Off-target effects of bacillus Calmette-Guérin vaccination on immune responses to SARS-CoV-2: implications for protection against severe COVID-19

Abstract

Background and objectives: Because of its beneficial off-target effects against non-mycobacterial infectious diseases, bacillus Calmette-Guérin (BCG) vaccination might be an accessible early intervention to boost protection against novel pathogens. Multiple epidemiological studies and randomised controlled trials (RCTs) are investigating the protective effect of BCG against coronavirus disease 2019 (COVID-19). Using samples from participants in a placebo-controlled RCT aiming to determine whether BCG vaccination reduces the incidence and severity of COVID-19, we investigated the immunomodulatory effects of BCG on in vitro immune responses to SARS-CoV-2.

Methods: This study used peripheral blood taken from participants in the multicentre RCT and BCG vaccination to reduce the impact of COVID-19 on healthcare workers (BRACE trial). The whole blood taken from BRACE trial participants was stimulated with γ-irradiated SARS-CoV-2-infected or mock-infected Vero cell supernatant. Cytokine responses were measured by multiplex cytokine analysis, and single-cell immunophenotyping was made by flow cytometry.

Results: BCG vaccination, but not placebo vaccination, reduced SARS-CoV-2-induced secretion of cytokines known to be associated with severe COVID-19, including IL-6, TNF-α and IL-10. In addition, BCG vaccination promoted an effector memory phenotype in both CD4⁺ and CD8⁺ T cells, and an activation of eosinophils in response to SARS-CoV-2.

Conclusions: The immunomodulatory signature of BCG's off-target effects on SARS-CoV-2 is consistent with a protective immune response against severe COVID-19.

Keywords: BCG; COVID‐19; SARS‐CoV‐2; T cell; cytokine; immunoregulation.

Figure 1

Schematic of study design. Created with BioRender.com.

Figure 2

Whole‐blood cytokine responses to SARS‐CoV‐2. (a) Violin and Tukey boxplots of cytokine concentration (in pg mL⁻¹) following stimulation of whole blood, taken prior to randomisation, with γ‐irradiated SARS‐CoV‐2 (iSARS) or γ‐irradiated uninfected Vero cell supernatant (iVero). Centre lines indicate medians; box limits indicate 25–75th percentiles; whiskers extend to 1.5 times the interquartile range from the 25th and 75th percentiles; and outliers are represented by dots. Stimulation effect measured by the Wilcoxon signed‐rank test comparing the iSARS with the iVero response (n = 50). Asterisks in the box plots depict significance (*P < 0.05, **P < 0.01 and ***P < 0.001). (b) The results from unsupervised hierarchical clustering of whole‐blood cytokine responses to a range of pathogens and TLR agonist ligands are shown. Clustering was done using Spearman’s correlation as the measure of similarity between cytokine–stimulant pairs. Red indicates a strong negative correlation, whereas blue indicates a strong positive correlation. The data shown are from samples taken prior to randomisation in participants with no missing cytokine–stimulant pair results (n = 38). BCG, bacillus Calmette–Guérin; iSARS, γ‐irradiated SARS‐CoV‐2; and Th, T helper.

Figure 3

Immune cell activation in response to SARS‐CoV‐2. (a–d) Violin and Tukey boxplots of immune cell populations following stimulation of whole blood, taken prior to randomisation, with γ‐irradiated SARS‐CoV‐2 (iSARS) or γ‐irradiated uninfected Vero cell supernatant (iVero). Centre lines indicate medians; box limits indicate 25th–75th percentiles; whiskers extend to 1.5 times the interquartile range from the 25th and 75th percentiles; and outliers are represented by dots. Data are presented as (a) proportion (%) of CD45⁺, proportion of parent (granulocyte or monocyte as indicated) or expression [median fluorescence intensity (MFI)]; (b) proportion of total T cells or indicated T‐cell subpopulation; (c) proportion of CD69⁺ and PD‐1⁺ among parent populations and MFI of CD69 and PD‐1 among CD69⁺ and PD‐1⁺ cells for each parent population, respectively; and (d) proportion of each subset among NK cells (CD3⁻CD19⁻CD14⁻), proportion of CD69⁺ within NK cell subsets and MFI of CD25, CD127 and PD‐1 among CD56^lowCD16^high and CD56^lowCD16^low NK cells. Stimulation effect measured by the Wilcoxon signed‐rank test comparing the iSARS with the iVero response (n = 29). Asterisks in the box plots depict significance (*P < 0.05, **P < 0.01 and ***P < 0.001). NK, natural killer; Tconv, conventional T cell; Treg, regulatory T cell; and TEMRA, terminally differentiated effector memory T cells.

Figure 4

Effect of BCG vaccination on immune responses to γ‐irradiated SARS‐CoV‐2. Volcano and Tukey boxplots depicting differences in γ‐irradiated SARS‐CoV‐2 (iSARS) stimulation effect (iSARS‐iVero response) on whole‐blood (a, b) cytokine (n = 50), (c, d) immune cell population (n = 17) and (e, f) extracellular marker (n = 17) responses 3 months after randomisation. Differences in iSARS stimulation effect measured by the Wilcoxon signed‐rank test comparing the iSARS stimulation effect at 3 months (m3) post‐randomisation with the iSARS stimulation effect at baseline (d0). Volcano plots: blue dots show changes in the BCG‐vaccinated group, gold dots show changes in the placebo‐vaccinated group, and black dots show immune responses for which there was no change. Red line indicates P = 0.05. Boxplots: Centre lines indicate medians; box limits indicate 25–75th percentiles; whiskers extend to 1.5 times the interquartile range from the 25th and 75th percentiles; and outliers are represented by dots. Asterisks in the box plots depict significance (*P < 0.05, **P < 0.01 and ***P < 0.001). _f45, frequency of CD45⁺ cells; _fp, frequency of parent; _fm, frequency of mononuclear cells; _ft, frequency of T cells; BCG, bacillus Calmette–Guérin; CM, central memory; EM, effector memory; iSARS, γ‐irradiated SARS‐CoV‐2; iVero, γ‐irradiated uninfected Vero; MFI, median fluorescence intensity; NK, natural killer; Treg, regulatory T cell; and TEMRA, terminally differentiated effector memory T cells.