建立小鼠模式以探討慢性B型肝炎病毒引發的免疫反應

Abstract

根據統計，全球大約有4億人口受到B型肝炎病毒( human hepatitis B virus - HBV ) 的慢性感染。與健康的人相比，慢性B型肝炎患者，明顯有較高的機率發生肝臟病變，包括肝硬化甚至到肝癌。然而，至今對於B肝病毒所導致慢性肝炎的病理機轉，在研究上一直受到限制，主要原因是缺乏一個適當的動物疾病模式。小鼠雖然廣泛應用於各種疾病的研究，但慢性B型肝炎的小鼠模式很難被建立，目前推測主要原因是小鼠肝細胞缺乏B肝病毒的細胞表面受體，因此無法自然感染小鼠肝細胞。為了克服此問題，此研究利用腺相關病毒(adeno-associated virus，AAV )為載體，攜帶B型肝炎病毒基因體，進入小鼠肝細胞，在免疫健全的小鼠中產生一個HBV感染的模式。此AAV載體攜帶具有HBV聚合酶點突變的HBV基因體(AAV/HBVp-)，同時，我們也建立一個AAV載體，會產生HBV聚合酶蛋白質(AAV/ Pol)，其受到肝臟內特異性驅動子的轉錄調控。共同打入AAV/HBVp-和AAV/Pol，在肝臟和血清中會有HBV病毒和蛋白質的產生。第二個研究部分，我們單獨感染AAV/HBVp-於C57BL/6小鼠，結果顯示此複製缺陷的AAV/HBVp-會產生HBs抗原和HBe抗原於血清中超過1年。在免疫組織切片染色中，AAV/HBVp-感染的小鼠在肝臟會有HBc抗原產生，但其他器官則不會有。血清中的ALT仍然是正常的。有趣的是，我們發現HBs抗原表現量隨著時間變化的不同，與小鼠品系和感染時的年齡有關，在C57BL/6成鼠( > 8週齡)，AAV/HBVp-感染1週後，可產生高量的HBs抗原於血清中，但2-3週後HBs效價則大幅下降；相反的，AAV/HBVp-感染的幼鼠(3週齡)，血清中的HBs抗原量在4週的觀察期間則相對穩定。在BALB/c小鼠，AAV/HBVp-感染1週後，會有高量HBs抗原表現，但是第二週後則快速下降到幾乎測不到的HBs量，此現象在成鼠和幼鼠都可以觀察到。早期HBs抗原下降，在免疫缺陷的NSG小鼠則沒有被觀察到，因此推論，控制HBs抗原的量，在抗HBV免疫中扮演重要角色。另一個有趣的是，在感染的AAV/HBVp-的C57BL/6和BALB/c小鼠中，並沒有看到HBe抗原有類似HBs下降的趨勢，此可能的機制還不清楚，將保留於之後更進一步探討。本研究的第三部分，探討在C57BL/6小鼠其會導致早期HBs抗原下降是在成鼠而非幼鼠的可能機制。HBV清除能力與年齡相關的現象在人類的HBV感染也被觀察到，根據統計，95 % 以上的新生兒，如果受到HBV的感染則易形成帶原者，然而成人則是小於5 % 機會發展為慢性B型肝炎感染。比較AAV/HBVp- 感染成鼠和幼鼠後，免疫細胞數目和表現型態的差異，以流式細胞儀分析，成鼠的CD4+ T細胞活化程度比幼鼠多。ELISPOT分析證明，AAV/HBVp-感染後的成鼠與幼鼠相比，能夠產生較多的HBV特異性CD4+ T細胞和CD8+ T細胞，有趣的是，我們觀察到，與幼鼠相比，在成鼠中有較少的調節性T細胞(Treg)和較多的follicular T細胞( TFH )，而與AAV/HBVp-的感染無關，因為TFH細胞與germinal center (GC)的發育和B細胞產生抗體有關，我們假設早期在AAV/HBVp-感染的成鼠內，其HBs抗原下降可能與HBs抗體的產生有關。的確，我們在RAG-/-小鼠，其已先接受來自AAV/HBVp-慢性感染小鼠的脾臟和骨髓細胞，有觀察到明顯的HBs抗體產生，此結果顯示，仍有一些產生HBs抗體的B細胞或漿細胞存在於HBV帶原小鼠中。本研究建立之AAV/HBVp-感染的小鼠模式，可以提供一個便利的工具，以研究在慢性HBV感染的情況下，其宿主對HBV的免疫反應。此外，也可以利用此模式來發展免疫治療藥物，來對抗慢性B型肝炎。

About 400 million people worldwide are estimated to be chronically infected by hepatitis B virus (HBV). Compared to healthy people, these patients are at higher risks in developing progressed liver diseases, including cirrhosis and hepatocellular carcinoma. Due to the absence of appropriate animal models, it is difficult to study the pathogenic mechanisms that lead to hepatic inflammation and liver diseases in chronic HBV. Mice have been broadly used as animal models to study various diseases. However, it is difficult to establish an appropriate mouse model to study HBV, presumably due to the lack of HBV receptors on mouse hepatocytes. To overcome this problem, we used adeno-associated viral (AAV) vectors to deliver HBV into mouse hepatocytes to generate a HBV infection model in immunocompetent mice. This AAV vector carried a HBV genome with point mutations in the polymerase (AAV/HBVp-) open-reading frame and thus eliminate production of HBV polymerase protein. We also generated an AAV vector to produce HBV polymerase protein (AAV/Pol) under the transcriptional control of a liver-specific promoter. Coinjection of AAV/HBVp- and AAV/Pol produced HBV virions and proteins in the liver and circulation. However, the HBV titer in these mice was relatively low ( 1x105 copies /ml) and further efforts are needed to improve this HBV infection mouse model. In the second part of studies, we used AAV/HBVp- alone to infect C57BL/6 mice and demonstrated that it can produce HBsAg and HBeAg in the serum for more than one year in the infected mice. Immunohistochemistry analysis of AAV/HBVp--infected mice demonstrated HBcAg expression in the liver but not in other organs. The serum alanine aminotransferase (ALT) remained normal in these mice. Interestingly, we found that the level of HBsAg expression and its kinetics were mouse strain- and age-dependent. In adult (> 8-week of age) C57BL/6 mice, AAV/HBVp- infection produced high serum HBsAg at week 1 post infection but the titer droped quickly at 2-3 weeks after infection. In contrast, in young C57BL/6 mice infected by AAV/HBVp-, the serum HBsAg level was relatively stable during the four-week observation period. In BALB/c mice, AAV/HBVp- infection resulted in high serum HBsAg at week 1 but quickly droped to an almost undectable level at week 2 and the following weeks in both young an adult mice. The early decrease of serum HBsAg level was not observed in immunodeficient NSG mice, suggesting that the anti-HBV immunity might play a role in controlling the serum HBsAg level. Interestingly, we did not observe similar decrease of HBeAg in either BALB/c or C57BL/6 mice infected by AAV/HBVp-. The underlying mechanisms are not clear and remain to be further approached. The third part of this study is to investigate mechanisms that might cause the early HBsAg decrease in adult but not in young C57BL/6 mice. The age-dependent HBV clearance has also been observed in human HBV infection. More than 95 % of exposed neonates become chronic infection, while chronic infection is observed in less than 5 % of adult HBV infection. We compared the number and phenotypes of lymphocyte subpopulations in adult and young AAV/HBVp- infected mice. Flowcytometric analysis showed that there were more CD4+ and CD8+ T cells with activation phenotypes in adult mice than in young mice. ELISPOT analysis confirmed that AAV/HBVp- infection in adult mice produced more HBV-specific IFN-gama+ CD4+ and CD8+ T cells compared with that in young mice. Interestingly, we observed less T regulatory (Treg) cells and more T follicular helper (Tfh) cells in adult mice than in young mice which are independent of HBV infection. Since Tfh cells are important in germinal center formation and antibody production, we hypothesized that the early decrease of HBsAg in adult mice might be due to their production of more anti-HBs antibodies. Indeed, a significant amount of anti-HBs antibodies can be detected in Rag deficient mice adoptively transferred with the splenocytes and bone marrow cells from AAV/HBVp- infected mice. This result demonstrates that there are still some anti-HBs-producing B cells or plasma cells present in HBV carrier mice. In conclusion, the AAV/HBVp- use model generated in this study offers a convenient tool to study HBV immune responses in chronic HBV infection. This animal model could be useful in developing immunotherapeutic approaches to treat chronic HBV.