Mapping mutations to the SARS-CoV-2 RBD that escape binding by different classes of antibodies

  1. Allison J. Greaney
  2. Tyler N. Starr
  3. Christopher O. Barnes
  4. Yiska Weisblum
  5. Fabian Schmidt
  6. Marina Caskey
  7. Christian Gaebler
  8. Alice Cho
  9. Marianna Agudelo
  10. Shlomo Finkin
  11. Zijun Wang
  12. Daniel Poston
  13. Frauke Muecksch
  14. Theodora Hatziioannou
  15. Paul D. Bieniasz
  16. Davide F. Robbiani
  17. Michel C. Nussenzweig
  18. Pamela J. Bjorkman
  19. Jesse D. Bloom

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Abstract

Monoclonal antibodies targeting a variety of epitopes have been isolated from individuals previously infected with SARS-CoV-2, but the relative contributions of these different antibody classes to the polyclonal response remains unclear. Here we use a yeast-display system to map all mutations to the viral spike receptor-binding domain (RBD) that escape binding by representatives of three potently neutralizing classes of anti-RBD antibodies with high-resolution structures. We compare the antibody-escape maps to similar maps for convalescent polyclonal plasmas, including plasmas from individuals from whom some of the antibodies were isolated. While the binding of polyclonal plasma antibodies are affected by mutations across multiple RBD epitopes, the plasma-escape maps most resemble those of a single class of antibodies that target an epitope on the RBD that includes site E484. Therefore, although the human immune system can produce antibodies that target diverse RBD epitopes, in practice the polyclonal response to infection is skewed towards a single class of antibodies targeting an epitope that is already undergoing rapid evolution.

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  1. Version published to 10.1038/s41467-021-24435-8
  2. ScreenIT

    SciScore for 10.1101/2021.03.17.435863: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementConsent: Samples were obtained upon written consent from community participants under protocols approved by the Institutional Review Board of the Rockefeller University (DRO-1006).
    IRB: Samples were obtained upon written consent from community participants under protocols approved by the Institutional Review Board of the Rockefeller University (DRO-1006).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Antibodies were produced with a human IgG1 heavy chain and human IgK (C002, C110, C135) or human IgL2 (C105, C121, C144) constant regions.
    human IgK
    suggested: None
    C121
    suggested: (Leinco Technologies Cat# C121, RRID:AB_2828361)
    The PBD accessions for the antibody-S complex structures are: 6XCM and 6XCN for C105, 7C01 for LY-CoV016, 7K8S and 7K8T for C002, 7K8X and 7K8Y for C121, 7K90 for C144, 7K8Z for C135, and 7K8V for C11025, 28, 29.
    antibody-S
    suggested: None
    C002
    suggested: (GenWay Biotech Inc. Cat# GWB-C002D4, RRID:AB_10269310)
    As previously described, these libraries were sorted to eliminate variants that lose ACE2 binding prior to mapping the antibody-escape variants30.
    antibody-escape variants30
    suggested: None
    Incubations were performed with 400 ng/mL for each monoclonal antibody (C105, C144, C002, C121, C135, or C110) or with a sub-saturating dilution of polyclonal plasma such that the amount of fluorescent signal due to plasma antibody binding to RBD was approximately equal across plasma (COV-021, 1:500; COV-047, 1:200; COV-057, 1:50; COV-072, 1:200; COV-107, 1:80).
    C135
    suggested: None
    C110
    suggested: (Leinco Technologies Cat# C110, RRID:AB_2828322)
    COV-021
    suggested: None
    Labeled cells were washed with ice-cold PBS-BSA followed by secondary labeling for 1 h at 4°C in 2.5 mL 1:200 PE-conjugated goat anti-human-IgG (Jackson ImmunoResearch 109-115-098) to label for bound monoclonal antibody or 1:200 Alexa-647-conjugated goat anti-human-IgA+IgG+IgM (Jackson ImmunoResearch 109-605-064) to label for bound plasma antibodies, and 1:100 FITC-conjugated anti-Myc (Immunology Consultants Lab, CYMC-45F) to label for RBD surface expression.
    anti-human-IgG
    suggested: None
    anti-human-IgA+IgG+IgM
    suggested: None
    anti-Myc
    suggested: None
    A markdown rendering of the identification of these sites of strong escape is at https://github.com/jbloomlab/SARS-CoV-2-RBD_MAP_Rockefeller/blob/main/results/summary/call_strong_escape_sites.md. Comparison of mutation escape fractions to previously measured neutralization concentrations: In Figure 5C and Figure S6, mutation-level antibody-escape fractions measured in this study are compared to previously measured neutralization titers (inhibitory concentration 50%, IC50) of the same monoclonal antibodies and polyclonal plasma against some RBD point-mutants7.
    antibody-escape
    suggested: None
    For identifying contact sites to highlight in Figure 1B logo plots or to classify sites in Figure 2B as contact sites (within 4A of antibody) or antibody-proximal sites within 4–8A, the following PDBs were used: 6XCM and 6XCN for C105, 7K8S and 7K8T for C002, 7K8X and 7K8Y for C121, 7K90 for C144, 7K8Z for C135, and 7K8V for C110)25, 29.
    antibody-proximal
    suggested: None
    C105
    suggested: (Leinco Technologies Cat# C105, RRID:AB_2828317)
    7K8X
    suggested: None
    C144
    suggested: (Leinco Technologies Cat# C144, RRID:AB_2828501)
    Software and Algorithms
    SentencesResources
    The numerical IC50 values were extracted from figures in Weisblum et al. (2020)7 using the WebPlotDigitizer tool v4.4 (https://apps.automeris.io/wpd/).
    WebPlotDigitizer
    suggested: (WebPlotDigitizer, RRID:SCR_013996)
    The multidimensional scaling in Figure 1C and Figure 4A 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)
    The static structural views (Figure 2A, S5) in the paper were rendered in PyMOL using antibody-bound RBD structures.
    PyMOL
    suggested: (PyMOL, RRID:SCR_000305)

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

    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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  3. Version published to 10.1101/2021.03.17.435863v1 on bioRxiv