- 學位論文
腸病毒71型類病毒顆粒的表現、特性分析及其應用為抗腸病毒疫苗之評估
The Expression and Characterization of Enterovirus 71 Virus-Like Particles and following Evaluation as a Potential Vaccine
摘要
腸病毒71型(enterovirus 71)是目前已知69種血清型腸病毒中的一種，過去十幾年來一直威脅著害國內幼童健康，造成至少兩百人死亡及每年仍有數起嚴重案例的悲劇。研究腸病毒類病毒顆粒(virus-like particle, VLP)可以提供對病毒;結構及其蛋白質相關知識，以輔助目前仍停滯不前的預防、治療工作，並可當作具有潛力之疫苗。腸病毒71型外殼之組合類似小兒麻痺病毒組合的模式，以3CD水解蛋白酶將P1結構蛋白之前趨物切割成單一的結構蛋白再組合而成。我們在先前的研究中利用兩株重組桿狀病毒分別表現3CD水解蛋白酶與P1結構蛋白，確認3CD水解蛋白酶能夠切割P1結構蛋白成為個別的結構蛋白(VP1, VP3與VP0)，並且能夠在細胞內形成腸病毒71型類病毒顆粒。在本研究中，我們進一步把3CD水解蛋白酶與P1結構蛋白的基因放入同一個重組桿狀病毒(vBac-P1-3CD)中，利用單一感染的方式同時表現這兩種蛋白質來生產類病毒顆粒。從電子顯微鏡觀測經超微薄切片的感染細胞中發現，細胞內有類病毒顆粒堆積形成，且聚集方式與真實腸病毒71型顆粒相似。在結合蔗糖不連續密度梯度與氯化銫等密度梯度離心的超高速離心純化後，我們得到高純度(>90%)的類病毒顆粒，純化後的類病毒顆粒以電子顯微鏡觀察，確認顆粒大小、形狀與中空結構的特性均與預測的相似，並經由SDS-PAGE與Western-blot的分析確認類病毒顆粒的組成。接著我們利用免疫金染色確認VP1結構蛋白的確實存在於顆粒的表面，而利用市售的腸病毒71型檢測試劑的檢驗也再度證實純化後的顆粒就是腸病毒71型類病毒顆粒。 在確認腸病毒71型類病毒顆粒的表現與純化後，我們進一步利用小鼠免疫測試以評估類病毒顆粒做為疫苗的可行性。實驗中以類病毒顆粒、變性類病毒顆粒、熱處理去活化腸病毒為抗原進行小鼠免疫，比較不同抗原在體液性與細胞性免疫反應的差異。對BALB/c小鼠進行免疫後，類病毒顆粒組別的小鼠血清能夠測到高濃度的的IgG抗體與中和抗體，顯示類病毒顆粒能夠誘發強而且持久的體液性免疫反應。另外，在以類病毒顆粒為抗原的小鼠組別中，由免疫後小鼠取出的splenocytes細胞經過抗原刺激後有著明顯的T細胞增生現象，並且能夠分泌高濃度的細胞激素IFN-γ、IL-2與IL-4，這些結果顯示類病毒顆粒能夠誘發Th1與Th2免疫反應。更重要的是，母鼠經過類病毒顆粒免疫後，所生出的小鼠對於致命劑量(1000 LD50)的腸病毒感染具有明顯增強的抵抗力(存活率提升至89%)。相較之下，變性類病毒顆粒與熱處理去活化腸病毒雖然能夠誘發高濃度的IgG抗體與細胞性免疫反應，但是只能誘發較低濃度的中和抗體，在小鼠challenge實驗中也只能提供較低效率的保護。這些結果顯示保存類病毒顆粒的完整結構的重要性，也更進一步證實類病毒顆粒做為抗腸病毒71型疫苗的可行性。
並列摘要
Enterovirus 71 (EV71) is one of the 69 enterovirus serotypes within the picornavirus family and has identified as the major etiological agent responsible for the outbreaks of severe neurological diseases in the past decades. The study of virus-like particles (VLPs) could provide further insight into the virus structure and virus assembly pathway that are crucial for antiviral drug design and could serve as a potential vaccine. We have previously cloned the genes encoding P1 polyprotein and 3CD protease into two separate baculoviruses (Bac-P1 and Bac-3CD) and demonstrated that the co-expression of P1 and 3CD in insect cells resulted in the cleavage of P1 by 3CD into individual structural proteins (VP1, VP3 and VP0) and subsequently formation of EV71 VLP. In this study, we further cloned the two genes into one baculovirus (vBac-P1-3CD) and expressed P1 and 3CD proteins by single-infection. The formation and aggregation of VLP within the infected insect cells were visualized by ultrathin-sectioning and transmission electron microscopy (TEM). These particles measured 25-27 nm in diameter and appeared icosahedral, thus resembling the authentic enterovirus in size and appearance. The VLPs were further purified by sucrose and CsCl gradient ultracentrifugation, and the purity of the sample was greater than 90%. The purified VLPs comprised VP0, VP3 and VP1 of roughly equal molar ratios, and implied the similarity between the VLP and the authentic virus in composition. Nonetheless, after purification the size and morphology of VLPs appeared indistinguishable from those of poliovirus VLP. More importantly, the immunogold labeling confirmed the presence of VP1 epitopes on the VLP surface. After characterizing the properties of VLP, we further evaluated the potential of VLP as a vaccine by comparing the humoral and cellular immune responses elicited by the purified VLP, denatured VLP and heat-inactivated EV71 virus. After immunization of BALB/c mice, EV71 VLP induced potent and long-lasting humoral immune responses as evidenced by the high total IgG titer and neutralization titer. The splenocytes collected from the VLP-immunized mice exhibited significant cell proliferation and produced high levels of IFN-γ, IL-2 and IL-4 after stimulation, indicating the induction of Th1 and Th2 immune responses by VLP immunization. More importantly, the VLP immunization of mother mice conferred protection (survival rate up to 89%) to neonatal mice against the lethal (1000 LD50) viral challenge. Compared with the VLP immunization, immunization with denatured VLP and heat-inactivated EV71 elicited lower neutralization titers and conferred less effective protection to newborn mice, although they induced comparable levels of total IgG and cellular immune responses. These data collectively indicate the importance of the preservation of VLP structure and implicate the potential of VLP as a vaccine to prevent EV71 infection.