Human organ chip-enabled pipeline to rapidly repurpose therapeutics during viral pandemics
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Abstract
The rising threat of pandemic viruses, such as SARS-CoV-2, requires development of new preclinical discovery platforms that can more rapidly identify therapeutics that are active in vitro and also translate in vivo . Here we show that human organ-on-a-chip (Organ Chip) microfluidic culture devices lined by highly differentiated human primary lung airway epithelium and endothelium can be used to model virus entry, replication, strain-dependent virulence, host cytokine production, and recruitment of circulating immune cells in response to infection by respiratory viruses with great pandemic potential. We provide a first demonstration of drug repurposing by using oseltamivir in influenza A virus-infected organ chip cultures and show that co-administration of the approved anticoagulant drug, nafamostat, can double oseltamivir’s therapeutic time window. With the emergence of the COVID-19 pandemic, the Airway Chips were used to assess the inhibitory activities of approved drugs that showed inhibition in traditional cell culture assays only to find that most failed when tested in the Organ Chip platform. When administered in human Airway Chips under flow at a clinically relevant dose, one drug – amodiaquine - significantly inhibited infection by a pseudotyped SARS-CoV-2 virus. Proof of concept was provided by showing that amodiaquine and its active metabolite (desethylamodiaquine) also significantly reduce viral load in both direct infection and animal-to-animal transmission models of native SARS-CoV-2 infection in hamsters. These data highlight the value of Organ Chip technology as a more stringent and physiologically relevant platform for drug repurposing, and suggest that amodiaquine should be considered for future clinical testing.
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SciScore for 10.1101/2020.04.13.039917: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IACUC: Native SARS-CoV-2 in vitro infection assay: All work with native SARS-CoV-2 virus was performed in a Biosafety Level 3 laboratory and approved by our Institutional Biosafety Committee. Randomization Micrographs of four or five random areas were taken from chips for subsequent quantification of infiltrated neutrophils. Blinding not detected. Power Analysis not detected. Sex as a biological variable Hamster PK studies: Amodiaquine dihydrochloride dihydrate (Sigma, #A2799) was formulated at 10 mg/ml in 12% sulfobutylether-β-cyclodextrin in water at pH 5.0 and administered to LVG male hamsters (n=3) at 50 mg/kg by subcutaneous injection (dose volume of 5 ml/kg). Cell … SciScore for 10.1101/2020.04.13.039917: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IACUC: Native SARS-CoV-2 in vitro infection assay: All work with native SARS-CoV-2 virus was performed in a Biosafety Level 3 laboratory and approved by our Institutional Biosafety Committee. Randomization Micrographs of four or five random areas were taken from chips for subsequent quantification of infiltrated neutrophils. Blinding not detected. Power Analysis not detected. Sex as a biological variable Hamster PK studies: Amodiaquine dihydrochloride dihydrate (Sigma, #A2799) was formulated at 10 mg/ml in 12% sulfobutylether-β-cyclodextrin in water at pH 5.0 and administered to LVG male hamsters (n=3) at 50 mg/kg by subcutaneous injection (dose volume of 5 ml/kg). Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Two days post-infection, the supernatant was harvested and subjected to Western blot analysis using anti-HA1 antibody. anti-HA1suggested: NoneIncorporation of the SARS-CoV-2 S protein into the SARS-CoV-2pp was confirmed using Western Blot analysis with anti-SARS-CoV-2 S1 chimeric monoclonal antibody with combined constant domains of the human IgG1 molecule and mouse variable regions (40150-D001 Sinobiological, 1:500); a recombinant receptor binding domain (RBD) fragment from the S1 region was used as a control (BEI resources, NR-52306). anti-SARS-CoV-2suggested: Nonehuman IgG1suggested: NoneExperimental Models: Cell Lines Sentences Resources Quantitative reverse transcription-polymerase chain reaction (RT-qPCR): Total RNA was extracted from differentiated human Airway chips, pre-differentiated lung airway epithelial cells, or MDCK cells using TRIzol (Invitrogen). MDCKsuggested: CLS Cat# 602280/p823_MDCK_(NBL-2, RRID:CVCL_0422)Analysis of cytokines and chemokines: Vascular effluents from Airway Chips were collected and analyzed for a panel of cytokines and chemokines, including IL-6, IP-10, MCP-1, RANTES, interferon-β, using custom ProcartaPlex assay kits (Invitrogen). MCP-1suggested: NonePseudotyped virus production: HEK293T cells (5 × 105 cell per well) were seeded into 6-well plates. HEK293Tsuggested: NoneSARS-CoV-2pp or VSVpp was added to 5 × 103 Huh-7 cells (a human liver cell line) per well in the presence or absence of the test drugs or compounds. Huh-7suggested: CLS Cat# 300156/p7178_HuH7, RRID:CVCL_0336)Titer of stock was determined by plaque assay using Vero E6 cells. Vero E6suggested: RRID:CVCL_XD71)Software and Algorithms Sentences Resources After 5-7 days, the apical medium was removed while allowing air to fill the channel to establish an ALI, and the airway epithelial cells were cultured for 3-4 additional weeks while being fed only by constant flow of PneumaCult-ALI medium (StemCell) supplemented with 0.1% VEGF, 0.01% EGF, and 1mM CaCl2 from an Endothelial Cell Medium Kit (Cell Biological, M1168) through the bottom vascular channel. Cell Biologicalsuggested: NoneFluorescence imaging was carried out using a confocal laser-scanning microscope (SP5 X MP DMI-6000, Germany) and image processing was done using Imaris software (Bitplane, Switzerland) Imarissuggested: (Imaris, RRID:SCR_007370)Vero E6 cells (ATCC# CRL 1586) were cultured in DMEM (Quality Biological), supplemented with 10% (v/v) heat inactivated fetal bovine serum (Sigma), 1% (v/v) Quality Biologicalsuggested: NoneLuminescence was read on a BioTek Synergy HTX plate reader (BioTek Instruments Inc., Winooski, VT) using the Gen5 software (v7.07 Gen5suggested: (Gen5, RRID:SCR_017317)Illumina libraries were quantified by Qbit and Agilent Bioanalyzer prior to being run on an Illumina NextSeq500 using a high capacity flow cell. Agilent Bioanalyzersuggested: NoneResults from OddPub: Thank you for sharing your data.
Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:While drugs were administered at levels similar to their Cmax here to compare relative potencies, one caveat is that we did not quantify drug absorption or protein binding in this study. Importantly, by carrying out mass spectrometry measurements of drug levels in these devices, full PK profiles can be recapitulated in these Organ Chip models8, which should further aid clinical translation in the future. While animal models remain the benchmark for validation of therapeutics to move to humans, it is important to note that human Organ Chips are now being explored as viable alternatives to animal models38 and regulatory agencies are encouraging pharmaceutical and biotechnology companies to make use of data from Organ Chips and other microphysiological systems in their regulatory submissions39. These studies led to the identification of multiple approved drugs that could serve as prophylactics and therapeutics against viral pandemics. The anticoagulant drug, nafamostat, significantly extended the current treatment time window of oseltamivir from 2 to 4 days after infection by influenza virus, which could have great clinical relevance given that most patients do not begin treatment until days after they are infected. Similarly, while the human Organ Chip model successfully predicted the inability of chloroquine, hydroxychloroquine, and arbidol to work in animals4 and human patients1, 2, 40, in contrast with what was reported in cell lines41, 42, we successfully identified amodiaq...
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.
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Cathryn Sundback
Review 1: "Human-organ-chip-enabled pipeline to rapidly repurpose therapeutics during viral pandemics"
This study employs micro-fluidic devices to test candidate therapeutics blocking SARS-CoV-2 entry and demonstrates amodiaquine efficacy in preclinical models. The claims presented in this work are reliable but could be strengthened through more rigorous model characterization.
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Jeffrey T Borenstein, Ashley L Gard, Jennifer P Wang
Review 2: "Human organ chip-enabled pipeline to rapidly repurpose therapeutics during viral pandemics"
This study employs micro-fluidic devices to test candidate therapeutics blocking SARS-CoV-2 entry and demonstrates amodiaquine efficacy in preclinical models. The claims presented in this work are reliable but could be strengthened through more rigorous model characterization.
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Strength of evidence
Reviewers: Cathryn Sundback (Massachusetts General Hospital) | 📘📘📘📘📘
Jeffrey T Borenstein, Ashley L Gard (Draper), Jennifer P Wang (University of Massachusetts) | 📒📒📒 ◻️ ◻️ -
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SciScore for 10.1101/2020.04.13.039917: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization However , our results in the human Airway Chip are reminiscent of the findings indicating that while chloroquine was reported to show mild therapeutic effects in preliminary clinical studies with a small numbers of patients1,33 , it was not found to demonstrate any significant anti-viral activity in other studies , and that significant toxicity was observed , including in preliminary findings from a larger randomized , double-blinded , phase IIb clinical trial. Blinding not detected. Power Analysis not detected. Sex as a biological variable Toremifene , on the other hand , has been shown to be a relatively well tolerated chronic … SciScore for 10.1101/2020.04.13.039917: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization However , our results in the human Airway Chip are reminiscent of the findings indicating that while chloroquine was reported to show mild therapeutic effects in preliminary clinical studies with a small numbers of patients1,33 , it was not found to demonstrate any significant anti-viral activity in other studies , and that significant toxicity was observed , including in preliminary findings from a larger randomized , double-blinded , phase IIb clinical trial. Blinding not detected. Power Analysis not detected. Sex as a biological variable Toremifene , on the other hand , has been shown to be a relatively well tolerated chronic treatment ( > 5 years ) for breast cancer in males as well as females , and thus could be considered for both prophylaxis and treatment of COVID19454 . Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Two days post-infection , the supernatant was harvested and subjected to Western blot analysis using anti-HA1 antibody . anti-HA1suggested: NoneIncorporation of the CoV-2 S protein into the CoV2pp was confirmed using Western Blot analysis with anti-CoV2 S1 chimeric monoclonal antibody with combined constant domains of the human IgG1 molecule and mouse variable regions ( 40150-D001 Sinobiological , 1:500); a recombinant receptor binding domain ( RBD ) fragment from the S1 region was used as a control ( BEI resources , NR-52306) anti-CoV2suggested: None<div style="margin-bottom:8px"> <div><b>human IgG1</b></div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Experimental Models: Cell Lines</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Chips, which contain human lung epithelial cells that express high levels of ACE2 and TMPRSS2, were then used to assess the inhibitory activities of 7 clinically approved drugs (chloroquine, arbidol, toremifene, clomiphene, amodiaquine, verapamil, and amiodarone) that we found inhibit infection by viral pseudoparticles expressing SARS-CoV-2 spike protein in human Huh-7 cells, and others recently showed suppress infection by native SARS-CoV-2 in Vero cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div><b>Vero</b></div> <div>suggested: CLS Cat# 605372/p622_VERO, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_0059">CVCL_0059</a></div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">H1N1 infection also was accompanied by increased secretion of various inflammatory cytokines and chemokines , including IL-6 , IP-10 , RANTES , interferon-β , and MCP-1 , which could easily be measured in the effluent from the vascular channel ( Fig . 2E) .</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div><b>MCP-1</b></div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All of these drugs demonstrated dose-dependent inhibition of SARS-CoV-2pp entry in Huh-7 cells when added at 1 and 5 µM simultaneously with the virus and culturing for 72 hours ( Fig . 4A) , without producing any detectable cell toxicity in this model ( Fig .</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div><b>Huh-7</b></div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Confluent MDCK cell monolayers in 12-well plate were washed with PBS , inoculated with 1 mL of 10-fold serial dilutions of influenza virus samples , and incubated for 1 h at 37℃ .</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div><b>MDCK</b></div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">24 h later , HEK293T cells were transfected with 1.0 µg of pNL43.</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div><b>HEK293T</b></div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Software and Algorithms</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After 5-7 days , the apical medium was removed while allowing air to fill the channel to establish an ALI , and the airway epithelial cells were cultured for 3-4 additional weeks while being fed only by constant flow of PneumaCult-ALI medium ( StemCell ) supplemented with 0.1 % VEGF , 0.01 % EGF , and 1mM CaCl2 from an Endothelial Cell Medium Kit ( Cell Biological , M1168 ) through the bottom vascular channel .</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div><b>Cell Biological</b></div> <div>suggested: None</div> </div> </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Fluorescence imaging was carried out using a confocal laser-scanning microscope ( SP5 X MP DMI-6000 , Germany ) and image processing was done using Imaris software ( Bitplane , Switzerland)</td><td style="min-width:100px;border-bottom:1px solid lightgray"> <div style="margin-bottom:8px"> <div><b>Imaris</b></div> <div>suggested: (Imaris, <a href="https://scicrunch.org/resources/Any/search?q=SCR_007370">SCR_007370</a>)</div> </div> </td></tr></table>Results from Barzooka: We also found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).
Results from OddPub: Thank you for sharing your data.
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