登革病毒 ( DENV ) 是一種具有4種不同血清型的蚊媒病毒，每年大約會造成3.9億個感染病例。大部分的感染者只會出現輕微的症狀或甚至無徵狀，然而，少數一部分的人可能會發展成嚴重的登革出血熱以及導致登革休克症候群。直到現在，只有一支減毒登革疫苗Dengvaxia被核可上市，但是它對疾病的保護程度不一以及存在引發嚴重症狀的風險，讓它的使用仍然有很大的疑慮。我們認為，比起活體減毒疫苗，由重組蛋白組成的次單元疫苗可能會是更好的候選者。為了讓疫苗能產生對非結構性蛋白 ( NS ) 專一的抗體，並對受登革病毒感染的細胞進行補體依賴性細胞毒性作用 ( CDC )，我們選擇NS1作為疫苗的組成元素。然而在我們的先前研究中指出，抗NS1抗體會和人體內的內皮細胞以及血小板進行交叉反應並造成其失去功能，所以我們去除NS1位於C端的區域以產生出∆C NS1。另外，一致性外膜蛋白第三結構域 ( cEDIII ) 在四型登革病毒中具有相似的氨基酸序列，其所誘導的抗體對於四型登革病毒皆具有中和活性。在我們的先前研究中也發現將重組蛋白cEDIII-∆C NS1予以免疫小鼠，小鼠所產生的抗體不僅對四型登革病毒皆具有中和活性，也同時能藉由補體介導的毒殺作用殺死被登革病毒感染的細胞。而為了更進一步增強細胞免疫力，我們另外選擇了在其他研究中證實可以引發CD4+和CD8+ T細胞反應的NS3的C端區域。因此在本研究中，我們建構了由∆C NS1片段、cEDIII和NS3片段所組成的重組蛋白∆C NS1f-cEDIII-NS3cf和∆C NS1f-NS3cf作為疫苗的候選，並將重組蛋白轉染至HEK293F以及Drosophila S2表達系統中並純化出蛋白。我們將重組蛋白加上alum佐劑以皮下注射施打3劑至小鼠體內，並分析重組蛋白在受登革病毒感染的小鼠提供的保護效果。從結果得知，在抗體反應的細胞試驗中，疫苗免疫所誘導的抗體在∆C NS1f-cEDIII-NS3cf施打組別顯示出中和活性，∆C NS1f-NS3cf施打組別則無；而在補體介導的毒殺作用上兩個組別都顯示具保護力的結果；另外在小鼠的登革病毒感染模型中，有施打疫苗的組別，比起控制組別在出血時間以及血清中病毒量上都有顯著的下降。研究結果顯示，不管是從HEK293F或是Drosophila S2系統所純化的重組蛋白誘導的抗體不僅在體外試驗顯示抗病毒的效果，在感染登革病毒的小鼠模型中也能提供保護效果。這些結果代表這兩支重組蛋白疫苗皆可在登革疫苗的發展中作為有潛力的疫苗候選者。

Dengue virus (DENV) is a well-known mosquito-borne virus with four serotypes which cause over 390 million infection cases every year. Most of the infected patients have mild symptoms or even asymptomatic, while a small proportion of them develop severe dengue hemorrhagic fever and dengue shock syndrome. Up to date, there is only one licensed dengue vaccine, Dengvaxia. Its overall efficacy to disease and the risk of vaccine-induced severe adverse effect still need to be concerned. Compared to the live attenuated vaccines, we consider that recombinant protein subunit vaccine might be a better vaccine candidate. In order to induce the nonstructural (NS) protein-specific antibody (Ab) triggering complement-dependent cytolysis (CDC) to DENV-infected cells, we chose NS1 as a component of subunit vaccine. However, in our previous studies, anti-DENV NS1 Abs can lead to endothelial cell and platelet dysfunction by cross reactivity. We therefore modified the DENV NS1 by depleting the C-terminal region to generate a △C NS1. Also, Abs against consensus envelope protein domain III (cEDIII), of which the amino acid sequences are consensus among four serotypes of DENV, showed neutralizing activity against all the four serotypes of DENV. Our previous studies showed that Abs induced by recombinant protein cEDIII-ΔC NS1 can neutralize four serotypes of DENV and also cause complement-mediated cytolysis of DENV-infected cells. To enhance the cellular immunity, we further selected C-terminal region of NS3 protein, which was found to induce CD4+ and CD8+ T cell responses. In this study, we have constructed the recombinant proteins composing of △C NS1 fragment, cEDIII, and NS3c fragment as the vaccine candidate, and purified the recombinant proteins, ∆C NS1f-cEDIII-NS3cf and ∆C NS1f-NS3cf, from transfected HEK293F cells and stable clone Drosophila S2 cells. We immunized 3 doses of vaccine candidates in alum adjuvant and analyzed the protective effects of these recombinant proteins in DENV2-infected mice. Our results demonstrated that Abs in serum from ∆C NS1f-cEDIII-NS3cf-immunized mice showed neutralizing effects but not in serum from ∆C NS1f-NS3cf-immunized mice. Additionally, CDC induced by vaccine-immunized groups both showed protective results in vitro. Besides, bleeding time and viral titer of both ∆C NS1f-cEDIII-NS3cf- and ∆C NS1f-NS3cf-immunized mice were significantly reduced as compared with the control groups. In conclusion, this study reveals that recombinant proteins from both HEK293F and Drosophila S2 expression systems can not only induce Abs that show efficacy against virus in vitro but provide protection against DENV infection in the mouse model. These recombinant proteins are considered as potential candidates in DENV vaccine development.

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