cmx-001 and Ectromelia--Infectious

Orthopoxviruses continue to pose a significant threat to the population as potential agents of bioterrorism. An intentional release of natural or engineered variola virus (VARV) or monkeypox viruses would cause mortality and morbidity in the target population. To address this, antivirals have been developed and evaluated in animal models of smallpox and monkeypox. One such antiviral, brincidofovir (BCV, previously CMX001), has demonstrated high levels of efficacy against orthopoxviruses in animal models and is currently under clinical evaluation for prevention and treatment of diseases caused by cytomegaloviruses and adenoviruses. In this study we use the mousepox model of smallpox to evaluate the relationship between the magnitude of the infectious virus dose and an efficacious BCV therapy outcome when treatment is initiated concomitant with detection of ectromelia virus viral DNA (vDNA) in mouse buccal swabs. We found that vDNA could be detected in buccal swabs of some, but not all infected mice over a range of challenge doses by day 3 or 4 postexposure, when initiation of BCV treatment was efficacious, suggesting that detection of vDNA in buccal swabs could be used as a trigger to initiate BCV treatment of an entire potentially exposed population. However, buccal swabs of some mice did not become positive until 5 days postexposure, when initiation of BCV therapy failed to protect mice that received high doses of virus. And finally, the data suggest that the therapeutic window for efficacious BCV treatment decreases as the virus infectious dose increases. Extrapolating these findings to VARV, the data suggest that treatment should be initiated as soon as possible after exposure and not rely on a diagnostic tool such as the measurement of vDNA in buccal cavity swabs; however, consideration should be given to the fact that the behavior/disease-course of VARV in humans is different from that of ectromelia virus in the mouse.

Topics: Animals; Antiviral Agents; Cytosine; Disease Models, Animal; DNA, Viral; Ectromelia virus; Ectromelia, Infectious; Mice; Mouth Mucosa; Organophosphonates; Orthopoxvirus; Smallpox

Natural orthopoxvirus outbreaks such as vaccinia, cowpox, cattlepox and buffalopox continue to cause morbidity in the human population. Monkeypox virus remains a significant agent of morbidity and mortality in Africa. Furthermore, monkeypox virus's broad host-range and expanding environs make it of particular concern as an emerging human pathogen. Monkeypox virus and variola virus (the etiological agent of smallpox) are both potential agents of bioterrorism. The first line response to orthopoxvirus disease is through vaccination with first-generation and second-generation vaccines, such as Dryvax and ACAM2000. Although these vaccines provide excellent protection, their widespread use is impeded by the high level of adverse events associated with vaccination using live, attenuated virus. It is possible that vaccines could be used in combination with antiviral drugs to reduce the incidence and severity of vaccine-associated adverse events, or as a preventive in individuals with uncertain exposure status or contraindication to vaccination. We have used the intranasal mousepox (ectromelia) model to evaluate the efficacy of vaccination with Dryvax or ACAM2000 in conjunction with treatment using the broad spectrum antiviral, brincidofovir (BCV, CMX001). We found that co-treatment with BCV reduced the severity of vaccination-associated lesion development. Although the immune response to vaccination was quantifiably attenuated, vaccination combined with BCV treatment did not alter the development of full protective immunity, even when administered two days following ectromelia challenge. Studies with a non-replicating vaccine, ACAM3000 (MVA), confirmed that BCV's mechanism of attenuating the immune response following vaccination with live virus was, as expected, by limiting viral replication and not through inhibition of the immune system. These studies suggest that, in the setting of post-exposure prophylaxis, co-administration of BCV with vaccination should be considered a first response to a smallpox emergency in subjects of uncertain exposure status or as a means of reduction of the incidence and severity of vaccine-associated adverse events.

Topics: Animals; Antiviral Agents; Cytosine; Ectromelia virus; Ectromelia, Infectious; Female; Humans; Immunity; Mice; Mice, Inbred C57BL; Organophosphonates; Smallpox Vaccine; Vaccination; Virus Replication

The human population is currently faced with the potential use of natural or recombinant variola and monkeypox viruses as biological weapons. Furthermore, the emergence of human monkeypox in Africa and its expanding environs poses a significant natural threat. Such occurrences would require therapeutic and prophylactic intervention with antivirals to minimize morbidity and mortality of exposed populations. Two orally-bioavailable antivirals are currently in clinical trials; namely CMX001, an ether-lipid analog of cidofovir with activity at the DNA replication stage and ST-246, a novel viral egress inhibitor. Both of these drugs have previously been evaluated in the ectromelia/mousepox system; however, the trigger for intervention was not linked to a disease biomarker or a specific marker of virus replication. In this study we used lethal, intranasal, ectromelia virus infections of C57BL/6 and hairless SKH1 mice to model human disease and evaluate exanthematous rash (rash) as an indicator to initiate antiviral treatment. We show that significant protection can be provided to C57BL/6 mice by CMX001 or ST-246 when therapy is initiated on day 6 post infection or earlier. We also show that significant protection can be provided to SKH1 mice treated with CMX001 at day 3 post infection or earlier, but this is four or more days before detection of rash (ST-246 not tested). Although in this model rash could not be used as a treatment trigger, viral DNA was detected in blood by day 4 post infection and in the oropharyngeal secretions (saliva) by day 2-3 post infection - thus providing robust and specific markers of virus replication for therapy initiation. These findings are discussed in the context of current respiratory challenge animal models in use for the evaluation of poxvirus antivirals.

Topics: Animals; Benzamides; Biomarkers, Pharmacological; Cell Line; Cytosine; Disease Models, Animal; Drug Evaluation, Preclinical; Ectromelia virus; Ectromelia, Infectious; Female; Humans; Isoindoles; Mice; Mice, Hairless; Mice, Inbred C57BL; Monkeypox virus; Organophosphonates; Smallpox; Variola virus; Virus Replication

In 2001, Jackson et al. reported that murine IL-4 expression by a recombinant ectromelia virus caused enhanced morbidity and lethality in resistant C57BL/6 mice as well as overcame protective immune memory responses. To achieve a more thorough understanding of this phenomenon and to assess a variety of countermeasures, we constructed a series of ECTV recombinants encoding murine IL-4 under the control of promoters of different strengths and temporal regulation. We showed that the ECTV-IL-4 recombinant expressing the highest level of IL-4 was uniformly lethal for C57BL/6 mice even when previously immunized. The lethality of the ECTV-IL-4 recombinants resulted from virus-expressed IL-4 signaling through the IL-4 receptor but was not due to IL-4 toxicity. A number of treatment approaches were evaluated against the most virulent IL-4 encoding virus. The most efficacious therapy was a combination of two antiviral drugs (CMX001(®) and ST-246(®)) that have different mechanisms of action.

Topics: Animals; Antiviral Agents; Benzamides; Cytosine; Ectromelia virus; Ectromelia, Infectious; Female; Gene Expression Regulation; Interleukin-4; Isoindoles; Mice; Mice, Inbred C57BL; Organophosphonates; Promoter Regions, Genetic; Recombinant Proteins; Survival Analysis; Treatment Outcome

The intranasal lethal mousepox model employing the A/Ncr mouse strain is used to evaluate anti-orthopoxvirus therapies. These infections mimic large droplet transmission and result in 100% mortality within 7-10 days with as little as 1 PFU of ectromelia virus. Unlike the A/Ncr model, humans are less susceptible to lethal respiratory infections with variola virus and monkeypox virus as demonstrated by their lower mortality rates. In this study we show that a low dose intranasal infection of C57BL/6 mice results in 60-80% mortality and better models smallpox. Comparing CMX001 (HDP-cidofovir) efficacy in the A/Ncr strain and the C57BL/6 strain revealed that delayed treatment with CMX001 is more efficacious at preventing severe disease in the C57BL/6 strain. The increased efficacy of CMX001 in C57BL/6 over A/Ncr following an intranasal infection with ectromelia appears to be mediated by a stronger Th1 cell mediated response. Following footpad infection we show that the C57BL/6 strain has earlier and more robust transcriptional activity, Th1 cytokine secretions, antigen presenting activity and IFNgamma splenic CD8+ T cell responses as compared to the A/Ncr strain. As a result of the enhanced immune response in the C57BL/6 strain, non-lethal intradermal ectromelia infections can therapeutically protect up to 3 days following a homologous, lethal intranasal infection - much like how smallpox vaccination can protect humans for up to 4 days following intranasal variola infection.

Topics: Animals; Antiviral Agents; Cell Line; Chlorocebus aethiops; Cytokines; Cytosine; Disease Models, Animal; Ectromelia virus; Ectromelia, Infectious; Female; Mice; Mice, Inbred C57BL; Organophosphonates

The emergence of human monkeypox and the potential use of recombinant variola and monkeypox viruses as biological terrorist agents have necessitated the development of therapeutic and prophylactic therapies. The primary, or index, cases of smallpox and/or human monkeypox will likely be identified by a characteristic rash. Effective biomarkers will be required to monitor disease progression, guide the choice of therapeutic intervention strategies and evaluate their efficacies. To address this we have evaluated several biomarkers of disease in a lethal mousepox model.. The efficacy of a single dose of a hexadecyloxypropyl ester of cidofovir (CMX001) at 20, 25 and 30 mg/kg doses administered on days 4, 5, 6 and 7 post-infection was evaluated in A/Ncr mice intranasally infected with low doses of ectromelia virus (<20 plaque-forming units). Mice were monitored for weight loss, blood interferon-gamma levels, alanine aminotransferase (ALT), aspartate aminotransferase, viral DNA copies and neutrophilia levels to stage disease progression.. We have used these biomarkers to establish the optimal dosing regimen for treatment and reveal that a single dose of 25 mg/kg of CMX001 can be efficacious at treating lethal mousepox when administered on days 4 or 5 post-infection. This dose significantly reduces ALT, interferon-gamma and DNA copies found in the blood of infected animals.. A single dose regimen of CMX001 is efficacious at treating mousepox. Disease progression and antiviral efficacy can be monitored using several biomarkers that could readily be used in the case of a human monkeypox or smallpox outbreak.

Topics: Alanine Transaminase; Animals; Antiviral Agents; Aspartate Aminotransferases; Biomarkers; Cell Line; Cytosine; Disease Models, Animal; Disease Progression; DNA, Viral; Ectromelia virus; Ectromelia, Infectious; Female; Humans; Interferon-gamma; Mice; Organophosphonates; Treatment Outcome; Weight Loss

In the 21st century we are faced with the potential use of natural or recombinant VARV and MPXV as biological weapons, and the emergence of human MPXV. Such an occurrences would require therapeutic and prophylactic intervention with antivirals. Cidofovir, an antiviral approved for the treatment of cytomegalovirus retinitis in AIDS patients, has activity against poxviruses, but must be administered intravenously and is associated with nephrotoxicity. An ether-lipid analogue of CDV, CMX001 (HDP-CDV), has potent antiviral activity against a range of DNA viruses including poxviruses, excellent oral bioavailability and minimal nephrotoxicity. CMX001 and CDV are equally efficacious at protecting mice from mortality following high ectromelia virus doses (10,000 x LD(50)) introduced by the intra-nasal route or small particle aerosol. Using CMX001 at a 10mg/kg dose followed by 2.5mg/kg doses every other-day for 14 days provided solid protection against mortality and weight loss following an intra-nasal challenge of (100-200) x LD(50) of ectromelia virus. Furthermore, complete protection against mortality was achieved when administration was delayed until as late as 5 days post-infection, which is 3-4 days prior to the death of the untreated controls. This therapeutic window would be equivalent to intervening during the rash stage of ordinary smallpox.

Topics: Administration, Oral; Animals; Antiviral Agents; Cidofovir; Cytosine; Ectromelia virus; Ectromelia, Infectious; Ether; Female; Hepatocytes; Humans; Mice; Mice, Inbred A; Organophosphonates