艰难梭菌脂磷壁酸相关寡糖缀合物疫苗的设计、合成与生物活性研究

Infectious diseases with Clostridium difficile (CD) usually cause high rates of morbidity and mortality in the clinic, which have been becoming a rapidly growing problem in healthcare worldwide. Thus, the development of safe and efficient vaccine strategy for prevention and treatment of CD infections is currently hot topic. More recently, the cell surface polysaccharides from C. difficile have been structurally characterized and identified as promising antigenic epitopes for prophylactic vaccine development. This research project aims at developing of semi- and fully synthetic glycoprotein and glycolipid vaccines based on the artificial lipoteichoic acid (LTA)-based glycans of C. difficile as target antigens and recombinant ScpA193 protein and MPLA derivative as novel carriers. For this purpose, the structurally well-defined LTA oligosaccharide antigens with different chain lengths and functional groups will be first chemically synthesized using highly convergent glycosylation strategies, followed by incorporation with carrier molecules via a bifunctional activated glutaric ester as linker, to furnish the synthetic oligosaccharide-based conjugate vaccines. Moreover, the immunobiological studies on these synthetic conjugates will be detailedly investigated by mice immunization, antiserum titration and binding, antibody-mediated complement-dependent cytotoxicity, and the protection of mice against CD infections, respectively, so as to find the promising oligosaccharide antigen epitopes for the development of LTA-based vaccines against C. difficile infectious diseases. This research will not only yield novel semi-/fully synthetic glycoconjugates or promising lead compounds worthy further investigation, but also provide with the structure-activity relationships (SARs) of the synthetic glycans as target antigens to guide further optimization of anti-C. difficile vaccine design. The result will have a major impact on antibacterial research.

艰难梭菌感染疾病在临床上具有高的发病率和死亡率，是全球所面临地新的公共卫生问题。发展安全有效的艰难梭菌疫苗受到科研工作者的密切关注。近年来，随着艰难梭菌细胞表面多糖结构的解析与鉴定，基于这些多糖结构的寡糖缀合物疫苗的研制成为当前热点课题。本申请项目拟采用系列人工合成的艰难梭菌脂磷壁酸（LTA）相关寡糖链为抗原，结合最新发展的ScpA193蛋白和MPLA为载体分子，研发新型、高效和具有明确结构的抗艰难梭菌的半合成LTA寡糖蛋白缀合物疫苗和全合成LTA寡糖脂复合物疫苗。项目研究方法是采用化学法合成一系列具有明确结构的各种长度不一的LTA 寡糖半抗原，通过双活化酯连接臂与载体分子缀合，制备一系列具有确定糖抗原结构的新型LTA 寡糖缀合物，并进行相应的生物免疫学和抗菌活性的研究，阐明不同结构的糖缀合物疫苗间的构效关系，筛选最佳的LTA寡糖半抗原，从而为发现新型抗艰难梭菌糖缀合物疫苗奠定实验基础。

艰难梭菌（Clostridium difficile，CD）感染疾病在临床上具有高的发病率和死亡率，是全球所面临地新的公共卫生问题。发展安全有效的艰难梭菌疫苗受到科研工作者的密切关注。本项目选用艰难梭菌脂磷壁酸（LTA）为研究对象，通过化学法合成了一系列具有明确结构特征的各种长度不一的LTA寡糖半抗原，包括三糖、五糖和七糖半抗原；构建表达了系列C5a肽酶的功能结构域，对它们进行较详细的免疫活性研究，筛选到具有免疫增强活性的rsScpA193蛋白，可作为新型载体分子用于糖缀合物的制备；构建表达了具有潜在广谱保护作用的融合蛋白YAPO和YAPL，证实了它们在递呈寡糖半抗原免疫原性方面的免疫增强潜力；通过双活化酯连接臂将上述LTA寡糖半抗原与具有免疫增强活性的rsScpA193新型载体蛋白进行缀合，制备了系列具有确定结构特征的半合成LTA寡糖-rsScpA蛋白缀合物，同时与含有6’-氨基的新型单磷酰类脂A（MPLA）载体进行偶合，制备了系列具有确定结构特征的全合成LTA寡糖-MPLA糖脂复合物；对上述制备的LTA寡糖-rsScpA蛋白缀合物进行了初步的小鼠体内免疫活性评价，结果表明所获得抗血清中诱导产生了诱导了T细胞依赖的特异性IgG抗体免疫应答，且LTA五糖-rsScpA和七糖-rsScpA蛋白缀合物能够诱导产生更强的IgG抗体滴度。上述研究工作为后续开展抗血清与LTA菌体外结合和吞噬调理实验以及LTA菌体内冲击保护实验等免疫活性评价实验奠定了实验基础，且为进一步设计和优化下一代的新型半合成和全合成基于LTA结构的艰难梭菌糖缀合物疫苗提供实验依据。