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

Abstract

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.

Keywords: CMX001; antiviral; poxvirus; rabbitpox; smallpox treatment.

Figure 1

Clinical observations for evaluation of 1, 2 or 3 doses of CMX001 given every other day beginning at day 3 or 4 post infection. Animals were dosed at concentrations and schedules as outlined in Table 1. (A) Average weight change from weight at day of infection for animals that began treatment on day 3 post infection. Negative values indicated weight loss. (B) Average body temperatures for animals that began treatment on day 3 post infection. (C) Average clinical scores for animals that began treatment on day 3 post infection. (D) Average weight change from weight at day of infection for animals that began treatment on day 4 post infection. Negative values indicated weight loss. (E) Average body temperatures for animals that began treatment on day 4 post infection. (F) Average clinical scores for animals that began treatment on day 4 post infection. (G) Pictures of primary lesions (necrosis only) from representative animals at 7 dpi. Bars represent 1cm. Black circles denote site of intradermal inoculation.

Figure 2

Disease severity measurements for evaluation of 1, 2 or 3 doses of CMX001 given every other day beginning at day 3 or 4 post infection. Animals were dosed at concentrations and schedules as outlined in Table 1. (A) Average of maximum clinical scores for each animal per group over the course of the experiment. (B) Average of maximum percent weight loss from weight at day 0 for each animal per group over the course of the experiment. *p≤0.01 as compared to vehicle

Figure 3

Animals treated beginning at the appearance of secondary lesions. Groups of 12 animals were treated with CMX001 1, 2 or 3 times every other day beginning at the appearance of secondary lesions in the ears. Animals were dosed at concentrations and schedules as outlined in Table 2. (A) Average weight change over time for each group. Negative values represent weight loss. (B) Average body temperatures over time. Temperatures over 39.5 °C are considered a fever. (C) Average clinical score over time. Error bars represent SEM. (D) Photographs of primary lesions from each treatment group at 8 dpi. Bar represents 1cm.

Figure 4

Disease severity measurements when CMX001 treatment began at the appearance of secondary lesions. Animals were dosed at concentrations and schedules as outlined in Table 2. (A) Average of maximum clinical scores for each animal per group over the course of the experiment. (B) Average of maximum percent weight loss from weight at day 0 for each animal per group over the course of the experiment. (C) Average of maximum diameters of primary lesions of each animal per group over the course of the experiment.

Figure 5

Time line of disease in 8–10 week old New Zealand White rabbits infected via natural aerosol. Symptoms in red boxes represent clinical measurements that contribute to euthanasia guidelines. Clinical symptoms are indicated in blue boxes. Reproduced with permission from American Society for Microbiology [12].

Figure 6

Disease in rabbits lethally infected with RPV via natural aerosol. (A) Survival of naturally infected rabbits (sentinel), infected cage mates providing aerosolized RPV (index) and uninfected control animals (PBS). (B) Average weight change from weight at time of infection for each group. Negative values represent weight loss. (C) Average body temperatures over time. Temperatures over 39.5 °C are considered a fever. (D) Average clinical score over time. Error bars represent SEM. Reproduced with permission from American Society for Microbiology [12].

Figure 7

Presentation of disease in rabbits infected by aerosol transmitted RPV. Rabbits were infected as described in Figure 5. (A) Secondary lesions observed in the ears of animals exposed to RPV via aerosol. Arrows show locations of 2 representative lesions in the ear. (B) Photograph of a secondary lesion on the eye of an animal marked with arrow. (C) Normal profile of intradermally lethally infected RPV rabbit nose. (D) Profile of aerosol infected RPV rabbit showing severe swelling of nose and muzzle. Reproduced with permission from American Society for Microbiology [12].

Figure 8

Aerosol infected animals treated beginning at the appearance of secondary lesions. Groups of 3 animals were treated with CMX001 1, 2 or 3 times every other day beginning at the appearance of secondary lesions in the ears. Animals were dosed at concentrations and schedules as outlined in Table 3. (A) Average weight change over time for each group. Negative values represent weight loss. (B) Average body temperatures over time. Temperatures over 39.5 °C are considered a fever. (C) Average clinical score over time.