Cell-Free Glycoengineering of the Recombinant SARS-CoV-2 Spike Glycoprotein

  1. Johannes Ruhnau
  2. Valerian Grote
  3. Mariana Juarez-Osorio
  4. Dunja Bruder
  5. Reza Mahour
  6. Erdmann Rapp
  7. Thomas F. T. Rexer
  8. Udo Reichl

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Abstract

The baculovirus-insect cell expression system is readily utilized to produce viral glycoproteins for research as well as for subunit vaccines and vaccine candidates, for instance against SARS-CoV-2 infections. However, the glycoforms of recombinant proteins derived from this expression system are inherently different from mammalian cell-derived glycoforms with mainly complex-type N- glycans attached, and the impact of these differences in protein glycosylation on the immunogenicity is severely under investigated. This applies also to the SARS-CoV-2 spike glycoprotein, which is the antigen target of all licensed vaccines and vaccine candidates including virus like particles and subunit vaccines that are variants of the spike protein. Here, we expressed the transmembrane-deleted human β -1,2 N-acetlyglucosamintransferases I and II (MGAT1ΔTM and MGAT2ΔTM) and the β -1,4-galactosyltransferase (GalTΔTM) in E. coli to in-vitro remodel the N -glycans of a recombinant SARS-CoV-2 spike glycoprotein derived from insect cells. In a cell-free sequential one-pot reaction, fucosylated and afucosylated paucimannose-type N -glycans were converted to complex-type galactosylated N -glycans. In the future, this in-vitro glycoengineering approach can be used to efficiently generate a wide range of N -glycans on antigens considered as vaccine candidates for animal trials and preclinical testing to better characterize the impact of N -glycosylation on immunity and to improve the efficacy of protein subunit vaccines.

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  1. Version published to 10.3389/fbioe.2021.699025
  2. ScreenIT

    SciScore for 10.1101/2021.04.30.442139: (What is this?)

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

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    No key resources detected.

    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 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.

    About SciScore

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  3. Version published to 10.1101/2021.04.30.442139v1 on bioRxiv