In vitro efficacy of ST246 against smallpox and monkeypox

Abstract

Since the eradication of smallpox and the cessation of routine childhood vaccination for smallpox, the proportion of the world's population susceptible to infection with orthopoxviruses, such as variola virus (the causative agent of smallpox) and monkeypox virus, has grown substantially. In the United States, the only vaccines for smallpox licensed by the Food and Drug Administration (FDA) have been live virus vaccines. Unfortunately, a substantial number of people cannot receive live virus vaccines due to contraindications. Furthermore, no antiviral drugs have been fully approved by the FDA for the prevention or treatment of orthopoxvirus infection. Here, we show the inhibitory effect of one new antiviral compound, ST-246, on the in vitro growth properties of six variola virus strains and seven monkeypox virus strains. We performed multiple assays to monitor the cytopathic effect and to evaluate the reduction of viral progeny production and release in the presence of the compound. ST-246 had 50% effective concentrations of <or=0.067 microM against variola virus and <0.04 microM against monkeypox virus. In a dose-dependent manner, plaque size and comet tail formation were markedly reduced in the presence of the drug at low, noncytotoxic concentrations between 0.015 and 0.05 microM. Our in vitro phenotype data suggest that ST-246 inhibits variola and monkeypox viruses similarly by reducing the production and release of enveloped orthopoxvirus and support the development of ST-246 as an antiviral therapeutic compound for the treatment of severe systemic orthopoxvirus infections.

FIG. 1.

ST-246 dose-response curves. BSC-40 cells were infected with VARV (top panel) or MPXV (bottom panel) at an MOI of 0.1. The CPE was evaluated by plotting the absorbance at 570 nm of crystal violet-stained cell monolayers versus the concentration of ST-246. The results represent the means ± the standard deviations of three independent measurements.

FIG. 2.

Plaque size reduction assays. Confluent monolayers of BSC-40 cells were infected with 25 PFU of VARV (top panel) or MPXV (bottom panel). Infected cells were incubated with various concentrations of ST-246 (none, 0.5, 0.15, 0.05, 0.015, or 0.005 μM) in RPMI (2 ml) containing 2% FBS and 1% CMC. Plaque sizes were visualized by IHC staining.

FIG. 3.

Comet tail formation reduction assays. Confluent monolayers of BSC-40 cells were infected with 25 PFU of VARV (top panel) or MPXV (bottom panel). Infected cells were incubated at a fixed angle with various concentrations of ST-246 (none, 0.5, 0.15, 0.05, 0.015, or 0.005 μM) in RPMI (2 ml) containing 2% FBS. Comet tails were visualized by IHC staining.