呼吸道融合病毒 (respiratory syncytial virus, RSV) 與第三型人類副流感病毒 (human parainfluenza virus type 3) 是造成嬰幼兒急性呼吸道感染的主要病毒性病原，流行範圍廣及全世界，但目前仍未有良好的預防與治療措施。根據流行病學的研究統計，呼吸道病毒共同感染 (co-infection) 可能將增加疾病嚴重程度，然而現今對共同感染現象有諸多不清楚之處也缺乏合適的動物模式。於本研究中，首度以BALB/c 小鼠建立HPIV3 與 RSV 共同感染的動物模式，同時證明共同感染的確會增加肺部發炎狀態和嗜酸性白血球的浸潤，以及免疫反應偏向Th2 response 等疾病加劇的特徵。進一步，測試本實驗室先前以腺病毒載體構築表達RSV F 蛋白與HPIV3 F蛋白之雙價疫苗-Ad-RSV-F/HPIV3-F 之應用可行性。對BALB/c 小鼠進行免疫，確實誘發分別對RSV及HPIV3 專一之抗體；取免疫後之小鼠脾臟細胞經由抗原刺激後能夠促使其分泌細胞激素IFN-γ與IL-4，並且誘發T 細胞之增生，這些結果顯示雙價疫苗-Ad-RSV-F/HPIV3-F 引起的免疫反應包含體液性與細胞性免疫，亦綜合了Th1與Th2 的反應。另外，於小鼠免疫後再套用本研究中建立的HPIV3 與 RSV 共同感染動物模式以評估雙價疫苗-Ad-RSV-F/HPIV3-F 的實際保護效力，唯效果不如預想，該部分需要再進行檢討及修正。總結上述結果，本研究已完成HPIV3 與 RSV 共同感染動物模式之建立，而另一方面，雙價疫苗-Ad-RSV-F/HPIV3-F 雖然可以引發小鼠之免疫反應，但是免疫強度似乎不足提供對HPIV3 與 RSV 共同感染的保護力。

Respiratory syncytial virus (RSV) and human parainfluenza virus type 3 (HPIV3) are the major causes of acute respiratory infections in infants. Some clinical reports have already shown that co-infection might increase disease severity. Here, we would like to develop a model for HPIV3 and RSV co-infection in BALB/c mice and to know whether co-infection will cause more severe pathogenesis. Furthermore, the adenovirus expressing fusion (F) protein of RSV and fusion (F) protein of HPIV3 (Ad-RSV-F/HPIV3-F) was constructed and vaccinated to test its protective efficacy using co-infection of HPIV3 and RSV mouse model. The immune responses specific to RSV and HPIV3, and lung pathology of animals challenged with RSV and HPIV3 are investigated. The results demonstrated that HPIV3 and RSV co-infection does increase disease severity in BALB/c mice as evidenced by promotion of eosinophilia and Th2-skewed immune response. In addition, recombinant adenovirus expressing both RSV F and HPIV3 F proteins induced humoral and cellular immune responses. BALB/c mice immunized with Ad-RSV-F/HPIV3-F produced antibodies specific to RSV and HPIV3. The splenocytes collected from the Ad-RSV-F/HPIV3-F-immunized mice showed T cell proliferation and secreted IFN-γ and IL-4 after re-stimulation with antigens, indicating the induction of Th1 and Th2 immune responses. Unfortunately, this recombinant adenovirus expressing the RSV F protein and HPIV3 F protein was unable to evoke a protective immune response in BLAB/c upon challenge with RSV and HPIV3. Taken together, these results not only implicate the mechanism of pathogenesis of RSV and HPIV3 co-infection but also establish a pre-clinical model to evaluate the efficacy of bi-valent vaccine candidate against RSV and HPIV3 infection.

Abstract I
摘要 II
目錄 IV
一、緒論 1
1-1-1 RSV的流行病學 1
1-1-2 RSV 的病毒結構與抗原特性 1
1-1-3 RSV造成的症狀 3
1-1-4 引起嚴重症狀的風險因子 3
1-1-5 RSV 感染引起的發炎反應與動物模式 4
1-2-1 HPIV3的流行病學與造成的症狀 5
1-2-2 HPIV3 的病毒結構與抗原特性 5
1-2-3 HPIV3感染引起的免疫反應 6
1-3 共同感染 7
1-4-1 疫苗發展簡史 7
1-4-2 RSV 和 HPIV3 疫苗開發歷程 8
1-5 腺病毒載體 10
1-6 研究動機及目的 10
二、材料與方法 12
2-1 細胞與病毒的培養 12
2-1-1哺乳動物細胞 12
2-1-2基因改造 (genetically modified) 細胞 13
2-1-3呼吸道融合病毒 (Respiratory Syncytial Virus-B1, RSV-B1) 13
2-1-4第三型人類副流感病毒(Human Parainfluenza Virus 3-C243, HPIV3-C243) 13
2-1-5病毒力價測定 14
2-2 Ad-RSV-F/HPIV3-F 疫苗之生產和蛋白質表現分析 15
2-2-1 Ad-RSV-F/HPIV3-F增殖與純化 15
2-2-2 Ad-RSV-F/HPIV3-F 力價測定 16
2-2-3 SDS-PAGE 凝膠電泳和西方墨點法(Western blotting) 16
2-3 RSV 和 HPIV3 雙重感染之小鼠模式建立 (development of mice model for RSV and HPIV3 co-infection) 17
2-3-1小鼠感染 (HPIV3 and RSV challenge) 17
2-3-2小鼠血液採集與肺臟取得 17
2-3-3酵素免疫分析測定抗體力價 (antibody titer determination by ELISA) 18
2-3-4組織包埋切片與免疫組織化學染色(Immunohistochemistry, IHC) 18
2-3-5 mRNA isolation and reverse transcription 20
2-3-6 Quantitative polymerase chain reaction (qPCR) 21
2-4 分析Ad-RSV-F/HPIV3-F 疫苗引發之免疫反應 21
2-4-1以Ad-RSV-F/HPIV3-F免疫小鼠 21
2-4-2體液性免疫分析 22
2-4-2-1小鼠血液採集與抗體力價測定 22
2-4-3細胞性免疫分析 22
2-4-3-1 ELISPOT 23
2-4-3-2 T cell proliferation assay 24
2-5 測試Ad-RSV-F/HPIV3-F之保護效力 24
2-5-1小鼠注射疫苗後感染HPIV3及RSV 24
2-5-2組織包埋切片與免疫組織化學染色 25
2-5-3 Quantitative polymerase chain reaction (qPCR) 25
2-5-4分析小鼠肺部T細胞浸潤之狀態 25
2-6 統計分析 26
三、結果 27
3-1 RSV 和 HPIV3 雙重感染之小鼠模式建立 (development of mice model for RSV and HPIV3 co-infection) 27
3-1-1 測試感染HPIV3後96小時再感染RSV是否加重肺部發炎 27
3-1-2 以感染兩病毒間的時間間隔做為改變條件觀察是否加重肺部發炎 28
3-1-2-1 同一時間感染HPIV3及RSV 28
3-1-2-2 感染HPIV3後間隔5小時再感染RSV 28
3-1-2-3 感染HPIV3後間隔24小時再感染RSV 29
3-1-2-4 Th1/Th2 response 分析 29
3-2 分析Ad-RSV-F/HPIV3-F 疫苗引發之免疫反應 30
3-2-1 製備複製缺陷型腺病毒載體雙價疫苗 (Ad-RSV-F/HPIV3-F) 30
3-2-2 西方墨點法(western blotting)確認RSV F 和HPIV3 F 蛋白之表現 30
3-2-3 體液性免疫分析 31
3-2-4 細胞性免疫分析–ELISPOT 31
3-2-5 細胞性免疫分析–T細胞增生 (proliferation) 32
3-3 測試Ad-RSV-F/HPIV3-F之保護效力–lung pathology 32
3-4 測試Ad-RSV-F/HPIV3-F之保護效力–viral clearance 33
四、討論 34
4-1 RSV 和 HPIV3 雙重感染之小鼠模式建立 34
4-2 Ad-RSV-F/HPIV3-F 疫苗 35
符號及縮寫檢索表 40
參考文獻 41

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