The SARS-CoV-2 receptor-binding domain elicits a potent neutralizing response without antibody-dependent enhancement

  1. Brian D. Quinlan
  2. Huihui Mou
  3. Lizhou Zhang
  4. Yan Guo
  5. Wenhui He
  6. Amrita Ojha
  7. Mark S. Parcells
  8. Guangxiang Luo
  9. Wenhui Li
  10. Guocai Zhong
  11. Hyeryun Choe
  12. Michael Farzan

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Abstract

The SARS-coronavirus 2 (SARS-CoV-2) spike (S) protein mediates entry of SARS-CoV-2 into cells expressing the angiotensin-converting enzyme 2 (ACE2). The S protein engages ACE2 through its receptor-binding domain (RBD), an independently folded 197-amino acid fragment of the 1273-amino acid S-protein protomer. Antibodies to the RBD domain of SARS-CoV (SARS-CoV-1), a closely related coronavirus which emerged in 2002-2003, have been shown to potently neutralize SARS-CoV-1 S-protein-mediated entry, and the presence of anti-RBD antibodies correlates with neutralization in SARS-CoV-2 convalescent sera. Here we show that immunization with the SARS-CoV-2 RBD elicits a robust neutralizing antibody response in rodents, comparable to 100 µg/ml of ACE2-Ig, a potent SARS-CoV-2 entry inhibitor. Importantly, anti-sera from immunized animals did not mediate antibody-dependent enhancement (ADE) of S-protein-mediated entry under conditions in which Zika virus ADE was readily observed. These data suggest that an RBD-based vaccine for SARS-CoV-2 could be safe and effective.

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  1. ScreenIT

    SciScore for 10.1101/2020.04.10.036418: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIACUC: Immunizations and sera collection: All rats used in these studies were handled and maintained in accordance with NIH guidelines and approved by Institutional Animal Care and Use Committee (IACUC) of Scripps Research (Protocol 18-025).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableFemale Sprague Dawley rats were immunized with incremental increasing doses of antigen over seven days starting at day 0, and boosted with a similar regimen at day 30.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    hACE2 expression was confirmed by SARS1-PV and SARS2-PV entry assays and by immunofluorescence staining using mouse monoclonal antibody recognizing c-Myc.
    c-Myc
    suggested: None
    After washing, cells were stained with anti-rabbit IgG-Alexa647 antibody for 45 minutes at 4°C, and mean fluorescence intensities were measured for each well by flow cytometry.
    anti-rabbit IgG-Alexa647
    suggested: None
    Measurement of antibody-dependent enhancement: The ability of anti-SARS-CoV-2 RBD immune plasma to mediate antibody-dependent enhancement (ADE) was measured using HEK293T cells or HEK293T cells stably expressing human ACE2 (293T-ACE2 cells), transfected using the calcium phosphate transfection method to express the rat ortholog of FcγRI (CD64).
    anti-SARS-CoV-2
    suggested: None
    ACE2
    suggested: None
    CD64
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    293T-hACE2 cells were selected and maintained with medium containing puromycin (Sigma).
    293T-hACE2
    suggested: None
    Protein Production: Expi293 cells (Thermo-Fisher) were transiently transfected using FectoPRO (Polyplus) with plasmids encoding SARS-CoV2 RBD with a human or rabbit-Fc fusion or a C-terminal C-tag (-EPEA).
    Expi293
    suggested: RRID:CVCL_D615)
    One hour later, 104 ACE2-239T cells were added along with DEAE-Dextran (final concentration 5 µg/ml), and media was exchanged 6 hours later with fresh media without rat sera.
    ACE2-239T
    suggested: None
    Measurement of antibody-dependent enhancement: The ability of anti-SARS-CoV-2 RBD immune plasma to mediate antibody-dependent enhancement (ADE) was measured using HEK293T cells or HEK293T cells stably expressing human ACE2 (293T-ACE2 cells), transfected using the calcium phosphate transfection method to express the rat ortholog of FcγRI (CD64).
    HEK293T
    suggested: None
    The human monocytic cell line K562 (ATCC CCL-243), which endogenously expresses FcγRII, was also used for ADE assays.
    K562
    suggested: ATCC Cat# CCL-243, RRID:CVCL_0004)
    Experimental Models: Organisms/Strains
    SentencesResources
    Female Sprague Dawley rats were immunized with incremental increasing doses of antigen over seven days starting at day 0, and boosted with a similar regimen at day 30.
    Sprague Dawley
    suggested: None
    Software and Algorithms
    SentencesResources
    Statistical analyses: The statistical significance of differences between preimmune and immune sera, or ACE2-Ig, in their abilities to neutralize, bind the S protein, and mediate ADE was analyzed by two-way ANOVA calculated using GraphPad Prism 8.0.
    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 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: 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.
    • No funding statement was detected.
    • No protocol registration statement was detected.

    About SciScore

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  2. Version published to 10.1101/2020.04.10.036418v1 on bioRxiv