Calcium Phosphate-based Adjuvant Formulation Optimization And Effectiveness Evaluation

Aluminum adjuvant is currently the most widely used human vaccine adjuvant in the world.It can combine multiple source antigens to enhance the body’s immune response and has good safety.However,with the development of recombinant subunit vaccines and inactivated vaccines,the immunogenicity of some vaccines is weak.Aluminum adjuvants cannot provide strong enough immune stimulation.And aluminum adjuvants can hardly activate Th1 immunity.Response,the immune enhancement effect on cellular immunity is limited.So it is necessary to develop adjuvants with stronger immune stimulating effects.Calcium phosphate has good biological safety and has also been confirmed to have adjuvant effect.This study optimized and evaluated the adjuvant formulation based on calcium phosphate.In this study,six calcium phosphate adjuvants with different ratios of phosphorus to calcium were first prepared.Through physical and chemical characterization,the calcium phosphate adjuvant with a phosphorus to calcium ratio of 1 with complete calcium ion precipitation and the highest protein adsorption rate was selected as the subsequent optimization object.The first step was to modify the metal ion part.We used aluminum ions and manganese ions to prepare 8 new adjuvants.Then we conducted physical and chemical properties such as particle morphology,particle size,and protein adsorption rate.Three immunogens of OVA,VZV gE,and CR-T3-B were used to evaluate the immunostimulatory effect.It was found that the existence of manganese ions can significantly improve the immunostimulatory effect of calcium phosphate adjuvant.Manganese phosphate adjuvant can induce high-titer binding and Neutralize antibodies.And it produced higher titers of IgG 2 antibodies,effectively activating Th1 immune responses.Next,we modified the phosphate part,introduced six organic phosphonic acids to replace inorganic phosphate,and prepared a total of 18 calcium-manganese organic-inorganic hybrid adjuvants.After physical and chemical properties were tested,we used OVA,VZV gE and CR-T3-B to evaluate the effectiveness of adjuvants.The test results show that the introduction of zoledronic acid and risedronic acid to replace inorganic phosphoric acid can improve the adjuvant effect.The antibody binding and neutralization titers are significantly higher than aluminum adjuvants,and the level of IgG 2 antibodies is also better.Finally,this study conducted a preliminary exploration of the adjuvant mechanism through cytokine detection and T cell subpopulation analysis.The results showed that compared with aluminum adjuvants,manganese phosphate,adjuvants wirth zoledronic acid or risedronic acid can induce higher titers of IL-2,IFN-γ.And manganese phosphate and adjuvants with risedronic acid can stimulate more CD4+,CD8+T cells production.