Experimental design. Each group consisted of six adult cynomolgus macaques (Macaca fascicularis). For group I animals, MVA-BN (10⁸ PFU) was administered by s.c. injection on days −28 and 0; for group II animals, an MVA-BN primer dose (2 × 10⁶ PFU) was administered by s.c. injection on day −10 and Elstree-RIVM was administered i.c. with a bifurcated needle on day 0; for group III animals, Elstree-RIVM was administered i.c. with a bifurcated needle on day 0; for group IV, Elstree-BN was administered i.c. with a bifurcated needle on day 0; and group V consisted of nonvaccinated animals. Fifteen weeks after the (last) vaccination, all animals were challenged i.t. with MPXV strain MSF#6 in the trachea by intubation.

Body temperature profiles after respiratory inoculation with 10⁶ (a) or 10⁷ (b) PFU of MPXV strain MSF#6. The macaques were not vaccinated or had been vaccinated with two doses of MVA-BN, one low dose of MVA-BN followed by Elstree-RIVM, Elstree-RIVM alone, or Elstree-BN. Each macaque had an active temperature transponder in its peritoneal cavity. The animals exhibited differences in their baseline temperatures (37.7 ± 0.9°C) and slight shifts in their day-night temperature cycles. Therefore, the data are expressed as percentages of the average temperature change per day per group with (upper limit) 95% confidence intervals. The dotted line denotes a 1°C temperature increase above the average baseline.

Monkeypox viral loads in plasmas and throat swabs from vaccinated and control animals at different time points after respiratory inoculation with 10⁶ PFU (left graphs) or 10⁷ PFU of MPXV (right graphs). The data are expressed as average log₁₀ values per group plus SD. (a) Viral loads in throat swabs, as measured by real-time quantitative PCR; (b) plasma viremia, as measured by real-time quantitative PCR; (c) viral loads in throat swabs, as measured by quantitative virus isolation in Vero cell monolayers. †, natural death; X, euthanasia. (d) AUCs of the viral loads in plasmas and throat swabs from vaccinated and control animals over a 28-day follow-up after respiratory inoculation with 10⁶ PFU (left graph) or 10⁷ PFU of MPXV (right graph). The data are expressed as average log₁₀ values per group plus SD.

Reactivities and immunogenicities of different smallpox vaccines. (a) Sizes (areas) of vaccine-induced pocks measured on day 7 at the site of s.c. inoculation of MVA (groups I and II) or i.c. inoculation of Estree-RIVM (groups II and III) or Elstree-BN (group IV). (b, left side) Induction of MVA-specific IFN-γ-secreting cells, as measured by an ELISPOT assay. PBMC were isolated before vaccination, 4 and 9 weeks after the (last) vaccination, and at the moment of challenge (week 15). The data are expressed as the average numbers of specific IFN-γ-secreting cells in 300,000 PBMC per group ± SD. (b, right side) Induction of MVA-specific lymphoproliferation, as determined by fluorescence-activated cell sorting after a BrdU incorporation assay. PBMC were isolated before vaccination and 3 weeks after the (last) vaccination. The data are expressed as the average SI per group ± SD. (c) Development of specific plasma IgG responses in samples collected on different days after vaccination, as measured by ELISA. The data are expressed as average titers per group ± SD. (d) Vaccinia virus-specific neutralizing plasma antibody titers in samples collected on different days after vaccination, as measured by a plaque reduction assay. The moments of vaccination are indicated with the respective symbols. The data are expressed as average 50% plaque reduction titers (PRNT) per group ± SD.

Histological lesions and ultrastructural detection of MPXV in lung and skin of a nonvaccinated macaque. (a) Edge of typical cutaneous lesion characterized by epidermal hyperplasia, orthokeratotic hyperkeratosis, ballooning degeneration of keratinocytes (arrowhead) with necrosis, and infiltration by inflammatory cells towards the center (arrow). (b) Necrosis of pulmonary alveolar wall, indicated with arrowheads, and flooding of alveolar lumen with fibrin, edema fluid, foamy macrophages, degenerative neutrophils, and necrotic cellular debris. (c) Mature (arrowhead) and immature poxvirus particles in keratinocyte. Bar, 5,000 nm. (d) Poxvirus particles in necrotic alveolar wall. Bar, 5,000 nm. (Inset) Higher magnification of mature poxvirus particle with characteristic dumbbell structure of the core (left) and immature poxvirus particle (right). Sections were stained with hematoxylin and eosin (a and b) or with uranyl acetate and lead citrate (c and d). Original magnification, ×10 (a) or ×40 (b).