Single‐dose immunisation with a multimerised SARS‐CoV‐2 receptor binding domain (RBD) induces an enhanced and protective response in mice

  1. Ralf Salzer
  2. Jordan J. Clark
  3. Marina Vaysburd
  4. Veronica T. Chang
  5. Anna Albecka
  6. Leo Kiss
  7. Parul Sharma
  8. Andres Gonzalez Llamazares
  9. Anja Kipar
  10. Julian A. Hiscox
  11. Andrew Owen
  12. A. Radu Aricescu
  13. James P. Stewart
  14. Leo C. James
  15. Jan Löwe

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Abstract

The COVID‐19 pandemic, caused by the SARS‐CoV‐2 coronavirus, has triggered a worldwide health emergency. Here, we show that ferritin‐like Dps from hyperthermophilic Sulfolobus islandicus, covalently coupled with SARS‐CoV‐2 antigens via the SpyCatcher system, forms stable multivalent dodecameric vaccine nanoparticles that remain intact even after lyophilisation. Immunisation experiments in mice demonstrated that the SARS‐CoV‐2 receptor binding domain (RBD) coupled to Dps (RBD‐S‐Dps) elicited a higher antibody titre and an enhanced neutralising antibody response compared to monomeric RBD. A single immunisation with RBD‐S‐Dps completely protected hACE2‐expressing mice from serious illness and led to viral clearance from the lungs upon SARS‐CoV‐2 infection. Our data highlight that multimerised SARS‐CoV‐2 subunit vaccines are a highly efficacious modality, particularly when combined with an ultra‐stable scaffold.

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  1. Version published to 10.1002/1873-3468.14171
  2. ScreenIT

    SciScore for 10.1101/2021.05.18.444622: (What is this?)

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

    Table 1: Rigor

    EthicsIACUC: Mouse immunisation (Fig. 3): Six weeks-old C57BL/6J mice (Jackson) were used in immunisation experiments, which were conducted in accordance with the E7 moderate severity limit protocol and the UK Home Office Animals (Scientific Procedures) Act (ASPA, 1986), and approved by the UKRI Animal Welfare and Ethical Review Body.
    Sex as a biological variablenot detected.
    RandomizationAnimals were randomly assigned into multiple cohorts and given 25 μg antigen (RBD-S-DPS or RBD-SpyT2) & 10 μg CpG or PBS via subcutaneous injection.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The Ag-S-Dps complexes were detected using the HisProbe-HRP (Thermo Fisher Scientific, TFS) and human transferrin was used as a loading control and detected using transferrin antibodies from chicken and chicken-HRP conjugated antibodies (Thermo Fisher Scientific, TFS, cat. #PA1-9525 and cat. # 31401).
    human transferrin
    suggested: (Thermo Fisher Scientific Cat# PA1-9525, RRID:AB_1088031)
    transferrin
    suggested: (Thermo Fisher Scientific Cat# PA1-9525, RRID:AB_1088031)
    chicken-HRP
    suggested: None
    Plates were washed with MPBST and bound antibodies were detected with goat anti-mouse IgG-HRP (Jackson Immunoresearch, #115-035-071)
    anti-mouse IgG-HRP
    suggested: None
    , B cells (CD45R/B220+) and macrophages (Iba1+), using the horseradish peroxidase (HRP) method and the following primary antibodies: rabbit anti-SARS-CoV NP (Rockland, 200-402-A50)
    anti-SARS-CoV NP
    suggested: (RayBiotech Cat# MD-05-0425, RRID:AB_951740)
    Briefly, after deparaffination, sections underwent antigen retrieval in citrate buffer (pH 6.0; Agilent) (anti-SARS-CoV-2, -CD8, -CD45R, -Iba1) or Tris-EDTA buffer, pH 9.0 (anti-CD3, -CD4) for 20 min at 98 °C and for 20 min at 37 °C respectively, followed by incubation with the primary antibody overnight at 4 °C (anti-SARS-CoV-2), 60 min at RT (anti-CD3, -CD8, -CD45R, -Iba1) or 60 min at 37 °C (anti-CD3, -CD4).
    anti-SARS-CoV-2
    suggested: None
    anti-CD3
    suggested: None
    This was followed by blocking of endogenous peroxidase (peroxidase block, Agilent) for 10 min at RT and incubation with the secondary antibody, EnVision+/HRP, Rabbit and Rat respectively (Agilent) for 30 min at RT (anti-SARS-CoV, -CD8, -CD45R, -Iba1) or the Omni-Map anti Rb HRP (Ventana) for 16 min at 37 °C (anti-CD3, -CD4), followed by EnVision FLEX DAB+ Chromogen in Substrate buffer (Agilent; anti-SARS-CoV-2, -CD8, -CD45R, -Iba1) for 10 min at RT or the DAB-Map-Kit (Ventana; anti-CD3, -CD4), all in an autostainer (Dako or Ventana).
    anti-SARS-CoV
    suggested: None
    anti Rb HRP
    suggested: None
    -CD8
    suggested: (Christine Clayton, Centre for Molecular Biology (ZMBH, University of Heidelberg Cat# CC_RBP10, RRID:AB_2890154)
    Experimental Models: Cell Lines
    SentencesResources
    Briefly, virions were produced in HEK 293T cells by transfection with 1 μg of the plasmid encoding SARS CoV-2 Spike protein (pCAGGS-SpikeΔc19), 1 μg pCRV GagPol and 1.5 μg GFP-encoding plasmid (CSGW)
    HEK 293T
    suggested: None
    Spike-pseudotyped neutralisation assays with mouse sera: HEK 293T-hACE2-TMPRSS2 cells were described previously (Papa et al., 2021).
    HEK 293T-hACE2-TMPRSS2
    suggested: None
    Cell culture and virus: UK strain of SARS-CoV-2 (hCoV-2/human/Liverpool/REMRQ0001/2020; PANGO lineage B), was used and grown to P4 in Vero E6 cells (Patterson et al., 2020).
    Vero E6
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Mouse immunisation (Fig. 3): Six weeks-old C57BL/6J mice (Jackson) were used in immunisation experiments, which were conducted in accordance with the E7 moderate severity limit protocol and the UK Home Office Animals (Scientific Procedures) Act (ASPA, 1986), and approved by the UKRI Animal Welfare and Ethical Review Body.
    C57BL/6J
    suggested: None
    Mice carrying the human ACE2 gene under the control of the keratin 18 promoter (K18-hACE2; formally B6.Cg-Tg(K18-ACE2)2Prlmn/J) were purchased from Jackson Laboratories.
    B6.Cg-Tg(K18-ACE2)2Prlmn/J
    suggested: RRID:IMSR_JAX:034860)
    Recombinant DNA
    SentencesResources
    Briefly, virions were produced in HEK 293T cells by transfection with 1 μg of the plasmid encoding SARS CoV-2 Spike protein (pCAGGS-SpikeΔc19), 1 μg pCRV GagPol and 1.5 μg GFP-encoding plasmid (CSGW)
    pCAGGS-SpikeΔc19
    suggested: None
    Software and Algorithms
    SentencesResources
    In vitro human plasma stability assay: The in vitro stability of RBD-S-Dps was studied in clotted human plasma (MD Biomedicals, cat. #2930149).
    MD Biomedicals
    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: 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.

  3. Version published to 10.1101/2021.05.18.444622 on bioRxiv