Li Hui, Li Rong, Zhong Sen, Ren Hong, Zou Yongsheng, Chen Xuanshi, Shi Xiaoling, Wang Mingyong, Long Han'an, Luo Yuebei
The Affiliated hospital of Luzhou Medical College, Luzhou, Sichuan 646000, China.
Vaccine. 2006 Feb 27;24(9):1315-23. doi: 10.1016/j.vaccine.2005.09.025. Epub 2005 Sep 27.
DNA vaccination has emerged as a powerful approach in the search for a more efficacious vaccine against tuberculosis (TB). In this study, we evaluated the immunogenicity and protective efficacy of Mtb8.4/hIL-12 chimeric gene vaccine. The Mtb8.4/hIL-12 chimeric gene was amplified by PCR and cloned into the eukaryotic expression vector pCI-neo. C57BL/6N mice were vaccinated with Mtb8.4/hIL-12 chimeric gene vaccine for three times at 3 weeks intervals. Four weeks after the final inoculation, three mice per group were sacrificed to assess cytokine response and CTL induction and the other five mice per group were challenged intravenously in a lateral tail vein with 1 x 10(6) CFU of virulent Mycobacterium tuberculosis H37Rv. Spleen and the left lung were harvested from each mouse at 4 weeks after infection and homogenized in sterile saline. Serial dilutions of organ homogenates were plated on L-J agar and incubated 37 degrees C until colonies were visible 4 weeks later. Protective efficacies in each experiment were expressed as reduced CFU and were compared with the negative control group. The right lung was obtained from each mouse and immediately inflated with and stored in 10% formalin saline. Tissues were embedded in paraffin, sectioned and stained with hematoxylin and eosin. Mtb8.4/hIL-12 chimeric gene vaccine induced the secretion of more of Th1 cytokines, but not IL-4 and enhanced CTL activity. Mice immunized with Mtb8.4/hIL-12 chimeric gene vaccine had fewer and smaller tubercles than control groups. As expected, control mice had the highest bacterial loads in both lung and spleen. Immunization with Mtb8.4/hIL-12 chimeric gene vaccine could remarkably reduced CFU counts in organs. When it was used to construct the chimeric gene vaccine, hIL-12 could improve the immune efficacy of Mtb8.4 gene vaccine.
DNA疫苗已成为寻找更有效抗结核（TB）疫苗的一种有力方法。在本研究中，我们评估了Mtb8.4/hIL-12嵌合基因疫苗的免疫原性和保护效力。通过PCR扩增Mtb8.4/hIL-12嵌合基因，并将其克隆到真核表达载体pCI-neo中。C57BL/6N小鼠每隔3周用Mtb8.4/hIL-12嵌合基因疫苗接种3次。末次接种后4周，每组处死3只小鼠以评估细胞因子反应和CTL诱导，每组另外5只小鼠经尾静脉外侧静脉注射1×10(6)CFU强毒结核分枝杆菌H37Rv进行攻毒。感染后4周从每只小鼠采集脾脏和左肺，在无菌盐水中匀浆。将器官匀浆的系列稀释液接种于L-J琼脂平板，37℃孵育至4周后可见菌落。每次实验中的保护效力以CFU减少量表示，并与阴性对照组进行比较。从每只小鼠获取右肺，立即用10%甲醛生理盐水充气并保存。组织用石蜡包埋、切片，并用苏木精和伊红染色。Mtb8.4/hIL-12嵌合基因疫苗诱导分泌更多的Th1细胞因子，但不包括IL-4，并增强了CTL活性。用Mtb8.4/hIL-12嵌合基因疫苗免疫的小鼠比对照组有更少、更小的结核结节。正如预期的那样，对照小鼠在肺和脾脏中的细菌载量最高。用Mtb8.4/hIL-12嵌合基因疫苗免疫可显著降低器官中的CFU计数。当用于构建嵌合基因疫苗时，hIL-12可提高Mtb8.4基因疫苗的免疫效力。
