Short-Term Instantaneous Prophylaxis and Efficient Treatment Against SARS-CoV-2 in hACE2 Mice Conferred by an Intranasal Nanobody (Nb22)

  1. Xilin Wu
  2. Yaxing Wang
  3. Lin Cheng
  4. Fengfeng Ni
  5. Linjing Zhu
  6. Sen Ma
  7. Bilian Huang
  8. Mengmeng Ji
  9. Huimin Hu
  10. Yuncheng Li
  11. Shijie Xu
  12. Haixia Shi
  13. Doudou Zhang
  14. Linshuo Liu
  15. Waqas Nawaz
  16. Qinxue Hu
  17. Sheng Ye
  18. Yalan Liu
  19. Zhiwei Wu

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Abstract

Current COVID-19 vaccines need to take at least one month to complete inoculation and then become effective. Around 51% of the global population is still not fully vaccinated. Instantaneous protection is an unmet need among those who are not fully vaccinated. In addition, breakthrough infections caused by SARS-CoV-2 are widely reported. All these highlight the unmet needing for short-term instantaneous prophylaxis (STIP) in the communities where SARS-CoV-2 is circulating. Previously, we reported nanobodies isolated from an alpaca immunized with the spike protein, exhibiting ultrahigh potency against SARS-CoV-2 and its variants. Herein, we found that Nb22, among our previously reported nanobodies, exhibited ultrapotent neutralization against Delta variant with an IC 50 value of 0.41 ng/ml (5.13 pM). Furthermore, the crystal structural analysis revealed that the binding of Nb22 to WH01 and Delta RBDs both effectively blocked the binding of RBD to hACE2. Additionally, intranasal Nb22 exhibited protection against SARS-CoV-2 Delta variant in the post-exposure prophylaxis (PEP) and pre-exposure prophylaxis (PrEP). Of note, intranasal Nb22 also demonstrated high efficacy against SARS-CoV-2 Delta variant in STIP for seven days administered by single dose and exhibited long-lasting retention in the respiratory system for at least one month administered by four doses, providing a strategy of instantaneous short-term prophylaxis against SARS-CoV-2. Thus, ultrahigh potency, long-lasting retention in the respiratory system and stability at room-temperature make the intranasal or inhaled Nb22 to be a potential therapeutic or STIP agent against SARS-CoV-2.

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  1. Version published to 10.3389/fimmu.2022.865401
  2. Version published to 10.1101/2021.09.06.459055v2 on bioRxiv
  3. ScreenIT

    SciScore for 10.1101/2021.09.06.459055: (What is this?)

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

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    For flow cytometric analysis, the cells were resuspended in 500 µl PBSF buffer (PBS+2% FBS) and analyzed using ACEA NovoCyte TM (Agilent Biosciences), non-transfected 293T cells served as a negative control.
    293T
    suggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)
    Recombinant DNA
    SentencesResources
    The Nb-Fcs were finally cloned into the pcDNA3.4 eukaryotic expression vector (Invitrogen), which were transfected into 293F cells (cat.# R79007, Thermo Scientific) to produce Nb-Fcs.
    pcDNA3.4
    suggested: RRID:Addgene_131198)
    Briefly, pseudovirus of SARS-CoV-2 variants was produced by co-transfection of pNL4-3.
    pNL4-3
    suggested: None
    Luc.R-E-, an HIV-1 NL4-3 luciferase reporter vector that comprises defective Nef, Env and Vpr (HIV AIDS Reagent Program), and pCDNA3.1 (Invitrogen) expression vectors encoding the spike proteins of respective variants into 293T cells (ATCC).
    pCDNA3.1
    suggested: RRID:Addgene_79663)
    The eluted proteins were further purified by Superdex 75 (GE Healthcare, USA) and stored in 20 mM Tris-HCl, 150 mM NaCl, pH 7.5. 2.6 Expression and purification of Nb22 for crystal structural analysis: The VHH gene for Nb22 was amplified by PCR and cloned into a pET21a vector with BamH I and Xho I restriction sites.
    pET21a
    suggested: RRID:Addgene_26640)
    Software and Algorithms
    SentencesResources
    Half-maximal inhibitory concentrations (IC50) of the evaluated nanobodies were determined by luciferase activity 48 h following exposure to virus-antibody mixture, and analyzed by GraphPad Prism 8.01 (GraphPad Software Inc.). 2.3 Immunofluorescence and flow cytometric analysis: Immunofluorescence and flow cytometric analysis were conducted following our previously published protocol [27], with minor modifications.
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    For flow cytometric analysis, the cells were resuspended in 500 µl PBSF buffer (PBS+2% FBS) and analyzed using ACEA NovoCyte TM (Agilent Biosciences), non-transfected 293T cells served as a negative control.
    Agilent Biosciences
    suggested: None
    The structure was elucidated using the molecular replacement (MR) method in PHASER program [29] with the structure of SARS-CoV-2 RBD (PDB code: 7CJF) [30] as the initial searching model.
    PHASER
    suggested: (Phaser, RRID:SCR_014219)
    The model was built into the modified experimental electron density using COOT [31] and further refined in PHENIX [32].
    COOT
    suggested: (Coot, RRID:SCR_014222)
    PHENIX
    suggested: (Phenix, RRID:SCR_014224)
    Structural figures were prepared by PyMOL.
    PyMOL
    suggested: (PyMOL, RRID:SCR_000305)
    Epitope and paratope residues, as well as their interactions, were identified by PISA (http://www.ebi.ac.uk/pdbe/prot_int/pistart.html) at the European Bioinformatics Institute. 2.8.
    PISA
    suggested: (PISA, RRID:SCR_015749)
    Quantification and statistical analysis: All statistical analyses were carried out 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 did not find any issues relating to the usage of bar graphs.

    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

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  4. Version published to 10.1101/2021.09.06.459055v1 on bioRxiv