Table 1_Bacillus subtilis spores displaying Toxoplasma gondii GRA12 induce immunity against acute toxoplasmosis.doc

BackgroundToxoplasma gondii (T. gondii) is a widely prevalent intracellular parasite that infects almost all warm-blooded animals and causes serious public health problems. The drugs currently used to treat toxoplasmosis have the disadvantage of being toxic and prone to the development of resistance, and the only licensed vaccine entails a risk of virulence restoration. The development of a safe and effective vaccine against T. gondii is urgently needed. Bacillus subtilis (B. subtilis) has been used as a potential vaccine expression vector for the treatment and prevention of various diseases. T. gondii GRA12 is a key virulence factor that resists host innate immunity and exhibits good antigenicity with several excellent B and T cell epitopes.
MethodsA recombinant spore named rBS-GRA12 was constructed by fusing the T. gondii GRA12 protein to the B. subtilis coat protein B (CotB). rBS-GRA12 spores were identified by PCR, western blotting, immunofluorescence assays, amylase activity, and ultrastructural analysis. Immunological experiments were then conducted to assess the immunoprotective effects of rBS-GRA12. Groups of mice immunized with rBS-GRA12 (106, 108, or 1010 colony-forming units), GRA12 protein emulsified with Freund’s adjuvant (FA+GRA12), Freund’s adjuvant alone (FA), phosphate buffered saline (PBS), or wild-type B. subtilis spores (WT). Splenocyte proliferation, antibodies, and cytokine expression levels were used to assess immune responses induced by the immunizations. All groups were inoculated with T. gondii RH strain, and survival times and parasite loads in tissues were used to assess protective effects against T. gondii infection.
ResultsAmylase activity assays confirmed the generation of recombinant B. subtilis. PCR, western blotting and immunofluorescence assays confirmed that the rBS-GRA12 spores expressed GRA12. Observation of rBS-GRA12 spores via transmission and scanning electron microscopy indicated that GRA12 expression had no effect on spore morphology or structure. Splenocyte proliferation was significantly greater in all three rBS-GRA12 groups than in the FA+GRA12 group, and IgG and IgG2a subclass titers were higher. Substantial production of interferon gamma (IFN-γ), interleukin (IL)-12, and an increase in IL-4 production were evident in the rBS-GRA12-108 group. Secretory sIgA levels were significantly elevated in all three rBS-GRA12 groups than in the FA+GRA12 group and the control groups. Brain and liver tissues parasite loads were significantly lower in the three rBS-GRA12 groups than in any other group. Compared to all other groups, mice in the three rBS-GRA12 groups exhibited longer survival times when challenged with acute T. gondii infection.
ConclusionMice immunized with rBS-GRA12 exhibited higher levels of cellular, humoral, and mucosal immune responses than control mice. These results provide a new perspective for the development of T. gondii vaccines.

背景
刚地弓形虫（Toxoplasma gondii，简称T. gondii）是一种广泛流行的胞内寄生虫，可感染几乎所有温血动物，引发严重的公共卫生问题。当前用于治疗弓形虫病的药物存在毒性较强、易诱发耐药性产生的缺陷，而唯一获批的疫苗还存在毒力返强的风险。因此，研发安全有效的抗弓形虫疫苗迫在眉睫。枯草芽孢杆菌（Bacillus subtilis，简称B. subtilis）已被用作潜在的疫苗表达载体，用于多种疾病的防治。弓形虫GRA12是一种关键毒力因子，能够抵抗宿主固有免疫，且具备良好的抗原性，包含多个优质的B细胞与T细胞表位。
方法
本研究将弓形虫GRA12蛋白与枯草芽孢杆菌衣壳蛋白B（CotB）融合，构建了一株命名为rBS-GRA12的重组芽孢。通过聚合酶链式反应（PCR）、蛋白质印迹（western blotting）、免疫荧光实验、淀粉酶活性检测以及超微结构分析，对rBS-GRA12芽孢进行鉴定。随后开展免疫学实验，评估rBS-GRA12的免疫保护效果。实验将小鼠分为若干组，分别以rBS-GRA12（10^6、10^8或10^10菌落形成单位（CFU））、弗氏佐剂乳化的GRA12蛋白（FA+GRA12）、单用弗氏佐剂（FA）、磷酸盐缓冲液（PBS）以及野生型枯草芽孢杆菌芽孢（WT）进行免疫。通过检测脾细胞增殖水平、抗体水平以及细胞因子表达量，评估各免疫方案诱导的免疫应答。所有小鼠均接种弓形虫RH株，通过记录存活时间以及组织内寄生虫载量，评估其抗弓形虫感染的保护效果。
结果
淀粉酶活性实验证实了重组枯草芽孢杆菌的成功构建。PCR、蛋白质印迹以及免疫荧光实验均证实rBS-GRA12芽孢可表达GRA12蛋白。通过透射电镜与扫描电镜观察rBS-GRA12芽孢，结果显示GRA12的表达对芽孢的形态与结构无影响。三个rBS-GRA12免疫组的脾细胞增殖水平均显著高于FA+GRA12组，且IgG及IgG2a亚类抗体滴度更高。在rBS-GRA12-10^8组中，可检测到γ干扰素（IFN-γ）、白细胞介素-12（IL-12）的大量分泌，以及白细胞介素-4（IL-4）水平的升高。三个rBS-GRA12组的分泌型免疫球蛋白A（sIgA）水平均显著高于FA+GRA12组与各对照组。三个rBS-GRA12组小鼠的脑组织与肝脏组织内的寄生虫载量均显著低于其他所有组别。与其他所有组别相比，三个rBS-GRA12组小鼠在感染急性弓形虫后存活时间更长。
结论
接种rBS-GRA12的小鼠相较于对照组小鼠，展现出更高水平的细胞免疫、体液免疫以及黏膜免疫应答。本研究结果为抗弓形虫疫苗的研发提供了新的视角。