A single-dose inhalable rAAV-vectored vaccine against mpox virus

Zhouguang Jiao; Zhihui Zheng; Yue Xue; Hongjie Wang; Meng Zou; Peijie Qu; Guangzhi Zhang; Jiabo Ding; Yang Liu; Wenjun Liu; Lei Sun; Pengtao Jiao

doi:10.1016/j.xcrm.2026.103019

. 2026 Sep 15;7(9):103019. doi: 10.1016/j.xcrm.2026.103019

A single-dose inhalable rAAV-vectored vaccine against mpox virus

Zhouguang Jiao ^1,^2,^7,^∗, Zhihui Zheng ¹, Yue Xue ², Hongjie Wang ^2,³, Meng Zou ¹, Peijie Qu ¹, Guangzhi Zhang ⁴, Jiabo Ding ⁴, Yang Liu ⁵, Wenjun Liu ^5,⁶, Lei Sun ^6,^∗∗, Pengtao Jiao ^4,^∗∗∗

PMCID: PMC13589484 PMID: 42743913

Summary

In response to the global mpox outbreak, an inhalable vaccine platform based on rAAV/DJ vectors is developed. This platform represents a needle-free, single-dose, highly immunogenic defense against monkeypox virus (MPXV) and related orthopoxviruses (OPXVs). Two multivalent vaccines, MV-A (encoding M1R/A35R) and MV-B (encoding A29L/B6R), are engineered, and MV-A, MV-B, and their combination (MV-C) are assessed preclinically. The vector integrates the mucosal adjuvant cholera toxin subunit B (CTB) at the 3′ terminus of antigen genes, enabling the sustained production of self-assembled antigen-adjuvant nanoparticles. A single dose induces potent activation of antigen-presenting cells and pulmonary T cell responses, generating lung tissue-resident memory T cells. MV-C induces strong, durable MPXV-specific antibodies in mice and non-human primates, with sera and bronchoalveolar lavage fluid showing high neutralizing activity. Single pulmonary immunization with MV-C establishes a protective mucosal barrier in MPXV-susceptible CAST/EiJ mice and confers protection against OPXVs via respiratory or genital routes. These vaccines hold promise for combating future MPXV variants.

Keywords: mpox, rAAV vector, inhalable vaccine, mucosal adjuvant, mice, non-human primates

Graphical abstract

Highlights

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MV-C is an inhalable rAAV/DJ-based vaccine encoding MPXV antigens fused with adjuvant CTB
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Single-dose vaccination elicits durable antibodies with broad cross-neutralizing activity
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Pulmonary immunization induces mucosal antibodies and lung tissue-resident memory T cells
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MV-C protects against MPXV and VACV-WR challenge via respiratory and genital routes

Jiao et al. develop an inhalable, single-dose rAAV/DJ-vectored mpox vaccine. The optimal formulation, MV-C, elicits durable mucosal and systemic immunity, inducing neutralizing antibodies and tissue-resident memory T cells, and confers protection against broad orthopoxvirus challenge.

Introduction

Monkeypox virus (MPXV) is an enveloped virus with a double-stranded DNA genome that belongs to the Orthopoxvirus genus of Poxviridae family, together with variola virus (VARV), vaccinia virus (VACV), ectromelia virus (ECTV), and cowpox virus (CPXV). Mpox is a zoonotic viral disease that is caused by the MPXV. MPXV infection can cause the typical clinical symptoms including fever, headache, lymphadenopathy, myalgia, asthenia, and skin rash in humans.¹

Since the first human case of MPXV infection was reported in the Democratic Republic of the Congo in the 1970s,² MPXV has generally spread in West and Central Africa.³^,⁴^,⁵ However, in May 2022, a global mpox outbreak erupted in the UK and rapidly spread beyond its natural reservoirs in Africa to numerous non-endemic regions worldwide. This pandemic was driven by the MPXV clade IIb, which was the predominant strain and demonstrated enhanced human-to-human transmissibility.⁶ Consequently, mpox was declared a Public Health Emergency of International Concern (PHEIC) by the World Health Organization (WHO) in July 2022, and the PHEIC was discontinued in May 2023 as cases declined.⁷^,⁸ Recently, following a surge in cases in Africa and the emergence of the clade Ib subtype, the WHO reinstated mpox as a PHEIC on August 14, 2024. By April 30, 2026, over 184,531 cases of mpox and 506 deaths had been reported across 144 countries since 2022.⁸ Vaccination is the most effective strategy for controlling and preventing transmissible diseases. Currently, three live vaccines (MVA-BN [Jynneos], LC16m8, and ACAM2000) are available for vaccination against MPXV infection. All of these three vaccines are developed on the base of live attenuated VACV, which shares a high amino acid identity with MPXV in terms of surface antigens.⁹ Persistent challenges regarding the long-term efficacy, safety profiles, production capacity, and supply chain limitations of existing vaccines continue to hinder global accessibility and impede the response to the ongoing global mpox outbreak.¹⁰^,¹¹^,¹²^,¹³^,¹⁴^,¹⁵ Therefore, the development of novel, potent, durable, and safe mpox vaccines is essential for the prevention and control of current transmissible mpox.

MPXV has two distinct infectious forms, the intracellular mature virus (IMV) and the extracellular enveloped virus (EEV), each of which has a unique set of surface antigens.¹⁶ Previous studies have identified candidate antigens of MPXV for vaccine development, including M1R (homologous to VACV L1R) and A29L (homologous to VACV A27L) in IMV, and A35R (homologous to VACV A33R) and B6R (homologous to VACV B5R) in EEV.¹⁷^,¹⁸ These antigens have been widely used for developing vaccines against poxvirus.¹⁹^,²⁰^,²¹ Compared with vaccines based on single antigen, combination of these antigens has demonstrated significantly improved protection against poxviruses in mice or monkeys, particularly when combining antigens from both IMV and EEV forms.²²^,²³^,²⁴^,²⁵^,²⁶

Adeno-associated viruses (AAVs), a genus of the non-enveloped, single-stranded DNA family Parvoviridae (subfamily Parvovirinae, genus Dependoparvovirus), are widely utilized as gene delivery vectors in gene therapy due to their high transduction efficiency, low immunogenicity, and sustained transgene expression.²⁷ Based on the above properties, AAVs have been applied for the development of safe and long-acting vaccines.²⁸^,²⁹^,³⁰ Given that mucosal contact and inhalation of virus-containing bioaerosols are critical pathways for MPXV human-to-human transmission,³¹^,³² and that the mucosal immune system in the body is interconnected (common mucosal immune system),³³ this has prompted our interest in developing a single-dose, inhalable mucosal vaccine against mpox using AAV viral vectors, which would contribute to the establishment of systemic mucosal immune barriers in the host.

In this study, we constructed inhalable candidate mpox vaccine A (MV-A) and mpox vaccine B (MV-B), which are based on the recombinant AAV/DJ (rAAV/DJ) vector, a chimeric AAV2/AAV8/AAV9 vector known for its broad tissue tropism and enhanced gene delivery efficiency.³⁴^,³⁵ The vaccines were also formulated with the mucosal adjuvant cholera toxin subunit B (CTB) to enhance mucosal immune responses.³⁶ Given that MPXV exists in two distinct forms (IMV and EEV), each vaccine contains two antigenic genes, with each antigenic gene derived from a different viral form. Specifically, MV-A contains a gene encoding the fusion protein M1R-A35R-CTB and MV-B contains a gene encoding the fusion protein A29L-B6R-CTB. Additionally, to identify the optimal candidate inhalable mpox vaccine, this study evaluated the immune responses and protective efficacy induced by MV-A, MV-B, and their combined formulation (MV-C), to assess synergistic immune responses. Our findings indicate that a single-dose pulmonary immunization (i.e., intratracheal delivery, so termed to emphasize pulmonary deposition) with MV-C elicited strong immune responses in rodents and non-human primates (NHPs). Notably, this vaccination established a mucosal immune barrier in mice effectively protecting against poxvirus challenge via either the respiratory or genital tract routes. Collectively, the results in this study demonstrate that the rAAV vector-based inhalable mpox vaccine offers the advantages of favorable biosafety, ease of administration, and long-lasting immunogenicity. This vaccine platform can serve as a countermeasure against both the current outbreak and potential future resurgence of mpox.

Results

Construction and characterization of rAAV/DJ-vectored mpox vaccines

To develop inhalable mpox vaccines with strong mucosal immunogenicity and potent immune protection, candidate vaccines MV-A and MV-B were constructed by incorporating antigens encoded with the N-terminal signal peptide tPA and the C-terminal mucosal adjuvant CTB³⁶ into the rAAV/DJ vector (Figure 1A). Vaccine MV-A expressed antigen-adjuvant fusion proteins of M1R-A35R-CTB, whereas MV-B expressed antigen-adjuvant fusion proteins of A29L-B6R-CTB. The four antigens corresponded to the following amino acid residues: M1R (1–185), A35R (58–181), A29L (1–110), and B6R (21–275). To enhance the stability of the expressed fusion antigens, the antigenic proteins lacked transmembrane domains and intracellular components. In addition, to prevent structural disruption induced by unpaired cysteine residues, the following mutations were introduced: Cys62Ala (C62A) in A35R and Cys140Glu (C140E) in B6R (Figure 1B).

Subsequently, the expressions of the four antigenic proteins were analyzed in culture supernatants and lysates from A549 cells infected with MV-A or MV-B, both of which expressed the antigens of expected molecular weight (Figure S1A). Given CTB’s inherent ability to self-assemble into pentamers, these expressed antigenic proteins were further evaluated using native polyacrylamide gel electrophoresis. As designed, both MV-A and MV-B produced fusion proteins that self-assembled into pentameric complexes of the expected molecular weight (Figure 1C). Morphologically, transmission electron microscopy (TEM) revealed the formation of well-dispersed antigen-adjuvant nanoparticles (NPs), resembling the pentameric structure predicted using AlphaFold 2 (Figure 1D).

To determine the in vivo biodistribution and duration of the expressed antigens following pulmonary delivery of the candidate rAAV/DJ-based mpox vaccine, an rAAV/DJ vector with reporters luciferase and green fluorescent protein (GFP) (rAAV/DJ-Luc-GFP) was employed as a model viral vector. In vivo bioluminescence imaging and fluorescence microscopy of the tissue sections confirmed reporter expression in the lungs following pulmonary delivery of rAAV/DJ-Luc-GFP (Figures S1B–S1D). The high levels of luciferase expression persisted in the lung tissues for up to 1 year after administration (Figure 1E). These findings indicate that the rAAV/DJ-based mpox vaccines developed in this study express antigen-adjuvant NPs for extended periods in vivo.

Single-dose rAAV/DJ-based mpox vaccines elicited high and durable antibody responses

The abovementioned findings prompted an investigation of antigen-specific and neutralizing antibody responses induced by a single dose of the mpox vaccine. Prior to this assessment, the biosafety of MV-A, MV-B, and MV-C (an equal mixture of MV-A and MV-B) was first assessed. Immunization of BALB/c mice with these three vaccines via pulmonary delivery at a dose of 1 × 10¹¹ viral genomes (vg) per mouse did not induce considerable hematological alterations (Figure S2A). Furthermore, obvious histopathological lesions were not observed in the examined organs of the heart, liver, spleen, lung, or kidney from any immunized mice (Figure S2B). Moreover, the plasma sC5b-9 levels remained unchanged after immunization (Figure S2C).

After confirming the high safety profile of in vivo immunization, vaccine-induced antibody responses in BALB/c mice were subsequently evaluated (Figure 2A). All three vaccines induced antigen-specific IgG, IgG1, and IgG2a antibodies in the serum within 14 days of immunization. Peak titers were achieved by 42–56 days, which remained high throughout the 1-year monitoring period. No significant decline was observed in any vaccine group (Figure 2B; Figures S3A and S3B). Higher antibody levels were consistently maintained in the MV-C group when compared with those in the MV-A and MV-B groups. Specifically, MV-C induced higher levels of antigen-specific IgG2a antibodies than MV-A (Figure S3B), implying a more potent Th1-biased humoral response. Further analysis of the IgG2a/IgG1 ratio (a marker for Th1/Th2 bias) indicated that both MV-B and MV-C elicited strong Th1-biased antibody responses. Their IgG2a/IgG1 ratios exhibited a biphasic trend, initially decreasing to a nadir by 70 days after immunization or earlier, followed by a gradual increase (Figure 2C). This finding suggests that MV-B and MV-C induced Th1-biased humoral responses in both the early and late phases of immunization. In contrast, IgG2a/IgG1 ratios were low in MV-A (<1.0) throughout the study.

Single-dose rAAV/DJ-based mpox vaccines elicited high and durable antibody responses in BALB/c mice

(A) Schematic illustration of immunization and sampling in mice.

(B) Serum IgG titers against MPXV antigens determined by ELISA on days 0, 14, 28, 42, 70, 126, 210, 273, and 365 post-immunization (n = 6). The horizontal dotted line indicates the limit of detection.

(C) Heatmap visualization of the IgG2a/IgG1 titer ratio. The label for each row represents vaccines and coated antigens for ELISA.

(D) BALF IgA and IgG titers against MPXV antigens determined by ELISA on day 21 post-immunization (n = 5).

(E) Measurement of 50% microneutralization titer (MN₅₀) against VACV-VTT in the serum (n = 6) and BALF (n = 5), with sampling on day 70 for serum and day 21 for BALF post-immunization. The horizontal dotted line indicates the limit of detection.

(F) Measurement of MN₅₀ against MPXV in the serum (n = 6) and BALF (n = 5), with sampling on day 70 for serum and day 21 for BALF. The horizontal dotted line indicates the limit of detection.

Data in (B) are shown as mean ± SEM. Data in (D) are shown as mean ± SEM, and statistical significance was assessed using the Mann-Whitney test. Data in (E) and (F) are shown as mean ± SEM, and statistical significance was assessed using the Kruskal-Wallis test with Dunnett’s multiple comparison test. ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; ns, not significant. See also Figures S2 and S3.

To determine mucosal immune responses, a separate cohort of mice was vaccinated. Bronchoalveolar lavage fluid (BALF), nasal lavage fluid (NLF), oral lavage fluid (OLF), and genital lavage fluid (GLF) were collected on day 21 after immunization. High levels of MPXV antigen-specific IgA as well as IgG were detected in the BALF from all three mpox vaccine-immunized mice (Figure 2D). High levels of MPXV antigen-specific IgA were also detected in the NLF, OLF, and GLF from immunized mice (Figure S3C). Furthermore, neutralizing antibody titers in the serum and BALF of immunized mice were measured against a panel of live orthopoxviruses (OPXVs) using standard neutralization assays. Compared with the Sham group (empty rAAV/DJ vectors), the serum and BALF from vaccinated mice exhibited significantly higher neutralizing activities against VACV Tian Tan strain (VACV-VTT), VACV Western Reserve (VACV-WR), ECTV, and MPXV, indicating that all three vaccines induced the production of antibodies with a broad antiviral spectrum (Figures 2E and 2F; Figures S3D and S3E). The neutralizing titers of the MV-B group against MPXV were slightly higher than those of the MV-A group. The MV-C group consistently exhibited the highest neutralizing activity in both the serum and BALF against all tested strains, highlighting its superior mucosal and systemic protective potential. Moreover, mouse serum collected 1 year after immunization retained full neutralizing activity against VACV-VTT, VACV-WR, and ECTV (Figure S3F), consistent with the sustained long-lasting antigen-specific antibody responses (Figure 2B; Figures S3A and S3B). Notably, at 1 year after MV-C immunization, high levels of MPXV antigen-specific antibodies could still be detected in the BALF, NLF, OLF, and GLF of mice, and the BALF still exhibited high neutralizing activity against VACV-WR, VACV-VTT, and ECTV (Figures S3G–S3I). This result further underscores the long-term protective potential of the vaccines, particularly MV-C.

Single-dose rAAV/DJ-based mpox vaccines elicited comprehensive immune cell responses

Encouraged by the substantial and durable antibody responses, the pulmonary immune cell responses induced by the developed mpox vaccines were further investigated. Given the superior immunoprotective potential of MV-C observed above, its cellular tropism was initially analyzed by single-cell RNA sequencing (scRNA-seq) 21 days after respiratory inoculation (Figure S4). The intention was to comprehend the in vivo infection and expression dynamics of the rAAV/DJ vector used in vaccine development. MV-C predominantly infected macrophages, accounting for approximately 65% of MV-C-expressing cells, as inferred from transcriptomic analysis (Figures S4C and S4D). Moreover, macrophages exhibited the most pronounced transcriptional alterations, with nearly 2,000 differentially expressed genes (DEGs) identified compared with MV-C-negative cells (Figure S4E).

Owing to the critical roles of antigen-presenting cells (APCs) in initiating the adaptive immune response, APC activation was next assessed on day 21 post-immunization using flow cytometry. Compared with the Sham group, CD86, a crucial co-stimulatory molecule, was markedly upregulated in both dendritic cells (DCs) and macrophages across all vaccinated groups. The most pronounced increase was observed in the MV-C group (Figure 3A). scRNA-seq data corroborated APC activation, revealing substantial enrichment of pathways associated with “cell activation,” “lymphocyte activation,” and “immune response” in DCs and macrophages from MV-C-immunized mice (Figures S5A and S5B). Furthermore, transcriptomic profiling showed elevated expression of a panel of genes vital for APC function, which included co-stimulatory molecules (e.g., Cd86, Cd80, and Cd40) and homing-related genes (e.g., Ccr7, Cd74, and H2-Ab1) (Figures S5C and S5D). This finding underscores MV-C’s ability to drive strong APC activation and migration.

Single-dose rAAV/DJ-based mpox vaccines induced comprehensive immune cell responses in pulmonary tissues (containing mediastinal lymph nodes)

(A) CD86 expression on DCs and macrophages in the lungs of immunized mice (n = 4).

(B) The proportion of naive CD4⁺ T cells (CD4⁺CD44⁻CD62L⁺) and CD4⁺ tissue-resident memory T (Trm) cells (CD4⁺CD44⁺CD69⁺CD11a⁺) among CD4⁺ T cells in the lung on day 21 post-immunization (n = 4).

(C) The proportion of naive CD8⁺ T cells (CD8⁺CD44⁻CD62L⁺) and CD8⁺ Trm cells (CD8⁺CD44⁺CD69⁺CD103⁺) among CD8⁺ T cells in the lung on day 21 post-immunization (n = 4).

(D) The proportion of germinal center B (GC B) cells (CD19⁺GL7⁺IgD⁻) among CD19⁺ cells in the lung on day 21 post-immunization (n = 4).

(E) The proportion of IFN-γ⁺ Trm cells with or without MPXV antigen stimulation among CD8⁺ T cells in the lung on day 70 post-immunization (n = 4).

(F) Multifunctional CD8⁺ T cells in the lung on day 70 post-immunization (n = 4).

(G) The proportion of plasma cells among CD45⁺ cells in the lung on day 70 post-immunization (n = 4).

Data in (B) (CD4⁺ naive T cells), (C), (D), (E) (except for the PBS-treated samples in the left panel), (F), and (G) are shown as mean ± SEM, and statistical significance was assessed using one-way ANOVA with Dunnett’s multiple comparison test. Data in (B) (right) and (E) (the PBS-treated samples in the left panel) are shown as mean ± SEM, and statistical significance was assessed using the Kruskal-Wallis test with Dunnett’s multiple comparison test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; ns, not significant. See also Figures S4–S9.

As the terminal effectors of adaptive immunity, lymphocyte responses in the respiratory tract were further assessed during the antibody-elevation phase (day 21 post-vaccination) following mpox vaccination. Flow cytometric analysis of T cells revealed that differentiation was enhanced after vaccination, with decreased proportions of CD4⁺ naive and CD8⁺ naive T cells and increased proportions of CD4⁺ and CD8⁺ tissue-resident memory T (Trm) cells (Figures 3B and 3C). The highest percentage of CD8⁺ Trm cells was present in the MV-C group (Figure 3C). The scRNA-seq data further demonstrated MV-C-mediated CD8⁺ T cell activation, showing significant mobilization of effector and/or memory T cells, with upregulated expression of genes including Ifng and Gzmb (Figures S6A–S6C). Parallel B cell analysis indicated elevated frequencies of germinal center B cells (GC B cells) after immunization, with the GC B cell frequency of MV-C being the highest (Figure 3D). Consistent with these findings, light-sheet imaging and immunofluorescence of mediastinal lymph nodes (mLNs) revealed vaccine-driven lymphoid expansion (Figure S7A) and high levels of B cell proliferation markers (Figure S7B) in the MV-C group.

Integrated analysis of scRNA-seq and single-cell BCR immune repertoire sequencing (scBCR-seq) data further demonstrated MV-C’s strong activation of B cells (Figures S6D–S6G and S8). Mechanistically, GC B cells exhibited upregulated expression of differentiation-associated genes, including Ighg1 and Aicda (Figure S6F), which encode key drivers of recombination and somatic hypermutation.³⁷ Consistently, B cells expressing the IgG1 antibody isotype were generated during GC differentiation (Figure S8B). In addition, plasma cell differentiation might produce numerous antigen-driven “larger clonotypes” (defined as 21–100 cells sharing the same clonotype across samples)³⁸ owing to clone expansion and differentiation (Figure S8C). Two immunodominant clonotypes, IGH: CVRLGAYW and IGH: CARGEAWFAYW, emerged in plasma cells as a result of antigen-driven differentiation and selection processes under MV-C stimulation (Figures S8D–S8E). This finding highlights the vaccine’s ability to induce repertoire selection of focused B cells.

Furthermore, to assess vaccine-induced MPXV antigen-specific cellular immune responses, respiratory lymphocyte responses during the antibody plateau phase (70 days post-immunization) were determined using flow cytometry. All three candidate vaccines elicited significantly higher levels of CD4⁺ and CD8⁺ Trm cells than the Sham group. Restimulation with vaccine-encoded antigens ex vivo further augmented Trm cell differentiation, confirming their antigen specificity (Figures S9A and S9B). The efficacy of MV-C was consistently the highest. Moreover, MV-C induced the generation of interferon gamma (IFN-γ)-secreting CD8⁺ Trm cells (Figure 3E) and multifunctional CD8⁺ T cells capable of secreting IFN-γ and/or tumor necrosis factor alpha (TNF-α) (Figure 3F). These findings highlight MV-C’s superior capacity in driving cellular immunity. In addition, when compared with the Sham group, higher levels of plasma cells (Figure 3G) and GC B cells were maintained in the vaccinated groups (Figure S9C). This observation underscores the critical role of these vaccines in eliciting potent humoral immune responses. Of note, this type of vaccine-elicited immune response may not be identical in CAST/EiJ mice, which are susceptible to MPXV infection.

Single-dose MV-C elicited immune responses in NHPs

These findings collectively highlight MV-C as a potent vaccine that elicits both cellular and humoral immune responses against MPXV. To enhance its clinical relevance, NHPs were immunized with MV-C at a dose of 1 × 10¹² vg per animal (Figure 4A). High levels of MPXV antigen-specific antibody responses were detected in the serum (Figure 4B), lasting for at least 120 days. Serum collected 6 weeks post-immunization exhibited cross-neutralizing activity against a panel of OPXV species, including VACV-VTT, VACV-WR, ECTV, and MPXV (Figure 4C). Antigen-specific IgA and IgG were also detected in the BALF at 3 weeks after immunization (Figure 4D), and these levels correlated with strong mucosal neutralizing activity against MPXV (Figure 4E). These findings demonstrate MV-C’s ability to evoke systemic and mucosal immunity.

Single-dose rAAV/DJ-based mpox vaccines elicited high levels of antibody responses in nonhuman primates

(A) Schematic illustration of immunization and sampling in cynomolgus monkeys.

(B) Serum IgG titers against MPXV antigens determined by ELISA on days 0, 14, 28, 42, 56, and 120 post-immunization (n = 3).

(C) Measurement of MN₅₀ against VACV-VTT, VACV-WR, ECTV, and MPXV in the serum, with sampling on day 42 post-immunization (n = 3). The horizontal dotted line indicates the limit of detection.

(D) BALF IgA and IgG titers against MPXV antigens determined by ELISA on day 21 post-immunization (n = 3). The horizontal dotted line indicates the limit of detection.

(E) Measurement of MN₅₀ against MPXV in the BALF, with sampling on day 21 post-immunization (n = 3). The horizontal dotted line indicates the limit of detection.

Data in (B)–(E) are shown as mean ± SEM. The statistical significance in (C) and (E) was assessed using Mann-Whitney test. ∗p < 0.05.

Single-dose MV-C-vaccinated mice are protected from VACV-WR

As protective efficacy is a critical determinant for clinical translation, the immunoprotective capacity of the mpox vaccines developed here against VACV-WR in BALB/c mice was next evaluated. In the first cohort, in addition to four groups receiving pulmonary immunization (Sham, MV-A, MV-B, and MV-C), a group undergoing tail scarification with VACV-VTT was included as a positive control to benchmark the efficacy of the rAAV/DJ-based vaccines (Figure 5A). Five weeks after immunization, all mice were intranasally challenged with 1 × 10⁵ plaque-forming units (PFUs) of VACV-WR per mouse and euthanized 6 days post-infection (dpi). Progressive weight loss was noted in mice from the Sham group, with 4 of 5 mice exceeding the 25% weight-loss humane endpoint by 6 dpi. In contrast, in all vaccinated groups, the weight loss was transient, followed by recovery (Figure 5B). Compared with the Sham group, significantly lower levels of VACV-WR genomic copies were detected in the lungs of all vaccinated groups except MV-A. The viral burden was the lowest in the MV-C group (Figure 5C). Infectious VACV-WR was undetectable in the lungs of both MV-C- and VACV-VTT-immunized mice (Figure 5D).

Single-dose MV-C-vaccinated BALB/c mice are protected from VACV-WR

(A) Schematic illustration of immunization and intranasal VACV-WR challenge in mice.

(B) Weight loss of mice following intranasal VACV-WR challenge (n = 5).

(C) Viral genome copies in lung tissues determined by quantitative PCR (qPCR) at 6 days post-infection (dpi) (n = 5).

(D) Infectious viral load in lung tissues determined by plaque assay at 6 dpi (n = 5). The horizontal dotted line indicates the limit of detection.

(E) Schematic illustration of immunization and vaginal VACV-WR challenge in mice.

(F) Weight loss of mice following vaginal VACV-WR challenge (n = 5).

(G) Ovary viral genome copies determined by qPCR at 7 dpi (n = 5).

(H) Lung viral genome copies determined by qPCR at 7 dpi (n = 5).

(I) Ovary infectious viral load determined by plaque assay at 7 dpi (n = 5). The horizontal dotted line indicates the limit of detection.

(J) Lung infectious viral load determined by plaque assay at 7 dpi (n = 5). The horizontal dotted line indicates the limit of detection.

(K) Histopathology analysis of spleen tissue sections at 7 dpi. Scale bar, 100 μm.

Data in (B) are shown as mean ± SD and analyzed as area under the curve (AUC) using one-way ANOVA with Tukey’s test. Data in (C) and (D) are shown as mean ± SEM, and statistical significance was assessed using the Kruskal-Wallis test with Tukey’s test. Data in (F) are shown as mean ± SD and analyzed as AUC using t tests. Data in (G) and (H) are shown as mean ± SEM, and statistical significance was assessed using t tests. Data in (I) and (J) are shown as mean ± SEM, and statistical significance was assessed using Mann-Whitney test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; ns, not significant. See also Figures S10.

Furthermore, the protective efficacies of a lipid NP (LNP)-encapsulated quadrivalent mRNA mpox vaccine (LNP-MV) and MV-C were compared in another cohort of BALB/c mice. Compared with the two-dose LNP-MV regimen, a single MV-C immunization induced higher levels of MPXV antigen-specific antibodies in both the serum and BALF (Figures S10A–S10D). After intranasal challenge with VACV-WR (Figure S10E), the best protective efficacy was observed in the MV-C group, as evidenced by minimal body weight loss, undetectable infectious virus in the lungs, and minimal lung histopathological alterations (Figures S10F–S10H).

In addition, given the current significance of sexual transmission in the spread of OPXVs,³⁹ the protective efficacy of pulmonary MV-C immunization against vaginal VACV-WR challenge was next evaluated in a separate mouse cohort (Figure 5E). The findings indicated that mice in the Sham group exhibited progressive weight loss after the challenge, with 1 of 5 mice reaching the humane endpoint by day 7. In contrast, body weight was maintained in the MV-C-immunized mice without significant loss, and they survived the challenge (Figure 5F). Quantitative analysis of viral load at 7 dpi revealed significantly lower viral genomic copies in both ovarian and lung tissues of MV-C-immunized mice (Figures 5G and 5H). Critically, infectious VACV-WR was undetectable in ovarian and lung tissues of MV-C-immunized mice (Figures 5I and 5J). Histopathological examination of the spleen tissues confirmed the protective effect, with no significant pathological alterations in the MV-C group (Figure 5K). These data demonstrate that MV-C vaccination via pulmonary delivery elicits protective immunity across mucosal and systemic compartments, providing broad protection against diverse transmission modes of OPXVs.

Single-dose MV-C-vaccinated mice are protected from MPXV

To further evaluate the protective efficacy of rAAV/DJ-vectored mpox vaccines against authentic MPXV, the investigations were extended to MPXV-susceptible CAST/EiJ mice. In addition to four pulmonary-immunized groups (Sham, MV-A, MV-B, and MV-C), an intramuscular MV-C group (MV-C-i.m.) was included to facilitate the comparison of immunization routes. At 2 and 4 weeks after immunization, serum samples were collected for analyzing MPXV-specific antibodies. Five weeks after immunization, all mice were challenged with MPXV (clade IIb, WIBP-MPXV-001) via dual routes (intranasal, 6 × 10⁴ PFU; intraperitoneal, 2.4 × 10⁵ PFU; total 3 × 10⁵ PFU per mouse) and were euthanized at 6 dpi (Figure 6A). MV-C immunization induced high-titer MPXV-specific antibodies before the viral challenge. No significant differences were observed between the pulmonary and intramuscular routes of immunization (Figures 6B and 6C; Figures S11A and S10B). After the challenge, the Sham and MV-A groups gained weight progressively, whereas the MV-B, MV-C, and MV-C-i.m. groups maintained stable body weights (Figure 6D).

Single-dose MV-C-vaccinated CAST/EiJ mice are protected from MPXV

(A) Schematic illustration of immunization, sampling, and MPXV challenge in mice.

(B) Serum IgG titers against A35R and M1R determined by ELISA on days 14 and 28 post-immunization (n = 6–9).

(C) Serum IgG titers against B6R and A29L determined by ELISA on days 14 and 28 post-immunization (n = 6–9).

(D) Weight loss of mice following MPXV challenge (n = 6–9).

(E) Lung viral genome copies determined by qPCR at 6 dpi (n = 6–9). The horizontal dotted line indicates the limit of detection.

(F) Ovary viral genome copies determined by qPCR at 6 dpi (n = 6–9). The horizontal dotted line indicates the limit of detection.

(G) Lung infectious viral load determined by 50% tissue culture infectious dose (TCID₅₀) assay at 6 dpi (n = 6–9). The horizontal dotted line indicates the limit of detection.

(H) Ovary infectious viral load determined by TCID₅₀ assay at 6 dpi (n = 6–9). The horizontal dotted line indicates the limit of detection.

(I) Histopathology analysis of lung tissues at 6 dpi. Scale bar, 100 μm.

(J) Principal component analysis showing the antibody and viral load profiles across pulmonary-immunized groups (n = 6–8). Confidence ellipses represent 68% confidence intervals for group distributions.

Data in (B) and (C) are shown as mean ± SEM, and statistical significance was assessed using Kruskal-Wallis test with Dunnett’s multiple comparison test. Data in (D) are shown as mean ± SD. Data in (E)–(H) are shown as mean ± SEM, and statistical significance was assessed using Kruskal-Wallis test with Tukey’s test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; ns, not significant. See also Figure S11.

Virological analysis demonstrated that, compared with the Sham group, all vaccinated groups except MV-A showed a significant reduction in MPXV genomic copies in the lungs and ovaries (Figures 6E and 6F). Notably, viral genomes were undetectable in the lungs of MV-C and MV-C-i.m. mice, with MV-C showing the lowest ovarian viral burden. Furthermore, infectious MPXV was significantly reduced in the lungs of all vaccinated groups (Figure 6G) and in the ovaries of the MV-B, MV-C, and MV-C-i.m. groups (Figure 6H). Sterilizing immunity (undetectable virus) was achieved in both tissues of the MV-C and MV-C-i.m. groups. Consistent with the virological data, diffuse vacuolar degeneration and extensive megakaryocyte infiltration were observed in the histopathological analysis of spleens from the Sham group. The lesions were markedly milder in the MV-A and MV-B groups, while no significant pathology was noted in the MV-C and MV-C-i.m. groups (Figure S11C). Pulmonary histopathology indicated severe damage in the Sham group. Focal alveolar wall thickening was observed in the MV-A, MV-B, and MV-C-i.m. groups, while significant histopathological changes were almost absent in the MV-C group (Figure 6I). These observations are consistent with virological data indicating that pulmonary MV-C vaccination provides superior protection against MPXV. Moreover, principal component analysis (PCA) of MPXV antigen-specific antibody levels (week 4 post-immunization) and viral load (6 dpi) from pulmonary-immunized groups (Sham, MV-A, MV-B, and MV-C) confirmed MV-C’s superior protective effect. In the PCA space, MV-C formed a distinct cluster, separated from the other groups, exhibiting the maximal Euclidean distance from Sham (Figure 6J). To evaluate the contributions of MPXV antigen-specific antibodies induced by pulmonary immunization to protective immunity, the mice were stratified into four categories based on viral load: healthy, mild infection, moderate infection, and severe infection. Antigen-specific antibody isotypes were ranked by their importance in determining health status using random forest analysis (Figure S11D). Antibodies against the IMV-associated antigens M1R and A29L were identified as the top predictors of protection against MPXV, suggesting their critical roles in immune defense.

Discussion

In addition to the VACV-based attenuated live vaccines already approved for clinical use, mpox vaccine candidates are being developed at various stages of preclinical or clinical studies, including protein subunit vaccines⁴⁰^,⁴¹ and mRNA vaccines.¹⁷^,⁴²^,⁴³^,⁴⁴^,⁴⁵ Safety, high efficacy, and long-lasting protection are the key factors in developing an ideal mpox vaccine. Certain MPXV antigens, including the EEV antigens A35R and B6R and the IMV antigens M1R, A29L, E8L, and H3L, have been shown to induce neutralizing antibodies. The rAAV/DJ vector is a promising tool for gene therapy owing to its broad tropism and efficiency. In this study, three inhalable candidate mpox vaccine formulations, MV-A, MV-B, and MV-C, were designed and developed using this vector. Furthermore, immune responses and protective efficacy induced by these vaccines against OPXVs were systematically evaluated, and the potential protective mechanism was elucidated.

Notably, the mpox vaccines developed in this study are not a simple repetition of previous work. Instead, their development is a crucial attempt to formulate an inhalable mpox vaccine and is based on our previous design of an inhalable dry powder vaccine against COVID-19,³⁶ incorporating structure-based design strategies. As reported previously, full-length antigenic proteins might lead to poor induction of specific antibodies and reduced immune protection.¹⁷^,⁴² Therefore, for the antigen design, only the soluble fragments of the antigenic proteins were selected to preserve their antigenicity and stability. Removal of free cysteine residues reduces the likelihood of covalent disulfide bond formation with other proteins, thereby mitigating the risk of compromising the spatial structure of the antigenic proteins. Specific point mutations were further introduced to replace the free cysteine residues at position 62 of A35R (C62A) and position 140 of B6R (C140E). Finally, the self-assembling mucosal adjuvant CTB was introduced at the C terminus of the dual-antigenic protein, enabling the expressed fusion antigen-adjuvant protein to self-assemble post-translationally into antigen-adjuvant NPs. This strategy of vaccine construction leverages the advantage of nano-vaccines inducing potent immune response.⁴⁰ Indeed, data in this study have demonstrated the strong capability of the inhalable mpox vaccine to induce mucosal, humoral, and cellular immune responses, particularly for the MV-C vaccine formulation.

Given the innovative design of our vaccine, an extreme dual-route challenge model was employed to evaluate protection against MPXV. In this study, CAST/EiJ mice were simultaneously challenged with MPXV via the intranasal and intraperitoneal routes, simulating a clinical scenario in which multiple organs are concurrently exposed to MPXV under extreme conditions. The MV-C vaccine provided potent immune protection against the challenge, even when the challenge was administered via both routes, and effectively prevented the systemic dissemination of MPXV. Unexpectedly, the Sham and MV-A groups (which exhibited more severe disease) showed mild increases in body weight after infection, a finding that appears to be linked to MPXV-induced peritoneal effusion. In fact, during necropsy, peritoneal effusion was observed exclusively in these two groups. Substantial fluid was present in the peritoneal cavity of all Sham group mice, while visible effusion was noted in some MV-A group mice. The absence of weight loss, or even weight gain, in clade II MPXV-challenged CAST/EiJ mice has been documented in published studies.⁴⁶^,⁴⁷^,⁴⁸ Hence, this phenomenon may be a typical feature of MPXV infection in these mice. Furthermore, this study revealed a difference in antibody-mediated protection between antigens from IMV and EEV. Antibodies against IMV M1R/A29L provided greater protection against MPXV than those against EEV A35R/B6R, which is consistent with the results of a previous study on mRNA MPXV vaccines,⁴⁴ potentially reflecting underlying virological mechanisms governing MPXV pathogenesis in vivo.

In summary, an inhalable mpox vaccine was developed in this study using the rAAV/DJ platform, and MV-C was identified as the optimal vaccine formulation. A single pulmonary immunization with MV-C conferred potent protection against respiratory and genital tract challenges with multiple OPXVs by establishing a mucosal immune barrier. Given the critical role of CD8⁺ Trm cells in antiviral immunity and in viral clearance,⁴⁹ the high levels of CD8⁺ Trm induced by MV-C likely played a crucial role in the vaccination-established mucosal immune barrier. The vaccines developed in this study hold promise for deployment against future potential monkeypox variants exhibiting enhanced respiratory transmissibility. Furthermore, the potent systemic antibody responses observed after intramuscular administration of MV-C indicate its potential for broader application; future studies will focus on optimizing this route. Crucially, prolonged gene expression of the rAAV/DJ vector enables durable immunity after a single-dose immunization, eliminating the need for boosters. This needle-free and self-administrable immunization enhances vaccine accessibility, particularly in low-resource regions. Vaccination rates can, therefore, be increased, strengthening the prevention and control of mpox outbreaks at the community level. Finally, this platform can be translated to develop potent vaccines against other infectious diseases.

Limitations of the study

In this study, the protective efficacy of the vaccine was evaluated only in rodents, specifically BALB/c and CAST/EiJ mice. Moreover, the challenge experiments were conducted using MPXV clade IIb; the efficacy of this vaccine against a more virulent MPXV clade, such as clade Ib in CAST/EiJ mice,⁵⁰ remains to be determined. Further assessment in NHPs would provide more clinically relevant data on protective efficacy. In addition, the rectal route represents another important route of exposure in the sexual transmission of MPXV. Therefore, it is important to assess the protective efficacy of the vaccine against MPXV infection via the rectal route. Last, although neutralizing antibodies in mouse sera persist for more than 1 year post-immunization, whether the vaccine confers durable long-term protection in vivo warrants examination in future studies. Furthermore, whether this vaccine could induce immune tolerance in humans warrants discussion, although no evidence of immune tolerance was observed in mice.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Zhouguang Jiao (jiao_zg@126.com).

Materials availability

All reagents generated in this study are available from the lead contact with a completed materials transfer agreement.

Data and code availability

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The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive⁵¹ in National Genomics Data Center,⁵² China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences, (GSA: CRA028032) and are publicly accessible at https://ngdc.cncb.ac.cn/gsa/browse/CRA028032. All summary datasets supporting the findings of this study are contained within the article and its supplemental information.
•
This study does not report original code.
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All other raw data underlying the findings of this study are available from the corresponding author upon reasonable request.

Acknowledgments

This work was supported by Beijing Natural Science Foundation (no. L232068), the Shenzhen Medical Research Fund (no. E24010010), the National Natural Science Foundation of China of China (nos. 82402134 and 82241082), the Fundamental Research Funds for the Central Universities (no. 3332024071), CAMS Innovation Fund for Medical Sciences (CIFMS) (no. 2021-I2M-1-036), and the National Science and Technology Infrastructure of China (project no. National Pathogen Resource Center-NPRC-32). We acknowledge the Wuhan Institute of Biological Products Co., Ltd. for sharing the clade IIb MPXV. We acknowledge the support provided by Professor Lingjun Zhan (Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences) for mouse immunization. We acknowledge the support provided by Professor Jing Xue (Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences) for the quantification of MPXV load using probe-based qPCR. We acknowledge the support provided by Professor Hongqi Liu (Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences) for the MPXV challenge in CAST/EiJ mice. We also thank Professor Dongsheng Zhou (Beijing Institute of Microbiology and Epidemiology) for his valuable assistance in non-human primate immunization.

Author contributions

Conceptualization, Z.J. and P.J.; methodology, Z.J., Z.Z., L.S., and P.J.; investigation, Z.J., P.J., Z.Z., Y.X., H.W., and M.Z.; formal analysis, Z.J., P.J., Z.Z., M.Z., P.Q., Y.X., H.W., G.Z., Y.L., and L.S.; supervision, Z.J., W.L., Y.L., L.S., and J.D.; writing – original draft, Z.J. and P.J.; writing – review and editing, Z.J., L.S., and P.J.

Declaration of interests

Patent applications have been filed on MV-A, MV-B, and MV-C. Z.J., Z.Z., M.Z., and P.Q. are the co-inventors.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies

Rabbit anti-MPXV M1R	Sino Biological	Cat# 40904-T62; RRID:AB_3677032
Rabbit anti-MPXVA35R	Sino Biological	Cat# 40886-T62; RRID:AB_3677033
Rabbit anti-MPXV A29L	Sino Biological	Cat# 40891-T62;
Rabbit anti-MPXV B6R	Sino Biological	Cat# 40902-T62; RRID:AB_3677031
Anti-mouse MerTK PE	Biolegend	Cat# 151505; RRID:AB_2617036
Anti-mouse CD16/CD32	Biolegend	Cat# 101301; RRID:AB_312800
Anti-mouse CD3 AF700	Biolegend	Cat# 100215; RRID:AB_493696
Anti-mouse CD45 APC	Biolegend	Cat# 103112; RRID:AB_312977
Anti-mouse CD11c BV785	Biolegend	Cat#117335; RRID:AB_11219204
Anti-mouse B220 AF647	Biolegend	Cat#103229; RRID:AB_492875
Anti-mouse CD31 FITC	Biolegend	Cat#160211; RRID:AB_2910329
Anti-mouse CD64 PE/Dazzle™ 594	Biolegend	Cat#139319; RRID:AB_2566558
Anti-mouse CD4 FITC	Biolegend	Cat# 100510; RRID:AB_312713
Anti-mouse CD8a BV650	Biolegend	Cat#100741; RRID:AB_11124344
Anti-mouse CD11a PerCP-Cy5.5	Biolegend	Cat# 162907; RRID:AB_3097463
Anti-mouse IFN-γ PE	Biolegend	Cat# 505808; RRID:AB_315402
Anti-mouse CD62L PE/Dazzle™ 594	Biolegend	Cat#104447; RRID:AB_2566162
Anti-mouse CD44 APC	Biolegend	Cat#103011; RRID:AB_312962
Anti-mouse CD86 FITC	Biolegend	Cat#105005; RRID:AB_313148
Anti-mouse CD69 APC-Cy7	Biolegend	Cat#104525; RRID:AB_10683447
Anti-mouse CD103 Pacific Blue	Biolegend	Cat#121417; RRID:AB_2128620
Anti-mouse TNF-α BV421	Biolegend	Cat#506327; RRID:AB_10900823
Anti-mouse CD19 PerCP-Cy5.5	Biolegend	Cat#115533; RRID:AB_2259869
Anti-mouse IgD APC-Cy7	Biolegend	Cat#405715; RRIDAB_10660304
Anti-mouse GL7 AF488	Biolegend	Cat#144611; RRID:AB_2563284
Anti-mouse CD138 PE/Dazzle™ 594	Biolegend	Cat#142527; RRID:AB_2566494
Anti-monkey IgG HRP	Abcam	Cat# ab112767; RRID:AB_10866625
Anti-mouse IgG HRP	Abcam	Cat# ab6789; RRID:AB_955439
Anti-mouse IgG1 HRP	Abcam	Cat# ab97240; RRID:AB_10695944
Anti-mouse IgG2a HRP	Abcam	Cat# ab97245; RRID:AB_10680049
Anti-mouse IgA HRP	Abcam	Cat# ab97235; RRID:AB_10681186
Goat anti-monkey IgA HRP	Abclonal	Cat# AS116
Rabbit anti-mouse B220	Thermo Fisher Scientific	Cat# 14-0452-82; RRID:AB_467254
Rat anti-mouse Ki67	Abcam	Cat# ab15580; RRID:AB_443209

Bacterial and virus strains

mpox virus clade IIb (MPXV clade IIb)	Wuhan Institute of Biological Products Co., Ltd.	WIBP-MPXV-001
Vaccinia virus Western Reserve (VACV-WR)	Institute of Microbiology, Chinese Academy of Sciences	N/A
Vaccinia virus Tian Tan strain (VACV-VTT)	Institute of Microbiology, Chinese Academy of Sciences	N/A
Ectromelia virus (ECTV)	Institute of Microbiology, Chinese Academy of Sciences	ATCC number: VR-1374

Chemicals, peptides, and recombinant proteins

Trypsin-EDTA	Viva Cell	Cat# C3530-0500
Phosphate buffered saline (PBS)	Servicebio	Cat# G4202
Dulbecco’s modified eagle medium (DMEM)	Corning	Cat# 10-013-CV
Fetal bovine serum (FBS)	GIBCO	Cat# 16000-044
Penicillin/streptomycin	Viva cell	Cat# C3421-0100
3,3′,5,5′-tetramethytlbenzidine (TMB)	Bioss	Cat# C04-03001
ELISA Coating Buffer	Acmec	Cat# AC10810
Bovine serum albumin	Beyotime	Cat# ST2249
Tween 20	Solarbio	Cat# T8220
D-luciferin potassium salt	MREDA	Cat# M049254
Hoechst 33342	Coolaber	Cat# SL7130
OCT compound	Servicebio	Cat# G6059
Brefeldin	MCE	Cat# HY-16592
CUBIC-L	TCI	Cat# T3740
CUBIC-R+(M)	TCI	Cat# T3741
Mounting solution	TCI	Cat# M3294
Recombinant M1R protein	Vazyme	Cat# RM2152-00
Recombinant A35R protein	Vazyme	Cat# RM2165-00
Recombinant A29L protein	Vazyme	Cat# RM2143-00
Recombinant B6R protein	Vazyme	Cat# RM2169-00
4% paraformaldehyde	Biosharp	Cat# BL539A
Red blood cell lysis buffer	TIANGEN	Cat# RT122-01
Staining buffer	Biosharp	Cat# BL1136A

Critical commercial assays

MiniBEST Viral RNA/DNA Extraction Kit	Takara	Cat# 9766
Premix Ex Taq™ (Probe qPCR) Kit	Takara	Cat# RR390

Deposited data

Raw and analyzed data	This paper	GSA: CRA028032

Experimental models: Cell lines

Vero E6	Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences	N/A
BSC-1	Institute of Microbiology, Chinese Academy of Sciences	N/A
A549	Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences	N/A

Experimental models: Organisms/strains

BALB/c	Beijing HFK Bioscience Co., Ltd.	N/A
CAST/EiJ	Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences	N/A
Cynomolgus monkey	Wincon Theracells Biotechnologies Co., Ltd.	N/A

Oligonucleotides

VACV-WR E9L qPCR Forward Primer:5′-CGGCTAAGAGTTGCACATCCA-3′	Sangon Biotech	N/A
VACV-WR E9L qPCR Reverse Primer:5′-CTCTGCTCCATTTAGTACCGATTCT-3′	Sangon Biotech	N/A
VACV-WR E9L qPCR Probe: FAM-AGGACGTAGAATGATCTTGTA-MGB	Sangon Biotech	N/A
MPXV F3L qPCR Forward Primer:5′-CTCATTGATTTTTCGCGGGATA-3′	Sangon Biotech	N/A
MPXV F3L qPCR Reverse Primer:5′-GACGATACTCCTCCTCGTTGGT-3′	Sangon Biotech	N/A
MPXV F3L qPCR Probe: FAM-CATCAGAATCTGTAGGCCGT-MGB	Sangon Biotech	N/A

Software and algorithms

GraphPad Prism	GraphPad Software, LLC	Version 8.0.1
R software	Open source software R	Version 4.4.2
PyMOL	Schrodinger	Version 2.5.0
De Novo FCS Express software	De Novo Software	Version 7.22.0006
Omicsmart	GENE DE NOVO	https://www.omicsmart.com/

Open in a new tab

Experimental models and study participant details

Cell lines

Vero E6, BSC-1, and A549 cells were cultured in Dulbecco’s modified Eagle medium (DMEM, high glucose; Corning) supplemented with 100 U/mL of Penicillin-Streptomycin solution, and 10% fetal bovine serum (FBS, GIBCO) in a 5% CO₂ environment at 37°C. Trypsin-EDTA (0.25%, Viva Cell) was used to detach cells for subculturing every 2–3 days. Cell lines were tested to be mycoplasma-free on a regular basis for mycoplasma.

Viruses

Mpox virus clade IIb (WIBP-MPXV-001) was obtained from Wuhan Institute of Biological Products Co., Ltd., Wuhan, China. The virus stock was propagated and titered in Vero E6 cells.

Vaccinia virus Western Reserve (VACV-WR), vaccinia virus Tian Tan strain (VACV-VTT), and ectromelia virus (ECTV, ATCC number: VR-1374) were obtained from Institute of Microbiology, Chinese Academy of Sciences. The virus stock was propagated and titered in BSC-1 cells.

Animals

Female BALB/c mice (6–8 weeks old) were purchased from the Beijing HFK Bioscience Co., Ltd. Female CAST/EiJ mice (5–8 weeks old) were obtained from the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences. All procedures involving mice in this study were conducted following the guidelines approved by the Institutional Animal Care and Use Committee of the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences (approval No. LHQ24002), and the Institute of Biophysics, Chinese Academy of Sciences (approval No. ABSL-2-2024056), the Hebei University (approval No. HBU2026M011), and the Institute of Animal Science, Chinese Academy of Agricultural Sciences (approval No. IAS2026-142).

A total of three female cynomolgus monkeys in good health (negative for tuberculosis), including two 6-year-olds and one 7-year-old, were obtained from the Wincon Theracells Biotechnologies Co., Ltd. and were also housed there. All procedures involving monkeys in this study were conducted following the guidelines approved by the Institutional Animal Care and Use Committee of the Wincon Theracells Biotechnologies Co., Ltd. (approval No. W00307). All of the experiments complied with the relevant ethical regulations.

Method details

Construction and preparation of rAAV/DJ-based vaccines

AAV/DJ serotype was chosen as the vector to express the recombinant MPXV antigens (IMV-derived antigens M1R and A29L, and EEV-derived antigens A35R and B6R) and CTB in this study. The amino acid sequences of MPXV antigen proteins used in this study were based on the sequence of the newly identified MPXV isolate MPXV_USA_2022_MA001 (GenBank: ON563414.2). Two combinations of MPXV antigen proteins were chosen and fused with CTB individually, which was referred as mpox vaccine A (MV-A, the fused fragment of M1R, A35R and CTB) and mpox vaccine B (MV-B, the fused fragment of A29L, B6R and CTB). The genes of fused fragment of MV-A or MV-B were cloned into the plasmid of pAAV-CAG-MCS-WPRE, in which a tPA signal peptide with KOZAK sequence was added to the 5′ of the fused genes to increase protein expression and secretion. The recombinant AAV/DJ expressing MPXV antigens (MV-A or MV-B) were packaged at Vigene Biosciences Inc (Shandong, China). The three-dimensional structures of antigen-adjuvant fusion proteins were predicted using AlphaFold2 and visualized via PyMOL v2.5.0.

A recombinant AAV/DJ vector co-encoding GFP and luciferase (rAAV/DJ-Luc-GFP), separated by a P2A self-cleaving peptide sequence, was used as a model vector to evaluate gene expression patterns of rAAV/DJ vectors in vivo. The rAAV/DJ-CAG-MCS-WPRE vector, which does not encode any gene product, was purchased from OBiO Technology Company (Shanghai, China).

Construction and preparation of mRNA vaccines

The quadrivalent mpox mRNA vaccine (designated LNP-MV), which encapsulates mRNAs encoding clade IIb MPXV antigens A35R, M1R, A29L and B6R, was commissioned from Genscript following established microfluidic LNP preparation protocols.⁴⁵^,⁵³ mRNA was synthesized via in vitro transcription from linearized DNA templates, treated with DNase I to remove template DNA, and purified by lithium chloride precipitation and ethanol washing. LNPs were prepared by microfluidic mixing of mRNA-containing citrate buffer (pH 4.0) and ethanol-dissolved lipids (SM-102:cholesterol:DMG-PEG2000:DSPC = 50:38.5:1.5:10 molar ratio) at a 3:1 aqueous-to-ethanol volume ratio. The formulation was dialyzed against Tris-HCl/sucrose buffer to remove residual ethanol, concentrated, sterile-filtered and stored at −80°C.

In vitro detection of MPXV antigens expressed by MV-A and MV-B

A549 cells were infected (MOI = 1 × 10⁵) with MV-A or MV-B, and the empty non-encoding rAAV vector was used as a control. The culture supernatants were collected at 72 h post-infection and concentrated 50-fold using a 10-kDa centrifugal concentrator (Millipore, USA). The concentrated supernatants and whole cell lysates were prepared for subsequent western blot (WB) analysis. Protein samples (lysates and supernatants) were separated via PAGE under both denaturing (SDS-PAGE) and non-denaturing (native PAGE) conditions. Denaturing electrophoresis was performed using standard Laemmli buffer system containing sodium dodecyl sulfate (SDS) and β-mercaptoethanol, while non-denaturing electrophoresis was conducted in the absence of SDS and reducing agents. Following electrophoresis, separated proteins in gels were electrophoretically transferred onto polyvinylidene difluoride (PVDF) membranes using a semidry blotting apparatus (15 V, 80 min). After transfer, the membranes were blocked with 5% skim milk in TBS buffer containing 0.1% Tween 20 (TBST) at 37°C for 2 h and rinsed once with TBST. Subsequently, the membranes were incubated with the respective primary antibodies (rabbit polyclonal antibodies against M1R (Sino Biological, 40904-T62), A35R (Sino Biological, 40886-T62), A29L (Sino Biological, 40891-T62) and B6R (Sino Biological, 40902-T62) at 37°C for 1 h. After three washes with TBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (goat anti-rabbit IgG) at 37°C for 1 h. Following further three washes, target bands were detected by an ECL system (CLINX, China).

Characterization of antigen-adjuvant nanocomplexes

The concentrated supernatants from MV-A or MV-B infected A549 cells were analyzed by TEM to characterize the ultrastructure of antigen-adjuvant nanocomplexes.³⁶^,⁵⁴ Briefly, a 15 μL aliquot of concentrated supernatant was added and adsorbed onto a carbon-coated copper grid for 5 min, then negatively stained with 1% uranyl acetate. Samples were observed using a JEM-1400 TEM (JEOL, Japan), with images acquired by a 12 M-B charge-coupled device camera (AMT BioSprint, Canada).

Determination of rAAV/DJ-protein expression in vivo

To evaluate the rAAV/DJ-protein expression in vivo, the rAAV/DJ vector with reporters luciferase and GFP was delivered into the lungs at a dose of 1 × 10¹¹ vg per mouse through a liquid aerosol delivery device for mice (Beijing Huironghe Technology).³⁶^,⁵⁵ Briefly, mice were anesthetized with pentobarbital sodium (75 mg/kg, i.p.) and placed supine. The tongue was gently moved aside with forceps, and a laryngoscope was used to visualize the tracheal opening. The microsprayer (Figure S12A) was then inserted through the larynx to the region of the tracheal bifurcation for pulmonary delivery. Luciferase expression was monitored at multiple time points post rAAV/DJ delivery. Specifically, mice were injected with D-luciferin potassium salt (150 mg/kg, MREDA) intraperitoneally, and then the bioluminescence signals generated by luciferase expressed from the rAAV/DJ vector were captured using a Living Imaging System (IVIS Lumina III, PerkinElmer).⁵⁴ Furthermore, on day 8 post-delivery, a subset of mice was euthanized, and their major organs were collected to assess the spatial distribution of the luciferase expression post rAAV/DJ delivery via bioluminescence imaging. Then mouse lungs were fixed with 10% neutral buffered formalin for 48 h, followed by immersion in 30% sucrose solution. Subsequently, the tissues were embedded in OCT compound (Servicebio, G6059) and processed for cryosectioning. The 10-μm sections were stained with Hoechst 33342 (Coolaber, SL7130) to label nuclei, and GFP expression in the tissue sections was then evaluated using fluorescence microscopy.

Immunization, sample collection, and live virus challenge

i) Immunogenicity and safety evaluations in BALB/c mice

Female BALB/c mice (6–8 weeks old) were intratracheally immunized once with MV-A (1 × 10¹¹ vg in 50 μL per mouse), MV-B (1 × 10¹¹ vg in 50 μL per mouse), or MV-C (5 × 10¹⁰ vg of MV-A and 5 × 10¹⁰ vg of MV-B in 50 μL per mouse), using a liquid aerosol delivery device for mice (Beijing Huironghe Technology). Mice in the control group received intratracheal administration of equal volume of PBS (50 μL per mouse). Mice in the LNP-MV group received an intramuscular injection of LNP-MV at a dose of 8 μg on day 0, and a booster dose was administered on day 21.

For safety evaluation, anti-coagulated blood was collected from mice at 2- and 21-days post immunization for hematology analysis. In addition, plasma collected on day 2 post-immunization was used to evaluate terminal complement activation. On day 21 post-immunization, mice were euthanized and their major organs were fixed in 10% neutral buffered formalin for histopathology analysis.

For analysis of MPXV antigen-specific antibodies, non-anti-coagulated blood was collected at the indicated time points for serum separation. Furthermore, BALF (0.6 mL), NLF (0.2 mL), OLF (0.15 mL), and GLF (0.15 mL) were collected from separate batches of mice at day 21, 28 or 1 year post-immunization. Antigen-specific antibodies in serum, BALF, NLF, OLF and GLF were determined by ELISA, while neutralizing antibody levels of BALF and serum collected at selected time points post-immunization were evaluated using a live virus neutralization assay.

ii) Differentiation detection of APCs and lymphocytes in BALB/c mice

Female BALB/c mice were intratracheally immunized once with MV-A, MV-B, or MV-C via a liquid aerosol delivery device for mice (Beijing Huironghe Technology) at a dose of 1 × 10¹¹ vg of rAAV/DJ vector per mouse. In addition, mice in the Sham group received intratracheal administration of an equal volume of empty non-encoding rAAV vector (1×10¹¹ vg in a volume of 50 μL per mouse). Mice were sacrificed at 21- and 70-day post-immunization. The lung tissues including mLNs from each mouse were harvested for preparation of single-cell suspension, followed by flow cytometric analysis.

iii)Single-cell RNA sequencing, BCR immune repertoire sequencing, and immunofluorescence analysis in BALB/c mice

Female BALB/c mice were intratracheally immunized once with MV-C or empty AAV vector at a dose of 1 × 10¹¹ vg per mouse. At 21 days post immunization, mice were euthanized, lung tissues containing mLNs were harvested for preparation of single-cell suspension. In each group, two pooled samples (each sample consisting of tissues pooled from two mice) were prepared and then sequenced by Gene Denovo Biotechnology. Briefly, the four single-cell suspensions were then loaded onto the 10× Genomics GemCode Single-cell instrument to generate single-cell Gel Bead-In-EMulsion (GEMs). Following encapsulation, GEMs were incubated, and mRNA with the polyA tail was reverse transcribed to generate barcoded full-length cDNA. The cDNA amplification, library preparation, and sequencing (including single-cell RNA and B cell receptor repertoire analysis) were performed. Overall, cells with ≥300 detected genes were chosen/retained for downstream analysis, resulting in 9,799 and 10,836 high-quality cells from the Sham group, and 10,215 and 9,933 cells from the MV-C group. All procedures were performed on Omicsmart (https://www.omicsmart.com/home.html#/) to analyze the single-cell RNA sequencing data, including cell cluster identification, visualization of UMAP dimensionality reduction plots, analysis of relative proportions of cell populations, acquisition of gene expression data, and Gene Ontology enrichment analysis.

For light-sheet imaging analysis, mice were euthanized on day 21 post immunization. mLNs were collected and fixed in 10% neutral buffered formalin, followed by processing for light-sheet imaging and germinal center immunofluorescence analysis.

iv)VACV challenge in BALB/c mice

Female BALB/c mice were intratracheally immunized once with MV-A, MV-B, or MV-C at a dose of 1 × 10¹¹ vg per mouse via a liquid aerosol delivery device for mice (Beijing Huironghe Technology), while mice in the Sham group received intratracheal administration of an equal volume of empty AAV vector (1×10¹¹ vg in a volume of 50 μL per mouse). Mice in the LNP-MV group received an intramuscular injection of LNP-MV at a dose of 8 μg on day 0, and a booster dose was administered on day 21. In addition, mice in the VACV-VTT group were immunized by depositing VACV-VTT stock solution (10⁷ PFU per mL) at the base of the tail and subsequently scratching the skin over the deposited droplet 20 times using the needle of an insulin syringe. After three days, pustules or scabs were observed at the scarification site, indicating successful VACV-VTT immunization.

At 35 days post-immunization, mice in all six groups were challenged intranasally with 50 μL of VACV-WR virus suspension containing 1 × 10⁵ PFU. Starting on the day of challenge, body weight was recorded daily. On day 6 post-challenge, all mice were euthanized, and lung tissues were collected for determination of infectious virus by plaque assay and/or viral genome copy numbers by qPCR. In a separate cohort of mice from the MV-C, Sham, and LNP-MV groups, lung tissues were also harvested for histopathological examination. In another separate cohort of mice from the MV-C and Sham groups, the mice were challenged with two doses (one hour apart) of 50 μL of VACV-WR virus containing 2 × 10⁵ PFU at 35 days post-immunization after vaginal mucus was cleared using sterile cotton swabs. Body weight was recorded daily starting on the day of infection. On day 7 post-challenge, all mice were euthanized, and lung and ovary tissues were collected for analysis of infectious virus by plaque assay and viral genome copy numbers by qPCR. In addition, spleen tissues were harvested for histopathological examination.

v)MPXV challenge in CAST/EiJ mice

Female CAST/EiJ mice were intratracheally immunized once with MV-A, MV-B, or MV-C at a dose of 1 × 10¹¹ vg per mouse via a liquid aerosol delivery device for mice (Beijing Huironghe Technology), while mice in the Sham group received intratracheal administration of an equal volume of empty AAV vector (1 × 10¹¹ vg in a volume of 50 μL per mouse). In addition, another group of mice was immunized with the same dose of MV-C via intramuscular injection (i.m.). At 14- and 28-day post-immunization, peripheral blood was collected from mice in each group and serum were separated for the determination of antigen-specific antibody titers using ELISA. On day 35 post-immunization, all mice were challenged via both intranasal and intraperitoneal routes with 3 × 10⁵ PFU of MPXV (25 μL intranasally and 100 μL intraperitoneally). Starting on the day of challenge, body weight was recorded daily. On day 6 post-challenge, all mice were euthanized for dissection. Portions of lung and ovary tissues were collected for analysis of infectious virus by TCID₅₀ assay and viral genome copy numbers by quantitative PCR (qPCR), while additional portions of lung and spleen tissues were fixed in 10% neutral buffered formalin for histopathology analysis.

vi)Immunogenicity evaluation in non-human primates

One day prior to immunization, serum and BALF samples were collected. Fifteen mL of normal saline was used to perform bronchoalveolar lavage to collect BALF from the lung of each animal. The cynomolgus macaques were intratracheally immunized once with MV-C at a dose of 1 × 10¹² vg of rAAV/DJ vector per animal via a liquid aerosol delivery device for monkeys (Beijing Huironghe Technology). The immunization procedure was similar to that for mice: monkeys were anesthetized with Zoletil 50 (5 mg/kg) before immunization, and the microsprayer used is shown in Figure S12B. At various time points post-immunization, peripheral blood samples were collected and serum were isolated, and on day 21 post-immunization, BALF was collected. Antigen-specific antibody titers in both serum and BALF were determined by ELISA, while levels of neutralizing antibody in BALF and serum collected at 6 weeks post immunization were evaluated using a live virus neutralization assay.

MPXV antigen ELISA

Binding properties of antibodies in serum, BALF, NLF, OLF, and GLF from BALB/c mice, CAST/EiJ mice and monkeys to MPXV antigens were determined by ELISA. Polystyrene 96-well flat bottom plates (Corning) were coated with 2 μg mL⁻¹ of recombinant antigen proteins (M1R, A35R, A29L, and B6R were purchased from Vazyme) overnight at 4°C, and blocked in PBS containing 0.05% Tween 20 (PBST) and 5% bovine serum albumin (Beyotime) at 37°C for 2 h. Serum and BALF were subjected to 3-fold serial dilution, and 100 μL of diluted serum or BALF was added to each well of the coated and blocked ELISA plates. After incubating at 37°C for 1 h, the plates were washed 5 times with PBST. Then, 100 μL of horseradish peroxidase-conjugated secondary antibodies were added to each well accordingly, including goat anti-mouse IgG (Abcam, ab6789, 1:10,000), goat anti-mouse IgG1 (Abcam, ab97240, 1:10,000), goat anti-mouse IgG2a (Abcam, ab97245, 1:10,000), goat anti-mouse IgA (Abcam, ab97235, 1:5,000), goat anti-monkey IgG (Abcam, ab112767, 1:20,000) and rabbit anti-monkey IgA (ABclonal, AS116, 1:5,000). After incubation at 37°C for 45 min, plates were again washed five times with PBST. Each well in the plates was developed with 100 μL TMB substrate (Bioss, C04-03001) for 8 min at room temperature, and the reaction was terminated with 100 μL of 2 M H₂SO₄. The absorbance at 450 nm and 630 nm was measured by a microplate reader (Thermo). Absorbance value in each well was calculated by subtracting the absorbance at 630 nm from that at 450 nm of the same well. Endpoint titers were defined as the reciprocal of the highest dilution at which the sample yielded an absorbance more than 2.1 times the background absorbance.¹⁷ Antibody titers below the limit of detection were assigned a value of one-third of the detection limit.

Neutralization assays

The neutralization assays were performed using a TCID₅₀-based microneutralization protocol.³⁶ The VACV-VTT, VACV-WR, and ECTV neutralization assays were conducted in the biosafety level-2 (BSL-2) laboratory, while the MPXV neutralization assay was performed in the biosafety level-3 (BSL-3) laboratory. All samples (serum and BALF) were heat-inactivated at 56°C for 30 min to eliminate any complement activity. In brief, the 3-fold serial dilutions of the serum (mouse serum starting at 40×, monkey serum starting at 20×) or BALF (mouse BALF starting at 20×, monkey BALF starting at 2×) were mixed with an equal volume of the virus suspension of VACV-VTT, VACV-WR, ECTV, or MPXV, which was prepared to deliver 100 TCID₅₀ per well, in a 96-well plate, followed by a 1.5 h incubation at 37°C in a 5% CO₂ incubator. Then the sample-virus mixtures were added to the 96-well plates seeded with Vero E6 cells and incubated for 1 h at 37°C. After incubation, the sample-virus mixtures were then removed and replaced with 200 μL of DMEM containing 2% FBS. The plates seeded with Vero E6 cells were then incubated at 37°C in 5% CO₂ for three days. Cytopathic effect (CPE) in each well was assessed microscopically, and the neutralizing titer was determined as the reciprocal of the highest sample dilution that produced 50% CPE. The geometric mean titer was then calculated based on these data. Neutralizing titers below the limit of detection were assigned a value of one-third of the detection limit.

Flow cytometry analysis

Analysis of activated APC

At 21-days post-immunization, the activation of APC in lungs (containing mLNs) of immunized mice was measured using flow cytometry. As described in our previous study,⁵⁶ after treated with red blood cell lysis buffer, the prepared single-cell suspensions with the counts of approximately 2 × 10⁶ per mouse were incubated in blocking solution containing 0.25% FcBlock (Biolegend, 101301) in staining buffer (Biosharp, BL1136A) for 15 min. The collected cells were then stained with the following antibodies at 4°C for 30 min: anti-CD45-APC (Biolegend, 103112, 1:150), anti-CD11c-BV785 (Biolegend, 117335, 1:150), anti-MerTK-PE (Biolegend, 151505, 1:150), anti-CD64-PE/Dazzle 594 (Biolegend, 139319, 1:150), and anti-CD86-FITC (Biolegend, 105005, 1:150). After washing with PBS containing 0.5% BSA, cells were analyzed using LSRII Fortessa (BD Biosciences, USA), and the data were analyzed using De Novo FCS Express 7 software.

Analysis of T cells

At 21 and 70 days post-immunization, the T cell responses in lungs (containing mLNs) of immunized mice were determined using flow cytometry. After being treated with red blood cell lysis buffer, the prepared single-cell suspensions were blocked (at 4°C for 15 min) and stained (at 4°C for 30 min) with the following indicated antibodies. (1) To evaluate Trm cell levels,³⁶ cells were stained with the following antibodies (the gating strategy is shown in Figure S12C): anti-CD3-AF700 (Biolegend, 100215, 1:150), anti-CD4-FITC (Biolegend, 100510, 1:150), anti-CD8a-BV650 (Biolegend, 100741, 1:150), anti-CD44-APC (Biolegend, 103011, 1:150), anti-CD69-APC-Cy7 (Biolegend, 104525, 1:150), anti-CD103-Pacific Blue (Biolegend, 121417, 1:150), anti-CD11a-PerCP-Cy5.5 (Biolegend, 162907, 1:150), anti-CD62L-PE/Dazzle 594 (Biolegend, 104447, 1:150), and anti-IFN-γ-PE (Biolegend, 505808, 1:150). (2) To evaluate levels of multi-functional T cells, cells were stained with the following antibodies: anti-CD3-AF700 (Biolegend, 100215, 1:150), anti-CD4-FITC (Biolegend, 100510, 1:150), anti-CD8a-BV650 (Biolegend, 100741, 1:150), and anti-IFN-γ-PE (Biolegend, 505808, 1:150), and anti-TNF-α-BV421 (Biolegend, 506327, 1:150). Note that to evaluate the levels of antigen-specific Trm induced 70 days post-immunization, single-cell suspensions were prepared and subsequently stimulated with the designated antigenic protein combinations (A35R + M1R or B6R + A29L) at a final concentration of 3 μg mL⁻¹ for 18 h. For intracellular cytokine staining, cells were incubated with 5 μg mL⁻¹ brefeldin A (MCE, HY-16592) during the last 6 h of stimulation prior to flow cytometric analysis. The stained cells were washed with PBS containing 0.5% BSA and measured using LSRII Fortessa (BD Biosciences), and the data were analyzed using De Novo FCS Express 7 software.

Analysis of B cells

At 21- and 70-day post-immunization, the B cell responses in lungs (containing mLNs) of immunized mice were measured using flow cytometry. After treated with red blood cell lysis buffer, the prepared single-cell suspensions were blocked and stained with the following antibodies: anti-CD45-APC (Biolegend, 103112, 1:150), anti-CD19-PerCP-Cy5.5 (Biolegend, 115533, 1:150), anti-GL7-AF488 (Biolegend, 144611, 1:150), anti-IgD-APC-Cy7 (Biolegend, 405715, 1:150), and anti-CD138-PE/Dazzle 594 (Biolegend, 142527, 1:150). The stained cells were washed with PBS containing 0.5% BSA and measured using LSRII Fortessa (BD Biosciences), and the data were analyzed using De Novo FCS Express 7 software.

Fluorescence analysis

Light-sheet imaging

To visualize B cell follicles and measure the size of mLNs, B cells were labeled with B220-AF647 antibody (Biolegend, 103229, 1:150). mLNs were stained with CD31-FITC antibody (Biolegend, 160211, 1:150), processed with tissue-clearing reagent (TCI, T3740), and were captured in mounting solution (TCI, M3294) for imaging by LiTone XL Light-sheet Microscope (Light Innovation Technology Limited). Laser power and gain were kept consistent across groups. Image reconstructions and measurements of mLN length and width were performed using LitScan v3.3.0 software (Light Innovation Technology Limited). The mLN volume was calculated as 0.5×length×width² based on the published methods.⁵⁷

Immunofluorescence analysis

To evaluate B cell proliferation, the fixed mLNs were sent to Beijing Laike Biological Technology Co., Ltd. for subsequent histological processing. Specifically, mLNs fixed for 48 h underwent dehydration, paraffin embedding, and sectioning into about 4-μm thick tissue sections. After deparaffinization and antigen retrieval, the sections were incubated with primary antibodies (rabbit anti-mouse B220, eBioscience, 14-0452-82; rat anti-mouse Ki67, Abcam, ab15580), followed by fluorescently labeled secondary antibodies (Cy3 conjugated goat anti-rabbit IgG, Servicebio, GB21303; FITC conjugated goat anti-rat IgG, Servicebio, GB22302). Nuclei were counterstained with DAPI. Finally, immunofluorescence signals were visualized and imaged using a fluorescence microscope.

Plaque assay

Lung and ovary tissues from VACV-WR challenged BALB/c mice were weighed and homogenized in DMEM. After centrifugation, the supernatant was collected. Viral titers were determined by plaque assay on Vero E6 cells. Briefly, the supernatant was subjected to 10-fold serial dilutions, starting at a 1:10 dilution. Each dilution was added to confluent monolayers of Vero E6 cells plated in 12-well plates and incubated at 37°C for 1 h for adsorption. Following adsorption, the inoculum was removed. Cells were overlaid with 1 mL of DMEM containing 1% carboxymethyl cellulose and cultured for 72 h at 37°C. After incubation, cells were fixed with 4% formaldehyde for 2 h and stained with 0.5% crystal violet overnight. The plaques were captured and counted using an ELISpot reader and BioSpot image analysis software. Viral load (PFU/g) was calculated based on the number of plaques and normalized by the weight of the organ sample. Virus titers below the limit of detection were assigned a value of one-tenth of the detection limit.

TCID₅₀ assay

Lung and ovary tissues from MPXV challenged CAST/EiJ mice were weighed and homogenized in 0.15 mL DMEM per tissue sample. Vero E6 cells were seeded into 96-well plates at 25,000 cells per well in DMEM supplemented with 10% FBS and 1% Penicillin-Streptomycin, and incubated at 37°C with 5.0% CO₂. Virus titrations were performed using endpoint titration in Vero E6 cells. Tissue homogenates were serially diluted 3-fold (lung) or 5-fold (ovary) in 96-well plates (starting at 5× and 50× dilutions for lung and ovary homogenates, respectively). The media of Vero E6 cells in the 96-well plates was aspirated and replaced with 100 μL of each serially diluted sample. The plates were re-incubated at 37°C with 5.0% CO₂ for three days. CPE was inspected. TCID₅₀ was calculated using the Reed-Muench formula. Virus titers below the limit of detection were assigned a value of one-third (lung) or one-fifth (ovary) of the detection limit.

Determination of viral load in tissue samples by probe-based qPCR

Lung and ovary tissues from challenged mice were weighed and homogenized in PBS, and viral DNA was isolated using MiniBEST Viral RNA/DNA Extraction Kit (TaKaRa, 9766). Then, levels of viral DNA for VACV-WR and MPXV were determined by probe-based qPCR with Premix Ex Taq (Probe qPCR) Kit (TaKaRa, RR390) on an ABI 7500 instrument. For the detection of VACV-WR DNA, as described in a previous study,¹⁷ one set of primers and probes was used to detect the E9L gene of the VACV-WR viral genome, with sequences as follows: E9L-F, 5′-CGGCTAAGAGTTGCACATCCA-3’; E9L-R, 5′- CTCTGCTCCATTTAGTACCGATTCT-3’; E9L-probe, 5′-FAM-AGGACGTAGAATGATCTTGTA-MGB-3’. For the detection of MPXV DNA, as described in a previous study,⁵⁸ one set of primers and probes were used to detect a region of the F3L of the MPXV viral genome, with sequences as follows: F3L-F, 5′-CTCATTGATTTTTCGCGGGATA-3’; F3L-R, 5′-GACGATACTCCTCCTCGTTGGT-3’; F3L-probe, 5′-FAM-CATCAGAATCTGTAGGCCGT-MGB-3’. Viral DNA levels in lung and ovary tissues were expressed as genome copy numbers per gram after comparison with a standard curve produced using serial 10-fold dilutions of VACV-WR DNA or MPXV DNA. The quantification cycle values ≥ 34 were considered negative.

Histopathology analysis

The fixed tissues were dehydrated, embedded in paraffin, and sectioned. Then the tissue sections were deparaffinized, stained with hematoxylin and eosin (H&E), and examined microscopically.

Quantification and statistical analysis

All statistical details for individual experiments, including exact sample sizes (n) and specific statistical tests, are provided in the corresponding figure legends. By default, the sample size n throughout this study denotes the number of biologically independent animals per experimental group; the only exception is Figure S7A, where n refers to the number of independent lymph node samples.

To evaluate differences in specific antibody levels induced by pulmonary immunization with various vaccines and their protective efficacy, PCA was performed on log-transformed specific antibody levels (28 days post-immunization) and viral load data from lung and ovary tissues of CAST/EiJ mice in the MPXV challenge experiment using R v4.4.2. Based on viral load in tissue samples, the above mice were classified into four categories: healthy, mild infection, moderate infection, and severe infection. The correlation between antibody isotypes and health status was analyzed via random forest algorithms in R v4.4.2 to assess the importance of each antibody isotype. Additionally, heatmaps and volcano plots were generated using relevant R packages in R v4.4.2.

For other data, GraphPad Prism 8.0.1 and R v4.4.2 were used for plotting and statistical analysis. Values are expressed as mean ± SEM or mean ± SD, as specified in each figure legend. Statistical significance was calculated using one-sided unpaired t-tests and one-way ANOVA with Dunnett’s multiple comparison test or Tukey’s test when the data followed a normal distribution and exhibited homogeneity of variance. Otherwise, nonparametric analyses were performed using the Mann-Whitney test (for two-group comparisons) or the Kruskal-Wallis test (for multi-group comparisons). Significance levels were defined as follows: ns (not significant), p ≥ 0.05; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

Published: September 15, 2026

Footnotes

Supplemental information can be found online at https://doi.org/10.1016/j.xcrm.2026.103019.

Contributor Information

Zhouguang Jiao, Email: jiao_zg@126.com.

Lei Sun, Email: sunlei362@im.ac.cn.

Pengtao Jiao, Email: jiaopengtao@caas.cn.

Supplemental information

Document S1. Figures S1–S12

mmc1.pdf^{(12MB, pdf)}

Document S2. Article plus supplemental information

mmc2.pdf^{(44.7MB, pdf)}

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Figures S1–S12

mmc1.pdf^{(12MB, pdf)}

Document S2. Article plus supplemental information

mmc2.pdf^{(44.7MB, pdf)}

Data Availability Statement

•
The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive⁵¹ in National Genomics Data Center,⁵² China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences, (GSA: CRA028032) and are publicly accessible at https://ngdc.cncb.ac.cn/gsa/browse/CRA028032. All summary datasets supporting the findings of this study are contained within the article and its supplemental information.
•
This study does not report original code.
•
All other raw data underlying the findings of this study are available from the corresponding author upon reasonable request.

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PERMALINK

A single-dose inhalable rAAV-vectored vaccine against mpox virus

Zhouguang Jiao

Zhihui Zheng

Yue Xue

Hongjie Wang

Meng Zou

Peijie Qu

Guangzhi Zhang

Jiabo Ding

Yang Liu

Wenjun Liu

Lei Sun

Pengtao Jiao

Summary

Graphical abstract

Highlights

Introduction

Results

Construction and characterization of rAAV/DJ-vectored mpox vaccines

Figure 1.

Single-dose rAAV/DJ-based mpox vaccines elicited high and durable antibody responses

Figure 2.

Single-dose rAAV/DJ-based mpox vaccines elicited comprehensive immune cell responses

Figure 3.

Single-dose MV-C elicited immune responses in NHPs

Figure 4.

Single-dose MV-C-vaccinated mice are protected from VACV-WR

Figure 5.

Single-dose MV-C-vaccinated mice are protected from MPXV

Figure 6.

Discussion

Limitations of the study

Resource availability

Lead contact

Materials availability

Data and code availability

Acknowledgments

Author contributions

Declaration of interests

STAR★Methods

Key resources table

Experimental models and study participant details

Cell lines

Viruses

Animals

Method details

Construction and preparation of rAAV/DJ-based vaccines

Construction and preparation of mRNA vaccines

In vitro detection of MPXV antigens expressed by MV-A and MV-B

Characterization of antigen-adjuvant nanocomplexes

Determination of rAAV/DJ-protein expression in vivo

Immunization, sample collection, and live virus challenge

i) Immunogenicity and safety evaluations in BALB/c mice

ii) Differentiation detection of APCs and lymphocytes in BALB/c mice

iii)Single-cell RNA sequencing, BCR immune repertoire sequencing, and immunofluorescence analysis in BALB/c mice

iv)VACV challenge in BALB/c mice

v)MPXV challenge in CAST/EiJ mice

vi)Immunogenicity evaluation in non-human primates

MPXV antigen ELISA

Neutralization assays

Flow cytometry analysis

Analysis of activated APC

Analysis of T cells

Analysis of B cells

Fluorescence analysis

Light-sheet imaging

Immunofluorescence analysis

Plaque assay

TCID50 assay

Determination of viral load in tissue samples by probe-based qPCR

Histopathology analysis

Quantification and statistical analysis

Footnotes

Contributor Information

Supplemental information

References

Associated Data

Supplementary Materials

Data Availability Statement

TCID₅₀ assay