CBA/CaJ小鼠长期携带慢性乙肝固有特征的实用模型研究

Persistence of the covalently closed circular DNA of hepatitis B virus (HBV cccDNA) is the hallmark of chronic Hepatitis B (CHB) in humans but also a status as yet difficult to mimic in mice. In particular, it remains an imminent task to set up a practical model for daily research in commonly used strains of immunocompetent and non-transgenic mice. To this end, we have tested the feasibility and efficiency of direct transduction in vivo of HBV cccDNA by hydrodynamic injections (HDI) and established a model in C57BL/6 mice which expressed HBV antigens for 10 weeks. We now seek to further prolong the cccDNA-bearing time by choosing CBA/CaJ mice as the new recipient hosts for their previously unknown total tolerance with HBV at all ages, in spite of the fact that the virus was expressed from a non-HBV cccDNA vector pAAV/HBV1.2 (Chou et al, PNAS 2015). The target of this project is to reach a chronic course for over 26 weeks (6 months) as reported with specific modification and improvement by direct HBV cccDNA transduction. A single injection of 2 μg HBV cccDNA in CBA/CaJ mice will be followed by examinations of viral markers in serum and liver tissues. Direct detection of the presence and persistence of HBV cccDNA in the nuclei will be performed by rolling circular amplification (RCA) combined with in situ PCR (IS-PCR). We will use pegylated interferon (PEG-IFNα2a/2b) to test the model for its performance and practicality in drug evaluation and development. We will also test a newly discovered anti-HBV tetrapeptides of our own as well as recently reported antibody-based drug leads which were shown to be able to induce specific and non-cytotoxic degradation of HBV cccDNA (Lucifora et al, Science. 2014). The advantage and significance of the model would be its simplicity and easiness suitable for practical usage in basic research and drug development.

乙肝病毒共价闭环DNA（HBV cccDNA）的长期存在是慢性乙肝难以治愈的根本原因，现有小鼠模型难以模拟这一本质特征。对此，我们以直接注射HBV cccDNA的方法，在C57BL/6小鼠中建立了携带10周的模型。而最近发现，各年龄组的CBA/CaJ小鼠对HBV均具有此前未知的超强的耐受性，携带可达半年以上，但所用载体仍非cccDNA。根据这一重大机遇，本研究将直接以cccDNA转导该系小鼠，以建立简易、高效并可推广的实用模型。将采用已建立的全套实验技术体系，系统检测病毒学和病理学等各项指标，追踪病毒携带时限和表达水平，并以滚环扩增合并原位PCR的方法，对细胞核内的cccDNA进行直观特异和敏感的监测与鉴定。将以已知的长效干扰素、自主研发的“乙肝四肽”和新近报道的特异性抗体等3类药物，切实进行实用性检验和应用示范。这一模型的建立和推广，对乙肝药物和机理研究具有极为重要的科学意义和应用价值。

乙肝病毒共价闭环DNA（HBV cccDNA）的长期存在是慢性乙肝难以治愈的根本原因，现有小鼠模型难以模拟这一本质特征。本研究直接以HBV cccDNA转导CBA/CaJ小鼠，采用已建立的全套实验技术体系，系统检测病毒学和病理学等各项指标，追踪病毒携带时限和表达水平，对细胞核内cccDNA进行了直观特异和敏感的监测与鉴定，以核苷类似物、长效干扰素、多肽类、特异性抗体、中草药单体和化合物单体等6类药物，进行了实用性检验和应用示范，此外本研究对HBV cccDNA的制备、HBV DNA和cccDNA拷贝数检测方法进行了优化。本研究成功筛选到了HBeAg(-)病毒株，并回复突变获得了与之相匹配的HBeAg(+)病毒株，建立了一对能够长期携带慢性乙肝固有的本质特征cccDNA，又具有健全的免疫机能，同时具备实用性和可推广性的简易、高效小鼠模型，该模型应用于慢性乙肝感染急性期和慢性期的药物筛选和评价中在个别时间点存在显著性差异。这一对新模型的建立和推广，对乙肝药物和机理研究具有极为重要的科学意义和应用价值。