A potent bispecific nanobody protects hACE2 mice against SARS-CoV-2 infection via intranasal administration

  1. Xilin Wu
  2. Lin Cheng
  3. Ming Fu
  4. Bilian Huang
  5. Linjing Zhu
  6. Shijie Xu
  7. Haixia Shi
  8. Doudou Zhang
  9. Huanyun Yuan
  10. Waqas Nawaz
  11. Ping Yang
  12. Qinxue Hu
  13. Yalan Liu
  14. Zhiwei Wu

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Abstract

No abstract available

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  1. Version published to 10.1016/j.celrep.2021.109869
  2. ScreenIT

    SciScore for 10.1101/2021.02.08.429275: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: All animal experimental procedures without infection were approved by the Committee on the Use of Live Animals by the Ethics Committee of Nanjing University.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableEvaluating the efficacy of Nbs in SARS-CoV-2 infected hACE2 mice: A total of 31 8-week-old male transgenic hACE2 mice (C57BL/6J) (cat.# T037630, GemPharmatech Co., Ltd., Nanjing, China) were challenged with SARS-CoV-2 as previously reported35 with following modifications.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The membrane was first blocked and then incubated overnight at 4 °C or 37 °C for one hour with diluted plasma or antibody, followed by incubation with the secondary antibody of either anti-human IgG or anti-rabbit IgG conjugated with an IRDye 800CW (cat.# 926-32232,
    anti-human IgG
    suggested: (LI-COR Biosciences Cat# 926-32232, RRID:AB_10806644)
    ELISA analysis: Anti-sera titer and antibody characterization or antibody quantification in vivo were examined by ELISA as reported in our previously published method33 with modifications.
    Anti-sera
    suggested: None
    Following washing, goat anti-llama IgG (H+L) secondary antibody with HRP (Novus, cat.#
    goat
    suggested: None
    IgG ( H+L ) secondary antibody with HRP ( Novus
    suggested: None
    After washing, an anti-M13 bacteriophage antibody with HRP (1:10000 dilution, cat.# 11973-MM05T-H, Sino Biological) was added and incubated at 37 °C for 1 h.
    anti-M13
    suggested: (Sino Biological Cat# 11973-MM05T-H, RRID:AB_2857928)
    Cells were permeabilized with 0.2% Triton X-100 and incubated with cross-reactive rabbit anti-SARS-CoV-N IgG (Sino Biological, Inc) for 1 h at room temperature before the addition of HRP-conjugated goat anti-rabbit IgG (H+L) antibody (Jackson ImmunoResearch) and further incubated at room temperature.
    anti-SARS-CoV-N IgG
    suggested: None
    anti-rabbit IgG
    suggested: None
    250 μg SNB02 (Y-Clone, China), an anti-SFTSV antibody constructed by Nb fused with human Fc1 (Nb-Fc)13, was intranasally injected 1 h after infection and served as an isotype treated control (Isotype).
    Y-Clone , China
    suggested: None
    anti-SFTSV
    suggested: None
    The neutralization potency of Nb15-Fc (A), Nb22-Fc (B), Nb31-Fc (C), SNB02 (isotype control antibody) (D) was detected based on authentic SARS-CoV-2 plaque reduction neutralization test.
    SNB02
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Luc.R-E-, an HIV-1 NL4-3 luciferase reporter vector that contains defective Nef, Env and Vpr (HIV AIDS Reagent Program), and pCDNA3.1 (Invitrogen) expression vectors encoding the respective spike proteins (MN988668.1 for SARS-CoV-2, AAP13567.1 for SARS-CoV, AFS88936.1 for MERS-CoV) into 293T cells (ATCC)
    293T
    suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
    HEK293T-ACE2 cells (cat.# 41107ES03, Yeasen Biotech Co., Ltd. China) for SARS-CoV-2 and SARS-CoV, Huh7 cells (ATCC) for MERS-CoV (approximately 1.5×10 4 per well) were then added in duplicate to the virus-antibody mixture.
    HEK293T-ACE2
    suggested: None
    Huh7
    suggested: CLS Cat# 300156/p7178_HuH7, RRID:CVCL_0336)
    The mixtures were then transferred to 96-well plates seeded with Vero E6 cells and incubated at 37 °C for 1 h.
    Vero E6
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Evaluating the efficacy of Nbs in SARS-CoV-2 infected hACE2 mice: A total of 31 8-week-old male transgenic hACE2 mice (C57BL/6J) (cat.# T037630, GemPharmatech Co., Ltd., Nanjing, China) were challenged with SARS-CoV-2 as previously reported35 with following modifications.
    C57BL/6J
    suggested: None
    Transgenic hACE2 mice typically clear virus within five days after SARS-CoV-235.
    Transgenic hACE2
    suggested: None
    Images were obtained by OLYMPUS IX73 using HCImage Live (×64) software and analyzed by ImageJ (NIH). 15. Pharmacokinetics of Nbs in vivo: Purified Nbs were injected intranasally (i.n.), intraperitoneally (i.p.) or intravascularly into BALB/c (Qing Long Shan Animal Center, Nanjing, China) at a dose of 10-20 mg/kg.
    BALB/c
    suggested: None
    Software and Algorithms
    SentencesResources
    Luc.R-E-, an HIV-1 NL4-3 luciferase reporter vector that contains defective Nef, Env and Vpr (HIV AIDS Reagent Program), and pCDNA3.1 (Invitrogen) expression vectors encoding the respective spike proteins (MN988668.1 for SARS-CoV-2, AAP13567.1 for SARS-CoV, AFS88936.1 for MERS-CoV) into 293T cells (ATCC)
    HIV AIDS Reagent Program
    suggested: None
    Half-maximal inhibitory dilution (ND50) of the evaluated sera or half-maximal inhibitory concentrations (IC50) of the evaluated Nbs were determined by luciferase activity 48 h after exposure to virus-antibody mixture, and analyzed by GraphPad Prism 8.01 (GraphPad Software Inc.). 9.
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Evaluating the efficacy of Nbs in SARS-CoV-2 infected hACE2 mice: A total of 31 8-week-old male transgenic hACE2 mice (C57BL/6J) (cat.# T037630, GemPharmatech Co., Ltd., Nanjing, China) were challenged with SARS-CoV-2 as previously reported35 with following modifications.
    GemPharmatech
    suggested: (GemPharmatech, RRID:SCR_017239)
    Images were obtained by OLYMPUS IX73 using HCImage Live (×64) software and analyzed by ImageJ (NIH). 15. Pharmacokinetics of Nbs in vivo: Purified Nbs were injected intranasally (i.n.), intraperitoneally (i.p.) or intravascularly into BALB/c (Qing Long Shan Animal Center, Nanjing, China) at a dose of 10-20 mg/kg.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    Statistics: All statistical analyses were performed using GraphPad Prism 8.01 software (GraphPad) or OriginPro 8.5 software (OriginLab).
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    OriginPro
    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.

    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 10.1101/2021.02.08.429275 on bioRxiv