| 摘要: | 寨卡病毒 (ZKV) 是一種新出現的病毒，已在全球範圍內引起疫情。該病毒與兒科先天性神經障礙和成人吉蘭-巴雷綜合徵有關。另一方面，柯薩奇病毒 A16 (CA16) 是手足口病 (HFMD) 的主要病原體之一。全球 5 歲以下兒童受 CA16 HFMD 影響最大。儘管 CA16 感染的臨床症狀通常是輕微的，但已記錄到嚴重的並發症，例如無菌性腦膜炎甚至死亡。目前，沒有有效的疫苗或抗病毒藥物可用。因此，迫切需要 ZKV 和 CA16 治療。在這項研究中，單鏈可變片段 (scFv) 是使用噬菌體展示技術開發的，該技術針對 ZKV 包膜 (ZKVE) 蛋白和 CA16 感染的裂解物中的蛋白質，包括遺傳相關的腸道病毒 71 (EV71)。在白色母雞中檢測到針對聯合截斷 ZKVE 蛋白（ZKVEF3-146 和 ZKVEF2-251）和 CA16 感染裂解物中的蛋白的體液免疫反應。給雞接種疫苗並從蛋黃中分離出多克隆 IgY 抗體。在第二次、第三次和第五次免疫後，使用 ELISA 檢測到高水平的抗 ZKVE 和抗 CA16 IgY 抗體。 CA16 感染裂解物中截短的 ZKVE 蛋白或蛋白的滴度持續了近 9 或 10 週。針對 ZKVE 蛋白構建了兩個抗體文庫，針對 CA16 感染的裂解物構建了另外兩個，分別包含 5.3 × 106 或 4.5 × 106 和 4.0 × 106 或 5.0 × 106 轉化體。經過四或五輪淘選後，篩選出一種有效的 scFv 抗體，該抗體對截短的 ZKVE 蛋白和 ELISA 板上 CA16 感染的裂解物中的蛋白具有良好的結合活性。我們隨機篩選了表達 ZKVE scFv 抗體的 26 個克隆和顯示 CA16 scFv 抗體的 13 個克隆，並將它們分為兩組，即短接頭和長接頭。其中，四個表現出對 ZKVE 蛋白的特異性結合能力，一個克隆靶向 CA16 感染的裂解物中的病毒蛋白。此外，篩選出的抗 CA16 scFv 克隆顯示出對 CA16 病毒的中和能力，並與 EV71 感染的裂解物中的蛋白質發生交叉反應。有趣的是，多克隆-IgY 不僅表現出對 CA16 感染裂解物中蛋白質的特異性結合，而且還表現出顯著的中和活性。然而，抗 CA16 IgY 不與 EV71 感染的裂解物中的蛋白質發生交叉反應。這些數據表明，多克隆和單克隆 scFv 抗體具有治療 ZKV 的診斷或治療潛力，並且還可以在體外試驗中提供針對 CA16 病毒感染的保護；因此，這些抗體可能是治療脆弱幼兒 CA16 感染的潛在候選者。
Zika virus (ZKV) is a newly emerging virus that has caused worldwide outbreaks. The virus has been associated to congenital neural disorders in pediatrics and to Guillain–Barré syndrome in adults. On the other hand, Coxsackievirus A16 (CA16) is one of the major etiological agents of hand, foot, and mouth disease (HFMD). Children aged less than 5 years are the most affected by CA16 HFMD globally. Although clinical symptoms of CA16 infections are usually mild, severe complications, such as aseptic meningitis or even death, have been recorded. Currently, no effective vaccines or antiviral drugs are available. As a result, an urgent need for ZKV and CA16 treatments exists. In this study, single chain variable fragment (scFv) was developed using a phage display technology, which targets the ZKV envelope (ZKVE) protein and proteins in the CA16-infected lysate, including genetically-related enterovirus 71 (EV71). The humoral immune response in white female chickens was detected against the combined truncated ZKVE proteins (ZKVEF3-146 and ZKVEF2-251) and proteins in CA16-infected lysate. Chickens were vaccinated and polyclonal-IgY antibodies were isolated from yolks. High-level titers of anti-ZKVE and anti-CA16 IgY antibodies were detected using an ELISA after the second, third, and fifth immunizations. The titers for truncated ZKVE proteins or proteins in the CA16-infected lysate persisted for almost 9 or 10 weeks. Two antibody libraries were constructed against ZKVE proteins and another two against CA16-infected lysate that contained 5.3 × 106 or 4.5 × 106 and 4.0 × 106 or 5.0 × 106 transformants, respectively. After selection with four or five cycles of panning, an effective scFv antibody showing favorable binding activity to truncated ZKVE proteins and proteins in the CA16-infected lysate on ELISA plates was screened. We randomly screened 26 clones that expressed ZKVE scFv antibodies and 13 clones displaying CA16 scFv antibodies and classified them into two groups, namely short and long linkers. Of these, four showed specific binding abilities toward ZKVE proteins, and one clone targeted viral proteins in the CA16-infected lysate. Additionally, the screened anti-CA16 scFv clone showed a neutralizing capability against the CA16 virus and cross-reacted with proteins in EV71-infected lysate. Interestingly, polyclonal-IgY not only showed specific binding against proteins in the CA16-infected lysate but also showed significant neutralizing activity. However, anti-CA16 IgY did not cross-react with proteins in the EV71-infected lysate. These data suggest that the polyclonal and monoclonal scFv antibodies have diagnostic or therapeutic potential for treating ZKV and also can provide protection against CA16 viral infection in in vitro assays; thus, these antibodies may be potential candidates for treating CA16 infection in vulnerable young children. |
