B型肝炎病毒之小鼠耐受模式

Abstract

目前全球約有三億五千萬人口感染慢性B型肝炎，欲治療慢性B型肝炎必須先了解其致病機制，以助發展適當的治療方法。鑑於目前仍無適當的動物模式可供作研究造成B型肝炎病毒持續性感染的成因，我的研究主題即是在免疫健全的小鼠建立B型肝炎病毒持續感染的模式且進一步研究造成持續感染的機制，期望有助於發展適當的治療策略。 由於B型肝炎病毒無法直接感染小鼠，我以高壓注射法(hydrodynamic injection)自小鼠尾靜脈打入一個具複製能力的B型肝炎病毒基因體，促使B型肝炎病毒基因體進入小鼠肝細胞內。在注射質體pAAV/HBV1.2後，約有40%的C57BL/6小鼠在注射後26週仍無法清除體內的B型肝炎抗原，在其血清內仍可偵測到表面抗原、e抗原及成熟病毒顆粒；此外其肝臟內也能偵測到B型肝炎病毒複製時的中間產物、RNA、表面抗原及核心抗原但無明顯的發炎反應且無法產生具保護力的表面抗體。上述特性與人類慢性B型肝炎健康帶原者的免疫耐受期(immune tolerant phase)相似。 同時我也發現小鼠的品系及載體的序列，為決定高壓注射後B型肝炎病毒是否能於小鼠體內持續表現的重要因子。AAV載體有助pAAV/HBV1.2持續存在肝細胞中，而C57BL/6小鼠對B型肝炎抗原的免疫反應較BALB/c小鼠弱，因而在注射pAAV/HBV1.2後傾向持續表現B型肝炎抗原，即造成帶原；而BALB/c小鼠在注射pAAV/HBV1.2後，可於其體內偵測到較強的表面抗體反應及核心抗原的細胞免疫反應，且將B型肝炎病毒清除。 我進一步發現在沒有核心抗原或具核心抗原特異性的免疫反應存在時，不論是C57BL/6小鼠或是對B型肝炎抗原免疫反應較強的BALB/c小鼠都無法有效清除表面抗原及產生具保護力的表面抗體。此結果暗示在此動物模式中，具核心抗原特異性的免疫反應對於清除B型肝炎病毒之感染具決定性，且能幫助感染者產生具保護力之表面抗體。同時也發現核心抗原存在時，有較多的棘細胞(dendritic cells)及自然殺手細胞(natural killer cells)浸潤於肝組織內，且可偵測到較高量的IL-12。由此結果推測核心抗原會吸引且活化肝臟內淋巴球藉此清除B型肝炎病毒感染。 我們初步發現具核心抗原特異性免疫反應與B型肝炎病毒之持續感染具相關性，未來希望能將此動物模式應用於研究核心抗原如何啟動抗病毒機制以清除B型肝炎病毒感染，進一步釐清造成持續性感染的成因；希望藉此能發展出適當的治療策略。

There are approximately 350 million people chronically infected with hepatitis B viruses (HBV) worldwide. To develop an appropriate treatment for chronic hepatitis B, it is imperative to have a suitable animal model for studying the pathogenesis of HBV. Our goal is to establish a persistent HBV model in immunocompetent mice for studying the mechanism responsible for HBV persistence, which may facilitate the development of appropriate treatments for chronic hepatitis B. Due to the inability of HBV to infect mice directly, a replication-competent HBV plasmid, pAAV/HBV1.2, was injected hydrodynamically into the tail veins of mice, which mimicked the infection of HBV to hepatocytes. In 40% of the injected C57BL/6 mice, HBV surface antigenemia, viral replication, transcription and protein expression in the liver tissues persisted for more than 6 months but minimal inflammation and no anti-HBs antibody production were detected. Overall, the status of the long-term HBsAg-positive carrier mice in our study is similar to that of healthy human HBV carriers in the immune tolerant phase. Mouse genetic background and vector backbone were found to be the most important factors for determining HBV persistence after hydrodynamic injection. The AAV vector helped the stabilization of pAAV/HBV1.2 in the hepatocytes of injected mice. C57BL/6 mice exhibited milder immune responses against HBV antigens as compared with those of BALB/c mice and tended to express HBV antigens persistently after hydrodynamic injection of pAAV/HBV1.2. After hydrodynamic injection of pAAV/HBV1.2, all the BALB/c mice developed protective levels of anti-HBs antibodies and a strong cellular immune response against HBcAg and could clear HBV persistence. In the absence of expression of HBcAg or HBcAg-specific immunity, C57BL/6 mice and a portion of BALB/c mice failed to clear HBV infections and produce protective immunity efficiently, which suggests that HBcAg-specific immune responses play an important role in the clearance of HBV persistence and also in the generation of protective immunity. Large numbers of dendritic cells and natural killer cells and IL-12 mRNA were found in response to the expression of HBcAg in the livers of mice injected hydrodynamically with pAAV/HBV1.2. Based on this observation, I propose that HBcAg can recruit and activate intrahepatic lymphocytes, which facilitates the clearance of HBV infection. We already demonstrated the correlation of HBcAg-specific immunity and HBV persistence in this model. Further studies are needed to elucidate how HBcAg triggers the antiviral defense and the detailed mechanisms for persistent infection of HBV in this model. Based on these studies, hopefully we will develop an appropriate treatment for chronic infection of HBV in the future.