Antibody-dependent enhancement (ADE) of SARS-CoV-2 infection in recovered COVID-19 patients: studies based on cellular and structural biology analysis

  1. Fan Wu
  2. Renhong Yan
  3. Mei Liu
  4. Zezhong Liu
  5. Yingdan Wang
  6. Die Luan
  7. Kaiyue Wu
  8. Zhigang Song
  9. Tingting Sun
  10. Yunping Ma
  11. Yuanyuan Zhang
  12. Qimin Wang
  13. Xiang Li
  14. Ping Ji
  15. Yaning Li
  16. Cheng Li
  17. Yanling Wu
  18. Tianlei Ying
  19. Yumei Wen
  20. Shibo Jiang
  21. Tongyu Zhu
  22. Lu Lu
  23. Yongzhen Zhang
  24. Qiang Zhou
  25. Jinghe Huang

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Abstract

Antibody-dependent enhancement (ADE) has been reported in several virus infections including dengue fever virus, severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronavirus infection. To study whether ADE is involved in COVID-19 infections, in vitro pseudotyped SARS-CoV-2 entry into Raji cells, K562 cells, and primary B cells mediated by plasma from recovered COVID-19 patients were employed as models. The enhancement of SARS-CoV-2 entry into cells was more commonly detected in plasma from severely-affected elderly patients with high titers of SARS-CoV-2 spike protein-specific antibodies. Cellular entry was mediated via the engagement of FcγRII receptor through virus-cell membrane fusion, but not by endocytosis. Peptide array scanning analyses showed that antibodies which promote SARS-CoV-2 infection targeted the variable regions of the RBD domain. To further characterize the association between the spike-specific antibody and ADE, an RBD-specific monoclonal antibody (7F3) was isolated from a recovered patient, which potently inhibited SARS-Cov-2 infection of ACE-2 expressing cells and also mediated ADE in Raji cells. Site-directed mutagenesis the spike RBD domain reduced the neutralization activity of 7F3, but did not abolish its binding to the RBD domain. Structural analysis using cryo-electron microscopy (Cryo-EM) revealed that 7F3 binds to spike proteins at a shift-angled pattern with one “up” and two “down” RBDs, resulting in partial overlapping with the receptor binding motif (RBM), while a neutralizing monoclonal antibody that lacked ADE activity binds to spike proteins with three “up” RBDs, resulting in complete overlapping with RBM. Our results revealed that ADE mediated by SARS-CoV-2 spike-specific antibodies could result from binding to the receptor in slightly different pattern from antibodies mediating neutralizations. Studies on ADE using antibodies from recovered patients via cell biology and structural biology technology could be of use for developing novel therapeutic and preventive measures for control of COVID-19 infection.

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  1. ScreenIT

    SciScore for 10.1101/2020.10.08.20209114: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: Study design and participants: The study was conducted under a clinical protocol approved by the Investigational Review Board in the Shanghai Public Health Clinical Center (Study number: YJ-2020-S018-02).
    Consent: All participants signed an informed consent approved by the IRB.
    RandomizationThe fused cells were counted on five random fields in each well.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Mouse anti-human CD32 monoclonal antibody (clone number FLI8.26) was purchased from BD Pharmingen (USA).
    Mouse anti-human CD32 monoclonal antibody
    suggested: None
    anti-human CD32
    suggested: None
    Biolayer interferometry binding assay: The kinetics of monoclonal antibody binding to SARS-CoV-2 RBD protein was measured by biolayer interferometry binding assay on a FortéBio OctetRED96 instrument using anti-human IgG (AHC) biosensors as previously described38 The assay followed sequential steps at 30°C as follows.
    anti-human IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Materials: The human primary embryonic kidney cell line (HEK293T) (CRL-3216™) and Raji cells were obtained from the American Type Culture Collection (
    HEK293T
    suggested: None
    Raji cells and K562 cells were cultured in RPMI 1640 medium with 10% fetal bovine serum (FBS), and the other cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% FBS.
    K562
    suggested: None
    HEK293 cells expressing SARS-CoV-2 RBD protein was purchased from GenScript Company (Nanjing, China).
    HEK293
    suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
    Pseudoviruses of SARS-CoV-2, SARS-CoV, Bat-SL-RS3367 and WIV1 coronaviruses were generated by cotransfection of 293T cells with pNL4-3.
    293T
    suggested: None
    Neutralization assay: Neutralization activity of plasma and antibodies was measured by the inhibition of pseudovirus infection with 293 T/ACE2 cells as previously described18.
    T/ACE2
    suggested: RRID:CVCL_YZ65)
    Briefly, HEK-293T cells expressing the SARS-CoV-2 S protein on the cell membrane were used as effector cells, while Raji cells were used as target cells.
    HEK-293T
    suggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)
    Raji
    suggested: CLS Cat# 300359/p25072_RAJI, RRID:CVCL_0511)
    Software and Algorithms
    SentencesResources
    The stacks were motion corrected with MotionCor2 40 and binned 2-fold, resulting in a pixel size of 1.087 Å/pixel.
    MotionCor2
    suggested: (MotionCor2, RRID:SCR_016499)
    After 2D classification with Relion, good particles were selected and subjected to two cycles of heterogeneous refinement without symmetry using cryoSPARC 47.
    cryoSPARC
    suggested: (cryoSPARC, RRID:SCR_016501)
    Then the datasets of three similar RBD-mAb sub-complexes were combined and subjected to focused refinement with Relion.
    Relion
    suggested: (RELION, RRID:SCR_016274)
    The fitted atomic models were further manually adjusted with Coot 51.
    Coot
    suggested: (Coot, RRID:SCR_014222)
    Statistical analysis: Statistical analyses were carried out using GraphPad Prism 7.0.
    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: We detected the following sentences addressing limitations in the study:
    Limitations: In this study, plasma and antibodies was measured by an in vitro cell-based pseudovirus assay to evaluate the enhancement of SARS-CoV-2 infection of immune cells. Whether such enhancement of virus infection results in disease severity needs to be validated in appropriate animal models. Implication for SARS-CoV-2 vaccine research and therapies: Although several SARS-Cov-2 vaccines have been undergoing phase III clinical trials, the potential ADE of coronavirus infection still remains a safety concern for any vaccine candidates. The observation of enhancement of SARS-CoV-2 infection mediated by plasma and antibodies from recovered COVID-19 patients in this study does not indicate that vaccine candidates would necessarily induce ADE or disease severity. However, these results suggest that vaccine candidates should be evaluated for induction of ADE in addition to induction of neutralizing antibodies. A vaccine that can induce high titers of neutralizing antibodies should be safer than one inducing low titers since 1) most the newly invaded virions are neutralized before the ADE occurs and 2) neutralizing antibodies mediate ADE only at the suboptimal neutralizing concentration. Furthermore, these results also suggested that plasma and antibodies from patients who recovered from COVID-19 should be tested for potential ADE effect before clinical usage.

    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

    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.

  2. Version published to 10.1101/2020.10.08.20209114v1 on medRxiv