Christian Doppler Laboratory for Immunomodulation, Department of Pathophysiology and Allergy Research, Medical University of Vienna, Vienna, Austria.
Department of Molecular Biology, University of Salzburg, Salzburg, Austria.
J Allergy Clin Immunol. 2018 Jan;141(1):293-299.e6. doi: 10.1016/j.jaci.2017.02.044. Epub 2017 Apr 26.
Recombinant fusion proteins of flagellin and antigens have been demonstrated to induce strong innate and adaptive immune responses. Such fusion proteins can enhance the efficacy of allergen-specific immunotherapy.
We sought to characterize different fusion proteins of flagellin and the major birch pollen allergen Bet v 1 for suitability as allergy vaccines.
A truncated version of flagellin (NtCFlg) was genetically fused to the N- or C-terminus of Bet v 1. Toll-like receptor (TLR) 5 binding was assessed with HEK293 cells expressing TLR5. Upregulation of CD40, CD80, CD83, and CD86 on monocyte-derived dendritic cells from allergic patients was analyzed by using flow cytometry. The T cell-stimulatory capacity of the fusion proteins was assessed with naive and Bet v 1-specific T cells. IgE binding was tested in inhibition ELISAs and basophil activation tests. Mice were immunized with the fusion proteins in the absence and presence of aluminum hydroxide. Cellular and antibody responses were monitored. Murine antibodies were tested for blocking capacity in basophil activation tests.
Both fusion proteins matured monocyte-derived dendritic cells through TLR5. Compared with Bet v 1, the fusion proteins showed stronger T cell-stimulatory and reduced IgE-binding capacity and induced murine Bet v 1-specific antibodies in the absence of aluminum hydroxide. However, only antibodies induced by means of immunization with NtCFlg fused to the C-terminus of Bet v 1 inhibited binding of patients' IgE antibodies to Bet v 1.
Bet v 1-flagellin fusion proteins show enhanced immunogenicity, reduced allergenicity, and intrinsic adjuvanticity and thus represent promising vaccines for birch pollen allergen-specific immunotherapy. However, the sequential order of allergen and adjuvant within a fusion protein determines its immunologic characteristics.
已证实鞭毛蛋白与抗原的重组融合蛋白可诱导强烈的先天和适应性免疫反应。此类融合蛋白可增强过敏原特异性免疫疗法的疗效。
我们旨在研究鞭毛蛋白与主要桦树花粉过敏原 Bet v 1 的不同融合蛋白，以确定其作为变应原疫苗的适用性。
采用基因融合技术，将截短型鞭毛蛋白（NtCFlg）融合到 Bet v 1 的 N 端或 C 端。通过转染 TLR5 的 HEK293 细胞评估 TLR5 结合情况。采用流式细胞术分析融合蛋白对变应性患者来源树突状细胞 CD40、CD80、CD83 和 CD86 的上调作用。采用幼稚 T 细胞和 Bet v 1 特异性 T 细胞评估融合蛋白的 T 细胞刺激能力。通过抑制 ELISA 和嗜碱性粒细胞激活试验检测 IgE 结合情况。在无和有氢氧化铝存在的情况下，用融合蛋白对小鼠进行免疫。监测细胞和抗体应答。在嗜碱性粒细胞激活试验中检测鼠源抗体的阻断能力。
两种融合蛋白均通过 TLR5 使单核细胞来源的树突状细胞成熟。与 Bet v 1 相比，融合蛋白具有更强的 T 细胞刺激作用和更低的 IgE 结合能力，且在无氢氧化铝的情况下诱导小鼠产生 Bet v 1 特异性抗体。然而，只有通过用融合蛋白 NtCFlg 免疫 C 端融合到 Bet v 1 而诱导的抗体可抑制患者 IgE 抗体与 Bet v 1 的结合。
Bet v 1-鞭毛蛋白融合蛋白具有增强的免疫原性、降低的变应原性和内在佐剂活性，因此是桦树花粉变应原特异性免疫疗法有前途的疫苗。然而，融合蛋白中过敏原和佐剂的顺序决定了其免疫学特征。
