介白素-21預防慢性B型肝炎病毒的感染：B淋巴細胞所扮演角色

Abstract

根據統計，全球大約有4億人口受到慢性B型肝炎病毒(Human hepatitis B virus, HBV)感染，且B型肝炎病毒感染會引起急性及慢性肝臟疾病，甚至進展為肝硬化及肝癌。臨床顯示，清除HBV病毒的能力與年紀有相關，感染大人，只有5 %會形成慢性感染，然而，感染新生兒90 %則會發展為慢性HBV感染，影響HBV感染走向急性或慢性感染，與宿主能否引發病毒專一性的細胞性免疫反應有關，然而，最終產生anti-HBs抗體是清除HBV的關鍵。慢性B型肝炎患者，血清中無法測得anti-HBs抗體；然而，越來越多臨床證據顯示，在慢性B型肝炎患者，可能還是會產生anti-HBs抗體，且有抑制HBV病毒的作用。因此，本論文第一部分主要探討B細胞在HBV感染時扮演的保護角色。 實驗使用腺相關病毒(Adeno-associated virus, AAV)載體，攜帶聚合酶缺陷B型肝炎病毒基因體(AAV/HBVp-)，在小鼠肝臟表現HBV mRNA及相關蛋白，以模擬人類B型肝炎感染時，在肝臟產生抗原所引發的免疫反應相關機制。首先，我們用AAV/HBVp-感染不同品系3週齡的幼鼠及8-12週齡的成鼠，發現在感染早期，成鼠比幼鼠較能有效的清除HBsAg。然而，清除HBsAg的效果無法長期維持。進一步探討感染初期成鼠清除HBsAg的可能機制，首先利用免疫共沉澱方式分析感染後，不同時間點肝臟中HBsAg表現的情形，發現肝臟仍持續表現HBsAg，沒有減少。利用不同免疫缺陷小鼠進行實驗，證實只要沒有B細胞的存在，HBsAg就無法被清除；另外，我們也分析感染小鼠第三週，血清HBsAg表現下降時間點，是否有免疫複合物的產生。結果顯示HBV感染小鼠血清中都有偵測到HBs-anti-HBs複合物的存在，證實HBV的感染會產生anti-HBs抗體，只是與血清中的HBsAg結合，以免疫複合物形式存在。 從第一部分的結果，我們提出一個假說，HBV感染早期引發的anti-HBs抗體的量，會影響HBV感染的病程。在急性感染病人，產生的anti-HBs量較高，可達到完全清除病毒的目的，痊癒後血液中可以測到anti-HBs抗體。而慢性感染病人，可能因為感染初期產生的anti-HBs量較低，無法有效清除病毒，因此造成慢性感染。B細胞抗體反應需要T follicular helper cell (Tfh)T細胞的幫忙，Tfh會進入二級淋巴器官的Germinal center (GC)與B細胞進行交互作用。其中Tfh細胞表現的IL-21，對GC B細胞分化成漿細胞(plasma cell)的過程、以及維持GC的反應很重要。因此本論文第二部分探討，以IL-21治療早期HBV感染，以及治療已形成慢性B型肝炎感染，對清除病毒的影響。 我們發現以IL-21治療早期感染的小鼠，能夠有效清除血清中HBsAg及HBeAg，長達一年，甚至在成鼠的治療可測到anti-HBs抗體的存在。以IL-21治療慢性B型肝炎小鼠，也能觀察到有效清除血清HBV抗原，特別是HBeAg有持續下降的現象。利用免疫組織染色和北方墨點法，證實IL-21治療，也能有效清除肝臟中的HBV。但IL-21清除HBV的機制和B細胞無關，因為治療B細胞基因剔除小鼠也會造成血清HBsAg的下降。流式細胞分析顯示，IL-21治療會造成肝臟免疫細胞大量增加，同時也有CD8+T細胞的活化及GC B細胞增加的現象。然而，脾臟免疫細胞則沒有顯著影響。 本篇研究發現，慢性B型肝炎病毒感染，會產生anti-HBs抗體，只是在帶原者與HBsAg結合，形成免疫複合物。另外，以IL-21治療HBV，能夠透過活化肝臟免疫反應，達到有效且長期清除HBV病毒的目的。

Hepatitis B virus (HBV) infects approximately 400 million people and can lead to the development of severe liver disease. The chance of clearing HBV infection is age dependent. Only 5% of adult-acquired infections lead to chronic infection, whereas more than 90% of exposed neonates will develop chronic diseases. Current evidence suggests that virus specific CD4+ and CD8+ T cell responses play a central role in the outcome and pathogenesis of HBV; whereas antibodies to HBsAg might be the key to HBV clearance. It is know that in chronic HBV patients anti-HBs antibody is not present in the circulation. However, there is growing evidence suggest that anti-HBs antibody might still be produced in chronic HBV, and play a role in suppressing viruses. Therefore, in the first part of the study, we investigated the role of B cell in HBV infection. We used recombinant adeno-associated viral vectors to deliver a HBV genome carrying mutations in the polymerase gene (AAV/HBVp-) and thus not being able to replicate, but otherwise normal in protein and mRNA production. Our results showed that adult mice (8-to 12-week-old) were more effective than young mice (3-week-old) in clearing serum HBsAg in early infection, but the suppressive effect did not persist. Immunoprecipitation analysis revealed that HBsAg expression in the liver was not reduced. Infection of AAV/HBVp- in different immunodeficiency mouse strains, showed that B cells play a critical role in reducing serum HBsAg in early infection. Moreover, HBsAg-containing immune complexes can be identified in the serum of mice at the time of HBsAg decrease. Together, these result that anti-HBs antibodies are present in the excess of HBsAg in the form of immune complex with HBsAg. We hypothesize that the level of anti-HBsAb in early infection may influence the outcome of HBV infection, for those who produce low level of anti-HBs antibody fail in clearance of HBV and eventually develop chronic HBV disease. The generation of Ab-forming cells occurs during a germinal center (GC) reaction, in which T follicular helper T cells (Tfh) cells are essential for GC formation. Interleukin-21 (IL-21) is the most critical Tfh-derived stimulator for differentiation GC B cells into memory B cells or plasma cells, which together sustain long-term humoral immunity. In the second part of the study, I addressed whether IL-21 treatment can promote HBV clearance in early infection and in persistent infection. IL-21 treatment in early infection significantly reduced all viral proteins in the serum and the suppressive response last for at least one year, resulting in appearance of anti-HBs antibody in most adult mice. Even in mice with established chronic HBV infection, IL-21 treatment still significantly decreased all HBV proteins, in particular the HBeAg, for at least one year. Immunohistologic staining and northern blot analysis of the liver tissue from IL-21 treated mice showed significantly reduction of HBV core protein and mRNAs in liver. However, mechanistic studies revealed that the anti-HBV effect of IL-21 was not related to anti-HBV antibodies since the therapeutic effect of IL-21 maintained in B cell-deficient μMT-/- mice. Flow cytometry analysis showed a significant increase in the absolute number of several lymphocyte subpopulations in the liver, and among them CD8+T cells showed increased activation phenotypes. The percentage of GC B cells in liver, but no in spleen, was also significantly increased. Together, this study showed evidence that anti-HBs antibody was present in the form of immune complexes in mice that eventually developed chronic infection, and IL-21 treatment was effective in controlling chronic HBV infection by inducing anti-HBV immune responses in the liver.