Evolution of Antibody Immunity to SARS-CoV-2

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1. 

   ScreenIT

   Mar 1, 2021

   SciScore for 10.1101/2020.11.03.367391: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	not detected.
   Randomization	The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment.
   Blinding	The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	Authentication: This laboratory-developed test has been authorized by New York State under an Emergency Use Authorization (EUA) for use by authorized laboratories.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   The Elecsys antinSARSnCoV-2 assay uses a recombinant protein representing …

   SciScore for 10.1101/2020.11.03.367391: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	not detected.
   Randomization	The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment.
   Blinding	The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	Authentication: This laboratory-developed test has been authorized by New York State under an Emergency Use Authorization (EUA) for use by authorized laboratories.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   The Elecsys antinSARSnCoV-2 assay uses a recombinant protein representing the N antigen for the determination of antibodies against SARSnCoVn2.	SARSnCoVn2 suggested: None
   Plates were washed 6 times with washing buffer and then incubated with anti-human IgG, IgM or IgA secondary antibody conjugated to horseradish peroxidase (HRP) (Jackson Immuno Research 109-036-088	anti-human IgG suggested: (Jackson ImmunoResearch Labs Cat# 109-036-088, RRID:AB_2337594) IgA suggested: None
   The enriched B cells were incubated in FACS buffer (1× PBS, 2% FCS, 1 mM EDTA) with the following anti-human antibodies (all at 1:200 dilution): anti-CD20-PECy7 (BD Biosciences, 335793), anti-CD3-APC-eFluro 780 (Invitrogen, 47-0037-41)	anti-human suggested: (GenWay Biotech Inc. Cat# 18-202-335793-0.1 mg, RRID:AB_1981874) anti-CD20-PECy7 suggested: None anti-CD3-APC-eFluro 780 suggested: None
   The frequency distributions of human V genes in anti-SARS-CoV-2 antibodies from this study was compared to 131,284,220 IgH and IgL sequences generated by 54 and downloaded from cAb-Rep55, a database of human shared BCR clonotypes available at https://cab-rep.c2b2.columbia.edu/.	anti-SARS-CoV-2 suggested: None
   Slides were washed 3 times in PBS and then incubated in secondary antibody and 4′,6-diamidino-2-phenylindole (1ug/mL) for 1 hour at room temperature.	4′,6-diamidino-2-phenylindole suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Briefly, 293T cells were transfected with pNL4-3ΔEnv-nanoluc and pSARS-CoV-2-SΔ19.	293T suggested: None
   Software and Algorithms
   Sentences	Resources
   Plasma samples were assayed on the Pylon 3D analyzer (ET HealthCare, Palo Alto, CA) as previously described 4.	ET HealthCare suggested: None
   The average of its signal was used for normalization of all of the other values on the same plate with Excel software before calculating the area under the curve using Prism V8.4 (GraphPad).	Excel suggested: None Prism suggested: (PRISM, RRID:SCR_005375)
   The half-maximal inhibitory concentration for plasma (NT50) or monoclonal antibodies (IC50) was determined using four-parameter nonlinear regression (least squares regression method without weighting; constraints: top=1, bottom=0) (GraphPad Prism).	GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)
   High-dimensional data analysis of flow cytometry data: viSNE and FlowSOM analyses were performed on B cells using the Cytobank platform (https://cytobank.org). viSNE analysis was performed using equal sampling of 4893 cells from each FCS file, with 7500 iterations, a perplexity of 30, and a theta of 0.5.	FlowSOM suggested: (FlowSOM, RRID:SCR_016899) Cytobank suggested: (Cytobank, RRID:SCR_014043)
   Single CD3−CD8−CD14−CD16−CD20+Ova−RBD-PE+RBD-AF647+ B cells were sorted into individual wells of 96-well plates containing 4 μl of lysis buffer (0.5× PBS, 10 mM DTT, 3,000 units/ml RNasin Ribonuclease Inhibitors (Promega, N2615) per well using a FACS Aria III and FACSDiva software (Becton Dickinson) for acquisition and FlowJo for analysis.	FACSDiva suggested: (BD FACSDiva Software, RRID:SCR_001456) FlowJo suggested: (FlowJo, RRID:SCR_008520)
   Sequence analysis was performed using MacVector.	MacVector suggested: (MacVector, RRID:SCR_015700)
   7k) was created with R pheatmap package (	pheatmap suggested: (pheatmap, RRID:SCR_016418)
   Probes were assessed by NCBI BLAST to exclude off target binding to other cellular transcripts.	BLAST suggested: (BLASTX, RRID:SCR_001653)
   Data presentation: Figures arranged in Adobe Illustrator 2020.	Adobe Illustrator suggested: (Adobe Illustrator, RRID:SCR_010279)

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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.

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   Read the original source
2. 

   ScreenIT

   Nov 6, 2020

   SciScore for 10.1101/2020.11.03.367391: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement

   Informed consent was obtained from all participants.

   Randomization

   The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment.

   Blinding

   The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment.

   Power Analysis

   not detected.

   Sex as a biological variable

   not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   7,8,27-31 . 9,10,12,34-37, . a–d, Results of serological assays measuring plasma reactivity to RBD (a,b,c) and N protein (d) at the initial 1.3 and 6.2 month follow-up visit, respectively. a, Anti-RBD IgM. b, Anti-RBD IgG. …

   SciScore for 10.1101/2020.11.03.367391: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement

   Informed consent was obtained from all participants.

   Randomization

   The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment.

   Blinding

   The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment.

   Power Analysis

   not detected.

   Sex as a biological variable

   not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   7,8,27-31 . 9,10,12,34-37, . a–d, Results of serological assays measuring plasma reactivity to RBD (a,b,c) and N protein (d) at the initial 1.3 and 6.2 month follow-up visit, respectively. a, Anti-RBD IgM. b, Anti-RBD IgG. c, Anti-RBD IgA d, Anti-N total antibodies.	Anti-RBD IgM. suggested: None Anti-RBD IgG suggested: None Anti-RBD IgA suggested: None
   Positive and negative controls were included for validation 1. e, Relative change in plasma antibody levels between 1.3 and 6.2 months for anti-RBD IgM, IgG, IgA and anti-N total Ig, respectively. f-i, Relative change in antibody levels between 1.3 and 6.2 months plotted against the corresponding antibody levels at 1.3 months. i, Anti-N total antibodies.	anti-RBD IgM, IgG suggested: None anti-N total Ig, suggested: None Anti-N total antibodies. suggested: None
   The Elecsys anti‐SARS‐CoV-2 assay uses a recombinant protein representing the N antigen for the determination of antibodies against SARS‐CoV‐2.	SARS‐CoV‐2 suggested: None
   Plates were washed 6 times with washing buffer and then incubated with anti-human IgG, IgM or IgA secondary antibody conjugated to horseradish peroxidase (HRP) (Jackson Immuno Research	anti-human IgG suggested: None IgA suggested: None
   The enriched B cells were incubated in FACS buffer (1× PBS, 2% FCS, 1 mM EDTA) with the following anti-human antibodies (all at 1:200 dilution): anti-CD20-PECy7 (BD Biosciences, 335793), anti-CD3-APC- eFluro 780 (Invitrogen, 47-0037-41)	anti-human suggested: (GenWay Biotech Inc. Cat# 18-202-335793-0.1 mg, RRID:AB_1981874) anti-CD20-PECy7 suggested: None anti-CD3-APC- eFluro 780 suggested: None
   2: Correlations of plasma antibody measurements. a, Normalized AUC for IgG anti-RBD plotted against Pylon IgG anti-RBD index values at 1.3 months. b, Normalized AUC for IgG anti-RBD plotted against Pylon IgG anti-RBD index values at 6.2 months. c, Normalized AUC for IgM anti-RBD plotted against Pylon IgM anti-RBD index values at 1.3 months. d, Normalized AUC for IgM anti-RBD plotted against Pylon IgM anti-RBD index values at 6.2 months. e, Normalized AUC for IgM anti-RBD at 6.2 months plotted against IgM anti-RBD at 1.3 months. f, Normalized AUC for IgG anti-RBD at 6.2 months plotted against IgG anti-RBD at 1.3 months. g, Normalized AUC for IgA anti-RBD at 6.2 months plotted against IgA anti-RBD at 1.3 months. h, COI values for anti-N total Ig titres at 6.2 months plotted against anti-N total Ig titres at 1.3 months. i, Anti-RBD IgG titres at 1.3 months plotted against anti-N total Ig titres at 1.3 months. j, Anti-RBD IgG titres at 6.2 months plotted against anti-N total Ig titres at 6.2 months. k, Anti-RBD IgM titres at 1.3 months plotted against anti-N total Ig titres at 1.3 months. l, Anti-RBD IgM titres at 6.2 months plotted against anti-N total Ig titres at 6.2 months. m, Anti-RBD IgA titres at 1.3 months plotted against anti-N total Ig titres at 1.3 months. l	IgM suggested: None anti-RBD suggested: None anti-N suggested: None
   Repertoire ** IGLV4−69 IGKV1−16 IGKV2−26 * IGLV1−36 IGKV1−6 IGLV7−43 **** IGKV2−40 IGLV4−3 IGKV1−43 IGLV2−18 ** Extended Data Fig. 4: Frequency distributions of human V genes. a, Two-sided binomial tests were used to compare the frequency distributions of human V genes of anti-SARS-CoV-2 antibodies from donors at 1.3 months to 6.2 months 1. b, Two-sided binomial tests were used to compared the frequency distributions of human V genes of anti-SARS-CoV-2 antibodies from this study to sequence from C. Soto et al. 49. b, For each patient, the number of IgG heavy chain sequences (black) analyzed from six individuals at month 1.3 (left panel) or month 6.2 post- infection (right panel).	anti-SARS-CoV-2 suggested: None
   Software and Algorithms
   Sentences	Resources
   Plasma samples were assayed on the Pylon 3D analyzer (ET HealthCare, Palo Alto, CA) as previously described 4.	ET HealthCare suggested: None
   The average of its signal was used for normalization of all of the other values on the same plate with Excel software before calculating the area under the curve using Prism V8.4 (GraphPad).	Excel suggested: None Prism suggested: (PRISM, RRID:SCR_005375) GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)
   Single CD3−CD8−CD14−CD16−CD20+Ova−RBD- PE+RBD-AF647+ B cells were sorted into individual wells of 96-well plates containing 4 μl of lysis buffer (0.5× PBS, 10 mM DTT, 3,000 units/ml RNasin Ribonuclease Inhibitors (Promega, N2615) per well using a FACS Aria III and FACSDiva software (Becton Dickinson) for acquisition and FlowJo for analysis.	FACSDiva suggested: (BD FACSDiva Software, RRID:SCR_001456) FlowJo suggested: (FlowJo, RRID:SCR_008520)
   Sequence analysis was performed using MacVector.	MacVector suggested: (MacVector, RRID:SCR_015700)
   8k) was created with R pheatmap package (	pheatmap suggested: (pheatmap, RRID:SCR_016418)
   Tomographic tilt series and large-area montages were acquired automatically using the SerialEM software package were collected at 1° intervals as samples were tilted +/- 62°.	SerialEM suggested: (SerialEM, RRID:SCR_017293)
   Tomograms were calculated, analyzed and modeled using the IMOD software package 56,57 on MacPro and iMac Pro computers (Apple, Inc).	IMOD suggested: (IMOD, RRID:SCR_003297)

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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.

   ---

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   Read the original source
3. 

   Version published to 10.1101/2020.11.03.367391 on bioRxiv

   Nov 5, 2020