One dose of mRNA vaccine produces strong and lasting antibody and cell immunity against chickenpox virus in mice

All mice were handled in accordance with the Good Animal Practice Requirements of the Animal Ethics Procedures and Guidelines of the People’s Republic of China, and the protocol was reviewed and approved by the Animal Ethics Committee of Chengdu Kanghua Biological Products Co. Ltd (Permit No. AEC-CDKH2025-0014).

The coding sequence (CDS) of gE derived from the sequence of the VZV vOKa strain was codon optimized and synthesized, then cloned into a plasmid vector with backbone sequence elements of a high copy origin of replication, a kanamycin resistance gene, a T7 promoter, 5′- and 3′ UTR, and a 100-nucleotide poly(A) tail. The plasmid DNAs were sequence verified, linearized by digestion with BspQ I downstream of poly(A) and purified by DNA Clean Beads (N411-01; Vazyme Biotech Co., Ltd., Nanjing, China). Following linearization, gE mRNA (named as KH014) sequence was synthesized with a T7 High Yield RNA Transcription Kit (TR101-01; Vazyme Biotech Co., Ltd., Nanjing, China) and N1-methyl-pseudouridine-5’-triphosphate (m1Ψ) instead of Uridine-5’-Triphosphate (UTP). Synthesized mRNA was further capped using a Vaccinia Capping System (DD4109-01; Vazyme Biotech Co., Ltd., Nanjing, China) and purified via RNA Clean Beads (N412-01; Vazyme Biotech Co., Ltd., Nanjing, China). The length and integrity of gE mRNA was analyzed using capillary electrophoresis. RNA characteristics including concentration, sequence length, and integrity were assessed by ultraviolet spectrophotometer, agarose gel electrophoresis and capillary electrophoresis, respectively.

For the preparation of LNP, lipids were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5 (SM-102: DSPC: cholesterol: PEG2000), and mRNA-gE-LNPs were dissolved at a charge ratio of N/P=6 in a 50 mM sodium citrate buffer (pH=4) solution. LNPs were formulated using a microfluidic mixer (Micro & Nano Technology, China) by mixing the aqueous and organic solutions at a ratio of 3:1. The LNP solution was concentrated by ultrafiltration using the Consieve TFF Ultrafiltration Cassettes (UFCLA0300001P; Hangzhou Cobetter Filtration Equipment Co. Ltd, Hangzhou, China), according to the manufacturer’s instructions. The mRNA-LNP was dispensed into appropriate cryoprotectant in vials, and then were lyophilized. LNP formulations were tested for particle size by Dynamic Light Scattering, mRNA encapsulation and concentration by RiboGreen Assay, RNA integrity by capillary electrophoresis (Agilent Fragment Analyzer).

In vitro expression of KH014 mRNA-LNP was assessed by ELISA kit (RAS-A185; Acrobiosystems Inc., Beijing, China). HepG2 cells were seeded into 12-well culture plates and cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., MA, USA) and 1% penicillin-streptomycin at 37˚C in the presence of 5% CO₂. Once confluency was reached approximately 80%, cells were transfected with LNPs containing 2 μg KH014 mRNA or empty LNP. After 24 h of incubation at 37 °C, the supernatants and cells were harvested together, and then lysed by three cycles of freeze-thawing. The gE protein expression levels in the supernatant clarified by centrifugation at 1,000 g/rpm for 10 min were detected using ELISA kit according to the manufacturer’s protocol.

Female specific pathogen-free BALB/c mice between 6 and 8 weeks of age were purchased from Chengdu GemPharmatech Co. Ltd. and housed in individually ventilated cages (IVCs). For mRNA immunization groups, mice were received intramuscular injections of single or two doses of KH014 on days 0 and/or 21. Each dose contained 1μg, 5μg, 10 μg, or 20 μg of mRNA for LNPs. Additionally, the positive group was immunized intramuscularly with two doses of 0.1 human dose of Shingrix^® at a 3-week interval (17, 20). Serum samples were collected from the submandibular (facial) vein of mice for antibody response analysis. Mice were euthanized by cervical dislocation at the indicated time points after the final vaccination. Spleens were collected and processed to obtain single-cell suspension for cellular immune responses analysis.

gE proteins (GLE-V52H3; Acrobiosystems Inc., Beijing, China) were coated into 96-well plates (42592; Corning Incorporated Life Sciences, Shanghai, China) at a concentration of 0.1 μg/well and incubated overnight at 4 °C. The plates were washed three times with PBS containing 0.1% Tween-20 (PBST) and blocked for two hours at room temperature (RT) with 2% bovine serum albumin (BSA). Serially diluted serum samples (ranging from 625,000 to 80,000,000) were added and incubated for 2 h at room temperature. After triple washing with PBST solution, goat anti-mouse IgG HRP-conjugated antibody (Abcam, ab205719, 1:5000 dilution) was added and incubated at 37 °C for 40 min. Then, the plates were washed with PBST and TMB substrate was added to each well. The reaction was stopped with sulfuric acid, and absorbance was measured at 450/620 nm using a microplate reader. Endpoint titers were defined as the highest serum dilution yielding an optical density (OD) > 2.1× background.

Vazyme Testing Technology Co., Ltd. (Nanjing, China) conducted microneutralization assays (21). MRC-5 cells were seeded in 96-well plates and incubated overnight (37 °C, 5% CO₂). Heat-inactivated sera (56 °C, 30 min) underwent serial 3-fold dilutions from 1:30, generating eight concentrations in duplicate. Diluted virus was added to samples/viral controls, neutralized (1 h, 37 °C), then transferred to cell monolayers with controls (50 μL/well, duplicate). After 2 h incubation, medium was replaced. Following 48 h incubation, cells were fixed, stained with fluorescent antibodies, and analyzed using CTL. Neutralization activity (ID₅₀) was calculated using the Reed-Muench method by comparing infected cells to virus controls, with ≥30 as positive cutoff (21).

For the ELISpot assay, splenocytes were isolated and resuspended in RPMI-1640 medium supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc., MA, USA) and penicillin-streptomycin (15140122; Gibco; Thermo Fisher Scientific, Inc., MA, USA) at a final concentration of 10⁷ cells/mL. IL-2 and IFN-γ positive splenocytes were performed according to the manufacturer’s instructions for the ELISpot assay (MabTech AB, Stockholm, Sweden). Briefly, splenocytes (3×10⁵ cells/well) were seeded in the IFN-γ/IL-2 antibodies precoated plates and stimulated at 37 °C for 48 h using a protein gE peptide pool at a concentration of 2 μg/mL. In the positive control well, PMA + ionomycin was used as a stimulus. After five times washing, detection antibodies were added and incubated at room temperature for 2 h. Finally, fluorophore-conjugated reagents and fluorescence enhancer were added to each well. Plates were read and analyzed in an FluoroSpot reader system (iSpot Spectrum, AID, Strassberg, Germany).

Splenocyte suspensions were incubated in a 96-well plate (10⁶ cells/well) with 200 μL of RPMI 1640 medium supplemented with 10% FBS, 1% penicillin-streptomycin, and 1× protein transport inhibitor cocktail (Invitrogen). The VZV gE peptide mix (2 µg/mL final concentration) or concanavalin A was added to each well and incubated for 5 h at 37 °C under 5% CO₂. During the final 2 h, 25 µL each of GolgiPlug and GolgiStop were added to all samples. After centrifugation at 4 °C for 10 min, cells were stained using the LIVE/DEAD fixable violet dead cell stain kit (Invitrogen) following the manufacturer’s instructions. Following two washes, Fc receptors were blocked with anti-mouse CD16/32 antibody (553142; BD Biosciences) for 10 min. Subsequently, cells were stained with surface antibodies including FITC anti-mouse CD3 antibody (100204, Biolegend), APC anti-mouse CD4 antibody (100412, Biolegend), and Brilliant Violet 650 anti-mouse CD8a antibody (100742, Biolegend) for 30 min. After surface staining, cells were fixed and permeabilized using Cytofix/Cytoperm Soln Kit (554714, BD Biosciences) according to the manufacturer’s instructions. Intracellular cytokine expression was assessed using Brilliant Violet 421 anti-mouse IFN-γ (505830; BioLegend), PE anti-mouse TNF-α (506306; BioLegend), PerCP/Cyanine5.5 anti-mouse IL-4 (504123; BioLegend) and PE/Cyanine7 anti-mouse IL-2 (503832; BioLegend). For the analysis of memory T cells, the cells were incubated with PerCP/Cyanine5.5 anti-mouse CD3 antibody (100218, Biolegend), FITC anti-mouse CD4 antibody (100204, Biolegend), PE anti-mouse CD62L antibody (161204, Biolegend), and APC anti-mouse CD44 antibody (103012, Biolegend) for 30 min. Intracellular IL-2 and IFN-γ expression in memory T cells was detected using Brilliant Violet 421 anti-mouse IFN-γ (505830; BioLegend) and PE/Cyanine7 anti-mouse IL-2 (503832; BioLegend). Cells were washed with intracellular staining perm/wash buffer and resuspended in 1× PBS for flow cytometry analysis (CytoFLEX, Beckman Coulter).

Tissue samples were fixed in 10% formalin, embedded in paraffin, and sectioned at 5 μm. Sections were stained with hematoxylin and eosin (HE) and scanned using 3DHISTECH.

For hematology parameters, blood was collected in potassium EDTA tubes and analyzed using an Advia 120 Haematology Analyser for the determination of white blood cell count, red blood cell count, platelet count, neutrophil count, percentage of lymphocyte, percentage of neutrophile, percentage of monocyte, percentage of eosinophile, and percentage of eosinophil.

Data are presented as means ± SD. One-way or two-way analysis of variance (ANOVA) was applied for multiple comparisons, and Student’s t-test for single comparisons. Statistical analyses were performed using GraphPad Prism 9.5.0 (GraphPad Software Inc., La Jolla, CA, USA).

Since glycoprotein E (gE) is a dominant immunogen of VZV and has been widely used as a key antigen candidate for VZV vaccine development, the KH014 mRNA vaccine was designed to encode the full-length gE antigen. Using algorithms such as LinearDesign and GenScript, a codon-optimized gE mRNA sequence was generated based on the wild-type VZV Oka strain (GenBank: AY253715.1↗). The optimized gE mRNA was synthesized via in vitro transcription (IVT) incorporating N¹-methylpseudouridine (m¹Ψ) modification. After transcription, a 5’ cap-1 structure was enzymatically added to produce fully mature mRNA. The IVT products and capped mRNA were verified using agarose gel electrophoresis (Supplementary Figure 1A), revealing a single band and confirming good purity. Further analysis by capillary electrophoresis of RNA revealed a single peak at 2100 nt (Supplementary Figure 1C), indicating its integrity. To evaluate in vitro expression, naked and LNP-encapsulated gE mRNAs were transfected into HepG2 cells. Western blotting revealed a distinct band at ~60 kDa in both cell lysates and supernatants (Supplementary Figure 1B). As expected, ELISA detected a higher protein expression level from LNP-encapsulated mRNA in the cell lysates (Supplementary Figure 1F), suggesting the encoded gE protein can be secreted outside transfected cells. Then, the quality control of KH014 mRNA vaccine was conducted by examining the size distribution and zeta potential with dynamic light scattering. We discovered that KH014 mRNA vaccine displayed a good uniformity of the particle sizes (~94 nm) (Supplementary Figure 1D), polydispersity index (0.16) (Supplementary Figure 1E). Zeta-potential measurements indicated that KH014 had a surface charge of -3.89 (Supplementary Figure 1E). By calculating the ratio between the quality of detected mRNA and the input dose, the encapsulation efficiency of the vaccine was obtained, and the result was 94.6% (Supplementary Figure 1E).

To evaluate immunogenicity and effectiveness, mice were immunized according to the scheme in Figure 1A with two doses of KH014 mRNA vaccine, Shingrix^®, or PBS via intramuscular injection. The serum samples from the mice were collected at day 0 (pre-vaccination), 15, and 35 post-first immunization. Anti-gE IgG binding antibodies in the sera were quantified using ELISA. As shown in Figures 1B, neither vaccine elicited detectable gE-specific IgG antibodies at baseline (day 0) or day 15, but both induced substantially elevated levels by day 35. Although KH014 mRNA vaccine generated slightly higher gE-specific binding antibody levels at day 35 than Shingrix^®, this difference was not statistically significant.

Neutralizing antibody titers are another key indicator of vaccination efficacy and important for the varicella vaccine (21). We thus measured neutralizing antibody titers using a neutralizing assay at day 0, 15, and 35 post-first immunization (Figure 1C). The results showed that mice immunized with KH014 mRNA vaccine produced neutralizing antibody titers significantly higher than those of the Shingrix^® group by 2.25-fold at day 35. Notably, at day 15, three sera from the KH014 mRNA vaccine group exhibited measurable neutralizing activity, whereas none from the Shingrix^® group did, suggesting that KH014 elicited faster and stronger neutralizing antibody responses.

Given that cellular immune responses play a key role in preventing VZV reactivation, IFN-γ and IL-2 secretion was quantified by ELISpot assay. As shown in Figure 2A, the number of IFN-γ-secreting cells after gE stimulation was 11.6-fold higher in KH014 mRNA group than in the Shingrix^® group. Consistent with IFN-γ spot, the number of IL-2 spots induced by KH014 also higher than Shingrix^® (Figure 2B). Subsequently, gE-specific T-cell responses were assessed by intracellular cytokine staining (ICS) assay to quantify IFN-γ, IL-2 and TNF-α-producing CD4⁺ (Figures 2C–2E, 2G–2I) and CD8⁺ T (Supplementary Figure 2) cells at day 14 after boost immunization. Compared with Shingrix^®, KH014 vaccination elicited higher frequencies of Th1-type CD4⁺ T cells in spleens. For Th2-prone cytokines (IL-4), there was no significant difference between the KH014 and Shingrix^® groups (Figure 2F). Assessment of cytokines-producing CD8⁺ T cell frequencies by ICS also demonstrated higher induction levels with KH014 than with Shingrix^® (Supplementary Figure 2).

Effective vaccines are expected to establish adaptive immunological memory (22). To determine whether KH014 vaccination induces long-lasting immunity, we investigated the presence of gE-specific CD4⁺ central (CD44⁺CD62L⁺) and effector (CD44⁺CD62L^–) memory T cells in the spleens of mice, collected at day 14 after boost immunization. As shown in Figure 3, there were no significant differences in the proportions of CD4⁺CD44⁺CD62L⁺ and CD4⁺CD44⁺CD62L^– cells among all groups (Figure 3A, B), suggesting that the different vaccines administered to the mice had no significant effect on memory T cells. However, KH014 induced significantly higher frequencies of gE-specific central and effector memory CD4⁺ T cell responses, particularly IFN-γ-producing CD4⁺ memory T cells, compared to Shingrix^® (Figure 3C, D). These results highlight the ability of our proposed KH014 vaccine candidates to elicit a potent T cell and long-lasting immune responses against VZV in the mouse model.

Given that vaccination with KH014 provided potent humoral and cellular immune responses, we investigated whether a single-dose administration of the vaccine would confer protection. BALB/c mice were vaccinated with escalating dosages (1 μg, 5 μg, 10 μg, and 20 μg) of KH014 mRNA vaccine (Figure 4A). As a positive control, mice received two intramuscular 5-μg doses of Shingrix^® on days 0 and 21. By ELISA, serum samples collected at day 35 were evaluated for gE-specific IgG. All four dosages of KH014 induced potent levels of gE-specific antibody following a single-dose immunization (Figure 4B). Furthermore, administration of KH014 at doses below 10 μg stimulated the production of gE-specific IgG titers in a dose-dependent manner. With regards to the cellular immune response, splenocytes were subjected to enzyme-linked immunospot (ELISpot) and intracellular cytokine staining (ICS) assays at day 35. ELISpot assays revealed that KH014 administered at dosage of 5 μg or 10 μg elicited a significantly higher level of IFN-γ or IL-2-producing T cells than Shingrix^® (Figures 4C, D). Among the various immunization dosage groups evaluated, the 5-µg mRNA dose induced the highest number of IFN-γ and IL-2 spots. Flow cytometry further demonstrated that KH014 administered at ≥ 5 µg doses elicited substantially higher proportions of CD4⁺ T cells expressing one or two cytokines than Shingrix^®, while responses in the 1 µg group were comparable (Figures 4E–J). However, frequencies of cytokine-producing CD8⁺ T cells (except IL-2-producing CD8⁺ T cells) induced by all KH014 immunization groups were moderate elevated compared to Shingrix^®-treated animals (Supplementary Figure 3). Collectively, these findings indicate that even a single low dose of KH014 effectively activates both humoral and gE-specific T-cell immunity, with 5 μg or 10 µg doses producing stronger responses than Shingrix^®.

Ideal prophylactic vaccines are expected to generate long-lasting protection. To evaluate whether a single dose of the KH014 vaccine candidate could induce long-term persistence of humoral and cellular immune responses, mice were received a single intramuscular injection of 10 µg KH014. The positive control group of Shingrix^® was similarly designed as the previous mouse study (Figure 5A). Following vaccination, the body weight of mice was monitored weekly. As shown in Supplementary Figure 4A, mice in the Shingrix^® group exhibited a transient loss of body weight 7 days following booster immunization, which attribute to the high reactogenicity of Shingrix^® in line with clinical observation. Furthermore, several routine blood parameters and histopathological examination of major organs were assessed to evaluate the safety properties of KH014 in mice at day 35. No significant changes were observed in blood levels of routine blood parameters including white blood cell count, red blood cell count, platelet count, neutrophil count, percentage of neutrophile, percentage of monocyte, percentage of eosinophile, and percentage of eosinophil (Supplementary Figure 4C). The H&E staining results showed no obvious pathological changes in any of the organs (Supplementary Figure 4B). These results support the safety of the novel KH014 in mice.

Anti-gE binding antibodies were detected by ELISA from sera of mice collected on 0, 3, 5, 7, and 10 weeks (Figure 5B). Serum IgG binding antibody levels induced by a single 10 µg dose of KH014 were lower than those in Shingrix^® vaccinated animals at weeks 5 and 7. However, the antibody response to KH014 increased over time, reaching a level comparable to that of Shingrix^® by weeks 10 (Figure 5B). We next examined the cellular response to immunization with single dose of KH014 at 5, 7, and 10 weeks. Splenocytes from all groups were isolated and subjected to ELISpot assay (Figure 5C, D). We observed greater numbers of gE-specific IFN-γ/IL-2 spot-forming T cells in the KH014 group than in the Shingrix^® group at 5, 7, and 10 weeks. Polyfunctional T cell responses were analyzed by ICS. Similarly, flow cytometry results showed that the KH014 induced significantly higher numbers of CD4⁺ T cells across all timepoints, including gE protein-specific IFN-γ⁺ (Figure 5E), IL-2⁺ (Figure 5F), and TNF-α⁺ (Figure 5G) single-positive CD4⁺ T cells, as well as IFN-γ⁺IL-2⁺ (Figure 5H), IFN-γ⁺TNF-α⁺ (Figure 5I), and IL-2⁺TNF-α⁺ (Figure 5J) double-positive CD4⁺T cells, compared to those for the Shingrix^®. While a statistically significant difference was observed exclusively in the frequencies of gE protein-specific IFN-γ⁺ single-positive and IFN-γ⁺TNF-α⁺ double-positive CD8⁺ T cells among stimulated splenocytes derived from KH014 immunized mice (Supplementary Figure 5). Altogether, these results confirm that a single dose regimen of KH014 mRNA vaccine induced superior long-term immunogenicity compared to the licensed subunit vaccine in the mouse model.

Given the limitations of the two-dose Shingrix^® regimen, including systemic adverse reactions, high cost, and reduced adherence, we evaluated the humoral and cellular immune responses elicited by heterologous immunization strategies combining the KH014 mRNA vaccine and Shingrix^®. Mice were assigned to different immunization groups: one received the conventional two-dose regimen of Shingrix^®; another received a heterologous regimen beginning with 10 μg KH014 or 5 μg Shingrix^® followed by a booster (Shingrix^®>KH014 and KH014>Shingrix^®); and the last received only a single dose of KH014 (1xKH014). As a negative control, mice received 100 μL phosphate buffered saline (PBS) on days 0 and 21 (Figure 6A). We performed microscopic histopathological observation using muscle tissues at the injection sites on days 35. H&E staining of muscles at the administration site revealed slight intramuscular/perimuscular inflammatory in the Shingrix^® group and KH014>Shingrix^® groups. The PBS, 1xKH014 and Shingrix^®>KH014 groups exhibited no inflammation at injection sites (Supplementary Figure 6).

ELISA results demonstrated that anti-gE IgG binding antibody titers were significantly higher in both homologous and heterologous prime-boost immunization groups compared to the single-dose KH014 group, while the IgG levels in homologous and heterologous prime-boost regimens were comparable (Figure 6B). To further investigate the effect of heterologous prime-boost schedules on immune response types, the cytokines (IFN-γ and IL-2) and polyfunctional T cell responses were measured using ELISpot, and flow cytometry, respectively. Compared to the two-dose Shingrix^® regimen, both the single-dose KH014 group and the KH014>Shingrix^® group exhibited a significantly higher number of gE-specific IFN-γ/IL-2 spot-forming T cells (SFCs) (Figures 6C, D). In contrast, priming with Shingrix^® followed by a KH014 booster resulted only in a slight elevation of SFCs compared to the Shingrix^® control. Further analysis of T-cell-mediated immunity revealed that the KH014>Shingrix^® group induced the highest proportions of gE-specific CD4⁺ T cells, including IFN-γ⁺, IL-2⁺, and TNF-α⁺ single-positive subsets, as well as IL-2⁺IFN-γ⁺, IL-2⁺TNF-α⁺, and IFN-γ⁺TNF-α⁺ double-positive subsets (Figures 6E–J). The proportions of these single- and double-cytokine-producing CD4⁺ T cells in the single-dose KH014 and Shingrix^®>KH014 groups were comparable to those in the two-dose Shingrix^® control group. Frequencies of cytokine-producing CD8⁺ T cells were also assessed by flow cytometry (Supplementary Figure 7). The proportions of single- and double-cytokine-producing CD8⁺ T cells, except IL-2 producing CD8⁺ T cells (Supplementary Figure 7B), were all higher in the heterologous prime-boost immunization groups and single-dose KH014 group than that in the two-dose Shingrix^® control group, in which these responses were minimal or undetectable in some mice.

To further evaluate a single-dose administration and heterologous prime-boost immunization strategy for inducing gE-specific memory immune response, the proportion of gE-specific CD4⁺ central memory (CD44⁺CD62L⁺) and effector memory (CD44⁺CD62L^–) T cells induced by different immunization strategies were then assessed by flow cytometry (Figure 7). According to the flow cytometry analysis, a single-dose KH014 induced significantly higher frequencies of gE-specific IFN-γ-producing CD4⁺ central memory T cells (Figure 7A) and IL-2-producing CD4⁺ effector memory T cells (Figure 7D), compared to Shingrix^®. As for the heterologous prime-boost immunization strategy, mice primed with KH014 and boosted with Shingrix^® were induced significantly higher frequencies of gE-specific CD4⁺ effector memory T cell response (Figures 7C, D), but mice in Shingrix^®>KH014 group displayed higher proportions of gE-specific CD4⁺ central memory T cell than those in Shingrix^® vaccinated animals (Figures 7A, B). Overall, these findings highlight KH014 mRNA vaccine boosted with Shingrix^® strategy produced superior cell-mediated immune, and also further underscores the potential of a single-dose KH014 vaccination.

The original contributions presented in the study are included in the article/. Further inquiries can be directed to the corresponding author. 1