Application And Mechanism Of Calcium Phosphate Nanoparticles As Vaccine Adjuvant

Nano-sized calcium phosphate particles have been widely used in medical treatment and diagnosis fields such as the restoration of teeth/bone due to their good safety and biological characteristics.There are also many preclinical studies that have confirmed that calcium phosphate nanoparticles have the good adjuvant effect on different immunogens.The aim of this study was to investigate the effect of calcium phosphate nanoparticles as a vaccine adjuvant and to explore the mechanism of its role as an adjuvant.In order to improve its adjuvant effect,this study also optimized its traditional preparation formula.In addition,this study also investigated whether its role as an adjuvant in adsorbing particulate antigens would affect the epitope structure of antigens as a candidate adjuvant.First,in this study,eight calcium phosphate nanoparticles with different phosphorus-calcium ratios(P/Ca=0.15/0.30/0.45/0.60/0.75/0.90/1.00/1.10)were prepared by co-precipitation method.The sizes of these particles are all between 10 nm and 100 nm.With the increase of P/Ca,the particle morphology gradually changes from a spherical shape to a long rod shape.When P/Ca reaches 0.60,almost all of the added calcium precipitates.When the P/Ca is 0.45,the OVA adsorption rate of the particles is the highest.When the pH is 6,the nanoparticles with P/Ca of 1.00 began to dissolve.In addition,except for the positive charge of particles with P/Ca of 0.45,the rest of the particles are negatively charged.Second,using varicella-zoster virus glycoprotein E as an immunogen,it was confirmed in vivo that calcium phosphate nanoparticles have an adjuvant effect.At the same time,the mechanism of action of calcium phosphate nanoparticles as a vaccine adjuvant was explored through in vitro cell experiments,and it was presumed that it may be by promoting the proliferation of dendritic cells and phagocytosing antigens,or by promoting the production of TNF-a by macrophages.Subsequently,in order to improve the adjuvant effect of calcium phosphate nanoparticles,this study changed the source of phosphorus in traditional methods for the preparation of calcium phosphate nanoparticles,using bisphosphonates instead of Na2HPO4·12H2O to prepare two novel particles:calcium zoledronate and calcium risedronate.It was also demonstrated in the in vivo evaluation in mice that these two kinds of particles have superior adjuvant effects.Finally,to investigate whether calcium phosphate nanoparticle as an adjuvant will affect the epitope structure of antigen when adsorbing particle antigen,this study used HPV45 VLP as a mimic antigen.Therefore,this study established a set of immunochemical quality analytical methods for HPV45 VLP to monitor of antigenic epitopes,including competition ELISA,surface plasmon resonance assays and double antibody sandwich ELISA.The results showed that calcium phosphate nanoparticles,calcium risedronate and calcium zoledronate did not affect the integrity of the HPV45 VLP epitope.In summary,after a series of experimental verification,we have further recognized calcium phosphate nanoparticles as a vaccine adjuvant,which also provides a certain foundation for further research and transformation.