Identification of B-cell Epitopes for Monoclonal ntibodies against Dengue Virus

引起登革熱、登革出血熱、登革休克症候群的登革病毒為斑蚊所媒介傳染的人類病原體，此病毒在分類上具有四種血清並且盛行於熱帶和亞熱帶地區。全球每年粗估約有五千萬到一億的感染案例，其中約百分之ㄧ的病人甚至會再進一步惡化成登革出血熱的症狀。登革病毒之套膜蛋白是參與並且誘發宿主免疫反應的重要抗原，而且此蛋白對於病毒自身和宿主進行膜融合以及細胞表面受體的辨認扮演重要角色。套膜蛋白之膜外區域可以再進一步區分為三個功能區塊：區塊一、區塊二、區塊三。根據過往及我們實驗室關於抗原決定位的研究指出，若干能對抗黃病毒家族之專一暨中和性抗體大多數偏好辨認套膜蛋白上的區塊三，而此區塊的胺基酸替換會造成這類單株抗體失去了結合能力；除此之外，對於那些中和能力稍弱並且對於其他種類黃病毒有交叉反應的單株抗體則多會結合到套膜蛋白的區塊二。另一方面，許多過往研究也指出人類宿主血清裡倘若有弱中和性的抗體之存在似乎會惡化二次感染之登革熱病人其病情，此種機制已被稱為「抗體依賴性免疫加強反應」假說。而對於登革病毒套膜蛋白上之功能區塊在病理發生機制所扮演的角色仍然未被清楚定位。因此我們篩選了若干株由我們實驗室所生產的抗體並藉此來釐清功能區塊和中和性抗體之間的交互作用以冀望最終能改善未來對於此疾病之預防及治療方法。在本篇研究中，我們利用了酵素聯結免疫吸附測試、西方墨點法、套膜蛋白質表現、定量式反轉錄聚合酶連鎖反應等方法來分析弱中和性、叉反應性和專一暨中和性單株抗體與其所辨認的功能區塊之關聯性。此外，我們發現偏好辨認區塊三的抗體常對於登革病毒會具備強中和能力，而偏好辨認區塊一和區塊二的抗體則具弱中和能力或是不具有中和能力，更甚者還提高了病毒對於宿主的感染率。Dengue virus (DEN), the human pathogen leading to dengue fever (DF), is epidemic in tropical and subtropical areas around the world. The DEN envelope protein (E), the primary viral protein inducing 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) (Modis et al., 2003). According to previous and our laboratory’s epitope mapping studies, many type-specific neutralizing antibodies against individual flaviviruses were localized to EDIII and alteration of specific residues in EDIII contributed to the loss of binding of neutralizing monoclonal antibodies (MAbs). Furthermore, subneutralizing MAbs that cross-react with other flaviviruses mainly bound to EDI-II. On the other hand, the presence of subneutralizing DEN cross-reactive serum antibodies in human hosts seems to cause an increase in the severity of secondary DEN infections via antibody-dependent enhancement (ADE). he roles of B-cell epitopes on the EDs in DEN pathogenesis, however, are still blurred. Accordingly, we screened a panel of MAbs from our laboratory to define the interaction between EDs and cross-reactive and neutralizing MAbs. Identification of neutralizing and pathologic epitopes will be useful for improving therapeutics to these diseases. In this study, we identified a number of cross-reactive and type-specific MAbs with their recognized EDs through ELISA, western blot analysis and quantitative real-time PCR (QRT-PCR). Additionally, we found that MAbs which recognized EDIII usually displayed strong neutralizing activity while MAbs which bound to EDI-II were likely to cause sub-/non-neutralizing or enhancing effects on DEN infections frequently.Ackowledgements……………………………………………1文摘要 ……………………………………………………3bstract ……………………………………………………4ntroduction……………………………………………………6aterials and Methods…………………………………………18esults ……………………………………………………24iscussion……………………………………………………29igures ……………………………………………………35ables ……………………………………………………48eferences……………………………………………………53ppendixes……………………………………………………6