台灣人常見第一型白血球抗原所呈獻流感病毒CD8+ T細胞保守抗原決定區之鑑定

Abstract

直至今日，流行性感冒病毒仍然是人類全球健康的重大威脅，每年都會導致病患輕度至重度呼吸道疾病。由於抗原漂變(antigenic drift)經常發生在抗體的辨識位點上，使得疫苗誘導產生的抗體對於流感病毒不同亞型或病毒株的交叉保護能力很差。因此，開發既長效又廣效的疫苗是有必要的。 CD8+ T細胞與抗體的作用機制不同，是通過辨識第一型主要組織相容性複合體(major histocompatibility complex class-1 [MHC-I])所呈獻的病毒蛋白衍生之序列具有保守性的胜肽(conserved virus-derived peptides)，來提供交叉保護的作用。第一型主要組織相容性複合體，在人類又稱為白血球抗原(human leukocyte antigen [HLA])，辨識它即是CD8+ T細胞活化的第一步。有鑒於第一型主要組織相容性複合體和病毒的抗原決定區(epitope)有著緊密的關聯，鑑定人類第一型白血球抗原限制性(HLA class I-restricted)保守抗原決定區對於T 細胞疫苗的開發至關重要。 目前已發表的許多關於流感病毒的抗原決定區鑑定之研究，但關於台灣人常見的第一型白血球抗原限制性保守抗原決定區的資訊知之甚少。因此，本研究旨在鑑定由CD8+ T細胞辨識之台灣人常見第一型白血球抗原所呈獻的流感病毒保守抗原決定區，這這是開發通用的流感T細胞疫苗重要的一環。為了無誤差的鑑定特定第一型白血球抗原限制性保守抗原決定區，我們使用表現單一白血球抗原基因型的細胞株(monoallelic HLA cell lines)，利用轉染或感染的方式送入病毒蛋白至細胞中，再以免疫胜肽組(immunopeptidomics)搭配逆相高效液相層析法(reversed phase high performance liquid chromatography [RP-HPLC])與液相層析串聯質譜儀(LC-MS/MS)來分析白血球抗原限制性保守抗原決定區。 目前我們已建立八種分別表達單一台灣人常見的第一型白血球抗原的細胞株，且也已經使用建立之表現HLA-A* 11:01的293T細胞鑑定出流感病毒蛋白M1、NP與PB1衍生之胜肽。除此之外，為了提高胜肽鑑定的敏感度，我們也運用抗原呈獻細胞(antigen presenting cells)外加γ干擾素(IFN-γ)的方式提升293T細胞第一型白血球抗原的表達量，來達到提高鑑定出的胜肽數量。目前發現以48小時的γ干擾素可造成表達量上升至原本的兩倍之效果。若能夠結合增加使用的細胞數以及添加γ干擾素，對於提升抗原決定區之胜肽鑑定是很有幫助的。

Influenza virus remains a significant threat of human global health, causing mild to severe respiratory diseases annually. Vaccine-induced antibodies have poor cross-protectivity against different viral strains or subtypes because of antigenic drift and shift of antibodies. It is necessary to develop a long-lasting, universal vaccine. Unlike antibodies, CD8+ T cells can provide cross-protectivity by recognizing conserved virus-derived peptides presented by major histocompatibility complex class I (MHC-I), also named human leukocyte antigen (HLA) in human. It is essential to identify conserved HLA-restricted epitopes for development of T cell vaccine. There have been many studies published which identified influenza virus-derived epitopes, but little information is known about those restricted by common HLA class I alleles of Taiwanese. Therefore, this study aimed to identify Taiwanese common HLA class I restricted influenza A virus CD8+ T cell conserved epitopes, which are a critical stepping stone for development of universal influenza T cell vaccine. To unbiasedly identify the epitopes for a particular HLA allele, we utilized monoallelic HLA cells with the overexpression of viral antigens and infection approaches, and then analyzed the HLA-restricted peptides by mass spectrometry-based immunopeptidomics. Currently, we have generated 8 individual single-HLA allele cell lines that express 8 common Taiwanese HLA alleles, and we identified one M1- and NP-derived peptide, and two PB1-derived peptides using monoallelic HLA-A* 11:01 expressing 293T cells. To improve epitope mapping sensitivity, we treated antigen presenting cells with IFN-γ for 48 hours and the HLA expression levels increased two times. By simultaneous increase of the input cell number and IFN-γ treatment, we achieved more sensitive epitope mapping.