腸病毒 A71 (Enterovirus A71; EV-A71)，是主要造成手足口疾病(Hand-Foot-Mouth Disease; HFMD)最大宗的病原體，並從1997年開始，時常於亞太地區如越南、泰國、台灣、韓國等地造成流行。腸病毒 A71 感染通常發生於年齡低於十歲的幼童，在一些重症的病患會有神經及心臟相關的併發症，甚至造成死亡。目前，自2015年起，中國有已有三個商品化的腸病毒 A71 去活性單價疫苗，分別由中國醫學科學院(Institue Of Medical Biology; CAMS)、北京科興(Sinovac Biotech)與武漢生物製品研究所(Wuhan Institute Of Biological Products; 北京微谷(Vigoo Biologicals)成立)製造。 此外，臺灣國家衛生研究院(National Health Research Institutes; NHRI)也有研發另外兩個疫苗，並由高端(Medigen)和國光(Adimmune)生產。上述五個疫苗展現了廣效的血清交叉保護效果對抗多種腸病毒 A71 基因型亞種能力，表示這些疫苗在未來大部分國家中可能具有潛力扮演管控腸病毒 A71 相關手足口疾病的角色。於此同時，中和抗體不只對於人體提供保護，免於腸病毒 A71 的嚴重感染及致死，也提供疫苗的品質及保護效力的評估。在我們先前的研究中發現，我們自行研發的老鼠單株抗體(MAb NHRI2016-1)相較於市售抗體(MAb979) ，擁有對抗多種腸病毒 A71 基因型亞種(A, B4, B5, C4)更高的中和效價。此外，MAb NHRI2016-1所辨識的抗原決定區為結構型的。因此，可藉由產生不被抗體辨識的突變病毒株來找出結合位點，此理論在未來對於設計一個有效的疫苗，或發展腸病毒 A71 抗原偵測與定量的分析方法顯得格外重要。我們先前已發展出一株不被MAb NHRI2016-1抗體辨識的突變病毒株，此突變株於非結構蛋白以及結構蛋白上有共有十九個核苷酸的改變。因為抗體的中和及辨識與非結構蛋白無關，因此我們著重在探討結構性蛋白的改變，包含五個胺基酸突變 (3個在VP1，2個在VP3)以及一個同義突變(VP3)。同時在本研究中，我們進一步利用反向基因體學針對病毒外鞘蛋白產生出多個突變病毒株，並且確認VP3-S64R為MAb NHRI2016-1的結合點位。

Enterovirus A71 (EV-A71), which is the major pathogen of hand-foot-mouth disease (HFMD), has frequently caused epidemics in the Asia-Pacific region, including Vietnam, Thailand, Taiwan, and Korea since 1997. EV-A71 infections commonly occur in children under the age of 10 and some severe cases will have neurological and cardiac complications or even mortality in young children. There are three licensed EV-A71 inactivated monovalent vaccines produced by the Institute of Medical Biology (CAMS), Sinovac Biotech, and the Wuhan Institute of Biological Products available in China since 2015. In addition, two more vaccine products are developed by National Health Research Institutes (NHRI), Taiwan and produced by two manufactures, Medigen and Adimmune. These five vaccines showed broad cross-neutralizing serological responses against several EV-A71 genotypes, suggesting that these vaccines could play potential roles in controlling EV-A71-associated HFMD epidemics in most countries. Meanwhile, neutralizing antibodies play significant roles not only in protection against severe EV-A71 infections and related mortality in humans, but also in assessment of the quality and protective efficacy of vaccines. We previously found that our in-house mouse monoclonal antibody (MAb NHRI2016-1) has higher neutralization titers against EV-A71 genotypes A, B4, B5, C4 compared to the commercial MAb979. Moreover, the MAb NHRI2016-1 can recognize conformational epitopes. Therefore, it is vital to generate escape mutants in order to map the binding epitope, which is critical to rationally design an effective vaccine and develop assays for detection and quantification of EV-A71 antigens. Previously, we generated a MAb-escape mutant, which harbored 19 nucleotide changes in both non-structural and structural proteins. Because mutations happened in non-structural proteins were not responsible for antibody recognition and neutralization. Therefore, we only focused on mutations located at structural proteins, including five amino acid mutations (three in VP1 (viral capsid protein 1), two in VP3) and one synonymous mutation in VP3. In this study, we further employed reverse genetics to generate multiple mutant viruses targeting the viral capsid protein and confirmed that VP3-S64R is the binding site of the MAb NHRI2016-1.

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