A mouse model of lethal infection for evaluating prophylactics and therapeutics against Monkeypox virus

Abstract

Monkeypox virus (MPXV) is an orthopoxvirus closely related to variola, the etiological agent of smallpox. In humans, MPXV causes a disease similar to smallpox and is considered to be an emerging infectious disease. Moreover, the use of MPXV for bioterroristic/biowarfare activities is of significant concern. Available small animal models of human monkeypox have been restricted to mammals with poorly defined biologies that also have limited reagent availability. We have established a murine MPXV model utilizing the STAT1-deficient C57BL/6 mouse. Here we report that a relatively low-dose intranasal (IN) infection induces 100% mortality in the stat1(-)(/)(-) model by day 10 postinfection with high infectious titers in the livers, spleens, and lungs of moribund animals. Vaccination with modified vaccinia virus Ankara (MVA) followed by a booster vaccination is sufficient to protect against an intranasal MPXV challenge and induces an immune response more robust than that of a single vaccination. Furthermore, antiviral treatment with CMX001 (HDP-cidofovir) and ST-246 protects when administered as a regimen initiated on the day of infection. Thus, the stat1(-)(/)(-) model provides a lethal murine platform for evaluating therapeutics and for investigating the immunological and pathological responses to MPXV infection.

FIG. 1.

Dose response to MPXV challenge in female and male C57BL/6 stat1^−/− mice. Male and female mice were inoculated with 4.7 to 4,700 PFU of MPXV and monitored for death (A and C) and weight loss (B and D). The higher doses of 4,700 and 470 PFU induced significant weight loss from approximately 4 days p.i. * indicates 1 death at day 21 (75% survival). n = 5 mice per group.

FIG. 2.

Day 8 tissue titers. Liver, spleen, and lung titers from mice sacrificed on day 8 p.i. n = 5 mice per group.

FIG. 3.

Histopathology (H and E) of the cerebrum, optic nerve, nasal turbinates, and nasal cavity. (A) Ventral cerebrum and optic nerve from C57BL/6 stat1^−/− mice. Acute leptomeningitis (arrow) characterized by neutrophil and macrophage infiltration and fibrin deposition was observed with infected mice of both strains (magnification, ×200). (B) Cerebrum from the C57BL/6 stat1^−/− strain presented with mild acute leptomeningitis (arrow) characterized by neutrophil and macrophage infiltration, which was observed with infected mice of both strains (magnification, ×100). (C) Nasal turbinate from the C57BL/6 stat1^−/− strain presented with fibrinopurulent exudate (arrow) adherent to nonulcerated respiratory epithelium in infected mouse (magnification, ×200). (D) Nasal turbinate from the C57BL/6 stat1^−/− strain presented with fibrinopurulent rhinitis with inflammatory cells extending throughout the respiratory submucosa. Fibrinopurulent exudate (circled E) occludes the lumen of much of nasal cavity (magnification, ×200). Pictures are typical results from experiments in which n = 7.

FIG. 4.

Protection of single and double MVA-vaccinated male and female stat1^−/− mice following MPXV infection. (A) Survival of C57BL/6 stat1^−/− mice following a primary MVA vaccination at 56 days before MPXV challenge (d-56) or following a primary vaccination with a booster vaccination at 28 days before infection (d-56, d-28). (B) Weight change in vaccinated and infected animals (n = 21, 7, 9, 19, and 8 for stat1 MVA d-56, d-28; stat1 NI NT; stat1 placebo; stat1 MVA d-56; and B6 MVA d-56, d-28, respectively). (C and D) Infected and noninfected CD8⁺ and CD4⁺ intracellular IFN-γ levels from blood taken 7 days before infection. (E and F) Antibody responses at day −30 and day −7 following the primary MVA vaccination and following a booster vaccination. NI indicates not infected; NT indicates no treatment; d-56 indicates MVA primary vaccination at 56 days before infection; d-28 indicates MVA booster vaccination at 28 days before infection; pl indicates placebo; an MOI of 0 is mock infected; and B6 indicates C57BL/6 wild type; OD₄₉₀, optical density at 490 nm.

FIG. 5.

CMX001 and ST-246 partially protect against lethal MPXV challenges. C57BL/6 stat1^−/− mice were intranasally infected with 5,000 PFU of MPXV and treated with 10 mg/kg of CMX001 on the day of infection followed by every-other-day dosing of 2.5 mg/kg until 14 days p.i., or C57BL/6 stat1^−/− mice were treated with 100 mg/kg of ST-246 daily for 10 days starting on the day of infection. Survival curves and weight changes are shown (A and B, respectively).Vehicle (veh) mice received saline gavages according to the CMX001 dosing regimen or according to the ST-246 dosing regimen (CMC veh). NI indicates noninfected mice. Arrows indicate rechallenge with MPXV at day 38 p.i. n = 5 per group.

FIG. 6.

Anti-orthopoxvirus serum neutralization in mice treated with CMX001 compared to those treated with ST-246. Serum from mice 28 day p.i. was used to determine the 50% serum neutralization values of VACV-infected BSC-1 cells. The dashed line indicates the 50% neutralization value.