A Nucleic Acid-Based Orthopoxvirus Vaccine Targeting the Vaccinia Virus L1, A27, B5, and A33 Proteins Protects Rabbits against Lethal Rabbitpox Virus Aerosol Challenge.

In the age of COVID, nucleic acid vaccines have garnered much attention, at least in part, because of the simplicity of construction, production, and flexibility to adjust and adapt to an evolving outbreak. Orthopoxviruses remain a threat on multiple fronts, especially as emerging zoonoses. In response, we developed a DNA vaccine, termed 4pox, that protected nonhuman primates against monkeypox virus (MPXV)-induced severe disease. Here, we examined the protective efficacy of the 4pox DNA vaccine delivered by intramuscular (i.m.) electroporation (EP) in rabbits challenged with aerosolized rabbitpox virus (RPXV), a model that recapitulates the respiratory route of exposure and low dose associated with natural smallpox exposure in humans. We found that 4pox-vaccinated rabbits developed immunogen-specific antibodies, including neutralizing antibodies, and did not develop any clinical disease, indicating protection against aerosolized RPXV. In contrast, unvaccinated animals developed significant signs of disease, including lesions, and were euthanized. These findings demonstrate that an unformulated, nonadjuvanted DNA vaccine delivered i.m. can protect against an aerosol exposure. IMPORTANCE The eradication of smallpox and subsequent cessation of vaccination have left a majority of the population susceptible to variola virus or other emerging poxviruses. This is exemplified by human monkeypox, as evidenced by the increase in reported endemic and imported cases over the past decades. Therefore, a malleable vaccine technology that can be mass produced and does not require complex conditions for distribution and storage is sought. Herein, we show that a DNA vaccine, in the absence of a specialized formulation or adjuvant, can protect against a lethal aerosol insult of rabbitpox virus.

[The rapid ELISA method for detection of orthopoxviruses].

INTRODUCTION: Following the successful eradication of smallpox, mass vaccination against this disease was discontinued in 1980. The unvaccinated population continues to be at risk of infection due to military use of variola virus or exposure to monkeypox virus in Africa and non-endemic areas. In cases of these diseases, rapid diagnosis is of great importance, since the promptness and effectiveness of therapeutic and quarantine measures depend on it. The aim of work is to develop a kit of reagents for enzyme-linked immunosorbent assay (ELISA) for fast and highly sensitive detection of orthopoxviruses (OPV) in clinical samples. MATERIALS AND METHODS: The efficiency of virus detection was evaluated by single-stage ELISA in the cryolisate of CV-1 cell culture samples infected with vaccinia, cowpox, rabbitpox, and ectromelia viruses, as well as in clinical samples of infected rabbits and mice. RESULTS: The method of rapid ELISA was shown to allow the detection of OPV in crude viral samples in the range of 5.0 1025.0 103 PFU/ml, and in clinical samples with a viral load exceeding 5 103 PFU/ml. CONCLUSIONS: The assay involves a minimum number of operations and can be performed within 45 minutes, which makes it possible to use it in conditions of a high level of biosecurity. Rapid ELISA method was developed using polyclonal antibodies, which significantly simplifies and reduces the cost of manufacturing a diagnostic system.

Evaluation of Rapid Dot-Immunoassay for Detection Orthopoxviruses Using Laboratory-Grown Viruses and Animal's Clinical Specimens.

The aim of the work was an experimental evaluation of the characteristics of the kit for the rapid immunochemical detection of orthopoxviruses (OPV). The kit is based on the method of one-stage dot-immunoassay on flat protein arrays using gold conjugates and a silver developer. Rabbit polyclonal antibodies against the vaccinia virus were used as capture and detection reagents. The sensitivity of detection of OPV and the specificity of the analysis were assessed using culture crude preparations (monkeypox virus, vaccinia virus, rabbitpox virus, cowpox virus, and ectromelia virus), a suspension from a crust from a human vaccination site as well as blood and tissue suspensions of infected rabbits. It has been shown that the assay using the kit makes it possible to detect OPV within 36 min at a temperature of 18-40 °C in unpurified culture samples of the virus and clinical samples in the range of 103-104 PFU/mL. Tests of the kit did not reveal cross-reactivity with uninfected cell cultures and viral pathogens of exanthematous infections (measles, rubella and chicken pox). The kit can be used to detect or exclude the presence of a virus threat in samples and can be useful in various aspects of biosecurity. The simplicity of analysis, the possibility of visual accounting the and interpretation of the results make it possible to use the test in laboratories with a high level of biological protection and in out-of-laboratory conditions.

Rabbitpox in New Zealand White Rabbits: A Therapeutic Model for Evaluation of Poxvirus Medical Countermeasures Under the FDA Animal Rule.

The elimination of smallpox as an endemic disease and the obvious ethical problems with clinical challenge requires the efficacy evaluation of medical countermeasures against smallpox using the FDA Animal Rule. This approach requires the evaluation of antiviral efficacy in an animal model whose infection recapitulates the human disease sufficiently well enough to provide predictive value of countermeasure effectiveness. The narrow host range of variola virus meant that no other animal species was sufficiently susceptible to variola to manifest a disease with predictive value. To address this dilemma, the FDA, after a public forum with virologists in December 2011, suggested the development of two animal models infected with the cognate orthopoxvirus, intradermal infection of rabbits and intranasal infection of mice, to supplement the non-human primate models in use. In this manuscript, we describe the development of an intradermal challenge model of New Zealand White rabbits with rabbitpox virus (RPXV) for poxvirus countermeasure evaluation. Lethality of RPXV was demonstrated in both 9 and 16-weeks old rabbits with an LD50 < 10 PFU. The natural history of RPXV infection was documented in both ages of rabbits by monitoring the time to onset of abnormal values in clinical data at a lethal challenge of 300 PFU. All infected animals became viremic, developed a fever, exhibited weight loss, developed secondary lesions, and were euthanized after 7 or 8 days. The 16-weeks RPXV-infected animals exhibiting similar clinical signs with euthanasia applied about a day later than for 9-weeks old rabbits. For all animals, the first two unambiguous indicators of infection were detection of viral copies by quantitative polymerase chain reaction and fever at 2 and 3 days following challenge, respectively. These biomarkers provide clinically-relevant trigger(s) for initiating therapy. The major advantage for using 16-weeks NZW rabbits is that older rabbits were more robust and less subject to stress-induced death allowing more reproducible studies.

The efficacy and pharmacokinetics of brincidofovir for the treatment of lethal rabbitpox virus infection: a model of smallpox disease.

Brincidofovir (BCV) has broad-spectrum in vitro activity against dsDNA viruses, including smallpox, and is being developed as a treatment for smallpox as well as infections caused by other dsDNA viruses. BCV has previously been shown to be active in multiple animal models of smallpox. Here we present the results of a randomized, blinded, placebo-controlled study of the efficacy and pharmacokinetics of a novel, "humanized" regimen of BCV for treatment of New Zealand White rabbits infected with a highly lethal inoculum of rabbitpox virus, a well characterized model of smallpox. Compared with placebo, a dose-dependent increase in survival was observed in all BCV-treatment groups. Concentrations of cidofovir diphosphate (CDV-PP), the active antiviral, in rabbit peripheral blood mononuclear cells (PBMCs) were determined for comparison to those produced in humans at the dose proposed for treatment of smallpox. CDV-PP exposure in PBMCs from rabbits given BCV scaled to human exposures at the dose proposed for treatment of smallpox, which is also currently under evaluation for other indications. The results of this study demonstrate the activity of BCV in the rabbitpox model of smallpox and the feasibility of scaling doses efficacious in the model to a proposed human dose and regimen for treatment of smallpox.

Orthopoxvirus inhibitors that are active in animal models: an update from 2008 to 2012.

Antiviral agents are being sought as countermeasures for the potential deliberate release of smallpox (variola) and monkeypox viruses, for the treatment of naturally acquired monkeypox virus infections, and as therapy for complications due to smallpox (live-attenuated vaccinia virus) vaccination or accidental infection after exposure to vaccinated persons. Reviews of the scientific literature spanning 1950-2008 have documented the progress made in developing small-animal models of poxvirus infection and identifying novel antiviral agents. Compounds of considerable interest include cidofovir, CMX001 and ST-246® (tecovirimat; SIGA Technologies, NY, USA). New inhibitors have been identified since 2008, most of which do not exhibit the kind of potency and selectivity required for drug development. Two promising agents include 4'-thioidoxuridine (a nucleoside analog) and mDEF201 (an adenovirus-vectored interferon). Compounds that have been effectively used in combination studies include vaccinia immune globulin, cidofovir, ST-246 and CMX001. In the future there may be an increase in experimental work using active compounds in combination.

Viral skin diseases of the rabbit.

This article describes the viral skin diseases affecting the domestic rabbit, the most important being myxomatosis. Transmission and pathogenesis, clinical signs, diagnosis, treatment, and control are described and the article will be of interest to veterinary practitioners who treat rabbits. Shope fibroma virus, Shope papilloma virus, and rabbitpox are also discussed.

Efficacy of CMX001 as a post exposure antiviral in New Zealand White rabbits infected with rabbitpox virus, a model for orthopoxvirus infections of humans.

CMX001, a lipophilic nucleotide analog formed by covalently linking 3-(hexdecyloxy)propan-1-ol to cidofovir (CDV), is being developed as a treatment for smallpox. In the absence of human cases of smallpox, new treatments must be tested for efficacy in animal models. Previously, we demonstrated the efficacy of CMX001 in protecting New Zealand White rabbits from mortality following intradermal infection with rabbitpox virus as a model for smallpox, monkeypox and for treatment of adverse reactions to smallpox vaccination. Here we extend these studies by exploring different dosing regimens and performing randomized, blinded, placebo-controlled studies. In addition, because rabbitpox virus can be transmitted via naturally generated aerosols (animal to animal transmission), we report on studies to test the efficacy of CMX001 in protecting rabbits from lethal rabbitpox virus disease when infection occurs by animal to animal transmission. In all cases, CMX001 treatment was initiated at the onset of observable lesions in the ears to model the use of CMX001 as a treatment for symptomatic smallpox. The results demonstrate that CMX001 is an effective treatment for symptomatic rabbitpox virus infection. The rabbitpox model has key similarities to human smallpox including an incubation period, generalized systemic disease, the occurrence of lesions which may be used as a trigger for initiating therapy, and natural animal to animal spread, making it an appropriate model.

Efficacy of CMX001 as a prophylactic and presymptomatic antiviral agent in New Zealand white rabbits infected with rabbitpox virus, a model for orthopoxvirus infections of humans.

CMX001, a lipophilic nucleotide analog formed by covalently linking 3-(hexdecyloxy)propan-1-ol to cidofovir (CDV), is being developed as a treatment for smallpox. CMX001 has dramatically increased potency versus CDV against all dsDNA viruses and, in contrast to CDV, is orally available and has shown no evidence of nephrotoxicity in healthy volunteers or severely ill transplant patients to date. Although smallpox has been eliminated from the environment, treatments are urgently being sought due to the risk of smallpox being used as a bioterrorism agent and for monkeypox virus, a zoonotic disease of Africa, and adverse reactions to smallpox virus vaccinations. In the absence of human cases of smallpox, new treatments must be tested for efficacy in animal models. Here we first review and discuss the rabbitpox virus (RPV) infection of New Zealand White rabbits as a model for smallpox to test the efficacy of CMX001 as a prophylactic and early disease antiviral. Our results should also be applicable to monkeypox virus infections and for treatment of adverse reactions to smallpox vaccination.