矿化减毒活疫苗—— 一种新型肺炎链球菌减毒活疫苗的构建及其免疫保护机制研究

Immunogenicity and thermal stability is the focus of vaccine development. New advances in biomimetic mineralization have created the vaccine products,such as yeast cells and virus coated with an artificial mineral,which improved their immunogenicity and thermal stability. However，the techniques have not been used in the development of live vaccines. Therefore, we intend to rationally integrate calcium ion high-affinity peptides onto the surface-expressed protein of live attenuated pneumococcal vaccine SPY1-△lytA,which is lack of autolysis activity, by reverse genetics so that calcium phosphate mineralization could be biologically induced onto vaccine surfaces under physiological conditions, generating a mineral exterior.The mineral phase of vaccine can be observed by scanning electron microscopy, transmission electron microscopy and energy dispersive X-ray spectroscopy; the thermal stability of vaccine can be evaluated by observation of the growth of the heat -treated strain and the protective effect;the immunogenicity of the mineralized vaccine can be evaluated by comparing its protective effect with that of unmineralized vaccine; the mechanism of the increase of the immunogenicity and protective effect induced by mineralization can be preliminarily clarified by a series of experiment in vitro and in vivo. This research will not only provide theoretical and experimental basis for further development of highly efficient and stable streptococcus pneumoniae vaccines but also provide a novel idea for developing other kinds of live vaccines which are potent and stable.

免疫原性和热稳定性是疫苗研发的重点。近年来采用仿生矿化技术在活酵母和病毒表面形成一矿化层后，其热稳定性和免疫原性均得以提高，但尚未见到对活细菌疫苗进行矿化的报道。我们前期获得了一株保护效果优良的肺炎链球菌活菌疫苗SPY1，为提高其热稳定性并进一步增强其免疫原性，本项目拟依据反向遗传学原理，采用DNA重组技术，将对无机钙离子具有高亲和力的肽段，插入无自溶活性的SPY1菌株（SPY1-△lytA)的表面蛋白中，使其在细菌表面表达，于富钙环境中培养，使该疫苗菌株 “穿上一层矿化外衣”。通过电镜、能量色散X射线光谱仪等检测疫苗菌株矿化情况及矿物成分；通过热处理后菌株的生长情况和保护效果评价其热稳定性；比较矿化前后疫苗菌株的保护效果、通过体内外实验阐明矿化前后疫苗菌株免疫原性差异产生的机制。本项目的完成，不但为肺炎链球菌活菌疫苗的进一步开发奠定基础，同时也为研制高效稳定的其他活菌疫苗提供新的思路。

研究背景：免疫原性和热稳定性是疫苗研发的重点，近年来采用仿生矿化技术在活酵母和病毒表面形成一矿化层后，其热稳定性和免疫原性均得以提高，但尚未见到对活细菌疫苗进行矿化的报道。我们对前期获得的一株保护效果优良的肺炎链球菌活菌疫苗SPY1进行矿化处理，以提高其热稳定性并进一步增强其免疫原性。.研究内容：①初步探讨了SPY1△lytACaPi免疫保护效果及机制，尽管前期通过原位矿化制备的SPY1△lytACaPi矿化效果不稳定；②构建多株在不同部位整合有多种矿化肽的SPY1△ lytA菌株，对其进行矿化处理；③ΔA146PLY是肺炎链球菌溶血素（Ply）的无溶血活性突变体，已被本课题组证明是一种安全高效的肺炎链球菌候选蛋白疫苗。通过原位矿化对该蛋白进行矿化处理，并研究生物矿化对该蛋白的稳定性和免疫原性的影响。.研究结果：①原位矿化制备的SPY1△lytACaPi的动物实验显示，无论是粘膜免疫还是皮下免疫，均提示矿化菌的免疫保护作用得以提高，表明CaPi既可作为细菌的保护外壳，也具有佐剂效应，可以诱导体液免疫和Th17细胞免疫。②成功构建了多株在不同部位整合有多种矿化肽的SPY1△ lytA菌株，先后采用多种方法进行矿化处理，反复进行了多次实验，依然没有获得理想矿化效果的菌株。③通过原位生物矿化技术对其进行生物矿化后，ΔA146PLY蛋白可以形成具有矿化外壳的近球形的纳米颗粒，该纳米颗粒具有一定的抗蛋白酶K降解能力和高温耐受性，高温处理并不影响其免疫原性。.科学意义：本研究提示，对于表面积较大的细菌进行矿化处理有难度，而对于含酸性氨基酸较多的蛋白质进行矿化处理则比较容易。对蛋白质进行矿化处理，可提高其耐酶性和耐热性，且免疫原性不受影响。本项目为稳定性疫苗的制备提供了有价值的实验依据和理论参考。