Structural basis for broad sarbecovirus neutralization by a human monoclonal antibody

  1. M. Alejandra Tortorici
  2. Nadine Czudnochowski
  3. Tyler N. Starr
  4. Roberta Marzi
  5. Alexandra C. Walls
  6. Fabrizia Zatta
  7. John E. Bowen
  8. Stefano Jaconi
  9. Julia di iulio
  10. Zhaoqian Wang
  11. Anna De Marco
  12. Samantha K. Zepeda
  13. Dora Pinto
  14. Zhuoming Liu
  15. Martina Beltramello
  16. Istvan Bartha
  17. Michael P. Housley
  18. Florian A. Lempp
  19. Laura E. Rosen
  20. Exequiel Dellota
  21. Hannah Kaiser
  22. Martin Montiel-Ruiz
  23. Jiayi Zhou
  24. Amin Addetia
  25. Barbara Guarino
  26. Katja Culap
  27. Nicole Sprugasci
  28. Christian Saliba
  29. Eneida Vetti
  30. Isabella Giacchetto-Sasselli
  31. Chiara Silacci Fregni
  32. Rana Abdelnabi
  33. Shi-Yan Caroline Foo
  34. Colin Havenar-Daughton
  35. Michael A. Schmid
  36. Fabio Benigni
  37. Elisabetta Cameroni
  38. Johan Neyts
  39. Amalio Telenti
  40. Gyorgy Snell
  41. Herbert W. Virgin
  42. Sean P.J. Whelan
  43. Jesse D. Bloom
  44. Davide Corti
  45. David Veesler
  46. Matteo Samuele Pizzuto

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Abstract

The recent emergence of SARS-CoV-2 variants of concern (VOC) and the recurrent spillovers of coronaviruses in the human population highlight the need for broadly neutralizing antibodies that are not affected by the ongoing antigenic drift and that can prevent or treat future zoonotic infections. Here, we describe a human monoclonal antibody (mAb), designated S2×259, recognizing a highly conserved cryptic receptor-binding domain (RBD) epitope and cross-reacting with spikes from all sarbecovirus clades. S2×259 broadly neutralizes spike-mediated entry of SARS-CoV-2 including the B.1.1.7, B.1.351, P.1 and B.1.427/B.1.429 VOC, as well as a wide spectrum of human and zoonotic sarbecoviruses through inhibition of ACE2 binding to the RBD. Furthermore, deep-mutational scanning and in vitro escape selection experiments demonstrate that S2×259 possesses a remarkably high barrier to the emergence of resistance mutants. We show that prophylactic administration of S2×259 protects Syrian hamsters against challenges with the prototypic SARS-CoV-2 and the B.1.351 variant, suggesting this mAb is a promising candidate for the prevention and treatment of emergent VOC and zoonotic infections. Our data unveil a key antigenic site targeted by broadly-neutralizing antibodies and will guide the design of pan-sarbecovirus vaccines.

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    SciScore for 10.1101/2021.04.07.438818: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: Sample donors: Samples from a SARS-CoV-2 recovered individual, designated as donor X (male, 52 years-old), was obtained 75 days after symptoms onset under study protocols approved by the local Institutional Review Boards (Canton Ticino Ethics Committee, Switzerland, the Ethical committee of Luigi Sacco Hospital, Milan, Italy).
    Consent: The donor provided written informed consent for the use of blood and blood components (e.g. PBMCs, sera or plasma).
    IACUC: Housing conditions and experimental procedures were approved by the ethical committee of animal experimentation of KU Leuven (license P065-2020).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableSample donors: Samples from a SARS-CoV-2 recovered individual, designated as donor X (male, 52 years-old), was obtained 75 days after symptoms onset under study protocols approved by the local Institutional Review Boards (Canton Ticino Ethics Committee, Switzerland, the Ethical committee of Luigi Sacco Hospital, Milan, Italy).
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    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)
    One day post-transfection, cells were infected with VSV (G*ΔG-luciferase) and after 2 h, infected cells were washed four times with DMEM before adding medium supplemented with anti-VSV-G antibody (I1-mouse hybridoma supernatant diluted 1 to 50, from CRL-2700, ATCC).
    G*ΔG-luciferase
    suggested: None
    anti-VSV-G
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cell lines: Cell lines used in this study were obtained from ATCC (HEK293T, Vero and Vero-E6), ThermoFisher Scientific (Expi CHO cells, FreeStyle™ 293-F cells and Expi293F™ cells) or were generated via lentiviral transduction (Expi CHO-S, HEK293T-ACE2).
    HEK293T
    suggested: None
    293-F
    suggested: None
    For neutralization, stable 293T cells expressing ACE257 in DMEM supplemented with 10% FBS, 1% PenStrep were seeded at 40,000 cells/well into clear bottom white walled 96-well plates and cultured overnight at 37°C.
    293T
    suggested: RRID:CVCL_H376)
    Vero E6 cells were seeded into black-walled, clear-bottom 96-well plates at 2 x 104 cells/well and cultured overnight at 37 °C.
    Vero E6
    suggested: None
    Cell-surface mAb-mediated S1 shedding: CHO cells stably expressing wild-type SARS-CoV-2 S were resuspended in wash buffer (PBS 1 % BSA, 2 mM EDTA) and treated with 10 µg/mL TPCK-trypsin (Worthington Biochem) for 30 min at 37°C.
    CHO
    suggested: None
    After a 15-minute incubation, Jurkat cells stably expressing FcγRIIIa receptor (V158 variant) or FcγRIIa receptor (H131 variant) and NFAT-driven luciferase gene (effector cells) were added at an effector to target ratio of 6:1 for FcγRIIIa and 5:1 for FcγRIIa.
    Jurkat
    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
    Experimental Models: Organisms/Strains
    SentencesResources
    The following sequences were retrieved from GISAID and NCBI: A021 (AAV97986.1); HKU3 (QND76020.1); WIV1 (AGZ48831.1); Rs3367 (AGZ48818.1); Anlong-112 (ARI44804.1); RsSHC014 (AGZ48806.1); Rs4081 (AGZ48798.1); YN2013 (AIA62330.1); Rs4874 (ATO98205.1); Rs4255 (ATO98193.1); Rs4231 (ATO98157.1); Rs4084 (ATO98132.1); ZXC21 (AVP78042.1); SC2018 (QDF43815.1); ZC45 (AVP78031.1); Rp/Shaanxi2011 (AGC74165.1); Rm1/2004 (ABD75332.1); Rf1-2004 (ABD75323.1); Rf4092 (ATO98145.1); BM48-31/BGR/2008 (YP_003858584.1); LYRa11 (AHX37558.1); RaTG13 (QHR63300.2); PC4-127 (AAU93318.1); CS24 (ABF68959.1); SARS-CoV2 (YP_009724390.1); LYRa3 (AHX37569.1); Cp/Yunnan2011 (AGC74176.1); SARS coronavirus Urbani (AAP13441.1); As6526 (ATO98108.1); BtkY72 (APO40579.1); RmYN02 (EPI_ISL_412977); Pangolin_Guangdong-2019 (EPI_ISL_410721); Pangolin-Guanxi-2017 (EPI_ISL_410539).
    Pangolin_Guangdong-2019
    suggested: None
    Pangolin-Guanxi-2017
    suggested: None
    VSV-based pseudotype virus production and neutralization assay: SARS-CoV-2 S (CAD0240757.1), RaTG13 S (QHR63300.2), Pangolin-Guangdong S(QLR06867.1), Pangolin-Guanxi S (QIA48623.1)
    RaTG13 S
    suggested: None
    Software and Algorithms
    SentencesResources
    Sarbecovirus sequence analysis and SARS-CoV-2 mutant counts: Alignment and phylogenetic tree of the strains within the sarbecovirus subgenus was generated using MEGA 7.0.26 and CLC Main workbench 21.0.3 (Qiagen).
    MEGA
    suggested: (Mega BLAST, RRID:SCR_011920)
    Variants were then extracted as compared to the reference with R 4.0.2 (https://www.r-project.org/) using Biostrings 2.56.0.
    https://www.r-project.org/
    suggested: (R Project for Statistical Computing, RRID:SCR_001905)
    Biostrings
    suggested: (Biostrings, RRID:SCR_016949)
    After 30 min incubation, absorbance at 405 nm was measured by a plate reader (Biotek) and data plotted using Prism GraphPad.
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Measurements were done in duplicate, RLU values were converted to percentage of neutralization and plotted with a nonlinear regression curve fit in Graph Prism.
    Graph Prism
    suggested: None
    Data were plotted in Prism.
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    Response values were exported and plotted using GraphPad Prism.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Particle images from each dataset were subjected to Bayesian polishing72 using Relion before merging them to perform another round of non-uniform refinement in cryoSPARC followed by per-particle defocus refinement and again non-uniform refinement.
    cryoSPARC
    suggested: (cryoSPARC, RRID:SCR_016501)

    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: Please consider improving the rainbow (“jet”) colormap(s) used on page 27. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    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.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. Version published to 10.1101/2021.04.07.438818 on bioRxiv