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標題: | 發展新型B型肝炎疫苗來克服不反應現象及研究慢性B型肝炎病毒與肝細胞癌的免疫致病機轉 Development of novel HBV vaccines to overcome nonresponsiveness and studies of immunopathogenic mechanisms of chronic HBV and HBV-associated hepatocellular carcinoma |
作者: | Ho-Yuan Chou 周和源 |
指導教授: | 陶秘華 |
關鍵字: | B型肝炎病毒,不反應現象,細胞激素,熱感應凝膠,免疫耐受性,肝細胞癌,免疫致病機轉, Hepatitis B virus,non-responsiveness,hydrogel,granulocyte-macrophage colony-stimulating factor,immune tolerance,immunopathogenesis,hepatocellular carcinoma, |
出版年 : | 2011 |
學位: | 博士 |
摘要: | B型肝炎病毒感染為人類最重要的感染疾病之一,世界上約有三分之一以上的人曾被B型肝炎病毒所感染,有三億五千萬的人成為慢性肝炎帶原者,其中百分之十五到四十都有罹患B型肝炎相關疾病的風險,包括肝硬化及肝細胞癌。利用從病人血清分離或重組DNA技術所製造的B型肝炎表面抗原疫苗來進行免疫注射,是目前主要控制B型肝炎傳染以及散播的方式,但是百分之五到十的一般人卻無法藉由這些疫苗的免疫注射產生有效的B型肝炎表面抗原的抗體。這些人仍有被B型肝炎病毒感染的風險,因此需要發展更有效的疫苗來保護他們。而對於慢性B型肝炎感染的病人,目前的兼具抗病毒和免疫調節特性的藥物如干擾素及抑制B肝病毒的藥物如核苷、核苷酸衍生物都因為免疫的不反應或是抗藥的突變種,使得效果受到限制。依據目前的研究報告,一般皆認為,B型肝炎的細胞性免疫反應和肝臟相關疾病的進展是有關連的,但是真正的致病機轉並未清楚闡明。因此,本論文主要的研究目標如下:(1)開發更有效的B型肝炎疫苗來克服表面抗原不反應現象。(2)研究B型肝炎病毒引起免疫耐受性及造成肝細胞癌的致病機轉,希望藉此能對於慢性B型肝炎病毒感染的治療方法提供更明確的方向。
B型肝炎表面抗原的不反應現象和人類組織相容性抗原 (human leukocyte antigen) 及缺陷的輔助T細胞反應有關。某些組織相容性抗原,不易引發B型肝炎表面抗原的抗體反應。我們使用可以增強在啟動免疫反應時扮演重要角色的樹突細胞(Dendritic cells)呈現抗原能力的細胞激素(GM-CSF)當作疫苗的佐劑,來提供B型肝炎抗原呈現給輔助T細胞更好的環境,進而幫助B細胞來產生更強的抗體反應。我們應用新發展的PEG500-PLGA2880-PEG500高分子製備的熱感應凝膠(Hydrogel)製作新型的B型肝炎疫苗:在低溫(4℃)下凝膠呈液態時,混入B型肝炎表面抗原和細胞激素。當注射到動物,熱感應凝膠在溫度提高(37℃)後,會變成固態的膠狀,因此可以緩慢釋放B型肝炎抗原和細胞激素,達到讓抗原和細胞激素持續且同時刺激免疫反應的效果。我們的實驗結果顯示,在一般小鼠體內,可引起更強的抗體及T細胞增生反應。這樣的效果,必須是抗原與細胞激素同時局部的釋放才能達到。更重要的是,我們證明了在B型肝炎疫苗不反應小鼠(B10.M mice),僅需要注射一次新型疫苗,就可以克服不反應現象,引起顯著的抗體和T細胞免疫反應。而傳統的B型肝炎疫苗重覆注射在不反應小鼠也無法引發反應。進一步分析發現,經由熱感應凝膠所釋放的GM-CSF,吸引了在啟動免疫反應時扮演重要角色的樹突細胞,移動到疫苗注射部位附近的淋巴結,使淋巴結中樹突細胞的數量增多,共同刺激分子(costimulatory molecules, MHC II, CD40, CD80 and CD86)表現也更為成熟。根據以上結果,我們所製作的新型疫苗除了可以改善B型肝炎疫苗不反應現象,降低不反應現象的發生機會之外,也非常有潛力應用於其他種類疫苗的研發。 臨床以及實驗證據顯示,B型肝炎病毒特異性的免疫反應對於B型肝炎病毒所造成的肝臟損傷扮演很重要的角色,但是在研究B型肝炎致病機轉最主要的阻礙,在於缺乏對B型肝炎病毒可引發免疫反應的動物模式。我們實驗室最近發展出可以藉由腺相關病毒載體攜帶B型肝炎病毒基因,進入免疫健全小鼠的肝臟細胞。這些小鼠可持續製造B型肝炎病毒,血清學反應以及缺陷的特異性免疫反應與B型肝炎病毒慢性感染的病人類似。更重要的是,在大於十二個月後,所有的小鼠都產生肝臟腫瘤,並且大部分腫瘤都是肝細胞癌。因此,我們利用這個小鼠模式,來研究B型肝炎免疫耐受性的機轉,以及宿主免疫反應在B型肝炎病毒引起肝細胞癌所扮演的角色。我們的結果顯示,在施打AAV/HBV的小鼠,B型肝炎表面抗原在血液中的量和表面抗原特異性的CD8+ T細胞數目呈現負相關。當施打較低劑量的AAV/HBV後再進行DNA免疫,發現可以克服這些小鼠的免疫耐受性,在血清中偵測到表面抗原特異性的抗體反應。另一方面,我們沒有發現在施打AAV/HBV的小鼠的肝臟和脾臟中,調節性T細胞 (Tregs)有明顯增加的現象,調節性T細胞對於B型肝炎免疫耐受性的引發和維持會再進一步的探討。另一方面,我們發現計畫性死亡(PD-1)在施打AAV/HBV的小鼠的肝臟而非脾臟的CD8+ T細胞表現明顯增加。此結果表示PD-1和其接受器(PD-L1, B7-H1),與AAV/HBV造成的免疫耐受性有關連。因此,我們利用B7-H1基因剃除小鼠來進一步證實推論:結果發現,B型肝炎表面抗原的表現在施打AAV/HBV的B7-H1基因剃除小鼠明顯減少,而且B型肝炎表面抗原特異性的CD8+ T細胞也明顯增加。顯示在我們的AAV/HBV小鼠中,B7-H1對CD8+ T細胞的免疫耐受性的引發非常重要。最後,我們利用一系列的免疫缺陷小鼠,包括I-A (CD4+ T)、CD8a (CD8+ T)和CD1d (NKT) 基因剃除小鼠,來探討AAV/HBV造成的肝細胞癌的免疫致病機轉。我們發現 (1) 施打AAV/HBV的CD1d基因剔除小鼠的血清中麩胺酸丙酮酸轉胺酶 (ALT),在施打之後九個月有較明顯的上升,且在施打後九個月就陸續產生肝細胞癌,比野生型小鼠的肝細胞癌發生提早三個月發生。所有CD1d基因剔除的小鼠在施打AAV/HBV的十二到十五個月後都產生了肝細胞癌。(2) 和野生型小鼠比較,施打AAV/HBV的CD8 Hepatitis B virus (HBV) infection remains one of the most important infectious diseases for human. More than a third of the world's population has been infected with HBV and more than 350 million are chronic carriers, of whom 15-40% is at risk of developing HBV-associated liver diseases, including cirrhosis and hepatocellular carcinoma (HCC). Vaccination with HBsAg derived from the plasma of HBV carriers or produced by recombinant DNA technology is the main strategy for effective control of the infection and viral transmission, but fails to induce adequate anti-HBs antibodies (Abs) in 5-10% healthy subjects, a phenomenon known as HBsAg non-responsiveness. These individuals remain at risk of HBV infection and thus there is a need for a more immunogenic vaccine for these non-responders. For those chronically infected HBV patients, therapeutic effects of current anti-HBV drugs, including interferon and nucleoside and nucleotide analogues, are hindered by unresponsiveness or emergence of drug-resistance mutants. It is generally believed that HBV-specific cellular responses are closely associated with disease progression in chronic HBV patients, but the exact pathogenic mechanisms remain unresolved. The major purposes of the present study are (1) to develop a potent HBV vaccine to overcome HBsAg non-responsiveness and (2) to investigate the possible immunopathogenic mechanisms associated with chronic HBV and HBV-associated HCC, which may have a major impact on the development of new treatment strategies. The phenomenon of HBsAg non-responsiveness has been known to be closely related to human leukocyte antigen (HLA)-linked genes and impaired helper T (Th) cell responses to HBsAg in these subjects. We hypothesized that granulocyte-macrophage colony-stimulating factor (GM-CSF), a potent adjuvant in enhancing the antigen (Ag) presentation activity of APCs, might help generate Th cell responses in non-responders, subsequently providing help for B cells to produce anti-HBs Abs. A thermosensitive biodegradable co-polymer (hydrogel) system was used to co-deliver HBsAg and GM-CSF to achieve maximal local cytokine activity at the injection site. In responder mouse strains, hydrogel-formulated HBsAg plus GM-CSF vaccine (Gel/HBs+GM) elicited much higher anti-HBs Ab titers and Th cell proliferative responses than a commercial aluminum-formulated HBsAg vaccine or free HBsAg. The adjuvant effect of the Gel/HBs+GM vaccine was dependent upon the local release of GM-CSF. More importantly, the Gel/HBs+GM vaccine elicited high HBsAg-specific Ab titers and Th cell responses in B10.M mice, a mouse strain that does not respond to the current HBsAg vaccine because of its H-2 haplotype. Analysis of the draining lymph nodes of Gel/HBs+GM-treated mice revealed an elevated number of CD11c+ dendritic cells (DCs) showing enhanced expression of MHC class II and a variety of costimulatory molecules. These results demonstrate that hydrogel-formulated GM-CSF might represent a simple and effective method to generate next generation HBV vaccines for inducing anti-HBs Abs in non-responders. Clinical and experimental evidence suggests that antiviral immune responses play an important role for hepatocyte injury in chronic HBV infection. However, understanding the immunopathogenic mechanisms of chronic HBV is hindered by the lack of a convenient small animal model not being born tolerant to HBV and having convenient reagents for immune assays. Our lab has recently generated a chronic HBV disease model in immune competent mice by adeno-associated viral (AAV) vectors carrying the HBV genome. These AAV/HBV-transduced mice displayed similar serological profiles and impaired HBV-specific T cell responses to that observed in chronic HBV patients. Moreover, the majority of AAV/HBV-transduced mice developed HCC over one year after AAV/HBV transduction. Taking advantage of this HBV disease model, we proposed to investigate mechanisms involved in HBV-associated immune tolerance and the role of hepatic lymphocyte subpopulations in the AAV/HBV-associated HCC. Our results showed an inverse relationship between the circulating HBsAg level and the number of HBs-specific T cells in AAV/HBV-transduced mice. In mice transduced with lower doses of AAV/HBV, DNA immunization can effectively restore HBV-specific Ab and T cell responses. As for CD4+CD25+ regulatory T (Tregs), no significant increase of this suppressor T cell population was observed in AAV/HBV-transduced mice. In contrast, expression of programmed death-1 (PD-1) was significantly increased on hepatic but not splenic CD8+ T cells, suggesting that this inhibitory receptor was involved in induction of HBV immune dysfunction in AAV/HBV mice. This conclusion was further supported by experiments using programmed death 1 ligand 1 (PD-L1, B7-H1) knockout mice, in these mice AAV/HBV-transduction induced a much stronger HBV-specific CD8+ T cells response accompanied with decreased levels of serum HBsAg. Finally, a panel of gene knockout mice, I-A KO (deficient of CD4+ T cells), CD8 KO (deficient of CD8+ T cells), and CD1d KO (deficient of natural killer T was used to investigate the immunopathogenic mechanisms of HCC developed in AAV/HBV-transduced mice. CD1d KO mice showed increased serum alanine aminotransferase (ALT) from 9 months after AAV/HBV transduction, and developed liver tumors at the age of 9 month-old; at least three months earlier than similarly treated wild-type mice. All AAV/HBV-transduced CD1d KO mice developed macroscopically visible liver tumor nodules between 12 and 15 months after AAV/HBV transduction. AAV/HBV-transduced CD8 KO mice had lower tumor incidence and smaller tumor size compared with similarly treated wild-type mice, suggesting that CD8+ T cells play a critical but not essential role in AAV/HBV-associated HCC. The detailed mechanisms of tumor formation associated with these immune cells need to be further investigated in the future. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/36974 |
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