2014-01-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/660506摘要：腸病毒七十一型是腸病毒中最主要造成口手足症以及神經方面嚴重併發症的病毒，並導致幼兒的死亡。若能有疫苗將可以防止腸病毒病毒誘導的發病率和死亡率。最近，我們發表以腸病毒類病毒顆粒為疫苗，已經成功完成彌猴動物試驗。我們發現無活性的腸病毒或腸病毒類病毒顆粒與氫氧化鋁混合後肌肉注射彌猴都可以得到相當好的抗體濃度，而且都可以中和七十一型腸病毒，並具有交叉保護作用。黏膜疫苗可以有效地誘導分泌 IgA 抗體在黏膜表面，從而防止或限制腸病毒進入體內。然而到目前為止很少有關腸病毒黏膜疫苗的研究。在疫苗的研發中，尋找無毒的疫苗佐劑為增強疫苗免疫反應的研究重點。許多 Toll-like receptor (TLR)的配體(ligand)可以做為全身性和黏膜的佐劑，我們將與子計畫二合作，使用第二 b 型忌熱性腸毒素 B 次單元 (LTIIb-B5)、鞭毛蛋白 (FliC)以及其它的 Toll 樣受體的配體當佐劑來開發腸病毒黏膜疫苗。另外，Flt3L是一種細胞生長激素，對很多免疫細胞的發育有重要的作用，近年一些研究也指出，在週邊組織施打 Flt3L 可以增加樹突細胞的數目，以及促進其成熟。而 PELC 是一種微小的乳膠狀佐劑，它可以吸附抗原並幫助抗原停留在鼻腔黏膜較久的時間，進而提高抗原在鼻腔黏膜的濃度，可使樹突狀細胞有足夠的時間去吞噬抗原。因此在本研究中： Aim 1: 我們將利用無活性的腸病毒七十一型為疫苗基礎，加上各種 TLR ligand (包括 LTIIb-B5 是 TLR2/1 的配體; poly I:C 是 TLR3 的配體; MPLA 是 TLR4 的配體; FliC 是TLR5 的配體; imiquimod 是 TLR7/8 的配體; CpG 是 TLR9 的配體)或 cholera toxin 作為佐劑合併由鼻腔投予小鼠並觀察後續誘發之免疫反應。 Aim 2: 我們將利用無活性的腸病毒七十一型為疫苗基礎，加上 Flt3L 或 PELC 作為佐劑合併由鼻腔投予小鼠並觀察後續誘發之免疫反應。 Aim 3: 我們將依據 Aim 1 和 Aim 2 的研究結果，選擇效果最好的 TLR ligand 合併 Flt3L或 PELC 作為佐劑來開發腸病毒黏膜疫苗。 在評估體液免疫反應中，我們將測定腸病毒特異性 IgG 和 IgA 的量，以及對於抑制腸病毒感染的能力做為指標；另外，評估細胞免疫反應中，將測試脾藏細胞中的 T 細胞和 B 細胞是否產生記憶免疫反應。所以，本計畫主要的目的是以無活性的腸病毒七十一型為疫苗，並利用 TLR ligand、Flt3L 或 PELC 作為佐劑以增加抗原細胞呈現腸病毒的機會，預期將能引發較強的黏膜免疫反應，在腸病毒疫苗發展上相當值得研究，未來將有助於腸病毒的預防。<br> Abstract: Enterovirus 71 (EV71) is an etiologic agent responsible for seasonal epidemics of hand-foot-and-mouth disease and causes significant mortality among young children. Due to the high risk of poliomyelitis-like paralysis and fatal encephalitis, an effective vaccine to EV71 could potentially prevent virus-induced morbidity and mortality. Recently, we first demonstrated a potential EV71 vaccine candidate based on virus-like particles (VLP). Whatever the inactivated EV71 or EV71 VLP vaccine adjuvanted with alum was intramuscular given to macaque monkeys, all these monkeys developed both specific humoral and cellular immune responses to EV71. Mucosal vaccines can efficiently induce secretory IgA at mucosal surfaces, thereby preventing or limiting infection at the site of enteroviruses entry. Until now there is only a little study about EV71 vaccine through mucosal. In developing vaccines, adjuvants are often used to augment the effects of vaccines by stimulating the immune system to respond more vigorously, and thus providing increased immunity to disease. Since Toll-like receptor (TLR) ligands can act as both the systematic and mucosal adjuvants. We like to use FliC or LTIIb-B5 (cooperate with Sub-project 2) as adjuvant to study the EV71 mucosal vaccine. Fms-like tyrosine kinase 3 ligand (Flt3L) is an agent that has been known as an immunological adjuvant to enhance global responses to vaccinations. PELC is an W/O/W nanoemulsion adjuvant. Adsorption of antigens to PELC can help retain the antigens at the intranasal site at a higher concentration, therefore providing a sufficient uptake time for DCs. Therefore in this project: Aim 1: We plan to use TLR ligands (include LTIIb-B5, an agonist to TLR2/1; poly I:C, an agonist to TLR3; MPLA, an agonist to TLR4; FliC, an agonist to TLR5; imiquimod, an agonist to TLR7/8; CpG, a ligand for TLR9) as an adjuvant to study the EV71 mucosal vaccine. Aim 2: We plan to use Flt3L or PELC as adjuvant to study the EV71 mucosal vaccine. Aim 3: According to the results from Aim 1 and Aim 2, we like to combine TLR ligand with Flt3L and/or PELC as adjuvants to study the EV71 mucosal vaccine. In this plan, mice will be intranasal immunized with inactivated virus with the following adjuvants: saline (negative control), cholera toxin (positive control), TLR ligand, Flt3L or PELC. Each mouse will intranasal immunized at 0, 3 and 6 weeks. To evaluate the humoral immune responses, the anti-EV71 IgG and IgA will be titrated, and the neutralization assay would be done. To evaluate the cellular immune responses, spleens would be harvested to test the splenocyte proliferation and cytokines assay. Therefore, our studies in this project may provide useful information for the developing of EV71 mucosal vaccine.腸病毒七十一型黏膜疫苗佐劑鼻腔內Toll 樣受體配體Enterovirus 71 (EV71)mucosal vaccineadjuvantToll-like receptor (TLR)ligand