利用乳酸菌穿梭載體系統開發腸病毒 71 型 DNA 疫苗

腸病毒在溼熱的環境下容易散播並造成感染，地處亞熱帶的台灣，每年都會出現大小不一的疫情，尤其是在夏天最為高峰。感染腸病毒 71 型 (Enterovirus 71, EV71) 後會造成手足口症，甚至導致中樞神經癱瘓及死亡，在幼童中有極高的死亡率。於 1998 年時，台灣爆發首次 EV71 大流行，但迄今仍未有疫苗可供施打。研究已證實 EV71 的主要抗原決定基位於病毒外殼蛋白中的VP1，因此在本篇研究中將發展可表現 VP1 之 DNA 疫苗，並利用 Lactobaillus casei 作為疫苗的運送載體兼免疫佐劑，以生產出 EV71 的口服 DNA 疫苗。我們使用的載體是乳酸菌穿梭載體 pMN5，其可於哺乳類細胞中表現目標基因，也可於乳酸菌中複製。目前已成功建構可分別在胞內及胞外表現 VP1 的重組質體 pMN5-VP1 及 pMN5-sVP1，並且在 HEK293T 細胞中以西方轉漬法確認 VP1 之表現。在黏膜免疫系統中，樹突細胞及巨噬細胞皆為主要的抗原呈現細胞，本研究中發現 L. casei 可被 RAW264.7 巨噬細胞及 DC2.4 樹突細胞所吞噬，同時能刺激細胞產生 TNF-α 及 IL-6，亦能使 DC2.4 細胞表面的成熟標誌 CD40、CD80、CD86 及 MHC II 之表現量上升，顯示L. casei確實具有免疫佐劑的作用。本研究已成功建構可表現 EV71 VP1 之乳酸菌穿梭載體，未來將進一步以帶有 pMN5-VP1 或 pMN5-sVP1 之 L. casei，在動物實驗中評估以其作為 EV71 口服 DNA 疫苗之潛力。

關鍵字

腸病毒71型； DNA 疫苗；乳酸菌穿梭載體系統；黏膜免疫；佐劑

並列摘要

Enteroviruses easily spread and cause infectious under hot and humid environment. In Taiwan, several enteroviruses epidemics occur throughout the year but usually peak in summer months. Enterovirus 71 (EV71) infection causes hand, foot, and mouth disease (HFMD) in infants and children, and it may also cause central nerve system collapse and even death. Since 1998, EV-71 has become an emerging infectious agent in Taiwan, but there is still no vaccine available against EV-71 so far. Previous studies have shown that the epitopes on the capsid protein VP1 can elicit the majority of neutralizing antibodies in animal models. Hence in this study, we attempt to develop an oral EV-71 DNA vaccine that expresses VP1 and is delivered by Lactobacillus casei, which functions both as a vector and an adjuvant. The Lactobacillus shuttle vector pMN5 can express target genes in mammalian cells and replicate in Lactobacillus. We have constructed pMN5-VP1 and pMN5-sVP1, which express VP1 intracellularly and extracellularly, respectively. The expression of VP1 by these constructs in HEK293T cells was confirmed by Western blotting. Dendritic cells and macrophages are major antigen-presenting cells in the mucosal immune system. Our results showed that L. casei could be phagocytosed by RAW264.7 macrophages and DC2.4 dendritic cells, and meanwhile L. casei could stimulate these cells to produce high levels of TNF-α and IL-6. Furthermore, L. casei also stimulated DC2.4 cells to express the maturation markers CD40, CD80, CD86 and MHC II, indicating that L. casei can function as an adjuvant. In this study, we have successfully constructed Lactobacillus shuttle vectors expressing EV71 VP1, and the potential of using L. casei carrying pMN5-VP1 or pMN5-sVP1 as oral DNA vaccines against EV71 will require further investigation in animals.

並列關鍵字

Enterovirus 71 ； DNA vaccine ； Lactobacillus shuttle vector system ； mucoal immunity ； adjuvant

參考文獻

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利用乳酸菌穿梭載體系統開發腸病毒 71 型 DNA 疫苗

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