Curin M, Weber M, Thalhamer T, Swoboda I, Focke-Tejkl M, Blatt K, Valent P, Marth K, Garmatiuk T, Grönlund H, Thalhamer J, Spitzauer S, Valenta R
Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria; Christian Doppler Laboratory for Allergy Research, Division of Immunopathology, Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.
Clin Exp Allergy. 2014 Jun;44(6):882-94. doi: 10.1111/cea.12294.
The major cat allergen Fel d 1 represents one of the most important respiratory allergens. Aim of this study was to engineer recombinant Fel d 1 derivatives with reduced IgE reactivity and preserved T cell epitopes for vaccination and tolerance induction.
Seven recombinant mosaic proteins were generated by reassembly of non-IgE-reactive peptides of Fel d 1 which contained the sequence elements for induction of allergen-specific blocking IgG antibodies and T cell epitopes. Mosaic proteins were expressed in Escherichia coli using codon-optimized synthetic genes and compared with Fel d 1 regarding structural fold by circular dichroism, IgE-binding capacity, activation of allergic patients' basophils and ability to induce allergen-specific blocking IgG antibodies upon immunization.
Although each of the mosaic proteins had lost the alpha-helical fold typical for Fel d 1, a strong reduction in IgE reactivity as well as allergenic activity in basophil activation assays was only obtained for three constructs, two reassembled fragments (Fel d 1 MB, Fel d 1 MC) and a fusion of the latter two (Fel d 1 MF) in which the cysteines of Fel d 1 MC were replaced by serines. Immunization of rabbits with Fel d 1 MB, MC and MF induced high levels of IgG antibodies that inhibited IgE reactivity of cat-allergic patients to Fel d 1 in a comparable manner as IgG induced with the wild-type allergen.
We report the development of hypoallergenic reassembled Fel d 1 proteins suitable for vaccination and tolerance induction in cat-allergic patients.
主要猫过敏原Fel d 1是最重要的呼吸道过敏原之一。本研究的目的是构建重组Fel d 1衍生物，其IgE反应性降低且保留T细胞表位，用于疫苗接种和诱导耐受性。
通过重新组装Fel d 1的非IgE反应性肽段生成7种重组嵌合蛋白，这些肽段包含诱导过敏原特异性阻断性IgG抗体和T细胞表位的序列元件。使用密码子优化的合成基因在大肠杆菌中表达嵌合蛋白，并通过圆二色性比较其与Fel d 1的结构折叠、IgE结合能力、对过敏患者嗜碱性粒细胞的激活以及免疫后诱导过敏原特异性阻断性IgG抗体的能力。
尽管每种嵌合蛋白都失去了Fel d 1典型的α螺旋结构，但只有三种构建体，即两个重新组装的片段（Fel d 1 MB、Fel d 1 MC）以及后两者的融合体（Fel d 1 MF，其中Fel d 1 MC的半胱氨酸被丝氨酸取代）在嗜碱性粒细胞激活试验中IgE反应性和致敏活性显著降低。用Fel d 1 MB、MC和MF免疫兔子诱导产生了高水平的IgG抗体，这些抗体抑制猫过敏患者对Fel d 1的IgE反应性，其效果与野生型过敏原诱导产生的IgG相当。
我们报道了适用于猫过敏患者疫苗接种和诱导耐受性的低变应原性重组Fel d 1蛋白的研发。
