st-246 and Vaccinia

Orthopoxviruses such as variola and monkeypox viruses continue to threaten the human population. Monkeypox virus is endemic in central and western Africa and outbreaks have reached as far as the U.S. Although variola virus, the etiologic agent of smallpox, has been eradicated by a successful vaccination program, official and likely clandestine stocks of the virus exist. Moreover, studies with ectromelia virus (the etiological agent of mousepox) have revealed that IL-4 recombinant viruses are significantly more virulent than wild-type viruses even in mice treated with vaccines and/or antivirals. For these reasons, it is critical that antiviral modalities are developed to treat these viruses should outbreaks, or deliberate dissemination, occur. Currently, 2 antivirals (brincidofovir and tecovirimat) are in the U.S. stockpile allowing for emergency use of the drugs to treat smallpox. Both antivirals have advantages and disadvantages in a clinical and emergency setting. Here we report on the efficacy of a recombinant immunoglobulin (rVIG) that demonstrated efficacy against several orthopoxviruses in vitro and in vivo in both a prophylactic and therapeutic fashion. A single intraperitoneal injection of rVIG significantly protected mice when given up to 14 days before or as late as 6 days post challenge. Moreover, rVIG reduced morbidity, as measured by weight-change, as well as several previously established biomarkers of disease. In rVIG treated mice, we found that vDNA levels in blood were significantly reduced, as was ALT (a marker of liver damage) and infectious virus levels in the liver. No apparent adverse events were observed in rVIG treated mice, suggesting the immunoglobulin is well tolerated. These findings suggest that recombinant immunoglobulins could be candidates for further evaluation and possible licensure under the FDA Animal Rule.

Topics: Animals; Antiviral Agents; Benzamides; Cell Line; Chlorocebus aethiops; Cytosine; Female; Humans; Immunoglobulins; Isoindoles; Mice; Mice, Inbred BALB C; Organophosphonates; Orthopoxvirus; Smallpox; Smallpox Vaccine; Vaccines, DNA; Vaccinia

The Orthopoxvirus (OPV) genus of the Poxviridae family contains several human pathogens, including Vaccinia virus (VACV), which have been implicating in outbreaks of a zoonotic disease called Bovine Vaccinia in Brazil. So far, no approved treatment exists for OPV infections, but ST-246 and Cidofovir (CDV) are now in clinical development. Therefore, the objective of this work was to evaluate the susceptibility of five strains of Brazilian VACV (Br-VACV) to ST-246 and Cidofovir. The susceptibility of these strains to both drugs was evaluated by plaque reduction assay, extracellular virus's quantification in the presence of ST-246 and one-step growth curve in cells treated with CDV. Besides that, the ORFs F13L and E9L were sequenced for searching of polymorphisms associated with drug resistance. The effective concentration of 50% (EC

Topics: Antiviral Agents; Benzamides; Brazil; Cidofovir; Humans; Isoindoles; Phylogeny; Vaccinia; Vaccinia virus

Whole-body bioimaging was used to study dissemination of vaccinia virus (VACV) in normal and in immune deficient (nu(-)/nu(-)) mice protected from lethality by postchallenge administration of ST-246. Total fluxes were recorded in the liver, spleen, lungs, and nasal cavities of live mice after intranasal infection with a recombinant IHD-J-Luc VACV expressing luciferase. Areas under the flux curve were calculated for individual mice to assess viral loads. Treatment for 2 to 5 days of normal BALB/c mice with ST-246 at 100 mg/kg starting 24 h postchallenge conferred 100% protection and reduced viral loads in four organs compared to control mice. Mice also survived after 5 days of treatment with ST-246 at 30 mg/kg, and yet the viral loads and poxes were higher in these mice compared to 100-mg/kg treatment group. Nude mice were not protected by ST-246 alone or by 10 million adoptively transferred T cells. In contrast, nude mice that received T cells and 7-day treatment with ST-246 survived infection and exhibited reduced viral loads compared to nonreconstituted and ST-246-treated mice after ST-246 was stopped. Similar protection of nude mice was achieved using adoptively transferred 1.0 and 0.1 million, but not 0.01 million, purified T cells or CD4(+) or CD8(+) T cells in conjunction with ST-246 treatment. These data suggest that ST-246 protects immunocompetent mice from lethality and reduces viral dissemination in internal organs and poxvirus lesions. Furthermore, immune-deficient animals with partial T cell reconstitution can control virus replication after a course of ST-246 and survive lethal vaccinia virus challenge.

Topics: Adoptive Transfer; Animal Structures; Animals; Antiviral Agents; Benzamides; Disease Models, Animal; Female; Genes, Reporter; Immunocompromised Host; Isoindoles; Luciferases; Mice; Mice, Inbred BALB C; Mice, Nude; Staining and Labeling; Survival Analysis; T-Lymphocytes; Treatment Outcome; Vaccinia; Vaccinia virus; Viral Load; Whole Body Imaging

Progressive vaccinia (PV) is a rare but potentially lethal complication that develops in smallpox vaccine recipients with severely impaired cellular immunity. We describe a patient with PV who required treatment with vaccinia immune globulin and who received 2 investigational agents, ST-246 and CMX001. We describe the various molecular, pharmacokinetic, and immunologic studies that provided guidance to escalate and then successfully discontinue therapy. Despite development of resistance to ST-246 during treatment, the patient had resolution of PV. This case demonstrates the need for continued development of novel anti-orthopoxvirus pharmaceuticals and the importance of both intensive and timely clinical and laboratory support in management of PV.

Topics: Adult; Antibodies, Viral; Antiviral Agents; Benzamides; Cytosine; Drug Resistance, Viral; Humans; Immunoglobulins; Isoindoles; Male; Organophosphonates; Smallpox Vaccine; Treatment Outcome; Vaccinia; Vaccinia virus

As part of a program to identify new compounds that have activity against orthopoxviruses, a number of 4'-thionucleosides were synthesized and evaluated for their efficacies against vaccinia and cowpox viruses. Seven compounds that were active at about 1 microM against both viruses in human cells but that did not have significant toxicity were identified. The 5-iodo analog, 1-(2-deoxy-4-thio-beta-d-ribofuranosyl)-5-iodouracil (4'-thioIDU), was selected as a representative molecule; and this compound also inhibited viral DNA synthesis at less than 1 microM but only partially inhibited the replication of a recombinant vaccinia virus that lacked a thymidine kinase. This compound retained complete activity against cidofovir- and ST-246-resistant mutants. To determine if this analog had activity in an animal model, mice were infected intranasally with vaccinia or cowpox virus and treatment with 4'-thioIDU was given intraperitoneally or orally twice daily at 50, 15, 5, or 1.5 mg/kg of body weight beginning at 24 to 120 h postinfection and was continued for 5 days. Almost complete protection (87%) was observed when treatment with 1.5 mg/kg was begun at 72 h postinfection, and significant protection (73%) was still obtained when treatment with 5 mg/kg was initiated at 96 h. Virus titers in the liver, spleen, and kidney were reduced by about 4 log(10) units and about 2 log(10) units in mice infected with vaccinia virus and cowpox virus, respectively. These results indicate that 4'-thioIDU is a potent, nontoxic inhibitor of orthopoxvirus replication in cell culture and experimental animal infections and suggest that it may have potential for use in the treatment of orthopoxvirus infections in animals and humans.

Topics: Animals; Antiviral Agents; beta-Galactosidase; Cell Line; Cell Proliferation; Cell Survival; Cowpox; Cytopathogenic Effect, Viral; DNA, Viral; Drug Resistance, Viral; Humans; Mice; Mice, Inbred BALB C; Nucleosides; Orthopoxvirus; Poxviridae Infections; Structure-Activity Relationship; Vaccinia

The re-emerging threat of smallpox and the emerging threat of monkeypox highlight the need for effective poxvirus countermeasures. Currently approved smallpox vaccines have unacceptable safety profiles and, consequently, the general populace is no longer vaccinated, leading to an increasingly susceptible population. ST-246, a small-molecule inhibitor of poxvirus dissemination, has been demonstrated in various animal models to be safe and effective in preventing poxviral disease. This suggests that it may also be used to improve the safety of the traditional smallpox vaccine provided that it does not inhibit vaccine-induced protective immunity. In this study, we compared the immune responses elicited by the smallpox vaccine alone or in combination with ST-246 in mice. Normal lesion formation following dermal scarification with the attenuated New York City Board of Health strain (Dryvax), commonly referred to as a vaccine "take", was not inhibited although severe lesions and systemic disease due to vaccination with the virulent Western Reserve (VV-WR) strain were prevented. The vaccine given with ST-246 did not affect cellular immune responses or neutralizing antibody titers although anti-vaccinia ELISA titers were slightly reduced. Vaccination in combination with ST-246 provided equivalent short- and long-term protection against lethal intranasal challenge with VV-WR when compared to vaccine alone. These results suggest that ST-246 does not compromise protective immunity elicited by the vaccine and provide the basis for future studies examining the efficacy of ST-246 in preventing or treating adverse events due to vaccination.

Topics: Animals; Antibodies, Viral; Benzamides; Cytokines; Drug Therapy, Combination; Female; Immunoglobulin G; Isoindoles; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Neutralization Tests; Smallpox Vaccine; Time Factors; Vaccination; Vaccinia; Vaccinia virus; Virus Replication

Orthopoxviruses, such as variola and monkeypox viruses, can cause severe disease in humans when delivered by the aerosol route, and thus represent significant threats to both military and civilian populations. Currently, there are no antiviral therapies approved by the U.S. Food and Drug Administration (FDA) to treat smallpox or monkeypox infection. In this study, we showed that administration of the antiviral compound ST-246 to rabbits by oral gavage, once daily for 14 days beginning 1h postexposure (p.e.), resulted in 100% survival in a lethal aerosolized rabbitpox model used as a surrogate for smallpox. Furthermore, efficacy of delayed treatment with ST-246 was evaluated by beginning treatment on days 1, 2, 3, and 4 p.e. Although a limited number of rabbits showed less severe signs of the rabbitpox disease from the day 1 and day 2 p.e. treatment groups, their illness resolved very quickly, and the survival rates for these group of rabbits were 88% and 100%, respectively. But when the treatment was started on days 3 or 4 p.e., survival was 67% and 33%, respectively. This work suggests that ST-246 is a very potent antiviral compound against aerosolized rabbitpox in rabbits and should be investigated for further development for all orthopoxvirus diseases.

Topics: Administration, Oral; Aerosols; Animals; Antiviral Agents; Benzamides; Chemoprevention; Female; Isoindoles; Lung; Rabbits; Survival Analysis; Time Factors; Vaccinia; Vaccinia virus

Topics: Antiviral Agents; Benzamides; Bioterrorism; Child, Preschool; Fathers; Humans; Immunization Programs; Indoles; Isoindoles; Kaposi Varicelliform Eruption; Male; Military Personnel; Public Health Practice; Smallpox Vaccine; United States; Vaccinia

ST-246 is a low-molecular-weight compound (molecular weight = 376), that is potent (concentration that inhibited virus replication by 50% = 0.010 microM), selective (concentration of compound that inhibited cell viability by 50% = >40 microM), and active against multiple orthopoxviruses, including vaccinia, monkeypox, camelpox, cowpox, ectromelia (mousepox), and variola viruses. Cowpox virus variants selected in cell culture for resistance to ST-246 were found to have a single amino acid change in the V061 gene. Reengineering this change back into the wild-type cowpox virus genome conferred resistance to ST-246, suggesting that V061 is the target of ST-246 antiviral activity. The cowpox virus V061 gene is homologous to vaccinia virus F13L, which encodes a major envelope protein (p37) required for production of extracellular virus. In cell culture, ST-246 inhibited plaque formation and virus-induced cytopathic effects. In single-cycle growth assays, ST-246 reduced extracellular virus formation by 10 fold relative to untreated controls, while having little effect on the production of intracellular virus. In vivo oral administration of ST-246 protected BALB/c mice from lethal infection, following intranasal inoculation with 10x 50% lethal dose (LD(50)) of vaccinia virus strain IHD-J. ST-246-treated mice that survived infection acquired protective immunity and were resistant to subsequent challenge with a lethal dose (10x LD(50)) of vaccinia virus. Orally administered ST-246 also protected A/NCr mice from lethal infection, following intranasal inoculation with 40,000x LD(50) of ectromelia virus. Infectious virus titers at day 8 postinfection in liver, spleen, and lung from ST-246-treated animals were below the limits of detection (<10 PFU/ml). In contrast, mean virus titers in liver, spleen, and lung tissues from placebo-treated mice were 6.2 x 10(7), 5.2 x 10(7), and 1.8 x 10(5) PFU/ml, respectively. Finally, oral administration of ST-246 inhibited vaccinia virus-induced tail lesions in Naval Medical Research Institute mice inoculated via the tail vein. Taken together, these results validate F13L as an antiviral target and demonstrate that an inhibitor of extracellular virus formation can protect mice from orthopoxvirus-induced disease.

Topics: Administration, Oral; Amino Acid Sequence; Animals; Antiviral Agents; Benzamides; Cytopathogenic Effect, Viral; Drug Evaluation, Preclinical; Ectromelia virus; Ectromelia, Infectious; Female; Indoles; Isoindoles; Liver; Lung; Membrane Proteins; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Molecular Weight; Orthopoxvirus; Poxviridae Infections; Sequence Alignment; Spleen; Vaccinia; Viral Envelope Proteins; Viral Plaque Assay; Virus Assembly