登革病毒是盛行於熱帶及亞熱帶地區感染人類的病原體，主要的傳染途徑是經由帶有病毒的病媒蚊叮咬，後而易引起登革熱、登革出血熱和登革休克症候。登革病毒的套膜蛋白[envelope (E) protein]具有結合細胞表面受器及引發病毒與宿主間膜融合的功能，同時亦是能誘發人體免疫反應的重要抗原；套膜蛋白的膜外區域(E domain)可以再進一步區分為三個功能區塊：區塊一、區塊二與區塊三(EDI, II, III)。為了探究登革病毒中和性抗體之抗原決定位及未來研發登革的DNA疫苗，本研究篩選了若干株由我們實驗室所生產的抗體，以釐清第四型登革病毒與中和性抗體之間的交互作用。作法上，我們先利用免疫光染色法與西方墨點法來確認單株抗體的專一性，並且找到若干株單株抗體能對抗病毒的套模蛋白或非結構性蛋白一[Non-structural (NS)-1]。我們更進一步利用蝕斑減少中和試驗[Plaque reduction neutralization test (PRNT)]，與保護試驗檢測這些單株抗體對於登革第四型的中和能力。我們亦製造了兩個DNA載體，分別含有第四型登革病毒的功能區塊一、二及三，並將此載體利用基因槍施打於小鼠身上，希望能藉此比較功能區塊一、二及功能區塊三對於誘發免疫反應的能力。我們亦施打第四型登革病毒DNA疫苗，pCB8D4-2J，及我們製作的載體，進一步比較並評估該製造的載體是否有成為DNA疫苗的潛力。在本實驗中我們t辨識出九株單株抗體能專一辨識第四型登革病毒與九株會變其他型登革病毒的單株抗體；其中有十四株抗體是辨識套模蛋白，兩株是辨識非結構性蛋白一。在辨識套模蛋白中，有七株單株抗體是辨識區塊一、二，另有兩株抗體能辨識區塊三。最後找到兩株能辨識登革第四型功能區塊一、二，並在細胞實驗上能阻止病毒感染，但只有一株抗體能在動物模式上提供保護。同時我們製造的DNA疫苗，雖然能夠表現其蛋白質於細胞且被抗體所辨識，但是在誘發對抗登革病毒免疫的能力上，仍不及pCB8D4-2J；顯示該DNA疫苗需多次免疫方能有效誘導出對抗登革病毒的抗體。

Dengue virus (DENV), the human pathogen leading to dengue fever (DF), dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), is epidemic in tropical and subtropical areas around the world. The envelope protein (E) of DENV, which could induce protective immunity, is critical for membrane fusion and mediates binding to cellular receptors. The ectodomain of the E monomer could further be divided into three domains assigned to domain I, domain II and domain III (EDI, EDII and EDIII). In order to confirm the target of neutralizing antibodies and develop the DNA vaccines of DENV, we generated a large panel of MAbs against DENV4 in our laboratory. In our study, we further these MAbs against DENV4. At first, we use the immunofluorescence and Western blotting to identify the specificity of those MAbs. Several MAbs could recognize the envelop protein (E protein) or non-structural protein 1 (NS-1). We further demonstrated their neutralizing activity of DENV4 by plaque reduction neutralization test (PRNT) and protection assay. We have identified 9 serotype-specific MAbs and 9 cross-reactive MAbs against DENV4, and 14 MAbs could recognize E protein and 2 MAbs recognized NS-1 protein. Among these MAb recognized E protein, seven MAbs could recognize EDI-II and 2 MAbs recognized EDIII. Finally, we identified 2 MAbs, which recognized EDI-II of DENV4, displayed neutralizing activity in vitro, but only one MAb could reveal protective activity in vivo. We also constructed two DNA vectors which contained the EDI-II and EDIII of DENV4, and immunized the mice for identification the neutralizing antibodies. We used the DNA vaccine of DENV4, pCB8D4-2J, to immunize the mice to determine the neutralizing avtivity of EDI-II or EDIII DNA vaccine by PRNT and protection assay for imitating immune responses against DENV4 in vitro and in vivo. Although the two DNA vaccines, EDI-II and EDIII, could be detected the protein by IFA, their humoral immunity against DENV4 still weaker than pCB8D4-2J. It might result from less immunization, so we need to immunize mice more times.

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