Effects of a multistrain probiotic formulation on immune response and microbiome modulation following influenza vaccination in adults: a randomised, triple-blind, placebo-controlled trial

Background Seasonal influenza remains a major global health burden, despite widespread vaccination. Vaccine responsiveness varies considerably between individuals and is increasingly recognised to be influenced by host factors, including the gut microbiome. Probiotics have been proposed as a strategy to modulate microbiome–immune interactions, although their effects on vaccine responses and the underlying microbial mechanisms remain incompletely understood. Objective To investigate the effects of a probiotic formulation on immune responses and gut microbiome dynamics following seasonal influenza vaccination in healthy adults. Methods In this randomised, triple-blind, placebo-controlled, multicentre trial, 120 healthy adults received either a 14-strain probiotic formulation (14 bacterial strains; 8 × 10 9 CFU/day, 40 mg vitamin C, and 2.5 μg vitamin B 12 ) or placebo for six weeks, commencing three weeks before influenza vaccination and continuing for three weeks afterwards. The primary endpoint was change in strain-specific haemagglutination inhibition (HAI) antibody titres. Secondary endpoints included seroprotection and seroconversion rates, microneutralization activity, serum immune biomarkers and shotgun metagenomic analysis of the gut microbiome. Results Vaccination induced the expected increases in HAI antibody titres in both groups, with no significant differences between the active and placebo groups. The probiotic formulation was, however, associated with selective changes in the gut microbiome, including enrichment of the butyrate-producing species Roseburia inulinivorans and alterations in predicted microbial metabolic pathways, without affecting overall microbiome diversity. Participants who achieved seroprotection exhibited distinct compositional and functional microbiome responses to vaccination. These patterns included community-level changes that occurred predominantly in the active group, alongside taxonomic and functional differences associated with seroprotection. Conclusions Although supplementation with the probiotic formulation did not increase influenza antibody responses, it did alter the gut microbiome response to vaccination, with selective changes in microbial composition and predicted functional potential while maintaining overall community diversity. Importantly, seroprotection was associated with distinct compositional and functional microbiome adaptations, and community-level changes associated with seroprotection occurred predominantly in the active group. Collectively, these findings support a microbiome-conditioning model in which the probiotic formulation shapes the gut microbiome response during vaccination, rather than acting as a direct immunostimulant. Future studies are required to determine whether these microbiome adaptations contribute causally to vaccine responsiveness. Clinical Trial Registration https://clinicaltrials.gov/study/NCT06103994 , identifier NCT06103994