IgE Fc介导的靶向树突状细胞的DNA疫苗治疗过敏性疾病的研究

IgE mediated allergy has shown an increasing prevalence and severity in the past decade, however, safe and effective vaccines for prevention and treatment of allergic disease are still lacking. Allergen DNA vaccine have been shown to have efficient inhibitory effects on IgE response in animal experiment, but the immune responses of the DNA vaccine were relatively low, which has been a common problem for DNA vaccine. The current proposal is based on the fact that IgE has an extremely high affinity for IgE Fc receptor (FcεRI) which is expressed on the surface of dendritic cells (DC), and there are higher levels of FcεRI on the DC of allergic patient than that of normal individual. We designed DC targeting DNA vaccines via Fcε in order to present the allergen specifically and efficiently to induce specific IgE immune tolerance. We plan to couple the human Fcε-poly lysine fusion protein (EPL), which has been successfully prepared in our preliminary work, with the plasmid that contain DC-specific promoter and peanut allergen gene. In vitro experiments will be carried out to analyze the EPL-DNA uptake and presentation by DC cell lines and human primary DC. The human FcεRI transgenic mice will be applied to analyze the prevention and therapeutic effects of EPL-DNA on peanut sensitization animal model. This study proposes a new strategy for the treatment of allergic disease by targeting DC, and hope to establish a new platform for the treatment of infection, tumor and immune-related diseases.

近年来，变态反应性疾病高发而严重，一直缺乏有效而安全的预防和治疗性疫苗。变应原DNA疫苗在动物实验水平显示了对IgE应答的有效抑制，但存在DNA疫苗免疫效率低的普遍问题。本研究基于IgE与树突状细胞(DC)表面高亲和力IgE Fc受体(FcεRI)极高亲和力结合的特性，以及过敏患者体内FcεRI在DC高水平表达的特点，设计Fcε介导的靶向DC的DNA疫苗，以期特异而高效地呈递抗原、诱导IgE特异性免疫耐受。本研究利用在前期工作中已成功制备的人IgE Fc-多聚赖氨酸融合蛋白(简称EPL)，与含有DC特异性启动子及花生致敏原基因的质粒偶联，体外实验分析DC细胞系及诱导分化的人DC对EPL-DNA的摄取、抗原表达及呈递，并利用人FcεRI转基因小鼠分析EPL-DNA对花生致敏动物模型的预防及治疗作用。本研究提出靶向DC治疗过敏性疾病的新策略，并有望建立治疗感染、肿瘤及免疫相关性疾病的新平台。

近年来，变态反应性疾病高发而严重，一直缺乏有效而安全的预防和治疗性疫苗。变应原DNA疫苗在动物实验水平显示了对IgE应答的有效抑制，但存在DNA疫苗免疫效率低的普遍问题。本研究基于IgE与树突状细胞(DC)表面高亲和力IgE Fc受体(FcεRI)极高亲和力结合的特性，以及过敏患者体内FcεRI在DC高水平表达的特点，设计Fcε介导的靶向DC的DNA疫苗，以期特异而高效地呈递抗原、诱导IgE特异性免疫耐受。本研究首先基于OptiCHO体系，构建了含有人IgE Fc段-多聚赖氨酸融合基因(EPL)的真核表达载体，转染OptiCHO细胞系后获得了高表达EPL的稳定转染细胞系；高效表达后的EPL经抗人IgE抗体亲和层析柱纯化，鉴定后证实获得了对FcεRI和DNA均具有良好结合活性和亲和力的融合蛋白。进而体外培养并诱导正常人和花生过敏患者的DC，发现DC可以有效摄取EPL-DNA并呈递该DNA所编码的抗原。与此相平行，我们制备了多聚赖氨酸预处理的DNA 疫苗，发现多聚赖氨酸处理可以有效改变DNA的构象，促进DC对DNA的摄取和呈递；应用于小鼠花生过敏模型，发现多聚赖氨酸预处理的DNA对花生过敏模型具有良好的预防和治疗作用，降低花生抗原Arah2特异性IgG1、IgG2a和IgE的水平，其效果显著优于单纯DNA作用组；机制分析发现，多聚赖氨酸预处理的DNA疫苗皮下注射后可以促进皮肤Langerhans细胞的迁移，促进Treg和IL-10产生。在此基础上，利用人FcεRI Tg+人IgE KI转基因小鼠，发现EPL-DNA皮下免疫该小鼠可以有效预防和治疗花生抗原引起的免疫反应，效果优于单纯DNA组和多聚赖氨酸预处理的DNA组，其机制在于特异性地抑制IgE应答，而对过敏原特异性IgG1和IgG2a的影响较小，提示基于Fcε靶向DC的DNA疫苗可以有效调控针对过敏原的过敏反应。综上，本研究成功制备了EPL融合蛋白并获得了EPL-DNA疫苗，在体外细胞培养水平和转基因小鼠水平发现EPL-DNA疫苗可以被DC有效摄取和呈递，对花生过敏模型具有良好的预防和治疗作用，为下一步的临床应用奠定了基础。本研究提出的靶向DC的DNA疫苗策略，不仅适用于过敏性疾病，还有望建立治疗感染、肿瘤及免疫相关性疾病的新平台。