摘要:子計劃一: 建構以 HBc 蛋白形成類病毒顆粒為基礎之可誘發黏膜性免疫反應的表面抗原決定區域疫苗-以 RSV 疫苗為例呼吸道融合病毒為引起呼吸道感染重症的主要感染原之一,然而迄今臨床上仍無安 全的疫苗可用。這是因為 RSV 減毒疫苗誘發 Th2 過敏反應,導致血管內膜發炎。所以 發展誘發 Th1 免疫為主的疫苗為現今主要策略。已知 HBc 類病毒顆粒可以做為口服疫 苗,且其主要誘發 Th1 免疫反應,而且目前仍無結合 HBc 類病毒顆粒的 RSV 疫苗被發 展出來。因此,我們希望藉由這樣的策略,將 RSV 主要的 F 抗原上兩段具中和病毒能 力的抗原決定區域插入 HBc 三個區域;透過蛋白質表現純化,利用電顯檢視類病毒顆 粒是否形成。然後以小鼠為模式,利用注射、口服和噴鼻等途徑,測試各式顆粒誘發免 疫反應的能力和形式。子計劃二: 重組鞭毛蛋白與第二 b 型忌熱性腸毒素 B 次單元用於 H5N1 禽流感疫苗黏 液免疫佐劑開發高病原性禽流感 H5N1 病毒能造成人類高致死率的呼吸道感染疾病,開發能引發黏 膜性免疫反應的禽流感疫苗更是需要。因為刺激類鐸受體可增強全身性與黏膜性免疫, 本計畫以能引發 TLR-5 的鞭毛蛋白 (FliC)與能引發 TLR-2/1 的第二 b 型忌熱性腸毒素 B 次單元 (LTIIb-B5)來開發 H5N1 黏膜疫苗,將以三劑鼻腔免疫方式研究應用於血凝素次 單位疫苗開發,或是配合去活化與次單位疫苗以兩劑 prime-boost 交叉免疫,以評估黏 膜蛋白佐劑的功能。同時將以 sortase A 蛋白酶進一步結合成 FliC/H5HA 與 H5HA/LTIIb-B5 融合巨分子進行鼻腔免疫。本研究成果可提供發展新型黏液免疫力的 H5N1 禽流感疫苗。子計劃三: 腸病毒七十一型類病毒顆粒黏膜疫苗之開發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(EV71 VLP)為疫苗,已經成功完成彌猴動物試驗。我 們發現腸病毒類病毒顆粒與氫氧化鋁混合後肌肉注射彌猴可以得到相當好的抗體濃 度,而且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 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。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。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和腸病毒類病毒顆粒為疫苗基礎,加上Alum 或 Flt3L 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: 將依據吳夙欽教授的計畫〝重組鞭毛蛋白與第二 b 型忌熱性腸毒素 B 次單元用 於 H5N1 禽流感疫苗黏液免疫佐劑開發〞的研究結果,選擇鞭毛蛋白 (FliC)或第二 b 型 忌熱性腸毒素 B 次單元 (LTIIb-B5)作為佐劑來開發腸病毒黏膜疫苗。Aim 3: 我們將依據 Aim 1 和 Aim 2 的研究結果,選擇 Flt3L 合併 FliC 或 LTIIb-B5 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 IgA 和 IgG 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和 腸病毒類病毒顆粒為疫苗,並利用 Flt3L 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。子計劃四: 黏膜疫苗研發的核心計畫黏膜免疫反應與一般經由注射的免疫反應有些不同,主要誘發的抗體為 IgA,而且 又比全身性注射的免疫反應較容易誘發耐受性。但是,黏膜給予的疫苗由於較方便,所 以如果能夠找到一個較好的方法來誘發黏膜反應,將能夠讓疫苗更容易推廣和普及。在 此一研究計劃中我們將建立各種免疫評估方法來研究此一黏膜疫苗的效果,所以在此一 核心計畫中我們將分別建立方法來測定在此整合計畫中所使用的各種疫苗在致敏後所 誘發的免疫反應。由於黏膜的免疫反應會牽涉到免疫調控,因此經由黏膜途徑進入的抗 原會遇到究竟是會誘發一個好的免疫反應,或是會誘發免疫耐受性,一直是在製備黏膜 疫苗時最重要而且需要突破的部份。在此一整合計畫中,我們將建立各個子計畫所使用的抗原蛋白的抗體測定方法,其中包括利用抗體濃度和測定不同抗體分泌性 B 細胞的頻 率,同時也進行抗原特異性的 T 細胞增殖反應,並分析這些 T 細胞的細胞激素製造情形。 經由這些相關的免疫分析,我們能夠更清楚地了解黏膜疫苗的功效和特性,並從中研發 出一些能夠應用到未來黏膜疫苗的應用上。Aim 1:我們將建立各種測定方法來測定各個子計畫中黏膜疫苗的免疫反應,主要包括 IgA 和 IgG 的抗體測定,抗原特異性的 T 細胞增殖反應。此外,為了進一步了解這些黏 膜疫苗注射後的免疫走向,我們也將分別測定這些抗原刺激的 T 細胞製造細胞激素的情 形。尤其是了解在幫忙製造 IgA 的 T 細胞是否會分泌較高量的 IFN-g 或是 TGF-b,由於 TGF-b 是在 B 細胞由 IgM 轉變成 IgA 的重要細胞激素;但是 TGF-b 本身又是扮演一個 具有免疫調控角色的細胞激素。所以,在這些黏膜疫苗注射中這些細胞激素的製造情形 也將會影響到這些黏膜疫苗的未來應用。Aim 2:我們將整合在各個子計畫中所使用來增強黏膜免疫反應的方法和分子,讓這些 相關的黏膜免疫促進分子和佐劑,可以更方便地應用到各個子計畫中。目前的子計畫中 分別有使用 Flt-3 ligand,GM-CSF,Flagellin 等物質來增強黏膜的免疫反應,同時利用 不同的疾病抗原來進行黏膜疫苗的研發。由於,要找到更多的新穎分子可以來幫忙刺激 黏膜反應,我們也將建立樹突細胞的培養,來研究那些分子能夠更有效地刺激樹突細胞 而達到活化免疫反應的目的。Aim 3:此一核心計畫實驗室也將在未來三年內繼續研發能夠促進黏膜免疫反應的分 子,可以應用到這些相關的子計畫,目前能夠應用到促進黏膜的免疫反應上。目前的黏 膜疫苗研發主要是著重於鼻腔和呼吸道的疫苗研發,因此研發 nanoparticles 的方法來給 予這些疫苗抗原也是一個很重要的研究。在子計畫中,在此一計畫中,主要是要研發能夠誘發較佳免疫反應的黏膜疫苗,因此本計畫將是此一 研究計畫的核心計畫。在此一核心實驗室中我們將要建立各種疫苗的免疫反應測定方 法,其中主要包括血中抗原特異性抗體濃度測定、抗原特異性的 T 細胞增殖反應、T 細 胞製造細胞激素的情形、抗原特異性抗體分泌的 B 細胞頻率測定和不同細胞激素製造的 T 細胞頻率。同時,也要建立樹突細胞培養的平台來分析用在黏膜疫苗中來增進免疫反 應的物質,以找出更多有效刺激黏膜免疫反應的物質。此外,此一核心計畫也將配合子 計畫中的研究內容,來找出更好的
Abstract: Respiratory Syncytial Virus (RSV) has been recognized as the most important viral pathogen leading to severe respiratory tract diseases. Until now there is no safe vaccine in clinical use.One of the important factor for RSV vaccine is the requirement of inducing TH1-biased immune response. Since HBc-based VLP vaccine strategy has not been reported for RSV, oral immunization with HBc VLP was proven to induce TH1 response. Taken together, we are going to develop a HBc VLP-based mucosal immune system against RSV. In this study, we have inserted two major neutralizing epitopes of RSV F protein into HBc gene. After protein expression and purification, HBc VLP formation would be confirmed by EM examination. Different immune routes and strategies would be applied to test the immunity of different VLP constructs, including oral and intranasal injection. Respective immune markers would be tested to determine what kind of mucosal immune responses occurred.Highly pathogenic avian influenza (HPAI) H5N1 viruses can cause severe respiratory diseases in humans with a relatively high mortality rate around 60%. Development of H5N1 mucosal vaccines is thus particularly interesting for generating protective mucosal immunity. Since Toll-like receptor (TLR) ligands can act as both the systematic and mucosal adjuvants, we propose to investigate (a) flagellin (FliC), a TLR-5 ligand and (b) type IIb heat labile enterotoxin B subunit (LTIIb-B5), a TLR-2/1 ligand as the mucosal adjuvants for H5N1 vaccine development. Three-dose immunization with H5N1 HA subunit vaccine will be conducted in mice using intranasal immunization. Two-dose prime-boost immunization regimens with the inactivated H5N1 vaccine followed by subunit H5HA vaccine will be also studied using both mucosal adjuvants. The fusion antigens of FliC/H5HA and H5HA/LTIIb-B5 proteins through sortase A-mediated posttranslational fusion will be also investigated for intranasal immunization. Our findings may provide useful information for developing mucosal H5N1 vaccines.Enterovirus 71 (EV71) is an etiologic agent responsible for seasonal epidemics ofhand-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). When the VLP vaccine adjuvanted with alum was intramuscular given to macaque monkeys, these monkeys developed both specific humoral and cellular immune responses to EV71. Hence, VLP-based EV71 vaccine is a potential vaccine against EV71 infection. 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. Fms-like tyrosine kinase 3 ligand (Flt3L) is an agent that has been known as an immunological adjuvant to enhance global responses to vaccinations. Since Toll-like receptor (TLR) ligands can act as both the systematic and mucosal adjuvants. Flagellin (FliC), a TLR-5 ligand and type IIb heat labile enterotoxin B subunit (LTIIb-B5), a TLR-2/1 ligand both can as the mucosal adjuvants for vaccine development. Therefore in this project:Aim 1: We plan to use Flt3L as an adjuvant to study the EV71 mucosal vaccine.Aim 2: According to the result of the plan “Recombinant Flagellin and Type IIb Heat Labile Enterotoxin B Subunit as Mucosal Adjuvants for H5N1 Vaccine Development” from Prof. Suh-Chin Wu. We like to use FliC or LTIIb-B5 as adjuvant to study the EV71 mucosal vaccine.Aim 3: According to the result from Aim 1 and Aim 2, we like to combine Flt3L with FliC or LTIIb-B5 as adjuvant to study the EV71 mucosal vaccine.In this plan, mice will be intranasal immunized with inactivated virus or EV71 VLP with the following samples: saline, aluminum hydroxide, Flt3L, FliC or LTIIb-B5. Each mouse will intranasal immunized at 0, 3, 6, and 12 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.Mucosal immune response is somewhat different from systemic immune response by predominantly producing IgA antibody. In addition, the phenomenom of mucosal tolerance has been documented in many studies. It is critical to explore the methods which are able to induce better immune response instead of mucosal tolerance. In this study, we like to establish the methods of assaying both cellular and humoral immune response to support all thesub-projects in the integrated project. We expect to establish the methods in assessing both humoral and cellular immune responses induced by mucosal vaccine immunization.Aim 1: We like to establish the method of ELISA to determine both IgA, IgG1 and IgG2a antibody against antigenic proteins such as influenza antigen, EV71 and RSV core antigen. In addition, we also like to establish the method of antigen-specific T cell proliferative response. Cytokine profile of antigen-stimulated T cells will also be assayed to further understand the immunogenic nature of mucosal vaccine. Transforming growth factor-b (TGF-b) has been found to play the critical role in class switch from IgM to IgA, assay of TGF-b production is very important for the assessment of efficacy of mucosal vaccine. Further, ELISpot will be used to determine the frequency of antigen-specific B cells and T cells.Aim 2: We aim to identify the major compoents which can induce good mucosal immune responses with antigenic protein. In this integrated project, we aim to apply the components or reagents to enhance the mucosal immune responses. In the sub-project certain molecules such as flagellin, Flt-3 ligand and GM-CSF will be used to enhance mucosal response of mucosal vaccines used in the sub-project. In addition, we also like to establish the culture of dendritic cells for the screening the potential compounds with the ability to enhance immune responses. Aim 3: Since most of the vaccine antigens will be delivered through mucosal route, to find the tool in increasing the uptake of mucosal antigen is also critical for the development of mucosal vaccine. We like to apply the nanoparticle to deliver the mucosal vaccine to local action site to induce higher immune response with mucosal vaccine. In addition to the development of effector molecules as the adjuvant for mucosal vaccine, we also aim to study the effect of nanoparticle for enhancing mucosal immune response.In this project, we plan to set up the methods to determine antigen-specific antibodies of different isotypes, antigen-specific T cell proliferative response and also cytokine profile of antigen-stimulated T cells. In addition, we also aim to establish the methods of ELISpot to determine the frequency of antigen-specific B cells and T cells. The dendritic cells culture for the screening the potential mucosal immunity enhancing reagents will be also established in this core sub-project. We believe the core sub-project here could provide the best supportive role for the integrated project of mucosal vaccine development.