Division of Allergology, Paul-Ehrlich-Institut, Langen, Germany.
Mol Immunol. 2010 Nov-Dec;48(1-3):341-50. doi: 10.1016/j.molimm.2010.07.006. Epub 2010 Oct 20.
Toll-like receptor ligands are immune-modulatory components linking innate and adaptive immune responses and are considered to be promising vaccine components. Objective of this study was to investigate the adjuvant activity of Listeria monocytogenesis-derived TLR5-ligand flagellin A (flaA) genetically fused to ovalbumin (Ova, major chicken white egg allergen) in a murine in vitro system. Recombinant flaA, rOva, and a fusion protein of rflaA and rOva (rflaA:Ova) were over-expressed in Escherchia coli and purified by FPLC. LPS depletion was confirmed by LAL test. TLR5-binding was evaluated by human and murine TLR5-transgenic HEK 293 cells. The immune-modulatory effect of rflaA:Ova and rflaA:Ova modified by reduction and alkylation on purified BALB/c bone marrow-derived myeloid (mDC) and plasmacytoid dendritic cells (pDC) was investigated by flow cytometry and intracellular cytokine staining (ICS). Dose-dependent IL-8 secretion from transgenic HEK 293 cells confirmed binding of rflaA and rflaA:Ova molecules to human and murine TLR5. Recombinant flaA showed similar biological reactivity to TLR5-ligand fliC derived from Salmonella typhimurium applied as positive control. Compared to rflaA, both rflaA:Ova preparations induced higher expression of maturation markers (CD40, CD69, CD80, and CD86) on mDC, whereas only CD69 and CD40 were upregulated on pDC. Moreover, IL-6 and IL-10 production by mDC was enhanced upon stimulation with rflaA:Ova constructs in comparison to an equimolar mixture of both proteins whereas pDC did not show secretion of the investigated cytokines. Any immunological effects of LPS can be excluded by depletion of endotoxins and the lack of IL-10 production upon proteinase K digestion of rflaA:Ova. In summary, the rflaA:Ova fusion proteins showed an enhanced immune modulating capacity in comparison to rflaA or the mixture of rflaA and antigen. Since the rflaA:Ova fusion proteins induce strong IL-10 induction they are considered as potential vaccine candidates to improve allergen-specific immunotherapy.
Toll 样受体配体是连接先天免疫和适应性免疫反应的免疫调节成分，被认为是有前途的疫苗成分。本研究的目的是在体外研究李斯特菌衍生的 TLR5 配体鞭毛蛋白 A (flaA)与卵清蛋白 (Ova，主要鸡白蛋过敏原) 基因融合物在小鼠中的佐剂活性。重组 flaA、rOva 和 rflaA 和 rOva 的融合蛋白 (rflaA:Ova) 通过 FPLC 进行过表达和纯化。通过 LAL 试验证实 LPS 耗尽。通过人源和鼠源 TLR5 转基因 HEK 293 细胞评估 TLR5 结合。通过流式细胞术和细胞内细胞因子染色 (ICS) 研究 rflaA:Ova 和 rflaA:Ova 经还原和烷基化修饰对纯化的 BALB/c 骨髓来源髓样 (mDC) 和浆细胞样树突状细胞 (pDC) 的免疫调节作用。从转基因 HEK 293 细胞中 IL-8 的剂量依赖性分泌证实了 rflaA 和 rflaA:Ova 分子与人源和鼠源 TLR5 的结合。重组 flaA 显示出与作为阳性对照的鼠伤寒沙门氏菌衍生的 TLR5 配体 fliC 相似的生物学反应性。与 rflaA 相比，rflaA:Ova 制剂均可诱导 mDC 上更高水平的成熟标志物 (CD40、CD69、CD80 和 CD86) 的表达，而仅 CD69 和 CD40 在 pDC 上上调。此外，与两种蛋白质的等摩尔混合物相比，rflaA:Ova 构建物刺激 mDC 时可增强 IL-6 和 IL-10 的产生，而 pDC 未显示所研究细胞因子的分泌。通过 LPS 的内毒素耗竭和 rflaA:Ova 蛋白消化时缺乏 IL-10 的产生，可以排除任何 LPS 的免疫作用。总之，与 rflaA 或 rflaA 和抗原混合物相比，rflaA:Ova 融合蛋白显示出增强的免疫调节能力。由于 rflaA:Ova 融合蛋白可诱导强烈的 IL-10 诱导，因此它们被认为是改善过敏原特异性免疫疗法的潜在疫苗候选物。
