表位嵌合型类病毒颗粒疫苗对慢性HBV感染的治疗作用与机制

Chronic hepatitis B virus (HBV) infection is a major global public health issue. Chronic HBV infection (CHB) can cause chronic hepatitis and places patients at high risk of death from liver cirrhosis (LC) and hepatocellular carcinoma (HCC). It is estimated that there are approximately 248 million individuals worldwide that are chronically infected with HBV. Worldwide, approximately 78,000 people die each year because of the acute or chronic consequences of HBV infection. The currently available drugs to treat hepatitis B virus (HBV) infection include interferons and nucleos(t)ide analogs, which can only induce disease remission and are inefficient for the functional cure of patients with chronic HBV infection (CHB). In our previous studies, we found a novel mAb E6F6 which could profoundly suppress the levels of HBsAg and HBV DNA in HBV mouse models. We have also identified the E6F6 binding epitope, displayed the epitope on a virus-like-particle carrier and therefor constructed a novel epitope-chimeric therapeutic vaccine against HBV infection. We have demonstrated that the new vaccine had significant anti-HBV effects, good immunogenicity and safety in various animal models. However, how such vaccine induce immune responses of high potency remains poorly understood. Based on these findings, we will perform a systematical analysis of immune responses induced by this vaccine in HBV mouse models, employing state of the advanced techniques such as Time of Flight Mass Cytometry (CyTOF), to identify the key cell populations and molecules that mediate the effective immune responses and anti-HBV effects induced by the new vaccine. The knowledge obtained from this study will facilitate the design of new therapeutic vaccines against persistent viral infections.

慢性乙型肝炎病毒感染是全球性的公共卫生问题，可导致慢性乙型肝炎、肝硬化和肝癌。据估计世界范围内有2.48亿慢性乙肝感染者，每年约78万人死于与HBV感染相关的各种疾病。现有治疗药物包括干扰素、核苷/核苷酸类似物，能控制疾病进展，但并不能有效实现功能性治愈。我们在前期研究中发现了能在HBV小鼠模型中高效清除HBV和HBsAg的E6F6抗体，成功鉴定出其识别表位，并将其展示于类病毒颗粒载体上，构建出了新型表位嵌合型治疗性乙肝疫苗。我们已经在多种动物模型中证明该疫苗具有显著的Anti-HBV治疗效果，良好的免疫原性和安全性，但是对该疫苗在机体中诱导的免疫应答过程和分子机制尚不清楚。本项目拟在HBV小鼠模型中，通过质谱流式细胞技术等先进工具解析该疫苗引发高效免疫应答并介导HBV清除的关键细胞亚群及效应因子，为针对持续性病毒感染的新型治疗性疫苗的设计提供理论依据。

慢性乙型肝炎病毒感染的治疗药物有限，治愈率低，仍然存在较大挑战。本项目在前期研究的基础上，利用多种动物模型对自主发展的表位嵌合类病毒颗粒乙肝治疗性疫苗CRT3-Seq13的治疗作用，应答情况和免疫机制进行了系统研究。我们的研究发现CRT3-Seq13免疫可在小鼠、大鼠、兔和食蟹猴等多种动物中靶向诱导针对sA表位的E6F6-like抗体，在HBV携带动物中可以诱导明显的外周和肝内HBsAg的降低。在联合治疗的探索中，我们发现ETV和TDF与CRT3-Seq13联合使用并不会造成相互干扰，且与TDF联用能够进一步提升CRT3-Seq13降低HBsAg效果。为了提升治疗效果，我们研发了具有自主知识产权的利塞磷酸锌铝复合佐剂(FH002C)，经过动物实验证实能够显著提升CRT3-Seq13的抗HBV效果，并进一步在重组新冠疫苗中进行了应用。为了深入解析CRT3-Seq13免疫应答和持续性HBV感染的免疫耐受机制，我们发展了E6F6的BCR转基因小鼠。在这一小鼠模型中，我们采用质谱流式和单细胞测序等技术揭示了外周高水平的HBsAg持续刺激可导致E6F6-B细胞上BCR信号转导的持续激活，在缺乏CD40-CD40L作为共刺激分子时，E6F6-B细胞分化为短寿型浆细胞，最终因为内质网压力而凋亡。浆细胞的快速减少最终导致了宿主外周Anti-HBs抗体水平的快速降低以及慢性感染的持续。针对HBsAg的免疫耐受作用，我们采用能降低HBsAg水平的治疗性抗体与CRT3-Seq13进行联合治疗，证明提前降低HBsAg有利于进一步提升CRT3-Seq13的治疗应答。上述研究结果系统揭示了慢性HBV感染导致体液免疫应答耐受的免疫机制和CRT3-Seq13的治疗效应和机制，为发展乙肝创新治疗策略奠定了基础。