不定型流感嗜血杆菌OMP P6和PD优势T-B联合表位鉴定及其MAP候选疫苗的保护性

Haemophilus influenzae (Hi) is a common pathogen in children, causing community-acquired pneumoniae, otitis media and upper respiratory infections. Most common Hi infections are caused by non-typeable Hi (NTHi) strains whereas the current Hi vaccine (Hib) provides no protection because NTHi strains are noncapsulated, and there is no other vaccine for NTHi available. Outer-membrane protein P6 (OMP6) and PD are two important surface antigens in NTHi and have showed to be protective in animal models. However, they are not very immunogenic when tested in human trials. The immunogenicity of these two proteins can be improved through screening effective antigen epitopes and making multi-antigen presentation. In the current proposal, the T and B cell (T-B) common epitopes for OMP6 and PD were analyzed by software predictions. We will screen these epitopes through a phage display library and an overlap peptide library. Positive epitopes will be further validated by their ability to induce Th1 and Th2 cytokines in mice, measured by flow cytometry and ELISA. MAP will then be made from selected epitopes and tested in animal models for its protection. Successful completion of this project will pave the road for developing a MAP vaccine for NTHi and have significant implications for vaccine development for other bacteria infections.

流感嗜血杆菌(Hi)是社区获得性肺炎、儿童中耳炎和上感的常见病原，当前不定型Hi(NTHi)已成为主流菌型，现有Hib疫苗对其无保护性，至今尚无NTHi疫苗产品。外膜蛋白P6和PD是NTHi的优势表面抗原，在动物中具有良好保护性，但在人中免疫原性弱，而通过筛选有效的抗原表位可研制出高效安全的基因工程疫苗。本项目中，我们预测了NTHi外膜P6和PD的T-B联合表位，采用噬菌体展示、重复肽文库及ELISA技术，确定P6、PD中有效T-B联合表位；采用流式细胞及ELISA法检测不同抗原表位诱导Th1型和Th2型细胞因子的水平及其在患者中的抗体滴度，确定诱导抗感染免疫应答的Th1型T-B联合表位；制备P6和PD有效T-B联合表位的多靶点MAP候选疫苗，并通过动物实验研究其保护性。本研究为NTHi MAP疫苗产业化开发提供依据，具有较高学术价值、创新性和应用前景。

流感嗜血杆菌(Hi)是社区获得性肺炎、儿童中耳炎和上感的常见病原，当前不定型Hi(NTHi)已成为主流菌型，现有Hib疫苗对其无保护性，至今尚无NTHi疫苗产品。外膜蛋白P6和PD是NTHi的优势表面抗原，在动物中具有良好保护性，但在人中免疫原性弱，而通过筛选有效的抗原表位可研制出高效安全的基因工程疫苗。本项目中，我们预测了NTHi外膜P6和PD的T-B联合表位，通过生物合成12条表位肽，其中来自P6蛋白有4条，来自PD抗原有8条，免疫小鼠后检测血清中相应表位肽抗体水平，以及以这些表位肽为抗原检测NTHi感染患儿恢复期血清中相应抗体水平，根据免疫后小鼠中相应抗体的增高幅度、NTHi感染儿恢复期其抗体上升的幅度，初步筛选出6条免疫原性较高的表位。本研究健康对照组中，新生儿期P6、PD及其相应表位肽抗体水平最低，1月龄后抗体水平迅速增加，到7月-3岁达最高峰，4-6岁依旧维持在较高水平。NTHi感染后7～14天患儿血清中相应表位肽抗体水平明显增高，以抗P6-61、P6-123和PD-167抗体增高幅度最大。将筛选出的这三条表位重复串联后偶联BSA，联合氟氏佐剂免疫小鼠后，分析血清中细胞因子水平，并再次以同一抗原刺激小鼠脾脏细胞进行增殖活性研究和脾脏细胞产生细胞因子能力分析，结果发现P6-123和PD-167两条表位肽再次以8μg/ml～16μg/ml刺激后脾脏细胞增殖活性上升最明显高于对照组，且其上清液中Th1型细胞因子与对照相比有明显增高。该结果提示P6-123和PD-167是NTHi外膜蛋白中能有效诱导抗感染免疫应答的Th1型T-B联合表位，重复串联并与BSA偶联后免疫小鼠能产生一定的免疫保护。本研究为NTHi MAP疫苗产业化开发提供依据，具有较高学术价值、创新性和应用前景。

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