Prevalent, protective, and convergent IgG recognition of SARS-CoV-2 non-RBD spike epitopes in COVID-19 convalescent plasma

  1. William N. Voss
  2. Yixuan J. Hou
  3. Nicole V. Johnson
  4. Jin Eyun Kim
  5. George Delidakis
  6. Andrew P. Horton
  7. Foteini Bartzoka
  8. Chelsea J. Paresi
  9. Yuri Tanno
  10. Shawn A. Abbasi
  11. Whitney Pickens
  12. Katia George
  13. Daniel R. Boutz
  14. Dalton M. Towers
  15. Jonathan R. McDaniel
  16. Daniel Billick
  17. Jule Goike
  18. Lori Rowe
  19. Dhwani Batra
  20. Jan Pohl
  21. Justin Lee
  22. Shivaprakash Gangappa
  23. Suryaprakash Sambhara
  24. Michelle Gadush
  25. Nianshuang Wang
  26. Maria D. Person
  27. Brent L. Iverson
  28. Jimmy D. Gollihar
  29. John Dye
  30. Andrew Herbert
  31. Ralph S. Baric
  32. Jason S. McLellan
  33. George Georgiou
  34. Jason J. Lavinder
  35. Gregory C. Ippolito

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Abstract

Although humoral immunity is essential for control of SARS-CoV-2, the molecular composition, binding epitopes and effector functions of the immunoglobulin G (IgG) antibodies that circulate in blood plasma following infection are unknown. Proteomic deconvolution of the circulating IgG repertoire (Ig-Seq 1 ) to the spike ectodomain (S-ECD 2 ) in four convalescent study subjects revealed that the plasma response is oligoclonal and directed predominantly (>80%) to S-ECD epitopes that lie outside the receptor binding domain (RBD). When comparing antibodies directed to either the RBD, the N-terminal domain (NTD) or the S2 subunit (S2) in one subject, just four IgG lineages (1 anti-S2, 2 anti-NTD and 1 anti-RBD) accounted for 93.5% of the repertoire. Although the anti-RBD and one of the anti-NTD antibodies were equally potently neutralizing in vitro , we nonetheless found that the anti-NTD antibody was sufficient for protection to lethal viral challenge, either alone or in combination as a cocktail where it dominated the effect of the other plasma antibodies. We identified in vivo protective plasma anti-NTD antibodies in 3/4 subjects analyzed and discovered a shared class of antibodies targeting the NTD that utilize unmutated or near-germline IGHV1-24, the most electronegative IGHV gene in the human genome. Structural analysis revealed that binding to NTD is dominated by interactions with the heavy chain, accounting for 89% of the entire interfacial area, with germline residues uniquely encoded by IGHV1-24 contributing 20% (149 Å 2 ). Together with recent reports of germline IGHV1-24 antibodies isolated by B-cell cloning 3,4 our data reveal a class of shared IgG antibodies that are readily observed in convalescent plasma and underscore the role of NTD-directed antibodies in protection against SARS-CoV-2 infection.

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  1. ScreenIT

    SciScore for 10.1101/2020.12.20.423708: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableAt 12h before infection, twelve-month-old female BALB/c mice (n=5/group) were injected intraperitoneally with 200μg/mouse of mAb or PBS.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    ELISA: The methods for enzyme-linked immunosorbent assay to measure titers of anti-SARS-CoV-2 IgG plasma antibodies have been previously described4.
    anti-SARS-CoV-2 IgG
    suggested: None
    Antibody expression and purification: Cognate VH and VL antibody sequences of interest were ordered as gBlocks (Integrated DNA Technologies) and cloned into a customized pcDNA 3.4 vector containing a human IgG1 Fc region.
    VL
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Viral titers in the lung tissue were measured by plaque assay on Vero E6 cells.
    Vero E6
    suggested: RRID:CVCL_XD71)
    Experimental Models: Organisms/Strains
    SentencesResources
    At 12h before infection, twelve-month-old female BALB/c mice (n=5/group) were injected intraperitoneally with 200μg/mouse of mAb or PBS.
    BALB/c
    suggested: None
    Software and Algorithms
    SentencesResources
    Sequences with ≥ 2 reads were clustered into clonal lineages defined by 90% CDRH3 amino acid identity using USEARCH10. LC-MS/MS search databases were prepared as previously described7, using custom Python scripts (available upon request).
    Python
    suggested: (IPython, RRID:SCR_001658)
    Movies were collected using SerialEM at 22,500X magnification with a corresponding calibrated pixel size of 1.1 Å2/ pixel.
    SerialEM
    suggested: (SerialEM, RRID:SCR_017293)
    Micrographs were then imported into cryoSPARC v2.15.0 for CTF-estimation, particle picking, 2D classification, ab initio reconstruction, heterogenous 3D refinement and homogenous refinement14.
    cryoSPARC
    suggested: (cryoSPARC, RRID:SCR_016501)
    The model was built further and iteratively refined using a combination of Coot, Phenix, and ISOLDE18-20.
    Coot
    suggested: (Coot, RRID:SCR_014222)
    Statistics: GraphPad Prism version 9.0.0 (GraphPad Software Inc., La Jolla, CA, USA) was used to conduct statistical analyses.
    Statistics: GraphPad Prism
    suggested: None
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    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 38. 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 10.1101/2020.12.20.423708 on bioRxiv