暴露N端对趋化因子LTN黏膜佐剂作用的影响

Exposed N-terminus of chemokine LTN is important for its mucosal adjuvant effects

我们前期构建了编码CVB3结构蛋白VP1的真核表达质粒(pVP1),通过滴鼻免疫诱导了一定水平的细胞和体液免疫应答。为了进一步增强其免疫效果,我们在该黏膜疫苗中引入新型黏膜佐剂LTN,发现当两种质粒共免疫时可显著增强pVP1诱导的CVB3特异性血清IgG和黏膜IgA水平,促进脾脏及黏膜局部IFN-γ+T细胞产生,显著减轻CVB3感染小鼠心肌病理损伤。随后我们对该疫苗体系进行了优化,使用双顺反子形式将编码VP1和LTN的基因构建在同一质粒上,通过滴鼻免疫小鼠后同样获得了免疫增强作用;当尝试将LTN N端融合至VP1蛋白,以该融合质粒pVP1-LTN滴鼻免疫小鼠后发现其增强免疫应答的能力显著下降;为排除融合蛋白空间构象改变及空间位阻对LTN佐剂功能的影响,我们在蛋白连接处引入柔性接头肽段(G4S)3,构建了融合质粒pVP1-IRES-LTN,发现该疫苗增强全身及黏膜局部特异性抗体细胞免疫应答的能力也非常有限,与pVP1-LTN相比并无明显差异,因此不能有效清除心肌病毒和减轻心肌损伤,提示融合蛋白的空间位阻或构像改变并非是影响LTN佐剂效应的关键因素,而暴露的LTN N端对其增强全身及黏膜局部免疫应答强度、提高心肌炎的免疫保护作用十分关键。

We previously reported that intransal DNA vaccine encoding CVB3 structure VP1 could induce specific cellular and humoral immune responses.To further enhance the immunoprotection of pVP1,chemokine LTN was introduced as a novel mucosal adjuvant.When monocistronic pLTN was intranasally administrated with pVP1,significantly enhanced levels of CVB3 specific serum IgG and fecal IgA,increased splenic and mucosal IFN-γ＋ T cell frequency,and improved myocardial histophathogical changes were evidenced.To optimize the vaccine system,we constructed a bicistronic DNA encoding VP1 and LTN simultaneously and also witnessed the immunoopotentiating effects.However,when we fused N-terminus of LTN to VP1 and obtained pVP1-LTN,no expected enhanced immune responses were achieved.To exclude the influences of conformational changes and steric effects of fusion protein on LTN adjuvant function,we inserted a flexible adaptor linker（G4S）3 between VP1 and LTN and got pVP1-linker-LTN vaccine,but no improved immune-enhancing effects were showed,indicating that the exposed N-terminus is critical to LTN adjuvant functions.