The data presented in this study clearly demonstrate that ST-246 is efficacious for prophylactic, postexposure, and therapeutic (after rash onset) treatment of monkeypox virus infection in prairie dogs. Animals inoculated with an infectious dose of virus equivalent to 65× the LD50
and administered antiviral treatment initiated on the day of infection or by 3 days postinfection were asymptomatic. Furthermore, all infected animals that began antiviral treatment following rash onset survived infection, whereas three infected animals that did not receive antiviral treatment became severely ill and died. These 3 animals (PD5, PD8, and PD15) underwent an asymptomatic period of 8 days until symptoms consistent with an upper respiratory infection were observed. The disseminated rash observed on PD8 consisted of variously sized, vesicular lesions on multiple areas of the face and body. Although a pustular rash was not observed on PD15, its death on day 10 likely preceded rash eruption. Previous studies by Hutson et al. have shown that rash onset with a Congo Basin strain of MPXV typically occurs on days 9 to 13 p.i. (12
). The rash observed on PD5 was a petechial rash and was present in addition to bleeding from the nose. These symptoms are reminiscent of symptoms from human cases of hemorrhagic smallpox, which is a more virulent form of smallpox than ordinary smallpox. Each of these 3 prairie dogs was losing substantial weight at the time of their deaths, and PCR and culture analysis of tissues harvested at necropsy demonstrated that virus had spread to vital target organs, including liver, kidney, and spleen, which probably contributed to their deaths. Unexpectedly, PD6 in this group was asymptomatic; and although random samples were DNA positive, no infectious virus was detected from swab samples, and it did not seroconvert by day 30, suggesting that it was not successfully infected at the time of inoculation.
Similarly, prairie dogs in the rash onset treatment group were asymptomatic for 8 days and then developed upper respiratory symptoms identical to those in the vehicle control group. After antiviral treatment was initiated at rash onset, symptoms generally resolved in 10 days. Specifically, 3 of the 4 prairie dogs (PD2, PD12, and PD14) developed a rash on the same day (10
) as the vehicle control “untreated” animal, PD8. Rash progressed as expected in two (PD14 and PD2) of these animals and had resolved by day 16 or 22, respectively. In contrast, the macular rash observed on PD12 did not progress and quickly resolved by day 14. These findings are quite remarkable compared to historic control prairie dogs infected with doses as high as 8 × 105
PFU of the same MPXV strain as used in this study; in these animals lesions did not resolve until 24 to 27 days postinoculation (9
). PD12 also did not lose weight or produce measurable levels of antiorthopoxvirus antibodies. Interestingly, PD3 developed late-onset rash on day 24, developed the greatest number of lesions (50+), and produced the highest levels of antibodies. Hutson et al. also observed one prairie dog that experienced a late onset of infection with lesions developing on day 27 p.i. and ultimately succumbing to infection (9
). However, for PD3, weight loss and other observable symptoms were not dissimilar from symptoms of the other animals in this group. Moreover, once ST-246 treatment began, the lesions dried and resolved over a period similar to that of the therapeutically treated animals. Differences in rash onset and progression in these animals are difficult to explain. Since these prairie dogs are wild-caught, outbred animals, there is individual variability in their host responses to viral infection and in their responses to drug treatment, both of which could influence disease progression.
Because the prairie dogs in the vehicle-only control group either died early in the study or were not infected, a comparison can be made of the clinical, virological, and molecular data only between therapeutically treated animals and vehicle control animals through day 12. Up to that point, concentrations of MPXV DNA and infectious virus followed the same trends for both groups. In animals treated therapeutically, MPXV DNA and infectious virus reached peak values for all sample types, including blood, at or within 4 days of rash onset and then decreased with initiation of ST-246. Viral DNA was detected from 6 days prior to 20 days post-rash onset, and infectious virus was recovered from 6 days prior to 16 days post-rash onset. The greatest amounts of viral DNA and viable virus were obtained from oral secretions, followed equally by ocular and anal secretions. The absence of an infected survivor from the vehicle control group prevented a comparison between the molecular findings and the duration of recovery for animals treated therapeutically and untreated animals. This analysis would have been beneficial for more adequately describing the impact that ST-246 has on viral clearance and disease recovery in this animal model.
Animals treated prophylactically (day 0) or postexposure (day 3) were protected from observable illness and death. None of the animals in either of these groups presented visible symptoms, including rash development. Instead, they appeared healthy, remained active, and maintained or increased body weight. While on drug treatment, viral DNA was largely undetected in blood, and little to no viral shedding was detected in ocular and anal secretions. Oral secretions yielded the greatest numbers of infectious virus, with peak concentrations measuring 2 to 3 logs less than those of untreated and therapeutically treated animals. Ocular and anal secretions yielded lower or undetectable concentrations of virus, and detection was limited to fewer days postinoculation. These results could be considered consistent with the proposed mechanism that ST-246 inhibits production and release of EV from infected tissues. A reduction in EV, which is considered responsible for spread of the virus within a host, may have limited the infection to localized areas near the intranasal inoculation site, such as the nasal and oral cavities, thereby lessening the duration and morbidity of illness. When prophylactic or postexposure use of drug was discontinued, although animals remained asymptomatic, virus could be measured in secretions and excretions. Interestingly, trends in the range of days that infectious virus was detected differed between the two groups. Detection in animals treated prophylactically began near the end (day 12) of antiviral treatment, increased by 2 to 3 logs following treatment cessation, and persisted until day 24. Detection in animals treated postexposure was generally restricted to days 8 to 12, with the exception of one animal, PD7, which shed virus until day 26. While both prophylactic and postexposure treatments were successful for preventing illness, virus was not ultimately cleared by the end of treatment.
An unanticipated observation was the recovery of infectious virus from animals following cessation of drug exposure. One explanation could have been the presence of drug-resistant viruses. Highly drug-resistant CPXV variants, acquired through passaging of virus in the presence of drug, have been previously described from wild-type CPXV (27
). We evaluated for the presence of drug resistance in select, twice-passaged virus isolates from prairie dog samples by a comet reduction assay. ST-246 antiviral activity could be demonstrated by reductions in plaque size and comet formation which are both produced by release of EV. The sensitivity of this assay was shown previously to be approximately equivalent to a standard EC50
). Additionally, isolates obtained postcessation or near the end of therapy, evaluated for ST-246 resistance without additional passaging, showed drug susceptibility similar to that of wild-type MPXV-ROC-358. Our results do not suggest that significant drug resistance was found in monkeypox viruses isolated postcessation of drug treatment. However, a prophylaxis, postexposure, or therapeutic regimen extending beyond 14 days, such as 21 days, may eliminate posttreatment viral shedding. This will need to be studied in the context of the development of an immunologic response to infection.
Comparison of antibody production between the three treatment groups suggests that timing of treatment initiation and the duration of treatment influence the humoral immune response. Prophylactic, postexposure, and therapeutic treatment resulted in three different antibody response profiles as measured by ELISA. Three of the prairie dogs treated therapeutically began producing measurable levels of Ig within 2 to 4 days following rash onset, and levels sharply increased to peak levels by 16 to 23 days post-rash onset. On the other hand, prairie dogs treated prophylactically produced measurable levels of Ig 4 to 10 days following rash onset, and peak levels were approximately half those of the therapeutic animals. Finally, prairie dogs treated postexposure produced measurable levels of Ig within 2 to 4 days post-rash, and peak levels were approximately a fourth of the levels of the therapeutic animals, with the exception of one animal. Differences in the immune profiles could be related to the availability of immunodominant EV epitopes, such as A33R and B5R; this has not yet been tested.
In several prairie dogs, clearance of infectious virus was temporally associated with antibody production. In the prophylactic group, PD11 and PD16 produced measurable levels of antibodies and eliminated infectious virus (oral swabs) 4 to 6 days earlier than PD10 and PD17. Similarly, in the postexposure group, PD1, PD4, and PD13 produced antibodies 6 to 8 days earlier than PD7 and cleared infectious virus from oral secretions by day 12. Antiorthopoxvirus antibodies were not measured in PD7 until day 20, and virus was not eliminated until day 26. Although cell-mediated responses were not addressed here, the temporal association of clearance of infectious virus and development of anti-orthopoxvirus antibodies suggests that the humoral immune response is important for virus clearance in the prairie dog model of MPXV infection.
Overall, prophylactic and postexposure ST-246 treatment prevented illness, and therapeutic ST-246 treatment prevented death in prairie dogs infected with a virulent strain of MPXV. We used the intranasal route of inoculation in order to more closely resemble a natural route of infection. Disease progression, including an asymptomatic period followed by systemic rash, closely resembled MPXV infection in humans, reaffirming the utility of the prairie dog system as a model for human systemic OPXV infection. These characteristics make the prairie dog a suitable model for investigating and identifying antiviral therapeutics and treatment regimens for monkeypox or smallpox in humans. Our results support future investigations with ST-246 to further characterize postexposure treatment modalities in the prairie dog model. These studies may be important for providing information that can be used for the identification of a safe and efficacious antiviral compound meeting the standards for federal approval.