Engineered receptor binding domain immunogens elicit pan-sarbecovirus neutralizing antibodies outside the receptor binding motif

  1. Blake M. Hauser
  2. Maya Sangesland
  3. Evan C. Lam
  4. Kerri J. St. Denis
  5. Jared Feldman
  6. Ashraf S. Yousif
  7. Timothy M. Caradonna
  8. Ty Kannegieter
  9. Alejandro B. Balazs
  10. Daniel Lingwood
  11. Aaron G. Schmidt

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Abstract

Effective countermeasures are needed against emerging coronaviruses of pandemic potential, similar to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Designing immunogens that elicit broadly neutralizing antibodies to conserved viral epitopes on the major surface glycoprotein, spike, such as the receptor binding domain (RBD) is one potential approach. Here, we report the generation of homotrimeric RBD immunogens from different sarbecoviruses using a stabilized, immune-silent trimerization tag. In mice, we find that a cocktail of these homotrimeric sarbecovirus RBDs elicits antibodies to conserved viral epitopes outside of the ACE2 receptor binding motif (RBM). Importantly, these responses neutralize all sarbecovirus components even in context of prior SARS-CoV-2 imprinting. We further show that a substantial fraction of the neutralizing antibodies elicited after vaccination in humans also engages non-RBM epitopes on the RBD. Collectively, our results suggest a strategy for eliciting broadly neutralizing responses leading to a pan-sarbecovirus vaccine.

Author summary

Immunity to SARS-CoV-2 in the human population will be widespread due to natural infection and vaccination. However, another novel coronavirus will likely emerge in the future and may cause a subsequent pandemic. Humoral responses induced by SARS-CoV-2 infection and vaccination provide limited protection against even closely related coronaviruses. We show immunization with a cocktail of trimeric coronavirus receptor binding domains induces a neutralizing antibody response that is broadened to related coronaviruses with pandemic potential. Importantly, this broadening occurs in context of an initial imprinted SARS-CoV-2 spike immunization showing that preexisting immunity can be expanded to recognize other related coronaviruses. Our immunogens focused the serum antibody response to conserved epitopes on the receptor binding domain outside of the ACE2 receptor binding motif; this contrasts with current SARS-CoV-2 therapeutic antibodies, which predominantly target the receptor binding motif.

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  1. Version published to 10.1101/2020.12.07.415216v2 on bioRxiv
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    SciScore for 10.1101/2020.12.07.415216: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIACUC: All experiments were conducted with institutional IACUC approval (MGH protocol 2014N000252).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableIn this study, female mice aged 6-10 weeks were used.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    This included the following mouse-specific antibodies: CD3-BV786 (BioLegend), CD19-BV421 (BioLegend), IgM-BV605 (BioLegend), IgG-PerCP/Cy5.5 (BioLegend).
    CD19-BV421
    suggested: None
    IgG-PerCP/Cy5.5
    suggested: None
    HRP-conjugated rabbit anti-mouse IgG antibody (Abcam) at a concentration of 1:20,000 in PBS and a volume of 150 μL was used as a secondary antibody.
    HRP-conjugated rabbit anti-mouse IgG antibody
    suggested: None
    anti-mouse IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Lentiviral particles were produced via transient transfection of 293T cells.
    293T
    suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
    Afterwards, 10,000 293T-ACE2 cells (33) in 20 μL of media containing 15 μg/mL polybrene was added.
    293T-ACE2
    suggested: RRID:CVCL_YZ65)
    Experimental Models: Organisms/Strains
    SentencesResources
    Immunizations: C57BL/6 mice (Jackson Laboratory) received 20 μg of protein adjuvanted with 50% w/v Sigma adjuvant in 100 μL of inoculum.
    C57BL/6
    suggested: None
    Software and Algorithms
    SentencesResources
    Constructs were codon optimized by Integrated DNA Technologies, cloned into pVRC, and sequence confirmed by Genewiz.
    Genewiz
    suggested: (GENEWIZ, RRID:SCR_003177)
    FCS files were analyzed using FlowJo (version 10).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    GraphPad Prism was used to fit nonlinear regressions to the data, which allowed IC50 values to be calculated using the interpolated 50% inhibitory concentration.
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Statistical Analysis: Statistical analyses and curve fitting were performed using GraphPad Prism (version 9).
    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.

    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 10.1101/2020.12.07.415216v1 on bioRxiv