Antibodies to the SARS-CoV-2 receptor-binding domain that maximize breadth and resistance to viral escape

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   ScreenIT

   Apr 10, 2021

   SciScore for 10.1101/2021.04.06.438709: (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
   B-cell isolation and recombinant mAb production: Discovery and initial characterization of six antibodies in our panel was previously reported (S309 and S3044,17, S2X35, S2H13 and S2H1417, and S2E128), and six new antibodies are first described here (S2H97, S2X16, S2H58, S2D106, S2X58, S2X227).	S2H1417 suggested: None S2E128 suggested: None
   Epitope classes shown in Fig. 1a are defined as in Piccoli et al.17 Briefly, the classification of these epitope classes results from Octet binning experiments using structurally characterized antibodies, structural insights to define the recognition of open-only RBD …

   SciScore for 10.1101/2021.04.06.438709: (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
   B-cell isolation and recombinant mAb production: Discovery and initial characterization of six antibodies in our panel was previously reported (S309 and S3044,17, S2X35, S2H13 and S2H1417, and S2E128), and six new antibodies are first described here (S2H97, S2X16, S2H58, S2D106, S2X58, S2X227).	S2H1417 suggested: None S2E128 suggested: None
   Epitope classes shown in Fig. 1a are defined as in Piccoli et al.17 Briefly, the classification of these epitope classes results from Octet binning experiments using structurally characterized antibodies, structural insights to define the recognition of open-only RBD and ability of antibodies to interfere with RBD binding to ACE2.	ACE2 suggested: None
   Cells were washed, and secondarily labeled with 1:200 PE-conjugated goat anti-human-IgG antibody (Jackson ImmunoResearch 109-115-098) to label for bound antibody, and 1:100 FITC-conjugated chicken anti-Myc-tag (Immunology Consultants Lab, CYMC-45F) to label for RBD surface expression.	anti-human-IgG suggested: None anti-Myc-tag suggested: None
   We sorted approximately 7.5e6 RBD+ cells per library on a BD FACSAria II, collecting yeast cells from the antibody-escape sort bin (fractions of library falling into antibody escape bin given in Extended Data Fig.	antibody-escape sort bin suggested: None
   Sorted cells were recovered overnight, plasmids were extracted from the pre-sort and antibody-escape populations, and variant-identifier barcode sequences were PCR amplified and sequenced on an Illumina HiSeq 25003,28.	antibody-escape populations , suggested: None
   As previously described3,23, sequencing counts pre- and post-selection were used to estimate the “escape fraction” for each library variant, which reflects the fraction of yeast expressing a variant that fall into the antibody-escape FACS bin.	antibody-escape FACS bin . suggested: None
   The interactive visualizations of antibody escape maps (https://jbloomlab.github.io/SARS-CoV-2-RBD_MAP_Vir_mAbs) were created using dms-view42.	dms-view42 suggested: None
   Although we have various follow-up binding data illustrating reduced affinity binding for some “escaped” sarbecovirus RBDs, these follow-up experiments were not conducted systematically for all antibody/RBD combinations, and therefore would bias breadth estimates.	antibody/RBD suggested: None
   These antibodies and their citations include: S2X259, accompanying paper Tortorici et al. 2021; LY-CoV55521; COV2-2196 and COV2-213033; REGN10933, REGN10987, and LY-CoV01623; and all other COV2 antibodies and CR30223.	CR30223 suggested: None
   2c incorporate the authentic SARS-CoV-2 neutralization IC50s as reported in this study (Fig. 1a), together with the live SARS-CoV-2 neuralization IC50s for the COV2 antibodies reported by Zost et al.10.	COV2 suggested: None
   goat anti-human IgG secondary antibody (Southern Biotech, 2040-04) was added and incubated for 1 h at room temperature.	goat anti-human IgG secondary antibody suggested: None anti-human IgG suggested: (SouthernBiotech Cat# 2040-04, RRID:AB_2795643)
   Experimental Models: Cell Lines
   Sentences	Resources
   Lenti-X 293T cells (Takara, 632180) were seeded in 10-cm dishes at a density of 1e5 cells/cm2 and the following day transfected with 5 μg of spike expression plasmid with TransIT-Lenti (Mirus, 6600) according to the manufacturer’s instructions.	293T suggested: None
   VeroE6 cells were used for VSV-SARS-CoV-2, VSV-SARS-CoV-1, and VSV-GD-Pangolin-CoV.	VeroE6 suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
   BHK-21 cells stably expressing ACE2 were used for VSV-GX-Pangolin-CoV and VSV-WIV1.	BHK-21 suggested: ATCC Cat# CRL-6282, RRID:CVCL_1914)
   SARS-CoV-2 HexaPro Spike protein for cryoEM analysis was produced in HEK293F cells grown in suspension using FreeStyle 293 expression medium (Life Technologies) at 37°C in a humidified 8% CO2 incubator rotating at 130 r.p.m.	HEK293F suggested: None
   Escape clones were plaque-purified on Vero cells in the presence of mAb, and plaques in agarose plugs were amplified on MA104 cells with the mAb present in the medium.	Vero suggested: None
   Software and Algorithms
   Sentences	Resources
   Percent neutralization data were analyzed and graphed using Prism (GraphPad, v9.0.1)	Prism suggested: (PRISM, RRID:SCR_005375) GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)
   The geometric mean from at least two independent experiments was calculated using Excel (Microsoft, Version 16.45)	Excel suggested: None
   The gene sequence phylogeny was inferred using RAxML version 8.2.1249, with the GTRGAMMA substitution model and a partition model with separate parameters for first, second, and third codon positions.	RAxML suggested: (RAxML, RRID:SCR_006086)
   2 was performed using the Python scikit-learn package.	Python suggested: (IPython, RRID:SCR_001658) scikit-learn suggested: (scikit-learn, RRID:SCR_002577)
   3D refinements were carried out using non-uniform refinement64 along with per-particle defocus refinement in CryoSPARC.	CryoSPARC suggested: (cryoSPARC, RRID:SCR_016501)
   Mutant RBD:S309 complexes were constructed from the fully glycosylated WT RBD:S309 complex using PyMOL 2.3.5 (Schrödinger, LLC).	PyMOL suggested: (PyMOL, RRID:SCR_000305)
   The glycosylated proteins were parameterized with the Amber ff14SB74 and GLYCAM_06j-175 force fields.	Amber suggested: (AMBER, RRID:SCR_016151)
   Non-bonded interactions were treated with the Particle Mesh Ewald method79 using a real-space cutoff of 1.0 nm and the OpenMM default relative error tolerance of 0.0005, with grid spacing selected automatically.	OpenMM suggested: (OpenMM, RRID:SCR_000436)

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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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   Version published to 10.1101/2021.04.06.438709v1 on bioRxiv

   Apr 8, 2021