猪痘病毒SPV009蛋白抑制宿主免疫应答的分子机制

As a potential vector for commercial vaccines, the safety and immunity of swinepox virus (SPV) should be further enhanced. Previous studies showed that, the virulence of the SPV009 gene deleted SPV NT1501 strain was decreased more than 50 times, while the levels of cellular immune responses and humoral immune responses were significantly increased. In this study, the effect of SPV009 protein and the SPV△009 virus on the activity of NK cells, the expression of complement components, MHC molecule, cytokines and co-stimulators were investigated to evaluate the inhibitory effect of SPV009 protein on host innate immunity and acquired immunity. On this basis, the key domains and the signal pathways of SPV009 protein inhibiting the host immunity were analyzed, and the interactions of SPV009 protein with MHC or TAP were also identified. Based on the above studies, the molecular mechanism of the SPV009 protein for suppression of host immune responses was elucidated. The results provide a theoretical guidance for the design and development of more safe and more efficient recombinant swinepox virus vector vaccines, and also provided references for other live vector vaccines development.

猪痘病毒（SPV）作为商业化活载体疫苗研发的潜在载体，其安全性和免疫效力需进一步提升。前期研究显示，猪痘病毒NT1501株缺失SPV009基因后，其毒力下降50倍以上，同时诱导的细胞免疫和体液免疫应答水平均显著升高，但机制不明。本研究以SPV009基因缺失重组猪痘病毒SPV△009以及SPV009蛋白为研究对象，通过检测其对NK细胞活性以及对C3b等补体成分、MHC分子、细胞因子、共刺激因子表达的影响，评价SPV009蛋白对宿主先天性免疫和获得性免疫应答的抑制作用。在此基础上，分析SPV009蛋白抑制宿主免疫应答的关键结构域以及相关信号通路，并验证其与MHC、TAP等效应分子的互作。通过上述研究，阐明猪痘病毒SPV009蛋白抑制宿主免疫应答的分子机制，为研发更加安全高效的重组猪痘病毒活载体疫苗提供理论指导，也为其他活载体疫苗的开发提供借鉴。

猪痘病毒（SPV）是一种理想的商业化疫苗载体，本项目构建了SPV009基因缺失的猪痘病毒SPV△009。与wtSPV相比，SPV△009 的毒力下降至少50倍。通过检测SPV△009对NK细胞杀伤活性、C3b补体成分以及IFN-α、IFN-β先天性免疫细胞因子的影响，证明SPV009蛋白可抑制宿主的天然免疫应答。通过验证SPV009蛋白抑制Th1型、Th2型细胞因子、MHC I、MHC II类分子的表达，证明SPV009蛋白可抑制宿主的获得性免疫应答。通过对SPV009蛋白进行截短表达，验证其第40-100位氨基酸片段是其抑制宿主免疫应答的关键结构域，该结构域包含1个E3泛素连接酶功能区，是SPV009蛋白抑制宿主天然免疫应答的关键结构域。通过对SPV009蛋白E3泛素连接酶功能区（SVRKPFSQW）内的赖氨酸残基（K59）、谷氨酰胺残基（Q63）进行定点突变，检测MHC I、MHC II类分子表达量的变化，验证了赖氨酸残基（K59）是影响猪痘病毒SPV009蛋白泛素连接酶活性的关键氨基酸。上述研究成果初步，阐明猪痘病毒SPV009蛋白抑制宿主免疫应答的分子机制，为研发更加安全高效的重组猪痘病毒活载体疫苗提供理论指导，也为其他活载体疫苗的开发提供借鉴。