Plant-based production of SARS-CoV-2 antigens for use in a subunit vaccine

  1. Jordan Demone
  2. Maryam Nourimand
  3. Mariam Maltseva
  4. Mina Nasr-Sharif
  5. Yannick Galipeau
  6. Marc-André Langlois
  7. Allyson M. MacLean

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Abstract

The COVID-19 pandemic has brought to the forefront an urgent need for the rapid development of highly efficacious vaccines, particularly in light of the ongoing emergence of multiple variants of concern. Plant-based recombinant protein platforms are emerging as cost-effective and highly scalable alternatives to conventional protein production. Viral glycoproteins, however, are historically challenging to produce in plants. Herein, we report the production of plant-expressed wild-type glycosylated SARS-CoV-2 Spike RBD (receptor-binding domain) protein that is recognized by anti-RBD antibodies and exhibits high-affinity binding to the SARS-CoV-2 receptor ACE2 (angiotensin-converting enzyme 2). Moreover, our plant-expressed RBD was readily detected by IgM, IgA, and IgG antibodies from naturally infected convalescent, vaccinated, or convalescent and vaccinated individuals. We further demonstrate that RBD binding to the ACE2 receptor was efficiently neutralized by antibodies from sera of SARS-CoV-2 convalescent and partially and fully vaccinated individuals. Collectively, these findings demonstrate that recombinant RBD produced in planta exhibits suitable biochemical and antigenic features for use in a subunit vaccine platform.

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  1. ScreenIT

    SciScore for 10.1101/2021.10.17.464700: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: Patient Samples and Collection: Use of human samples for this study was approved by the University of Ottawa Ethics Review Board: Certificates H-04-20-5727, H-04-21-6643 and H-07-20-6009.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    In initial experiments, aliquots of each fraction were visualized via immunoblotting (anti-His antibody; Cat: SAB2702218
    anti-His
    suggested: None
    In conjunction, titration curves of conformation-dependent monoclonal IgM (Absolute Antibody, Ab01680-15.0), IgA (Absolute Antibody, Ab01680-16.0), and IgG (Absolute Antibody, Ab01680-10.0) CR3022 antibodies were used as reference material to assess protein folding.
    titration curves of conformation-dependent monoclonal IgM ( Absolute Antibody , Ab01680-15.0) , IgA ( Absolute Antibody , Ab01680-16.0)
    suggested: None
    IgG ( Absolute Antibody , Ab01680-10.0 ) CR3022 antibodies
    suggested: None
    After blocking, plates were washed thrice with PBS-T, and followed by addition of 100 μL of the respective diluted serum samples and CR3022 antibodies.
    CR3022
    suggested: None
    The plates were incubated for two hours on a shaker at room temperature, washed thrice with PBS-T followed by the addition of 50 μL of the respective secondary-HRP antibody at specified dilutions (1:4000 secondary anti-human IgG-HRP (NRC anti-hIgG#5-HRP
    anti-human IgG-HRP
    suggested: None
    Surrogate Neutralization ELISA (snELISA) assay for evaluation of neutralization in serum samples: The described methodology was adapted from the surrogate neutralization ELISA assay as shown in Abe et al. 2020, for the evaluation of the relative inhibition of neutralizing antibodies to RBD protein from binding to soluble ACE2.
    ACE2
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    RBD Production in mammalian cells: For comparison, a mammalian RBD was produced in HEK 293F cells and purified.
    HEK 293F
    suggested: RRID:CVCL_6642)
    Experimental Models: Organisms/Strains
    SentencesResources
    (Mount Sinai, NYC) encoding the Whuhan-Hu-1 RBD (MN908947) sequence coding for the amino acid 319-541 and fused with the N-terminal SARS-CoV-2 spike secretory signal and a C-terminal hexa-histidine tag was transfected into 293F cells cultivated in Freestyle 293 expression media (Thermo Fisher, #12338018) at 37°C, 7% CO2, while shaking (125rpm).
    Whuhan-Hu-1 RBD
    suggested: None
    Recombinant DNA
    SentencesResources
    The construct was codon-optimized for N. benthamiana expression and was synthesized by GenScript into the pHREAC vector (Peyret et al., 2019) via BsaI sites
    pHREAC
    suggested: RRID:Addgene_134908)
    Lectin-binding human chaperone calreticulin (NP_004334.1) was codon-optimized for N. benthamiana and synthesized in the pHRE vector (Peyret et al., 2019).
    pHRE
    suggested: RRID:Addgene_134909)
    Transient expression in Nicotiana benthamiana: Prior to infiltration, pHREAC-RBD and pHRE-calreticulin were freshly transformed into Agrobacterium tumefaciens strains AGL1 and Gv2260, respectively, via electroporation.
    pHREAC-RBD
    suggested: None
    pHRE-calreticulin
    suggested: None
    Software and Algorithms
    SentencesResources
    Dissociation constant (KD) was determined using 4-parameter curve fitting with GraphPad Prism 9.1.2 software.
    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 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

    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 10.1101/2021.10.17.464700v1 on bioRxiv