高致病性禽流感 (HPAI) H5N1 病毒由於在鳥禽類以及人造成大量感染吸引了高度的關注，市場上需要更有效且安全的黏膜疫苗對抗H5N1病毒。在本篇研究中，我們使用LTIIb-B5 (第二型b型忌熱型腸毒素B次單元) 重組蛋白與H5HA-LTIIb-A (H5HA結合第二型b型忌熱型腸毒素A次單元)融合蛋白作為抗H5N1病毒的黏膜疫苗。透過對Balb/c品種小鼠執行三劑鼻腔免疫注射，我們期望小鼠表現體液性與細胞性的免疫反應，進而發展出保護免疫力。實驗結果顯示， H5HA-LTIIb-A與H5HA-LTIIb-A+LTIIb-B5兩個組別在血清以及肺部支氣管沖洗液(BAL)中引發了大量針對H5HA IgG、IgA的專一性抗體。這兩個免疫小鼠組別的抗體中和能力皆有增加，且與抗H5HA IgG、IgA抗體有高度的相關性。H5HA-LTIIb-A與H5HA-LTIIb-A+LTIIb-B5小鼠組別在脾臟中發現大量的細胞因子如: IFN-γ、IL-4與IL-17。然而頸部淋巴結中，卻只有H5HA-LTIIb-A小鼠組別檢測出了高量的細胞因子。病毒攻毒實驗中，H5HA-LTIIb-A與H5HA-LTIIb-A+LTIIb-B5兩個組別對抗H5N1病毒的能力與其他控制組相比提供大量的保護力且有顯著的差異。在本篇研究中，H5HA-LTIIb-A被證實是個有效且有用的佐劑與抗原。H5HA-LTIIb-A 提供了一個新的方向研究發展黏膜佐劑疫苗。

The highly pathogenic avian influenza (HPAI) H5N1 virus can cause serious infections in both avian and human. The markets need a more effective and safe vaccine against H5N1 virus infections. In this study, we have constructed a recombinant HA (rH5HA) fusion protein that contains an E.coli type IIb heat-labile enterotoxin A subunit (LTIIb-A) (H5HA-LTIIb-A) and another H5HA-LTIIb-A fusion proteins that has additional LTIIb-B5 subunits (H5HA-LTIIb-A fusion proteins plus LTIIb-B5 subunits). A three-dose intranasal immunization was administrated to BALb/c mice to evaluate the anti-H5N1 humoral and cellular immune responses as well as the protection against live virus challenge. Our results showed that both of the H5HA-LTIIb-A fusion proteins and H5HA-LTIIb-A fusion proteins plus LTIIb-B5 subunits induced significantly higher levels of H5HA-specific IgG and IgA in sera and bronchoalveolar lavage fluids (BAL) when compared with the PBS and H5HA groups. The neutralizing ability of these two immunization groups is also higher than the PBS and H5HA groups. High levels of cytokines, such as IFN-γ, IL-4 and IL-17, were detected in the spleen in the H5HA-LTIIb-A fusion proteins with LTIIb-B5 subunits group as well as the group without LTIIb-B5 subunits. However, it was only in the H5HA-LTIIb-A fusion proteins group that elevated levels of cytokines were detected in the cervical lymph node. In the live viral challenge study, both the groups of H5HA-LTIIb-A and H5HA-LTIIb-A plus LTIIb-B5 subunits provided 100% protection against the H5N1 influenza virus. The survival rate of these two immunization groups is better than PBS and H5HA group, which have a survival rate of 0% and 80% respectively. This study shows that H5HA-LTIIb-A and the H5HA-LTIIb-A plus LTIIb-B5 subunits are potent immunogens.

Abstract……………………………………………………………………………I
摘要…………………………………………………………………………………II
致謝………………………………………………………………………………… IV
1.簡介………………………………………………………………………………1
1.1. 簡介流感病毒…………………………………………………………………1
1.2. 黏膜免疫、鼻腔注射免疫與佐劑……………………………………………2
1.3. 類鐸受體(Toll-like receptor ligands)及其佐劑之應用…………………………3
1.4. 簡介忌熱型腸毒素(heat-labile enterotoxin)及其佐劑之應用 ………………4
2. 研究目標…………………………………………………………………………8
3. 材料與方法………………………………………………………………………10
3.1. LTIIb-B5 (第二型b型忌熱型腸毒素B次單元)重組蛋白克隆、表現以及純化……………………………………………………………………………………..10
3.2. H5HA重組蛋白與H5HA-LTIIb-A (H5HA結合第二型b型忌熱型腸毒素A次單元) 重組蛋白克隆、表現以及純化………………………………………………11
3.3. 血球凝集試驗…………………………………………………………………..11
3.4. 胎球蛋白結合試驗……………………………………………………………..12
3.5. 類鐸受體試驗…………………………………………………………………..12
3.6. 免疫小鼠及樣品採集………………………………………………………….13
3.7. T-細胞細胞激素分析…………………………………………………………...14
3.8. 抗H5HA酵素結合免疫吸附(ELISA)試驗…………………………………..14
3.9. 中和抗體試驗………………………………………………………………….15
3.10. 病毒攻毒試驗………………………………………………………………...15
3.11. (NTHU1)H5HA-LTIIb-A與(NTHU2)H5HA-LTIIb-A之克隆……………...16
4. 結果……………………………………………………………………………...17
4.1. H5HA重組蛋白與H5HA-LTIIb-A (H5HA結合第二型b型忌熱型腸毒素A次單元)重組蛋白的純化與定性分析…………………………………………………17
4.2. 利用類鐸受體2/1功能性試驗檢測LTIIb-B5 (第二型b型忌熱型腸毒素B次單元) 重組蛋白與H5HA-LTIIb-A (H5HA結合第二型b型忌熱型腸毒素A次單元)重組蛋白 ……………………………………………………………………………18
4.3. 在血清與肺部支氣管沖洗液(BAL)中檢測針對H5HA抗原的IgG、IgA抗體……………………………………………………………………………………..19
4.4. 利用H5N1假性病毒檢測血清與肺部支氣管沖洗液(BAL)中和抗體能力...20
4.5. T-細胞反應試驗檢測脾臟與頸部淋巴結(CLNs)中的細胞因子……………...21
4.6. 病毒攻毒實驗確認H5HA-LTIIb-A (H5HA結合第二型b型忌熱型腸毒素A次單元)之保護力……………………………………………………………………24
4.7. (NTHU1)H5HA-LTIIb-A與(NTHU2)H5HA-LTIIb-A之克隆………………..
5. 討論………………………………………………………………………………26
6. 參考文獻…………………………………………………………………………31
7. 圖檔………………………………………………………………………………36

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