SciScore for 10.1101/2022.01.25.477753: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">Contamination: The absence of mycoplasma contamination in cells was routinely tested.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Blots were probed with the following antibodies: rabbit anti-eIF4E2/4EHP (Proteintech, 12227-1-AP), rabbit anti-DDX6 (Proteintech, 14632-1-AP), rabbit anti-CNOT9 (Proteintech, 22503-1-AP), mouse anti-Flag M2 (Sigma-Aldrich, F1804), rabbit anti-GIGYF2 (Proteintech, 24790-1-AP)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-eIF4E2/4EHP</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-DDX6</div><div>suggested: (Proteintech Cat# 14632-1-AP, RRID:AB_2091264)</div></div><div style="margin-bottom:8px"><div>anti-CNOT9</div><div>suggested: (Proteintech Cat# 22503-1-AP, RRID:AB_11232413)</div></div><div style="margin-bottom:8px"><div>anti-Flag</div><div>suggested: (Sigma-Aldrich Cat# F1804, RRID:AB_262044)</div></div><div style="margin-bottom:8px"><div>F1804</div><div>suggested: (Sigma-Aldrich Cat# F1804, RRID:AB_262044)</div></div><div style="margin-bottom:8px"><div>anti-GIGYF2</div><div>suggested: (Proteintech Cat# 24790-1-AP, RRID:AB_2879727)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Flp-In T-REx 293 cells (Thermo Fisher Scientific) were grown in similar conditions supplemented with 100μg/ml zeocin and 15μg/ml blasticidin.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Flp-In T-REx 293</div><div>suggested: RRID:CVCL_U427)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The full-length cDNA encompassing the coding region of human 4EHP, obtained by RT-PCR using total RNA from HEK293T cells, were cloned into the pCI-Neo vector (Promega) at the XhoI-NotI sites in frame with a sequence encoding a V5 tag inserted at the NheI-XhoI sites.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">CRISPR-Cas9-mediated genome editing of HEK293 cells was performed according to Ran et al. (53).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293</div><div>suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The cell line inducibly expressing Flag-NSP2 was generated by co-transfecting pcDNA5-FRT-TO-FH-Nsp2 (Addgene plasmid 157683) and pOG44 (Thermo Fisher Scientific) with a 1:10 ratio.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA5-FRT-TO-FH-Nsp2</div><div>suggested: RRID:Addgene_157683)</div></div><div style="margin-bottom:8px"><div>pOG44</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Truncated versions of NSP2 were generated by replacing the sequence encoding full-length NSP2 by PCR-amplified fragments at the BamHI-XhoI sites of the pcDNA5-FRT-TO-FH vector.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA5-FRT-TO-FH</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The mCherry2-NSP2 expressing plasmids were created by cloning a PCR-amplified NSP2 fragment into the mCherry2-C1 (Addgene plasmid 54563) and mCherry2-N1 vectors (Addgene plasmid 54517) using EcoRI and BamHI restriction enzymes.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>mCherry2-NSP2</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>mCherry2-C1</div><div>suggested: RRID:Addgene_54563)</div></div><div style="margin-bottom:8px"><div>mCherry2-N1</div><div>suggested: RRID:Addgene_54517)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To generate the pCI-λNV5-GIGYF2 vector, a fragment containing the GIGYF2 sequence was obtained by PCR from the pcDNA4/TO/GFP-GIGYF2 vector (Addgene plasmid 141189) (34) and inserted into the pCI-λNV5 at the XhoI-NotI sites.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCI-λNV5-GIGYF2</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pcDNA4/TO/GFP-GIGYF2</div><div>suggested: RRID:Addgene_141189)</div></div><div style="margin-bottom:8px"><div>pCI-λNV5</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A similar strategy was used to generate the V5-tagged fragments of GIGYF2 using primers to isolate the following domains encompassing residues: 1-267; 257-495; 485-752; 742-1,085; 1,075-1,320. pCI-eGFP-GIGYF2 was generated by inserting the eGFP sequence, PCR-amplified from pEGFP-C1 (Clontech), at the NheI-XhoI sites of the pCI-Neo vector and then by adding the fragment encoding GIGYF2 at the XhoI-NotI sites.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCI-eGFP-GIGYF2</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pEGFP-C1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The full-length cDNA encompassing the coding region of human 4EHP, obtained by RT-PCR using total RNA from HEK293T cells, were cloned into the pCI-Neo vector (Promega) at the XhoI-NotI sites in frame with a sequence encoding a V5 tag inserted at the NheI-XhoI sites.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCI-Neo</div><div>suggested: RRID:Addgene_16574)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To generate the pCI-λNV5-GW182SD vector, a fragment encoding the residues 1382-1690 was cloned by PCR as a XhoI-NotI fragment from the pFRT/TO/FLAG/HA-DEST TNRC6C vector (Addgene plasmid 19885) (52) and inserted into the pCI-λNV5 at the XhoI-NotI sites.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCI-λNV5-GW182SD</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pFRT/TO/FLAG/HA-DEST TNRC6C</div><div>suggested: RRID:Addgene_19885)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For recombinant GST-fused protein expression, full-length 4EHP and GIGYF2742-1,085 coding sequences contained in PCR-amplified BamHI-NotI and XhoI-NotI fragments, respectively, were cloned into a pGEX-6P-1 vector (Amersham) in frame with the GST coding sequence.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pGEX-6P-1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The pET-28b vector (EMD Biosciences) was used to express NSP2 as a recombinant protein fused to an His6 tag at the N-terminus.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pET-28b</div><div>suggested: RRID:Addgene_47327)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To generate KO HEK293 cells, we transfected 700,000 cells with the pSpCas9(BB)-2A-Puro plasmid.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pSpCas9 ( BB)-2A-Puro</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For the reporter containing the 3’UTR of Ifnb1, 20 ng of psiCHECK2-RLuc-Ifnb1 3’ UTR reporter (31), or empty psiCHECK2 (Promega), was added along with 100 ng of vectors encoding Flag-NSP2 or Flag (empty vector).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>psiCHECK2</div><div>suggested: RRID:Addgene_40763)</div></div></td></tr></table>

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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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About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

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SciScore for 10.1101/2022.01.25.477789: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IACUC: The mouse and hamster studies were carried out in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals under the approval of the Institutional Animal Care and Use Committee of Xiamen University.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">Rhesus monkey immunizations: Ten rhesus monkeys were allocated randomly into two groups (three females and two males per group).</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">Rhesus monkey immunizations: Ten rhesus monkeys were allocated randomly into two groups (three females and two males per group).</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">Microscopic evaluation of pathological lung lesions was performed blindly by pathologists following a semiquantitative scoring system with the inclusion of three indicators (49): (i) alveolar septum thickening and consolidation; (ii) hemorrhage, exudation, pulmonary edema, and mucous; and (iii) recruitment and infiltration of inflammatory immune cells.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Two weeks after boosting, serum samples were collected for antibody analyses, including measurement of anti-spike IgG, anti-RBD IgG, pseudovirus neutralizing antibody, and authentic neutralizing antibody titers.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-spike IgG</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-RBD IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In brief, single-cell suspensions were obtained from mouse spleen (106 cells per well) through grinding in 70 μm cell strainers and were seeded in anti-mouse IFN-γ antibody precoated ELISpot plates.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-mouse IFN-γ</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The mixtures (150 μL per well) were added to a monolayer of Vero cells in a 96-well plate and incubated at 37 °C supplying with 5% CO2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Organisms/Strains</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Experimental animals: BALB/c and C57BL/6 mice were purchased from Shanghai SLAC Laboratory Animal Co.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>C57BL/6</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Mouse immunizations: Six to eight-week-old BALB/c mice (n=6 per group) were immunized with STFK vaccines at 0.01, 0.1, 1, or 10 μg per dose in 150 μL through intramuscular injection following a two-dose schedule at weeks 0 and 3.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BALB/c</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The tag-free STFK and STFK variants were constructed into a pGS01b vector, modified from the pCGS3 vector containing glutamine synthetase (GS) selection marker (Sigma Aldrich).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pGS01b</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pCGS3</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data were acquired using the SerialEM software on an FEI Tecnai F30 transmission electron microscope (ThermoFisher Scientific) operated at 300 kV and equipped with a Gatan K3 direct detector.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SerialEM</div><div>suggested: (SerialEM, RRID:SCR_017293)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Image processing and 3D reconstruction: Drift and beam-induced motion correction were performed with MotionCor2 (38) to produce a micrograph from each movie.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MotionCor2</div><div>suggested: (MotionCor2, RRID:SCR_016499)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">We initially fitted the templates into the corresponding final cryo-EM maps using Chimera (43), and further corrected and adjusted them manually by real-space refinement in Coot (44).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Coot</div><div>suggested: (Coot, RRID:SCR_014222)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The resulting models were then refined with phenix.real_space_refine in PHENIX (45).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PHENIX</div><div>suggested: (Phenix, RRID:SCR_014224)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The final atomic models were validated with Molprobity (46, 47).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Molprobity</div><div>suggested: (MolProbity, RRID:SCR_014226)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All statistical analyses were conducted in GraphPad Prism 8 software.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

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Results from OddPub: Thank you for sharing your data.

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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: 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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About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

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SciScore for 10.1101/2022.01.26.477860: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">A pool of DNA constructs containing a randomized 18 bp barcode sequence (N18) was cloned into the NotI and AscI sites upstream of the LexA promoter sequence via restriction digestion, ligation and transformation into chemically competent E. coli.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">15 µg of total cellular protein was resolved by SDS-PAGE, transferred to a PVDF membrane, and probed using an anti-his antibody (ref).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-his</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">W303 cells were transformed with the p416LexA-UbMpro(C145A)-his construct and the resulting yeast cells were grown to exponential phase in SD-ura media at 30°C. 125 nM β-estradiol was added when indicated and cells were grown for an additional eight hours.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>W303</div><div>suggested: Coriell Cat# GM03889, RRID:CVCL_W303)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Organisms/Strains</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The Gal4, Gal80 and Pdr5 genes were disrupted to create the following strain: W303 HO::Gal1-GFP-v5-His3; gal4::trp1; gal80::leu2 pdr5::natMX.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Gal4 , Gal80</div><div>suggested: RRID:BDSC_51280)</div></div><div style="margin-bottom:8px"><div>HO::Gal1-GFP-v5-His3; gal4::trp1; gal80::leu2 pdr5::natMX</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Construction of WT Ub-Mpro vector (p416LexA_UbMpro(WT)_B112): The Ubiqutin-Mpro gene fusion was constructed using overlapping PCR of the yeast ubiquitin gene and SARS-CoV-2 Mpro gene (Jin, Du et al. 2020) and was inserted into the pRS416 vector after digestion with SpeI and BamHI.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pRS416</div><div>suggested: RRID:Addgene_158144)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">(Addgene plasmid # 58434; http://n2t.net/addgene:58434 ; RRID:Addgene_58434))(Ottoz, Rudolf et al. 2014) and inserted between the SacI and SpeI sites upstream of the ubiquitin-Mpro gene.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div></div><div>detected: RRID:Addgene_58434)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Addgene plasmid # 58437; http://n2t.net/addgene:58437; RRID:Addgene_58437))(Ottoz, Rudolf et al. 2014) and inserted into the KpnI site.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div></div><div>detected: RRID:Addgene_58437)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Barcode association: To associate barcodes with Mpro variants, we digested the p416-UbMpro(lib)-B112 plasmid upstream of the N18 sequence and downstream of the Mpro sequence with NotI and SalI enzymes (NEB).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>p416-UbMpro(lib)-B112</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The randomized 18 bp barcode sequence (N18) was cloned between the NotI and AscI sites upstream of the LexA promoter sequence in the p416LexA-Ub-Mpro(WT)-B112 vector with the goal of the WT sequence being represented by approximately 100 barcodes.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>p416LexA-Ub-Mpro(WT)-B112</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The resulting DBD-MproCS-AD fusion gene was inserted between the EcoRI and SacI sites downstream of the CUP promoter in the integrative bidirectional pDK-ATC plasmid (kindly provided by D. Kaganovich)(Amen and Kaganovich 2017).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pDK-ATC</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The mCherry gene was subsequently cloned into the XhoI/BamHI sites downstream of the TEF promoter in the opposite orientation to create the plasmid pDK-CUP-DBD-MproCS-AD-TEF-mCherry.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pDK-CUP-DBD-MproCS-AD-TEF-mCherry</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Bulk Split transcription factor (TF) competition experiment: Barcoded WT UbMpro (p416LexA-UbMpro(WT)-N18) plasmid was mixed with the barcoded UbMpro library (p416LexA-UbMpro(lib)-N18) at a ratio of 20-fold WT to the average library variant.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>p416LexA-UbMpro(WT)-N18</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The CyPet gene was amplified by PCR from the pCyPet-His vector (pCyPet-His was a gift from Patrick Daugherty (Addgene plasmid # 14030 ; http://n2t.net/addgene:14030 ; RRID:Addgene_14030)) with a forward primer containing the BamHI site (BamHI_F) and a reverse primer containing the extending MproCS overhang sequence.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div></div><div>detected: RRID:Addgene_14030)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The YPet gene was amplified by PCR from the pYPet-His vector (pYPet-His was a gift from Patrick Daugherty (Addgene plasmid # 14031 ; http://n2t.net/addgene:14031 ; RRID:Addgene_14031)) with a forward primer containing the extending MproCS overhang sequence and a reverse primer containing the XhoI site (XhoI_R).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div></div><div>detected: RRID:Addgene_14031)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The resulting CyPet-MproCS-YPet gene was inserted between the BamHI and XhoI sites downstream of the TEF promoter in the integrative bidirectional pDK-ATG plasmid (kindly provided by D. Kaganovich)(Amen and Kaganovich 2017).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pDK-ATG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Analysis of Mpro expression and Ubiquitin removal by Western Blot: To facilitate analysis of expression levels of Mpro and examine effective removal of Ubiquitin, a his tag was fused to the C-terminus of Mpro to create the plasmid p416LexA-UbMpro-his6-B112.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>p416LexA-UbMpro-his6-B112</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">W303 cells were transformed with the p416LexA-UbMpro(C145A)-his construct and the resulting yeast cells were grown to exponential phase in SD-ura media at 30°C. 125 nM β-estradiol was added when indicated and cells were grown for an additional eight hours.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>p416LexA-UbMpro(C145A)-his</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">PacBio circular consensus sequences (CCS) were generated from the raw reads using SMRTLink v.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SMRTLink</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Illumina sequence reads were filtered for Phred scores > 10 and strict matching of the sequence to the expected template and identifier sequence.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Phred</div><div>suggested: (Phred, RRID:SCR_001017)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The figures were generated using Matplotlib (Hunter 2007), PyMOL and GraphPad Prism version 9.3.1.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Matplotlib</div><div>suggested: (MatPlotLib, RRID:SCR_008624)</div></div><div style="margin-bottom:8px"><div>PyMOL</div><div>suggested: (PyMOL, RRID:SCR_000305)</div></div><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

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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: We detected the following sentences addressing limitations in the study:

However, there are a couple of caveats that should be kept in mind when utilizing these data sets. For example, we do not fully understand how Mpro’s biochemical function relates to viral fitness. Having some Mpro function is essential to the virus, so mutations that destroy Mpro function will form non-functional viruses. Function-fitness relationships tend to be non-linear (Heinrich and Rapoport 1974, Kacser and Fell 1995, Jiang, Mishra et al. 2013) and it may be likely that Mpro function must be decreased by a large amount in order to cause measurable changes in viral replication efficiency. This relationship between Mpro function and SARS-CoV-2 fitness would need to be determined in order to translate our functional scores to fitness scores. Additionally, our TF and FRET screens quantify cleavage at one defined site (Nsp4/5) and it may be important to analyze all sites in order to fully understand the selection pressures acting on Mpro. Another important caveat is that our fitness landscape captures single amino acid changes and therefore does not provide information on the potential interdependence or epistasis between double and higher order mutations. Information regarding epistasis will be important for accurately predicting the impacts of multiple mutations on fitness. Despite these caveats, the similarity in fitness landscapes for the TF and FRET screens with the yeast growth screen suggests that all three capture fundamental and general aspects of Mpro selection. In...

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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: 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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About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

</footer>

SciScore for 10.1101/2022.01.26.477856: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After incubation for 1h at room temperature and washing, the ACE-2 mFc Tag was revealed using a peroxidase-conjugated anti-mouse secondary antibody (1:1000 dilution).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-mouse</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">SARS-CoV-2 clinical isolates (D614G variant; GenBank accession number MW322968) , Alpha (GenBank accession number MW633280) , Beta (GenBank accession number MW580244) , Gamma (Gene accession number pending), Delta (Gene accession number pending) and Omicron (Gene accession number pending) were isolated from SARS-CoV-2 RT-PCR confirmed patients by inoculating Vero cells with sputum sample or nasopharyngeal swabs in the biosafety level-3 (BSL-3) facility of the Pitié-Salpêtrière University Hospital.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero</div><div>suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Hundred μl of a Vero E6 cell suspension (3 × 105 cells/ml) were then added to the mixture and incubated at 37°C under an atmosphere containing 5% CO2 until microscopy examination on day 4 to assess cytopathogenic effect (CPE), as previously described (Vanhove et al</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero E6</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">IC50s were analyzed by nonlinear regression using a four-parameter dosage-response variable slope model with the GraphPad Prism 8.0.2 software (GraphPad Software, USA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

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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).

---

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: We found the following clinical trial numbers in your paper:<br><table><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Identifier</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Status</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Title</td></tr><tr style="background-color:#FF0000"><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT0492830</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Trial number did not resolve on clinicaltrials.gov. Is the number correct?</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NA</td></tr></table>

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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: 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.

---

<footer>

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

</footer>

SciScore for 10.1101/2022.01.24.477625: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Library Design: A library of antibodies directed against SARS-CoV-2 Spike (S) protein was developed using paired antibody sequences, meaning antibody sequences for which the heavy and light chain are both known, from the Coronavirus Antibody Database, CoV-AbDab57.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CoV-AbDab57</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">CrossMAb, antibody, hFc-ACE2, and Lentiviral plasmids were maxi prepped in the same fashion.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>hFc-ACE2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Two microliters of mouse anti-hexa His antibody (BioLegend) were added to the 10 mL sample and incubated for 1 h at room temperature.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-hexa</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Viral transfections were done in HEK293T cells using calcium phosphate transfection reagent.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The resultant 10mL was added to plated HEK cells from which the medium had been removed.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK</div><div>suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Neutralization: The target cells used for infection in viral neutralization assays were from a HeLa cell line stably overexpressing the SARS-CoV-2 receptor, ACE2, as well as the protease known to process SARS-CoV-2, TMPRSS2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HeLa</div><div>suggested: CLS Cat# 300194/p772_HeLa, RRID:CVCL_0030)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">ACE2/TMPRSS2/HeLa cells were plated one day prior to infection at 5,000 cells per well.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ACE2/TMPRSS2/HeLa</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Equimolar aliquots of each scFv plasmid were pooled and the resultant pool was amplified using primers which annealed to the hexa-his Tag (reverse primer) or signal peptide (forward primer) and had a 50bp overlap with the pPNL6 vector digested with NheI and BamHI.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>scFv</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Yeast were resuspended in electroporation buffer (10 mM Tris Base, 250 mM sucrose, 2mM MgCl2) containing the gel extracted library amplification and digested pPNL6 vector.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pPNL6</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Light chain (LC) and LC-ACE2 fusion proteins: Antibody sequences were cloned into the CMV/R plasmid backbone for expression under a CMV promoter.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CMV/R</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">hCoV spike proteins constructs: Spike proteins from six hCoVs were cloned into a pADD2 vector between the rBeta-globin intron and β-globin poly(A).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pADD2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Lentivirus plasmids: Plasmids encoding the full-length spike proteins with native signal peptides were cloned into the background of the HDM-SARS2-Spike-delta21 plasmid (Addgene Plasmid #155130).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HDM-SARS2-Spike-delta21</div><div>suggested: RRID:Addgene_155130)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">They are: pHAGE-Luc2- IRS-ZsGreen (NR-52516), HDM-Hgpm2 (NR-52517), pRC-CMV-Rev1b (NR-52519), and HDM- tat1b (NR-52518).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pHAGE-Luc2- IRS-ZsGreen</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The EtOH solution was then added to a geneJET plasmid miniprep column and the regular wash steps and elution steps were followed.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>geneJET</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Digested FAbs were purified by SP/AKTA using 50 mM NaOAc, pH5 with gradient NaCl elution (using 50 mM NaOAc + 1M NaCl, pH5).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pH5</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The plasmids were added to D10 medium in the following ratios: 10 µg pHAGE-Luc2-IRS-ZsGreen, 3.4 µg FL Spike, 2.2 µg HDM-Hgpm2, 2.2 µg HDM-Tat1b, 2.2 µg pRC-CMV-Rev1b in a final volume of 1000 µL.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pRC-CMV-Rev1b</div><div>suggested: RRID:Addgene_164443)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The sequence alignment was again produced using MUSCLE31 and sequence identity was calculated using Geneious</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Geneious</div><div>suggested: (Geneious, RRID:SCR_010519)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The sequences used were: 6VXX, UniRef90_U5NJG5, _L7UP8, _A0A7U3W1C7, _K9N5Q8, _A0A2I6PIW5, _A0A3Q8AKM0, _U5WHZ7, _A0A5H2WTJ3, _A0A0U1WJY8, _A0A166ZND9, _A0A678TRJ7, _A0A2R4KP93, _A0A2Z4EVK1, _A0A7R6WCE7, _E0ZN36, _A0A6M3G9R1, _F1DAZ9, _A0A0U1UYX4, _A0A2R3SUW7, _A0A2Z4EVN5, _A0A2Z4EVN2, _U5LMM7, _A0A5Q0TVR4, _E0XIZ3, _A0A023Y9K3, _A0A2R4KP86, _A0A088DJY6, _A0A7G6UAJ9, _S4X276, _A0A4Y6GL90, _A3EXG6, _F1BYL9, _E0ZN60, _A0A0K1Z054, _A0A0U1WHI2, and NCBI accession numbers: YP_009047204.1, QLR06867.1, AAK32191.1, AGZ48828.1, AAT84362.1, QHR63300.2, ABD75513.1, YP_003767.1.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>NCBI</div><div>suggested: (NCBI, RRID:SCR_006472)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The sequences were first aligned using the MUSCLE algorithm, and then two phylogenetic trees were made, both using PhyML 3.3.20180621.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MUSCLE</div><div>suggested: (MUSCLE, RRID:SCR_011812)</div></div><div style="margin-bottom:8px"><div>PhyML</div><div>suggested: (PhyML, RRID:SCR_014629)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The sequences were then analyzed by sequence alignment using SnapGene software.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SnapGene</div><div>suggested: (SnapGene, RRID:SCR_015052)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">FAb Production from IgGs: 1/10 volume of 1M Tris, pH 8 was added to IgGs at ∼2 mg/mL in PBS. 2 µL of a 1 mg/mL stock of Lys-C (stock stored at -70C) was added for each mg of human IgG1 and digested for 1 hour at 37 °C with moderate rotation.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>IgGs</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Final data analysis was done in Prism.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Prism</div><div>suggested: (PRISM, RRID:SCR_005375)</div></div></td></tr></table>

---

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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About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

</footer>

SciScore for 10.1101/2022.01.24.477037: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">The 293 cells were co-transfected with split GFP vectors, and fluorescence intensity was determined by 5 random areas with the same exposure, followed by ImageJ analysis.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibodies against GSK-3β (610202, BD Transduction Laboratories), pS9-GSK-3β (9323S, Cell Signaling), pY216-GSK-3β (612312, BD Transduction Laboratories), Flag (F-3165, Sigma), Snail (L70G2, Cell Signaling), β-catenin (610154, BD Transduction Laboratories), and Tubulin (LF-PA0146, AbFrontier) were obtained from the commercial vendors</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pS9-GSK-3β ( 9323S , Cell Signaling) , pY216-GSK-3β ( 612312</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>Snail ( L70G2 , Cell Signaling) ,</div><div>suggested: (Cell Signaling Technology Cat# 3895, RRID:AB_2191759)</div></div><div style="margin-bottom:8px"><div>β-catenin</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>Tubulin ( LF-PA0146 , AbFrontier )</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Phosphorylation status of N protein was determined by anti-flag antibody and mobility shift on a Phos-tag gel (Wako) as described previously (38).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-flag</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cells were then washed three times with PBS containing 0.1% Tween 20 followed by incubation with anti-mouse-Alexa Fluor-488 (for green) or anti-rabbit-Alexa Fluor-594 (for red) secondary antibody.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-mouse-Alexa</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-rabbit-Alexa</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The 293 cells were co-transfected with split GFP vectors, and fluorescence intensity was determined by 5 random areas with the same exposure, followed by ImageJ analysis.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293</div><div>suggested: NCI-DTP Cat# NCI-293TT, RRID:CVCL_1D85)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The expression vector pGBW-m4134490 (plasmid number 152580) having codon optimized N of SARS-CoV, pGBW-m4134909 (plasmid number 151901) having N of human coronavirus 229E,</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pGBW-m4134490</div><div>suggested: RRID:Addgene_152580)</div></div><div style="margin-bottom:8px"><div>pGBW-m4134909</div><div>suggested: RRID:Addgene_151901)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">pGBW-m4134899 (plasmid number 151902) having N of human coronavirus OC43 and pGBW-m4134901 (plasmid number 151922) having N of human coronavirus HKU1 229E were obtained from Addgene.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pGBW-m4134899</div><div>suggested: RRID:Addgene_151902)</div></div><div style="margin-bottom:8px"><div>pGBW-m4134901</div><div>suggested: RRID:Addgene_151922)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Those N expression vectors were subcloned into pcDNA3.1 with C-terminal flag or EGFP tag.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3.1</div><div>suggested: RRID:Addgene_79663)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibodies against GSK-3β (610202, BD Transduction Laboratories), pS9-GSK-3β (9323S, Cell Signaling), pY216-GSK-3β (612312, BD Transduction Laboratories), Flag (F-3165, Sigma), Snail (L70G2, Cell Signaling), β-catenin (610154, BD Transduction Laboratories), and Tubulin (LF-PA0146, AbFrontier) were obtained from the commercial vendors</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pS9-GSK-3β</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">TCF/LEF and E-cadherin reporter assay: For TCF/LEF and E-cadherin reporter assay, the cells were transfected with 50 ng of the Super-Top or E-cad(−108)-Luc reporter vector (30,39) and 1 ng of pSV40-Renilla expression vector in combination with N or N-mutant as indicated.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>E-cad(−108)-Luc reporter</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pSV40-Renilla</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The 293 cells were co-transfected with split GFP vectors, and fluorescence intensity was determined by 5 random areas with the same exposure, followed by ImageJ analysis.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ImageJ</div><div>suggested: (ImageJ, RRID:SCR_003070)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">: Amino acid sequences of N for SARS-CoV-2 (P0DTC9), SARS-CoV (P59595), HKU1 (Q0ZME3), HCoV-OC43 (P33469), HCoV-229E (PP15130), HCoV-NL63 (Q6Q1R8) were obtained from UniProt (https://www.uniprot.org/).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>UniProt</div><div>suggested: (UniProtKB, RRID:SCR_004426)</div></div><div style="margin-bottom:8px"><div>https://www.uniprot.org/</div><div>suggested: (Universal Protein Resource, RRID:SCR_002380)</div></div></td></tr></table>

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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).

---

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.

---

<footer>

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

</footer>

SciScore for 10.1101/2022.01.21.477288: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IRB: The study protocol was approved by the University of Washington Human Subjects Division Institutional Review Board (STUDY00010350)</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Two hours after infection, the cells were washed 5 times with DMEM and grown in DMEM supplemented with 10% FBS and 1% PenStrep along with an anti-VSV-G antibody (I1-mouse hybridoma supernatant diluted 1:25, from CRL-2700, ATCC).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-VSV-G</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HEK-293T cells and HEK-293T cells stably expressing the human ACE2 receptor (HEK-ACE2)38 were grown in DMEM supplemented with 10% FBS and 1% PenStrep at 37°C and 5% CO2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK-293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Vero cells stably expressing the human protease TMPRSS2 (Vero-TMPRSS2) were grown in DMEM supplemented with 10% FBS, 1% PenStrep, and 8 µg/mL puromycin at 37°C and 5% CO2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Recombinant protein expression and purification: To produce the SARS-CoV-2 spike ectodomain containing the E406W mutation, 125 mL of Expi293 cells were grown to density of 2.5 × 106 cells per mL and transfected with 125 µg of DNA using PEI MAX diluted in Opti-MEM.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Expi293</div><div>suggested: RRID:CVCL_D615)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Neutralization assays with vaccine-elicited sera and monoclonal antibodies: For neutralization assays using vaccine-elicited sera, HEK-ACE2 cells were seeded in 96-well poly-D-lysine coated plates at a density of 30,000 cells per well and grown overnight until they reached approximately 80% confluency.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK-ACE2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Neutralizations were conducted as described above with one modification: prior to the addition of the virus-antibody mixture, Vero-TMPRSS2 cells were washed 3 times with DMEM.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero-TMPRSS2</div><div>suggested: JCRB Cat# JCRB1818, RRID:CVCL_YQ48)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The spike ectodomain was codon optimized, stabilized with the hexapro mutations39 and mutation of the furin cleavage site (682RRAR685 to 682GSAS685), and inserted into the pCDNA3.1 vector containing a C-terminal foldon followed by an avi tag and an octa-histidine tag.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCDNA3.1</div><div>suggested: RRID:Addgene_79663)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The construct encoding the E406W RBD was generated by performing around-the-horn mutagenesis using a pCMVR vector encoding the wildtype SARS-CoV-2 RBD containing an N-terminal mu-phosphatase signal peptide and a C-terminal avi tag and octa-histidine tag.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMVR</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Reference-free 2D classification was performed using cryoSPARC to select for well-defined particle images42.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>cryoSPARC</div><div>suggested: (cryoSPARC, RRID:SCR_016501)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">These selected particles were then used for 3D classification with 50 iterations (angular sampling 7.5° for 25 iterations followed by 1.8° with local search for 25 iterations) using Relion and a previously reported closed model for the SARS-CoV-2 spike ectodomain (PBD: 6VXX) as the initial model without imposing any symmetry.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Relion</div><div>suggested: (RELION, RRID:SCR_016274)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Luminescence readings from the neutralization assays were normalized and analyzed using GraphPad Prism 9.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

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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: 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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At this point in the article the information that the audience has been met with the introduction that the black liquid being referenced can be used for faces and feet, overhyping jargon, and $110 price tag for what is essentially dirt.

What kind of proof is available about not only the improved brain function, heart health, and toxin eliminator, but also that it can simply work for both all-ages humans and animals?

Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

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Reply to the reviewers

Manuscript number: RC-2021-01129

Corresponding author(s): Koji Kikuchi

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Reviewer #1

Evidence, reproducibility and clarity (Required):

In this manuscript, Kikuchi et al describe the characterization of MAP7D2 and MAP7D1, two MAP7 family members in mouse with specific expression patterns. Focusing mostly on MAP7D2, they assess its expression pattern across the body and find that it is mostly expressed in certain neuronal subsets. They then characterize the MT-related properties of MAP7D2 based on previous knowledge of other MAP7 family members. They show that MAP7D2 binds MTs (via the N-terminus), determine the binding affinity, and show that it can stimulate MT polymerization (or stabilization) both in vitro and in vivo. Using a specific antibody, they localize MAP7D2 to centrosomes, midbody and neurites in N1-E115 cells. Functionally, they show that loss of MAP7D1/2 mildly affects microtubule stability as judged by acetyl-tubulin staining, and properties of these cells that rely on cytoskeletal elements such as cell migration and neurite growth. Interestingly, there might be a feedback loop regulating MAP7D1/2 expression, as knockdown of MAP7D1 upregulates MAP7D2.

Overall, the experiments and conclusions are very solid and convincing, such that I would not ask for further experiments. This is in part because the experiments are largely based on previous characterizations of other MAP7 family members, which are largely confirmed. The presentation of the data is also very clear.

Significance (Required):

I see the value of the study in the fact that it provides solid and specific research tools for MAP7D1/2 which could be very useful for the microtubule/neuronal cytoskeleton community.

Response: We thank the reviewer very much for appreciating the content of our manuscript.

\*Referees cross-commenting***

Reviewers 2 and 3 criticize that the evidence for an effect of MAP7D1/2 on MT dynamics is weak. I would agree in that ac-tub stainings and in vitro experiments are rather indirect. The experiments suggested by reviewer 2 should clarify this (esp. nocodazole should be easy). I also agree that an experiment addressing the potential involvement of kinesin-1 would help, the involvement of which seems to have been omitted by the authors. A kinesin-binding deficient mutant would add another MAP7D1/2 tool and increase the value for the community.

Response: As for the reviewer’s suggestions listed above, please refer to our responses to the comments of Reviewer #2.

Reviewer #2

Evidence, reproducibility and clarity (Required):

In this study, the authors investigate 2 members from the MAP7 family Map7D2 and Map7D1. They first address the tissue distribution of Map7D2, by northern blotting using a variety of rat tissues. To complement their analysis, they also raised an antibody to look at the protein distribution. From their studies, they concluded that Map7D2 is abundantly expressed in the brain and testis. The authors went on to perform a series of functional assays. First, they biochemically demonstrated that rat Map7D2 directly binds to MTs by MT co-sedimentation assay. The MT binding domain was mapped to the N-terminal half. They performed MT turbidity assay to demonstrate enhanced MT polymerisation in the presence of Map7D2, suggesting that this Map stabilises MTs. The authors went on to characterise in detail the subcellular localisation of Map7D2 which was predominantly present in the centrosome and partially localised to MTs including within neurites from N1-E115 cells. Kikuchi et al. further revealed the overlap in expression between Map7D2 and another family member, Map7D1. The authors continued these studies by a series of functional studies in N1-E115 cells where they performed single or combined knock-downs of Map7D2 and Map7D1 and studied the levels of acetylated and detyrosinated tubulins and the effect of the knock-downs on migration and neurite extension. The main conclusion from this work was that Map7D2 and Map7D1 facilitate MT stabilization through distinct mechanisms which are important in controlling cell motility and neurite outgrowth. Map7D2 is proposed to stabilise MTs by direct binding whereas Map7D1 does it indirectly by affecting acetylation.

Major comments:

The main conclusion from this work that Map7D2 and Map7D1 facilitate MT stabilization and that this is necessary for correct migration and neurite extension has not been convincingly demonstrated. In my opinion, a more detailed study of MT properties to demonstrate a role in MT stabilisation would greatly benefit the work, eg. experiments using MT destabilising agents such as nocodazole. In addition, a series of experiments aiming to study MT dynamics would help to understand the function of these MT regulators. The authors proposed an elevation in microtubule dynamics to explain the increase in migration and neurite extension but no experimental proof was provided.

Response: According to the reviewer’s suggestion, we plan to assess the role of MT stabilization in greater detail by analyzing the sensitivity to the MT-destabilizing agent, nocodazole.

To study MT dynamics, methods such as analyzing the velocity and direction of an EB1-GFP comet are commonly used. We have previously analyzed the roles of Map7 and Map7D1 in MT dynamics using HeLa cells stably expressing EB1-GFP (Kikuchi et al., EMBO Rep., 2018). However, no such tools have been developed for analyzing MT dynamics in N1-E115 cells, which were used in this study. In addition, it is difficult to analyze MT dynamics by transient expression of EB1-GFP because of the low plasmid transfection efficiency. Therefore, we instead plan to assess the effect on MT dynamics by measuring the EB1 comet length by immunofluorescence, referring to Fig. 7D in EMBO J. 32:1293–1306, 2013.

Moreover, considering the possibility that the Map7D2 dynamics are altered when MT stability is changed, e.g., before and after differentiation induction, we analyzed the Map7D2 dynamics at the centrosome by fluorescence recovery after photobleaching (FRAP) using N1-E115 cells stably expressing EGFP-rMap7D2. We found that the dynamics were altered between the proliferative and differentiated states (see the figure below). Compared to the proliferative state, the recovery rate of EGFP-Map7D2 was reduced (lower left panel), and the immobile fraction of Map7D2 was increased in the differentiated state (lower right panel). As these data suggest that the increase in immobile Map7D2 may enhance MT stabilization, we will present them in a new figure in our manuscript along with the results of the above two experiments.

It has been previously demonstrated that loss of MAP7D2 leads to a decrease in axonal cargo entry to axons resulting in defects in axon development and neuronal migration. The C-terminus is necessary for this function as it mediates interaction with Kinesin-1 (Pan et al., 2019). Such mechanisms could also explain the defects in migration and neurite growth that the authors observed. This possibility has not been considered but instead, the subtle changes in total α-tubulin led to suggest MT stabilisation as a key function without proof of causation. Could the authors provide some further experimental evidence to demonstrate that stability is the main contributor to the phenotypes observed? Eg. by rescuing migration and neurite phenotypes with a variant of MAP7D2 which cannot bind kinesin1.

Response: The reviewer states “Such mechanisms could also explain the defects in migration and neurite growth that the authors observed;” however, our results showed that loss of Map7D2 elevated the rates of both cell motility and neurite outgrowth (original Fig. 5). In contrast, it has been reported in several papers that when Kinesin-1 function is impaired, both cell motility and neurite outgrowth are reduced (Curr. Biol., 23: 1018–1023, 2013; Mol. Cell. Biol., 39: e00109–19, 2019; etc.). Therefore, it is likely that the phenotypes we observed are independent of the functions associated with Kinesin-1 in N1-E115 cells. It is indeed possible that the experiment suggested by the reviewer may reveal relationships between Map7D2 and kinesin-1 in terms of cell motility and neurite outgrowth, however, it is difficult to conduct such an experiment because transient expression of Map7D2 induces MT bundling, as shown in original Fig. 2F. Based on the above, we plan to add a discussion of the relationship between Map7D2 and Kinesin-1.

A key conclusion proposed by the authors is that Map7D2 and Map7D1 facilitate MT stabilization through distinct mechanisms. Such different roles in MT stabilisation are important in controlling cell motility and neurite outgrowth. In my opinion, their data does not fully support this statement and the findings using MT readouts do not match the defects in migration and neurite growth. Loss of Map7D2 leads to a very subtle phenotype on α-tubulin, while Map7D1 decreases both α-tubulin and acetylated tubulin, but Map7D1 seems to have a milder or similar effect on migration and neurite growth than Map7D2. Furthermore, it would be expected that the combined loss of function would lead to a stronger phenotype in cell migration when compared to the single loss of functions due to their distinct roles on MT stability, however, this seems not to be the case.

Response: The fact that no stronger phenotype was observed may be because, besides Map7D2 and Map7D1, other molecules are involved in MT stabilization. Another possible explanation is that the increases in both cell motility and neurite outgrowth caused by decreased MT stabilization are offset by Kinesin-1 dysfunction. We plan to add a discussion of the above two possibilities.

Minor comments:

1) In the first result section, the author refers to Fig. S3 to suggest the expression of MAP7D2 in the cerebral cortex, however, there are no transcripts in the cerebral cortex according to the figure. Similarly, the immunofluorescence analysis done by the authors shows marginal expression of MAP7D2 in the cerebral cortex.

Response: According to the reviewer’s comment, we have changed the order of the data shown in Fig. 1C, top panels. The data from the olfactory bulb, cerebellum, and hippocampus, in which Map7D2 expression was detected in the database, were arranged in the top three rows, and the data from the cerebral cortex, in which Map7D2 expression was not detected in the database, were moved to the bottom row as a negative control. In addition, we have revised the relevant part of the Results section as follows: “Based on RNA-seq CAGE, RNA-Seq, and SILAC database analysis (Expression Atlas, https://www.ebi.ac.uk/gxa/home/), Map7D2 expression was detected in the cerebellum, hippocampus, and olfactory bulb, and not in the cerebral cortex (Fig. S3). We further confirmed Map7D2 expression in the above four brain tissue regions of postnatal day 0 mice by immunofluorescence. Among these regions, Map7D2 was the most highly expressed in the Map2-negative area of the olfactory bulb, i.e., the glomerular layer (Fig. 1C). Weak signals were detected in the cerebellum, and marginal signals were observed in the hippocampus and cerebral cortex (Fig. 1C).” (page 5, lines 4–11)

2) The authors use γ-Tubulin as a housekeeping gene in Fig. 3D, since Map7D2 is enriched in centrosomes this may not be the most appropriate choice.

Response: γ-Tubulin is abundant in both the cytosol and the nuclear compartments of cells (Sig. Transduct. Target Ther. 3: 24, 2018). As it has been used for similar purposes in several other studies (Cancer Res., 61: 7713–7718, 2001; J. Biol. Chem., 291: 23112–23125, 2016; etc.), we considered it acceptable for use as a loading control for immunoblotting.

3) According to the authors, knockdown of Map7D2 leads to a decrease in the intensity of α-tubulin and Map7D1 (Fig. 4C and D). This data doesn't agree with the previous statement made by the authors where they show that Map7D2 knockdown or knockout did not affect Map7D1 expression by Western Blot Analysis (Fig. S2C and S5B)

Response: The immunoblotting results indicate that the total amount of Map7D1 in the cells is not affected by loss of Map7D2. In contrast, the immunofluorescence results indicate that the amount (distribution) of Map7D1 localized around the centrosome is decreased by loss of Map7D2, presumably due to a reduction in the number of MT structures that can serve as scaffolds for Map7D1. We plan to add this interpretation in the Results section.

4) Line 6 page 7 "Endogenous Map7D2 expression is suppressed in N1-E115 cells stably expressing EGFP-rMap7D2 and was restored by specific knock-down of EGFP-rMap7D2 using gfp siRNA (Fig. 3D)". No quantifications and stats are shown. Also, endogenous Map7D2 after knock-down of EGFP-rMap7D2 is not comparable to the control.

Response: According to the reviewer’s suggestion, we have quantified the amount of endogenous Map7D2 or EGFP-rMap7D2, normalized it to the amount of γ-tubulin, and calculated relative values to endogenous Map7D2 in the parental control. The amount of endogenous Map7D2 was decreased to 53% in N1-E115 cells stably expressing EGFP-rMap7D2, suggesting that EGFP-rMap7D2 expression suppressed endogenous Map7D2 expression. In this cell line, the total amount of Map7D2 (EGFP-rMap7D2 + endogenous Map7D2) was increased, however, when EGFP-rMap7D2 was depleted using sigfp in this cell line, endogenous Map7D2 was expressed to the same level as EGFP-rMap7D2 before knock-down. Together with the finding that Map7d1 knock-down increased the amount of Map7D2, these findings indicate that the amount of Map7D2 in the cells is regulated in response to the amount of Map7D1 and exogenous Map7D2. We have added this interpretation in the Results section. (page 7, lines 8–15)

In addition, we have changed the legend of the original Fig. 3D to clarify the quantification method, as follows: “(D) Generation of N1-E115 cells stably expressing EGFP-rMap7D2. To check the expression level of EGFP-rMap7D2, lysates derived from the indicated cells were probed with anti-GFP (top panel) and anti-Map7D2 (middle panel) antibodies. The blot was reprobed for γ-tubulin as a loading control (bottom panel). The amount of endogenous Map7D2 or EGFP-rMap7D2 was normalized to the amount of γ-tubulin, and the value relative to endogenous Map7D2 in the parental control was calculated.” (page 22, lines 18–20)

5) Line 8 page 7 "These results suggest that the expression of Map7D2 was influenced by changes in that of Map7D1" This statement seems in the wrong place, after the Map7D2 and EGFP-rMap7D2 experiment. Instead for clarity, it would be better placed after line 5 where the authors explain the effect of Map7D1 knock-down on the levels of Map7D2.

Response: According to the reviewer’s suggestion, we have rephrased the relevant sentence as “Interestingly, Map7d1 knock-down upregulated Map7D2 expression, as confirmed with three different siRNAs (Fig. S2C), suggesting that Map7D2 expression is affected by changes in Map7D1 expression, not by off-target effects of a particular siRNA.” (page 7, lines 7, 8)

6) Line 8 page 8 "Although the physiological role of the C-terminal region of Map7D2 is currently unknown..." This statement seems not adequate as there are several studies reporting the role of the C-terminal region of Map7D2 in Kinesin1- mediated transport. The authors mention such studies in the discussion.

Response: According to the reviewer’s suggestion, we plan to add a discussion of the relationship between Map7D2 and kinesin-1.

7) Line 6 page 9 " Further, the knock-down of either resulted in a comparable reduction of MT intensity (Fig. 4C and D) ..." This is not visible and/or justified by the images provided and would benefit from some sort of quantification at other regions such as neurites.

Response: Considering the cell motility, quantification of α-tubulin/Ace-tubulin/Map7D1/Map7D2 intensities in neurites is not appropriate. Instead, we have added arrowheads indicating α-tubulin/Ace-tubulin/Map7D1/Map7D2 in Fig. 4C, for better understanding.

8) In Fig. 2B, a band corresponding to his6-rMAP7D2 of molecular weight >97 kDa co-sedimented with the microtubules. However, the cloned rMAP7D2 had a molecular weight of 84.82 kDa and the addition of 6XHis-Tag would add another 2-3 kDa, therefore, the final protein band observed should be less than 90 kDa. It would be beneficial if the authors could specify the molecular weight of the purified protein after the addition of the V5-his tag and/or if there was addition of amino acids due to cloning strategy.

Response: In Fig. 2B, we used full-length GST-tagged rMap7D2, like in Fig. 2E and D; therefore, we have corrected His6-rMap7D2 as GST-rMap7D2. We apologize for the mistake.

9) In Fig. 2C, there is misalignment of the western blot with the panel or text underneath.

Response: We thank the reviewer for pointing this out; we have corrected the misalignment of the CBB staining in Fig. 2C.

10) In Fig. 3C the inset from the first panel seems to correspond to a different focal plane than the main image.

Response: We have revised the relevant part of the figure legend as follows: “In C, images of differentiated cells were captured by z-sectioning, because the focal planes of the centrosome and neurites are different. Each inset shows an enlarged image of the region indicated with a white box at each focal plane. Arrowheads indicate the centrosomal localization of Map7D2.”

11) In Fig. 4A, the cell type is not specified and is referred as "indicated cells", also the material and methods section seems to omit the specific cells used.

Response: We have added “in N1-E115 cells treated with each siRNA” in the legend of Fig. 4A.

12) Fig. S6 is not mentioned in the results.

Response: We apologize for having referred to Fig. S6 only in the Discussion section in the original manuscript. We plan to describe the findings shown in the original Fig. S6 to the Results section and renumber the figures accordingly.

Significance (Required):

MTs play essential roles in practically every cellular process. Their precise regulation is therefore crucial for cellular function and viability. MAPs are specialised proteins that interact with MTs and regulate their behaviour in different manners. Understanding their precise function in different cellular contexts is of utmost importance for many biological and biomedical fields.

MAPs are well known for their ability to promote MT polymerization, bundling and stabilisation in vitro (Bodakuntla et al., 2019). Several members of the Map7 family have been shown to regulate microtubule stability. For instance, MAP7 can prevent nocodazole-induced MT depolymerization and maintain stable microtubules at branch points in DRG neurons (Tymanskyj & Ma, 2019). Ensconsin, the Drosophila Map, is required for MT growth in mitotic neuroblasts by regulating the mean rate of MT polymerization (Gallaud et al., 2014). However, this family of Maps seems to have diverse functions encompassing a variety of mechanisms, as exemplified by a series of studies demonstrating the involvement of MAP7 family proteins in the recruitment and activation of kinesin1 (Hooikaas et al., 2019; Pan et al., 2019) and in microtubule remodelling and Wnt5a signalling (Kikuchi et al., 2018). Further understanding of this family of Maps and how its members differ in their function is important and will help to advance the field.

Response: We appreciate the reviewer’s comments. We believe that our revision plan will greatly improve the quality of our manuscript.

Reviewer #3

Evidence, reproducibility and clarity (Required):

Summary:

Microtubule Associated Proteins (MAPs) are important regulators of microtubule dynamics, microtubule organization and vesicular transport by modulating motor protein recruitment and processivity. In the current manuscript the authors have characterized 2 members of the MAP7 protein family, MAP7D1 and MAP7D2. The authors characterized MAP7D2 expression pattern in the brain and its microtubule binding properties in vitro and in cells. In cells both proteins localize to the centrosome and to microtubules and upon depletion centrosome localized microtubules seem reduced, and cell migration and neurite outgrowth are increased. Surprisingly, they find that microtube acetylation (a common marker for stable microtubules) is reduced upon MAP7D1 depletion but not MAP7D2 depletion. Based on this finding the authors conclude that these proteins have a distinct mechanism in stabilizing MTs to affect cell migration and neurite outgrowth; MAP7D2 stabilizes by binding to MTs, whereas MAP7D1 stabilizes MTs by acetylation.

Main comments:

- Both MAP7 proteins show strong localization to the centrosome and to a lesser degree to MTs. Knockdown of either protein leads to reduced MTs around the centrosome, which lead the authors to conclude the MAP7s are stabilizing the MTs. However, the effect could just as well be an indirect effect due to a function of these MAPs at the centrosome. To address this authors could e.g. quantify microtubule properties in postmitotic cells. In addition, antibody specificity should be tested using knockdown of knockout cells, as this centrosome localization was not observed in Hela cells (Hooikaas, 2019; Kikuchi, 2018). Maybe this localization is specific to rat MAP7s or to the cell line used.

Response: We think that this comment partly overlaps with the comments raised by Reviewer #2. We plan to assess the role of MT stabilization in greater detail by analyzing the sensitivity to the MT-destabilizing agent, nocodazole, and the effect on MT dynamics by measuring the EB1 comet length by immunofluorescence.

Regarding the reviewer’s concern about antibody specificity, we had carefully confirmed the antibody specificity, as shown in Fig. S2 of the original manuscript. Subsequently, Map7D2 localization was confirmed in N1-E115 cells stably expressing EGFP-rMap7D2, as shown in Fig. 3D, E of the original manuscript. In addition, we are currently conducting analyses using Map7d1-egfp knock-in mice, which confirmed that Map7D1 localizes around the centrosome in cortical neurons, as shown below (we would like to disclose these unpublished data to the reviewers only). Therefore, it is thought that the localization pattern of Map7D2 and Map7D1 differs depending on the cell type and cell line. We plan to add this interpretation to the Results section.

- Centrosome nucleated microtubules are typically highly dynamic and little modified. Therefore is the Ac-tub staining at the centrosome really MTs? I cannot identify MTs in the fluorescent images in 4C. Maybe authors could consider ac-tub/alpha-tub ratio in non centrosomal region (e.g. neurites). Moreover, as both Acetylation and detyrosination are associated with long-lived/stable MTs, it is surprising that only acetylated tubulin goes down on WB. Does this suggest that long-lived MTs are still present to normal level? If so, can one still argue that the loss of acetylation is the cause of the lower MT levels? This should at least be discussed.

Response: As for the reviewer’s statement “Centrosome nucleated microtubules are typically highly dynamic and little modified. Therefore is the Ac-tub staining at the centrosome really MTs?”, it has been previously reported that tubulin acetylation is observed around the centrosome in some cell lines (J. Neurosci., 30: 7215–7226, 2010; PLoS One, 13: e0190717, 2018; etc.). N1-E115 is one of the cell lines in which tubulin acetylation is observed around the centrosome.

It is not surprising that “only acetylated tubulin goes down on WB,” as it has been previously reported that acetylated and detyrosinated tubulins are sometimes not synchronous (J. Neurosci., 23: 10662–10671, 2003; J. Neurosci., 30: 7215–7226, 2010; J. Cell Sci., 132: jcs225805, 2019., etc.). For instance, Montagnac et al. (Nature, 502: 567–570, 2013) showed that defects in the α-tubulin acetyltransferase αTAT1-clathrin-dependent endocytosis axis reduce only tubulin acetylation, resulting in a shift from directional to random cell migration. Although the details of the molecular function of Map7D1 are beyond the main purpose of this study, we plan to add a discussion of the reduced tubulin acetylation by Map7d1 knock-down based on the above.

- MAP7D1 and MAP7D2 depletion leads to subtle defect in cell migration and neurite outgrowth, which the author suggest is caused by reduced MT stability. However, MAP7 proteins have well characterized functions in kinesin-1 transport, and thus the phenotypes may well be caused by defects in kinesin-1 transport. Ideally the authors would do rescue experiments with FL or just the MT binding N-termini to separate these functions. Moreover this is needed to substantiate the claim of the authors that MAP7D1 effect on MT stability is not mediated by direct binding.

Response: As this comment largely overlaps with the comments raised by Reviewer #2, please refer to our responses to the comments of Reviewer #2.

- The authors do not refer well to published work. Several papers have published very similar work (especially to Fig1+2) and it would help the reader much if this would be discussed/compared along the results section and not briefly mention these in the results section. In addition, authors overstate the novelty of their results e.g. page 3: these proteins are not "functionally uncharacterized" nor are their expression patter and biochemical properties analyzed for the first time in this manuscript; page 8 "Although the physiological role of the C-terminal region of Map7D2 is currently unknow, ..." There is a clear function for the C-terminus for the recruitment/activation of kinesin-1.

Response: According to the reviewer’s suggestion, we plan to add a comparison with data on the Map7 family members presented in previous papers in the Results section and rephrase the relevant part regarding the physiological role of the C-terminal region of Map7D2.

Minor comments

- P6 Map7D3 also binds with its N-terminus to MTs, like other MAP7s (Yadav et al)

Response: According to the reviewer’s comment, we have revised this as “Map7D3 binds through a conserved region on not only the N-terminal side, but also the C-terminal side (Sun, 2011; Yadav et al., 2014).” (page 6, lines 4, 5)

- P7 "As Map7D2 has the potential to functionally compensate for Map7D1 loss" where is this based on?

Response: For clarity, we have rephrased this as “As Ma7D2 expression was upregulated upon suppression of Map7D1 expression, Map7D2 has the potential to functionally compensate for Map7D1 loss.” (page 7, line 17, 18)

- Fig2F quality of black-white images is low potentially due to conversion issues

Response: We thank the reviewer for pointing out these conversion issues, and we have made the necessary corrections.

Significance (Required):

At this stage the conceptual advance is limited. Part of the findings are not novel. The finding that MAP7s depletion have a different effect on MTs acetylation may be interesting to cytoskeleton researchers, although the potential mechanism has not been addressed experimentally or textually.

However, their conclusion that this leads to reduced MTs and then to cellar migration and neurite formation defects is not sufficiently supported by experimental evidence.

Response: We appreciate the reviewer’s comments. We believe that our revision plan will greatly improve the quality of our manuscript.

\*Referees cross-commenting***

I completely agree with reviewer #2: At this stage the paper's conclusions are not sufficiently supported by the data. Important will be to further characterize the effect om the MTs (do they really have a different effect) and to look at the possible involvement of the motor recruitment. Maybe that a 3 to 6 months revision time would have been more accurate.

Response: Please refer to our responses to the comments of Reviewer #2.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

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Referee #2

Evidence, reproducibility and clarity

In this study, the authors investigate 2 members from the MAP7 family Map7D2 and Map7D1. They first address the tissue distribution of Map7D2, by northern blotting using a variety of rat tissues. To complement their analysis, they also raised an antibody to look at the protein distribution. From their studies, they concluded that Map7D2 is abundantly expressed in the brain and testis. The authors went on to perform a series of functional assays . First, they biochemically demonstrated that rat Map7D2 directly binds to MTs by MT co-sedimentation assay. The MT binding domain was mapped to the N-terminal half. They performed MT turbidity assay to demonstrate enhanced MT polymerisation in the presence of Map7D2, suggesting that this Map stabilises MTs. The authors went on to characterise in detail the subcellular localisation of Map7D2 which was predominantly present in the centrosome and partially localised to MTs including within neurites from N1-E115 cells. Kikuchi et al. further revealed the overlap in expression between Map7D2 and another family member, Map7D1. The authors continued these studies by a series of functional studies in N1-E115 cells where they performed single or combined knock-downs of Map7D2 and Map7D1 and studied the levels of acetylated and detyrosinated tubulins and the effect of the knock-downs on migration and neurite extension. The main conclusion from this work was that Map7D2 and Map7D1 facilitate MT stabilization through distinct mechanisms which are important in controlling cell motility and neurite outgrowth. Map7D2 is proposed to stabilise MTs by direct binding whereas Map7D1 does it indirectly by affecting acetylation.

Major comments:

The main conclusion from this work that Map7D2 and Map7D1 facilitate MT stabilization and that this is necessary for correct migration and neurite extension has not been convincingly demonstrated. In my opinion, a more detailed study of MT properties to demonstrate a role in MT stabilisation would greatly benefit the work, eg. experiments using MT destabilising agents such as nocodazole. In addition, a series of experiments aiming to study MT dynamics would help to understand the function of these MT regulators. The authors proposed an elevation in microtubule dynamics to explain the increase in migration and neurite extension but no experimental proof was provided.

It has been previously demonstrated that loss of MAP7D2 leads to a decrease in axonal cargo entry to axons resulting in defects in axon development and neuronal migration. The C-terminus is necessary for this function as it mediates interaction with Kinesin-1 (Pan et al., 2019). Such mechanisms could also explain the defects in migration and neurite growth that the authors observed. This possibility has not been considered but instead, the subtle changes in total -tubulin led to suggest MT stabilisation as a key function without proof of causation. Could the authors provide some further experimental evidence to demonstrate that stability is the main contributor to the phenotypes observed? Eg. by rescuing migration and neurite phenotypes with a variant of MAP7D2 which cannot bind kinesin1.

A key conclusion proposed by the authors is that Map7D2 and Map7D1 facilitate MT stabilization through distinct mechanisms. Such different roles in MT stabilisation are important in controlling cell motility and neurite outgrowth. In my opinion, their data does not fully support this statement and the findings using MT readouts do not match the defects in migration and neurite growth. Loss of Map7D2 leads to a very subtle phenotype on -tubulin, while Map7D1 decreases both -tubulin and acetylated tubulin, but Map7D1 seems to have a milder or similar effect on migration and neurite growth than Map7D2. Furthermore, it would be expected that the combined loss of function would lead to a stronger phenotype in cell migration when compared to the single loss of functions due to their distinct roles on MT stability, however, this seems not to be the case.

Minor comments:

1. In the first result section, the author refers to Fig. S3 to suggest the expression of MAP7D2 in the cerebral cortex, however, there are no transcripts in the cerebral cortex according to the figure. Similarly, the immunofluorescence analysis done by the authors shows marginal expression of MAP7D2 in the cerebral cortex.
2. The authors use -Tubulin as a housekeeping gene in Fig. 3D, since Map7D2 is enriched in centrosomes this may not be the most appropriate choice.
3. According to the authors, knockdown of Map7D2 leads to a decrease in the intensity of -tubulin and Map7D1 (Fig. 4C and D). This data doesn't agree with the previous statement made by the authors where they show that Map7D2 knockdown or knockout did not affect Map7D1 expression by Western Blot Analysis (Fig. S2C and S5B)
4. Line 6 page 7 "Endogenous Map7D2 expression is suppressed in N1-E115 cells stably expressing EGFP-rMap7D2 and was restored by specific knock-down of EGFP-rMap7D2 using gfp siRNA (Fig. 3D)". No quantifications and stats are shown. Also, endogenous Map7D2 after knock-down of EGFP-rMap7D2 is not comparable to the control.
5. Line 8 page 7 "These results suggest that the expression of Map7D2 was influenced by changes in that of Map7D1" This statement seems in the wrong place, after the Map7D2 and EGFP-rMap7D2 experiment. Instead for clarity, it would be better placed after line 5 where the authors explain the effect of Map7D1 knock-down on the levels of Map7D2.
6. Line 8 page 8 "Although the physiological role of the C-terminal region of Map7D2 is currently unknown..." This statement seems not adequate as there are several studies reporting the role of the C-terminal region of Map7D2 in Kinesin1- mediated transport. The authors mention such studies in the discussion.
7. Line 6 page 9 " Further, the knock-down of either resulted in a comparable reduction of MT intensity (Fig. 4C and D) ..." This is not visible and/or justified by the images provided and would benefit from some sort of quantification at other regions such as neurites.
8. In Fig. 2B, a band corresponding to his6-rMAP7D2 of molecular weight >97 kDa co-sedimented with the microtubules. However, the cloned rMAP7D2 had a molecular weight of 84.82 kDa and the addition of 6XHis-Tag would add another 2-3 kDa, therefore, the final protein band observed should be less than 90 kDa. It would be beneficial if the authors could specify the molecular weight of the purified protein after the addition of the V5-his tag and/or if there was addition of amino acids due to cloning strategy.
9. In Fig. 2C, there is misalignment of the western blot with the panel or text underneath.
10. In Fig. 3C the inset from the first panel seems to correspond to a different focal plane than the main image.
11. In Fig. 4A, the cell type is not specified and is referred as "indicated cells", also the material and methods section seems to omit the specific cells used.
12. Fig. S6 is not mentioned in the results.

Significance

MTs play essential roles in practically every cellular process. Their precise regulation is therefore crucial for cellular function and viability. MAPs are specialised proteins that interact with MTs and regulate their behaviour in different manners. Understanding their precise function in different cellular contexts is of utmost importance for many biological and biomedical fields.

MAPs are well known for their ability to promote MT polymerization, bundling and stabilisation in vitro (Bodakuntla et al., 2019). Several members of the Map7 family have been shown to regulate microtubule stability. For instance, MAP7 can prevent nocodazole-induced MT depolymerization and maintain stable microtubules at branch points in DRG neurons (Tymanskyj & Ma, 2019). Ensconsin, the Drosophila Map, is required for MT growth in mitotic neuroblasts by regulating the mean rate of MT polymerization (Gallaud et al., 2014). However, this family of Maps seems to have diverse functions encompassing a variety of mechanisms, as exemplified by a series of studies demonstrating the involvement of MAP7 family proteins in the recruitment and activation of kinesin1 (Hooikaas et al., 2019; Pan et al., 2019) and in microtubule remodelling and Wnt5a signalling (Kikuchi et al., 2018). Further understanding of this family of Maps and how its members differ in their function is important and will help to advance the field.

Author Response:

Reviewer #2 (Public Review):

This manuscript by Barton and colleagues explores the roles of the conserved Eco1 transacetylase in modulating cohesin function in meiosis in budding yeast. Numerous studies in mitotically dividing cells have shown that the Eco1 family of transacetylases acetylate the Smc3 subunit of cohesin and that this acetylation renders cohesin on chromosomes resistant to removal by the Wapl (Wpl1 in budding yeast) family of proteins. Cohesins play critical roles in both sister chromatid cohesion and chromatin organization (through the formation of intrachromosomal loops). How cohesins are regulated by Eco1 in meiosis to accommodate meiotic chromosome structures such as the synaptonemal complex, chromatin domains around centromeres, repair of programmed meiotic double strand DNA breaks in prophase, and sequential removal of cohesins - first at arms in meiosis I and centromeres at meiosis II - is largely unexplored. Thus, this manuscript is exploring important new areas.

The authors show that Eco1 persists thru prophase I (longer than it does in vegetative cell cycles), that it is not necessary for cohesin loading at centromeres but is needed to counteract Wpl1 to protect centromeric cohesion, that it is critical for the establishment of chromatin loops on meiotic chromosome arms and that it is critical for protection of the arm cohesin from removal by Wpl1. The authors also provide evidence that, in meiosis, Wpl1 exhibits underappreciated functions in cohesin loading or cohesion establishment in addition to its recognized role in cohesin removal.

The experiments demonstrate that Eco1 is necessary for sharp cohesin boundaries that flank the centromeres and suggest this might be a replication-independent function of Eco1 (the boundaries form in clb5, clb6 cells with no DNA replication phase) but it is unclear if the detectable, but diminished, boundaries in clb5,clb6 cells were formed in the replication-free meiosis or presist from the S-phase associated loading and cohesion establishment from the preceding mitotic cycle.

Entry into meiosis occurs from G1 when there is no cohesin on the chromosomes and boundaries are not present, therefore this would only be a concern if there were persistent mitotic cells in G2 (i.e. after DNA replication). Our flow cytometry shows that the cells used in the experiment were unreplicated, so even if mitotic cells were present, they would not have been through S phase.

Nevertheless, we addressed this point by analysis of pre-S phase meiotic cells (ime1/ime4 block) and by anchoring away Eco1 in unreplicated cells.

Immunofluorescence imaging assays are used to observe the behavior of sister chromatids in meiosis I and meiosis II as a function of Eco1 activity. In wild-type cells sister chromatids co-orient in meiosis I and move to the same pole of the spindle. In mammalian cells and fission yeast this co-orientation requires cohesin while studies in budding yeast have suggested the co-orientation is cohesin-independent. Here, the authors show that when Eco1 is depleted, the sisters often move to opposite poles at meiosis I, and suggest that cohesin (and Eco1) is indeed required for sister co-orientation. An alternate possibility is that the sisters have lost their association in meiotic prophase (due to cohesin failures) before attaching to microtubles and segregating randomly - often to opposite poles.

We agree with this point, but would argue that the “alternative possibility” (which our data support) still leads to the conclusion that cohesin and Eco1 are required for sister co-orientation. A prior study (Monje-Casas et al., 2007) had suggested that monopolin could link sister kinetochores even without cohesin. We now show that this is not the case, which we believe to be an important conclusion.

Our results indicate that establishment of monoorientation requires the cohesin that is localized at centromeres. WPL1 deletion in eco1-aa rescues centromeric cohesion (Figure 2F, Figure 8E), but not chromosome arm cohesion (Figure 2H) or sister chromatid segregation in meiosis II (Figure 8F), indicating that pericentromeric cohesion must still be defective.

For clarity, please note that the relevant data is not immunofluorescence, but live cell imaging (now shown in Figure 8) so these conclusions are based on observation of single chromosomes in individual live cells from prophase I until anaphase II.

In summary the authors show that Eco1 has distinct roles on chromosome arms and centromeres and probably in both replication-linked and replication-independent events, acts to modulate cohesin location and function in meiosis.

Reviewer #3 (Public Review):

This paper investigates the meiotic roles of two regulators of cohesin, the cohesin destabilizer Wpl1 and the cohesin acetyltransferase Eco1. The authors provide evidence that Eco1 antagonizes Wpl1 to allow stabilization of centromeric cohesin, which is important to establish meiotic chromosome segregation patterns. In addition, Eco1 regulates the stable anchoring of cohesin at boundaries to promote defined chromosome loop formation in meiotic prophase.

The study uses a combination of calibrated ChIP-seq analysis, and chromosome conformation capture techniques to convincingly show that loop formation is altered in wpl1 depletion and eco1 depletion mutants. Well-established cytological techniques are used to demonstrate different effects on chromosome cohesion along arms and at centromeres, and to show that Eco1 is important for establishing the meiotic segregation pattern. The paper is well written and the data largely support the conclusions. As such, this paper is expected to be of substantial interest to the field.

One notable weakness is the poor definition of the eco1 anchor-away allele (eco1-aa), on which much of the eco1 phenotypic analysis is based. The presented data indicate that addition of the FRB-GFP tag alone causes most of the phenotypes, regardless of nuclear depletion. It is well possible that the tag creates a meiosis-specific loss-of-function allele, although it is surprising that the tag does not have mitotic defects even though Eco1 presumably has the same substrate (the cohesin subunit Smc3) in both situations. Encouragingly, some of the phenotypes could be confirmed using a non-acetylatable smc3 mutant. However, the tag may also create neomorphic effects that may contribute to the Wpl1-independent effects and the apparent stronger defects of the eco1-aa allele compared to the non-acetylatable smc3 mutant.

Available evidence suggests that eco1-aa is a loss of function allele.

Reviewer #3 (Public Review):

This paper investigates the meiotic roles of two regulators of cohesin, the cohesin destabilizer Wpl1 and the cohesin acetyltransferase Eco1. The authors provide evidence that Eco1 antagonizes Wpl1 to allow stabilization of centromeric cohesin, which is important to establish meiotic chromosome segregation patterns. In addition, Eco1 regulates the stable anchoring of cohesin at boundaries to promote defined chromosome loop formation in meiotic prophase.

The study uses a combination of calibrated ChIP-seq analysis, and chromosome conformation capture techniques to convincingly show that loop formation is altered in wpl1 depletion and eco1 depletion mutants. Well-established cytological techniques are used to demonstrate different effects on chromosome cohesion along arms and at centromeres, and to show that Eco1 is important for establishing the meiotic segregation pattern. The paper is well written and the data largely support the conclusions. As such, this paper is expected to be of substantial interest to the field.

One notable weakness is the poor definition of the eco1 anchor-away allele (eco1-aa), on which much of the eco1 phenotypic analysis is based. The presented data indicate that addition of the FRB-GFP tag alone causes most of the phenotypes, regardless of nuclear depletion. It is well possible that the tag creates a meiosis-specific loss-of-function allele, although it is surprising that the tag does not have mitotic defects even though Eco1 presumably has the same substrate (the cohesin subunit Smc3) in both situations. Encouragingly, some of the phenotypes could be confirmed using a non-acetylatable smc3 mutant. However, the tag may also create neomorphic effects that may contribute to the Wpl1-independent effects and the apparent stronger defects of the eco1-aa allele compared to the non-acetylatable smc3 mutant.

SciScore for 10.1101/2022.01.20.477107: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IRB: EXPERIMENTAL MODEL AND SUBJECT DETAILS: Blood, PBMCs and HLA typing: Whole blood from COVID-19 convalescent donors, healthy donors, or vaccine recipients was collected under protocol approved by the UMass Chan Medical School Institutional Review Board of the University of Massachusetts and informed consent was obtained from all subjects.<br>Consent: EXPERIMENTAL MODEL AND SUBJECT DETAILS: Blood, PBMCs and HLA typing: Whole blood from COVID-19 convalescent donors, healthy donors, or vaccine recipients was collected under protocol approved by the UMass Chan Medical School Institutional Review Board of the University of Massachusetts and informed consent was obtained from all subjects.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For blocking of antigen-stimulation assays, in-house produced antibodies to HLA-DR (LB3.1), HLA-DQ (SPVL-3), HLA-DP (B7/21), or HLA-ABC (w6/32), were added at a final concentration of 10 μg/mL.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HLA-DR</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>HLA-DP</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>HLA-ABC</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cells were collected, washed, and stained using a standard protocol which included: staining for dead cells with Live/Dead Fixable Aqua Dead Cell Stain Kit™ (Life Technologies, Thermo Fisher Scientific, Waltham, MA); blocking of Fc receptors with human Ig (Sigma-Aldrich, St. Louis, MO); staining with the mix of DP4-PE and APC tetramers (final concentration of 2-4 μg/mL each) at 37°C for 2 hours; surface staining antibodies CD3-APC-H7, CD4-PerCP-Cy5.5, CD8-APC-R700, CD14-BV510, CD19-BV510, CD56-BV510 were added for the last 20 minutes of incubation, followed by washes and resuspension in buffer for data acquisition.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CD19-BV510</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>CD56-BV510</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Partially-match HLA cell lines: EBV-transformed LG2 cell line (10984, IPD-IMGT/HLA), 9068 cell line (BM9, IHWG), and mouse DP4-transfected MN605 cell line (M12C3-DPA1*0103/DPB1*0401; (Williams et al., 2018); kindly provided by Dr. S. Kent, UMMS), were maintained in RPMI 1640 medium supplemented with L-glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 μg/mL) and 10% FBS at 37°C/5% CO2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MN605</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After 6 hours incubation, cells were collected, washed, and stained using a standard protocol, which included: staining for dead cells with Live/Dead Fixable Aqua Dead Cell Stain Kit™ (Life Technologies, Thermo Fisher Scientific, Waltham, MA); blocking of Fc receptors with human Ig (Sigma-Aldrich, St. Louis, MO); surface staining with mouse anti-human CD3-APC-H7 (SK7), CD4-PerCPCy5.5 (RPA-T4), CD8-APC-R700 (RPA-T8), CD14-BV510 (MϕP9), CD19-BV510 (SJ25C1), CD56-BV510 (NCAM16.2); fixation and permeabilization using BD Cytofix/Cytoperm™; and intracellular staining with mouse anti-human IFN-γ-V450 (B27), TNF-α-PE-Cy7 (MAb11), IL-2-BV650 (5344.111), (all from BD Biosciences, San Jose, CA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BD Cytofix/Cytoperm™</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data were acquired using a BD LRSII flow cytometer equipped with BD FACSDiva software (BD Biosciences, San Jose, CA) and analyzed using FlowJo v.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BD FACSDiva</div><div>suggested: (BD FACSDiva Software, RRID:SCR_001456)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Polyfunctional analysis was performed in FlowJo, defining Boolean combinatorial gates for all the markers in the CD3+/CD4+/CD8-population.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>FlowJo</div><div>suggested: (FlowJo, RRID:SCR_008520)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">These results were visualized in SPICE software v6.0 (Roederer et al., 2011). t-SNE analysis was done in concatenated samples (control, SARS-CoV-2, peptide 163 and peptide 164) from 3 donors using the available plugin in FlowJo.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SPICE</div><div>suggested: (SPICE, RRID:SCR_016603)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All primers sequences shown in STAR Methods.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>STAR</div><div>suggested: (STAR, RRID:SCR_004463)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">These files were processed using MIGEC v1.2.9 pipeline: Checkout-batch for de-multiplexing and UMI tag extraction, Histogram for MIG (molecular identifier groups) statistics, and Assemble-batch to perform UMI-guided assembly (Shugay et al., 2014); followed by MiXCR v3.0.13: analyze amplicon pipeline, to align, assemble and export clonotypes (Bolotin et al., 2015).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MIGEC</div><div>suggested: (migec, RRID:SCR_016337)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HLA-peptide binding prediction was performed with NetMHCIIpan v4.0 server (Reynisson et al., 2020).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>NetMHCIIpan</div><div>suggested: None</div></div></td></tr></table>

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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: We detected the following sentences addressing limitations in the study:

There are several limitations of our study. We mapped the specificity of the cross-reactive response by following IFN-γ-secreting cells, but non-IFN-γ-secreting populations could also contribute to the response. In expanded T cell lines we observed higher frequencies of T cells staining with DP4-163/164 tetramer than responding to the same peptide in IFN-γ ELISPot essays, indicating that some T cells can recognize the epitope but not secrete IFN-γ. We observed the cross-reactive T cell response to involve mostly CD4+ T cells. This might be due to in vitro culture conditions that favor CD4+ over CD8+ T cell populations, or an intrinsic bias of cross-reactive T cells because of the different patterns of pMHC-TCR interaction for MHC-I and MHC-II proteins. We studied a relatively small group of 27 individuals exposed to SARS-CoV-2 antigens by infection or vaccination, mostly over 40 years of age. Younger individuals with more frequent previous exposures to HCoVs might show a different pattern of response. Our initial screen for immunodominant epitopes involved only three donors, all of whom recognized 163/164, but other immudominant cross-reactive epitopes might have escaped our attention, including those recognized by other MHC proteins. For all of the donors, previous HCoV infection was inferred but not observed, and we did not attempt to determine which donors were exposed previously to which of the HCoVs. In conclusion, we identified a pan-coronavirus epitope that dominates t...

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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 32 and 36. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

---

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.

---

<footer>

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

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test

SciScore for 10.1101/2022.01.20.477056: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IRB: Ethical statement: The study was approved by the ethical review board in Gothenburg (Dnr: 2021-02252).</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Levels of human anti-SARS-CoV-2 IgG antibodies in convalescent serum samples: Serum samples from SARS-CoV-2 convalescent individuals (n=24) were obtained from the department of Clinical Microbiology, Sahlgrenska University Hospital, Gothenburg, Sweden.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-SARS-CoV-2 IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Anti-SARS-CoV-2 antibody reactivity assay: The antibody reactivity towards glycosidase treated proteins were assessed using an enzyme-linked immunosorbent assay (ELISA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-SARS-CoV-2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The comparison between anti-RBD IgG levels and antibody reactivity towards recombinant RBD was done with Pearson correlation coefficient, assuming normal distribution.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-RBD IgG</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Lec3.2.8.1 cells (a mutated CHO cell line kindly received from Prof. P Stanley (Chen & Stanley, 2003)) were cultured under the same conditions.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CHO</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The HEK293 derivate HEK293F cell line (Cat nr R79007, Thermo Fisher Scientific) were cultured in Freestyle 293 medium.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293F</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Expression of recombinant S protein constructs: The receptor-binding domain of the SARS-CoV-2 spike protein (amino acids 319-541) was produced in three cell lines using an expression vector obtained through BEI Resources, NIAID, NIH, which is vector pCAGGS containing the SARS-CoV-2, Wuhan-Hu-1 spike glycoprotein gene RBD with C-terminal Hexa-Histidine tag (NR-52309) (Table EV1).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCAGGS</div><div>suggested: RRID:Addgene_127347)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Glycan database search and data processing: The acquired data were analyzed using Proteome Discoverer version 2.4 (Thermo Fisher Scientific).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Proteome Discoverer</div><div>suggested: (Proteome Discoverer, RRID:SCR_014477)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The SARS-CoV-2 IgG II Quant assay is a chemiluminescent microparticle immunoassay (CMIA) used for quantitative determination of IgG antibodies to SARS-CoV-2 in human serum and plasma on the ARCHITECT System (Abbott Laboratories, Chicago, IL).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Abbott Laboratories</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All statistical analyses were performed using the Graphpad Prism software version 9.3.1</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Graphpad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">(GraphPad Software Inc, San Diego, CA, USA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

---

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.

---

<footer>

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

</footer>

SciScore for 10.1101/2022.01.19.476940: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">Contamination: To generate HEK-293T cells that transiently expressed wild type and mutated spike protein (ATCC; mycoplasma-free low passage stock), the cells were transfected with Spike protein expressing pLP plasmids using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s protocol and stained for their spike protein expression 72 hours after the transfection as described in Staining and Flow cytometry Analysis.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">ACE2 CAR construct: CAR constructs consisting of CD8 alpha signal peptide, extracellular domain of ACE2 molecule or single chain variable fragment (scFv) of anti-CD19 or anti-Spike protein antibodies, CD8 hinge domain, CD8 transmembrane domain, 4-1BB (CD137) intracellular domain and CD3ζ domain were designed with Snapgene and synthesized via Genscript.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-CD19</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-Spike protein</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>CD8 transmembrane domain, 4-1BB (CD137</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Anti-CD3 antibody single-chain variable fragment, His-Tag, and Hemagglutinin (HA) Tag sequences were obtained from Addgene plasmid #85437.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-CD3</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>His-Tag</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>Hemagglutinin (HA</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Activation of CAR CD8 T cells was determined with CD25 staining using CD25-APC antibody (Biolegend).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CD25-APC</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">CAR expressions of ACE2 CAR and anti-Spike CAR and ACE2 expression of ACE2-293 cells were determined with SARS-CoV-2 S1 protein, Mouse IgG2a Fc Tag (Acro Biosystems) incubation followed with APC Goat anti-mouse IgG2a Fc Antibody (Invitrogen) staining and RFP expression.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-Spike CAR</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>Mouse IgG2a</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">CAR expression of anti-CD19 CAR was determined with Human CD19 (20-291) Protein, Fc Tag, low endotoxin (Super affinity) (Acro) followed by a secondary staining with APC conjugated anti-human IgG Fc Antibody (Biolegend) and RFP expression.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-CD19 CAR</div><div>suggested: (Miltenyi Biotec Cat# 130-115-965, RRID:AB_2811310)</div></div><div style="margin-bottom:8px"><div>Human CD19 (20-291) Protein, Fc Tag, low endotoxin (Super affinity)</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For Spike protein flow cytometry analysis, the cells were stained with Biotinylated Human ACE2 / ACEH Protein, Fc,Avitag (Acro Biosystems), then stained with APC anti-human IgG Fc Antibody clone HP6017 (Biolegend).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Detection reagent was prepared using Human CD3 epsilon Protein, Mouse IgG2a Fc Tag (Acro) and Phycoerythrin-conjugated Goat anti-Mouse IgG2a Cross-Adsorbed Secondary Antibody (Fisher) for ACE2-Bite and APC anti-human IgG Fc Antibody clone HP6017 (Biolegend) for ACE2-Fc were added to the wells and incubated for another hour at room temperature.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Human CD3 epsilon Protein, Mouse IgG2a Fc Tag (Acro)</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">VSVG and Spike Protein pseudotyped lentivirus production: The lentiviruses pseudotyped with vesicular stomatitis virus G protein envelope were generated with HEK293T cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To measure viral titers of VSV-G pseudotyped lentiviruses, virus preparations were serially diluted on Jurkat cells and 3 days post-infection, infected cells were measured using flow cytometry and the number of cells transduced with 1 mL of virus supernatant was calculated as infectious units per milliliter.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Jurkat</div><div>suggested: KCB Cat# KCB 94018YJ, RRID:CVCL_0065)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To generate HEK-293T cells that transiently expressed wild type and mutated spike protein (ATCC; mycoplasma-free low passage stock), the cells were transfected with Spike protein expressing pLP plasmids using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s protocol and stained for their spike protein expression 72 hours after the transfection as described in Staining and Flow cytometry Analysis.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK-293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All engineered and wild-type HEK-293 and T2 cells were cultured in complete RPMI 1640 medium (RPMI 1640 supplemented with 10% FBS; Atlanta Biologicals, Lawrenceville, GA), 8% GlutaMAX (Life Technologies), 8% sodium pyruvate, 8% MEM vitamins, 8% MEM nonessential amino acid, and 1% penicillin/streptomycin (all from Corning Cellgro).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK-293</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>T2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To generate T2s and 293s with stable Spike overexpression, wild-type T2 and 293 cells were transduced with 3 MOI of Spike protein overexpressing VSVG lentivirus and proliferated.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293</div><div>suggested: NCI-DTP Cat# NCI-293TT, RRID:CVCL_1D85)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">CAR expressions of ACE2 CAR and anti-Spike CAR and ACE2 expression of ACE2-293 cells were determined with SARS-CoV-2 S1 protein, Mouse IgG2a Fc Tag (Acro Biosystems) incubation followed with APC Goat anti-mouse IgG2a Fc Antibody (Invitrogen) staining and RFP expression.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ACE2-293</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Spike protein constructs: Human codon optimized wild-type full-length SARS-CoV-2 Spike protein sequence was synthesized by MolecularCloud (MC_0101081) and then cloned into pLP/VSVG plasmid from Thermo Fisher under CMV promoter after removing the VSVG sequence via EcoRI-EcoRI restriction digestion. 5’-ACGACGGAATTCATGTTCGTCTTCCTGGTCCTG-3’ and 5’-ACGACGGAATTCTTAACAGCAGGAGCCACAGC-3’ primers were used to generate wild-type SARS-CoV-2 Spike protein sequence without the Endoplasmic Reticulum Retention Signal (ERRS, last 19 amino acids of Spike) (Ou et al., 2020).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pLP/VSVG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">E484K and N501Y mutated spike protein sequences without ERRS domain were obtained from VectorBuilder plasmids pRP[Exp]-CMV-human beta globin intron>S(E484K,deltaC19)/3xFLAG and pRP[Exp]-CMV-human beta globin intron>S(N501Y,deltaC19)/3xFLAG, respectively.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pRP[Exp]-CMV-human</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Since wild-type Spike protein did not have the FLAG tag and efficiently incorporated into the lentiviruses, FLAG Tags in each construct were removed to have the same amino acid sequences among all Spike constructs with the exception of the necessary mutations, via PCR amplification with 5’-ACGACGGAATTCATGTTCGTTTTCCTTGTTCTGTTGC-3’ and 5’-ACGACGGAATTCTTAGCAACATGATCCGCAAGAGCA-3’ primers and cloned into the same pLP expression plasmid.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pLP</div><div>suggested: RRID:Addgene_22310)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, the lentivector plasmids containing the constructs were co-transfected with vesicular stomatitis virus G protein, pLP1, and pLP2 plasmids into HEK293T cells at 80–90% confluency using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s protocol.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pLP1</div><div>suggested: RRID:Addgene_22614)</div></div><div style="margin-bottom:8px"><div>pLP2</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">ACE2 extracellular domain, CD8a signal peptide, CD8 hinge, CD8 transmembrane domain, 4-1BB intracellular domain and CD3ζ domain sequences were obtained from Ensembl Gene Browser and codon optimized with SnapGene by removing the restriction enzyme recognition sites that are necessary for subsequent molecular cloning steps, while preserving the amino acid sequences.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Ensembl Gene Browser</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Anti-CD19 and anti-Spike scFv amino acid sequences were obtained from Addgene plasmids #79125 and #155364, respectively, reverse translated to DNA sequences and codon optimized with Snapgene 5.2.4.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Snapgene</div><div>suggested: (SnapGene, RRID:SCR_015052)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Samples were acquired on a BD FACSymphony A5 analyzer and data were analyzed using FlowJo (BD Biosciences).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>FlowJo</div><div>suggested: (FlowJo, RRID:SCR_008520)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Geometric means of PE fluorescence in different titrations were used to generate the titration curve and the area under the curve was calculated using GraphPad Prism 9.0 software (GraphPad Software).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Statistical Analyses and Reproducibility: All statistical analyses were performed and graphs were prepared using GraphPad Prism V9 software.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

---

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: Please consider improving the rainbow (“jet”) colormap(s) used on pages 37, 38, 39, 41 and 42. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

---

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.

---

<footer>

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

</footer>

In other words, adding a svelte:error tag wouldn't help much.

annotation meta: may need new tag: client/server cooperation?

annotation meta: may need new tag: demonstrating....

Reminds me of that really long series of things on a contamination theory of obesity.

SciScore for 10.1101/2022.01.14.22268980: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">Consent: Whole blood samples: The 60 samples used for figures 6, S1, S2 and S3 were routine care residues from patients of the Toulouse hospital, where all patients give, by default, their consent for any biological material left over to be used for research purposes after all the clinical tests requested by doctors have been duly completed.<br>Field Sample Permit: Those were anonymized within 24 hours of collection, transferred from the hospital to the research lab, and kept at room temperature until being used for HAT assays within 24 hours (i.e. less than 48 hours after blood samples were collected).</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">Authentication: Ethical statement: RBCs from O-blood donors were obtained from the Toulouse branch of the Etablissement Français du Sang (EFS), with whom the project was validated under agreement n° 21PLER2020-025.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Polyclonal anti-human Igs secondary antibodies, all conjugated to Alexa-488, were from Jackson laboratories, and purchased from Ozyme (France)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human Igs secondary</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Anti RBD monoclonal antibodies: CR3022 (ter Meulen et al. 2006) and EY6A (Zhou et al. 2020) were obtained using antibody-expression plasmids, as previously described (Townsend et al. 2021).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti RBD</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>antibody-expression</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The activity of those aliquots of working stocks was then evaluated at regular intervals by performing titrations of the IH4-RBD reagent against either the CR3022 monoclonal antibody diluted to a final concentration of 100 ng/ml, or various immune sera diluted to give similar endpoint to those obtained with CR3022.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CR3022</div><div>suggested: (Imported from the IEDB Cat# CR3022, RRID:AB_2848080)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For titration experiments, we prepared stocks containing RBCs and the appropriate amount of the reagent to be kept constant (either the IH4-RBD reagent at 1 µg/ml or IH4 alone at 0.5 µg/ml for plasma titrations, or the appropriate antibody dilution for IH4-RBD titrations).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>IH4</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">One drop (i.e. ca. 30 µl) of either the pan-specific anti-Ig-GAM secondary fluorescent antibodies, as well as anti-IgG, -IgA or -IgM, all diluted 1/200 in PFN was added to each of the four wells for each sample, and the cells resuspended by gentle shaking of the plates.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-Ig-GAM</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">One drop (i.e. ca. 30 µl) of anti-human secondary antibody conjugated to alexa-488, diluted 1/200 in PFN was added to each of the wells, and the cells resuspended by gentle shaking of the plates.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human secondary</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>alexa-488</div><div>suggested: (Bioss Cat# bsm-4579M-Alexa488, RRID:AB_11071160)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Covid-19 sera 186, 197 and 203 were obtained from the virology department of the Toulouse hospital; plasmas 79 and 206 were from whole blood samples used in our previous paper describing the Jurkat-S&R-flow test (Maurel Ribes et al. 2021) IH4-RBD (Wuhan and Delta) and IH4 alone were produced by transient transfection of HEK-293T cells, and purified from the supernatant by HIS-tag affinity purification, as previously described (Townsend et al. 2021)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK-293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Jurkat-S&R-flow: Briefly, Jurkat-S and Jurkat-R cell lines, obtained and grown as previously described (Maurel Ribes et al. 2021), were resuspended in their own tissue culture medium at a concentration of 2.2 106 cells/ mL before pooling equal volumes of the two.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Jurkat-R</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The values used as results are those for specific staining, i.e. the difference between the GMFI (geometric mean fluorescent index) measured on the Jurkat cells expressing the SARS-CoV-2 spike protein and the control Jurkat cells expressing the mCherry fluorescent protein.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Jurkat</div><div>suggested: KCB Cat# KCB 94018YJ, RRID:CVCL_0065)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Post-acquisition analysis of all the samples was performed using the Flowjo software (version 10.7.1).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Flowjo</div><div>suggested: (FlowJo, RRID:SCR_008520)</div></div></td></tr></table>

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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: 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.

---

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

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About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

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JD

SciScore for 10.1101/2022.01.11.475922: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Western blot: Western blot was performed using an anti-SARS-COV-2 S antibody following a protocol described previously (56).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-SARS-COV-2 S</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Alkaline phosphatase conjugated anti-Rabbit IgG (1:5000) (Sigma-Aldrich, St. Louis, MO) was used as a secondary antibody.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-Rabbit IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To measure binding of a full-length S protein to monoclonal antibodies, the antibody was immobilized to anti-human IgG Fc Capture (AHC) biosensor (ForteBio, Fremont, CA) following a protocol recommended by the manufacturer.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Control sensors with no ACE2 or antibody were also dipped in the S protein solutions and the running buffer as references.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ACE2</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, Expi293F cells transfected with monomeric ACE2 or dimeric ACE2 expression construct and the supernatant of the cell culture was collected.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Expi293F</div><div>suggested: RRID:CVCL_D615)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, to produce S-expressing cells, HEK293T cells were transfected by polyethylenimine (PEI; 80 μg) with either 5 or 10 μg of the full-length SARS-CoV2 (G614</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To prepare for infection, 7.5×103 of HEK 293 cells, stably transfected with a full-length human ACE2 expression construct, in 15 μl culture medium were plated into a 384-well white-clear plate coated with poly-D-Lysine to enhance the cell attachment.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK 293</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pseudotyped virus particles were produced in 293T/17 cells (ATCC) by co-transfection of plasmids encoding codon-optimized SARS-CoV-2 full-length S constructs, packaging plasmid pCMV DR8.2, and luciferase reporter plasmid pHR’ CMV-Luc.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T/17</div><div>suggested: ATCC Cat# CRL-11268, RRID:CVCL_1926)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The 293T cell line stably overexpressing the human ACE2 cell surface receptor protein was kindly provided by Drs.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T</div><div>suggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The S gene was fused with a C-terminal twin Strep tag (SGGGSAWSHPQFEKGGGSGGGSGGSSAWSHPQFEK) and cloned into a mammalian cell expression vector pCMV-IRES-puro (Codex BioSolutions, Inc, Gaithersburg, MD).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMV-IRES-puro</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pseudotyped virus particles were produced in 293T/17 cells (ATCC) by co-transfection of plasmids encoding codon-optimized SARS-CoV-2 full-length S constructs, packaging plasmid pCMV DR8.2, and luciferase reporter plasmid pHR’ CMV-Luc.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMV DR8.2, and luciferase reporter</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pHR’</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Automated data collection was carried out using SerialEM version 3.8.6 (59) at a nominal magnification of 105,000× and the K3 detector in counting mode (calibrated pixel size, 0.83 Å) at an exposure rate of 13.362 electrons per pixel per second.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SerialEM</div><div>suggested: (SerialEM, RRID:SCR_017293)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Image processing and 3D reconstructions: Drift correction for cryo-EM images was performed using MotionCor2 (60), and contrast transfer function (CTF) was estimated by Gctf (61) using motion-corrected sums without dose-weighting.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MotionCor2</div><div>suggested: (MotionCor2, RRID:SCR_016499)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Density maps were corrected from the modulation transfer function of the K3 detector and sharpened by applying a temperature factor that was estimated using post-processing in RELION.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>RELION</div><div>suggested: (RELION, RRID:SCR_016274)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Several rounds of manual building were performed in Coot (64).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Coot</div><div>suggested: (Coot, RRID:SCR_014222)</div></div></td></tr></table>

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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.

---

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 32, 26, 28 and 30. 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.

---

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

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About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

</footer>

SciScore for 10.1101/2022.01.11.475901: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IRB: The use of HEK293 cells in this study was approved by the University of Waterloo Research Ethics Board (ORE#42425). 2.2 Plasmids, transfections, and treatments: Plasmids expressing ORF8 protein (YP_009724396.1) tagged at the C-terminus with 3 x DYKDDDK tag (Ex-NV229-M14), ORF10 protein (YP_009725255.1) tagged at the C-terminus with 3 x hemagglutinin tag (Ex-NV231-M07), and M protein (YP_009724393.1) tagged at the C-terminus with green fluorescent protein (Ex-NV225-M03) were from GeneCopoeia (Rockland, MD, U.S.A).<br>Field Sample Permit: All work was performed in accordance with a Health Canada approved Cannabis Tracking and Licencing System Research License held by the University of Waterloo (PI: Dr. Robin Duncan).</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, HEK293 cells were seeded (1 × 104 cells) in 96-well plates and transfected with the respective plasmids after 24 h, then treated a few hours after transfection with either CBD or vehicle for 24 h.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The control plasmid was pCMV-3Tag-3A (pCMV) (Agilent Technologies, Santa Clara, CA, U.S.A.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMV</div><div>suggested: RRID:Addgene_16459)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Live cells were maintained at 37° C while fluorescence was recorded at 469/525 nm for the detection of pSIVA and at 531/647 nm for the detection of PI.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pSIVA</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cells were grown in 24 well plates and transfected with either pCMV-3Tag-3A, or plasmids expressing ORF8, ORF10, or M protein, and then treated with either 2 μM CBD or vehicle overnight for 14 h, so that analyses were performed prior to measures of effects on cell number and apoptosis markers.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMV-3Tag-3A</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Analyses were performed using Prism GraphPad 9 software.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

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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).

---

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 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 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.
• No funding statement was detected.
• No protocol registration statement was detected.

---

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

---

<footer>

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

</footer>

牛了牛了终于实现pdf带tag批注自由

SciScore for 10.1101/2022.01.10.475532: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, the constructs were transiently transfected into HEK293F cells using polyethylenimine (PEI).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293F</div><div>suggested: RRID:CVCL_6642)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All MAbs were 4-fold serially diluted and tested by pseudovirus neutralization assay with human ACE2-overexpressing HEK 293T cells (293T-Hace2) following our previous protocol44.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK 293T</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">) S glycoprotein ectodomain (residues M1-Q1208) with proline substitutions at K986 and V987, a “GSAS” substitution at the furin cleavage site (R682–R685) was cloned into vector pcDNA 3.1+.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA 3.1+</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A gene encoding human ACE2 PD domain (Q18-D615) with an N-terminal interleukin-10 (IL-10) signal peptide and a C-terminal His tag was cloned into vector pcDNA 3.436.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA 3.436</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, HEK 293T cells in 10-cm dish were co-transfected using PEI (polysciences) with 10 μg of Pcmv-Dr8.91 packaging plasmid, 10 μg of recombinant Plvx-IRES-ZsGreen1 plasmid containing luciferase reporter gene, and 2 μg of recombinant Pvax1 plasmids encoding SARS-CoV-2 S proteins.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Plvx-IRES-ZsGreen1</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Then, the S trimer proteins were biotinylated using the EZ-Link™ Sulfo-NHS-LC-LC-Biotin kit (Thermo Fisher) and then purified by Zeba™ spin desalting columns (Thermo Fisher)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Zeba™</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data were analyzed by non-linear regression using GraphPad Prism 8 to calculate half inhibitory concentration (IC50).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cryo-EM 3D reconstruction: For each dataset, the motion correction of image stack was performed using the embedded module of Motioncor2 in Relion 3.136,52,53 and CTF parameters were determined using CTFFIND454 before further data processing.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Motioncor2</div><div>suggested: (MotionCor2, RRID:SCR_016499)</div></div><div style="margin-bottom:8px"><div>Relion</div><div>suggested: (RELION, RRID:SCR_016274)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In addition, after obtaining the 3.5-Å-resolution map, we performed focused 3D classification on the S3H3-SD1 region of protomer 2 (highlighted by dotted orange ellipsoid), leading to a dataset of 101,192 particles, which was further local refined on the S3H3-SD1 region in cryoSPARC, deducing a 3.61-Å-resolution map of this region.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>cryoSPARC</div><div>suggested: (cryoSPARC, RRID:SCR_016501)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The atomic models were validated using Phenix.molprobity command in Phenix.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Phenix</div><div>suggested: (Phenix, RRID:SCR_014224)</div></div><div style="margin-bottom:8px"><div>molprobity</div><div>suggested: (MolProbity, RRID:SCR_014226)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Interaction interface analyses were conducted through PISA server58.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PISA</div><div>suggested: (PISA, RRID:SCR_015749)</div></div></td></tr></table>

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Results from OddPub: Thank you for sharing your data.

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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 33, 34 and 31. 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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Results from scite Reference Check: We found no unreliable references.

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Author Response

Reviewer #1 (Public Review):

This manuscript addresses a major issue facing consumers of structure-organism pair data: the landscape of databases is very difficult to navigate due to the way data is made available (many resources do not have structured data dumps) and the way data is standardized (many resources' structured data dumps do not standardize their nomenclature or use stable entity identifiers). The solution presented is a carefully constructed pipeline (see Figure 1) for importing data, harmonizing/cleaning it, automating decisions about exclusions, and reducing redundancy. The results are disseminated through Wikidata to enable downstream consumption via SPARQL and other standard access methods as well as through a bespoke website constructed to address the needs of the natural products community. The supplemental section of the manuscript provides a library of excellent example queries for potential users. The authors suggest that users may be motivated to make improvements through manual curations on Wikidata, through semi-automated and automated interaction with Wikidata mediated by bots, or by addition of importer modules to the LOTUS codebase itself.

Despite the potential impact of the paper and excellent summary of the current landscape of related tools, it suffers from a few omissions and tangents:

1. It does not cite specific examples of downstream usages of structure-organism pairs, such as an illustration on how this information in both higher quantity and quality is useful for drug discovery, agriculture, artificial intelligence, etc. These would provide a much more satisfying bookend to both the introduction and conclusion.

Thank you for this remark. We deliberately decided not to insist too heavily on the application examples of the LOTUS outputs. Indeed we are somehow biased by our main investigation field, natural products chemistry, and expect that the dissemination of specialized metabolites occurrences will benefit a wide range of scientific disciplines (ecology, drug discovery, chemical ecology, ethnopharmacology, etc.)

However, Figure 5 was established to illustrate how the information available through LOTUS is quantitatively (size) and qualitatively (color classes) superior to what is available through single natural products resources.

As added in the introduction, one of the downstream usages of those pairs is for example to perform taxonomically informed scoring as described in https://doi.org/10.3389/fpls.2019.01329. Obtaining an open database of natural products’ occurrences to fuel such taxonomically informed metabolite annotation tools was the initial impulse for us to build LOTUS. These metabolite annotation strategies, tailored for specialized metabolites, have been shown to offer appreciable performance improvements for current state-of-the-art computational metabolite annotation tools. Since metabolite annotation is still regularly cited as “the major bottleneck” in metabolomics in the scientific literature over the last 15 years (https://europepmc.org/article/med/15663322, https://doi.org/10.1021/acs.analchem.1c00238), any tangible improvement in this field is welcome. With LOTUS we offer a reliable and reusable structures-organisms data source that can be exploited by the community to tackle such issues of importance.

Other possible usages are suggested in the conclusion, but benchmarking or even exemplifying such uses is clearly out of the scope of this paper, each one of them being an article per se.

The additional queries are written in our first answer (see “essential revisions”) and demonstrate the impact of LOTUS on accelerating the initial bibliographic survey of chemical structures occurrences over the tree of life.

This query (https://w.wiki/4VGC) can be compared to a literature review work, such as https://doi.org/10.1016/j.micres.2021.126708. In seconds, it allows retrieving a table listing compounds reported in given taxa and limits the search by years.

1. The mentions of recently popular buzzwords FAIR and TRUST should be better qualified and be positioned as a motivation for the work, rather than a box to be checked in the modern publishing climate.

It is true that the modern publishing system certainly suffers from some drawbacks (also critically mentioned within the paper). However, after consultation of all authors, we believe that because LOTUS checks both boxes of FAIR and TRUST, we would rather stick to these two terms. In our view, rules 1 (Don’t reinvent the wheel) and 5 (put yourself in your user’s shoes) of https://doi.org/10.1371/journal.pcbi.1005128 apply here. Both terms are indeed commonly (mis-)used but we felt that redefining other complicated terms would not help the reader/user.

1. The current database landscape really is bad; and the authors should feel emboldened to emphasize this in order to accentuate the value of the work, with more specific examples on some of the unmaintained databases

We perfectly agree with this statement and it is the central motivation of the LOTUS initiative to improve this landscape. It was a deliberate choice not to emphasize how bad the actual landscape is, but rather to focus on better habits for the future. We do not want to start devaluing other resources and elevate our initiative at the cost of others. We also believe that an attentive look at the complexity of the LOTUS gathering, harmonization, and curation speaks for itself and describes the huge efforts required to access properly formatted natural products occurrence data.

If the reviewer and editors insist, although not in our scope, we are happy to list a series of specific (but anonymized) examples of badly formatted entries, of wrong structures-organisms associations, or poorly accessible resources.

1. While the introduction and supplemental tables provide a thorough review of the existing databases, it eschews an important more general discussion about data stewardship and maintenance. Many databases in this list have been abandoned immediately following publication, have been discontinued after a single or limited number of updates, or have been decommissioned/taken down. This happens for a variety of reasons, from the maintainer leaving the original institution, from funding ending, from original plans to just publish then move on, etc. The authors should reflect on this and give more context for why this domain is in this situation, and if it is different from others.

We do agree with the reviewer and added a “status” column in the table https://github.com/lotusnprod/lotus-processor/blob/main/docs/dataset.csv We chose 4 possible statuses:

• Maintained (self-explanatory)
• Unmaintained: the database did not see any update in the last year.
• Retired: the authors stated they will not maintain the database anymore.
• Defunct: the database is not accessible anymore

As for question 3 above, we decided not to focus too heavily on the negative points and resume the current situation in the previous table. Reasons for the databases publishing being in this situation are multiple, and we think they are well summarized in https://doi.org/10.1371/journal.pcbi.1005128 (Rule 10: Maintain, update, or retire), already cited in the manuscript introduction.

1. Related to data stewardship: the LOTUS Initiative has ingested several databases that are no longer maintained as well as several databases with either no license or a more restrictive license than the CC0 under which LOTUS and Wikidata are distributed. These facts are misrepresented in Supplementary Table 1 (Data Sources List), which links to notes in one of the version controlled LOTUS repositories that actually describes the license. For example, https://gitlab.com/lotus7/lotus-processor/-/blob/8b60015210ea476350b36a6e734ad6b66f2948bc/docs/licenses/biofacquim.md states that the dataset has no license information. First, the links should be written with exactly what the licenses are, if available, and explicitly state if no license is available. There should be a meaningful and transparent reflection in the manuscript on whether this is legally and/or scientifically okay to do - especially given the light that many of these resources are obviously abandoned.

This point is a very important one. We did our best to be as transparent as possible in our initial table. Following the reviewer’s suggestion, we updated it to better reflect the licensing status of each resource (https://github.com/lotusnprod/lotus-processor/blob/main/docs/dataset.csv). Therefore, we removed the generic “license” header, which could indeed be misleading, and replaced it with ”licensing status”, filled with the attributed license type and hyperlink to its content). It remains challenging since some resources changed their copyright in the meantime. We remain at the editor and reviewers’ disposal for any further improvement.

Moreover, as stated in the manuscript, we took care of collecting all licenses and contacted authors of resources whose license was not perfectly explicit to us, therefore accomplishing our due diligence. Additionally, we contacted legal offices in our University and explained our situation. We did everything that we had been advised.

1) To the best of our knowledge, the dissemination of the LOTUS initiative data falls under the Right to quote for scientific articles, as we do not share the whole information, but only a very small part.

2) We do not redistribute original content. What comes out of LOTUS has undergone several curation and validation steps, adding value to the original data. The 500 random test entries, provided in their original form for the sake of reproducibility and testing, are the only exception.

Many scientific authors forget about the importance of proper licensing. While it might be deliberate to restrict the use, inappropriate license choice (or omission) is too often due to a lack of information on its implication.

All authors of the utilized resources can freely benefit from our curation. We are sharing with the community the results of our work, while always citing the original reference.

Concerning the possible evolution of licensing, it remains a real challenge. While we tried to “freeze” the license status when we accessed the data, some resources updated their licensing since then. This can be tracked in the git history of the table (https://github.com/lotusnprod/lotus-processor/blob/main/docs/dataset.csv). Discrepancies between our frozen licensing (at the time of gathering) and actual license can therefore occur. Initiatives such as https://archive.org/web could help solving this issue, coming with other legal challenges.

1. The order of sections of the manuscript results in several duplicated, but not further substantiated explanations. Most importantly, the methods should be much more specific throughout and the results/discussion should more heavily cross-link to it, as a reader who examines the paper from top to bottom will be left with large holes of misunderstanding throughout.

As our paper focuses a lot on the methods, the barrier between results & methods becomes thinner. We took into account the reviewers’ suggestions and added some additional cross-links for the reader to be able to quickly access related methods.

1. The work presented was done in a variety of programming languages across a variety of repositories (and even version control systems), making it difficult to give a proper code review. It could be argued that the most popular language in computational science at the moment is Python, with languages like R, Bash, and in some domains, still, Java maintaining relevance. The usage of more esoteric languages (again, with respect to the domain) such as Kotlin hampers the ability for others to deeply understand the work presented. Further, as the authors suggest additional importers may implemented in the future, this restricts what external authors may be able to contribute.

Scientific software has indeed always been written in multiple languages. To this day, scientists have used all kinds of languages adapted both to their needs and their knowledge. Numpy uses Fortran libraries and many projects published in biology and chemistry recently are in Java, R, Python, C#, PHP, Groovy, Scala… We understand that some authors are more comfortable with one language or another. But R syntax is for example much more distant from Python's syntax than Kotlin can be. We needed a highly performant language for some parts of the pipeline and R, Bash, or Python were not sufficient. We decided to use Kotlin as it provides an easier syntax than Java while staying 100% compatible with it.

The advantage of the way LOTUS is designed is that importers are language-agnostic. As long as the program can produce a file or write to the DB in the accepted format, it can be integrated into the pipeline. This was our goal from the beginning, to have a pipeline that can have its various parts replaced without breaking any of the processes.

1. As a follow up to the woes of point 4., 5., and 7., the manuscript fails to reflect on the longevity of the LOTUS Initiative. Like many, will the project effectively end upon publication? If not, what institutions will be maintaining it for how long, how actively, and with what funding source? If these things are not clear, it only seems fair to inform the reader and potential user.

LOTUS is an initiative that aims to improve knowledge management and sharing in natural products research. Our first project, which is the object of the current manuscript, is to provide a free and open resource of natural products occurrences for the scientific community. Its purpose is not to be a database by itself, but instead to provide through Wikidata and associated tools a way to access natural products knowledge. The objective was not to create yet another database (https://doi.org/10.1371/journal.pcbi.1005128), but instead to remove this need and give our community the tools and the power to act on its knowledge. This way, as everything is on Wikidata, the initiative is not “like many”. This also means that this project should not be considered and evaluated exactly like a classical DB. Once the initial curation, harmonization, and dissemination jobs have been done, they should ideally not be run again. The community should switch to Wikidata as a point of access, curation, and addition of data. If viewed with such arguments in mind, yes, LOTUS can live long!

Wikimedia is a public not-for-profit organization, whose financial development appears to indicate solid health https://en.wikipedia.org/wiki/Wikimedia_Foundation#Finances.

In terms of funding sources, we would like to refer to https://elifesciences.org/articles/52614#sa2 , which stated the following in response to a similar question: "Wikidata is sustained by funding streams that are different from the vast majority of biomedical resources (which are mostly funded by the NIH). Insulation from the 4-5 year funding cycles that are typical of NIH-funded biomedical resources does make Wikidata quite unique." The core of the Wikidata funding streams are donations to the Wikipedia ecosystem. These donations - with a contributor base of millions of donors from almost any country in the world, chipping in at an average order of magnitude of around 10 dollars - are likely to continue as long as that ecosystem is useful to the community of its users. See <https://wikimediafoundation.org/about/financial-reports for details>.

1. Overall, there were many opportunities for introspection on the shortcomings of the work (e.g., the stringent validation pipeline could use improvement). Because this work is already quite impactful, I don't think the authors will be opening themselves to unfair criticism by including more thoughtful introspection, at minimum, in the conclusions section.

We agree with the reviewer and therefore, list again the major limitations of our processing pipeline:

First, our processing pipeline is heavy. It includes many dependencies and requires a lot of time for understanding. We are aware of this issue and tried to simplify it as much as possible while keeping what we considered necessary to ensure high data quality. Second, it can sometimes induce errors. Those errors, ranging from unnecessary discarded correct entries to more problematic ones can be attributed to various parameters, reflecting the variety of our input. We will therefore try listing them, keeping in mind that the list won’t be exhaustive. For each detected issue, we tried fixing it at best, knowing it will not lead to an ideal result, but hopefully increase data quality gradually.

● Compounds

○ Sanitization (the three steps below are performed automatically since we observed a higher ratio of incorrect salts, charged or dimerized compounds. However, this also means that true salts, charged or dimeric compounds were erroneously “sanitized”.)

■ Salt removals

■ Charged molecules

■ Dimers

○ Translation (both processes below are pretty error-prone)

■ Name to structure

■ Structure to name

● Biological organisms

○ Synonymy

■ Lotus (https://www.wikidata.org/wiki/Q3645698, https://www.wikidata.org/wiki/Q16528).

This is also one of the reasons why we decided to call the resource Lotus, as it illustrates part of the problem.

■ Iris (https://www.wikidata.org/wiki/Q156901, https://www.wikidata.org/wiki/Q2260419)

■ Ficus variegata (https://www.wikidata.org/wiki/Q502030, https://www.wikidata.org/wiki/Q5446649)

○ External and internal dictionaries are not exhaustive, impacting translation

○ Some botanical names we use might not be the accepted ones anymore because of the tools we use and the pace taxonomy is renaming taxa.

● References

○ The tool we favored, Crossref, returns a hit whatever the input. This generates noise and incorrect translations, which is why our filtering rules focus on reference types.

● Filtering rules:

○ Limited validation set, requires manual validation

○ Validates some incorrect entries (False positives)

○ Does not validate some correct entries (False negatives)

Again, our processing pipeline removes entries we do not yet know how to process properly.

Our restrictive filters but substantial contribution to Wikidata in terms of structure-organisms pairs data upload should hopefully incentivize the community to contribute by further adding its human validated data.

We updated the conclusion part of the manuscript accordingly. See https://github.com/lotusnprod/lotus-manuscript/commit/a866a01bad10dfd8b3af90e2f30bb3ae51dd7b9e.

Reviewer #2 (Public Review):

Rutz et al. introduce a new open-source database that links natural products structures with the organisms they are present in (structure-organism pairs). LOTUS contains over 700,000 referenced structure-organism pairs, and their web portal (https://lotus.naturalproducts.net/) provides a powerful platform for mining literature for published data on structure-organism pairs. Lotus is built within the computer-readable Wikidata framework, which allows researchers to easily contribute, edit and reuse data within a clear and open CC0 license. In addition to depositing the database into Wikidata, the authors provide many domain-specific resources, including structure-based database searches and taxon-oriented searches.

Strengths:

The Lotus database presented in this study represents a cutting-edge resource that has a lot of potentials to benefit the scientific community. Lotus contains more data than previous databases, combines multiple resources into a single resource.

Moreover, they provide many useful tools for mining the data and visualizing it. The authors were thoughtful in thinking about the ways that researchers could/would use this resource and generating tools to make it ways to use. For example, their inclusion of structure-based searches and multiple taxonomy classification schemes is very useful.

Overall the authors seem conscientious in designing a resource that is updatable and that can grow as more data become available.

Weaknesses/Questions:

1) Overall, I would like to know to what degree LOTUS represents a comprehensive database. LOTUS is clearly, the best database to date, but has it reached a point where it is truly comprehensive, and can thus be used for a metanalysis or as a data source for research questions. Can it truly replace doing a manual literature search/review?

As highlighted by the reviewer, even if LOTUS might be the most comprehensive natural products occurrences ressources at the moment, TRUE or FULL comprehensive quality of such resource will always be limited to the available data in the litterature. And the community is far from fully describing the metabolome of living beings. We however hope that the LOTUS infrastructure will offer a good place to start this ambitious and systematic description process.

1) Yes it can serve as data source for research questions, as exemplified in the query table

2) No, it cannot and must not replace manual literature search. Manual literature search is the best but at an enormous cost. If the outcome of such search can be made available to the whole community (eg. via Wikidata), the value of such would be even bigger. However, LOTUS can expedite a decent part of a manual litterature search and liberate time to complement this search. See our comment to the editors “To further showcase the possibilities opened by LOTUS, and also answer the remark on the comprehensiveness of our resource, we established an additional query (https://w.wiki/4VGC).This query is comparable to a literature review work, such as: https://doi.org/10.1016/j.micres.2021.126708. In seconds, it allows retrieving a table listing compounds reported in given taxa and limits the search by years.”

We added these examples in the manuscript (see https://github.com/lotusnprod/lotus-manuscript/commit/a6ee135b83e56e8e2041d09d7ce2d5b913c1029d)

2) Data Cleaning & Validation. The manuscript could be improved by adding more details about how and why data were excluding or included in the final upload. Why did only 30% of the initial 2.5 million get uploaded? Was it mostly due to redundant data or does the data mining approach result in lots of missed data?

The reason for this “low” yield is that we highly favored quality over quantity (as in the F-score equation, ß being equal to 0.5, so more importance is given to the precision than the recall). Of course there is redundancy, but the rejected entries are mostly because of too low confidence level according to our developed rules. It is not fully discarded data as we keep it for further curation (ideally including the community) before uploading to Wikidata. We adapted the text accordingly.

3) Similarly, more information about the accuracy of the data mining is needed. The authors report that the test dataset (420 referenced structure-organisms pairs) resulted in 97% true positives, what about false negatives? Also, how do we know that 420 references are sufficiently large to build a model for 2.5M datapoints? Is the training data set is sufficiently large to accurately capture the complexities of such a large dataset?

False negatives are 3%, which is, in our opinion, a fair amount of “loss” given the quality of the data. We actually manually checked 500+ documented pairs, which is more or less the equivalent of a literature review. We were careful in sampling the entries in the right proportions, but we cannot (and did not) state they are enough. We cannot model it either, since the 2.5M+ points have absolutely different distributions, in terms of databases, quality, etc. Only “hint” is the similar behaviour among all subsets. (the 420 + 100 entries) were divided between 3 authors, which obtained similar results.

4) Data Addition and Evolution: The authors have outlined several mechanisms for how the LOTUS database will evolve in the future. I would like to know if/how their scripts for data mining will be maintained if they will continue to acquire new data for the database. To what extent does the future of LOTUS depend on the larger natural products community being aware of the resource and voluntarily uploading to it? Are there mechanisms in place such as those associated with sequencing data and NCBI?

Programs have been not only maintained but also updated with new possibilities (as, for example: the addition of a “manual mode” allowing user to run the LOTUS processing pipeline on a set of their own entries and make them Wikidata-ready (https://github.com/lotusnprod/lotus-processor/commit/f49e4e2b3814766d5497f9380bfe141692f13f23). We will of course do our best to keep on maintaining it, but as no one in academia can state he/she will maintain programs forever. However the LOTUS initiative hopefully embraces a new way of considering database dynamics. If the repository and website of the LOTUS initiative shut down tomorrow, all the work done will still be available to anyone on Wikidata. Of course, future data addition strongly relies on community involvement. We have already started to advocate for the community to start taking part of it, in the form of direct upload to Wikidata, ideally. At the time, there are no mechanisms in place to push publishing of the pairs on Wikidata (as for sequencing, mass spec data), but we will be engaged in pushing forward this direction. The initiative needs stronger involvement of the publishing sector (also reviewers) to help change those habits.

5) Quality of chemical structure accuracy in the database. I would imagine that one of the largest sources of error in the LOTUS database would be due to variation in the quality of chemical structures available. Are all structure-organism pairs based on fully resolved NMR-based structures are they based on mass spectral data with no confirmational information? At what point is a structural annotation accurate enough to be included in the database. More and more metabolomics studies are coming out and many of these contain compound annotations that could be included in the database, but what level (in silico, exact mass database search, or relative to a known standard) are required.

This is a very interesting point and some databases have this “tag” (NMR, cristal, etc.). We basically rely on original published articles, included in specialized databases. If poorly reported structures have been accepted for publication, labelled as “identified” (and not “annotated”) and the authors publishing the specialized databases overlooked it, we might end up with such structures.

Here, the Evidence Ontology (http://obofoundry.org/ontology/eco.html) might be a good direction to look at and further characterize the occurrences links in the LOTUS dataset.

Reviewer #3 (Public Review):

Due to missing or incomplete documentation of the LOTUS processes and software, a full review could not be completed.

Some parts of LOTUS were indeed not sufficiently described and we improved both our documentation and accessibility to external users a lot. We thank the reviewer for insisting on this point as it will surely improve the adoption of our tool by the community.

• My tagnote structure for my tag doesnt work on repeated trying 1248hrs

This seems inconsistent (since we do specify a folder for Drafts), but I confirmed that it still gets stored in Sent even after unchecking this. I guess because Gmail adds the Sent tag on the server, whereas with a draft, it is initiated client-side so the client has to be responsible for adding that tag.

.imp inline tag和双链在我的认知里面 第一次完成了联合

SciScore for 10.1101/2022.01.05.475037: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">Consent: Written informed consent was obtained from all study subjects.<br>IRB: This study was approved by the Institutional Review Board of The University of Hong Kong/Hospital Authority Hong Kong West Cluster (Ref No. UW 21-120 452).</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">Briefly, 6-10-week-old male and female hamsters were obtained from the Chinese University of Hong Kong Laboratory Animal Service Centre through the HKU Centre for Comparative Medicine Research.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">The hamsters were randomized from different litters into experimental groups.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Serially diluted plasma from healthy individuals or previously published monoclonal antibodies against SARS-CoV-2 (B8) were used as negative controls.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SARS-CoV-2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Two consecutive staining steps were conducted: the first one used an antibody and spike cocktail incubation of 30 min at 4 °C; the second staining involved staining with anti-His-APC and anti-His-FITC antibodies (Abcam) at 4 °C for 30 min to detect the His tag of the RBD.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-His-APC</div><div>suggested: (Miltenyi Biotec Cat# 130-101-322, RRID:AB_2800415)</div></div><div style="margin-bottom:8px"><div>anti-His-FITC</div><div>suggested: (Miltenyi Biotec Cat# 130-092-675, RRID:AB_1103226)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cells were further permeabilized with 0.2% Triton X-100 and incubated with cross-reactive rabbit sera anti-SARS-CoV-2-N for 1 hour at RT before adding an Alexa Fluor 488 goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody (Life Technologies).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-SARS-CoV-2-N</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-rabbit IgG</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cell lines: HEK293T cells, HEK293T-hACE2 cells and Vero-E6-TMPRSS2 cells were maintained in DMEM containing 10% FBS, 2 mM L-glutamine, 100 U/mL/mL penicillin and incubated at 37 □in a 5% CO2 setting 51</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>HEK293T-hACE2</div><div>suggested: RRID:CVCL_A7UK)</div></div><div style="margin-bottom:8px"><div>Vero-E6-TMPRSS2</div><div>suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, The pseudovirus was generated by co-transfection of 293T cells with pVax-1-S-COVID19 and pNL4-3Luc_Env_Vpr, carrying the optimized spike (S) gene (QHR63250) and a human immunodeficiency virus type 1 backbone, respectively 54</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The antibody-virus mixtures were subsequently added to pre-seeded HEK 293T-ACE2 cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK 293T-ACE2</div><div>suggested: RRID:CVCL_A7UK)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Mixtures were then transferred to 96-well plates pre-seeded with 1×104/well Vero E6 cells and incubated at 37°C for 24 hours.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero E6</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, The pseudovirus was generated by co-transfection of 293T cells with pVax-1-S-COVID19 and pNL4-3Luc_Env_Vpr, carrying the optimized spike (S) gene (QHR63250) and a human immunodeficiency virus type 1 backbone, respectively 54</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pVax-1-S-COVID19</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pNL4-3Luc_Env_Vpr</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">https://www.ncbi.nlm.nih.gov/igblast/).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>https://www.ncbi.nlm.nih.gov/igblast/</div><div>suggested: (IgBLAST, RRID:SCR_002873)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Sequences were aligned using Clustal W in the BioEdit sequence analysis package (Version 7.2)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BioEdit</div><div>suggested: (BioEdit, RRID:SCR_007361)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Half-maximal (IC50) or 90% (IC90) inhibitory concentrations of the evaluated antibody were determined by inhibitor vs. normalized response --4 Variable slope using GraphPad Prism 8 or later (GraphPad Software Inc.)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The fluorescence density of SARS-CoV-2 infected cells were scanned using a Sapphire Biomolecular Imager (Azure Biosystems) and the neutralization effects were then quantified using Fiji software (NIH)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Fiji</div><div>suggested: (Fiji, RRID:SCR_002285)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The structure alignment, cartoon representations, labeling of amino acids in RBD (from PDB 7K45) were generated by PyMOL.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PyMOL</div><div>suggested: (PyMOL, RRID:SCR_000305)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Quantification and statistical analysis: Statistical analysis was performed using PRISM 8.0 or later.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PRISM</div><div>suggested: (PRISM, RRID:SCR_005375)</div></div></td></tr></table>

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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: We detected the following sentences addressing limitations in the study:

Limitations of the study: ZCB11 probably represents the broadest breadth among bNAbs reported thus far with comparable potency against all current SARS-CoV-2 VOCs including Omicron and OmicronR346K. We are still in the process in determining its mode of action by solving structures of the RBD-ZCB11 Fab complex. Such information will be useful to guide vaccine design as mentioned because the frequency of elite vaccine remains low (2/34 in this study). To understand the frequency of ZCB11-like bNAb among BNT162b2-vaccinees, we need to investigate other elite responders who show equally potent bNAb responses. More ZCB11-like bNAbs should be also discovered to improve current antibody-based cocktail immunotherapy. For animal challenge experiments, we have done a single dose efficacy experiment. Different doses and routes of administration or antibody combination will be tested in future experiments to provide useful information to support clinical development of ZCB11 and ZCB11-like bNAb.

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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: 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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About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

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SciScore for 10.1101/2022.01.04.474979: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">Contamination: Cells were frequently tested for mycoplasma contamination and consistently tested negative.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After 15 additional minutes, cells were incubated in blocking buffer (permeabilization buffer supplemented with 1 % BSA), and further incubated in blocking buffer containing anti-Strep mouse antibody (1:1000 dilution), and anti-Sirt5 rabbit antibody (1:1000 dilution).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-Strep</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-Sirt5</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The next day, the cells were washed with PBS three times and incubated in the blocking buffer containing anti-mouse IgG donkey antibody conjugated with Alexa 488 (1:500 dilution, Thermo Fisher)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-mouse IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">anti-rabbit IgG donkey antibody conjugated with Alexa 555 (1:500 dilution, Thermo Fisher), and for counter-staining, DAPI (1 μg/ml</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-rabbit IgG donkey antibody</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-rabbit IgG</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A549 cells stably expressing ACE2 (A549-ACE2) were a gift from O. Schwartz (Pasteur Institute, Paris).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>A549</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Early passage HEK293T cells were transfected with 1.5 μg of dCas9-KRAB-MeCP2 repressor plasmid (Addgene #110824) and 0.5 μg of Piggyback Transposase (gift from Maxim Greenberg), using PEI 25K transfection reagent (Polysciences Inc, Cat. 23966-1), according to the manufacturer’s instructions.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">We combined 10 pmol of Streptococcus pyogenes NLS-Sp.Cas9-NLS (SpCas9) nuclease (Aldevron, USA, Cat. 9212) with 30 pmol of total synthetic sgRNA (10 pmol each sgRNA) to form ribonucleoproteins (RNPs) in 20 μL of total volume with SE Buffer for A549-ACE2 cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>A549-ACE2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Transfection, Strep affinity purification, and Flag immunoprecipitation in HEK-293T cells: HEK-293T cells were plated in six-well plates or 10-cm dishes.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK-293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Sample Preparation for Proteomic Analysis: HEK-293T Sirt5-KD cells were transfected with plasmids expressing Nsp14-strep in the presence or absence of SIRT5 with 3 biological replicates for each condition.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Sirt5-KD</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">SARS-CoV-2 stocks were propagated in Vero-E6 cells, and their sequences were verified by next-generation sequencing.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero-E6</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For infection experiments, A549-ACE2 or Calu3 cells were seeded into 12- or 24-well plates and rested for at least 24 hours prior to infection.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Calu3</div><div>suggested: RRID:CVCL_EQ19)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Infections of HCT-8 cells with HCoV-OC43 were performed similarly.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HCT-8</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Plasmids expressing GFP and Nsp14 proteins (from SARS-CoV-2, SARS-CoV and HCoV-OC43) with a C-terminus strep tag were a gift from Nevan Krogan (1, 2), and are also available on Addgene (pLVX-EF1alpha-SARS-CoV-2-nsp14-2xStrep-IRES-Puro, Addgene #141380).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pLVX-EF1alpha-SARS-CoV-2-nsp14-2xStrep-IRES-Puro</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Mammalian expression plasmids for SIRT5 and SIRT5-H158Y with a myc-his tag in a pCDNA 3.1 vector were available in the Verdin lab (26).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCDNA 3.1</div><div>suggested: RRID:Addgene_20407)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Early passage HEK293T cells were transfected with 1.5 μg of dCas9-KRAB-MeCP2 repressor plasmid (Addgene #110824) and 0.5 μg of Piggyback Transposase (gift from Maxim Greenberg), using PEI 25K transfection reagent (Polysciences Inc, Cat. 23966-1), according to the manufacturer’s instructions.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>dCas9-KRAB-MeCP2</div><div>suggested: RRID:Addgene_110821)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Nsp14 is cytotoxic, and we used 0.5 μg of Nsp14-strep plasmid for a six-well plate and 4 μg for a 10-cm dish.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Nsp14-strep</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Other co-transfecting plasmids, such as pcDNA-SIRT5, were used at the same concentration except when specifically mentioned.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA-SIRT5</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Protein purification and enzymatic assays: Nsp10 and Nsp14 proteins from the Wuhan strain of SARS-CoV-2 (NC_045512.2) were codon-optimized, ordered as Gblocks (IDT), and cloned into a pVFT1S expression vector using a HiFi DNA Assembly kit (NEB)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pVFT1S</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Methyltransferase assays were performed in reaction buffer (50 mM Hepes, pH 7.0, 6 mM KCl, 2 mM DTT, 1 mM MgCl2, and 0.1 mg/ml BSA) in presence of 0.1 mM NAD+ and 10 μM SAM.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SAM</div><div>suggested: (SAM, RRID:SCR_010951)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Analyses were run using GraphPad Prism version 9.1.2 for macOS (GraphPad Software, USA, www.graphpad.com).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A genome index was constructed using GRCh38 genome build with Gencode v38 annotation of the transcriptome, and Genbank MT246667.1 for the sequence of SARS-CoV-2, USA/WA-1/2020 isolate.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Gencode</div><div>suggested: (GENCODE, RRID:SCR_014966)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Differential gene expression analysis was done with DEseq2, which was also used to generate normalized gene counts (71).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>DEseq2</div><div>suggested: (DESeq2, RRID:SCR_015687)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Over-representation of biological gene sets in the gene clusters was investigated using the R clusterProfiler package and enricher function (72).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>clusterProfiler</div><div>suggested: (clusterProfiler, RRID:SCR_016884)</div></div></td></tr></table>

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Results from OddPub: Thank you for sharing your code and data.

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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: 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.

---

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

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About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

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Kaffee

SciScore for 10.1101/2022.01.03.22268599: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">Consent: NSCLC patient cohort: Peripheral blood samples from NSCLC patients were collected at Winship Cancer Institute following written informed consent approved by the Institutional Review Board at Emory University.<br>IRB: NSCLC patient cohort: Peripheral blood samples from NSCLC patients were collected at Winship Cancer Institute following written informed consent approved by the Institutional Review Board at Emory University.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">MesoScale Discovery Assay: V-PLEX COVID-19 Respiratory Panel 2 Kit (K15372U panel 2) were used to measure the IgG, IgM and IgA antibody against the antigens SARS-CoV-2 Spike, receptod binding domain (RBD),</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>antigens SARS-CoV-2 Spike , receptod binding domain ( RBD) ,</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Following this, 50 uL per well of 1X MSD SULFO-TAG™ Anti-Human IgG Antibody was added and incubated for one hour at room temperature, shaking at a speed of 700 rpm.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-Human IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cells were incubated with either an anti-SARS-CoV spike primary antibody directly conjugated to Alexaflour-647 (CR3022-AF647) or an anti-SARS-CoV spike primary antibody directly conjugated to biotin (CR3022-biotin) for at least 4 hours at room temperature.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CR3022-AF647</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-SARS-CoV spike</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>CR3022-biotin</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Viruses and cells: VeroE6 cells were obtained from ATCC (clone E6, ATCC, #CRL-1586) and cultured in complete DMEM medium consisting of 1x DMEM (VWR, #45000-304), 10% FBS, 25mM HEPES Buffer (Corning Cellgro), 2mM L-glutamine, 1mM sodium pyruvate, 1x Non-essential Amino Acids, and 1x antibiotics.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>VeroE6</div><div>suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">VeroE6-TMPRSS2 cells were generated and cultured as previously described18. nCoV/USA_WA1/2020 (WA/1), closely resembling the original Wuhan strain and resembles the spike used in the mRNA-1273 and Pfizer-BioNTech vaccine, was propagated from an infectious SARS-CoV-2 clone as previously described19.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>VeroE6-TMPRSS2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Using VeroE6-TMPRSS cells, the B.1.1.529 variant was plaque purified directly from the nasal swab, propagated once in a 12-well plate, and expanded in a confluent T175 flask to generate a working stock.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>VeroE6-TMPRSS</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Focus Reduction Neutralization Assay: FRNT-mNG assays were performed on VeroE6 cells and FRNT assays were performed on Vero-TMPRSS2 cells as previously described18,21,22.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero-TMPRSS2</div><div>suggested: JCRB Cat# JCRB1818, RRID:CVCL_YQ48)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Statistical analysis: Statistical analysis was conducted using Graphpad Prism V9 and R 4.1.2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Graphpad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

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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.

---

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.

---

<footer>

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

</footer>

SciScore for 10.1101/2021.12.30.474561: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">Field Sample Permit: All animal experiments were approved by the ANSES/EnvA/UPEC Ethics Committee (CE2A-16) and authorized by the French ministry of Research under the number APAFIS#25384-2020041515287655 v6, in accordance with the French and European regulations.<br>IRB: All animal experiments were approved by the ANSES/EnvA/UPEC Ethics Committee (CE2A-16) and authorized by the French ministry of Research under the number APAFIS#25384-2020041515287655 v6, in accordance with the French and European regulations.<br>Euthanasia Agents: SARS-CoV-2 virus infection: At day of infection (day post-infection DPI-0), mice were infected via intra-nasal inoculation of SARS-CoV-2 (10 µL each nostril, 104 TCID50 in total) in Dulbecco’s modified Eagle medium, under isoflurane anesthesia.<br>IACUC: The score for clinical symptoms was attributed following an IACUC approved clinical scoring system, and included the following criteria: body weight, posture/fur, activity/ mobility, eye closure, respiratory rate.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">Animals: K18-hACE2 C57BL/6 transgenic mice (males, 10-week-old), which expresses human ACE2 driven by a human cytokeratin 18 (K18) promoter (Jackson Laboratory, https://www.jax.org/strain/034860) were housed in an animal facility of biosafety level 3 (BSL3) at the French National Veterinary School in Maisons–Alfort, with water and food ad libitum.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">K18-hACE2 transgenic mice were randomly divided into the following groups (6 mice/group): vehicle, melatonin 10mg/kg (MLT10)</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">For all analyses, we imaged four fields from at least two sections per mouse and analysis was performed blinded using ImageJ.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">Sample sizes were designed to give statistical power while minimizing animal use.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">Contamination: Cell lines were checked regularly for any mycoplasma contamination. hCMEC/D3 cells: For Mpro-induced cell death assay, hCMEC/D3 cells were cultivated and transfected as described previously 29.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Mouse BD Fc Block™) for 15 min at 37°C before incubation with primary antibodies Alexa Fluor 647 Rat Anti-mouse CD31 (BD Pharmigen cat:563608) for CD31 cell labeling and Alexa Fluor 647 Rat IgG2a,k Isotype control (BD Pharmigen cat:557690)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-mouse CD31</div><div>suggested: (Bio-Rad Cat# MCA2388P647, RRID:AB_871974)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Sections were blocked with 3% BSA in PBS containing 0.1% Triton X-100 for 6h at room temperature, and incubation with primary antibodies (collagen IV: Bio-Rad, #134001, 1:200; caveolin-1: Cell Signaling Technology, #3267, 1:400) was performed at 4 °C overnight, while incubation with secondary antibodies was performed in blocking solution at room temperature for 2h.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>1:200; caveolin-1: Cell Signaling Technology , #3267</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After RNAscope® assays, a classical immunofluorescence was performed using the Anti-Collagen IV primary antibody (Anti-Collagen IV, Abcam: ab6586, 1:500°) and a secondary Alexa488 Fluor-conjugated antibody (1:500; Molecular Probes, Invitrogen) and DAPI (ACDBio) was used to stain the nuclei.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-Collagen IV</div><div>suggested: (Abcam Cat# ab6586, RRID:AB_305584)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Staining was performed as described above using primary antibodies against GFP (Abcam, #ab13970, 1:2000) and DAPI (1:2000), and imaged using a fluorescence microscope (DMI6000B, Leica).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GFP</div><div>suggested: (Abcam Cat# ab13970, RRID:AB_300798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, Lumi4-Tb-labelled ACE2 cells were incubated with d2-labelled anti-FLAG tag antibody (2 μg/mL, 1h at room temperature; 61FG2DLF, Cisbio Bioassays), followed by addition of non-labelled RBD (5 nM) or melatonin (100 µM) and TR-FRET signal was immediately read during 1h.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-FLAG</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>melatonin</div><div>suggested: (Novus Cat# NB100-62745, RRID:AB_964468)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cell culture and transfection: HEK293T cells: HEK293T (RRID:CVCL 0063) cells were obtained from Sigma-Aldrich and authenticated by the provider.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">SNAP-tagged ACE2, expressed in HEK293 cells, were fluorescently labelled by incubating cells with a SNAP suicide substrate conjugated to the long-lived fluorophore Terbium cryptate (Tb;</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293</div><div>suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, Lumi4-Tb-labelled ACE2 cells were incubated with d2-labelled anti-FLAG tag antibody (2 μg/mL, 1h at room temperature; 61FG2DLF, Cisbio Bioassays), followed by addition of non-labelled RBD (5 nM) or melatonin (100 µM) and TR-FRET signal was immediately read during 1h.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ACE2</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Organisms/Strains</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Animals: K18-hACE2 C57BL/6 transgenic mice (males, 10-week-old), which expresses human ACE2 driven by a human cytokeratin 18 (K18) promoter (Jackson Laboratory, https://www.jax.org/strain/034860) were housed in an animal facility of biosafety level 3 (BSL3) at the French National Veterinary School in Maisons–Alfort, with water and food ad libitum.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>C57BL/6</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">K18-hACE2 transgenic mice were randomly divided into the following groups (6 mice/group): vehicle, melatonin 10mg/kg (MLT10)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>K18-hACE2</div><div>suggested: RRID:IMSR_GPT:T037657)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, after withdrawing heparin from the medium, we transfected the cells using Lipofectamine 3000 (Thermo Fisher Scientific) and the following plasmids: pCAG-GFP, pCAG-p.Cys145Ala-Mpro-HA or pCAG-Mpro-HA (100 ng per well on 96-well plates).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCAG-GFP</div><div>suggested: RRID:Addgene_11150)</div></div><div style="margin-bottom:8px"><div>pCAG-p.Cys145Ala-Mpro-HA</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pCAG-Mpro-HA</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Analysis was performed blinded using ImageJ.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ImageJ</div><div>suggested: (ImageJ, RRID:SCR_003070)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Molecular dynamics simulations were computed with GROMACS 2020.5 67.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GROMACS</div><div>suggested: (GROMACS, RRID:SCR_014565)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All statistical comparisons were performed using Prism 9 (GraphPad).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

---

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 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 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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About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

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SciScore for 10.1101/2021.12.30.21268554: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">, Omicron variant RBD, full-length wild type spike and the Omciron variant RBD were cloned into pVRC vector containing an HRV 3C-cleavable C-terminal SBP-His8X tag and sequence confirmed by Genwiz.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pVRC</div><div>suggested: RRID:Addgene_49746)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibodies with manufacturer and clone information is detailed under the STAR Methods section.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>STAR</div><div>suggested: (STAR, RRID:SCR_004463)</div></div></td></tr></table>

---

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: Please consider improving the rainbow (“jet”) colormap(s) used on page 29. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

---

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.

---

<footer>

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

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But this seems to conflict with https://hyp.is/NO4vMmzVEeylBfOiPbtB2w/kit.svelte.dev/docs

The load function is reactive, and will re-run when its parameters change, but only if they are used in the function.

which seems to imply that load is not just run once for the component statically, but rather, since it can be reactive to:

url, params, fetch, session and stuff

may be sufficiently like a per-instance callback, that it could be used instead of onMount?

SciScore for 10.1101/2021.12.25.21268392: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IRB: Ethics committee approval: The CoCo Study and the analysis conducted for this article were approved by the Internal Review Board of Hannover Medical School (institutional review board no. 8973_BO-K_2020,</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Binding is further revealed by an anti-tag peroxidase-labelled antibody and colorimetric quantification.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-tag peroxidase-labelled</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pre-incubation of the Spike-protein with serum or plasma of convalescent patients or vaccinees prevents subsequent binding to ACE2 to various degrees, depending on the amount of neutralizing antibodies present.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ACE2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Plates were washed three times with PBST and incubated with an HRP-conjugated anti-His-tag antibody (clone HIS 3D5, provided by Helmholtz Zentrum München) for 1⍰h at 37⍰°C.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-His-tag</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Next, without washing, an antibody mix of anti-CD3-AF532 (UCHT1; #58-0038-42; Lot # 2288218; Invitrogen; 1:50), anti-CD4-BUV563 (RPA-T4; #741353; Lot # 9333607; BD Biosciences; 1:200), anti-CD8-SparkBlue 550 (SK1; #344760; Lot #B326454; Biolegend; 1:200), anti-CD45RA (HI100, #740298, Lot # 0295003; BD Biosciences; 1:200), anti-CCR7 (G043H7; #353230; Lot # B335328; Biolegend; 1:50), anti-CD38 PerCP-eF710 (HB7; #46-0388-42; Lot # 2044748; Invitrogen; 1:100) and Zombie NIR(tm)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-CD3-AF532</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-CD4-BUV563</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-CD8-SparkBlue</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-CD45RA</div><div>suggested: (BD Biosciences Cat# 740298, RRID:AB_2740037)</div></div><div style="margin-bottom:8px"><div>anti-CCR7</div><div>suggested: (Fluidigm Cat# 3159003, RRID:AB_2714155)</div></div><div style="margin-bottom:8px"><div>anti-CD38</div><div>suggested: (Thermo Fisher Scientific Cat# 46-0388-42, RRID:AB_1834399)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, the rhabdoviral pseudotyped particles were produced in 293T cells transfected to express the desired SARS-CoV-2-S variant inoculated with VSV*DG-FLuc, a replication-deficient VSV vector that encodes for enhanced green fluorescent protein and firefly luciferase (FLuc) instead of VSV-G protein (kindly provided by Gert Zimmer, Institute of Virology and Immunology, Mittelhäusern, Switzerland)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The assay was performed in 96-well plates in which Vero cells were inoculated with the respective pseudotyped particles/plasma mixtures.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero</div><div>suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">READER (AESKU.GROUP, Wendelsheim, Germany) and the Gen5 2.01 Software for analysis.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Gen5</div><div>suggested: (Gen5, RRID:SCR_017317)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Statistics: Statistical analysis was done using GraphPad Prism 8.4 (GraphPad Software, USA) and SPSS 20.0.0 (IBM SPSS Statistics, USA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div><div style="margin-bottom:8px"><div>SPSS</div><div>suggested: (SPSS, RRID:SCR_002865)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Ethics committee approval: The CoCo Study and the analysis conducted for this article were approved by the Internal Review Board of Hannover Medical School (institutional review board no. 8973_BO-K_2020,</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CoCo</div><div>suggested: (CoCo, RRID:SCR_010947)</div></div></td></tr></table>

---

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: Please consider improving the rainbow (“jet”) colormap(s) used on pages 17 and 20. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

---

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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About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

</footer>

SciScore for 10.1101/2021.12.26.474192: (What is this?)

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

Table 1: Rigor

NIH rigor criteria are not applicable to paper type.

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HRP-conjugated secondary antibodies against the T7-tag were diluted at 1:5,000 or 1:75,00 in the blocking buffer and incubated for 1 hour at room temperature.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>T7-tag</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A validated SARS-CoV-2 antibody-negative human serum control and a validated NIBSC SARS-CoV-2 plasma control were obtained from the National Institute for Biological Standards and Control, UK) and an uninfected cells control were also used to ensure that virus neutralization by antibodies was specific.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Control, UK</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A 96-well ELISA plate (R&D system) was coated with the RBD protein or the HEK-293T cell lysate at an amount of approximately 3-5 ng per well in a coating buffer (15 mM sodium carbonate, 35 mM sodium bicarbonate, pH 9.6) overnight at 4°C, with subsequent blockage with a blocking buffer (DPBS, v/v 0.05% Tween 20, 5% milk) at room temperature for 2 hours.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK-293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pseudotyped SARS-CoV-2 neutralization assay: The 293T-hsACE2 stable cell line (Cat# C-HA101, Lot# TA060720C) and pseudotyped SARS-CoV-2 (Wuhan-Hu-1 strain, D614G, Alpha, Beta, Lambda, Delta and Omicron) particles with luciferase reporters were purchased from the Integral Molecular.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T-hsACE2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The Nb–virus mixes (220 μl total) were incubated at 37°C for 1 h, after which they were added dropwise onto confluent Vero E6 cell (ATCC® CRL-1586™, for Munich) or Vero E6-TMPRSS2-T2A-ACE2 cells (BEI cat# NR- 54970, for Delta) monolayers in the six-well plates.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero E6</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>Vero E6-TMPRSS2-T2A-ACE2</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The monomeric Nbs 2-31 and 2-45 were also cloned into a pET-22b(+) vector at the BamHI and XhoI sites for periplasmic expression.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pET-22b(+)</div><div>suggested: RRID:Addgene_12651)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The PCR fragment was then inserted into the 2-45 pET-21b(+) vector at the same restriction sites to produce the heterodimer 2-45-(GGGGS)3-2-31.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pET-21b(+)</div><div>suggested: RRID:Addgene_112204)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Crystallographic analysis of psNbs with RBD: The receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein (GenBank: QHD43416.1), used in the crystallographic study, was cloned into a customized pFastBac vector (69), and fused with an N-terminal gp67 signal peptide and C-terminal His6 tag (70).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pFastBac</div><div>suggested: RRID:Addgene_1925)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The MS data obtained from different RBD-specific VHH isolations were analyzed by AugurLlama to identify high-affinity Nbs for each RBD (15).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>AugurLlama</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data were processed by Prism 9 (GraphPad) to fit into a 4PL curve and to calculate the logIC50 (half-maximal inhibitory concentration).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Automated data collection was carried out using serialEM (46) at a nominal magnification of 64,000x with a physical pixel size of 1.329 Å/pixel (0.6645 Å/pixel at super-resolution).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>serialEM</div><div>suggested: (SerialEM, RRID:SCR_017293)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Image processing was performed on-the-fly using CryoSPARC Live version 3.2 (47–49).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CryoSPARC</div><div>suggested: (cryoSPARC, RRID:SCR_016501)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Models were manually corrected in Coot (version 9.6.0) between rounds of read-space refinement in Phenix.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Coot</div><div>suggested: (Coot, RRID:SCR_014222)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Maps colored by local resolution were generated using RELION 3.1 (57).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>RELION</div><div>suggested: (RELION, RRID:SCR_016274)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Iterative model building and refinement were carried out in COOT (74) and PHENIX (75), respectively.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PHENIX</div><div>suggested: (Phenix, RRID:SCR_014224)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antigens were clustered at 70% sequence identity with a minimum length coverage of 15% using CD-HIT (78), the Bio.align pairwise sequence alignment module and CATH domain identifiers.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CD-HIT</div><div>suggested: (CD-HIT, RRID:SCR_007105)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibody hit rate calculation: We constructed a multiple sequence alignment for each antigen cluster using MAFFT (79) and selected a representative structure with highest structure coverage and resolution.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MAFFT</div><div>suggested: (MAFFT, RRID:SCR_011811)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Conservation scores range from 0 to ln(20) = 2.99, higher is more conserved.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Conservation</div><div>suggested: (Conservation, RRID:SCR_016064)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The simulation was run starting from the RBD structure (PDB 6lzg) using Gromacs 2020 version with the CHARMM36m force field (82).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Gromacs</div><div>suggested: (GROMACS, RRID:SCR_014565)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Sequences were aligned by MUSCLE (83) with default parameters.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MUSCLE</div><div>suggested: (MUSCLE, RRID:SCR_011812)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The phylogenetic tree was then constructed by the MEGA (84) using the maximized likelihood estimation method.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MEGA</div><div>suggested: (Mega BLAST, RRID:SCR_011920)</div></div></td></tr></table>

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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: 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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About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

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SciScore for 10.1101/2021.12.25.474155: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In vitro transcription and purification: The sequences of S1hp, S1.5hp, and S2hp were cloned from a gBlock containing the 5’ 1,000 nt of SARS-CoV-2 genome into the pUC19 vector using restriction sites EcoRI and KpnI.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pUC19</div><div>suggested: RRID:Addgene_50005)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, the coding sequences for NCAP and its truncations with N-terminal 6xHis-SUMO tag were cloned into the pEC-28a vector.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pEC-28a</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The gel images were obtained using an Amersham™ Typhoon™ scanner (Cytiva) and were quantified using Quantity One (BIO-RAD, version 4.6.9) or ImageJ.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Quantity One</div><div>suggested: (Quantity One 1-D Analysis Software, RRID:SCR_014280)</div></div><div style="margin-bottom:8px"><div>ImageJ</div><div>suggested: (ImageJ, RRID:SCR_003070)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The data were fitted and plotted using Prism (GraphPad, version 8).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Prism</div><div>suggested: (PRISM, RRID:SCR_005375)</div></div><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Statistical analyses: Unpaired Student’s t-tests with two tails were performed in Excel (Microsoft).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Excel</div><div>suggested: None</div></div></td></tr></table>

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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).

---

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.

---

<footer>

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

</footer>

SciScore for 10.1101/2021.12.28.474244: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IACUC: All animal procedures (including surgery, anesthesia, and euthanasia, as applicable) used in the current study were submitted to the Institutional Animal Care and Use Committee of the CIPHE approved by the French authorities (CETEA DSV – APAFIS#26484-2020062213431976 v6).<br>IRB: Ethics approval was given on February 5, 2020, by the French Ethics Committee CPP-Ile-de-France VI (ID RCB: 2020-A00256-33).<br>Consent: The study was conducted with the understanding and consent of each participant or their surrogate covering the sampling, storage, and use of biological samples.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">Vaccination and infection of hCD40/K18-hACE2 transgenic mice: Mice of 8 to 12 weeks of age of both sexes received two intraperitoneal injections of the CD40.CoV2 vaccine (10 µg) plus polyinosinic-polycytidylic acid (Poly-IC; Oncovir) (50 µg) or poly(IC) alone three weeks apart.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Materials availability: The CD40.CoV2 vaccine generated in this study was deposited in GenBank: anti-human CD40 12E12 antibody IgG4 H chain (GenBank ID: AJD85779.1 residues 20-467) fused to SARS_CoV_2RBD (GenBank ID: UEP92470.1 residues 17-240) followed by EPEA (C-tag) and the anti-human CD40 12E12 antibody kappa L chain (GenBank ID: AJD85780.1 residues 21-236) fused sequentially to a linker (GenBank ID: AJD85777.1 residues 699-725), nucleocapsid phosphoprotein, partial [Severe acute respiratory syndrome coronavirus 2] (GenBank ID: QWE63393.1 residues 95-230), linker residues AR, Chain A, Spike protein S1 [Severe acute respiratory syndrome coronavirus 2] (GenBank ID: 7M8J_A residues 113-237), linker residues TR, and Sequence 12 from patent US 8518410</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>C-tag</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">[IgG] Kits, MesoScale Discovery, Rockville, MD, USA) were used on all available plasma samples to measure plasma IgG antibodies to SARS-CoV-2, SARS-CoV, MERS-CoV, and HCoVs.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>plasma IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibody concentrations were quantified using a reference standard (ACE2 Calibration Reagent) and are expressed as units/mL (one unit per mL concentration of ACE2 Calibration Reagent corresponds to neutralizing activity of 1 µg/mL monoclonal antibody to SARS-CoV-2 Spike protein).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SARS-CoV-2 Spike protein) .</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The flow cytometry panel included a viability marker, CD3, CD4, and CD8 to determine the T-cell lineage, and IFN-γ, TNF, and IL-2 antibodies.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CD3</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>CD4</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>CD8</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>TNF</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>IL-2 antibodies</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>IL-2</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Infectious stocks were grown by inoculating Vero E6 cells and collecting supernatants upon observation of the cytopathic effect.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero E6</div><div>suggested: RRID:CVCL_XD71)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Organisms/Strains</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Heterozygous K18-hACE C57BL/6J mice (strain: 2B6.Cg-Tg (K18-ACE2)2Prlmn/J) were obtained from The Jackson Laboratory.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>C57BL/6J</div><div>suggested: RRID:MGI:3589388)</div></div><div style="margin-bottom:8px"><div>2B6.Cg-Tg ( K18-ACE2)2Prlmn/J</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For the AIM assay, PBMCs were stimulated in vitro with various concentrations of the CD40.CoV2 vaccine or an equimolar combination of 15-mer overlapping peptide pools covering the full-length sequence of vaccine antigens (vS1 + vS2 + vRBD + vN2) referred to as vOLPmix.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>vS1 + vS2 + vRBD + vN2</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Distributions were plotted using SPICE version 5.22, downloaded from http://exon.niaid.nih.gov/spice (Roederer et al., 2011)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SPICE</div><div>suggested: (SPICE, RRID:SCR_016603)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Statistical analysis: Graphpad Prism software version 8 was used for nonparametric statistics and plots, as described in the figure legends.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Graphpad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

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Results from OddPub: Thank you for sharing your data.

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Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

Our study had several limitations. These included the absence of characterization of cross-neutralizing antibodies in vaccinated mice against the B.1.1.529 Omicron variant, which emerged during the writing of this manuscript. However, we could expect that our vaccine would elicit T-cell responses against N epitopes because of the high homology (100%) between vaccine sequences and this VOC. Due to the limited availability of hCD40/K18-hACE2 mice, we did not evaluate various VOC challenges in mice. Finally we favored the analysis of T cell responses using samples from recovered individuals instead of in vivo preclinical models. Although these responses may be dependent on the “clinical history” of patients and their HLA haplotypes, they are less biased than those that would be observed in an animal model. Our results show that the in-vitro vaccine responses are directed against all vaccine proteins, which confirmed the broad HLA coverage of the vaccine sequences. In conclusion, it is becoming urgent to develop a “pan-sarbecovirus vaccine”. The development of a new protein-based vaccine with expected improved tolerability suitable for people with specific vulnerabilities and children would extend the portfolio of current vaccines and be instrumental in controlling the circulation of the virus and the emergence of new variants. By selecting a narrow range of immunodominant epitopes, presented by a wide variety of HLA alleles and less prone to genetic variations across sarbecoviru...

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Results from TrialIdentifier: We found the following clinical trial numbers in your paper:<br><table><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Identifier</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Status</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Title</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT04842682</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Recruiting</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Dose Escalation Trial of CD40.HIVRI.Env Vaccine Combined or …</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT04262921</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Recruiting</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">French COVID Cohort</td></tr></table>

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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: 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.

---

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

---

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About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

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"Export" annotations

The costs here tend to be overstated (and stated by people without a strong background in either browsers or languages, I've found).

I remember someone mentioning to Boris Zbarsky (and expecting him to agree) that IE compatibility was a source of bloat in Gecko. Of course, he corrected them.

SciScore for 10.1101/2021.12.23.473975: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">In addition, purified PCR products were inserted into pcDNA3.1, transformed into E. coli and sequencing was performed on randomly picked colonies.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cells were washed and incubated with anti-FLAG-APC and anti-Histidine6-PE antibodies on ice for 20 mins, followed by washing.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-FLAG-APC</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-Histidine6-PE</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Expression and purification of soluble proteins: HEK 293 cells were transfected with mammalian expression vectors encoding the relevant protein using polyethylenimine and cells incubated for approximately 3 days to allow accumulation of the secreted protein in the medium.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK 293</div><div>suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Flow cytometry: Vero-E6 cells were cultured in DMEM with 10% (v/v) FBS.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero-E6</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Organisms/Strains</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">cDNA encoding a fusion protein comprising of an N-terminal CD5 secretory leader sequence followed by the Wuhan-Hu-1 CoV-2 RBD (residues 319-541), linker region and FLAG epitope tag together with a C-terminal transmembrane domain and short intracellular domain fragment of platelet-derived growth factor receptor-β was synthesised (GeneArt Gene Synthesis) and inserted into the pHypermut2 vector23 This was transfected into DT40 cells by electroporation and stable transfectants derived by growth in puromycin.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Wuhan-Hu-1 CoV-2 RBD</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">cDNA encoding a fusion protein comprising of an N-terminal CD5 secretory leader sequence followed by the Wuhan-Hu-1 CoV-2 RBD (residues 319-541), linker region and FLAG epitope tag together with a C-terminal transmembrane domain and short intracellular domain fragment of platelet-derived growth factor receptor-β was synthesised (GeneArt Gene Synthesis) and inserted into the pHypermut2 vector23 This was transfected into DT40 cells by electroporation and stable transfectants derived by growth in puromycin.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pHypermut2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In addition, purified PCR products were inserted into pcDNA3.1, transformed into E. coli and sequencing was performed on randomly picked colonies.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3.1</div><div>suggested: RRID:Addgene_79663)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, movies were corrected for motion using MotionCor 2.1.425 and the contrast transfer function parameters were estimated using GCTF 1.1826.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GCTF</div><div>suggested: (GCTF, RRID:SCR_016500)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Initial rigid-body docking of the crystal structure (PDB ID 6M0J) was performed using UCSF Chimera 1.1528 and further model building was performed in Coot 0.9.629.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Coot</div><div>suggested: (Coot, RRID:SCR_014222)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After manual rebuilding real-space refinement of the coordinates was performed using Phenix 1.19.230.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Phenix</div><div>suggested: (Phenix, RRID:SCR_014224)</div></div></td></tr></table>

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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: Please consider improving the rainbow (“jet”) colormap(s) used on page 26. 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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Results from scite Reference Check: We found no unreliable references.

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About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

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SciScore for 10.1101/2021.12.21.473733: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IACUC: All animal work was performed under the guidelines of Yale University Institutional Animal Care and Use Committee (IACUC) with approved protocols (Chen-2018-20068; Chen-2020-20358; Wilen-2018-20198).</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">M. musculus (mice), 6-12 weeks old females, of C57BL/6J and BALB/c strains, were used for immunization.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">Six to eight-week-old K18-hACE2 littermate-controlled mice, mixed gender (male / female) mice were divided randomly into three groups, and administered with 20 mg/kg (of mice body weight) Clone 2, Clone 6 or placebo / control, via intraperitoneal (IP) injection.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">Replication, randomization, blinding and reagent validations: Replicate experiments have been performed for all key data shown in this study.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">Contamination: All cell lines tested negative for mycoplasma.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Plasmid construction: The cDNA sequences of the paired variable heavy and light chain region of anti-RBD antibody clones were synthesized as gBlocks (IDT) and cloned by the Gibson assembly (NEB) into human IgG1 heavy chain and light chain expression plasmids, pFUSEss-CHIg-hG1(InvivoGen, pfusess-hchg1) and pFUSE2ss-CLIg-hK (InvivoGen, pfuse2ss-hclk), respectively.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pfusess-hchg1</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pFUSE2ss-CLIg-hK</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">, containing cDNA sequence of variable region of light chain of anti-RBD antibody clones and the regions overlapping with corresponding flanking sequences of EcoRI and BsiWI restriction sites pFUSE2ss-CLIg-hK, were ordered from IDT.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-RBD</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">IgG1 antibodies were expressed in Expi293FTM cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>IgG1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">) bispecific antibody is a human IgG1-like bispecific antibody, generated based on CrossMab-KiH bispecific constructs, including pFUSE2ss-knobLight-hK, pFUSE2ss-knobheavy-hG1, pFUSE2ss-HoleLight-hK, pFUSE2ss-HoleHeavy-hG1.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pFUSE2ss-knobheavy-hG1</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pFUSE2ss-HoleLight-hK</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pFUSE2ss-HoleHeavy-hG1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">BLI): Antibody binding kinetics for anti-spike mAbs were evaluated by BLI on an Octet RED96e instrument (FortéBio) at room temperature.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-spike</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After that, the biosensors were associated with indicated concentrations of the antibodies (from 50 nM to 0.78125 nM with 2-fold dilutions, where the kinetic buffer was served as the negative control) for 200 s, then dissociated in the kinetic buffer for 1000 s. (2) 25ng/ul of Clone13A-IgG1 antibodies were captured on a AHC biosensor (ForteBio).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Clone13A-IgG1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The anti-S antibodies and HRP-conjugated goat anti-mouse IgG (Sigma, 12-349) in PBS supplemented with 0.1% saponin and 0.1% bovine serum albumin.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-S</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-mouse IgG</div><div>suggested: (Millipore Cat# 12-349, RRID:AB_390192)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">15x106 293FT cells were seeded in a 150 mm plates one day before in 20 ml D10 media.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293FT</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, 293T cells were seeded in 150 mm plates, and transfected with 21 µg pHIVNLGagPol, 21 µg pCCNanoLuc2AEGFP, and 7.5 µg of a SARS-CoV-2 SΔ19 or SARS-CoV-2 SA SΔ19 plasmid utilizing 198 µl PEI.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The pseudovirus neutralization assays were performed on 293T-hACE2 cell line 71.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T-hACE2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">5x105 Vero-E6 cells were plated per well of a 96-well plate.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero-E6</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibody-virus complexes were added to Vero-TMPRSS2 cell monolayers in 96-well plates and incubated at 37°C for 1 h.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero-TMPRSS2</div><div>suggested: JCRB Cat# JCRB1818, RRID:CVCL_YQ48)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The replication-competent SARS-CoV-2 (USA-WA1/2020) virus was produced in Vero E6 cells, and the titer was determined by plaque assay using WT VeroE6.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero E6</div><div>suggested: RRID:CVCL_XD71)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Organisms/Strains</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">M. musculus (mice), 6-12 weeks old females, of C57BL/6J and BALB/c strains, were used for immunization.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>C57BL/6J</div><div>suggested: RRID:IMSR_JAX:000664)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Clone 5-11 were mAbs chosen from RBD-his tag protein immunized BALB/c mice.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BALB/c</div><div>suggested: RRID:IMSR_ORNL:BALB/cRl)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The K18-hACE2 mice (B6.Cg-Tg(K18-ACE2)2Prlmn/J) were purchased from the Jackson Laboratory and bred in house using a trio breeding scheme.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>K18-hACE2</div><div>suggested: RRID:IMSR_GPT:T037657)</div></div><div style="margin-bottom:8px"><div>B6.Cg-Tg(K18-ACE2)2Prlmn/J</div><div>suggested: RRID:IMSR_JAX:034860)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In vivo efficacy testing of humanized Clone13A to authentic SARS-CoV-2 virus: 10-12-week-old littermate-controlled female and male K18hAce2Tg+ mice were pretreated with 20 mg/kg of either control hIgG1 (purchased from BioXCell) or clone 13A mAb (produced by the Chen lab) administered IP in 300 uL of DPBS.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>K18hAce2Tg+</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Parallelly, pFUSE2-CLIg-hK is a cloning plasmid that expresses the constant region of the human kappa light chain and contains multiple cloning sites to enable cloning of the light chain variable region.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pFUSE2-CLIg-hK</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Four plasmids were employed: pFUSE2ss-knobLight-hK, pFUSE2ss-knobheavy-hG1, pFUSE2ss-HoleLight-hK, pFUSE2ss-HoleHeavy-hG1.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pFUSE2ss-knobheavy-hG1</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pFUSE2ss-HoleLight-hK</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pFUSE2ss-HoleHeavy-hG1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The pFUSE2ss-knobLight-hK is pFUSE2ss-CLIg-hK with no further editing.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pFUSE2ss-CLIg-hK</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The gBlock (pPR024), containing constant region of heavy chain with two knob mutations and the regions overlapping with corresponding flanking sequences of NsiI and NheI restriction sites in pFUSEss-CHIg-hG1 was ordered from IDT, and then cloned into NsiI and NheI restriction enzymes digested pFUSEss-CHIg-hG1 backbone by the Gibson assembly (NEB).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pPR024</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The pFUSE2ss-HoleLight- hK was generated by replacing the constant region of Light chain (CL) in pFUSE2ss-CLIg-hK with CH1 region of heavy chain in pFUSEss-CHIg-hG1 vector.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pFUSEss-CHIg-hG1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The gBlock (pPR023), containing cDNA sequence of constant region of light chain, CH2 and CH3 with “hole” mutations, and regions overlapping with corresponding flanking sequences of NsiI and NheI restriction sites in pFUSEss-CHIg-hG1 was ordered from IDT, and cloned into NsiI and NheI restriction enzymes digested pFUSEss-CHIg-hG1 backbone through Gibson assembly (NEB).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pPR023</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The pVP21-SA and pVP28-Indian were generated based on pcDNA3.1-pSARS-CoV-2-S, which was derived by insertion of a synthetic human codon- optimized cDNA (Geneart) encoding a WA1 SARS-CoV-2 S protein.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pVP28-Indian</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pcDNA3.1-pSARS-CoV-2-S</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For pVP28-Indian, four gBlocks, contains mutations in Indian variant regions overlapping with corresponding flanking sequences of NheI and BamHI restriction sites pcDNA3.1-pSARA-CoV-2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3.1-pSARA-CoV-2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For the HIV1-based SARS-CoV-2 spike pseudotyped virus generation, WT pcDNA3.1-pSARS-CoV-2-S, pVP21-SA-variant, and pVP28-Indian variant lacking the C-terminal 19 codons were employed.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pVP21-SA-variant</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A pair of forward and reverse primers were utilized to amplify fragments lacking the C- terminal 19 codons with pVP21-SAvariant and pVP28-Indian variant as template separately.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pVP21-SAvariant</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The amplified fragments were gel-purified and cloned into pVP21-SAvariant backbone and pVP28-Indianvariant backbone, digested with BbvCI and BamHI.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pVP28-Indianvariant</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Recombinant antibody generation: The top-ranked enriched IgG clones were selected and cDNAs of relative variable region of paired heavy- and light-chain were codon-optimized and cloned separately into human IgG1 heavy chain and light chain expression vectors, containing the human IgG1 constant regions (pFuse plasmids)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pFuse</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The variable region of Clone 6 heavy chain was cloned into pFUSE2ss-knobheavy-hG1vector.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pFUSE2ss-knobheavy-hG1vector</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The variable region of Clone 6 light chain was cloned into pFUSE2ss-knobLight-hK vector.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pFUSE2ss-knobLight-hK</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Plasmid expression a C-terminally truncated SARS-CoV-2 S protein (pSARS-CoV-2Δ19) was obtained from Dr Bieniasz’ lab.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pSARS-CoV-2Δ19</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, 293T cells were seeded in 150 mm plates, and transfected with 21 µg pHIVNLGagPol, 21 µg pCCNanoLuc2AEGFP, and 7.5 µg of a SARS-CoV-2 SΔ19 or SARS-CoV-2 SA SΔ19 plasmid utilizing 198 µl PEI.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCCNanoLuc2AEGFP</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">It was inserted into a piggybac transposon (Matt Wilson of Baylor College of Medicine, along with the transposase plasmid pCMV-piggybac) that had been modified to encode a CMV-IRES-bsdr cassette; resultant plasmid was named pT-PB-SARS-CoV-2-Spike- IRES-Blasti.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMV-piggybac</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pT-PB-SARS-CoV-2-Spike- IRES-Blasti</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">This too was inserted into piggybac transposon to make pT-PB-SARS-CoV-2-UK Spike-IRES- Blasti.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pT-PB-SARS-CoV-2-UK</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">hACE2 was subsequently introduced by VSV G-mediated HIV-based transduction using pHIV-CMV-hACE2-IRES-Puro to produce HOS-3734, which cell lines maintained in selection using 10 μg/mL puromycin (Sigma-Aldrich).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pHIV-CMV-hACE2-IRES-Puro</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">TZMbl cells stably expressing wild type S/UK variant S were created by co-transfecting TZMbl cells with pT-PB-SARS-CoV-2- Spike-IRES-Blasti or pT-PB-SARS-CoV- 2-UK Spike-IRES-Blasti, respectively, along with pCMV-piggybac and resistant cells selected with 10 μg/mL blasticidin (Invivogen).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pT-PB-SARS-CoV-2-</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pT-PB-SARS-CoV-</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The control TZMbl cell line not expressing S was generated by co-transfecting pCMV- piggybac with pT-pB-IRES-Blasti and selecting for blasticidin-resistant TZMbl cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMV-</div><div>suggested: RRID:Addgene_99510)</div></div><div style="margin-bottom:8px"><div>pT-pB-IRES-Blasti</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibody humanization: In order to humanize the antibody, we first determine the six CDR loops from murine variable domains by using the online free program “IGBLAST” (https://www.ncbi.nlm.nih.gov/igblast/).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>https://www.ncbi.nlm.nih.gov/igblast/</div><div>suggested: (IgBLAST, RRID:SCR_002873)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The 50% inhibitory concentration (IC50) was calculated with a four-parameter logistic regression using GraphPad Prism 8.0 (GraphPad Software Inc.)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data were analyzed with non-linear regression using GraphPad Prism to determine the neutralization curve and the IC50 values calculated.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Image processing and 3D reconstruction using cryoSPARC 78 produced similar results.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>cryoSPARC</div><div>suggested: (cryoSPARC, RRID:SCR_016501)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The final map of each body was corrected for K3 detector modulation and sharpened by a negative B-factor estimated by RELION 80, and then merged in Chimera for deposition.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>RELION</div><div>suggested: (RELION, RRID:SCR_016274)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The initial models were subsequently manually rebuilt in COOT 83, followed with iterative cycles of refinement in Refmac5 84 and PHENIX 85.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>COOT</div><div>suggested: (Coot, RRID:SCR_014222)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The cryo-EM structures and homology models were analyzed in Pymol.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Pymol</div><div>suggested: (PyMOL, RRID:SCR_000305)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Genomic sequencing raw data are deposited to Gene Expression Omnibus (GEO) with the accession code (GSE174635).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Gene Expression Omnibus</div><div>suggested: (Gene Expression Omnibus (GEO, RRID:SCR_005012)</div></div></td></tr></table>

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Results from OddPub: Thank you for sharing your code and data.

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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.

---

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.

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Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 54. 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.

---

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

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About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

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SciScore for 10.1101/2021.12.23.474009: (What is this?)

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

Table 1: Rigor

NIH rigor criteria are not applicable to paper type.

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The resulting PCR product containing the S197TAG substitution and carrying the C-terminal cleavable His6-tag was treated with NdeI/HindIII restriction endonucleases and then cloned into a pRBC_14-3-3γ/His6SUMO_mBAD43-204 vector 30 pretreated with the same endonucleases.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pRBC_14-3-3γ/His6SUMO_mBAD43-204</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The C-terminal His6-tag enabled the use of the phosphoserine-incorporating E.coli system [BL21 ΔserB(DE3) cells transformed by the pKW2-EFSep plasmid (chloramphenicol resistance) and the target N plasmid] having the Release Factor 1 (responsible for translation termination at TAG codons) 51.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pKW2-EFSep</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The SARS-CoV N-containing pGEX plasmid (ampicillin resistance) was kindly provided by Prof. Fulvio Reggiori (University of Groningen, The Netherlands)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pGEX</div><div>suggested: RRID:Addgene_18100)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Untagged full-length human 14-3-3γ (Uniprot ID P61981) cloned into a pET21 vector (ampicillin resistance) 52, the surface entropy reduction mutants 53 of the C-terminally truncated human 14-3-3σ (residues 1-231), i.e. Clu1 (159KKE161 → 159AAA161) and Clu3 (75EEK77 → 75AAA77), each containing an N-terminal 3C protease cleavable His6-tag and cloned into the pET28 vector (kanamycin resistance) have been described previously 37.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pET21</div><div>suggested: RRID:Addgene_90313)</div></div><div style="margin-bottom:8px"><div>pET28</div><div>suggested: RRID:Addgene_21766)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To ensure phosphorylation of SARS-CoV-2 N, its S197L mutant, or SARS-CoV N in E.coli cells, target proteins were co-expressed with the catalytically active subunit of mouse protein kinase A (PKA) encoded in a separate pACYC-PKA plasmid (chloramphenicol resistance), essentially as described earlier 26.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pACYC-PKA</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Crystallization and X-ray data collection: Crystallization was performed using 14-3-3σ surface entropy reduction mutants Clu1 or Clu3 (storage buffer 20 mM Tris-HCl pH 7.6, 150 mM NaCl, 0.1 mM EDTA, 2 mM DTT, 2 mM MgCl2) mixed with either pS197 or pT205 phosphopeptide at a 1:5 protein:peptide molar ratio with the final protein concentration of 11.5 mg/ml.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pS197</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pT205</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The Mw distributions were calculated in Astra 8.0 software (Wyatt Technology) using dn/dc=0.185.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Astra</div><div>suggested: (ASTRA, RRID:SCR_016255)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To determine KD values, the binding curves were approximated using the quadratic equation 56 in Origin 9.0 (OriginLab Corporation, Northampton, MA, USA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>OriginLab Corporation</div><div>suggested: (Origin, RRID:SCR_014212)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Peptide residues were manually built into difference electron density maps in Coot 59 and the overall structures were refined using Buster 2.10.4 60, which used NCS information (with pruning), TLS and all-atom individual isotropic B-factor restrained refinement.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Coot</div><div>suggested: (Coot, RRID:SCR_014222)</div></div><div style="margin-bottom:8px"><div>Buster</div><div>suggested: (BUSTER, RRID:SCR_015653)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The refined structures were validated using the comprehensive validation algorithm in Phenix 1.10 62.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Phenix</div><div>suggested: (Phenix, RRID:SCR_014224)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Structural illustrations were prepared using PyMOL 2.20 (Schrodinger, Inc.).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PyMOL</div><div>suggested: (PyMOL, RRID:SCR_000305)</div></div></td></tr></table>

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Results from OddPub: Thank you for sharing your data.

---

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.

---

<footer>

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

</footer>

SciScore for 10.1101/2021.12.24.474091: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IRB: The present study was approved by the Institutional Review Board of the National Institute of Infectious<br>Consent: All volunteers provided written informed consent prior to the enrollment.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Nucleocapsid antibody was analyzed by cobas e411 plus (Roche) with Elecsys Anti-SARS-CoV-2 (Roche), and <1 was evaluated as seronegative according to the manufacturer’s instructions.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-SARS-CoV-2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To obtain comparable titers of IgA to those of IgG, we normalized the IgA titers using a converting unit, which was calculated from the binding curves of CR3022 monoclonal antibodies prepared as human IgG1 and human IgA1 isotypes (20, 43).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>human IgA1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The cells were washed with the medium and stained with subsequent antibodies/reagents using the Brilliant Stain Buffer Plus (BD Biosciences) for 30 min at room temperature: FITC-conjugated anti-IgA (polyclonal rabbit F(ab’)2, Dako), BV421-conjugated anti-IgG (G18-145, BD Biosciences)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-IgA</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-IgG</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The virus-plasma mixtures were placed on VeroE6/TMPRSS2 cells (JCRB1819) seeded in 96-well plates and cultured at 37 °C with 5% CO2 for 5 days.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>VeroE6/TMPRSS2</div><div>suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The plasmid vectors were transfected into 293T cells followed by infection with 0.5 MOI of G-complemented VSVΔG/Luc 24 h after the transfection (45), and then the uninfected viruses were washed out.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, the human codon-optimized DNA sequence encoding amino acids 331-529 of the SARS-CoV-2 spike (GenBank: MN994467) with an N-terminal signal peptide sequence (MIHSVFLLMFLLTPTESYVD) and C-terminal avi-tag and histidine-tag were cloned into the pCAGGS vector.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCAGGS</div><div>suggested: RRID:Addgene_127347)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To biotinylate RBD proteins, the RBD expression vectors were co-transfected into Expi293F cells together with the secreted BirA-Flag plasmid (Addgene) in the presence of 100 μM biotin.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BirA-Flag</div><div>suggested: RRID:Addgene_64395)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Blood was collected in Vacutainer CPT tubes (BD Biosciences), followed by centrifugation at 1800 × g for 20 min.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BD Biosciences</div><div>suggested: (BD Biosciences, RRID:SCR_013311)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Statistical analysis: The numerical data were statistically analyzed and visualized with GraphPad Prism 9 software (GraphPad)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Flow cytometry data were analyzed using FlowJo software (BD Biosciences).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>FlowJo</div><div>suggested: (FlowJo, RRID:SCR_008520)</div></div></td></tr></table>

---

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: We detected the following sentences addressing limitations in the study:

A primary limitation of our study is the lack of longitudinal analysis after the third dose of vaccines in our cohort. Therefore, we cannot determine the extents to which the numbers of cross-neutralizing Bmem subset correlate with the magnitudes of cross-neutralizing antibody responses following the third vaccine dose. Owing to the paucity of Bmem subsets, detailed phenotypic and transcriptomic characterization of the cross-neutralizing Bmem subset is hampered in this study. Finally, we focused on the neutralizing antibodies but non-neutralizing antibodies also confer the protection in vivo at least in animal models. The analysis on non-neutralizing and protective antibodies against the variants may generate the distinct outcome owing to the epitope difference.

---

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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WARC Format

The WARC format is the raw data from the crawl, providing a direct mapping to the crawl process. Not only does the format store the HTTP response from the websites it contacts (WARC-Type: response), it also stores information about how that information was requested (WARC-Type: request) and metadata on the crawl process itself (WARC-Type: metadata).

For the HTTP responses themselves, the raw response is stored. This not only includes the response itself, what you would get if you downloaded the file, but also the HTTP header information, which can be used to glean a number of interesting insights.

In the example below, we can see the crawler contacted http://102jamzorlando.cbslocal.com/tag/nba/page/2/ and received a HTML page in response. We can also see the page was served from the nginx web server and that a special header has been added, X-hacker, purely for the purposes of advertising to a very specific audience of programmers who might look at the HTTP headers!

WARC/1.0
WARC-Type: response
WARC-Date: 2013-12-04T16:47:32Z
WARC-Record-ID: 
Content-Length: 73873
Content-Type: application/http; msgtype=response
WARC-Warcinfo-ID: 
WARC-Concurrent-To: 
WARC-IP-Address: 23.0.160.82
WARC-Target-URI: http://102jamzorlando.cbslocal.com/tag/nba/page/2/
WARC-Payload-Digest: sha1:FXV2BZKHT6SQ4RZWNMIMP7KMFUNZMZFB
WARC-Block-Digest: sha1:GMYFZYSACNBEGHVP3YFQNOSTV5LPXNAU

HTTP/1.0 200 OK
Server: nginx
Content-Type: text/html; charset=UTF-8
Vary: Accept-Encoding
Vary: Cookie
X-hacker: If you're reading this, you should visit automattic.com/jobs and apply to join the fun, mention this header.
Content-Encoding: gzip
Date: Wed, 04 Dec 2013 16:47:32 GMT
Content-Length: 18953
Connection: close


...HTML Content...

.readwise .tagging How to tag chapters and sections

SciScore for 10.1101/2021.12.21.473668: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IACUC: Toxicology: Animal experimental procedures were reviewed and approved by the University of Illinois Institutional Animal Care and Use Committee (</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">Toxicity of each peptide was assessed in 5 female and 5 male CD-1 IGS mice (8 weeks old).</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">Animals with sex- and age-matched littermates were randomly included in experiments.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">Animal experiments were carried out in a blinded fashion whenever feasible.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">For the number of animals needed to achieve statistically significant results, we conducted a priori power analysis.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After this, cells were stained with antibodies including CD45-EF450 (1:2000, eBioscience #48-0451-82) and CD31-APC (1:100, eBioscience #17-0311-82) for 45 minutes at 4°C.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CD45-EF450</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>CD31-APC</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Anti-SARS-CoV-2 Monoclonal Antibodies: Sequences for monoclonal antibodies that had received Emergency Use Authorization from the U.S. Food and Drug Administration were pulled from the KEGG database (Accession No. REGN10933, D11938; REGN10987, D11939; VIR-7831, D12014; LY-CoV555, D11936).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-SARS-CoV-2</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>D12014; LY-CoV555, D11936</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>LY-CoV555</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The SARS-CoV-2 were propagated in Vero E6 cells (CRL-1586; American Type Culture Collection).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero E6</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HEK293T cells were grown to 80% confluency before transfection with pCMV3-SARS-CoV-2-spike (Sino Biological) using Lipofectamine®3000 (Invitrogen).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cell culture: The 2019n-CoV/USA_WA1/2019 isolate as well as the P.1 variant of SARS-CoV-2 and ACE-2 expressing A549 cell lines were obtained from BEI Resources.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ACE-2</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>A549</div><div>suggested: NCI-DTP Cat# A549, RRID:CVCL_0023)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Vero E6 (CRL-1586; American Type Culture Collection) Vero CCL81 (American Type Culture Collection),</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero CCL81</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Assessment of ACE2 mutants: Expi293F cells transfected with pCEP4-myc-ACE2 plasmids were collected 24 h post-transfection (600 × g, 60 s), washed with ice-cold Dulbecco’s phosphate buffered saline (PBS) containing 0.2% bovine serum albumin (BSA), and stained with 1:50 RBD-sfGFP expression medium (prepared as previously described 22 and 1:250 anti-myc Alexa 647 (clone 9B11, Cell Signaling Technology) in PBS-BSA.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Expi293F</div><div>suggested: RRID:CVCL_D615)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Organisms/Strains</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Hemizygous K18-hACE mice with c57BL/6J background (strain#034860: B6.Cg-Tg(K18-ACE2)2Prlmn/J) were purchased from The Jackson Laboratory.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>K18-hACE</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>B6.Cg-Tg(K18-ACE2)2Prlmn/J</div><div>suggested: RRID:IMSR_JAX:034860)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For in vivo studies, 10 mg/kg sACE22.v2.4-IgG1 (wt), sACE22.v2.4-IgG1 and control peptide buffer were intravenously administrated into K-18 hACE-2 mice for 30 minutes prior to SARS-CoV-2 pseudo-entry virus (106 pfu) i.p. injection for 1 dpi.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>hACE-2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pharmacokinetics: 8-week-old CD-1 IGS mice (3 female and 3 male per time point) were IV administered protein solutions.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CD-1 IGS</div><div>suggested: RRID:MGI:5461217)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To characterize different routes side-by-side, peptides were administered to C57BL/6 mice, 3 males per time point.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>C57BL/6</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HEK293T cells were grown to 80% confluency before transfection with pCMV3-SARS-CoV-2-spike (Sino Biological) using Lipofectamine®3000 (Invitrogen).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMV3-SARS-CoV-2-spike</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">8his-tagged monomeric sACE2 (ACE2 a.a. 1-615; pcDNA3-sACE2(WT)-8his, Addgene No. 149268, and pcDNA3-sACE2v2.4-8his, Addgene No. 149664), and human IgG1-Fc fused dimeric sACE22 (ACE2 a.a. 1-732; pcDNA3-sACE2-WT(732)-IgG1,</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3-sACE2</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pcDNA3-sACE2v2.4-8his</div><div>suggested: RRID:Addgene_149664)</div></div><div style="margin-bottom:8px"><div>pcDNA3-sACE2-WT(732)-IgG1</div><div>suggested: RRID:Addgene_154104)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Addgene No. 154104, and pcDNA3-sACE2v2.4(732)-IgG1, Addgene No. 154106) are previously described.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3-sACE2v2.4</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Human codon-optimized S (GenBank No. YP_009724390.1) was subcloned into pCEP4 (Invitrogen) from pUC57-2019-nCoV-S(Human) (distributed by Molecular Cloud on behalf of Haisheng Yu, Chinese Academy of Medical Sciences) with a N-terminal HA leader (MKTIIALSYIFCLVFA), myc-tag (EQKLISEEDL), and linker (GSPGGA) upstream of the mature polypeptide (a.a. V16-T1273).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCEP4</div><div>suggested: RRID:Addgene_16479)</div></div><div style="margin-bottom:8px"><div>pUC57-2019-nCoV-S(Human</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Assessment of ACE2 mutants: Expi293F cells transfected with pCEP4-myc-ACE2 plasmids were collected 24 h post-transfection (600 × g, 60 s), washed with ice-cold Dulbecco’s phosphate buffered saline (PBS) containing 0.2% bovine serum albumin (BSA), and stained with 1:50 RBD-sfGFP expression medium (prepared as previously described 22 and 1:250 anti-myc Alexa 647 (clone 9B11, Cell Signaling Technology) in PBS-BSA.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCEP4-myc-ACE2</div><div>suggested: RRID:Addgene_141185)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In vitro binding assays to S variants: Expi293F cells were transfected with pCEP4-myc-S plasmids as described above.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCEP4-myc-S</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">H and L chains were cloned with CD5 leader sequences into pcDNA3.1(+) and expressed in Expi293F cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3.1</div><div>suggested: RRID:Addgene_79663)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Images were taken with a Zeiss microscope and analyzed by Zen software (Zeiss)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Zen</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For sACE22.v2.4 without any tags or fusion partner (corresponding to ACE2 residues 19-732, protein provided by Orthogonal Biologics, Inc.), mice were IV administered (tail vein, 0.5 mg/kg) protein twice daily for 5 consecutive days (days 0-4) and sacrificed day 7.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Orthogonal Biologics</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Mutations were introduced using PyMOL (https://pymol.org/2/) and the systems were solvated using TIP3P water and 150 mM NaCl using PACKMOL 71.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PyMOL</div><div>suggested: (PyMOL, RRID:SCR_000305)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Protein residues and glycosylation sites were parameterized using AMBER ff14SB 72 and GLYCAM06 73 force fields.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>AMBER</div><div>suggested: (AMBER, RRID:SCR_016151)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Anti-SARS-CoV-2 Monoclonal Antibodies: Sequences for monoclonal antibodies that had received Emergency Use Authorization from the U.S. Food and Drug Administration were pulled from the KEGG database (Accession No. REGN10933, D11938; REGN10987, D11939; VIR-7831, D12014; LY-CoV555, D11936).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>KEGG</div><div>suggested: (KEGG, RRID:SCR_012773)</div></div></td></tr></table>

---

Results from OddPub: Thank you for sharing your code and data.

---

Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

Another limitation is our focus on intravenous delivery, whereas intratracheal or inhalation delivery may be more readily applied in patients, especially in the outpatient setting. It would be useful in future studies to use varying doses and delivery route combinations for defined disease stages to identify the optimal combination prior to initiating human efficacy trials. Finally, we chose to fuse the ACE2 peptide to an unmodified Fc from IgG1 (isoallotype nG1m1), whereas others have considered fusions to Fc mutants or IgG4 to dampen interactions with FcγR subtypes that might contribute to inflammation24, 68. We instead reasoned that IgG1 effector functions will be necessary for optimum in vivo protection. In summary, we show for the first time that an engineered decoy ACE2 peptide demonstrating much higher affinity for the SARS-CoV-2 Spike protein is efficacious in vivo against multiple SARS-CoV-2 variants. This peptide prevented viral entry into cells and the peptide’s efficacy against a variant of concern prevented lung endothelial injury and ARDS and significantly reduced mortality. The results show the potential of this engineered peptide to treat COVID-19 patients and others with inadequate antibody titer to protect against emerging, more virulent SARS-CoV-2 variants.

---

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).

---

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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About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

</footer>

SciScore for 10.1101/2021.12.21.473774: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IRB: The study protocol was approved by the Human Research Ethics Committees of the Northern Sydney Local Health District, the University of New South Wales, NSW Australia (ETH00520), CALHN Human Research Ethics Committee, Adelaide, Australia<br>Consent: Written informed consent was obtained from all participants before enrolment.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">Archival serum or plasma collected from 25 healthy donors prior to the pandemic with an age range of 24-73 years, and a male to female ratio of 1:2.4 was used as controls.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Expression of surface Fc-receptors was assessed by flow cytometry using monoclonal antibodies (mAbs) against Fcγ receptor RI (CD64)-FITC, FcγRIII (CD16)-PE, CD14-PerCP</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CD64)-FITC</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Endpoint titre and Isotyping of anti-SARS-CoV-2 Spike and anti-RBD antibodies in sera of patients with SARS-CoV-2 infection: Anti-SARS-CoV-2 Spike and RBD IgG antibody in sera was quantified using a modified direct ELISA [27].</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-SARS-CoV-2</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>RBD IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For the endpoint titre of total anti-Spike or anti-RBD antibodies, 50μL of horseradish peroxidase (HRP)-conjugated mouse anti-human detection antibody added per well (Jackson ImmunoResearch, USA) (1:5000 dilution in 5% skim milk).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-Spike</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-RBD</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-human detection</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For isotyping of the anti-Spike or anti-RBD antibodies, 50μL/well of HRP-conjugated immunoglobulin subtype or IgG subclass specific detection antibodies were added.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>IgG subclass specific detection antibodies</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">These include 1:6000 dilution of anti-total human IgG (Jackson Immunoresearch), 1:3000 dilution of anti-human IgA (α-chain-specific) (Sigma) or 1:3000 dilution of anti-human IgM (μ-chain-specific) (Sigma) antibody subtypes and 1:6000 dilution of anti-human IgG1, IgG2, IgG3 or IgG4 (Southern Biotech, USA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-total human IgG</div><div>suggested: (Cell Signaling Technology Cat# 13123, RRID:AB_2617178)</div></div><div style="margin-bottom:8px"><div>anti-human IgA ( α-chain-specific ) ( Sigma )</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-human IgM ( μ-chain-specific ) ( Sigma )</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-human IgG1</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>IgG2</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>IgG3</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>IgG4</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To assess whether the uptake of the microbeads opsonised with the plasma of patients with acute disease was via Fc-receptor (antibody-dependent), and other heat labile opsonins such as complements (eg C3b), either the Fc-receptors on the THP-1 cells were pre-blocked using the universal Fc receptor blocking agent (Miltenyi, USA) [23], or the plasma heat inactivated at 56°C for 30 minutes, as described [28].</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>antibody-dependent) ,</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Surface plasmon resonance: Surface plasmon resonance (SPR) was performed using a Biacore T200 (Cytiva, USA) to determine the binding characteristics of the anti-Spike polyclonal antibodies in patient plasma to SARS-CoV-2 Spike antigen.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SARS-CoV-2 Spike antigen .</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Friedman’s test with pairwise Dunn’s tests were used to compare the repeated measures of Spike p-score, Spike and RBD end point titre, neutralisation titre and antibody affinity across the timepoints V1, V2 and V3.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>V3</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To rank the variables of statistical importance that associated with Spike p-score (dependent variable), multiple linear regression analysis was performed using disease severity, age, gender, DPS, anti-Spike/RBD endpoint antibody titre, anti-Spike/RBD antibody subtypes and neutralisation titre as independent variables.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-Spike/RBD</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To assess whether the uptake of the microbeads opsonised with the plasma of patients with acute disease was via Fc-receptor (antibody-dependent), and other heat labile opsonins such as complements (eg C3b), either the Fc-receptors on the THP-1 cells were pre-blocked using the universal Fc receptor blocking agent (Miltenyi, USA) [23], or the plasma heat inactivated at 56°C for 30 minutes, as described [28].</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>THP-1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Retroviral SARS-CoV-2 Spike pseudovirus were generated in 293T cells by co-transfecting expression plasmids containing SARS-CoV-2 Spike and MLV gag/pol and luciferase vectors using Calphos transfection kit (Takara Bio, USA) as described [20].</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T</div><div>suggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, pseudovirus were incubated for one hour with heat inactivated (56 °C for 30 minutes) patient serum prior to infecting 293T-ACE2 cells (kindly provided by A/Prof Jesse Bloom) by a two hour spinoculated at 800xg in 96-well white flat bottom plates in triplicates (Sigma-Aldrich, USA)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T-ACE2</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Biotinylated recombinant SARS-CoV-2 Spike and RBD proteins: SARS-CoV-2 Wuhan-Hu-1 (GenPept: QJE37812) RBD protein (amino acid residues 319–541) and Spike protein (amino acid residue 15-1213) were cloned into pCEP4 mammalian expression vector containing N-terminal human Ig kappa leader sequence and C-terminal Avi-tag and His-tag (Invitrogen, USA)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCEP4</div><div>suggested: RRID:Addgene_16479)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After a two hours incubation, cells were washed once with 1mL of cold PBS containing 0.5% FBS and 0.005% of sodium azide and gentle centrifugation at 335xg for five minutes at 4°C, fixed in 400μL of 1% paraformaldehyde and kept at 4°C in the dark until acquisition of data using BD FACSCalibur™ Flow cytometer.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BD FACSCalibur™</div><div>suggested: (BD FACSCalibur Flow Cytometry System, RRID:SCR_000401)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A total of 2×104 events were acquired and the proportions of cells that phagocytosed the beads (% of cells that took up the beads) and their fluorescent intensities (amounts of beads taken up per cell) were analysed using BD FlowJo version 10.5.0 software.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>FlowJo</div><div>suggested: (FlowJo, RRID:SCR_008520)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All the images were Airyscan processed with Zen Black Edition (Zeiss Software).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Zen</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Relative Luminescence Unit (RLU) in cell lysates was measured using CLARIOstar microplate reader (BMG Labtech, Australia), percentage neutralisation of Spike pseudovirus determined and the fifty percent inhibitory (ID50) calculated using non-linear regression model (GraphPad Prism version 9.0) [29]</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Kinetic constants, including association constant (Ka), equilibrium constant (KD) (affinity) and dissociation constant (Ka) (avidity), were calculated using BIAcore evaluation software version 4.1 [30].</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BIAcore</div><div>suggested: (Biacore T100 System, RRID:SCR_019679)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Statistical analysis: All data were analysed with Prism Software (version 9.0, GraphPad, USA)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Prism</div><div>suggested: (PRISM, RRID:SCR_005375)</div></div></td></tr></table>

---

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.

---

<footer>

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

</footer>

SciScore for 10.1101/2021.12.19.473391: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IRB: donors: Convalescent plasma, Ad26.COV2.S, and some BNT162b2 samples were obtained from the HAARVI study approved by the University of Washington Human Subjects Division Institutional Review Board (STUDY00000959).</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">Authentication: None of the cell lines used were authenticated or tested for mycoplasma contamination.<br>Contamination: None of the cell lines used were authenticated or tested for mycoplasma contamination.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Infected cells were then washed an additional five times with DMEM prior to adding media supplemented with anti-VSV-G antibody (I1-mouse hybridoma supernatant diluted 1:25, from CRL-2700, ATCC) to reduce parental background.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-VSV-G</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cell lines: Cell lines used in this study were obtained from ThermoFisher Scientific (HEK293T and Expi293F) or were kindly gifted by Florian</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, HEK-293T cells seeded in poly-D-lysine coated 100 mm dishes at ∼75 % confluency were washed five times with Opti-MEM and transfected using 24 µg of the S glycoprotein plasmid with Lipofectamine 2000 (Life Technologies).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK-293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pseudotyped VSV neutralization assay: To evaluate neutralization of D614G, Delta (B.1.617.2), and Omicron (B.1.1.529) pseudotypes by plasma of vaccinees or previously infected individuals, Vero-TMPRSS2 cells in DMEM supplemented with 10% FBS, 1% PenStrep, and 8 ug/mL puromycin were seeded at 60-70% confluency into white clear-bottom 96 well plates (Corning) and incubated at 37°C.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero-TMPRSS2</div><div>suggested: JCRB Cat# JCRB1818, RRID:CVCL_YQ48)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Media was removed from the cells and 40 µL from each well (containing plasma and pseudovirus) was transferred to the 96-well plate seeded with Vero-TMPRESS2 cells and incubated at 37°C for 2 h.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero-TMPRESS2</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The SARS-CoV-2-RBD-Avi construct was synthesized by GenScript into pcDNA3.1-with an N-terminal mu-phosphatase signal peptide and a C-terminal octa-histidine tag, flexible linker, and avi tag (GHHHHHHHHGGSSGLNDIFEAQKIEWHE).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3.1-with</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">fold-on trimerization motif, and an 8× His tag in the pCMV vector.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMV</div><div>suggested: RRID:Addgene_16459)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The plasmids encoding the SARS-CoV-2 Omicron (B.1.1.529) S variant was generated by overlap PCR mutagenesis of the wildtype plasmid, pcDNA3.1(+)-spike-D19 (80)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3.1</div><div>suggested: RRID:Addgene_79663)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Prism (GraphPad) nonlinear regression with “[inhibitor] versus normalized response with a variable slope” was used to determine ID50 values from curve fits with 2-3 repeats.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Prism</div><div>suggested: (PRISM, RRID:SCR_005375)</div></div><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

---

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.

---

<footer>

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

</footer>

SciScore for 10.1101/2021.12.20.473401: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">Field Sample Permit: Experiments involving animals were conducted using protocols approved by the National Research Council Canada Animal Care Committee and in accordance with the guidelines set out in the OMAFRA Animals for Research Act, R.S.O. 1990, c. A.22. b)<br>IACUC: Experiments involving animals were conducted using protocols approved by the National Research Council Canada Animal Care Committee and in accordance with the guidelines set out in the OMAFRA Animals for Research Act, R.S.O. 1990, c. A.22. b)</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">The denatured-induced unfolding of VHHs was considered to be reversible based on their small size as shown to be the case for small proteins and numerous times for VHHs 56–59, 88, 89. d) Serum stability: Three female Syrian hamsters were injected intraperitoneally (IP) with 1 mg of 1d or VHH-72 VHH-Fc diluted in 200 µL PBS.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Reactions were stopped by adding 50 µL 1 M H2SO4 to wells, and absorbance were subsequently measured at 450 nm using a Multiskan™ FC photometer (Thermo Fisher). b) Binding to cognate anti-spike glycoprotein polyclonal antibody: The four spike glycoprotein antigens were passively adsorbed as described above.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-spike glycoprotein</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After blocking with PBSC, wells were emptied, washed five times with PBST and incubated at room temperature for 1 h with 100 µL of 1 µg/mL anti-SARS-CoV-2 spike rabbit polyclonal antibody (Sino Biological, Beijing, China, Cat#40589-T62) in PBSCT.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-SARS-CoV-2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Following 1 h incubation at room temperature, wells were washed 10 times with PBST and incubated with HRP-conjugated polyclonal goat anti-llama IgG heavy and light chain antibody (Bethyl Laboratories, Montgomery, TX, Cat#A160-100P) for 1 h at room temperature.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-llama IgG heavy and light chain</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To determine the presence of antibodies that block the binding of S to ACE2 (surrogate for neutralization) in the immune sera of llamas, 400 ng of chemically biotinylated SARS-CoV-2 S was mixed with 1 × 105 Vero E6 cells in the presence of 2-fold dilutions of sera (pre immune, day 21 and day 28 sera) in a final volume of 150 µL.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ACE2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Then plates were washed 10 times with PBST and binding of VHHs to S1-Fc was detected with rabbit anti-6xHis Tag antibody HRP Conjugate (Bethyl Laboratories, Cat#A190-114P), diluted at 10 ng/mL in PBST and added at 100 µL/well.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-6xHis</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After 1 h incubation at room temperature, strips were washed 10 times with PBST and the binding of VHH-Fcs to denatured S was probed by incubating strips with 1 mL of 100 ng/mL anti-human Ig Fc antibody-peroxidase conjugate (Jackson ImmunoResearch, Cat#016-030-084) at room temperature for 1 h.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human Ig Fc antibody-peroxidase conjugate</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After 1 h incubation at room temperature, plates were washed 10 times with PBST and the ACE2-Fc binding was detected using 1 µg/mL goat anti-human IgG (Fc specific) HRP conjugate antibody (Sigma, Cat#A0170) in 100 µL PBSCT.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human IgG</div><div>suggested: (Sigma-Aldrich Cat# A0170, RRID:AB_257868)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Immune cell infiltrate was detected using rabbit polyclonal antibodies against CD3 (1:500, Dako, Cat#A0452), and Iba-1 (ionized calcium binding adaptor protein, 1:5000, Dako, Cat#019-19741)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CD3</div><div>suggested: (Agilent Cat# A0452, RRID:AB_2335677)</div></div><div style="margin-bottom:8px"><div>Iba-1 ( ionized calcium binding adaptor protein</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Mouse anti-SARS-CoV-2 N monoclonal antibody (1:5000, R&D Systems, Cat#MAB10474) was used for the detection of SARS-CoV-2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Mouse anti-SARS-CoV-2 N monoclonal antibody</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-SARS-CoV-2 N</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Conversely, a binding response was seen during the second injection for VHHs that did not compete with ACE2. c) ACE2 competition assay by flow cytometry: Experiments were performed as described in 4.3c, except that biotinylated S/Vero E6 cells were mixed with VHHs or VHH-Fcs instead of sera.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>S/Vero E6</div><div>suggested: RRID:CVCL_JX48)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pseudotyped and live virus neutralization assays: a) Pseudotyped virus neutralization assays: (i) Generation of SARS-CoV-2 spike pseudotyped lentiviral particles (LVP): HEK293T cells were plated in a 100-mm tissue culture dish and transfected the next day at about 75% confluency with a combination of a lentiviral transfer vector encoding eGFPLuc (addgene#119816), the packaging plasmid psPAX2 (addgene#12260), and a plasmid encoding the viral glycoprotein of interest SARS-CoV-2 Spike-ΔS1/S2-Δ20 expressed in pcDNA3.1+.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The pellet was then resuspended in PBS buffer at 1/10 of the original supernatant volume with gentle up-and-down pipetting, aliquoted and stored at −80°C. (ii) Viral neutralization assays: HEK293T-hACE2 cell line (BEI Resources, Manassas, VA, Cat#NR-52511) were seeded in poly-L-Lysine (PLL)-coated white, clear bottom 384-wells plate (NUNC, Thermo Fisher) at a density of 9,000 cells/well in 45 µL of media (DMEM without phenol red supplemented with 5% [v/v] FBS) and incubated for 24 h at 37°C, 5% CO2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T-hACE2</div><div>suggested: RRID:CVCL_A7UK)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Quantitative microneutralization assay was performed on Vero E6 cells with SARS-CoV-2 strains hCOV-19/Canada/ON-VIDO-01/2020, NR-53565; hCOV- 19/England/204820464/2020, NR-54000; or hCOV-19/South Africa/KRISP-EC-K005321 /2020, NR-54008.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero E6</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Virus titer was determined by plaque assay on Vero cells. b) Immunohistochemistry: Lungs were immersed in 10% neutral buffered formalin and fixed for 1 week at room temperature and then transferred into 70% ethanol.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">VH/VHH genes were amplified using semi-nested PCR and cloned into the phagemid vector pMED1, followed by transformation of E. coli TG1 (Lucigen, Middleton, WI, Cat#60502-02) to construct two libraries with sizes of 1 × 107 and 2 × 107 independent transformants for Green and Red, respectively.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pMED1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Expression and purification of VHHs and VHH-Fcs: a) Expression and validation of VHHs: Positive VHHs were cloned into a modified pET expression vector (pMRo.BAP.H6) for their production in BL21(DE3) E.coli as monomeric soluble protein 84.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pET</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pMRo.BAP.H6</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For the VHH-72 benchmark 29, the sequence of the VHH was synthesized as a GeneBlock (Integrated DNA Technologies, Coralville, IA) flanked by SfiI sites for cloning into pMRo.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pMRo</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Production of VHHs in mammalian cells in fusion with human IgG1 Fc (VHH-Fcs): Codon-optimized genes for bivalent VHH-Fcs were synthesized and cloned into pTT5 (GenScript; Piscataway, NJ.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pTT5</div><div>suggested: RRID:Addgene_52326)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pseudotyped and live virus neutralization assays: a) Pseudotyped virus neutralization assays: (i) Generation of SARS-CoV-2 spike pseudotyped lentiviral particles (LVP): HEK293T cells were plated in a 100-mm tissue culture dish and transfected the next day at about 75% confluency with a combination of a lentiviral transfer vector encoding eGFPLuc (addgene#119816), the packaging plasmid psPAX2 (addgene#12260), and a plasmid encoding the viral glycoprotein of interest SARS-CoV-2 Spike-ΔS1/S2-Δ20 expressed in pcDNA3.1+.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>psPAX2</div><div>suggested: RRID:Addgene_12260)</div></div><div style="margin-bottom:8px"><div>pcDNA3.1+</div><div>suggested: RRID:Addgene_117272)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After a final wash, cells were resuspended in 100 µL PBSB and data were acquired on a CytoFLEX S flow cytometer (Beckman Coulter, Brea, CA) and analyzed by FlowJo software (FlowJo LLC, v10.6.2</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>FlowJo</div><div>suggested: (FlowJo, RRID:SCR_008520)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Proteins were buffer exchanged using Amicon® Ultra-15 Centrifugal Filter Units (Millipore-Sigma</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Millipore-Sigma</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">IC50 was determined from non-linear regression [Inhibitor] vs. response, Variable slope (four parameters) using GraphPad Prism version 9 (La Jolla, CA). 4.11.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

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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: 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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About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

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SciScore for 10.1101/2021.12.18.473303: (What is this?)

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

Table 1: Rigor

NIH rigor criteria are not applicable to paper type.

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibodies and Virtual screening for anti-nsp5 compounds are described in the Supplementary Methods Cells and virus: HEK-293T cells (purchased from ATCC, # CRL-11268) and A549 cells stably expressing the human ACE2 receptor (A549-ACE2, kindly provided by O. Schwartz, Institut Pasteur) were grown in complete Dulbecco’s modified Eagle’s medium (Gibco) supplemented with 10% fetal bovine serum (FBS).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-nsp5</div><div>suggested: (Grupo de la Dra. Susana Lopez; Instituto de Biotecnologia, UNAM. Cat# NSP5 (C-6, RRID:AB_2802098)</div></div><div style="margin-bottom:8px"><div>A549-ACE2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Immunoblot membranes were incubated with primary antibodies directed against nsp5 [31], NS3/4A (ab21124, Abcam), FLAG-tag (F3165, Sigma) or GAPDH (MA5-15738, Invitrogen), and revealed with HRP-linked secondary antibodies (Jackson ImmunoResearch) using ECL2 substrate (Pierce).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>FLAG-tag</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>GAPDH</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>MA5-15738 , Invitrogen) ,</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibodies and Virtual screening for anti-nsp5 compounds are described in the Supplementary Methods Cells and virus: HEK-293T cells (purchased from ATCC, # CRL-11268) and A549 cells stably expressing the human ACE2 receptor (A549-ACE2, kindly provided by O. Schwartz, Institut Pasteur) were grown in complete Dulbecco’s modified Eagle’s medium (Gibco) supplemented with 10% fetal bovine serum (FBS).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>A549</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Transfected HEK-293T cells were trypsinized at 6 hpt and distributed in the 384-well plates (2×104 cells per well in 50 µL).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK-293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antiviral activity and cell viability assays: A549-ACE2 cells seeded in 384-well plates (1.5×103 cells per well) were incubated with the compound of interest at the indicated concentration in DMEM-2% FBS 2 h prior to infection.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>A549-ACE2</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">These plasmids were used as templates to amplify the sequence encoding nsp5 (WT or C145A), and the resulting amplicons were subcloned into the pcDNA3.1 plasmid.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3.1</div><div>suggested: RRID:Addgene_79663)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The pLEX expression vectors for SARS-CoV-2, IBV and hCoV-229E nsp5 are described in [26] and were kindly provided by N. Heaton (Duke University).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pLEX</div><div>suggested: RRID:Addgene_117987)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The Hepatitis C Virus (HCV) NS3/4A expression vector was generated by subcloning a synthetic gene which was codon optimized for expression in human cells (Genscript) into the pCI plasmid.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCI</div><div>suggested: RRID:Addgene_74230)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To generate the reverse-Nanoluciferase reporter plasmid Rev-Nluc-CoV, a synthetic codon-optimized nucleotide sequence encoding a permuted Nanoluciferase with the NGSVRLQSSLK linker between the N- and the C-terminal domains was cloned into the pLV-CMV-eGFP lentiviral vector (Duke vector core, Duke University) in place of the eGFP insert.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>reverse-Nanoluciferase reporter</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pLV-CMV-eGFP</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Viral protease activity assays: HEK-293T cells were seeded in 96-well white opaque plates (Greiner Bio-One, 3×104 cells per well) one day before being transfected with 5 ng of the Rev-Nluc-CoV reporter plasmid and 95 ng of the pcDNA3.1-nsp4-5-6 WT, pcDNA-3.1-nsp4-5-6 C145A, pLEX-nsp5 or an empty control pCI plasmid, using polyethyleneimine (PEI-max, #24765-1 Polysciences Inc).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3.1-nsp4-5-6 WT</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pcDNA-3.1-nsp4-5-6 C145A</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pLEX-nsp5</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To measure NS3/4A activity, 5 ng of the Rev-Nluc-HCV reporter plasmid was co-transfected with 145 ng of the pCI-NS3/4A WT or pCI-NS3/4A S139A, using the same protocol.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCI-NS3/4A</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pCI-NS3/4A S139A</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In the 384-well plate setting, ∼107 HEK-293T cells were transfected in 50 cm2 dishes with 1 µg of the Rev-Nluc-CoV plasmid and 15 µg of the nsp4-5-6 WT or nsp4-5-6 C145A expression plasmids.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Rev-Nluc-CoV</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The Luna Universal One-Step RT-qPCR Kit (New England Biolabs) was used, with SARS-CoV-2 specific primers targeting the N gene region (5’-TAATCAGACAAGGAACTGATTA-3’ and 5’-CGAAGGTGTGACTTCCATG-3’) and with the following cycling conditions: 55 °C for 10 min, 95 °C for 1 min, and 40 cycles of 95 °C for 10 sec, followed by 60 °C for 1 min, in an Applied Biosystems QuantStudio 6 thermocycler.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SARS-CoV-2</div><div>suggested: (BioLegend Cat# 946101, RRID:AB_2892515)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">ChEMBL protocols [56]); Removal of chemical duplicates (Open Babel [57]); Selection of dominant tautomers (pH 7.4, ChemAxon Calculator Plugins version 19.0.9, https://chemaxon.com); Selection of species populating more than 25% or, if none, species that are present at more than 75% of the most frequent one, or at least the three most frequent ones; Generation of stereoisomers and low energy ring conformers generation (CORINA classic [58] version 3.4, https://www.mn-am.com);</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ChEMBL</div><div>suggested: (ChEMBL, RRID:SCR_014042)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Molecules were kept in the sdf format for docking with FlexX [40] and converted into pdbqt files using MGLTools scripts [63]for docking with Smina [39].</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MGLTools</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">We used Smina (version Dec 2020 based on Autodock Vina 1.1.2, exhaustiveness set to 12, 10 best score poses saved) to perform docking targeting all three PDB structures.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Autodock</div><div>suggested: (AutoDock, RRID:SCR_012746)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">It also comprised 5RH8, which gave the best results with FlexX, allowing to diversify the screening with FlexX, while providing a basis for comparison between the two programs.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>FlexX</div><div>suggested: (FlexX, RRID:SCR_000186)</div></div></td></tr></table>

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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: We found the following clinical trial numbers in your paper:<br><table><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Identifier</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Status</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Title</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT04960202</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Recruiting</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">EPIC-HR: Study of Oral PF-07321332/Ritonavir Compared With P…</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT05011513</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Recruiting</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Evaluation of Protease Inhibition for COVID-19 in Standard-R…</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT05047783</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Recruiting</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Masitinib in Patients With Symptomatic Mild to Moderate COVI…</td></tr></table>

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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: 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.

---

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

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<footer>

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

</footer>

SciScore for 10.1101/2021.12.16.472155: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IRB: Mouse studies: Ethical approval: All the experimental procedures were performed in accordance with the guide for the use of laboratory animals of the University of Sao Paulo and approved by the institutional ethics committee under the protocol number 105/2021. SARS-CoV-2: SARS-CoV-2 was isolated from a COVID-19 positive-tested patient.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">SARS-CoV-2 experimental infection and treatments: Female K18-hACE2 mice, aged 8 weeks, were infected with 2×104 PFU of SARS-CoV-2 (in 40 μL) by intranasal route.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">A total of 10 photomicrographs in 40X magnification per animal were randomly obtained using a microscope Novel (Novel L3000 LED, China) coupled to a HDI camera for images capture.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Expression and purification of Spike RBD of SARS-CoV-2: A codon-optimized gene encoding for SARS-CoV-2 (331 to 528 amino acids, QIS60558.1) was expressed in Expi293 cells (Thermo Fisher Scientific) with human serum albumin secretion signal sequence and fusion tags (6xHistidine tag, Halo tag, and TwinStrep tag) as described before 1.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Expi293</div><div>suggested: RRID:CVCL_D615)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HUVEC single cell donor (Lonza, cat#C2517A) cells were transduced at room temperature with ACE2 using a BacMam viral vector at a concentration of 2e9 VG/ml (Montana Molecular #C1120G Pseudo SARS-CoV-2 D614G Green Reporter) followed by incubation at 36°C for 24 hours.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HUVEC</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">SARS-Cov-2 tested in A549-ACE2 cells: A549-ACE2 cells were plated in Corning black walled clear bottom 96 well plates 24 hours before infection for confluency.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>A549-ACE2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">SARS-Cov-2 tested in Calu-3 cells: Calu-3 (ATCC, HTB-55) cells were pretreated with test compounds for 2 hours prior to continuous infection with SARS-CoV-2 (isolate USA WA1/2020) at a MOI=0.5.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Calu-3</div><div>suggested: ATCC Cat# HTB-55, RRID:CVCL_0609)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Supernatant from the Caco-2 cells are collected on day 3 post-infection and titrated on Vero 76 cells for virus titer as before.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Caco-2</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>Vero 76</div><div>suggested: KCLB Cat# 21587, RRID:CVCL_0603)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HCoV 229E antiviral assay: HCoV 229E, (a gift from Ralph Baric, UNC, Chapel Hill) was propagated on Huh-7 cells and titers were determined by TCID50 assay on Huh-7 cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Huh-7</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The virus was propagated and titrated in Vero E6 cells in a biosafety level 3 laboratory (BSL3) at the Ribeirao Preto Medical school (Ribeirao Preto, Brazil).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero E6</div><div>suggested: RRID:CVCL_XD71)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Penicillin 10,000 U/mL; Streptomycin10,000 μ Vero cells in DMEM (FBS 2%) incubated at 37 °C with 5% CO2 for 48 h.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Organisms/Strains</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">K18-hACE2 mice: To evaluate the effects of vandetanib in vivo, we infected the K18-hACE2 humanized mice (B6.Cg-Tg(K18-ACE2)2Prlmn/J)7, 8, 9.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>B6.Cg-Tg(K18-ACE2)2Prlmn/J)7</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">K18-hACE2 mice were obtained from The Jackson Laboratory and were breed in the Centro de Criação de Animais Especiais</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>K18-hACE2</div><div>suggested: RRID:IMSR_GPT:T037657)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">MHV-A59 with nano-Luciferase: The MHV-A59 G plasmid was engineered to replace most of the coding sequence for orf4a and 4b with nano-luciferase (nLuc).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>The MHV-A59 G</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A sequence verified G-nLuc plasmid was used with MHV-A59 wild type A, B, C, D, E and F plasmids to recover virus expressing nLuc, using our previously described molecule clone (Systematic assembly of a full-length infectious cDNA of mouse hepatitis virus strain A59 4.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>G-nLuc</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The total septal area and total area were analyzed with the aid of the Pro Plus 7 software (Media Cybernetics, Inc., MD, USA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Pro Plus</div><div>suggested: (Image-Pro Plus, RRID:SCR_016879)</div></div></td></tr></table>

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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: We found the following clinical trial numbers in your paper:<br><table><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Identifier</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Status</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Title</td></tr><tr style="background-color:#FF0000"><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT0427541464</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Trial number did not resolve on clinicaltrials.gov. Is the number correct?</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NA</td></tr></table>

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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: 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.

---

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

---

<footer>

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

</footer>

SciScore for 10.1101/2021.12.15.472864: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The IgG antibodies and Fabs were purified with a CaptureSelect™ CH1-XL Matrix column (Thermo Fisher Scientific) for affinity purification and a HiLoad Superdex 200 pg column (Cytiva) for size exclusion chromatography.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Sequences of the antibodies against SARS-CoV-2 were included in further analysis.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SARS-CoV-2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">11 antibody (BioLegend, 901524), PE-conjugated goat anti-human IgG polyclonal antibodies (Southern Biotech, 2040-09), and propidium iodide (Invitrogen, P1304MP) for 20 minutes on ice.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human IgG</div><div>suggested: (SouthernBiotech Cat# 2040-09, RRID:AB_2795648)</div></div><div style="margin-bottom:8px"><div>2040-09</div><div>suggested: (SouthernBiotech Cat# 2040-09, RRID:AB_2795648)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pseudovirus production and neutralization assays: Plasmids encoding SARS-CoV, SARS-CoV-2, or other variants of the spike protein, with the ER retrieval signal removed were co-transfected with MLV-gag/pol and MLV-CMV-Luciferase plasmids into HEK293T cells to generate pseudoviruses.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Right before the end of the incubation, HeLa-hACE2 cells were suspended with culture medium at a concentration of 2 x 105/ml.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HeLa-hACE2</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Expression and purification of SARS-CoV-2 RBD: The receptor-binding domain (RBD) (residues 333-529) of the SARS-CoV-2 spike (S) protein (GenBank: QHD43416.1), was cloned into a customized pFastBac vector (Ekiert et al., 2011), and fused with an N-terminal gp67 signal peptide and C-terminal His6 tag (Yuan et al., 2020).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pFastBac</div><div>suggested: RRID:Addgene_1925)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">YYDRxG pattern search: Over 205,000 antibody sequences including heavy and light chains were retrieved from GenBank using Biopython program (Cock et al., 2009), supplemented with sequences reported in previous publications (Cho et al., 2021; Gaebler et al., 2021; Robbiani et al., 2020; Z. Wang, J. C. C. Lorenzi, et al., 2021; Z. Wang, F. Muecksch, et al., 2021; Z. Wang, F. Schmidt, et al., 2021), and then subjected to repertoire analysis using PyIR program (Soto et al., 2020) implemented with IgBLAST (Ye et al., 2013).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Biopython</div><div>suggested: (Biopython, RRID:SCR_007173)</div></div><div style="margin-bottom:8px"><div>PyIR</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>IgBLAST</div><div>suggested: (IgBLAST, RRID:SCR_002873)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Sequence analysis and surface conservation: Sequences of sarbecoviruses were retrieved from GenBank using Biopython program, aligned using MUSCLE program (Edgar, 2004) built in European Bioinformatics Institute (EBI) web services (Madeira et al., 2019) and scored for surface conservation in ConSurf server (Ashkenazy et al., 2016; Landau et al., 2005).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MUSCLE</div><div>suggested: (MUSCLE, RRID:SCR_011812)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The conserved surface of SARS-CoV-2 RBD was visualized using the PyMOL program (Schrödinger, LLC).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PyMOL</div><div>suggested: (PyMOL, RRID:SCR_000305)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Iterative model building and refinement were carried out in COOT (Emsley & Cowtan, 2004) and PHENIX (Acta Crystallographica Section D: Biological CrystallographyAdams et al., 2010), respectively.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>COOT</div><div>suggested: (Coot, RRID:SCR_014222)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Epitope and paratope residues, as well as their interactions, were identified with the PISA program (Krissinel & Henrick, 2007) using calculated buried surface area (BSA >0 Å2) as the criterion.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PISA</div><div>suggested: (PISA, RRID:SCR_015749)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Binding curves were fitted with four-parameter non-linear regression analysis to calculate the apparent equilibrium binding constant (KDApp) in GraphPad Prism 9.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

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Results from OddPub: Thank you for sharing your data.

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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.

---

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.

---

<footer>

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

</footer>

SciScore for 10.1101/2021.12.14.472668: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">Field Sample Permit: Human blood collection and isolation of platelets: Platelet studies using human samples were conducted according to the principles of the Declaration of Helsinki and approved by St Thomas’s Hospital, London, UK Research Ethics Committee (Ref. 07/Q0702/24).<br>IRB: Human blood collection and isolation of platelets: Platelet studies using human samples were conducted according to the principles of the Declaration of Helsinki and approved by St Thomas’s Hospital, London, UK Research Ethics Committee (Ref. 07/Q0702/24).<br>Consent: All volunteers were screened prior to entering the study and gave written informed consent.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">Of these, 25 were male and 16 were female; the average age was 77 for men and 84 for women.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After washing 3 times with PBS, the plate was permeabilized with 0.5% Triton (Sigma) for 10 min, blocked with 1% BSA for 1 hr at room temperature (RT) and stained with anti-GFP antibody (1:1000; Abeam #ab6556) or anti-V5 tag antibody (1:500; Invitrogen #37-7500) for 2 hr at RT, and Cell Mask (1:2000; Thermo Fisher Scientific) for 30 min at RT.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-V5</div><div>suggested: (SICGEN Cat# AB0096, RRID:AB_2333109)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Blots were then incubated (4°C overnight) with primary antibodies against Spike, tubulin or TMEM16F.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>antibodies against Spike, tubulin</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Membranes were incubated for 1 hr at room temperature with anti-rabbit HRP-conjugated antibody (1:5,000) or anti-mouse HRP-conjugated antibody (1:10,000).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-rabbit</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-mouse</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibodies: Immunofluorescence analysis was performed for actin (AlexaFluor 488 Phalloidin, A12379, ThermoFisher Scientific) and GFP (ab6556, Abcam).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>actin</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>GFP</div><div>suggested: (Abcam Cat# ab6556, RRID:AB_305564)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Immunoblots were performed with primary antibodies against Spike (Genetex, #GTX632604; 1:1,000), tubulin (Cell Signaling, #3873S; 1:10,000) and TMEM16F (Sigma-Aldrich, #HPA038958; 1:1,000).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>antibodies against Spike (Genetex,</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>, tubulin (Cell Signaling,</div><div>suggested: (Cell Signaling Technology Cat# 86298, RRID:AB_2715541)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pseudotyped viral particles were produced as follows: HEK293T cell (2.5 × 106) were seeded in a 100 mm-dish and co-transfected by calcium phosphate with 10 μg pLVTHM/GFP (Addgene 12247), 7.5 μg psPAX2 (Addgene 12260), and 6 μg pMD2.G (Addgene 12259) or pEC120-S-D19-V5, for a total of 13.5 μg DNA for each dish.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To assess pseudoparticle infectivity, HEK-293T cells were bulk transfected with a plasmid expressing human ACE2 and then seeded in a 96-well plate (3×103 cells per well).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK-293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For platelet aggregation on Spike-expressing cells, Vero cells were kept in culture in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% of foetal bovine serum (FBS) and transfected using FuGENE HD (Promega) transfectant reagent as suggested by the manufacturer either with a plasmid encoding the Green Fluorescent Protein (GFP; pZac-GFP) or a plasmid encoding the full-length SARS-CoV-2 Spike protein with V5 tag (pSARS-COV-2-S).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The modified DNA segment was obtained by recombinant PCR using primers Fw 5’ ACGCGTCGACTTTTGTGGCAAAGGTT, Re 5’ CCCAAGCTTGGGACGCGTCGTTACGTAGAATCGAGACCGAGGAGAGGGTTAGGGATAGGCTTACCACCACCTCCACCGCAGCATGATCCGCATGAGC and cloned into the pEC117-Spike-V5 vector33.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pEC117-Spike-V5</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pseudotyped viral particles were produced as follows: HEK293T cell (2.5 × 106) were seeded in a 100 mm-dish and co-transfected by calcium phosphate with 10 μg pLVTHM/GFP (Addgene 12247), 7.5 μg psPAX2 (Addgene 12260), and 6 μg pMD2.G (Addgene 12259) or pEC120-S-D19-V5, for a total of 13.5 μg DNA for each dish.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pLVTHM/GFP</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>psPAX2</div><div>suggested: RRID:Addgene_12260)</div></div><div style="margin-bottom:8px"><div>pMD2.G</div><div>suggested: RRID:Addgene_12259)</div></div><div style="margin-bottom:8px"><div>pEC120-S-D19-V5</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, PRP was incubated with VSV-G or Spike pseudoparticles (1:10) for 10 min at 37°C, followed by stimulation with collagen (30 μg/ml) for 20 min (350 rpm, 37°C).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>VSV-G</div><div>suggested: RRID:Addgene_138479)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Analysis was performed using the ImageJ software (Fiji).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Fiji</div><div>suggested: (Fiji, RRID:SCR_002285)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Analysis was performed using ImageJ software (Fiji).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ImageJ</div><div>suggested: (ImageJ, RRID:SCR_003070)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Analysis was performed using the FlowJo software v.10 (TreeStar Inc).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>FlowJo</div><div>suggested: (FlowJo, RRID:SCR_008520)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">PRP was incubated with VSV-G (1:10), Spike pseudoparticles (1:10), His-tag recombinant SARS-CoV-2 Spike Protein S1/S2 (S-ECD) (1 ng/ml; ThermoFisher Scientific; aa11-1208; RP-87680) or Flag-tag recombinant SARS-CoV-2 Spike RBD (1 ng/ml; Bio-Techne, 10689-CV-100) for 10 min at 37°C, followed by stimulation with thrombin (0.5 units).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ThermoFisher Scientific</div><div>suggested: None</div></div></td></tr></table>

---

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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About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

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lista multiple

color==concept; tag==value from a dominion

ok, good answer

SciScore for 10.1101/2021.12.14.472513: (What is this?)

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

Table 1: Rigor

<table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>

Table 2: Resources

<table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The primary antibodies used were rabbit anti-ACE2 (1:1000, ab15348, Abcam)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-ACE2</div><div>suggested: (Abcam Cat# ab15348, RRID:AB_301861)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">, rabbit anti-TACE (1:1000, 3976S, Cell Signaling Technology, MA, USA), rabbit anti-ADAM10 (1:1000, 14194S, Cell Signaling Technology), rabbit anti-Flag-tag (1:1000, PM020, MBL, MA, USA)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-TACE</div><div>suggested: (Leinco Technologies Cat# T460, RRID:AB_2831960)</div></div><div style="margin-bottom:8px"><div>anti-ADAM10</div><div>suggested: (LSBio (LifeSpan Cat# LS-C122598-1000, RRID:AB_10800328)</div></div><div style="margin-bottom:8px"><div>anti-Flag-tag</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">, mouse anti-tubulin (1:1000, CP06, Millipore), and mouse anti-VSVM (1:1000, 23H12, Absolute antibody).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-tubulin</div><div>suggested: (LSBio (LifeSpan Cat# LS-C76623-1000, RRID:AB_10633290)</div></div><div style="margin-bottom:8px"><div>CP06</div><div>suggested: (Millipore Cat# CP06, RRID:AB_2617116)</div></div><div style="margin-bottom:8px"><div>anti-VSVM</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The Secondly antibodies used were HRP-linked donkey anti-rabbit IgG antibody (NA934; GE Healthcare, Piscataway, NJ, USA) and HRP-linked donkey anti-mouse IgG antibody (NA931V; GE Healthcare)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-rabbit IgG</div><div>suggested: (GE Healthcare Cat# NA934, RRID:AB_772206)</div></div><div style="margin-bottom:8px"><div>HRP-linked donkey anti-mouse IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cells were then incubated with anti-SARS-CoV-2 nucleocapsid (1:1000, GTX135357, GeneTex, CA, USA) primary antibody for 16 h at 4 °C and detected with anti-rabbit-Alexa488 (1:200, A11008, Invitrogen, CA, USA) secondary antibodies for 40 min at RT.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-SARS-CoV-2 nucleocapsid ( 1:1000 , GTX135357</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-rabbit-Alexa488</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>A11008</div><div>suggested: (Molecular Probes Cat# A-11008, RRID:AB_143165)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A704, Calu-3, VeroE6, HEC50B, and Caco-2 cells were maintained in Eagle’s minimum essential medium (EMEM; 055-08975,</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Calu-3</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>Caco-2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">OUMS-23, IGROV1, and 293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; 041-30081,</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HEC50B cells infected with pseudotype viruses were selected with 300 μg/mL hygromycin for at least 1 week.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEC50B</div><div>suggested: JCRB Cat# JCRB1145, RRID:CVCL_2929)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For the DSP assay using 293FT cells, DSP8-11 expressing effector cells expressing S protein and DSP1-7 expressing target cells expressing CD26 or ACE2 alone or together with TMPRSS2 were seeded in 10 cm cell culture plates (4 × 106 cells/10 mL) one day prior to the assay (S1a,b Fig).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293FT</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After an overnight incubation at 37 °C in 5% CO2, cells were treated with protease inhibitors for 1 h and added with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.01 for HEC50B and HEC50B-TMPRSS2 cells, and MOI of 0.1 for VeroE6, Calu-3, and A704 cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEC50B-TMPRSS2</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>A704</div><div>suggested: NCI-DTP Cat# A704, RRID:CVCL_1065)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Organisms/Strains</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">OVTOKO (JCRB1048), OVISE (JCRB1043), HEC50B (JCRB1145), VeroE6-TMPRSS2 (JCRB1819)[67], and OUMS-23 (JCRB1022) cells were obtained from the Japanese Collection of Research Bioresources Cell Bank (Osaka,</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>VeroE6-TMPRSS2 (JCRB1819)</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">VeroE6-TMPRSS2 (JCRB1819) cells were cultured in DMEM containing 10% FBS and 1 mg/mL G418.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>VeroE6-TMPRSS2</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To establish stable cell lines expressing the S protein of SARS-CoV, SARS-CoV-2, or MERS-CoV, recombinant pseudotype lentiviruses were produced in 293T cells with psPAX2 packaging plasmid, vesicular stomatitis virus (VSV)-G-expressing plasmid and lentiviral transfer plasmid expressing S protein.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>VSV)-G-expressing</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">recombinant pseudotype lentivirus expressing TMPRSS2 was produced using 293T cells with psPAX2 packaging plasmid and VSV-G-expressing plasmid.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>psPAX2</div><div>suggested: RRID:Addgene_12260)</div></div><div style="margin-bottom:8px"><div>VSV-G-expressing</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">IGROV1 cells (SCC203) were purchased from Merck (Darmstadt, Germany) and Caco-2 cells (RCB0988) were obtained from the RIKEN BioResource Research Center (Tsukuba, Japan).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>RIKEN BioResource</div><div>suggested: (RIKEN BioResource Center, RRID:SCR_003250)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Statistical analysis: Statistical analyses were performed in Microsoft Excel 2016 (</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Microsoft Excel</div><div>suggested: (Microsoft Excel, RRID:SCR_016137)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">(Microsoft, Redmond, WA, USA) and GraphPad Prism 8 (GraphPad Software, San Diego, CA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>

---

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 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 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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About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

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Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

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Reply to the reviewers

Description of the planned revisions

From Review Commons: Please find below our point-by-point reply that explains what revisions, additional experimentations and analyses are planned to address the points raised by the referees

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Reviewer #1 (Evidence, reproducibility and clarity (Required)):

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Comment #1: In this manuscript, the authors follow up on an interesting finding that varA null mutants of V. cholerae form spherical cells in stationary phase. The authors determine that this cell rounding is due to weakening of the cell wall via less production of tetrapeptide cross links. Mutation of the regulator csrA and the enzyme aspA lead to a model in which a varA mutant cell lacks aspartate leading to low cross-linked cell wall that is unable to hold the typical curved V. cholerae shape. The data are robust, and the manuscript is clearly written.

Authors’ reply #1: We very much appreciate the reviewer’s accurate summary and the appraisal of the robust data and the clearly written manuscript.

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Comment #2: I think the finding is quite interesting, even though it is not clear to me if this observed cell morphology has a biological function or if it is an artifact of completly removing VarA. However, this manuscript builds the foundation to further test this question.

Authors’ reply #2: We agree with the reviewer. However, it is worth mentioning that this two-component system (TCS) has been first described in 1998 yet the input signal (or repression of the signal under certain conditions) still remains elusive. Maybe, this isn’t too surprising, given that studies on V. cholerae are strongly biased towards its pathogenic lifestyle, while the varA/varS system is highly conserved among Gram-negative bacteria including non-pathogenic environmental V. cholerae strains. These strains can live under very diverse conditions of slow or fast growth, including long starvation periods. Unfortunately, we still lack significant insight into this part of the V. choleraebiology. We therefore believe that the current study is very important, as the elucidation of the molecular mechanism of the observed shape transition within the varA mutant will foster fresh hypotheses on the role that the system plays in V. cholerae and what signals might be sensed.

We would also like to remind the editor and reviewer(s) that a plethora of studies have been published based on varA and varS (and csrA) deletion mutants of V. cholerae with various readouts ranging from transcriptomics to quorum sensing defects, impairment of virulence, etc. Thus, the argument that the complete removal of varA might cause an artifact seems equally valid for previous work by others and, maybe, even the vast majority of studies in which TCS are investigated for which the sensed signal has yet to be identified.

In conclusion, we propose to address this valid point of critique in the revised manuscript by clearly stating the caveat of the gene deletion(s). However, as the reviewer correctly stated, “this manuscript builds the foundation to further test this question.”

Comment #3: The data all support the conclusions, but I do think the authors could have really confirmed their model by connecting mutations in csrA and aspA to restoration of high cross-linked cell well similar to the WT strain as done in Fig. 2. As it stands, this is still somewhat hypothetical and has not been directly demonstrated, although I do think their model is correct and these experiments will be conformation of it.

Authors’ reply #3: We thank the reviewer for their comment and the assumption that our model might be correct. It is very unlikely that the csrA suppressor mutant(s) or the ∆varA∆aspA mutant maintain the low level of cross-links and the high level of dipeptides that we observed for the ∆varA mutant. Indeed, it would be unclear how the cells could restore the Vibrio shape that we visualized in the phase contrast image under such conditions. However, as this point seems very important to the reviewer (see also comment #7 below), we will perform the suggested cell wall analysis of these mutants and include the new data in the revised manuscript.

Comment #4: I also have a few other suggestions to improve the manuscript, but in sum I think it is a well-done research study that will be interesting to research in V. cholerae and other gamma proteobacteria.

Authors’ reply #4: Once again, we thank the reviewer for their kind words.

Major comments:

Comment #5: 1. The enrichment for suppressors is very creative and connected the varA impact on cell morphology to misregulation of csrA as 10/10 mutants were ultimately linked to this gene. However, insertion in aspA should also suppress this phenotype, and I am curious why this gene was not identified in the transposon suppressor screen.

Authors’ reply #5: This is a very relevant and important comment. The reason why we did not isolate ∆varA-aspA::Tn mutants is most likely due to a growth defect that we observed for the double ∆varA∆aspA mutant compared to the ∆varA-csrA suppressor mutant(s). In the figure on the right, respective growth curves are shown [∆varA∆aspA in orange and the ∆varA-csrA suppressor mutant ∆varA-Tn A in gray]. Any ∆varA-aspA::Tn mutant is therefore expected to be outcompeted by the ∆varA-csrA suppressor mutants during the enrichment process. We will include this information and the corresponding data (e.g., final growth curves after 3 biologically independent experiments) in the revised manuscript.

[figure not shown in online form]

Comment #6: 2. The authors should complement at least one of their varA/csrA mutants with csrA.

Authors’ reply #6: Agreed. We are in the process of performing the suggested experiment and will include the results in the revised manuscript.

Comment #7: 3. The changes in cell wall structure are not directly connected to the genetic identification of csrA and aspA. Yes, I agree their model makes sense, but to really nail it down they should analyze the cell wall composition in the varA/csrA and varA/aspA double mutants and show it returns to WT levels of crosslinking.

Authors’ reply #7: As mentioned above, we will perform those cell wall analyses (see also authors’ reply #3 above), as requested.

Comment #8: 4. Does deletion of aspA in the WT or varA mutant impact the growth rate?

Authors’ reply #8: This is indeed the case in the ∆varA background but not in the WT background (as shown under authors reply #5). These data will be included in the revised manuscript.

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Minor comments

Comment #9: 5. Are the round cells able to divide? The data in Fig. S2 would suggest they can based on the increase in CFUs from hour 6 to hour 8, but the authors never comment on this point and it might be worth addressing in the discussion.

Authors’ reply #9: We never observed diving round cells. Indeed, the increase of the optical density and CFUs from 6h to 8h is most likely based on those bacteria that have not yet changed their cellular morphology and therefore keep dividing (see below the imaging/quantification for this timeframe taken from Fig. 2). What we observed though is that upon dilution into fresh medium, the round cells start to elongate and then divide resulting in newborn Vibrio shaped cells. We will include these new data in the revised manuscript.

[figure not shown in online form]

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Comment #10: 6. Fig. S2-Why does the OD600 increase from 8 to 24 hours but the CFUs decrease in the varA mutant?

Authors’ reply #10: This is an interesting observation that might reflect the presence of dead but not yet lysed cells in these cultures. Indeed, while it looks as if the OD600 values are still increasing for the ∆varA mutant at 24h, we cannot exclude at this point that the OD600 values increased during the 16h-time interval and went again down at 24h (e.g., like shifting the WT peak to later time points/the right of the X-axis). Notably, the purpose of this figure was mostly to i) indicate the slower growth of the ∆varA mutant while ii) emphasizing that late during growth (e.g., 24h here) the strain can still reach similar OD600 values as well as CFUs/ml as the WT strain. We will change Fig. S2 to better emphasize these two points in the revised manuscript.

Comment #11: 7. Lines 309-A little bit more detail here would help the reader. Are the authors examining whole cell lysates or lysates from specific cellular components? I am actually very surprised this worked as there are so many proteins in crude cell lysates.

Authors’ reply #11: Indeed, these are whole cell lysates, which were prepared as described in the methods section (lines 482 onwards; “SDS-PAGE, Western blotting and Coomassie blue staining”). We fully agree that there are many proteins in the crude cell lysates and realized that we might not have explained well enough that only the gel region containing the overproduced band in the ∆varA strain and the same location in the WT sample were analyzed by mass spectrometry (even though we were referring to the “gel pieces” in line 498 onwards). Please accept our sincerest apologies for this neglect. During the revision, we will ensure that this information is explicitly stated and that these details are included in the main text and the methods section.

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Comment #12: 8. Lines 320-321-I don't think there is evidence that CsrA enhances aspA RNA translation, merely that CsrA enhances AspA protein production. It is likely through increasing translation, but this cannot be concluded without direct evidence.

Authors’ reply #12: We fully agree and thank the reviewer for this important comment. Indeed, we meanwhile know that the aspA mRNA levels also increase in the ∆varA mutant strain (which might or might not be linked to enhanced translation). We will add these transcript level data to the revised manuscript and discuss all possibility that could explain the AspA overproduction.

Comment #13: 9. Line 348-350-I do not understand the logic of this sentence stating that the "..until now, the signal that abrogates VarA phosphorylation..." as this manuscript does not contribute to our understanding of the VarS signal.

• *Authors’ reply #13: We apologize that this sentence or the logic behind it wasn’t clear. As this is a combined result and discussion section, the aim of the sentence was to put the observed shape transition of the bacteria into a broader context, which required us to mentioned that the input signal is still unknown. We will make sure that this becomes more obvious in the revised manuscript by rephrasing this sentence.

Comment #14: 10. I am curious if the total volume of the round versus curved cells is constant at 20 hours. This should be easy to determine using ImageJ and worth reporting.

Authors’ reply #14: We are not entirely sure how this question is relevant for the study (e.g., for this report on the observed shape transition phenotype it doesn’t matter if the cells maintain the same volume or not). However, given the importance for the reviewer, we will perform these volume measurements on our images and add a sentence to the revised manuscript on the analysis’ outcome (plus include the data as a supplementary panel).

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Reviewer #1 (Significance (Required)):

Comment #15: Understanding changes to cell morphology and their biological implications is a growing area of microbiology. This study makes a new contribution to this area by demonstrating a round, spherical form of V. cholerae that is driven by alterations to the cell that decrease cell-wall cross linking.

Authors’ reply #15: Once again, we thank the reviewer for this summary and for placing our study into context. We agree that cell morphological changes and the underlying molecular mechanism(s) are an exciting and growing area of microbiology.

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

Comment #16: In this manuscript, Rocha et al studied the effect of the VarA response regulator on cell shape of Vibrio cholerae. VarA is part of a two-component system that also includes the histidine kinase VarS. It has previously been shown that VarA activates the expression of three redundantly acting regulatory RNAs called CsrB, CsrC, and CsrD. All three Csr RNAs share the same regulatory principle, which is to sequester the activity of the RNA-binding protein CsrA. CsrA in turn can bind hundreds of mRNA species in the cell, which in the majority of cases results in reduced translation of these mRNAs (in addition to various other modes of action that have been reported). Here, the authors discovered that deletion of the varA gene results in an abnormal, spherical cell shape in stationary phase grown V. cholerae cells. Biochemical analysis revealed an unusual peptidoglycan composition in varA-deficient cells, showing increased levels of dipeptides, a reduction of tetrapeptides, and an overall decrease in peptide-cross linkage. Interestingly, the varA phenotype was complemented by the addition of conditioned medium from wild-type cells, which are likely to provide peptidoglycan building blocks in trans. The authors further discover that varA-deficiency results in AspA over-production, which could be linked to the activity of CsrA. The authors speculate that high AspA levels deplete the cell of aspartate, which is required to produce peptidoglycan precursors. The manuscript is interesting, well-written, and the rationale of the experiments is easy to follow.

Authors’ reply #16: We thank the reviewer for this excellent summary and the kind words on the quality of the manuscript.

Comment #17: However, I have two major points of criticism, which reduce my enthusiasm for this work. First, the molecular pathways that links varA-deficiency to increased AspA levels is incomplete: please clarify how CsrA activates AspA levels and if this phenotype is linked to direct binding of CsrA to the aspA mRNA and if so how is activation is achieved at the molecular level.

Authors’ reply #17: We thank the reviewer for the comment. However, we never had the intention to decipher the entire pathway and it is indeed possible that intermediate regulators might be involved. Notably, the first part of the signaling pathway (VarA -> CsrB,C,D -> CsrA) seemed well established in the literature and we truly believe that our work supports this part of the pathway (given the numerous csrA suppressor mutants that we obtained in the varA-minus background). For the link between CsrA and AspA, we indeed do not provide direct evidence. Nonetheless, we discuss recent work in Salmonella by mentioning “Interestingly, previous studies identified the aspA mRNA amongst hundreds of direct CsrA targets in Salmonella using the CLIP-seq technique to identify protein-RNA interactions (32)”. Notably, this finding by Holmqvist et al. (2016, EMBO J.) has been reproduced for E. coli by Potts et al. (2017, Nat. Commun.; see Supplementary Data file 1, CsrA CLIP-seq data; with three highly significant peaks corresponding to aspA mRNA binding), an information that we will add to the revised manuscript. Of course, neither Salmonella nor E. coli belongs to the same genus as V. cholerae (though, of course, all are gamma-Proteobacteria). Thus, to accommodate the reviewer’s comment, we will revise the manuscript to include the caveat that direct CsrA binding of the aspA mRNA has been shown in both Salmonella and E. coli but that it is still feasible that intermediate regulatory proteins might be involved in the case of V. cholerae. We will also revise the model to show such potential intermediate steps between CsrA and AspA.

Comment #18: Second, I am not convinced about the biological relevance of the findings. The authors speculate in the discussion section that the VarA-pathway could modulate cell shape under physiological conditions, however, I am not sure such conditions exist given that VarA activity is not only controlled by VarS, but rather integrates information from multiple histidine kinases. I have a several additional comments, which I listed below.

Authors’ reply #18: We regret the referee’s personal opinion that our findings might not be of biological relevance.

However, we respectfully disagree with the notion that such physiological conditions would never occur just because several histidine kinases can feed into VarA signaling. Indeed, as discussed above under authors’ reply #2, the (VarS/)VarA-CsrA pathway is highly conserved in Vibrio species and other proteobacteria. Yet, for V. choleraemost studies have focused on virulence-inducing conditions, while the species’ environmental lifestyle has been vastly neglected in the past. Indeed, even our own work on several V. cholerae’s phenotypes (natural competence for transformation [Meibom*, Blokesch* et al., 2005, Science]; T6SS production in pandemic strains [Borgeaud et al., 2015, Science]; pilus-mediated aggregation [Adams et al., 2019, Nat. Microbiol]; etc.) has remained unknown for decades, given the chitin dependency for their induction – a substrate not commonly studied in lab settings. Interestingly, several of these findings have also initially been considered biologically irrelevant, “artifacts”, or even non-reproducible by reviewers in the past, while nowadays all these phenotypes have been extensively reproduced by many different research groups and are well accepted in the field as biologically highly relevant. Thus, we truly believe that one should be open to new phenotypes and, as reviewer #1 rightfully acknowledged, consider that “this manuscript builds the foundation to further test this question.”

It should also be noted that the (VarS/)VarA-CsrA system has been studied for >15 years based on deletion strains, as we did in this study, and the readouts of these studies have been well accepted in the field without provision of the physiological conditions that would mimic the situation of these knock-out strains.

Collectively, we truly believe that there are still many understudied physiological conditions for V. cholerae; however, finding the right conditions could take years and is therefore beyond the scope of the current study.

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Major points:

Comment #19: - Figs. 1 and S1: I think it is interesting that the varS mutant strain does not share the cell shape phenotype with the varA mutant. As pointed out by the authors, this result indicates that varA activity is controlled by another histidine kinase. While I believe it might be beyond the scope of this manuscript to determine which other histidine kinases signal towards VarA, I think it would be useful to measure and compare CsrB/C/D levels in WT, DvarA, and DvarS cells.

Authors’ reply #19: Thanks for this comment. We fully agree that finding the secondary histidine kinase is beyond the scope of this study. In the revised manuscript, we will, however, include the CsrB/C/D levels of the WT, ∆varA, and ∆varS strains, as suggested by the reviewer.

Comment #20: - Figs. 1, S1, and 4C: The regulatory logic implied by these results suggest that deletion of varA results in reduced CsrB/C/D levels, which in turn leads to higher activity of CsrA in the cell. Thus, it would be useful to test if A) over-production of CsrB, CsrC, or CsrC can rescue the phenotype of an varA mutant and if B) combined deletion of csrB/C/D will phenocopy the mutation of varA.

Authors’ reply #20: These are also very good suggestions. Notably, this has been done in the past for the V. cholerae system (Lenz et al., 2005, Mol. Microbiol.). Indeed, after receiving the reviewer’s comment, we immediately asked these authors to kindly share their csrB,C,D overproduction plasmids as well as the triple knock-out strain with us (as all of these constructs have been extensively verified in their published work). Unfortunately, we are not entirely sure whether it will be possible to receive these constructs any time soon, as we were told that such shipment might take >1 year (though, upon further discussion, this timeframe was lowered to ~3 months). If we manage to receive these published constructs in a reasonable timeframe, we will certainly perform the suggested experiments.

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Comment #21: - Figs. 4B & 5C: I was somewhat surprised by these results. Given that AspA overproduction is suggested to cause cell shape abnormalities in the varA mutant, I would have expected additional transposon insertion in aspA. The fact that mutations only occurred in csrA could indicate that additional (CsrA-controlled) could be involved in the phenotype.

Authors’ reply #21: See authors’ reply #5 above, where we explain that the ∆varA∆aspA strain has a slight growth disadvantage. For this reason, any ∆varA-aspA::Tn transposon mutant would likely be outcompeted by the csrAsuppressor mutants in our genetic screen.

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Minor points:

Comment #22: - Throughout study: italicize gene names

Authors’ reply #22: Gene names have been italicized in the initial manuscript; however, strain names - such as strain ∆varA – haven’t been italicized, in accordance with several of our previous publications. However, for the revision, we will italicize all strain names to accommodate the reviewer’s request.

Comment #23: - Figs. 1C and S3C: please quantify the results of these western blots and indicate how many replicates were performed.

Authors’ reply #23: We apologize for this oversight – indeed, all Western Blot were performed three independent times, as is good scientific practice. We will add this information into the methods section of the revised manuscript.

Concerning the quantification: the primary claim of these figures is that the HapR protein is still produced in the ∆varA mutant in the different pandemic strain backgrounds, while the luxO-mutated strains have a significant defect in HapR production (as we have previously reported; Stutzmann and Blokesch, 2016, mSphere). These data are qualitatively very clear in the Western Blots and can be considered as “black or white” results.

However, for the revision we will quantify the bands’ intensities of the performed Western blots and provide these quantitative data, as requested by the reviewer.

Comment #24: - Fig. 5A and B: in order to properly quantify the levels of AspA in the cell (and link them to CsrA activity in the transposon mutants), I think it would be better to add a tag to the chromosomal aspA gene and perform quantitative Western blot analysis.

Authors’ reply #24: We respectfully disagree. Firstly, this is not a subtle difference that we observe in these cell lysates/the corresponding stained gel bands but a rather strong difference when WT is compared to the mutants (see, for instance, a copy of panel 5B below as a kind reminder). Together with the genetic experiments that follow afterwards, the link seems very solid to us. Secondly, adding a tag could change the proteins abundance (change of the protein’s production/degradation dynamics) and/or activity, which could cause more confusion than needed (and a loss of the spherical cell shape if the enzyme loses its activity through the tagging).

However, as mentioned above under authors’ reply#12, we meanwhile observed that the aspA mRNA levels also increase in the ∆varA mutant. Thus, we will provide qRT-PCR data in the revised manuscript (and discuss all options on how the increase of the transcript and subsequently the protein might be caused, as mentioned above under authors’ reply #12), which we truly believe will fulfill the reviewer’s request for quantification.

[figure not shown in online form]

Reviewer #2 (Significance (Required)):

Comment #25: I think this manuscript starts with an interesting observation, which is that varA mutant cells of V. cholerae display an aberrant cell shape. The manuscript also provides several important findings explaining the molecular basis of this phenotype.

Authors’ reply #25: Once again, we thank the reviewer for the kind words.

Comment #26: However, as pointed out in my report, I think the manuscript is yet incomplete in connecting this information to identify the underlying regulatory mechanism.

Authors’ reply #26: As mentioned above, the focus of this study was never on the elucidation of the entire regulatory pathway. Instead, we aimed at deciphering the molecular mechanism behind an observed phenotype - that is, the cell wall modification in the varA-deficient strain that leads to the bacterium’s spherical shape, which can be restored to the WT Vibrio shape by peptidoglycan precursor cross-feeding from neighboring cells – followed by the identification of several regulators and enzymes that trigger these phenotypes. Overall, we consider this a very complete study. However, as mentioned above, we will certainly discuss in the revised manuscript that the step between CsrA and AspA could be indirect in V. cholerae, in contrast to what was experimentally shown for Salmonella and E. coli.

**Referee Cross-commenting**

Comment #27: As pointed out in my review, I think this manuscript is well written and easy to follow. However, I agree with reviewer #1 that the underlying phenotype is most likely an artifact, which limits the biological relevance of this study. In addition, I am missing the molecular mechanism that connects CsrA with AspA production in V. cholerae.

Authors’ reply #27: See authors’ reply #18 above. We disagree that there is any strong indication that the observed phenotype is an artifact. Given that it is state-of-the-art to study TCS by deleting their genes, our study isn’t any more prone to being an artifact than any other study on TCSs.

We truly believe that it is also important to not take reviewer #1’s comment out of context by stating “I agree with reviewer #1 that the underlying phenotype is most likely an artifact”. Indeed, he/she provided a rather encouraging statement in which he/she mentions the possibility of an artifact but also clearly states that this study is interesting and builds the foundation to further investigate the newly observed phenotype(s): “I think the finding is quite interesting, even though it is not clear to me if this observed cell morphology has a biological function or if it is an artifact of completly removing VarA. However, this manuscript builds the foundation to further test this question.”

Moreover, whether there is a direct (as in Salmonella and E. coli) or indirect connection between CsrA and AspA production is not a key aspect of the current study, as discussed above.

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