CAS OpenIR
乳液颗粒化策略在疫苗佐剂中的应用
夏宇飞
Thesis Advisor马光辉
2018-07
Degree Grantor中国科学院研究生院
Degree Name博士
Degree Discipline生物化工
Keyword乳液颗粒化,疫苗佐剂,粘膜免疫响应,Pickering乳液,肿瘤免疫治疗
Abstract

随着疫苗产业的长足发展,开发一种安全、有效的疫苗佐剂成为研究的重点和难点。针对现有临床应用佐剂在功能化修饰、免疫刺激物的多级装载以及重现天然病原体柔性等方面的缺陷,本研究提出乳液颗粒化策略,并对佐剂进行合理化设计,使其兼具油乳佐剂和颗粒佐剂的优势。利用内向乳液颗粒化技术构建内部为乳滴,外部有坚硬高分子外壳的核壳复合结构,实现对抗原以及功能化成分(全反式维甲酸,retinoic acid, RA)的多级装载,同时引发有效的系统性免疫与粘膜免疫应答,拓宽疫苗的免疫保护广度。另一方面,通过外向乳液颗粒化策略建立颗粒稳定的乳液(Pickering乳液),开发其柔性和流动性,增大与免疫细胞的接触面积,动态地促进免疫识别和应答,提升疫苗的免疫响应深度。论文具体工作展开如下: 1. 内向乳液颗粒化策略构建硬乳液颗粒实现RA与抗原的协同递送与时空耦合性释放,促进溶酶体逃逸和交叉递呈。硬乳液颗粒(253.3 ± 35.2 nm)呈现核壳复合结构,内部为包埋RA的角鲨烯乳滴,外部为具备正电荷的PLGA/DDAB混合壳层,并实现了高RA包埋率(83.6 ± 4.3%)和抗原吸附率(88.5 ± 4.2%)。在溶酶体内产生 “胞内熟化现象”,颗粒间聚并,产生更大粒径且更小比表面积的微囊,展现出更加优异的RA缓释性能。并且,其正电荷的表面产生“质子海绵效应”,导致溶酶体膜破裂而释放抗原。从而在胞内实现RA长效而持久的缓释,并促进抗原在细胞质途径中的交叉递呈。 2. 利用硬乳液颗粒构建“免疫车票”策略,通过肌肉注射的方式,同时引发系统性免疫和粘膜免疫应答。硬乳液颗粒首先募集树突状细胞(dendritic cells, DCs),并提升其对抗原的摄取及交叉递呈。通过实现抗原和RA的时空耦合性联合递送,促进RA/RAR信号通路,同时抑制RA的免疫抑制效果。其诱导DCs产生肠粘膜归巢受体CCR9,提升细胞中RALDH2酶的活性,诱导RA的分泌,进一步引发外周免疫细胞沿着体内趋化因子CCL25浓度梯度向肠粘膜免疫组织的归巢,强化粘膜免疫应答。进而,在OVA和EV71疫苗中,促进了抗原特异性IgG抗体滴度以及杀伤性T细胞的活化。并在肠粘膜淋巴组织中激活了最高水平的递呈抗原的DCs、抗原特异性杀伤T细胞以及抗原特异性粘膜抗体IgA的响应,具备同时激活系统性和肠粘膜免疫应答的佐剂效应。 3. 利用外向乳液颗粒化策略构建模拟病原体柔性和流动性的Pickering乳液(PPAS)。PPAS具备颗粒吸附的表面,为抗原吸附提供了巨大的比表面积,大大提升了抗原的载量,模拟出了病原体表面抗原密集而重复的排列,有利于其更好地刺激免疫应答。并且,其可以在细胞膜表面发生柔性形变,增大了与免疫细胞表面的接触面积。在接触区域内,PPAS上的抗原可以在缝隙中流动,从而动态地激活免疫细胞的识别和摄取,具备提升免疫效果的佐剂潜力。与此同时,PPAS具备良好的室温储存和冻融稳定性,利于大规模生产和临床应用。 4. PPAS增强抗原内吞以及交叉递呈,通过募集并激活APCs,以及促进淋巴结成熟来提升免疫应答。PPAS强化抗原摄取以及共刺激分子CD86的表达。在溶酶体的酸性条件下,PPAS表面羧基发生质子化而使其表面电荷翻转呈正电荷,致使溶酶体内膜不稳定而破裂,实现溶酶体逃逸,促进抗原的交叉递呈。PPAS在注射部位形成抗原储库效应,有效募集APCs并促进其活化和交叉递呈。并且,刺激APCs向淋巴结归巢,实现抗原在淋巴结中大量而长效的富集,促生发中心活化,引发高效适应性免疫响应。 5. PPAS在OVA、H1N1流感疫苗以及MUC1抗肿瘤短肽疫苗中展现出优异的预防性和治疗性免疫佐剂效果。与商品化佐剂相比,PPAS有效激活体内抗原特异性抗体IgG的表达,以及抗原特异性杀伤T细胞的活化。在预防性效果表征中,PPAS得到了最高的H1N1流感病毒和E.G7/OVA淋巴瘤细胞的攻毒保护率。在肿瘤治疗方面, PPAS可以有效调动脾细胞中IFN-g分泌T细胞的活化和增值,显著杀伤肿瘤,并抑制MUC1/B16黑色素瘤以及E.G7/OVA淋巴瘤细胞的生长,提升荷瘤小鼠生存率。;With the rapid development of vaccine industry, accumulating research interest has been concentrated into the design of safe and efficient adjuvant. However, the limitations on the functionalization, multi-component loading, and recapitalizing the dynamic remodeling of natural pathogens hampered the clinical applications of these adjuvants. To address this, this work developed emulsion particulate strategy to combine the advantages of emulsion and particulate adjuvant. Through intrinsic emulsion particulate strategy, hierarchical structures were constructed with oily core and solid shell to co-encapsulate antigens and immune potentiator (retinoic acid, RA), which simultaneously provoke systemic and mucosal immunity to broaden the breadth of the immune responses. In addition, through extrinsic emulsion particulate strategy, particle-stabilized emulsion (Pickering emulsion) was developed to mimic the pliability and lateral mobility of the natural pathogens, which would increase the contact area and dynamically activate the immune recognition to deepen the depth of the immune responses. The thesis was unfolded as follow:1. By intrinsic emulsion particulate strategy, solid emulsion capsules were prepared to facilitate the spatial-temporal delivery of antigen and RA, and induced evident endosomal escape and cross-presentation of antigens. The core-shell structure was constructed with RA-loaded squalene on the inside, covering with the positive-charged PLGA/DDAB shells (253.3 ± 35.2 nm), which harbored high RA encapsulation efficiency (83.6 ± 4.3%), and antigen adsorption (88.5 ± 4.2%). Within the lysosomes, the capsules experienced “intracellular ripening”, that aggregated into larger sizes with smaller specific surface area, and manifested enhanced RA control-release behavior. Meanwhile, the positive charged surfaces demonstrated “proton sponge effect”, which caused the rupture of lysosomes for cytosolic delivery of antigens. Accordingly, the capsules elicited long-lasting release of RA and the cross-presentation of antigens.2. Through solid emulsion capsules, “immunoticket” strategy was developed to simultaneously activate systemic and mucosal immune responses via intramuscular injections. The solid emulsion capsules stimulated the recruitment, antigen uptake and cross-presentation of dendritic cells (DCs). Through spatial-temporal delivery of RA and antigen, RA/RAR signal was activated to stimulate the expression of gut-homing receptor, CCR9. In addition, the enzyme activity of RALDH2 was boosted to secret RA, which caused the gut-tropism of other immunocytes to home to the intestines along the concentration gradient of chemokine CCL25, and provoked the mucosal immunity. Meanwhile, RA-involved regulatory T responses were attenuated. In OVA and EV71 vaccinations, the capsules elicited potent antigen-specific IgG titer, cytotoxic T lymphocyte engagement and antigen-specific mucosal antibody IgA secretion, which proved to stimulate both systemic and mucosal immune responses.3. Through extrinsic emulsion particulate strategy, PLGA nanoparticle-stabilized emulsion (Pickering emulsion, PPAS) was developed to mimic the elasticity and lateral mobility of the natural pathogens. With the particle-adsorbed surfaces, PPAS manifested large specific surface area and increased antigen loading capability to recapitalize the condense array of surface antigens on the pathogen. On the cellular membrane, PPAS demonstrated force-dependent deformation to increase the contact area. Within the contact zone, antigens laterally moving to dynamically activate the recognition and internalization of DCs for enhanced immune responses. And, PPAS also harbored high storage and freeze-thaw stability for large-scale production and clinical applications.4. PPAS stimulated the internalization and cross-presentation of antigens, and boosted the recruitment and activation of APCs, which facilitated the mature of lymph nodes for enhanced immune responses. Under the acidic environment, the carboxyl groups on the surface was protonated to demonstrate positive charges, which caused the rupture of lysosomes for cytosolic delivery, and subsequently cross-presentation of the antigens. In addition, PPAS formed antigen depot at the injection sites to potently recruit and activate antigen presenting cells (APCs), and provoke the homing of APCs to the draining lymph nodes. The primed APCs maintained antigen persistence and germinal center engagement within the lymph nodes for enhanced adaptive immune activations.5. In OVA, H1N1 and MUC1 vaccinations, PPAS demonstrated robust prophylactic and therapeutic adjuvant activity. Compared with commercial adjuvants, PPAS increased antigen-specific IgG titer and cytotoxic T lymphocytes activation. In prophylactic vaccinations, PPAS induced highest survival rates in H1N1 virus challenge model and E.G7/OVA tumor challenge model. In therapeutic evaluations, PPAS stimulated the actrivation and proliferation of IFN-g secreting cells for tumor lysis, and delayed the tumor growth and onset of E.G7/OVA tumor and MUC1/B16 melanoma. 

Language中文
Document Type学位论文
Identifierhttp://ir.ipe.ac.cn/handle/122111/26902
Collection中国科学院过程工程研究所
研究所(批量导入)
Recommended Citation
GB/T 7714
夏宇飞. 乳液颗粒化策略在疫苗佐剂中的应用[D]. 中国科学院研究生院,2018.
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