Supplementary Material for: A Dendritic Cells-Targeting Nano-Vaccine by Coupling Polylactic-Co-Glycolic Acid-Encapsulated Allergen with Mannan Induces Regulatory T Cells

<b><i>Background:</i></b> The efficacy of allergen-specific immunotherapy (AIT) is mainly depended on the tolerogenic immune responses elicited. Properly conjugated nano-vaccine has the advantages of both specific targeting and continuous and on-demand release of allergen. <b><i>Objectives:</i></b> The aim of this study is to investigate the effects of a dendritic cells (DCs)-targeting nano-vaccine for AIT. <b><i>Methods:</i></b> The nano-vaccine was produced by coupling polylactic-co-glycolic acid (PLGA)-encapsulated ovalbumin (OVA) with mannan. Allergen capture, human monocytes-derived DCs (hMoDCs) activation, and T cells responses were assessed by flow cytometry, confocal microscopy, quantitative real-time PCR, ELISA, and Cytometric Bead Array. Balb/c mice were immunized with the nano-vaccines, and the immune responses were analyzed. <b><i>Results:</i></b> OVA-PLGA nanoparticle (NP) displayed favorable safety profile. OVA-mannan-PLGA NP was captured more efficiently by hMoDCs than OVA-PLGA NP, which was mediated mainly through DC-specific intercellular adhesion molecule 3-grabbing nonintegrin. A tolerogenic phenotype of hMoDCs was induced by OVA-mannan-PLGA NP, but not OVA-PLGA NP, and increased number of regulatory T (Treg) cells was generated subsequently in in vitro coculture. Immunization of Balb/c mice with OVA-mannan-PLGA NP resulted in lower serum level of OVA-specific immunoglobulins and less production of pro-inflammatory cytokines in splenocytes culture than the mice immunized with OVA-PLAG NP, PLGA NP, or OVA, while the number of splenic Treg cells was higher in OVA-mannan-PLGA group than in other groups. Moreover, preimmunization with OVA-mannan-PLGA NP significantly inhibited the Th2 immune response induced by OVA sensitization. <b><i>Conclusions:</i></b> The biocompatible PLGA-encapsulated OVA coupling with mannan has augmented ability for tolerance induction and could be developed as a novel vaccine for AIT.

**<i>背景：</i>** 过敏原特异性免疫治疗（allergen-specific immunotherapy, AIT）的疗效主要依赖于其所诱导的耐受型免疫应答。经合理偶联的纳米疫苗兼具特异性靶向与持续按需释放过敏原的双重优势。
**<i>目的：</i>** 本研究旨在探究树突状细胞（dendritic cells, DCs）靶向纳米疫苗用于AIT的效果。
**<i>方法：</i>** 本研究将聚乳酸-羟基乙酸共聚物（polylactic-co-glycolic acid, PLGA）包裹的卵清蛋白（ovalbumin, OVA）与甘露聚糖偶联，制备得到目标纳米疫苗。通过流式细胞术、共聚焦显微镜、实时荧光定量PCR、酶联免疫吸附试验（enzyme-linked immunosorbent assay, ELISA）及流式微球阵列（Cytometric Bead Array），分别评估了过敏原摄取、人单核细胞来源树突状细胞（human monocytes-derived DCs, hMoDCs）活化及T细胞应答情况。此外，使用该纳米疫苗免疫Balb/c小鼠，并对其免疫应答进行分析。
**<i>结果：</i>** OVA-PLGA纳米颗粒（nanoparticle, NP）展现出良好的安全性。相较于OVA-PLGA纳米颗粒，OVA-甘露聚糖-PLGA纳米颗粒可被hMoDCs更高效地摄取，且该摄取过程主要通过DC特异性细胞间黏附分子3捕获非整合素（DC-specific intercellular adhesion molecule 3-grabbing nonintegrin）介导。OVA-甘露聚糖-PLGA纳米颗粒可诱导hMoDCs产生耐受表型，而OVA-PLGA纳米颗粒无此效果；体外共培养实验中，该纳米疫苗可进一步诱导产生更多的调节性T（regulatory T, Treg）细胞。与OVA-PLGA纳米颗粒、PLGA纳米颗粒或OVA免疫组小鼠相比，经OVA-甘露聚糖-PLGA纳米颗粒免疫的小鼠，其血清中OVA特异性免疫球蛋白水平更低，脾细胞培养上清中促炎细胞因子的分泌量更少；同时，OVA-甘露聚糖-PLGA组小鼠的脾脏Treg细胞数量显著高于其余各组。此外，预先用OVA-甘露聚糖-PLGA纳米颗粒免疫，可显著抑制OVA致敏所诱导的Th2型免疫应答。
**<i>结论：</i>** 经甘露聚糖偶联的生物相容性PLGA包裹OVA制剂，其耐受诱导能力得到增强，有望开发为用于AIT的新型疫苗。