每年全球約有百分之二十的人口感染人類流行性感冒病毒而造成嚴重的疾病甚至死亡。到目前為止，最有效預防流感病毒感染的方法是每年接受季節性流感病毒疫苗。然而，流感病毒本身為了逃脫宿主體內抗體的辨認，容易在抗原性決定位點產生突變而造成抗原性改變。因此，評估每年度流感疫苗施打後的效力及探討流感病毒抗原性差異，對於了解流感病毒的演化與每年疫苗的研發及製備是非常重要的。在本研究中，首先建立一中和抗體免疫分析平台─結合微量中和試驗(microneutralization test)及酵合免疫吸附分析法(Enzyme-linked immunosorbent assay, ELISA)測定血清之中和抗體，並以此方法測定2006-2007及2007-2008年疫苗前後的血清中之中和抗體效價，以評估流感疫苗之效力。我們發現微量中和試驗結合ELISA之方法比傳統血球凝集抑制法較能較專一的測得具有中和該病毒能力的抗體，且所測得之疫苗前後血清中抗體效價上升倍數改變較為顯著。此外，在這二季流感疫苗的效力評估中，我們也發現，相較於H3N2及B型流感病毒，H1N1亞型疫苗病毒株能引發較佳之中和抗體反應，而且在不同年齡族群的疫苗反應分析中，幼兒組及成人組之抗體反應皆較老人組之反應佳。另外，我們也以此免疫分析平台來探討近年在台灣所分離出之H1N1亞型病毒株之間的抗原差異性。我們挑選了十八個血清樣本，發現這些血清中對於一些2006年所分離出來的病毒株的中和抗體效價明顯較低，顯示了這些流感病毒株之間的抗原性已有明顯的改變，即使這些病毒株在流感病毒血球凝集素(Hemagglutinin，HA)基因片段的演化樹分析上皆屬於A/Solomon Islands/3/2006這株疫苗株的群系裡。因此，我們更進一步藉著分析這些病毒株表面血球凝集素的胺基酸序列差異及利用反轉基因系統，來探討直接影響此抗原性差異的決定位點，我們發現在胺基酸第90個位置的突變，會影響H1N1病毒的抗原性。總之，在此研究中，我們建立評估流感疫苗效力之中和抗體免疫分析平台及探討流感病毒之抗原性差異，並發現了在HA基因上一個多株抗體所辨識的新中和性位點，這些結果不論對於了解此病毒之突變及演化或是未來每年疫苗株的篩選，都提供了相當重要的資訊。

Approximately 20% of the human population was infected by influenza viruses with significant morbidity and mortality each year. Until now, vaccination is the most efficient method for prevention of this infectious disease; however, it is considered that influenza viruses may escape the antibody neutralization through mutations in the antigenic sites. Thus, evaluating the seasonal influenza vaccine efficacy and characterizing the antigenic diversity between circulating viruses are important for the surveillance of influenza evolution and vaccine development. In this study, we set up the microneutralization test (NT) with the detection by ELISA to investigate the neutralizing antibody titers of the pre- and post-vaccination sera from healthy workers who received 2006/2007 and 2007/2008 WHO recommended vaccines. We found microNT-ELISA assay showed higher capability in several folds of magnitude than traditional hemagglutination inhibition (HI) test after vaccination. In addition, the antibody titers against influenza H1 virus were higher than H3 virus and influenza B virus in most HI test and micro-NT ELISA, indicating that H1 vaccine strain had higher antigenicity. Moreover, this microNT-ELISA test was used to investigate the antigenic changes of influenza H1N1 viruses among clinical isolates circulating in recent years in Taiwan. We found the NT titers of sera against different clinical isolates in 2006 were varied. This suggested that the antigenicity among influenza viruses in Taiwan has changed progressively during recent years, even though those viruses were belonged to the same A/Solomon Islands/3/2006 (H1N1)-like lineage based on the sequence analysis of hemagglutinin (HA) gene. Furthermore, the HA sequences analysis and reverse genetics were applied to investigate the drift of neutralizing epitope of these circulating H1N1 viruses. We found that the amino acid substitution (arginine to lysine) at position 90 may affect the neutralizing antigenicity of influenza H1N1 viruses. In conclusions, a novel neutralizing epitope was identified and examination of the antigenic variations of new influenza viruses may provide more surveillance information of the virus evolution and may contribute to the selection of vaccine strains in the future.

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