Fusion proteins of flagellin and the major birch pollen allergen Bet v 1 show enhanced immunogenicity, reduced allergenicity, and intrinsic adjuvanticity
Fusion proteins of flagellin and the major birch pollen allergen Bet v 1 show enhanced immunogenicity, reduced allergenicity, and intrinsic adjuvanticity
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DOI：
10.1016/j.jaci.2017.02.044
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发表时间：
2018-01-01
影响因子：
14.2
通讯作者：
Bohle, Barbara
中科院分区：
文献类型：
--
作者：
Kitzmueller, Claudia;Kalser, Julia;Bohle, Barbara
背景：鞭毛蛋白和抗原的重组融合蛋白已被证明能诱导强烈的先天性和获得性免疫反应。目的：研究鞭毛蛋白与主要的桦树花粉变应原Bet v1的不同融合蛋白作为过敏疫苗的可行性。方法：将截短的鞭毛蛋白(NtCF1g)与Bet v1的N端或C端进行基因融合，用表达TLR5的HEK293细胞检测Toll样受体(TLR)5的结合情况。用流式细胞术分析CD40、CD80、CD83和CD86对过敏患者单核细胞来源的树突状细胞的上调作用。用NAIVE和Bet v1特异性T细胞检测融合蛋白对T细胞的刺激作用。通过抑制ELISAs和嗜碱性粒细胞活化试验检测IgE结合情况。在无氢氧化铝和有氢氧化铝存在的情况下，用融合蛋白免疫小鼠。监测细胞和抗体反应。结果：两种融合蛋白均通过TLR5成熟单核细胞来源的树突状细胞。与Bet v1相比，融合蛋白具有更强的T细胞刺激活性和较低的IgE结合能力，并在无氢氧化铝条件下诱导小鼠产生Bet v1特异性抗体。结论：Bet v1鞭毛蛋白融合蛋白具有较强的免疫原性、较低的致敏原性和内在佐剂作用，有望成为白桦花粉变应原特异性免疫治疗的疫苗。然而，融合蛋白中变应原和佐剂的顺序决定了其免疫学特性。
Background: 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.Objective: We sought to characterize different fusion proteins of flagellin and the major birch pollen allergen Bet v 1 for suitability as allergy vaccines.Methods: A truncated version of flagellin (NtCF1g) 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.Results: 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 NtCF1g fused to the C-terminus of Bet v 1 inhibited binding of patients' IgE antibodies to Bet v 1.Conclusion: 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.