FliC’s Hypervariable D3 Domain Is Required for Robust Anti-Flagellin Primary Antibody Responses

Abstract

Bacterial flagellin is a well-known agonist of the innate immune system that induces proinflammatory responses through the TLR5 and Naip5/6 recognition pathways. Several clinical trials investigating flagellin fusion proteins have demonstrated promising results for inducing protective immunity toward influenza virus, which has been largely attributed to flagellin's ability to activate TLR5. Our laboratory previously demonstrated that the Salmonella enterica serovar Typhimurium flagellin protein, FliC, induces Ab responses in mice through a third pathway that is independent of TLR5, Casp1/11, and MyD88. In this study, we further define the structural features of FliC that contribute to this unknown third pathway. By destroying the Naip5/6 and TLR5 recognition sites, we demonstrate that neither were required for the TLR5-, inflammasome-and MyD88-independent Ab responses toward FliC. In contrast, deletion of FliC's D3 or D0/D1 domains eliminated primary anti-flagellin Ab responses. For optimal primary and secondary anti-flagellin Ab responses we show that TLR5, inflammasome recognition, and the D3 domain of FliC are essential for flagellin's robust immunogenicity. Our data demonstrate that the D3 domain of FliC influences immunogenicity independent of the known innate recognition sites in the D0/D1 domains to augment Ab production. Our results suggest full-length FliC is critical for optimal immunogenicity and Ab responses in flagellin-based vaccines. ImmunoHorizons, 2019, 3: 422-432.

Deimmunization of flagellin for repeated administration as a vaccine adjuvant

“…To determine Ab production in mucosal secretions, two-weeks after the third vaccination, we collected BALF and determined FlaB-specific Ab responses by Western blot analysis. Since it has been reported that deletions of hypervariable region of Salmonella typhimurium FliC abrogates antibody-mediated neutralization [ 16 , 17 ], we employed FlaB ΔD2D3 as a control protein to investigate the role of 19 amino acids on the Ab production as well as Ag–Ab interaction profiles. As shown in Fig.…”

“…To develop deimmunized FlaB, we targeted B-cell epitopes by using in silico prediction and structure modeling tools. It has been reported that deletions of hypervariable region of Salmonella typhimurium FliC abrogates antibody-mediated neutralization [ 16 ]. Our computational analyses also found that the most probable B-cell epitope would be located within hypervariable D2D3 regions (170–284 amino acid) of the FlaB molecule.…”

“…The helical D0 and D1 domains are relatively well conserved while D2 and D3 domains are variable among different flagellated bacteria across genus and species. The D2 and D3 domains are exposed outward and induce specific antibody responses [ 16 , 17 ]. V. vulnificus , a halophilic pathogenic bacterium, possesses a total of six flagellin genes and among the six flagellin genes, flaB is the major subunit contributing to the flagellum biogenesis and the function, indicating FlaB should have been conserved physico-chemically stable throughout the long history of natural evolution [ 18 ].…”

Bacterial Flagellar Filament: A Supramolecular Multifunctional Nanostructure

“…Flagella elicit the activation of host inflammatory responses via flagellin interaction with specific molecules through a variety of signaling pathways. Flagellins act as potent agonists of the innate immune system inducing proinflammatory responses, given their ability to stimulate the extracellular Toll-like receptor 5 (TLR5), the intracellular NOD-like receptor (NLR) family 4 (NLRC4)-NAIP 5/6 inflammasome, and an unknown additional third pathway recently proposed that is independent of TLR5, Casp1/11, and MyD88 ( Figure 7 ) [ 201 , 202 , 203 ].…”

“…Mice immunized with FliC and different truncated versions made antibodies that showed reactivity to the D0/D1 and D2/D3 domains, indicating that both components of the molecule are antigenic. The D3 domain of FliC influences immunogenicity independently of the known innate recognition sites in the D0/D1 domains to augment antibody production, suggesting that full-length FliC and the preservation of all four domains would be critical for optimal immunogenicity and primary and secondary antibody responses in flagellin-based vaccines [ 203 ]. The nature of the third pathway is poorly understood and requires further investigation.…”