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稳定的抗原和安全、有效的佐剂是疫苗研究的重点和难点。针对灭活口蹄疫病毒（inactivated foot and mouth disease virus, iFMDV）抗原稳定性差、极易发生裂解导致免疫活性降低，目前常用商业化的ISA 206乳液佐剂难以有效激发细胞免疫，且会进一步降低iFMDV稳定性的问题，本课题在深入研究iFMDV抗原体外稳定性对体内免疫活性的影响规律基础上，提出以具有良好生物相容性的正电荷固体脂质纳米颗粒（cationic solid lipid nanoparticles，cSLN）和表面修饰Zn2+的壳聚糖纳米颗粒（CP-PEI-Zn）为佐剂，设计可以在保护抗原结构稳定的同时，有效提升抗原免疫应答效率的新佐剂和新的抗原运载方式。主要研究内容与结果如下：（1） 研究了iFMDV体外稳定性对体内免疫活性的影响及其规律。分别通过疏水层析、聚乙二醇沉淀、疏水层析结合分子排阻层析、以及疏水层析结合聚乙二醇沉淀等四种工艺纯化制备iFMDV，发现第四种工艺所制备的抗原具有最好的稳定性，免疫小鼠的抗体水平也最高。进一步改变第四种纯化工艺制备抗原的溶液环境以准确调控iFMDV的稳定性，在抗原纯度和免疫剂量相同条件下开展细胞和动物实验，进一步证实了抗原越稳定，越能有效激活骨髓源树突状细胞（BMDC），促进iFMDV特异性IgG和IgM抗体的分泌。（2） 以山嵛酸甘油酯为脂质材料，以双十二烷基二甲基溴化铵（DDAB）为阳离子脂质体，通过O/W乳化法，制备出粒径250 nm左右，分散性良好、带有正电荷的cSLN，用于静电吸附146S（即颗粒完整的iFMDV）。发现低离子强度有利于cSLN对146S的吸附，却也会导致146S的快速裂解。在10 mM PB，0.075 M NaCl（pH 8.0）的最适负载条件下，cSLN对146S的吸附效率为87.8%，负载量为70.2 mg/mg。透射电镜可以观测到cSLN表面完整的146S颗粒，且负载于cSLN上的146S裂解温度Tm提高了1.2°C；高效液相层析尺寸排阻色谱（HPSEC）分析从cSLN表面洗脱下来的146S，证实146S颗粒结构的完整性。上述结果显示cSLN作为iFMDV佐剂有利于保持抗原结构的稳定。（3） 通过细胞和动物实验评价了cSLN作为iFMDV佐剂的效果。cSLN的运载使BMDC对146S的摄取比例提高了6倍以上，BMDC表面共刺激因子及MHCI表达水平显著提高。小鼠实验结果显示，cSLN显著促进FMDV的体液免疫和细胞免疫，特异性抗体（IgG，IgG2a和IgG1）的分泌水平以及记忆T细胞的增殖活化，均优于目前广泛应用的ISA 206油乳佐剂。（4） 基于iFMDV表面存在大量组氨酸可以与过渡金属离子配位结合的机理，制备表面修饰Zn2+的交联壳聚糖纳米颗粒（CP-PEI-Zn），提出通过iFMDV-Zn2+配位结合运载抗原的新模式。HPSEC分析证实了CP-PEI-Zn负载146S主要通过与Zn2+的配位作用，而PEI修饰的壳聚糖（CP-PEI）则通过静电作用负载146S。146S@CP-PEI-Zn的Tm略高于146S@CP-PEI，表明配位结合更有利于146S的稳定。动物实验结果表明，CP-PEI-Zn和CP-PEI用于iFMDV佐剂，在诱导特异性抗体反应、B淋巴细胞活化、T细胞增殖和效应记忆T细胞分化方面均优于ISA 206乳液佐剂。相比于CP-PEI，CP-PEI-Zn对效应记忆T细胞的增殖和细胞因子的分泌有更强的促进作用，表明壳聚糖颗粒与146S配位结合方式诱导了更强烈的细胞免疫应答。综上所述，本研究所制备的正电荷固体脂质纳米颗粒和壳聚糖微球稳定了抗原结构并增强了抗原的体液免疫和细胞免疫水平，并且生物安全性好，制备简单，成本低廉，有广阔的应用前景。;Much efforts have been devoted to improving stability of antigen and design of safe and effective adjuvant for vaccine. Inactivated foot and mouth disease virus (iFMDV), also known as 146S, is extremely unstable and easily dissociated, leading to severe decreace in its immunogenicity. The stability of iFMDV in the most widely employed commercial ISA 206 emulsion adjuvant was reported even worse, meanwhile, the ISA 206 was not efficient in stimulation of cellular immunity. To address these problems, the present work proposed biocompatible cationic solid lipid nanoparticles (cSLN) and zinc ion-chelated chitosan particles (CP-PEI-Zn) as potential vaccine adjuvant for iFMDV, on the basis of in-depth study of effect of antigen stability on its in vivo immunoactivity. The new adjuvant and the novel antigen delivery pattern were favorable for structural integrity of iFMDV and effectively improved the efficiency of the immune response of the antigen.The main results and findings are as follows: (1) The influence of iFMDV stability on its in vivo immune activity was investigated. Four different purification processes, including hydrophobic chromatography, PEG 6000 precipitation, hydrophobic chromatography combined with size exclusion chromatography, as well as hydrophobic chromatography combined with PEG 6000 precipitation were developed to purify iFMDV. The iFMDV purified from the fourth process showed the highest stability and induced strongest immune response in immuninzed mice. To make more accurate investigation on influence of antigen stability and its immunogenicity, the stability of iFMDV purified from the fourth process was manipulated by changing the buffer solution to ensure an identical antigen and inoculation dosage. Results showed that more stable iFMDV could activate bone marrow derived dendritic cells (BMDC) and stimulate the secretion of FMDV specific IgG, IgM antibodys more effectively, which further confirmed the positive correlation between iFMDV in vitro statality and its in vivo immunogenicity.(2) The cSLN was prepared by O/W emulsion method with matrix lipid compritol 888 ATO and modified by cationic lipid didodecyldimethylammonium bromide (DDAB). The cSLN with diamter about 250 nm has good dispersibility and positive surface zeta potential to load negatively charged iFMDV via electrostatic adsorption. It was found that the buffer with low ion strength could enhance antigen adsorption but accelerate the dissociation of iFMDV. Then the adsorption condition was optimized to protect the structure of iFMDV and strengthen the loading capacity of cSLN. In the buffer containing 10 mM PB and 0.075 M NaCl (pH 8.0), cSLN could adsorb 87.8% initially added antigen, whose loading capacity reached 70.2 mg/mg. Loading iFMDV onto cSLN by electrostatic attraction did not destruct iFMDV particle structure as measured by high performance size exclusion chromatography (HPSEC) and transmission electron microscopy (TEM). The Tm related to iFMDV dissociation increased for 1.2°C after loading on cSLN, indicating a good biocompatibility of cSLN for this unstable antigen.(3) The adjuvant effect of cSLN was evaluated by both celluar and animial experiments. The cSLN loaded iFMDV enhanced antigen uptake by 6-fold and activation of bone-marrow-derived dendritic cells (BMDCs) with augmented expression of CD86, CD40, and MHC I. In the animal trials, cSLN significantly enhanced both humoral and cellular immune response in immunized Balb/c mice, with higher FMDV specific IgG, IgG2a and IgG1 antibodys titers as well as more memory T cells than that induced by widely used ISA 206 emulsion adjuvant.(4) Based on the principle that the histidine (His) residuals rich in iFMDV could coordinatedly interact with transition metal ions, zinc chelated chitosan particles (CP-PEI-Zn) were prepared to deliver iFMDV via the coordination interaction between iFMDV and Zn2+. The iFMDV loaded on CP-PEI-Zn showed better thermal stability than that on CP-PEI, as revealed by a slightly higher transition temperature (Tm). After subcutaneous immunization in female Balb/C mice, antigens loaded on CP-PEI and CP-PEI-Zn all induced higher specific antibody titers, better activation of B lymphocytes, and more effector-memory T cells proliferation than the free antigen and iFMDV adjuvanted with ISA 206 emulsion did. Moreover, CP-PEI-Zn showed superior efficacy to CP-PEI in promoting the proliferation of effector-memory T cells and secretion of cytokines, indicating a more potent cellular immune response. In summary, the cationic solid lipid nanoparticles and chitosan particles facilitated both strong cellular and humoral immune responses and stabilized iFMDV after loading. In addition, they are biocompatible, easily preparaed and low-cost, reflecting their potential to be a promising adjuvant for the iFMDV vaccine.
|李帅. 口蹄疫疫苗的稳定、免疫活性与颗粒佐剂的研究[D]. 中国科学院大学,2020.|
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