Immunological Mechanism And Security Evaluation Of Chitosan Microspheres Vaccine Adjuvant

Chitosan, a kind of alkaline polysaccharide, has abundant reserves and wide variety of sources in nature. It becomes an ideal biomedical material as itself and derivatives are non-toxic and biocompatible. Microspheres prepared from chitosan and its derivatives, not only are biological compatibility as good as chitosan, but also have advantages of microspheres such as APC-targeting, antigen releasing control, effective protection of a ntigen from degradation and inactivation. C hitosan has become a worldwide hot topic as vaccine adjuvant in recent years because of its unique physicochemical and biological characteristics. In this thesis, high pressure homogeneous technique was used to prepare uniform-sized N-(2-hydroxy) propyl-3-trimethylammonium chitosan chloride microspheres(HTCC MP) with controllable sizes. The prepared microspheres were used to load H5N1 split vaccines, and the effectivity and security were evaluated as parenteral vaccine adjuvant.In detail, this thesis mainly contained the following issues. The first part was to prepare HTCC MP by the high pressure homogeneous technique. The effect of key parameters in preparation process on the uniformity and structure of HTCC MP w ere systematically investigated. And uniform-sized HTCC MP were successfully prepared. The second part was to evaluate the effectivity of HTCC microspheres as H5N1 split vaccines adjuvant in vitro and in vivo. Firstly, immune effects of HTCC microspheres were investigated in bone marrow-derived dendritic cells. The results suggested that the HTCC microspheres could promote APCs activation and antigen internalization, and effectively enhanced the immune response. Then the effect of HTCC microspheres as H5N1 split vaccines adjuvant on humoral and cellular immunity was evaluated. The results suggested that HTCC microspheres could significantly enhance the specific antibody in the serum of immunized mice and powerfully improve CD4+CD8+ T lymphocyte subgroup percentage compared with aluminum adjuvant. It was suggested that HTCC microspheres could more effectively induce immune response than aluminum adjuvant.The third part was to evaluate the security evaluation of HTCC microspheres as parenteral vaccine adjuva nt in mice, SD rats, guinea pigs and rabbits. The security evaluation included acute toxicity test and abnormal toxicity test in mice, multiple toxicity test in SD rats, allergy test in guinea pigs, muscle stimulation test on rabbits’ thigh muscles and hemolytic test in vitro. Results showed that the security of HTCC microspheres was good. No toxicity, irritation and hemolysis were found after injection of HTCC microspheres. The HTCC microspheres could be used for intramuscular injection.The above results showed that the HTCC microspheres were safe and effective vaccine adjuvant for H5N1 split vaccine. And the HTCC MP adjuvants have strong potential for clinic applications.