B型肝炎病毒與宿主間的免疫調控: 以小鼠模型篩選抗單株抗體病毒株以及研究宿主對B型肝炎病毒的先天性免疫反應

Abstract

長久以來，B型肝炎病毒被認為無法由傳統已知的先天性免疫反應辨認，因此B型肝炎病毒有”隱形病毒”之稱。在B型肝炎病毒感染後，有95%成人6個月內都能將病毒清除，說明後天性免疫反應的活化及功能均可正常發揮，因此先天性免疫反應仍應存在。然而，仍有5%的成人在感染病毒後演變成慢性帶原，最終可能導致肝癌發生。根據GLOBOCAN資料庫統計，2020年原發性肝癌全球發生率為4.7%，而其中有56%的肝癌是由B肝病毒慢性帶原造成，肝臟移植提供肝癌病患一個選擇，而移植前後給予B肝病毒陽性病患B型肝炎免疫球蛋白，能夠直接中和病毒避免B肝病毒再次感染，然而其昂貴及有潛在感染風險的缺點，使新型抗B肝病毒表面抗原抗體持續研發，以取代傳統的B型肝炎免疫球蛋白。 本篇研究分為二個主題，分別是找尋一種新型B肝病毒表面抗原抗體的抗原表位，以及探討B型肝炎病毒與先天性免疫反應之間相互調控的機制。 主題一:以人肝嵌合鼠篩選抗單株抗體突變病毒以分析抗體之抗原表位 Lenvervimab為一進入第三期臨床試驗的B肝病毒表面抗原人類單株抗體，而抗體的抗原表位(epitope)對於病毒突變與抗體治療效果息息相關。本篇文章利用人肝嵌合鼠進行抗體篩選找出Lenvervimab可能的抗原表位。研究結果顯示，人肝嵌合鼠的B肝病毒表面抗原在注射抗體初期迅速下降，但隨著注射次數增加其逐漸回升，進一步分析人肝嵌合鼠血清與肝臟內的病毒序列發現，表面抗原胺基酸在140與164位置發生突變，140由蘇胺酸(Threonine)突變成異白胺酸（Isoleucine），而164則由麩胺酸（Glutamic acid）變異為甘胺酸（Glycine），經由體外細胞感染模型證實，兩種突變株對sLenvervimab產生強弱不一的抗性，說明表面抗原胺基酸140及164兩個位點對於sLenvervimab辨認有重要角色。除了小鼠體內篩選系統之外，利用原態膠體電泳法（Native-PAGE）發現表面抗原胺基酸160從離胺酸（Lysine）突變為天門冬醯胺酸（Asparagine）亦使sLenvervimab無法辨認，最後也經由體外細胞感染模型證實160位點對於sLenvervimab結合的重要性。 本次實驗結果利用人肝嵌合鼠體內篩選系統，以及原態膠體電泳法找到B肝病毒變異株對Lenvervimab結合力的影響，並利用體外細胞感染模型驗證其抵抗性，藉此找到Lenvervimab可能的抗原結合位，解決了一個臨床重要的問題。 主題二:以高壓注射小鼠探討B型肝炎病毒與宿主先天性免疫反應的相互調控 本篇研究利用B型肝炎病毒質體高壓注射小鼠，在注射後不同時間點收集小鼠肝臟進行染色。本次實驗搭配細胞激素或趨化因子的RNA原位雜交化學技術，以及病毒蛋白的免疫組織化學染色，在同一個肝臟切片分析病毒與免疫反應間的交互作用及空間分布。結果顯示，腫瘤壞死因子(TNFα)的表現無論在控制組或野生型(wild-type)與B型肝炎病毒核心蛋白去除組(core-null)幾乎偵測不到；然而，和控制組相比，野生型B型肝炎病毒注射後48小時肝臟內的免疫細胞數量顯著增加，而且單核細胞趨化蛋白-1 (MCP-1; CCL2)表現顯著提升。研究發現單核細胞趨化蛋白-1集中表現在一小部分的肝臟細胞，以及聚集的免疫細胞，但將病毒核心蛋白去除後，肝臟內的免疫細胞數量與單核細胞趨化蛋白-1表現則與控制組沒有差別。這個結果說明表現單核細胞趨化蛋白-1的少部分肝臟細胞及免疫細胞可能為觸發後續免疫反應的重要媒介。 本次研究結果顯示，B型肝炎病毒，尤其是病毒核心蛋白，可能會引發一小部分特定的肝臟細胞和免疫細胞表現單核細胞趨化蛋白-1，吸引周邊血液中的單核球進入肝臟，進而幫助清除B型肝炎病毒，然而，這些特定的肝臟細胞與免疫細胞為何及其特性需要進一步釐清。

Topic 1: Mapping the conformational epitope of an anti-HBs antibody by in vivo selection of HBV escape variants Background & Aims Hepatitis B immunoglobulin (HBIG) has been routinely applied in the liver transplantation setting to block hepatitis B virus (HBV) reinfection of grafts. However, new monoclonal anti-HBV surface (HBs) antibodies have been developed to replace HBIG. The epitopes of such monoclonal antibodies may impact the emergence of escape variants and deserve study. Approach & Results The conformational epitope of sLenvervimab, a surrogate form of Lenvervimab, which is a monoclonal anti-HBs antigen (HBsAg) antibody currently under phase III trial, was investigated by selecting escape mutants from a human liver chimeric mouse. HBV-infected chimeric mice treated with sLenvervimab monotherapy showed an initial decline in circulating HBsAg levels, followed by a quick rebound in one month. Sequencing of circulating or liver HBV DNA revealed emerging variants, with replacement of amino acid E164 or T140. E164 HBV variants strongly resisted sLenvervimab neutralization in cell culture infection, and the T140 variant moderately resisted sLenvervimab neutralization. Natural HBV variants with amino acid replacements adjacent to E164 were constructed and examined for sLenvervimab neutralization effects. Variants with K160 replacement also resisted neutralization. These data revealed the conformational epitope of sLenvervimab. Conclusions Selection of antibody-escape HBV variants in human chimeric mice works efficiently. Analysis of such emerging variants helps to identify anchor amino acid residues of the conformational epitope that are difficult to discover by conventional approaches. Topic 2: The innate immune response between hepatitis B virus and host Background & Aims Hepatitis B virus is generally considered as a stealth virus that does not trigger conventional innate immune response in host. However, the adaptive immune response activates subsequently and clears virus infection in the liver. Therefore, it is interesting to investigate whether and how the innate immune response is stimulated by HBV during acute infection stage by the HDI-HBV model. Approach & Results The HBV innate immune mechanism was studied using in situ hybridization and immunohistochemistry co-staining in mouse liver exposed to hydrodynamic injection of HBV DNA. Spatial and single-cell distribution facilitates analysis of HBV interactions with different cells in the liver. The BALB/c mouse was exposed to vector, wild-type HBV, or core-null HBV using hydrodynamic injection. The mice were sacrificed at 24, 36, 48, or 72 hours after injection and the liver was collected for staining. The cells with TNFα expression were scarcely observed in vector control and both HBV group. In contrast, the CCL2 expression significantly increased in a small subset of immune cells in wild-type HBV mouse compared to that of vector control at 48 hr after hydrodynamic injection. Meanwhile, the number of immune cells was also increased dramatically in wild-type HBV mouse. However, the phenomenon disappeared in mouse injected with core-null HBV. These results revealed a role of a subset of immune cells expressing CCL2 as a candidate triggering subsequent immune responses. Conclusions HBV might induce a small proportion of hepatocytes to attract immune cells to express CCL2, thereby further attracting circulating monocytes to help clear HBV in the liver. The HBV core protein may be an essential viral factor, but the properties of these responsive hepatocytes require further study.