A型流感病毒(IAV)會透過飛沫傳染在人類社會中引起嚴重呼吸道感染流行病，季節性的流感每年造成5~15%的人口感染，以及500000人口的死亡，造成極大的危害。由於A型流感有很高的基因突變與抗原轉移的特性，導致目前傳統的滅活病毒疫苗無法廣效並持久進行保護，所以設計一款能夠廣效並持久進行保護的疫苗成為一大挑戰。目前市面上有兩種疫苗設計方式來解決這個挑戰，其一是針對A型流感高度保守的抗原設計的廣效性疫苗，其二就是活性減毒流感疫苗。相比於滅活疫苗，活性減毒流感疫苗除了能活化IgG外，同時能夠活化IgA以及刺激更好的T細胞適應性免疫。目前活性減毒疫苗設計朝向能夠刺激更高的干擾素(IFN)活化進行，因為干擾素以被證實可以更強的活化適應性免疫。我們實驗室最近研究發現一個IAV新的免疫逃脫機制，主要透過流感的非結構蛋白(NS1)攔截三個宿主偵測流感RNA的sensor(RIG-I, TLR3和TLR7) 活化TRAF3到第一型干擾素的路徑，進而降低第一型干擾素的產生。我們實驗室對NS1結合TRAF3的位點進行突變後，產生了能夠活化更高IFN的減毒病毒株(PR/8 NS1 E152A/E153A)。因為此病毒株在動物實驗中顯示有減毒及誘發高干擾素特性，所以我們推估PR/8 NS1 E152A/E153A病毒株有作為疫苗候選株的潛力，於是我們設計動物疫苗實驗來測試PR/8 NS1 E152A/E153A的保護效果。實驗結果顯示，鼻腔施打的PR/8 NS1 E152A/E153A疫苗在疫苗接種階段小鼠體重沒有顯著變化，顯示作為疫苗的安全性，且在後續對同源病毒(PR/8)感染有完整的保護力。這之後我們也測試了PR/8 NS1 E152A/E153A疫苗對兩種不同的異源病毒的保護力，這兩種異源病毒分別是H1N1 WSN毒株與抗原相差更遠的H3N2 X31毒株。結果顯示PR/8 NS1 E152A/E153A疫苗對H1N1 WSN毒株有完整保護力，對H3N2 X31則是有部分保護力。因為我們先前已在小鼠的體內與體外試驗中觀察到PR/8 NS1 E152A/E153A活化高干擾素的特性，但IAV的主要宿主是人類肺臟上皮細胞，所以我們要測試這個PR/8 NS1 E152A/E153A病毒株是否在肺臟上皮細胞(A549)中活化更高干擾素，結果顯示，PR/8 NS1 E152A/E153A感染的A549，比PR/8 WT感染的A549有更高的IFN-β和IFN-λ表達，在病毒NS1中，則是PR/8 WT多於PR/8 NS1 E152A/E153A。我們也嘗試著以TRAF3-相互作用基序(TRAF3-interaction motif)作為基礎，做更多的NS1突變，看是否能做出可以活化更高干擾素的NS1突變。利用報告基因測試來檢測新的NS1突變株是否有更好的干擾素活化效果，結果顯示新設計的NS1突變蛋白量表達不好，所以目前還沒辦法下定論。根據這篇論文的實驗結果，我們觀察到PR/8 NS1 E152A/E153A賦予小鼠免疫力抵抗同源或異源病毒的感染。同時在人類肺臟上皮細胞(A549)中觀察到有更高的第一型和第三型干擾素活化。未來我們會著重PR/8 NS1 E152A/E153A病毒株誘發小鼠適應性免疫的IgA與T細胞分析，以及受PR/8 NS1 E152A/E153A病毒株感染後的小鼠適應性免疫相關RNA大規模並行測序(NGS)，確認其作為疫苗候選株的可能性。

Influenza A virus (IAV) is a major pathogen causing respiratory tract diseases in humans yearly and represents a severe threat to global health. Due to the characteristics of high gene mutation, the current traditional inactivated influenza vaccines cannot provide broad and lasting protection. Therefore, designing a vaccine that can provide broad and lasting protection has become a significant challenge. The development of a live attenuated influenza vaccine is one of the feasible options because live attenuated influenza vaccine can activate IgA, in addition to IgG activation, and stimulate better T cell immunity. The current design of live attenuated vaccines aims to produce higher interferon (IFN) activation, as interferons are critical for activating innate and adaptive immunity. Our lab has uncovered a novel NS1-mediated immune evasion by which NS1 employs a conserved TRAF3-interacting motif (TIM) (a.a. 150-153; FTEE) to target TRAF3 and interrupt three RNA sensing pathways to the TRAF3-type I IFN axis. A recombinant mutant virus with NS1 E152A/E153A mutations exhibits attenuated pathogenicity in mice. We performed vaccination experiments to examine whether PR8 NS1 E152/153A could be a vaccine candidate. The experimental results showed that the PR8 NS1 E152/153A had a protective effect on lethal dose infection of both homologous and heterologous viruses.
Further, Elisa and real-time PCR were performed to confirm whether PR8 NS1 E152/153A also could activate higher IFN in human lung epithelial cells (A549). The results showed that NS1 E152A/E153A induced higher IFN-β expression than PR8 WT in A549 cells. In addition, NS1 E152A/E153A also induced more IFN-λexpression in A549 cells. Additionally, our ongoing work is to design more NS1 TIM mutations to test their effects on IFN induction. Together, the current study confirms the potential of PR8 NS1 E152/153A as a vaccine candidate.

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