Development of humanized transgenic mice for HBV/HIV co-infection studies

摘要 更好地了解乙型肝炎病毒的先天免疫反应和适应性免疫反应之间的相互作用 人类免疫缺陷病毒（HIV-1）引起的免疫系统损伤中的乙型肝炎病毒（HBV）对于 开发新的抗病毒免疫疗法。携带人类免疫系统并受到影响的小鼠 HIV-1 在研究 HBV/HIV 共感染的体内实验模型中具有广阔的前景。尽管目前 人源化小鼠模型取得进展，物种差异阻碍了人类的有效“协作” 小鼠非免疫细胞外壳中的免疫系统。我们建议创建一个新模型，该模型具有 淋巴组织发育最重要的一组人类基因，即淋巴毒素β受体 (hLTβR)、两种参与 T 细胞和 B 细胞区域建立的趋化因子 (hCCL21、hCXCL13) 和一种胸腺 基质淋巴细胞生成素（hTSLP）。在此应用中，我们建议用 hLTβR、hCCL21 替换小鼠基因， hCXCL13 和 hTSLP 开发适合未来 HBV/HIV 双重感染研究的新模型。我们将聘用 Easi-CRISPR 基因编辑是我们最近开发的高度稳健的方法，用于创建拟议的小鼠 型号。在该菌株中，我们期望有效生成人类滤泡辅助细胞，这是一个关键组成部分 建立针对乙型肝炎病毒的适应性免疫。研究乙肝病毒免疫反应的能力 HIV-1 感染的存在将成为 HBV/HIV 合并感染建模的新一步。所有的基因操作都会 在 NOG 背景上已经具有人 IL-34 转基因的小鼠品系上进行。理性的 NOG-hIL34 转基因小鼠的优点是它能有效支持人类组织驻留巨噬细胞（包括 库普弗细胞）并引入组织人为因素的淋巴组织，这将为 滤泡辅助 T 细胞的发育和对 HBV 抗原的适应性免疫反应。我们将验证 新的人源化毒株控制 HIV-1 感染的能力以及感染对免疫反应的影响 HBsAg。这样的模型还将提供一种开发治疗方法和解决问题的方法。 HBV/HIV 共感染的病理生物学范式以前不可能在体内研究。 我们的近期目标是改进现有模型，为研究人员创建一个新的、宝贵的工具， 可以支持广泛的 HBV/HIV 合并感染研究。通过测试人体免疫系统的功效 通过观察所有四种修饰中的发育和功能，我们可以确定是否将所有基因型组合成一个 小鼠品系。作为下一步的开发，我们将引入TK-NOG背景进行高效移植 人类肝细胞。我们的长期目标是促进 HBV/HIV-1 先天性和适应性免疫的研究 合并感染。

ABSTRACT A better understanding of the interplay between innate and adaptive immune responses to hepatitis B virus (HBV) in human immunodeficiency virus (HIV-1)-induced immune system impairment is crucial for the development of new antiviral immune therapeutics. Mice that carry human immune system and are affected by HIV-1 have great promise in in vivo experimental models for studying HBV/HIV co-infection. Despite current progress in humanized mouse models, species differences preclude efficient “collaboration” of the human immune system in a shell of mouse non-immune cells. We propose to create a new model that possesses the most crucial set of human genes for lymphoid tissue development, namely the lymphotoxin beta receptor (hLTβR), two chemokines involved in T- and B-cell zone establishment (hCCL21, hCXCL13), and a thymic stromal lymphopoietin (hTSLP). In this application, we propose replacing mouse genes with hLTβR, hCCL21, hCXCL13, and hTSLP to develop a new model suitable for future HBV/HIV coinfection studies. We will employ Easi-CRISPR gene editing, our recently developed and highly robust method, to create the proposed mouse model(s). In this strain, we expect efficient generation of human follicular helper cells, which are a key component for the establishment of adaptive immunity to HBV. The ability to study the immune response to HBV in the presence of HIV-1 infection will be a new step in modeling HBV/HIV coinfection. All genetic manipulations will be performed on a strain of mice that already has human IL-34 transgene on a NOG background. The rational for NOG-hIL34 transgenic mice is that it efficiently supports human tissue-resident macrophages (including Kupffer cells) and introduces lymphoid tissue organizing human factors, which will provide a strong support for development of follicular helper T cells and the adaptive immune response to HBV antigens. We will validate the ability of the new humanized strain to control HIV-1 infection and the effects of infection on immune responses to HBsAg. Such a model will also provide a means for the development of therapeutics and address pathobiological paradigms of HBV/HIV coinfection previously not possible to study in vivo. Our immediate goal is to improve the existing model to create a new, invaluable tool for researchers that can support a wide range of HBV/HIV coinfection studies. By testing the efficacy of human immune system development and function in all four modifications, we can determine whether to combine all genotypes into one mouse strain. As the next step in development, we will introduce TK-NOG background for efficient transplanting of human hepatocytes. Our long-term goal is to facilitate studies of innate and adaptive immunity in HBV/HIV-1 coinfection.