COVID-19 patient serum less potently inhibits ACE2-RBD binding for various SARS-CoV-2 RBD mutants

  1. Daniel Junker
  2. Alex Dulovic
  3. Matthias Becker
  4. Teresa R. Wagner
  5. Philipp D. Kaiser
  6. Bjoern Traenkle
  7. Katharina Kienzle
  8. Stefanie Bunk
  9. Carlotta Struemper
  10. Helene Haeberle
  11. Kristina Schmauder
  12. Natalia Ruetalo
  13. Nisar Malek
  14. Karina Althaus
  15. Michael Koeppen
  16. Ulrich Rothbauer
  17. Juliane S. Walz
  18. Michael Schindler
  19. Michael Bitzer
  20. Siri Göpel
  21. Nicole Schneiderhan-Marra

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Abstract

As global vaccination campaigns against SARS-CoV-2 proceed, there is particular interest in the longevity of immune protection, especially with regard to increasingly infectious virus variants. Neutralizing antibodies (Nabs) targeting the receptor binding domain (RBD) of SARS-CoV-2 are promising correlates of protective immunity and have been successfully used for prevention and therapy. As SARS-CoV-2 variants of concern (VOCs) are known to affect binding to the ACE2 receptor and by extension neutralizing activity, we developed a bead-based multiplex ACE2-RBD inhibition assay (RBDCoV-ACE2) as a highly scalable, time-, cost-, and material-saving alternative to infectious live-virus neutralization tests. By mimicking the interaction between ACE2 and the RBD, this serological multiplex assay allows the simultaneous analysis of ACE2 binding inhibition to the RBDs of all SARS-CoV-2 VOCs and variants of interest (VOIs) in a single well. Following validation against a classical virus neutralization test and comparison of performance against a commercially available assay, we analyzed 266 serum samples from 168 COVID-19 patients of varying severity. ACE2 binding inhibition was reduced for ten out of eleven variants examined compared to wild-type, especially for those displaying the E484K mutation such as VOCs beta and gamma. ACE2 binding inhibition, while highly individualistic, positively correlated with IgG levels. ACE2 binding inhibition also correlated with disease severity up to WHO grade 7, after which it reduced.

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  1. Version published to 10.1038/s41598-022-10987-2
  2. ScreenIT

    SciScore for 10.1101/2021.08.20.21262328: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: Sample collection and execution of this study was approved by the Ethics committee of the Eberhard Karls University Tübingen and the University Hospital Tübingen under the ethical approval numbers 188/2020A and 764/2020BO2 to Prof. Dr. Michael Bitzer.
    Consent: All participants signed the broad consent of the Medical Faculty Tübingen for sample collection.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line AuthenticationAuthentication: Commercial assay formats: For further validation, one sample from each individual (n=186) were analyzed with the commercially available in-vitro diagnostic test SARS-CoV-2 NeutraLISA (Euroimmun).

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    Confirmed constructs were expressed in Expi293 cells (28, 46).
    Expi293
    suggested: RRID:CVCL_D615)
    Recombinant DNA
    SentencesResources
    Expression and Purification of SARS-CoV-2 RBD mutants: The expression plasmid pCAGGS, encoding the receptor-binding domain (RBD) of SARS-CoV-2 spike protein (amino acids 319-541), was kindly provided by F.
    pCAGGS
    suggested: RRID:Addgene_18926)
    Amplificates were inserted into the pCDNA3.4 expression vector using XbaI and NotI restriction sites.
    pCDNA3.4
    suggested: RRID:Addgene_131198)
    Software and Algorithms
    SentencesResources
    Data analysis: Data collection and assignment to metadata was performed with Microsoft Excel 2016.
    Microsoft Excel
    suggested: (Microsoft Excel, RRID:SCR_016137)
    Data analysis and visualization was performed with Graphpad Prism (version 9.1.2).
    Graphpad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Figures were edited with Inkscape (version 0.92.4).
    Inkscape
    suggested: (Inkscape, RRID:SCR_014479)

    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:
    Our ACE2 RBD competition assay has limitations similar to other protein-based in vitro neutralization assays, such as only accounting for the Nabs that block the RBD-ACE2 interaction site through steric hindrance, and not for Nabs that interfere with cell entry mechanisms as would be analyzed in a VNT. Furthermore, the binding assay is also more prone to non-specific binding events. However, a major advantage of the multiplex ACE2 RBD competition assay over VNTs is the speed of response toward viral evolution such as emerging variants of concern. The bead-based format of the assay is also highly reproducible and not susceptible to changes in experimental conditions, as is the case for cell culture based VNTs. The plate format of the assay also enables automation and high-throughput screening. Our ACE2 RBD competition assay only requires recombinant expressed RBD proteins which can be quickly and easily produced. Additionally, this assay has the possibility of introducing artificial mutants to screen for possible escape variants that could arise in the future. Similar to other neutralization studies, our study is limited by the availability of appropriate control samples. Additionally, as the majority of this study population were admitted to the intensive care unit, the more serious grades of COVID-19 infection are heavily overrepresented in our population. Our samples are also highly variable in their longitudinal nature, with no consistent time points. However, this large v...

    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.

    Results from scite Reference Check: We found no unreliable references.

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  3. Version published to 10.1101/2021.08.20.21262328 on medRxiv