Effect of SARS-CoV-2 spike mutations on animal ACE2 usage and in vitro neutralization sensitivity

  1. Weitong Yao
  2. Danting Ma
  3. Haimin Wang
  4. Xiaojuan Tang
  5. Chengzhi Du
  6. Hong Pan
  7. Chao Li
  8. Hua Lin
  9. Michael Farzan
  10. Jincun Zhao
  11. Yujun Li
  12. Guocai Zhong

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Abstract

The emergence of SARS-CoV-2 variants poses greater challenges to the control of COVID-19 pandemic. Here, we parallelly investigated three important characteristics of seven SARS-CoV-2 variants, including two mink-associated variants, the B.1.617.1 variant, and the four WHO-designated variants of concerns (B.1.1.7, B.1.351, P.1, and B.1.617.2). We first investigated the ability of these variants to bind and use animal ACE2 orthologs as entry receptor. We found that, in contrast to a prototype variant, the B.1.1.7, B.1.351, and P.1 variants had significantly enhanced affinities to cattle, pig, and mouse ACE2 proteins, suggesting increased susceptibility of these species to these SARS-CoV-2 variants. We then evaluated in vitro neutralization sensitivity of these variants to four monoclonal antibodies in clinical use. We observed that all the variants were partially or completely resistant against at least one of the four tested antibodies, with B.1.351 and P.1 showing significant resistance to three of them. As ACE2-Ig is a broad-spectrum anti-SARS-CoV-2 drug candidate, we then evaluated in vitro neutralization sensitivity of these variants to eight ACE2-Ig constructs previously described in three different studies. All the SARS-CoV-2 variants were efficiently neutralized by these ACE2-Ig constructs. Interestingly, compared to the prototype variant, most tested variants including the variants of concern B.1.1.7, B.1.351, P.1, and B.1.617.2 showed significantly increased (up to ∼15-fold) neutralization sensitivity to ACE2-Ig constructs that are not heavily mutated in the spike-binding interface of the soluble ACE2 domain, suggesting that SARS-CoV-2 evolves toward better utilizing ACE2, and that ACE2-Ig is an attractive drug candidate for coping with SARS-CoV-2 mutations.

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  1. Version published to 10.1101/2021.01.27.428353v3 on bioRxiv
  2. ScreenIT

    SciScore for 10.1101/2021.01.27.428353: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    In brief, SARS-CoV-2 Spike variant-pseudotyped luciferase reporter viruses were pre-diluted in DMEM (2% FBS, heat-inactivated) containing titrated amounts of the ACE2-Ig or one of the three anti-SARS-CoV-2 antibodies.
    anti-SARS-CoV-2
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cells: 293T and HeLa cells were kindly provided by Stem Cell Bank, Chinese Academy of Sciences, confirmed mycoplasma-free by the provider, and maintained in Dulbecco’s Modified Eagle Medium (DMEM, Life Technologies) at 37 °C in a 5% CO2-humidified incubator.
    HeLa
    suggested: None
    HeLa-based stable cells expressing human ACE2 (HeLa-hACE2) were maintained under the same culture condition as HeLa, except that 3 μg/mL of puromycin was added to the growth medium.
    HeLa-based
    suggested: None
    293F cells for the production of ACE2-Ig protein and SARS-CoV-2 antibodies were generously provided by Dr. Yu J.
    293F
    suggested: None
    SARS-CoV-2 pseudovirus infection of 293T cells expressing ACE2 orthologs: Pseudovirus infection assay was performed according to our previous study11.
    293T
    suggested: None
    Virus-inhibitor mixtures were incubated at 37 °C for 30min, then added to HeLa-hACE2 cells in 96-well plates and incubated overnight at 37 °C.
    HeLa-hACE2
    suggested: None
    Software and Algorithms
    SentencesResources
    GraphPad Prism 6.0 software was used for figure preparation and statistical analyses.
    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 10.1101/2021.01.27.428353v2 on bioRxiv
  4. Version published to 10.1101/2021.01.27.428353v1 on bioRxiv