Comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against MERS-CoV

Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV) is the causative agent of a severe respiratory disease associated with more than 2468 human infections and over 851 deaths in 27 countries since 2012. There are no approved treatments for MERS-CoV infection although a combination of lopinavir, ritonavir and interferon beta (LPV/RTV-IFNb) is currently being evaluated in humans in the Kingdom of Saudi Arabia. Here, we show that remdesivir (RDV) and IFNb have superior antiviral activity to LPV and RTV in vitro. In mice, both prophylactic and therapeutic RDV improve pulmonary function and reduce lung viral loads and severe lung pathology. In contrast, prophylactic LPV/RTV-IFNb slightly reduces viral loads without impacting other disease parameters. Therapeutic LPV/RTV-IFNb improves pulmonary function but does not reduce virus replication or severe lung pathology. Thus, we provide in vivo evidence of the potential for RDV to treat MERS-CoV infections.

Conflict of interest statement

A.C.S. received a contract from Gilead Sciences to support the in vitro and in vivo efficacy studies reported herein. These authors are employees of Gilead Sciences and hold stock in Gilead Sciences: Alison Hogg, Darius Babusis, Michael O. Clarke, Jamie E. Spahn, Laura Bauer, Scott Sellers, Danielle Porter, Joy Y. Feng, Tomas Cihlar, and Robert Jordan.

Figures

Fig. 1. RDV and IFNb have superior antiviral activity to LPV and RTV.

Graphs depict mean % inhibition of MERS-CoV replication (left Y-axis) and % cytotoxicity (right Y-axis) of antivirals. Calu-3 cells were infected in sextuplicate with MERS-CoV nanoluciferase (nLUC) at a multiplicity of infection (MOI) of 0.08 in the presence of a dose response of drug for 48 h, after which replication was measured through quantitation of MERS-CoV–expressed nLUC. Cytotoxicity was measured in similarly treated but uninfected cultures via Cell-Titer-Glo assay. Representative data are shown from four independent experiments.

Fig. 2. Prophylactic RDV reduces MERS-CoV replication and disease.

a Percent starting weight of 9–12-week-old male and female Ces1c−/−hDPP4 mice prophylactically administered subcutaneous vehicle or remdesivir (RDV, 25 mg/kg) BID the day prior to infection with either 5E + 04 (vehicle n = 14, RDV n = 14) or 5E + 05 (vehicle n = 14, RDV n = 15) plaque-forming units (pfu) MERS M35C4. Asterisks indicate statistically significant differences (P < 0.05) as determined by two-way ANOVA and Tukey’s multiple comparison test. b Percent survival of each cohort and survival analysis by Mantel–Cox test (P < 0.05, N per group noted in a). c Lung hemorrhage scored on a scale of 0–4, where 0 is a normal pink healthy lung and 4 is a completely dark red lung. On 4 dpi, N = 4/group, and on 6 dpi the remaining animals are plotted. Asterisks indicate statistically significant differences (P < 0.05) as determined by two-way ANOVA and Tukey’s multiple comparison test. d MERS-CoV lung titer on 4 (N = 4) and 6 dpi (all remaining animals). Asterisks indicate statistically significant differences (P < 0.05) as determined by two-way ANOVA and Sidek’s multiple comparison test. For a, c, d, the boxes encompass the 25th to 75th percentile, the line is at the median, while the whiskers represent the range. e Hematoxylin (nuclei, blue) and immunostaining for MERS-CoV antigen (brown) in lung tissue sections from 4 dpi. All photos were taken with the same magnification. The black bar indicates 100 µM scale. Images from representative mice for each group are shown.

Fig. 3. Remdesivir prophylaxis reduces features of acute lung injury.

The histological features of acute lung injury were blindly scored using the American Thoracic Society Lung Injury Scoring system creating an aggregate score for the following phenotypes: neutrophils in the alveolar and interstitial space, hyaline membranes, proteinaceous debris filling the air spaces, and alveolar septal thickening. Three randomly chosen high power (×60) fields of diseased lung were assessed per mouse. Representative images are shown for mock infected as well as those administered prophylactic vehicle or RDV and infected with either 5E + 04 or 5E + 05 pfu mouse adapted MERS-CoV. The numbers of mice scored per group: Vehicle 5E + 04 pfu MERS-CoV N = 5, Vehicle 5E + 05 pfu MERS-CoV N = 3, RDV 5E + 04 pfu MERS-CoV N = 5, RDV 5E + 05 pfu MERS-CoV N = 5. Symbols identifying example features of disease are indicated in the figure. All images were taken at the same magnification. The black bar indicates 100 µm scale. For the graph, the boxes encompass the 25th to 75th percentile, the line is at the median, while the whiskers represent the range. Statistical significance was determined by Mann–Whitney test.

Fig. 4. Prophylactic LPV/RTV + IFNb does not improve disease outcomes.

a Percent starting weight (Left) of 12–14-week-old female Ces1c−/−hDPP4 mice infected with 5E + 04 pfu MERS M35C4 and treated BID with either vehicle (n = 9) or remdesivir (RDV, 25 mg/kg, n = 9) subcutaneously beginning −1 dpi. Asterisks indicate statistically significant differences (P < 0.05) as determined by two-way ANOVA and Tukey’s multiple comparison test. (Middle) MERS-CoV lung titer on 2 (N = 3) and 6 dpi (all remaining animals). Asterisks indicate statistically significant differences (P < 0.05) as determined by Mann–Whitney test. (Right) WBP was used to assess pulmonary function in mice. PenH is a surrogate measure of airway resistance or bronchoconstriction. Asterisks indicate statistical differences by two-way ANOVA with Sidek’s multiple comparison test. b Percent starting weight (left), virus lung titer (middle), and pulmonary function metric PenH (right) of cohorts of mice similar in age and sex and infected similarly with MERS-CoV as in b but treated with vehicle (n = 9), LPV/RTV + IFNb low (1× human equivalent) (n = 9), LPV/RTV + IFNb high (25× human equivalent) (n = 9), or IFNb high only (n = 9). Oral vehicle or lopinavir/ritonavir (160/40 mg/kg) were administered orally once daily beginning the −1 dpi. IFNb treatment was initiated 2 h prior to infection and every other day thereafter. To control for dosing effects, vehicle-treated mice received both LPV/RTV vehicle and subcutaneous PBS to mirror IFNb injections. Likewise, IFNb only group received oral vehicle to mirror that seen in orally dosed groups. Similar statistical tests performed on a were performed on b. For the box and whisker plots, the boxes encompass the 25th to 75th percentile, the line is at the median, while the whiskers represent the range.

Fig. 5. Therapeutic RDV reduces replication and pathology.

Percent starting weight of 10–12-week-old female Ces1c−/−hDPP4 mice infected with 5E + 04 pfu MERS M35C4 and treated with a subcutaneous vehicle for RDV (N = 13) or remdesivir (RDV, 25 mg/kg, N = 14) BID beginning 1 dpi or b vehicle for LPV/RTV-IFNb (N = 15), LPV/RTV-IFNb low (N = 16) or LPV/RTV-IFNb high (N = 16) beginning 1 dpi. Oral vehicle or lopinavir/ritonavir (160/40 mg/kg) was administered orally once daily. IFNb low (1x human equivalent dose of 1.6 MIU/kg) and high (25x human equivalent dose of 40 MIU/kg) or PBS vehicle were administered via subcutaneous injection every other day. Asterisks indicate statistical differences by two-way ANOVA with Tukey’s multiple comparison test. c Lung hemorrhage 6 dpi for all animals in a, b scored on a scale of 0–4, where 0 is a normal pink healthy lung and 4 is a diffusely discolored dark red lung. d MERS-CoV lung titer 6 dpi in mice as described in a, b. Asterisks indicate statistical significance (N group described in a and b, P < 0.05) by one-way ANOVA with Kruskal–Wallis test for (c, d). Data for a–d are compiled from two independent experiments. For the box and whisker plots, the boxes encompass the 25th to 75th percentile, the line is at the median, while the whiskers represent the range. e Representative photomicrographs of MERS-CoV antigen (brown) and hematoxylin stained nuclei (blue) in mouse lung tissue sections from 6 dpi. The black bar is 100 µM.

Fig. 6. Therapeutic RDV and LPV/RTV-IFNb improve pulmonary function.

Whole-body plethysmography (WBP) was used to assess pulmonary function in mice. Representative WBP data for one of the two studies depicted in Fig. 5 are shown. All groups were N = 4 per day. EF50 is the flow rate at 50% expired volume. PenH is a surrogate measure of airway resistance. Rpef is the fraction of expiration before peak expiratory flow is reached. Altered EF50, PenH, and Rpef indicate bronchoconstriction or airway obstruction. Asterisks indicate statistical differences by two-way ANOVA with Sidek’s multiple comparison test in a and two-way ANOVA with Tukey’s multiple comparison test in b. The boxes encompass the 25th to 75th percentile, the line is at the median, while the whiskers represent the range.

Fig. 7. Therapeutic RDV but not LPV/RTV-IFNb diminishes signs of ALI.

a Representative images of the histological features of acute lung injury 6 dpi comparing a mock-infected mouse to the therapeutic treatment groups described in Figs. 5 and 6. Symbols identifying example features of disease are indicated in the figure. b American Thoracic Society Lung Injury Score derived as described in Fig. 3. The numbers of animals per group quantitated: vehicle RDV N = 7, RDV N = 7, vehicle LPV/RTV-IFNb N = 9, LPV/RTV-IFNb low N = 7, LPV/RTV-IFNb high N = 8. c Diffuse alveolar damage score quantitating the degree of cellular sloughing, necrosis, and breakdown of barrier epithelium and vascular leakage. For both b and c, scores were blindly assessed in three random high power (×60) fields of diseased lung tissue sections. d Quantitation of cleaved caspase-3 antigen staining in lung tissue sections from studies described in Figs. 5–7. Cleaved caspase-3 is a marker of cell death. The numbers of animals per group quantitated for all groups was N = 5/group. For the box and whisker plots, the boxes encompass the 25th to 75th percentile, the line is at the median, while the whiskers represent the range. For b–d, asterisks indicate statistical significance by one-way ANOVA and Kruskal–Wallis multiple comparison test.