Both 293T and DC 2.4 cells were maintained in DMEM (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco) and penicillin–streptomycin (100 U/ml, Gibco). Pseudovirus pSG-cmv-flu-chikv (22) was stored at −80°C and titrated using the 50% tissue culture infectious dose (TCID₅₀) assay in 293T cells.

The cDNAs encoding wild-type (WT) or codon-optimized (OP) structural protein C-E3-E2-6K-E1 genes from CHIKV strain LR2006 OPY1 (23) were cloned into plasmid ABOP-028 (GENEWIZ), which contained 5′ and 3′ untranslated regions (UTRs) and a poly A tail. The mRNA was synthesized in vitro using T7 polymerase-mediated transcription from the linearized plasmid DNA template.

Formulation was performed as described previously for the COVID-19 vaccine (24). Briefly, lipids were dissolved in ethanol containing an ionizable lipid, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and PEG-lipid (molar ratios of 50:10:38.5:1.5). The lipid mixture was run through a T-mixer with mRNA dissolved in 20 mM citrate buffer (pH = 4.0) at a ratio of 1:2. The generated mRNA-LNPs were diafiltrated against PBS (pH = 7.4) in a dialysis cassette with 20 kD MWCO overnight, passed through a 0.22-μm filter, and stored at 2°C–8°C until use. The product was characterized for particle size and distribution using a particle size analyzer (Malvern Panalytical), RNA concentration using HPLC (Thermo Fisher Scientific), and encapsulation using RiboGreen reagent (Thermo Fisher Scientific).

DC 2.4 cells were seeded in 24-well plates at 500,000 cells/well and then transfected with 1 μg of mRNA to form cell monolayers using Lipofectamine 2000 Transfection Reagent (Thermo Fisher Scientific). After 6 h, the medium was replaced with DMEM supplemented with 3% FBS. Following 24 h post-transfection, cells were fixed with ice-cold acetone for 10 min and incubated with a polyclonal mouse anti-CHIKV antibody (prepared in our laboratory) and then FITC-labeled goat anti-mouse IgG (Abcam). Nuclei were counterstained with DAPI (Thermo Fisher Scientific). Fluorescence was observed using a fluorescent microscope (Olympus).

After 16–26 h post-transfection, cells were lysed with RIPA buffer plus proteinase inhibitor (Sigma), clarified by centrifugation at 12,000 × g, and then 5 × SDS loading buffer was added, and cells were kept at 95°C for 10 min. The lysates were run on a 10% NuPAGE Bis-Tris gels (Invitrogen) followed by transferring proteins to a nitrocellulose membrane. The membrane was incubated with a polyclonal mouse anti-CHIKV antibody (prepared in our laboratory) and HRP-labeled goat anti-mouse IgG (Abcam). DC 2.4 cells with lysing buffer and E2 protein were used as negative and positive controls (ProSpec), respectively. Blots were developed using ECL reagents (GE).

Female C57BL/6 mice were obtained from the Institute for Laboratory Animal Resources at the National Institutes for Food and Drug Control (NIFDC). For neutralizing antibody responses, mice aged 6–8 weeks were injected via an intramuscular (im) route with 100 μl of the indicated vaccine or negative control. Serum was collected from the submandibular vein before and/or post-prime immunization. For cellular immune responses, mice aged 6–8 weeks were immunized via the im route with 100 μl of the indicated vaccine or negative control. Spleen tissues were collected after immunization. Housing and experimentation of mice were performed strictly in accordance with the guidelines set by the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC). The study protocol was approved by the Animal Care and Use Committee of the NIFDC.

The vaccines used in this experiment were the mRNA vaccine and Ad5 vaccine (developed in our laboratory). Both vaccines contain the prototype gene of structural protein. Mice were immunized using different vaccination regimens: same vaccination routes (2×Ad5, 2×mRNA), different vaccination routes (Ad5 + mRNA), and negative control groups. The prime vaccination day was set as day 0, and the boost was performed on day 14. Serum and spleen tissues were collected before and after prime immunization.

The neutralization assay using pseudovirus was similar to that used for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as described previously (25). Serial threefold diluted heat-inactivated serum obtained from C57BL/6 mice was incubated with 400 TCID₅₀ of the pseudovirus for 1 h at 37°C. The 293T cells were added to serum–virus complexes in 96-well plates at 50,000 cells/well and incubated for approximately 48 h, together with the virus control and cell control in wells. The supernatant was then removed and luciferase substrate (Perkin Elmer) was added to each well, followed by incubation for 2 min. Luminescence was measured for pseudovirus titration. The 50% inhibitory dilution (EC₅₀) was defined as the serum dilution at which the relative light units (RLUs) were reduced by 50% compared with the virus control after subtraction of the background RLUs in control cells.

Spleens were isolated from mice and dispersed in a 40-μm cell strainer with mouse lymphocyte separation medium (Dakewe). The tissues were centrifuged at 800 × g for 20 min and covered with 1 ml of RPMI-1640 medium (Hyclone). After centrifugation, splenocytes were resuspended in serum-free medium (Dakewe). Interferon gamma (IFN-γ)- or interleukin-2 (IL-2)-positive cells were assessed using precoated enzyme-linked immunospot (ELISpot) kits (MabTech) according to the manufacturer’s protocol. Briefly, 96-well plates were blocked with RPMI-1640 medium containing 10% FBS for at least 2 h at 24°C. The splenocytes were transferred to the wells at 500,000 cells/well and stimulated at 37°C for 18 h with Ad5 containing entire structural proteins C-E3-E2-6K-E1. Phorbol ester (PMA)/Ionomycin (Dakewe) and RPMI-1640 media were used as the positive and blank control, respectively. The plates were incubated with anti-mouse IFN-γ or IL-2 antibody for 2 h and streptavidin-horseradish peroxidase for 1 h at ~25°C. The visualized immunoprecipitate revealed that the following treatment with TMB substrate solution was subsequently imaged and quantified using an ImmunoSpot S6 Universal instrument (Cellular Technology Limited).

Splenocytes were transferred into wells at 500,000 cells/well and stimulated at 37°C for 8 h with Ad5 containing entire structural proteins C-E3-E2-6K-E1. Brefeldin A (BioLegend) was then added to block cytokine secretion and incubation continued for 4 h. Following two washes with PBS, splenocytes were incubated with the following antibodies against lineage markers: PE anti-mouse CD3e, FITC anti-mouse CD4, and PerCP/Cy 5.5 anti-mouse CD8a (all from BioLegend). After two washes with PBS, cells were fixed, permeabilized with Cytofix/Cytoperm (BD Biosciences), and incubated with the following antibodies against intracellular markers: APC anti-mouse IFN-γ and PE/Cy7 anti-mouse IL-2 (all from BioLegend). Cells were analyzed using a FACS Lyric analyzer (BD Biosciences).

Data were plotted and analyzed using GraphPad Prism 8.0 and presented as geometric means ± geometric standard deviation. Neutralization antibody levels were compared by two-way analysis of variance (ANOVA), and an unpaired t-test was used for comparison of cellular immune levels. A p-value of <0.05 was considered statistically significant (*p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, no significance).

The structural proteins C-E3-E2-6K-E1 of WT CHIKV virus strain LR2006 OPY1 were selected as the target antigens for the mRNA coding sequence. Moreover, 5′ and 3′ UTRs were added to increase mRNA stability and regulation of translation. A poly(A) tail of ~100–130 bp was synthesized to follow the 3′ UTR. Transcription of the linearized DNA template to mRNA was mediated by the T7 promoter, and the complete mRNA was obtained by adding the Cap-1 structure (N7^mGpppG^m) to the 5′ end of the RNA using a vaccinia capping enzyme. We also constructed an mRNA-OP using OP sequences (26) ( Figure 1A ) and a sequence optimization strategy for mRNA vaccines as previously described (24). The resulting mRNA was transfected into DC 2.4 cells. Immunofluorescence assays using anti-CHIKV structural protein polyclonal antibodies indicated that the mRNA-WT and -OP translated targeted proteins were expressed intracellularly and on the cell membrane ( Figure 1B ). Cell lysates were collected, immunoblotting assays were performed using anti-CHIKV structural protein polyclonal antibodies, and specific protein expression was detected in both mRNA-WT and -OP transfected cell samples ( Figure 1C ). mRNA was encapsulated in LNPs for diafiltration and purification; formulation with an average particle size of ~79 nm and an encapsulation rate of over 90% were obtained ( Table S1 , Figure S1 ).

In the single-dose immunization regimen, C57BL/6 mice received an im injection of 1, 5, or 10 μg WT or OP vaccine, with PBS solution as the negative control. Serum was collected before immunization and on days 7, 14, 28, 42, 56, and 70 post-immunization for neutralizing antibody testing using a pseudovirus assay ( Figures 2A–C ). For the 1-μg WT dose, the EC₅₀ antibody titer was maintained at a low level approaching negative control; the EC₅₀ antibody titer exhibited a high agreement between that of the 5- and 10-μg doses (p = 0.9958), which reached ~20,000 on day 42 post-immunization and remained steady. At the 1-µg OP dose, antibody was not induced; at the 10 μg dose, the EC₅₀ antibody titer reached a peak of 26,833 on day 42 post-immunization, which was approximately twice that of the 5-μg dose, although the EC₅₀ antibody titer showed no significant difference with the 10-μg dose of OP (p = 0.0827). The neutralizing antibody titers of the WT vaccine were better than those of the OP vaccine at the 5-μg dose, which suggests that base changes in the target genes may have affected humoral immunity.

In the booster immunization regimen, WT and OP vaccines were given at a dose of 10 μg and boosted with an equal dose of vaccine on day 14 post-initial immunization. Serum was collected before immunization and on days 7, 14, 28, 42, 56, and 70 post-prime immunization for neutralizing antibody testing ( Figure 2D ). Neutralizing antibody levels for both vaccines increased substantially on day 28 post-immunization, with a greater than sixfold increase compared with that of single-dose immunization. Furthermore, EC₅₀ antibody titers reached over 100,000, peaking on day 42 post-immunization for the WT and OP vaccines. Neutralizing antibody titers were significantly higher following booster immunization than single-dose immunization (WT: p < 0.0001; OP: p = 0.0028). The results indicate that booster immunization with both WT and OP vaccines induced relatively high levels of neutralizing antibodies in mice.

C57BL/6 mice were immunized with a single dose of 10 μg of WT or OP vaccine, with PBS solution as the negative control. Splenocytes were harvested from the mice on days 7, 14, and 28 after immunization ( Figure 2A ) and stimulated with Ad5 containing CHIKV structural proteins; ELISpot and intracellular cytokine staining (ICS) assays were performed.

The levels of IFN-γ and IL-2 secretion by splenocytes were analyzed by the ELISpot assay ( Figure 3 ). The WT and OP vaccines induced strong IFN-γ (WT: p = 0.0025; OP: p = 0.0002) and IL-2 (WT: p < 0.0001; OP: p = 0.0009) levels on day 7 post-immunization compared with the negative control group. There was no statistically significant difference in the levels of cytokine-secreting cell number between the WT and OP vaccine groups (IFN-γ: p = 0.1484; IL-2: p = 0.8606). Furthermore, both vaccines displayed stable cellular responses within 28 days post-immunization.

Both mRNA vaccines induced CD4⁺ and CD8⁺ T-cell responses ( Figure 4 ). However, the WT vaccine induced IFN-γ CD4⁺ T-cell response on day 7, but not on day 14 and day 28. Interestingly, IFN-γ CD4⁺ T cells were not clearly detected or were detected at very low levels in the OP vaccine group. In contrast, both vaccines induced IL-2 CD4⁺ T-cell responses with the OP vaccine group having a lower T-cell number on day 28 (p = 0.0010) than the WT vaccine. The OP vaccine induced a higher IFN-γ CD8⁺ T-cell response than the WT group. The latter showed a very low level of IFN-γ CD8⁺ T-cell responses. The IL-2 T-cell response showed a similar trend with the OP group having a higher IL-2 CD8⁺ T-cell response although the differences between the two groups were not significant on day 14 (p = 0.3622) and day 28 (p = 0.2874).

To compare the immune responses of the different prime-boost regimens, we used the Ad5 CHIKV vaccine constructed in our previous study, which was obtained by cloning the complete CHIKV structural protein genes (C-E3-E2-6K-E1) into a shuttle plasmid. We employed homologous recombination with the plasmid using the AdEasy adenovirus packaging system, transfecting Ad293 cells and assembling them into a mature recombinant adenovirus, which was amplified and purified to obtain the Ad5 vaccine expressing the CHIKV antigen.

As illustrated for the immunization regimen in Figure 5 , mice were immunized using different vaccination regimens: prime-boosted homologously with mRNA vaccine, or with Ad5 vaccine, or primed with Ad5 vaccine followed by heterologous boosting with mRNA vaccine at a 14-day interval. The overall scheme for group design and immunization is shown in Table 1 . A total of 10⁷ infectious units (IFU) of Ad5 vaccine or 10 μg of mRNA vaccine was used for vaccination.

Serum was collected on day 0 before immunization and on days 14, 28, 42, 56, and 70 post-prime immunization ( Figure 6 ). The EC₅₀ antibody titers of all immunization groups peaked on day 42 post-prime immunization. The EC₅₀ antibody titer of the 2×mRNA immunization group was 149,428, which was significantly higher than that of the Ad5 + mRNA group (p = 0.0004) and the 2×Ad5 group (p < 0.0001), and it was maintained above 100,000 thereafter. There was a statistically significant difference in the titers between the Ad5+mRNA and 2×Ad5 groups (p = 0.0014), with peak antibody titers of 43,679 and 13,335, respectively. No significant increase in neutralizing antibody titers was observed with heterologous immunization compared to the homologous regimen.

Splenocytes were harvested on days 21 and 28 post-prime immunization, and ELISpot and ICS assays were performed. The ELISpot assay ( Figure 7 ) showed that the 2×Ad5 group failed to induce IFN-γ (day 21: p = 0.0519; day 28: p = 0.0561), but inducing IL-2 response (day 21: p = 0.0117; day 28: p = 0.0003), while IFN-γ and IL-2 were induced in the other two groups (2×mRNA and Ad5+mRNA). On day 21 post-prime immunization, the cytokine levels in the 2×mRNA group were higher than those in the other two groups with a statistically significant difference compared with the Ad5 + mRNA group (IFN-γ: p = 0.0041; IL-2: p = 0.0002). On day 28 post-initial immunization, the cytokine levels in the Ad5+mRNA group were significantly higher, while they decreased in the other two groups. Moreover, cytokine levels in the Ad5+mRNA group showed a statistically significant difference from those in the 2×Ad5 group (IFN-γ: p = 0.0003; IL-2: p < 0.0001); IL-2 levels were higher than those in the 2×mRNA group (p = 0.0004), while the Ad5+mRNA group showed similar levels of IFN-γ induction to those of the 2×mRNA group (p = 0.0564).

ICS results ( Figure 8 ) showed that CD4⁺ and CD8⁺ T cells in the Ad5+mRNA group exhibited higher levels of induced IFN-γ on both day 21 and day 28 post-prime immunization, while IFN-γ in the other two groups was induced primarily by CD8⁺ T cells. CD4⁺ and CD8⁺ T cells in all three immunization groups induced comparable levels of IL-2 on day 28 post-prime immunization.