Flagellin’s hypervariable D2/D3 domain is required for robust anti-flagellin primary antibody responses (VAC11P.1065)

Abstract

Abstract Bacterial flagellin is a well-known agonist of the innate immune system. Several clinical trials investigating flagellin fusion proteins have demonstrated promising results for inducing protective immunity towards influenza virus, which has been largely attributed to flagellin activation of TLR5. Our lab recently demonstrated that the Salmonella enterica serovar Typhimurium flagellin protein, FliC, induces antibody responses in mice through a third pathway that is independent of TLR5, Casp1, and MyD88. Herein, we define the structural features of FliC that contribute to this third pathway. By destroying the Naip5/6 (D0 domain) or TLR5 (D1 domain) recognition sites, we demonstrate that neither affected the TLR5-, inflammasome- and MyD88-independent antibody responses towards FliC. In contrast, deletion of the D2/D3 domain eliminated primary anti-flagellin antibody responses. Optimal primary and secondary anti-flagellin antibody responses required TLR5- and inflammasome-mediated recognition, and FliC’s D2/D3 domain. We demonstrate FliC’s D2/D3 domain is required for induction of primary antibody responses and robust secondary antibody responses. Our results indicate that FliC’s D2/D3 domain is essential for flagellin’s robust immunogenicity and suggest that the D2/D3 domain interacts with the host immune system to augment antibody production. Thus, the D2/D3 domain of FliC may also be critical for optimal immunogenicity and antibody responses in flagellin based vaccines.