臺灣博碩士論文加值系統

登革病毒感染已成為全球性的重要衛生議題，為有效防治登革熱的危害，除從公共衛生著手外，疫苗的研發也刻不容緩。目前愈來愈多的證據顯示：登革病毒非結構性蛋白1 (nonstructural protein 1，NS1) 的DNA質體 (plasmid)能誘發宿主產生保護力，所以非常有潛力發展成有效的疫苗以供預防使用。本實驗室之前以第二型登革病毒（PL046）之非結構性蛋白質（dengue 2 nonstructural protein1, pD2NS1）基因作為疫苗，以肌肉注射方式接種C3H小鼠，然後再以同型致死劑量之病毒注射接種過基因疫苗的小鼠，結果發現：以pD2NS1基因疫苗免疫的小鼠，確實較對照組具有保護小鼠免受病毒攻擊的能力。且以ELISA分析其週邊血也發現有NS1特異性抗體存在，因此推論體液型免疫反應可能參與pD2NS1的保護機轉。本實驗室進一步發現pD2NS1疫苗在主要組織相容性抗原複合體第一型 (major histocompatibility complex class I, MHC class I) 基因缺陷的小鼠中無法提供有效的免疫保護力，而在脾臟細胞被動轉移 (splenocyte adaptive transfer) 實驗發現：將pD2NS1免疫過捐贈鼠的脾臟細胞注射到接受鼠體內後，再注射登革病毒，能有效提高存活率。然而血清被動轉移實驗中，只能延長接受鼠發病的天數，無法降低死亡率。因此推論pD2NS1造成的保護機轉中，細胞免疫反應 (cellular immune response)可能重於體液型免疫反應，且MHC class I分子相較於class II分子可能扮演較重要角色。本研究主要是利用基因轉殖 (transgenic)或剔除 (KO) 鼠為實驗動物模式，再深入探討pD2NS1疫苗保護機轉中的細胞型免疫反應，並區別細胞型與體液型免疫反應間重要性的差異。另一方面也將探討登革病毒感染小鼠後可能的免疫反應。
我們以pD2NS1接種第一型及第二型輔助性T細胞雙基因轉殖鼠(BALB/cTh1&Th2 transgenic mice)，發現該鼠脾臟CD4+細胞有非常顯著的上升；並且能誘發第二型輔助性T細胞 (T help 2 cell) 反應。若將B細胞缺陷小鼠以pD2NS1疫苗免疫後，再以病毒注射這群的小鼠，則發現疫苗仍能誘發保護效果。在另一組實驗中，直接將病毒注入第一型及第二型輔助性T細胞雙基因轉殖鼠(BALB/cTh1&Th2 transgenic mice)中，藉由流式細胞儀 (FACS) 方式每週分析脾臟細胞的變化連，實驗結果顯示CD4+細胞在感染初期顯著增加，在感染後第一週出現最大值，晚期則降低，在感染後第四週出現最小值；B細胞則從感染初期就開始減少，之後逐漸回升，在第五週左右回歸到正常值；但CD8+細胞在感染初期先緩緩減少於感染後第四週卻轉而增加。此外，Th2反應在感染早期增加且於注射病毒後第三週左右見到最高峰；然而Th1反應感染早期先下降，到第四週才上升到最高峰。所有的細胞反應於感染後第五週都回歸正常值。在B細胞缺陷小鼠模式中，將登革病毒接種於6週大小鼠，結果發現：於登革病毒靜脈注射時，相較於基因正常小鼠 (C57BL/6 mice)，B細胞缺陷小鼠之死亡率可高達100%。由以上實驗結果，我們認為：pD2NS1基因疫苗的保護機轉中細胞型免疫反應較體液型免疫反應重要在宿主對抗登革病毒感染時，B細胞是不可或缺的；但在無B細胞的狀況下，pD2NS1疫苗仍然能提供保護力。

Dengue viruses cause a severe public health problem throughout the tropical and subtropical areas of the world. Accumulating data indicate that dengue nonstructural protein 1 (NS1) can serve as vaccine protecting mice against dengue infection. We previously demonstrated that mice vaccinated with NS1 DNA (pD2NS1) and subsequently challenged by lethal DEN-2 exhibited a delay onset of paralysis, a marked decrease of morbidity, and a significant enhancement of survival. Moreover, by using MHC class I and II knockout mice as vaccination/challenge models, we found CD8+ T cells are essential for pD2NS1 vaccination-induced protection. To further investigate the virus-induced immune responses and protective mechanisms involved in pD2NS1 immunization, we established similar vaccination/challenge models by using Th1 and Th2 double transgenic and B-cell deficient mice in this study. Mice deficient in B cells immunized with pD2NS1 still protected from subsequent viral challenge, indicating that NS1-specific antibody can be dispensable in DEN-2 NS1 DNA-mediated protection. However, compared with wild type littermates, unimmunized B-cell deficient mice showed much higher mortality after lethal viral challenge, suggesting that humoral immune response still plays certain role for host against DEN infection. In Th1 and Th2 double transgenic mice immunized with pD2NS1, there is a significant increase of CD4+ subpopulation and enhancement of Th2-type response. Similar results of increasing CD4+ cells and Th2 immunity were found in these pD2NS1-immunized mice followed by viral challenge. These results may help us to understand host immunity against DEN infection and provide further insight into vaccine development.

目 錄
項目 頁數
正文目錄 ……………………………………………………………...…. I
中文摘要 ……………………………………………………………..… V
英文摘要 …………………………………………………………...… VIII
第一章 緒言………………………………………………….………….. 1
第二章 材料與操作方法……………………………………….……….. 7
壹、質體的大量製備…………………………….…………............. 7
貳、登革病毒 (dengue virus, PL046) 的增殖............……..……… 9
參、測定病毒效價...................................…………………..…...… 9
肆、非結構性蛋白 (NS1 protein) 之產製與純化...........….......…. 10
伍、第一及第二型輔助性T細胞雙基因轉殖鼠的篩檢..…..….... 14
陸、B細胞缺陷小鼠的分析.............................…………..…….… 16
柒、T細胞增殖反應..................................……………..……..….. 17
捌、以流式細胞儀分析老鼠脾臟細胞表面分子的變化.....……... 18
玖、pD2NS1基因疫苗誘發細胞型免疫反應.............…………… 19
拾、pD2NS1基因疫苗免疫B細胞缺陷小鼠.............…………… 20
拾壹、被基因疫苗免疫後小鼠再遭登革病毒感染的細胞型
免疫反應...........................................................….…….…... 20
拾貳、宿主感染登革病毒後細胞型免疫反應.......….......……….. 21
拾參、病毒感染B細胞缺陷小鼠.........................………...……… 21
拾肆、血清被動轉移試驗.............….........…………………..…… 22
第三章 結果………………………………………………………..…… 24
壹、pD2NS1基因疫苗保護機轉之評估..……………………....... 24
一、pD2NS1基因疫苗保護機轉中細胞型免疫反應
二、pD2NS1基因疫苗保護機轉中B細胞重要性
三、被基因疫苗免疫後小鼠再遭登革病毒感染的細胞型免疫反應
貳、宿主受登革病毒感染後免疫反應之析…………………….... 27
一、細胞型免疫反應的評估
二、B細胞在宿主對抗登革病毒感染時的重要性探討
參、血清被動轉移試驗..............................…………..………….... 30
第四章 討論………………………………………………….…………. 31
第五章 結論...........................................................................…............... 36
第六章 圖表………………………………………………..…………… 38
圖1.第一型及第二型輔助性T細胞雙基因轉殖鼠篩檢結果..... 38
圖2. B細胞缺陷小鼠淋巴細胞分析........................................…. 39
圖3. pD2NS1基因疫苗保護機轉中T細胞增殖反應 ……..…. 40
圖4. pD2NS1基因疫苗保護機轉中細胞型免疫反應的分析…. 41
圖5. pD2NS1基因疫苗保護機轉中Th1&Th2反應的分析….... 42
圖6. pD2NS1基因疫苗保護機轉中B細胞的重要性分析……. 43
圖7. pD2NS1基因疫苗免疫後再遭登革病毒感染的T細胞
增殖反應……………………………….……..…………....... 44
圖8. pD2NS1基因疫苗免疫後再遭登革病毒感染的細胞型
免疫反應……………………………........……..........…........ 45
圖9. pD2NS1基因疫苗免疫後再遭登革病毒感染的Th1&Th2反應分析…………………………………….…..................… 46
圖10.小鼠遭登革病毒感染後T細胞增殖反應的結果…..….…. 47
圖11.1小鼠遭登革病毒感染後CD4+細胞免疫反應趨勢
分析之一………………………………………………….. 48
圖11.2小鼠遭登革病毒感染後CD4+細胞免疫反應趨勢
分析之二………………………………………………….. 49
圖12.1小鼠遭登革病毒感染後CD8+細胞免疫反應趨勢
分析之一………………………………………………….. 50
圖12.2小鼠遭登革病毒感染後CD8+細胞免疫反應趨勢
分析之二…………………………………………………. 51
圖13.1小鼠遭登革病毒感染後B細胞免疫反應趨勢
分析之一..………………………………………………... 52
圖13.2小鼠遭登革病毒感染後B細胞免疫反應趨勢
分析之二………………………………………………….. 53
圖14.1小鼠遭登革病毒感染後Th1免疫反應趨勢
分析之一....……………………………………………….. 54
圖14.2小鼠遭登革病毒感染後Th1免疫反應趨勢
分析之二.……………………………………………....…. 55
圖15.1小鼠遭登革病毒感染後Th2免疫反應趨勢
分析之一...…………………………………………....….. 56
圖15.2小鼠遭登革病毒感染後Th2免疫反應趨勢
分析之二.....……………………………………...…..….. 57
圖16.1小鼠遭登革病毒感染後細胞型免疫反應趨勢
分析之一.………………………….……………..…..…... 58
圖16.2 小鼠遭登革病毒感染後細胞型免疫反應趨勢
分析之二.………………………...…………………......... 59
圖17.小鼠遭登革病毒感染的體液型免疫反應分析............…... 60
第七章 參考文獻……………………………………………………….. 61

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