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BCG vaccination stimulates integrated organ immunity by feedback of the adaptive immune response to imprint prolonged innate antiviral resistance

Nature Immunology volume 25, pages 41–53 (2024)Cite this article

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Abstract

Bacille Calmette–Guérin (BCG) vaccination can confer nonspecific protection against heterologous pathogens. However, the underlying mechanisms remain mysterious. We show that mice vaccinated intravenously with BCG exhibited reduced weight loss and/or improved viral clearance when challenged with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 B.1.351) or PR8 influenza. Protection was first evident between 14 and 21 d post-vaccination and lasted ∼3 months. Notably, BCG induced a biphasic innate response and robust antigen-specific type 1 helper T cell (TH1 cell) responses in the lungs. MyD88 signaling was essential for innate and TH1 cell responses, and protection against SARS-CoV-2. Depletion of CD4+ T cells or interferon (IFN)-γ activity before infection obliterated innate activation and protection. Single-cell and spatial transcriptomics revealed CD4-dependent expression of IFN-stimulated genes in lung myeloid and epithelial cells. Notably, BCG also induced protection against weight loss after mouse-adapted SARS-CoV-2 BA.5, SARS-CoV and SHC014 coronavirus infections. Thus, BCG elicits integrated organ immunity, where CD4+ T cells feed back on tissue myeloid and epithelial cells to imprint prolonged and broad innate antiviral resistance.

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Fig. 1: BCG vaccination protects mice against challenge with SARS-CoV-2.
Fig. 2: BCG vaccination induced a biphasic pattern of innate activation.
Fig. 3: BCG vaccination induced a robust TH1 cell profile at 21 d post-vaccination.
Fig. 4: Protection against SARS-CoV-2 B.1.351 conferred by BCG vaccination is dependent on MyD88 signaling.
Fig. 5: CD4+ T cells and IFN-γ production play a critical role in the heterologous protection mediated by BCG vaccination.
Fig. 6: TH1 cells and IFN-γ drive the activation of myeloid cells and lung ECs.
Fig. 7: Spatial transcriptomics of CD4-dependent innate antiviral program in lungs.
Fig. 8: Model depicting integrated organ immunity induced by BCG, leading to nonspecific protection in the lungs.

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Data availability

The scRNA-seq data generated in the present study are publicly accessible in the Gene Expression Omnibus under accession no. GSE244126. GeoMx data generated in this study are publicly accessible under GEO accession code GSE247145. Source data are provided with this paper.

Code availability

Computer code is available upon reasonable request.

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Acknowledgements

Work in the laboratory of B.P. is supported in part by grants and contracts from the NIH (R01 AI048638, U19 AI057266, U19 AI167903 and ACC 75N93022C00052), the Bill and Melinda Gates Foundation, Open Philanthropy, the Violetta L. Horton and Soffer Endowments, and other anonymous donors to B.P. We thank P. Jagannathan for his help in obtaining the BCG TICE vaccine. We thank the Stanford Shared FACS Facility for technical support. Data were collected on instruments in the Shared FACS Facility purchased by the Parker Institute for Cancer Immunotherapy or obtained using National Institutes of Health (NIH) S10 Shared Instrument Grant (Symphony, grant no. 1S10OD026831-01). We thank the Stanford Functional Genomics Facility for technical assistance, D. Wagh and E. Kim for library preparation and J. Coller for data analysis. The sequencing data were generated with instrumentation purchased with NIH funds (grant nos. S10OD025212 and 1S10OD021763) at the Stanford Genomics Facility. We thank the Stanford Human Immune Monitoring Core for technical assistance in performing Luminex assays. We thank B. Franco from Stanford Veterinary Service Center for technical support. We acknowledge the pathological services provided by HistoWitz, Inc. and pathological analysis provided by J. Feldstein at HistoWitz, Inc. Diagrams in Figs. 1a, 4a, 5a,c, 8 and Extended Data Fig. 2a were created with BioRender.com.

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Authors and Affiliations

  1. Institute for Immunity, Transplantation and Infection, Stanford University School of Medicine, Stanford University, Stanford, CA, USA

    Audrey Lee, Shengyang Wu, Zhuoqing Fang, Chunfeng Li, Harold Hui, Victor Lujan, Prabhu S. Arunachalam & Bali Pulendran

  2. Department of Pediatrics, Emory Vaccine Center, Emory National Primate Research Center, Emory University School of Medicine, Atlanta, GA, USA

    Katharine Floyd & Mehul S. Suthar

  3. Department of Pathology, Stanford University School of Medicine, Stanford University, Stanford, CA, USA

    Tze Kai Tan, Garry P. Nolan & Bali Pulendran

  4. Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA

    Tze Kai Tan

  5. Department of Epidemiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Heather M. Froggatt, John M. Powers, Sarah R. Leist, Kendra L. Gully, Miranda L. Hubbard & Ralph S. Baric

  6. NanoString Technologies, Seattle, WA, USA

    David Scoville, Alistaire D. Ruggiero, Yan Liang & Anna Pavenko

  7. Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford University, Stanford, CA, USA

    Garry P. Nolan & Bali Pulendran

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Contributions

A.L. and B.P. designed the study, interpreted the data and wrote the manuscript. A.L. led and planned the research and analyzed the data. K.F. and M.S.S. performed SARS-CoV-2 mouse experiments including challenge, viral load quantification and sample analysis. S.W. performed mouse injections and helped with tissue harvesting and processing. Z.F. contributed to scRNA-seq analysis. T.K.T. helped with CyTOF staining and data acquisition. D.S., A.D.R., Y.L. and A.P. contributed to and performed GeoMx. H.H. and V.L. helped with tissue harvesting and processing. H.M.F. and K.L.G. vaccinated mice for mouse-adapted coronavirus challenge. H.M.F., J.M.P., S.R.L. and M.L.H. performed viral infections, tissue harvesting and sample analysis for mouse-adapted coronavirus challenge. J.M.P. developed the mouse-adapted SARS-CoV-2 BA.5 virus. C.L. propagated the PR8 virus. R.S.B. provided advice and guidance to SARS-CoV-2 challenge experiments. P.S.A. provided guidance and advice. G.P.N. provided CyTOF instrument.

Corresponding author

Correspondence to Bali Pulendran.

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Competing interests

B.P. has served or is serving on the External Immunology Network of GSK and on the scientific advisory boards of Sanofi, Medicago, CircBio and Boehringer-Ingelheim. R.S.B. serves on the Scientific Advisory Board of Takeda, Vaxart, Inc. and Invivyd, and has collaborations with Janssen Pharmaceuticals, Gilead, Chimerix and Pardes Biosciences. S.R.L. and R.S.B. are listed on a patent for the SARS-CoV-2 MA10 virus (US 11225508 B1, ‘Mouse-adapted SARS-CoV-2 viruses and methods of use thereof’). G.P.N is a co-founder and stockholder of Ionpath Inc., a co-founder and stockholder of Akoya Biosciences, Inc. and an inventor on patent US9909167., a Scientific Advisory Board member for Akoya Biosciences, Inc., received research grants from Pfizer, Inc., Vaxart, Inc., Celgene, Inc. and Juno Therapeutics, Inc. and is a co-founder of Scale Biosciences Inc. M.S.S. has served in an advisory role for Ocugen, Inc. The remaining authors declare no competing interests.

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Nature Immunology thanks Rino Rappuoli, Maria Rescigno and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Jamie Wilson, in collaboration with the Nature Immunology team.

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Extended data

Extended Data Fig. 1 BCG delivered via multiple routes of vaccination conferred protection against SARS-CoV-2 and influenza A PR8.

a, Representative immunohistochemisry images depicting staining of SARS-CoV-2 nucleocapsid protein (NP). Blue circles represent expression of SARS-CoV-2 NP in focal alveoli, alveolar macrophages, alveolar pneumocytes and rare bronchiolar epithelial cells. BCG IV: Intravenous BCG vaccinated. b, Left, weight loss of intranasal BCG vaccinated mice followed 3 days post-SARS-CoV-2 challenge. Right, SARS-CoV-2 RNA-dependent RNA polymerse (RdRp) viral load as fold-change over mock-infected, in the lungs and nasal turbinates. Statistical analysis was performed by two-tailed Mann-Whitney test. c, Survival plot and weight loss of mice following PR8 infection. Mice were vaccinated via various routes including intravenous (IV), intranasal (IN), intramuscular (IM), subcutaneous (SC). BCG IV and IN, data combined from 2 independent experiments, n = 11 (BCG IV), n = 12 (BCG IN); BCG IM, data combined from 3 independent experiments (n = 16 for naïve and n = 17 for BCG IM). BCG SC, data from one independent experiment (n = 5). Survival analysis was performed using log-rank (Mantel-Cox) test. Statistical analysis for weight loss was performed by Two-way ANOVA with Sidak’s multiple comparisons test. **, P < 0.01.

Extended Data Fig. 2 BCG vaccination protects mice from weight loss against various strains of mouse-adapted Sarbecoviruses.

a, Experimental outline and weight loss of mice following challenge. b, Gross lung discoloration score at day 2 or 4 post-challenge. c, Lung viral titer at day 2 or 4 post-challenge, as assessed by plaque assay. Data representative of one independent experiment; In a, b, c, for SHC014 MA15, n = 8 (BCG); For SARS-CoV MA15 n = 9 (BCG); n = 7 for all other groups. Mean±SEM values are plotted. Statistical analysis was performed by Two-way ANOVA with Sidak’s multiple comparisons test. *, P < 0.05; **, P < 0.01; ***, P < 0.005; ****, P < 0.0001.

Extended Data Fig. 3 BCG induced prolonged serum cytokine and lung innate activation.

a, Extended data of kinetics of serum cytokine responses measured by Luminex. Data representative of two independent experiments; n = 10 (day 0), n = 4 (day 21), n = 5 (all other time points). b, Flow cytometry gating strategy of lung populations (Top) and cell frequencies (CD45+ live cells) of innate cells in lungs (Bottom). Data is representative of two independent experiments for day 21 and one representative experiment for other timepoints (n = 5-8). Data representative of two independent experiments at day 21 (n = 4) and one independent experiment for all other timepoints; n = 9 (day 0), n = 5 for all other timepoints. Statistical analysis was performed by One-way ANOVA with Tukey’s multiple comparisons test (a), One-way ANOVA with Dunnett’s multiple comparison test for each timepoint compared to day 0 (b). *, P < 0.05; **, P < 0.01; ***, P < 0.005; ****, P < 0.0001.

Extended Data Fig. 4 BCG induced systemic innate activation and bone marrow stem and progenitor expansion.

a, Flow cytometry gating strategy, cell frequencies (CD45+ live cells), and CD86 MFI of innate cells in spleen. Data is representative of two independent experiments for day 21 and one representative experiment for other timepoints; n = 4 for day 21, n = 8 (day 0), n = 5 for all other timepoints. b, Flow cytometry gating strategy and frequencies of bone marrow cells (% of live cells). Data from one representative experiment. Abbreviations, LSK: Lin−Sca-1+c-Kit+ cells, LT-HSC: Long-term HSC. ST-HSC: Short-term HSC. MPP: Multipotent progenitor. One-way ANOVA with Dunnett’s multiple comparison test for each timepoint compared to day 0 (a and b). *, P < 0.05; **, P < 0.01; ***, P < 0.005; ****, P < 0.0001.

Extended Data Fig. 5 Extended data on CyTOF analysis in the spleen.

UMAP embedding of the cell clusters identified by FlowSOM clustering (left), heatmap depicting the normalized values of key surface markers across identified clusters (right). (bottom) Frequencies of CD4+ T cell and Ly6Chi monocyte in the spleen and normalized and scaled values of intracellular markers detected by CyTOF. The box plots show median, first and third quartiles and the whiskers show 1.5x interquartile range (IQR) on either side. Statistical analysis was performed by two-tailed Wilcoxon rank-sum test. *, P < 0.05; ***, P < 0.005.

Extended Data Fig. 6 Differential immune responses to BCG vaccination in knock-out (KO) mice.

a and b, Serum cytokine responses in wild-type (WT) and Myd88−/− mice vaccinated with BCG intravenously at baseline, 6 h and 18 days post-vaccination (a), and day 3 post-SARS-CoV-2 challenge (b). c and d, Serum cytokine responses in wild-type (WT) and ASC−/− (c) and STING−/− (d) mice vaccinated with BCG intravenously. Data representative of two independent experiments for Myd88−/− and ASC−/− (n = 6). Two-way ANOVA with Tukey’s multiple comparison test (panel b, e, f). Multiple Mann-Whitney with Holm-Sidak multiple comparisons test (panel c, d). *, P < 0.05; **, P < 0.01; ****, P < 0.0001.

Extended Data Fig. 7 SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) level in CD4 and/or CD8 antibody depleted mice.

Viral load was measured at day 3 post-challenge by quantitative PCR and is represented as fold-change over mock-infected mice (mean±SEM). Data representative of one independent experiment, n = 6. Statistical analysis by two-tailed Mann-Whitney test; ns, not significant.

Extended Data Fig. 8 Extended data from scRNA-seq analysis of lungs in mice at day 0 and 21 post-vaccination.

a, Top variable genes defining cell clusters identified from scRNA-seq data. b, Distribution of cells identified day 0 and 21 post-vaccination on the UMAP embedding. c, Overrepresentation analysis and enrichment of Gene Ontology (GO) biological processes across identified cell clusters, using differentially expressed genes (DEGs; log2FC cutoff > 0.25; FDR cutoff < 0.05). Enrichment was performed using hypergeometric distribution with BH correction.

Extended Data Fig. 9 Protein level expression of interferon-stimulated gene (Mx1) and IFN-γ in lung cells, and CD4+ T cells, respectively.

a, Mx1 median fluorescent intensity (MFI) of lung myeloid and alveolar type II epithelial cells detected using Mx1-GFP reporter mice at various timepoints post-vaccination with BCG intravenously (n = 3-5). Data representative of two independent experiments in lung myeloid cells and one representative experiment in alveolar type II cells; n = 5 for day 21, n = 4 for all other timepoints. b, Frequency of IFN-g+ cells (% of cell population of interest) in vivo, as measured by flow cytometry staining 6 h following Brefeldin A injection in mice. Data from one representative experiment; n = 7 for BCG, n = 4 for Unimmunized. Statistical analysis was performed by Two-way ANOVA with Dunnet’s test (a; myeloid cells) Sidak’s multiple comparisons test (b), and with two-tailed Mann-Whitney test (a; alveolar type II cells). *, P < 0.01; ****, P < 0.0001.

Extended Data Fig. 10 Extended data showing impaired activation of lung myeloid cells following CD4+ T cell or IFN-γ depletion.

CD86 median fluorescence intensity (MFI) of DCs in the lungs at day 21 post-BCG IV vaccination following CD4 depletion (top) and IFN-g depletion (bottom). Data representative of one independent experiment; n = 5 (BCG+Isotype), n = 6 (Unvaccinated and BCG+anti-CD4), n = 9 (BCG) in CD4 depletion; n = 4 (Unvaccinated) and n = 5 (rest of the groups) in IFN-g depletion. Statistical analysis was performed by One-way ANOVA with Tukey’s multiple comparison test. *, P < 0.05; **, P < 0.01; ***, P < 0.005; ****, P < 0.0001.

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Supplementary Table 1

Metal-conjugated antibody panel used for CyTOF.

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Lee, A., Floyd, K., Wu, S. et al. BCG vaccination stimulates integrated organ immunity by feedback of the adaptive immune response to imprint prolonged innate antiviral resistance. Nat Immunol 25, 41–53 (2024). https://doi.org/10.1038/s41590-023-01700-0

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