Department of Parasitology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangdong, People's Republic of China.
Parasitol Res. 2014 Aug;113(8):3095-103. doi: 10.1007/s00436-014-3975-9. Epub 2014 Jun 7.
Human clonorchiasis caused by Clonorchis sinensis (C. sinensis) has been increasingly prevalent in recent years so that an effective measure is essential and urgent to control the infectious disease. Oral delivery of antigens from C. sinensis may be an important approach to effectively induce both systemic and local immune responses to anti-infection of the parasite. In the current study, we used Bacillus subtilis (B. subtilis) spores as a delivery vehicle to introduce leucine aminopeptidase 2 of C. sinensis (CsLAP2), an excretory/secretory antigen with high immunogenicity, expressing on their surface. SDS-PAGE, western blotting, and flow cytometry indicated that CsLAP2 was successfully expressed on the surface of B. subtilis spores (CotC-CsLAP2 spores). BALB/c mice were treated with spores intragastrically. On day 31 after the treatment, we found that mice intragastrically treated with CotC-CsLAP2 spores exhibited higher IgG, IgG1, IgG2a, and IgA level in sera as well as higher sIgA level in bile and intestinal lavage fluid compared to mice orally administrated with spores not expressing CsLAP2 (CotC spores) and naïve mice. The peak titer of IgG/IgA presented on day 31/49 after oral administration. IgG1 level was lower than IgG2a in group administrated with CotC-CsLAP2 spores. sIgA-secreting cells were obviously observed in intestinal epithelium of mice orally treated with CotC-CsLAP2 spores. After incubated with CotC-CsLAP2, the levels of IFN-γ, IL-6, IL-10, IL-17A, and TNF significantly increased in the supernatant of splenocytes isolated from mice orally treated with CotC-CsLAP2 spores, while there was no statistically significant difference of IL-4 level representing Th2 response among the groups. Our study demonstrated that oral administration of CsLAP2 delivered by B. subtilis spore elicited obvious systemic and local mucosal immunity. Secretory IgA and Th1-Th17 cellular immunity might involved in mechanisms of the immune response.
由华支睾吸虫（Clonorchis sinensis，C. sinensis）引起的人体华支睾吸虫病近年来日益流行，因此，采取有效措施控制这种传染病迫在眉睫。口服华支睾吸虫抗原可能是一种重要的方法，可以有效地诱导针对寄生虫感染的全身和局部免疫反应。在本研究中，我们使用枯草芽孢杆菌（Bacillus subtilis，B. subtilis）孢子作为载体，将具有高度免疫原性的分泌/排泄抗原亮氨酸氨肽酶 2（CsLAP2）表达在其表面。SDS-PAGE、western blot 和流式细胞术表明，CsLAP2 成功地表达在枯草芽孢杆菌孢子的表面（CotC-CsLAP2 孢子）。BALB/c 小鼠经灌胃给予孢子。在治疗后第 31 天，我们发现经灌胃给予 CotC-CsLAP2 孢子的小鼠血清中 IgG、IgG1、IgG2a 和 IgA 水平以及胆汁和肠灌洗液中的 sIgA 水平均高于未表达 CsLAP2 的孢子（CotC 孢子）和未处理的对照组小鼠。口服给药后第 31/49 天达到 IgG/IgA 峰值。与 CotC-CsLAP2 孢子处理组相比，IgG1 水平低于 IgG2a。在经 CotC-CsLAP2 孢子处理的小鼠肠上皮中明显观察到分泌 sIgA 的细胞。与 CotC-CsLAP2 孵育后，经灌胃给予 CotC-CsLAP2 孢子的小鼠脾细胞上清液中 IFN-γ、IL-6、IL-10、IL-17A 和 TNF 的水平显著升高，而 Th2 反应的代表性细胞因子 IL-4 水平在各组之间无统计学差异。本研究表明，口服给予枯草芽孢杆菌孢子递送的 CsLAP2 可引起明显的全身和局部黏膜免疫。分泌型 IgA 和 Th1-Th17 细胞免疫可能参与了免疫反应的机制。
