Design And Exploration Of Novel Ajuvants Based On H5N1 Influenza Vaccine

Highly pathogenic avian H5N1 influenza is a serious infectious disease. Currently, most of the commercial H5N1 vaccines are inactivated vaccines. While, in the intranasal immunization, these inactivated vaccine could not overcome the nasal mucosal barrier and usually generate relative weak immunity. Moreover, in the intramuscular immunization, these inactivated H5N1 vaccines could not induce optimal immune response, even with commercial Alum adjuvant and always require large antigen dosage. Therefore, the thesis employed two biodegradable high molecular polymer, chitosan and poly(lactic-co-glycolic) acid (PLGA), to prepare thermal-sensitive hydrogel and uniform non-spherical PLGA particles as the intranasal and intramuscular adjuvant respectively, which could enhance the immune responses. For hydrogel adjuvant, the relationship between the physiochemical properties and immune effects and the underlying mechanism were explored. Then, toxicity and stability of the hydrogel formulation were investigated. For particle adjuvant, a simple method was developed to prepare non-spherical PLGA particles and explore their shape effects. This thesis was divided into the following parts.(1) The synthesis of quaternized chitosan (HTCC) was magnified and the thermal-sensitive HTCC hydrogel was well prepared by ionic gelation with glycerol phosphate sodium (GP). By adjusting the quaternize degree (QD) of HTCC, the influence of hydrogel cationic character and rheological behavior on the corresponding immune responses was investigated. Results showed there was competition between cationic character and thermal-sensitivity. As a consequence, the hydrogel with a moderate QD of 41% had both the good cationic character and good thermal-sensitivity, resulting in the induction of the best humoral immunity. In-vivo animal experiments showed that the IgG titer induced by 41% hydrogel was 5.5 times higher than the pure antigen intranasal group (Ag IN) and comparable to the pure antigen intramuscular group (Ag IM). The nasal IgA titer of hydrogel group was 170 times higher than Ag IM.(2) The safety and toxicity of the hydrogel were systematically investigated. The tests of acute toxicity, repetitive dose toxicity, abnormal toxicity, allergy reaction, pyrogen and hemolysis were carried out in the animal models of mice, rats, rabbits and guinea pigs. Results showed that the HTCC hydrogel was well tolerated and had very low toxicity when used as an intranasal vaccine adjuvant.(3) Furthermore, we chose the import chitosan glutamine salt (CS-Glu) which has presented file to the Food and Drug Administration (FDA) to prepare the CS-Glu hydrogel. Through the orthogonal experiment, the influence of CS-Glu concentration, GP concentration and antigen dose on the immune responses were investigated and optimized along with the formulation stability. Results showed that the antigen dose had a dominant effect on the immune effects and the influences of CS-Glu and GP were comparable. At last, the CS-Glu hydrogel adjuvant was optimized, which could induce the value of 120 for HI titer and IgA titer 178 times higher than Ag IM group. This hydrogel could store at 25± 1 ℃ for 6 days and 2～8 ℃ for more than 2 months。(4) There is lack of researches about the influence of particle shape on the biological outcomes because of the difficulty in the preparation of non-spherical particles.In order to resolve this problem, a novel method was developed based on the premix membrane emulsification technique to prepare uniform non-spherical particles. By employing an inorganic reagent, phosphate buffer saline (PBS), as the deformation initiator, non-spherical PLGA particles were prepared. The shape, morphology and size of the particles could be well controlled in this method and it is easy to scale up the production. Then, the non-spherical particles were used as H5N1 vaccine adjuvants and the shape effects were studied. The preliminary results showed that the spherical particles could potentiate humoral immunity while the rod particles could promote the activation of antigen specific T cells and increase the antigen specific memory T cells.In summary, by using the high molecular polymers of chitosan and PLGA, two adjuvants of H5N1 inactivated vaccine were developed. The influence of the physicochemical properties, the rheological behavior and the shape on the corresponding immune responses was deeply investigated. Moreover, the safety and formulation stability were studied and optimized, which could establish a foundation for clinical application.