Characterization of SARS-CoV-2 variants B.1.617.1 (Kappa), B.1.617.2 (Delta) and B.1.618 on cell entry, host range, and sensitivity to convalescent plasma and ACE2 decoy receptor

  1. Wenlin Ren
  2. Xiaohui Ju
  3. Mingli Gong
  4. Jun Lan
  5. Yanying Yu
  6. Quanxin Long
  7. Yu Zhang
  8. Jin Zhong
  9. Guocai Zhong
  10. Xinquan Wang
  11. Ailong Huang
  12. Rong Zhang
  13. Qiang Ding

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Abstract

Recently, highly transmissible SARS-CoV-2 variants B.1.617.1 (Kappa), B.1.617.2 (Delta) and B.1.618 were identified in India with mutations within the spike proteins. The spike protein of Kappa contains four mutations E154K, L452R, E484Q and P681R, and Delta contains L452R, T478K and P681R, while B.1.618 spike harbors mutations Δ145-146 and E484K. However, it remains unknown whether these variants have altered in their entry efficiency, host tropism, and sensitivity to neutralizing antibodies as well as entry inhibitors. In this study, we found that Kappa, Delta or B.1.618 spike uses human ACE2 with no or slightly increased efficiency, while gains a significantly increased binding affinity with mouse, marmoset and koala ACE2 orthologs, which exhibits limited binding with WT spike. Furthermore, the P618R mutation leads to enhanced spike cleavage, which could facilitate viral entry. In addition, Kappa, Delta and B.1.618 exhibits a reduced sensitivity to neutralization by convalescent sera owning to the mutation of E484Q, T478K, Δ145-146 or E484K, but remains sensitive to entry inhibitors-ACE2-lg decoy receptor. Collectively, our study revealed that enhanced human and mouse ACE2 receptor engagement, increased spike cleavage and reduced sensitivity to neutralization antibodies of Kappa, Delta and B.1.618 may contribute to the rapid spread of these variants and expanded host range. Furthermore, our result also highlighted that ACE2-lg could be developed as broad-spectrum antiviral strategy against SARS-CoV-2 variants.

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  1. ScreenIT

    SciScore for 10.1101/2021.09.03.458829: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: This study was approved by the Institution Review Board of Tsinghua University (20210040)
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line AuthenticationContamination: Cells were tested routinely and found to be free of mycoplasma contamination.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    Cell culture: HEK293T (American Tissue Culture Collection, ATCC, Manassas, VA, CRL-3216), Vero E6 (Cell Bank of the Chinese Academy of Sciences, Shanghai, China) and A549 (ATCC) cells were maintained in Dulbecco’s modified Eagle medium (DMEM) (Gibco, NY, USA) supplemented with 10% (vol/vol
    Vero E6
    suggested: None
    A549
    suggested: None
    Surface ACE2 binding with RBD-His assay: HeLa cells were transduced with lentiviruses expressing the ACE2 variants for 48 h.
    HeLa
    suggested: None
    Production of SARS-CoV-2 pseudotyped virus, determination of viral entry efficiency and analysis of spike protein cleavage: Pseudoviruses were produced in HEK293T cells by co-transfecting the retroviral vector pTG-MLV-Fluc, pTG-MLV-Gag-pol, and pcDNA3.1 expressing SARS-CoV-2 spike gene or VSV-G (pMD2.G (Addgene #12259)) using VigoFect (
    HEK293T
    suggested: None
    For neutralization experiments, S protein bearing pseudotyped virion particles were pre-incubated for 30 min at 37°C with diluted plasma samples obtained from convalescent COVID-19 patients, before the mixtures were inoculated onto HeLa-ACE2 cells.
    HeLa-ACE2
    suggested: None
    Recombinant ACE2-lg protein expression and purification: ACE2-lg, a recombinant Fc fusion protein of soluble human ACE2 (residues Gln18-Ser740) was expressed in 293F cells and purified using protein A affinity chromatography as described in our previous study28.
    293F
    suggested: None
    Recombinant DNA
    SentencesResources
    Plasmids: The cDNAs encoding the ACE2 orthologs were synthesized by GenScript and cloned into the pLVX-IRES-zsGreen1 vector (Catalog No. 632187, Clontech Laboratories, Inc) with a C-terminal FLAG tag.
    pLVX-IRES-zsGreen1
    suggested: None
    (Addgene #12259) and psPAX2 (Addgene #12260) and the transfer vector with VigoFect DNA transfection reagent (Vigorous) into HEK293T cells.
    psPAX2
    suggested: RRID:Addgene_12260)
    Production of SARS-CoV-2 pseudotyped virus, determination of viral entry efficiency and analysis of spike protein cleavage: Pseudoviruses were produced in HEK293T cells by co-transfecting the retroviral vector pTG-MLV-Fluc, pTG-MLV-Gag-pol, and pcDNA3.1 expressing SARS-CoV-2 spike gene or VSV-G (pMD2.G (Addgene #12259)) using VigoFect (
    pTG-MLV-Fluc
    suggested: None
    pTG-MLV-Gag-pol
    suggested: None
    pcDNA3.1
    suggested: RRID:Addgene_79663)
    VSV-G
    suggested: RRID:Addgene_138479)
    pMD2 . G
    suggested: None

    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: Please consider improving the rainbow (“jet”) colormap(s) used on pages 25, 26 and 27. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    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

    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 10.1101/2021.09.03.458829v1 on bioRxiv