Title

發展抗B型肝炎病毒大型表面蛋白之細胞內抗體作為治療慢性B型肝炎的藥物

Translated Titles

Intrabody directed against HBV LHBsAg as potential therapeutic agents for CHB

DOI

10.6342/NTU.2006.02420

Authors

吳佩玲

Key Words

B型肝炎病毒 ; B型肝炎 ; 慢性B型肝炎感染 ; 病毒複製 ; 抗病毒 ; 抗病毒藥物 ; 療法 ; 治療 ; 藥物開發 ; 抗體 ; 抗原 ; 細胞內抗體 ; 胞內抗體 ; 表面抗原 ; 大型表面蛋白 ; 單鏈 ; 單鏈抗體 ; hepatitis B virus ; HBV ; chronic hepatits B ; CHB ; viral replication ; antiviral ; antiviral agent ; therapy ; drug development ; antibody ; antigen ; viral antigen ; intracellular antibody ; intrabody ; surface antigen ; large surface antigen ; LHBsAg ; HBsAg ; preS1 ; MA18/7 ; single-chain ; variable domain ; single-chain variable domain ; scFv

PublicationName

臺灣大學微生物學研究所學位論文

Volume or Term/Year and Month of Publication

2006年

Academic Degree Category

碩士

Advisor

陶秘華

Content Language

英文

Chinese Abstract

當今全世界約有三億五千萬人患有慢性B型肝炎,由慢性B型肝炎所引發之肝硬化及肝癌等相關疾病,每年造成的全球死亡人數高達一百多萬,嚴重危害人類的健康。目前治療慢性B型肝炎藥物有免疫調節劑及抗病毒藥物兩類,它們在臨床上雖皆具一定療效,但前者會爲病患帶來諸多不適的副作用,而後者則面臨著引起病毒抗藥性的挑戰。因此,研發對抗B型肝炎病毒的新型藥物,遂成為刻不容緩的研究課題。 基於與抗原間的結合極具專一性及親和力,單株抗體素被廣泛應用於疾病的診斷,近年來更逐漸成為新藥研發的主流之一。過去,抗體藥物的研發方向多著重於調控細胞外蛋白質之生物功能,而涵蓋多數致病因子的細胞內蛋白質反被忽略。隨著抗體工程技術的進展,一個原應分泌至細胞外的抗體分子,經過適當修飾後,可以停留在細胞內部與細胞內抗原結合,進而干擾抗原之正常生理作用。有鑑於此,本研究欲探討此類細胞內抗體(intrabody)作為治療慢性B型肝炎之藥物的可行性。在諸多B型肝炎病毒之病毒蛋白中,大型表面蛋白(LHBsAg)對病毒之複製有舉足輕重的影響,故本研究選其作為細胞內抗體之抗原標的。 過去,我們已將一個能有效辨認大型表面蛋白preS1區域之單株抗體—MA18/7—轉成單鏈抗體型態(single-chain Fv)。本研究中,我們證實該表現質體轉染進入細胞後確能順利表現出MA18/7scFv蛋白質,以作為細胞內抗體。接著,我們分別在兩種B型肝炎病毒體外複製之模式系統中檢驗MA18/7細胞內抗體對病毒複製的影響:一、將病毒基因組DNA與細胞內抗體表現質體DNA共同轉染入肝細胞株;二、以腺病毒載體攜帶病毒基因組DNA進入已穩定表現細胞內抗體之肝細胞株。初步結果發現:在系統一中,MA18/7細胞內抗體對B型肝炎病毒表面蛋白(HBsAg)的分泌量無顯著影響。至於在系統二中,MA18/7細胞內抗體卻能明顯抑制肝細胞分泌B型肝炎病毒大型表面蛋白,然而B型肝炎病毒DNA的總釋放量,以及子代病毒外套膜之存在與否皆不因MA18/7細胞內抗體有所改變。造成此間差異之原因究竟為何,尚需後續實驗加以驗證。 此外,本研究亦計畫修飾MA18/7細胞內抗體之蛋白組成,使之專一地分佈於細胞內特定胞器中,以期提高其尋找目標抗原的成功率,進而加強其抑制能力。目前我們已完成了分布在細胞核或細胞質中的兩種MA18/7細胞內抗體的構築,並藉由免疫螢光染色法證實其分佈位置確如預期。 最後,本研究更進一步探討前述抑制作用之機制。我們分別利用螢光蛋白標籤以及免疫螢光染色法兩種方法證實,MA18/7細胞內抗體與其目標抗原—大型表面蛋白—在細胞內的分佈位置相疊合,初步證明兩者之間可能存有交互作用。 綜合而言,本研究初步發現,抗B型肝炎病毒大型表面蛋白之MA18/7細胞內抗體在蛋白質層次上對病毒有顯著抑制效果。另一方面,雖然抗體與抗原間之交互作用已被證實,惟其詳盡機制尚待日後研究方能解答。不論答案為何,以細胞內抗體作為治療慢性B型肝炎藥物確為值得發展的方向。

Topic Category 醫藥衛生 > 基礎醫學
醫學院 > 微生物學研究所
Reference
  1. 1. Sprinzl, M.F., et al. Transfer of hepatitis B virus genome by adenovirus vectors into cultured cells and mice: crossing the species barrier. J Virol (2001) 75: 5108-18.
    連結:
  2. 2. Chisari, F.V. Rous-Whipple Award Lecture. Viruses, immunity, and cancer: lessons from hepatitis B. Am J Pathol (2000) 156: 1117-32.
    連結:
  3. 3. Lee, W.M. Hepatitis B virus infection. N Engl J Med (1997) 337: 1733-45.
    連結:
  4. 4. Ganem, D. and A.M. Prince Hepatitis B virus infection--natural history and clinical consequences. N Engl J Med (2004) 350: 1118-29.
    連結:
  5. 5. Dane, D.S., C.H. Cameron, and M. Briggs Virus-like particles in serum of patients with Australia-antigen-associated hepatitis. Lancet (1970) 1: 695-8.
    連結:
  6. 6. Ganem, D. Assembly of hepadnaviral virions and subviral particles. Curr Top Microbiol Immunol (1991) 168: 61-83.
    連結:
  7. 7. Robinson, W.S. and L.I. Lutwick The virus of hepatitis, type B (first of two parts). N Engl J Med (1976) 295: 1168-75.
    連結:
  8. 8. 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: 4597-601.
    連結:
  9. 9. Galibert, F., et al. Nucleotide sequence of the hepatitis B virus genome (subtype ayw) cloned in E. coli. Nature (1979) 281: 646-50.
    連結:
  10. 10. Pasquetto, V., et al. Cytokine-sensitive replication of hepatitis B virus in immortalized mouse hepatocyte cultures. J Virol (2002) 76: 5646-53.
    連結:
  11. 12. Summers, J. and W.S. Mason Replication of the genome of a hepatitis B--like virus by reverse transcription of an RNA intermediate. Cell (1982) 29: 403-15.
    連結:
  12. 15. Tuttleman, J.S., C. Pourcel, and J. Summers Formation of the pool of covalently closed circular viral DNA in hepadnavirus-infected cells. Cell (1986) 47: 451-60.
    連結:
  13. 16. Lambert, C. and R. Prange Dual topology of the hepatitis B virus large envelope protein: determinants influencing post-translational pre-S translocation. J Biol Chem (2001) 276: 22265-72.
    連結:
  14. 17. Lambert, C., S. Mann, and R. Prange Assessment of determinants affecting the dual topology of hepadnaviral large envelope proteins. J Gen Virol (2004) 85: 1221-5.
    連結:
  15. 19. Hildt, E., et al. The hepatitis B virus large surface protein (LHBs) is a transcriptional activator. Virology (1996) 225: 235-9.
    連結:
  16. 21. Eble, B.E., et al. Multiple topogenic sequences determine the transmembrane orientation of the hepatitis B surface antigen. Mol Cell Biol (1987) 7: 3591-601.
    連結:
  17. 23. Bruss, V., et al. Post-translational alterations in transmembrane topology of the hepatitis B virus large envelope protein. Embo J (1994) 13: 2273-9.
    連結:
  18. 25. Prange, R. and R.E. Streeck Novel transmembrane topology of the hepatitis B virus envelope proteins. Embo J (1995) 14: 247-56.
    連結:
  19. 26. Prange, R. and R.E. Streeck Novel transmembrane topology of the hepatitis B virus envelope proteins. Embo J (1995) 14: 247-56.
    連結:
  20. 27. Chisari, F.V. and C. Ferrari Hepatitis B virus immunopathogenesis. Annu Rev Immunol (1995) 13: 29-60.
    連結:
  21. 28. Zoulim, F. Antiviral therapy of chronic hepatitis B. Antiviral Res (2006).
    連結:
  22. 29. Zoulim, F. Therapy of chronic hepatitis B virus infection: inhibition of the viral polymerase and other antiviral strategies. Antiviral Res (1999) 44: 1-30.
    連結:
  23. 30. Lin, O.S. and E.B. Keeffe Current treatment strategies for chronic hepatitis B and C. Annu Rev Med (2001) 52: 29-49.
    連結:
  24. 31. Pawlotsky, J.M. Virology of hepatitis B and C viruses and antiviral targets. J Hepatol (2006) 44: S10-3.
    連結:
  25. 32. Locarnini, S., et al. Management of antiviral resistance in patients with chronic hepatitis B. Antivir Ther (2004) 9: 679-93.
    連結:
  26. 33. Zoulim, F. Mechanism of viral persistence and resistance to nucleoside and nucleotide analogs in chronic hepatitis B virus infection. Antiviral Res (2004) 64: 1-15.
    連結:
  27. 34. Mailliard, M.E. and J.L. Gollan Emerging therapeutics for chronic hepatitis B. Annu Rev Med (2006) 57: 155-66.
    連結:
  28. 35. Puoti, M., et al. Hepatitis B virus co-infection in human immunodeficiency virus-infected subjects. AIDS Rev (2002) 4: 27-35.
    連結:
  29. 36. Feld, J. and S. Locarnini Antiviral therapy for hepatitis B virus infections: new targets and technical challenges. J Clin Virol (2002) 25: 267-83.
    連結:
  30. 37. Feld, J., J.Y. Lee, and S. Locarnini New targets and possible new therapeutic approaches in the chemotherapy of chronic hepatitis B. Hepatology (2003) 38: 545-53.
    連結:
  31. 38. Bottcher, B., et al. Peptides that block hepatitis B virus assembly: analysis by cryomicroscopy, mutagenesis and transfection. Embo J (1998) 17: 6839-45.
    連結:
  32. 39. Lu, X., et al. Aberrant trafficking of hepatitis B virus glycoproteins in cells in which N-glycan processing is inhibited. Proc Natl Acad Sci U S A (1997) 94: 2380-5.
    連結:
  33. 40. Stocks, M.R. Intrabodies: production and promise. Drug Discov Today (2004) 9: 960-6.
    連結:
  34. 41. Stocks, M. Intrabodies as drug discovery tools and therapeutics. Curr Opin Chem Biol (2005) 9: 359-65.
    連結:
  35. 42. Antman, K.H. and D.M. Livingston Intracellular neutralization of SV40 tumor antigens following microinjection of specific antibody. Cell (1980) 19: 627-35.
    連結:
  36. 43. Wirtz, P. and B. Steipe Intrabody construction and expression III: engineering hyperstable V(H) domains. Protein Sci (1999) 8: 2245-50.
    連結:
  37. 44. Lobato, M.N. and T.H. Rabbitts Intracellular antibodies as specific reagents for functional ablation: future therapeutic molecules. Curr Mol Med (2004) 4: 519-28.
    連結:
  38. 45. Lobato, M.N. and T.H. Rabbitts Intracellular antibodies and challenges facing their use as therapeutic agents. Trends Mol Med (2003) 9: 390-6.
    連結:
  39. 46. Cardinale, A., et al. Intracellular targeting and functional analysis of single-chain Fv fragments in mammalian cells. Methods (2004) 34: 171-8.
    連結:
  40. 47. Biocca, S. and A. Cattaneo Intracellular immunization: antibody targeting to subcellular compartments. Trends Cell Biol (1995) 5: 248-52.
    連結:
  41. 48. Cattaneo, A. and S. Biocca The selection of intracellular antibodies. Trends Biotechnol (1999) 17: 115-21.
    連結:
  42. 49. Marasco, W.A., W.A. Haseltine, and S.Y. Chen Design, intracellular expression, and activity of a human anti-human immunodeficiency virus type 1 gp120 single-chain antibody. Proc Natl Acad Sci U S A (1993) 90: 7889-93.
    連結:
  43. 50. Goncalves, J., et al. Functional neutralization of HIV-1 Vif protein by intracellular immunization inhibits reverse transcription and viral replication. J Biol Chem (2002) 277: 32036-45.
    連結:
  44. 51. Maciejewski, J.P., et al. Intracellular expression of antibody fragments directed against HIV reverse transcriptase prevents HIV infection in vitro. Nat Med (1995) 1: 667-73.
    連結:
  45. 53. Shaheen, F., et al. Targeting human immunodeficiency virus type 1 reverse transcriptase by intracellular expression of single-chain variable fragments to inhibit early stages of the viral life cycle. J Virol (1996) 70: 3392-400.
    連結:
  46. 56. Duan, L., et al. Potent inhibition of human immunodeficiency virus type 1 replication by an intracellular anti-Rev single-chain antibody. Proc Natl Acad Sci U S A (1994) 91: 5075-9.
    連結:
  47. 57. Wu, Y., et al. Binding of intracellular anti-Rev single chain variable fragments to different epitopes of human immunodeficiency virus type 1 rev: variations in viral inhibition. J Virol (1996) 70: 3290-7.
    連結:
  48. 58. Duan, L., et al. Intracellular immunization against HIV-1 infection of human T lymphocytes: utility of anti-rev single-chain variable fragments. Hum Gene Ther (1995) 6: 1561-73.
    連結:
  49. 59. Kubota, S., et al. Nuclear preservation and cytoplasmic degradation of human immunodeficiency virus type 1 Rev protein. J Virol (1996) 70: 1282-7.
    連結:
  50. 60. Steinberger, P., et al. Functional deletion of the CCR5 receptor by intracellular immunization produces cells that are refractory to CCR5-dependent HIV-1 infection and cell fusion. Proc Natl Acad Sci U S A (2000) 97: 805-10.
    連結:
  51. 61. Visintin, M., et al. The intracellular antibody capture technology (IACT): towards a consensus sequence for intracellular antibodies. J Mol Biol (2002) 317: 73-83.
    連結:
  52. 62. Lecerf, J.M., et al. Human single-chain Fv intrabodies counteract in situ huntingtin aggregation in cellular models of Huntington's disease. Proc Natl Acad Sci U S A (2001) 98: 4764-9.
    連結:
  53. 63. Wagner, R.W. and W.M. Flanagan Antisense technology and prospects for therapy of viral infections and cancer. Mol Med Today (1997) 3: 31-8.
    連結:
  54. 64. Hannon, G.J. RNA interference. Nature (2002) 418: 244-51.
    連結:
  55. 65. Tuschl, T. Expanding small RNA interference. Nat Biotechnol (2002) 20: 446-8.
    連結:
  56. 66. Miller, T.W. and A. Messer Intrabody applications in neurological disorders: progress and future prospects. Mol Ther (2005) 12: 394-401.
    連結:
  57. 67. zu Putlitz, J., A. Skerra, and J.R. Wands Intracellular expression of a cloned antibody fragment interferes with hepatitis B virus surface antigen secretion. Biochem Biophys Res Commun (1999) 255: 785-91.
    連結:
  58. 68. Yamamoto, M., et al. Intracellular single-chain antibody against hepatitis B virus core protein inhibits the replication of hepatitis B virus in cultured cells. Hepatology (1999) 30: 300-7.
    連結:
  59. 69. Wu, C.J., et al. In vivo electroporation of skeletal muscles increases the efficacy of Japanese encephalitis virus DNA vaccine. Vaccine (2004) 22: 1457-64.
    連結:
  60. 70. Kuttner, G., et al. Characterization of neutralizing anti-pre-S1 and anti-pre-S2 (HBV) monoclonal antibodies and their fragments. Mol Immunol (1999) 36: 669-83.
    連結:
  61. 71. Cardinale, A., I. Filesi, and S. Biocca Aggresome formation by anti-Ras intracellular scFv fragments. The fate of the antigen-antibody complex. Eur J Biochem (2001) 268: 268-77.
    連結:
  62. 72. Accardi, L., et al. Intracellular anti-E7 human antibodies in single-chain format inhibit proliferation of HPV16-positive cervical carcinoma cells. Int J Cancer (2005) 116: 564-70.
    連結:
  63. 73. Sprinzl, M.F., et al. Transfer of hepatitis B virus genome by adenovirus vectors into cultured cells and mice: crossing the species barrier. J Virol (2001) 75: 5108-18.
    連結:
  64. 74. Guidotti, L.G., et al. High-level hepatitis B virus replication in transgenic mice. J Virol (1995) 69: 6158-69.
    連結:
  65. 75. Tsui-Fang Hung Pseudoreceptors for hepatitis B virus infection. (Thesis for Master’s degree) (2005)
    連結:
  66. 76. Kay, M.A., D. Liu, and P.M. Hoogerbrugge Gene therapy. Proc Natl Acad Sci U S A (1997) 94: 12744-6.
    連結:
  67. 77. Ragot, T., P. Opolon, and M. Perricaudet Adenoviral gene delivery. Methods Cell Biol (1997) 52: 229-60.
    連結:
  68. 78. Ren, S. and M. Nassal Hepatitis B virus (HBV) virion and covalently closed circular DNA formation in primary tupaia hepatocytes and human hepatoma cell lines upon HBV genome transduction with replication-defective adenovirus vectors. J Virol (2001) 75: 1104-16.
    連結:
  69. 80. Kalderon, D., et al. A short amino acid sequence able to specify nuclear location. Cell (1984) 39: 499-509.
    連結:
  70. 81. Lanford, R.E., P. Kanda, and R.C. Kennedy Induction of nuclear transport with a synthetic peptide homologous to the SV40 T antigen transport signal. Cell (1986) 46: 575-82.
    連結:
  71. 82. Fischer-Fantuzzi, L. and C. Vesco Cell-dependent efficiency of reiterated nuclear signals in a mutant simian virus 40 oncoprotein targeted to the nucleus. Mol Cell Biol (1988) 8: 5495-503.
    連結:
  72. 83. Cormack, B.P., R.H. Valdivia, and S. Falkow FACS-optimized mutants of the green fluorescent protein (GFP). Gene (1996) 173: 33-8.
    連結:
  73. 84. Haas, J., E.C. Park, and B. Seed Codon usage limitation in the expression of HIV-1 envelope glycoprotein. Curr Biol (1996) 6: 315-24.
    連結:
  74. 85. Biocca, S., et al. Redox state of single chain Fv fragments targeted to the endoplasmic reticulum, cytosol and mitochondria. Biotechnology (N Y) (1995) 13: 1110-5.
    連結:
  75. 86. Arafat, W., et al. Antineoplastic effect of anti-erbB-2 intrabody is not correlated with scFv affinity for its target. Cancer Gene Ther (2000) 7: 1250-6.
    連結:
  76. 11. Pollack, J.R. and D. Ganem Site-specific RNA binding by a hepatitis B virus reverse transcriptase initiates two distinct reactions: RNA packaging and DNA synthesis. J Virol (1994) 68: 5579-87.
  77. 13. Will, H., et al. Replication strategy of human hepatitis B virus. J Virol (1987) 61: 904-11.
  78. 14. Wang, G.H. and C. Seeger Novel mechanism for reverse transcription in hepatitis B viruses. J Virol (1993) 67: 6507-12.
  79. 18. Bruss, V. and K. Vieluf Functions of the internal pre-S domain of the large surface protein in hepatitis B virus particle morphogenesis. J Virol (1995) 69: 6652-7.
  80. 20. Le Seyec, J., et al. Infection process of the hepatitis B virus depends on the presence of a defined sequence in the pre-S1 domain. J Virol (1999) 73: 2052-7.
  81. 22. Prange, R., R. Nagel, and R.E. Streeck Deletions in the hepatitis B virus small envelope protein: effect on assembly and secretion of surface antigen particles. J Virol (1992) 66: 5832-41.
  82. 24. Ostapchuk, P., P. Hearing, and D. Ganem A dramatic shift in the transmembrane topology of a viral envelope glycoprotein accompanies hepatitis B viral morphogenesis. Embo J (1994) 13: 1048-57.
  83. 52. Gargano, N., et al. Human recombinant antibody fragments neutralizing human immunodeficiency virus type 1 reverse transcriptase provide an experimental basis for the structural classification of the DNA polymerase family. J Virol (1996) 70: 7706-12.
  84. 54. Levy-Mintz, P., et al. Intracellular expression of single-chain variable fragments to inhibit early stages of the viral life cycle by targeting human immunodeficiency virus type 1 integrase. J Virol (1996) 70: 8821-32.
  85. 55. Mhashilkar, A.M., et al. Inhibition of HIV-1 Tat-mediated LTR transactivation and HIV-1 infection by anti-Tat single chain intrabodies. Embo J (1995) 14: 1542-51.
  86. 79. Lenhoff, R.J. and J. Summers Coordinate regulation of replication and virus assembly by the large envelope protein of an avian hepadnavirus. J Virol (1994) 68: 4565-71.