Dynamic single-cell RNA sequencing reveals BCG vaccination curtails SARS-CoV-2 induced disease severity and lung inflammation

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   ScreenIT

   Mar 18, 2022

   SciScore for 10.1101/2022.03.15.484018: (What is this?)

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

   Table 1: Rigor

   Ethics	IACUC: Experimental procedure involving live animals were carried out in agreement with the protocol (#HA20M310) approved by the Institutional Animal Care and Use Committee (IACUC) at The Johns Hopkins University School of Medicine. Euthanasia Agents: Animals were randomly assigned to be euthanized by isoflurane overdose at end points (day 4 and day 7) following SCV2 infection.
   Sex as a biological variable	Animals: In vivo experiments involving BCG vaccination and SCV2 infection were carried out using male golden Syrian hamsters (Mesocricetus auratus).
   Randomization	Individual seed-stock vial was randomly picked from frozen stock and was subsequently propagated in 7H9 medium before immunization.
   Bl…

   SciScore for 10.1101/2022.03.15.484018: (What is this?)

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

   Table 1: Rigor

   Ethics	IACUC: Experimental procedure involving live animals were carried out in agreement with the protocol (#HA20M310) approved by the Institutional Animal Care and Use Committee (IACUC) at The Johns Hopkins University School of Medicine. Euthanasia Agents: Animals were randomly assigned to be euthanized by isoflurane overdose at end points (day 4 and day 7) following SCV2 infection.
   Sex as a biological variable	Animals: In vivo experiments involving BCG vaccination and SCV2 infection were carried out using male golden Syrian hamsters (Mesocricetus auratus).
   Randomization	Individual seed-stock vial was randomly picked from frozen stock and was subsequently propagated in 7H9 medium before immunization.
   Blinding	Protein expression was scored by a board-certified pulmonary pathologist blinded to cohort status or treatment group.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Following antibodies were used for cell surface staining: anti-CD3 (Bio-Rad, #MCA1477PB, CD3-12), anti-CD4 (BioLegend, #100451, GK1.5), anti-CD11b (Novus, #NB110-89474PECY7, Poly), and anti-RT1D (BioLegend, #110211, 14-4-4S).	anti-CD4 suggested: (BioLegend Cat# 100451, RRID:AB_2564591) anti-CD11b suggested: None anti-RT1D suggested: None
   Primary antibody for CD3 (1:200 dilution; Cat# RM-9107-S1, Thermo Fisher Scientific) was incubated for 30m at 4 °C and detection was achieved using UltraView DAB detection kit (Roche).	CD3 suggested: (Thermo Fisher Scientific Cat# RM-9107-S1, RRID:AB_149924)
   Experimental Models: Cell Lines
   Sentences	Resources
   Vero C1008 [Vero 76, clone E6, Vero E6] (ATCC CRL-1586) cells were used for viral growth and determination of virus stock titers.	Vero C1008 suggested: ECACC Cat# 85020206, RRID:CVCL_0574)
   Vero E6 cells were grown in MEM with 10% fetal bovine serum (FBS), L-glutamine, and penicillin-streptomycin at 37°C with 5% CO2.	Vero E6 suggested: None
   Software and Algorithms
   Sentences	Resources
   Cells were passed through a 70 μm filter and washed twice using ice-cold PBS followed by RBC lysis using ACK lysis buffer (Thermo Fisher Scientific: A1049201) at room temperature for 5 minutes.	Thermo Fisher Scientific suggested: (Thermo Fisher Scientific, RRID:SCR_008452)
   Following antibodies were used for cell surface staining: anti-CD3 (Bio-Rad, #MCA1477PB, CD3-12), anti-CD4 (BioLegend, #100451, GK1.5), anti-CD11b (Novus, #NB110-89474PECY7, Poly), and anti-RT1D (BioLegend, #110211, 14-4-4S).	Poly suggested: None
   Cells were washed three times using FACS buffer and acquired using BD LSRII with FACSDiva Software.	FACSDiva suggested: (BD FACSDiva Software, RRID:SCR_001456)
   Data analyses was carried out using FlowJo (v10) (TreesStar).	FlowJo suggested: (FlowJo, RRID:SCR_008520)
   Differentially expresses genes (DEGs) and pathway analysis: The FindMarker function in Seurat package was performed to identify differentially expressed genes between BCG-WT + SCV2 and SCV2 groups in a particular cell cluster.	Seurat suggested: (SEURAT, RRID:SCR_007322)
   GO term enrichment analysis of the DEGs was performed using Metascape (www.metascape.org) (63).	Metascape suggested: (Metascape, RRID:SCR_016620)

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   Results from OddPub: Thank you for sharing your data.

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   Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

   Despite being a well-defined animal model of COVID-19 with human-like pathology, one key limitation of using golden Syrian hamsters is the limited immunological resources such as well-characterized and validated antibodies (anti-CD8 Abs for example) and the relative paucity of immunologic data on hamster immune responses for correlation with our scRNAseq data. Along these lines, it was surprising that no CD8+ T cells were observed across the hamster groups even though they have been well-described in human scRNAseq studies of BAL samples in SCV2 (57, 58). While BCG vaccination is routinely used in most countries as a TB prevention, the vaccine is generally given intradermally, and recent human studies showing protection by BCG against COVID-19 disease also used intradermal BCG (13). Like other animal studies using BCG in animal models of SCV2 (28) we used the intravenous route based on literature that IV BCG is far more potent against tuberculosis in non-human primates, and evidence that IV BCG reprograms bone marrow hematopoietic stem cells towards a more protective state against bacterial challenge (22). Nevertheless, SCV2 animal studies using percutaneous BCG have shown significant immunologic benefits at the level of flow cytometry (29), and we anticipate that many of the scRNAseq shifts which we observed in IV BCG vaccinated hamsters would be present following intradermal BCG albeit potentially at a lower level. In summary, our study reveals that BCG vaccination reduces ...

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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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   Version published to 10.1101/2022.03.15.484018v1 on bioRxiv

   Mar 15, 2022