Potent neutralization of SARS-CoV-2 variants of concern by an antibody with an uncommon genetic signature and structural mode of spike recognition

  1. Kevin J. Kramer
  2. Nicole V. Johnson
  3. Andrea R. Shiakolas
  4. Naveenchandra Suryadevara
  5. Sivakumar Periasamy
  6. Nagarajan Raju
  7. Jazmean K. Williams
  8. Daniel Wrapp
  9. Seth J. Zost
  10. Lauren M. Walker
  11. Steven C. Wall
  12. Clinton M. Holt
  13. Ching-Lin Hsieh
  14. Rachel E. Sutton
  15. Ariana Paulo
  16. Rachel S. Nargi
  17. Edgar Davidson
  18. Benjamin J. Doranz
  19. James E. Crowe
  20. Alexander Bukreyev
  21. Robert H. Carnahan
  22. Jason S. McLellan
  23. Ivelin S. Georgiev

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Abstract

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  1. Version published to 10.1016/j.celrep.2021.109784
  2. ScreenIT

    SciScore for 10.1101/2021.05.16.444004: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: The studies were reviewed and approved by the Institutional Review Board of Vanderbilt University Medical Center.
    Consent: The sample was obtained after written informed consent was obtained.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    In brief, synthesized antibody-encoding DNA (∼2 μg per transfection) was added to OptiPro serum free medium (OptiPro SFM), incubated with ExpiFectamine CHO Reagent and added to 800 µl of ExpiCHO cell cultures into 96-deep-well blocks using a ViaFlo 384 liquid handler (Integra Biosciences).
    antibody-encoding DNA
    suggested: None
    The secondary antibody, goat anti-human IgG conjugated to peroxidase, was added at 1:10,000 dilution in 1% milk in PBS-T to the plates, which were incubated for one hour at room temperature.
    anti-human IgG
    suggested: None
    Briefly, antibodies were biotinylated using NHS-PEG4-biotin (Thermo Fisher Scientific, cat# A39259) according to manufacturer protocol.
    NHS-PEG4-biotin
    suggested: None
    After subtracting the background signal, the signal obtained for binding of the biotin-labeled reference antibody in the presence of the unlabeled tested antibody was expressed as a percentage of the binding of the reference antibody in the presence of 10 µg/mL of the anti-dengue antibody DENV 2D22, which served as a no-competition control.
    anti-dengue
    suggested: None
    Real-time Cell Analysis (RTCA) Neutralization Assay Screen: To screen for neutralizing activity in the panel of recombinantly expressed antibodies, we used a high-throughput and quantitative RTCA assay and xCelligence RTCA HT Analyzer (ACEA Biosciences) that assesses kinetic changes in cell physiology, including virus-induced cytopathic effect (CPE).
    CPE
    suggested: None
    Plates were washed three times with PBS-T, and bound ACE2 was detected using HRP-conjugated anti-mouse Fc antibody and TMB substrate.
    anti-mouse
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    HCoV-NL63 and HCoV-229E alpha coronavirus spike proteins were purchased from Sino Biological.
    HCoV-NL63
    suggested: RRID:CVCL_RW88)
    For antibody expression, microscale transfection were performed (∼1 ml per antibody) of CHO cell cultures using the Gibco ExpiCHO Expression System and a protocol for deep 96-well blocks (Thermo Fisher Scientific).
    CHO
    suggested: None
    A suspension of 18,000 Vero-E6 cells in 50 μL of cell culture medium was seeded in each well, and the plate was placed on the analyzer.
    Vero-E6
    suggested: None
    Triplicate wells containing virus only (maximal CPE in the absence of antibody) and wells containing only Vero cells in medium (no-CPE wells) were included as controls.
    Vero
    suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
    Each spike protein mutant was transfected into HEK-293T cells and allowed to express for 22 hrs.
    HEK-293T
    suggested: None
    Plaque reduction neutralization test (PRNT): The virus neutralization with live authentic SARS-CoV-2 virus (USA-WA1) was performed in the BSL-3 facility of the Galveston National Laboratory using Vero E6 cells (ATCC CRL-1586) following the standard procedure.
    Vero E6
    suggested: None
    Software and Algorithms
    SentencesResources
    ) sequencing core at an appropriate target concentration for 10X Genomics library preparation and subsequent sequencing.
    Genomics
    suggested: (UTHSCSA Genomics Core, RRID:SCR_012239)
    FACS data were analyzed using FlowJo.
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    In brief, synthesized antibody-encoding DNA (∼2 μg per transfection) was added to OptiPro serum free medium (OptiPro SFM), incubated with ExpiFectamine CHO Reagent and added to 800 µl of ExpiCHO cell cultures into 96-deep-well blocks using a ViaFlo 384 liquid handler (Integra Biosciences).
    Integra Biosciences
    suggested: None
    RTCA IC50 values were determined by nonlinear regression analysis using Prism software.
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    For antibodies, the inhibitory concentration at 50% (IC50) values were calculated in GraphPad Prism software by plotting the midway point between the upper and lower plateaus of the neutralization curve among dilutions.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    SerialEM was used to collect movies at 29,000X nominal magnification with a calibrated pixel size of 0.81 Å/pixel.
    SerialEM
    suggested: (SerialEM, RRID:SCR_017293)
    Cryogenic electron microscopy (Cryo-EM): Motion correction, CTF estimation, particle picking, and preliminary 2D classification were performed using cryoSPARC v3.2.0 live processing (Punjani et al., 2017).
    cryoSPARC
    suggested: (cryoSPARC, RRID:SCR_016501)
    The model was iteratively refined and completed using a combination of Phenix, Coot, and ISOLDE (Adams et al., 2002; Croll, 2018; Emsley and Cowtan, 2004).
    Phenix
    suggested: (Phenix, RRID:SCR_014224)
    Coot
    suggested: (Coot, RRID:SCR_014222)
    RMSD calculation for the differences in angle of antigen approach for different antibodies: The SARS-CoV-2 spike receptor binding domain coordinates present in each antibody-antigen complex were aligned in PyMOL (The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.) using an all-atom alignment with 5 cycles of outlier rejection of atom pairs having an RMSD greater than 2.
    PyMOL
    suggested: (PyMOL, RRID:SCR_000305)

    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: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    NCT04625725Active, not recruitingPhase III Double-blind, Placebo-controlled Study of AZD7442 …
    NCT04723394RecruitingPhase III Study of AZD7442 for Treatment of COVID-19 in Outp…
    NCT04518410RecruitingACTIV-2: A Study for Outpatients With COVID-19
    NCT04501978RecruitingACTIV-3: Therapeutics for Inpatients With COVID-19

    Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 58 and 53. 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.

    Results from scite Reference Check: We found no unreliable references.

    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.

  3. Version published to 10.1101/2021.05.16.444004v1 on bioRxiv