The FDA-approved drug ivermectin inhibits the replication of SARS-CoV-2 in vitro

Abstract

Although several clinical trials are now underway to test possible therapies, the worldwide response to the COVID-19 outbreak has been largely limited to monitoring/containment. We report here that Ivermectin, an FDA-approved anti-parasitic previously shown to have broad-spectrum antiviral activity in vitro, is an inhibitor of the causative virus (SARS-CoV-2), with a single addition to Vero-hSLAM cells 2 h post-infection with SARS-CoV-2 able to effect ~5000-fold reduction in viral RNA at 48 h. Ivermectin, therefore, warrants further investigation for possible benefits in humans.

Introduction

Ivermectin is an FDA-approved broad-spectrum anti-parasitic agent (Gonzalez Canga et al., 2008) that in recent years we, along with other groups, have shown to have anti-viral activity against a broad range of viruses (Gotz et al., 2016; Lundberg et al., 2013; Tay et al., 2013; Wagstaff et al., 2012) in vitro. Originally identified as an inhibitor of the interaction between the human immunodeficiency virus-1 (HIV-1) integrase protein (IN) and the importin (IMP) α/β1 heterodimer responsible for IN nuclear import (Wagstaff et al., 2011), Ivermectin has since been confirmed to inhibit IN nuclear import and HIV-1 replication (Wagstaff et al., 2012). Other actions of ivermectin have been reported (Mastrangelo et al., 2012), but ivermectin has been shown to inhibit nuclear import of host (eg. (Kosyna et al., 2015; van der Watt et al., 2016)) and viral proteins, including simian virus SV40 large tumor antigen (Tag) and dengue virus (DENV) non-structural protein 5 (Wagstaff et al., 2012, Wagstaff et al., 2011). Importantly, it has been demonstrated to limit infection by RNA viruses such as DENV 1-4 (Tay et al., 2013), West Nile Virus (Yang et al., 2020), Venezuelan equine encephalitis virus (VEEV) (Lundberg et al., 2013) and influenza (Gotz et al., 2016), with this broad-spectrum activity believed to be due to the reliance by many different RNA viruses on IMPα/β1 during infection (Caly et al., 2012; Jans et al., 2019). Ivermectin has similarly been shown to be effective against the DNA virus pseudorabies virus (PRV) both in vitro and in vivo, with ivermectin treatment shown to increase survival in PRV-infected mice (Lv et al., 2018). Efficacy was not observed for ivermectin against Zika virus (ZIKV) in mice, but the authors acknowledged that study limitations justified re-evaluation of ivermectin's anti-ZIKV activity (Ketkar et al., 2019). Finally, ivermectin was the focus of phase III clinical trial in Thailand in 2014–2017, against DENV infection, in which a single daily oral dose was observed to be safe and resulted in a significant reduction in serum levels of viral NS1 protein, but no change in viremia or clinical benefit was observed (see below) (Yamasmith et al., 2018).



Ivermectin is a potent inhibitor of the SARS-CoV-2 clinical isolate Australia/VIC01/2020. Vero/hSLAM cells were infected with SARS-CoV-2 clinical isolate Australia/VIC01/2020 (MOI = 0.1) for 2 h before addition of vehicle (DMSO) or Ivermectin at the indicated concentrations. Samples were taken at 0–3 days post-infection for quantitation of viral load using real-time PCR of cell-associated virus (A) or supernatant (B). IC50 values were determined in subsequent experiments at 48 h post-infection using the indicated concentrations of Ivermectin (treated at 2 h post-infection as per A/B). Triplicate real-time PCR analysis was performed on the cell-associated virus (C/E) or supernatant (D/F) using probes against either the SARS-CoV-2 E (C/D) or RdRp (E/F) genes. Results represent mean ± SD (n = 3). 3 parameter dose-response curves were fitted using GraphPad prism to determine IC50 values (indicated). G. Schematic of ivermectin's proposed antiviral action on coronavirus. IMPα/β1 binds to the coronavirus cargo protein in the cytoplasm (top) and translocates it through the nuclear pore complex (NPC) into the nucleus where the complex falls apart and the viral cargo can reduce the host cell's antiviral response, leading to enhanced infection. Ivermectin binds to and destabilizes the Impα/β1 heterodimer thereby preventing Impα/β1 from binding to the viral protein (bottom) and preventing it from entering the nucleus. This likely results in reduced inhibition of the antiviral responses, leading to a normal, more efficient antiviral response.




To further determine the effectiveness of ivermectin, cells infected with SARS-CoV-2 were treated with serial dilutions of ivermectin 2 h post-infection and supernatant and cell pellets collected for real-time RT-PCR at 48 h (Fig. 1C/D). As above, a >5000 reduction in viral RNA was observed in both supernatant and cell pellets from samples treated with 5 μM ivermectin at 48 h, equating to a 99.98% reduction in viral RNA in these samples. Again, no toxicity was observed with ivermectin at any of the concentrations tested. The IC50 of ivermectin treatment was determined to be ~2 μM under these conditions. Underlining the fact that the assay indeed specifically detected SARS-CoV-2, RT-PCR experiments were repeated using primers specific for the viral RdRp gene (Fig. 1E/F) rather than the E gene (above), with nearly identical results observed for both released (supernatant) and cell-associated virus.

Methods

Cell culture, viral infection, and drug treatment
Vero/hSLAM cells (Ono et al., 2001) were maintained in Earle's Minimum Essential Medium (EMEM) containing 7% Fetal Bovine Serum (FBS) (Bovogen Biologicals, Keilor East, AUS) 2 mM L-Glutamine, 1 mM sodium pyruvate, 1500 mg/L sodium bicarbonate, 15 mM HEPES and 0.4 mg/ml geneticin at 37 °C, 5% CO2. Cells were seeded into 12-well tissue culture plates 24 h before infection with SARS-CoV-2 (Australia/VIC01/2020 isolate) at an MOI of 0.1 in infection media (as per maintenance media but containing only 2% FBS) for 2 h. Media containing inoculum was removed and replaced with 1 mL fresh media (2% FBS) containing Ivermectin at the indicated concentrations or DMSO alone and incubated as indicated for 0–3 days. At the appropriate time point, cell supernatant was collected and spun for 10 min at 6,000 g to remove debris and the supernatant was transferred to fresh collection tubes. The cell monolayers were collected by scraping and resuspension into 1 mL fresh media (2% FBS). Toxicity controls were set up in parallel in every experiment on uninfected cells.

Generation of SARS-CoV-2 cDNA
RNA was extracted from 200 μL aliquots of sample supernatant or cell suspension using the QIAamp 96 Virus QIAcube HT Kit (Qiagen, Hilden, Germany) and eluted in 60 μl. Reverse transcription was performed using the BioLine SensiFAST cDNA kit (Bioline, London, United Kingdom), the total reaction mixture (20 μl), containing 10 μL of RNA extract, 4 μl of 5x TransAmp buffer, 1 μl of Reverse Transcriptase and 5 μl of Nuclease free water. The reactions were incubated at 25 °C for 10 min, 42 °C for 15 min and 85 °C for 5 min.
2.3. Detection of SARS-CoV-2 using a TaqMan Real-time RT-PCR assay
TaqMan RT-PCR assay was performed using 2.5 μl cDNA, 10 μl Primer Design PrecisonPLUS qPCR Master Mix 1 μM Forward (5′- AAA TTC TAT GGT GGT TGG CAC AAC ATG TT-3′), 1 μM Reverse (5′- TAG GCA TAG CTC TRT CAC AYT T-3′) primers and 0.2 μM probe (5′-FAM- TGG GTT GGG ATT ATC-MGBNFQ-3′) targeting the BetaCoV RdRp (RNA-dependent RNA polymerase) gene or Forward (5′-ACA GGT ACG TTA ATA GTT AAT AGC GT -3′), 1 μM Reverse (5′-ATA TTG CAG CAG TAC GCA CAC A-3′) primers and 0.2 μM probe (5′-FAM-ACA CTA GCC ATC CTT ACT GCG CTT CG-286 NFQ-3′) targeting the BetaCoV E-gene (Corman et al., 2020). Real-time RT-PCR assays were performed on an Applied Biosystems ABI 7500 Fast real-time PCR machine (Applied Biosystems, Foster City, CA, USA) using cycling conditions of 95 °C for 2 min, 95 °C for 5 s, 60 °C for 24 s. SARS-CoV-2 cDNA (Ct~28) was used as a positive control. Calculated Ct values were converted to fold-reduction of treated samples compared to control using the ΔCt method (fold changed in viral RNA = 2^ΔCt) and expressed as % of DMSO alone sample. IC50 values were fitted using 3 parameter dose-response curves in GraphPad prism.

Funding

This work was supported by a National Breast Cancer Foundation Fellowship, Australia (ECF-17-007) for KMW and a National Health and Medical Research Council (NHMRC), Australia Senior Principal Research Fellow (SPRF) (APP1103050) for DAJ. buy ivermectin | buy ivermectin India | buy ivermectin | buy ivermectin India | ivermectin tablet for humans | ivermectin tablet price ||ivermectin 12 mg tablet price in India | ivermectin buy online | where to buy ivermectin for humans | ivermectin dosage | where to buy ivermectin UK | ivermectin uses | ivermectin | Stromectol |buy ivermectin online | buy ivermectin online UK | buy ivermectin online NZ | buy ivermectin online south Africa | buy ivermectin online Malaysia | Buy Stromectol (ivermectin) Online at Lowest Price | Buy Ivermectin for Covid 19 Over the Counter | Buy Ivermectin for Humans and Ivermectin 3mg | Ivermectin Online Prescription | Buy Ivermectin Online (@buyivermectin) | order/ Buy Ivermectin Online Nz | No Prescription
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