Study On Hepatitis B Vaccine Based On Polvlactic Acid Microparticles Adjuvant

Treatment of chronic hepatitis B(CHB)is full of challenge.Compared with traditional antiviral therapy,vaccine therapy is an important potential approach for the prevention and treatment of CHB.However,how to prime effective cellular immune response to meet the therapeutic requirements and endow a better protective effect is a great challenge.This dissertation proposes that biocompatible polylactic acid(PLA)microparticles(MPs)which rationally loaded with antigen and immunopotentiator according to the delivery demand to act as therapeutic hepatitis B vaccine.Meanwhile,PLA MPs were used as adjuvants to explore vaccines with reduced immunization dose.In detail,this thesis mainly includes the following issues:1)Aim at the immunopotentiator need to function at specific cell locations,DP and DP-D MPs were successfully fabricated according to the release demand of antigen and immunopotentiator,and evaluated the immune response at cellular level.The introduction of cationic lipid didodecyldimethylammonium bromide(DDAB)into PLA MPs could assist them adsorb antigen gently and efficiently(adsorption rates were all above 85%);meanwhile DMXAA could release into the cytoplasm through the proton sponge effect,then interact with its receptor,activate downstream pathway.At the molecular level,the expression of IRF-7 and IFN-? in the STING pathway of DP-D increased by 12 and 8.2 times respectively compared with the antigen group.The MPs vaccines could facilitate cell uptake and lysosome escape,increase the expression of costimulatory and MHC molecules of dendritic cells(DCs),promote cytokines secretion,thus activating DCs and prime subsequent immune responses efficiently.2)The humoral and cellular immune responses of PLA MPs vaccines on healthy mice were evaluated and the immunization mechanism was explored as vaccine adjuvant.MPs vaccines can induce the expression of inflammatory cytokines and chemokines such as IL-1? and CXCL-10 at the injection site,and recruit immune cells such as DCs and macrophages to the injection site.After being transported to lymph nodes by APCs,they can promote DCs,B and Tfh cells activation in lymph nodes.In healthy mice immunization experiment,high levels of HBsAg-specific IgG and IgG2a/IgGl ratio were induced,the number of Thl splenocytes,and the secretion of IFN-y together with other cytokines were also elevated,indicating the MPs induce both humoral and cellular immune responses.3)The chronic hepatitis B(CHB)model mice were successfully constructed,and the immune and therapeutic effect of the MPs vaccines were investigated.CHB model mice were constructed by tail vein injection of recombinant AAV/1.3HBV virus.The results indicated that DP and DP-D induced high-level HBsAg-specific IgG,promoted the splenocytes proliferation and activation as well as the secretion of Thl cytokines.The HBsAg-specific CTL responses were improved and memory immune responses for protection were maintained.The serum HBsAg seroconversion rate of the two MPs vaccines both achieved 50%and the expression of HBcAg in the liver significantly reduced,indicating a favorable therapeutic effect.4)The long-term immunoprotection effect of two dose PLA MPs vaccines instead of three dose aluminum vaccines were verified.The MPs vaccines produced comparable antibodies compared to aluminum adjuvant after two dose immunization schedule,and the antibodies maintained at a high level for half a year.When rechallenging with HBsAg,the mice can quickly produce antibodies.The type and corresponding concentrations of lyoprotectants were optimized,and the stability of lyophilized vaccines at room temperature was investigated.The results clarified that the MPs vaccine adjuvant has the capacity of reducing the immunization dose and can be lyophilized,thus possessing the potential for clinical transformation.In summary,the PLA MPs vaccines designed in this dissertation possess great potential for application in hepatitis B vaccines.