接种疫苗是预防H5N1禽流感爆发的有效手段，H5N1流感疫苗免疫原性较差，抗原用量高，需要添加佐剂以提高疫苗效果。壳聚糖微球作为颗粒型佐剂具有良好的生物相容性和免疫刺激性。然而，壳聚糖微球的制备及其作为疫苗佐剂的应用还存在以下的需求与问题：1）传统的壳聚糖微球制备方法（如机械搅拌法、凝聚法等）所制备的微球粒径不均一，产品的稳定性与重复性差；2）与包埋相比，抗原吸附过程简单，在细胞内易于释放抗原，而高抗原吸附率要求制备阳离子性、网络多孔的壳聚糖微球，以增大抗原与微球的接触面积与结合力；3）交叉提呈是细胞免疫应答的前提，有效的交叉提呈需要壳聚糖微球具备足够的质子泵作用以及pH敏感性，以促进抗原从溶酶体中释放；然而通常以戊二醛进行化学交联的壳聚糖微球中大量氨基被交联占用因而降低了微球的质子泵作用，且不具备pH敏感性。基于以上问题，本论文旨在开发一种新型壳聚糖季铵盐凝胶微球，有效提高H5N1流感疫苗的免疫效果并降低抗原用量。本文具体结论如下：1）采用快速膜乳化—热固化相结合的新型微球制备方法制备出粒径可控（807 nm，2.5 μm和4.3 μm）和季铵取代度可控（20% - 83%）的壳聚糖季铵盐凝胶微球，该方法不使用化学交联剂，凝胶微球呈阳离子性网状多孔结构。凝胶微球可以有效吸附H5N1裂解疫苗，吸附率高达90%。值得注意的是，其抗原释放行为具有明显的pH依赖性，而基于化学交联的壳聚糖季铵盐微球则对pH不敏感。2）研究发现壳聚糖季铵盐凝胶微球作为H5N1裂解疫苗的佐剂具有多重免疫增强机制：a：在注射部位形成“抗原储库”，募集炎症细胞；b：提高抗原胞吞率，基于其阳离子性和pH敏感性有效促进抗原从溶酶体途径中释放，为抗原交叉提呈提供基础；c：激活树突细胞，促进抗原提呈。而化学交联微球不能有效促抗原从溶酶体中释放，其对树突细胞的激活程度显著弱于凝胶微球。体内动物实验结果显示，凝胶微球可以显著提高小鼠体液与细胞免疫应答，诱导较为平衡的Th1与Th2混合型免疫应答。组间进行比较，结果发现凝胶微球组诱导的血凝抑制抗体滴度分别是铝佐剂组和化学交联微球组的1.9倍与2.0倍，具有杀伤功能的CD8+ T细胞增殖水平分别是铝佐剂和化学交联微球组的1.7和1.9倍，脾细胞中Th1型细胞因子IFN-γ的分泌水平分别是铝佐剂和化学交联微球组的3.0和2.1倍。3）粒径与季铵取代度是壳聚糖季铵盐凝胶微球的两个重要参数，对凝胶微球的佐剂效应有重大影响。实验结果阐明了季铵取代度接近中间值（41%和60%）、粒径较小（807 ± 74 nm）的凝胶微球具备较为平衡的亲疏水性、表面电荷以及可质子化的氨基，可使凝胶微球的佐剂效应最大化，显著提高疫苗的免疫原性，并且与铝佐剂相比，凝胶微球有效节约75%的抗原用量。4）除裂解疫苗外，从溶酶体途径中逃逸同样是提高DNA疫苗免疫效果的关键所在。实验尝试以pcDNA3.1-GFP质粒为模型，进行凝胶微球作为DNA疫苗佐剂的研究。结果发现凝胶微球可以有效吸附紧缩质粒；更重要的是，在血清存在的条件下，凝胶微球的体外基因转染率是商业化转染试剂的2倍，说明壳聚糖季铵盐凝胶微球在生理条件下更稳定，具有作为DNA疫苗载体的潜力。综上所述，壳聚糖季铵盐凝胶微球具有多重免疫增强机制，可有效促进抗原提呈，并显著提高H5N1裂解疫苗的体液与细胞免疫应答，且具有作为基因疫苗载体的潜力，因此壳聚糖季铵盐凝胶微球是一种具有应用前景的疫苗佐剂。

Vaccination is one of the potent tools to avoid the H5N1 influenza pandemic. However, the poor immunogenicity and high antigen dosage requirement of H5N1 split vaccine demand the incorporation of adjuvants to enhance the vaccine efficacy. Chitosan microparticles (MPs) as the particulate adjuvants have attracted much attention due to their biocompatibility and immunostimulatory. Neverthless, the preparation and application of chitosan MPs as adjuvants are still needed to be improved: 1) The size distribution of chitosan particles is wide and difficult to control. 2) Chitosan particles with positive surface and porous structure are highly needed to adsorb antigen; 3) Robust “proton sponge effect” and pH sensitivity of chitosan particles are demanded to improve the antigen cross-presentation and enhance the cellular immune responses. In this paper, we promoted a novel method (premix membrane emulsion technology combaining with thermal-solidification) to prepare the quaternized chitosan hydrogel microparticles (Gel MPs) without using any chemical cross-linking agent. Then the adjuvanticity of Gel MPs as adjuvants of the H5N1 split vaccine was investigated. The main coclusions were attached below:1) The Gel MPs with well defined quaternization degrees (QDs, 20% - 80%) and particle sizes (800 nm - 5 μm) were prepared by the premix membrane emulsification technique combining with the thermal-solidification method. Gel MPs with porous structure were high water content, pH sensitive and positive charged. Gel MPs proved to adsorb antigen efficiently and showed pH-dependent antigen release profile. Chitosan MPs based on chemical cross-linking (GC MPs) were not pH sensitive.2) The potential multiple mechanisms of Gel MPs as adjuvants were confirmed: A: form an antigen depot and recruit the inflammatory cells at the injection site; B:improve antigen uptake by DCs and enhance the endosomal escape of antigen into cytoplasm; C: dramatically activate DCs. In comparison to GC MPs, Alum adjuvants and LPS, Gel MPs exhibited higher humoral and cellular immune enhancement with a mixed Th1/Th2 immunity.3) To maximize the adjuvanticity of Gel MPs, the effects of QDs (range from 20% to 80%) and sizes (range from 800 nm to 5 μm) on the adjuvant activity of Gel MPs were investigated. Results suggested that Gel MPs with relatively smaller size (807 nm) and moderate quaternization degree (41% and 60%) were favorable for a maximum immune response. Most importantly, they induced significantly stronger immune responses at lower antigen doses (known as antigen sparing effect) compared to the Alum adjuvant.4) Considering the ability of Gel MPs to significantly improve the endosomal escape of antigen, the potential of Gel MPs to increase the efficacy of DNA vaccines was investigated. Gel MPs could efficiently adsorb and condense the pDNA. In vitro transfection assasy exhibited that Gel MPs were superior as the gene delivery systems than the commercial transfection agents at the prensence of serum, suggesting that Gel MPs were promising adjuvants of DNA vaccines for clinical use.In this thesis, the Gel MPs without chemical cross-linking agent proved to induce robust humoral and cellular immune responses of H5N1 split vaccines based on the potential multiple mechanisms. Moreover, Gel MPs proved to be efficient as the delivery system of DNA vaccines. These data strongly demonstrated that Gel MPs had the capacity to function as a promising vaccine adjuvant.