臺灣博碩士論文加值系統

黃質病毒科 (Flaviviridae) 是一具有單鏈正股RNA的病毒家族，其中的黃質病毒屬 (Flavivirus)涵蓋了許多的法定傳染病病原體，例如：四型登革熱病毒 (Dengue virus)、茲卡病毒 (Zika virus)、 西尼羅河病毒 (West Nile virus)和日本腦炎病毒 (Japan encephalitis virus)等，這些病毒大多藉由蚊蟲類的叮咬完成傳染，登革熱病毒每年會造成約400萬人口的感染，且超過全球四分之一的人口都居住在登革熱病毒的流行區，然而目前並無登革熱的有效療法，因此疫苗開發的議題十分重要。
在2015年，一項研究發現了一個以前未知的表位，被稱為套膜二聚體表位 (EDE)，此表位分布於成熟病毒顆粒上的套膜二聚體上，針對此EDE的單株抗體 (mAbs)能在登革病毒血清複合物中廣泛作用，並且能夠完全中和在昆蟲細胞或原代人類細胞中產生的病毒。在後續的研究中，發現EDE抗體結合到envelope domain I至domain III的多個抗原決定基。對這種具有強效中和能力和交叉反應性抗體的揭示了疫苗開發的全新策略的可能性，因此EDE的發現可能使未來疫苗中生成強效且廣效的中和抗體成為一個現實的目標，甚至可能實現使用單一通用免疫抗原的可能性。
因此本研究使用胜肽合成將EDE表位序列與先前實驗室所研究的bc loop進行排列組合共4個實驗組別，並以此作為免疫抗原進行實驗，利用佐劑與實驗組胜肽進行混合施打BALB/c小鼠，並測定其體外中和試驗及體內動物保護力試驗，結果顯示各週血清對DENV-1及DENV-3的中和能力較DENV-2及 DENV-4高，在動物保護力試驗方面，將各組中具有高中和能力之個體血清進行混合並施打觀測其保護效力，可以發現實驗組小鼠的病毒血症有下降的趨勢。總結， EDE作為免疫抗原雖然能誘導免疫反應，但其中和能力仍具有保護性不平衡的現象，而在後續小鼠實驗中，雖使用較佳個體血清能有效抑制病毒，但由於其個體血清的不一致性揭示了距離製作一成功的疫苗還需一段努力。

Flaviviridae is a family of viruses with single-strand positive-sense RNA. The genus Flavivirus covers many notifiable infectious disease pathogens, such as: Dengue virus type 1-4, Zika virus, West Nile virus, Japanese encephalitis virus. Most of these viruses are transmitted through mosquito bites. Dengue virus infects about 4 million people every year. And more than a quarter of the world's population lives in areas where dengue virus is endemic. However, there is currently no effective treatment for dengue fever, so the issue of vaccine development is very important.
In 2015, one previous study identified a previously unknown epitope, the envelope dimer epitope (EDE), that bridges two envelope protein subunits that make up the 90 repeating dimers on the mature virion. The mAbs to EDE were broadly reactive across the dengue serocomplex and fully neutralized virus produced in either insect cells or primary human cells. In subsequent studies, it was found that the EDE antibody binds to multiple epitopes in envelope domain I to domain III, The description of such potent and cross-reactive antibodies indicates a way for the development of subunit vaccines containing the desired epitope and possibly heterologous prime-boost strategies to recapitulate responses seen in natural sequential infections. The discovery of the EDE might make the generation of potent and broadly neutralizing antibodies a realistic goal for future vaccines and might even allow the possibility of a single universal immunogen.
Therefore, this study used peptide synthesis to combine the EDE epitope sequence and the bc loop into four experimental groups, and used these as immune antigens for experiments. The adjuvant was mixed with the experimental group peptides and administered to BALB/c mice. The in vitro neutralization test and the in vivo animal protection test were also measured. The results showed that the neutralizing ability of DENV-1, DENV-2 and DENV-3 in each week's serum was higher than DENV-4, but it also showed an imbalance in the protective efficacy. Subsequently, through the in vivo protective efficacy of the serum of the experimental group was measured in mice. The results showed that administration the higher NT50¬ level individual serum can reduce the viremia level in mice. In summary, although EDE can induce the specific immune responses, but there is a phenomenon of protective imbalance. In subsequent experiments on mice, although the use of high NT50¬ level individual serum can effectively inhibit the virus. However, the inconsistencies in individual serum indicate that we are still a long way from producing a successful vaccine.

中文摘要.......................III
英文摘要.......................V
前言.......................1
一、黃質病毒簡介.......................2
二、黃質病毒結構介紹 3
三、抗體依賴性增強作用 (Antibody-Dependent Enhancement, ADE).......................4
四、黃質病毒的疫苗發展.......................5
五、套膜蛋白 (Envelope protein)介紹.......................7
六、 抗體表位：套膜二聚體表位 (Envelope Dimer Epitope, EDE).......................8
七、抗體表位：保守序列bc loop介紹.......................10
八、研究動機.......................11
九、本論文主要探討.......................12
材料與方法.......................13
一、細胞培養.......................14

二、病毒株製備.......................15

三、本論文之Peptide序列設計.......................16

四、抗體製備.......................17

五、酵素結合免疫吸附分析法 (Enzyme-linked immunosorbent assay, ELISA).......................19

六、病毒斑減少中和試驗 (50% Focus Reduction Neutralization Test, FRNT50).......................20

七、病毒效價測定.......................20

八、AG129小鼠被動免疫保護力試驗.......................22

九、病毒血症測試 (Viremia Test).......................22
十、抗體依賴性增強試驗 (Antibody-Dependent Enhancement).......................23
實驗結果.......................25
一、選擇EDE抗體表位並設計實驗組胜肽.......................26

二、將實驗組peptide免疫BALB/c小鼠.......................29

三、體外實驗：抗體中和試驗FRNT50測定 (In vitro 50% Focus Reduction Neutralization Test, FRNT50).......................31

四、體內實驗：AG129小鼠保護力試驗 (In vivo AG129 mice Protective Efficacy test).......................32

討論.......................34

結論.......................40

圖示.......................46
附圖一、實驗組序列設計簡圖及實驗組序列總覽.......................47

附圖二、各實驗組胜肽序列結構及B cell epitope prediction結果圖.......................49

附圖三、使用實驗組peptide進行BALB/c小鼠免疫試驗的疫苗施打實驗進程表.......................50

附圖四、利用ELISA檢測BALB/c小鼠血清抗體含量.......................51

附圖五、利用ELISA檢測BALB/c小鼠血清抗體含量.......................52

附圖六、利用FRNT50檢測免疫流程中BALB/c小鼠混合血清抗體之中和登革病毒的能力.......................54

附圖七、利用FRNT50檢測免疫流程中BALB/c小鼠個體血清抗體之中和登革病毒的能力.......................56

附圖八、利用被動免疫AG129小鼠的形式測試實驗組抗體在DENV-1、DENV-2、DENV-3與DENV-4中的保護效力.......................59

1. van Leur, S.W., et al., Pathogenesis and virulence of flavivirus infections. Virulence, 2021. 12(1): p. 2814-2838.

2. Messina, J.P., et al., Global spread of dengue virus types: mapping the 70 year history. Trends Microbiol, 2014. 22(3): p. 138-46.

3. Kok, B.H., et al., Dengue virus infection - a review of pathogenesis, vaccines, diagnosis and therapy. Virus Res, 2023. 324: p. 199018.

4. Chen, W.J., Dengue outbreaks and the geographic distribution of dengue vectors in Taiwan: A 20-year epidemiological analysis. Biomed J, 2018. 41(5): p. 283-289.

5. Hou, J., W. Ye, and J. Chen, Current Development and Challenges of Tetravalent Live-Attenuated Dengue Vaccines. Front Immunol, 2022. 13: p. 840104.

6. Kularatne, S.A. and C. Dalugama, Dengue infection: Global importance, immunopathology and management. Clin Med (Lond), 2022. 22(1): p. 9-13.

7. Guzman, M.G. and E. Harris, Dengue. Lancet, 2015. 385(9966): p. 453-65.

8. Zeyaullah, M., et al., Preparedness for the Dengue Epidemic: Vaccine as a Viable Approach. Vaccines (Basel), 2022. 10(11).

9. Cordero-Rivera, C.D., et al., The importance of viral and cellular factors on flavivirus entry. Curr Opin Virol, 2021. 49: p. 164-175.

10. Salles, T.S., et al., History, epidemiology and diagnostics of dengue in the American and Brazilian contexts: a review. Parasit Vectors, 2018. 11(1): p. 264.

11. Nanaware, N., et al., Dengue Virus Infection: A Tale of Viral Exploitations and Host Responses. Viruses, 2021. 13(10).

12. Pang, X., R. Zhang, and G. Cheng, Progress towards understanding the pathogenesis of dengue hemorrhagic fever. Virol Sin, 2017. 32(1): p. 16-22.

13. Pan, Y., et al., Flaviviruses: Innate Immunity, Inflammasome Activation, Inflammatory Cell Death, and Cytokines. Front Immunol, 2022. 13: p. 829433.

14. Carbaugh, D.L. and H.M. Lazear, Flavivirus Envelope Protein Glycosylation: Impacts on Viral Infection and Pathogenesis. J Virol, 2020. 94(11).

15. Rastogi, M., N. Sharma, and S.K. Singh, Flavivirus NS1: a multifaceted enigmatic viral protein. Virol J, 2016. 13: p. 131.

16. Carpio, K.L. and A.D.T. Barrett, Flavivirus NS1 and Its Potential in Vaccine Development. Vaccines (Basel), 2021. 9(6).

17. Urakami, A., et al., An Envelope-Modified Tetravalent Dengue Virus-Like-Particle Vaccine Has Implications for Flavivirus Vaccine Design. J Virol, 2017. 91(23).

18. Li, A., et al., A Zika virus vaccine expressing premembrane-envelope-NS1 polyprotein. Nat Commun, 2018. 9(1): p. 3067.

19. Sankhala, R.S., et al., Zika-specific neutralizing antibodies targeting inter-dimer envelope epitopes. Cell Rep, 2023. 42(8): p. 112942.

20. Sarker, A., N. Dhama, and R.D. Gupta, Dengue virus neutralizing antibody: a review of targets, cross-reactivity, and antibody-dependent enhancement. Front Immunol, 2023. 14: p. 1200195.

21. Shukla, R., et al., Antibody-Dependent Enhancement: A Challenge for Developing a Safe Dengue Vaccine. Front Cell Infect Microbiol, 2020. 10: p. 572681.

22. Rathore, A.P.S. and A.L. St John, Cross-Reactive Immunity Among Flaviviruses. Front Immunol, 2020. 11: p. 334.

23. Moi, M.L., et al., Development of an antibody-dependent enhancement assay for dengue virus using stable BHK-21 cell lines expressing Fc gammaRIIA. J Virol Methods, 2010. 163(2): p. 205-9.

24. Paradkar, P.N., et al., Unfolded protein response (UPR) gene expression during antibody-dependent enhanced infection of cultured monocytes correlates with dengue disease severity. Biosci Rep, 2011. 31(3): p. 221-30.

25. Rey, F.A., et al., The bright and the dark side of human antibody responses to flaviviruses: lessons for vaccine design. EMBO Rep, 2018. 19(2): p. 206-224.

26. Plevka, P., et al., Maturation of flaviviruses starts from one or more icosahedrally independent nucleation centres. EMBO Rep, 2011. 12(6): p. 602-6.

27. Huang, C.H., et al., Dengue vaccine: an update. Expert Rev Anti Infect Ther, 2021. 19(12): p. 1495-1502.

28. Ma, E. and G. Cheng, Host immunity and vaccine development against Dengue virus. Infect Med (Beijing), 2022. 1(1): p. 50-58.

29. Hadinegoro, S.R., et al., Efficacy and Long-Term Safety of a Dengue
Vaccine in Regions of Endemic Disease. N Engl J Med, 2015. 373(13): p. 1195-206.

30. Khetarpal, N. and I. Khanna, Dengue Fever: Causes, Complications, and Vaccine Strategies. J Immunol Res, 2016. 2016: p. 6803098.

31. Sirivichayakul, C., et al., Long-term Safety and Immunogenicity of a Tetravalent Dengue Vaccine Candidate in Children and Adults: A Randomized, Placebo-Controlled, Phase 2 Study. J Infect Dis, 2022. 225(9): p. 1513-1520.

32. López-Medina, E., et al., Efficacy of a Dengue Vaccine Candidate (TAK-003) in Healthy Children and Adolescents 2 Years after Vaccination. J Infect Dis, 2022. 225(9): p. 1521-1532.

33. Kallás, E.G., et al., Live, Attenuated, Tetravalent Butantan-Dengue Vaccine in Children and Adults. N Engl J Med, 2024. 390(5): p. 397-408.

34. Zhang, X., et al., Structures and Functions of the Envelope Glycoprotein in Flavivirus Infections. Viruses, 2017. 9(11).

35. de Alwis, R., et al., Identification of human neutralizing antibodies that bind to complex epitopes on dengue virions. Proc Natl Acad Sci U S A, 2012. 109(19): p. 7439-44.

36. Slon Campos, J.L., J. Mongkolsapaya, and G.R. Screaton, The immune response against flaviviruses. Nat Immunol, 2018. 19(11): p. 1189-1198.

37. Dejnirattisai, W., et al., A new class of highly potent, broadly
neutralizing antibodies isolated from viremic patients infected with dengue virus. Nat Immunol, 2015. 16(2): p. 170-177.

38. Barba-Spaeth, G., et al., Structural basis of potent Zika-dengue virus antibody cross-neutralization. Nature, 2016. 536(7614): p. 48-53.

39. Rouvinski, A., et al., Recognition determinants of broadly neutralizing human antibodies against dengue viruses. Nature, 2015. 520(7545): p. 109-13.

40. Rouvinski, A., et al., Covalently linked dengue virus envelope glycoprotein dimers reduce exposure of the immunodominant fusion loop epitope. Nat Commun, 2017. 8: p. 15411.

41. Wang, Y., et al., Substitution of the precursor peptide prevents anti-prM antibody-mediated antibody-dependent enhancement of dengue virus infection. Virus Res, 2017. 229: p. 57-64.

42. Dejnirattisai, W., et al., Cross-reacting antibodies enhance dengue virus infection in humans. Science, 2010. 328(5979): p. 745-8.

43. Smith, S.A., et al., The potent and broadly neutralizing human dengue virus-specific monoclonal antibody 1C19 reveals a unique cross-reactive epitope on the bc loop of domain II of the envelope protein. mBio, 2013. 4(6): p. e00873-13.

44. Deng, Y.Q., et al., A broadly flavivirus cross-neutralizing monoclonal antibody that recognizes a novel epitope within the fusion loop of E protein. PLoS One, 2011. 6(1): p. e16059.

45. Yen, L.C., et al., Neutralizing antibodies targeting a novel epitope on envelope protein exhibited broad protection against flavivirus without risk of disease enhancement. J Biomed Sci, 2023. 30(1): p. 41.

46. Nicholls, C.M.R., M. Sevvana, and R.J. Kuhn, Structure-guided paradigm shifts in flavivirus assembly and maturation mechanisms. Adv Virus Res, 2020. 108: p. 33-83.

47. Kielian, M. and F.A. Rey, Virus membrane-fusion proteins: more than one way to make a hairpin. Nat Rev Microbiol, 2006. 4(1): p. 67-76.

48. Li, L., et al., Potent neutralizing antibodies elicited by dengue vaccine in rhesus macaque target diverse epitopes. PLoS Pathog, 2019. 15(6): p. e1007716.

49. Abbink, P., et al., Therapeutic and protective efficacy of a dengue antibody against Zika infection in rhesus monkeys. Nat Med, 2018. 24(6): p. 721-723.

50. Fernandez, E., et al., Human antibodies to the dengue virus E-dimer epitope have therapeutic activity against Zika virus infection. Nat Immunol, 2017. 18(11): p. 1261-1269.