Long-term effects of BCG vaccination on telomere length and telomerase activity

Abstract

This study explores the effects of Bacillus Calmette-Guérin (BCG) vaccination on telomere maintenance, an aging-related process, in immune cells. While BCG reduces systemic inflammation and enhances innate immune responsiveness by inducing trained immunity, its effects on other immune aging hallmarks, such as telomere shortening, are not fully understood. We assessed telomere length in two independent human cohorts before and three months after BCG vaccination. Telomere shortening was consistently observed after BCG, but not after placebo vaccination. Trained immunity non-responders were likelier to lose telomere length, but only among males. Higher pre-vaccination testosterone levels were associated with greater telomere loss in males. In vitro, BCG training activated telomerase, particularly in females, and this was partially prevented by exogenous testosterone. These findings suggest BCG vaccination influences telomere dynamics in a sex-specific manner, contributing to understanding BCG's broader effects on aging-related processes.

Keywords: Age; Cell biology; Immunology.

Graphical abstract

Figure 1

Transcriptional changes upon in vitro BCG training revealed an impact on telomere maintenance (A) Gene set enrichment analysis performed after RNA sequencing upon in vitro BCG training of healthy PBMCs (n = 5) compared to non-trained controls. (B and C) Overrepresentation analysis using the 3384 genes that were upregulated (B) and 2271 genes that were downregulated (C) by BCG. The top 20 significant (adjusted p < 0.05) biological processes with the highest fold enrichment are depicted. Processes related to telomere maintenance are underlined.

Figure 2

BCG vaccination led to shorter telomeres in young, healthy individuals 3 months after vaccination (A–F) Average telomere length (TL) per chromosome end in whole blood, quantified by qPCR, (A) in all young individuals (aged 18–35, n = 80), (B) young males (n = 40), or C. young females (n = 40) before, 14 days or 90 days after vaccination. Average TL in (D) all older individuals (aged 50–71, n = 20), (E) older males (n = 9), or (F) older females (n = 11) before, 14 days, or 90 days after vaccination. (G and H) Sex comparisons of (G) average TL before vaccination and (H) fold change in telomere length in three months. (I) Age group comparison of average TL before vaccination and fold change in TL in 3 months. Statistical analyses were performed using Wilcoxon matched-pairs signed rank test (A–F) or Mann-Whitney test (G–I). ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001. Error bars indicate standard deviation.

Figure 3

Telomere shortening was linked to the trained immunity response, particularly in males (A and B) Average telomere length (TL) per chromosome end in whole blood in young (aged 18–35) (A) responders (n = 40) and (B) non-responders (n = 40) before, 14 days, or 90 days after vaccination. (C and D) Average TL of (C) male (n = 20) and (D) female (n = 20) non-responders before, 14 days, or 90 days after vaccination. (E and F) Comparisons of the fold change in TL in three months for responders and non-responders among (E) males and (F) females. (G–I) (G) Average TL comparison of responders and non-responders before vaccination. Correlation of the change in IL-6 response and TL change over 3 months in (H) all, (I) male, and (J) female responders. Statistical analyses were performed using Wilcoxon matched-pairs signed rank test (A–D), Mann-Whitney test (E–G), or Spearman’s rank correlation (H–J). ∗p ≤ 0.05, ∗∗p ≤ 0.01. r: Spearman’s rank correlation coefficient. Error bars indicate standard deviation in A-B and E–G. Dashed lines in H-J indicate 95% confidence intervals.

Figure 4

Telomere shortening 3 months after BCG vaccination in an independent validation cohort (A–D) Average telomere length (TL) per chromosome end in whole blood was assessed for different treatment arms: (A) placebo (n = 5), (B) single-dose BCG (n = 13), (C) double-dose BCG (n = 13), and (D) BCG revaccination (n = 16). (E) Average TL of all participants vaccinated with placebo and assessed before and 90 days after vaccination (n = 18). (F) Average TL of all participants vaccinated with one single-dose BCG and assessed before and 90 days after vaccination (n = 29). Statistical analyses were performed using the paired t test. ∗p ≤ 0.05, ∗∗p ≤ 0.01.

Figure 5

BCG-induced telomerase activation that can be inhibited by testosterone (A and B) (A) Average telomere length (TL) per chromosome end and (B) telomerase activity, represented as activity relative to RPMI control, at day 6 of in vitro BCG training of healthy PBMCs (n = 11–12). BCG concentration was 5 μg/mL for A. (C) Correlation of TL change in three months after BCG vaccination and basal testosterone levels in young males of the 300BCG cohort with available hormone measurements. (D–F) (D) Basal testosterone levels of male responders and non-responders of the 300BCG cohort. Correlation of TL change in three months with the basal testosterone levels in male (E) responders and (F) non-responders. (G) Relative telomerase activity of PBMCs from healthy female donors trained with 5 μg/mL BCG with or without 50 μM testosterone (n = 7). (H) Relative telomerase activity of BCG-trained female PBMCs compared to non-trained controls at different time points during the training protocol (n = 9). (I) IL-6 and TNF production by female PBMCs trained with BCG with or without testosterone upon restimulation with 10 ng/mL LPS (n = 8–9). Statistical analyses were performed using Wilcoxon matched-pairs signed rank test (A–B, G–I), Mann-Whitney test (D), or Spearman’s rank correlation (C, E–F). r: Spearman’s rank correlation coefficient, TST: testosterone, h: hour, d; day. ∗p ≤ 0.05, ∗∗p ≤ 0.01. Error bars indicate standard deviation in (A–B, D, G, and I). Dashed lines in (C and E–F) indicate 95% confidence intervals.

Figure 6

Schematic representation of telomeric regulation by BCG vaccination BCG leads to telomere damage due to the induction of initial acute inflammation, potentially through increased oxidative stress. On the other hand, BCG can induce long-term telomerase activation, likely linked to the establishment of trained immunity, and limit the telomeric damage induced by acute inflammation. Testosterone inhibits BCG-induced telomerase activation.