Ethics statement

All animal care and viral-related experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Shenzhen Bay Laboratory (BACG202101) and the ethics committee of Changchun Veterinary Research Institute (Approval number: IACUC of AMMS-11-2024-028).

Cells, viruses, and plasmids

Vero E6 cells (Cat. No# CRL-1586) and HEK293T cells (Cat. No# CRL-3216) were obtained from the American Type Culture Collection (ATCC). All cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with GlutaMAX (Gibco, Cat. No# 10566016) supplemented with 10% fetal bovine serum (FBS) (PAN-Biotech, Cat. No# ST220622) and 100 units/mL of penicillin-streptomycin. The MPXV virus (Genbank: PP778666.1) used in this study was isolated from a patient in Guangzhou, China. The virus was propagated in Vero E6 cells that were cultured in DMEM (Sigma-Aldrich) containing 10% fetal bovine serum (Invitrogen), 50 U/ml of penicillin, and 50 µg/ml of streptomycin. All experiments with infectious MPXV virus were conducted in a biosafety level 3 laboratory. The plasmids encoding MPXV surface proteins or CTL-specific antigens were constructed and managed within our laboratory.

Animals

BALB/c mice and HLA-A*02:01/DR1 transgenic mice were chosen for vaccination and/or challenge experiments. All mice were housed in standard ventilated cages (5–6 mice per cage) under specific pathogen-free (SPF) conditions. The animal room was maintained on a 12-h light/dark cycle (lights on at 7:00 A.M.) at an ambient temperature of 22 ± 2 °C and a relative humidity of 50–60%. Food and water were provided ad libitum, and bedding was replaced twice weekly. All animal care and experimental procedures were conducted following the approval of the Institutional Animal Care and Use Committee (IACUC) of Shenzhen Bay Laboratory and the ethics committee of Changchun Veterinary Research Institute.

Analysis of functional MPXV HLA-I epitopes and identification of epitope-enriched fragments

The NetMHCpan 4.1 server (http://www.cbs.dtu.dk/services/NetMHCpan/) was employed to predict potential HLA-I epitope locations within all structural and nonstructural proteins of the MPXV strain (GenBank: ON563414.2). All potential epitopes with predicted affinity values (IC50) less than 20 nM were included for further analysis. Epitope-enriched fragments were characterized as fragments of MPXV proteins containing over 20 such epitopes per 100 amino acids.

Generation of modified mRNA

The mRNAs were synthesized in vitro through T7 polymerase-mediated DNA-dependent RNA transcription, incorporating 1-methylpseudoUTPs in place of UTPs. To enhance the stability and translation efficiency of the mRNAs, modifications including a 5′-UTP cap and a 3′ poly-A tail were introduced using the ScriptCap™ Cap 1 Capping System and A-Plus™ Poly (A) Polymerase Tailing Kit. For surface antigens, the mRNA templates were constructed so that the mRNA sequences comprised a signal peptide from tissue plasminogen activator (MDAMKRGLCCVLLLCGAVFVSAS), the viral extracellular region of the surface protein, and a His tag (HHHHHH) inserted before the stop codon in sequential order. In the case of MPX-EPs, epitope-enriched peptides were linked using A-A-Y motif linkers, with a ubiquitin sequence featuring a G76A amino acid modification at the N-terminus and a V5 tag sequence (GKPIPNPLLGLDST) at the C-terminus.

Validation of naked mRNA expression

To verify the in vitro expression from naked mRNA, HEK293T cells were transfected with each mRNA using TransIT®-mRNA Reagent (Mirus Bio, Cat. No# MIR-2250). HEK293T cells were seeded in 12-well plates 24 h prior to transfection, and the medium was changed to Opti-MEM (Gibco, Cat. No# 31985062) 2 h before transfection. Each well was transfected with 0.5 μg of mRNA mixed with TransIT®-mRNA Reagent. For surface antigens, 72 h post-transfection, supernatants were collected for Western blot analysis. The antibodies used in the Western blot analysis included a horseradish peroxidase (HRP)-conjugated anti-His antibody (Sino Biological, Cat. No# 105327-MM02T-H, 1:2000).

For MPX-EPs, MG132 (APEXBIO, Cat. No# A2585) was added to the cell culture medium 48 h post-transfection. Six hours later, the cells were collected, and treated with trypsin-EDTA (Gibco, Cat. No# 25300120) to create single cells, fixed, and permeabilized using Cytofix/Cytoperm reagent (BD Biosciences, Cat. No# BDB554714) following the manufacturer’s instructions. The cells were stained with anti-V5 tag mouse mAb (Cell Signaling Technology, Cat. No# 80076S, 1:1000) and subsequently with goat anti-mouse IgG H&L (Cy3®) (Abcam, Cat. No# ab97035, 1:1000). Mock-transfected cells and cells not treated with MG132 served as two different controls. The stained cells were analyzed using flow cytometry (BD FACSAria III cell analyzer).

Preparation and characterization of LNP

LNP (Lipid Nanoparticle) formulations were prepared following a modified protocol derived from Precision Nanosystems. Initially, ionizable lipid (ALC-0315), DSPC, cholesterol, and PEGylated lipid (ALC-0159) were dissolved in ethanol with a weight ratio of 48%/10%/40%/2%. This lipid mixture was combined with citrate buffer (100 mM, pH 5.0) containing mRNA at a concentration of 174 μg/mL using microfluidic cartridges. The aqueous solution and the ethanol solution were mixed rapidly at an aqueous-to-ethanol ratio of 3/1 by volume with a weight ratio of 40/1 (total lipids/mRNA). The resulting formulations were concentrated using 30-kDa Amicon Ultra Centrifugal Filters, passed through a 0.22-μm filter, and stored at 4 °C until needed. The concentrations of all mRNA formulations were verified using the Quant-iT RiboGreen RNA Kit (Invitrogen, Cat. No# R11490). Each formulation exhibited an endotoxin level below 1 EU/ml. For the transmission electron microscope test, the prepared LNPs@mRNA was dialyzed against Milli-Q water for 2 h. Then the dialyzed LNPs@mRNA was diluted 100 times using Milli-Q water and 10 μL of the sample was dropped onto a carbon-coated copper grid. Samples were dried under a vacuum to remove water. Tests were carried out on a Hitachi HT-7700. For dynamic light scattering analysis and zeta potential analysis, dialyzed LNPs@mRNA was diluted 10 times using Milli-Q water, and tests were carried out on Zetasizer Nano-ZS90 (Malvern Instruments, UK).

Animal immunization

1. a.

   BALB/c mice (female, 6–8 weeks) were randomly allocated into groups (n = 5). First, mice were intramuscularly (I.M.) immunized with mRNA-LNP for each MPXV surface protein (12 μg/100 μl/mouse), or PBS (control), and boosted at 3 weeks with the same immunogens (I.M.). 14 days after the 2nd immunization, sera were collected to detect neutralizing antibodies of MPXV.

2. b.

   BALB/c mice (female, 6–8 weeks) were randomly allocated into groups (n = 12). First, mice were intramuscularly (I.M.) immunized with mRNA-LNP mixtures of MPXV surface antigens (12 μg/100 μl/mouse), or PBS (control), and boosted at 3 weeks with the same immunogens (I.M.). 14 days after the 2nd immunization, sera were collected from 6 mice per group to detect neutralizing antibodies. The rest immunized mice were then subjected to challenge experiments, described below.

3. c.

   HLA-A*02:01/DR1 transgenic mice (female, 4–5 weeks) were randomly allocated into groups (n = 11). MPX-EPs diluted in PBS with 8.7% sucrose were injected intramuscularly (I.M., 3 μg per mouse) following a prime and boost regimen with an interval of 3 weeks, PBS with 8.7% sucrose served as the control. The immunized mice were further processed for subsequent experiments as described below.

4. d.

   HLA-A*02:01/DR1 transgenic mice (female, 4–5 weeks) were randomly allocated into groups (n = 25) and vaccinated via the intramuscular route with either the MPX-m-Mix, which contained MPX-EPs (3 μg per mouse) and a surface antigen mRNA mix (12 μg per mouse). The MPX-p-Mix, which was formulated with an equal-ratio mixture of 12 recombinant surface antigens (A29, M1, B6, A35, H3, A17, A30, H2, A28, A21, G2, and I2, 12 μg per mouse), the MPX-EPs-derived synthetic peptide pool (3 μg per mouse) and Alum (Alhydrogel® adjuvant 2%, 150 μg/mouse, InvivoGen) or PBS with 8.7% sucrose as buffer controls. The same doses were administered again for a booster immunization at 3 weeks post-priming vaccination. Serum samples were collected from 5 mice per group at 21 days, 40 days, 90 days, and 150 days after the boost vaccination and analyzed as described below. Lymph nodes were collected from 5 mice per group at 7 days after the boost vaccination and analyzed as described below. Another 5 mice per group were sacrificed at 21 days and 150 days post-booster vaccination to analyze T cell immune responses as described below. The remaining mice were further processed for subsequent challenge experiments as described below.

ELISA analysis of MPXV-specific IgG and subtypes

To detect MPXV-specific antibodies in immunized sera or BALF collected from immunized mice, plates were coated with purified MPXV at a concentration of 1 μg/ml in PBS, followed by sequential addition of serially diluted serum samples and HRP-conjugated anti-mouse IgG (1:5000) antibodies (Invitrogen, Cat. No# A16066), HRP-conjugated anti-mouse IgG1 (1:5000) antibodies (Invitrogen, Cat. No# PA1-74421), HRP-conjugated anti-mouse IgG2a (1:5000) antibodies (Invitrogen, Cat. No# A-10685), HRP-conjugated anti-mouse IgG2c (1:5000) antibodies (Abcam, Cat. No# ab97255), HRP-conjugated anti-mouse IgG2b (1:5000) antibodies (Invitrogen, Cat. No# SA5-10266), HRP-conjugated anti-mouse IgG3 (1:2000) antibodies (Invitrogen, Cat. No# M32607), or HRP-conjugated anti-mouse IgA (1:1000) antibodies (Proteintech, Cat. No#SA00012-7) for 1 h at 37 °C. The plates were sequentially incubated with the substrate TMB (3,3′,5,5′-tetramethylbenzidine) (Sigma, Cat. No# T0440) and then H₂SO₄ (1N) to stop the reaction. The absorbance at 450 nm was measured on a microplate reader.

Plaque reduction neutralization test (PRNT)

The neutralizing activity of serum samples from immunized mice against the authentic MPXV was evaluated using a plaque formation-based neutralization assay. The Vero E6 cells were seeded at a density of 1.0 × 10⁵ cells per well and cultured for 2 h at 37 °C. Serial dilutions of the serum samples were prepared in 96-well plates by a factor of two and then incubated with a viral solution containing ~00 PFU of MPXV at 37 °C for 2 h. The serum-virus mixture was added to the Vero E6 cells. Plaque formation was examined 3 or 4 days post-infection, and neutralizing titers were calculated from the serial dilutions of sera that exhibited plaque formation reduction.

ELISpot assays of IFN-γ and IL-4

ELISpot assays were conducted utilizing mouse IFN-γ (Abcam, Cat. No# ab64029) and IL-4 ELISpot kits (Mabtech, Cat. No# 3311-4HPW-10) following the manufacturer’s instructions. The enumerated splenocytes were ex vivo restimulated with a mixture of 15-amino-acid overlapping peptide pool (at 2 μg/ml per peptide) or the DMSO control. Subsequently, spots were counted using an ELISpot reader (iSpot).

Profiling of lymphocytes in the spleens, the lungs, and the lymph nodes

The spleens and lungs from immunized HLA-A*02:01/DR1 transgenic mice (n = 5 under animal immunization c. and n = 5 under animal immunization d.) were harvested on Day 21 post-second immunization. The spleens and lungs were briefly lysed, and red blood cells were removed by suspension in ammonium-chloride-potassium buffer. The splenocytes were then washed and resuspended in RPMI medium 1640 supplemented with 10% fetal bovine serum. Cells were first stained with Ghost Dye™ Red 780 (TONBO Biosciences, Cat. No# 13-0865-T100) to exclude dead cells, and then stained with a cocktail of the following fluorescently labeled antibodies: anti-CD45-Alexa Fluor™ 700 (Invitrogen, Cat. No# 56-0451-82), anti-CD8-PerCP-Cyanine5.5 (TONBO Biosciences, Cat. No# 65-0081-U100), anti-CD44-APC (Biolegend, Cat.No# 103012), anti-CD62L-BV421 (Biolegend, Cat.No# 104436), and anti-CD107a-BV711 (BioLegend, Cat. No# 121631). For ICS, the cells underwent additional fixation and permeabilization using the Cytofix/Cytoperm reagent (BD Biosciences, Cat. No# BDB554714) according to the manufacturer’s instructions and staining with the anti-IFN-γ-PE (TONBO Biosciences, Cat. No# 50-7311-U100), anti-TNF-α-BV421 (BioLegend, Cat. No# 506328), anti-Granzyme B-FITC (BioLegend, Cat. No# 372206), and anti-CD107a-BV711 (BioLegend, Cat. No# 121631). The lung cells were washed and resuspended in RPMI medium 1640 supplemented with 10% fetal bovine serum. Cells were first stained with Ghost Dye™ Red 780 (TONBO Biosciences, Cat. No# 13-0865-T100) to exclude dead cells, and then stained with a cocktail of the following fluorescently labeled antibodies: anti-CD45-Alexa Fluor™ 700 (Invitrogen, Cat. No# 56-0451-82), anti-CD8-PerCP-Cyanine5.5 (TONBO Biosciences, Cat. No# 65-0081-U100), anti-CD69-FITC (Biolegend, Cat.No# 104506), anti-CD103-AF594 (Biolegend, Cat.No# 121428). The inguinal lymph nodes from immunized mice (n = 5, HLA-A*02:01/DR1 transgenic mouse under animal immunization d.) were harvested at day 7 post-second immunization and pooled. The nodes were homogenized into single-cell suspensions using a syringe plunger and passed through a 70 µm cell strainer in complete RPMI 1640 media containing 10% fetal bovine serum. Cells were washed and resuspended in fresh RPMI-10% FBS media in a 96-well round-bottomed plate for staining. Cells were first stained with Ghost Dye™ Red 780 (TONBO Biosciences, Cat. No# 13-0865-T100) for dead cells, and then stained with a cocktail of the following fluorescently labeled antibodies: anti-CD45-Alexa Fluor™ 700 (Invitrogen, Cat. No# 56-0451-82), anti-CD4-FITC (Tonbo Biosciences, Cat. No# 35-0042-U100), anti-CD185-Brilliant Violet 605™ (BioLegend, Cat. No# 145513), anti-PD-1-Brilliant Violet 421™ (BioLegend, Cat. No# 135218), anti-B220-PerCP-Cyanine5.5 (TONBO Biosciences, Cat. No# 65-0452-U100), anti-CD95-PE (BioLegend, Cat. No# 152608), anti-GL-7-APC (BioLegend, Cat. No# 144618) in the cell staining buffer (BioLegend, Cat. No# 420201) and incubated for 20 min in the dark at room temperature. Cells were then washed and resuspended in cell stain buffer and were analyzed using BD Aria III cell analyzer. The resulting data were processed using FlowJo software V_10.

Profiling of lung tissue-resident immune responses

HLA-A*02:01/DR1 transgenic mice were vaccinated intramuscularly with two doses of MPX-p-Mix or MPX-m-Mix at a 3-week interval. Twenty-one days post boost vaccination, lung cells were stained with anti-CD45-Alexa Fluor^TM 700, CD8-PerCP-Cyanine5.5, CD69-FITC and CD103-AF594. The population of CD45⁺CD8⁺CD69⁺CD103⁺ T cells was sorted by flow cytometry and stimulated in vitro with a control peptide pool (Con-Pool) from unrelated antigens or a mixture of 15-amino-acid overlapping peptides (Pep-Pool, at 2 μg/ml per peptide) for 48 h. The supernatant was collected for the cytokine detection using a Mul-Analyte Flow Assay Kit (BioLegend, Cat. No# 740150, for INF-γ, TNF-α and GM-CSF), and ELISA kits (Invitrogen, Cat. No# BMS601 for IL-2; Invitrogen, Cat. No# BMS6029 for granzyme B), according to the manufacturer’s instructions.

HLA tetramer preparation and staining assay

Predicted functional peptides (Supplementary Table 3) matched with HLA-A*02:01were synthesized and used for peptide-specific tetramer preparation following the instructions of the HLA-A*02:01 Tetramer Kit-PE (MBL International Corporation, Cat. No# TB-7300-K1). Cells of spleens or lungs isolated from immunized HLA-A*02:01/DR1 transgenic mice were washed and stained with Ghost Dye™ Red 780 (TONBO Biosciences, Cat. No# 13-0865-T100) for dead cells. Surface markers were stained with CD45-Alexa Fluor™ 700 (Invitrogen, Cat. No# 56-0451-82), CD8-PerCP-Cyanine5.5 (TONBO Biosciences, Cat. No# 65-0081-U100) and Peptide-tetramer-PE mixture, for 20 min at room temperature. The stained cells were pelleted and washed three times before being analyzed by flow cytometry (BD Aria III cell analyzer).

Validation of epitope-enriched regions

HEK293T cells stably expressing the pHAGE_EF1a_ICADCR, pHAGE_EF1a_IFPGZB, and pHAGE_EF1aHLA vectors (HLA-A*02:01 were separately overexpressed in HEK293T cells)⁵⁶ were transfected with plasmids encoding the computationally predicted epitope-enriched regions of each MPXV protein. At 36 h post-transfection, 1 million HEK293T cells were cocultured with 0.2 million CD8⁺ T cells from MPX-EPs immunized mice for 12 h, after which IFP-positive target cells were sorted and analyzed with FlowJo V_10 software.

MPXV challenge studies

Immunized, plasma, or T cells transfused mice were challenged with MPXV in the following experiments to evaluate immune protection efficacy. (1). BALB/c mice (male and female, 3 weeks) randomly allocated into groups (n = 6) were transfused with 200 μl of the plasma collected from Mix-12, Mix-8, Mix-4, or PBS vaccinated mice (under animal immunization b.). Two hours later, the transfused mice were intranasally inoculated with MPXV at the dose of 1.0 × 10⁶ PFU. Mice were sacrificed at 4 days post-infection to collect lungs for viral load quantification by a plaque assay. (2). BALB/c mice (male and female, 3 weeks) randomly allocated into groups (n = 6) were transfused with 2.0 × 10⁷ CD8⁺ or CD4⁺ T cells sorted from immunized mice collected from Mix-12, Mix-8, Mix-4, or PBS vaccinated mice (under animal immunization b.). Six hours later, the transfused mice were intranasally inoculated with MPXV at the dose of 1.0 × 10⁶ PFU. Mice were sacrificed at 4 days post-infection to collect lungs for viral load quantification by a plaque assay. (3). MPX–EPs (3 μg) or PBS immunized HLA-A*02:01/DR1 transgenic mice (n = 6 under animal immunization c.) were injected intraperitoneally with an anti-mouse anti-CD8-α (IgG2b) mAb (Bio X Cell, Cat. No# BE0061, 100 μg/mouse) or IgG2b isotype control mAb (Bio X Cell, Cat. No# BE0090, 100 μg/mouse) on Days-1 and 0 of the challenge. Peripheral blood cells were collected for the evaluation of CD8⁺ T-cell depletion by flow cytometry on the day of the challenge. The mice were intranasally inoculated with MPXV (1.0 × 10⁷ PFU), and half of the mice were sacrificed at 4 days post-infection. The lung tissues were collected for viral load detection by a plaque assay and pathological analysis. (4). Mice immunized using the synergistic strategy (n = 5, HLA-A*02:01/DR1 transgenic mouse under animal immunization d.) were intranasally inoculated with MPXV (1.0 × 10⁷ PFU). The challenged mice per group were sacrificed at 4 days post-infection to collect lung tissues for viral loads and pathological analysis. All procedures adhered to institutional guidelines for animal use and care.

Histopathological assay

Lung tissues collected from challenged mice were immediately fixed in 10% neutral-buffered formalin without inflation, and embedded in paraffin. Approximately 5-μm sections were cut and mounted on slides. Histopathological changes caused by MPXV infection were examined by standard hematoxylin and eosin (H&E) staining and viewed under a light microscope. H&E-stained lung tissue sections were blindly examined and scored by trained histo-pathologists.

Statistics

Statistical analyzes were conducted using Prism 8.0 (GraphPad software). Student’s t-test was used for pairwise comparisons. Group comparisons were executed using one-way ANOVA, followed by Tukey’s multiple comparison post-hoc tests. Specific details regarding the statistical methods and results were provided in the figure legends.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.