Abstract: ANTIVIRAL AGENTS Moxidectin and Ivermectin Inhibit SARS-CoV-2 Replication in Vero E6 Cells but Not in Human Primary Bronchial Epithelial Cells Nilima Dinesh Kumar,a,b Bram M. ter Ellen,b Ellen M. Bouma,b Berit Troost,b Denise P. I. van de Pol,b Heidi H. van der Ende-Metselaar,b Djoke van Gosliga,c Leonie Apperloo,d Orestes A. Carpaij,e Maarten van den Berge,e Martijn C. Nawijn,d Ymkje Stienstra,f Izabela A. Rodenhuis-Zybert,b Jolanda M. Smitb Department of Biomedical Sciences of Cells & Systems, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands a Department of Medical Microbiology and Infection Prevention, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands b c Department of Pediatrics, Beatrix Children’s Hospital, University Medical Center Groningen, University of Groningen, GRIAC Research Institute, Groningen, The Netherlands Department of Pathology and Medical Biology, University Medical Center Groningen, University of Groningen, GRIAC Research Institute, Groningen, The Netherlands d Department of Pulmonary Diseases, University Medical Center Groningen, University of Groningen, GRIAC Research Institute, Groningen, The Netherlands e Department of Internal Medicine/Infectious Diseases, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands f Nilima Dinesh Kumar, Bram M. ter Ellen and Ellen M. Bouma contributed equally to this work, and Izabela A. Rodenhuis-Zybert and Jolanda M. Smit contributed equally to this work. Author order was determined based on contribution to preparing the manuscript. Antiviral therapies are urgently needed to treat and limit the development of severe COVID-19 disease. Ivermectin, a broad-spectrum anti-parasitic agent, has been shown to have anti-SARS-CoV-2 activity in Vero cells at a concentration of 5 m M. These limited in vitro results triggered the investigation of ivermectin as a treatment option to alleviate COVID-19 disease. However, in April 2021, the World Health Organization stated the following: “The current evidence on the use of ivermectin to treat COVID-19 patients is inconclusive.” It is speculated that the in vivo concentration of ivermectin is too low to exert a strong antiviral effect. Here, we performed a head-to-head comparison of the antiviral activity of ivermectin and the structurally related, but metabolically more stable moxidectin in multiple in vitro models of SARS-CoV-2 infection, including physiologically relevant human respiratory epithelial cells. Both moxidectin and ivermectin exhibited antiviral activity in Vero E6 cells. Subsequent experiments revealed that these compounds predominantly act on the steps following virus cell entry. Surprisingly, however, in human-airway-derived cell models, both moxidectin and ivermectin failed to inhibit SARS-CoV-2 infection, even at concentrations of 10 m M. These disappointing results call for a word of caution in the interpretation of anti-SARS-CoV-2 activity of drugs solely based on their activity in Vero cells. Altogether, these findings suggest that even using a high-dose regimen of ivermectin, or switching to another drug in the same class, is unlikely to be useful for treatment of SARS-CoV-2 in humans. KEYWORDS moxidectin, ivermectin, antiviral, SARS-CoV-2, ALI, in vitro W ithin less than 1.5 years, the pandemic SARS coronavirus 2 (SARS-CoV-2) has infected over 153 million individuals and resulted in over 3.2..