一种新型肺炎链球菌矿化纳米蛋白疫苗——疫苗的构建、热稳定性及其免疫保护效应研究

Streptococcus pneumoniae, leading to serious invasive infection, is the primary reason of deaths caused by bacterial disease which can be prevented by vaccine in children. However, the newly available pneumococcal conjugated vaccines still have shortcomings including serotype replacement and high prices, highlighting the urgency of development of novel vaccines, of which the focus is mucosal immunization with protein vaccine. In our previous research, either combination or fusion of △A146Ply, a mutant of pneumococcal protein Ply and protein DnaJ could induce protection by mucosal immunization without the aid of adjuvant. And the vaccine could elicit protection effect which was equal or better compared with that induced by the commercially available 23-valent polysaccharide vaccine, implying its potential of further development. However, the long-acting protection was still not satisfying. The mineralized nanoparticles preparation not only effectively enhances the immunological effect and memory of vaccine protein, but also improves the vaccine thermostability. Thus, in this study, We will utilize the biomineralization technique to insert the biomineralized peptides into appropraite positions in vaccine protein, making the formation of mineralized nanoparticles of vaccine protein through the deposition effect of mineral substance. The vaccine practicability will be evaluated in terms of thermostability and protection induced by mucosal immunization, and the activation of dendrtic cells, B cells and T cells by vaccine will be explored to investigate the protective mechanism, providing experimental foundation for further development of novel pneumococcal vaccines.

肺炎链球菌（Streptococcus pneumoniae，S.pn）可导致严重的侵袭性感染，是疫苗可预防性儿童细菌疾病的首要致死病因，但最新上市的S.pn结合疫苗仍存在血清型转换、价格昂贵等缺陷，急需开发新型疫苗，其中蛋白疫苗粘膜免疫是研发热点。课题组前期发现，S.pn溶血素Ply的突变体△A146Ply与DnaJ蛋白联合或融合，在无佐剂辅助的条件下粘膜免疫小鼠，即可诱导产生与市售23价多糖疫苗相当或更优的保护效果，有进一步开发的潜质。该疫苗长效保护作用尚不理想。矿化纳米制剂不仅能有效增强疫苗蛋白的免疫效应和免疫记忆，还可提高其热稳定性。本研究拟利用生物矿化肽的无机物沉积效应，将其插入疫苗蛋白的适当位置，从而使疫苗蛋白形成矿化纳米颗粒，从热稳定性和粘膜免疫保护作用方面评价纳米制剂的实用性，并从对DCs、B细胞和T细胞的激活等方面探索其免疫保护机制，为新型S.pn疫苗的研发奠定实验基础。

蛋白疫苗是新一代疫苗的发展热点，然而蛋白疫苗作为蛋白质，具有不耐热，易降解的缺点，其运输和保存都需要冷链运输，所需花费几乎占了疫苗成本的80%。因此研发疫苗蛋白的耐热耐水解制剂具有重要的实用价值。本研究以具有良好保护效应的肺炎链球菌疫苗蛋白为研究对象，主要为溶血素Ply、其无溶血活性突变体△A146Ply和DnaJ蛋白，采用矿化肽偶联的方式进行生物矿化，获得了具有一定耐热性和水解酶抗性的纳米颗粒。该研究结果显示，本项目采用的方法适用于不同氨基酸序列的疫苗蛋白。进一步的研究显示，矿化的纳米颗粒较可溶性蛋白对抗原提呈细胞的激活效应更好，对B细胞和T细胞免疫的诱导效应更强，对肺炎链球菌定植和致死性感染的保护效应也有所提高。同时我们发现未连接矿化肽的蛋白也可被生物矿化而表现出耐热性和水解酶抗性，并保留原有的免疫原性和免疫保护效应。. 我们还进一步探索了△A146Ply和DnaJ蛋白免疫保护效应机制，发现△A146Ply也具有皮下佐剂效应，其与DnaJ融合后皮下免疫小鼠，可有效激活小鼠的免疫保护效应，该效应依赖于TLR2和TLR4，TLR2主要参与调节Th1型免疫的激活，而TLR4则部分参与B细胞免疫和T细胞免疫的整体激活。同时，我们还筛选鉴定出了DnaJ蛋白的B细胞优势抗原表位。. 综上，本研究结果为耐热耐水解的蛋白药物制剂制备提供了有价值的数据支撑，同时也为肺炎链球菌△A146Ply和DnaJ蛋白的疫苗应用提供了更全面的实验支持。