用佐剂调节肺组织定居记忆T细胞的平衡是解决呼吸道合胞病毒疫苗安全性问题的新策略

Respiratory syncytial virus (RSV) is the most important pathogen of lower respiratory tract infection of infants and young children worldwide. Key reason of no licensed vaccines is the problem about the safety of vaccines. Children that were administered the FI-RSV vaccine exhibited a more severe pulmonary inflammatory illness, which is called vaccine-enhanced disease (VED). Immune memory is the conceptual basis for efficacious vaccines. Tissue-resident memory T cells (TRM) is a new subset of memory T cells, which permanently resides in peripheral non lymphoid tissue, such as the lung, with the strongest functions. TRM plays an important role in immune protection and immunoregulation for inflammatory response in local tissues. Our previous studies found that unbalanced immune memory response induced by RSV vaccine was important pathogenesis of VED; Adjuvants could promote RSV vaccine to induce lung TRM, and the TRM was involved in pulmonary inflammation following RSV challenge. So, we ask whether adjuvants prevent VED by regulating the balance of the vaccine-induced TRM. In this study, we will investigate the regulating effect of adjuvants on the balance of TRM in mice immunized with novel adjuvants and inactivated RSV vaccine, and study the role of TRM with certain balanced state in preventing VED through adoptive transferring TRM or inhibiting development of TRM with inhibitors. In addition, we will in vivo and in vitro investigate the mechanism by which adjuvants regulate the balance of lung TRM, and the mechanism by which activated TRM inhibits excessive inflammation through testing cells and molecules in pulmonary microenvironment, and removing alveolar macrophages. The results will provide a novel strategy to solve the security problem of RSV vaccines.

呼吸道合胞病毒（RSV）是婴幼儿下呼吸道感染的最重要病原，目前尚无疫苗上市的关键原因是安全性问题，接种疫苗者在感染RSV时发生更严重的肺部炎症疾病，即疫苗增强性疾病（VED）。免疫记忆是疫苗的理论基础，组织定居记忆T细胞（TRM）是定居于肺等非淋巴组织的记忆细胞新亚群，其功能强大，对该组织有重要免疫保护和调节炎症作用。我们前期研究发现记忆应答不平衡是VED的重要发病机制；佐剂促进RSV疫苗诱导TRM，且TRM与肺部炎症相关。那么佐剂是否通过调节疫苗诱导的TRM平衡来预防VED呢？本项目拟用多种新型佐剂与灭活疫苗免疫小鼠，研究佐剂对TRM平衡的调节作用；通过过继转移及抑制剂阻止TRM形成等方法，研究不同平衡状态TRM在预防VED中的作用；通过分析肺微环境中细胞和分子成分及删除肺泡巨噬细胞等方法，体内外研究佐剂调节肺TRM平衡，及TRM激活后抑制过分炎症的机制；为解决疫苗安全性问题提供新思路。

选取三大类佐剂：四种TLR配体型佐剂，Notch信号激动剂和抑制剂，及铝盐佐剂；单独或适当组合后与FI-RSV联合免疫小鼠，评价佐剂对疫苗增强性疾病的调节作用。结果发现：有的佐剂可部分抑制疫苗增强性疾病，有的抑制效果良好（如LPS），有的反而有增强作用（如CpG）；而CpG与L685,458联合作为佐剂效果非常理想，显著抑制了疫苗增强性疾病。.以L685,458+CpG作为重点佐剂研究了FI-RSV 疫苗是否诱导组织定居记忆T 细胞（TRM），以及佐剂对TRM平衡的调节，结果显示：CpG+L685,458促进TRM的形成，且促进诱导CD8+TRM亚群，该TRM经体内外刺激后可迅速表达活性分子，表明佐剂可以调节TRM的亚群和活性。.佐剂对TRM的调节是否通过调节肺微环境中固有免疫细胞而实现呢？结果发现:不同佐剂均导致记忆相的巨噬细胞和DC增加，CpG+L685,458促进诱导CD103+DC，而Al(OH)3促进CD11b+DC亚群，且FI-RSV+CpG+L685,458组DC和巨噬细胞经体内外刺激后活性更强，与TRM的活性一致，提示佐剂通过调节肺部微环境中的固有免疫记忆细胞的亚群和活性来调节TRM。.用过继Th1细胞调节TRM类型或用FTY-720抑制TRM形成，均显著减轻了肺部炎症病理。单独过继转移来自FI-RSV+Al(OH)3组的TRM，RSV感染后呈现TH17优势应答和中性粒细胞增加，病毒复制未被抑制；该TRM与本组记忆DC联合过继即可产生增强的炎症，而与来自FI-RSV+CpG+L685,458组的记忆DC联合过继则可减轻炎症，但两种组合均未明显抑制病毒复制，但将TRM换为来自FI-RSV+CpG+L685,458组的，则病毒显著被抑制，表明不同佐剂调节的记忆DC与TRM协同产生不同效果，且DC在调节炎症中发挥重要作用，而TRM亚群在抑制病毒复制中发挥重要作用。另外用组蛋白甲基转移酶抑制剂抑制记忆DC形成可抑制疫苗增强性炎症。所有这些结果表明：TRM的平衡状态影响RSV疫苗增强性疾病的严重程度和病毒复制，且不同佐剂通过对记忆DC与TRM亚群和活性的调节而影响疫苗增强性疾病。.本研究的重要结果将指导我们科学合理地设计疫苗佐剂，以提高疫苗安全性。