Novel Adjuvant Of H5N1 Split Vaccine Based On The Quaternized Chitosan Hydrogel Microparticles

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 quatemized 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-5μm) 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 Thl/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 5μm) on the adjuvant activity of Gel MPs were investigated. Results suggested that Gel MPs with relatively smaller size (807nm) 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.