Nasal delivery of single-domain antibody improves symptoms of SARS-CoV-2 infection in an animal model

  1. Kei Haga
  2. Reiko Takai-Todaka
  3. Yuta Matsumura
  4. Chihong Song
  5. Tomomi Takano
  6. Takuto Tojo
  7. Atsushi Nagami
  8. Yuki Ishida
  9. Hidekazu Masaki
  10. Masayuki Tsuchiya
  11. Toshiki Ebisudani
  12. Shinya Sugimoto
  13. Toshiro Sato
  14. Hiroyuki Yasuda
  15. Koichi Fukunaga
  16. Akihito Sawada
  17. Naoto Nemoto
  18. Kazuyoshi Murata
  19. Takuya Morimoto
  20. Kazuhiko Katayama

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Abstract

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the disease COVID-19 can lead to serious symptoms, such as severe pneumonia, in the elderly and those with underlying medical conditions. While vaccines are now available, they do not work for everyone and therapeutic drugs are still needed, particularly for treating life-threatening conditions. Here, we showed nasal delivery of a new, unmodified camelid single-domain antibody (VHH), termed K-874A, effectively inhibited SARS-CoV-2 titers in infected lungs of Syrian hamsters without causing weight loss and cytokine induction. In vitro studies demonstrated that K-874A neutralized SARS-CoV-2 in both VeroE6/TMPRSS2 and human lung-derived alveolar organoid cells. Unlike other drug candidates, K-874A blocks viral membrane fusion rather than viral attachment. Cryo-electron microscopy revealed K-874A bound between the receptor binding domain and N-terminal domain of the virus S protein. Further, infected cells treated with K-874A produced fewer virus progeny that were less infective. We propose that direct administration of K-874A to the lung could be a new treatment for preventing the reinfection of amplified virus in COVID-19 patients.

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  1. Version published to 10.1371/journal.ppat.1009542
  2. ScreenIT

    SciScore for 10.1101/2021.04.09.439147: (What is this?)

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

    Table 1: Rigor

    NIH rigor criteria are not applicable to paper type.

    Table 2: Resources

    Antibodies
    SentencesResources
    PVDF membranes were treated with 6xHis Tag Monoclonal Antibody (3D5) HRP (Invitorogen, USA) for 6xHis-tagged proteins, and ANTI-FLAG M2-peroxidase (HRP)-conjugated
    M2-peroxidase
    suggested: (Sigma-Aldrich Cat# A8592, RRID:AB_439702)
    Then, serial diluted recombinant ACE2 (from 1 μg/ml to 0.06 μg/mL) (ab151852, abcam), subsequent rabbit anti ACE2 antibody (HPA000288, Atlas Antibodies) and HRP conjugated anti rabbit IgG (7074S, Cell signaling) were incubated.
    subsequent rabbit anti ACE2 antibody
    suggested: None
    anti ACE2
    suggested: None
    anti rabbit IgG
    suggested: None
    To detect K-874A binding to immobilized S protein, serial diluted K-874A (1 μg/ml to 0.002 μg/ml) in 1%BSA/PBS was incubated, and K-874A which bound to S protein was detected by HRP-conjugated anti VHH antibody (#128-035-232, Jackson immuno Research).
    anti VHH
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    SARS-CoV-2 binding on and early replication in Vero/TMPRSS2 cells: 2.5×104 TCID50 (MOI=50) of SARS-CoV-2 was incubated with 150 μg/ml of VHHs for 2 hours at 37°C and then 24 hours at 4°C and inoculated to 5.0×104 VeroE6/TMPRSS2 cells for an hour.
    VeroE6/TMPRSS2
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Software and Algorithms
    SentencesResources
    The raw Illumina paired-end reads that passed through the Q30 filter were merged using PEAR software [21].
    PEAR
    suggested: (PEAR, RRID:SCR_003776)
    The VHHs-encoding sequences were translated based on standard genetic code using MEGA X software [22].
    MEGA X
    suggested: None
    Equal numbers of ZsGreen-HiBiT-expressing cells and LgBiT-expressing cells were plated on a 96-well plate (1603101, ThermoFisher Science).
    ThermoFisher Science
    suggested: None
    Individual movies were subjected to per-frame drift correction by MotionCor2 [26].
    MotionCor2
    suggested: (MotionCor2, RRID:SCR_016499)
    The contrast transfer function parameters of each micrograph were estimated using CTFFIND4 [27] and the flowing 2D and 3D classification, 3D refinement, and local resolution calculation were performed with RELION3.1 software [28].
    RELION3.1
    suggested: None
    The extracted density was used to generate the mask using “Mask creation” in RELION 3.1.
    RELION
    suggested: (RELION, RRID:SCR_016274)
    The homology models of K-874A, RBD and NTD were rigid-body-fitted into the map using the COOT [31] and then refined using PHENIX [32].
    COOT
    suggested: (Coot, RRID:SCR_014222)
    PHENIX
    suggested: (Phenix, RRID:SCR_014224)

    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.

    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 10.1101/2021.04.09.439147 on bioRxiv