臺灣博碩士論文加值系統

流感病毒在全球廣為流行，其中以A型的變異性最高且最嚴重，近來發現會嚴重病徵像肺炎、ARDS等案例，致死率相當高；而目前已上市疫苗主要抗原性是針對病毒表面的HA與NA，但對不斷產生突變的A型流感病毒來說無法做出有效的預測及防治，因此在研究上希望以改進疫苗的廣效性及安全性作為設計重點。近來研究發現，流感病毒表面的M2離子通道的extracelluar片段(M2e)在各分型中具有高度相似性，有動物實驗指出M2e可促使動物產生產生專一性抗體，並能防禦後續不同型別的病毒感染；然而，M2e的促進免疫能力並沒有傳統HA疫苗來得高，所以需要搭配佐劑使用，但是佐劑主成分通常來自細菌鞭毛或毒素，可能使接種者引發過敏反應；為此，我們利用國衛院冷治湘博士開發出的平台，接上來自奈瑟氏雙球菌N端40個殘基可以促進脂化修飾我們的目標蛋白，實驗證實脂化修飾可以替代傳統佐劑作用且較為安全；此外，為了更加強化M2e的抗原性，實驗室利用來自諾羅病毒的外殼蛋白P單元所聚合成的P粒子來做抗原載體；在單獨表現的P單元呈雙元體存在，並可進一步聚合成12聚體，即P粒子，該粒子的圈環位置上可用來呈現目標重組蛋白抗原。本實驗利用建構脂化修飾諾羅病毒P粒子載體，藉此呈現M2e抗原，轉殖入大腸桿菌中，利用調控大腸桿菌培養液pH值、葡萄糖濃度，來探討IPTG誘導目標蛋白結果，結果顯示在中性環境下含2%葡萄糖的M9培養液，可誘導出相當程度的蛋白表現於細菌內涵體中，並利用LC-MS/MS質譜儀比對目標蛋白序列；針對內涵體部分，我們比較不同濃度的尿素及鹽酸胍對目標蛋白溶出情形，結果顯示鹽酸胍效果較佳。並進一步透過管柱純化，以透析方式去除變性劑，讓P粒子重新聚合，並去除內毒素後，再進行細胞動物免疫相關實驗。在細胞實驗部分利用樹突狀細胞活化來測試確定具有脂化效果；另外在免疫小鼠的血清中確認未使用佐劑免疫的脂化修飾P粒子疫苗可誘發較高的特異性血清抗體反應，其效果等同使用佐劑於非脂化修飾蛋白組別；再者，由小鼠血清中抗體可偵測到IgG2a抗體的存在，意即能誘導老鼠免疫系統產生TH1的細胞免疫反應；最後，再利用免疫小鼠血清，與H1N1及H5N1兩株活病毒株進行抗體中和試驗，感染MDCK後再利用流式細胞儀偵測病毒核蛋白表現量，結果指出小鼠血清可中和不同株型的A型流感活病毒，降低MDCK細胞的感染率，達到廣效型的保護；因此利用脂化修飾諾羅病毒P粒子夾帶流感抗原來作為疫苗使用，有潛力做為新一代流感疫苗。

Influenza A virus (IAV) can cause diverse clinical symptoms and highly pathogenic strains may cause fatal symptoms such as acute respiratory distress syndrome (ARDS) etc. Commercial influenza vaccine is usually designed upon specific viral hemagglutinin (HA) and neuraminidase (NA); however the protection from seasonal flu vaccine is of limit used due to high viral mutation. Therefore, the universal vaccine strategies aim to overcome strain-specific limitations. Recent researches suggest that the extracellular domain of M2 ion channel, M2e, is highly conserved among different subtypes of IAVs. It could induce specific antibody response and offer protection against virus challenge in animal model. Due to its low immunogenicity, it should be combined with adjuvant or presented by viral capsid protein as carrier. For this purpose, we used P particles composed of the P domain of structural protein VP1 of norovirus as an antigen-presenting cassette, because the loop structure of P particle can carry foreign proteins. In addition, we also added a lipidation signal peptide, the 40 amino acids in the N-terminal from Neisseria meningitides (from Dr. Leng), as a self-adjuvanting platform to enhance P particle immunogenicity. First, we constructed the plasmid which encodes the lipidated P particle with M2e antigen, Lipo-PMP (LPM). To obtain best induction condition, we compared the effects of pH, and glucose concentration in medium to assess the expression and location of target protein after IPTG induction. Our data showed that LPM was mainly expressed in inclusion bodies using 2% glucose-contained M9 medium after induction. To further purify LPM, we utilized different denature reagents, and we found that guanidine HCl could dissolve more recombinant protein. After denaturation, we separated LPM by liquid chromatography and performed dialysis for functional protein purification. After purification, we used the dendritic cells activity assay to determine the function of lipoprotein. With mouse immunization, we found that the specific antibody titers in lipoprotein immunized mice without adjuvant are comparable to those in the immunized group with adjuvant. We also found that IgG2a specific antibody was increased in the serum of LPM-immuned mouse, indicating that LPM could induce Th1 cell-immune pathway. Finally, with micro-neutralization test, we found that sera from lipo-immuned mouse could neutralize H1N1/H5N1 activated virus, and reduce virus infection. Therefore, Lipidated P particle containing influenza virus antigen could serve as a potential universal influenza vaccine.

致謝..........i
中文摘要........ii
英文摘要..........iii
目錄...........iv

第一章 介紹...........1
1.1 流行性感冒病毒結構介紹..........1
1.2 流感病毒感染機制.........1
1.3 傳染途徑...........2
1.4 病毒致病能力........2
1.5 A型流感病毒流行病學..........2
1.6 A型流感臨床症狀..........3
1.7 流感診斷與治療..........4
1.8 現今流感疫苗發展..........4
1.9病毒表面M2離子通道蛋白之研究..........5
1.10諾羅病毒結構與複製..........6
1.11 諾羅病毒傳染途徑與致病機轉..........7
1.12 諾羅病毒類病毒顆粒及P粒子之發展與應用..........8
1.13 諾羅病毒類病毒顆粒(Norwalk virus-like particle；NV VLP)介紹..........8
1.14 諾羅病毒 P 粒子 (P particle)的發展與應用..........9
1.15脂化修飾的功能..........9
1.16脂化修飾的機轉..........9
1.17脂化修飾技術的發展與應用..........10
1.18 研究動機與目的..........10
第二章 實驗材料..........11
第三章 實驗方法..........27
3.1 流感病毒M2e基因於脂化諾羅病毒蛋白P粒子基因之建構..........27
3.2 重組脂化諾羅病毒蛋白Ｐ粒子之置備與純化..........29
3.3 免疫C57BL/6 小鼠..........34
3.4免疫小鼠相關血清實驗..........35
第四章 實驗結果..........38
4.1 流感病毒M2e基因於脂化諾羅病毒蛋白P粒子基因之建構..........38
4.2 重組脂化諾羅病毒蛋白Ｐ粒子之置備與純化..........39
4.3 脂化修飾諾羅病毒P粒子疫苗之動物免疫血清相關分析..........42
第五章 實驗討論..........45
第六章 參考文獻..........49
第七章 結果附圖..........53
第八章 補充附錄..........66

參考文獻
1. Air, G.M., Sequence relationships among the hemagglutinin genes of 12 subtypes of influenza A virus. Proc. Natl Acad. Sci. USA, 1981. 78: p. 7639-7643.
2. Immunologic studies on the influenza A virus nonstructural protein NS1. The Journal of Experimental Medicine, 1982. 156(1): p. 243-254.
3. Wu, Y., et al., Bat-derived influenza-like viruses H17N10 and H18N11. Trends in Microbiology. 22(4): p. 183-191.
4. Das, K., et al., Structures of influenza A proteins and insights into antiviral drug targets. Nat Struct Mol Biol, 2010. 17(5): p. 530-8.
5. Chen, H., Avian flu: H5N1 virus outbreak in migratory waterfowl. Nature, 2005. 436: p. 191-192.
6. Kuiken, T., Host species barriers to influenza virus infections. Science, 2006. 312: p. 394-397.
7. Lin, Y.P., Avian-to-human transmission of H9N2 subtype influenza A viruses: relationship between H9N2 and H5N1 human isolates. Proc. Natl Acad. Sci. USA, 2000. 97: p. 9654-9658.
8. Belser, J.A., et al., Ocular tropism of influenza A viruses: identification of H7 subtype-specific host responses in human respiratory and ocular cells. J Virol, 2011. 85(19): p. 10117-25.
9. Matrosovich, M., et al., The surface glycoproteins of H5 influenza viruses isolated from humans, chickens, and wild aquatic birds have distinguishable properties. J. Virol., 1999. 73: p. 1146-1155.
10. Ito, T., Molecular basis for the generation in pigs of influenza A viruses with pandemic potential. J. Virol., 1998. 72: p. 7367-7373.
11. Holmes, E.C., Whole-genome analysis of human influenza A virus reveals multiple persistent lineages and reassortment among recent H3N2 viruses. PLoS Biol., 2005. 3: p. e300.
12. Garten, R.J., Antigenic and genetic characteristics of swine-origin 2009 A(H1N1) influenza viruses circulating in humans. Science, 2009. 325: p. 197-201.
13. Johnson, N.P. and J. Mueller, Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull. Hist. Med., 2002. 76: p. 105-115.
14. Seo, S.H., E. Hoffmann, and R.G. Webster, Lethal H5N1 influenza viruses escape host anti-viral cytokine responses. Nature Med., 2002. 8: p. 950-954.
15. Liu, J., Structures of receptor complexes formed by hemagglutinins from the Asian Influenza pandemic of 1957. Proc. Natl Acad. Sci. USA, 2009. 106: p. 17175-17180.
16. WHO Global Alert and Response. 2011.
17. Cowling, B.J., et al., Comparative epidemiology of human infections with avian influenza A H7N9 and H5N1 viruses in China: a population-based study of laboratory-confirmed cases. Lancet, 2013. 382(9887): p. 129-37.
18. Russell, R.J., H1 and H7 influenza haemagglutinin structures extend a structural classification of haemagglutinin subtypes. Virology, 2004. 325: p. 287-296.
19. Yu, H., et al., Human infection with avian influenza A H7N9 virus: an assessment of clinical severity. Lancet, 2013. 382(9887): p. 138-45.
20. Claas, E.C., Human influenza A H5N1 virus related to a highly pathogenic avian influenza virus. Lancet, 1998. 351: p. 472-477.
21. Hatta, M., Growth of H5N1 influenza A viruses in the upper respiratory tracts of mice. PLoS Pathog., 2007. 3: p. 1374-1379.
22. de Jong, M.D., et al., Fatal outcome of human influenza A (H5N1) is associated with high viral load and hypercytokinemia. Nat Med, 2006. 12(10): p. 1203-7.
23. Koopmans, M., Transmission of H7N7 avian influenza A virus to human beings during a large outbreak in commercial poultry farms in the Netherlands. Lancet, 2004. 363: p. 587-593.
24. Davenport, F.M., R. Rott, and W. Schäfer, PHYSICAL AND BIOLOGICAL PROPERTIES OF INFLUENZA VIRUS COMPONENTS OBTAINED AFTER ETHER TREATMENT. The Journal of Experimental Medicine, 1960. 112(5): p. 765-782.
25. Behzadi, M.A., M. Ziyaeyan, and A. Alborzi, A diagnostic one-step real-time reverse transcription polymerase chain reaction method for accurate detection of influenza virus type A. Archives of Medical Science : AMS, 2016. 12(6): p. 1286-1292.
26. Krammer, F. and P. Palese, Advances in the development of influenza virus vaccines. Nat Rev Drug Discov, 2015. 14(3): p. 167-82.
27. Huang, W.-T., et al., Design of a robust infrastructure to monitor the safety of the pandemic A(H1N1) 2009 vaccination program in Taiwan. Vaccine, 2010. 28(44): p. 7161-7166.
28. Sugrue, R.J. and A.J. Hay, Structural characteristics of the M2 protein of influenza a viruses: Evidence that it forms a tetrameric channe. Virology, 1991. 180(2): p. 617-624.
29. Holsinger, L.J. and R. Alams, Influenza virus M2 integral membrane protein is a homotetramer stabilized by formation of disulfide bonds. Virology, 1991. 183(1): p. 32-43.
30. Rossman, J.S., et al., Influenza virus m2 ion channel protein is necessary for filamentous virion formation. J Virol, 2010. 84(10): p. 5078-88.
31. Liu, W., P. Zou, and Y.H. Chen, Monoclonal antibodies recognizing EVETPIRN epitope of influenza A virus M2 protein could protect mice from lethal influenza A virus challenge. Immunol Lett, 2004. 93(2-3): p. 131-6.
32. Hutchinson, E.C., et al., Erratum: Conserved and host-specific features of influenza virion architecture. Nature Communications, 2015. 6: p. 6446.
33. Zou, P., W. Liu, and Y.H. Chen, The epitope recognized by a monoclonal antibody in influenza A virus M2 protein is immunogenic and confers immune protection. Int Immunopharmacol, 2005. 5(4): p. 631-5.
34. Deng, L., et al., M2e-Based Universal Influenza A Vaccines. Vaccines, 2015. 3(1): p. 105-136.
35. Haynes, J.R., Influenza virus-like particle vaccines. Expert Review of Vaccines, 2009. 8(4): p. 435-445.
36. WHO. Pandemic (H1N1) 2009 - update 109. WHO Global Alert and Response, 2009.
37. Xi, J.N., et al., Norwalk virus genome cloning and characterization. Science, 1990. 250(4987): p. 1580.
38. Lambden, P.R., et al., Sequence and genome organization of a human small round-structured (Norwalk-like) virus. Science, 1993. 259(5094): p. 516.
39. Thorne, L.G. and I.G. Goodfellow, Norovirus gene expression and replication. Journal of General Virology, 2014. 95(2): p. 278-291.
40. Karst, S.M., Pathogenesis of Noroviruses, Emerging RNA Viruses. Viruses, 2010. 2(3).
41. Jones, M.K., et al., Human norovirus culture in B cells. Nature protocols, 2015. 10(12): p. 1939-1947.
42. Robilotti, E., S. Deresinski, and B.A. Pinsky, Norovirus. Clinical Microbiology Reviews, 2015. 28(1): p. 134-164.
43. Prasad, B.V.V., M.E. Hardy, and M.K. Estes, Structural Studies of Recombinant Norwalk Capsids. The Journal of Infectious Diseases, 2000. 181(Supplement_2): p. S317-S321.
44. Jiang, X., et al., Expression, self-assembly, and antigenicity of the Norwalk virus capsid protein. Journal of Virology, 1992. 66(11): p. 6527-6532.
45. Green, K.Y., et al., Comparison of the reactivities of baculovirus-expressed recombinant Norwalk virus capsid antigen with those of the native Norwalk virus antigen in serologic assays and some epidemiologic observations. Journal of Clinical Microbiology, 1993. 31(8): p. 2185-2191.
46. Tan, M., et al., Noroviral P particle: structure, function and applications in virus-host interaction. Virology, 2008. 382(1): p. 115-23.
47. Tan, M. and X. Jiang, The P Domain of Norovirus Capsid Protein Forms a Subviral Particle That Binds to Histo-Blood Group Antigen Receptors. Journal of Virology, 2005. 79(22): p. 14017-14030.
48. Tan, M. and X. Jiang, Norovirus P particle: a subviral nanoparticle for vaccine development against norovirus, rotavirus and influenza virus. Nanomedicine (Lond), 2012. 7(6): p. 889-97.
49. Chen, H.W., et al., A novel technology for the production of a heterologous lipoprotein immunogen in high yield has implications for the field of vaccine design. Vaccine, 2009. 27(9): p. 1400-9.
50. Tseng, C.L. and C.H. Leng, Influence of medium components on the expression of recombinant lipoproteins in Escherichia coli. Appl Microbiol Biotechnol, 2012. 93(4): p. 1539-52.
51. Bollati, M., et al., Structure and functionality in flavivirus NS-proteins: Perspectives for drug design. Antiviral Research, 2010. 87(2): p. 125-148.
52. Molinari, N.A., The annual impact of seasonal influenza in the US: measuring disease burden and costs. Vaccine, 2007. 25: p. 5086-5096.
53. Joseph, U., et al., The ecology and adaptive evolution of influenza A interspecies transmission. Influenza and Other Respiratory Viruses, 2017. 11(1): p. 74-84.
54. Kim, Y.-I., et al., Genetic and phylogenetic characterizations of a novel genotype of highly pathogenic avian influenza (HPAI) H5N8 viruses in 2016/2017 in South Korea. Infection, Genetics and Evolution.
55. Onuma, M., et al., Characterizing the temporal patterns of avian influenza virus introduction into Japan by migratory birds. Journal of Veterinary Medical Science, 2017. advpub.
56. Nizolenko, L.P., A.G. Bachinsky, and S.I. Bazhan, Evaluation of Influenza Vaccination Efficacy: A Universal Epidemic Model. BioMed Research International, 2016. 2016: p. 5952890.
57. Worobey, M., G.-Z. Han, and A. Rambaut, A synchronized global sweep of the internal genes of modern avian influenza virus. Nature, 2014. 508(7495): p. 254-257.
58. Cho, K.J., et al., Crystal structure of the conserved amino-terminus of the extracellular domain of matrix protein 2 of influenza A virus gripped by an antibody. J Virol, 2015.
59. Nieva, J.L., V. Madan, and L. Carrasco, Viroporins: structure and biological functions. Nat Rev Micro, 2012. 10(8): p. 563-574.
60. Francis, D.M. and R. Page, Strategies to Optimize Protein Expression inE. coli. 2010: p. 5.24.1-5.24.29.
61. Kwok, Y., et al., Rapid isolation and characterization of bacterial lipopeptides using liquid chromatography and mass spectrometry analysis. Proteomics, 2011. 11(13): p. 2620-2627.
62. Diehl, S. and M. Rincón, The two faces of IL-6 on Th1/Th2 differentiation. Molecular Immunology, 2002. 39(9): p. 531-536.
63. Tailleux, L., et al., Stabilization of Influenza Vaccine Enhances Protection by Microneedle Delivery in the Mouse Skin. PLoS ONE, 2009. 4(9): p. e7152.
64. Kim, M.-C., et al., Microneedle patch delivery to the skin of virus-like particles containing heterologous M2e extracellular domains of influenza virus induces broad heterosubtypic cross-protection. Journal of Controlled Release, 2015. 210: p. 208-216.
65. Ai, W., et al., Optimal Method to Stimulate Cytokine Production and Its Use in Immunotoxicity Assessment. International Journal of Environmental Research and Public Health, 2013. 10(9): p. 3834-3842.
66. Luckheeram, R.V., et al., CD4+T Cells: Differentiation and Functions. Clinical and Developmental Immunology, 2012. 2012: p. 1-12.
67. Gauger, P.C. and A.L. Vincent, Serum Virus Neutralization Assay for Detection and Quantitation of Serum-Neutralizing Antibodies to Influenza A Virus in Swine, in Animal Influenza Virus, E. Spackman, Editor. 2014, Springer New York: New York, NY. p. 313-324.
68. Wingfield, P.T., Overview of the Purification of Recombinant Proteins. 2015: p. 6.1.1-6.1.35.
69. Du, L., Y. Zhou, and S. Jiang, Research and development of universal influenza vaccines. Microbes and Infection, 2010. 12(4): p. 280-286.