Respiratory mucosal vaccination of peptide-poloxamine-DNA nanoparticles provides complete protection against lethal SARS-CoV-2 challenge

  1. Si Sun
  2. Entao Li
  3. Gan Zhao
  4. Jie Tang
  5. Qianfei Zuo
  6. Larry Cai
  7. Chuanfei Xu
  8. Cheng Sui
  9. Yangxue Ou
  10. Chang Liu
  11. Haibo Li
  12. Yuan Ding
  13. Chao Li
  14. Dongshui Lu
  15. Weijun Zhang
  16. Ping Luo
  17. Ping Cheng
  18. Yuwei Gao
  19. Changchun Tu
  20. Bruno Pitard
  21. Joseph Rosenecker
  22. Bin Wang
  23. Yan Liu
  24. Quanming Zou
  25. Shan Guan

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Abstract

No abstract available

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  1. Version published to 10.1016/j.biomaterials.2022.121907
  2. ScreenIT

    SciScore for 10.1101/2022.05.29.493866: (What is this?)

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

    Table 1: Rigor

    EthicsIACUC: All animal studies were approved by the Laboratory Animal Welfare and Ethics Committee of Third Military Medical University and were performed in accordance with the institutional and national policies and guidelines for the use of laboratory animals.
    Sex as a biological variableBone marrow derived dendritic cells (BMDC) maturation study: Bone marrow cells were isolated from the femurs of female BALB/c mice and cultured in RPMI 1640 complete medium (Gibco, USA) supplemented with 10% FBS, 1% penicillin/streptomycin, 10 ng/mL of Interleukin-4 (IL-4) and Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF).
    RandomizationAnimals were randomly divided into groups and conceded an adaption time of at least 7 days before the beginning of the experiments.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line AuthenticationAuthentication: All cell lines used in current study were obtained from original providers who authenticated the cell lines using morphology, karyotyping and PCR-based approaches.
    Contamination: All cell lines tested negative for mycoplasma contamination.

    Table 2: Resources

    Antibodies
    SentencesResources
    In order to detect the TEM/TCM and TRM cells, the cell samples were stained with the following indicated antibodies in FACS buffer: anti-CD62L (161204, BioLegend), anti-CD44 (25-0441-82, BioLegend), anti-CD69 (104506, BioLegend) and anti-CD103 (121416, BioLegend).
    anti-CD62L ( 161204
    suggested: None
    anti-CD44
    suggested: (Thermo Fisher Scientific Cat# 25-0441-82, RRID:AB_469623)
    anti-CD69
    suggested: (BioLegend Cat# 104506, RRID:AB_313109)
    anti-CD103
    suggested: (BioLegend Cat# 121416, RRID:AB_2128621)
    After incubation at 37 °C, 5% CO2 for 24 h, the plates were washed with PBS and incubation with biotinylated anti-mouse IFN-γ or IL-4 antibody for 2 h at RT.
    anti-mouse IFN-γ
    suggested: None
    IL-4
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cell lines: MH-S cell line (Mice alveolar macrophages cells), DC2.4 cell line (Mouse bone marrow-derived dendritic cells), BEAS-2B (human bronchial epithelial cells) cell line and Calu-3 (human lung cancer cells) cell line were obtained from ATCC (Manassas, VA,
    MH-S
    suggested: ATCC Cat# CRL-2019, RRID:CVCL_3855)
    DC2.4
    suggested: Millipore Cat# SCC142, RRID:CVCL_J409)
    BEAS-2B
    suggested: NCBI_Iran Cat# C561, RRID:CVCL_0168)
    Calu-3
    suggested: KCLB Cat# 30055, RRID:CVCL_0609)
    ACE2-293T cells (ACE2-expressing cell line, constructed by hygromycin B screening) were purchased from PackGene (LV-2058, Guangzhou, China).
    ACE2-293T
    suggested: None
    Another part was serially diluted in DMEM and added into Vero E6 cells in 96-well plates.
    Vero E6
    suggested: None
    Serum or BALF were incubated with 10 μl of Luc-SARS-Cov-2 pseudotyped virus (LV-2058, PackGene, China) for 60 min, then added to the HEK293T cells stably expressing ACE2 to incubate in a standard incubator (37°C, 5% CO2) for 72 h.
    HEK293T
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Bone marrow derived dendritic cells (BMDC) maturation study: Bone marrow cells were isolated from the femurs of female BALB/c mice and cultured in RPMI 1640 complete medium (Gibco, USA) supplemented with 10% FBS, 1% penicillin/streptomycin, 10 ng/mL of Interleukin-4 (IL-4) and Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF).
    BALB/c
    suggested: None
    The SARS-CoV-2 challenge model was based on a novel mouse-adapted SARS-CoV-2 strain, C57MA14 (NCBI GenBank number: OL913104.1, details can be found in: https://www.ncbi.nlm.nih.gov/nuccore/2167992552), that causes severe respiratory symptoms, and mortality to BALB/c mice.
    C57MA14
    suggested: None
    Recombinant DNA
    SentencesResources
    Reporter Vectors (pFLuc, E1320) was purchased from Promega (Madison, USA)
    pFLuc
    suggested: None
    Plasmids encoding SARS-CoV-2 S protein (pSpike) and pVax were kindly provided by Advaccine Biopharmaceuticals Co., Ltd (Suzhou, China)
    pVax
    suggested: RRID:Addgene_141350)
    To evaluate the in vivo transfection of PP-sNp, pDNA/PP-sNp complexes encoding firefly luciferase (i.e., pFLuc/PP-sNp) were prepared. pFLuc/PP-sNp and naked-pFLuc were incubated with cells for 4 h in Opti-MEM I Reduced Serum Medium, then were replaced by fresh complete medium.
    pDNA/PP-sNp
    suggested: None
    pFLuc/PP-sNp
    suggested: None
    To examine the maturation of BMDCs in vitro, BMDCs (1 × 106 mL-1) were co-cultured with pSpike/PP-sNp and naked-pSpike only for 24 h, respectively. Subsequently, FITC anti-mouse CD11c (117305, Biolegend)
    pSpike/PP-sNp
    suggested: None
    Each anesthetized mouse intratracheally received 50 μL of pSpike/PP-sNp formulation containing 15 μg pSpike. pVax/PP-sNp and phosphate buffered saline (PBS) was adopted as a mock control and a negative control, respectively.
    pVax/PP-sNp
    suggested: None
    Software and Algorithms
    SentencesResources
    The trajectories of the nanoparticles were precisely quantified from the videos by software (TrackMate plugin in FIJI (ImageJ)), then the trajectory data was used to calculate the MSD and the corresponding diffusion coefficients (De) in MATLAB through the following equations, as implemented in MSD Analyzer.
    MATLAB
    suggested: (MATLAB, RRID:SCR_001622)
    Data are analyzed with FlowJo software V10.
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Statistics and analysis: Statistical analyses were performed using the GraphPad Prism 8 (GraphPad Software, USA).
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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: We detected the following sentences addressing limitations in the study:
    There are several limitations that we did not address in this study and will be useful topics for future studies, including the absence of data on the neutralization and protection efficiency elicited by pSpike/PP-sNp against emerging SARS-CoV-2 variants of concern. Similar to those cases of authorized COVID-19 vaccines62, the neutralizing activity of NAb induced by the pSpike/PP-sNp vaccine may suffer a significant decrease within several months/years after vaccination, more boost doses may be necessary. Besides, immunization and challenge studies with larger animals such as non-human primates should be carried out to confirm the extent of protective mucosal immunity conferred by pSpike/PP-sNp. Another limitation relates to the intratracheal dosing which is not appropriate to be applied in humans when considering its poor compliance. Most of the relevant studies chose the intranasal inoculation because of its noninvasive and convenient features, but there are still huge concerns and uncertainties regarding intranasal route of vaccination. For example, negative perception for nasal vaccines was generated from reported cases of Bell’s palsy after intranasal dosing of influenza vaccines63, 64. Alternatively, the noninvasive nebulized formulations seem to be one of the most appropriate approaches in delivering mucosal vaccines to the human airway. However, the nebulized DNA formulations still face many challenges as indicated by a previous study showing that as little as 10% of ...

    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

    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 10.1101/2022.05.29.493866v1 on bioRxiv