Lukschal Anna, Wallmann Julia, Bublin Merima, Hofstetter Gerlinde, Mothes-Luksch Nadine, Breiteneder Heimo, Pali-Schöll Isabella, Jensen-Jarolim Erika
Department of Comparative Medicine, Messerli Research Institute of the University of Veterinary Medicine Vienna, the Medical University of Vienna and the University of Vienna, Vienna, Austria.
Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.
Allergy Asthma Immunol Res. 2016 Mar;8(2):124-31. doi: 10.4168/aair.2016.8.2.124. Epub 2015 Oct 16.
In the celery-mugwort-birch-spice syndrome, a significant proportion of IgE is directed against high molecular weight (HMW) glycoproteins, including the celery allergen Api g 5. BIP3, a monoclonal antibody originally raised against birch pollen, recognizes HMW allergens in birch and mugwort pollens, celery, and Apiaceae spices. Our aim was to generate mimotopes using BIP3 for immunization against the HMW allergens relevant in the celery-mugwort-birch-spice cross reactivity syndrome.
Mimotopes were selected from a random-peptide display library by BIP3 and applied in IgE inhibition assays. The 3 phage clones with the highest inhibitory capacity were chosen for immunization of BALB/c mice. Mouse immune sera were tested for IgG binding to blotted birch pollen extract and used for inhibiting patients' IgE binding. Furthermore, sera were tested for binding to Api g 5, to horseradish peroxidase (HRP) as a second glycoprotein, or to non-glycosylated control allergen Phl p 5 in ELISA, and the specific Api g 5-specific IgG titers were determined.
Three rounds of biopanning resulted in phage clones exhibiting 7 different sequences including 1 dominant, 1-6-cyclo-CHKLRCDKAIA. Three phage clones had the capacity to inhibit human IgE binding and induced IgG to the HMW antigen when used for immunizing BALB/c mice. The induced BIP3-mimotope IgG reached titers of 1:500 specifically to Api g 5, but hardly reacted to glycoprotein HRP, revealing a minor role of carbohydrates in their epitope.
The mimotopes characterized in this study mimic the epitope of BIP3 relevant for Api g 5, one of the cross-reactive HMW allergens relevant in the celery-mugwort-birch-spice syndrome. BIP3 mimotopes may be used in the future for hyposensitization in this clinical syndrome by virtue of good and specific immunogenicity.
在芹菜 - 艾蒿 - 桦树 - 香料综合征中，相当一部分IgE针对高分子量（HMW）糖蛋白，包括芹菜过敏原Api g 5。BIP3是一种最初针对桦树花粉产生的单克隆抗体，可识别桦树和艾蒿花粉、芹菜及伞形科香料中的HMW过敏原。我们的目的是利用BIP3产生模拟表位，用于针对芹菜 - 艾蒿 - 桦树 - 香料交叉反应综合征中相关的HMW过敏原进行免疫。
通过BIP3从随机肽展示文库中筛选模拟表位，并应用于IgE抑制试验。选择抑制能力最强的3个噬菌体克隆用于免疫BALB/c小鼠。检测小鼠免疫血清与印迹桦树花粉提取物的IgG结合情况，并用于抑制患者的IgE结合。此外，在酶联免疫吸附测定（ELISA）中检测血清与Api g 5、作为第二种糖蛋白的辣根过氧化物酶（HRP）或非糖基化对照过敏原Phl p 5的结合情况，并测定特异性Api g 5特异性IgG滴度。
三轮生物淘选产生了具有7种不同序列的噬菌体克隆，其中包括1个优势序列1-6-cyclo-CHKLRCDKAIA。3个噬菌体克隆具有抑制人IgE结合的能力，用于免疫BALB/c小鼠时可诱导产生针对HMW抗原的IgG。诱导产生的BIP3模拟表位IgG对Api g 5的特异性滴度达到1:500，但与糖蛋白HRP几乎无反应，表明碳水化合物在其表位中作用较小。
本研究中鉴定的模拟表位模拟了与Api g 5相关的BIP3表位，Api g 5是芹菜 - 艾蒿 - 桦树 - 香料综合征中相关的交叉反应性HMW过敏原之一。由于具有良好的特异性免疫原性，BIP3模拟表位未来可能用于该临床综合征的脱敏治疗。
