A bispecific monomeric nanobody induces spike trimer dimers and neutralizes SARS-CoV-2 in vivo

  1. Leo Hanke
  2. Hrishikesh Das
  3. Daniel J. Sheward
  4. Laura Perez Vidakovics
  5. Egon Urgard
  6. Ainhoa Moliner-Morro
  7. Changil Kim
  8. Vivien Karl
  9. Alec Pankow
  10. Natalie L. Smith
  11. Bartlomiej Porebski
  12. Oscar Fernandez-Capetillo
  13. Erdinc Sezgin
  14. Gabriel K. Pedersen
  15. Jonathan M. Coquet
  16. B. Martin Hällberg
  17. Ben Murrell
  18. Gerald M. McInerney

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Abstract

Antibodies binding to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike have therapeutic promise, but emerging variants show the potential for virus escape. This emphasizes the need for therapeutic molecules with distinct and novel neutralization mechanisms. Here we describe the isolation of a nanobody that interacts simultaneously with two RBDs from different spike trimers of SARS-CoV-2, rapidly inducing the formation of spike trimer–dimers leading to the loss of their ability to attach to the host cell receptor, ACE2. We show that this nanobody potently neutralizes SARS-CoV-2, including the beta and delta variants, and cross-neutralizes SARS-CoV. Furthermore, we demonstrate the therapeutic potential of the nanobody against SARS-CoV-2 and the beta variant in a human ACE2 transgenic mouse model. This naturally elicited bispecific monomeric nanobody establishes an uncommon strategy for potent inactivation of viral antigens and represents a promising antiviral against emerging SARS-CoV-2 variants.

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  1. Version published to 10.1038/s41467-021-27610-z
  2. ScreenIT

    SciScore for 10.1101/2021.03.20.436243: (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 variableAlpaca immunization, library generation and nanobody isolation: One adult female alpaca (Funny) at PreClinics, Germany, was immunized four times in a 60-day immunization schedule.
    Cell Line AuthenticationContamination: All cell lines used for the experiments were negative for mycoplasma as determined by PCR.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    Cells and viruses: HEK293T cell (ATCC-CRL-3216) and Vero E6 cells (ATCC-CRL-1586) were maintained in DMEM (Gibco) supplemented with 10% fetal calf serum and 1% penicillin-streptomycin in a humidified incubator with 5% CO2 at 37°C.
    HEK293T
    suggested: None
    Infectious SARS-CoV-218 and the B.1.3511 isolate were propagated in Vero E6 cells and titrated by plaque assay.
    Vero E6
    suggested: RRID:CVCL_XD71)
    Pseudotyped viruses sufficient to generate 100,000 relative light units (RLU) were incubated with serial dilutions of nanobody for 60 min at 37 °C. 15,000 HEK293T-ACE2 cells were then added to each well, and the plates were incubated for 48 h at 37 °C.
    HEK293T-ACE2
    suggested: None
    Flow cytometry: HEK293T-hACE2 cells were trypsinized and fixed in 4% formaldehyde in PBS for 20 min.
    HEK293T-hACE2
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    SARS-CoV-2 challenge experiments: K18-hACE2 transgenic mice were purchased from Jackson laboratories and maintained as a hemizygous line.
    K18-hACE2
    suggested: RRID:IMSR_GPT:T037657)
    Software and Algorithms
    SentencesResources
    Fluorescence was quantified using a BD FACSCelesta and the FlowJo software package.
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Extracted particles were imported into cryoSPARC v3.0.135 for 2D classification, 3D classification and non-uniform 3D refinement.
    cryoSPARC
    suggested: (cryoSPARC, RRID:SCR_016501)
    Structure figures and EM density-map figures were generated with UCSF ChimeraX41 and COOT, respectively.
    COOT
    suggested: (Coot, RRID:SCR_014222)

    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: Please consider improving the rainbow (“jet”) colormap(s) used on page 35. 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.
    • Thank you for including a protocol registration statement.

    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 10.1101/2021.03.20.436243 on bioRxiv