許多新一代的候選疫苗成分含有純化的重組蛋白或合成胜肽，比去活化微生物疫苗更能提供安全且便於製造等優點。有許多案例指出，這些以蛋白質或胜肽為基礎的疫苗，其免疫效價較低並且需要搭配佐劑(adjuvant)來引起具有保護性且持久的免疫反應。在許多佐劑中，油包水(water-in-oil)型乳化液已經被評估可以引發長期的免疫反應，但是這種類型的乳化液可能會在施打的部位造成副作用。相反的，有文獻指出水包油(oil-in-water)型乳化液例如MF59可以在施打部位迅速地創造具有免疫活性(immunocompepetent)的環境。在此研究中，我們準備不同非離子性(nonionic)的乳化劑包括PEG-b-PLACL, Tween®80 以及 Span®85，這些乳化劑所組成的乳化劑系統呈現了不同的親水-疏水平衡值(hydrophilic-lipophilic balance value)，並且利用適化的製程進行製造疫苗配方。此外，我們還嘗試檢驗這些乳化劑的成分和製造過程對於所形成的乳液或疫苗配方之相關物化特性分析(包含穩定度、導電度、顯微鏡之鏡檢方面、粒徑分佈、體外釋放以及體內分佈)與佐劑活性的評估(包含B-cell和T-cell反應)的影響。這些乳化劑經由適化的製作過程，所得到的是具有奈米顆粒等級之均質乳液，而我們也發現乳化劑的成份和乳液的製程對於疫苗配方的物化性質和免疫生成性(immunogenicity)有顯著的影響。此研究結果將對於抗原遞送系統的設計和疫苗的研發方面提供一些實驗證據和研發策略。

Many new vaccine candidates comprising purified recombinant proteins or synthetic peptides offer substantial advantages over inactivated microorganism vaccines in terms of safety and ease of manufacturing. But protein/peptide-based vaccines in most cases have low immunogenicity and need adjuvant to elicit protective and long-lasting immune responses. Water-in-oil emulsions have been evaluated and achieved long-term protective immune responses. However, this type of emulsions are crowded in the oily phase and causing local reactions at the site of injection. On contrary, it is well documented that the oil-in-water emulsions like MF59 can quickly induce and create immunocompetent environment at the site of injection. In this study, we prepared different emulsion formulations using non-ionic surfactants (PEG-b-PLACL, Tween®80 and Span®85) with various hydrophilic-lipophilic balance values of the surfactant systems under the optimized manufacturing procedure. Furthermore, we attempted to examine the effects of the surfactant components and manufacturing process of the prepared emulsion formulations on the physicochemical characteristics (including stability, electrolytic conductivity, microscopic aspects, size distribution, in vitro release and in vivo distribution) and adjuvant activity (including B-cell immunoassays and T-cell immunoassays). After the optimization of emulsification procedure, the homogeneous nanoparticles were obtained in the emulsions and we also found that the physicochemical properties and the immunogenicity of formulated vaccines are strongly influenced by the compositions and manufacture process. These results were obtained in this study with potential features provide experiment evidences and suggest strategies for the design of effective antigen delivery and vaccine development.

TABLE of CONTENTS
中文摘要 i
ABSTRACT ii
ACKNOWLEDGEMENTS iii
TABLE of CONTENTS iv
LIST of FIGURES vi
LIST of TABLES vii
CHAPTER I. INTRODUCTION 1
1.1. Background 1
1.1.1. Vaccinology and diseases control 1
1.1.2. Concepts of vaccine adjuvants 2
1.1.3. Emulsion-type adjuvants 5
1.1.4. Influenza 8
1.1.5. Human cervical cancer 10
1.2. Motivation of the study 11
CHAPTER II. MATERIALS AND METHODS 13
2.1. Materials 13
2.1.1. Chemicals and reagents 13
2.1.2. Buffers and media 14
2.1.3. Antibody 15
2.1.4. Enzyme 15
2.1.5. Experimental animals 15
2.1.6. Kit reagents 15
2.1.7. Vaccine preparation 16
2.1.8. Consumables 16
2.1.9. Instruments 17
2.2. Methods 19
2.2.1. Synthesis of PEG-b-PLACL 19
2.2.2. Polymer characterizations 21
2.2.3. Preparation of emulsion-formulated vaccines 22
2.2.4. Physicochemical characterizations of the emulsions 23
2.2.5. The immune response of ovalbumin combined with the different emulsion formulations 24
2.2.6. Immunogenicity of inactivated trivalent influenza vaccine formulated with the different emulsion formulations 28
2.2.7. Immunogenicity of the RAH peptide formulated with the different emulsion formulations. 30
CHAPTER III. RESULTS AND DISCUSSION 33
3.1. Synthesis and characterization of PEG-b-PLACL 33
3.2. Preparation and physicochemical characterizations of the emulsions 36
3.3. The immune response of ovalbumin combined with the different emulsion formulations. 43
3.4. Immunogenicity of inactivated trivalent influenza vaccine formulated with the different emulsion formulations. 46
3.5. Immunogenicity of the RAH peptide formulated with the different emulsion formulations. 48
CHAPTER IV.CONCLUSIONS 50
CHAPTER V.REFERENCE 52
FIGURES 56
TABLES 74

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