壳聚糖递送的结核基因疫苗诱导的肺部黏膜免疫及在抗结核免疫保护中的意义

The mucosal immunity induced by chitosan-delivered gene vaccine and its significance in protection against tuberculosis

诱导肺局部黏膜免疫对于早期控制结核分枝杆菌(Mycobacterium tuberculosis)十分关键。在我们过去构建的结核基因疫苗pHSP65pep有效诱导脾脏IFN-γ+Th1细胞应答的基础上,为更好诱导呼吸道和肺局部黏膜免疫,采用天然生物多糖壳聚糖(chitosan)作为黏膜递送介质递送结核基因疫苗。制备壳聚糖-pHSP65pep结核黏膜疫苗,滴鼻免疫BALB/c小鼠,检测全身和黏膜T细胞免疫及抗体应答;并以大剂量BCG攻毒,以肺脏HE染色及菌落计数检测免疫保护效果。结果显示,壳聚糖黏膜递送虽不能增加血清HSP65特异性IgG抗体产生,但显著提高了肺灌洗液的SIgA水平;同时显著增强了肺黏膜局部pHSP65pep疫苗诱导的IFN-γ+CD4+Th1细胞应答。攻毒结果证实,壳聚糖递送显著增强了结核基因疫苗的免疫保护效果。表明壳聚糖黏膜递送可显著增强结核基因疫苗诱导的特异性肺黏膜Th1应答和SIgA水平,提高免疫保护效果。提示呼吸道黏膜免疫对于结核保护性免疫具有关键意义。

Induction of local（pulmonary） mucosal immunity plays a critical role in preventing Mycobacterium tuberculosis（M.tb） dissemination at the early infection stage.On the basis of our previously constructed muti-T-epitope DNA vaccine-pHSP65pep which effectively induced splenic IFN-γ＋ Th1 response,chitosan,a natural cationic polysaccharide was employed as mucosal delivery media to enhance the specific mucosal immunity.The pHSP65-pep DNA was complexed with chitosan,and intranasally administered to BABL/c mice four times,using naked gene vaccine as control.Systemic and mucosal T cells response as well as antibody response was followed,and after challenging mice with lethal dose of BCG,the protection was evaluated by assessing H E staining of lung tissue and local bacterial loads.It was found that chitosan delivery did not change the specific serum IgG antibody induction,but significantly enhanced SIgA level in the lung lavage（P0.05）.Chitosan significantly increased pulmonary CD4＋ IFN-γ＋ and CD8＋ IFN-γ＋ T cell frequency induced by pHSP65pep,which was much higher than the splenic counterpart（P0.05）;Better protection was achieved by chitosan-pHSP65pep immunization as compared with naked gene vaccine.Our study indicated that chitosan delivery remarkably strengthened gene vaccine-induced specific mucosal Th1 response and SIgA level which was positively correlated with immunological protection against M.tb,and local mucosal immunity is very critical for the protection against M.tb infection.