A Bacteriophage-Based, Highly Efficacious, Needle- and Adjuvant-Free, Mucosal COVID-19 Vaccine

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1. Version published to 10.1128/mbio.01822-22

   Aug 30, 2022
2. 

   ScreenIT

   May 2, 2022

   SciScore for 10.1101/2022.04.28.489809: (What is this?)

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

   Table 1: Rigor

   Ethics	IACUC: All animal experiments were approved by the Institutional Animal Care and Use Committee of the Catholic University of America (Washington, DC) (Office of Laboratory Animal Welfare assurance number A4431-01) and the University of Texas Medical Branch (Galveston, TX) (Office of Laboratory Animal Welfare assurance number A3314-01). Field Sample Permit: The SARS-CoV-2 virus challenge studies were conducted in the animal BSL-3 (ABSL-3) suite at UTMB.
   Sex as a biological variable	not detected.
   Randomization	Five-week-old female BALB/c (Jackson Laboratory) or hACE2 transgenic mice AC70 (Taconic Biosciences) were randomly grouped (5-10 animals per group) and allowed to acclimate for 14 days.
   Blindin…

   SciScore for 10.1101/2022.04.28.489809: (What is this?)

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

   Table 1: Rigor

   Ethics	IACUC: All animal experiments were approved by the Institutional Animal Care and Use Committee of the Catholic University of America (Washington, DC) (Office of Laboratory Animal Welfare assurance number A4431-01) and the University of Texas Medical Branch (Galveston, TX) (Office of Laboratory Animal Welfare assurance number A3314-01). Field Sample Permit: The SARS-CoV-2 virus challenge studies were conducted in the animal BSL-3 (ABSL-3) suite at UTMB.
   Sex as a biological variable	not detected.
   Randomization	Five-week-old female BALB/c (Jackson Laboratory) or hACE2 transgenic mice AC70 (Taconic Biosciences) were randomly grouped (5-10 animals per group) and allowed to acclimate for 14 days.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   The copies of the phage-packaged NP protein were quantified by Western blotting using the commercial rabbit anti-NP antibody (Sino Biological) and NP protein standard (ThermoFisher Scientific) as previously described (Zhu et al., 2021).	anti-NP suggested: None
   ELISA determination of IgG, IgG subtypes, and IgA antibodies: ELISA plates (Evergreen Scientific) were coated with 100 µL (1 µg/mL) per well of SARS-CoV-2 Secto protein (Sino Biological), SARS-CoV-2 Secto-β protein, SARS-CoV-2 RBD-untagged protein (Sino Biological), SARS-CoV-2 NP (Sino Biological), or SARS-CoV-2 E protein (1 to 75 amino acids) (ThermoFisher Scientific) in coating buffer [0.05 M sodium carbonate-sodium bicarbonate (pH 9.6)] at 4°C for overnight incubation.	SARS-CoV-2 E protein ( 1 to 75 amino acids ) suggested: None
   Then, the secondary antibody was added at 1:10,000 dilution in PBS-1% BSA buffer (100 µL per well) using either goat anti-mouse IgG-HRP, goat anti-mouse IgG1-HRP, goat anti-mouse IgG2a-HRP, or goat anti-mouse IgA-HRP (Thermo Fisher Scientific).	anti-mouse IgG-HRP suggested: None anti-mouse IgG1-HRP suggested: None anti-mouse IgG2a-HRP suggested: None anti-mouse IgA-HRP suggested: None
   Virus neutralization assay: Neutralizing antibody titers in mouse immune sera against SARS-CoV-2 US-WA-1/2020 or its Beta, Delta, or Omicron variants were quantified by using Vero E6 cell–based microneutralization assay in the BSL-3 suite as previously described (Zhu et al., 2021).	SARS-CoV-2 suggested: None US-WA-1/2020 suggested: None
   To measure T-cell phenotypes, the above overnight (16 h) stimulated splenocytes were similarly blocked with anti-mouse CD16/32 antibodies (BioLegend) and stained with Fixable Viability Dye eFluor™ 506 (eBioscience) followed by APC anti-mouse CD3e (eBioscience),	anti-mouse CD16/32 suggested: None anti-mouse CD3e suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   The CHO cell growth and Spike recombinant plasmid transfection were performed according to the ExpiCHO expression system User Guide (MAN0014337, ThermoFisher website).	CHO suggested: None
   After incubation, 100 µL of the mixture in individual wells was transferred to Vero E6 cell monolayer grown in 96-well microtiter plates containing 100 µL of MEM/2% fetal bovine serum (FBS) medium in each well and was cultured for 72 h at 37°C before assessing the presence or absence of cytopathic effect (CPE)	Vero E6 suggested: None
   Experimental Models: Organisms/Strains
   Sentences	Resources
   Five-week-old female BALB/c (Jackson Laboratory) or hACE2 transgenic mice AC70 (Taconic Biosciences) were randomly grouped (5-10 animals per group) and allowed to acclimate for 14 days.	BALB/c suggested: None
   Software and Algorithms
   Sentences	Resources
   After Coomassie Blue R-250 (Bio-Rad) staining and destaining, the displayed S-trimer protein bands on SDS-PAGE gels were scanned and quantified by ChemiDoc MP imaging system (Bio-Rad) and ImageJ.	ImageJ suggested: (ImageJ, RRID:SCR_003070)
   Splenocytes were then seeded into 24 well tissue culture plates at a density of 2.0 × 106 cells/well (4 wells/mouse) and stimulated with either SARS-CoV-2 S-trimer (10-100 µg/mL) or SARS-CoV-2	SARS-CoV-2 suggested: (Active Motif Cat# 91351, RRID:AB_2847848)
   The percent of BrdU positive cells in CD3 positive populations were calculated using FACSDiva software.	FACSDiva suggested: (BD FACSDiva Software, RRID:SCR_001456)
   Cytokines in the supernatants were then measured by using Bio-Plex Pro mouse cytokine 23-plex assay (Bio-Rad Laboratories).	Bio-Rad Laboratories suggested: None
   Bacterial diversity and community composition were evaluated using QIIME v1.8 (Caporaso et al., 2010), and taxonomy assignment of the representative sequence for each OTU was completed using the RDP classifier algorithm and the SILVA reference database (v123) (Quast et al., 2013).	QIIME suggested: None SILVA suggested: (SILVA, RRID:SCR_006423)
   Statistics and software: Statistical analyses were performed by GraphPad Prism 9.0 software using one-way or two-way analysis of variance (ANOVA) with Tukey’s post hoc test or multiple t-test according to the generated data.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)
   Photo credit: the mouse and immune cell images were created with BioRender.com.	BioRender suggested: None
   The figure data were organized by Photoshop CS6 (Adobe).	Photoshop suggested: None

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   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).

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   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.

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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   Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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   Results from JetFighter: We did not find any issues relating to colormaps.

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   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.

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   Results from scite Reference Check: We found no unreliable references.

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3. 

   Version published to 10.1101/2022.04.28.489809v1 on bioRxiv

   Apr 29, 2022