Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta , Edmonton, Alberta T6G 2N8, Canada.
Mol Pharm. 2012 Apr 2;9(4):946-56. doi: 10.1021/mp200553x. Epub 2012 Mar 20.
This work investigates the formulation and in vivo efficacy of dendritic cell (DC) targeted plasmid DNA loaded biotinylated chitosan nanoparticles for nasal immunization against nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) as antigen. The induction of antigen-specific mucosal and systemic immune response at the site of virus entry is a major challenge for vaccine design. Here, we designed a strategy for noninvasive receptor mediated gene delivery to nasal resident DCs. The pDNA loaded biotinylated chitosan nanoparticles were prepared using a complex coacervation process and characterized for size, shape, surface charge, plasmid DNA loading and protection against nuclease digestion. The pDNA loaded biotinylated chitosan nanoparticles were targeted with bifunctional fusion protein (bfFp) vector for achieving DC selective targeting. The bfFp is a recombinant fusion protein consisting of truncated core-streptavidin fused with anti-DEC-205 single chain antibody (scFv). The core-streptavidin arm of fusion protein binds with biotinylated nanoparticles, while anti-DEC-205 scFv imparts targeting specificity to DC DEC-205 receptor. We demonstrate that intranasal administration of bfFp targeted formulations along with anti-CD40 DC maturation stimuli enhanced magnitude of mucosal IgA as well as systemic IgG against N protein. The strategy led to the detection of augmented levels of N protein specific systemic IgG and nasal IgA antibodies. However, following intranasal delivery of naked pDNA no mucosal and systemic immune responses were detected. A parallel comparison of targeted formulations using intramuscular and intranasal routes showed that the intramuscular route is superior for induction of systemic IgG responses compared with the intranasal route. Our results suggest that targeted pDNA delivery through a noninvasive intranasal route can be a strategy for designing low-dose vaccines.
这项工作研究了负载树突状细胞 (DC) 靶向质粒 DNA 的生物素化壳聚糖纳米粒的配方和体内疗效，用于针对严重急性呼吸综合征冠状病毒 (SARS-CoV) 核衣壳 (N) 蛋白的鼻内免疫。在病毒进入部位诱导抗原特异性粘膜和全身免疫反应是疫苗设计的主要挑战。在这里，我们设计了一种非侵入性受体介导的基因传递策略，用于鼻腔常驻 DC。使用复凝聚过程制备负载 pDNA 的生物素化壳聚糖纳米粒，并对其大小、形状、表面电荷、质粒 DNA 负载和对核酸酶消化的保护进行了表征。负载 pDNA 的生物素化壳聚糖纳米粒用双功能融合蛋白 (bfFp) 载体进行靶向，以实现 DC 选择性靶向。bfFp 是一种重组融合蛋白，由截短的核心链霉亲和素与抗 DEC-205 单链抗体 (scFv) 融合而成。融合蛋白的核心链霉亲和素臂与生物素化纳米粒结合，而抗 DEC-205 scFv 赋予 DC DEC-205 受体靶向特异性。我们证明，鼻内给予 bfFp 靶向制剂和抗 CD40 DC 成熟刺激物可增强针对 N 蛋白的粘膜 IgA 以及全身 IgG 的量。该策略导致检测到增强的 N 蛋白特异性全身 IgG 和鼻内 IgA 抗体水平。然而，裸 pDNA 鼻内递送后未检测到粘膜和全身免疫反应。通过肌肉内和鼻内途径进行靶向制剂的平行比较表明，与鼻内途径相比，肌肉内途径更有利于诱导全身 IgG 反应。我们的结果表明，通过非侵入性鼻内途径进行靶向 pDNA 递送可以成为设计低剂量疫苗的策略。
