High quality reagents for innate immunity research, vaccine development and drug discovery

		Bespoke flagellin fusion protein synthesis We offer cloning, expression and purification of bespoke flagellin fusion proteins for vaccine research and development. Your antigen of interest may be fused either to the C-terminus, or within a central domain replacing the D3 loop. The protein is expressed in mammalian systems for maximum purity and structural authenticity.
		Potential applications of our flagellin fusion proteins include use in the development of vaccines targeting:
		Bacterial and viral surface protein antigens Bacterial and viral protein T-cell epitopes Tumour cell-derived mutant cell-surface antigens (for elicitation of antibodies) Tumour cell-derived mutant T-cell epitopes (for elicitation of cellular immunity) Allergens, such as pollen and dust-mite protein antigens Other antigens where there is an advantage in being able to deliver intranasally or to other mucosal surfaces Other antigens where there is an advantage in being able to administer without a separate adjuvant or carrier
		Your antigen fused to flagellin We will be happy to attempt synthesis of a fusion protein comprising (generally) up to 250 amino acids of a sequence of your choice. This may comprise one antigen of interest for elicitation of humoral responses, or a peptide comprising multiple T-cell epitopes of your choosing. Please email us with your requirements. Please click here to download a flyer with further information regarding our flagellin fusion proteins. Background Flagellin is the principal structural protein of bacterial flagella, the helical appendages that enable bacterial motility [1]. Almost uniquely among proteins, flagellin contains regions that are sufficiently conserved across bacterial species to be recognised by two distinct pattern recognition receptors (PRRs) of the mammalian innate immune system. Flagellin may bind Toll-like receptor 5 (TLR5) on the surface of immune cells, triggering signalling pathways that result in the production of pro-inflammatory cytokines and other immune responses [2]. Alternatively, flagellin that enters the cytosol may be recognised by the NAIP / NLRC4 inflammasome, resulting in the processing of pro-IL1β to the active form of IL1β [3]. As these responses make flagellin a potent activator of dendritic cells and adaptive immune responses more generally, it has received much interest as a vaccine adjuvant and carrier in both pre-clinical models and clinical trials [4]. Caithness Biotech offers recombinant flagellin proteins, covalently fused to bespoke antigens of interest, for studies of innate immune signalling, host-pathogen interactions, immunoassays, and as an adjuvant and carrier for vaccine development. Uniquely, our flagellin proteins are expressed in mammalian cells to maximise authenticity of the partner antigen structure and minimise the presence of contaminating bacterial stimulants of other TLRs. Advantages of combined adjuvant and carrier Subunit vaccines benefit from improved potential for safety and ease of manufacture in comparison to live-attenuated vaccines, but often require two additional components for vaccine efficacy - an adjuvant and a carrier. The adjuvant serves to stimulate innate immune receptors to trigger DC activation, maturation and antigen presentation. The carrier protein contains peptide sequences which are readily presented on MHC molecules of DCs to generate the T-cell help necessary to support effective immune responses to antigens, such as polysaccharides, which may not otherwise efficiently induce T-cell help. Flagellin proteins are capable of serving as both adjuvant and carrier for protein antigens covalently attached to them as fusion partners [4]. This linkage ensures that antigen, adjuvant and carrier all enter the same endosomal compartment at the same time, a process that has been shown to generate far more robust immune responses than antigens simply admixed with carrier or adjuvant [5]. A proven adjuvant with low toxicity Systemic exposure to some TLR-stimulants, such as the TLR4 agonist lipopolysaccharide (LPS), can result in the damaging systemic inflammatory response syndrome (SIRS), caused by the excessive release of pro-inflammatory cytokines from myeloid cells. However, the systemic response to flagellin, even at high doses, does not seem to induce such a response. It is thought this may be because of the profile of TLR5 expression (mainly on epithelial cells and DCs) and lower induction of the key inflammatory cytokines IL1β and TNFα [6]. Flagellin proteins, either in the native form or as recombinant fusion proteins, have also been administered to human volunteers in dozens of small scale trials since the 1960s, with either minor or no side effects reported [7,8]. Thus, the flagellin-fusion platform has a long track record of use with low toxicity. Capacity to induce diverse immune responses The administration of flagellin fusion proteins to mice has been shown to induce a mixed antibody response including induction of IgM, IgG1, IgG2a/IgG2c and IgA antibodies specific for the partner antigens [9]. Flagellin activates DCs in vivo and contains its own CD4+ T-cell epitopes [10]. It furthermore promotes CD4+ T-cell responses to peptides of its fusion partners [11], stimulating both TH1 and TH2 based responses to the partner antigen [10][11]. Flagellin also contains CD8+ T cell epitopes, and promotes CD8+ T-cell responses to partner antigens when these are covalently attached to the flagellin molecule [5,11,12]. Flagellin fusion proteins have been shown to induce protective immunity in diverse pre-clinical models of viral or bacterial challenge [9]. Diverse potential routes of administration Flagellin, and its fusion proteins, have been shown to be capable of inducing protective immunity when given subcutaneously, intraperitoneally, intramuscularly and intranasally [13]. The latter option raises the possibility of needle-free vaccination with the additional advantage of inducing mucosal immunity, including an IgA response, which is limited in response to most other subunit vaccines. Advantages of our flagellins Caithness Biotech recombinant flagellins are based on the FljB protein of Salmonella enterica subsp. enterica serovar Typhimurium. We offer the full-length native protein, the protein lacking the D3 hypervariable domain, and fusion proteins placing your antigen of interest at either the C-terminus, or in place of the D3 domain. We test every batch using a HEK-293 cell TLR5 reporter assay to ensure potent activation of TLR5 by each product. Uniquely, our proteins are expressed in mammalian cells to maximise purity and minimise presence of contaminating bacterial stimulants of other TLRs, such as TLR2 and TLR4. References [1] Samatey FA, et al. Structure of the bacterial flagellar protofilament and implications for a switch for supercoiling. Nature 410:331-7 (2001) [2] Smith KD, et al. Toll-like receptor 5 recognizes a conserved site on flagellin required for protofilament formation and bacterial motility. Nat Immunol 4:1247-53 (2003) [3] Zhao Y, et al. The NLRC4 inflammasome receptors for bacterial flagellin and type III secretion apparatus. Nature 477:596-600 (2011) [4] Huleatt JW, et al. Vaccination with recombinant fusion proteins incorporating Toll-like receptor ligands induces rapid cellular and humoral immunity. Vaccine 25:763-75 (2007) [5] Bates JT, et al. Enhanced antigen processing of flagellin fusion proteins promotes the antigen specific CD8+ T cell response independently of TLR5 and MyD88. J Immunol 186:6255-6262 (2011) [6] Burdelya LG, et al. An agonist of toll-like receptor 5 has radioprotective activity in mouse and primate models. Science 320:226-30 (2008) [7] Rowley MJ, Mackay IR. Measurement of antibody-producing capacity in man. I. The normal response to flagellin from Salmonella adelaide. Clin Exp Immunol 5:407-418 (1969) [8] Taylor DN, et al. Induction of a potent immune response in the elderly using the TLR-5 agonist, flagellin, with a recombinant hemagglutinin influenza-flagellin fusion vaccine (VAX125, STF2.HA1 SI). Vaccine 29:4897-902 (2011) [9] [5222] Hajam IA, et al. Bacterial flagellin - a potent immunomodulatory agent. Exp Mol Med 49:e373 (2017) [10] McSorley SJ, et al. Bacterial flagellin is an effective adjuvant for CD4+ T cells in vivo. J Immunol 169:3914-3919 (2002) [11] Cuadros C, et al. Flagellin fusion proteins as adjuvants or vaccines induce specific immune responses. Infect Immun 72:2810-2816 (2004) [12] Huleatt JW, et al. Vaccination with recombinant fusion proteins incorporating Toll-like receptor ligands induces rapid cellular and humoral immunity. Vaccine 25:763-75 (2007) [13] Song L, et al. Efficacious recombinant influenza vaccines produced by high yield bacterial expression: A solution to global pandemic and seasonal needs. PLoS One. 3:e2257 (2008)

 

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Caithness Biotechnologies Ltd., 72 Boston Road, Leicester, UK, LE4 1HB. Telephone: +44 (0) 116 326 3802 | email: contact@caithnessbiotechnologies.com