were performed to enumerate RSV F_85-93 specific CD8⁺ T cells and cytokine-producing cells as described previously.25^,27 In brief, freshly isolated splenocytes (2 × 10⁶) were left untreated, stimulated with individual peptide (RSV F_85-93 KYKNAVTEL, 1 μg/sample, Genscript), or stimulated with cell stimulation cocktail (Thermo Fisher, Cat no. 00-4970-93) for 6 h at 37 °C in 5% CO₂ in the presence of brefeldin A (BD Pharmingen, San Diego, California, USA). TruStain FcX^TM (anti-mouse CD16/32, BioLegend, Cat no. 101320) was used to inhibit non-specific binding of surface staining antibodies. Then, cells were subsequently stained with a viability dye (Zombie aqua, BioLegend, Cat no. 423102) to determine live/dead cells. Next, cell surface staining including tetramer staining was performed with PE-H-2K^d F_85-93 tetramer (NIH tetramer facility, Atlanta, Georgia, USA), BV605-CD3 (BD Biosciences, Cat # 564009), eFluor 450-CD4 (RM4-5, Thermofisher, Cat no. 48-0042-82), APC-Cy7-CD8 (BD Biosciences, Cat no. 557654) for 30 min at 4 °C, followed by permeabilization using Cytofix/Cytoperm (Fisher Scientific, Cat no. BD 555028). The following intracellular stains were used to quantify cytokine production, FITC-IFN-γ (BioLegend, Cat no. 503806), PE/Cyanine7-IL-2 (BioLegend, Cat # 503832), Alexa Fluor-TNF-α (BioLegend, Cat no. 506314). Fluorescence was measured using Symphony 5 (BD Immunocytometry Systems) and analyzed using FlowJo software (Tree Star, Ashlan, Oregon, USA).

Statistical analyses were conducted using GraphPad Prism software (San Diego, California, USA). Comparisons between 2 groups were assessed with a 2-tailed Student’s t test, while comparisons across more than 2 groups were evaluated using 1-way analysis of variance (ANOVA) followed by Tukey’s test to account for multiple comparisons. Data values below limits of detection were assigned a value of half the limit of detection. Statistical significance was defined as P < 0.05.

A novel mRNA-based RSV vaccine candidate, VER-027, was rationally designed to express the prefusion-stabilized F glycoprotein from both RSV genotypes A and B. The prefusion conformation of the F protein is well-established as the optimal immunogen for eliciting potent neutralizing antibody responses.11 To maintain the prefusion state, four stabilizing substitutions (S155C, S190F, V207L, and S290C) were introduced into the F protein sequence. Each mRNA component was synthesized via T7 RNA polymerase–driven in vitro transcription (IVT) from DNA templates containing a T7 promoter, 5′ untranslated region (UTR), the prefusion F gene, 3′ UTR, and a 120-nt polyadenylated tail (Fig. 1A and B). Transient transfection of 293FT cells with an equal amount of the resulting mRNAs demonstrated robust expression of the RSV F proteins. Western blot analysis of cell lysates collected 24 h post-transfection confirmed that the engineered constructs predominantly expressed the prefusion conformation, as evidenced by a distinct migration pattern compared to wild-type F (pre-F_A vs. wt-F_A; Fig. 1C). For vaccine formulation, the pre-F_A and pre-F_B mRNAs were co-encapsulated in a proprietary ionizable lipid nanoparticle (LNP) system (STLNP^®) to generate VER-027. Prior to in vivo immunogenicity assessment, formulation integrity and functionality were validated by transfecting 293FT cells with VER-027, which reproduced the protein expression profiles observed with individual mRNA transfections (Fig. 1D).

Following confirmation of the size and in vitro expression of the VER-027 vaccine, we proceeded to manufacture the formulation and assess its immunogenicity in mice. VER-027 was formulated to harbor equal amounts of the 2 subgroup pre-F mRNAs. Here, we selected the BALB/c mouse model because it is well established in our laboratory and is known for its documented susceptibility to RSV infection, providing reproducible immune responses that facilitate evaluation of both humoral and cell–mediated protection.26^,28^,29 These characteristics make it a genetically uniform, well-characterized, and reliable system for preclinical assessment of RSV vaccine efficacy and safety. Groups of BALB/c mice were primed and boosted with VER-027 at doses of 2, 10, or 20 µg total mRNAs, administered 3 wk apart, as illustrated in Fig. 2. The RSV F protein mediates viral entry and cell-cell fusion, forming syncytia, and undergoes a transition from a metastable prefusion (pre-F) to a stable postfusion (post-F) state, making it the main target of neutralizing antibodies (nAb).30 Multiple studies have demonstrated that pre-F specific IgG responses play a crucial role in protection against RSV infection, with the pre-F conformation eliciting higher levels of neutralizing antibodies compared to the post-F form, suggesting greater potential for effective immunity.31^,32 Thus, we hypothesized that VER-027 would elicit robust humoral immune responses, supporting its potential as a promising new mRNA vaccine candidate. To examine this, we measured RSV pre-F–specific IgG responses at weeks 2, 5, and 7 after the prime, as well as neutralizing antibody titers following challenge with both RSV A and B strains. As shown in Fig. 3, serum samples from mice vaccinated with VER-027 exhibited significantly higher levels of RSV pre-F specific IgG antibodies compared to those from mice vaccinated with control LNP or naïve mice. While a clear difference in pre-F–specific IgG levels was observed between the 2 µg and the 10 or 20 µg groups only at two weeks post-prime, no significant differences were detected among the vaccinated groups at 5 or 7 wk following priming.

Protection against RSV infection has been shown to correlate positively with elevated serum neutralizing antibody (nAb) titers, while higher nAb levels are inversely associated with the risk of progression from infection to lower respiratory tract illness (LRTI) in children.33^,34 Accordingly, we next quantified serum nAb titers to evaluate whether VER-027 vaccination can elicit subgroup-specific responses against both RSV A and RSV B, as detailed in the Methods. Stored serum samples were heat-inactivated at 56 °C for 30 min and nAb titers were assessed against the mkate2 A2 strain (Fig. 4A) or the recombinant mkate2 BAF BAG strain expressing RSV F and G protein from Buenos Aires B strain (Fig. 4B), generated using reverse genetics. As shown in Fig. 4, all VER-027 immunized mice exhibited robust nAb titers compared with naive or control LNP immunized mice. Week 2 post-prime nAb titers were excluded from analysis due to levels being below the reliable detection threshold (data not shown). Boost vaccination led to a significant increase in nAb titers across all dose groups. Collectively, these results clearly demonstrated that VER-027 elicited robust humoral immune responses, including RSV pre-F–specific IgG and nAbs against both RSV A and RSV B subgroups.

In addition to humoral immunity, cellular immune responses are critical for protection against RSV infection, with RSV-specific CD8⁺ T cells playing a key role in viral clearance and recovery.24^,35–37 To evaluate RSV-specific cellular immune responses, we performed MHC class I tetramer staining for the RSV F_85–93 epitope, along with intracellular cytokine staining following in vitro restimulation with the F_85–93 peptide. The RSV F_85-93, an immunodominant cytotoxic T lymphocyte (CTL) epitope within the RSV F protein, exhibits K^d-restricted specificity.38 By employing an MHC class I tetramer containing the F_85-93 epitope (KYKNAVTEL) synthesized at the NIH tetramer facility (Atlanta, Georgia, USA), we successfully visualized and quantified F_85-93-specific CD8⁺ T cell response in RSV challenged BALB/c mice. Importantly, this tetramer was applicable to both RSV A and B strains, as the epitope sequence was identical in our challenge strains (mkate2 RSV A2 and RSV B1). The representative flow cytometry gating strategy for surface markers, shown in Fig. S1, was used to sequentially identify single, viable CD3⁺ T cells and further delineate the CD8⁺ T cell population, within which F_85–93-specific cells were quantified using the MHC class I tetramer. As shown in Fig. 5, all VER-027 vaccinated mice increased F_85–93-specific CD8⁺ T cell responses, ranging from 1.4% to 1.7% of total CD8⁺ T cells following challenge with either RSV A or RSV B strains. In contrast, naïve mice and those immunized with control LNP exhibited only minimal frequencies (< 0.1%) of F_85–93-specific CD8⁺ T cells. Consistent with humoral responses, no dose-dependent differences were observed among mice receiving 2, 10, or 20 µg of VER-027. Although day 4 p.i. is generally considered early for the detection of RSV-induced CD8⁺ T cell responses, a substantial population of F_85–93-specific CD8⁺ T cells was readily detected in VER-027–immunized mice. These findings suggest that VER-027 induces a rapid and robust RSV F-specific cellular immune response against both RSV subgroups.

Next, we quantified cytokine production by CD8⁺ T cells, specifically IFN-γ, TNF-α, and IL-2, to evaluate their functional activity (Fig. 6). Upon re-stimulation with the RSV F_85-93 peptide, naïve and control LNP-vaccinated mice exhibited minimal cytokine production, whereas VER-027 vaccinated mice showed a significantly higher frequency of CD3⁺CD8⁺ T cells producing these cytokines. The hierarchy of cytokine production followed the pattern IFN-γ > TNF-α > IL-2. Tetramer⁺ CD8⁺ T cells were not directly gated in this assay. However, our analyses indicate that only approximately 10% of tetramer⁺ CD8⁺ T cells produce TNF or IFN-γ upon F_85-93 peptide restimulation, corresponding to ∼0.1%–0.2% cytokine-positive cells among ∼1.5% tetramer⁺ CD8⁺ T cells (data not shown). Accordingly, the frequency of cytokine-producing cells detected by intracellular cytokine staining is expected to be lower than the total tetramer⁺ population. In addition, these analyses were performed using splenocytes rather than lung-derived lymphocytes, which typically exhibit lower frequencies of effector CD8⁺ T cells at the time point analyzed. Furthermore, the F_85–93 epitope is a subdominant CD8⁺ T cell epitope in BALB/c mice, whereas M2_82–90 is immunodominant, which may further contribute to the relatively modest magnitude of the observed antigen-specific response. Since splenocytes were restimulated with a CTL-restricted epitope, RSV F_85-93 specific CD4⁺ T cell responses were minimal, indicating a lack of cross-reactivity between RSV F_85-93 specific CD8⁺ and CD4⁺ T cell responses. In Fig. S2, CD8⁺ T cells exhibited distinct intracellular cytokine staining profiles across the three restimulation conditions, as illustrated by representative gating of negative control, positive control, and F_85–93 peptide–stimulated samples. While CD4⁺ T cells did not generate F_85–93–specific cytokine responses (data not shown), they demonstrated a high frequency of cells producing IFN-γ, TNF-α, and IL-2 following PMA/ionomycin restimulation. These results indicate that CD4⁺ T cells are healthy and fully capable of producing cytokines, and that the absence of an F_85–93–specific response is not due to T cell dysfunction, but rather reflects the MHC class I–restricted nature of this epitope.

Extensive analyses of recent clinical isolates from multiple global regions reveal the co-circulation of RSV-A and RSV-B subgroups, with strain predominance fluctuating over time.39^,40 Because both subgroups contribute to ongoing transmission and disease burden, these findings emphasize the necessity of developing vaccines that provide broad protection against both RSV-A and RSV-B. To the best of our knowledge, VER-027 represents the first mRNA vaccine candidate designed to target both RSV A and B subgroups concurrently. Protective efficacy was evaluated by assessing lung viral loads following intranasal challenge with either the RSV mKate2 A2 (subgroup A) or RSV B1 (subgroup B) strain. All immunized mice were challenged at 63 days post-boost, a time point corresponding to the memory phase of vaccine-induced immunity. This design allowed us to specifically evaluate the durability and effectiveness of VER-027 in mediating long-term protective immune responses. Naive mice and those immunized with control LNP were included as controls, and left lungs were harvested 4 d p.i. While we initially hypothesized that vaccine efficacy would vary in a dose-dependent manner, plaque assay analysis of RSV subgroup A challenge (Fig. 7A) and RT-qPCR quantification following RSV subgroup B challenge (Fig. 7B) demonstrated that all vaccinated groups, irrespective of administered dose, exhibited complete protection in contrast to naive or control LNP-immunized mice, which displayed high viral loads. This complete protection significantly correlated with (1) high pre-F specific IgG production, (2) robust anti-RSV neutralization activity for both A and B subgroups, and (3) significant F_85-93-specific, functionally active CD8⁺ T cell responses.

Data are available from the corresponding authors upon reseanable request.