生產與評估H7N9禽流感病毒血球凝集素單株抗體與多株抗體的特性

Generating and characterizing of mono- and polyclonal antibodies against hemagglutinin of avian H7N9 influenza A virus

洪浥瑋(Yi-Wei Hong)

指導教授 ： 王聖帆

高雄醫學大學/健康科學院/醫學檢驗生物技術學系博士班/碩士(2017年)

摘要

自2013年起於中國沿海地帶的城市傳出人類感染一種新型的基因重配(Gene reassortment) A 型H7N9禽流感病毒，該病毒的致死率高(約18-25%)。流感病毒感染方式主要利用其套膜蛋白上的血球凝集素(Hemagglutinin;HA)與宿主細胞表面的醣受體包括α-2,3 或α-2,6 linked sialic acid。因此，血球凝集素(Hemagglutinin)被視為抗病毒藥物發展的重要標的。本研究之目的欲開發生產anti-HA7的單株與多株抗體並鑑定抗體的特性以及應用於H7N9病毒之偵測。抗原方面，由於HA屬於醣基化的蛋白，所以我們以A/Shanghai/2/2013(H7N9)病毒株的HA基因序列，採用昆蟲桿狀系統(Baculovirus；簡稱Bac)來產生的HA7重組蛋白(HA7 recombinant proetein; rHA7)，包括: (a)表現HA7的桿狀病毒(HA7-Bac)、(b)純化過的HA7單體(Monomer- rHA7，簡稱Mono- rHA7)與(c) 經過純化的HA7三聚體(Trimer- rHA7，簡稱Tri-rHA7)蛋白。以這些蛋白來免疫老鼠及兔子產生單株與多株抗體，並比較這些抗原引起中和性抗體能力的差異性。我們使用反向遺傳學技術(Reverse-genetics；簡稱RG)生產出H7N9-RG病毒以及利用轉染(Transfection)的方式將pNL-Luc-E−R− 與pHA7及pNA9 質體送入HEK293T 細胞來生產H7N9假病毒(H7N9 pseudotyped virus)，並且利用穿透式點子顯微鏡觀察H7N9假病毒其結構與臨床分離株相似度。利用血球凝集試驗(Hemagglutination;HA)證明這些病毒具有功能性，能凝集火雞的紅血球(RBC)。我們也評估不同的rHA7抗原的特性，發現HA7-Bac在不含佐劑的免疫情況下就能以發中和性抗體的產生；此外，這三種不同的HA7抗原在含有佐劑的免疫條件下均能引發較高的中和性抗體，其中以三聚體的rHA7的抗原性最好。接著，利用融合瘤技術與限制性稀釋法來製備單株抗體，並以血球凝集抑制試驗(Hemagglutination inhibition; HI)用於評估抗體中和病毒的能力。本研究總共篩選出6個具有病毒中和性的單株抗體。利用噬菌體呈現技術(Phage display)鑑定出單株抗體之抗原決定位，結果發現有5個單株抗體(1-7C、2-2A、2-9D、2-12H、6-4D)辨識到相同的HA7上的抗原決定位-78IERS84；另一個單株抗體5-5B之抗原決定位為- 186YSL192。這些抗體之決定位都分佈於HA1(H7)球狀區域。從H7N9禽流感病毒的流行株之HA7胺基酸序列比對發現，這兩個抗體所辨識的中和性抗原決定位點均專一性的表現於HA7。最後，我們利用所產生的單株與多株抗體來建構antigen-captured ELISA來偵測H7N9禽流感病毒。結果發現，其偵測靈敏度為7.8ng/mL，而且能夠專一性的偵測出不同序列稀釋下的H7N9-RG 或H7N9 pesudotyped virus，不會辨識到其它流感病毒包括:H1N1、 H3N2與B型流感病毒。
本研究結果顯示昆蟲桿狀系統產生出的rHA7蛋白能引發中和性抗體的產生；我們也篩選出6個單株抗體並標定到2個重要的中和性抗原決定位，而這些抗體亦可運用於H7N9禽流感病毒之偵測。這些資訊能提供未來對於H7N9禽流感病毒的免疫治療、臨床診斷與抗病毒藥物發展的標的。

關鍵字

H7N9禽流感單株抗體 ； 多株抗體 ； 昆蟲桿狀病毒表現系統 ； 捕捉病毒抗原酵素免疫分析法

並列摘要

Numerous outbreaks of human infection from gene reassorted avian influenza virus H7N9 have been reported in the coastal areas of China since 2013. The death rate of the virus is about 18-25%. Influenza virus infection is initiated via interacting its envelope hemagglutinin (HA) with the α-2,3 or α-2,6 linked sialic acid receptors on host cell. Accordingly, HA of influenza virus is an important candidate for the development of antiviral drugs. The aim of this study is to develop mono- and polyclonal antibodies against HA7 and futher to characterize their properies and applications. Since HA is a glycosylated protein, we used the HA sequence of the A / Shanghai / 2/2013 (H7N9) strain to produce recombinant HA7 proeteins (rHA7) including (a) baculovirus surface expression of HA7 (recombinant baculovirus, HA7-Bac) (b) purified monomer (Monomer-HA7, referred to as Mono-rHA7), and (c) trimer (Trimer-rHA7, referred to as Tri-rHA7) protein through baculovirus system. These proteins were used as antigens to immunize mice and rabbits and to produce mono- and polyclonal antibodies as well as comparing thier immunogenicity for inducing neutrazling antibodies.
We firstly produced H7N9-RG reassotant strain containg A/PR/8/34(H1N1) internal six gene fragments with HA7 and NA9 envelope and psedutyped virus by transfecting pNL-Luc-E-R- with pHA7 and pNA9 plasmids into HEK293T cells. The transmission electron microscopy (TEM) observation found that its structure is similar with the H7N9 influenza clinical isolates. We conducted hemagglutination test (HA) to confirm the hemagglutination ability of H7N9 pseudotyped virus using 0.5% turkey red blood cells (RBC). Through the evaluation of viral neutralzing antigenicity of different rHA7, we found that all three rHA7 antigens can efficiently trigger the production of neutralizing antibodies, among which HA7 trimer is the best, and that HA7-Bac is able to facilitate the production even without adjuvanted immunity. We therefore used trimeric rHA7 to immunize mice and to generate monoclonal antibodies using the hybridoma tech and the limited dilution assay. Total six monoclonal antibodies with virus neutralization abiltiy were screened. The antigenic determinants of the monoclonal antibodies were identified using phage display. Five monoclonal antibodies (1-7C, 2-2A, 2-9D, 2-12H, and 6-4D) recognized same linear epitope-"IERS" at amino acid position 78,79, 80 and 84. Only monoclonal antibody 5-5B recognized a conformational epitope-"YSL" at amino acid position 186,188 and 192. These antibody recognition domains were located in HA1 (H7) globular region. Sequence alignment indicates that these two eitopes were highly conserved in HA7 compared with recent human influenza A virus. Futher, the antigen-captured ELISA was established using our generated mono- and polyclonal antibodies to detect H7N9-RG virus. The detection sensitivity is 7.8ng/mL. Other human influenza viruses including H1N1, H3N2 and B virus were not detected.
In this study we genetaed six monoclonal clonal antibodies through immunizing rHA7 from baculovirus-insect based system. We charaterized their properties and identified two novel neutralizing epitopes specific on HA of ciculating H7N9 influenza viruses. These information is valuable for the future of H7N9 avian influenza virus immunotherapy, clinical diagnosis, and antiviral drug developmentor.

並列關鍵字

Avian influenza A H7N9 ； Monoclonal ； Polyclonal ； Baculovirus-insect system ； Antigen captured ELISA

參考文獻

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