Ethics and biosafety statement

Twenty-four mature male Cynomolgus macaques (Macaca fascicularis) aged 3.5–7 years with a body weight of 4–14.6 kg and originating from Mauritian AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care International)-certified breeding centers were used in this study. All animals were housed in the IDMIT infrastructure facilities (CEA, Fontenay-aux-Roses) under Biosafety Level 3 containment (animal facility authorization E92-032-02, Prefecture des Hauts de Seine, France) and in compliance with European Directive 2010/63/EU, French regulations, and the Standards for Human Care and Use of Laboratory Animals of the Office for Laboratory Animal Welfare (OLAW, assurance number F22-00556, United States). The protocols were approved by the institutional ethics committee “Comité d’Ethique en Expérimentation Animale du Commissariat à l’Energie Atomique et aux Energies Alternatives” (CEtEA n° 44) under statement number A22_037. The study was authorized by the ‘Research, Innovation and Education Ministry under registration number (APAFIS#38039–2022072015031466 v2).

Virus production

Monkeypox virus (MPXV) IIb strain MPXV/France/IRBA2211i/2022 (Genbank access number ON755039) used for infections and the neutralization assays was produced in Vero cells (African green monkey kidney, ATCC CCL-81) grown in Dulbecco’s modified Eagle medium (DMEM) and 10% fetal calf serum (FCS) at 37 °C in a 5% CO₂ atmosphere. Supernatants and lysed cells from MPXV-infected Vero cells were frozen/thawed three times. The virus was pelleted by two rounds of ultracentrifugation (30,000 x g, 2 h) through a 40% sucrose cushion. MPXV was titrated on Vero cell monolayers using plaque-forming assays in 24-well plates. The MPXV strain MSF#10 2001 Zaïre⁶⁵ used for neutralization assays on clade Ia was kindly provided by Dr Hermann Meyer from the Institute of Microbiology of the Bundeswehr. The strain was produced and titrated in the same way. Vaccinia virus (VACV) strain Lister clone 107 (VACV-107, GenBank access number DQ121394), used for the neutralization assays, was produced and titrated in the same way.

The MVA vaccine used for the efficacy study in NHP is MVATGN33.1 or MVA-N33, a MVA subclone, isolated in Transgene’s laboratories, from the MVA strain provided by Professor A. Mayr in February 1995 (Virus “MVA” II/85, passage 575 on chicken embryo fibroblasts (CEF) (GenBank access number EF675191.1), and was used for immunization. Crude harvests containing infected cells and culture supernatants were recovered and stored at −20 °C until use. Before purification, viral particles were released from the thawed suspension by homogenization using a homogenizing mixer equipped with an in-line chamber. Large cellular debris was then eliminated by depth filtration using filters with 5 µm pores. The clarified viral suspension was subsequently concentrated and subjected to diafiltration in formulation buffer (50 g/L saccharose, 50 mM NaCl, 10 mM Tris, 10 mM glutamate-Na, pH 8.0) using tangential flow and fiber microfiltration filters with 0.2 µm pores. Finally, the purified virus was further concentrated using the same tangential flow filtration system, aliquoted, and stored at −80 °C until use. Titration of MVA was performed on DF-1 cells (ATCC: CRL-3586) monolayers using plaque-forming assays in six-well plates. MVA-GFP, used for the neutralization assays, was produced and titrated in the same way.

DNA and protein sequences analysis

Sequences were extracted from GenBank⁶⁶. Clades were confirmed using Nextclade⁶⁷. Nucleic and amino acid sequences of five proteins of interest were extracted from GB files for sequence ON755039.1 and MSF#10. They were generated using Prokka⁶⁸ using standard parameters and gb file from NC_063383.1 as reference. Both DNA and protein sequences were aligned using MAFFT⁶⁹ version 7.520 then visualized using Jalview⁷⁰ version 2.11.3.2 with colors indicating identity percentage. The phylogenetic tree was created using the whole DNA sequences of each sample and reference sequence of VACV Copenhagen as outgroup (OP868847.1). IQ-TREE2⁷¹ was used to generate the tree with ModelFinder⁷² and standard parameters. Visualization was produced using a homemade Python script using ETE3⁷³ module, available in the following GitHub repository: https://github.com/Mossy-Frog/Phylogeny_visualization.git.

NHP challenge

Non-human primates (NHPs) were split into groups of n = 2 or n = 4 and exposed to MPXV IIb at 1 x 10⁷ plaque-forming unit (pfu)/NHP by the rectal route (n = 4), 1 × 10⁶ pfu/NHP by the intradermal (ID) and rectal routes (ratio 1:1) (n = 2), 1 x 10⁶ pfu/NHP by the ID route (n = 2), or 1 x 10⁶ pfu/NHP by the rectal route (n = 2). Briefly, animals were anesthetized with ketamine (5 mg/kg) associated with medetomidine (0.05 mg/kg) for viral challenge. For the ID challenge, the virus was diluted in 1 mL and administered by 10 injections in the back. For the rectal challenge, animals were placed on their ventral side to raise the rectum. The virus preparation was diluted in 3 mL saline buffer and slowly applied to the rectum using a nasogastric tube. The lower part of the animals was kept elevated for several minutes until no reflux was observed. For the MVA efficacy study, eighteen NHPs were similarly challenged by the IR route at 1 × 10⁷ pfu/NHP.

MVA immunization

For the efficacy study, MVATGN33.1 (Transgene) was administered at a dose of 1 x 10⁸ pfu in 0.5 mL by the subcutaneous route. The MVA pre-exposure prophylaxis group (MVA-PrEP) (n = 4) received two doses 28 days apart, with the first dose received three months before the viral challenge. The post-exposure prophylaxis (MVA-PEP) group (n = 4) received one dose of MVA vaccine four days after the challenge.

MVA-BN vaccine has been administered at the human clinical dose of 0.5 mL containing a minimum of 5 x 10⁷ IU (Infectious Unit). The two animals used for immunogenicity study received two doses 28 days apart.

Clinical follow-up

Animals were observed every day, and clinical exams were performed at baseline and each blood draw, first when awake and then following anesthetization using ketamine (5 mg/kg) and medetomidine (0.05 mg/kg). Behavior was observed and qualified with and without human positive (alimentary reward distribution) or negative reinforcement (squeeze-back mobilization). Before handling, the animals were monitored and scored extensively, and food and water consumption were recorded, as well as behavioral abnormalities. Anorexia was scored at two levels according to fruit or pellet consumption. General observations were recorded using a dedicated grid adapted to the monitoring of general behavior, skin lesions, and mucosal lesions (mouth, eyes, and anorectal). Detected lesions were identified and scored depending on their location, severity, and number. Rectal bleeding and exudate were also reported and scored. Body weight, lymphadenomegaly, and rectal temperature were also recorded. The compilation of these observations was graded as a longitudinal follow-up. Blood cell counts, hemoglobin, and hematocrit were determined from EDTA blood using a DXH800 analyzer (Beckman Coulter).

IgG and IgA antibody quantification assay

Cynomolgus macaque serum and seminal plasma samples were screened for quantitative measurement of IgG antibodies to antigens from MPXV and VACV using the V-PLEX Orthopoxvirus Panel 1 (IgG) Kit (Meso Scale Discovery, Rockville, USA: MSD) according to the manufacturer’s instructions. The V-PLEX Orthopoxvirus Panel 1 (IgG) Kit contains MPXV antigens A29, A35, B6, E8, and M1 and VACV antigens A27, A33, B5, D8, and L1. The plates were blocked with 50 μL blocker A (1% BSA in MilliQ water) solution for at least 30 min at room temperature (RT) with shaking at 700 rpm using a digital microplate shaker. During blocking, heat-inactivated serum samples were diluted 1:500 or 1:50,000 in diluent buffer, and heat-inactivated seminal plasma samples were diluted at 1:10, 1:50, or 1:250. Each plate contained duplicate wells of a seven-point calibration curve with serial dilutions of a reference standard and a blank well. Plates were then washed three times with 150 μL MSD kit wash buffer and blotted dry. 50 μL of the diluted samples was added, and the plates were incubated at RT for at least 2 h with shaking at 700 rpm. Plates were again washed three times, 50 μL SULFO-Tagged anti-human IgG antibody were added to each well, and the plates were incubated at RT for at least 1 h with shaking at 700 rpm. Plates were then washed three times, and 150 μL MSD GOLD Read Buffer B was added to each well. The plates were read immediately using a MESO QuickPlex SQ 120 machine. The electro-chemiluminescence (ECL) signal was recorded, and the results were expressed as arbitrary units (AU)/mL. For measurement of IgA, heat-inactivated sera were diluted 1:100 in diluent buffer, and heat-inactivated rectal fluid samples were used undiluted. The same protocol as for IgG quantification was followed with detection done using a SULFO-tagged anti-human IgA antibody. Analyses were performed using Discovery Workbench 4.0.12.

Evaluation of the MVA-specific neutralizing antibody response

MVA-neutralizing antibodies in serum samples were titrated according to the standard plaque reduction neutralization test (PRNT)⁷⁴. Briefly, serum samples were subjected to serial two-fold dilutions in PBS in a total volume of 1 mL. The samples were mixed with 50 µL MVA-GFP (MVA expressing green fluorescent protein) to contain 1750 pfu/mL in the final mix, which was incubated for 1 h at 37 °C. The mixture was then diluted 10-fold, and 250 µL of the mixture was added in triplicate to DF1 cell (ATCC: CRL-3586) monolayers in six-well plates. Plates were incubated for 30 min at RT, and 2 mL of DMEM containing 5% FCS, 40 µg/L gentamicin, 2 mM glutamine, and 10 g/L agarose was then added. After solidification, plates were incubated at 37 °C in 5% CO₂. Seventy-two hours later, GFP-positive plaques were counted. The neutralization titer is expressed as the reciprocal serum dilution that led to a 50% reduction in the number of plaques compared to the negative control (serum from naïve NHP). Vaccinia immune globulin (CNJ-016) was used in each experiment as a positive control.

Evaluation of VACV-specific and MPXV-specific neutralizing antibody responses

VACV-neutralizing antibodies in serum samples were titrated according to this protocol⁷⁵. Serum samples were first incubated at 56 °C for 1 h to inactivate complement and virus and then subjected to two-fold serial dilutions in DMEM containing 0.5% FCS and ATB (100 IU penicillin/ml and 100 IU/ml streptomycin). The samples were mixed with an equal volume of VACV-107 containing approximately 35 pfu in 0.1 ml and incubated for 1 h at 37 °C. The mixture was then added in quadruplicate to Vero cell monolayers in DMEM containing 0.5% FCS and ATB. Forty-six hours later, viral plaques were counted. The neutralization titer is expressed as the reciprocal serum dilution that led to a 50% reduction in the number of plaques compared to the negative control (pool of inactivated sera from naïve NHPs). The threshold of the titration was 10. Standardized serum from smallpox-vaccinated humans (EDQM Y0000502 human vaccinia immunoglobulin) was used in each experiment as a positive control.

For the quantification of MPXV-neutralizing antibodies without complement, serum samples were first incubated at 56 °C for 1 h to inactivate complement and the virus and then subjected to two-fold serial dilutions in DMEM containing 0.5% FCS and ATB. Five hundred microliters of each dilution of the samples was mixed with 500 µL MPXV containing approximately 35 PFU in 0.1 ml and the mixture was incubated for 1 h at 37 °C. Then, 200 µL of each dilution was added in quadruplicate to Vero cell monolayers in 24-well plates. Plates were incubated for 2 h at 37 °C in 5% CO₂, and then 600 µL DMEM containing 2.5% FCS, 1% ATB, and 0.8% carboxymethyl cellulose was added. Plates were incubated for 72 h at 37 °C in 5% CO₂, and the viral plaques were counted. The neutralization titer is expressed as described for VACV neutralization.

Virus quantification by PCR in fluids and organs

Rectal, nasal, and seminal fluid and cutaneous swab samples were extracted using the QIAamp DNA Blood mini kit (Qiagen) with small modifications. Briefly, pre-extraction inactivation was performed in a BSL-3 facility by adding 200 µl of each sample to a tube containing 200 µl Qiagen buffer AL and 10 µL extraction/inhibition control DNA SPC 10-2 (Yakima Yellow-BHQ-1, Eurogentec), followed by 20 µL protease. The tubes were vortexed and incubated for 10 min at 70 °C to also inactivate the virus. Extraction was then performed as recommended, with elution in less than 100 µL of AE buffer.

Organ samples were extracted using the QIAamp DNA mini kit (Qiagen). Less than 25 mg of each organ (10 mg for spleen) was added to 80 µL PBS with two 3 mm tungsten balls (Qiagen), and the tissue was dissociated using a Tissue Lyser II® (Roche Diagnostics) with a 4 min 30 Hz cycle. One hundred microliters ATL buffer, 10 µL extraction and inhibition/control DNA SPC 10-2 (Yakima Yellow-BHQ-1, Eurogentec), and 20 µL proteinase K were added. Samples were vortexed and incubated at 56 °C, with the duration depending on the dissolution of the organs (1–12 h). Extraction was then performed as recommended, with elution in less than 100 µL AE buffer.

For the pan-MPXV PCR assays, each reaction consisted of 5 μl extracted DNA and 15 μl iTaq Universal Probes Supermix (BioRad), the Supermix containing 0.4 µM of each primer (Eurogentec: F: GGA-AAA6TGT-AAA-GAC-AAC-GAA-TAC-AG; R: GCT-ATC-ACA-TAA-TCT-GGA-AGC-GTA), and 0.2 µM probe (Eurogentec: Quasar 705 – AAG-CCG-TAA-TCT-ATG-TTG-TCT-ATC-GTG-TCC – BHQ®-3) in the pan-MPXV PCR and 1X SPC Mix (Yakima Yellow-BHQ®-1, Eurogentec). All assays were performed on a CFX96 thermocycler (BioRad). The data and results were analyzed and reported using BioRad CFX Maestro v2.3.

Detection and titration of the virus in cell culture

Samples were frozen/thawed three times and centrifuged at 1,500 x g for 5 min. All rectal fluids were homogenized and then passed through a 1.2 µm filter (Sartorius Ministart 17593 K). Filtered rectal fluid, nasal and seminal fluid, and cutaneous swab samples were diluted to various serial five-fold dilutions (from pure to 1/12,500, depending on the DNA titer measured for each sample) in DMEM containing 0.5% FCS, antibiotics, and antifungals (gentamycin: 2.5 µg/ml, Mycostatin: 10 U/mL, penicillin: 100 UI/ml, streptomycin: 100 µg/ml) to inhibit bacterial and fungal contamination. Monolayers of Vero cells in 24-well plates were inoculated with 200 μl diluted supernatants in quadruplicate for isolation and titration. Plates were incubated at 37 °C in 5% CO₂ for 2 h, and then 600 µL DMEM containing 2.5% FCS, 1% ATB (100 IU penicillin/mL, and 100 IU/mL streptomycin), and 1.6% carboxymethyl cellulose was added. Plates were incubated at 37 °C in 5% CO₂ and observed daily for cytopathic effects from days 3 to 7. Samples were considered negative for infectious virus in the absence of the appearance of lytic plaques by day 14.

Organs were dissociated in DMEM containing 0.5% FCS and ATB (100 IU penicillin/mL, and 100 IU/mL streptomycin) with two 3 mm tungsten balls (Qiagen) in a Tissue Lyser II® (Roche Diagnostics) with a 4 min, 30 Hz cycle. Dissociated samples were frozen/thawed three times and centrifuged at 1500 x g for 5 min. Monolayers of Vero cells in 24-well plates were inoculated with 200 µL of the diluted supernatants in quadruplicate for isolation and titration. The plates were incubated at 37 °C in 5% CO² for 2 h, and then 600 µL DMEM containing 2.5% FCS, 1% ATB (100 IU penicillin/mL, and 100 IU/mL streptomycin), and 1.6% of carboxymethyl cellulose was added. Plates were incubated at 37 °C in 5% CO₂ and observed daily for cytopathic effects from days 3 to 7. Samples were considered negative for infectious virus in the absence of the appearance of lytic plaques by day 14.

Histopathology

At necropsy, organs were fixed by immersion in 10% formalin solution for 48 h. Formalin-fixed samples were paraffin-embedded (FFPE) using a vacuum inclusion processor (Excelsior, ThermoScientific), cut into 4 µm (Microtome RM2255, Leica) slices, mounted on coated glass slides (Superfrost + , ThermoScientific), and stained with hematoxylin and eosin (H&E) using an automated staining processor (Autostainer ST5020, Leica). Each slide was then scanned using Axioscan microscope scanner (Zeiss) and observed using Zen software version 3.7 (Zeiss) by a certified veterinary pathologist (DVM, DESV-AP).

Immunohistochemistry

Serial sections of rectal mucosa and skin tissue were stained for CD3, CD20, CD68, calprotectin, and vaccinia virus. Briefly, the slides were deparaffinized and stained using a Ventana Ultra instrument (Roche) with CC1 antigen retrieval and 32 min incubation with the primary antibody. See the following Table 2 for the references.

In situ hybridization

Serial sections were stained for monkeypox, UBC, and DapB using a Ventana Ultra device and the following probes with the RNAScope® 2.5 Assay (ACD^TM Bio), see Table 3.

Briefly, slides were deparaffinized and pretreated at 97 °C for 24 min in Cell Conditioning Buffer (Roche), followed by six amplification cycles, and then stained with purple chromogen (Discovery Purple Kit, Roche). Slides were then counterstained with Hematoxylin II (Roche).

Intracellular staining of PBMCs

T-cell responses were characterized by measuring the proportion of Peripheral Blood Mononuclear Cells (PBMCs) expressing IL-2, IL-17a, IFN-γ, TNF-α, CD137, and CD154 upon stimulation with MPXV IIb virus (see above) or MVATGN33.1 (Transgene). CD3, CD4, and CD8 antibodies were used as lineage markers. The Table 4 below recapitulates the references of the antibodies used.

One million PBMCs were cultured in complete medium (RPMI1640 Glutamax+, [Gibco], supplemented with 10% FBS and 5% penicillin-streptomycin) supplemented with co-stimulatory antibodies (FastImmune CD28/CD49d, Becton Dickinson). Then, cells were stimulated with the virus at a final concentration of 0.3 pfu/cell. Brefeldin A was added to each well 5 hours later to a final concentration of 10 μg/mL, and the plate was incubated at 37 °C in 5% CO₂ for an additional 13 h. Cells were then washed, stained with a viability dye (LIVE/DEAD Fixable Blue Dead Cell Stain Kit, Thermo Fisher), and fixed and permeabilized with BD Cytofix/Cytoperm reagent. Permeabilized cells were stored at −80 °C before the staining procedure. Antibody staining was performed in a single step following thawing. After 30 minutes of incubation at 4 °C in the dark, cells were washed in BD Perm/Wash buffer, and the data were acquired using the ZE5 flow cytometer (Bio-Rad) and analyzed using FlowJo v10 software. The gating strategy used to quantify each cytokine production in CD4 and CD8 cells is described (Fig. S18).

Data and statistical analysis

Data were analyzed using Prism v10 (GraphPad). Comparisons were performed using a Kruskal-Wallis test, followed by a non-parametric two-sided Mann-Whitney test. For the statistical analysis of the area under the curve for the virus in rectal fluid, the value of 0 was replaced by 1 for representation and statistics. Correlation analysis was done using a two-tailed non-parametric Spearman correlation test.

Reporting summary

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