Complete Mapping of Mutations to the SARS-CoV-2 Spike Receptor-Binding Domain that Escape Antibody Recognition

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1. Version published to 10.1016/j.chom.2020.11.007

   Jan 1, 2021
2. 

   Version published to 10.1101/2020.09.10.292078v2 on bioRxiv

   Sep 28, 2020
3. 

   ScreenIT

   Sep 16, 2020

   SciScore for 10.1101/2020.09.10.292078: (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

   Antibodies
   Sentences	Resources
   Induced cultures were washed and incubated with 400 ng/mL antibody for 1 h at room temperature with gentle agitation, followed by secondary labeling with 1:100 FITC-conjugated anti-Myc to label for RBD expression and 1:200 PE-conjugated goat anti-human-IgG (Jackson ImmunoResearch 109-115-098) to label for bound antibody.	anti-Myc suggested: (Jackson ImmunoResearch Labs Cat# 109-115-098, RRID:AB_2337675) anti-human-IgG suggested: None
   We then computed the “escape fraction” for each barcoded variant in each antibody-selected library, which we define as Ev = F × (nvpost/Npost)/ nvpre/Npre)/ where F is the …

   SciScore for 10.1101/2020.09.10.292078: (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

   Antibodies
   Sentences	Resources
   Induced cultures were washed and incubated with 400 ng/mL antibody for 1 h at room temperature with gentle agitation, followed by secondary labeling with 1:100 FITC-conjugated anti-Myc to label for RBD expression and 1:200 PE-conjugated goat anti-human-IgG (Jackson ImmunoResearch 109-115-098) to label for bound antibody.	anti-Myc suggested: (Jackson ImmunoResearch Labs Cat# 109-115-098, RRID:AB_2337675) anti-human-IgG suggested: None
   We then computed the “escape fraction” for each barcoded variant in each antibody-selected library, which we define as Ev = F × (nvpost/Npost)/ nvpre/Npre)/ where F is the total fraction of the library that escapes antibody binding (these fractions are given as percentages in the bottom two rows of Figure S1C), nvpost and nvpre are the counts of variant v in the RBD library after and before enriching for antibody-escape variants with a pseudocount of 0.5 added to all counts, and and are the total counts of all variants before and after the antibody-escape enrichment.	antibody-escape enrichment. suggested: None
   We then computationally applied two filters to remove variants that fail to express properly folded RBD and so escape antibody binding for that trivial reason rather than antibody-specific escape mutations.	antibody-specific escape mutations. suggested: None
   Cells were stained with recombinant biotinylated ACE2 (ACROBiosystems, AC2-H82E6) and serial dilutions of rCR3022 antibody for 1 h at room temperature, washed with FACS buffer, resuspended in a 1:200 dilution of PE-conjugated streptavidin (ThermoFisher, S866) and APC-conjugated Goat Anti-Human IgG (Jackson Labs, 109-115-098), and incubated on ice for 1 h.	ACE2 suggested: None AC2-H82E6 suggested: None Anti-Human IgG suggested: (Jackson ImmunoResearch Labs Cat# 109-115-098, RRID:AB_2337675)
   SARS-CoV-2 antibodies were added to each of three wells with the respective antibody at 2.5 μg/mL in a 5 μL/well volume (final 0.1 μg/mL concentration of biotinylated antibody) without washing of unlabeled antibody and then incubated for 1 h at ambient temperature.	SARS-CoV-2 suggested: None
   Sequence analysis of the gene encoding spike protein from spike protein-expressing VSV escape mutants: To identify escape mutations present in spike protein-expressing VSV antibody-selected escape variants, the escape viruses isolated after RTCA escape screening were propagated in 6-well culture plates with confluent Vero E6 cells in the presence of 10 μg/mL of the corresponding antibody.	VSV suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   293T-ACE2 cells (BEI NR-52511) were seeded at 1.25e4 cells per well in 50 μL D10 in poly-L-lysine coated 96-well plates (Greiner 655930).	293T-ACE2 suggested: RRID:CVCL_YZ65)
   For the neutralization assays, the ACE2-293T cells were plated as described above for viral titering.	ACE2-293T suggested: None
   293T cells were seeded at 6e5 cells per well in a 6-well plate.	293T suggested: None
   S2Pecto protein was expressed in FreeStyle 293 cells (ThermoFisher) or Expi293 cells (ThermoFisher).	Expi293 suggested: RRID:CVCL_D615)
   The S6Pecto protein was expressed in FreeStyle293 cells and isolated on a StrepTrap HP column following the addition of BioLock Biotin Blocking Solution (IBA Lifesciences) to the culture supernatant.	FreeStyle293 suggested: ATCC Cat# PTA-5080, RRID:CVCL_D603)
   Eighteen thousand (18,000) Vero E6 cells in 50 μL of cell culture medium were seeded per each well, and plates were placed on the analyzer.	Vero E6 suggested: None
   Software and Algorithms
   Sentences	Resources
   The multidimensional scaling in Figure 2D that projects the antibodies into a two-dimensional space of escape mutations was performed using the Python scikit-learn package.	Python suggested: (IPython, RRID:SCR_001658) scikit-learn suggested: (scikit-learn, RRID:SCR_002577)
   Compensation and gating was performed using FlowJo v10.7.	FlowJo suggested: (FlowJo, RRID:SCR_008520)
   Images were collected using a Gatan US4000 4k × 4k CCD camera on a FEI TF20 (TFS) transmission electron microscope operated at 200 keV and controlled with SerialEM (Mastronarde, 2005).	SerialEM suggested: (SerialEM, RRID:SCR_017293)
   Image processing was performed using the cryoSPARC software package (Punjani et al., 2017).	cryoSPARC suggested: (cryoSPARC, RRID:SCR_016501)

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

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

   While these analyses come with the caveat that no experimental measures of the effects of mutations fully capture how they affect true viral fitness, it is nonetheless informative to assess how mutations that escape antibody binding impact the key biochemical functions of the RBD. Remarkably, combining the escape maps with these functional measurements predicts which mutations are selected when spike-expressing virus is grown in the presence of individual antibodies. The selected viral escape mutations are consistently those that have large effects on antibody escape but little negative impact on ACE2 binding and RBD folding, and are also accessible by single-nucleotide mutations. Furthermore, one of the antibodies was highly resistant to viral escape–and we showed this could be explained by the fact that the virus has no escape mutations from this antibody that are both tolerable for RBD function and accessible by single-nucleotide changes. Therefore, complete measurements of both the antigenic and functional consequences of viral mutations provide the phenotypic data necessary to assess both the likelihood of viral escape under antibody pressure and the specific mutations that arise when escape occurs. One immediate implication of our results is that counter to prevailing wisdom, antibody cocktails do not have to target distinct regions of the RBD in order to resist viral escape. Simple inspection of the escape maps reveals pairs of antibodies targeting the RBD’s ACE2-bindi...

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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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4. 

   Version published to 10.1101/2020.09.10.292078v1 on bioRxiv

   Sep 10, 2020