INTRODUCTION
The genetically diverse coronavirus (CoV) family, currently composed of four genogroups [1 (alpha), 2 (beta), 3 (gamma), and 4 (delta)], infects birds and a variety of mammals. Thus far, only CoV groups 1 and 2 are known to infect humans. Although CoV replication machinery exhibits substantial proofreading activity, replication of viral genomic RNA is inherently error-prone, driving the existence of genetically related yet diverse quasi-species (
1). Most CoV strains are narrow in their host range, but zoonotic CoVs have a proclivity to jump into new host species (
2). Severe acute respiratory syndrome CoV (SARS-CoV) and Middle East respiratory syndrome CoV (MERS-CoV) are recent examples of newly emerging CoV that caused severe disease in immunologically naïve human populations. SARS-CoV emerged in Guangdong, China in 2002 and, with the aid of commercial air travel, spread rapidly throughout the globe, causing more than 8000 cases with 10% mortality (
2). In 2012, it was discovered that MERS-CoV evolved to infect humans through bats by way of an intermediate camel host, causing more than 1700 cases with almost 40% mortality and, like SARS-CoV, air travel has fueled global spread to 27 countries (
2). MERS-CoV is endemic in the Middle East, and serologic studies in the Kingdom of Saudi Arabia and Kenya suggest fairly frequent infections in humans (>45,000 persons) (
3,
4). The SARS-CoV epidemic ended over a decade ago, but several SARS-like CoVs have been isolated from bats that efficiently use the human angiotensin-converting enzyme 2 receptor, replicate to high titer in primary human airway cells, and are resistant to existing therapeutic antibodies and vaccines (
5,
6). With increasing overlap of human and wild animal ecologies, the potential for novel CoV emergence into humans is great (
2). Broad-spectrum CoV therapies capable of inhibiting known human CoV would address an immediate unmet medical need and could be an invaluable treatment in the event of novel CoV emergence in the future.
Currently, there are no approved specific antiviral therapies for CoV in humans. Attempts made to treat both SARS-CoV and MERS-CoV patients with approved antivirals (that is, ribavirin and lopinavir-ritonavir) and immunomodulators (that is, corticosteroids, interferons, etc.) have not been effective in randomized controlled trials (
7). Clinical development of effective CoV-specific direct-acting antivirals (DAAs) has been elusive, although there are several conserved druggable CoV enzyme targets including 3C-like protease, papain-like protease, and nonstructural protein 12 (nsp12) RNA-dependent RNA polymerase (RdRp) (
7). In 2016, Warren
et al. reported the in vivo antiviral efficacy of a small-molecule monophosphoramidate prodrug of an adenosine analog, GS-5734, against Ebola virus in nonhuman primates (
8). Because the mechanism of action of GS-5734 for Ebola virus is the inhibition of the viral RdRp and previous work had suggested weak activity of the nucleoside component of GS-5734 against SARS-CoV (
9), we sought to assess the antiviral potency and breadth of activity of GS-5734 against a diverse panel of human and zoonotic CoV.
DISCUSSION
Emerging viral infections represent a critical global health concern because specific antiviral therapies and vaccines are usually lacking. To maximize the potential public health benefit of therapeutics against emerging viruses, they should be efficacious against past (that is, SARS-CoV), current (that is, MERS-CoV), and future emerging viral threats. Knowledge of the spectrum of therapeutic efficacy is essential for making informed clinical decisions, especially in the early stages of an outbreak. Because zoonotic CoV emergence is driven by an amalgamation of human, wild animal, and viral factors, it is difficult to gauge zoonotic CoV emergence potential based on viral genome sequences alone (
2). Here, we provide an example of a successful public-private partnership that combines metagenomics, synthetic biology, primary human cell culture models, drug metabolism, PKs, and in vivo models of viral pathogenesis to demonstrate broad-spectrum activity of a drug candidate against a virus family prone to emergence (fig. S8). Although we demonstrated broad-spectrum efficacy against human and zoonotic CoV from multiple CoV genogroups, we have not yet assessed antiviral efficacy for all CoV genogroups, which is a limitation of our current study. Nevertheless, our panel of reconstructed human and zoonotic bat CoV was essential to determine whether GS-5734 would be efficacious against highly divergent emerged (SARS-CoV), emerging (MERS-CoV), and circulating zoonotic strains with pandemic potential (that is, WIV1 and SHC014) (
5,
6,
16,
17). In the future, the rapid development of vaccines, therapies, and diagnostics for emerging viruses will be dependent on the reconstruction and the in vitro and in vivo adaptation of these viruses in the laboratory.
Here, we report the broad-spectrum antiviral efficacy of a small molecule against multiple genetically distinct CoV in vitro and in vivo. Current vaccine and human monoclonal antibody approaches have proven to be effective but typically have limited breadth of protection due to antigenic diversity in the CoV spike glycoprotein (
5,
6). Conversely, RdRp-targeting therapies such as GS-5734 are more likely to be broadly active against past, current, and future CoV due to the inherent genetic conservation of the CoV replicase. As evidenced by the failure of the nucleoside prodrug balapiravir to translate in vitro efficacy into in vivo efficacy in mice and humans, antiviral drug candidates should be thoroughly evaluated in the most biologically relevant models of pathogenesis to maximize clinical translatability (
18). Cell type–specific differences in the active transport and/or metabolism of nucleoside analogs may affect the antiviral profile (
19). Thus, we aimed to expand on previous in vitro studies of SARS-CoV and MERS-CoV antiviral efficacy that were limited to monkey kidney cancer cell lines (
8,
9). Similar in cellular complexity and physiology to the human conducting airway, the HAE cell culture contains mucus-secreting cells, basolateral cells, and some of the main target cells of SARS-CoV (ciliated epithelial cells) and MERS-CoV (nonciliated epithelial cells) in vivo (
11,
20). With our HAE cell antiviral efficacy data, we provide strong evidence that GS-5734 will be taken up and metabolized in cells targeted by multiple human and zoonotic CoV in the human lung.
Preclinical in vivo antiviral efficacy studies provide insight into the PK/pharmacodynamic (PD) relationship of a drug from which effective dosing regimens can be extrapolated for human clinical trial. To maximize the utility of preclinical PK/PD studies, the use of animal models that accurately recapitulate human disease is essential. Multiple aspects of the human disease are captured by the mouse-adapted SARS-CoV (SARS-CoV MA15) model used herein, including high-titer virus replication limited to the lung, the development of ARDS, age-related exacerbation of disease, and death. In contrast to humans infected with SARS-CoV where viral titers peak 7 to 10 days after the onset of symptoms, virus titers in the lungs of SARS-CoV MA15–infected C57BL/6 mice rapidly increase 4 to 5 logs and peak at 2 dpi, concurrent with maximal damage to the conducting airway epithelium and alveoli (
14). After 2 dpi, virus titers wane, and the remainder of the disease course is driven by immunopathology. Thus, the 7 to 10 days before peak replication in humans is compressed into the first 48 hours of our mouse model. Similar to humans, disease severity in SARS-CoV–infected mice is directly correlated with lung viral load, which can be modulated through increasing dose of input virus (
14,
21). With both prophylactic (−1 dpi) and therapeutic (+1 dpi) dosing of GS-5734, we demonstrate a reduction in replication below a disease-causing threshold. Therapeutic treatment beginning at 2 dpi reduced lung viral loads yet did not improve disease outcomes, suggesting that antivirals initiated after virus replication and immunopathology have reached their tipping point were not clinically beneficial. This result is not surprising given the precedent set by the influenza antiviral oseltamivir, where treatment efficacy diminishes with time after the onset of symptoms (
22). Like SARS-CoV, MERS-CoV titers in the respiratory tract peak in the second week after the onset of symptoms (
23). Thus, the window in which to administer antiviral treatment after the onset of symptoms but before achieving peak virus titers should be prolonged in humans as compared to experimentally infected mice. Unfortunately, the differences in SARS-CoV pathogenesis among mice and humans noted above limit our ability to determine the time at which treatment no longer will provide a clinical benefit in humans. Nevertheless, our studies provide data that strongly support the testing of GS-5734 in nonhuman primates and suggest that therapeutic treatment of MERS-CoV–infected humans with GS-5734 will help diminish virus replication and disease if administered early enough during the course of infection.
Currently, there are no approved antiviral treatments for SARS-CoV or MERS-CoV that specifically target the virus. Multiple therapeutic approaches against SARS-CoV and MERS-CoV are currently in development including immunomodulation, vaccination, DAAs, and host-targeted antivirals (
7). Known antivirals, such as ribavirin and lopinavir-ritonavir, and immunomodulators, such as interferon and corticosteroids, have been used to treat both SARS-CoV and MERS-CoV patients, but none were proven effective in randomized controlled trials (
7). Cell culture studies in multiple cell lines have demonstrated antiviral effects of several U.S. Food and Drug Administration–approved drugs (ritonavir, lopinavir, nelfinavir, mycophenolic acid, and ribavirin), but contradictory results and experimental incongruities make the interpretation difficult (
24–
29). Small molecules targeting SARS-CoV and MERS-CoV have been assessed in cancer cell lines in vitro, but their antiviral efficacy against other human or zoonotic CoV remains unknown (
16,
30). Very few small molecules have been assessed in CoV animal models of viral pathogenesis, and some have even been shown to exacerbate disease (for example, ribavirin and mycophenolic acid) (
31,
32). Although the
Ces1c−/− mice used herein foster increased drug stability, they are not suitable for MERS-CoV efficacy studies because the murine ortholog of the MERS-CoV receptor, dipeptidyl peptidase 4 (DPP4), does not facilitate MERS-CoV infection (
33). Thus, our in vivo studies were limited to SARS-CoV, and future studies assessing MERS-CoV efficacy in double-transgenic humanized DPP4/
Ces1c−/− mice are planned. Human safety testing for GS-5734 is ongoing, and the drug has already been used to treat a small number of Ebola virus–infected patients under the “compassionate use” clause (
34). Overall, our work provides evidence that GS-5734 may protect CoV-infected patients from progression to severe disease and could prophylactically protect health care workers in areas with existing endemic MERS-CoV and that its broad-spectrum activity may prove valuable when a novel CoV emerges in the future.
MATERIALS AND METHODS
Study design
The primary goal of this study was to determine whether the small-molecule nucleoside analog GS-5734 exhibited broad-spectrum antiviral activity against the CoV family. Using multiple in vitro models including human primary cells, we measured the antiviral effect of GS-5734 on multiple CoV, encompassing much of the inherent family-wide genetic diversity. Data presented for studies in human primary cultures are representative of those from three human donors. Cytotoxicity was assessed in the 2B4 cell line and in two human primary lung cell types. Experiments were performed in triplicate unless otherwise stated. Drug effects were measured relative to vehicle controls. The secondary goal of this study was to assess antiviral efficacy in vivo within mouse models of severe CoV disease. The in vivo efficacy studies were intended to gain the data required to justify further testing in nonhuman primates and collectively inform future human clinical trials. Mice were age- and sex-matched and randomly assigned into groups before infection and treatment. The prophylactic and therapeutic in vivo studies presented in the main text were repeated at least once. Pathology and SARS-CoV antigen scoring were performed in a blinded manner. Exclusion criteria for in vivo studies were as follows: If a given mouse unexpectedly did not lose weight after infection and their virus lung titers were more than 2 log10 lower the mean of the group, this indicated that infection was inefficient and all data related to that mouse were censored. Primary data are located in table S3.
Viruses
SARS-CoV expressing GFP (GFP replaces ORF7) and MERS-CoV expressing RFP (RFP replaces ORF3) were created from molecular complementary DNA clones as described (
11,
20). To create SARS-CoV and MERS-CoV expressing nLUC, the genes for GFP and RFP were replaced with nLUC and isolated as referenced above. Recombinant human CoV NL63 and recombinant bat CoV for strains HKU3, HKU5, WIV1, and SHC014 were created as described (
5,
6,
12,
16,
17).
GS-5734
GS-5734 was synthesized at Gilead Sciences Inc., and its chemical identity and purity were determined by nuclear magnetic resonance, high-resolution mass spectrometry, and high-performance liquid chromatography (HPLC) analysis (
9). GS-5734 was solubilized in 100% DMSO for in vitro studies and in vehicle containing 12% sulfobutylether-β-cyclodextrin in water (with HCl/NaOH) at pH 5 for in vivo studies. GS-5734 was made available to the University of North Carolina (UNC) at Chapel Hill under a materials transfer agreement with Gilead Sciences.
In vitro efficacy and cytotoxicity in 2B4 cells
The human lung epithelial cell line 2B4 was maintained in Dulbecco’s modified Eagle’s medium (Gibco), 20% fetal bovine serum (HyClone), and 1× antibiotic-antimycotic (Gibco) (
10). Twenty-four hours after plating 5 × 10
4 cells per well, fresh medium was added. In triplicate, cells were infected for 1 hour with MERS-nLUC diluted in growth medium (MOI of 0.08), after which virus was removed, cultures were rinsed once, and fresh medium containing dilutions of GS-5734 or vehicle was added. DMSO (0.05%) was constant in all conditions. At 48 hours postinfection (hpi), virus replication was measured by nLUC assay (Promega), and cytotoxicity was measured via CellTiter-Glo (Promega) assay and then read on a SpectraMax plate reader (Molecular Devices). The IC
50 value was defined in GraphPad Prism 7 (GraphPad) as the concentration at which there was a 50% decrease in viral replication using ultraviolet-treated MERS-nLUC (100% inhibition) and vehicle alone (0% inhibition) as controls. CC
50 value was determined through comparison of data with that from cell-free (100% cytotoxic) and vehicle-only (0% cytotoxic) samples.
In vitro efficacy and toxicity in HAE cells
HAE cell cultures were obtained from the Tissue Procurement and Cell Culture Core Laboratory in the Marsico Lung Institute/Cystic Fibrosis Research Center at UNC. Before infection, HAE were washed with phosphate-buffered saline (PBS) and moved into air-liquid interface medium containing various doses of GS-5734 ranging from 10 to 0.00260 μM (final DMSO, <0.05%) (
11). HAE cultures were infected at an MOI of 0.5 for 3 hours at 37°C, after which virus was removed, and cultures were washed with PBS and then incubated at 37°C for 48 hours. Fluorescent images of MERS-RFP were taken at 48 hpi after nuclear staining with Hoechst 33258. Virus replication/titration was performed as previously described (
11). Similar data were obtained using cells from three different patient donors. Cytotoxicity was measured via CellTiter-Glo (Promega) in duplicate HAE cell cultures treated with 10 or 0.1 μM GS-5734 or DMSO at 0.05%.
In vivo pharmacokinetic analysis in plasma after GS-5734 administration in Ces1c−/− mice and marmosets
Mice were subcutaneously administered with GS-5734 (25 mg/kg), after which plasma was isolated from triplicate mice at 0.25, 0.5, 1, 2, 4, 6, 8, and 12 hours after administration. Three male marmosets were administered a single dose of GS-5734 intravenously at 10 mg/kg, after which plasma was isolated at 0.083, 0.25, 0.5, 1, 2, 4, 8, and 24 hours after administration. For both mouse and marmoset, 25 μl of plasma was treated and analyzed as described in the Supplementary Materials and Methods “Stability of GS-5734 in WT or Ces1c−/− mouse plasma.” Plasma concentrations of GS-5734, alanine metabolite (Ala-Met), and nucleoside monophosphate were determined using 8- to 10-point calibration curves spanning at least three orders of magnitude with quality control samples to ensure accuracy and precision and prepared in normal mouse plasma. Analytes were separated by a 75 × 2 mm (4-μm) Synergi Hydro-RP 30A column (Phenomenex) using a multistage linear gradient from 0.2 to 99% acetonitrile in mobile phase A at a flow rate of 260 μl/min.
Quantitation of GS-5734 metabolites in the lung after GS-5734 administration in Ces1c−/− mice and marmosets
Mice were dosed with GS-5734 (25 or 50 mg/kg), as described above. Lungs from triplicate mice were isolated at 1, 2, 6, 12, and 24 hours after administration and snap-frozen. Four male marmosets were dosed with GS-5734, as described above, and lungs were isolated at 2 and 24 hours after administration and were snap-frozen. On dry ice, frozen lung samples were pulverized and weighed. Dry ice–cold extraction buffer containing 0.1% potassium hydroxide and 67 mM EDTA in 70% methanol and containing 0.5 μM chloroadenosine triphosphate as internal standard was added and homogenized. After clarifying centrifugation at 20,000g for 20 min, supernatants were dried in a centrifuge evaporator. Dried samples were then reconstituted with 60 μl of mobile phase A, containing 3 mM ammonium formate (pH 5) with 10 mM dimethylhexylamine in water, and centrifuged at 20,000g for 20 min, with final supernatants transferred to HPLC injection vials. An aliquot of 10 μl was subsequently injected onto an API 5000 LC/MS/MS system for analysis performed using a similar method, as described for intracellular metabolism studies.
Prophylactic and therapeutic efficacy of GS-5734 against SARS-CoV in Ces1c−/− mice
Male and female (25- to 28-week-old) mice genetically deleted for carboxylesterase 1C (
Ces1c−/−) (stock 014096, The Jackson Laboratory) were anesthetized with ketamine/xylazine and infected with 10
4 PFU/50 μl (prophylactic studies) or 10
3 PFU/50 μl (therapeutic studies) SARS-CoV MA15. Animals were weighed daily to monitor virus-associated weight loss and to determine the appropriate dose volume of GS-5734 or vehicle. GS-5734 or vehicle was administered subcutaneously BID, 12 hours apart. On 2 and 5 dpi (prophylactic) or 2 and 4 or 6 dpi (therapeutic), animals were sacrificed by isoflurane overdose, and the large left lobe was frozen at −80°C for viral titration via plaque assay, as described (
14). The inferior right lobe was placed in 10% buffered formalin and stored at 4°C until histological analysis. Aberrations in lung function were determined by WBP (Data Sciences International), as described (
15).
Biocontainment and biosafety
Reported studies were initiated after the UNC Institutional Biosafety Committee approved the experimental protocols under the Baric Laboratory Safety Plan (20167715). SARS-CoV is a select agent. All work for these studies was performed with approved standard operating procedures for SARS-CoV, MERS-CoV, and other related CoVs in facilities conforming to the requirements recommended in the Biosafety in Microbiological and Biomedical Laboratories, the U.S. Department of Health and Human Services, the U.S. Public Health Service, the U.S. Centers for Disease Control and Prevention, and the National Institutes of Health.
Animal care
Efficacy studies were performed in animal biosafety level 3 facilities at UNC Chapel Hill. All works were conducted under protocols approved by the Institutional Animal Care and Use Committee at UNC Chapel Hill (protocol #13-288; continued on #16-284) according to guidelines set by the Association for the Assessment and Accreditation of Laboratory Animal Care and the U.S. Department of Agriculture.
Statistics
All statistical calculations were performed in GraphPad Prism 7. Specific tests to determine statistical significance are noted in each figure legend.
Acknowledgments
We would like to thank A. West and D. T. Scobey for excellent technical expertise. Funding: This study was funded by the Antiviral Drug Discovery and Development Center (5U19AI109680), grants from the NIH (AI108197 and AI109761), and the Cystic Fibrosis and Pulmonary Diseases Research and Treatment Center (BOUCHE15RO and NIH P30DK065988). Travel of M.R.D. to Gilead Sciences Inc. to discuss this project was paid for by Gilead Sciences. In addition, compound formulation and pharmacokinetic and metabolism studies were performed and paid for by Gilead Sciences. Author contributions: A.C.S., J.Y.F., T.P.S., T.C., R.J., and R.B. designed the in vitro efficacy studies. A.C.S., T.P.S., and S.R.L. executed and analyzed the in vitro efficacy studies. T.P.S., J.Y.F., R.J., T.C., R.S.B., and A.S.R. designed the in vivo efficacy studies. T.P.S. executed and analyzed the in vivo efficacy studies. R.B. scored the pathology and virus lung antigen staining. R.L.G. and K.P. performed qRT-PCR. V.D.M. and L.E.G. performed WBP for some in vivo studies. J.B.C. and M.R.D. designed and performed the pilot studies initially demonstrating efficacy against CoV. C.A.P., R.B., Y.P., D.B., and A.S.R., designed, executed, and analyzed the metabolism, pharmacokinetics, stability, or toxicity studies. M.O.C., D.S., R.L.M., J.E.S., and I.T. were responsible for the synthesis, scale-up, and formulation of small molecules. T.P.S., A.C.S., J.Y.F., T.C., R.S.B., M.R.D., D.B., R.J., and A.S.R. wrote the manuscript. Competing interests: The authors affiliated with Gilead Sciences are employees of the company and may own company stock. M.O.C., J.Y.F., R.J., R.L.M., A.S.R., and D.S. are listed as inventors on international application no. PCT/US2016/052092 filed by Gilead Sciences Inc., directed to methods of treating coronaviridae virus infections. All other authors declare that they have no competing interests. Data and materials availability: GS-5734 was made available to UNC under a materials transfer agreement with Gilead Sciences.