TLR7激动剂通过受训免疫促进慢性HBV感染清除的抗病毒效应及其机制研究

Hepatitis B virus infection is hard to clear cause there is still no sufficient method to reconstruct host specific immunity on patients with chronic hepatitis B. Toll-like receptors could induce the innate immune response which enhance the specific immunity facilitating the pathogen clearance. Our preliminary studies showed that the TLR7 ligand injection could increase the IFN-γ production of splenocytes for a period of time, and affect the differentiation of CD8+T cells in liver and spleen. In the HBV mouse model, pre-activation with TLR7 agonist could accelerate HBsAg clearance. Moreover, the frequency and cytokines production of HBV-specific CD8+T cells in TLR7 mice were increased. Based on these data, we speculated that pre-treatment with TLR7 agonist might induce the trained immunity in host immune system, which may facilitate the clearance of subsequent HBV infection. To address this hypothesis, we will enroll the immunological technology (e.g. in situ cells perfusion and isolation, adoptive immunotherapy ) to demonstrate the effect of TLR7 pre-activation on host immune status in the mouse model with acute or chronic HBV infection. Furthermore, we want to explore the effect and mechanism of TLR7 agonist on elimination of HBV infection. These findings hopefully would elucidate the immune mechanism of TLR7-mediated HBV clearance upon trained immunity, and provide the experimental and theoretical evidences for clinical application of TLR7 ligands in the immunotherapy of chronic HBV infection.

慢性乙型肝炎（CHB）患者目前仍缺乏有效手段重建宿主特异性免疫应答，导致乙肝病毒（HBV）难以清除。Toll样受体（TLR）可诱导天然免疫应答促进机体对病原体感染的特异性免疫。我们前期研究发现小鼠注射TLR7配体可增强脾脏淋巴细胞分泌IFN-γ和影响肝脾内CD8+T细胞亚群的分化；采用HBV质粒建模的结果显示，TLR7预活化后的小鼠可加快表面抗原（HBsAg）的清除，其肝内HBV特异性CD8+T细胞的频数与功能也显著增加。由此我们推测，TLR7的预活化可能诱导宿主产生受训免疫，从而促进后续HBV感染的清除。本项目拟采用原位细胞灌注分离、过继免疫等技术，在HBV急、慢性感染小鼠模型中阐明TLR7预活化对宿主免疫状态的影响，并进一步研究其诱导HBV感染清除的效应及机制。本研究有望从受训免疫这一新视角，揭示TLR7介导HBV清除的免疫机制，为TLR7配体用于慢乙肝免疫治疗提供理论依据。

慢性乙型肝炎（CHB）功能性治愈需要有效重建宿主特异性免疫应答，但目前单一抗病毒治疗无法清除乙肝病毒（HBV），联合/序贯免疫治疗是潜在的治愈策略。Toll样受体（TLR）可诱导天然免疫应答促进机体对病原体感染的特异性免疫。在抗病毒治疗有应答的患者中，治疗前外周血单个核细胞TLR7表达水平显著升高；故本研究利用HBV质粒构建小鼠模型，结果显示TLR7预活化后可促进小鼠血清表面抗原（HBsAg）的清除，其肝内HBV特异性CD8+T细胞的频数与功能也显著增加，肝组织RNA-Seq结果提示多条免疫应答激活的通路富集。进一步体外实验结果显示TLR7激动剂可增强脾脏淋巴细胞非特异性免疫应答，诱导肝脾内CD8+T细胞亚群的分化，且脾脏巨噬细胞可促进细胞因子产生；脾脏荧光染色提示TLR7预活化后巨噬细胞增多，且向M1极化；脾脏RNA-Seq结果提示代谢及免疫多条通路富集。.综上，本研究发现TLR7激动剂可通过趋化巨噬细胞并诱导其M1极化，使天然免疫受训，从而促进后续HBV感染的清除。本研究从受训免疫这一新视角，揭示TLR7介导HBV清除的免疫新机制，同时为TLR7激动剂用于慢乙肝免疫治疗提供了充分依据。