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基于抗原装载方式的均一 PLGA 纳微球疫苗递送与佐剂系统的研究
刘婍
学位类型博士
导师马光辉
2016-07
学位授予单位中国科学院研究生院
学位授予地点北京
学位专业生物化工
关键词Plga 纳微球佐剂 表面偶联 内部包埋 免疫应答
摘要

生物降解聚合物纳微球作为疫苗递送与佐剂系统,具有诸多优势,近年来备受研究和关注。纳微球对抗原的装载方式主要通过表面携载和内部包埋,困扰纳微球表面偶联抗原的主要问题是纳微球表面活性集团少,很难实现对抗原的安全、高效偶联,而限制纳微球佐剂包埋抗原发挥免疫效应的主要问题在于纳微球无法实现抗原在胞内的可控释放,造成抗原提呈及后续免疫反应水平不足。因此,本论文采用不同的装载方式,合理地设计、构建高效的纳微球疫苗佐剂,针对性地提出了基于多巴胺修饰的仿病原体纳微球和胞内pH敏感型纳微球疫苗制剂两大新的策略,系统考察了免疫应答水平与佐剂效应,为今后新型纳微球佐剂的设计和构建奠定基础。具体研究内容包括以下四个方面:1)纳微球表面携载抗原作为疫苗佐剂的研究。选用FDA批准的聚乳酸-羟基乙酸(PLGA)为材料,以乙肝表面抗原(HBsAg)为抗原,采用O/W乳液-溶剂去除法结合快速膜乳化技术,制备了粒径约为900 nm、尺寸均一的PLGA纳微球。采用仿生材料多巴胺进行自聚镀层修饰,在纳微球表面成功携载抗原和免疫刺激剂,其尺寸与病原体相似,结构和成分上也能很好地模拟病原体。经细胞毒性实验和初步安全性评价可知生物相容性良好。该纳微球能很好地模拟病原体的侵染过程,在注射部位诱导炎性细胞因子和趋化因子的产生,同时招募单核细胞、中性粒细胞,以及抗原提呈细胞中的巨噬细胞和DCs,;并能有效地活化DCs,诱导强烈的抗原特异性细胞免疫应答,同时提升抗原特异性抗体的分泌水平。2)纳微球包埋抗原作为疫苗佐剂的构建。设计了胞内pH敏感型纳微球,选用PLGA为材料,以OVA为模型抗原,采用W/O/W复乳-溶剂去除法结合快速膜乳化技术,通过调节纳微球的结构由实心转变为“薄皮大腔”,并在内水相中添加碳酸氢铵,制备得到粒径约为900 nm、尺寸均一的胞内pH敏感型纳微球。在保证所包埋抗原结构稳定的基础上,纳微球在体外模拟DCs胞内内体、溶酶体和胞质的环境中,表现出了pH敏感响应释放抗原的特性。3)包埋抗原的胞内pH敏感型纳微球作为疫苗佐剂的免疫效果评价。纳微球在免疫系统中可控、快速释放抗原显著增强了其作为疫苗佐剂的免疫效果。纳微球通过pH敏感响应释放抗原,显著提高抗原的交叉提呈水平(是常规PLGA纳微球组的3.3倍)。并通过促进DCs活化与成熟,从而有效诱导淋巴细胞的活化。在效应免疫阶段,产生了强有力的T细胞杀伤效应,尤其是诱导脾细胞分泌IFN-γ和颗粒酶B的水平比常规PLGA纳微球组分别提高了105%和79%,并诱导更高水平的记忆性T细胞反应,提供了比铝佐剂更强的血清抗体保护。4)双重免疫刺激剂共递送的纳微球复合疫苗佐剂的构建及佐剂效应评价。为了进一步增强免疫效果,在胞内pH敏感型纳微球的基础上,将抗原装载在内水相中,油相共包埋咪喹莫特(IMQ)后,复乳萃取法结合快速膜乳化工艺制备得到粒径均一的纳微球,利用多巴胺镀层修饰在纳微球表面携载CpG,构建了双重免疫刺激剂共递送的复合纳微球疫苗佐剂。不仅通过进一步增强胞内的交叉提呈效应,还具有活化模式识别受体发挥双重免疫刺激的功能,双管齐下,更高水平地增强了抗原特异性免疫应答(包括更高水平的体液免疫应答、细胞免疫应答和免疫记忆)。由于纳微球复合佐剂生物安全性良好,作为新型疫苗佐剂应用于临床,具有值得期待的前景。综上所述,本论文通过探索性研究,根据纳微球对抗原的主要装载方式,并结合应用分子免疫刺激剂,设计和构建了极具发展潜力的新型纳微球疫苗递送与佐剂系统,获得了良好的佐剂效应,为今后开发安全、有效的疫苗佐剂提供了全新的思路。

其他摘要

Biodegradable polymer-based micro/nanoparticles (MPs/NPs) could function as effective adjuvants. Antigen loading by NPs could be mainly through surface conjugation and encapsulation, with some particular issues currently hampering their development. The surface inherent chemical inactivity of polymeric NPs such as poly (D,L-lactic-co-glycolic acid) (PLGA), raised a big challenge for antigen loading by surface conjugation. And for encapsulating antigens, the NPs couldn’t realize the adjustable antigen intracellular release resulting in weak immune response. Therefore, this thesis proposed pathogen-mimicking polymeric NPs based on dopamine polymerization and pH-responsive polymeric NPs with rapid antigen release behavior focusing on these challenges. In detail, this thesis mainly included the following issues:1) NPs conjugated antigen on the surface as antigen delivery and adjuvant system. Uniform-sized PLGA NPs of ~900 nm were fabricated using a premixed membrane emulsification technique followed by O/W emulsion-diffusion-evaporation method. The mussel-inspired biomimetic polydopamine (pD) not only served as a coating to NPs but also functionalized NP surfaces, and incorporated hepatitis B surface antigen (HBsAg) and TLR9 agonist unmethylated cytosine-guanine (CpG) motif with the pD surface. The NPs possessed pathogen-mimicking manners owing to their size, shape, and surface molecular immune-activating properties given by CpG. The biocompatibility and biosafety of these pathogen-mimicking NPs were confirmed. Pathogen-mimicking NPs might hold great potential as vaccine delivery and adjuvant system due to their ability to: 1) enhance cytokine secretion and immune cell recruitment at the injection site; 2) significantly activate and maturate dendritic cells (DCs); 3) induce stronger humoral and cellular immune responses in vivo.2) With the respect of antigen encapsulated in NPs, pH-responsive PLGA NPs with rapid antigen intracellular release behavior in APCs were fabricated. Using the W/O/W emulsion-diffusion-extraction method combined with the premixed membrane emulsification technique, uniform-sized pH-responsive PLGA NPs with rapid antigen release behavior by adjusting the ratio of the inner water phase and oil phase and adding NH4HCO3 in the inner water phase were successfully prepared. The NPs, had thin shells and large inner space.NH4HCO3 co-encapsulated with antigen (ovalbumin, OVA) could regulate release in endosomes and lysosomes, acting as an antigen release promoter in DCs. And antigens loaded in the NPs retained activity from the analysis results of fluorescent and CD spectra.3) The adjuvanticity of pH-responsive PLGA NPs with rapid antigen intracellular release behavior were further investigated including antigen presentation, DC activation and maturation, and the subsequent in vivo immune response. After uptake by DCs, antigens encapsulated in pH-responsive PLGA NPs could escape from lysosomes into the cytoplasm and be crosspresented (increased about 330% than normal PLGA NPs). Moreover, the NPs promoted DC activation and maturation. Mouse immunization with the NPs induced greater lymphocyte activation, more antigen-specific CD8+ T cells, stronger cytotoxic capacity (IFN-γ and granzyme B, increased respectively about 105% and 79% than normal NPs), enhanced antigen-specific IgG antibodies, and higher serum IgG2a/IgG1, indicating excellent cellular immunity. The NPs also improved generation of memory T cells to protect against reinfection.4) Based on the aforementioned investigations, the dual-immunopotentiator-loaded polymeric NPs were constructed and their adjuvanticity was further evaluated. By incorporating imiquimod (IMQ) and conjugating CpG onto the surface of pH-responsive PLGA NPs, the efficacy of NPs exhibited great enhancement in activating antigen-presenting cells, enhancing crosspresentation and eliciting PRRs recognition. Dual-immunopotentiator-loaded NPs showed superior adjuvanticity including potent humoral and cellular immune responses. Considering adjuvanticity and safety profiles, the as-constructed NPs were promising robust vaccine adjuvant.In conclusion, these results indicated that PLGA NPs-based adjuvant with superior activity could be achieved by manipulating antigen loading modes and combining molecular immunopotentiators with NPs.These might have significant implications for rational vaccine design, and break new ground for the development of vaccine adjuvants in the future.. 

语种中文
文献类型学位论文
条目标识符http://ir.ipe.ac.cn/handle/122111/22904
专题研究所(批量导入)
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刘婍. 基于抗原装载方式的均一 PLGA 纳微球疫苗递送与佐剂系统的研究[D]. 北京. 中国科学院研究生院,2016.
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