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

Abstract

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.

Fig. 1

Groups of SKH1 mice were infected IN with ECTV inoculums of 14000, 1400, 140, 14, and 1.4 PFU. Survival curves (A) and weight change (B) are shown. LD₅₀ = 101 PFU. Weight change error bars have been removed for clarity. Data points after day 18 did not change and are therefore not shown in (A). N = 5 animals per group.

Fig. 2

Mice were infected IN with 1000 (1 × LD₅₀) PFU of ECTV-GFP. (A) Mouse head cut laterally indicating anatomical positions. (B) Infected mouse heads were cut laterally and examined for GFP. First column shows mouse head section under white light; second column shows GFP + areas; third column is overlay.

Fig. 3

Groups of SKH1 mice were infected IN with 1000 (1 × LD₅₀) PFU of ECTV-GFP. GFP + foci could be detected in the skin from day 5 and increased in size until day 9. Upper row shows GFP + skin; lower row shows the same sample of skin under white light. No white light lesion was seen at day 5 (not shown). Scale bar = 1 mm. Typical lesions (from the flanks) for the specified day are shown.

Fig. 4

C57BL/6 mice were infected by the IN route with 450 (4.5 × LD₅₀) PFU of ECTV and oropharyngeal secretions and blood were sampled for viral DNA using qPCR. Saliva was sampled on days 1–7 p.i. Blood samples were taken on days 0–5 p.i. NI indicates non-infected controls. This is a representative experiment out of a total of 2. N = 5 animals per group.

Supplementary Fig. S1

Groups of SKH1 mice were infected via aerosol with ECTV inoculums of 15,000, 1500, 150, 15, 1.5 PFU. Survival curves (A) and weight change (B) are shown. LD50 = 85 PFU. Data points after day 18 did not change and are therefore not shown in (A). Data points after day 18 are not shown in (A). Weight change error bars have been removed for clarity.

Supplementary Fig. S2

Histological examination of day 8 p.i. lesions from sacrificed SKH1 mice infected IN with 1000 PFU of ECTV. Arrow indicates an A-type inclusion body. Hematoxylin and eosin stain.

Supplementary Fig. S3

C57BL/6 mice were infected IN with 1000 (10 × LD50) PFU of ECTV. (A) NI veh mice were noninfected and received vehicle, NI CMX mice were non-infected and received CMX001, Inf veh mice were infected and received vehicle, 1 dose d4 and 1 dose d6 were infected and received a single 20 mg/kg dose of CMX001 on day 4 or day 6 p.i., respectively. (B) Infected mice were administered by oral gavage 20 mg/kg of CMX001 at days 0, 4, 5, 6, 7, 8, or 9 p.i. Weight change is shown. Weight change error bars have been removed for clarity. N = 5 animals per group.

Supplementary Fig. S4

C57BL/6 mice were infected by the IN route with 300 (3 × LD50) PFU of ECTV. Mice were administered by oral gavage 100 mg/kg of ST-246 at days 0, 4, 5, 6, 7, 8 or 9 p.i. Upon initiation of therapy, mice were treated daily for 14 days. Weight change is shown. NI veh: group infected and treated with vehicle; NI ST-246: group not infected and treated with ST-246; Inf veh: group infected and treated with vehicle; “d” represents the day p.i. that treatment was initiated. Weight change error bars have been removed for clarity. N = 5 animals per group.