Design of the SARS-CoV-2 RBD vaccine antigen improves neutralizing antibody response

  1. Thayne H. Dickey
  2. Wai Kwan Tang
  3. Brandi Butler
  4. Tarik Ouahes
  5. Sachy Orr-Gonzalez
  6. Nichole D. Salinas
  7. Lynn E. Lambert
  8. Niraj H. Tolia

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Abstract

The receptor binding domain (RBD) of the SARS-CoV-2 spike protein is the primary target of neutralizing antibodies and is a component of almost all current vaccines. Here, RBD immunogens were created with stabilizing amino acid changes that improve the neutralizing antibody response, as well as characteristics for production, storage, and distribution. A computational design and in vitro screening platform identified three improved immunogens, each with approximately nine amino acid changes relative to the native RBD sequence, and four key changes conserved between immunogens. The changes are adaptable to all vaccine platforms and compatible with mutations in emerging variants of concern. The immunogens elicit higher levels of neutralizing antibodies than native RBD, focus the immune response to structured neutralizing epitopes, and have increased production yields and thermostability. Incorporating these variant-independent amino acid changes in next-generation COVID vaccines may enhance the neutralizing antibody response and lead to longer duration and broader protection.

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  1. Version published to 10.1126/sciadv.abq8276
  2. ScreenIT

    SciScore for 10.1101/2021.05.09.443238: (What is this?)

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

    Table 1: Rigor

    EthicsIACUC: Mouse immunizations: Mouse immunogenicity studies were performed under the guidelines and approval of the Institutional Animal Care and Use Committee (IACUC) at the National Institutes of Health.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Neutralizing epitopes were probed using an Ace2-Fc(IgG1) fusion (0.2 ug/well) or a human IgG1 antibody containing the CR3022 variable domain (0.05 ug/well).
    human IgG1
    suggested: (Imported from the IEDB Cat# CR3022, RRID:AB_2848080)
    100 μl primary antibody was added to each well at the indicated concentration: Ace2 – 3.1 ng/mL, REGN10933, CR3022, and S309 – 1.5 ng/mL, and P2B-2F6 – 7.5 ng/mL.
    Ace2
    suggested: None
    S309
    suggested: None
    P2B-2F6
    suggested: None
    CR3022
    suggested: (Imported from the IEDB Cat# CR3022, RRID:AB_2848080)
    Primary antibody was incubated 1 hour at room temperature then plates were washed three times with PBST and 200 μl 1:5000 peroxidase-conjugated anti-human IgG was added (Jackson ImmunoResearch Laboratories, Inc. Cat.# 109-035-098).
    anti-human IgG
    suggested: (Jackson ImmunoResearch Labs Cat# 109-035-098, RRID:AB_2337586)
    100 μl primary antibody was added to each well at the indicated concentration: Ace2 – 3.1 ng/mL, REGN10933, CR3022, and S309 – 1.5 ng/mL, and P2B-2F6 – 7.5 ng/mL.
    Ace2
    suggested: None
    S309
    suggested: None
    P2B-2F6
    suggested: None
    CR3022
    suggested: (Imported from the IEDB Cat# CR3022, RRID:AB_2848080)
    Experimental Models: Cell Lines
    SentencesResources
    After 1 hour incubation at room temperature, 20,000 HEK293 cells overexpressing Ace2 were added to each well.
    HEK293
    suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
    Experimental Models: Organisms/Strains
    SentencesResources
    Five 5-6 week old female CD-1 mice (Charles River Laboratories) per group were immunized with 10 μg antigen each.
    CD-1
    suggested: None
    Recombinant DNA
    SentencesResources
    RRID:Addgene_99845)45.
    detected: RRID:Addgene_99845)
    The Ace2-Fc fusion was expressed from pcDNA3: pcDNA3-sACE2(WT)-Fc(IgG1) was a gift from Erik Procko (Addgene plasmid # 145163; http://n2t.net/addgene:145163; RRID:Addgene_145163)47.
    pcDNA3
    suggested: RRID:Addgene_15475)
    detected: RRID:Addgene_145163)
    pcDNA3-sACE2
    suggested: None
    The Ace2-Fc fusion was expressed from pcDNA3: pcDNA3-sACE2(WT)-Fc(IgG1) was a gift from Erik Procko (Addgene plasmid # 145163; http://n2t.net/addgene:145163; RRID:Addgene_145163)47.
    pcDNA3
    suggested: RRID:Addgene_15475)
    detected: RRID:Addgene_145163)
    pcDNA3-sACE2
    suggested: None
    Software and Algorithms
    SentencesResources
    Extinction coefficients were calculated using the ExPASy ProtParam tool46.
    ExPASy ProtParam
    suggested: None
    This model was then edited in COOT to incorporate the amino acid changes present in immunogen 3 and subsequent rounds of refinement and model building were performed with COOT and PHENIX Refine53.
    COOT
    suggested: (Coot, RRID:SCR_014222)
    The final model was evaluated with MolProbity, which showed good geometry, with 96.0% of the residues as Ramachandran favored and 0% outlier residues (Extended Data Table 2)54.
    MolProbity
    suggested: (MolProbity, RRID:SCR_014226)
    % inhibition values were measured for each serum dilution in triplicate and average values were plotted in GraphPad Prism 8.
    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.
    • 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

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  3. Version published to 10.1101/2021.05.09.443238v1 on bioRxiv