A Mouse Model of Lethal Infection for Evaluating Prophylactics and Therapeutics against Monkeypox Virus

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.During the early smallpox-free epoch, the orthopoxviruses were of minor bioterroristic concern due to the largely vaccinated population; however, this has changed with the increased risk of bioterrorism, and variola virus (VARV) and monkeypox virus (MPXV) are considered to have significant potential to become bioterror agents (36, 37). VARV, the etiological agent of smallpox, is officially stored at two WHO secure laboratories in the United States and Russia; however, there is concern that covert stocks exist. Furthermore, we are currently faced with the possibility of intentional release of wild-type or genetically modified VARV. Of most concern would be viruses encoding human interleukin-4 (IL-4), which could significantly increase virulence, as demonstrated with the mousepox/ectromelia virus (ECTV) model (18). As a result of the cessation of routine vaccination and the high number of individuals that are contraindicated for vaccination, the human population lacks solid “herd immunity” to naturally circulating orthopoxviruses. One such virus that is of particular concern is MPXV, due to its ability to infect humans, its mortality rate of approximately 10% (depending on the strain), its propensity to infect a large number of species, its apparently increasing transmissibility in the human population, and its reportedly expanding host range (36). One such example of increasing host range was observed during 2003 in the United States, where imported African rodents transmitted MPXV to native prairie dogs, which acted as an “amplification reservoir” that allowed for the transmission of MPXV to humans (14).To date, MPXV animal models for efficacy testing of prophylactics and therapeutics have been restricted to nonhuman primates and nonmurine small animal models, such as the 13-lined ground squirrel (Spermophilus tridecemlineatus) (49, 55), the black-tailed prairie dog (Cynomys ludovicianus) (14, 17, 22, 60), and the African dormouse (Graphiurus kelleni) (50). Because the ground squirrel and the prairie dog are difficult to propagate, have low fecundity rates, and have complex husbandry requirements, they must be obtained from their natural habitat and therefore have unknown health statuses (16, 58). Conversely, the African dormouse has many characteristics similar to those of laboratory mice and can be easily propagated in a research vivarium. The disadvantage to this model is that there are few commercially available reagents for characterizing the animals'' response to infection, and their biology is poorly understood (50).Suckling white mice have been shown to be highly susceptible to MPXV inoculations by various routes. Eight-day-old white mice developed disease and died following intraperitoneal or intranasal (IN) inoculations with 1.2 × 10⁶ PFU. Injection into the footpad also induced severe disease and death following 6 × 10² PFU inoculations. Disease symptoms included flabbiness; loss of appetite; and following footpad infections, edema of the foot. Similar symptoms were observed following inoculation by the oral route, which induced 40% lethality. Intradermal inoculations with MPXV resulted in 50% death. The intranasal route of infection was determined to induce the highest level of lethality, causing 100% death in mice as old as 15 days, compared to only 14% and 60% lethality in 12-day-old mice infected orally or via the footpad, respectively (24, 51). Recently, Osorio et al. showed that 4-week-old SCID-BALB/c mice are susceptible to 10⁵ PFU intraperitoneal (IP) MPXV inoculations, resulting in a mean day of death of 9 days postinfection (p.i.) (33). Unfortunately the IP route of infection does not model the natural transmission route of MPXV.Several factors make young (<15 days old) white mice a poor choice for studying MPXV. First, mice do not become fully immunocompetent until approximately 4 or 5 weeks old; therefore, the opportunity to study the immune response to infection is hampered. Moreover, immunoimmature animals cannot be used as models to study MPXV infections in immunocompetent humans. Second, a functioning immune system works in synergy with antiviral therapies to provide protection against viral challenge; thus, antiviral efficacy cannot be properly evaluated. Third, the relatively short susceptible time window of birth to 15 days old makes large-scale experiments impractical. Fourthly, young immunoimmature mice cannot be used to study vaccination efficacy. To this end, we sought to identify adult mice that are susceptible to lethal MPXV challenges and can be used for antiviral and vaccination efficacy studies.In the present study we found that most common strains of adult immunocompetent laboratory mice are resistant to MPXV. We also found that type 1 and type 2 interferon (IFN) receptor-null mice were resistant. Because strains lacking STAT1, a key protein involved in type 1 and 2 IFN signaling networks, have been shown to be sensitive to a wide number of viral and bacterial infections (13, 15, 30, 46, 52-54), we evaluated their sensitivities to MPXV challenges. We found that C57BL/6 mice lacking stat1 (C57BL/6 stat1^−/−) were highly sensitive to MPXV and that 129 mice lacking stat1 were sensitive but to a lesser degree than the C57BL/6 stat1^−/− animals. In this report, we show that the disease course in MPXV-infected C57BL/6 stat1^−/− mice, that is, weight loss and death by day 10 postinfection, is similar to that observed in wild-type mice infected with ECTV, the etiological agent of mousepox (11). Further, we reveal that antiviral therapy with CMX001 or ST-246 protects mice to a degree similar to that of vaccination with Dryvax or modified vaccinia virus Ankara (MVA), supporting the use of the C57BL/6 stat1^−/− as a model to evaluate orthopoxvirus prophylactics and therapeutics.