SARS-CoV-2 infection induces germinal center responses with robust stimulation of CD4 T follicular helper cells in rhesus macaques

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   ScreenIT

   Jul 11, 2020

   SciScore for 10.1101/2020.07.07.191007: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IACUC: Rhesus Macaques: Eight colony-bred Indian origin rhesus macaques (Macaca mulatta) were housed at the California National Primate Research Center and maintained in accordance with American Association for Accreditation of Laboratory Animal Care guidelines and Animal Welfare Act/Guide.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	Sex distribution within experimental groups was as follows; Infected (n=3 females, n=1 male); Infected + Convalescent Plasma (n=2 males); Infected + Normal Plasma (n=2 males).
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Pooled plasma had a …

   SciScore for 10.1101/2020.07.07.191007: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IACUC: Rhesus Macaques: Eight colony-bred Indian origin rhesus macaques (Macaca mulatta) were housed at the California National Primate Research Center and maintained in accordance with American Association for Accreditation of Laboratory Animal Care guidelines and Animal Welfare Act/Guide.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	Sex distribution within experimental groups was as follows; Infected (n=3 females, n=1 male); Infected + Convalescent Plasma (n=2 males); Infected + Normal Plasma (n=2 males).
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Pooled plasma had a nAb titer of 1:1149, binding antibody titers for SARS-CoV-2 antigen were as follows; anti-S1-IgG, 24.5	anti-S1-IgG suggested: None
   Normal plasma was collected prior to the COVID-19 pandemic and was negative for SARS-Cov2 antibody.	SARS-Cov2 suggested: None
   BAMA for IgG and IgM antibodies to S1, S2 and N proteins: A customized BAMA was developed to simultaneously measure antibodies to the following recombinant SARS CoV-2 proteins (all from SinoBiologicals, Wayne, PA): S1 (#40591-V08H), S2 extracellular domain (#40590-V08B) and nucleocapsid (N; #40588-V08B).	IgM suggested: None S1 suggested: None #40591-V08H suggested: None #40590-V08B suggested: None
   The following humanized (IgG1) monoclonal antibodies were used to estimate concentrations of IgG, IgM, and IgA antibodies in the rhesus serum standard: anti-S1 RBD (Genscript #HC2001), anti-S2 (SinoBiologicals #40590-D001) and anti-NC (Genscript #HC2003).	IgG1 suggested: (Sino Biological Cat# 40590-D001, RRID:AB_2857932) anti-S1 RBD suggested: None anti-S2 suggested: None anti-NC suggested: None
   Human and rhesus IgG antibodies were both detected in these calibration assays using biotinylated affinity-purified goat anti-human IgG γ chain polyclonal antibody (SBA #2048-08).	rhesus IgG suggested: None anti-human IgG suggested: (SouthernBiotech Cat# 2048-08, RRID:AB_2795689)
   In subsequent BAMA assays for SARS CoV-2-specific rhesus macaque IgG antibodies, biotinylated mouse anti-monkey IgG γ chain monoclonal antibody (SBA cat#4700-08) was used as the secondary antibody.	anti-monkey IgG suggested: (SouthernBiotech Cat# 4700-08, RRID:AB_2796070)
   IgM antibodies were detected using biotinylated affinity-purified goat anti-human IgM µ chain polyclonal antibody (SBA#2020-08) which cross-reacts well with macaque IgM.	anti-human IgM suggested: (SouthernBiotech Cat# 2020-08, RRID:AB_2795605) SBA#2020-08 suggested: None
   Macaque IgA antibodies were detected with the antibodies described below. IgG, IgM or IgA antibodies had to be increased 3-fold over the day 0 value to be considered significant ELISA for SARS-specific IgA and antibodies to RBD: These assays were done using methods similar to those described (52) and Immulon 4 microtiter plates (VWR, Radnor, PA) coated with 100ng per well of S1, S2, N or RBD protein (SinoBiological #40592-VNAH).	IgA suggested: None
   5) anti-rhesus IgA monoclonal antibodies, which do not cross-react with human IgA and, when combined, appear to recognize all allotypes of rhesus macaque IgA (Kozlowski, personal observation).	anti-rhesus IgA suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Vero cells (ATCC CCL-81) were used for viral isolation and stock expansion.	Vero suggested: None
   Software and Algorithms
   Sentences	Resources
   Fluorescence was measured using a BD Biosciences FACSymphony™ with FACSDiva™ version 8.0.1 software.	FACSDiva™ suggested: (BD FACSDiva Software, RRID:SCR_001456)
   Compensation, gating and analysis were performed using FlowJo (Version 10)	FlowJo suggested: (FlowJo, RRID:SCR_008520)
   Polyfunctionality plots were generated using SPICE (v 6) (51).	SPICE suggested: (SPICE, RRID:SCR_016603)
   A BioRad Bioplex 200 and BioManager software were used to measure fluorescent intensity and construct standard curves for interpolation of antibody concentrations in test samples.	BioManager suggested: None
   Statistics: Statistical analyses were performed using GraphPad Prism 8.4.2.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)

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

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

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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

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   Results from JetFighter: 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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   Version published to 10.1101/2020.07.07.191007v1 on bioRxiv

   Jul 8, 2020