日本血吸虫疫苗分子的筛选和病毒样颗粒疫苗的构建

Schistosomiasis remains a major public health problem worldwide. A protective vaccine represents an important strategy for long-term control of schistosomiasis. However, the schistosome antigens involved in the protective immunity are largely unknown, and the vaccine construction approaches still need to be improved. The secreted proteins play important roles in the host-parasite interactions and are the pool of diagnostic and vaccine candidates. In the previous study, we successfully established a GST-fusion protein-based high-throughput screening technology containing 204 Schistosoma japonicum putative secreted proteins, and identified dozens of diagnostic candidates. In this study, we will utilize this technology to identify vaccine candidates. The antigens which are involved in humoral protective immunity induced by radiation-attenuated cercariae will be selected via this high-throughput screening technology. The potential Th1-type epitopes of the selected antigens will be identified by a combination of in silico analysis with experimental validation. The vaccine candidates will be cloned into the L1 major capsid protein of human papillomavirus type 16 and expressed as chimeric proteins. The virus-like particle of chimeric proteins will be assembled in vivo or in vitro. The immunogenicity and protective efficacy of the virus-like particle vaccines will be evaluated in mice. This study will demonstrate the roles of secreted proteins in the protective immunity against schistosome infection and provide a potential novel approach for the schistosomiasis vaccine development.

血吸虫病是严重危害人类身体健康的重大寄生虫病。疫苗是预防血吸虫感染最理想的方法，但目前与血吸虫免疫保护相关的关键抗原尚需进一步鉴定，疫苗构建方法也有待改进。分泌蛋白在血吸虫与宿主的相互作用中发挥多方面的作用，是重要的抗原库。申请人前期克隆了204个日本血吸虫分泌蛋白，并通过高通量筛选技术鉴定到了数个诊断分子。本课题将利用此技术，比较辐照致弱尾蚴免疫和正常尾蚴感染小鼠后免疫应答差异，鉴定与体液免疫保护相关的抗原；获得的阳性抗原，再通过软件预测T细胞表位并经实验验证，进一步筛选能刺激CD4+T细胞分泌IFN-γ的疫苗候选分子。疫苗候选分子将嵌合表达在人乳头瘤病毒16主要衣壳蛋白L1上，在体内或体外模拟病毒颗粒组装条件，制备日本血吸虫病毒样颗粒疫苗，并在小鼠中评估该疫苗的保护效果与机制。本课题预期结果不仅能阐明分泌蛋白在血吸虫免疫保护中的作用，而且为血吸虫病疫苗的研制提供新思路。

日本血吸虫病仍然是严重危害我国人民健康的重大寄生虫病，疫苗是预防血吸虫感染的有效方法，但尚需鉴定高效的疫苗分子和创新疫苗构建方法。本研究中，我们建立了致弱尾蚴免疫和正常尾蚴感染小鼠模型，利用GST融合日本血吸虫分泌蛋白抗原筛选技术，比较了两种模型中不同分泌蛋白的特异性抗体水平，从中鉴定到10个抗原的抗体在致弱尾蚴免疫血清中显著升高。我们对其中6个抗原进行了重组表达，并在小鼠中评价了其免疫保护研究。这6个抗原均能诱导较强的免疫应答，但免疫保护水平和对照组相比无显著差异。为了提高抗原的免疫保护效果，我们选择其中一个抗原SjSP-13，将其嵌合表达在HPV 16 L1蛋白中，在酵母中表达制备重组蛋白，并通过自组装得到SjSP-13/ HPV-16 L1拟病毒颗粒。透射电镜表明，拟病毒颗粒大小在50nm左右。我们在小鼠中比较了拟病毒颗粒和SjSP-13单分子重组蛋白的免疫原性和诱导的免疫保护效果。拟病毒颗粒免疫组SjSP-13特异抗体滴度为766,000 ± 38,807，高于重组蛋白组(662,000 ± 50,040)，但差异不显著。尾蚴攻击感染实验表明，拟病毒颗粒免疫组平均虫荷为23.4 ± 1.3，显著低于重组蛋白组(29.8 ± 1.3)和弗氏佐剂组(32.4 ± 1.6) (p<0.05)；拟病毒颗粒组平均肝脏虫卵EPG (Eggs per gram)为22,500 ± 866，显著低于重组蛋白组(33,300 ± 2,267)和弗氏佐剂组(37,300 ± 2,131) (p<0.05)；重组蛋白组的成虫负荷和虫卵EPG与弗氏佐剂组相比无显著差异。以上结果表明，SjSP-13/HPV-16 L1 拟病毒颗粒诱导的免疫保护效果显著高于SjSP-13单分子重组蛋白，提示构建病毒样颗粒疫苗是提高血吸虫抗原免疫保护效果的有效方法。