2014-03-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/647361摘要：今年春季在大陸爆發 H7N9禽流感病毒流行，其之所以引起極大的關注，主要是因為 H7N9會快速造成下呼吸道的嚴重病徵。目前僅有神經胺酸酶抑制劑，如克流感及樂瑞莎，可有效抑制流感病毒。然而，一些 H7N9重症病人在接受克流感治療後，會對克流感產生抗藥性；而具有抗藥性 R292K 基因突變的病毒株也可自一些尚未治療的病人檢體中分離。所以，發展其它的治療策略，如治療性人類單株抗體，是極重要的。本整合型計畫擬建立一篩選抗 H7N9的人類單株抗體技術平臺，並針對篩選抗體做進一步的功能性分析。本子計畫將會負責(1)建立類流感顆粒及假病毒的抗體篩選及抗體確效的中和實驗系統，(2)篩選廣效性單株抗體及鑑定中和性抗原位點。第一年，我們將會製造類流感顆粒供 DCB進行抗體篩選，並將篩選出的抗體以假病毒進行中和實驗。假病毒平台將可做為中和性抗原位點的鑑定及未來新興流感病毒抗體篩選平台。第二年，我們將會以中和實驗決定篩選出的抗體是否只針對 H7N9或是對其它季節性流感病毒也具有中和能力；我們也會鑑定主宰這些抗體中和能力的抗原位點。這些研究成果不僅可幫助我們了解哪些抗原性位點是 H7N9或廣效性中和性抗體的重要標的，對於未來治療性人類抗體的開發與流感疫苗的設計將提供許多有效的資訊。<br> Abstract: A recent outbreak of a novel avian-origin re-assortant influenza A (H7N9) virus in China has raised great concerns owing to the rapidly progressive lower respiratory tract infections in infected individuals. The only class of antiviral drug used in treatment of H7N9 infection is neuraminidase inhibitor, including oseltamivir and zanamivir. Because oseltamivir treatment of H7N9 severe cases is associated with generation of resistant viruses, and some of the isolated H7N9 clinical strains from treatment-naive patients harboring R292K mutations, which has been reported to be associated with oseltamivir resistance, development of other therapeutic strategy is essential in clinical management of H7N9 infection. In the integrated proposal, we plan to set-up a platform in development and characterization of therapeutic human antibody against H7N9. Dr. Wu from the Development Center for Biotechnology (DCB) will be responsible for amplification of immunoglobulin variable region genes from each isolated memory B cells of H7N9-infected patients by RT-PCR to generate human monoclonal antibody. The current proposal will be responsible for (1) generation of H7N9 virus-like particles and pseudoviruses for antibody screening and confirmation, (2) screening and epitope-mapping of broadly neutralizing antibody. Four specific aims are proposed and we plan to accomplish two specific aims every year. In the first year, we plan to generate H7N9 virus-like particles as antibody screening platform and to confirm the antibody neutralizing activity by using pseudoviruses and H7N9 viruses. The correlation between neutralizing activity and binding affinity of the screened antibodies to H7N9 viruses and pseudoviruses will be determined by hemagglutination inhibition and microneutralization assays. The established pseudovirus platform can then be used in epitope mapping for the cross-neutralizing activity of the screened antibodies and can also be immediately applied to antibody detection and antibody screening against emerging novel influenza viruses in the future. In the second year, we will determine whether the screened antibodies are specific to H7N9 or have broadly neutralizing activity to seasonal influenza viruses by using H7N9 viruses and seasonal influenza viruses collected in our laboratory since 1999. The epitopes critical for neutralization will be determined by peptide screening, which will be conducted by Dr. Tseng in the third proposal, and by generating pseudoviruses and recombinant viruses expressing wild-type and various mutant HA proteins in this proposal. In summary, our study will provide a platform in generation of therapeutic human antibody against H7N9. The study results will shed light on the epitopes critical for neutralizing activity against H7N9 and even to other seasonal influenza viruses, and all these information will be useful in the development of therapeutic broadly neutralizing human antibody and in the design of influenza vaccine in the future.