Hazebrouck S, Ah-Leung S, Bidat E, Paty E, Drumare M-F, Tilleul S, Adel-Patient K, Wal J-M, Bernard H
INRA, UR 496, Unité d'Immuno-Allergie Alimentaire, CEA/iBiTeC-S/SPI, Gif-sur-Yvette, France.
Clin Exp Allergy. 2014 Apr;44(4):602-10. doi: 10.1111/cea.12261.
Goat's milk (GM) allergy associated with tolerance to cow's milk (CM) has been reported in patients without history of CM allergy and in CM-allergic children successfully treated with oral immunotherapy. The IgE antibodies from GM-allergic/CM-tolerant patients recognize caprine β-casein (βcap) without cross-reacting with bovine β-casein (βbov) despite a sequence identity of 91%. In this study, we investigated the non-cross-reactive IgE-binding epitopes of βcap.
Recombinant βcap was genetically modified by substituting caprine domains with the bovine counterparts and by performing site-directed mutagenesis. We then evaluated the recognition of modified βcap by IgE antibodies from 11 GM-allergic/CM-tolerant patients and 11 CM-allergic patients or by monoclonal antibodies (mAb) raised against caprine caseins. The allergenic potency of modified βcap was finally assessed by degranulation tests of humanized rat basophil leukaemia (RBL)-SX38 cells.
Non-cross-reactive epitopes of βcap were found in domains 44-88 and 130-178. The substitutions A55T/T63P/L75P and P148H/S152P induced the greatest decrease in IgE reactivity of GM-allergic/CM-tolerant patients towards βcap. The pivotal role of threonine 63 was particularly revealed as its substitution also impaired the recognition of βcap by specific mAb, which could discriminate between βcap and βbov. The modified βcap containing the five substitutions was then unable to trigger the degranulation of RBL-SX38 cells passively sensitized with IgE antibodies from GM-allergic/CM-tolerant patients.
Although IgE-binding epitopes are spread all over βcap, a non-cross-linking version of βcap was generated with only five amino acid substitutions and could thus provide new insight for the design of hypoallergenic variants.
在无牛奶过敏史的患者以及经口服免疫疗法成功治疗的牛奶过敏儿童中，均有报道称存在与牛奶耐受相关的羊奶过敏情况。尽管山羊β-酪蛋白（βcap）与牛β-酪蛋白（βbov）的序列同一性达91%，但来自羊奶过敏/牛奶耐受患者的IgE抗体可识别山羊β-酪蛋白，而不与牛β-酪蛋白发生交叉反应。在本研究中，我们调查了βcap的非交叉反应性IgE结合表位。
通过用牛对应结构域替换山羊结构域以及进行定点诱变对重组βcap进行基因改造。然后，我们评估了11例羊奶过敏/牛奶耐受患者和11例牛奶过敏患者的IgE抗体或针对山羊酪蛋白产生的单克隆抗体（mAb）对改造后的βcap的识别情况。最后通过人源化大鼠嗜碱性粒细胞白血病（RBL）-SX38细胞的脱颗粒试验评估改造后βcap的变应原效力。
在44-88和130-178结构域中发现了βcap的非交叉反应性表位。A55T/T63P/L75P和P148H/S152P替换导致羊奶过敏/牛奶耐受患者对βcap的IgE反应性最大程度降低。苏氨酸63的关键作用尤为明显，因为其替换也损害了特异性mAb对βcap的识别，而该mAb可区分βcap和βbov。然后，含有这五个替换的改造后βcap无法触发用来自羊奶过敏/牛奶耐受患者的IgE抗体被动致敏的RBL-SX38细胞的脱颗粒。
尽管IgE结合表位遍布βcap，但仅通过五个氨基酸替换就产生了一种非交叉连接形式的βcap，从而可为低变应原性变体的设计提供新的思路。
