High genetic barrier to SARS-CoV-2 polyclonal neutralizing antibody escape

  1. Fabian Schmidt
  2. Yiska Weisblum
  3. Magdalena Rutkowska
  4. Daniel Poston
  5. Justin DaSilva
  6. Fengwen Zhang
  7. Eva Bednarski
  8. Alice Cho
  9. Dennis J. Schaefer-Babajew
  10. Christian Gaebler
  11. Marina Caskey
  12. Michel C. Nussenzweig
  13. Theodora Hatziioannou
  14. Paul D. Bieniasz

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

No abstract available

Article activity feed

  1. Version published to 10.1038/s41586-021-04005-0
  2. ScreenIT

    SciScore for 10.1101/2021.08.06.455491: (What is this?)

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

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    RandomizationA set of twenty-seven plasmas samples from SARS-CoV-2 infected individuals with high neutralizing activity who had not been vaccinated2, termed the “RU27” plasma panel were used in VSV-SARS-CoV-2 selection procedures, while this panel plus a second set of 21 randomly selected plasmas (selected at random with blinding to neutralization titer or any demographic characteristic) from the same convalescent cohort formed the “Ran21” plasma panel 2.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The antibody/pseudotype virus mixture was then added to HT1080/ACE2.cl14 cells.
    antibody/pseudotype
    suggested: None
    The study visits and blood draws were reviewed and approved by the Institutional Review Board of the Rockefeller University (IRB no. DRO-1006, ‘Peripheral Blood of Coronavirus Survivors to Identify Virus-Neutralizing Antibodies’).
    Antibodies’
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Specifically, virus stocks were harvested 48 hours after transfection of 293T cells with pHIV-1 GagPol and pCCNano/LucGFP (Fig.1) or pNL4-3ΔEnv-nanoluc (all other Figs) along with a spike expression plasmid, filtered and stored at −80°C.
    293T
    suggested: None
    The antibody/pseudotype virus mixture was then added to HT1080/ACE2.cl14 cells.
    HT1080/ACE2.cl14
    suggested: None
    Selection of antibody resistant rVSV/SARS-CoV-2 variants: To select plasma-resistant spike variants, rVSV/SARS-CoV-2/GFP1D7 and rVSV/SARS-CoV-2/GFP2E1 were passaged to generate diversity, and populations containing 106 PFU were incubated with plasma (diluted 1:50 to 1:400) for 1h at 37°C before inoculation of 2×105 293T/ACE2cl.22 cells in 6-well plates.
    293T/ACE2cl.22
    suggested: None
    The antibody/recombinant virus mixture was then added to 293T/ACE2.cl22 cells.
    293T/ACE2.cl22
    suggested: None
    Recombinant DNA
    SentencesResources
    SARS-CoV-2 pseudotyped reporter virus: Plasmids pSARS-CoV-2-SΔ19 and pSARS-CoV-SΔ19 expressing C-terminally truncated SARS-CoV-2 (NC_045512) and SARS-CoV spike proteins have been described previously19 and were used to construct the SARS-CoV-2(1-RBD) and SARS-CoV(2-RBD) expression plasmids in which RBD-encoding sequences were reciprocally exchanged.
    pSARS-CoV-SΔ19
    suggested: None
    A panel of plasmids expressing spike proteins from SARS-CoV-2 VOC and VOI were constructed in the context of pSARS-CoV-2-SΔ19 (R683G) 19.
    pSARS-CoV-2-SΔ19
    suggested: None
    Spike sequences were codon-modified to maximize homology with the human codon-usage optimized of the pSARS-CoV-2 expressing plasmid VG40589-UT (Sinobiological).
    pSARS-CoV-2
    suggested: None
    The 19aa truncated CDS of bCoV-RaTG13 (QHR63300), pCoV-GD (CoV_EPI_ISL_410721), and pCoV-GX (CoV_EPI_ISL_410542) were synthesized by GeneART and subcloned into pCR3.1 using NheI and XbaI and Gibson assembly, and referred to as pCR3.1-bCoV-RaTG13-SΔ19, pCR3.1pCoV-GD-SΔ19 and pCR3.1-pCoV-GX-SΔ19, respectively.
    pCoV-GD
    suggested: None
    pCoV-GX
    suggested: None
    pCR3.1
    suggested: RRID:Addgene_106457)
    pCR3.1-bCoV-RaTG13-SΔ19
    suggested: None
    pCR3.1pCoV-GD-SΔ19
    suggested: None
    pCR3.1-pCoV-GX-SΔ19
    suggested: None
    Specifically, virus stocks were harvested 48 hours after transfection of 293T cells with pHIV-1 GagPol and pCCNano/LucGFP (Fig.1) or pNL4-3ΔEnv-nanoluc (all other Figs) along with a spike expression plasmid, filtered and stored at −80°C.
    pHIV-1
    suggested: None
    pCCNano/LucGFP
    suggested: None
    pNL4-3ΔEnv-nanoluc
    suggested: None
    Software and Algorithms
    SentencesResources
    The half-maximal neutralizing titer (NT50) was determined using four-parameter nonlinear regression (least squares regression method without weighting) (GraphPad Prism).
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    For analysis of the Illumina sequencing data, adapter sequences were removed from the raw reads and low-quality reads (Phred quality score <20) using BBDuk.
    Phred
    suggested: (Phred, RRID:SCR_001017)
    RBD-specific variant frequencies, P-values, and read depth were compiled using Python running pandas (1.0.5), numpy (1.18.5), and matplotlib (3.2.2).
    Python
    suggested: (IPython, RRID:SCR_001658)
    matplotlib
    suggested: (MatPlotLib, RRID:SCR_008624)
    For comparison of SARS-CoV-2 with sarbecoviruses, amino acid sequences were aligned with Clustal Omega.
    Clustal Omega
    suggested: (Clustal Omega, RRID:SCR_001591)
    Viral Genome Analysis Pipeline, https://cov.lanl.gov/content/index) 31was divided by the average frequency of change at any reside and projected in the SARS-CoV-2 spike structure PDB 6VXX 32 as relative change frequency using BioStructMap 33,34.
    BioStructMap
    suggested: None

    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 14. 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 10.1101/2021.08.06.455491v1 on bioRxiv