Comprehensive mapping of mutations in the SARS-CoV-2 receptor-binding domain that affect recognition by polyclonal human plasma antibodies
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SciScore for 10.1101/2020.12.31.425021: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources After the serum incubations, the libraries were secondarily labeled with 1:100 FITC-conjugated anti-MYC antibody (Immunology Consultants Lab, CYMC-45F) to label for RBD expression and 1:200 Alexa-647- or DyLight-405-conjugated goat anti-human-IgA+IgG+IgM (Jackson ImmunoResearch 109-605-064 or 109-475-064, respectively) to label for bound serum antibodies. anti-MYCsuggested: Noneanti-human-IgA+IgG+IgMsuggested: NoneBeads (manufacturer-reported binding … SciScore for 10.1101/2020.12.31.425021: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources After the serum incubations, the libraries were secondarily labeled with 1:100 FITC-conjugated anti-MYC antibody (Immunology Consultants Lab, CYMC-45F) to label for RBD expression and 1:200 Alexa-647- or DyLight-405-conjugated goat anti-human-IgA+IgG+IgM (Jackson ImmunoResearch 109-605-064 or 109-475-064, respectively) to label for bound serum antibodies. anti-MYCsuggested: Noneanti-human-IgA+IgG+IgMsuggested: NoneBeads (manufacturer-reported binding capacity of 10-40 ug/mL anti-RBD antibodies) were incubated with human serum at a 3:1 ratio beads:serum (150 uL beads + 50 uL serum), rotating overnight at 4°C. anti-RBDsuggested: NoneA magnet was used to separate antibodies that bind RBD from the supernatant, and the supernatant (the post-RBD antibody depletion sample) was removed. post-RBDsuggested: NoneDilution series of the “synthetic” sera comprised of the anti-RBD antibody rREGN10987 (Hansen et al., 2020) or anti-NTD antibody 4A8 (Chi et al., 2020) and pooled pre-pandemic human sera from 2017-2018 (Gemini Biosciences; nos. 100–110, lot H86W03J; pooled from 75 donors) were performed such that the anti-spike antibody was present at a highest concentration of 0.25 μg/mL. anti-NTDsuggested: Noneanti-spikesuggested: NoneExperimental Models: Cell Lines Sentences Resources 6e5 293T cells per well were seeded in 6-well plates in 2 mL D10 growth media (DMEM with 10% heat-inactivated FBS, 2 mM l-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin). 293Tsuggested: NoneSoftware and Algorithms Sentences Resources Depletion of RBD-binding antibodies from polyclonal sera: Magnetic beads conjugated to the SARS-CoV-2 RBD (AcroBiosystems, MBS-K002) were prepared according to the manufacturer’s protocol. AcroBiosystemssuggested: (ACRObiosystems, RRID:SCR_012550)Results from OddPub: Thank you for sharing your code and data.
Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:There are several limitations to our study. Most importantly, we only examined mutations to the RBD. While we and others (Piccoli et al., 2020; Steffen et al., 2020) have shown that RBD-binding antibodies contribute the majority of the serum neutralizing activity of most convalescent human sera and plasma, antibodies also target other regions of the spike. For example, mutations and deletions in the NTD can affect serum antibody neutralization (Andreano et al., 2020; Kemp et al., 2020b; Liu et al., 2020a; McCarthy et al., 2020; Voss et al., 2020), and are certainly of great importance. In addition, we only mapped samples from 11 individuals at two time points. Given the substantial inter- and intra-individual heterogeneity, mapping more samples may identify additional sites of importance. On a technical level, we assayed binding of antibodies to isolated RBD expressed by yeast, which implies several limitations. First, we are unable to map the effects of mutations that alter the spike’s overall conformation or affect antibodies spanning quaternary epitopes (Barnes et al., 2020a). Second, our mapping likely overestimates the contributions of antibodies that bind epitopes that are more accessible on isolated RBD than in the context of full spike (e.g., F456). Finally, the N-linked glycans on yeast-expressed proteins are more mannose-rich than those on mammalian-expressed proteins (Hamilton et al., 2003), which could affect measurements of how N-linked glycans affect antibody bi...
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.
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SciScore for 10.1101/2020.12.31.425021: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources We then used deep sequencing to measure the frequency of each RBD mutation in the initial population and the antibody-escape FACS bin. antibody-escape FACS bin.suggested: NoneAfter the serum incubations, the libraries were secondarily labeled with 1:100 FITC-conjugated anti-MYC antibody (Immunology Consultants Lab, CYMC-45F) to label for … SciScore for 10.1101/2020.12.31.425021: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources We then used deep sequencing to measure the frequency of each RBD mutation in the initial population and the antibody-escape FACS bin. antibody-escape FACS bin.suggested: NoneAfter the serum incubations, the libraries were secondarily labeled with 1:100 FITC-conjugated anti-MYC antibody (Immunology Consultants Lab, CYMC-45F) to label for RBD expression and 1:200 Alexa-647- or DyLight-405-conjugated goat anti-human-IgA+IgG+IgM (Jackson ImmunoResearch 109-605-064 or 109-475-064, respectively) to label for bound serum antibodies. anti-MYCsuggested: Noneanti-human-IgA+IgG+IgMsuggested: NoneA magnet was used to separate antibodies that bind RBD from the supernatant, and the supernatant (the post-RBD antibody depletion sample) was removed. post-RBDsuggested: NoneDilution series of the “synthetic” sera comprised of the anti-RBD antibody rREGN10987 (Hansen et al., 2020) or anti-NTD antibody 4A8 (Chi et al., 2020) and pooled pre-pandemic human sera from 2017-2018 (Gemini Biosciences; nos. 100–110, lot H86W03J; pooled from 75 donors) were performed such that the anti-spike antibody was present at a highest concentration of 0.25 μg/mL. anti-NTDsuggested: Noneanti-spikesuggested: NonePre-pandemic serum alone, without anti-RBD antibody depletion, was used as a negative control, averaged over 2 replicates. anti-RBDsuggested: NoneSerum is a unique identifier for each serum mapped, PID is the patient ID from (Crawford et al., 2020a), subject is the simpler patient identifier used for patients in the current study, the selection serum dilution indicates the reciprocal dilution at which each selection was performed (i.e., 500 is a 1:500 dilution of serum) and the 4 rightmost columns indicate the percentage of each population of cells that fell into the antibody-escape selection gate for the duplicate mutant libraries (lib1 and lib2) and for cells expressing unmutated RBD and incubated with the same dilution of serum as the mutant libraries (WT 1x) or 10-fold less serum (WT 0.1x). antibody-escapesuggested: NoneExperimental Models: Cell Lines Sentences Resources 6e5 293T cells per well were seeded in 6-well plates in 2 mL D10 growth media (DMEM with 10% heat-inactivated FBS, 2 mM l-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin). 293Tsuggested: NoneSoftware and Algorithms Sentences Resources Depletion of RBD-binding antibodies from polyclonal sera Magnetic beads conjugated to the SARS-CoV-2 RBD (AcroBiosystems, MBS-K002) were prepared according to the manufacturer’s protocol. AcroBiosystemssuggested: (ACRObiosystems, RRID:SCR_012550)There are no corresponding raw FACSDiva gating plots for expt_36 (subject K (day 29)) in Figure S2. FACSDivasuggested: (BD FACSDiva Software, RRID:SCR_001456)Results from OddPub: Thank you for sharing your data.
Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
There are several limitations to our study. Most importantly, we only examined mutations to the RBD. While we and others (Piccoli et al., 2020; Steffen et al., 2020) have shown that RBD-binding antibodies contribute the majority of the serum neutralizing activity of most convalescent human sera and plasma, antibodies also target other regions of the spike. For example, mutations and deletions in the NTD can affect serum antibody neutralization (Andreano et al., 2020; Kemp et al., 2020b; Liu et al., 2020a; McCarthy et al., 2020; Voss et al., 2020), and are certainly of great importance. In addition, we only mapped samples from 11 individuals at two time points. Given the substantial inter- and intra-individual heterogeneity, mapping more samples may identify additional sites of importance. On a technical level, we assayed binding of antibodies to isolated RBD expressed by yeast, which implies several limitations. First, we are unable to map the effects of mutations that alter the spike’s overall conformation or affect antibodies spanning quaternary epitopes (Barnes et al., 2020a). Second, our mapping likely overestimates the contributions of antibodies that bind epitopes that are more accessible on isolated RBD than in the context of full spike (e.g., F456). Finally, the N-linked glycans on yeast-expressed proteins are more mannose-rich than those on mammalian-expressed proteins (Hamilton et al., 2003), which could affect measurements of how N-linked glycans affect anti...
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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