進階搜尋


下載電子全文  
系統識別號 U0026-0609201023564100
論文名稱(中文) 合併使用腸病毒七十一型疫苗與五合一疫苗之可行性探討
論文名稱(英文) Investigation on the feasibility of a combined vaccination with enterovirus 71 and a pentavalent vaccine
校院名稱 成功大學
系所名稱(中) 微生物及免疫學研究所
系所名稱(英) Department of Microbiology & Immunology
學年度 98
學期 2
出版年 99
研究生(中文) 陳君瑋
研究生(英文) Jyun-Wei Chen
學號 s4696104
學位類別 碩士
語文別 中文
論文頁數 58頁
口試委員 口試委員-黎煥耀
口試委員-林貴香
指導教授-余俊強
中文關鍵字 腸病毒七十一型  疫苗  交叉抗原性 
英文關鍵字 enterovirus 71  vaccine  cross-antigenicity 
學科別分類
中文摘要 腸病毒七十一型(EV71)自從1998年在台灣爆發流行之後,每隔二至三年出現流行高峰。目前臨床上除了針對臨床症狀給與支持性治療外,並未有良好的抗病毒藥物,疫苗亦仍待開發中。我們希望探討合併使用目前臨床上已廣泛使用且極為有效的五合一疫苗(PentaV)和經福馬林去活化EV71病毒顆粒疫苗(IEV)的可行性。以實驗小鼠進行接種,藉中和性試驗監測小鼠血清中抗EV71及抗小兒痲痹病毒(Poliovirus, PV)中和性抗體(NT)效價,還有分析血清中抗破傷風類毒素與白喉類毒素的抗體濃度。結果發現,在接種三劑後,IEV 與 IEV+PentaV 免疫之小鼠血清含有超過 28 抗 EV71 的 NT 效價,而 PentaV 與 IEV+PentaV 免疫之小鼠血清含有超過 26 抗 PV1 的 NT 效價;另外在PentaV 與 IEV+PentaV 免疫之小鼠血清中亦含有高濃度抗破傷風類毒素與白喉類毒素的抗體。證實兩種疫苗同時接種確實可以獲得良好免疫力且不會產生互相干擾。另外,經由 IEV 疫苗株(C2)免疫所得之血清除了對原病毒株具有很好的效價外對其他流行病毒株(包括 C2, B4, C4 與 B5)亦有相當高的 NT 效價(28 左右)。因此所使用的疫苗株可提供針對廣泛基因型病毒的免疫力。這樣的結果可以提供開發腸病毒七十一型疫苗的策略參考。
英文摘要 Enterovirus 71 (EV71) vaccine, if available, is logically to be administrated with the vaccines currently used, such as the commercial pentavalent vaccine which containing the inactivated poliovirus (PV) type 1, 2, and 3. In this study we sought to test the immunity and efficacy of an inactivated EV71 vaccine and determine whether it would interfere with the immunity of a pentavalent vaccine after co-immunization. Inactivated EV71 vaccine (C2 genogroup) elicited a genogroup-specific anti-EV71 neutralization titer (NT) of 28, indicating the potent immunogenicity of the vaccine formula. Pups from inactivated EV71 vaccine-immunized dams were resistant to EV71 challenge. Pentavalent vaccine elicited anti-PV, anti-diphtheria toxoid and anti-tetanus toxoid antibodies in a level similar to those in mice co-immunized with the pentavalent and inactivated EV71 vaccines. Similarly, anti-EV71 NT levels were comparable between mice immunized with the inactivated EV71 only or the two vaccines together. These results indicated that co-immunization did not interfere with the immunogenicity of the both EV71 and pentavalent vaccines. The immune sera of inactivated EV71-vaccinated mice showed broad neutralization activity for other genogroups, including B4, C4, and B5.
論文目次 中文摘要I
英文摘要II
致謝III
目錄IV
圖目錄VII
第一章 緒論1
一、 腸病毒七十一型病毒學1
二、 腸病毒七十一型的分子流行病學與演化3
三、 腸病毒七十一型感染的管理與治療4
四、 腸病毒七十一型的疫苗發展5
五、 小兒痲痹疫苗發展之借鏡9
第二章 研究動機與特異目標10
第三章 材料與方法
一、 細胞培養12
二、 病毒培養12
三、 間接免疫螢光染色法13
四、 疫苗製備13
五、 病毒蛋白定量15
六、 小鼠免疫方法、流程與實驗組別16
七、 中和性試驗17
八、 酵素結合免疫吸附分析法18
九、 小鼠脾臟細胞體外抗原刺激18
十、 細胞增生酵素結合免疫吸附分析法19
十一、 小鼠免疫接種保護力實驗19
十二、 統計分析20
第四章 結果
一、 純化病毒與確認病毒身分21
二、 免疫後小鼠體液免疫反應變化趨勢21
三、 IEV免疫後小鼠血清抗流行病毒株的中和性抗體分析23
四、 免疫後小鼠細胞免疫反應23
五、 免疫母鼠後仔鼠保護力測試23
第五章 討論25
參考文獻 30
Figure 1. Confirmation of the viruses used for immunization35
Figure 2. Fractions profile of sucrose gradient purification of EV7136
Figure 3. Inactivated EV71 vaccine alone or a combined vaccination with a pentavalent vaccine induced comparable anti-EV71 antibodies37
Figure 4. Inactivated EV71 vaccine did not interfere with the anti-PV antibody response of the pentavalent vaccine38
Figure 5. Inactivated EV71 vaccine did not interfere with the anti-tetanus and anti-diphtheria toxoid antibody responses of the pentavalent vaccine39
Figure 6. Neutralization antibody against clinical isolates of EV71 after immunization with a formaldehyde-inactivated whole virus vaccine40
Figure 7. Inactivated EV71 vaccine alone and a combined vaccination with a pentavelent vaccine induced comparable cellular immune response41
Figure 8. Inactivated EV71 vaccination maintain the high survival rates of one-day-old pups challenged by lethal dose of EV7142
附錄 I 產品說明 ELISA- anti diphtheria antibody 43
附錄 II 產品說明 ELISA- anti tetanus antibody 51
參考文獻 1. Oberste, M.S., Penaranda, S., Maher, K. & Pallansch, M.A. Complete genome sequences of all members of the species Human enterovirus A. J Gen Virol 85, 1597-1607 (2004).
2. Sarkany, Z. & Polgar, L. The unusual catalytic triad of poliovirus protease 3C. Biochemistry 42, 516-522 (2003).
3. Nayak, A., Goodfellow, I.G. & Belsham, G.J. Factors required for the Uridylylation of the foot-and-mouth disease virus 3B1, 3B2, and 3B3 peptides by the RNA-dependent RNA polymerase (3Dpol) in vitro. J Virol 79, 7698-7706 (2005).
4. Goodfellow, I.G., Polacek, C., Andino, R. & Evans, D.J. The poliovirus 2C cis-acting replication element-mediated uridylylation of VPg is not required for synthesis of negative-sense genomes. J Gen Virol 84, 2359-2363 (2003).
5. Neznanov, N., et al. Poliovirus protein 3A inhibits tumor necrosis factor (TNF)-induced apoptosis by eliminating the TNF receptor from the cell surface. J Virol 75, 10409-10420 (2001).
6. Choe, S.S., Dodd, D.A. & Kirkegaard, K. Inhibition of cellular protein secretion by picornaviral 3A proteins. Virology 337, 18-29 (2005).
7. Dodd, D.A., Giddings, T.H., Jr. & Kirkegaard, K. Poliovirus 3A protein limits interleukin-6 (IL-6), IL-8, and beta interferon secretion during viral infection. J Virol 75, 8158-8165 (2001).
8. Deitz, S.B., Dodd, D.A., Cooper, S., Parham, P. & Kirkegaard, K. MHC I-dependent antigen presentation is inhibited by poliovirus protein 3A. Proc Natl Acad Sci U S A 97, 13790-13795 (2000).
9. Oberste, M.S., Maher, K., Kilpatrick, D.R. & Pallansch, M.A. Molecular evolution of the human enteroviruses: correlation of serotype with VP1 sequence and application to picornavirus classification. J Virol 73, 1941-1948 (1999).
10. Brown, B.A., Oberste, M.S., Alexander, J.P., Jr., Kennett, M.L. & Pallansch, M.A. Molecular epidemiology and evolution of enterovirus 71 strains isolated from 1970 to 1998. J Virol 73, 9969-9975 (1999).
11. Tee, K.K., et al. Evolutionary genetics of human enterovirus 71: origin, population dynamics, natural selection, and seasonal periodicity of the VP1 gene. J Virol 84, 3339-3350 (2010).
12. Bible, J.M., Pantelidis, P., Chan, P.K. & Tong, C.Y. Genetic evolution of enterovirus 71: epidemiological and pathological implications. Rev Med Virol 17, 371-379 (2007).
13. Shih, S.R., et al. Genetic analysis of enterovirus 71 isolated from fatal and non-fatal cases of hand, foot and mouth disease during an epidemic in Taiwan, 1998. Virus Res 68, 127-136 (2000).
14. Chang, L.Y. Enterovirus 71 in Taiwan. Pediatr Neonatol 49, 103-112 (2008).
15. Wang, S.M. & Liu, C.C. Enterovirus 71: epidemiology, pathogenesis and management. Expert Rev Anti Infect Ther 7, 735-742 (2009).
16. Wang, S.M., et al. Therapeutic efficacy of milrinone in the management of enterovirus 71-induced pulmonary edema. Pediatr Pulmonol 39, 219-223 (2005).
17. Li, Z.H., et al. Ribavirin reduces mortality in enterovirus 71-infected mice by decreasing viral replication. J Infect Dis 197, 854-857 (2008).
18. Hung, H.C., et al. Inhibition of enterovirus 71 replication and the viral 3D polymerase by aurintricarboxylic acid. J Antimicrob Chemother 65, 676-683 (2010).
19. Stephenson, J.R. Understanding dengue pathogenesis: implications for vaccine design. Bull World Health Organ 83, 308-314 (2005).
20. van Drunen Littel-van den Hurk, S., Mapletoft, J.W., Arsic, N. & Kovacs-Nolan, J. Immunopathology of RSV infection: prospects for developing vaccines without this complication. Rev Med Virol 17, 5-34 (2007).
21. Yu, C.K., et al. Neutralizing antibody provided protection against enterovirus type 71 lethal challenge in neonatal mice. J Biomed Sci 7, 523-528 (2000).
22. Foo, D.G., Alonso, S., Chow, V.T. & Poh, C.L. Passive protection against lethal enterovirus 71 infection in newborn mice by neutralizing antibodies elicited by a synthetic peptide. Microbes Infect 9, 1299-1306 (2007).
23. Wu, C.N., et al. Protection against lethal enterovirus 71 infection in newborn mice by passive immunization with subunit VP1 vaccines and inactivated virus. Vaccine 20, 895-904 (2001).
24. Lu, C.Y., et al. Incidence and case-fatality rates resulting from the 1998 enterovirus 71 outbreak in Taiwan. J Med Virol 67, 217-223 (2002).
25. Chen, L.C., et al. Enterovirus 71 infection induces Fas ligand expression and apoptosis of Jurkat cells. J Med Virol 78, 780-786 (2006).
26. Wang, S.M., et al. Pathogenesis of enterovirus 71 brainstem encephalitis in pediatric patients: roles of cytokines and cellular immune activation in patients with pulmonary edema. J Infect Dis 188, 564-570 (2003).
27. Yang, K.D., et al. Altered cellular but not humoral reactions in children with complicated enterovirus 71 infections in Taiwan. J Infect Dis 183, 850-856 (2001).
28. Tung, W.S., Bakar, S.A., Sekawi, Z. & Rosli, R. DNA vaccine constructs against enterovirus 71 elicit immune response in mice. Genet Vaccines Ther 5, 6 (2007).
29. Foo, D.G., et al. Identification of neutralizing linear epitopes from the VP1 capsid protein of Enterovirus 71 using synthetic peptides. Virus Res 125, 61-68 (2007).
30. Chen, H.F., Chang, M.H., Chiang, B.L. & Jeng, S.T. Oral immunization of mice using transgenic tomato fruit expressing VP1 protein from enterovirus 71. Vaccine 24, 2944-2951 (2006).
31. Chung, Y.C., et al. Immunization with virus-like particles of enterovirus 71 elicits potent immune responses and protects mice against lethal challenge. Vaccine 26, 1855-1862 (2008).
32. Chiu, C.H., Chu, C., He, C.C. & Lin, T.Y. Protection of neonatal mice from lethal enterovirus 71 infection by maternal immunization with attenuated Salmonella enterica serovar Typhimurium expressing VP1 of enterovirus 71. Microbes Infect 8, 1671-1678 (2006).
33. Wu, S.C., Liu, C.C. & Lian, W.C. Optimization of microcarrier cell culture process for the inactivated enterovirus type 71 vaccine development. Vaccine 22, 3858-3864 (2004).
34. Arita, M., et al. An attenuated strain of enterovirus 71 belonging to genotype a showed a broad spectrum of antigenicity with attenuated neurovirulence in cynomolgus monkeys. J Virol 81, 9386-9395 (2007).
35. Cherkasova, E.A., et al. Long-term circulation of vaccine-derived poliovirus that causes paralytic disease. J Virol 76, 6791-6799 (2002).
36. Chumakov, K. & Ehrenfeld, E. New generation of inactivated poliovirus vaccines for universal immunization after eradication of poliomyelitis. Clin Infect Dis 47, 1587-1592 (2008).
37. Wang, Y.F., et al. A mouse-adapted enterovirus 71 strain causes neurological disease in mice after oral infection. J Virol 78, 7916-7924 (2004).
38. Lee, M.S. & Chang, L.Y. Development of enterovirus 71 vaccines. Expert Rev Vaccines 9, 149-156 (2010).
39. Kung, S.H., et al. Genetic and antigenic analyses of enterovirus 71 isolates in Taiwan during 1998-2005. Clin Microbiol Infect 13, 782-787 (2007).
40. Mizuta, K., et al. Cross-antigenicity among EV71 strains from different genogroups isolated in Yamagata, Japan, between 1990 and 2007. Vaccine 27, 3153-3158 (2009).
41. Ong, K.C., Devi, S., Cardosa, M.J. & Wong, K.T. Formaldehyde-inactivated whole-virus vaccine protects a murine model of enterovirus 71 encephalomyelitis against disease. J Virol 84, 661-665 (2010).
論文全文使用權限
  • 同意授權校內瀏覽/列印電子全文服務,於2020-01-01起公開。
  • 同意授權校外瀏覽/列印電子全文服務,於2020-01-01起公開。


  • 如您有疑問,請聯絡圖書館
    聯絡電話:(06)2757575#65773
    聯絡E-mail:etds@email.ncku.edu.tw