Respiratory mucosal vaccination of peptide-poloxamine-DNA nanoparticles provides complete protection against lethal SARS-CoV-2 challenge
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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
Ethics IACUC: 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 variable 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). Randomization Animals were randomly divided into groups and conceded an … 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
Ethics IACUC: 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 variable 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). Randomization Animals were randomly divided into groups and conceded an adaption time of at least 7 days before the beginning of the experiments. Blinding not detected. Power Analysis not detected. Cell Line Authentication Authentication: 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 Sentences Resources 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 ( 161204suggested: Noneanti-CD44suggested: (Thermo Fisher Scientific Cat# 25-0441-82, RRID:AB_469623)anti-CD69suggested: (BioLegend Cat# 104506, RRID:AB_313109)anti-CD103suggested: (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-γsuggested: NoneIL-4suggested: NoneExperimental Models: Cell Lines Sentences Resources 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-Ssuggested: ATCC Cat# CRL-2019, RRID:CVCL_3855)DC2.4suggested: Millipore Cat# SCC142, RRID:CVCL_J409)BEAS-2Bsuggested: NCBI_Iran Cat# C561, RRID:CVCL_0168)Calu-3suggested: 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). ACE2-293Tsuggested: NoneAnother part was serially diluted in DMEM and added into Vero E6 cells in 96-well plates. Vero E6suggested: NoneSerum 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. HEK293Tsuggested: NoneExperimental Models: Organisms/Strains Sentences Resources 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/csuggested: NoneThe 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. C57MA14suggested: NoneRecombinant DNA Sentences Resources Reporter Vectors (pFLuc, E1320) was purchased from Promega (Madison, USA) pFLucsuggested: NonePlasmids encoding SARS-CoV-2 S protein (pSpike) and pVax were kindly provided by Advaccine Biopharmaceuticals Co., Ltd (Suzhou, China) pVaxsuggested: 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-sNpsuggested: NonepFLuc/PP-sNpsuggested: NoneTo 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-sNpsuggested: NoneEach 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-sNpsuggested: NoneSoftware and Algorithms Sentences Resources 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. MATLABsuggested: (MATLAB, RRID:SCR_001622)Data are analyzed with FlowJo software V10. FlowJosuggested: (FlowJo, RRID:SCR_008520)Statistics and analysis: Statistical analyses were performed using the GraphPad Prism 8 (GraphPad Software, USA). GraphPadsuggested: (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.
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- No protocol registration statement was detected.
Results from scite Reference Check: We found no unreliable references.
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