Study On Preparation And Immune Protection Of Biomineralized Streptococcus Pneumoniae Protein ΔA146Ply Nanoparticle

Objective: Streptococcus pneumonia（S.pn）is one of the main causes of invasive bacterial disease worldwide.Vaccines are an effective way to control the infections.Because of limitations of vaccines used in clinic,it is necessary to develop a new novel vaccine for enhancing immune response and decreasing toxic and side effect.Protein-based vaccine is a hot spot of vaccine research,because it has high conservatism and simple preparation,and it can also effectively activate B,T cell immunity.However,protein vaccine needs adjuvant to enhance immunogenicity during the immunization.Current studies have revealed that nanoparticle form is easier to induce stronger immune response.Therefore,we aim to fabricate biomineralized protein nanoparticles based on pneumococcal protein ΔA146Ply with biomineralization related peptides PA44,NW,N6,W6 and evaluate the application value by observing its immunoprotective effect and safety.Methods: Protein ΔA146Ply-PA44,ΔA146Ply-NW,ΔA146Ply-N6,ΔA146Ply-W6 and ΔA146Ply were expressed and removed endotoxin.Von kossa dye was used to determine the ability to chelate calcium phosphate of protein ΔA146Ply-PA44,ΔA146Ply-NW,ΔA146Ply-N6 and ΔA146Ply-W6.In the weak alkaline solution rich in Ca²⁺ and PO₄^3-,protein ΔA146Ply-PA44,ΔA146Ply-NW,ΔA146Ply-N6 and ΔA146Ply-W6 biomineralized by chelating calcium phosphate.The biomineralization products were analyzed by means of transmission electron microscopy（TEM）and X-ray photoelectron spectroscopy（XPS）.Mice bone marrow-derived dendritic cells（BMDCs）were stimulated with soluble protein ΔA146Ply,ΔA146Ply-PA44,ΔA146Ply-NW,ΔA146Ply-N6,ΔA146Ply-W6 and biomineralized protein nanoparticles respectively to assess the effect for antigen presentation by detecting secreting cytokines.In vivo,mice were subcutaneously immunized with different kinds of soluble proteins and biomineralized protein nanoparticles.ELISA was used to detect antibody titers,spleen cytokines,septicemia model and colonization model after the last immunization.Mouse survival rate,lung colonization,pulmonary inflammation were observed to evaluate the protective effect of biomineralized protein nanoparticles.The hemolysis experiment,the body weight of the immunized mice and the immunization site was used to evaluate the safety of the biomineralized protein nanoparticles.Results: Successfully preparation of protein ΔA146Ply-PA44,ΔA146Ply-NW,ΔA146Ply-N6,ΔA146Ply-W6 and ΔA146Ply with more than 90% purity.Protein ΔA146Ply-PA44,ΔA146Ply-NW,ΔA146Ply-N6 and ΔA146Ply-W6 have the ability to chelate calcium phosphate and form nanometer scale particles with different density and diameter（ΔA146Ply-PA44 @ CaP,ΔA146Ply-NW @ CaP,ΔA146Ply-N6@CaP,ΔA146Ply-W6@CaP）.Four kinds of biomineralized protein nanoparticles can activate BMDCs in vitro to induce higher level of cytokine secretion（IL-6 and TNF-α）compared with ΔA146Ply.ΔA146Ply-PA44@CaP and ΔA146Ply-W6@CaP can induce higher level of IL-6 and TNF-α secretion compare with the corresponding soluble protein.In vivo experiments,all biomineralized protein nanoparticles immunized groups produced higher specific antibody titers,spleen cytokines（IFN-γ,IL-4 and IL-17A）compared with ΔA146Ply immunized group.Splenocytes of ΔA146Ply-PA44@Ca P,ΔA146Ply-NW@CaP,ΔA146Ply-W6@CaP immunized groups secreted higher IL-4 compared with adjuvant-containing ΔA146Ply immunized group.ΔA146Ply-PA44@CaP and ΔA146Ply-W6@CaP immunized groups showed a protective effection comparable to positive control（PPV23）in the sepsis model.All biomineralized protein nanoparticles immunized groups in the colonization model showed lower lung colonization,mildest pulmonary infiltration compared with soluble proteins immunized groups and adjuvant-containing soluble proteins immunized groups.In the safety experiment,all the soluble protein and the biomineralized protein nanoparticles used in this experiment did not cause hemolysis.There was no difference in the body weight among the immunized groups.There was also no significant difference in the organs weights after the immunization.Biomineralized protein nanoparticles did not cause nodules or lumps at the immune site as aluminum adjuvant did.Conclusion: Calcium phosphate-based biomineralized protein nanoparticles can effectively enhance antigen presentation of protein vaccine,promote humoral immunity and cellular immunity,and induce protective effection in host,which is equvalent to commercially available vaccine.There is no nodule in immunized situation which is better than aluminum adjuvant.Therefore,biomineralized protein nanoparticle is a valuable development direction of protein vaccine formulations.