The cellular characterisation of SARS-CoV-2 spike protein in virus-infected cells using Receptor Binding Domain-binding specific human monoclonal antibodies

  1. Conrad En-Zuo Chan
  2. Ching-Ging Ng
  3. Angeline Pei-Chew Lim
  4. Shirley Lay-Kheng Seah
  5. De-Hoe Chye
  6. Steven Ka-Khuen Wong
  7. Jie-Hui Lim
  8. Vanessa Zi-Yun Lim
  9. Soak-Kuan Lai
  10. Pui-San Wong
  11. Kok-Mun Leong
  12. Yi-Chun Liu
  13. Richard J Sugrue
  14. Boon-Huan Tan

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Abstract

A human monoclonal antibody panel (PD4, PD5, PD7, SC23 and SC29) was isolated from the B cells of convalescent patients and used to examine the S protein in SARS-CoV-2- infected cells. While all five antibodies bound conformational-specific epitopes within SARS-CoV-2 Spike (S) protein, only PD5, PD7, and SC23 were able to bind to the Receptor Binding Domain (RBD). Immunofluorescence microscopy was used to examine the S protein RBD in cells infected with the Singapore isolates SARS-CoV-2/0334 and SARS-CoV-2/1302. The RBD-binders exhibited a distinct cytoplasmic staining pattern that was primarily localised within the Golgi complex and was distinct from the diffuse cytoplasmic staining pattern exhibited by the non-RBD binders (PD4 and SC29). These data indicated that the S protein adopted a conformation in the Golgi complex that enabled the RBD recognition by the RBD-binders. The RBD-binders also recognised the uncleaved S protein indicating that S protein cleavage was not required for RBD recognition. Electron microscopy indicated high levels of cell-associated virus particles, and multiple cycle virus infection using RBD-binder staining provided evidence for direct cell-to-cell transmission for both isolates. Although similar levels of RBD-binder staining was demonstrated for each isolate, the SARS-CoV-2/1302 exhibited slower rates of cell-to-cell transmission. These data suggest that a conformational change in the S protein occurs during its transit through the Golgi complex that enables RBD recognition by the RBD-binders, and suggests that these antibodies can be used to monitor S protein RBD formation during the early stages of infection.

Importance

The SARS CoV-2 spike (S) protein receptor binding domain (RBD) mediates the attachment of SARS CoV-2 to the host cell. This interaction plays an essential role in initiating virus infection and the S protein RBD is therefore a focus of therapeutic and vaccine interventions. However, new virus variants have emerged with altered biological properties in the RBD that can potentially negate these interventions. Therefore an improved understanding of the biological properties of the RBD in virus-infected cells may offer future therapeutic strategies to mitigate SARS CoV-2 infection. We used physiologically relevant antibodies that were isolated from the B cells of convalescent COVID19 patients to monitor the RBD in cells infected with SARS CoV-2 clinical isolates. These immunological reagents specifically recognise the correctly folded RBD and were used to monitor the appearance of the RBD in SARS CoV-2-infected cells and identified the site where the RDB first appears.

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  1. Version published to 10.1101/2021.12.06.471528v2 on bioRxiv
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    SciScore for 10.1101/2021.12.06.471528: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: Institutional Ethics Review Board (IRB no 0008/2020) approval was sought before using the de-identified nasopharyngeal samples for virus isolation.
    IACUC: Hence, all the experiments with live SARS-CoV-2, including primary virus isolation were conducted in the biosafety level 3 laboratory located at the DSO National Laboratories, with protocols (Protocol numbers BSL3-2020000001 and BSL3-2020000008) approved by DSO’s Institutional Biosafety Committee and the Ministry of Health.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Antibodies and specific reagents: The rabbit polyclonal antibody to S protein (polyS) (Sino Biological, Singapore) and anti-N (Thermo Fisher Scientific) were purchased.
    anti-N
    suggested: (Thermo Fisher Scientific Cat# PA5-19574, RRID:AB_10984909)
    The giantin rabbit polyclonal antibody was obtained from Dr Lu Lei (NTU).
    giantin
    suggested: None
    Antibody binding was detected using goat anti-Human IgG Fc Secondary Antibody conjugated to horse radish peroxidase (Thermo Fisher Scientific), diluted 1:5000 in blocking solution and incubated for 1 hr at RT.
    anti-Human IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    The SARS-CoV-2 viruses were isolated in the African green monkey kidney epithelial (Vero E6) cells (CCL-81, American Type Culture Collection, Virginia, USA) and maintained in Minimum Essential Medium (MEM) supplemented with 10% fetal bovine serum (FBS) (Invitrogen, Carlsbad, California, USA) and 1% penicillin and streptomycin (Invitrogen).
    Vero E6
    suggested: None
    This was performed on non-treated and decanoyl-RVKR-cmk-treated Vero cells following the manufacturer’s instructions
    Vero
    suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
    Recombinant DNA
    SentencesResources
    The codon-optimized full-length S gene (nucleotide residues 1 to 1273) was assembled as previously described[19] and cloned into the recombinant pCAGGS vector to create pCAGGS/S.
    pCAGGS
    suggested: RRID:Addgene_127347)
    Bulk preparation of pCAGGS/S was performed using the plasmid midiprep kit (Qiagen)
    pCAGGS/S
    suggested: None
    Software and Algorithms
    SentencesResources
    The nucleotide or amino acid sequences were assembled and aligned with the T-Coffee program which used both the Clustal W and Lalign methods for multiple sequence alignment [25].
    T-Coffee
    suggested: (T-Coffee, RRID:SCR_011818)
    Multiple-aligned amino acid sequences of S proteins were generated using the Boxshade program of Expasy.
    Boxshade
    suggested: (BOXSHADE 3.21, RRID:SCR_007165)
    The images recorded were examined and processed using Zen ver 2.3 software (
    Zen
    suggested: None

    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 found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 45. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    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 10.1101/2021.12.06.471528v1 on bioRxiv