Remdesivir Reduces Clinical Signs in Rhesus Macaques upon Prophylactic and Therapeutic Treatment.

To assess the efficacy of remdesivir to alleviate clinical signs of MERS-CoV infection, 18 rhesus macaques were randomly assigned to three groups of six animals. Three animals in the control group were treated with 1 mL/kg vehicle solution 24 h before MERS-CoV inoculation, and three animals were treated at 12 h post MERS-CoV inoculation. Another group of six rhesus macaques was treated prophylactically 24 h before MERS-CoV inoculation with 5 mg/kg remdesivir, and one group of six animals was treated therapeutically at 12 h postinoculation with MERS-CoV with 5 mg/kg remdesivir. Treatment was continued once daily until 6 d postinoculation (dpi), when animals were euthanized and necropsied (Fig. 1).

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Fig. 1.

Study outline. To test the prophylactic and therapeutic efficacy of remdesivir treatment in the rhesus macaque model of MERS-CoV infection, three groups of six rhesus macaques were inoculated with MERS-CoV strain HCoV-EMC/2012; one group was administered 5 mg/kg remdesivir starting at 24 h before inoculation (black circles), and one group was administered 5 mg/kg remdesivir starting at 12 h after inoculation (red circles). One group of six control animals was i.v.-administered 1 mL/kg vehicle solution, with three animals receiving vehicle solution according to the prophylactic treatment schedule, and three animals receiving it according to the therapeutic treatment schedule. Treatment was continued once daily until 6 dpi, when all animals were euthanized. At 0, 1, 3, 5, and 6 dpi, clinical examinations were performed to monitor the health status of the animals.

After inoculation with MERS-CoV on day 0, all animals were closely observed for signs of disease, and clinical scores were assigned according to a previously determined scoring sheet. All vehicle-treated animals displayed signs of disease, starting as early as 1 dpi, such as decreased appetite and ruffled fur; all vehicle-treated animals had respiratory signs such as increased respiration for 4 (n = 1) or 5 (n = 5) d after inoculation. The animals treated prophylactically with remdesivir did not show any respiratory signs of disease, but decreased appetite, possibly due to daily anesthesia, was noted in five of six animals. The animals treated therapeutically with remdesivir all displayed reduced appetites, and five out of six animals had increased respiration rates at 2 (n = 2), 3 (n = 2), or 4 (n = 1) d after inoculation. These observations are reflected in the clinical scores of the animals, with clinical scores in the prophylactically treated animals being statistically significantly lower than in vehicle-treated control animals at 2 to 6 dpi, and in the therapeutically treated animals at 2 to 4 dpi (Fig. 2A).

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Fig. 2.

Clinical findings in rhesus macaques inoculated with MERS-CoV and treated with remdesivir. Three groups of six rhesus macaques were inoculated with MERS-CoV strain HCoV-EMC/2012; one group was i.v.-administered 1 mL/kg vehicle solution (vehicle control; gray circles), one group was administered 5 mg/kg remdesivir starting at 24 h before inoculation (prophylactic remdesivir; black squares), and one group was administered 5 mg/kg remdesivir starting at 12 h after inoculation (therapeutic remdesivir; red triangles). After inoculation, the animals were observed twice daily for clinical signs of disease and scored using a predetermined clinical scoring system (A). On 0, 1, 3, 5 and 6 dpi, clinical examinations were performed during which respiration rate was determined (B), and radiographs were taken. Radiographs were used to score individual lung lobes for severity of pulmonary infiltrates by a clinical veterinarian according to a standard scoring system (0: normal; 1: mild interstitial pulmonary infiltrates; 2: moderate pulmonary infiltrates perhaps with partial cardiac border effacement and small areas of pulmonary consolidation; 3: serious interstitial infiltrates, alveolar patterns and air bronchograms); the cumulative X-ray score is the sum of the scores of the six individual lung lobes per animal; scores shown are from 6 dpi (C). Asterisks indicate statistically significant difference in a two-way (A and B) or one-way (C) ANOVA with Dunnett’s multiple comparisons; black asterisks indicate statistical significance between the vehicle control and prophylactic remdesivir groups, and red asterisks indicate statistical significance between the vehicle control and therapeutic remdesivir groups. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

On days 0, 1, 3, 5, and 6, clinical examinations were performed on the animals, and respiration rates were determined on anesthetized animals. There was a clear increase in respiration rates in the vehicle-treated animals (Fig. 2B), while respiration rates in prophylactically treated animals remained normal throughout the study. Although respiration rate was increased in therapeutically treated animals at 1 dpi, respiration was statistically significantly lower than in vehicle-treated controls at 3 and 6 dpi (Fig. 2B). On examination days, radiographs were collected from all animals and analyzed for the presence of infiltrates; from 3 dpi onward, lung infiltrates became visible on X-ray (SI Appendix, Fig. S1). At 6 dpi, there was statistically significantly less infiltration in the lungs of animals treated both prophylactically and therapeutically with remdesivir as compared to vehicle-treated control animals (Fig. 2C).

Reduced MERS-CoV Viral Lung Loads in Remdesivir-Treated Animals.

At 6 dpi, all animals were euthanized, and respiratory tissues were collected for quantitative analysis of the levels of viral RNA by qRT-PCR. Compared to vehicle-treated control animals, prophylactic remdesivir treatment resulted in significantly lower levels of MERS-CoV replication in the lungs, with lung viral loads 2.5 to 4 logs lower in each lung lobe (Fig. 3A). Although lung viral loads were, on average, lower in individual lung lobes after therapeutic treatment, this was statistically significant in only a few lung lobes, due to larger variation between animals in the therapeutically treated group (Fig. 3A). However, when all lung lobes were combined, the lung viral load in therapeutically treated animals was clearly lower than in vehicle-treated animals (Fig. 3B). Additionally, viral loads were significantly lower in trachea, bronchi, tonsils, and mediastinal lymph nodes of animals treated prophylactically and therapeutically with remdesivir than in vehicle-treated control animals (Fig. 3C and SI Appendix, Fig. S2); viral RNA was not detected in kidney tissue samples (SI Appendix, Fig. S2).

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Fig. 3.

Viral loads in respiratory tract tissues of rhesus macaques inoculated with MERS-CoV and treated with remdesivir. Three groups of six rhesus macaques were inoculated with MERS-CoV strain HCoV-EMC/2012; one group was i.v.-administered 1 mL/kg vehicle solution (vehicle control; gray circles), one group was administered 5 mg/kg remdesivir starting at 24 h before inoculation (prophylactic remdesivir; black squares), and one group was administered 5 mg/kg remdesivir starting at 12 h after inoculation (therapeutic remdesivir; red triangles). Treatment was continued once daily until 6 dpi, when all animals were euthanized and necropsies were performed. At necropsy, tissue samples were collected from all six lung lobes, RNA was extracted, and viral load was determined as TCID50 equivalents per gram tissue. Individual animals and lung lobes are indicated (A), and averages and SDs per group (B). Similarly, viral loads were determined in additional tissues from the respiratory tract of each animal (C). R: right; L: left. Asterisks indicate statistically significant differences in a two-way ANOVA with Dunnett’s multiple comparisons. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Reduced Gross and Histologic Lung Lesions upon Remdesivir Treatment.

Upon necropsy, the area of each lung lobe affected by gross lesions was estimated by a board-certified veterinary pathologist. Gross lung lesions were present in several lung lobes of all of the vehicle-treated control animals (Fig. 4A). In contrast, gross lung lesions were completely absent in the lungs of animals that received prophylactic remdesivir treatment. In animals treated therapeutically with remdesivir, there were obvious gross lesions present in five out of six animals; however, the total area of lungs affected by gross lesions was statistically significantly smaller than in vehicle-treated control animals (Fig. 4A).

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Fig. 4.

Pathological findings in the lungs of rhesus macaques inoculated with MERS-CoV and treated with remdesivir. Three groups of six rhesus macaques were inoculated with MERS-CoV strain HCoV-EMC/2012; one group was i.v.-administered 1 mL/kg vehicle solution (vehicle control; gray circles), one group was administered 5 mg/kg remdesivir starting at 24 h before inoculation (prophylactic remdesivir; black squares), and one group was administered 5 mg/kg remdesivir starting at 12 h after inoculation (therapeutic remdesivir; red triangles). Treatment was continued once daily until 6 dpi, when all animals were euthanized and necropsies were performed. At necropsy, the percentage of each lung lobe affected by gross lesions was estimated by a board-certified veterinary pathologist (A). Lung samples were collected and stained with H&E and analyzed for the presence of lesions by a board-certified veterinary pathologist. Each lung was given a score from 0 to 4 based on the abundance of lesions; the cumulative histology score is the sum of the scores of the six individual lung lobes per animal (B). One representative H&E image was chosen for each group (magnification: 100×) (C). Lung samples were also stained with a polyclonal α-MERS-CoV antibody; one representative image was chosen for each group (magnification: 200×) (D). Images in C and D were chosen as representative images of lung lesions and antigen expression, respectively, rather than being images from consecutive tissue slides. Asterisks indicate statistically significant differences in a two-way ANOVA with Dunnett’s multiple comparisons. **P < 0.01; ****P < 0.0001.

In addition, the severity of histologic lung lesions was assessed by assigning a score for each lung lobe. The resulting cumulative lung histology score was compared between treatment groups to assess differences in the severity of histologic lesions. Cumulative lung histology scores were significantly lower in animals treated prophylactically with remdesivir (Fig. 4B). The large variation between animals in the therapeutically treated group meant that the lower average histology score did not reach statistical significance (Fig. 4B).

Histologically, all of the vehicle-treated control animals developed some degree of pulmonary pathology when inoculated with MERS-CoV. Lesions were multifocal, frequently centered on terminal bronchioles, and consisted of minimal to marked, interstitial pneumonia, characterized by thickening of alveolar septae by edema fluid and fibrin and small to moderate numbers of macrophages and fewer neutrophils. Alveoli contained moderate numbers of pulmonary macrophages and neutrophils. In areas with moderate to marked changes, there was abundant alveolar edema and fibrin with multifocal formation of hyaline membranes, as well as abundant type II pneumocyte hyperplasia. Perivascular infiltrates of inflammatory cells multifocally within and adjacent to affected areas of the lung were also observed (Fig. 4C). In contrast, all animals treated prophylactically with remdesivir had essentially normal pulmonary tissue with no evidence of coronavirus infection (Fig. 4C). Animals treated with remdesivir therapeutically demonstrated various levels of severity of coronaviral pneumonia. In two out of six animals, no histologic evidence of pneumonia was detected. In three animals, multifocal, minimal to moderate interstitial pneumonia was observed like that described for the control animals; however, the lesions were less severe than in the controls and not as widely distributed throughout the lung lobes. Only one out of six animals had moderate interstitial pneumonia that was indistinguishable from the vehicle-treated control animals in severity and distribution.

Immunohistochemical analysis for the presence of MERS-CoV antigen showed small numbers of antigen-positive type I pneumocytes in all vehicle-treated control animals and in five out of six animals treated therapeutically with remdesivir; there was no difference in number or distribution of antigen-positive cells in animals where antigen was detected. MERS-CoV antigen could not be detected in any of the animals treated prophylactically with remdesivir (Fig. 4D).