日本血吸虫糖基分子优先诱导抗糖基IgG抗体产生的机制及其在疫苗选择中的运用

The effectiveness of vaccines is based largely on its ability to induce a long-lived protective adaptive immune response in advance before infection occurs. Although vaccines have successfully applied in the prevention of viral and bacterial infections, the search for an effective vaccine against scbistosomiasis, a parasitic disease currently affecting over 200 million people, remains a desirable but as yet challenging and elusive goal. Our results showed that the IgG antibodies induced by Schistosome japonica egg antigens (SEA) immunization mainly recognize glycan epitope but not protein epitope present in the SEA. The IgG antibodies against the protein epitope is induced only when deglycosylated SEA is used for immunization. Furthermore, the IgG antibodies present in infected mice also demonstrated a preferred recognition with the glycan molecules comparing to proteins. Therefore the glycan structures present in the schistosomes appear to possess a privileged right to stimulate the host to produce anti-glucan IgG. If this is true, it will indicate that the protective immunity induced during the schistosome infection may not be caused by protein antigens and thus the inappropriateness of testing protein-based molecule as vaccine candidates in prevention of schistosomiasis...In this study, we proposed to investigate if this observation is also true in cecariae and adult worms. Following cloning and expression of relevant glycoprotein, the monoclonal antibodies against protein part of the glycoprotein will be generated and will be used to purify the relevant molecules from the crude antigens from cercariae, egg or worms. Using the purifed antigens, we answer questions like if the response is long-lasting, if the response is carrier (protein)-dependent and its glycan structure basis and immune mechanisms. We hope our study will provide a new pathway by which a complex glycan can independently induce long-lasting adaptive immune response and to improve the vaccine design against any germs carrying complex glycans like schistosomes.

使未感染个体接触保护性抗原，诱导并预存与病原自然感染所诱导的相同的保护性免疫应答是疫苗作用的基础。尽管已有许多成功抗病毒和细菌的疫苗，但尚无有效的针对寄生虫如日本血吸虫的疫苗。我们实验室发现日本血吸虫卵抗原（SEA）免疫小鼠所诱导的IgG抗体，主要是针对糖基表位而非蛋白表位；去糖基后的SEA才得以诱导抗蛋白表位抗体。而血吸虫感染小鼠中的抗SEA IgG抗体也主要是针对糖基表位。因此SEA所携带的复杂糖基似乎优先诱导抗糖基IgG抗体，提示血吸虫自然感染所诱导的优势保护性应答可能非蛋白所致。本课题拟研究该现象是否也存在于针对尾蚴和成虫的抗体应答中，是否有记忆性，是否是蛋白载体依赖，是否依赖于特定的糖基结构及宿主免疫应答机制。并用纯化的尾蚴和成虫的糖基化抗原分子，验证其作为预防性及治疗性疫苗的效果，从而提出某些糖基分子可独立诱导具有记忆的免疫应答并将其应用于含有复杂糖基修饰病原类的疫苗设计中。

研究背景：我们实验室发现日本血吸虫感染的病人和小鼠及日本血吸虫卵抗原（SjEA）免疫小鼠所诱导的SjEA 特异性IgG抗体，主要是针对糖基表位的抗体(与蛋白表位相比)。由于针对蛋白的血吸虫疫苗一直效果不好，所以我们提出假说：血吸虫自然感染所诱导的优势保护性应答可能是糖复合物所致。并提出研究虫卵中糖基表位优势诱导IgG抗体的宿主机制，以及研究该现象的结构基础。.研究内容：首先利用经典的高碘酸处理及植物血凝素结合检测方法，明确虫卵SjEA所含的糖基是否都是高碘酸敏感类糖基。利用高碘酸和蛋白酶处理后的SjEA，竞争抑制感染后IgG和SjEA的结合，从而解析血吸虫感染中优势诱导的抗糖基IgG抗体是针对高碘酸敏感的糖基还是高碘酸不敏感的糖基。使用针对OVA的TCR转基因鼠DO11.10，去或不去蛋白的SjEA免疫小鼠，研究机体产生抗糖基抗体对相应T细胞克隆的依赖性及载体效应。最后通过比较IgG和高碘酸处理与未处理的SjEA的结合能力，筛选出可能的抗虫卵糖基的单克隆抗体。进而观察单抗是否丧失和O糖苷酶或N糖苷酶处理过的SjEA的结合能力，明确并获得抗虫卵糖基单克隆抗体2株。观察感染前注射这些抗虫卵糖基单克隆抗体是否提供小鼠抵抗血吸虫感染的能力。.重要结果、关键数据及科学意义：发现在日本血吸虫卵所含的糖基化修饰中，不仅有传统的高碘酸敏感糖基，还有高碘酸不敏感糖基，从而纠正了文献中将高碘酸处理等同为去糖基化的错误认识。实验结果还揭示了高碘酸不敏感糖基与高碘酸敏感糖基一样，都具有免疫原性。所获得针对高碘酸不敏感糖基与高碘酸敏感糖基的单克隆抗体各一株。最后通过注射单克隆抗体发现，识别高碘酸敏感和不敏感糖基的单克隆抗体都提供显著保护力，因此为在预防血吸虫这类富含糖基的微生物感染中，将糖基作为重要疫苗候选分子提供理论基础。