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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 顧家綺(Chia-Chi Ku) | |
| dc.contributor.author | Cheng-Yu Lin | en |
| dc.contributor.author | 林承右 | zh_TW |
| dc.date.accessioned | 2021-06-15T13:29:31Z | - |
| dc.date.available | 2020-08-26 | |
| dc.date.copyright | 2020-08-26 | |
| dc.date.issued | 2020 | |
| dc.date.submitted | 2020-08-10 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/51287 | - |
| dc.description.abstract | H5高致病性禽流感(HPAI)病毒於1996年開始出現禽傳人的病例以及其快速抗原漂移特性使其進化成多個分支，H5禽流感病毒相關研究顯得十分重要。從2013年起，新型2.3.4.4 H5禽流感病毒開始出現並傳播到東南亞和歐洲。儘管在亞洲實施了多年的疫苗接種、監測和生物安全防治，但自2003年起，H5禽流感病毒多次的爆發流行帶來禽畜經濟損失與未來造成人流感大流行的威脅，在在說明選擇適當的病毒株製成疫苗以因應當前的禽流感病毒流行，是迫切需要的研究課題。本研究目的主要有三大部份: (一) 建立雞禽接種禽流感病毒疫苗的實驗模式；(二)評估去活性病毒疫苗與類病毒顆粒疫苗在雞禽體內的致免疫性；(三)評估禽流感疫苗對於跨越不同分支禽流感病毒的保護力。 在這項研究中，我們使用了以反向遺傳學技術製備的分支2.3.4 H5N1安徽株疫苗株去活性病毒(rRG6)以及分支2.3.4.4 H5N2台灣株類病毒顆粒(VLP/H5N2)重組蛋白疫苗，對7日齡雞隻進行皮下注射；並且從抗原劑量、佐劑類型與疫苗接種時程等可能影響疫苗功效的因素設計實驗，收集免疫後血清進行血球凝集抑制 (HI)試驗。實驗結果顯示，以16 HAU 的rRG6或是含有至少100 ng HA 抗原的 VLP疫苗可以刺激雞宿主產生HI抗體陽性反應。乳化型佐劑71VG相對於鋁鹽能長期且高水平地增強rRG6的HI抗體反應。口蹄病毒表面蛋白VP3與71VG佐劑組合的VLP相比於單獨使用71VG、鋁鹽或VP3與鋁鹽混合佐劑，更能刺激雞禽產生更高的血凝抑制抗體效價。不論是rRG6 或是VLP佐劑疫苗，必須追加接種一劑才能誘導好的抗體陽轉反應。初次接種後第二週進行第二劑的補強接種(0/14時程)，雖然比第一週進行補強接種(0/7時程)延遲產生HI 抗體反應，但是整體的HI 抗體效價不但顯著提高，對於跨越其他分支H5病毒的HI抗體交叉反應也更好。在攻毒實驗方面，rRG6能完全保護雞隻免於致死劑量分支1 H5N1病毒感染而死亡 (N=4,存活率=100%)，但是對於分支2.3.4.4的病毒的保護只有50% (N=2)。雖然VLP疫苗無法顯著保護雞禽對於分支1 H5N1病毒的感染 (N=8,存活率=12.5%)，但是對於分支2.3.4.4 H5N2病毒有完全的保護力 (N=6, 存活率=100%)。進一步從H5禽流感病毒血球凝集抗原蛋白的親源分析結果，說明分支2.3.4 rRG6疫苗對於新分支2.3.4.4 H5 病毒提供極有限的保護力。 綜合上述的實驗結果，本研究成功建立以雞禽動物模式評估禽流感病毒疫苗效力以及發展應對新分支2.3.4.4 H5禽流感病毒疫苗的免疫學條件。對於接種疫苗後以流式細胞儀分析雞脾細胞特性包含細胞大小顆粒、泛B細胞標記Bu1以及表面抗體IgM及IgY的變化，進ㄧ步確立預測疫苗效力的分子標誌，是未來繼續需要努力的目標。 | zh_TW |
| dc.description.abstract | High pathogenic avian influenza (HPAI) virus appeared and began to transmit from avian to human since 1996. The property of rapid antigenic drift of field virus has made it evolved into multiple clades, highlighting the importance of H5 avian influenza virus studies to develop effective control strategies. A novel clade 2.3.4.4 of H5 viruses was emerged in 2013 and has extensively spread over Southeastern Asia and European since then. While routine vaccination, surveillance, and biosecurity measures have been adopted to control AIVs in some countries of Asia for years, several H5 AIV outbreaks in recent years have resulted in the great loss in the poultry industry as well as brought the threat of potential influenza pandemic in the future. It is an urgent need to develop an effective vaccine to control novel clade of H5 AIVs. Experiments conducted in my thesis research were designed to reach the following goals: (1) Establishment of chicken hosts as a model to evaluate AIV vaccines；(2) Evaluation of the immunogenicity induced by inactivated virus vaccine and virus-like-particle vaccine in chickens；(3) Evaluation of the protection of the AIV vaccine against different clades of H5 AIVs. In this study, inactivated recombinant clade 2.3.4 H5N1 virus (A/Anhui/01/2005/H5N1) made by reverse genetic method (rRG6) and the virus-like particle composed of HA of clade 2.3.4.4 H5N2 (A/goose/Taiwan/01004/2015) (VLP/H5N2) were used to subcutaneously immunize 7-day-old chickens. Serum samples periodically collected from chickens after immunization of different vaccine formula (e.g. antigen amount, types of adjuvant or timing for giving booster dose) were evaluated for vaccine-induced antibody response by hemagglutination inhibition assays. Results from these experiments showed that 16 HAU of rRG6 and VLP vaccine containing at least 100 ng of HA protein induced the highest HI antibody response in chickens. Compared with hydroxyl aluminum (Alum), the mineral oil-based MontanideTM ISA 71VG (71VG) enhanced a longer and higher level of HI antibody response in rRG6 immunized chickens. On the other hand, VLP adjuvanted with foot-and-mouth virus VP3 and 71VG combined adjuvant induced a better HI antibody response than that with 71VG, Alum, or VP3/Alum adjuvant. Booster dose for either type of vaccine was required to effectively induce seroconversion. While the booster dose given at two weeks after primary immunization (0/14 schedule) had a delayed generation of HI antibody compared with the booster dose given at one week (0/7 schedule), the former schedule significantly enhanced overall HI antibody response against vaccine strain as well as showed better HI reactivity against cross clade 1 H5 viruses. In viral challenge experiments, rRG6 fully protected immunized chickens from lethal dose infection of clade 1 H5N1 (N= 4, survival rate= 100 %) but only one of two rRG6-immunized chickens survived after infection with clade 2.3.4.4 H5N2. Although VLP/H5N2 vaccine did not protect immunized chickens from clade 1 H5N1 infection (N= 8, survival rate= 12.5 %), it conferred 100% protection for immunized chickens from infection of clade 2.3.4.4 H5N2 (N= 6, survival rate= 100 %). Further phylogenetic relationship analysis of HA of H5 viruses suggested that rRG6 vaccine made of clade 2.3.4 H5 viruses provided limited protection for chickens against novel clade 2.3.4.4 H5 viruses. In summary, this study has successfully established the methods to evaluate the effectiveness of AIV vaccine in chicken hosts and characterized the immunological factors for developing a good vaccine for clade 2.3.4.4 H5 viruses. Future work will be focused on the analysis of the vaccine-induced changes in the properties of chicken splenocytes (e.g. the size and granularity of cells, pan-B cell marker, Bu1, and surface antibody IgM and IgY) by flow cytometry. We hope to identify the molecular marker or markers that can be used to predict vaccine effectiveness. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T13:29:31Z (GMT). No. of bitstreams: 1 U0001-1008202013073500.pdf: 3228751 bytes, checksum: 462071347fbad043b6ee7216552c046b (MD5) Previous issue date: 2020 | en |
| dc.description.tableofcontents | Content 口試委員會審定書 i 致謝 ii 中文摘要 iii Abstract v Tables Content xiii Figure content xiii Ⅰ. Introduction 1 1.1 Development of avian B cells 1 1.1.1 Pre-bursal stage 1 1.1.2 Bursal stage 2 1.1.3 After-hatch stage 3 1.1.4 Rearrangement of immunoglobulin 3 1.1.5 Gene conversion 4 1.1.6 Isotypes of immunoglobulin 5 1.1.7 B cell response to antigen stimulation 6 1.2 Avian influenza virus 6 1.2.1 Highly pathogenic avian influenza virus (HPAI) 6 1.2.2 Emergence of HPAI H5 virus 7 1.2.3 Control strategies for AIV 8 1.2.4 Current vaccine for AIV 9 1.2.5 Vaccine efficacy of AIV vaccine 10 1.3 Adjuvant 11 1.3.1 Alum 11 1.3.2 MontanideTM ISA adjuvant 71VG 12 1.3.3 VP3 protein 13 Ⅲ. Materials Methods 17 3.1 Methods 17 3.1.1 Chickens 17 3.1.2 Vaccine antigen 17 3.1.3 Adjuvant 19 3.1.4 Immunization protocol 19 3.1.5 Splenocyte preparation 20 3.1.6 Flow Cytometry 20 3.1.7 Hemagglutination inhibition (HI)assays 21 3.1.8 Viral challenge experiments 22 3.1.9 Statistical analysis 22 3.2 Materials 23 3.2.1 List of antibodies 23 3.2.2 List of antigens for HI test 23 3.2.3 Buffer and solution 24 3.2.4 Chemical, reagent, and kits 26 Ⅳ. Results 27 4.1 Inactivated rRG6 (H5N1) vaccine 27 4.1.1 Adjuvant enhanced the antibody response induced by inactivated vaccine, rRG6 (H5N1) 27 4.1.2 Immunization of adjuvanted rRG6 (H5N1) vaccine stimulated HI antibody titers in dose-dependent response in chicken 28 4.1.3 Water-in-oil adjuvant, 71VG, enhanced a higher level of HI antibody response compared with alum in chickens after vaccinated with rRG6 (H5N1) vaccine 28 4.1.4 rRG6 (H5N1) vaccine induced antibody response to peak at day 28 to 42 and maintained until day 70 post primary immunization 29 4.2 Inactivated rH5N6 vaccine 31 4.2.1 Immunization of rH5N6 vaccine with 71VG stimulated HI antibody titers in dose-dependent response in chicken 31 4.3 VLP (H5N2) vaccine 31 4.3.1 Immunization of adjuvanted VLP (H5N2) vaccine stimulated HI antibody titers in dose-dependent response in chicken 31 4.3.2 VLP (H5N2) vaccine adjuvanted with VP3/Alum enhanced a higher antibody response than that with 71VG 32 4.3.3 Immunization of VLP (H5N2) vaccine stimulated HI antibody titers dose-dependently on HA antigen in chicken 33 4.3.4 VP3 protein enhanced the antibody response induced by VP3/71VG-adjuvanted VLP (H5N2) vaccine 35 4.4 Protection and cross-reactivity of vaccines 37 4.4.1 Protection of rRG6 (H5N1)-immunized chickens against H5N1/Vietnam/1203/2004 virus challenge 37 4.4.2 Protection of VLP (H5N2)- or rH5N6-immunized chickens against clade 2.3.4.4 virus challenge 38 4.4.3 Close phylogenetic relationship was observed between rRG6 (H5N1) and clade 1 virus, and VLP (H5N2) and clade 2.3.4.4 virus 38 4.4.4 Positive correlation of HI titers induced by rRG6 (H5N1) was observed in clade pair rRG6 (H5N1) and clade 1 virus 39 4.4.5 Positive correlation of HI titers was observed in clade pair VLP (H5N2) and clade 2.3.4.4 virus, and clade pair rH5N6 and clade 2.3.4.4 virus 40 4.5 Effect of booster immunization 41 4.5.1 Earlier and higher HI titers were induced in booster vaccination 41 4.5.2 Two dose vaccination of rRG6 (H5N1) induced cross-reactivity against clade 1 virus, H5N1 (Vietnam) 42 4.5.3 Two dose vaccination of VLP (H5N2) or rH5N6 vaccine induced cross-reactivity against clade 2.3.4.4 virus, GV20686 42 4.5.4 Similar phenotype of splenocytes was observed in chicken with different immunization schedules or unimmunized 43 Ⅴ. Discussion 45 5.1 Adjuvant effects on inactivated vaccine, rRG6 (H5N1) 46 5.2 Adjuvant effects on VLP (H5N2) 46 5.3 Effects of vaccine type on antibody response 48 5.4 Adjuvant effects on cell-mediated immune response 49 5.5 Selection of H5 viral strain for developing a better AIV vaccine 51 Ⅶ. Tables Figures 58 Ⅷ. Appendix 86 | |
| dc.language.iso | en | |
| dc.subject | 禽流感病毒 | zh_TW |
| dc.subject | 分支2.3.4.4 H5病毒 | zh_TW |
| dc.subject | 疫苗 | zh_TW |
| dc.subject | 雞禽 | zh_TW |
| dc.subject | 雞禽 | zh_TW |
| dc.subject | 疫苗 | zh_TW |
| dc.subject | 分支2.3.4.4 H5病毒 | zh_TW |
| dc.subject | 禽流感病毒 | zh_TW |
| dc.subject | clade 2.3.4.4 H5 virus | en |
| dc.subject | avian influenza virus | en |
| dc.subject | clade 2.3.4.4 H5 virus | en |
| dc.subject | vaccine | en |
| dc.subject | chicken | en |
| dc.subject | avian influenza virus | en |
| dc.subject | vaccine | en |
| dc.subject | chicken | en |
| dc.title | 藉由血球凝集抑制試驗評估針對分支2.3.4.4 H5禽流感病毒的疫苗成效 | zh_TW |
| dc.title | Evaluation of vaccine effectiveness against clade 2.3.4.4 H5 avian influenza viruses by hemagglutination inhibition assays | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 108-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 蕭培文(Pei-Wen Hsiao),李建國(Chien-Kuo Lee) | |
| dc.subject.keyword | 禽流感病毒,分支2.3.4.4 H5病毒,疫苗,雞禽, | zh_TW |
| dc.subject.keyword | avian influenza virus,clade 2.3.4.4 H5 virus,vaccine,chicken, | en |
| dc.relation.page | 88 | |
| dc.identifier.doi | 10.6342/NTU202002788 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2020-08-11 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 免疫學研究所 | zh_TW |
| 顯示於系所單位： | 免疫學研究所 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| U0001-1008202013073500.pdf 未授權公開取用 | 3.15 MB | Adobe PDF |
系統中的文件，除了特別指名其著作權條款之外，均受到著作權保護，並且保留所有的權利。