Sialic acid and fucose residues on the SARS-CoV-2 receptor binding domain modulate IgG reactivity

  1. Ebba Samuelsson
  2. Ekaterina Mirgorodskaya
  3. Kristina Nyström
  4. Malin Bäckström
  5. Jan-Åke Liljeqvist
  6. Rickard Nordén

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Abstract

The receptor binding domain (RBD) of the SARS-CoV-2 spike protein is a conserved domain and a target for neutralizing antibodies. We defined the carbohydrate content of recombinant RBD produced in different mammalian cells. We found a higher degree of complex type N-linked glycans, with less sialylation and more fucosylation, when the RBD was produced in Human embryonic kidney cells compared to the same protein produced in Chinese hamster ovary cells. The carbohydrates on the RBD proteins were enzymatically modulated and the effect on antibody reactivity was evaluated with serum samples from SARS-CoV-2 positive patients. Removal of all carbohydrates diminished antibody reactivity while removal of only sialic acids or terminal fucoses improved the reactivity. The RBD produced in Lec3.2.8.1-cells, which generate carbohydrate structures devoid of sialic acids and with reduced fucose content, exhibited enhanced antibody reactivity verifying the importance of these specific monosaccharides. The results can be of importance for the design of future vaccine candidates, indicating that it might be possible to enhance the immunogenicity of recombinant viral proteins.

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  1. ScreenIT

    SciScore for 10.1101/2022.01.20.477056: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: Ethical statement: The study was approved by the ethical review board in Gothenburg (Dnr: 2021-02252).
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Levels of human anti-SARS-CoV-2 IgG antibodies in convalescent serum samples: Serum samples from SARS-CoV-2 convalescent individuals (n=24) were obtained from the department of Clinical Microbiology, Sahlgrenska University Hospital, Gothenburg, Sweden.
    anti-SARS-CoV-2 IgG
    suggested: None
    Anti-SARS-CoV-2 antibody reactivity assay: The antibody reactivity towards glycosidase treated proteins were assessed using an enzyme-linked immunosorbent assay (ELISA).
    Anti-SARS-CoV-2
    suggested: None
    The comparison between anti-RBD IgG levels and antibody reactivity towards recombinant RBD was done with Pearson correlation coefficient, assuming normal distribution.
    anti-RBD IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Lec3.2.8.1 cells (a mutated CHO cell line kindly received from Prof. P Stanley (Chen & Stanley, 2003)) were cultured under the same conditions.
    CHO
    suggested: None
    The HEK293 derivate HEK293F cell line (Cat nr R79007, Thermo Fisher Scientific) were cultured in Freestyle 293 medium.
    HEK293F
    suggested: None
    Recombinant DNA
    SentencesResources
    Expression of recombinant S protein constructs: The receptor-binding domain of the SARS-CoV-2 spike protein (amino acids 319-541) was produced in three cell lines using an expression vector obtained through BEI Resources, NIAID, NIH, which is vector pCAGGS containing the SARS-CoV-2, Wuhan-Hu-1 spike glycoprotein gene RBD with C-terminal Hexa-Histidine tag (NR-52309) (Table EV1).
    pCAGGS
    suggested: RRID:Addgene_127347)
    Software and Algorithms
    SentencesResources
    Glycan database search and data processing: The acquired data were analyzed using Proteome Discoverer version 2.4 (Thermo Fisher Scientific).
    Proteome Discoverer
    suggested: (Proteome Discoverer, RRID:SCR_014477)
    The SARS-CoV-2 IgG II Quant assay is a chemiluminescent microparticle immunoassay (CMIA) used for quantitative determination of IgG antibodies to SARS-CoV-2 in human serum and plasma on the ARCHITECT System (Abbott Laboratories, Chicago, IL).
    Abbott Laboratories
    suggested: None
    All statistical analyses were performed using the Graphpad Prism software version 9.3.1
    Graphpad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    (GraphPad Software Inc, San Diego, CA, USA).
    GraphPad
    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 10.1101/2022.01.20.477056 on bioRxiv