腸病毒A71型是世界上造成手足口症的主要病原體之一，自1998年以來在台灣引起了好幾次大爆發。為了預防腸病毒A71型的感染，疫苗研發的重要性日漸上升。密碼子去最優化在先前研究已證明會影響病毒複製，但不會影響病毒蛋白序列，故病毒能保有相同的抗原特性。我們之前的研究中，建構了包含高保真性聚合酶點突變之密碼子去優化的腸病毒A71型，並證明了這些病毒展現了較低的生長速率，但抗原特性沒有變化。在本篇研究中，我們通過研究這幾株高保真性聚合酶點突變之密碼子去優化的腸病毒A71型引起的毒力、體液和細胞免疫反應，進一步評估其作為疫苗候選株的潛力。被高保真性密碼子去優化的病毒感染的ICR小鼠的體重變化與模擬感染的組別相似，且與感染後21天的毒力株相比，它們顯示出較輕微的臨床評分，亦沒有死亡，表明這些病毒的毒力降低。我們還檢查了各個器官中病毒感染後的進程，發現密碼子去優化和高保真性聚合酶點突變的組合能夠限制病毒在體內的複製。在組織學分析中，透過蘇木精-伊紅染色及免疫組織染色證實，在這些高保真性密碼子去優化的腸病毒A71型感染之小鼠中檢測到的組織病理學變化和病毒抗原比MP4病毒感染的小鼠還要少。這幾株高保真性密碼子去優化的腸病毒A71型產生的抗血清亦顯示出具有中和能力之強大的特異性IgG抗體。收集免疫後脾細胞，並用各種高保真性密碼子去優化的病毒蛋白刺激，我們量化在培養基中IL-1β、IL-2、IL-4、IL-6、TNF-α和IFN-γ的表現量，發現經高保真性密碼子去優化的腸病毒A71型免疫過的小鼠明顯高於陰性對照組，表明高保真性密碼子去優化病毒引起了Th1和Th2介導的免疫反應。更重要的是，這些病毒誘導的抗血清為新生小鼠提供了針對致命性攻擊的保護。總之，我們證明了透過免疫高保真性密碼子去優化的腸病毒A71型後，誘導了強大的體液和細胞免疫反應，並賦予了新生小鼠對抗致命性腸病毒攻擊的保護力，這表明了高保真性密碼子去優化之腸病毒A71型對於未來腸病毒A71型之疫苗開發的可行性。

Enterovirus A71 (EV-A71) is one of the major causative agents of hand, foot and mouth disease (HFMD) in the world, it caused several large outbreaks in Taiwan since 1998. To prevent EV-A71 infection, development of virus vaccine is urgent and important. Codon-deoptimization (CD) has been shown to impact virus replication, but it doesn’t affect the viral protein sequence and the virus retains the same antigenicity. Our previous study had constructed VP1 codon-deoptimized (VP1-CD) reverse genetics EV-A71 (rgEV-A71) containing high-fidelity (HF) 3D polymerase and demonstrated these CD-HF viruses showed slightly lower growth kinetics than wild type in vitro, but with no change in antigenicity. In this study, we further evaluated the potential of these rgEV-A71-CD-HF (CD-HF) viruses as a vaccine candidate by investigating the virulence, humoral and cellular immune responses elicited by the CD-HF viruses. ICR mice infected with CD-HF viruses showed similar body weight changes to the mock-infected group. They showed mild clinical score and no deaths when compared with virulence strain 21 days post-infection, indicating reduced virulence of these CD-HF viruses. We also examined the progression of virus infection in various organs, and found that a combination of codon-deoptimization and high-fidelity substitutions in 3D polymerase was able to limit the viral replication in vivo. In histology analysis, H&E and IHC staining confirmed that the histopathological changes and viral antigens were detected less in these CD-HF-infected mice than in mice infected with MP4 virulent strain virus. The antisera raised by rgEV-A71-CD-HF viruses showed strong specific IgG antibody with neutralize ability. We harvested the splenocytes post-immunization and stimulated with various CD-HF proteins. We quantified IL-1β, IL-2, IL-4, IL-6, TNF-α, and IFN-γ levels in culture supernatant. The levels in rgEV-A71-CD-HF-immunized mice were significantly higher than negative control, indicating elicitation of both Th1- and Th2-directed immune response by CD-HF viruses. More importantly, the antisera elicited by rgEV-A71-CD-HF conferred protection to neonatal mice against the lethal challenge. In conclusion, we demonstrated that immunization with CD-HF viruses induced potent humoral and cellular immune response and conferred protection to neonatal mice against lethal EV-A71 challenge, which suggest that the CD-HF viruses may be feasible for next generation EV-A71 vaccine development.

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