Circulating ACE2-expressing Exosomes Block SARS-CoV-2 Infection as an Innate Antiviral Mechanism

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   ScreenIT

   Dec 5, 2020

   SciScore for 10.1101/2020.12.03.407031: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement

   IRB: Human subject study and biosafety approvals: All research activities with human blood specimens of pre-COVID-19 and COVID-19 convalescent patients were implemented under NIH guidelines for human subject studies and the protocols approved by the Northwestern University Institutional Review Board (STU00205299 and #STU00212371) as well as the Institutional Biosafety Committee. IACUC: Human subject study and biosafety approvals: All research activities with human blood specimens of pre-COVID-19 and COVID-19 convalescent patients were implemented under NIH guidelines for human subject studies and the protocols approved by the Northwestern University Institutional …

   SciScore for 10.1101/2020.12.03.407031: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IRB: Human subject study and biosafety approvals: All research activities with human blood specimens of pre-COVID-19 and COVID-19 convalescent patients were implemented under NIH guidelines for human subject studies and the protocols approved by the Northwestern University Institutional Review Board (STU00205299 and #STU00212371) as well as the Institutional Biosafety Committee. IACUC: Human subject study and biosafety approvals: All research activities with human blood specimens of pre-COVID-19 and COVID-19 convalescent patients were implemented under NIH guidelines for human subject studies and the protocols approved by the Northwestern University Institutional Review Board (STU00205299 and #STU00212371) as well as the Institutional Biosafety Committee.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	Contamination: Cells were tested for mycoplasma contamination before culturing.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Flow cytometry: Cells were blocked with mouse serum IgG (Sigma, 15381) for 10 min at room temperature and then incubated with specific antibodies; AF-647 mouse anti-human ACE2 (R&D systems, FAB9332R),	anti-human ACE2 suggested: None
   The antibodies, ACE2 (R&D systems, AF933), CD81 (GeneTex, CTX101766), CD63 (Proteintech, 25682-1-AP), GRP94 (Proteintech, 1H10B7), TSG101 (Proteintech, 14497-1-AP) and β-actin (Sigma, A5441) were used as primary antibodies and horseradish peroxidase (HRP)-conjugated secondary antibodies were from Promega (Rabbit W401B and Mouse W402B), and the substrate ECL was detected by Pierce ECL2 solution (Thermo Fisher Scientific, 1896433A)	ACE2 suggested: (R and D Systems Cat# AF933, RRID:AB_355722) CD81 suggested: None CTX101766 suggested: None CD63 suggested: (Proteintech Cat# 25682-1-AP, RRID:AB_2783831) GRP94 suggested: (Proteintech Cat# 60012-1-Ig, RRID:AB_2119056) TSG101 suggested: (Proteintech Cat# 14497-1-AP, RRID:AB_2208090) 14497-1-AP suggested: (Proteintech Cat# 14497-1-AP, RRID:AB_2208090) β-actin suggested: (Sigma-Aldrich Cat# A5441, RRID:AB_476744)
   Results were normalized to the CR3022 antibody with known affinity to RBD of SARS-CoV-2 42.	CR3022 suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Cell culture: The parent ACE2− human embryonic kidney HEK-293 cells (HEK) or human cervical cancer HeLa cells (HeLa) are transduced with lentiviral pDual-ACE2 expression vector for stable ACE2 expression and production of ACE2+ exosomes. Dr. Thomas Gallagher of Stritch Medical School, Loyola University kindly provided HeLa and HeLa-ACE2 cells via the Hope group.	HEK-293 suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045) HeLa suggested: CLS Cat# 300194/p772_HeLa, RRID:CVCL_0030) HeLa-ACE2 suggested: None
   RBD bait that was incubated with PBS, or with ACE2− exosomes, non-fluorescent RBD bait (mock control) and ACE2− cells were used as controls.	ACE2− suggested: None
   Software and Algorithms
   Sentences	Resources
   After motion correction of the movies 38, exosomes were identified manually using ImageJ 39.	ImageJ suggested: (ImageJ, RRID:SCR_003070)
   Plasma was run in quadruplicate and reported as the average.	Plasma suggested: None
   Biotin conjugated RBD protein (ACROBiosystems, SPD-C82E9) were coupled with magnetic beads (CELLection Biotin Binder Kit, Thermo Fisher Sceintific, 11533D), and 5 μg of anti-ACE2 antibody (R&D systems, AF933) or isotope control IgG (R&D systems) were conjugated to dynabeads (Dynabeads Co-Immunoprecipitation Kit, Thermo Fisher Scientific, 14321D)	Thermo Fisher Sceintific suggested: None
   Statistical Analysis: GraphPad Prism 6.0 Software was used to perform statistical analyses and calculate the IC50.	GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)

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   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).

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   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.

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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

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   Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 16 and 21. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

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   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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   Version published to 10.1101/2020.12.03.407031 on bioRxiv

   Dec 4, 2020