The objective of the present work was to evaluate the efficacy of a novel antigen carrier using mannosylated gelatin nanoparticles with entrapped inactivated porcine reproductive and respiratory syndrome virus (PRRSV) in inducing T cell mediated immunity in vitro. Gelatin nanoparticles (GNP) were modified with mannose to form mannosylated gelatin nanoparticles (MnGNP), which can efficiently and specifically target monocyte derived dendritic cells (MoDCs). The inactivated PRRSV was encapsulated in the MnGNP and GNP, referred to as MnGNP-PRRSV and GNP-PRRSV, respectively. All these prepared nanometer particles were characterized for size, surface charge, drug encapsulation efficiency, and drug release. The efficacy of MnGNP in targeting MoDCs was investigated, as well as the subsequent MoDCs maturation and T cell mediated cytotoxicity. The developed MnGNP-PRRSV particle was characterized with a nanometric size of 302.67 ± 3.2 nm, surface charge of 23.81 ± 1.26 mV, and PRRSV encapsulation efficiency of 63.2 ± 1.85 %. The maximum uptake of MnGNP in MoDCs in vitro was 15.5 times higher than GNP with a shorter reaction time that peaked 4 h earlier. The uptake of MnGNP-PRRSV induced maturation of MoDCs and significantly enhanced expression of SWC-3a, CD80, CD1, SLA I, SLA II on MoDCs, compared to PRRSV (p < 0.001). The cytokine secretion of IL-1β, IL-6, IL-10, and IL-12 was also increased in MoDCs when treated with MnGNP-PRRSV, compared to PRRSV (p < 0.05). The matured MoDCs triggered T lymphocytes in autologous peripheral blood mononuclear cells (PBMCs) activation, proliferation, and differentiation into effector cytotoxic T lymphocyte, suggesting increased amount of activated T cells after MnGNP-PRRSV treatment. Additionally, the function of T cells to kill PRRSV infected cells was 83.98 ± 2.62 % when triggered by MnGNP-PRRSV, compared to 60 ± 4.7 % in PRRSV group (p < 0.001). These results indicate that MnGNP with entrapped inactivated PRRSV can effectively and specifically target dendritic cells for maturation and activation, and subsequently improve T cell activation, proliferation and function to kill PRRSV infected cells.

本工作的目的是评估使用甘露糖化明胶纳米颗粒和截留的灭活猪繁殖与呼吸综合征病毒 (PRRSV) 的新型抗原载体在体外诱导 T 细胞介导免疫的功效。. 明胶纳米粒子 (GNP) 用甘露糖修饰形成甘露糖化明胶纳米粒子 (MnGNP)，可有效且特异性地靶向单核细胞衍生的树突细胞 (MoDC)。灭活的 PRRSV 被封装在 MnGNP 和 GNP 中，分别称为 MnGNP-PRRSV 和 GNP-PRRSV。所有这些制备的纳米颗粒都对尺寸、表面电荷、药物包封效率和药物释放进行了表征。研究了 MnGNP 在靶向 MoDC 中的功效，以及随后的 MoDC 成熟和 T 细胞介导的细胞毒性。开发的 MnGNP-PRRSV 颗粒的特征在于纳米尺寸为 302.67 ± 3.2 nm，表面电荷为 23.81 ± 1.26 mV，PRRSV 封装效率为 63.2 ± 1.85%。MoDCs体外对MnGNP的最大摄取比 GNP 高 15.5 倍，反应时间更短，峰值提前 4 小时。与 PRRSV 相比，MnGNP-PRRSV 的摄取诱导 MoDCs 的成熟，并显着增强了 MoDCs 上 SWC-3a、CD80、CD1、SLA I、SLA II 的表达（p < 0.001）。与 PRRSV 相比，当用 MnGNP-PRRSV 处理时，MoDC 中 IL-1β、IL-6、IL-10 和 IL-12 的细胞因子分泌也增加（p< 0.05)。成熟的 MoDC 触发自体外周血单核细胞 (PBMC) 中的 T 淋巴细胞活化、增殖和分化为效应细胞毒性 T 淋巴细胞，表明 MnGNP-PRRSV 治疗后活化 T 细胞的数量增加。此外，当由 MnGNP-PRRSV 触发时，T 细胞杀死 PRRSV 感染细胞的功能为 83.98 ± 2.62 %，而 PRRSV 组为 60 ± 4.7 % ( p < 0.001)。这些结果表明，包埋了灭活 PRRSV 的 MnGNP 可以有效且特异性地靶向树突状细胞进行成熟和激活，进而提高 T 细胞的活化、增殖和杀死 PRRSV 感染细胞的功能。