研究促進腸病毒七十一型鼻黏膜疫苗免疫反應

Abstract

黏膜疫苗的優點在於施用方便且能在感染之初有效清除病原，但容易誘 發免疫耐受性而降低其效果；施用佐劑則可以透過營造有利於黏膜免疫反應發生的環境來突破耐受性的困境，甚至刺激黏膜抗體產生。一群病原相關分子模式與損傷相關分子模式已被認為能夠藉由與模式識別受體的交互作用活化先天免疫系統，進一步促進抗原專一免疫反應的發生。在這些分子模式中，我們鎖定三個先天免疫受體配基作為試驗目標: 酵母多醣為酵母菌細胞壁多醣成分，能與先天免疫細胞表面的類鐸受體2、樹突細胞相關之鈣型凝集素受體1 作用；殼聚醣與奈米氧化矽分別為真菌細胞壁成分與人工奈米化顆粒，兩者皆能活化先天免疫細胞內的NLRP3 發炎小體。在本篇文章中，我們探討的是酵母多醣(zymosan)、殼聚醣(chitosan)與奈米氧化矽(nano-SiO2)作為黏膜佐劑之潛力。在細胞試驗中，我 們證實酵母多醣能夠有效活化骨髓分化之樹突細胞，促進間白素-10、12 p40、12 p70 的分泌；除此之外，連續以酵母多醣與殼聚醣或奈米氧化矽處理能促進骨髓分化之樹突細胞與巨噬細胞的活化，使其產生高量的間白素-1β。在動物實驗中，我們也觀察到以抗原加上酵母多醣致敏能使BALB/c 小鼠的抗原專一免疫反應增強，包含血清中的免疫球蛋白G 濃度、鼻腔沖洗液與糞便中的免疫球蛋白A濃度、抗原再刺激之脾臟細胞分裂增生的能力與間白素-17 的分泌量相較於只以抗原致敏的組別皆顯著地提升；更重要的是，血清抗體與病毒中和試驗的結果支持以抗原加上酵母多醣、殼聚醣與奈米氧化矽致敏的小鼠血清能有效提供細胞保護力避免病毒感染。除此之外，這些多醣化物與奈米顆粒在細胞試驗與動物試驗中皆顯示為低毒性。綜合以上實驗結果，我們推論酵母多醣、殼聚醣與奈米氧化矽可以作為有潛力之腸病毒七十一型黏膜佐劑。

Mucosal vaccines benefit in convenience of application and pathogen clearance at the site of entry, but are limited in low efficiency to induce immune responses. Administration of an adjuvant could enhance immune responses by building the cytokine environment in favor of mucosal immunity rather than tolerance and promoting mucosal antibody production. To induce these antigen –specific responses, one effective approach is to activate the innate immunity. A range of pathogen associated molecular patterns (PAMP) and damage associated molecular patterns (DAMP) have been postulated to trigger innate immune responses by interacting with pattern recognition receptors (PRR). Of our interest are three innate receptor ligands in the following. Zymosan, a glucan derived from yeasts with β-1, 3-linkage, interacts with Toll-like receptor (TLR) 2/6 heterodimer and dectin-1 on the surface of innate immune cells. Another two include chitosan, a cationic polysaccharide abundant in the cell wall of fungi, and synthetic nanoparticle of silicon dioxide (nano-SiO2). Both of them could activate NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome in innate immune cells. In this study, we targeted at the potential immuno-inducibility of zymosan, chitosan and nano-SiO2 as mucosal adjuvants. At the cellular level, we showed that maturation of bone-marrow derived dendritic cells (BMDC) was promoted after treatment with zymosan by inducing IL-10, IL-12 p40 and IL-12 p70 production. Besides, activation of BMDC and bone-marrow derived macrophage (BMDM) were culminated in the induction of IL-1β secretion after sequential treatment with zymosan and chitosan/ nano-SiO2. Based on the in vitro results, we observed that BALB/c mice immunized with antigen plus zymosan exhibited elevated Enterovirus 71 (EV71)- specific immune responses, including the IgG titer in sera, the IgA titer in nasal wash and feces, the proliferation level and IL-17 production from the restimulated splenocytes increased significantly, compared with the antigen alone group. Furthermore, we confirmed minor toxicity of zymosan, chitosan and nano-SiO2 at low concentration both in vitro and in vivo. Taken together, our data suggested that zymosan, chitosan, and nano-SiO2 could be used as potential adjuvants in intranasal EV71 vaccine.