ST-246 Inhibits In Vivo Poxvirus Dissemination, Virus Shedding, and Systemic Disease Manifestation

Smallpox disease is marked by the dissemination of variola virus within the infected host and the appearance of skin lesions or pocks (reviewed in reference 3 and at http://whqlibdoc.who.int/smallpox/9241561106.pdf). The virus is transmitted between humans mainly by aerosol droplets; however, contact with variola virus-contaminated clothing or bedding may contribute to transmission. Once inhaled, variola virus appears to first infect the upper- or lower-respiratory-tract mucosa and spread to and replicate within the local lymph nodes. The virus then disseminates to the spleen and liver via a transient viremia and replicates in reticuloendothelial cells through an average incubation period of 10 to 12 days. During the high-fever prodrome period that follows, a second wave of viremia occurs and results in the dissemination of the virus to mucous membranes of the mouth and pharynx and to the dermal epithelium of the skin. This dissemination leads to the eruption of lesions over the tongue, mouth, and oropharynx and of rashes that start at the face and extremities and may eventually envelope the whole body. Studies performed for 2 to 3 weeks after the onset of fever indicated that infectious virus is excreted in the throat, urine, and conjunctiva of smallpox patients (20). Additionally, the level and duration of secretion was higher in clinically severe (those with hemorrhagic and confluent lesions) cases than less severe (those with discrete lesions) cases (20). Evidence from epidemiological studies further suggests that transmission from smallpox patients to their contacts occurs only from the time of the earliest appearance of rash, and that excretions from the mouth and nose are the most important source of infectious virus for transmission (8). The transmission rate of smallpox to unvaccinated contacts is estimated to be in the range of 37 to 88% (3). Smallpox was declared eradicated from the natural environment in 1980 as a result of a worldwide vaccination campaign conducted by the World Health Organization. Despite the potential threat of the intentional release of variola virus as a biological weapon in the future, routine mass vaccinations are not implemented due to adverse events associated with live vaccinia virus (VV) immunization (6). The occurrence of vaccine-associated adverse events, such as progressive vaccinia, eczema vaccinatum, generalized vaccinia, and postvaccinial encephalitis, suggests that uncontrolled VV dissemination in the vicinity of the vaccination site or to distal sites elsewhere on the body may have serious consequences in vaccinees, especially those that are immunocompromised. In addition, virus shed from the vaccination site and inadvertently transferred to a household contact with a history of atopic dermatitis may lead to a life-threatening case of eczema vaccinatum (26). VV, the prototypic member of the Orthopoxvirus genus, is used to study virus infection, replication, morphogenesis, and dissemination (reviewed in reference 19). After DNA replication in the cytoplasm, two infectious but structurally and functionally different forms of the virus are formed: intracellular mature virus (IMV or MV) and extracellular enveloped virus (EEV or EV). MVs remain confined to the cytoplasm and do not disseminate from the cell until cell lysis occurs. However, MVs that acquire a double membrane from early endosomes or the trans-Golgi network form intracellular enveloped virus (IEV) and get transported to the cell surface. After the fusion of its outer membrane with the plasma membrane, IEV may be either retained as cell-associated enveloped virus (CEV) or released from the cell surface as EV. In vitro, CEV is involved in the cell-to-cell spreading of VV, whereas EV is involved in virus dissemination to nonadjoining nearby or distal cells (2, 15). The 37-kDa protein p37, encoded by the VV F13L gene, plays a critical role in this process, since the deletion of the gene results in the blockade of MV envelopment and the inhibition of plaque formation (2). In vivo, EV is implicated in virus dissemination within the host and has been shown to be released from squamous epithelial cells of the nasal cavity in mice intranasally (i.n.) infected with the IHD-J strain of VV (15, 16). Despite the abundance of MVs in the cytoplasm of nasal squamous epithelial cells, no MVs are detected extracellularly and almost all free extracellular virions are EV, suggesting that the mechanism of virus release in vivo is by the exocytosis of EV, not by cell disruption (16). Although the original form of infectious VV released from infected hosts appears to be EV (not MV), due to the fragility of the EV membrane to withstand environmental factors, it is thought that the more physically stable MV released by the disruption of the EV outer membrane is responsible for the infection of susceptible hosts (22). As such, reducing the amount of EV released from epithelial cells of the respiratory tract might lower the risk of person-to-person transmission of VV by limiting the amount of shed EV available for conversion to MV, thus resulting in the infection of contacts. ST-246 is an orally bioavailable low-molecular-size (376 g/mol) compound that is selectively active against several orthopoxviruses, including VV and ectromelia, cowpox, camelpox, monkeypox, and variola viruses (4, 23, 28). In vitro, ST-246 treatment resulted in a 10-fold reduction of EV formation (without affecting MV production) and in the inhibition of plaque formation and virus-induced cytopathic effect (28). The analysis of ST-246-resistant cowpox virus variants in vitro has revealed that the antiviral activity of ST-246 is targeted against a product of the V061 gene, which is homologous to the VV F13L gene required for MV envelopment (28). In vivo, ST-246 treatment has been shown to protect mice (ectromelia, vaccinia, or cowpox viruses), rabbits (rabbitpox), and ground squirrels (monkeypox) from lethal orthopoxvirus infections (14, 17, 21, 28). ST-246 treatment additionally reduced virus replication in organs such as spleen and liver (17, 28). In nonhuman primates, ST-246 fully protected cynomolgus monkeys from lethal intravenous (i.v.) challenges with monkeypox and variola virus and significantly reduced viral load and lesion formation (9, 10). In this study, we extended on these previous reports and examined the impact of ST-246 treatment on the in vivo dissemination of virus from the site of inoculation to various distal organs in BALB/c mice infected with the Western Reserve strain of VV (VV-WR) by i.n., percutaneous, i.v., subcutaneous (s.c.), and intraperitoneal (i.p.) routes. We also investigated the effect of ST-246 treatment on the level of virus shedding from the nasal and lung cavities of mice challenged i.n. and from vaccine-induced lesions of ACAM2000-vaccinated mice.