The FDA-Approved Oral Drug Nitazoxanide Amplifies Host Antiviral Responses and Inhibits Ebola Virus

Jasenosky, Luke D.; Cadena, Cristhian; Mire, Chad E.; Borisevich, Viktoriya; Haridas, Viraga; Ranjbar, Shahin; Nambu, Aya; Bavari, Sina; Soloveva, Veronica; Sadukhan, Supriya; Cassell, Gail H.; Geisbert, Thomas W.; Hur, Sun; Goldfeld, Anne E.

Our data that NTZ broadly enhances antiviral sensing via the cytoplasmic RLR pathway and amplifies type I IFN-induced gene expression, including IFITM3 gene synthesis, which has previously been shown to inhibit EBOV infection (Huang et al., 2011) , led us to speculate that NTZ may overcome EBOV's capacity to evade host RNA sensing and to skirt triggering of the type I IFN response (Basler et al., 2000 (Basler et al., , 2003 Cardenas et al., 2006; Hartman et al., 2008; Leung et al., 2010; Luthra et al., 2013) , thus favoring host control of the virus. To test this hypothesis, we examined the ability of NTZ to inhibit infectious EBOV growth in human pulmonary epithelial A549 cells in vitro under biosafety level 4 conditions using an isolate of infectious EBOV from the original 1995 outbreak in the Democratic Republic of the Congo (Kikwit). Cells were pretreated with 0, 20, or 40 mM NTZ, which is not toxic to A549 cells (see Figure 3A ), for 4 h, and were then infected with EBOV at an MOI of 0.01. As shown in Figure 2 , in vehicle-only treated cells, EBOV growth increased by over 1.5 log units by 72 h post-infection. By contrast, over the same time frame, EBOV growth was completely inhibited in cells treated with 20 or 40 mM of NTZ (p < 0.001), dropping 2.5 log units from the initial inoculum titer in the cells treated with 40 mM NTZ (p < 0.001). We note that 20 and 40 mM are concentrations of NTZ that are either well below or equivalent to peak plasma levels ($39 mM) of its active metabolite TIZ that are observed in humans who have ingested 500 mg of NTZ twice a day in a typical oral regimen for cryptosporidial diarrhea (Romark, 2017) .