基于枯草芽孢为载体的口服CTB-Arah融合蛋白疫苗SIT作用机理的研究

Peanut allergy is one of the most dangerous food allergy, and there is no FDA approved treatment. The microbial derived adjuvant non-toxic cholera toxin B subunit (CTB) used in oral vaccines to enhance the protective immune response against infectious agents also induces antigen tolerance via induction of Tregs and IgA. Probiotic Bacillus subtilis (BS) spores, used as food supplements, are efficient oral mucosal vaccine carriers because of their safety, stability in gastrointestinal (GI) environment, and ease of genetic cloning. The major proteins Ara h1, Ara h2, Ara h3 and Arah h6 are the allergens in peanuts. We previously found that intragastrically (i.g.) delivered BS spores expressing CTB/Ara h2 fusion protein (BCA2 vaccine) significantly protected against peanut anaphylaxis in murine models, associated with increased IgA and Tregs, and reduced IgE and IL-4 levels. Tregs, vital to induction of tolerance to food proteins, also play a key role in mucosal IgA production, and maintenance of gut macrobiota diversity, involved in tolerance and immune homeostasis. This interaction has been referred to as “Treg-IgA-microbiota axis”. However, how this CTB-Arah oral vaccine works and the mechanisms on the "Treg-IgA-microbiota axis" to restore tolerance to peanut have not yet been determined. This study will investigate ①the potency and persistence of CTB/Arah(1/2/3/6) vaccine (BCA-M) induced protection against peanut anaphylaxis in murine models; ②the mechanisms on the "Treg-IgA-microbiota axis" to restore tolerance to peanut. This study will provide is a proof of concept efficacy data on this novel peanut vaccine.

花生是引起食物过敏最重要的过敏原（Ara h），但目前食物过敏尚无有效的治疗手段。微生物及其产物作为疫苗佐剂即可增强免疫反应，也可调节免疫应答；霍乱弧菌亚单位B（CTB）无毒副作用，作为疫苗佐剂成分可诱导免疫耐受；而用作食物添加剂益生菌的枯草杆菌芽孢，已成为安全高效的口服粘膜疫苗载体。我们前期结果发现使用表达CTB/Ara h2融合蛋白的枯草芽孢灌胃可对花生致敏小鼠产生显著耐受作用，但其机制尚不十分清楚。本项目将在建立花生过敏动物模型基础上，开展下列研究：①研究表达CTB/Ara h（1/2/3/6）融合蛋白枯草芽孢疫苗（BCA-M）诱导致敏小鼠免疫耐受的能力；检测免疫耐受的相关指标和生物标志物；②开展BCA-M疫苗诱导免疫耐受分子机制的研究。本课题将阐明BCA-M疫苗特异性免疫治疗（SIT）作用及其诱导免疫耐受的分子机制，为研制出针对花生过敏更好疗效的BCA-M疫苗提供新方法和理论依据。

花生是引起食物过敏最重要的过敏原（Ara h），但目前食物过敏尚无有效的治疗手段。本课题围绕研制安全、有效的花生过敏原新型疫苗并揭示其诱导免疫耐受的作用机制这一重大科学问题，首先，成功构建口服Ara h1/2/6融合蛋白疫苗（B.s-h126），并发现B.s-h126可抑制花生sIgE抗体和Th2 型细胞因子的分泌，且可能通过Treg-IgA轴达到SIT的抗原免疫耐受长期有效；进一步成功构建口服B.s-CTB-Ara h1/2/6疫苗（BCA-2），并发现BCA2能显著降低肠道食物过敏炎症、升高体温和Treg的细胞数量，且能刺激花生过敏患者的CD19+B细胞能显著增加Treg的数量，表明BCA2疫苗可能是通过tol B细胞使CD4+CD25-T细胞转变为CD4+CD25+Treg细胞，从而达到免疫耐受而降低花生过敏炎症。同时本项目还发现中药Formula-3能有效治疗食物过敏小鼠，可以通过增加拟杆菌和减少Firmicutes细菌性营养不良来改善食物过敏。此外，项目还参与了其它相关过敏性疾病的治疗研究，发现视网膜母细胞瘤细胞衍生的Twist蛋白促进Treg细胞发育而抑制过敏反应的发生；发现整合素αvβ6与雷西莫德配合使用可增加气道过敏的抗原特异性免疫耐受。总之，项目的研究结果对食物过敏等过敏性疾病的新型治疗疫苗或药物提供新方法和理论依据。