key: cord-0915645-fx05md78 authors: Miorin, Lisa; Mire, Chad E.; Ranjbar, Shahin; Hume, Adam J.; Huang, Jessie; Crossland, Nicholas A.; White, Kris M; Laporte, Manon; Kehrer, Thomas; Haridas, Viraga; Moreno, Elena; Nambu, Aya; Jangra, Sonia; Cupic, Anastasija; Dejosez, Marion; Abo, Kristine A.; Tseng, Anna E.; Werder, Rhiannon B.; Rathnasinghe, Raveen; Mutetwa, Tinaye; Ramos, Irene; de Aja, Julio Sainz; de Alba Rivas, Carolina Garcia; Schotsaert, Michael; Corley, Ronald B.; Falvo, James V.; Fernandez-Sesma, Ana; Kim, Carla; Rossignol, Jean-François; Wilson, Andrew A.; Zwaka, Thomas; Kotton, Darrell N.; Mühlberger, Elke; García-Sastre, Adolfo; Goldfeld, Anne E. title: The oral drug nitazoxanide restricts SARS-CoV-2 infection and attenuates disease pathogenesis in Syrian hamsters date: 2022-02-09 journal: bioRxiv DOI: 10.1101/2022.02.08.479634 sha: da4baabc6c51fc363da0fadf9be2b2cd0105f5e9 doc_id: 915645 cord_uid: fx05md78 A well-tolerated and cost-effective oral drug that blocks SARS-CoV-2 growth and dissemination would be a major advance in the global effort to reduce COVID-19 morbidity and mortality. Here, we show that the oral FDA-approved drug nitazoxanide (NTZ) significantly inhibits SARS-CoV-2 viral replication and infection in different primate and human cell models including stem cell-derived human alveolar epithelial type 2 cells. Furthermore, NTZ synergizes with remdesivir, and it broadly inhibits growth of SARS-CoV-2 variants B.1.351 (beta), P.1 (gamma), and B.1617.2 (delta) and viral syncytia formation driven by their spike proteins. Strikingly, oral NTZ treatment of Syrian hamsters significantly inhibits SARS-CoV-2-driven weight loss, inflammation, and viral dissemination and syncytia formation in the lungs. These studies show that NTZ is a novel host-directed therapeutic that broadly inhibits SARS-CoV-2 dissemination and pathogenesis in human and hamster physiological models, which supports further testing and optimization of NTZ-based therapy for SARS-CoV-2 infection alone and in combination with antiviral drugs. The ongoing COVID-19 pandemic has resulted in more than 396 million cases and 5.7 68 million deaths worldwide (Dong et al., 2020 ). An easily deployable, well-tolerated, inexpensive, 69 orally active drug that is safe in adults and children to treat or inhibit disease progression would 70 be a significant advance in the fight against COVID-19. Nitazoxanide (NTZ) is an FDA-approved 71 and well-tolerated oral therapy originally developed for parasitic infection that has been used for 72 treatment of Giardia-and Cryptosporidium-associated diarrhea in millions of adults and children To determine whether NTZ and its active metabolite TIZ inhibit SARS-CoV-2 replication, 102 we first performed antiviral assays in Vero E6 cells, which are well known to support productive 103 infection with SARS-CoV-2 (Matsuyama et al., 2020) (Suppl. Fig. 1A ). Vero E6 cells were thus In agreement with our findings using the USA-WA1/2020 strain in the antiviral assays above ( Given the robust antiviral activity of NTZ in Vero E6 cells, and based on their different 135 mechanisms of action, we next assessed NTZ's ability to synergize with RDV, which was a 136 standard of care drug treatment to shorten time of COVID-19 recovery (Beigel et al., 2020) . To 137 test this hypothesis, we performed combination assays in Vero E6 cells and found that NTZ 138 significantly increases the ability of RDV to inhibit viral infection (Fig. 1D ). The results of the 139 combination assay were then further analyzed using SynergyFinder to generate a synergy 140 landscape and combination score by the Loewe model (Fig. 1E ) (Ianevski et al., 2020) . In this 141 model, a synergistic interaction between drugs is indicated by a score greater than +10, while an 142 additive interaction has a score between -10 and +10, and an antagonistic interaction has a score 143 lower than -10. Notably, the landscape for the interaction of RDV with NTZ shows synergy scores 144 greater than +10 at low NTZ concentrations (6.26 M) (Fig. 1E) , denoting moderate synergy of 145 the two drugs in the inhibition of SARS-CoV-2 growth. These data are consistent with a study showing that NTZ synergistically enhances the ability of RDV to reduce cytopathic effect (CPE) (Bobrowski et al., 2021) . All together, these data show that in Vero E6 cells NTZ robustly inhibits 148 SARS-CoV-2 replication and forms an inhibitory synergistic compound pair with RDV. Validation of NTZ antiviral activity in human cell lines 151 We next evaluated the ability of NTZ to inhibit SARS-CoV-2 replication in the human cell 152 line A549 transduced with angiotensin-converting enzyme 2 (Ace2). After confirming that the 153 Ace2-A549 cells support SARS-CoV-2 replication (Suppl. Fig. 1B) , we pre-treated these cells with We next pretreated Ace2-A549 cells with 5 or 10 M NTZ for 4 hrs followed by infection Interestingly, the IC50 for NTZ in both human cell lines (Ace2-A549: 1.695 M; Ace2-170 HEK293T: 2.2 M) were lower than the IC50 in Vero E6 cells (4.444 M). Notably, Vero cells are 171 deficient in type I IFN production (Emeny and Morgan, 1979) due to a deletion on chromosome 12 resulting in loss of the type I IFN gene cluster (Osada et al., 2014) . To interrogate whether 173 signaling through the type I IFN receptor (IFNAR) plays a role in NTZ's ability to inhibit SARS- CoV-2 infection in human cells, we compared NTZ antiviral activity in wild-type and IFNAR knock-175 out (KO) Ace2-A549 cells. To verify that IFNAR signaling was abrogated in the knock-out cells, we showed that the IFN-dependent phosphorylation of STAT1 and STAT2 (Suppl. Fig. 3A ) and 177 transcription of the interferon stimulated gene (ISG) IFITM3 (Suppl. Fig. 3B ) were both abolished 178 in the IFNAR-KO Ace2-A549 cells as expected. In addition, we confirmed that Ace2 expression 179 was equivalent in the two cell lines (Suppl. Fig. 3A ). The wild-type and IFNAR-KO Ace2-A549 cells were then treated with 10 M NTZ or with 181 DMSO for 4 hrs followed by infection with NG-SARS-CoV-2 at an MOI of 0.1 or 0.01 for 48 hrs. Different SARS-CoV-2 variants have contributed to successive waves of the COVID-19 218 epidemic due to their increased transmissibility and virulence, as well as the waning of vaccine-219 mediated immunity (Harvey et al., 2021) . To determine NTZ's activity against three of the recently 220 emerged variants of concern (https://www.who.int/en/activities/tracking-SARS-CoV-2-variants/), we treated Vero E6 cells with NTZ for 4 hrs and then infected them with SARS-CoV-2 beta 222 (B.1.351), gamma (P.1), or delta (B.1617.2). As a control, cells were also infected with the original SARS-CoV-2 WA1 strain used in our studies described above. NTZ strongly inhibited the 224 replication of each variant tested similar to its ability to inhibit SARS-CoV-2 WA1 (Fig. 4A) In addition, animals from each group were sacrificed at 2, 5, and 14 dpi and lungs were At 2 dpi the interstitial score was significantly decreased (p=0.026) in the NTZ-treated animals 292 compared to the SARS-CoV-2-infected vehicle-treated animals (Fig. 6B ). This was reflected by 293 the absence of, or minimal, lymphohistiocytic and neutrophilic alveolar and/or septal infiltrate in 294 the group of NTZ-treated animals (Fig. 6A ). While at 2 dpi the airway score for NTZ-vs. vehicle-295 treated animals did not reach significance (Fig. 6C) , three of the four NTZ-treated animals 296 exhibited absence of discernible bronchiole epithelial pathology like that observed in naïve 297 animals, with a dramatic decline in neutrophilic and histiocytic luminal infiltrate compared to 298 vehicle-treated animals in the one animal that did display airway injury (Fig. 6A) . Histologically, 299 this was reflected by less severe and more segmental necrotizing bronchiolitis (see magnified 300 image in Suppl. Fig. 5 ). Blood vessel scores for NTZ-vs. vehicle-treated animals at 2 dpi also did 301 not reach significance, however again we note that three of the four NTZ-treated animals exhibited 302 an absence of any detectable perivascular infiltrate similar to naïve animals. NTZ therapy was halted at 4 dpi with 5 days of total treatment. 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