Research Of Hepatitis B Vaccines Based On Chitosan Derivative Nanoparticles

We evaluated several novel cationic derivatives of chitosan for carrying immunogenic non-particle hepatitis B virus core protein (HBCM) for vaccine candidates. In order to investigate the feasibility for vaccine carriers and the potential of inducing cross presentation via "endosomes to cytosol" mechanism, we screened a series of nanoparticles with Transmission Electron Microscopes (TEM), loading capability and release test in vitro and the experiments which could represent the endosome disruption ability: haemolysis assay and confocal microscopy of RAW 264. 7 cells after incubation with nanoparticles. Then we immunized C57BL/6 mice with the chosen candidate nanoparticles (N-trimethylaminoethylmethacrylate chloride—Methylmethacrylate (TM), Chitosan—Methylmethacrylate (CM),Chitosan—N-trimethylaminoethylmethacrylatechloride—Methylmethacrylate (CTM) andChitosan—N-dimethylaminoethylmethacrylatehydrochloride—Methylmethacrylate (CDM)) carrying HBCM and evaluated the immune responses. Those groups of mice were induced significantly higher immune response both in antigen-specific humoral responses and cellar responses than the group immunized with the naked protein solution, and among the candidates, CTM based vaccine (vaccine-CTM nanoparticles) showed the strongest immunogenecity. Both the enhanced immunoresponse and the results of in vitro experiments indicated that administration of nanoparticles based vaccine did induce cross presentation through the endosome to cytosol pathway and this antigen delivery strategy had great practical potential.Immunotherapeutic strategies stimulating both cellular and humoral immune responses to HBV antigens are essential to cure chronic HBV infection, and the traditional DNA vaccine is potentially capable of generating humoral immunity as well as cellular immunity while its efficiency is not satisfying. In the present report, several cationic derivatives of chitosan have been prepared as novel nanoparticle vaccine formulations in order to improve the performance of associated DNA vaccine. These novel chitosan derivatives showed higher solubility in physical condition, which increased the compatibility with hydrophilic molecules. These nanoparticles could protect the DNA molecules from DNase I degradation in vitro. The haemolysis assay and confocal microscopy indicated that these nanoparticles possessed membrane disruption ability and high potential efficiency of gene delivery. A pVAX (HBc) DNA vaccine was encapsulated into these candidate nanoparticles and administrated to C57BL/6 mice twice with 2 week' s interval. The humoral and cellular immune responses at different time point were evaluated by ELISA assay, ELISPOT and CTL specific lysis assay. It was demonstrated that the nanoparticle formulations significantly enhanced the immunogencicity of pVAX(HBc) in several parameters: elevated antibody production, higher level of IFN- Y secretion, and augmented specific cell lysis in immunized mice. All these results well revealed that the chitosan derivatives were practical for pVAX (HBc) DNA vaccine delivery.