The oral drug nitazoxanide restricts SARS-CoV-2 infection and attenuates disease pathogenesis in Syrian hamsters

Abstract

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.

SciScore for 10.1101/2022.02.08.479634: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Ethics	not detected.
Sex as a biological variable	not detected.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Cell Line Authentication	Contamination: All cell lines used in this study were regularly screened for mycoplasma contamination using the Universal Mycoplasma Detection Kit (ATCC, 30-1012K). Authentication: All viruses were validated by genome sequencing.

Table 2: Resources

Antibodies
Sentences	Resources
Cells were washed in PBS supplemented with 2 mM EDTA, permeabilized using Perm/Wash buffer (BD Biosciences), and subsequently stained for 1 h at room temperature with anti-NP mAb 1C7 antibody labeled with an Alexa488-fluorescent marker using the Alexa Fluor™ 488 Antibody …

SciScore for 10.1101/2022.02.08.479634: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Ethics	not detected.
Sex as a biological variable	not detected.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Cell Line Authentication	Contamination: All cell lines used in this study were regularly screened for mycoplasma contamination using the Universal Mycoplasma Detection Kit (ATCC, 30-1012K). Authentication: All viruses were validated by genome sequencing.

Table 2: Resources

Antibodies
Sentences	Resources
Cells were washed in PBS supplemented with 2 mM EDTA, permeabilized using Perm/Wash buffer (BD Biosciences), and subsequently stained for 1 h at room temperature with anti-NP mAb 1C7 antibody labeled with an Alexa488-fluorescent marker using the Alexa Fluor™ 488 Antibody Labeling Kit (Invitrogen).	anti-NP suggested: None
The plates were subsequently fixed using 5% formaldehyde and immuno-stained using a monoclonal anti-SARS-CoV-NP antibody (Creative-Biolabs; NP1C7C7).	anti-SARS-CoV-NP suggested: None
In brief, plates were blocked (3% skim-milk TBS with 0.1% Tween20 for 1 h), stained for 90 min with anti-NP antibody (mAb 1C7, diluted 1:1000 in 1% skim-milk TBS with 0.1% Tween20), and finally secondary-stained with anti-mouse-HRP (antibody diluted 1:5000 in 1% skim-milk TBS with 0.1% Tween20 for 45 mins).	anti-mouse-HRP suggested: (Kindle Biosciences Cat# R1005, RRID:AB_2800463)
In brief, SARS-CoV-2 spike protein monoplex IHC was conducted using a Chromomap DAB IHC kit (Roche, Basel, Switzerland) with CC1 antigen retrieval at 95°C for 64 minutes, primary incubation at 1:900 for 40 min at room temperature, rabbit anti-IgG1+IgG2a+IgG3 antibody (ab133469) at 37°C for 20 minutes (1:1,000), and ImmPRESS HRP Goat Anti-Rabbit IgG polymer pre-dilute detection (Vector Laboratories) at 37°C for 20 minutes.	anti-IgG1+IgG2a+IgG3 suggested: None Anti-Rabbit IgG suggested: None
Experimental Models: Cell Lines
Sentences	Resources
Cell lines: Vero E6 (ATCC, CRL-1586), A549 (ATCC, CRM-CCL-185)-derived, and HEK293T (ATCC	HEK293T suggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)
hACE2-A549 cells used in Fig.	hACE2-A549 suggested: None
IFNAR KO A549 cells were generated by CRISPR-Cas9 ribonucleoprotein (RNP) complex (IDT) transfection using the Nucleofector system (Lonza Bioscience).	A549 suggested: NCI-DTP Cat# A549, RRID:CVCL_0023)
Wild type and IFNAR KO A549 cells were then transduced with an Ace2 expression vector simultaneously using a pTRIP-SFFV-Blast-2A-myc-hACE2 construct, a kind gift from Nir Hacohen and Matteo Gentili (Massachusetts General Hospital and the Broad Institute), sequenced, and then functionally validated (Suppl. Fig. 3).	IFNAR KO suggested: RRID:CVCL_A8AL)
Viruses: SARS-CoV-2 isolate USA-WA1/2020 (BEI Resources NR-52281) stocks were grown in Vero E6 cells as previously described (Miorin et al., 2020). hCoV-19/Japan/TY7-503/2021 (P.1) was obtained from BEI Resources (NR-54982).	Vero E6 suggested: None
. B.1.351, B.1.617.2, and P.1 viral stocks were grown on Vero TMPRSS2 cells.	Vero TMPRSS2 suggested: None
Virus was reconstituted from lyophilized sample and then titered using Vero cells as described (Huang et al., 2020; Xie et al., 2020).	Vero suggested: None
In vitro antiviral growth assay: Vero E6, Ace2-A549, or Ace2-HEK293T cells were seeded in 96-well plates in DMEM (10% FBS) and incubated for 24 h at 37°C and 5% CO2.	Ace2-HEK293T suggested: None
Spike-induced syncytia assay: 30,000 Vero-TMPRSS2 cells were reverse transfected with 100 ng of pCAGGS-S plasmids in a black 96-wellplate using TransIT-LT1 Transfection reagent (Mirus) in complete growth medium (containing 10% FBS).	Vero-TMPRSS2 suggested: JCRB Cat# JCRB1818, RRID:CVCL_YQ48)
Recombinant DNA
Sentences	Resources
Wild type and IFNAR KO A549 cells were then transduced with an Ace2 expression vector simultaneously using a pTRIP-SFFV-Blast-2A-myc-hACE2 construct, a kind gift from Nir Hacohen and Matteo Gentili (Massachusetts General Hospital and the Broad Institute), sequenced, and then functionally validated (Suppl. Fig. 3).	pTRIP-SFFV-Blast-2A-myc-hACE2 suggested: None
The pCAGGS-S plasmids used in this assay have been previously described (Escalera et al., 2021).	pCAGGS-S suggested: None
Software and Algorithms
Sentences	Resources
The SARS-CoV-2 Delta variant (B.1.617.2 PV29995) was obtained from Dr. Viviana Simon (Mount Sinai Pathogen Surveillance Program)	Pathogen Surveillance Program suggested: None
The IC50 was determined using GraphPad Prism software.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)
NeonGreen fluorescent infected cells were evaluated using Flow-Jo software after gating on live cell populations.	Flow-Jo suggested: (FlowJo, RRID:SCR_008520)
Live focused single cells were gated and NeonGreen fluorescent positive cells were evaluated using Ideas software and pixels were exported for further analysis on Prism to graph the dot plots and compare NTZ-treated to mock by the Mann-Whitney two-tailed test.	Prism suggested: (PRISM, RRID:SCR_005375)
iAT2s were serially passaged approximately every 2 weeks by dissociation into single cells via the sequential application of dispase (2 mg/mL; Thermo Fisher Scientific; 17105-04) and 0.05% trypsin (Invitrogen; 25300054) and replated at a density of 400 cells/μL of Matrigel (Corning; 356231), as previously described (Jacob et al., 2019).	Thermo Fisher Scientific suggested: (Thermo Fisher Scientific, RRID:SCR_008452)

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

Results from rtransparent:

Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

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The oral drug nitazoxanide restricts SARS-CoV-2 infection and attenuates disease pathogenesis in Syrian hamsters

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