Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel betacoronavirus that was first identified in patients with unexplained pneumonia in Wuhan, Hubei, China in December 2020 [1
]. Within just four months, SARS-CoV-2 has disseminated globally to cause a pandemic of Coronavirus Disease 2019 (COVID-19) with more than 2,600,000 laboratory-confirmed cases including over 180,000 deaths and immeasurable socioeconomic disruption [3
]. The clinical severity of COVID-19 ranges from asymptomatic infection to fatal disease [4
]. Symptomatic COVID-19 patients commonly present with fever, myalgia, cough, dyspnea, fatigue, and radiological evidence of ground-glass lung opacities [4
]. Some patients also develop extrapulmonary manifestations, such as diarrhea, confusion, anosmia, ageusia, lymphopenia, thrombocytopenia, and deranged liver and renal function tests [4
]. Severe complications of COVID-19 include acute respiratory distress syndrome, multiorgan dysfunction syndrome, and cytokine storm [5
]. The overall case fatality rate of COVID-19 is about 7%, but may be up to 15%–20% among elderly and immunocompromised patients [3
Effective antivirals are essential for improving the clinical outcome of patients with severe COVID-19. As de novo development of novel antiviral agents would take years and inevitably lag behind the rapid expansion of the pandemic, repurposing of existing antiviral agents has been exploited to identify immediately available treatment options for COVID-19. Among existing antiviral agents, the most likely agents that may be active against SARS-CoV-2 would be those with known broad-spectrum antiviral activities and those with reported activities against coronaviruses. A number of such antiviral agents, such as remdesivir, lopinavir, chloroquine, and hydroxychloroquine, have been recently reported to have anti-SARS-CoV-2 activity in vitro [8
]. A recent preliminary report suggested that adult COVID-19 patients treated with intravenous remdesivir had a shorter median time to recovery and lower mortality rate than the patients treated with placebo [12
]. Additionally, immunomodulating agents, such as interferons (IFNs) and tocilizumab (humanized monoclonal antibody against interleukin-6 receptor), have been used in combination with other antivirals in ongoing clinical trials [3
]. However, while different types of IFNs are being tested in ongoing clinical trials for COVID-19, their comparative antiviral effects against SARS-CoV-2 and thus the optimal clinical choice of IFN for COVID-19 remains unknown. In this study, we conducted a primary screening to select the most potent anti-SARS-CoV-2 agents among 22 antiviral agents which have known broad-spectrum activities against coronaviruses and/or other viruses. We then thoroughly characterized the selected antiviral agents’ in vitro anti-SARS-CoV-2 activities. Our results identified host-based broad-spectrum antivirals targeting the IFN and lipogenesis pathways as potential anti-SARS-CoV-2 agents. These findings have important implications for rational design of animal studies and clinical trials for COVID-19.
In this study, we evaluated the anti-SARS-CoV-2 activity of antiviral agents with broad-spectrum antiviral activities against coronaviruses and/or other viruses. In our primary screening using a fixed antiviral agent concentration and virus inoculum, we identified recombinant IFNs and lipogenesis modulators to be the most potent anti-SARS-CoV-2 agents among 22 broad-spectrum antivirals. These findings have important implications for the choice of clinically available recombinant IFNs to be used in COVID-19 patients and development of lipogenesis modulators as potential anti-SARS-CoV-2 therapeutics.
IFNs are glycoproteins with strong antiviral activities that represent one of the first lines of host immune response against invading pathogens [20
]. These proteins are classified into three groups, types I, II, and III IFNs, based on the structure of their receptors on the cell surface [20
]. IFNs are known for their broad-spectrum antiviral activities against a wide range of DNA and RNA viruses, through inducing the expressions of interferon-stimulated genes in host cells, such as Protein Kinase R, oligoadenylate synthetase, and RNase L [20
]. These interferon-stimulated genes suppress viral replication by inhibiting multiple steps in a viral life cycle, including viral RNA transcription and viral protein translation. Recombinant IFN-α and IFN-β exhibited potent antiviral activity against SARS-CoV and MERS-CoV in vitro and in animal models [24
]. Recombinant IFN-γ exhibited limited anti-coronaviral activity in vitro, but might be synergistic with type I IFNs [17
]. In this study, we demonstrated the anti-SARS-CoV-2 activity of five clinically-approved preparations of recombinant IFNs, including Pegasys (pegylated IFN-α2a), Avonex (IFN-β1a), Rebif (IFN-β1a), Betaferon (IFN-β1b), and Immukin (IFN-γ1b). Among them, Betaferon exhibited the most potent anti-SARS-CoV-2 effect with the lowest EC50
of 31.2 IU/mL and the highest selectivity index of >1602.6. Importantly, the EC50
of Betaferon against SARS-CoV is below its achievable peak serum concentration (Cmax) with standard subcutaneous dosing of 16 million units of Betaferon (40 IU/mL). Notably, Betaferon was similarly found to be the most potent recombinant IFN for the highly virulent MERS-CoV and significantly improved the clinical, virological, and histopathological parameters of MERS-CoV-infected common marmosets [17
]. In SARS and MERS patients, the use of recombinant IFN-α and/or IFN-β treatment was generally well tolerated with minimal adverse effects [24
]. Although the clinical benefits of recombinant IFN treatment in SARS and MERS patients remain inconclusive, the apparent discrepancy between the in vitro and in vivo antiviral effects might be related to the delay in treatment commencement after symptom onset. Because SARS-CoV-2 is able to achieve more than 3 folds higher viral load than SARS-CoV within 48 h in human lung tissues by minimally eliciting the host IFN response, it would be important to supplement COVID-19 patients with recombinant IFNs, especially IFN-β1b, before cytokine storm develops, with other effective virus-targeting antivirals [60
]. Importantly, inhaled IFN-β is well tolerated and enhances both systemic and local innate immunity with upregulated antiviral gene expression and reduced proinflammatory cytokines in sputum [61
]. This treatment strategy might be especially useful when given early to COVID-19 patients who usually have the peak respiratory tract viral loads within the first week of symptom onset [62
In addition to the recombinant IFNs, we also identified antiviral agents that target the host lipogenesis pathways as potential anti-SARS-CoV-2 agents. AM580 is a selective retinoic acid receptor-α agonist which was recently identified to have broad-spectrum antiviral activities against various families of DNA and RNA viruses, including Coronaviridae, Flaviviridae, Orthomyxoviridae, Picornaviridae, and Adenoviridae [15
]. AM580 inhibits virus replication through interaction with sterol regulatory element-binding protein (SREBP) and downregulation of multiple SREBP proteolytic processes and SREBP-regulated lipid biosynthesis pathways, such as double-membrane vesicle formation by MERS-CoV [15
]. Our time-of-drug-addition assay showed that AM580 inhibited the post-entry events of the SARS-CoV-2 replication cycle, which corroborated with the hypothesized restrictive effects of AM580 on lipid biosynthesis in SARS-CoV-2 infection.
The oxysterol 25-hydroxycholesterol is a metabolite of cholesterol that is produced and secreted by macrophages and has multiple effects on lipid metabolism, especially lipid biosynthesis and immunity [63
]. 25-hydroxycholetserol has been shown to inhibit feline coronavirus, porcine epidemic diarrhea virus, and porcine transmissible gastroenteritis virus possibly through induction of intracellular cholesterol accumulation [64
]. Moreover, 25-hydroxycholesterol is active against various emerging RNA viruses, including Ebola virus, Nipah virus, Rift Valley fever virus, and Zika virus, and DNA and RNA viruses that cause chronic infections, such as human immunodeficiency virus, herpes simplex virus, and varicella zoster virus [18
]. Mechanistically, 25-hydroxycholesterol and its downstream metabolite 25-hydroxycholesterol-3-sulfate (25HC3S) block membrane fusion between virions and host cells through diametrical regulation of lipid metabolism and inflammatory response via LXR/SREBP-1 and IkappaBalpha/NF-kappaB signaling [18
]. Addition of 25HC3S to primary rat hepatocytes decreased nuclear LXR and SREBP-1 protein levels, downregulated their target genes, acetyl CoA carboxylase 1, fatty acid synthase, and SREBP-2 target gene HMG reductase, which are key enzymes involved in fatty acid and cholesterol biosynthesis [68
]. Interestingly, the expression of the interferon stimulating gene-encoded cholesterol-25-hydroxylase (CH25H) is upregulated by IFNs and Toll-like receptors to convert cholesterol into 25-hydroxycholesterol [69
]. Thus, combination treatment with IFNs may further enhance the antiviral effects of 25-hydroxycholesterol and should be further investigated.
Our study had limitations. First, we used a fixed antiviral agent concentration in our primary screening in order to identify the antiviral agents with the lowest EC50
among the 22 broad-spectrum antivirals. This might have overlooked antiviral agents that can inhibit SARS-CoV-2 at higher concentrations. For example, favipiravir has been shown to inhibit SARS-CoV-2 replication with an EC50
of 67 µM [8
]. Similarly, galidesivir, a broad-spectrum RNA-dependent RNA polymerase inhibitor, inhibited the 2003 SARS-CoV with an EC50
of 57.7 µM [36
]. Second, antiviral evaluation of the selected reagents should be performed in additional primary cells to comprehensively document their antiviral activities. Third, the combination effects of the host-based IFN-β1b, AM580, and 25-hydroxycholesterol with virus-based antivirals, such as remdesivir and lopinavir, should be further evaluated in vitro and/or in vivo. Targeting multiple steps in the viral replication cycle might help to enhance the therapeutic effects of these virus-based antivirals in COVID-19 patients. Indeed, during the revision of this manuscript, a multi-center, open-label, randomized phase 2 clinical trial comparing adult COVID-19 patients treated with triple combination antiviral therapy (IFN-β1b, lopinavir-ritonavir, and ribavirin) with those treated with lopinavir-ritonavir monotherapy was reported. The results showed that the combination therapy group had a significantly shorter median time from commencement of treatment to negative nasopharyngeal swab than the control monotherapy group (7 vs. 12 days) [70
]. Additional studies to evaluate the effects of combination therapies using the other antiviral agents identified in this study should be considered.