Design and immune response of a four-part mRNA vaccine against HSV-2 comparing combined and mixed formulations

gB2, gC2, gD2, and gE2 mRNAs were generated from DNA templates encoding full-length gB2 (GenBank accession no. YP_009137179.1), gC2 (GenBank accession no. WZW64493.1), gD2 (GenBank accession no. AAA45841.1), and gE2 (GenBank accession no. YP_009137220.1). DNA sequences were synthesized by Azenta Life Sciences (Burlington, MA, USA) and cloned into the mRNA platform backbone plasmid (T7 promoter – 5′ UTR – multiple cloning sites – 3′ UTR – poly(A) tail) (10). mRNAs were transcribed from the DNA templates using the EZ T7 High Yield In Vitro Transcription Kit (Enzynomics, Daejeon, Republic of Korea) with the SC101 Cap 1 analog (ST Pharm, Siheung, Republic of Korea), and uridine triphosphate (UTP) was replaced with N1-Methylpseudo-UTP (TriLink BioTechnologies, San Diego, CA, USA). Transcribed mRNAs were precipitated with lithium chloride and further purified using a cellulose-based method (11). RNA concentration and purity ratios were determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and RNA integrity was confirmed by denaturing agarose gel electrophoresis.

Lipid nanoparticle (LNP) components, including SM-102, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000), were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5. mRNAs were prepared in 50 mM citrate buffer (pH 4) at an N/P ratio of 6. mRNAs were encapsulated in LNPs by microfluidically mixing the aqueous and organic phases in a 3:1 ratio using an enCELL-Master (enParticle, Busan, Republic of Korea). For the co-formulation, gB2, gC2, gD2, and gE2 mRNAs were combined in equal amounts before being encapsulated in LNPs. For the admixed formulation, equal amounts of gB2, gC2, gD2, and gE2 mRNAs were separately encapsulated in LNPs and combined. mRNA–LNPs were buffer-exchanged into phosphate-buffered saline (PBS) and concentrated using an Amicon Ultra-15 Centrifugal Filter at 30 K. The size of the mRNA-loaded LNPs was measured using a Zetasizer Nano ZS (Malvern Panalytical Ltd., Malvern, UK).

The animal studies were approved by the Institutional Animal Care and Use Committee of the Catholic University of Korea (approval no. CUK-IACUC-2023-045). Female BALB/c mice, aged 5–6 weeks, were obtained from Koatech (Pyeongtaek, Republic of Korea) and housed under specific pathogen-free conditions with a standard light/dark cycle, 50–60% humidity, and a temperature of 21–25°C. All the experimental procedures complied with the guidelines of the Institutional Animal Care and Use Committee of the Catholic University of Korea. Mice were anesthetized by inhalation of 3–5% isoflurane in oxygen at a flow rate of 1–2 L/min for induction, and 1–3% isoflurane in oxygen at a flow rate of 1 L/min for maintenance. Mice were euthanized by gradual exposure to CO₂ at a flow rate of 30–70% of chamber volume per minute until cessation of respiration, in accordance with institutional animal guidelines.

In all the experiments, mRNA vaccines were injected twice into the hind gastrocnemius muscle of 6-9-week-old female mice at a two-week interval. Mice received a prime immunization at week 0, followed by a booster immunization at week 2. The standard immunization dose was 10 μg of mRNA per antigen. For the dose-dependent immunogenicity study, mice received doses of 1 μg, 5 μg, or 10 μg of mRNA per antigen.

Total IgG, IgG1, and IgG2a titers in serum were determined by ELISA. High-binding 96-well plates were coated with 250 ng/well of HSV-2 strain MS lysates in carbonate coating buffer (pH 9.5) at 4°C overnight and blocked with 1% bovine serum albumin (BSA) in PBS for 1 h at room temperature. Two-fold serial dilutions of serum in blocking buffer were added to the plates and incubated for 2 h at room temperature. Colorimetric detection was performed by incubating for 1 h with horseradish peroxidase (HRP)-conjugated antibodies: anti-mouse IgG (Bethyl Laboratories, Montgomery, TX, USA) at a 1:3,000 dilution, IgG1 (Bethyl Laboratories) at a 1:3,000 dilution, or IgG2a (Bio-Rad, Hercules, CA, USA) at a 1:2,000 dilution, followed by incubation with 3,3′,5,5′-tetramethylbenzidine (TMB) substrates. The reaction was stopped with 2N H₂SO₄, and the absorbance was measured at 450 nm. Endpoint titers were defined as the serum dilution factor at which the absorbance value was at least two-fold greater than that of the PBS group and not less than 0.1.

Serum samples were serially diluted two-fold, starting at a 1:10 dilution in serum-free medium. Diluted sera were incubated with 100 plaque-forming units (PFU) of HSV-2 strain MS (Korea Bank for Pathogenic Viruses, Seoul, Republic of Korea) for 1 h at 37°C. Vero cells (Korean Cell Line Bank, Seoul, Republic of Korea) were seeded the day before infection (1×10⁵ cells/well in 24-well plates or 5×10⁴ cells/well in 96-well plates), and infected with virus-serum mixtures for 1 h at 37°C, 5% CO₂, with gentle agitation. After adsorption, the inoculum was removed, and cells were overlaid with carboxymethylcellulose medium. Following incubation for 48 h at 37°C with 5% CO₂, cells were fixed with 4% paraformaldehyde and stained with crystal violet solution. Plaques were counted and neutralizing antibody titers were determined as the highest serum dilution that reduced the plaque count by 50% (PRNT₅₀).

Mice were pretreated with medroxyprogesterone 5 days before intravaginal challenge with 1×10⁵ PFU of HSV-2 strain MS. Following infection, mice were monitored daily for weight loss and survival and evaluated every two days for genital disease and clinical signs. Genital disease was scored 1 point each for redness/erythema, hair loss, and urinary staining, with a maximum score of 3. Clinical signs were scored on a scale of 0–4: 0, no visible signs of disease; 1, slight ruffling of the fur; 2, ruffled fur, reduced mobility; 3, ruffled fur, minimal mobility; and 4, ruffled fur, minimal mobility, huddled appearance, and hind limb weakness. Mice were euthanized if they lost more than 20% of their initial body weight and exhibited genital disease symptoms.

HSV-2 titer was determined by plaque assay. Vaginal swab samples and HSV-2 strain MS were serially diluted 10-fold. Vero cells were cultured in 96-well plates and infected with the diluted viruses or vaginal swab samples. After 1 h of incubation at 37°C with 5% CO₂, the medium was replaced with an overlay medium containing 1% carboxymethylcellulose. Following 48 h incubation, the cells were fixed and stained with a 0.05% crystal violet solution containing methanol and formaldehyde. Viral plaques were counted after overnight staining.

Total genomic DNA was extracted from HSV-2-infected mouse dorsal root ganglia (DRG) using the DNeasy Blood and Tissue Kit (Qiagen, Germantown, MD, USA) following the manufacturer's instructions. Real-time PCR was performed using SYBR Green and the CFX96 Real-Time System (Bio-Rad, Hercules, CA, USA). The primers targeting the HSV-2 US9 gene were as follows: Forward: 5′-GGCAGAAGCCTACTACTCGGAAA-3′; Reverse: 5′-CCATGCGCACGAGGAAGT-3′.

Sectioned tissues from the experimental animals were submerged in 10% neutral buffered formalin, dehydrated, paraffin-embedded, and sectioned at 4-µm thickness for histological examination. Histopathological images were obtained and evaluated using an Aperio ImageScope version 12.6 (Leica Biosystems Pathology Imaging, Buffalo Grove, IL, USA). The severity of the histological changes was determined using a 5-point score system as follows: 0, no abnormality detected (NAD); 1, minimal; 2, mild; 3, moderate; 4, moderately severe; and 5, severe. The distribution was recorded as focal, multifocal, or diffuse. Recruitment of inflammatory cells and morphological alterations in the tissues were assessed by hematoxylin and eosin (H&E) staining.

The gB2, gC2, and gD2 peptide pools (JPT Peptide Technologies, Berlin, Germany) consisted of 15-mer peptides spanning the entire antigen sequence with an 11-amino acid overlap. The gE2 peptides consisted of five 9-mer peptides that were predicted for MHC-I binding using the NetMHCpan-4.1b server and synthesized by Peptron, Inc. (Daejeon, Republic of Korea). Splenocytes were stimulated with the gB2, gC2, or gD2 peptide pools at a final concentration of 0.7–1 μg/mL per peptide. For gE2 peptides, a total concentration of 10 μg/mL was used for the ELISPOT assay, and 25 μg/mL for the flow cytometric analysis of intracellular cytokine staining (ICS).

Splenocytes secreting IFN-γ or IL-4 were measured using the ELISpot Flex (ALP) kit (Mabtech AB, Nacka, Sweden) according to the manufacturer's protocol. The day before splenocyte harvest, 96-well filter plates were coated with capture antibodies (anti-mouse IFN-γ: clone AN18; anti-mouse IL-4: clone 11B11) at 4°C overnight and blocked with Roswell Park Memorial Institute (RPMI) medium containing 10% FBS. Subsequently, 5×10⁵ splenocytes were added to the coated plates and stimulated with antigens (gB2 peptide pool, gC2 peptide pool, gD2 peptide pool, or gE2 peptides) for 48 h at 37°C with 5% CO₂. Biotinylated antibodies (anti-mouse IFN-γ: clone R4-6A2; anti-mouse IL-4: clone BVD6-24G2) were then added and incubated for 2 h at room temperature. Streptavidin-alkaline phosphatase (ALP) was linked to biotinylated antibodies during a 1-h incubation. Bromochloroindolyl phosphate/nitro blue tetrazolium (BCIP/NBT) substrate was added for color development, and the reaction was stopped by washing with tap water. The number of spots was counted using an AID ELISpot Reader with software 7.0 (AID GmbH, Strassberg, Germany).

1×10⁶ splenocytes were plated in 96-well plates and blocked with CD16/CD32 (clone 93) for 30 min at 4°C. After blocking, cells were stained with LIVE/DEAD Fixable Aqua Stain, CD8 BV605 (clone 53-6.7), CD44 FITC (clone NIM-R8), CD62L PE-Cy7 (clone MEL-14), CD127 APC (clone SB/199), and KLRG1 PE (clone 2F1/KLRG1) for 1 h at 4°C. Stained cells were then fixed with 4% paraformaldehyde and analyzed using a CytoFLEX flow cytometer with CytExpert software 2.4 (Beckman Coulter, Brea, CA, USA).

1×10⁶ splenocytes were stimulated with antigens (gB2 peptide pool, gC2 peptide pool, gD2 peptide pool, or gE2 peptides) in RPMI medium supplemented with 10% FBS and 1% antibiotics. Brefeldin A was added 1 h post-stimulation, followed by a 12-h incubation at 37°C with 5% CO₂. Following stimulation, cells were blocked with CD16/CD32 (clone 93) and subsequently stained with LIVE/DEAD Fixable Aqua Stain, CD4 APC-Cy7 (clone GK1.5), and CD8 BV605 (clone 53-6.7) for surface marker analysis. Cells were then permeabilized with Cytofix/Cytoperm for 30 min at 4°C and intracellularly labeled with IFN-γ APC (clone XMG1.2) or IFN-γ PE-Cy7 (clone XMG1.2), TNF-α FITC (clone MP6-XT22), and Granzyme B PE (clone QA16A02) for 1 h at 4°C. Cytokine-labeled cells were analyzed using a CytoFLEX flow cytometer with CytExpert software 2.4 (Beckman Coulter, Brea, CA, USA).

Graphs and statistical analyses were performed using GraphPad Prism (version 9.1.0). The Kruskal-Wallis test was used for multiple group comparisons of antibody titers, viral titers, and viral DNA copies. Dunn's multiple comparisons test was used as a post hoc analysis to determine adjusted p-values. A two-tailed Mann-Whitney U test was used when only two groups were present. Changes in antibody titers between the two time points within the same group were analyzed using a two-tailed Wilcoxon signed-rank test. One-way analysis of variance (ANOVA) was used for multiple group comparisons of CD8⁺ T cell subsets and vaginal histopathology. Tukey's multiple comparisons test was used for post hoc analysis to determine the adjusted p-values. T cell and splenocyte responses to HSV-2 peptide stimulation were analyzed using two-way ANOVA with Tukey's multiple comparisons test. Two-way ANOVA with Sidak's multiple comparisons test was used when only two groups were present. P-values are denoted as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant. Error bars represent 95% confidence intervals (CI) for antibody titers, viral titers, and viral DNA copies, and standard deviations (SD) for cellular immune responses, weight loss, genital disease, clinical signs, and vaginal histopathology.

To evaluate the humoral and cellular immune responses induced by gB2, gC2, gD2, and gE2 mRNAs, mice were immunized twice at a two-week interval with 10 μg of each mRNA, and sera and spleens were collected two weeks later (Figure 1A). Antibody analysis showed that gB2, gC2, and gD2 mRNA elicited high titers of total IgG, IgG1, and IgG2a antibodies that bound to the HSV-2 lysate, indicating strong antigen recognition. However, gE2 mRNA induced low levels of total IgG (Figure 1B) and neutralizing antibody titers (Figure 1C), suggesting a weaker humoral response. These results indicated that gB2, gC2, and gD2 contribute more effectively to viral neutralization and entry blockade.

Cellular immune responses were assessed in peptide-stimulated splenocytes. All four glycoprotein mRNAs induced significantly more IL-4-secreting cells than PBS did, although gE2 elicited weaker T-helper type 2 (Th2) responses (Figure 1D). T-helper type 1 (Th1) responses were evaluated by measuring cytokine-producing CD4⁺ and CD8⁺ T cells (12 –14). CD4⁺ T cells producing IFN-γ and TNF-α were detected in the gB2, gC2, and gD2 groups, while CD8⁺ T cells producing IFN-γ, TNF-α, and Granzyme B were observed in the gB2 and gE2 groups (Figures 1E, F). Although the frequency of IFN-γ⁺ or TNF-α⁺ CD4⁺ T cells appeared modest in absolute terms, it represented a multi-fold increase relative to unstimulated controls, with high statistical significance (p < 0.0001). Such fold increases were generally considered highly biologically significant (8, 9, 15 –18).

Additionally, the gB2 and gE2 groups showed increased frequencies of CD8⁺ T short-lived effector cells (SLEC) and effector memory T cells (TEM). These CD8⁺ TEM populations, though not antigen-specific ones, are likely to support long-term protection, given their role in controlling viral reactivation (Supplementary Figure S1). Overall, gB2, gC2, and gD2 mRNA elicited stronger Th2 responses and higher antibody levels than gE2 mRNA, suggesting more effective B cell activation. T cell analysis showed that gC2 and gD2 induced CD4⁺ Th1 responses, whereas gE2 primarily induced CD8⁺ type 1 cytotoxic T (Tc1) responses. Although CD4⁺ T cell responses to gE2 could not be fully assessed owing to limitations in peptide selection, their presence cannot be ruled out. Notably, gB2 mRNA induced both CD4⁺ and CD8⁺ T cell responses, reflecting its ability to generate broad cellular immunity.

The humoral immunogenicity of the quadrivalent vaccine, comprising mRNAs encoding gB2, gC2, gD2, and gE2, was evaluated using two different LNP formulation strategies: a co-formulated version, in which all antigen mRNAs were encapsulated in a single LNP, and an admixed version, in which each mRNA was encapsulated separately in individual LNPs (Figure 2A). Physicochemical characterization of the LNPs revealed that all four mRNAs had a high encapsulation efficiency (EE%) of approximately 90%. Both single and co-formulated LNPs exhibited a uniform size distribution, with Z-average diameters ranging from 86 to 117 nm, a range considered optimal for mRNA vaccine delivery (Supplementary Figure S2). Mice were immunized with 10 μg of each mRNA at a two-week interval, and sera were collected at 1 and 2 weeks post-second immunization (wpi) to measure total IgG and neutralizing antibody titers (Figure 2B). Both formulations induced substantial levels of HSV-2-specific IgG and neutralizing antibodies as early as 1 wpi, which further increased by week 2. Importantly, no significant differences in antibody titers were detected between the two formulation strategies (Figures 2C, D), indicating that the quadrivalent mRNA vaccine elicited comparable humoral immune responses, regardless of the LNP formulation method.

Four weeks after completing the two-dose immunization regimen, mice were intravaginally challenged with 1×10⁵ PFU of HSV-2 strain MS. Survival, weight loss, genital disease, and clinical signs were monitored over 28 days (Figure 3A). Vaginal swabs were collected on days 2 and 4 post-infection to determine viral titers, and the dorsal root ganglia (DRG) and vaginal tissues were harvested at the time of death or the study endpoint for HSV-2 DNA quantification and histopathological analysis. Uninfected and unvaccinated mice were used as baseline controls for histological comparison. By day 10, all the mice in the PBS group had succumbed, resulting in a 0% survival rate. All mice immunized with either the co-formulated or admixed quadrivalent mRNA vaccine survived until the study endpoint (Figure 3B). Vaccinated mice did not show severe weight loss (Figure 3C) or genital disease (Figure 3D). The PBS group, however, developed genital disease by day 5 post-infection (Figure 3D), followed by rapid weight loss (Figure 3C). Mild clinical signs, such as slight fur ruffling, were observed in some vaccinated mice between days 5 and 17 post-infection (Figure 3E). On days 2 and 4 post-infection, the PBS group exhibited active viral replication at the infection site, with vaginal viral titers exceeding the geometric mean of 10⁴ PFU/swab. No detectable virus was observed on day 4 in either quadrivalent mRNA vaccine group, indicating early suppression of HSV-2 infection (Figure 3F). Furthermore, HSV-2 DNA was detected in the DRG of all PBS-treated mice (8–10 days post-infection, the time of death), with viral loads exceeding 10³ copies. Unlike the PBS group, the vaccinated mice exhibited a markedly lower latent viral burden by day 28, with geometric mean levels below 10 copies. Specifically, viral DNA was detected in six of the seven mice in the co-formulated group (4–16 copies) and in three of the seven mice in the admixed group (21–58 copies), but this difference was not statistically significant (p > 0.05) (Figure 3G). These findings demonstrated that the quadrivalent mRNA vaccine markedly reduced HSV-2 latency in the DRG.

Histopathological analysis of the vaginal tissues revealed severe pathological changes in the PBS group (8–10 days post-infection, the time of death), including epithelial erosion, atrophic necrosis, vacuolation, and atrophic degeneration. These lesions were markedly reduced in both the vaccine groups at the study endpoint (4 weeks post-infection) (Figure 3H). The PBS group also showed extensive immune cell infiltration, suggesting tissue damage (Figure 3H). In contrast, the vaccine groups exhibited milder but persistent immune cell infiltration, possibly reflecting ongoing tissue repair (19 –22), residual responses to viral antigens (23 –25), or immune cells preventing the reactivation of HSV-2 (26 –30). Both quadrivalent mRNA vaccines, whether co-formulated or admixed, effectively suppressed viral replication, prevented disease, and reduced tissue damage and latent infection, with no significant difference in efficacy.

To investigate the dose-dependent immune response elicited by the quadrivalent mRNA vaccine, co-formulations containing 1 μg, 5 μg, or 10 μg of each antigen were administered to mice in two doses over two weeks. Antibody and cellular immune responses were assessed two weeks after the second dose (Figure 4A). Serum anti-HSV-2 IgG levels showed that the 1 μg dose generated substantially higher titers than the PBS control, whereas IgG titers in the 1 μg group were not significantly different from those in the 5 μg or 10 μg groups (Figure 4B). Neutralizing antibody titers increased with dose, with GMTs of 111, 364, and 1,267 in the 1 μg, 5 μg, and 10 μg doses, respectively. Although neutralizing antibody titers appeared to increase in a dose-dependent manner from 1 μg to 10 μg, no statistical significance was observed between the vaccinated groups (Figure 4C).

The cellular immune response was measured using the gD2 and gE2 peptides to stimulate CD4⁺ and CD8⁺ T cell responses, respectively. In IFN-γ ELISPOT assay, the 1 µg dose enhanced cellular responses, with a further increase observed at the 5 µg dose, but no additional enhancement detected at the 10 µg dose (Figure 4D). Consistent with the ELISPOT results, a dose-dependent trend was observed in CD4⁺ T cells producing IFN-γ or TNF-α (Figure 4E), as well as in CD8⁺ T cells producing IFN-γ, TNF-α, or Granzyme B (Figure 4F). Similarly, TEM and SLEC subsets among total CD8⁺ T cells increased at the 5 μg dose and showed no further increase at 10 μg (Supplementary Figure S3). The gating strategy and representative flow plots for cytokine-producing CD4⁺/CD8⁺ T cells and CD8⁺ SLEC/TEM subsets are shown in Supplementary Figure S4. Overall, the strongest neutralizing antibody response was achieved at the 10 μg dose, while cellular immunity peaked at 5 μg, indicating that this dose was sufficient to elicit maximal cellular immunity.

To assess the long-term immune-protective efficacy of the quadrivalent mRNA vaccine, mice were challenged 16 weeks after the second immunization (Figure 5A). As shown in Figures 5B, C, anti-HSV-2 IgG levels and neutralizing antibody titers measured at week 15 post-second immunization remained high, with only a minimal decline from week 4 (IgG GMT: 1,344,037 to 904,470; PRNT₅₀ GMT: 427 to 355), indicating durable humoral responses that likely contributed to early and effective control of viral replication. In addition to humoral immunity, splenocytes collected at the study endpoint (21 wpi) showed persistent T cell responses specific to all four vaccine antigens (gB2, gC2, gD2, and gE2), demonstrating the longevity of cellular immunity (Figure 5D).

All mice in the PBS group succumbed to the infection by day 9 after challenge, whereas all vaccinated mice survived until day 36 (5 weeks post-infection), the study endpoint (21 wpi) (Figure 5E). Vaccinated mice exhibited no significant weight loss (Figure 5F), genital disease (Figure 5G), or clinical signs (Figure 5H) during the observation period. Notably, even during the early phase of infection (days 2–4 after challenge), when viral replication is typically active (16, 31, 32), vaccinated mice maintained a stable body weight, suggesting early viral control. This was supported by vaginal viral titers measured on day 2 post-infection, which revealed high viral loads in PBS-treated mice (GMT, 1.2×10⁷), while no virus was detected in the vaccinated mice, indicating potent suppression of early viral replication (Figure 5I). Quantification of HSV-2 DNA in the DRG on day 36 (5 weeks post-infection) revealed that the viral genome was undetectable in vaccinated mice, suggesting that the vaccine effectively blocked neuroinvasion and the establishment of latency (Figure 5J). However, gD2 mRNA alone did not fully prevent viral replication or HSV-2 DRG infection (Supplementary Figure S5). When comparing protective efficacy with the trivalent vaccine, a difference was observed in vaginal viral titers at 2 days post-infection. Although this difference was not statistically significant, the quadrivalent vaccine showed undetectable virus, whereas virus was detected in 29% (2 of 7) of mice in the trivalent vaccine group (Supplementary Figure S6). Histological analysis confirmed the protective effects of the vaccination. PBS-treated mice (G1) exhibited severe epithelial degeneration and prominent infiltration of immune cells by days 8–9 post-infection, whereas vaccinated mice showed milder epithelial disruption and immune cell infiltration on day 36 (5 weeks post-infection). The presence of macrophages, neutrophils, and lymphocytes in vaginal tissues at this late time point may reflect ongoing immune surveillance and tissue remodeling (Figure 5K).

Collectively, these results indicated that the quadrivalent mRNA vaccine induced durable and comprehensive immune responses, providing full protection against HSV-2 by preventing genital herpes, suppressing viral replication, and blocking latent infections.