近年來，隨著全球暖化趨勢，登革熱成為全球重要傳染病之一。登革病人會出現許多症狀，輕微者出現紅疹和發燒等病癥，嚴重者可能會有危及生命的登革出血熱。登革是由登革病毒 (dengue virus) 所引起之症狀，登革病毒屬於黃熱病毒科，黃熱病毒屬。已知登革病毒的非結構性蛋白1 (NS1) 會引起促發炎細胞激素與血管通透性的增加，造成血管滲漏。宿主也會因NS1的分子擬態理論 (molecular mimicry) 導致內皮細胞、血小板受損以及功能不良。至今臨床上針對登革患者仍缺乏理想的抗登革病毒藥物。然而，越來越多研究開始重視治療型抗體 (therapeutic antibody)，例如抗NS1的抗體可以維持內皮細胞完整性及透過誘導補體介導的細胞毒殺作用 (CDC) 殺死被登革病毒感染的細胞。我們研究團隊先前篩選得到不會辨識至具交叉反應的抗原決定位，但皆能夠在體內及體外試驗提供對抗四型登革病毒感染保護力的鼠源單株抗體2E8及33D2。在小鼠動物模式中也證實被動給予鼠源單株抗體2E8和33D2，在不同血清型登革病毒感染情況下，都能提供治療效果。為了把過去的研究成果應用至臨床前試驗甚至臨床上，我們已針對降低原鼠源抗體的免疫原性 (immunogenicity)，完成抗體的人源化。首先，我們利用酵素免疫分析法、分光光度法以及膠體電泳分析，發現人源化單株抗體2E8和33D2在人體體溫攝氏37度中都非常穩定。接著，我們進一步證實對抗NS1的人源化抗體，並無抗體依賴性免疫加強反應 (ADE) 存在的風險。除此之外，我們也發現它們對於目標抗原NS1的結合能力不遜於鼠源抗體，亦能辨認在分別感染四種血清型登革病毒的細胞表面上所呈現的NS1。確認抗體能辨認細胞表面的NS1後，我們進行CDC試驗發現，人源化單株抗體2E8和33D2都能誘導CDC對抗登革病毒感染。為了評估人源化單株抗體的治療效果，我們使用可以表現出血症狀的登革病毒感染動物模式，並證實在感染後一天施打較高劑量人源化抗體2E8與33D2都能減緩包含局部皮下出血以及出血時間延長等登革病徵，而低劑量的人源化抗體33D2亦能提供相同的治療功效。總結，在這個研究中，我們證實抗NS1的人源化單株抗體在登革病毒感染下亦能提供良好的治療效果，且具有發展成治療性抗體藥物的發展潛力。

Recently, along with global warming, dengue becomes major threat to global public health. Dengue virus (DENV) belongs to the genus Flavivirus, family Flaviviridae and causes many symptoms including life-threatening dengue hemorrhagic fever. It has been known that DENV nonstructural protein 1 (NS1) could induce elevation of proinflammatory cytokines and vascular leakage. In addition, DENV NS1 may result in several responses such as endothelial cell damage and platelet dysfunction due to molecular mimicry. To date, there is no specific treatment for DENV infection; however, more and more studies have shown that therapeutic antibody (Ab) such as anti-DENV NS1 Ab can block the disruption of endothelial integrity and trigger complement-dependent cytotoxicity (CDC) to kill DENV-infected cells in vitro. Our previous studies demonstrated that anti-NS1 monoclonal antibody (mAb) can bind to DENV NS1 expressed on cell surface and induce CDC. Besides, passive transfer of mouse anti-NS1 mAbs 2E8 and 33D2 protected mice from all four serotypes of DENV challenge. However, the antigenicity of murine Ab causes a concern in human use. The anti-NS1 humanized mAbs, 2E8 and 33D2, are therefore generated and examined. Our results showed that the humanized mAbs have similar binding ability to NS1 as that of mouse mAbs and can recognize NS1 on the surface of cells infected by four different serotypes of DENV, respectively. Because these mAbs would be inoculated into human body, the stability of humanized mAbs at 37°C was analyzed by ELISA, spectrophotometer (Nanodrop 2000), and SDS-PAGE. The data indicated that the humanized Abs all have good stability. Furthermore, we confirmed that these two humanized mAbs do not have risk of Ab-dependent enhancement. After determining the surface NS1 binding activity, we examined whether anti-NS1 humanized mAbs cause a similar effect as murine mAbs, and the data showed that both humanized mAb 2E8 and 33D2 can elicit CDC of DENV (1-4)-infected cells. Importantly, we also evaluated the therapeutic efficacy attributed by humanized mAbs against DENV infection in vivo. Transfer of humanized mAbs 2E8 or 33D2 24 h after DENV infection both demonstrated therapeutic effects in STAT1-/- mice. Humanized mAb 33D2 has better therapeutic efficacy than mAb 2E8 since the in vivo results showed that lower-dose administration of mAb 33D2 can reduce DENV-induced skin hemorrhage and prolonged bleeding time. Collectively, the results from this study reveal a lead candidate of humanized anti-NS1 mAb that can act as a potential agent for anti-DENV therapy in the future.

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