Harnessing flagellin of <i>Ligilactobacillus agilis</i> as a surface display scaffold for an HIV-1 epitope

Lactic acid bacteria (LAB) are promising mucosal vaccine vectors due to their safety, immunostimulatory properties, and the availability of genetic tools for certain strains. Bacterial flagellin has attracted attention as both a versatile scaffold for antigen surface display and a potent adjuvant via Toll-like receptor 5 (TLR5) activation. Despite these advantages, LAB-derived flagellin remains largely unexplored as an antigen display platform. In this study, we demonstrate the potential of flagellin (FliC2) from <i>Ligilactobacillus agilis</i>, one of the few flagellated LAB species, as a scaffold for the surface display of the HIV-1 membrane-proximal external region (MPER) epitope. Recombinant <i>L. agilis</i> strains were engineered to express FliC2 with the MPER epitope inserted at various positions within its hypervariable domain, identifying optimal sites for effective surface display. To enhance the adjuvant activity of FliC2, specific amino acid substitutions were introduced into the TLR5 recognition site, resulting in improved TLR5-stimulating activity <i>in vitro</i>. Immunization of mice with MPER-displaying <i>L. agilis</i> strains induced MPER-specific IgA and IgG responses, demonstrating the efficacy of the <i>L. agilis</i> flagellin-based display platform in eliciting both mucosal and systemic immune responses. This study is the first to demonstrate LAB-derived flagellin as an antigen display scaffold, highlighting <i>L. agilis</i> flagellin as a promising platform for mucosal vaccine development.IMPORTANCELactic acid bacteria (LAB) are promising delivery vehicles of active molecules, and surface display systems are gaining interest for efficiently displaying heterologous peptides and proteins. Flagellin, a TLR5 agonist, has been widely used as an adjuvant and an antigen scaffold, making it a potentially valuable platform for such systems. However, the potential of LAB-derived flagellin as a surface display scaffold remains largely unexplored. This study demonstrates that flagellin from <i>Ligilactobacillus agilis</i>, a flagellated LAB species, effectively functions as an antigen display platform, eliciting mucosal and systemic immune responses. Our findings highlight the feasibility of LAB-derived flagellin as a versatile tool for surface display of heterologous peptides, expanding its potential applications in vaccine development and mucosal immunotherapy.