臺灣博碩士論文加值系統

B型肝炎病毒(HBV)在全球目前仍感染超過2億4千萬人，且每年超過60萬人死於急性或慢性B型肝炎。發展為慢性肝炎的病人，需要進行長期的治療，利用干擾素治療需完成1年療程，使用抗病毒藥物需要更長時間，且停藥後多數病人會復發，因此是否停藥成為一個難題。在過去的研究指出不論在HBV急性感染或是慢性感染中，T 細胞皆扮演重要的腳色，因此病人本身T 細胞免疫好壞可以做為一個用藥評估，但目前直接測量CD8 T 細胞免疫的方式，如：&;#37238;聯免疫斑點試驗或以流式細胞儀計數等方式由於成本、人力需求甚至組織相容性原限制性等因素仍不可行，因此轉而考慮目前臨床上常用的檢驗數據，如：HBV抗原、抗體或谷丙轉氨&;#37238;等是否可以直接或間接表示T 細胞 免疫將是一個有趣且有意義的研究。在2012年陳培哲教授與夏寧紹教授於Guts上所發表的論文，利用新型雙抗原酵素連結免疫吸附分析法(double antigen ELISA)對HBV抗核抗體進行絕對定量，指出HBV抗核抗體和谷丙轉氨&;#37238;量有良好相關性，且用藥前進行測定，可以做為用藥預後的良好指標，這樣的結果促使我們想到是否HBV抗核抗體的量可以做為一個T 細胞免疫的指標。因此我們想要研究哪些因素影響HBV抗核抗體產生量。首先在高壓注射質體表現HBV的老鼠模型中發現，HBV抗核抗體在第七天達到頂標後便會急速下降，這樣的抗體量變化有別於利用DNA疫苗的老鼠模型中可以持續上升的狀況。利用anti-CD4 抗體削除CD4 T細胞後，初步實驗的資料顯示不會影響HBV抗核抗體產生量。而利用Thioacetamide(TAA)使肝臟細胞破損，模仿一個在HBV感染後肝細胞受到CD8 T細胞攻擊受損的狀況，由實驗數據可以看到HBV抗核抗體產生量有明顯上升，但是在剔除CD4 T 細胞後仍可以觀察到這樣的現象，CD4 T細胞在高壓注射質體表現HBV的老鼠模型中，促進HBV抗核抗體產生的角色並不明顯。但將具有HBV特異性的總T細胞打入高壓注射質體表現HBV的老鼠後，可以觀察到抗體量有上升的現象。除了宿主本身的免疫系統因子外，利用HBV核心抗原突變(Y132A)使之不能形成病毒顆粒也會影響到HBV抗核抗體的產生。

More than 240 million people have chronic Hepatitis B virus (HBV) infection, and about 600,000 people die due to acute or chronic HBV infection every year. The therapeutic course of chronic HBV infection is very long. Viral relapse is common when anti-virus treatment is stopped. There is no indicator providing the doctor with confidence to know when to stop the therapeutic course. Clearance of hepatitis infection relies on the host immunity, so the quality of host immunity, particularly T cell immunity, should be a good indicator for prognosis before treatment. However, measurement of T cell immunity, such as counting cytokine-secreting cells by enzyme-linked immunospot (ELISPOT) or HLA-restriction cells by the tetramer staining, is expensive and labor-intensive. Therefore, development of a convenient and inexpensive indicator that appropriately represents the strength of T cell immunity is in pressing need.
In 2013 Yuan and his colleagues analyzed the anti-core antibody levels of chronic hepatitis B patients and healthy individuals. They found the anti-core antibody titer of chronic hepatitis B patients with elevated Alanine Aminotransferase (ALT) levels is significantly higher than that of those with normal ALT levels. This observation suggested that the level of anti-core antibody might be a good indicator of T cell immunity.
By using the hydrodynamic injection mouse model, we found the levels of anti-core antibody peaked at day 7 after hydrodynamic injection and dropped sharply to about 10 IU/ml. Compared to the hydrodynamic mouse model, the levels of anti-core antibody increased slowly in mice receiving DNA vaccine. To mimic liver damage in chronic hepatitis B infection, we used Thioacetamide(TAA) in the hydrodynamic mouse model. The anti-core antibody increased dramatically after TAA treatment. The result revealed that liver damage would affect the anti-core antibody response. Depletion of CD4 T cells had no impact on the anti-core antibody production in the primary response and in the TAA-induced liver damage in a hydrodynamic mouse model. To clarify the relationship between T cell immunity and the level of anti-core antibody, we adoptively transferred HBV-specific T cells to mice receiving hydrodynamical injection of the HBV-expressing plasmid. The clearance rate of HBV surface antigens in mice receiving HBV-specific T cells was faster than the control group. The anti-core antibody titer of mice to which HBV-specific T cells were adoptively transferred was higher at day 7 post transferring day. This result suggested that HBV-specific T cells including CD4 and CD8 populations were important in maintaining the anti-core antibody titer. Finally, hydrodynamic injection of the pAAV/HBV plasmid carrying Y132A mutation did not induce the anti-core antibody response. The previous study showed that the capsid structure was critical for the T cell-independent response. However, in a hydrodynamic model there was no antibody response by pAAV/HBV carrying Y132A mutation.

中文摘要 i
ABSTRACT iii
TABLE of CONTENTS vi
LIST of FIGURES x
1. INTRODUCTION 1
1.1. The history of Hepatitis B virus (HBV) 1
1.2. HBV genome structure, viron structure and core protein 1
1.2.1. HBV genome structure 1
1.2.2. Virion structure 2
1.2.3. The HBV Core protein 2
1.3. HBV epidemics and natural history 3
1.3.1. HBV epidemics 3
1.3.2. The natural history of HBV infection 4
1.4. HBV clearance 5
1.5. Hepatitis B therapy 8
1.6. Biomarkers of T cell immunity 9
1.7. Antibody production and maintenance 11
2. SPECIFIC AIM 14
3. Material and method 15
3.1. Plasmid 15
3.2. Hydrodynamic mouse model 15
3.3. Reagent and Antibody 16
3.4. Adenovirus production 17
3.5. HBV serological markers 18
3.6. Double antigen ELISA 18
3.7. Immunization and adoptive transfer 19
3.7.1. Immunization 19
3.7.2. Splenocytes harvest and B cell depletion 20
3.7.3. Adoptive transfer 20
3.8. Enzyme linked immunosorbent spot (ELISPOT) 21
3.9. Flow cytometry analysis 22
4. RESULTS 23
4.1. Dynamics of the HBV anti-core antibody production after pAAV/HBV1.2 hydrodynamic injection, pcDNA3.1 (+)/HBc immunization, or adenovirus/HBc infection. 23
4.2. Depletion of CD4 T cells did not affect anti-core antibody production 24
4.3. Adoptive transfer of HBV-specific T cells increased the level of anti-core antibody. 25
4.4. The increase of anti-core antibody resulted from liver damage 26
4.5. Depletion of CD4 T cell did not affect the anti-core antibody production induced by TAA treatment. 27
4.6. The nucleocapsid structure is critical for production of anti-core antibody in hydrodynamic mouse model. 28
5. Discussion 30
5.1. The role of CD4 T cell in anti-core antibody in a hydrodynamic HBV mouse model. 30
5.2. Increase of the anti-core antibody in mice receiving HBV-specific T cells 32
5.3. Increase of the anti-core antibody by TAA treatment 33
5.4. Capsid formation in anti-core antibody production 34
5.5. T cell immunity and anti-HBc antibody production 35
6. Reference 36
7. FIGURES 41

1.Blumberg, B.S. and H.J. Alter, A "new" antigen in leukemia sera. JAMA, 1965. 191(7): p. 541-546.
2.Blumberg, B.S., et al., A Serum Antigen (Australia Antigen) in Down''s Syndrome, Leukemia, and Hepatitis. Annals of Internal Medicine, 1967. 66(5): p. 924-931.
3.Blumberg, B.S., A.I. Sutnick, and W.T. London, Hepatitis and leukemia: their relation to Australia antigen. Bull N Y Acad Med, 1968. 44(12): p. 1566-86.
4.Prince, A.M., An antigen detected in the blood during the incubation period of serum hepatitis. Proc Natl Acad Sci U S A, 1968. 60(3): p. 814-21.
5.Prince, A.M., H. Fuji, and R.K. Gershon, IMMUNOHISTOCHEMICAL STUDIES ON THE ETIOLOGY OF ANICTERIC HEPATITIS IN KOREA. Am J Hyg, 1964. 79: p. 365-81.
6.Dane, D.S., C.H. Cameron, and M. Briggs, VIRUS-LIKE PARTICLES IN SERUM OF PATIENTS WITH AUSTRALIA-ANTIGEN-ASSOCIATED HEPATITIS. The Lancet, 1970. 295(7649): p. 695-698.
7.Robinson, W.S., DNA and DNA polymerase of a virus-like particle in hepatitis B. Dev Biol Stand, 1975. 30: p. 23-37.
8.Cattaneo, R., H. Will, and H. Schaller, Hepatitis B virus transcription in the infected liver. Embo j, 1984. 3(9): p. 2191-6.
9.Enders, G.H., D. Ganem, and H. Varmus, Mapping the major transcripts of ground squirrel hepatitis virus: the presumptive template for reverse transcriptase is terminally redundant. Cell, 1985. 42(1): p. 297-308.
10.Heermann, K.H., et al., Large surface proteins of hepatitis B virus containing the pre-s sequence. J Virol, 1984. 52(2): p. 396-402.
11.Heermann, K.H., et al., Immunogenicity of the gene S and Pre-S domains in hepatitis B virions and HBsAg filaments. Intervirology, 1987. 28(1): p. 14-25.
12.Kaplan, P.M., et al., DNA polymerase associated with human hepatitis B antigen. J Virol, 1973. 12(5): p. 995-1005.
13.Summers, J., A. O''Connell, and I. Millman, Genome of hepatitis B virus: restriction enzyme cleavage and structure of DNA extracted from Dane particles. Proc Natl Acad Sci U S A, 1975. 72(11): p. 4597-601.
14.Porterfield, J.Z., et al., Full-length hepatitis B virus core protein packages viral and heterologous RNA with similarly high levels of cooperativity. J Virol, 2010. 84(14): p. 7174-84.
15.Conway, J.F., et al., Visualization of a 4-helix bundle in the hepatitis B virus capsid by cryo-electron microscopy. Nature, 1997. 386(6620): p. 91-4.
16.Wynne, S.A., R.A. Crowther, and A.G. Leslie, The crystal structure of the human hepatitis B virus capsid. Mol Cell, 1999. 3(6): p. 771-80.
17.Bottcher, B., S.A. Wynne, and R.A. Crowther, Determination of the fold of the core protein of hepatitis B virus by electron cryomicroscopy. Nature, 1997. 386(6620): p. 88-91.
18.WHO:Media centre -Hepatitis B.
19.Dienstag, J.L., Hepatitis B Virus Infection. New England Journal of Medicine, 2008. 359(14): p. 1486-1500.
20.Stevens, C.E., et al., HBeAg and anti-HBe detection by radioimmunoassay: correlation with vertical transmission of hepatitis B virus in Taiwan. J Med Virol, 1979. 3(3): p. 237-41.
21.Liaw, Y.-F. and C.-M. Chu, Hepatitis B virus infection. The Lancet. 373(9663): p. 582-592.
22.Lok, A.S. and B.J. McMahon, Chronic hepatitis B. Hepatology, 2007. 45(2): p. 507-39.
23.Werle-Lapostolle, B., et al., Persistence of cccDNA during the natural history of chronic hepatitis B and decline during adefovir dipivoxil therapy. Gastroenterology, 2004. 126(7): p. 1750-8.
24.Hadziyannis, S.J. and D. Vassilopoulos, Hepatitis B e antigen—negative chronic hepatitis B. Hepatology, 2001. 34(4, Part 1): p. 617-624.
25.Wieland, S., et al., Genomic analysis of the host response to hepatitis B virus infection. Proc Natl Acad Sci U S A, 2004. 101(17): p. 6669-74.
26.Dunn, C., et al., Temporal analysis of early immune responses in patients with acute hepatitis B virus infection. Gastroenterology, 2009. 137(4): p. 1289-300.
27.Wang, H. and W.-S. Ryu, Hepatitis B Virus Polymerase Blocks Pattern Recognition Receptor Signaling via Interaction with DDX3: Implications for Immune Evasion. PLoS Pathog, 2010. 6(7): p. e1000986.
28.Yu, S., et al., Hepatitis B virus polymerase inhibits RIG-I- and Toll-like receptor 3-mediated beta interferon induction in human hepatocytes through interference with interferon regulatory factor 3 activation and dampening of the interaction between TBK1/IKKepsilon and DDX3. J Gen Virol, 2010. 91(Pt 8): p. 2080-90.
29.Wei, C., et al., The hepatitis B virus X protein disrupts innate immunity by downregulating mitochondrial antiviral signaling protein. J Immunol, 2010. 185(2): p. 1158-68.
30.Wang, X., et al., Hepatitis B virus X protein suppresses virus-triggered IRF3 activation and IFN-beta induction by disrupting the VISA-associated complex. Cell Mol Immunol, 2010. 7(5): p. 341-8.
31.Kumar, M., et al., Hepatitis B virus regulatory HBx protein binds to adaptor protein IPS-1 and inhibits the activation of beta interferon. J Virol, 2011. 85(2): p. 987-95.
32.Moss, B., et al., Live recombinant vaccinia virus protects chimpanzees against hepatitis B. Nature, 1984. 311(5981): p. 67-9.
33.Webster, G.J., et al., Incubation phase of acute hepatitis B in man: dynamic of cellular immune mechanisms. Hepatology, 2000. 32(5): p. 1117-24.
34.Thimme, R., et al., CD8(+) T cells mediate viral clearance and disease pathogenesis during acute hepatitis B virus infection. J Virol, 2003. 77(1): p. 68-76.
35.Yang, P.L., et al., Immune effectors required for hepatitis B virus clearance. Proc Natl Acad Sci U S A, 2010. 107(2): p. 798-802.
36.Guidotti, L.G., et al., Intracellular inactivation of the hepatitis B virus by cytotoxic T lymphocytes. Immunity, 1996. 4(1): p. 25-36.
37.Guidotti, L.G., et al., Cytotoxic T lymphocytes inhibit hepatitis B virus gene expression by a noncytolytic mechanism in transgenic mice. Proc Natl Acad Sci U S A, 1994. 91(9): p. 3764-8.
38.Guidotti, L.G., et al., Viral clearance without destruction of infected cells during acute HBV infection. Science, 1999. 284(5415): p. 825-9.
39.Penna, A., et al., Cytotoxic T lymphocytes recognize an HLA-A2-restricted epitope within the hepatitis B virus nucleocapsid antigen. J Exp Med, 1991. 174(6): p. 1565-70.
40.Rehermann, B., et al., The cytotoxic T lymphocyte response to multiple hepatitis B virus polymerase epitopes during and after acute viral hepatitis. J Exp Med, 1995. 181(3): p. 1047-58.
41.Bertoletti, A., et al., HLA class I-restricted human cytotoxic T cells recognize endogenously synthesized hepatitis B virus nucleocapsid antigen. Proc Natl Acad Sci U S A, 1991. 88(23): p. 10445-9.
42.Boni, C., et al., Characterization of hepatitis B virus (HBV)-specific T-cell dysfunction in chronic HBV infection. J Virol, 2007. 81(8): p. 4215-25.
43.Lopes, A.R., et al., Bim-mediated deletion of antigen-specific CD8 T cells in patients unable to control HBV infection. J Clin Invest, 2008. 118(5): p. 1835-45.
44.Grayson, J.M., et al., Role of Bim in regulating CD8+ T-cell responses during chronic viral infection. J Virol, 2006. 80(17): p. 8627-38.
45.Schurich, A., et al., Role of the coinhibitory receptor cytotoxic T lymphocyte antigen-4 on apoptosis-Prone CD8 T cells in persistent hepatitis B virus infection. Hepatology, 2011. 53(5): p. 1494-503.
46.Raziorrouh, B., et al., The immunoregulatory role of CD244 in chronic hepatitis B infection and its inhibitory potential on virus-specific CD8+ T-cell function. Hepatology, 2010. 52(6): p. 1934-47.
47.Ejrnaes, M., et al., Resolution of a chronic viral infection after interleukin-10 receptor blockade. J Exp Med, 2006. 203(11): p. 2461-72.
48.Lau, G.K.K., et al., Peginterferon Alfa-2a, Lamivudine, and the Combination for HBeAg-Positive Chronic Hepatitis B. New England Journal of Medicine, 2005. 352(26): p. 2682-2695.
49.Marcellin, P., et al., Peginterferon alfa-2a alone, lamivudine alone, and the two in combination in patients with HBeAg-negative chronic hepatitis B. N Engl J Med, 2004. 351(12): p. 1206-17.
50.Bohne, F., et al., T cells redirected against hepatitis B virus surface proteins eliminate infected hepatocytes. Gastroenterology, 2008. 134(1): p. 239-47.
51.Marcellin, P., et al., Tenofovir disoproxil fumarate versus adefovir dipivoxil for chronic hepatitis B. N Engl J Med, 2008. 359(23): p. 2442-55.
52.Marcellin, P., et al., Adefovir dipivoxil for the treatment of hepatitis B e antigen-positive chronic hepatitis B. N Engl J Med, 2003. 348(9): p. 808-16.
53.Flink, H.J., et al., Flares in chronic hepatitis B patients induced by the host or the virus? Relation to treatment response during Peg-interferon {alpha}-2b therapy. Gut, 2005. 54(11): p. 1604-9.
54.Webster, G.J., et al., Longitudinal analysis of CD8+ T cells specific for structural and nonstructural hepatitis B virus proteins in patients with chronic hepatitis B: implications for immunotherapy. J Virol, 2004. 78(11): p. 5707-19.
55.Loggi, E., et al., Virus-specific immune response in HBeAg-negative chronic hepatitis B: relationship with clinical profile and HBsAg serum levels. PLoS One, 2013. 8(6): p. e65327.
56.Liaw, Y.F., Clinical utility of hepatitis B surface antigen quantitation in patients with chronic hepatitis B: a review. Hepatology, 2011. 54(2): p. E1-9.
57.Yuan, Q., et al., Quantitative hepatitis B core antibody level may help predict treatment response in chronic hepatitis B patients. Gut, 2013. 62(1): p. 182-4.
58.Slifka, M.K., et al., Humoral immunity due to long-lived plasma cells. Immunity, 1998. 8(3): p. 363-72.
59.Ahuja, A., et al., Maintenance of the plasma cell pool is independent of memory B cells. Proc Natl Acad Sci U S A, 2008. 105(12): p. 4802-7.
60.DiLillo, D.J., et al., Maintenance of long-lived plasma cells and serological memory despite mature and memory B cell depletion during CD20 immunotherapy in mice. J Immunol, 2008. 180(1): p. 361-71.
61.Bernasconi, N.L., E. Traggiai, and A. Lanzavecchia, Maintenance of serological memory by polyclonal activation of human memory B cells. Science, 2002. 298(5601): p. 2199-202.
62.Benson, M.J., et al., Distinction of the memory B cell response to cognate antigen versus bystander inflammatory signals. J Exp Med, 2009. 206(9): p. 2013-25.
63.Huang, L.R., et al., An immunocompetent mouse model for the tolerance of human chronic hepatitis B virus infection. Proc Natl Acad Sci U S A, 2006. 103(47): p. 17862-7.
64.Lin, Y.J., et al., Hepatitis B virus nucleocapsid but not free core antigen controls viral clearance in mice. J Virol, 2012. 86(17): p. 9266-73.
65.Lin, Y.J., et al., Hepatitis B virus core antigen determines viral persistence in a C57BL/6 mouse model. Proc Natl Acad Sci U S A, 2010. 107(20): p. 9340-5.
66.Suda, T., et al., Structural impact of hydrodynamic injection on mouse liver. Gene Ther, 2007. 14(2): p. 129-37.
67.Li, A., et al., Novel double-antigen sandwich immunoassay for human hepatitis B core antibody. Clin Vaccine Immunol, 2010. 17(3): p. 464-9.
68.Milich, D.R. and A. McLachlan, The nucleocapsid of hepatitis B virus is both a T-cell-independent and a T-cell-dependent antigen. Science, 1986. 234(4782): p. 1398-401.
69.Jang, J.H., et al., Reevaluation of experimental model of hepatic fibrosis induced by hepatotoxic drugs: an easy, applicable, and reproducible model. Transplant Proc, 2008. 40(8): p. 2700-3.