Carbohydrate fatty acid monosulfateester enhances the immunogenicity ofinfluenza antigens via TLR4/2-dependent mechanisms

Adjuvants are critical for optimizing the efficacy of influenza subunit vaccines, yet currently approved formulations fail to elicit durable and broad protection. Here, we dissect the immunostimulatory mechanisms of a synthetic carbohydrate fatty acid monosulphate ester (CMS), a key immunogenic component of a squalane-in-water emulsion adjuvant currently in phase I clinical trials. Using human peripheral blood mononuclear cell (PBMC)-based assays, we show that CMS markedly enhances antigen‑specific T cell responses to influenza hemagglutinin (HA) peptides. While recombinant H7N9HA alone poorly activated antigen‑presenting cells, co‑formulation with CMS induced robust dendritic cell (DC) activation, cytokine secretion, and a balanced polyfunctional effector and regulatory T cell response. Bulk RNA sequencing of CMS‑stimulated DC revealed strong induction of toll‑like receptor (TLR)-driven innate inflammatory pathways. Furthermore, functional blockade experiments confirmed that CMS adjuvanticity depends on TLR4 and TLR2 receptors. These results define the molecular basis of CMS‑driven immune activation and highlight this adjuvant as a promising platform for next‑generation influenza vaccines targeting emerging pandemic strains.