Neutralizing antibodies targeting the SARS-CoV-2 receptor binding domain isolated from a naïve human antibody library

  1. Benjamin N. Bell
  2. Abigail E. Powell
  3. Carlos Rodriguez
  4. Jennifer R. Cochran
  5. Peter S. Kim

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Abstract

Infection with SARS-CoV-2 elicits robust antibody responses in some patients, with a majority of the response directed at the receptor binding domain (RBD) of the spike surface glycoprotein. Remarkably, many patient-derived antibodies that potently inhibit viral infection harbor few to no mutations from the germline, suggesting that naïve antibody libraries are a viable means for discovery of novel SARS-CoV-2 neutralizing antibodies. Here, we used a yeast surface-display library of human naïve antibodies to isolate and characterize three novel neutralizing antibodies that target the RBD: one that blocks interaction with angiotensin-converting enzyme 2 (ACE2), the human receptor for SARS-CoV-2, and two that target other epitopes on the RBD. These three antibodies neutralized SARS-CoV-2 spike-pseudotyped lentivirus with IC 50 values as low as 60 ng/mL in vitro . Using a biolayer interferometry-based binding competition assay, we determined that these antibodies have distinct but overlapping epitopes with antibodies elicited during natural COVID-19 infection. Taken together, these analyses highlight how in vitro selection of naïve antibodies can mimic the humoral response in vivo , yielding neutralizing antibodies and various epitopes that can be effectively targeted on the SARS-CoV-2 RBD.

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  1. ScreenIT

    SciScore for 10.1101/2021.01.07.425806: (What is this?)

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

    Table 1: Rigor

    NIH rigor criteria are not applicable to paper type.

    Table 2: Resources

    Antibodies
    SentencesResources
    Yeast scFv display levels were evaluated prior to sorting (data not shown) by incubating the yeast with a 1:1000 dilution of Chicken anti C-MYC Epitope Tag Primary Antibody from Exalpha Biologicals Inc. (Catalog No. ACMYC), followed by incubation with a 1:1000 dilution of Goat anti-Chicken IgY (H+L) Cross Adsorbed Secondary Antibody, Alexa Fluor Plus 647 from Thermo Fisher Scientific (Catalog No. A32933) RBD antigen binding during selection rounds 3 and 5 was evaluated using a 1:1000 dilution of rabbit anti-His FITC secondary antibody (Bethyl Laboratories).
    anti C-MYC
    suggested: None
    anti-Chicken IgY
    suggested: (Thermo Fisher Scientific Cat# A32933, RRID:AB_2762845)
    anti-His FITC
    suggested: None
    After a baseline step in assay buffer, RBD-coated biosensors were dipped in a 300 nM solution of ACE2 or assay buffer for 5 min before being dipped in a 100 nM solution of a given antibody.
    ACE2
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Recombinant protein expression and purification: IgGs, ACE2-huFc-Avi, and coronavirus RBDs were expressed and purified from Expi293F cells cultured in 33% Expi293 Expression / 66% FreeStyle Expression medium (Thermo Fisher Scientific) and grown in baffled polycarbonate shaking flasks (Triforest) at 37 °C and 8% CO2.
    Expi293F
    suggested: RRID:CVCL_D615)
    Briefly, 6 x 106 HEK293T cells cultured in D10 medium (DMEM, 10% fetal bovine serum (Gemini Bio-Products), 2 mM L-glutamine, 1% penicillin/streptomycin, and 10 mM HEPES [pH 7.0]) were transfected at ~50-70% confluency using calcium phosphate transfection20 with 5 lentiviral production plasmids: pHAGE_Luc packaging vector (10 μg), full-length SARS-CoV-2 spike (3.4 μg), HDM-Hgpm2 (2.2 μg), PRC-CMV-Rev1b (2.2 μg), and HDM-tat1b (2.2 μg)30.
    HEK293T
    suggested: None
    HeLa cells overexpressing ACE219 were plated at 5,000 cells per well in clear-bottom white-walled 96-well plates 1 day prior to infection in D10 medium.
    HeLa
    suggested: None
    Medium was removed from HeLa/ACE2 cells and replaced with virus/inhibitor mixtures which were then incubated for 48 h at 37 °C.
    HeLa/ACE2
    suggested: None
    Software and Algorithms
    SentencesResources
    Binding curves were fit using the “association then dissociation” equation in GraphPad Prism 8.4.1 to calculate KD.
    GraphPad Prism
    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: 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.

    About SciScore

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  2. Version published to 10.1101/2021.01.07.425806 on bioRxiv