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长期以来，传染性疾病一直是人类健康的重大威胁之一，目前接种疫苗仍然是控制它的有效手段。现被批准临床应用的佐剂大多无法有效激活细胞免疫应答，且油乳佐剂存在需添加表面活性剂，并难以实现功能化修饰等缺陷，因此研发一种安全有效的疫苗佐剂尤为重要。针对上述问题，本研究工作提出了将油乳佐剂与铝佐剂相结合，构建颗粒稳定的乳液（Pickering乳液）。利用Pickering乳液在免疫应答上的独特优势，如可以增加与抗原提呈细胞的接触面积，动态促进免疫识别和应答等，从而提升免疫应答效果。与此同时，铝佐剂是经美国食品与药品管理局（Food and Drug Administration, FDA）批准临床应用的无机颗粒佐剂，储存不易降解，且具有良好的生物相容性。因此在提升疫苗佐剂效果的同时，可以增强Pickering乳液的储存稳定性。此外，由于Pickering乳液特殊的表面结构，抗原可以吸附在其缝隙中，从而起到一定保护抗原的作用，使抗原获得较好的热稳定性。具体研究内容如下：1. 对目前市售的三种商品化铝佐剂进行筛选，以含有单磷酰脂质A（Monophosphoryl lipid A, MPLA）的角鲨烯为油相，铝佐剂为颗粒稳定剂，制备得到了可以在37℃下稳定储存4个月以上的Pickering乳液（油相中含有MPLA的Alum adjuvant-stabilized Pickering emulsion, ALMPE）。其粒径均一、分散性良好，同时具有良好的生物安全性。在OVA预防性疫苗中，ALMPE可以有效提高血清中特异性抗体IgG的滴度，促进脾细胞中IFN-γ分泌细胞的活化和增殖。装载MUC1和OVA抗原的ALMPE可以分别有效抑制B16/MUC1黑色素瘤和E.G7/OVA淋巴瘤细胞的生长，提高荷瘤小鼠的生存率。将重组疟疾抗原装载在含CpG的Pickering乳液（水相中含有CpG的Alum adjuvant-stabilized Pickering emulsion, ALPE-CpG）上，可以同时引发Th1和Th2型免疫响应，与弗氏佐剂组免疫效果相当，并且没有副反应发生。2. 以异丙醇铝为原料，采用醇盐水解-水热法制备勃姆石纳米氢氧化铝颗粒，优化了水热反应条件，制备得到了结晶度高且均一性良好的勃姆石纳米氢氧化铝颗粒。以其作为稳定剂，含有单磷酰脂质A的角鲨烯作为油相，通过超声破碎法制备Pickering乳液（油相中含有MPLA的Bothmite-stabilized Pickering emulsion, BMPE），考察了颗粒浓度、水相成分、超声时间及功率对Pickering乳液粒径和稳定性的影响。成功制备得到了粒径均一、稳定性强的Pickering乳液。在OVA模式抗原的免疫实验中，其可以同时引发机体产生体液/细胞免疫应答。在乙肝疫苗初步热稳定性实验中，也展现出了对抗原具有一定的保护作用，为疫苗在不发达国家的运输及应用提供了便利。;Infectious diseases have been one of the major threats to human health in the long term, and vaccination is an effective method to defend it. However, most of adjuvants are unable to effectively activate the cellular immune response. Moreover, the emulsion adjuvant also includes surfactants, and it is difficult to be modified. Therefore，it is important to develop a safe and effective vaccine adjuvant. This research aimed to combine the oil-in-water emulsion with Alum adjuvant to construct a particle-stabilized emulsion (Pickering emulsion), which would increase the contact area and dynamically activate the immune recognition to enhance the immune response. Besides, Alum adjuvant, as inorganic particle adjuvant, has been approved by FDA for clinical application, which has good biocompatibility and stability. The storage stability and immune efficiency of the Pickering emulsion could be enhanced by using Alum as particle stabilizer. In addition, due to the special surface structure of the Pickering emulsion, the antigen could be adsorbed in the gap, and be protected by the particles. The thesis was divided into the following parts.1. Three commercial aluminum adjuvants were investigated. Squalene containing monophosphoryl lipid A (MPLA) was used as the oil phase and alum adjuvant was used as the particle stabilizer to prepare the Pickering emulsion (Alum adjuvant-stabilized Pickering emulsion with MPLA in the oil phase, ALMPE), which could be stored stably at 37°C for more than 4 months. The prepared Pickering emulsion possessed uniform size, good dispersibility and biocompatibility. For the OVA prophylactic vaccine, ALMPE effectively activated the titer of specific antibody IgG in serum and promoted the activation and proliferation of IFN-γ secreting cells in splenocytes. In therapeutic evaluations, ALMPE loaded with MUC1 and OVA antigens effectively inhibited the tumor growth of B16/MUC1 melanoma and E.G7/OVA, and improved the survival rate of tumor-bearing mice. Loading the recombinant malaria antigen on ALPE-CpG (Alum adjuvant-stabilized Pickering emulsion with CpG in the water phase) simultaneously induced Th1 and Th2 type immune responses. For the recombinant malaria vaccine, ALPE-CpG simultaneously induced Th1 and Th2 type immune responses, and showed the same immune effect as Freund's adjuvant .2. The boehmite-type aluminium hydroxide nanoparticles were prepared from aluminum isopropoxide by hydrolysis-hydrothermal methods. The boehmite-type aluminium hydroxide nanoparticles were spherical with good dispersity, uniform size distribution and high crystallinity. Pickering emulsion was prepared by using boehmite-type aluminium hydroxide nanoparticles as stabilizer and squalene containing MPLA as oil phase. The effects of preparation conditions on size distribution and stability of Pickering emulsion were investigated, including the concentration of nanoparticles, water phase and dispersion condition. In the immunoassay of OVA model antigen, the Pickering emulsion simultaneously induced the humoral/cellular immune response. In the preliminary heat stability test of hepatitis B vaccine, it also showed a protective effect on the antigen, which provided convenience for the transportation and application of the vaccine in the underdeveloped countries.
|吴楠. Alum-Pickering 乳液疫苗佐剂的研究[D]. 中国科学院大学,2019.|
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