Effective antiviral treatment of systemic orthopoxvirus disease: ST-246 treatment of prairie dogs infected with monkeypox virus

Abstract

Smallpox preparedness research has led to development of antiviral therapies for treatment of serious orthopoxvirus infections. Monkeypox virus is an emerging, zoonotic orthopoxvirus which can cause severe and transmissible disease in humans, generating concerns for public health. Monkeypox virus infection results in a systemic, febrile-rash illness closely resembling smallpox. Currently, there are no small-molecule antiviral therapeutics approved to treat orthopoxvirus infections of humans. The prairie dog, using monkeypox virus as a challenge virus, has provided a valuable nonhuman animal model in which monkeypox virus infection closely resembles human systemic orthopoxvirus illness. Here, we assess the efficacy of the antiorthopoxvirus compound ST-246 in prairie dogs against a monkeypox virus challenge of 65 times the 50% lethal dose (LD(50)). Animals were infected intranasally and administered ST-246 for 14 days, beginning on days 0, 3, or after rash onset. Swab and blood samples were collected every 2 days and analyzed for presence of viral DNA by real-time PCR and for viable virus by tissue culture. Seventy-five percent of infected animals that received vehicle alone succumbed to infection. One hundred percent of animals that received ST-246 survived challenge, and animals that received treatment before symptom onset remained largely asymptomatic. Viable virus and viral DNA were undetected or at greatly reduced levels in animals that began treatment on 0 or 3 days postinfection, compared to control animals or animals treated post-rash onset. Animals treated after rash onset manifested illness, but all recovered. Our results indicate that ST-246 can be used therapeutically, following onset of rash illness, to treat systemic orthopoxvirus infections.

Figures

Fig. 1.

Percent fluctuations in weight. Prairie dogs were infected on day 0 with 3.8 × 105 PFU, and weights were measured every 2 days for a period of 30 days. The percent change in weight for each measurement was calculated from the starting weights on day 0 and is plotted as a function of days postinfection for each treatment group. Untreated animals (A) lost weight until death, except for animal PD6. Animals that received ST-246 treatment at rash onset (B) on day 10 (PD2, PD12, and PD14) or day 24 (PD3) lost weight during illness and regained weight upon recovery, except for PD12. Uninfected control animals (not shown) and animals that received ST-246 treatment beginning on day 0 (C) or day 3 (D) steadily increased in weight.

Fig. 2.

Quantitation of viral DNA in blood from prairie dogs infected with MPXV. Treatment with ST-246 reduces MPXV DNA in blood. Prairie dogs were infected on day 0 with 3.8 × 105 PFU, and blood was collected every 2 days over a period of 30 days. Animals began receiving vehicle alone on day 3 (A) or received ST-246 at rash onset (B), on day 0 (C), or on day 3 (D). DNA was extracted, and the E9L gene was amplified and quantitated by real-time PCR. Average values of genome copies per milliliter and standard deviations were plotted on a log scale as a function of day postinfection (A, C, and D) or day post-rash onset (B).

Fig. 3.

Quantitation of viral DNA in secretions from prairie dogs infected with MPXV. Early treatment with ST-246 greatly reduces MPXV DNA in secretions. Prairie dogs were infected on day 0 with 3.8 × 105 PFU, and swab samples were collected every 2 days over a period of 30 days. Animals began receiving vehicle alone on day 3 (A, B, and C) or ST-246 at rash onset (D, E, and F), on day 0 (G, H, and I), or on day 3 (J, K, and L). DNA was extracted from oral swabs (A, D, G, and J), ocular swabs (B, E, H, and K), and anal swabs (C, F, I, and L). The E9L gene was amplified and quantitated by real-time PCR. Average values of genome copies per milliliter and standard deviations were plotted on a log scale as a function of day postinfection (A, B, C, G, H, I, J, K, and L) or day post-rash onset (D, E, and F).

Fig. 4.

Quantitation of infectious virus in secretions from prairie dogs infected with MPXV. Treatment with ST-246 reduces infectious virus in secretions. Prairie dogs were infected on day 0 with 3.8 × 105 PFU, and swab samples were collected every 2 days over a period of 30 days. Animals began receiving vehicle alone on day 3 (A, B, and C) or ST-246 at rash onset (D, E, and F), on day 0 (G, H, and I), or on day 3 (J, K, and L). Infectious virus was recovered from oral swabs (A, D, G, and J), ocular swabs (B, E, H, and K), and anal swabs (C, F, I, and L) by tissue culture. The number of PFU was quantitated by plaque counts, and average values of PFU per milliliter and standard deviations were plotted on a log scale as a function of day postinfection (A, B, C, G, H, I, J, K, and L) or day post-rash onset (D, E, and F).

Fig. 5.

Quantitation of viral DNA and infectious virus in tissues of prairie dogs infected with MPXV. High concentrations of viral DNA and infectious virus were detected in tissues from prairie dogs that received vehicle treatment. Prairie dogs were infected with 3.8 ×105 PFU and administered vehicle daily from day 0 until death. PD5 and PD15 died on day 10, and PD8 died on day 12. Heart, liver, kidney, lung, skin, spleen, lymph node (LN), tongue, and eyelid were harvested for molecular analysis by RT-PCR and tissue culture. Average values for genome copies per milliliter (A) and PFU per gram of tissue (B) along with standard deviations are plotted on log scale as a function of tissue type.

Fig. 6.

Production of anti-OPXV antibodies in prairie dogs infected with MPXV. Initial detection and strength of antibody production in prairie dogs infected with MPXV produced different immune profiles dependent on ST-246 treatment regimen. Prairie dogs were infected with 3.8 × 105 PFU, and serum was collected from blood drawn every 2 days over a period of 30 days. Animals began receiving vehicle alone on day 3 (A) or ST-246 at rash onset (B), on day 0 (C), or on day 3 (D). Serum was diluted 1:100 and tested against whole, inactivated vaccinia virus, and antibody production was measured by ELISA. Test serum was considered positive if its absorbance value was above the COV, which was determined by averaging OD measurements of BSC-40 cell lysates and adding 2 standard deviations.

Fig. 7.

PD3 developed late-onset rash on day 24. Lesions, numbering greater than 50, were present on areas of the face, mouth, tongue, abdomen, back, extremities, and groin. Pustular lesions present on the tongue on day 24 (A) resolved by day 40 (B), and lesions present on the back formed scabs by day 28 (C) and mostly resolved by day 40 (D).

Fig. 8.

Evaluation of drug resistance in posttreatment virus isolates from prairie dogs by comet reduction. Approximately 50 PFU from swab isolates, VACV-IHDJ, and ST-246-resistant VACV-N267D was inoculated onto confluent monolayers of BSC-40 cells. Cultures were incubated with or without ST-246 for 2 (IHDJ and N267D) or 3 days (all PD isolates) and fixed with PBS containing formalin. Plaques and comets were visualized by IHC.

Fig. 9.

Evaluation of drug resistance in posttreatment virus isolates from prairie dog 5 by plaque reduction. Approximately 50 PFU from oral swab isolates, ST-246-resistant VACV-N267D, and wild-type MPXV-ROC-358 was inoculated onto confluent BSC-40 cells in the presence or absence of ST-246. Cultures were grown for 3 days and fixed with PBS containing formalin. Plaques were visualized by IHC.