FXa cleaves the SARS-CoV-2 spike protein and blocks cell entry to protect against infection with inferior effects in B.1.1.7 variant

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   ScreenIT

   Jun 10, 2021

   SciScore for 10.1101/2021.06.07.447437: (What is this?)

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

   Table 1: Rigor

   Ethics	IRB: The protocols for human specimen collection were approved by the institutional review board of City of Hope. Euthanasia Agents: Mice were euthanized using ketamine (100 mg/kg)/xylazine (10 mg/kg) when body weights dropped below 20% of their original body weights. IACUC: Experiments and handling of mice were conducted under federal, state, and local guidelines and with approvals from the Northern Arizona University (20-005) and City of Hope Animal Care and Use Committees.
   Sex as a biological variable	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	Contamination: All cell lines were routinely tested to confirm absence of …

   SciScore for 10.1101/2021.06.07.447437: (What is this?)

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

   Table 1: Rigor

   Ethics	IRB: The protocols for human specimen collection were approved by the institutional review board of City of Hope. Euthanasia Agents: Mice were euthanized using ketamine (100 mg/kg)/xylazine (10 mg/kg) when body weights dropped below 20% of their original body weights. IACUC: Experiments and handling of mice were conducted under federal, state, and local guidelines and with approvals from the Northern Arizona University (20-005) and City of Hope Animal Care and Use Committees.
   Sex as a biological variable	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	Contamination: All cell lines were routinely tested to confirm absence of mycoplasma using the MycoAlert Plus Mycoplasma Detection Kit from Lonza (Walkersville, MD).

   Table 2: Resources

   Antibodies
   Sentences	Resources
   SARS-CoV-2 viral nucleocapsid protein (NP) was detected using the anti-NP protein antibody (PA5-81794, Thermo Fisher) diluted 1:10000 in 0.1% tween-20/1%BSA/PBS solution as a primary antibody, followed by detecting with an anti-rabbit secondary antibody (ab6721, Abcam) at a 1:20,000 dilution.	SARS-CoV-2 viral nucleocapsid protein ( NP ) suggested: None SARS-CoV-2 viral nucleocapsid protein ( NP suggested: None PA5-81794 suggested: (Thermo Fisher Scientific Cat# PA5-81794, RRID:AB_2788968) anti-rabbit suggested: (Abcam Cat# ab6721, RRID:AB_955447)
   FXa-HRP conjugated anti-human Fc antibody (05-4220, Invitrogen) was used as a detecting antibody.	anti-human Fc suggested: (Thermo Fisher Scientific Cat# 05-4220, RRID:AB_2532922)
   The cells were then washed and incubated with an anti-S protein antibody for 20 minutes at room temperature, followed by staining with a FITC-labeled secondary antibody (111-605-045, Jackson ImmunoResearch).	anti-S protein suggested: None
   An HRP-conjugated anti-His tag antibody (ab1187, Abcam) was used as a detecting antibody.	An HRP-conjugated anti-His tag antibody suggested: None anti-His tag suggested: None
   IHC staining with an anti-FXa protein antibody (PIPA529118, Invitrogen), an anti-furin antibody (ab183495, Abcam), an anti-trypsin antibody (ab200997, Abcam), or an anti-plasmin antibody (LS-C150813-1, LSBio) as a primary antibody was performed by the Pathology Shared Resource Core at City of Hope Beckman Research Institute.	anti-FXa protein suggested: None anti-furin suggested: (Abcam Cat# ab183495, RRID:AB_2801581) anti-trypsin suggested: None anti-plasmin suggested: None
   H&E staining and IHC with anti-NP protein antibody (NB100-56576, Novus) as the primary antibody were performed by the Pathology Shared Resource Core at City of Hope Beckman Research Institute.	anti-NP protein antibody suggested: (Creative Diagnostics Cat# DPAB-DC4728, RRID:AB_2501850) anti-NP protein suggested: (Creative Diagnostics Cat# DPAB-DC4728, RRID:AB_2501850) NB100-56576 suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Monkey kidney epithelium-derived Vero cells, Vero E6 cells, human embryonic kidney-derived HEK293T cells, and Chinese hamster ovary (CHO) cells were cultured in DMEM with 10% FBS, penicillin (100 U/ml), and streptomycin (100 μg/ml).	Chinese hamster ovary (CHO) suggested: None
   To determine viral production, Vero cells were pre-seeded for 24 hours and infected with the supernatants collected from MA104 cells infected by VSV-SARS-CoV at 24 or 48 hpi.	Vero suggested: None
   Generation and purification of FXa: CHO cells were transduced with a pCDH lentiviral vector expressing FXa to produce the FXa-Fc fusion protein for functionality assays.	CHO suggested: CLS Cat# 603479/p746_CHO, RRID:CVCL_0213)
   Virus isolates were passaged in Vero E6 cells (ATCC CRL-1586) as previously described3.	Vero E6 suggested: None
   Assessment of binding between S protein and FXa using flow cytometry: HEK293T cells were transduced with lentiviral vector expressing FXa for 48 hours.	HEK293T suggested: None
   Experimental Models: Organisms/Strains
   Sentences	Resources
   In vivo infection model: 6-8-week-old K18-hACE2 mice were anesthetized with ketamine (80 mg/kg)/xylazine (8 mg/kg) and intranasally infected with 5×103 PFU wild type SARS-CoV-2 or B.1.1.7 variant in 25 μl DMEM, followed by intranasal treatment with PBS, FXa-Fc (200 μg), or Fc (200 μg) in 25 μl DMEM.	K18-hACE2 suggested: RRID:IMSR_GPT:T037657)
   Recombinant DNA
   Sentences	Resources
   Pull-down assay: HEK293T cells were transduced with a pCDH lentiviral vector expressing the full-length spike (S) protein for 48 hours.	pCDH suggested: RRID:Addgene_102325)
   Software and Algorithms
   Sentences	Resources
   Prism software v.8 (	Prism suggested: (PRISM, RRID:SCR_005375)

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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 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).

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

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

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   Version published to 10.1101/2021.06.07.447437v1 on bioRxiv

   Jun 8, 2021