流行性感冒病毒每年都造成全世界千萬的人口受到感染。而接種流感病毒疫苗是目前最有效預防病毒流行的方式。從1950年起，主要以雞胚胎蛋作為生產季節性流感疫苗的方法。但是這種方法受限於需要大量雞胚胎蛋 (Vaccine-quality eggs)作為生產疫苗使用。當流感大規模爆發時，流感疫苗製程所需的雞胚胎將不敷生產使用。因此，我們需要具能快速及大規模量產的策略來解決緊急的需求。目前，非雞胚胎的疫苗已被視為是做為流感病毒大流行時所採用的替代方法之一。其中，類病毒顆粒 (Virus-like particle, VLP)製程的流感疫苗比Protein Science Corporation製備的重組蛋白流感疫苗更具免疫效果。本研究計畫是要改善並找到最佳化的製程條件來建立可因應流感大流行時的類病毒顆粒流感疫苗生產平台。目前為止我們已經建立H7N9流感病毒類病毒顆粒的表達系統與生產平台，並發現維持氧氣含量是昆蟲細胞生產類病毒顆粒的重要參數，實驗結果顯示將此策略應用於5 L的培養系統中其HA titer可達1024 HAU/ 50 μl。另外，我們也建立類病毒顆粒的下游純化方法，為了簡化純化步驟及提高純化速度，我們不使用類病毒顆粒捕獲方式進行純化，而是以微量過濾、管柱層析、切向流過濾濃縮及透析和無菌過濾的順序將雜質去除並濃縮以達到純化的目標。以1.7 L的收穫液 (harvest)純化後的結果顯示，最終產物 (bulk) 的HA蛋白回收率約為58%，且總蛋白和DNA的去除率高，以電子顯微鏡及高效液相層析儀分析最終產物並未發現桿狀病毒殘留，純化後的VLP仍保留很好的抗原性。將純化後的VLP抗原搭配佐劑施打於小鼠測試此疫苗的免疫保護力，發現搭配AddaVax佐劑可引發高效價中和抗體（NT >1024）。H7N9-TW VLPs當做模板，製作H7N9第五波流感病毒及H3N2流感病毒的VLP並進行各項測試，期待本研究的結果有助於未來因應流感病毒大流行(pandemic)的準備。

Influenza viruses cause hundreds of thousands of respiratory diseases worldwide each year. Vaccination is considered the most effective way to prevent influenza annual epidemics or pandemic. Since 1950, chicken embryo eggs have been used as the main method for producing seasonal influenza vaccines. The main drawback of this platform is the lack of flexibility for scale-up. Thus, egg-based vaccine manufacturers cannot supply sufficient doses for pandemic use. As a result, strategies to decrease the response time and expand production capacity are urgently needed. Untraditional vaccine type (non-virion vaccine) methods have been considered as an alternative strategy against an influenza pandemic. In particular, virus-like particle (VLP) vaccines maintain the virus capsule structure and are non-infectious. In this study, an H7N9 influenza VLP production platform using insect cells was established, and the dissolved oxygen concentration was found to be the key factor affecting VLP production. The hemagglutinin (HA) titer was improved substantially (up 512 units/50 µl) in the 2 L culture system with well-control dissolvent oxygen condition. In addition, a downstream rapid purification method for the VLP platform utilizing microfiltration, chromatography, diafiltration, and sterile filtration steps with no VLP capture step was employed. The process is a fast and efficient chromatography-based purification strategy for purifying influenza VLPs. The overall recovery yield of HA protein was approximately 58%, and high removal rates of total protein and DNA residues were found, with no baculovirus observed in the final purification step. The vaccination formula for an immunity study, which involved purified VLP and Al(OH)3, was shown to be highly protective against H7N9 influenza with a low dose of antigen (0.2 g). This rapid bioprocess provides an effective VLP vaccine production system for preparation for an influenza pandemic.

Index
中文摘要 2
Abstract 3
List of Tables 9
List of Figures 11
Acknowledgments 13
Chapter 1. Introduction 14
1.1 Influenza 14
1.2 Influenza virus vaccine 15
1.3 Vaccine production type 16
1.3.1 Live-attune influenza virus vaccine (LAIVs) 16
1.3.2 Inactivated influenza virus (whole and split) vaccine 17
1.3.3 Recombinant HA vaccine 18
1.3.4 Universal influenza virus vaccine 18
1.3.5 Virus-like particles influenza virus vaccine 19
1.4 Upstream bioprocess for influenza vaccine 19
1.4.1 Common host cell Line 19
1.4.2 Inactivated influenza production system from egg platform. 20
1.4.3 Inactivated influenza production system from MDCK and Vero cells 21
1.4.4 VLP and HA protein production system from insect cells 21
1.5 Downstream purification process 22
1.6 The use of adjuvant 24
Chapter 2. Specific aims of this study 26
Chapter 3. Materials and Methods 27
3.1 Cell culture and media 27
3.2 Preparation of recombinant baculoviruses 27
3.3 Shake and spinner flask cultures of High FiveTM cells 28
3.4 Densitometric and western blot analysis 29
3.5 Assay of NA activity 29
3.6 Virological assays 30
3.7 Production of H7N9-TW VLPs in the bioreactor 30
3.8 Microfiltration of the H7N9-TW VLPs harvest 31
3.9 Design of experiment (DoE) for different chromatographic resins 31
3.10 Flow-through mode of chromatography 32
3.11 Concentration and diafiltration steps 33
3.12 Trypsin digestion and protein identification by LC-MS/MS 33
3.13 Negative-staining electron microscopy (EM) of influenza H7N9-TW VLPs 34
3.14 Mice study 34
3.15 Hemagglutination inhibition (HAI) titers 35
3.16 Neutralization titer (NT) assay 36
3.17 IgG assays using ELISPOT 36
Chapter 4. Results and discussion 37
4.1 upstream bioprocess 37
4.1.1 Evaluation of Influenza virus-like particle was produced by different expression promoter by the baculovirus expression system 37
4.1.2 Protein composition and functional analysis of influenza H7N9-TW VLPs 38
4.1.3 Establishment of influenza H7N9-TW VLPs production conditions 39
4.1.4 Optimization of H7N9-TW VLPs production conditions 40
4.1.5 Evaluation of the scale-up feasibility of the upstream bioprocess. 41
4.1.6 Brief summary 43
4.2 Downstream bioprocess 43
4.2.1 The influenza H7N9-TW VLPs harvested be removed cell debris by a microfiltration purification step 43
4.2.2 Optimization of H7N9-TW VLPs purification conditions by the DoE 45
4.2.3 Evaluation of the flow-through mode of anion-exchange chromatography 46
4.2.4 Evaluation of the scale-up feasibility of the downstream bioprocess. 48
4.2.5 brief summary 49
4.3 Vaccination 50
4.3.1 Quantification analysis of the H7N9-TW VLPs 50
4.3.2 Vaccination of mice study using H7N9-TW VLPs vaccine 51
4.3.3 Compare of adjuvant effect in mice study. 51
Chapter 5. Conclusions 54
Tables 56
Figures 72
Publication during my PhD study 85
1. Journal papers 85
2. Conference paper: 86
References 87

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