腸病毒A71 型(EV-A71)屬於微小病毒科中的腸病毒屬，為一神經趨性的病毒，易於嬰幼兒產生感染症狀，其症狀從輕微至嚴重皆有可能發生，包括常見的手足口症、疱疹性咽峽炎、無菌性腦膜炎至嚴重的腦幹腦炎、肺水腫、肺出血或是其他嚴重神經相關病症及死亡等。雖然有幾件疫苗已經開發並完成臨床測試，但截至目前為止，仍未有疫苗上市後之評估報告。除此之外，目前也尚未有一可針對腸病毒A71 型專一性的抗病毒藥物，可以有效的抑制病毒感染。因此於本篇研究中，則是希望可以透過抗體篩選資料庫平台，找出一抗病毒抗體，可以做為治療並避免發展成嚴重神經相關疾病。我們藉由具有較高抗腸病毒A71型抗體效價的自願者之周圍單核球細胞遺傳物質去氧核醣核酸(Deoxyribonucleic acid, DNA)，放大其抗體重鏈(heavy chain)及輕鏈(light chain)之可變區域(variable region)之基因序列，成功構築出人源單鏈抗體資料庫。並將此資料庫系統表現於哺乳類細胞株中，並藉由致死劑量之病毒感染，從存活的細胞中找到並放大出其表現的單鏈抗體基因。於本研究中，我們篩選出一候選單鏈抗體株scFv07，有表現此單鏈抗體之細胞可抑制EV-A71病毒之感染，使其病毒抗原表現量顯著降低。此外，我們亦將此單鏈抗體再透過基因工程重組成完整抗體，並發現具此抗體表現之細胞，能大幅降低病毒感染所造成之細胞凋亡。進一步，我們更發現宿主蛋白α-烯醇酶(α-enolase)為其專一標的，且於腸病毒A71型感染病患之血清中發現抗α-烯醇酶自體抗體之存在。最後，於動物實驗中，發現在腦部組織之核酸放大及染色結果顯示，此一專一性抗體可降低病毒RNA以及神經相關之病理現象，並且於此動物感染模式中，提升了小鼠存活率。總結於此研究中，我們建構了一於細胞內進行表現及透過感染模式直接篩選抗病毒單鏈抗體之人源資料庫，並透過此平台篩選出可有效抑制腸病毒A71型感染之重要候選者Clone 07。透過候選者之特性分析，探討其抗病毒機制或功能上所扮演的角色，未來期將可以應用於臨床治療上，有效抑制病毒感染，避免感染者發展成致命或不可復原的神經破壞相關疾病。

Enterovirus A71 (EV-A71) belongs to Enterovirus genus in Picornaviridae family. It is a neurotropic virus which may cause mild to severe neurological complications, especially in infant and children. The clinical manifestations include hand-foot-and-mouth disease, herpangina, aseptic meningitis, brain stem encephalitis, pulmonary edema/hemorrhage, and other severe neurological diseases. Although there are some clinical vaccine trials undergoing and approved, the postmarketing surveillance is still unavailable. Besides, there is also no antiviral drugs against EV-A71. Therefore, in this study, we aim to find the antiviral agent or strategy for treatment, preventing the disease progression to severe neurological complications. We successfully constructed a human single chain variable fragment (scFv) library expressed in mammalian cells and found the scFv expressed in survival cells after infection with lethal dose of viruses. After panning and screening we found one clone (clone07) which can inhibit the replication of EV-A71 when expressed intracellularly before infection in contrast to non-scFv expressed cells. We analyzed the sequence of complementarity-determining regions (CDRs) in variable region of heavy chain (VH) and light chain (VL), and expressed the scFv (scFv07) and recombinant intact antibody (07-human IgG1) to confirm the ability of viral inhibition. We identified that the host protein α-enolase (ENO1) is the target of our candidate antibody through immunoprecipitation assay, and ENO1 autoantibody can also be found in EV-A71 patient sera. Furthermore, we examined the antiviral effects in vivo. We found that 07-human IgG1 treatment significantly decreased the viral RNA and the level of neural pathology in brain tissue. In mouse model, it can also increase the survival rate after virus challenge. Collectively, through a promising intracellular scFv library expression and screening system, we found a potential scFv/antibody sequence which targets host protein and can inhibit the infection of EV-A71. The results indicate that the usage and application of this antibody may offer a potential treatment against EV-A71 infection.

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