Treatment With Lopinavir/Ritonavir or Interferon-β1b Improves Outcome of MERS-CoV Infection in a Nonhuman Primate Model of Common Marmoset

Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe disease in human with an overall case-fatality rate of >35%. Effective antivirals are crucial for improving the clinical outcome of MERS. Although a number of repurposed drugs, convalescent-phase plasma, antiviral peptides, and neutralizing antibodies exhibit anti-MERS-CoV activity in vitro, most are not readily available or have not been evaluated in nonhuman primates. We assessed 3 repurposed drugs with potent in vitro anti-MERS-CoV activity (mycophenolate mofetil [MMF], lopinavir/ritonavir, and interferon-β1b) in common marmosets with severe disease resembling MERS in humans. The lopinavir/ritonavir-treated and interferon-β1b-treated animals had better outcome than the untreated animals, with improved clinical (mean clinical scores ↓50.9%-95.0% and ↓weight loss than the untreated animals), radiological (minimal pulmonary infiltrates), and pathological (mild bronchointerstitial pneumonia) findings, and lower mean viral loads in necropsied lung (↓0.59-1.06 log10 copies/glyceraldehyde 3-phosphate dehydrogenase [GAPDH]; P < .050) and extrapulmonary (↓0.11-1.29 log10 copies/GAPDH; P < .050 in kidney) tissues. In contrast, all MMF-treated animals developed severe and/or fatal disease with higher mean viral loads (↑0.15-0.54 log10 copies/GAPDH) than the untreated animals. The mortality rate at 36 hours postinoculation was 67% (untreated and MMF-treated) versus 0-33% (lopinavir/ritonavir-treated and interferon-β1b-treated). Lopinavir/ritonavir and interferon-β1b alone or in combination should be evaluated in clinical trials. MMF alone may worsen MERS and should not be used.

Keywords: Kaletra; MERS; animal; common marmoset; coronavirus; interferon; lopinavir; mycophenolate; primate; treatment.

Figure 1.

Schedule of MERS-CoV inoculation, examination, treatment, and necropsy of the common marmosets. ^aK3 died unexpectedly during anesthesia at 24 hpi, which was likely related to the anesthesia procedure, as common marmosets are very small and fragile. Abbreviations: hpi, hours postinoculation; MERS-CoV, Middle East respiratory syndrome coronavirus; MMF, mycophenolate mofetil.

Figure 2.

Mean clinical scores of MERS-CoV-infected common marmosets at different time points after virus inoculation. Necropsies of the common marmosets were scheduled at 72 hpi or when the clinical score was ≥35. The number of animals remaining in the experiment is indicated above the bar at each time point. Student t test was performed for comparison when all 3 animals in each group were available. ^aK3 died unexpectedly during anesthesia at 24 hpi, which was likely related to the anesthesia procedure, as common marmosets are very small and fragile. Abbreviations: hpi, hours postinoculation; MERS-CoV, Middle East respiratory syndrome coronavirus; MMF, mycophenolate mofetil.

Figure 3.

Serial measurements of body weight of MERS-CoV-infected common marmosets. Mean percentage of weight loss at 24 and 72 hpi, compared with the baseline body weight at 0 hpi. The number of animals remaining in the experiment is indicated above the bar at each time point. ^aK3 died unexpectedly during anesthesia at 24 hpi, which was likely related to the anesthesia procedure, as common marmosets are very small and fragile. *P < .05. Abbreviations: hpi, hours postinoculation; MERS-CoV, Middle East respiratory syndrome coronavirus; MMF, mycophenolate mofetil.

Figure 4.

Representative radiological findings and macroscopic pathology of the lungs of MERS-CoV-infected common marmosets. Dorsal-ventral and lateral chest X-rays were performed immediately prior to necropsy. The circles represent areas of interstitial infiltration indicative of pneumonia. The arrows represent gross lesions in the necropsied lungs. A, Untreated common marmoset (U2) at 36 hpi. B, MMF-treated common marmoset (M2) at 36 hpi. C, Lopinavir/ritonavir-treated common marmoset (K1) at 72 hpi. D, Interferon-β1b-treated common marmoset (B2) at 72 hpi. Abbreviations: D, dorsal side; hpi; hours postinoculation; L, left side; MERS-CoV, Middle East respiratory syndrome coronavirus; MMF, mycophenolate mofetil.

Figure 5.

Representative photomicrographs of histopathology examination and immunohistochemical staining of necropsied lung tissues of MERS-CoV-infected common marmosets. Severe acute bronchointerstitial pneumonia centered on terminal bronchioles, with thickened alveolar interstitium and alveoli being filled with large amount of inflammatory cell infiltrate, edema, and hemorrhage in hematoxylin and eosin (H&E)–stained necropsied lung tissue of untreated (A) and MMF-treated (C) animals collected at 36 hpi (magnification, 100×). Abundant expression of MERS-CoV nucleocapsid protein was detected by mouse anti-MERS-CoV nucleocapsid protein (1:200) antibody (green) overlaid with counterstaining by propidium iodide (red) in the necropsied lung tissues of the untreated (B) and MMF-treated (D) animals (magnification, 100×). These pathological changes were seen extensively in multiple lobes of the necropsied lungs of the untreated and MMF-treated animals. Mild acute bronchointerstitial pneumonia with small amount of inflammatory cell infiltrate and preserved histological architecture in H&E-stained necropsied lung tissue of lopinavir/ritonavir-treated (E) animals collected at 72 hpi (magnification, 100×). Mild to moderate acute bronchointerstitial pneumonia with moderate amount of inflammatory cell infiltrate and hemorrhage in H&E-stained necropsied lung tissue of interferon-β1b-treated (G) animals collected at 72 hpi (magnification, 100×). Small amount of MERS-CoV nucleocapsid protein expression was detected in the necropsied lung tissues of the lopinavir/ritonavir-treated (F) and interferon-β1b-treated (H) animals (magnification, 100×). These pathological changes were only seen in lesions which were confined to 1–2 lobes of the necropsied lungs of the lopinavir/ritonavir-treated and interferon-β1b-treated animals. H&E-stained necropsied lung tissue (I) (magnification, 100×) without MERS-CoV nucleocapsid protein expression (J) of an uninfected control animal. Abbreviations: hpi; hours postinoculation; MERS-CoV, Middle East respiratory syndrome coronavirus; MMF, mycophenolate mofetil.

Figure 6.

Mean viral loads with standard deviation values in different tissues of MERS-CoV-infected common marmosets collected at the time of necropsy. *P <.05. ^aTwo of the 3 interferon-β1b-treated animals had undetected viral loads in necropsied kidney tissues, which accounted for a large standard deviation value and apparent lack of statistically significant difference from the mean viral load of the untreated animals. Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MERS-CoV, Middle East respiratory syndrome coronavirus; MMF, mycophenolate mofetil.