Supplementary Material for: Low-Allergenic Hydrolyzed Egg Induces Oral Tolerance in Mice

Background: Egg allergy is one of the most common food allergies in children. The standard therapy for egg allergy is strict avoidance. Yet, there is considerable clinical and scientific interest in primary or secondary prevention. A major drawback of oral tolerance (OT) induction protocols, however, is the possibility of severe side effects; thus, we have formulated a hypoallergenic egg product and demonstrate its in vivo capacity to modulate the immune system in the current study. Methods: Hydrolyzed egg (HE) was produced using a combination of moderate heat treatment and enzymatic hydrolysis. The capacity of HE to induce OT was tested in experimental models and compared to whole egg (WE). Delayed-type hypersensitivity (DTH) responses, immune markers and potential early markers of OT were analyzed. Results: Allergic responses, assessed by both DTH responses upon OVA challenge and serum OVA-specific IgE and IgG1, were decreased after treatment with HE and WE compared to the control group. Additionally, feeding WE and HE significantly decreased Th2 cytokine induction and cell proliferation, induced the activation of effector CD4+ T cells and increased numbers and percentages of ICOS+CD4+CD25+Foxp3+ cells. Furthermore, DO11.10 mouse experiments showed that HE contains other peptides than the OVA323-339 peptide that are able to induce tolerance to OVA. Conclusions: Altogether, results showed that HE induces OT in mice in a dose-dependent manner. Due to its low allergenicity compared to WE, it may represent a safer alternative for OT induction in at-risk subjects or oral immunotherapy in allergic patients.背景：鸡蛋过敏是儿童最常见的食物过敏之一。鸡蛋过敏的标准治疗方案为严格规避致敏原。然而，针对鸡蛋过敏的原发性与继发性预防，学界存在大量临床与科学研究兴趣。不过，口服耐受（oral tolerance, OT）诱导方案的主要弊端在于存在引发严重不良反应的风险；因此，本研究研发了一款低变应原性鸡蛋制品，并验证了其在体内调节免疫系统的能力。 方法：本研究采用温和热处理联合酶解工艺制备水解鸡蛋（hydrolyzed egg, HE）。在实验动物模型中检测了HE诱导口服耐受的能力，并与全蛋（whole egg, WE）进行对比。分析指标包括迟发型超敏反应（delayed-type hypersensitivity, DTH）应答、免疫标志物以及口服耐受的潜在早期标志物。 结果：相较于对照组，经HE与WE干预后，通过卵清蛋白（OVA）攻毒后的迟发型超敏反应应答、血清卵清蛋白（OVA）特异性IgE与IgG1水平评估的过敏反应均有所降低。此外，喂食WE与HE可显著降低Th2细胞因子的表达与细胞增殖水平，诱导效应性CD4+T细胞活化，并增加ICOS+CD4+CD25+Foxp3+细胞的数量与占比。进一步的DO11.10小鼠实验表明，HE中除OVA323-339肽段外，还含有其他可诱导卵清蛋白耐受的肽段。 结论：综合来看，实验结果证实HE可在小鼠体内以剂量依赖的方式诱导口服耐受。相较于WE，HE的变应原性更低，有望成为高危人群口服耐受诱导或过敏患者口服免疫治疗的更安全备选方案。