Deletion of Flagellin’s Hypervariable Region Abrogates Antibody-Mediated Neutralization and Systemic Activation of TLR5-Dependent Immunity

Abstract

TLRs trigger immunity by detecting microbe-associated molecular patterns (MAMPs). Flagellin is a unique MAMP because it harbors 1) an antigenic hypervariable region and 2) a conserved domain involved in TLR5-dependent systemic and mucosal proinflammatory and adjuvant activities. In this study, the contribution of the flagellin domains in TLR5 activation was investigated. We showed that TLR5 signaling can be neutralized in vivo by flagellin-specific Abs, which target the conserved domain. However, deletions of flagellin’s hypervariable region abrogated the protein’s intrinsic ability to trigger the production of neutralizing Abs. The fact that MAMP-specific Abs block TLR-mediated responses shows that this type of neutralization is a novel mechanism for down-regulating innate immunity. The stimulation of mucosal innate immunity and adjuvancy to foreign Ag was not altered by the hypervariable domain deletions. In contrast, this domain is essential to trigger systemic innate immunity, suggesting that there are distinct mechanisms for TLR5 activation in systemic and mucosal compartments. In summary, specific MAMP determinants control the production of neutralizing Abs and the compartmentalization of innate responses.

Antigen-Containing Liposomes Engrafted with Flagellin-Related Peptides Are Effective Vaccines That Can Induce Potent Antitumor Immunity and Immunotherapeutic Effect

“…Previous structure-function studies using truncated and/or mutated forms of flagellin indicate that the proinflammatory properties of flagellin are localized in the conserved N-and C-terminal regions (33)(34)(35)(36)(37)44), with conflicting data claiming an essential role for the central hypervariable domain (36). Although not previously reported, short peptides making up the regions of flagellin implicated in the interaction with TLR5 may also bind TLR5 themselves; however, short peptides generally lack significant secondary/tertiary structure, suggesting that any such interaction is likely to be of low affinity, potentially limiting their use in vaccine development.…”

“…Recent evidence suggests that a 14-aa a-helical motif within the N-terminal domain (aa 95-108) and a conserved C-terminal motif (around aa 408-438) of flagellin are essential for full activation of TLR5, with the N-region of flagellin (aa 88-97) having been predicted to physically interact with aa 555-559 of TLR5 (33)(34)(35)(36)(37)39). Our finding that liposomes engrafted with 9Flg and 42Flg (which contain aa 85-111 and aa 98-112 of flagellin, respectively) interact with TLR5-expressing cells, but that liposomes engrafted with 10Flg and 11Flg (which contain aa 405-425 and aa 408-438 of flagellin, respectively) exhibit only little binding, underscores the importance of the N-terminal aa 85-111 region and is consistent with the C-terminal region not playing a major role in the binding of flagellin to TLR5 (15,17,33,39).…”

“…The high-homology regions are defined by aa 79-117 (in N region) and aa 420-475 (in C region) (33,34). The threedimensional structure, coupled with mutagenesis studies of flagellin, showed that the ability of flagellin to interact with TLR5 resides in the first 117 aa of the N-terminal region, particularly the region defined by aa 79-117, but it may require contributions from C-region aa 408-439 (33)(34)(35)(36)(37)(38). Peptides spanning different regions conserved in flagellin and/or that have been implicated in binding to TLR5 can be designed (Table I), produced synthetically, and examined for their ability to interact with APCs.…”

“…pFlgs covering aa 79-117 of FliC in the conserved N-region may contain epitopes that can bind/stimulate TLR5 (34)(35)(36)(37)(38)(39). Therefore, two peptides overlapping by 4 aa were designed and denoted 41Flg (79-101) and 42Flg (98-117); two peptides denoted 9Flg (85-111) and 45Flg (98-111) were designed to cover, respectively, the central and N-parts of the conserved Nregion (Table I).…”

Salivary IgA enhancement strategy for development of a nasal-spray anti-caries mucosal vaccine

“…However, the very potent antigenicity of flagellin itself led to a concern that immunity to flagellin might affect the potency of this molecule and induce possible side effects when used as a mucosal adjuvant [64], although there were reports that prior immunity to flagellin does not impair its adjuvant activity and does not lead to serious systemic effects [15]. We tried to replace the main antigenicity region domains D2 and D3 of flagellin with rPAc and successfully obtained a soluble KFD-rPAc recombinant protein.…”