治療性H7N9禽流感病毒人源中和抗體之製作與驗證

Abstract

H7N9新型禽流感病毒在2013年春天自中國爆發感染人類病例，並向鄰國蔓延，包括台灣。截至2015年6月為止，總感染人數累積高達653人，其中227人死亡，死亡率為37%。H7N9屬於A型流感病毒，為RNA單股病毒，包含了8個基因，可轉譯出11種病毒蛋白，其中包括了表現在病毒膜上的兩種主要蛋白，即血球凝集素(hemagglutinin)以及神經氨酸酶(neuraminidase)。根據這兩種蛋白的基因序列及其抗原特型不同，可將A型流感病毒進一步分成兩大類，而H7N9病毒屬於第二群。目前對於H7N9的治療方式，仍是在感染初期給予神經氨酸酶抑制劑。而季節性流感疫苗所產生的中和性抗體，對於H7N9病毒的中和效果相當有限。此外，病人感染H7N9後病情惡化相當迅速，因此需要更有效率的治療方式。目前針對流感病毒最有效且最有潛力的治療方法之一便是人類治療性中和抗體，不僅可以單獨使用，亦可與神經氨酸酶抑制劑併用來達到加成性治療效果，這在感染後尚未產生H7N9病毒中和抗體前，尤其重要。 在2015年4月9日，一名感染H7N9之53歲台灣男性病患，從蘇州返回台灣之後，出現呼吸困難以及發燒的感染症狀。於4月20日到台大醫院就醫並住院接受治療之後康復出院。台大在與生物技術開發中心合作之下，我們分離此病人血液中的記憶B淋巴球和漿細胞，克隆它們的抗體基因和表達純化抗體。在約80支克隆的H7N9單株抗體中，有三支對於H7N9病毒有顯著的中和能力，且各有一支分別對H1N1及H3N2有交叉中和效果。因此，我的研究目地是針對這三支抗體的作用和抗原決定位點鑑定做了進一步的分析，以及建立偽病毒系統進行功能性探討。我們首先克隆和表達全長以及三聚體的H7，轉殖細胞後用流式細胞儀及免疫螢光染色來檢測抗體辨認程度。也表達和純化H7全長、片段和三聚體重組蛋白供酵素免疫分析法來判斷抗體對H7的抗原辨認位置。另外，建立抗體中和病毒類顆粒試驗，來鑑定抗體廣效中和能力，並希望能應用到動物實驗。總結，我們利用單B淋巴球抗體基因克隆方式成功篩選到H7中和抗體，可供做流感病毒治療性中和抗體。這種方法也可應用於其他種類病毒，透過給予抗體被動免疫的方式，提高防疫效果。

H7N9 is an emerging avian influenza A virus that can infect humans. As of June, a total of 653 human infections, resulting in 227 deaths, have occurred since March 2013. Influenza A virus is a negative-sense, single-stranded, segmented RNA virus, of which genome encodes 11 viral proteins, including hemagglutinin (HA) and neuraminidase (NA). The influenza A viruses can be further divided into two groups based on their genetic and antigenic differences; accordingly H7N9 belongs to the group 2. As other influenza viruses current recommended anti-H7N9 therapy relies on early treatment with NA inhibitors. In spite of prior seasonal flu vaccination the neutralizing antibodies (Abs) elicited in people offer little cross-protection against H7N9. Moreover, the often fast and deleterious clinical course of patients warrants more effective therapeutics against H7N9. One potentially effective therapy is to utilize therapeutic human neutralizing Abs, which can not only synergize the efficacy of NA inhibitors but surrogate the needed humoral immunity during the window when the host has not yet developed anti-influenza neutralizing Abs. For this, in collaboration with the Development Center for Biotechnology we have generated human anti-H7N9 neutralizing Abs by single B-cell cloning of Ab genes from the circulating plasmablasts of an infected 53-year-old male. He returned from Suchow, Jiangsu Province on April 9 in 2013 and later admitted to NTUH on April 20 due to progressive dyspnea for four days and prior fever for three days. So for we have cloned some 80 human monoclonal Abs, at least three of which showed significant neutralization specific to H7N9 as well as cross-reactive to H1N1 and H3N2 viruses, respectively. The reactivity of these neutralizing Abs was further investigated using full-length and trimeric H7-transfected cells by flow cytometry and immunofluorescence. To identify the residues where these human neutralizing Abs recognized on HA, we performed epitope mapping using the full-length and domain-specific recombinant proteins of H7 by ELISA. Moreover, we developed a WSN (A/WSN/1933) strain-based pseudovirus and reassortant assay to examine the functionality of strain-specific vs. broadly neutralizing H7 mAb using cytopathic plaque detection and reporter gene activity as readouts. Future directions include Ab engineering of H7 mAb for optimization and in vivo experiments for evaluation of Ab efficacy. In summary, our results demonstrate a feasible approach to develop an effective therapeutic against the emerging zoonotic influenza virus and perhaps other viruses through passive immunity.