Truncated flagellin lacking the hypervariable region: a structural basis for improved immune responses and adjuvanticity

Bacterial flagellins are recognized for their potent immunomodulatory properties and potential as vaccine adjuvants. They activate both innate and adaptive immune responses by interacting with Toll-like receptor 5 (TLR5) and the cytosolic NOD-like receptor protein 4 (NLRC4) inflammasome, thereby enhancing immune responses. This study investigates the impact of various truncated flagellin derivatives, derived from Escherichia coli (EHEC EDL933) and lacking specific domains, on TLR5 activation and their adjuvant properties. We generated several truncated flagellin mutants and assessed their ability to activate TLR5 in vitro and their immunoadjuvant effects in vivo. Our data show that only the FliCH7, FliCNC, FliCH7-FaeG, and FliCNC-FaeG proteins, which lack the hypervariable region (HVP) but retain both the amino- and carboxy-terminal regions, significantly enhanced TNF-α and IL-8 production compared to other flagellin derivatives. These findings underscore the essential roles of both conserved terminal regions in TLR5 activation. Notably, the FliCNC truncated mutant exhibited TLR5 activation comparable to that of native flagellin and induced higher antibody titers when co-administered with a model antigen or used as a fusion protein. Our results suggest that the HVR is not essential for flagellin’s immunoadjuvant activity and that its removal enhances flagellin’s ability to activate the innate immune system. This study provides valuable insights into optimizing flagellin derivatives for vaccine development, offering a more potent platform for enhancing immune responses against a range of pathogens.