A Single-dose Immunization Formulation Of Hbsag And The Evaluation Of Immune Effect

Hepatitis B virus(HBV)infection is the main reason for hepatitis B disease.Vaccination is one of the most effective methods for the prevention and treatment of hepatitis B virus infection.Currently,the most commonly used immunization schedule for hepatitis B vaccine is three injections given at 0th,1st and 6th month respectively,to elicit effective immune protection levels.The first and second vaccinations were given at intervals of 1 and 6 months after the first vaccination.The complex immunization program leads to a significant poor compliance and poor immunization coverage,especially in the remote area,which lead to poor immune protection.Conventional aluminum-adjuvant vaccines need cold-chain during the whole process,which greatly increases vaccine costs.It’s difficult for some poor families to pay for them,which further decline the immunization coverage.Therefore,it’s necessary to explore a new single-shot vaccine strategy with stable and inducing long-term immunity properties.Microsphere adjuvants are based on poly lactic acid(PLA)and sodium alginate,which is widely used in the field of vaccine.Nanoparticles can promote antigen uptake by antigen presenting cells(APCs)and cross-presentation via MHCI way,inducing potent humoral and cellular immune responses.Traditional preparation methods for microspheres encapsulating vaccine always involved severe conditions including intense shear and high temperature and so on,easy to result in denaturation of antigen.This further affected the encapsulation efficiency and greatly restricted scale up.This thesis mainly designed and fabricated HBsAg-DDAB/PLA-ALG MCs with core-shell structure,and the preparation parameters were optimized The HB s Ag-DD AB/PL A-ALG MCs were employed as single-shot vaccine.The detailed research contents are as follows:1)The DDAB/PLA NPs were prepared by modified nanoprecipitation method,and the hepatitis B surface antigen(HBsAg)-loaded DDAB/PLA NPs were successfully encapsulated into alginate MCs by modified spray-solidification technique.The response surface method was applied to optimize the preparation conditions.Based on the optimum conditions,the size of microcapsules was 24.25 μm,the Span value was 1.627,and the encapsulation efficiency of HBsAg was 68.4%.In vitro release profile exhibited a slow release rate of encapsulated HBsAg expecially in the phosphate buffered saline solution.2)The microcapsules immunized with single-shot induced a strong humoral response,which was comparable with traditional aluminum-based vaccine with three shots.Both the single-shot MCs vaccine formulation and the three-shot aluminum-based vaccine induced significantly higher IgG level than that of antigen alone.And significant higher cytokine levels(IL-2,IFN-γ,TNF-α,IL-10 and Granzyme B)were produced by splenocytes from mice immunized with the single-shot alginate MPs vaccine formulation comparing with three-shot HBsAg alone and aluminum-based vaccine.3)The mixture(NPs+MCs)was employed as single-shot vaccines in vivo immunize response.Data suggested that the mixed vaccine formulation effectively provided not only adequate initial antigen exposure but also long-term antigen persistence,eliciting potent humoral and cellular immune responses.The mixture also improved generation of immunological memory to protect against reinfection.In summary,the microcapsules encapsulating HBs Ag-loaded DDAB/PLA NPs developed in this study is a promising delivery system for single-shot vaccine formulations.