CAS OpenIR
基于聚乳酸微球佐剂的乙肝疫苗研究
卢婷
Thesis Advisor马光辉
2020-07-01
Degree Grantor中国科学院大学
Degree Name博士
Degree Discipline生物化工
Keyword聚乳酸微球,双十八烷基二甲基溴化铵,Sting刺激剂,慢性乙肝模型鼠,冻干
Abstract

慢性乙肝的治愈之路充满挑战。与传统的抗病毒疗法相比,疫苗疗法是慢性乙肝预防和治疗的重要潜力途径。但是如何使疫苗获得有效的细胞免疫效果满足治疗性需求,具有更好的保护效果,是当前面临的重要挑战。本课题提出生物相容性好的聚乳酸(PLA)微球合理装载抗原和免疫刺激剂,实现按需递送的功能,作为治疗性乙肝疫苗,同时以PLA微球为佐剂探索免疫针次减少的疫苗制剂。课题具体研究内容如下:1)针对免疫刺激剂5,6-二甲基黄酮-4-乙酸(DMXAA)在特定细胞位置发挥作用的需求,成功制备了按需释放抗原的DP微球及抗原/刺激剂的DP-D微球,并验证了其在细胞水平的效果。通过向PLA微球中引入阳离子脂双十八烷基二甲基溴化铵(DDAB),微球可以温和高效携载乙肝表面抗原(HBsAg,吸附率85%以上);并且借助质子海绵效应,促进溶酶体逃逸,使DMXAA释放到胞质中,与其受体结合,激活下游通路。在分子水平上,DP-D与抗原组相比,其STING通路的IRF-7和IFN-β表达分别提高了12和8.2倍。同时微球携载HBsAg后易于被树突状细胞(DCs)摄取及引起溶酶体逃逸,提高DCs表面共刺激分子及MHC分子的表达,促进细胞因子分泌,在高效促进DCs活化的同时诱导后续免疫应答。2)评价了PLA微球佐剂在健康鼠上的体液和细胞免疫效果,探索了其应用于疫苗时的作用机制。微球疫苗制剂诱导了注射部位IL-1β和CXCL-10等炎症因子和趋化因子的表达,同时招募DCs及巨噬等免疫细胞到注射部位,经APCs转运至淋巴结后,促进了淋巴结内DCs、B及滤泡辅助T细胞(Tfh细胞)的活化。在免疫健康鼠后,诱导产生了高水平的HBsAg特异性抗体IgG,提高IgG2a/IgG1比例、Th1细胞数量及IFN-γ等细胞因子的分泌,实现了体液和细胞免疫的双重提升。3)成功构建了慢性乙肝(CHB)模型鼠,并基于该评价模型开展了微球疫苗制剂的免疫及治疗评价。采用尾静脉注射重组乙肝腺病毒的方式构建CHB模型鼠。结果表明,DP、DP-D组诱导产生高水平的HBsAg特异性抗体IgG,可促进脾细胞增殖活化与Th1型细胞因子的分泌,提升了HBsAg特异性CTL反应,产生免疫记忆起到保护效果。两组模型鼠血清中HBsAg转阴率均达到50%,显著降低了肝脏中HBcAg的表达,取得了良好的治疗效果。4)验证了以PLA微球为佐剂,用两针代替三针铝佐剂疫苗的长效免疫保护效果。微球疫苗制剂两针免疫后产生与铝佐剂相当的抗体水平,且抗体半年内维持较高水平,在HBsAg再次入侵时可快速产生抗体。优化了该疫苗制剂的冻干保护剂种类及相应浓度,考察了冻干制剂在室温下的稳定性。总体结果明确了微球疫苗佐剂具有减少免疫针次及可冻干的特性,具备临床转化的潜力。综上,本课题设计的PLA微球疫苗制剂应用于乙肝疫苗有较大潜力;Treatment of chronic hepatitis B (CHB) is full of challenge. Compared with traditional antiviral therapy, vaccine therapy is an important potential approach for the prevention and treatment of CHB. However, how to prime effective cellular immune response to meet the therapeutic requirements and endow a better protective effect is a great challenge. This dissertation proposes that biocompatible polylactic acid (PLA) microparticles (MPs) which rationally loaded with antigen and immunopotentiator according to the delivery demand to act as therapeutic hepatitis B vaccine. Meanwhile, PLA MPs were used as adjuvants to explore vaccines with reduced immunization dose. In detail, this thesis mainly includes the following issues: 1) Aim at the immunopotentiator need to function at specific cell locations, DP and DP-D MPs were successfully fabricated according to the release demand of antigen and immunopotentiator, and evaluated the immune response at cellular level. The introduction of cationic lipid didodecyldimethylammonium bromide (DDAB) into PLA MPs could assist them adsorb antigen gently and efficiently (adsorption rates were all above 85%); meanwhile DMXAA could release into the cytoplasm through the proton sponge effect, then interact with its receptor, activate downstream pathway. At the molecular level, the expression of IRF-7 and IFN-β in the STING pathway of DP-D increased by 12 and 8.2 times respectively compared with the antigen group. The MPs vaccines could facilitate cell uptake and lysosome escape, increase the expression of costimulatory and MHC molecules of dendritic cells (DCs), promote cytokines secretion, thus activating DCs and prime subsequent immune responses efficiently. 2) The humoral and cellular immune responses of PLA MPs vaccines on healthy mice were evaluated and the immunization mechanism was explored as vaccine adjuvant. MPs vaccines can induce the expression of inflammatory cytokines and chemokines such as IL-1β and CXCL-10 at the injection site, and recruit immune cells such as DCs and macrophages to the injection site. After being transported to lymph nodes by APCs, they can promote DCs, B and Tfh cells activation in lymph nodes. In healthy mice immunization experiment, high levels of HBsAg-specific IgG and IgG2a / IgG1 ratio were induced, the number of Th1 splenocytes, and the secretion of IFN-γ together with other cytokines were also elevated, indicating the MPs induce both humoral and cellular immune responses. 3) The chronic hepatitis B (CHB) model mice were successfully constructed, and the immune and therapeutic effect of the MPs vaccines were investigated. CHB model mice were constructed by tail vein injection of recombinant AAV/1.3HBV virus. The results indicated that DP and DP-D induced high-level HBsAg-specific IgG, promoted the splenocytes proliferation and activation as well as the secretion of Th1 cytokines. The HBsAg-specific CTL responses were improved and memory immune responses for protection were maintained. The serum HBsAg seroconversion rate of the two MPs vaccines both achieved 50% and the expression of HBcAg in the liver significantly reduced, indicating a favorable therapeutic effect. 4) The long-term immunoprotection effect of two dose PLA MPs vaccines instead of three dose aluminum vaccines were verified. The MPs vaccines produced comparable antibodies compared to aluminum adjuvant after two dose immunization schedule, and the antibodies maintained at a high level for half a year. When rechallenging with HBsAg, the mice can quickly produce antibodies. The type and corresponding concentrations of lyoprotectants were optimized, and the stability of lyophilized vaccines at room temperature was investigated. The results clarified that the MPs vaccine adjuvant has the capacity of reducing the immunization dose and can be lyophilized, thus possessing the potential for clinical transformation. In summary, the PLA MPs vaccines designed in this dissertation possess great potential for application in hepatitis B vaccines. 

Language中文
Document Type学位论文
Identifierhttp://ir.ipe.ac.cn/handle/122111/49689
Collection中国科学院过程工程研究所
Recommended Citation
GB/T 7714
卢婷. 基于聚乳酸微球佐剂的乙肝疫苗研究[D]. 中国科学院大学,2020.
Files in This Item:
File Name/Size DocType Version Access License
基于聚乳酸微球佐剂的乙肝疫苗研究.pdf(4877KB)学位论文 限制开放CC BY-NC-SAApplication Full Text
Related Services
Recommend this item
Bookmark
Usage statistics
Export to Endnote
Google Scholar
Similar articles in Google Scholar
[卢婷]'s Articles
Baidu academic
Similar articles in Baidu academic
[卢婷]'s Articles
Bing Scholar
Similar articles in Bing Scholar
[卢婷]'s Articles
Terms of Use
No data!
Social Bookmark/Share
All comments (0)
No comment.
 

Items in the repository are protected by copyright, with all rights reserved, unless otherwise indicated.