高致病性禽流感病毒目前仍威脅著全球人類與禽類的健康，其中H5N1禽流感病毒從1997年被發現能傳染人類後，至今H5N1的突變株病毒依然可傳播至野外飛禽、家禽、海生哺乳動物(如海獅)、一般陸地哺乳動物(如貂、浣熊、貓)，以及人類。目前疫苗接種是預防 H5N1 和其他新型禽流感病毒感染的最主要保護策略，但一般的傳統肌肉注射疫苗無法產生具有對抗原專一性的IgA抗體於黏膜表面，需使用黏膜疫苗才有辦法於黏膜產生對抗原專一性的IgA抗體，然而黏膜免疫系統需仰賴黏膜佐劑突破黏膜免疫系統的耐受性。在本論文中，大腸桿菌第二b 型忌熱型腸毒素 之B 次單元 (LTIIb-B5) 作為黏膜佐劑，與 HA 蛋白一起施打於小鼠的鼻腔免疫。結果顯示：LTIIb-B5 佐劑在血清和支氣管肺泡灌洗液中，引發明顯更高的 IgG、IgA 和中和抗體，從而增強對致命性病毒的保護。 LTIIb-B5 也會在脾臟淋巴細胞和頸部淋巴結中引發更強的 Th17 細胞反應。小鼠被注射抗 IL-17A 單株抗體後，經過致命性病毒的感染測試，小鼠體內的IL-17A 耗竭導致死亡率從 0% 增加至 50%，代表 Th17 的細胞反應對保護性免疫有所關連。這些發現可能為H5N1次單元黏膜疫苗的開發提供有用的資訊。

Infections in humans caused by highly pathogenic avian influenza viruses continue to pose a significant global health threat. Vaccination represents the primary preventive measure against H5N1 and other emerging avian influenza virus infections. However, conventional intramuscular vaccines cannot generate antigen-specific IgA antibodies on mucosal surfaces. Mucosal vaccines are necessary to produce antigen-specific IgA antibodies. Mucosal immunization relies on mucosal adjuvants to overcome the tolerance of the mucosal immune system. This dissertation utilized E. coli type IIb heat labile enterotoxin B subunit (LTIIb-B5) as a mucosal adjuvant in conjunction with HA proteins for intranasal immunizations in a mouse model. The findings demonstrated that the LTIIb-B5 adjuvant induced notably elevated levels of IgG, IgA, and neutralizing antibodies in both sera and bronchoalveolar lavage fluids, thereby enhancing protection against lethal virus challenges. Additionally, LTIIb-B5 elicited a more robust Th17 cellular response in spleen lymphocytes and cervical lymph nodes. Depletion of IL-17A subsequent to vaccinations with anti-IL-17A monoclonal antibodies led to an increase in mortality from 0% to 50%, indicating a potential contribution of the Th17 cellular response to protective immunity. These results offer valuable insights for the development of mucosal H5N1 subunit vaccines.

中文摘要 i
Abstract ii
致謝 iii
Table of Contents iv
Abbreviations vii
1. Introduction 1
1.1 Overview of influenza A virus 1
1.2 Vaccination strategies for Influenza A virus 3
1.3 Mucosal immunization 4
1.4 Toll like receptor and innate immunity 5
1.5 Mucosal adjuvants and LTIIb-B5 6
1.6 Research aims 8
2. Materials and methods 9
2.1 Cloning, Expression, and Purification of Recombinant LTIIb-B5 9
2.2 Functional assays for Toll-like receptor (TLR) ligands 10
2.3 Expression and Purification of Recombinant HA Protein 10
2.4 Mouse immunization and sampling 11
2.5 Analysis of H5HA-specific antibody titers 12
2.6 Neutralization Assays 13
2.7 Analysis of secretory cytokines in SPL and CLN cells 13
2.8 Cell populations in SPL and CLN cells by flow cytometry 14
2.9 Virus Challenges 14
2.10 Pathological sections and H&E staining of lungs of mice 15
2.11 Statistical Analyses 15
3. Results 16
3.1 Expression, Purification, and Characterization of Recombinant LTIIb-B5 Proteins 16
3.1.1 Characterization of structure of recombinant LTIIb-B5 proteins 16
3.1.2 Characterization of function of recombinant LTIIb-B5 proteins 17
3.2 The humoral immune responses after H5HA protein intranasal immunizations with LTIIb-B5 proteins as a mucosal adjuvant 17
3.3 Protection immunity after intranasal immunization by Viral challenge 18
3.4 The influences on the lungs after intranasal immunization 19
3.5 Detecting T cell responses in SPLs and CLNs 19
3.6 Protection immunity of mice with IL-17A depletion and IL-17A knockout mice against H5N1 challenges 20
4. Discussion 22
5. References 26
6. Figures 37
Figure 1. 3D structure of LTIIb-B5 protein 37
Figure 2. The original sequence of LTIIb-B5 protein 38
Figure 3. Functional assays for Toll-like receptor ligands 39
Figure 4. Characterization of H5HA protein 40
Figure 5. Characterization of LTIIb-b5 protein 41
Figure 6. Analysis of the molecular weight of pentameric LTIIb-B5 protein 42
Figure 7. The 1H-15N HSQC spectrum of LTIIb-B5 43
Figure 8. TLR 2/1 functional assays for LTIIb-B5 and Pam3Csk4 44
Figure 9. The schedule of intranasal immunization in mice model and the H5HA-specific IgG and IgA titers 45
Figure 10. Neutralization test for antibodies from sera and BAL fluids of the immunized mice 46
Figure 11. Intranasal immunization was followed by H5N1 viral challenges 47
Figure 12. Illustrative images of lung tissue sections 48
Figure 13. T cell responses were assessed in both SPLs and CLNs 49
Figure 14. Flow analyses were conducted to identify the source of IL-17A in SPLs or CLNs 51
Figure 15. IL-17A depletion and its impact on protective immunity against influenza virus 52
Figure 16. The protective effect induced by LTIIb-B5 was reversed in
IL-17A knockout mice 54

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