BCG vaccine induces distinct metabolic shifts in monocytes in vitro and peripheral blood plasma in vivo: Implications for neonatal trained immunity

Introduction/Rationale: BCG protects infants against tuberculosis as well as unrelated infections via incompletely understood mechanisms, including metabolic reprogramming of innate immune cells. We sought to characterize age-specific shifts in extracellular metabolism of human monocytes (Mo) induced by BCG stimulation in vitro, determine how they contribute to metabolic reprogramming, and integrate observations in vitro with results from human newborns in vivo.

Methods: In a human in vitro Mo training platform, newborn (NB=10) and adult (AD=12) CD33+ Mo from US-based BCG-naive participants were stimulated with RPMI (control) or BCG for 24h, washed, cultured for 6 days and stimulated with LPS for 24h to assess trained immunity. Day 1 (D1) and D7 supernatants were subjected to mass-spectrometry-based global metabolomics and data integrated with those from an in vivo prospectively randomized newborn cohort from The Gambia (NCT03246230), where BCG was given early (birth) vs delayed (D7) and peripheral blood collected before and 1 or 7 days post-BCG (N=44-50/group).

Results: Arginine biosynthesis was highly and significantly enriched in vitro after both primary BCG stimulation and training in both NB and AD. NB BCG trained immunity was characterized by significant upregulation of female sex steroids and downregulation of most other lipid pathways compared to AD. In vivo, early vs delayed BCG resulted in significant downregulation of amino acid (AA) and upregulation of acylcholine pathways on D1, followed by increases in lysophospholipids and acylcholines and decreases in pyruvate, lactate and α-ketoglutarate on D7. In vivo-in vitro integration revealed overlapping signatures featuring decreases in key AA, phospholipids and acylglycines on D1, and sphingolipids, N-acetylglutamate, α-ketoglutarate and leucine/valine metabolites on D7.

Conclusion: Human in vitro modeling recapitulates distinct signatures of BCG primary and trained immunity in newborns that may contribute to BCG immunogenicity and off-target effects in vivo.