登革熱是全球重要的蚊媒病毒傳染病之一，每年約造成三億九千萬人感染。是經由登革病毒 (dengue virus) 所引起，它屬於黃熱病毒科的正向單股RNA病毒，會造成的病症可從無症狀、典型的登革熱到少數人發展成嚴重危及生命的登革出血熱以及登革休克症候群。儘管目前已有一個通過認證的減毒登革疫苗Dengvaxia，但此疫苗對於各型登革病毒的整體效能以及加重疾病的可能仍待確認。次單位疫苗(subunit vaccine) 能夠提供更安全的選擇。抗登革病毒的非結構性蛋白1 (NS1) 的抗體可以引發補體介導的細胞毒殺作用，並避免抗體依賴性免疫加強反應是更合適的登革疫苗候選者。我們實驗室先前研究顯示，NS1的C端區域會誘發導致內皮細胞失能和血小板功能障礙的交叉反應性自身抗體。因此，我們將此區域利用日本腦炎病毒 (JEV) 的相同區域替代並產生名為DJ NS1的嵌合蛋白。先前研究指出，以DJ NS1蛋白進行主動免疫，可以誘導小鼠產生良好的抗體反應及中等程度的毒殺性T淋巴球 (CTL)。為了加強細胞免疫反應，我們將DJ NS1與能夠引發顯著CD4+ 和CD8+ T細胞反應的登革非結構性蛋白3 (NS3) 結合。我們發現DJ NS1結合NS3能較有效地活化樹突狀細胞並增加抗原呈現相關受體分子的表現，也產生對於DJ NS1與NS3抗原具有特異性的CD4+ 和CD8+ T細胞活化反應以及抗體反應。此外我們也進一步地證實對於NS3特異性的T細胞具有產生干擾素伽瑪 (IFN-gamma 的能力並增加細胞毒殺活性標記的表現 (CD107a)。我們也進一步證實對於NS3特異性的T細胞，可藉由細胞介導的細胞毒殺功能將登革病毒感染的細胞殺死。為了評估疫苗所能提供的保護力，我們建立可以表現病毒血症的登革感染動物模式，並證實以DJ NS1結合NS3進行主動免疫確實能更全面性的壓制病毒複製並減緩包含出血時間延長以及出血問題等登革病徵。總結，在這個研究當中，我們證實了以DJ NS1結合NS3進行主動免疫能夠引發較優化的細胞免疫反應，並在登革感染情況下提供良好的保護效果。因此，DJ NS1結合NS3可做為一個有發展潛力的登革疫苗。

Dengue virus (DENV) has become a major global public health threat with four serotypes of the virus infecting over 390 million people annually. DENV causes a range of illness with a small proportion of infected patients developing severe dengue hemorrhagic fever or dengue shock syndrome. To date, there is only one licensed dengue vaccine; however, the overall efficacy and the risk of vaccine- enhanced severity of disease remain concerned. Recombinant protein subunit vaccines offer a safer alternative. To avoid antibody (Ab)-mediated enhancing effect of DENV infection, nonstructural protein 1 (NS1) is an appropriate candidate for Ab triggering complement-dependent cytolysis of DENV-infected cells. Our previous studies demonstrated that anti-DENV NS1 Abs can lead to endothelial cell damage and platelet dysfunction by cross-reactivity. Therefore, we modified the DENV NS1 by replacing the C-terminal region which contains cross-reactive epitopes with the corresponding region of JEV NS1 to generate a chimeric DJ NS1 protein as a vaccine candidate. Our preliminary data showed that active immunization with DJ NS1 can induce strong Ab response and moderate cytotoxic T lymphocyte (CTL) responses. To enhance cellular immunity, we further combined DJ NS1 with DENV NS3, which is immunodominant in CD4+ and CD8+ T cell responses. Combined immunization with DJ NS1 and NS3 can activate dendritic cells to up-regulate expression of CD40, CD80, CD86, MHC class I, and MHC class II. We also observed that immunization with DJ NS1 and NS3 can induce specific T cell activation and Ab responses. We further determined the NS3-specific CTL responses by increased IFN-gamma production and CD107a expression as well as the LDH release from target cells. Importantly, the protective effects attributed by DJ NS1 and NS3 immunization were demonstrated in DENV-infected mouse model including viral titers, soluble NS1 levels, mouse tail bleeding time, and hyperemia at local skin. Collectively, the results from this study reveal the humoral and cellular immune responses and the protective effects conferred by DJ NS1 and NS3 immunization in the mouse model of DENV infection, and provide a potential strategy for dengue vaccine design.

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