MPXV mRNA vaccine design and fusion protein expression

In this study, we developed potent MPXV trivalent mRNA vaccines that target the antigens of both the IMV and EEV forms and characterized their physicochemical properties. First, we generated four codon-optimized mRNAs through the in vitro transcription (IVT) process (Fig. 1A); these mRNAs encode different MPXV antigens derived from the MPXV Zaire-96-I-16 strain (clade I), the IFN-α protein, and red fluorescent protein (RFP). The first mRNA encodes the fusion protein AALI (A35R, A29L, L1R, and IFN-α linked by GSAGSAG), the second encodes AAL (A35R, A29L, and L1R), the third encodes IFN-α as a control, and the fourth encodes RFP as a reporter gene. Notably, we introduced secretion signal peptides (SP) at the N-terminus of the four encoded proteins to increase their immunogenicity. Structural modeling of AALI and AAL with AlphaFold 3 revealed that the domains of A35R, A29L, and L1R exhibited no obvious steric hindrance, and the fused AAL protein was independent of the IFN-α region (Fig. 1B), indicating that the domains of AAL and IFN-α do not affect each other. We subsequently used mannose-conjugated LNPs to encapsulate mRNAs and evaluated their physicochemical properties via transmission electron microscopy (TEM) and dynamic light scattering (DLS). The results displayed in Fig. 1C show that both the LNPs and LNPs-mRNAs (LNPs encapsulating mRNA) exhibit a spherical shape, with particle sizes ranging from 100 to 120 nm. Furthermore, these vaccine particles have a relatively uniform size distribution and are well dispersed in PBS, with a low polydispersity index (PDI) (Fig. 1D). By measuring the concentration of LNPs before and after demulsification of the mRNA in the LNPs, we found that the LNPs presented a high mRNA encapsulation efficacy of ~95 ± 3.1% (Fig. S1A). These results indicate that the MPXV mRNA vaccines AALI and AAL have promising properties for effective vaccine development.

A Two MPXV mRNAs encode fusion proteins: AALI (A35R, A29L, L1R, and IFN-α) and AAL (A35R, A29L, and L1R). mRNAs for IFN-α, RFP, and GFP were used as controls or markers. All sequences were codon optimized, fused with a signal peptide (SP), and inserted into the coding region. B Predicted 3D structures of the AALI and AAL fusion proteins: A29L (light green), A35R (red), L1R (blue), and IFN-α (cyan). C Characterization of LNPs and LNP-mRNA. TEM images of LNPs (left) and LNPs-mRNA (right); scale bar = 200 nm. D LNP (right) and LNPs-mRNA (left) particle sizes were measured via dynamic light scattering (DLS). E HeLa cells transfected with mRFP (RFP mRNA) or LNPs-mRFP were observed under a fluorescence microscope 24 h post-transfection. Images were captured at ×100 magnification. F In vivo bioluminescence imaging of mice 48 h after transfection with LNPs-mRFP. G HeLa cells were transfected with 2 μg of mRNA, and cell lysates collected at 24 h post-transfection were analyzed by SDS-PAGE and Western blotting with anti-A35R and anti-β-actin antibodies. Lane 1: markers; Lanes 2–4: LNPs-, AAL-, and AALI-treated groups. The red box indicates the target protein band. H, Microscopy images (×100) showing the growth status of HeLa cells (top) and RAW264.7 cells (bottom) treated with various LNPs concentrations. I The cell activity induced by LNPs was analyzed at 72 h. n = 3 biologically independent replicates. The data and error bars represent the means ± SDs. Statistical significance was calculated by one-way ANOVA with Tukey’s multiple comparison tests. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.001; ns, p > 0.05. J, K BMDCs were treated with AALI, AAL, IFN-α, LNPs, or PBS (control) for 48 h, then stained with antibodies against MHC I, MHC II, CD80, and CD86 for flow cytometry analysis. n = 3 biologically independent replicates. Data and error bars represent the means ± SDs. Statistical significance was calculated by one-way ANOVA with Tukey’s multiple comparison tests. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, p > 0.05.

Next, the effectiveness of LNPs-mRNAs in expressing target proteins as a vaccine was confirmed through the transfection of LNPs-mRFP (LNPs encapsulating RFP mRNA) into HeLa cells and BALB/c mice. Fluorescence microscopy and in vivo bioluminescence imaging demonstrated successful RFP expression in the LNPs-mRFP group, with no expression observed in the naked mRFP group, at both the cellular and whole-organism levels (Fig. 1E, F). These findings indicate that LNPs effectively protect mRNAs from degradation, increase their delivery into host cells, and enable the mRNA to express the target protein. Moreover, the expression of the fusion proteins AALI and AAL was confirmed through the transfection of an equivalent amount of mRNA into HeLa cells. The cells and their intracellular proteins were harvested and incubated with a primary antibody specific for A35R, followed by the application of either FITC-conjugated or HRP-conjugated secondary antibodies. This process was carried out to facilitate flow cytometry and Western blot analysis. As depicted in Fig. S1B, compared with the control group treated with PBS, the groups treated with AALI and AAL presented a marked increase in the expression of the A35R antigen. Additionally, all proteins of interest were successfully expressed and identified via Western blot analysis, with the mRNAs encoding AALI and AAL yielding robust protein expression levels, as illustrated in Fig. 1G. These findings suggest that the developed MPXV mRNA vaccines are capable of effectively expressing antigenic proteins both in vitro and in vivo.

To verify the ability of the mannose-conjugated LNPs to target DCs, we performed an LNPs-mRNA targeting assay. As shown in Fig. S1C, orange fluorescence indicated colocalization of the green fluorescent protein (GFP) in LNPs-mRNA with red fluorescent DCs, whereas no colocalization was detected in A549 cells. These findings indicate that mannose-modified LNPs can effectively deliver mRNA to DCs and express the target protein, which is consistent with the literature³⁷.

Safety and bioactivity of the MPXV mRNA vaccines

To determine the safety and bioactivity of the mRNA vaccines, we conducted the following experiments. Initially, the cytotoxicity of mannose-modified LNPs at various concentrations was assessed in HeLa and RAW264.7 cells. We found no significant differences in cytotoxicity between the LNPs- and PBS-treated groups at concentrations of 1.25, 12.5, 125, and 1.25 mM (Fig. 1H, I). Additionally, to evaluate the toxicity of LNPs-loaded mRNAs in mice, we conducted histopathological analysis and assessed indicators of liver and kidney function. As expected, no discernible pathological alterations were observed in the heart, liver, spleen, lung, or kidney of the mice in the AALI, AAL or IFN-α groups compared with those in the LNPs and PBS control groups, both in the short term (Fig. S1D, 14 days after the second immunization) and long term (Fig. S2A, 72 days after the third immunization). Furthermore, liver and kidney function indicators, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (T-BIL), albumin (ALB), alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT), total bile acid (TBA), creatinine (Crea), uric acid (UA), urea, and carbon dioxide (CO₂), remained within the normal range for all groups (Table S1, 14 days after the second immunization; Fig. S2B, 72 days after the third immunization)^39,40,41. These results indicate that the MPXV mRNA vaccines are safe.

After establishing the safety and efficacy of the MPXV mRNA vaccine, we investigated its biological activity in vitro. The splenocyte proliferation assay data in Fig. S2C revealed that the AALI, AAL, and IFN-α mRNA vaccines induced clusters of proliferating murine splenocytes. The results from the MTS assay (Fig. S2D) revealed significantly greater proliferation indices (PI) in the AALI, AAL, and IFN-α groups than in the LNPs and PBS control groups. Notably, the AALI group exhibited even greater PIs than the AAL group did (p < 0.5), suggesting that the AALI group had increased immune response capabilities.

DCs, crucial APCs, play pivotal roles in linking innate and adaptive immune responses⁴². We also treated bone marrow-derived DCs (BMDCs) with the mRNA vaccine and analyzed them using flow cytometry. Compared with treatment with LNPs or PBS, treatment with AALI, AAL, or IFN-α significantly increased the expression levels of the markers CD80, CD86, MHC I, and MHC II in BMDCs, suggesting increased maturation (Fig. 1J). Moreover, the AALI-treated BMDCs presented significantly higher expression levels of CD80 and CD86 than did the AAL-treated BMDCs. Additionally, the expression of MHC I was significantly greater in the AALI and IFN-α groups than in the AAL group (Fig. 1K). Collectively, these findings underscore the potential efficacy of our mRNA vaccines in effectively stimulating the proliferation of mouse splenic lymphocytes and inducing the maturation of BMDCs. Moreover, the combination of IFN-α as an adjuvant with AAL demonstrated promising immunostimulatory effects.

The mRNA vaccines induced antibodies specific to the MPXV and VACV antigens

Female BALB/c mice were immunized following a prime-boost-boost schedule, with 2-week intervals between each vaccination, and serum samples were collected 14 days after the final vaccination. To confirm whether the MPXV mRNA vaccine induced the production of MPXV-specific antibodies, we conducted enzyme-linked immunosorbent assays (ELISAs) with six recombinant proteins derived from both clade I MPXV (A29L, A35R, and L1R were derived from the hMpxV/DRC/INRB-0198V/2024 strain) and clade II MPXV (A29L from the MPXV-UK_P2-138 strain, A35R and L1R from the hMpxV/Portugal/INSA-PT0001/2022 strain). As expected, both MPXV mRNA vaccines elicited robust levels of antibodies against those antigens, whereas IFN-α and the LNPs did not induce any effective antibodies (Fig. 2A–F and Table S2). The IgG titers of AALI and AAL against A29L, A35R, and L1R from both clade I and clade II MPXV were >327680, >327680, and >327680, respectively. Interestingly, antisera from the AALI and AAL treatment groups also bound to the antigens A27L and A33R, which were derived from VACV, with high antibody titers (Fig. 2G, H and Table S3). The IgG titers induced by AALI and AAL against the three MPXV antigens were 81920 (A27L) and >327680 (A33R).

A–H The breadth of the total IgG antibody response elicited by the mRNA vaccine was determined by ELISAs of pooled antisera against clade IIb MPXV of A29L (A)-, A35R (B)-, and L1R (C)-, clade Ib MPXV of A29L (D)-, A35R (E)-, L1R (F)-, and VACV of A27L (G)- and A33R (H)-antigen proteins. All the data in (A–H) are shown as the means ± SDs (n = 3 biologically independent replicates). I Fifty percent live-virus neutralizing antibody titers were determined by PRNT for clade II MPXV. n = 3 biologically independent replicates. The data are presented as the means ± SDs. J–M Specific ASC responses to the A29L, AAL, and A35R proteins were measured with an ELISpot assay. J Frequencies of A29L-specific ASCs on days 0, 21, 35, 49 and 100. n = 3 biologically independent replicates. The data and error bars represent the means ± SDs. Statistical significance was calculated by one-way ANOVA with Tukey’s multiple comparison tests. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, p > 0.05. K, L, and M, Frequencies of AAL-, A29L- and A35R-specific ASCs on day 49. All the data and error bars in (K–M) represent the means ± SDs (n = 3 biologically independent replicates). Statistical significance was calculated by one-way ANOVA with Tukey’s multiple comparison tests. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, p > 0.05.

These findings indicate that the mRNA vaccines robustly induced broad antibody binding activity, potentially providing cross-protection. Given the trend observed for the antibody titer, AALI exhibited greater potency in inducing A29L- (from clade II and clade I MPXV), A35R- (from clade I MPXV), L1R- (from clade I MPXV) and A33R-specific antibodies than did AAL (Fig. 2A, D–F, H). Moreover, we measured antigen-specific antibodies of the IgG1 and IgG2a subclasses in antisera, which are essential for understanding B-cell somatic hypermutation and subclass switching⁴³. The results showed that the vaccines induced high levels of A29L-, A35R-, L1R-, A27L-, and A33R-specific IgG1 and IgG2a (Fig. S3A–J), which were consistent with the total IgG results. The elevated levels of the IgG1 and IgG2a subclasses suggest the activation of both Th1 and Th2 immune responses, which are essential for B-cell maturation and subclass switching.

The MPXV mRNA vaccines induce antibodies with high binding affinity for various antigens

We purified the serum antibodies induced by AALI and evaluated their binding affinities for the MPXV and VACV antigens via SPR (Fig. S3K). The results demonstrated that AALI-induced antibodies exhibited strong binding affinities for the clade II MPXV antigens A29L, A35R, and L1R, with KD values of 2.35 × 10⁻⁹ M, 8.40 × 10⁻⁸ M, and 6.0 × 10⁻⁸ M, respectively (Fig. S3L–N). Similarly, for clade I MPXV, the KD values for the A29L, A35R, and L1R antigens were 1.724 × 10⁻⁸ M, 6.361 × 10⁻⁹ M, and 2.406 × 10⁻⁸ M, respectively (Fig. S3O–Q). Additionally, the antibodies bound to the A27L protein derived from VACV, with a KD value of 2.23 × 10⁻⁷ M (Fig. S3R). These high-affinity interactions with both the MPXV and VACV antigens suggest that immunization with the MPXV mRNA vaccine induces broad cross-reactive protection against clade II MPXV, clade I MPXV, and VACV.

Neutralizing antibody responses were assessed via live-virus neutralization tests using a clade II MPXV strain. As shown in Fig. 2I, compared with the IFN-α, LNPs, and PBS groups, the AALI and AAL mRNA vaccines induced high neutralizing antibody titers after booster immunization, which prevented MPXV from infecting host cells. Furthermore, the median values of the AALI- and AAL-50% neutralizing antibody titers were 1:900 and 1:300, respectively. The fusion of AAL with the adjuvant-like protein IFN-α may strengthen humoral responses. These results demonstrate that the mRNA vaccines induced potent antibody responses with high binding affinities for both the MPXV and VACV antigens, effectively neutralizing and preventing clade II MPXV infection.

The MPXV mRNA vaccines induced antibody-secreting cell (ASC) responses

ELISpot assays were conducted to measure antigen-specific B-cell responses induced by the mRNA vaccines. The kinetics of MPXV antigen-specific ASCs were tracked before and after immunization, and the results are shown in Fig. S3S. The response strength was positively correlated with vaccination time, peaking on day 49. At this peak, the frequencies of AAL protein (expressed in Escherichia coli), A29L- and A35R-specific ASCs were significantly greater in the AALI- and AAL-immunized groups than in the IFN-α-, LNPs-, and PBS-immunized groups. Additionally, we found a statistically significant difference between the IFN-α group and the LNPs and PBS groups (Fig. 2J). Moreover, at the peak, the frequencies of the AAL-, A29L-, and A35R-specific ASCs induced by AALI were 69 ± 6, 134 ± 8 and 161 ± 17 per million, respectively, which were ~1.4-fold greater than the frequencies induced by AAL (p < 0.05, Fig. 2K–M). Notably, the vaccine-treated groups maintained robust A29L-specific ASC levels at 100 days post-third immunization, which remained significantly elevated compared with the pre-vaccination baseline, as shown in Fig. 2J. This sustained ASC presence beyond the typical plasmablast contraction phase (4–6 weeks post-immunization) strongly indicates the successful generation of memory B-cell pools—a key correlate of long-term immune protection⁴⁴. In summary, the AALI and AAL mRNA vaccines effectively induced antigen-specific B-cell responses and elicited serological memory. This memory is likely sustained by long-lived plasma cells and bolstered by memory B cells, which rapidly differentiate into ASCs upon re-exposure to specific antigens. In conclusion, these results suggest that MPXV mRNA vaccines induce B cells to differentiate into MPXV-specific antibody-secreting and memory cells, producing high-titer neutralizing antibodies that may provide cross-protection against MPXV and VACV.

The MPXV mRNA vaccines induced T-cell responses

MPXV infection is controlled primarily by antibodies, with T cells assisting in the humoral response, virus clearance, and spread prevention. Here, to investigate the cellular immunity induced by the developed mRNA vaccines, we investigated CD4⁺ T-cell and CD8⁺ T-cell immune responses. We isolated splenocytes from vaccinated mice on day 14 post-vaccination to explore the T-cell immune response induced by the vaccines in vivo. As depicted in Fig. 3A, B and Fig. S4A–D, AALI, AAL, and IFN-α, but not LNPs or PBS, significantly induced CD4⁺ T-cell and CD8⁺ T-cell proliferation. Furthermore, to investigate CD4⁺ T-cell differentiation, we used ELISpot assays to quantify the numbers of IFN-γ- and IL-4-secreting cells, which typically represent Th1 and Th2 cells, respectively (Fig. 3C). Compared with those in the LNPs and PBS groups, the numbers of IFN-γ- and IL-4-producing splenocytes in the mRNA vaccine and IFN-α groups were significantly greater (p < 0.05, Fig. 3D). Additionally, the AALI-treated group presented a significantly greater number of these cells than did the AAL-treated group (p < 0.05). Precise measurement of cytokine expression in helper T cells is crucial for understanding the immune responses elicited by vaccines. Thus, we also assessed the serum expression levels of cytokines, including IL-4, IL-6, IL-13, TNF-α, and IFN-γ, following vaccination via cytometric bead assays (Fig. 3E–I). The results were consistent with those obtained in the ELISpot assay, showing that the AALI and AAL mRNA vaccines significantly induced the upregulation of all these cytokines compared with LNPs alone and PBS. Notably, immunization with AALI and IFN-α induced greater expression of the investigated cytokines than did immunization with AAL. Taken together, these results show that immunization with the mRNA vaccines activated Th1 and Th2 cells, which are necessary for immune responses to clear virus infection.

A Splenocytes isolated from immunized mice in each group on day 14 were examined by flow cytometry. The clusters of CD3⁺CD4⁺ and CD3⁺CD8⁺ cells represented the CD4⁺ (above) and CD8⁺ T cells (below). B Summary data for the fractions of CD4⁺ and CD8⁺ T cells in different groups. The data and error bars represent the means ± SDs (n = 3 biologically independent replicates). Statistical significance was calculated by one-way ANOVA with Tukey’s multiple comparison tests. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, p > 0.05. C Splenocytes were plated in ELISpot wells, and IFN-γ and IL-4 cytokine-producing cells were detected. D Frequencies of IFN-γ- and IL-4 cytokine-producing cells on day 14. The data and error bars represent the means ± SDs (n = 3 biologically independent replicates). Statistical significance was calculated by one-way ANOVA with Tukey’s multiple comparison tests. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, p > 0.05. E–I Production of cytokines, including IL-4, IL-6, IL-13, TNF-α, and IFN-γ, in the serum of patients treated with the mRNA vaccines. All the data and error bars in (E–I) represent the means ± SDs (n = 3 biologically independent replicates). Statistical significance was calculated by one-way ANOVA with Tukey’s multiple comparison tests. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, p > 0.05. J Equal numbers of CFSE^high-labeled A35R-pretreated cells and CFSE^low-labeled BSA-pretreated splenocytes were mixed and injected into immunized mice. After 12 h, the splenocytes were collected to assess antigen-specific cytolytic responses using flow cytometry. K Infected cell-specific lysis in different groups. The data and error bars represent the means ± SDs (n = 3 biologically independent replicates). Statistical significance was calculated by one-way ANOVA with Tukey’s multiple comparison tests. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, p > 0.05.

The cytotoxic effects of CD8⁺ T cells are crucial for the elimination of infected cells and play an important role in the effective control of MPXV spread. To evaluate whether the developed mRNA vaccines could promote antigen-specific CD8⁺ T-cell responses, we intravenously administered a mixture of equal numbers of CFSE^high-labeled A35R-pretreated cells and CFSE^low-labeled BSA-pretreated splenocytes into immunized mice. Specific cytotoxic T lymphocyte (CTL) activity was then assessed via flow cytometry after 12 h. As shown in Figs. 3J and S4E, the fluorescence intensity of the A35R-pretreated cells surpassed that of the BSA-pretreated cells, underscoring the discernible difference in labeling. The number of residual A35R-pretreated cells (CFSE^high) in the mice immunized with AALI or AAL was markedly lower than that in the mice immunized with IFN-α, LNPs, or PBS (Fig. 3K). Furthermore, we found no significant differences between the IFN-α- and LNPs-treated groups (p > 0.05). These findings confirmed that the mRNA vaccines induced strong antigen-specific cytotoxic responses.

Immunization with the developed mRNA vaccines provides in vivo protection against MPXV and VACV challenge

To analyze the protective efficacy of the mRNA vaccines, 3 weeks after the third immunization, the mice were intranasally challenged with 4 × 10⁵ focus-forming units (FFUs) of the clade IIb MPXV hMpxV/China/SZ-SZTH42/2023 or hMpxV/China/SZ-SZTH41/2023 strain or 8 × 10⁴ FFUs of the VACV Tiantan strain⁴⁵, as shown in Fig. 4A. We separated the lung tissues and measured the viral load of MPXV SZTH42 on days 4 and 7 post-infection via real-time quantitative polymerase chain reaction (qPCR) analysis and a focus formation assay. As expected, the number of infectious viral particles in the mice immunized with AALI or AAL was significantly lower than that in the mice in the IFN-α- and LNPs-treated groups on day 4. Additionally, the lungs of the mice in the IFN-α group had fewer infectious viral particles than those in the LNPs group (Fig. 4B). Similar results were observed via qPCR analysis of the number of viral E9L gene copies in the mice in each group⁴⁶ (Fig. 4C). The relative viral load in the lungs of the mice in the AALI- and AAL-treated groups was significantly lower than that in the lungs of those in the mice in the IFN-α and LNPs groups at 4 days, indicating that the MPXV-specific antibodies induced by the mRNA vaccines effectively neutralized and inhibited infection in the lung tissue. Additionally, by 7 days post-infection, the viral load sharply decreased in the AALI and AAL groups but continued to increase in the IFN-α and LNPs groups. These findings suggest that the CTLs induced by AALI and AAL rapidly cleared MPXV-infected cells, reducing the spread of the virus. The greater reduction in MPXV load in the AALI group than in the AAL group at 7 days post-infection suggests that AALI offers slightly better protection.

A Mice were infected intranasally with the clade II MPXV SZTH41, SZTH42 strains or VACV Tiantan strain 21 days after the third immunization. Created in BioRender. gN, \. (2025) https://biorender.com/k8huiqq. B The viral load in the lung was detected on days 4 and 7 post-infection via a focus-forming assay. n = 3 biologically independent mice. Data are presented as means ± SD. C The viral copy numbers of MPXV SZTH42 in the lungs, which were derived from the same batch of lung tissue samples as shown in (B), were quantified by qPCR. n = 3 biologically independent mice. The data are presented as the means ± SDs. D (clade II MPXV SZTH42 strain), E (clade II MPXV SZTH41 strain), and F (VACV Tiantan strain) depict the percentage change in body weight monitored 7–14 days post-challenge. For D–F n = 6 biologically independent mice. G (clade II MPXV SZTH42 strain), H (clade II MPXV SZTH41 strain), and I (VACV Tiantan strain) present the survival curves of BALB/c mice following challenge. For G–I n = 6 biologically independent mice. J Histopathology of lung tissue on day 7 post-infection with the MPXV clade II strain SZTH42 (scale bar = 200 μm). The green arrows indicate macrophage, lymphocyte, or granulocyte infiltration in alveolar spaces; the red arrows highlight alveolar hemorrhage; the yellow arrows denote peribronchial infiltration; and the black arrows indicate bronchial epithelial shedding. Untreated and unchallenged naive mice served as controls. K Distribution of MPXV (yellow for A35R), lung-infiltrating CD8⁺ T cells (green for CD8) and lung-infiltrating macrophages (red for F4/80); scale bar = 100 μm. L Relative quantification of the fluorescence intensity of A35R, CD8, and F4/80. n = 3 biologically independent replicates. The data and error bars represent the means ± SDs. Statistical significance was calculated by one-way ANOVA with Tukey’s multiple comparison tests. *, p < 0.5; **, p < 0.01; ***, p < 0.001; ns, p > 0.05.

The AALI- and AAL-immunized mice experienced a reversible loss of body weight at 2 days and exhibited minimal overall weight loss, whereas the mice in the LNPs- and IFN-α-treated groups continued to lose weight, eventually losing more than 25% of their body weight (Fig. 4D–F). By monitoring the survival of the mice, we found that both AALI and AAL provided 100% cross-protection against the clade II MPXV SZTH42, MPXV SZTH41, and VACV Tiantan strains, as illustrated in Fig. 4G–I. In addition, histopathological analysis revealed the absence of visible pathological changes in the lungs of the AALI- and AAL-immunized mice. In stark contrast, the LNPs- and IFN-α-treated groups displayed severe alveolar damage and interstitial inflammatory infiltration (Fig. 4J) on day 7 post-infection. In conclusion, multiple findings demonstrate that our vaccines offer strong cross-protection against MPXV and VACV challenge in vivo.

To evaluate the inflammatory damage caused by MPXV infection, we examined lymphocyte infiltration into the lungs of the mice via immunohistochemistry (IHC). As expected, the common leukocyte antigen CD45 was widely detected in the lung sections of the IFN-α- and LNPs-treated mice but not in those of the uninfected naive mice (Fig. S4F). Compared with IFN-α and LNPs treatment, immunization with AALI or AAL before MPXV infection substantially reduced the number of lung-infiltrating CD45⁺ leukocytes (Fig. S4G), suggesting the mitigation of inflammatory lung damage. Additionally, immunofluorescence analysis of lung sections was performed using fluorescent antibodies targeting A35R, F4/80, and CD8 to assess the distribution of MPXV, infiltrating macrophages, and CD8⁺ T cells (Fig. 4K). As shown in Fig. 4L, the intense yellow fluorescence signals of A35R on day 7 post-infection indicated widespread MPXV distribution in the lungs of the IFN-α- and LNPs-treated mice. In contrast, A35R fluorescence was weaker in the lungs of the AALI- and AAL-vaccinated mice. Compared with the IFN-α and LNPs control groups, the AALI- and AAL-immunized groups presented significantly increased lung CD8⁺ T-cell distributions (p < 0.01), which was consistent with the CTL assay results. These CD8⁺ T cells effectively cleared virus-infected cells, leading to reduced viral loads, as indicated by decreased A35R fluorescence. In contrast, the IFN-α- and LNPs-treated mice presented greater macrophage staining intensities than did the AALI- and AAL-immunized mice (p < 0.05). This increased macrophage activity was associated with increased inflammation and delayed viral clearance in the IFN-α and LNPs groups⁴⁷. These findings underscore the robust protective efficacy of AALI and AAL immunization against MPXV challenge in vivo.

Single-cell transcriptional profiling of splenocytes isolated from mice vaccinated with the MPXV mRNA vaccine

To characterize the immunological features of BALB/c mice vaccinated with the AALI MPXV mRNA vaccine, we conducted droplet-based scRNA-seq using the 10× Genomics platform. Additionally, we performed single-cell TCR and BCR sequencing to comprehensively evaluate the transcriptomic profiles of spleens obtained from three AALI-treated and three LNPs-treated BALB/c mice, which served as controls (Fig. 5A). After employing a unified single-cell analysis pipeline, we sequenced a total of 44333 cells derived from the spleen across all the samples. Among these cells, 20,965 (47.3%) originated from the AALI-treated group, whereas 23,368 (52.7%) originated from the LNPs-treated group. By using graph-based clustering in uniform manifold approximation and projection (UMAP), we delineated the transcriptomes of 11 major cell types or subtypes on the basis of the expression patterns of canonical gene markers (Fig. 5B)^48,49. These cells included B cells (Cd79a⁺Ms4a⁺), CD4⁺T cells (Cd3d⁺Cd3e⁺Cd4⁺), CD8⁺T cells (Cd3d⁺Cd3e⁺Cd8a⁺Cd8b⁺), DCs (Siglech⁺Cd83⁺Cst3⁺), monocytes (Cd14⁺Lyz2⁺Csf1r⁺Ccr2⁺), natural killer cells (NK cells: Nkg7⁺Fcer1g⁺), plasma cells (Cd38⁺Mzb1⁺), hematopoietic stem and progenitor cells (HSPCs: Kit⁺Sox4⁺Gata2⁺Cd34⁺), erythrocytes (Gypa⁺Slc4a1⁺), endothelial cells (Pecam1⁺Igfbp7⁺) and neutrophils (Ly6g⁺S100a9⁺S100a8⁺) (Fig. 5C–E). Thus, we effectively delineated the splenic immune cell landscape in BALB/c mice receiving the developed mRNA vaccines.

A A schematic diagram of the overall study design. Splenocytes from AALI-treated (n = 3) and LNPs-treated mice (n = 3) were subjected to scRNA-seq gene expression profiling and TCR and BCR profiling analyses. Created in BioRender. gN, \. (2025) https://biorender.com/ac9szf7. B Cell populations were identified via UMAP projection, which revealed 11 major cell types across six samples. Each dot represents a single cell, colored according to its respective cell type. C Canonical cell markers were utilized to label clusters by cell identity in the UMAP plot, with data colored on the basis of expression levels via a log-scale legend. D Violin plots illustrating the expression distribution of selected canonical cell markers across 11 clusters, with rows representing marker genes and columns representing clusters. E Dot plot depicting cell type marker genes across 11 distinct cell types from spleen samples. F Average proportion of each cell type derived from the two groups. G Distribution of the average percentage of each immune cell type across the two conditions. n = 3 biologically independent replicates. The data and error bars represent the means ± SDs. Statistical significance was calculated by unpaired two-sided Student’s t-tests. *, p < 0.05; ns, p > 0.05.

Next, to gain further insight into the immune cell dynamics in MPXV mRNA-vaccinated mice, we employed scRNA-seq to analyze the distribution of major splenic immune cell populations. By quantifying the percentages of 11 key cell types in the spleens of vaccinated mice, we aimed to characterize the immune cell landscape post-vaccination (Fig. 5F). Our analysis revealed a noteworthy finding: the percentage of plasma cells in the spleens of the mice subjected to AALI was significantly greater (p < 0.05) than that in the LNPs control group on day 7 post-vaccination. This elevation in plasma cell abundance likely reflects increased production of protective neutralizing antibodies directed against MPXV, contributing to the efficacy of the vaccine. While the increases in monocyte and DCs percentages were not statistically significant (p > 0.05), these results should be considered potential trends given their biological context and the mechanisms of vaccine action, providing preliminary insights for future studies (Fig. 5G). These cells play pivotal roles in orchestrating the immune response by presenting antigens and activating other immune effectors. Interestingly, we also observed a postvaccination increase in neutrophils, which play key roles in both innate and adaptive immunity, including in pathogen killing, increasing antigen presentation, and modulating immune cell interactions⁵⁰. Collectively, these findings provide a comprehensive view of immune cell dynamics post-vaccination, highlighting the specific cell types that may be crucial for mediating protection against MPXV.

Expression profiles of B cells after vaccination

To characterize changes in individual B-cell subsets post-vaccination, we subclustered splenic B cells (23,824) and identified six subsets on the basis of B-cell markers and signature genes (Figs. 6A, B and  S5A, B): naive follicular B cells (Ighd⁺Sell⁺Fcer2a⁺), marginal zone B cells (MZ B, Ighd⁺Cd38⁺Cr2⁺), pre-B cells (Ms4a1⁺Vpreb3⁺), plasma cells (Mzb1⁺Jchain⁺Xbp1⁺), plasmablast cells (Mzb1⁺Cd38⁺Ms4a1⁺), and debris. Notably, the proportions of active B-cell subsets, including plasma B cells and MZ B cells, were greater in the AALI-treated groups than in the control groups, whereas the proportions of naive follicular B cells were significantly lower (Figs. 6C and  S5C).

A UMAP projection of B cells, with each dot representing a single cell and colored by cell type. B Violin plots illustrating the expression distribution of selected canonical cell markers across six clusters, with rows representing marker genes and columns representing clusters. C Distribution of the average percentage of each immune cell type across the two groups. n = 3 biologically independent replicates. The data and error bars represent the means ± SDs. Statistical significance was calculated by unpaired two-sided Student’s t-tests. *, p < 0.05; **, p < 0.001; ns, p > 0.05. D GO terms are labeled with names and IDs and are sorted by −log10 p values. E Volcano plot showing the DEGs between the AALI- and LNPs-treated samples. DEGs refer to genes with a Benjamini‒Hochberg adjusted p value ≤ 0.05. F Bar plot showing the percentages of IGHA, IGHD, IGHG, and IGHM expression in the two groups. n = 3 biologically independent replicates. The data and error bars represent the means ± SDs. Statistical significance was calculated by unpaired two-sided Student’s t-tests. *, p < 0.05; **, p < 0.001; ns, p > 0.05. G Bar plot showing the percentages of IGHG1, IGHG2B, IGHG2C, and IGHG3 expression in the two groups. n = 3 biologically independent replicates. The data and error bars represent the means ± SDs. Statistical significance was calculated by unpaired two-sided Student’s t-tests. *, p < 0.05; **, p < 0.001; ns, p > 0.05. H UMAP plot showing BCR detection. I UMAP plot showing the BCR clone status distribution. J Stacked bar plots showing the multiclonal state of BCR in each sample. K Circos plots showing BCR chain rearrangements in the two groups. Arc lengths indicate VDJ gene family frequencies; colored ribbons represent paired segment usage. L The CDR3 motifs in the IGH, IGK, and IGL chains that are unique to the AALI-treated group were predicted after excluding sequences that were shared with the LNPs-treated group. The label on the x-axis represents the position of the CDR3 amino acid, and the y-axis represents the bits.

To further investigate differential transcriptomic changes in B cells after vaccination, we compared the expression profiles of B cells between the vaccine-treated group and the control group. Initially, we employed Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses to illuminate the functional aspects of B cells (Fisg. 6D and  S5D). As anticipated, genes associated with “positive regulation of the MHC class I biosynthetic process” and “antigen processing and presentation” were enriched following MPXV mRNA vaccination, indicating that the presentation and conversion of antigens from the vaccine might be initiated. Additionally, genes related to “protein processing in the endoplasmic reticulum” and “positive regulation of peptide secretion” were enriched in B cells, indicating a heightened demand for protein synthesis and assembly during antibody production. The “Toll-like receptor signaling pathway” and “NOD-like receptor signaling pathway”, which play critical roles in regulating the adaptive immune response, were also enriched after vaccination. Genes associated with the “response to viral infection” were also notably enriched in B cells, suggesting that vaccination triggered a robust antiviral response, which was likely effective against MPXV infection. Furthermore, other biological pathways involved in host immune responses, including the “B-cell receptor signaling pathway” and “positive regulation of the inflammatory response”, were significantly enriched after MPXV mRNA vaccination. Next, we observed significant upregulation of differentially expressed genes (DEGs) in B cells, including Rpl37a, Jchain, Igkv4-91, Igkv8-19, Ighm, and Iglc1 (Fig. 6E), which are involved in antibody production, clonal expansion, and V(D)J gene utilization. These findings suggest that our vaccine activated B cells, potentially leading to distinctive and specific V(D)J rearrangements post-vaccination.

At 7 days after vaccination, B cells expressed genes encoding the constant regions of IgG, IgD, IgA, and IgM (Fig. S5E). Compared with the LNPs-treated samples, the AALI-treated samples presented a significant increase in IgG and a decrease in IgM expression (Fig. 6F), suggesting the occurrence of clonal expansion or a humoral immune response in the MPXV mRNA-vaccinated mice. Further analysis revealed a significant increase in the expression of the Ighg1 and Ighg2c subtypes post-vaccination, which was consistent with the ELISA results (Fig. 6G). Although Ighg2b and Ighg3 expression also tended to increase, the difference was not statistically significant (p > 0.5). The elevated expression of Ighg isotypes suggests B-cell responsiveness to Th1 and Th2 cells, which regulate class switching to IgG1 and IgG2, respectively. In conclusion, our scRNA-seq data revealed high levels of transcriptional specialization in B cells post-vaccination, validating differences in B-cell protective responses between the vaccine- and LNPs-treated groups.

Expansion of B cells and specific rearrangements of V(D)J genes after vaccination

B cells achieve substantial antigen recognition diversity through unique BCR specificity, enabling broad detection of antigens⁵¹. Only a minor subset undergoes antigen-driven clonal expansion, transitioning from single-cell clones (clone = 1) to expanded populations (clone ≥2)⁵². To assess the impact of the MPXV mRNA vaccine on BCR transcripts, we reconstructed BCR sequences and analyzed clonal expansion post-vaccination. Over 77% of the BCRs were detected in each cluster (Fig. 6H, I). To assess the impact of the MPXV mRNA vaccine on BCR transcripts, we reconstructed BCR sequences and analyzed clonal expansion post-vaccination. Notably, comparative analysis of BCR expanded clones between the AALI- and LNPs-treated groups revealed a greater proportion of multiclonal BCRs (clone ≥2) in the vaccine group than in the control group (Fig. 6J). Although there was no statistically significant difference (p > 0.5), the observed trend suggests that the AALI vaccine may have the potential to induce B-cell clonal expansion, as shown in Fig. S5F. Additionally, most of the top 20 clone types were present in the vaccine-treated samples (Fig. S5G). These findings indicate strong activation of B-cell activity and humoral immune responses, which is consistent with the increased antibody titers observed by ELISAs and focus reduction neutralization tests (FRNTs). To delve deeper into the biased V(D)J rearrangements within BCRs in response to specific stimuli, we conducted a comparative analysis of V(D)J gene utilization patterns across two distinct groups, focusing on the immunoglobulin heavy chain variable region (IGHV), immunoglobulin kappa chain variable region (IGKV), and immunoglobulin lambda chain variable region (IGLV) segments (as depicted in Fig. 6K). Our gene usage analysis revealed preferential selection of certain V(D)J genes, notably Ighv1-19, Ighv3-1, and Ighv9-3, among IGHVs; Igkv1-135 and Igkv10-96, among IGKVs; and Iglv1, among IGLVs, which were more prevalent than other germline counterparts. Intriguingly, after vaccination, we observed notable overrepresentation of Ighv1-5 and Igkv1-110, suggesting a potential role in the immune response to vaccination (as evident in Figs. 6K and S5H). Furthermore, our analysis revealed the clonal expansion of specific lineages, including Ighv1-72, Ighv8-4, and Igkv8-34, which was exclusive to mice treated with AALI (as shown in Figs. 6K and S6A). This exclusive expansion suggests that these genes encode antibodies specifically targeted against MPXV, emphasizing their potential importance in the development of an effective immune response against this virus.

CDR3 represents a hypervariable segment of the BCR due to diverse recombination processes and is considered the major determinant of antibody specificity, playing an essential role in the identification of and interaction with distinct antigenic peptides⁴⁶. To identify CDR3 consensus motifs, we analyzed the amino acid sequences of the IGH, IGK, and IGL chains in the CDR3s of the AALI- and LNPs-treated groups and then predicted the motifs using the motif-based sequence analysis tool Multiple Em for Motif Elicitation (MEME)⁵³. The top five highest-ranked IGH, IGK, and IGL motifs were obtained for each group of BCRs on the basis of sequence conservation and the E value (Table S4), and the results (E value < 0.05) are shown in Fig. S6B, C. We identified the unique CDR3 motifs in the BCRs from the immune and control groups (as shown in Table S4 for the AALI and LNPs groups, respectively). To specifically identify BCR clonotypes induced by the vaccine and minimize interference from the baseline immune repertoire, we excluded the common BCR sequences that were shared between the AALI- and LNPs-treated groups from the AALI-treated group (as shown in Table S4, labeled AALI-LNPs). Motif analysis was then performed solely on the post-immunization BCR sequences, as shown in Fig. 6L. The motifs “YAMDYW”, “CARGGY”, and “GYYYFDY” were found in the IGH chain, “CQQSSSYP” in the IGK chain, and “CALWYSNQ” in the IGL chain. These motifs are likely associated with vaccine-induced B-cell clonal expansion and selection, indicating a specific immune response to the administered antigen.

To gain insight into the BCR response to MPXV antigens, we performed molecular docking simulations of antibody motifs with conserved sequences and the AAL antigen. Specifically, we concatenated two conserved motifs: IGH sequences harboring motifs such as “GYYYFDY” or “CARGGY” and IGK sequences featuring the motif “CQQSSSYP” via a (GGGGS)3 linker. We then predicted the structures of these concatenated motifs using AlphaFold 3 and docked them with the AAL antigen using PDBePISA. The docking results demonstrated that these motifs were able to form hydrogen bonds and salt bridges with the AAL antigen, as illustrated in Fig. S6D, E and Tables S5–S6. These findings indicate that the identified motif sequences have the potential to bind to MPXV antigens.

Expression profiles of T cells in MPXV mRNA vaccine-treated mice

To identify alterations in T-cell subsets after vaccination, we subclustered T cells (11,474) from the spleen and identified 7 subsets on the basis of the expression and distribution of canonical T-cell markers (Figs. 7A, B and  S7A, B): naive CD4⁺ T cells (Cd4⁺Ccr7⁺Sell⁺), Th1 cells (Tbx21⁺Cd40lg⁺Ifng⁺), Th2 cells (Gata3⁺Maf⁺Ccr4⁺Cd44⁺), Th17 cells (Ccr6⁺Il23r⁺Rorc⁺Il17a⁺), CD8+ effector T cells (Cd8a⁺Cd8b1⁺Prf1⁺Lamp1⁺), Treg cells (Foxp3⁺Ctla4⁺Ikzf2⁺) and naive NK-T cells (Ccr7⁺Sell⁺Cd8a⁺Nkg7⁺Cd8b1⁺). The abundance of the Th1, Th2, Th17, and CD8 effector subsets increased in the AALI mRNA-vaccinated mice, although the differences were not statistically significant (p > 0.5), suggesting robust induction of the cellular immune response (Figs. 7C and  S7C). These T-cell composition changes align with findings from the ELISpot and flow cytometric assays.

A UMAP projection of T cells, with each dot representing a single cell and colored by cell type. B Violin plots depict the expression distribution of selected canonical cell markers across seven clusters, with marker genes in rows and clusters in columns. C Distribution of the average percentage of each immune cell type across the two groups. n = 3 biologically independent replicates. The data and error bars represent the means ± SDs. Statistical significance was calculated by unpaired two-sided Student’s t-tests. *, p < 0.05; **, p < 0.001; ns, p > 0.05. D GO terms are annotated with names and IDs, sorted by decreasing −log10 p values. DEGs denote genes with Benjamini‒Hochberg adjusted p values ≤ 0.05. E Volcano plot showing the DEGs between the AALI- and LNPs-treated samples. F Heatmap illustrating the expression of cytotoxic gene signatures in CD8⁺ T cells across the AALI- and LNPs-treated groups. The minimum signal is represented in blue, and the maximum signal is depicted in red. G Heatmap of the expression of the IFN response signature in T cells from all samples. The minimum signal is represented in blue, and the maximum signal is depicted in red. H Circos plots depicting the rearrangements of TCR chains in the two groups. Arc lengths reflect the relative frequency of VDJ gene families, whereas colored ribbons indicate the frequency of paired VDJ gene segments. I The CDR3 motifs for TRA and TRB chains that are unique to the AALI-treated group were predicted after sequences that were shared with the LNPs-treated group were excluded. The label on the x-axis represents the position of the CDR3 amino acid, and the y-axis represents the bits.

To characterize transcriptomic changes in T cells between the AALI-vaccinated groups and LNPs-treated groups, we compared the expression profiles of CD4⁺ and CD8⁺ T cells at 7 days after vaccination (Figs. 7D, E and  S7D, E). As expected, genes associated with “negative regulation by the host of viral transcription”, “defense response”, “regulation of T-cell differentiation” and “positive regulation of cell activation” were significantly enriched in CD4⁺ and CD8⁺ T cells after vaccination, indicating an ongoing host immune response against MPXV (Figs. 7E and  S7F–H). Genes associated with “regulation of cell motility”, “leukocyte migration involved in the inflammatory response”, and “positive regulation of peptide secretion” were enriched in CD8⁺ T cells (Fig. S7E). This enrichment suggests that CD8⁺ T cells may have a targeted killing effect on MPXV-infected cells. At the bulk cell level, we also observed increased expression of several typical cytotoxic genes, such as Nkg7, Gzma, Gzmb, Klrk1, Klrd1, Ctsw and Cst7, in effector-state T cells after vaccination (Fig. 7F). Similar to the GO and KEGG findings for B-cell subsets, genes associated with “NF-kappaB binding”, “antigen processing and presentation of peptide antigen”, and the “JAK-STAT signaling pathway” were enriched, indicating consistent activation of host immune responses after vaccination in splenic immune cells. Moreover, pathways involved in antiviral immune responses, such as “regulation of germinal center formation”, “regulation of B-cell differentiation”, and “positive regulation of the inflammatory response” were significantly enriched post-vaccination. Given the critical role of IFN in viral infection clearance, we compared a panel of proinflammatory and IFN-γ hallmark gene signatures in T cells (Fig. 7G). Our analyses revealed the activation of proinflammatory and IFN-γ response-related genes on day 7 post-vaccination, which was consistent with the enrichment of “cellular response to type II interferon” pathways identified in our GO analyses (Fig. 7D). In conclusion, our scRNA-seq analysis revealed a high degree of transcriptional specialization in both CD4⁺ and CD8⁺ T cells following vaccination. This finding corroborates the role of T cells in assisting B cells in producing antibodies and in the clearance of infected cells.

Clonal expansion of T cells and preferred usage of V(D)J genes after vaccination

Like B cells, T cells are also highly diverse, with each bearing a unique TCR that recognizes a specific antigen⁵⁴. Only a small subset of T cells can recognize antigens and undergo immune expansion, shifting from clone = 1 to clone ≥2. To elucidate the clonal relationships among T cells and analyze the utilization of V(D)J gene segments following vaccination, we performed TCR V(D)J sequencing and conducted a comprehensive analysis by integrating scRNA-seq with V(D)J-seq. In brief, more than 80% of the cells in all subsets had matched TCR information, except for the Cd8 effector and Th17 subsets (Fig. S7I). Notably, evident TCR clonal expansion (clone ≥2) was observed in the AALI-treated groups compared with the LNPs-treated groups (Fig. S7J, K). Moreover, the majority of the top 20 clone types were present in the vaccine-treated samples (Fig. S7L), suggesting that the mRNA vaccine could induce the amplification of AALI-specific T cells. To explore the dynamics and gene preferences of the TCR after vaccination, we compared the usage of V(D)J genes between the two groups. Notably, we observed a decrease in the diversity and distinct usage of V(D)J genes, particularly TRB genes, after vaccination (Fig. S7M). Compared with the LNPs-treated mice, the AALI-treated mice presented overrepresentation of Trav7d-2, Trav12n-2, Trbv13-3, and Trbv29, with Traj15 and Traj31 as the preferred TRAJ genes and Trbj1-1 as the preferred TRBJ gene (Fig. S8A). Additionally, the most distinct paired V‒J frequencies of TRA and TRB in the vaccine groups were Traj18/Trav11 and Trbvj2-4/Trbv12-2 + Trbv13-2, respectively (Fig. 7H). These findings suggest that AALI treatment elicits a unique TCR repertoire, potentially increasing the specificity and magnitude of the immune response to vaccination. To elucidate the differences in the TCR repertoire after immunization, we performed motif analysis of the CDR3 sequences of the TRA and TRB chains, which are critical elements for the identification of antigenic peptides, via MEME. The top five TRA and TRB motifs for each group were identified and ranked according to their sequence conservation and E values, as detailed in Table S7, and the results (E value < 0.05) are shown in Fig. S8B, C. Both the AALI- and LNPs-treated groups presented similar predominant motifs within their TCR repertoires, as shown in Table S7 for the AALI and LNPs groups, respectively. This observation indicates that these motifs may represent a baseline or conserved component of the TCR repertoire across treatment groups.

To specifically identify TCR clonotypes induced by the vaccine and minimize interference from the baseline immune repertoire, we excluded the common TCR sequences that were shared between the AALI- and LNPs-treated groups from the AALI-treated group (as shown in Table S7, labeled AALI-LNPs). This resulted in the unique motifs “CALSEG” and “GGNNKKLTF” in the TRA chain and “SSGGGGNT” in the TRB chain (Fig. 7I). The discovered motifs in the CDR3 sequences of the TCR after immunization suggest that the diversity of the vaccine-induced immune repertoire is increased and that these conserved amino acid regions in CDR3 play important roles in the immune response. Overall, increased T-cell clonality and skewed usage of TCR V(D)J genes post-vaccination suggest that AALI-associated V(D)J rearrangements occur in host T cells. Notably, the selective usage of dominant TRA and TRB genes, as well as the discovery of TRA and TRB motifs, indicate that various immunodominant epitopes may shape the molecular profile of T-cell responses, potentially aiding in vaccine design.