Division of Immunopathology, Department of Pathophysiology and Allergy Research, Center of Pathophysiology, Infectiology and Immunology, Vienna General Hospital (AKH), Medical University of Vienna, 1090 Vienna, Austria.
Mol Immunol. 2011 Jan;48(4):431-41. doi: 10.1016/j.molimm.2010.09.016. Epub 2010 Nov 18.
In order to reduce side effects in the course of allergen specific immunotherapy hypoallergenic allergen derivatives with reduced IgE reactivity have been made by genetic engineering. In contrast to other recombinant hypoallergenic allergen derivatives which showed reduced IgE reactivity, a recombinant trimer of the major birch pollen allergen Bet v 1 showed reduced allergenic activity despite preserved IgE reactivity. We studied rBet v 1 trimer by SDS-PAGE, mass spectrometry, circular dichroism and gel filtration. Furthermore we investigated IgE and IgG reactivity of the rBet v 1 trimer in solid and liquid phase assays and compared its allergenic activity with that of rBet v 1 wildtype using basophil activation assays. In solid phase immunoassays rBet v 1 trimer exhibited even stronger IgE reactivity than the rBet v 1 wildtype, whereas both proteins were equally well recognized by Bet v 1-specific IgG antibody probes. In fluid phase IgE experiments rBet v 1 trimer inhibited IgE reactivity to rBet v 1 wildtype but showed a more than 10-fold reduced allergenic activity compared to the rBet v 1 monomer. By analytical gel filtration it was demonstrated that, despite its monomeric appearance in SDS-PAGE the trimer occurred in fluid phase in the form of defined high molecular weight (>600 kDa) aggregates whereas rBet v 1 wildtype strictly appeared as monomeric protein. The results indicate that the hypoallergenic nature of the rBet v 1 trimer is due to formation of defined high molecular weight aggregates which may be responsible for an altered presentation of IgE epitopes in a form with reduced capacity to crosslink effector-cell bound IgE. We thus provide evidence for a novel mechanism for hypoallergenic activity.
为了减少变应原特异性免疫治疗过程中的副作用，通过基因工程制备了低 IgE 反应性的变应原衍生物。与其他显示低 IgE 反应性的重组低变应原变应原衍生物不同，尽管保留了 IgE 反应性，但主要桦树花粉过敏原 Bet v 1 的重组三聚体显示出降低的变应原活性。我们通过 SDS-PAGE、质谱、圆二色性和凝胶过滤研究了 rBet v 1 三聚体。此外，我们研究了 rBet v 1 三聚体在固相和液相测定中的 IgE 和 IgG 反应性，并使用嗜碱性粒细胞活化测定将其变应原活性与 rBet v 1 野生型进行了比较。在固相免疫测定中，rBet v 1 三聚体表现出比 rBet v 1 野生型更强的 IgE 反应性，而这两种蛋白质都被 Bet v 1 特异性 IgG 抗体探针同等识别。在液体相 IgE 实验中，rBet v 1 三聚体抑制了 rBet v 1 野生型的 IgE 反应性，但与 rBet v 1 单体相比，其变应原活性降低了 10 多倍。通过分析性凝胶过滤证明，尽管在 SDS-PAGE 中呈现单体形式，但三聚体在液体相中以定义的高分子量（>600 kDa）聚集体的形式存在，而 rBet v 1 野生型严格以单体蛋白的形式存在。结果表明，rBet v 1 三聚体的低变应原性质是由于形成了定义明确的高分子量聚集体，这可能导致 IgE 表位以交联效应细胞结合 IgE 能力降低的形式呈现。因此，我们为低变应原活性提供了一种新的机制。
