Gene Therapy and Hepatology Area, Center for Applied Medical Research (CIMA), University of Navarra, 31008 Pamplona, Spain.
Biomed Res Int. 2013;2013:864720. doi: 10.1155/2013/864720. Epub 2013 Sep 5.
The development of tools for efficient targeting of antigens to antigen presenting cells is of great importance for vaccine development. We have previously shown that fusion proteins containing antigens fused to the extra domain A from fibronectin (EDA), an endogenous TLR4 ligand, which targets antigens to TLR4-expressing dendritic cells (DC), are highly immunogenic. To facilitate the procedure of joining EDA to any antigen of choice, we have prepared the fusion protein EDAvidin by linking EDA to the N terminus of streptavidin, allowing its conjugation with biotinylated antigens. We found that EDAvidin, as streptavidin, forms tetramers and binds biotin or biotinylated proteins with a Kd ~ 2.6 × 10(-14) mol/L. EDAvidin favours the uptake of biotinylated green fluorescent protein by DC. Moreover, EDAvidin retains the proinflammatory properties of EDA, inducing NF- κβ by TLR4-expressing cells, as well as the production of TNF- α by the human monocyte cell line THP1 and IL-12 by DC. More importantly, immunization of mice with EDAvidin conjugated with the biotinylated nonstructural NS3 protein from hepatitis C virus induces a strong anti-NS3 T cell immune response. These results open a new way to use the EDA-based delivery tool to target any antigen of choice to DC for vaccination against infectious diseases and cancer.
开发高效靶向抗原呈递细胞的抗原工具对于疫苗的开发非常重要。我们之前已经证明，融合蛋白将抗原与纤维连接蛋白的外结构域 A（EDA）融合，EDA 是内源性 TLR4 配体，可将抗原靶向 TLR4 表达的树突状细胞（DC），具有高度的免疫原性。为了方便将 EDA 与任何所需抗原结合，我们通过将 EDA 连接到链霉亲和素的 N 端制备了融合蛋白 EDAvidin，使其能够与生物素化抗原缀合。我们发现 EDAvidin 像链霉亲和素一样形成四聚体，并以 Kd~2.6×10(-14)mol/L 的亲和力结合生物素或生物素化蛋白。EDAvidin 有利于 DC 摄取生物素化绿色荧光蛋白。此外，EDAvidin 保留了 EDA 的促炎特性，通过 TLR4 表达细胞诱导 NF-κβ，以及人单核细胞系 THP1 产生 TNF-α和 DC 产生 IL-12。更重要的是，用 EDAvidin 与丙型肝炎病毒的非结构 NS3 蛋白的生物素化形式免疫小鼠，可诱导强烈的抗 NS3 T 细胞免疫反应。这些结果为使用基于 EDA 的递药工具将任何所需抗原靶向 DC 以用于针对传染病和癌症的疫苗接种开辟了新途径。
