Functional properties of flagellin as a stimulator of innate immunity

Abstract

We report the development of a well-defined flagellin-based nanoparticle stimulator and also provide a new mechanism of action model explaining how flagellin-triggered innate immunity has evolved to favor localized rather than potentially debilitating systemic immune stimulation. Cell-free protein synthesis (CFPS) was used to facilitate mutational analysis and precisely orientated display of flagellin on Hepatitis B core (HBc) protein virus-like particles (VLPs). The need for product stability and an understanding of mechanism of action motivated investigations indicating that the D0 domain of flagellin is sensitive to amino acid sequence independent hydrolysis – apparently due to the need for structural flexibility during natural flagellin polymerization. When D0-stabilized flagellin was attached to HBc VLPs with the D0 domain facing outward, flagellin’s tendency to polymerize caused the VLPs to precipitate. However, attaching the D0 domain to the VLP surface produced a stable nanoparticle adjuvant. Surprisingly, attaching only 2 flagellins per VLP provided the same 1 pM potency as did VLPs with about 33 attached flagellins suggesting that the TLR5 receptor is highly effective in delivering its intracellular signal. These observations suggest that flagellin’s protease sensitivity, tendency to aggregate, and very high affinity for TLR5 receptors limit its systemic distribution to favor localized immune stimulation.

Chicken Immune Response after In Ovo Immunization with Chimeric TLR5 Activating Flagellin of Campylobacter jejuni

“…In S . Enteritidis FliC this region is embedded within flagellar filament and it is not surface exposed [ 32 ]. The incorporation of the TLR5 binding region from S .…”

Graphene in the Design and Engineering of Next‐Generation Neural Interfaces

“…Similarly to the case of neural stimulation, the performance of graphene‐based materials for recording applications can greatly improve by using structured or 3D films that increase the specific surface area of the electrode. This has been recently shown by recording in vivo brain activity using porous graphene‐related materials with a good signal‐to‐noise ratio.…”

“…However, recent studies have shown that when used in porous thin‐films processed from flakes or introduced in polymer composites, graphene‐based materials can exhibit dramatically improved performance for neural stimulation. For instance, porous graphene oxide electrodes reduced by laser treatment and by the Langmuir–Blodgett method have been reported to be able to stimulate neural tissues with outstanding charge injection values between 1 and 3 mC cm −2 . Furthermore, new promising materials are expected to emerge from the very active research field of the supercapacitors, in which graphene‐based materials exhibiting very large active surface area are being developed achieving very high double layer capacitance.…”

Fusion proteins of flagellin and the major birch pollen allergen Bet v 1 show enhanced immunogenicity, reduced allergenicity, and intrinsic adjuvanticity

“…To enhance its immunogenicity, we fused Bet v 1 to flagellin from Salmonella typhimurium (fliC) because the immunostimulatory capacity of this flagellin has been proved in various studies. 22 We substituted its hypervariable region, which is thought to promote production of neutralizing antibodies 23 that downregulate TLR5 signaling, with a short-turn motif to reduce its size and to keep the close proximity of the N-and C-termini necessary for TLR5 binding. Deletion of the hypervariable region did not abrogate the adjuvant effect because the resulting truncated flagellin, NtCFlg, still activated TLR5 and was fused to either the N-or C-terminus of Bet v 1, which was termed NtCFlg-Bet v 1 and Bet v 1-NtCFlg, respectively, to assess possible position effects.…”