疫苗候选抗原HBHA通过诱导IL-17发挥抗结核免疫保护效应

Heparin-binding hemagglutinin as a composition antigen of tuberculosis vaccine exerts protective immune effects by inducing IL-17

为探讨疫苗候选抗原—结核分枝杆菌肝素结合血凝素(HBHA)通过黏膜接种诱导的保护性免疫效应。动物实验均使用6~8周龄雌性C57BL/6小鼠。30只小鼠接受不同的免疫接种策略,采用随机数字表法分为对照组、早期分泌抗原靶蛋白-6(ESAT-6)滴鼻组、HBHA滴鼻组、BCG初免PBS对照组、BCG初免HBHA加强组,每组6只。通过检测免疫后小鼠血浆白细胞介素17A(IL-17A)等细胞因子水平;肺IL-17A mRNA相对表达量(RQ);肺三级淋巴结构的形成,及与其形成相关的趋化因子的检测;脾Th1、Th17细胞比例比较,从而分析免疫效应。BCG初免PBS对照组和BCG初免HBHA加强组(每组各30只小鼠),用BCG滴鼻模拟感染。在感染后预设的时间点做肺组织菌落计数,以评估细菌清除效率。多组间比较采用单因素方差分析,事后分析采用LSD检验;两组间比较采用独立样本t检验。结果显示,免疫后的小鼠血浆细胞因子IL-17A水平和肺IL-17A mRNA相对表达量,在BCG初免HBHA加强组[(14.76±4.73)pg/mL,RQ为(12.27±6.71)]中最高,比对照组[(5.57±2.95)pg/mL,RQ为(1.30±0.97)]明显升高(t=4.213,P<0.001;t=5.984,P<0.001),也显著高于BCG初免PBS对照组[(6.81±2.18)pg/mL,RQ为(1.44±1.16)](t=3.646 P=0.001;t=6.185 P<0.001)。脾脏Th17细胞比例,与BCG初免PBS对照组(0.38±0.38)%比较,BCG初免HBHA加强组(1.02±0.34)%显著增高(t=-0.280,P=0.048),该组在肺部诱导形成了三级淋巴结构,并在感染的早期降低了肺细菌负荷。综上,HBHA通过黏膜递送可有效增强BCG接种后的免疫保护效应,可能是一种有潜力的候选疫苗组分。

To explore the protective immune effect induced by mucosal delivery heparin-binding hemagglutinin(HBHA)-a candidate vaccine antigen of Mycobacterium tuberculosis.Female C57BL/6 mice were between 6 and 8 weeks of age before experimental use.Thirty mice received different immunization strategies and were randomly divided into the control group,the early secreting antigen target-6(ESAT-6)intranasal immunization group,the HBHA intranasal immunization group,the BCG priming PBS control group,or BCG priming HBHA boost group,6 mice in each group.In order to analyzed the immune effect,the concentrations of plasma Interleukin-17A(IL-17A)and other cytokines were measured by ELISA.Quantitative real-time PCR analyses were performed to detect the relative quantity(RQ)mRNA of IL-17A in the lung.The lung tissue sections were stained to detect the formation of the tertiary lymphoid structures.The chemokines contributed to formation of the tertiary lymphoid structures were also measured.Flow cytometry was used to detect the frequency of Th1 and Th17 cells in the system.Sixty mice in the BCG priming PBS control group and the BCG priming HBHA boost group were sacrificed at different time points after infection to count the lung bacterial burden.The concentrations of plasma IL-17A and relative quantity of lung IL-17A mRNA were highest in the BCG priming HBHA boost group[(14.76±4.73)pg/mL,RQ(12.27±6.71)],which was significantly higher than the control group[(5.57±2.95)pg/mL,RQ(1.30±0.97)](t=4.213,P<0.001;t=5.984,P<0.001),and also significantly higher than the BCG priming PBS control group[(6.81±2.18)pg/mL,RQ(1.44±1.16)](t=3.646 P=0.001;t=6.185 P<0.001).Compared with the BCG priming PBS control group(0.38±0.38)%the frequency of spleen Th17 cells were also significantly increased(t=-0.280,P=0.048)in the BCG-primary HBHA boost group(1.02±0.34)%.In addition,HBHA boosting could promote better formation of the tertiary lymphoid structures in the lung,and decrease the bacterial load on the early stage after BCG challenge.Collectively,mucosal delivery of HBHA can effectively enhance the protective effect after BCG vaccination,and it is a potential candidate vaccine component.