Intravenous administration of BCG protects mice against lethal SARS-CoV-2 challenge

  1. Kerry L. Hilligan
  2. Sivaranjani Namasivayam
  3. Chad S. Clancy
  4. Danielle O’Mard
  5. Sandra D. Oland
  6. Shelly J. Robertson
  7. Paul J. Baker
  8. Ehydel Castro
  9. Nicole L. Garza
  10. Bernard A.P. Lafont
  11. Reed Johnson
  12. Franca Ronchese
  13. Katrin D. Mayer-Barber
  14. Sonja M. Best
  15. Alan Sher

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Abstract

In addition to providing partial protection against pediatric tuberculosis, vaccination with bacille Calmette-Guérin (BCG) has been reported to confer nonspecific resistance to unrelated pulmonary pathogens, a phenomenon attributed to the induction of long-lasting alterations within the myeloid cell compartment. Here, we demonstrate that intravenous, but not subcutaneous, inoculation of BCG protects human-ACE2 transgenic mice against lethal challenge with SARS-CoV-2 (SCV2) and results in reduced viral loads in non-transgenic animals infected with an α variant. The observed increase in host resistance was associated with reductions in SCV2-induced tissue pathology, inflammatory cell recruitment, and cytokine production that multivariate analysis revealed as only partially related to diminished viral load. We propose that this protection stems from BCG-induced alterations in the composition and function of the pulmonary cellular compartment that impact the innate response to the virus and ensuing immunopathology. While intravenous BCG vaccination is not a clinically acceptable practice, our findings provide an experimental model for identifying mechanisms by which nonspecific stimulation of the pulmonary immune response promotes host resistance to SCV2 lethality.

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  1. Version published to 10.1084/jem.20211862
  2. ScreenIT

    SciScore for 10.1101/2021.08.30.458273: (What is this?)

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

    Table 1: Rigor

    EthicsIACUC: All animal studies were performed in accordance with institutional guidelines and were conducted in Assessment and Accreditation of Laboratory Animal Care–accredited Biosafety Level 2 and 3 facilities at the NIAID/National Institutes of Health (NIH) using a protocol (LPD-99E) approved by the NIAID Animal Care and Use Committee.
    Euthanasia Agents: Animals were anesthetized by isoflurane inhalation and 103 TCID50 was administered by intranasal instillation.
    Sex as a biological variableMice: Female, 7–9-week-old, B6.Cg-Tg(K18-ACE2)2Prlmn/J hemizygous and non-carrier control animals (JAX34860) were purchased from Jackson Laboratories (Bar Harbor, ME).
    RandomizationMice were housed under specific pathogen–free conditions with ad libitum access to food and water and were randomly assigned to experimental groups.
    BlindingFollowing infection, mice were monitored daily for weight change and clinical signs of disease by a blinded observer who assigned each animal a disease score based on the following criteria: 0) no observable signs of disease; 1) hunched posture
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Anti-CD4 (GK1.5),
    Anti-CD4
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Virology: The SARS-CoV-2 WA1/2020 strain (Pango lineage A), originally isolated at the Center for Disease Control and Prevention (Atlanta, GA) and representative of SARS-CoV-2 viruses circulating early during the pandemic, was obtained from BEI Resources and propagated in tissue culture in Vero CCL81 cells (ATCC).
    Vero CCL81
    suggested: None
    The SARS-CoV-2 USA/CA_CDC_5574/2020 isolate, an alpha variant of concern (Pango lineage B.1.1.7), was obtained from BEI resources and propagated in Vero cells overexpressing TMPRSS2, kindly provided by Dr Jonathan Yewdell (
    Vero
    suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
    Vero-TMPRSS2 cells were maintained in were maintained in DMEM medium supplemented with glutamax, 10% FBS and 250μg/ml Hygromycin B gold (InvivoGen).
    Vero-TMPRSS2
    suggested: JCRB Cat# JCRB1818, RRID:CVCL_YQ48)
    Determination of viral titers by TCID50 assay: Viral titers from lung and brain homogenate were determined by plating in triplicate on Vero E6 cells using 10-fold serial dilutions.
    Vero E6
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Mice: Female, 7–9-week-old, B6.Cg-Tg(K18-ACE2)2Prlmn/J hemizygous and non-carrier control animals (JAX34860) were purchased from Jackson Laboratories (Bar Harbor, ME).
    B6.Cg-Tg(K18-ACE2)2Prlmn/J
    suggested: RRID:IMSR_JAX:034860)
    Software and Algorithms
    SentencesResources
    All samples were collected on a FACSymphony A5 SORP™ flow cytometer (BD) and analyzed using FlowJo software (version 10, BD).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Statistical analyses: P-values were determined by Student’s unpaired t-test or Mann-Whitney test when comparing two groups, or by One-Way ANOVA with Tukey’s post-test or Kruskal-Wallis test with Dunn’s post-test when comparing three or more groups using GraphPad Prism software (v9).
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Weight change, viral copies, multiplex cytokine and flow cytometry measurements generated from WT and K18-hACE2 mice for the three experimental groups (PBS iv, BCG sc and BCG iv) from three independent experiments were compiled and principal component analysis (PCA) was performed and visualized in R version 4.1.0 with packages FactoMineR and factoextra.
    FactoMineR
    suggested: (FactoMineR, RRID:SCR_014602)
    The top 10 variables contributing to the first two principal components were identified for the cytokine and flow cytometry data and these measurements were normalized, clustered and visualized as a heatmap using the R package pheatmap.
    pheatmap
    suggested: (pheatmap, RRID:SCR_016418)
    Figure visualization: Figures were generated in Adobe Illustrator and R incorporating images from Biorender.com.
    Adobe Illustrator
    suggested: (Adobe Illustrator, RRID:SCR_010279)

    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

    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 10.1101/2021.08.30.458273v1 on bioRxiv