Schramm G, Kahlert H, Suck R, Weber B, Stüwe H T, Müller W D, Bufe A, Becker W M, Schlaak M W, Jäger L, Cromwell O, Fiebig H
Biochemische und Molekulare Allergologie, Forschungszentrum Borstel, Germany.
J Immunol. 1999 Feb 15;162(4):2406-14.
One problem of conventional allergen-specific immunotherapy is the risk of anaphylactic reactions. A new approach to make immunotherapy safer and more efficient might be the application of engineered allergens with reduced IgE-binding capacity but retained T cell reactivity. Using overlapping dodeca-peptides, the dominant T cell epitopes of the timothy grass pollen allergen Phl p 5b were identified. By site-directed mutagenesis outside these regions, point and deletion mutants were generated. Allergen variants were analyzed for IgE-binding capacity with sera of different grass pollen allergic patients by Western blotting, Dot blotting, and EAST inhibition test, and for histamine releasing capacity with peripheral blood basophils from different patients. The deletion mutants revealed significantly reduced IgE reactivity and histamine releasing capacity, compared with the wild-type Phl p 5b. Furthermore, in vivo skin prick tests showed that the deletion mutants had a significantly lower potency to induce cutaneous reactions than the wild-type Phl p 5b. On the other hand, T cell clones and T cell lines from different allergic patients showed comparable proliferation after stimulation with allergen variants and wild-type Phl p 5b. Considering their reduced anaphylactogenic potential together with their conserved T cell reactivity, the engineered allergens could be important tools for efficient and safe allergen-specific immunotherapy.
传统的变应原特异性免疫疗法存在一个问题，即有发生过敏反应的风险。一种使免疫疗法更安全、更有效的新方法可能是应用具有降低的IgE结合能力但保留T细胞反应性的工程化变应原。利用重叠十二肽，鉴定了梯牧草花粉变应原Phl p 5b的主要T细胞表位。通过在这些区域外进行定点诱变，产生了点突变体和缺失突变体。通过蛋白质印迹法、斑点印迹法和EAST抑制试验，用不同草花粉过敏患者的血清分析变应原变体的IgE结合能力，并用不同患者的外周血嗜碱性粒细胞分析组胺释放能力。与野生型Phl p 5b相比，缺失突变体的IgE反应性和组胺释放能力显著降低。此外，体内皮肤点刺试验表明，缺失突变体诱导皮肤反应的能力明显低于野生型Phl p 5b。另一方面，来自不同过敏患者的T细胞克隆和T细胞系在用变应原变体和野生型Phl p 5b刺激后显示出相当的增殖。考虑到它们降低的过敏反应潜力以及保守的T细胞反应性，工程化变应原可能是高效、安全的变应原特异性免疫疗法的重要工具。
