Calreticulin co-expression supports high level production of a recombinant SARS-CoV-2 spike mimetic in Nicotiana benthamiana

  1. Emmanuel Margolin
  2. Matthew Verbeek
  3. Ann Meyers
  4. Ros Chapman
  5. Anna-Lise Williamson
  6. Edward P Rybicki

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Abstract

An effective prophylactic vaccine is urgently needed to protect against SARS-CoV-2 infection. The viral spike, which mediates entry into cells by interacting with the host angiotensin-converting enzyme 2, is the primary target of most vaccines in development. These vaccines aim to elicit protective immunity against the glycoprotein by use of inactivated virus, vector-mediated delivery of the antigen in vivo , or by direct immunization with the purified antigen following expression in a heterologous system. These approaches are mostly dependent on the growth of mammalian or insect cells, which requires a sophisticated infrastructure that is not generally available in developing countries due to the incumbent costs which are prohibitive. Plant-based subunit vaccine production has long been considered as a cheaper alternative, although low expression yields and differences along the secretory pathway to mammalian cells have posed a challenge to producing certain viral glycoproteins. Recent advances that have enabled many of these constraints to be addressed include expressing the requisite human proteins in plants to support the maturation of the protein of interest. In this study we investigated these approaches to support the production of a soluble and putatively trimeric SARS-CoV-2 spike mimetic in Nicotiana benthamiana via transient Agrobacterium-mediated expression. The co-expression of human calreticulin dramatically improved the accumulation of the viral spike, which was barely detectable in the absence of the co-expressed accessory protein. The viral antigen was efficiently processed even in the absence of co-expressed furin, suggesting that processing may have occurred at the secondary cleavage site and was mediated by an endogenous plant protease. In contrast, the spike was not efficiently processed when expressed in mammalian cells as a control, although the co-expression of furin improved processing considerably. This study demonstrates the feasibility of molecular engineering to improve the production of viral glycoproteins in plants, and supports plant-based production of SARS-CoV-2 spike-based vaccines and reagents for serological assays.

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  1. ScreenIT

    SciScore for 10.1101/2020.06.14.150458: (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
    The recombinant spike was detected with 1:2000 of mouse monoclonal anti-histidine antibody (Serotech, MCA1396), which in turn was detected using 1:5000 goat anti-mouse IgG alkaline phosphatase conjugate (Sigma, A3562).
    anti-histidine
    suggested: (Bio-Rad Cat# MCA1396, RRID:AB_322084)
    anti-mouse IgG
    suggested: (Sigma-Aldrich Cat# A3562, RRID:AB_258091)
    A3562
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Recombinant protein production in mammalian cells: Protein expression as conducted in adherent HEK293 cells.
    HEK293
    suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)

    Results from OddPub: Thank you for sharing your data.

    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.
    • No funding statement was detected.
    • 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 10.1101/2020.06.14.150458v1 on bioRxiv