DataSheet_1_Preventive effects of Mycobacterium tuberculosis DNA vaccines on the mouse model with latent tuberculosis infection.pdf

BackgroundAbout a quarter of the world’s population with latent tuberculosis infection (LTBI) are the main source of active tuberculosis. Bacillus Calmette Guerin (BCG) cannot effectively control LTBI individuals from developing diseases. Latency-related antigens can induce T lymphocytes of LTBI individuals to produce higher IFN-γ levels than tuberculosis patients and normal subjects. Herein, we firstly compared the effects of M. tuberculosis (MTB) ag85ab and 7 latent DNA vaccines on clearing latent MTB and preventing its activation in the mouse LTBI model.
MethodsA mouse LTBI model was established, and then immunized respectively with PBS, pVAX1 vector, Vaccae vaccine, ag85ab DNA and 7 kinds of latent DNAs (including rv1733c, rv2660c, rv1813c, rv2029c, rv2628, rv2659c and rv3407) for three times. The mice with LTBI were injected with hydroprednisone to activate the latent MTB. Then, the mice were sacrificed for the bacterial count, histopathological examination, and immunological evaluation.
ResultsUsing chemotherapy made the MTB latent in the infected mice, and then using hormone treatment reactivated the latent MTB, indicating that the mouse LTBI model was successfully established. After the mouse LTBI model was immunized with the vaccines, the lung colony-forming units (CFUs) and lesion degree of mice in all vaccines group were significantly decreased than those in the PBS group and vector group (P<0.0001, P<0.05). These vaccines could induce antigen-specific cellular immune responses. The number of IFN-γ effector T cells spots secreted by spleen lymphocytes in the ag85ab DNA group was significantly increased than those in the control groups (P<0.05). In the splenocyte culture supernatant, IFN-γ and IL-2 levels in the ag85ab, rv2029c, and rv2659c DNA groups significantly increased (P<0.05), and IL-17A levels in ag85ab and rv2659c DNA groups also significantly increased (P<0.05). Compared with the PBS and vector groups, the proportion of CD4+CD25+FOXP3+ regulatory T cells in spleen lymphocytes of ag85ab, rv2660c, rv2029c, and rv3407 DNA groups were significantly reduced (P<0.05).
ConclusionsMTB ag85ab and 7 kinds of latent DNA vaccines showed immune preventive efficacies on a mouse model of LTBI, especially the rv2659c, and rv1733c DNA. Our findings will provide candidates for the development of new multi-stage vaccines against TB.

背景 全球约四分之一的潜伏性结核感染（latent tuberculosis infection, LTBI）人群是活动性结核的主要传染源。卡介苗（Bacillus Calmette Guerin, BCG）无法有效控制潜伏性结核感染个体发展为活动性结核病。潜伏相关抗原可诱导潜伏性结核感染个体的T淋巴细胞产生较结核患者与正常人群更高水平的γ干扰素（interferon-γ, IFN-γ）。本研究首先比较了结核分枝杆菌（Mycobacterium tuberculosis, MTB）ag85ab与7种潜伏型DNA疫苗在小鼠潜伏性结核感染模型中清除潜伏结核分枝杆菌及阻止其活化的效果。
方法 首先构建小鼠潜伏性结核感染模型，随后分别以磷酸盐缓冲液（phosphate buffered saline, PBS）、pVAX1空载体、卡介苗疫苗、ag85ab DNA疫苗以及7种潜伏型DNA疫苗（包含rv1733c、rv2660c、rv1813c、rv2029c、rv2628、rv2659c及rv3407）对小鼠进行三次免疫。向潜伏性结核感染小鼠注射氢化泼尼松以活化潜伏的结核分枝杆菌，随后处死小鼠，进行细菌菌落计数、组织病理学检查及免疫学评价。
结果 经化疗使感染小鼠体内的结核分枝杆菌进入潜伏状态，再经激素处理活化潜伏的结核分枝杆菌，证实小鼠潜伏性结核感染模型构建成功。经疫苗免疫后，各疫苗组小鼠的肺部菌落形成单位（colony-forming units, CFUs）及肺部损伤程度均显著低于PBS组与空载体组（P<0.0001, P<0.05）。上述疫苗均可诱导抗原特异性细胞免疫应答。ag85ab DNA疫苗组小鼠脾脏淋巴细胞分泌的γ干扰素效应性T细胞斑点数较对照组显著升高（P<0.05）。在脾细胞培养上清液中，ag85ab、rv2029c及rv2659c DNA疫苗组的γ干扰素与白细胞介素2（interleukin-2, IL-2）水平均显著升高（P<0.05）；ag85ab与rv2659c DNA疫苗组的白细胞介素17A（interleukin-17A, IL-17A）水平亦显著升高（P<0.05）。与PBS组及空载体组相比，ag85ab、rv2660c、rv2029c及rv3407 DNA疫苗组小鼠脾脏淋巴细胞中CD4+CD25+FOXP3+调节性T细胞的比例显著降低（P<0.05）。
结论 结核分枝杆菌ag85ab与7种潜伏型DNA疫苗在小鼠潜伏性结核感染模型中均展现出免疫预防效果，其中rv2659c与rv1733c DNA疫苗的效果尤为突出。本研究结果可为新型结核多阶段疫苗的研发提供候选靶点。