Buccal viral DNA as a trigger for brincidofovir therapy in the mousepox model of smallpox.

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.

[Evaluation of therapeutic-prophylactic effectiveness of chemical compound NIOC-14 against ectromelia virus in vivo].

AIM: Study pharmacodynamic parameters of anti-viral effectiveness of a chemical compound NIOC-14 in experiments in mice infected with ectromelia virus (EV). MATERIALS AND METHODS: EV (K-1 strain) was obtained from the State Collection of Viral Infections and Rickettsioses Causative Agents of the State Scientific Centre of Virology and Biotechnology "Vector". Outbred ICR mice were intranasally infected with EV at a dose of 10 LD50 per animal (10 x 50% lethal doses/animal) and per orally received NIOC-14 or ST-246 as a positive control. Chemical compound NIOC-14 (7-[N'-(4-trifluoromethylbenzoyl)-hidrazincarbonyl]-tricyclo[3.2.2.0(2,4)]non-8-en-6-carbonic acid) was synthesized in Novosibirsk Institute of Organic Chemistry (NIOC). Anti-pox preparation ST-246, developed by SIGA Technologies Inc. (USA), was synthesized in NIOC using the technique described by the authors. RESULTS: 50% effective doses against EV in vivo were shown not to differ significantly between the preparations NIOC-14 (3.59 µg/g mouse mass) and ST-246 (5.08 µg/g mouse mass). During determination of therapeutic window, administration of NIOC-14 to mice 1 day or 1 hour before EV infection, as well as 1, 2 and 4 days after EV infection and then for 9 days was found to ensure 100% animal survival. Administration of NIOC-14 as well as ST-246 resulted in the decrease relative to control of EV titers in lungs, nasal cavity, brains, liver, spleen, kidneys and pancreas. CONCLUSION: Anti-viral effectiveness of NIOC-14 against EV in vivo was thus comparable by all the studied pharmacodynamic parameters with anti-viral activity of anti-pox-virus preparation ST-246.

[A comparative study of the antiviral activity of chemical compounds concerning the orthopoxviruses experiments in vivo].

In the experiments using intranasal (i/n) infection of mice with the ectromelia virus (EV) in a dose 10 LD50/head (10 x 50% lethal doselhead) or with the monkaypox virus (MPXV) in a dose 10 ID50/head (10 x 50% infective dose/ head) it was demonstrated that the antiviral efficiency of chemical compounds - the condensed derivatives of pyrrolidin-2,5-dion, as well as their predecessors and the nearest analogues, synthesized in Novosibirsk Institute of Organic Chemistry of the Siberian Branch of the Russian Academy of Sciences (NIOCH SB RAS) was observed. As a positive control we used the antipoxvirus chemical preparation ST-246 available from SIGA Technologies Inc. (USA), synthesized in NIOCH SB RAS by the technique suggested by the authors. It was demonstrated that the compound NIOCH-14 (7-[N'-(4-Trifluoromethylbenzoil)-hydrazidecarbonil]-tricyclo[3.2.2.02,4]non-8-en-6-carbonic acid) possessed comparable with ST-246 antiviral activity concerning EV and MPXV on all indicators used. Therefore, at infection of mice with EV (strain K-1) and peroral administration of NIOCH-14 and ST-246 in a dose 50 mkg/g of mouse weight (12-14 g) within 10 days the survival rate and average life expectancy of mice authentically exceeded the control levels. EV titers in lungs through 6 days after infection in the same groups were lower than in the control. In addition to that, after 7 days of infection of mice with MPXV (strain V79-1-005) and daily peroral administration of NIOCH-14 and ST-246 in a dose 60 mkg/g of mouse weight (9-11 g) authentic decrease in a part of infected animals and MPXV titers in lungs was observed.

A single cidofovir treatment rescues animals at progressive stages of lethal orthopoxvirus disease.

BACKGROUND: In an event of a smallpox outbreak in humans, the window for efficacious treatment by vaccination with vaccinia viruses (VACV) is believed to be limited to the first few days post-exposure (p.e.). We recently demonstrated in a mouse model for human smallpox, that active immunization 2-3 days p.e. with either VACV-Lister or modified VACV Ankara (MVA) vaccines, can rescue animals from lethal challenge of ectromelia virus (ECTV), the causative agent of mousepox. The present study was carried out in order to determine whether a single dose of the anti-viral cidofovir (CDV), administered at different times and doses p.e. either alone or in conjunction with active vaccination, can rescue ECTV infected mice. METHODS: Animals were infected intranasally with ECTV, treated on different days with various single CDV doses and monitored for morbidity, mortality and humoral response. In addition, in order to determine the influence of CDV on the immune response following vaccination, both the "clinical take", IFN-gamma and IgG Ab levels in the serum were evaluated as well as the ability of the mice to withstand a lethal challenge of ECTV. Finally the efficacy of a combined treatment regime of CDV and vaccination p.e. was determined. RESULTS: A single p.e. CDV treatment is sufficient for protection depending on the initiation time and dose (2.5 - 100 mg/kg) of treatment. Solid protection was achieved by a low dose (5 mg/kg) CDV treatment even if given at day 6 p.e., approximately 4 days before death of the control infected untreated mice (mean time to death (MTTD) 10.2). At the same time point complete protection was achieved by single treatment with higher doses of CDV (25 or 100 mg/kg). Irrespective of treatment dose, all surviving animals developed a protective immune response even when the CDV treatment was initiated one day p.e.. After seven days post treatment with the highest dose (100 mg/kg), virus was still detected in some organs (e.g. lung and liver) yet all animals survived, suggesting that efficacious single CDV treatment requires a potent immune system. The combination of CDV and vaccination provided no additional protection over CDV alone. Yet, combining CDV and vaccination maintained vaccination efficacy. CONCLUSIONS: Altogether, our data substantiate the feasibility of single post-exposure antiviral treatment to face orthopoxvirus infection.

Evaluation of disease and viral biomarkers as triggers for therapeutic intervention in respiratory mousepox - an animal model of smallpox.

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.

Antibody inhibition of a viral type 1 interferon decoy receptor cures a viral disease by restoring interferon signaling in the liver.

Type 1 interferons (T1-IFNs) play a major role in antiviral defense, but when or how they protect during infections that spread through the lympho-hematogenous route is not known. Orthopoxviruses, including those that produce smallpox and mousepox, spread lympho-hematogenously. They also encode a decoy receptor for T1-IFN, the T1-IFN binding protein (T1-IFNbp), which is essential for virulence. We demonstrate that during mousepox, T1-IFNs protect the liver locally rather than systemically, and that the T1-IFNbp attaches to uninfected cells surrounding infected foci in the liver and the spleen to impair their ability to receive T1-IFN signaling, thus facilitating virus spread. Remarkably, this process can be reversed and mousepox cured late in infection by treating with antibodies that block the biological function of the T1-IFNbp. Thus, our findings provide insights on how T1-IFNs function and are evaded during a viral infection in vivo, and unveil a novel mechanism for antibody-mediated antiviral therapy.

Poxvirus interleukin-4 expression overcomes inherent resistance and vaccine-induced immunity: pathogenesis, prophylaxis, and antiviral therapy.

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.

Using biomarkers to stage disease progression in a lethal mousepox model treated with CMX001.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Efficacy of therapeutic intervention with an oral ether-lipid analogue of cidofovir (CMX001) in a lethal mousepox model.

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.

Oral 1-O-octadecyl-2-O-benzyl-sn-glycero-3-cidofovir targets the lung and is effective against a lethal respiratory challenge with ectromelia virus in mice.

Hexadecyloxypropyl-cidofovir (HDP-CDV) has been shown to be orally active against lethal infection with orthopoxviruses including, mousepox, cowpox, vaccinia and rabbitpox. The alkoxyalkyl group provides oral absorption and reduces greatly the amount of drug reaching the kidney, the site of CDV's dose limiting toxicity. However, the amount of HDP-CDV detected in lung, an important site of early poxvirus replication, is low and the reduction of viral titers in surviving animals is reduced moderately compared with the liver where poxvirus titers are virtually undetectable. We synthesized a novel glycerol ester of CDV, 1-O-octadecyl-2-O-benzyl-sn-glycero-3-CDV (ODBG-CDV), and compared its oral pharmacokinetics with that of HDP-CDV. Surprisingly, ODBG-CDV levels in lung are much higher and liver levels are reduced, suggesting that the compound is transported in small intestinal lymph instead the portal vein. ODBG-CDV has excellent in vitro activity in cells infected with ectromelia virus (ECTV). In mice infected with a lethal aerosol or intranasal challenge of ECTV, HDP-CDV and ODBG-CDV are equally effective in preventing death from disease. Other drugs esterified to 1-O-octadecyl-2-O-benzyl-sn-glycerol or 1-O-octadecyl-2-O-benzyl-sn-glycerol-3-phosphate may provide lung targeting for treatment of microbial or neoplastic diseases while reducing first pass removal by the liver during oral absorption.

The efficacy of cidofovir treatment of mice infected with ectromelia (mousepox) virus encoding interleukin-4.

Improved vaccines and therapies for virulent poxvirus infection are required, particularly in the light of recent threats of bioterrorism. Cidofovir (HPMPC) is an acyclic nucleoside analog with proven efficacy against poxviruses. Here, we evaluated HPMPC in mice given a recombinant ectromelia virus (ECTV) encoding interleukin-4 (ECTV-IL-4) that is highly immune suppressive. Mousepox-sensitive BALB/c mice given HPMPC for five consecutive days after infection were protected against the lethal effects of a control ECTV recombinant, although they suffered a chronic form of mousepox disease. High doses of the drug resulted in a milder localized disease. In contrast, HPMPC failed to protect mousepox-resistant C57BL/6 mice against ECTV-IL-4, although its lethal effects were delayed by five daily doses of 20 mg/kg or a single dose of 100 mg/kg. Higher daily doses further delayed mortality, although the majority of animals eventually succumbed to infection. It appears that HPMPC inhibited ECTV-IL-4 replication without clearance, with the virus having a lethal effect when the drug was removed. Resistance of ECTV-IL-4 to HPMPC treatment may relate to the virus's ability to inhibit antiviral cell-mediated immunity. Interestingly, ECTV-IL-4-mediated immune suppression was not accompanied by a reduction in systemic IFN-gamma expression, suggestive of an alternative or highly localized suppressive mechanism.

Efficacy of oral active ether lipid analogs of cidofovir in a lethal mousepox model.

Cidofovir (CDV) is a highly effective inhibitor of orthopoxvirus replication and may be used intravenously to treat smallpox or complications arising from the smallpox vaccine under an investigational new drug application (IND). However, CDV is absorbed poorly following oral administration and is inactive orally. To improve the bioavailability of CDV, others synthesized alkoxyalkanol esters of CDV and observed >100-fold more activity than unmodified CDV against cowpox, vaccinia, and variola virus (VARV) replication. These ether lipid analogs of CDV have high oral bioavailability in mice. In this study, we compared the oral activity of CDV with the hexadecyloxypropyl (HDP)-, octadecyloxyethyl-, oleyloxypropyl-, and oleyloxyethyl-esters of CDV in a lethal, aerosol ectromelia virus (ECTV) challenge model in A/NCR mice. Octadecyloxyethyl-CDV appeared to be the most potent CDV analog as a dose regimen of 5 mg/kg started 4 h following challenge completely blocked virus replication in spleen and liver, and protected 100% of A/NCR mice, although oral, unmodified CDV was inactive. These results suggest that this family of compounds deserves further evaluation as poxvirus antiviral.

[The virucidal properties of neoaquasept]. / Virulitsidnye svoistva neoakvasepta.

The paper provides the evidence suggesting that the disinfectant neoaquasept (NA) obtained by using sodium salt of dichlorisocyanuric acid has virucidal activity against ectromelia virus in BALB/c mice. Three concentrations of NA were tested. The recommended application rate and concentration of the disinfectant is 3 and 10 times higher. NA was demonstrated to decrease the degenerative changes of the liver and spleen and increases the survival of the animals infected with ectromelia virus. It is proposed to use the water disinfected with NA as a preventive agent in the spread foci of various infections.

Depression of protective mechanism during the early phase of a viral infection in tumor-bearing mice and prevention by PSK.

Effector mechanisms responsible for resistance against ectromelia virus including antiviral activity of non-immune macrophages, antiviral antibody, delayed footpad reaction to viral antigen, and interferon induction after viral infection were depressed in BALB/c mice bearing syngeneic Meth A tumor. The degree of viral growth correlated well with the depression of delayed footpad reaction, antibody production, and interferon induction. Therefore, modification of macrophage functions by a tumor-bearing state and treatment with PSK may contribute to this modification of antiviral resistance, at an early phase of infection. Cytotoxic activity may not be the principal effector, since the cytotoxicity was induced in normal and tumor-bearing mice to almost the same extent yet an extensive viral growth occurred only in the latter.