探討針對A型流感H7病毒之治療性單株抗體

Abstract

2013年於中國爆發的A型流感病毒H7N9由禽鳥類流感病毒重組生成，至今引起了五波大流行。在2017年的第五波爆發中出現了高致病性的病毒，死亡率高達50%。臨床上常用神經胺酸酶(neuraminidase, NA)抑制劑來治療流感病毒感染，但在高致病性的H7N9病毒中，發現了許多抗神經胺酸酶抑制劑的抗藥性突變，如NA上R292K的突變，因此必須研發新型的治療方式。針對流感病毒顆粒表面血球凝集蛋白(hemagglutinin, HA)的抗體，被認為可以抑制病毒進入宿主細胞的步驟，達到中和病毒感染力的效果。本篇研究目的為針對十支可中和2013 H7N9病毒的治療性抗體進行功能以及結合位點的探討。我們依據抗體的重鏈及輕鏈序列將抗體分成五組：A組包含CL-163099, CL-163110；B組包含CL-163084, CL-163118, CL-163121；C組包含CL-163095, CL-163098；D組包含CL-163109, CL-163219，E組為CL-163117。首先以微量中和試驗(Micro-neutralization test)初步測試十支抗體對於2013H7N9的中和力，IC50 範圍在0.1-0.3 μg/mL。另外也以免疫螢光染色(Immunofluorescence assay)的方式確認抗體可以結合2013H7N9的HA。接著針對抗體廣效性進行測試，發現十支抗體皆可結合2017H7N9, H7N3, H7N7的HA，也可中和2017年高致病性的H7N9重組病毒。進一步以溶菌斑減少中和試驗，確認在感染細胞前加入抗體，或是感染後才加入抗體，抗H7抗體皆可有效抑制病毒複製。為了找出抗體的結合位點，我們將抗體CL-163099與病毒共培養，A組第一代(抗體濃度60 ng/mL)在感染後48小時，病毒大量生成，第二代培養我們將抗體量提升至50 μg/mL，病毒仍能生長，我們將突變病毒的HA定序，發現病毒帶有G151E突變。而在B組，我們在第一代時看到了A156A/V突變，第二代(1 μg/mL)有R149K及A156V，並在第三代(5 μg/mL)及第四代(20 μg/mL)都帶有這兩個突變點，而在第五代我們將抗體量提升至50 μg/mL，病毒出現了del151-153+A156V突變。在HA的結構上，我們發現突變點集中在Antigenic site A 及Receptor binding site 140 loop上，這可能就是CL-163099的結合位。我們以定位點突變試驗合成帶有突變的HA，以免疫螢光染色及流式細胞儀測試是否會影響十支抗體的結合力。A組、B組及E組的抗體對於帶有R149K的HA失去結合力；帶有G151E的HA只有B組的三隻抗體可以結合；只有A組的抗體失去帶有A156V的HA的結合力；只有CL-163098仍可結合帶有兩個突變點R149K+A156V的HA；而所有抗體都無法結合上帶有del151-153或是del151+153+A156V的HA。接著我們製造了帶有突變點的重組病毒，測試其生長速度，分別帶有R149K、G151E及A156V的重組病毒生長曲線較平緩，低於野生株。帶有R149K+A156V的重組病毒於感染後24小時的病毒量高於野生株，帶有del151-153及del151-153+A156V的重組病毒於感染後0小時至36小時的病毒量都高於野生株，並於感染後36小時達複製高峰。最後測試了帶有突變的重組病毒是否影響抗體的中和效果，以50 ng/mL的濃度進行實驗，十支抗體可接近100%的抑制野生株的重組病毒。對於帶有突變點的重組病毒，前述實驗測定會影響結合力者，大部分皆失去或降低中和力。本篇研究所探討之十支抗H7抗體，可廣效結合2013H7N9, 2017H7N9, H7N3及H7N7，並且對於2013H7N9及2017H7N9有良好的中和效果。另外也透過突變病毒篩選，找到可掙脫抗體的突變點，並製造帶有突變的HA及重組病毒，以反向驗證不同的突變點對於不同組別抗體的影響。經過序列分析，發現此組抗體結合位在H7病毒具有高度保留性，顯示抗體結合處為H7病毒重要的抗原位，也因此我們的抗體可以有很好的中和效果，預期對於大部分的H7病毒都可以有保護力。

Novel ressortant influenza A virus H7N9, first detected in China in 2013, has caused five waves of outbreaks in human. Highly pathogenic avian influenza (HPAI) H7N9 with a multibasic cleavage site has been detected in the fifth wave and caused about 50% morality in infected patients. Neuraminidase inhibitors have been used to treat influenza virus infection but resistant virus has been reported, including HPAI H7N9. Therefore, it is nesseray to develop new strategy for H7N9 treatment. Among these neutralizing antibodies against influenza HA have been reported to block virus entry, fusion, or relase. In this study, function of ten anti-H7 antibodies have been characterized and the epitopes of anti-H7 antibodies have been determined. According to genetic features, we classified the ten anti-H7 antibodies into 5 groups, with Group A (CL-163099 and CL-163110), Group B (CL-163084, CL-163118, CL-163121), Group C (CL-163095, CL-163098), Group D (CL-163109, CL-163219), and Group E (CL-163117). Neutralizing activity of anti-H7 antibodies determined by micro neutralization test were in the range between 0.1-0.3 μg/mL. Anti-H7 antibodies can recognize 2013H7N9 HA, 2017H7N9 HA, H7N3 HA, and H7N7 HA. HPAI r2017H7N9 virus could be neutralized by anti-H7 antibodies. To determine the epitopes of these anti-H7 antibodies, escape mutants were selected by co-culture of virus and antibodies (CL-163099). Different mutants were observed in the duplicated experiments. G151E was detected in line I-A, while A156V, R149K+A156V were detected in line I-B. Del151-153+A156V were observed in the 5th passage with 50 μg/mL of CL-163099. To confirm whether the amino acid substitutions contribute to escape from anti-H7 antibodies, site-directed mutagenesis was performed in 2013H7N9-HA. Immunofluorescence assay and flow cytometry were performed to determine antibody binding ability. Group A, B, and E antibodies lost binding ability to 2013H7-HA with R149K mutation. Only Group B antibodies could bind 2013H7-HA with G151E mutation. A156V substitution weakly reduced the binding ability of Group A antibody. Only CL-163098 could bind 2013H7-HA with R149K+A156V. All antibodies can not recognize 2013H7-HA with del151-153 or del151-153+A156V. Furthermore, recombinant viruses with various mutations have been genetrated. The virus replication titers of rH7N9-R149K, rH7N9-G151E, rH7N9-A156V were lower than those of wt-rH7N9. The virus titer of rH7N9-R149K+A156V at 24 hours post infection (hpi), rH7N9-del151-153 at 36 hpi and rH7N9-del151-153+A156V at 36 hpi were all higher than those of wt-rH7N9. The ability of these antibodies to neutralize mutant virus was determined by plaque reduction assay. Those HA mutations reduced antibodies binding ability were confirmed to reduce antibodies neutralizing ability. In summary, we developed ten anti-H7 antibodies with great neutralizing ability against 2013H7N9 and 2017H7N9. We also did epitope mapping of these anti-H7 antibodies. Anti-H7 antibodies recognized critical and highly conserved regions of HA in H7 viruses.