基于菌影递送系统的梅毒螺旋体新型多表位疫苗的免疫保护效应及机制

High prevalence of syphilis and its serious damage to the adult and newborn's health urgently need to develop a highly effective vaccine against Treponema pallidum. Tp92, Tp0751 and Gpd are considered to be the most promise candidate vaccine molecules of outer membrane protein (OMP) of T. pallidum. However, neither current recombinant protein vaccines from foreign labs nor Tp92 and Gpd DNA vaccines build by us failed to induce complete protection when immunized with a single immunogen, which is maybe due to be a lack of synergy between immunogens, incorrect natural protein conformation, lack of a powerful antigen delivery system/adjuvant, ignoring the mucosal immunity, a homologous immunization strategy. Based our initial research, at epitope level, we plan to screen and identify immunodominant B, Th, or B-Th cell epitopes by immunological informatics,phage display technology and so on for constructing a concatemer composed of multiple immunodominant cell epitopes. Using a heterologous prime-boost vaccination strategy, challenge experiments were performed with T.pallidum in New Zealand rabbits immunized with bacterial ghosts (BG) multi-epitope DNA vaccine (prime) then a recombinant BG mulit-epitope vaccine (boost) to evaluate the degree of protection and immunological parameters were determined to elucidate the levels of the protective immune responses. Using the same vaccination strategy, other multivalent vaccines were firstly compared in protein-epitope, delivery systems then our and other lab's single immunogens were compared. Through this study, a new type multi-epitope vaccine against Treponema pallidum based on bacterial ghosts as a antigen delivery system which is able to induce high level mucosal and systemic Th1 bias humoral and cellar response and high effective protection is promising, laying the foundation for the development of syphilis vaccine for human use.

梅毒高发和严重危害成人与新生儿健康迫切需要高效疫苗研发。Tp92、Tp0751和Gpd是目前认为最有希望的梅毒螺旋体(Tp)外膜蛋白候选疫苗分子，但国外重组蛋白疫苗和我们首次构建的Tp92与Gpd核酸疫苗以单抗原均未能获完全保护，可能与目前免疫原单一、非天然构象、缺乏强效递送系统/佐剂、忽视黏膜免疫、同源免疫策略等有关。为此，本项目在我们前期研究基础上，深入到表位水平，拟先采用免疫信息学、噬菌体展示等技术，筛选鉴定各蛋白优势B/Th/B-Th细胞表位，构建含多个优势表位串联体的菌影多表位DNA疫苗和菌影多表位重组疫苗，先比较异源-同源免疫策略，再与以相同异源免疫的其它多价疫苗从蛋白-表位、递送系统比较，并与当前国内外单价疫苗比较，观察兔免疫保护性和探讨免疫保护机制，以期获得能诱导持久高水平黏膜和系统的Th1型偏向的体液免疫和细胞免疫应答和高效保护的新型梅毒疫苗，为人用梅毒疫苗研发奠定基础。

梅毒高发和严重危害成人与新生儿健康迫切需要高效疫苗研发。梅毒疫苗至今未能获得成功，除与Tp不能体外人工培养和基因操作、外膜蛋白稀少、缺乏内外毒素等特殊的生物学性状外，还与梅毒疫苗研究较其他病原体相对落后的疫苗方法学有关：单价重组蛋白为免疫原、未应用理想的抗原递送系统和佐剂、同源免疫接种策略、忽视黏膜免疫的保护作用。针对以上不足，本项目以目前候选的Tp外膜表面蛋白Tp0751、Tp92、TprF、Tp0155、Tp0483、TprI及TprC为靶点，（1）筛选鉴定了Tp0751、Tp92、TprF的B细胞和（或）T细胞表位；（2）分别应用基因裂解法和化学渗透压法构建了大肠埃希菌的空菌影(EBG)；（3）构建了黏附素Tp0751、Tp0155与Tp0483的单价以及Tp0751-TprI-TprC的三价原核菌影和真核菌影；（4）以重组菌影DNA初免-重组菌影蛋白加免的策略进行免疫，检测了各单价或多价蛋白的原核菌影和真核菌影及联合免疫小鼠，诱导特异性系统和粘膜的体液免疫和细胞免疫应答的水平。结果显示：（1）筛选出6个Tp92候选B细胞表位和3个人源性Th细胞表位及1个联合表位；2个Tp0751的B细胞表位和2个TprF的B细胞表位，未筛选出Tp0751与TprF的Th细胞表位。（2）电镜和DNA琼脂糖鉴定结果显示，基因裂解法和化学渗透压法均成功构建了空菌影EBG，裂解率达98%以上。（3）WB、电镜和DNA琼脂糖鉴定结果显示成功构建了Tp0751、Tp0155与Tp0483的单价以及Tp0751-TprI-TprC的三价原核菌影和真核菌影，各重组菌影的裂解率98%以上，各重组真核DNA对空菌影的装载率在70%以上。（4）各单价蛋白的原核菌影和真核菌影诱导高或较高水平的血清特异性IgG、IgG1、IgG2a和生殖道SIgA，小鼠脾细胞表达高或较高水平的IFN-γ，细胞增殖实验显示各实验组小鼠脾细胞高或较高增殖水平，上述指标的各初免-加免组较单独的真核菌影免疫组高。以上结果表明构建的各蛋白的重组菌影能诱导强或较强的系统和黏膜的Th1与Th2混合型免疫应答，本项目对研究新型的高效多价梅毒疫苗具有重要意义。