利用免疫胜肽組譜學鑑定三種台灣常見第一型人類白血球抗原呈現之流感病毒胜肽

Abstract

季節性流感每年在全球造成約十億人次感染，對公共衛生造成重大負擔。流感病毒的表面抗原，如HA和NA，經常發生細微的突變，稱為抗原漂移（antigenic drift），導致病毒株持續變異。此外，流感病毒基因組的重配現象，稱為抗原轉換（antigenic shift），可能產生新型病毒株，使病毒逃避免疫系統的識別，降低現有疫苗的效力。因此，現有疫苗的保護效果通常較為短暫，迫切需要開發具廣泛保護力的流感疫苗。 相較於依賴表面抗原的傳統疫苗，以T細胞為標的的疫苗策略可針對病毒內部較為保守的蛋白，提供與抗原變異無關的長效保護。研究也顯示，T細胞免疫在抵禦流感病毒感染中扮演關鍵角色。由於CD8⁺ T細胞的活化依賴於識別由第Ⅰ型主要組織相容性複合體（MHC I）呈現的抗原表位，鑑定與特定HLA等位基因相關的表位是發展T細胞疫苗的重要步驟。 目前T細胞表位的鑑定策略主要分為電腦預測與實驗鑑定。本研究使用表現單一白血球抗原基因型的細胞株 (monoallelic HLA cell lines) 以降低實驗誤差。這些細胞株過度表現目標抗原後，透過免疫沉澱法分離peptide–HLA -I複合物，並利用高效液相層析（High Performance Liquid Chromatography, HPLC）結合液相層析串聯質譜儀（LC-MS/MS）進行分析。 針對臺灣族群中常見的HLA-I等位基因，我們鑑定出HLA-A*11:01 (約占台灣人口47%) 呈現的20個流感病毒胜肽，且其中6個於A型流感病毒中具超過95%的保守性;於HLA-A*02:01和HLA-A*24:02中則分別鑑定出13與4條流感病毒胜肽，其中各有2與1條具高度保守性（>95%）。後續將以半年內感染流感者的人類外周血單核細胞（peripheral blood mononuclear cell, PBMC）進行免疫原性（Immunogenicity）測試，以得到能作為廣效型流感 T 細胞疫苗的候選抗原表位組合。

Seasonal influenza continues to cause approximately one billion infections worldwide each year, posing a significant healthcare burden. The surface antigens of influenza viruses, such as hemagglutinin (HA) and neuraminidase (NA), are prone to frequent minor mutations, known as antigenic drift, which leads to the continuous emergence of viral variants. In addition, reassortment of influenza viral genome, known as antigenic shift, may occur and generate novel viral strains, allowing them to evade immune recognition and reduce the effectiveness of existing vaccines. As a result, the protective effect of current vaccines is often short-lived, highlighting the urgent need for the development of broadly protective influenza vaccines. Unlike traditional vaccines that rely on frequently mutating surface antigens, T cell–based vaccine strategies target the more conserved internal proteins of the virus that are less affected by antigenic variation. Studies have also shown that T cell immunity plays a crucial role in the protection against influenza virus infection. Since CD8⁺ T cell activation depends on the recognition of antigenic epitopes presented by class I major histocompatibility complex, identifying allele-specific epitopes is a key step towards the development of T cell–based vaccines. Current strategies for T cell epitope identification can be classified into computational prediction and experimental validation. In this study, we aimed to use monoallelic class I HLA (HLA-I) cell lines that express a single HLA allele to reduce experimental complexity for immunopeptidomics analysis. Peptide–HLA-I complexes of these cells overexpressing desired antigens were isolated through immunoprecipitation and followed by high-performance liquid chromatography (HPLC) coupled with liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis. Focusing on HLA-I alleles common in the Taiwanese population, we identified 20 influenza-derived peptides presented by HLA-A*11:01, the most prevalent allele in Taiwan (approximately 47% of the population), of which 6 showed over 95% conservation among influenza A viruses. In HLA-A*02:01 and HLA-A*24:02, we identified 13 and 4 peptides, respectively, with 2 and 1 of them exhibiting high conservation (>95%). The immunogenicity of these peptides will be evaluated using peripheral blood mononuclear cells (PBMCs) from individuals who have been infected with influenza within the past six months, aiming to identify promising candidates for the development of a broadly protective T cell–based influenza vaccine.