Induction of adaptive immunity by flagellin does not require robust activation of innate immunity

Abstract

The ability of TLR agonists to promote adaptive immune responses is attributed to their ability to robustly activate innate immunity. However, it has been observed that, for adjuvants in actual use in research and vaccination, TLR signaling is dispensable for generating humoral immunity. Here, we examined the role of TLR5 and MyD88 in promoting innate and humoral immunity to flagellin using a prime/boost immunization regimen. We observed that eliminating TLR5 greatly reduced flagellin-induced cytokine production, except for IL-18, and ablated DC maturation but did not significantly impact flagellin's ability to promote humoral immunity. Elimination of MyD88, which will ablate signaling through TLR and IL-1b/IL-18 generated by Nod-like receptors, reduced, but did not eliminate flagellin's promotion of humoral immunity. In contrast, loss of the innate immune receptor for profilinlike protein (PLP), TLR11, greatly reduced the ability of PLP to elicit humoral immunity. Together, these results indicate that, firstly, the degree of innate immune activation induced by TLR agonists may be in great excess of that needed to promote humoral immunity and, secondly, there is considerable redundancy in mechanisms that promote the humoral immune response upon innate immune recognition of flagellin. Thus, it should be possible to design innate immune activators that are highly effective vaccine adjuvants yet avoid the adverse events associated with systemic TLR activation.Key words: Adjuvant . Cytokines . DC . Profilin-like protein . TLR Introduction TLR-mediated recognition of structural components of microbial pathogens plays a key role in the initiation of host defense. Specifically, these germ-line-encoded PRR recognize and initiate immune responses to a wide variety of microbial patterns, including those of bacteria, viruses, parasites, nucleic acids, carbohydrates, and lipids [1][2][3][4]. TLR agonists are classified into three broad categories such as nucleic acids (TLR 3,7,8,9), lipids/lipopeptides (TLR 4, 1/2, 2/6), and protein (TLR 5,11). Of particular importance to this study, TLR5 recognizes the bacterial protein flagellin [5], in its soluble/monomeric form, whereas TLR11 recognizes profilin-like protein (PLP), made by Toxoplasma gondii [6]. Activation of most TLR, including TLR5 and Eur. J. Immunol. 2009. 39: 359-371 DOI 10.1002 HIGHLIGHTS 359 Frontline TLR11, by their cognate ligands results in rapid nuclear translocation of the transcription factor NF-kB and, consequently, synthesis and secretion of a panel of pro-inflammatory cytokines. Another class of PRR thought to play an important role in innate immunity is the Nod-like receptors (NLR), which are expressed in the cytosol. Of particular relevance to this study, two NLR proteins, Ipaf and Naip5, have been reported to signal in response to flagellin that attains an intracellular location [7][8][9][10]. In contrast to TLR, the primary consequence of Ipaf signaling is not to induce transcription or protein synthesis but rather to activate caspase-1, which resul...

Salmonella enterica Serovar Enteritidis Core O Polysaccharide Conjugated to H:g,m Flagellin as a Candidate Vaccine for Protection against Invasive Infection withS.Enteritidis

“…However, since conjugate vaccines are extracellular antigens, activation of NLRC4/Ipaf may be negligible (41). Work in transgenic mice suggests that the immunogenicity and adjuvanticity of flagellin may be TLR independent (54). In these experiments, comparable antiflagellin antibody levels and a measurable adjuvant boost against coadministered ovalbumin were seen between TLR5-deficient and wild-type mice.…”

“…In these experiments, comparable antiflagellin antibody levels and a measurable adjuvant boost against coadministered ovalbumin were seen between TLR5-deficient and wild-type mice. In the absence of TLR5 signaling, high antiflagellin IgG levels may be due, in part, to partial activation of NLRC4/Ipaf (66) or highly efficient cross-linking of B-cell receptors due to the polymeric nature of the antigen or possibly through efficient antigen processing and presentation on major histocompatibility complex (MHC) molecules of flagellin epitopes (2,3,8,54). Furthermore, as anti-LPS antibody levels were similar between flagellin-and CRM 197 -based conjugates, in contrast to the powerful adjuvant boost that flagellin bestows on protein antigens, the comparative adjuvanticity toward polysaccharide antigens may be modest.…”

Immunogenicity of Recombinant Proteins Consisting of Plasmodium vivax Circumsporozoite Protein Allelic Variant-Derived Epitopes Fused with Salmonella enterica Serovar Typhimurium Flagellin

“…The innate immune agonist FliC activates different pattern recognition receptors (24)(25)(26). In our study, we investigated the activation of TLR-4, TLR-5, and MyD88 by immunizing wild-type (WT), as well as TLR-4, TLR-5, and MyD88 KO mice with His 6 -FliC-VK210.…”

Systemic Flagellin Immunization Stimulates Mucosal CD103+ Dendritic Cells and Drives Foxp3+ Regulatory T Cell and IgA Responses in the Mesenteric Lymph Node

“…Loss of TLR5 had a more profound effect on IgA than IgG responses. Sustained IgG titers after flagellin in TLR5 2/2 mice has been described, where NLRC4 can provide some compensation for its loss (32,42). The near absolute loss of IgA to sFliC in TLR5 2/2 mice suggests that TLR5 may have a nonredundant role in the generation of IgA in this response.…”

“…Flagellin is an important immunodominant and immunomodulatory protein with autoadjuvant properties, with a clear, although not exclusive (32), dependence on TLR5 for these activities (33). Previously, we showed that the immune response to flagellin within the same SLT can show significant plasticity (21).…”