Structural engineering of flagellin as vaccine adjuvant: quest for the minimal domain of flagellin for TLR5 activation

Abstract

Flagellin stimulates Toll-like receptor 5 (TLR5), triggering both innate and adaptive immune responses, making it a potential vaccine adjuvant. On mucosal surfaces, flagellin induces a strong release of cytokines, chemokines, and immunoglobulins. When used in its free monomeric form, flagellin has been shown to enhance immune responses when combined with vaccine antigens. Further research demonstrated that genetically linking flagellin to the antigen provides a more consistent immune boost. However, the bulky structure of flagellin presents challenges in designing the antigen-adjuvant construct, leading to ongoing research to determine the minimal flagellin domain necessary for its adjuvant effect. Early findings suggest that only the D0 and D1 domains are required for immune enhancement. Functional analysis revealed that the TLR5-binding region is located in the D1 domain, while TLR5 dimerization and signaling require the presence of D0. Further reductions in the size of the D0 and D1 domains may be possible as deeper studies aim to identify the key residues responsible for TLR5 activation and immune enhancement. Additionally, flagellin is being tested as a hapten carrier alongside its established adjuvant role. Recently, significant advancements in flagellin application have been observed as it progresses through clinical studies as an adjuvant, anti-radiation, and anti-cancer agent.

Keywords: Adjuvant; Dimerization; Flagellin; Hapten; TLR5.

Fig. 1

Flagellin acts as a danger signal by activating PRRs on dendritic cells, enhancing antigen presentation and upregulating cytokines and co-stimulatory molecules. This process amplifies both innate and adaptive immunity, ensuring a robust and coordinated immune response. Created in Biorender

Fig. 2

Epithelial cells recognize flagellin through surface TLR5, which leads to the activation of antigen-presenting cells (APCs). These APCs process and present the antigen to T-cells, subsequently activating B-cells. The activated B-cells produce IgA antibodies, which enter the circulation and also migrate to the lumen, where they capture and opsonize pathogens. Created in Biorender

Fig. 3

Crystal structure of flagellin. The previously solved 3D structure of the full-length flagellin (Protein Data Bank ID: 1UCU) as illustrated by the PyMol server is shown containing domains D0, D1, D2 and D3

Fig. 4

Crystal structure of flagellin D1 and the LRR region of TLR5 in 2:2 complex. Previously solved 3D structure of the zebrafish TLR5 (as a variable lymphocyte receptor hybrid protein) in complex with Salmonella flagellin (Protein Data Bank ID: 3V47) is illustrated by the PyMol server