Self-assembling Ferritin Nano-particles Enhance The Mucosal Immune Efficacy Of Anti-caries Vaccine

[Objective]Glucosyltransferase GtfB is the extracellular antigen of cariogenic bacteria Streptococcus mutans,and carboxyl terminated dextran binding zone Glu is one of the functional areas contained in GtfB.In this study,Glu-FTH is the nanoparticle anti caries polypeptide vaccine formed by fusion expression of Glu and ferritin(FTH).The fusion protein Glu-FTH was extracted from Escherichia coli protein expression system,and Glu-FTH was purified by nickel column affinity chromatography.The aim of this study was to observe the activation of Glu-FTH vaccine against dendritic cells(DCs)and the uptake of DC on Glu-FTH,and to evaluate the mucosal immune response and caries protection effect of Glu-FTH.[Methods]Glu-FTH was purified by Ni column affinity chromatography.The correctness of the size of the target protein was verified by SDS-PAGE and Western blot.Subsequently,transmission electron microscopy was used to observe the morphology of Glu-FTH and analyze its particle size distribution.6 weeks,female BALB/c mice were divided into Glu-FTH,Glu,FTH,Glu+Poly(I:C)and PBS group.They were immunized on the zeroth,fourteenth,twenty-eighth day respectively.The levels of specific antibodies in saliva and serum were detected by ELISA.To determine the type of immune response(type TH1 or TH2),the levels of cytokines in supernatant of splenocytes were also detected by ELISA.On day 18th,female Wistar rats were randomly divided into Glu group,Glu-FTH group,FTH group,Glu+Poly(I:C)group and PBS group.Rats were immunized with nasally.S.mutans UA159 was administrated to the oral cavity of rats on day 25-28.The numbers of Streptococcus mutans in dental plaque were detected.The protective effect of vaccine on dental caries was analyzed according to caries score.Serum and saliva was collected,and the levels of specific antibodies IgG and IgA were analyzed by ELISA.Flow cytometry was used to detect the ability of dendritic cells(DCs)to uptake nanoparticle vaccine,and to analyze the effect of nanoparticle vaccine on the maturation of DC.[Results]SDS-PAGE and Western blot proved that the size of purified protein Glu-FTH was correct.Transmission electron microscopy showed that Glu-FTH and FTH were spherical nanoparticles with diameters of 15.9 + 1.4nm and 14.1 + 1.lnm respectively.Compared with Glu,Glu-FTH significantly enhanced the level of IgA and IgG in saliva and serum of mice(P<0.05 or P<0.01).The level of specific antibodies in serum and saliva induced by Glu-FTH was higher than Glu combined with adjuvant Poly(I:C)(P<0.05 or P<0.01).Compared with mice immunized with Glu,the ratio of serum IgGl/IgG2a and the level of IL-4 secreted by spleen cells in Glu-FTH immunized mice were significantly higher.The rate of DC uptaking nanoparticle Glu-FTH is higher than that of DC uptaking Glu or nanoparticle FTH.Glu-FTH protein significantly increased the expression level of BMDC surface molecules CD80,CD86 and MHC-?.Both FTH and Glu increased the expression level of BMDC surface molecule CD80 and MHC-?(p<0.05).[Conclusion]Nanoparticle FTH as an antigen Glu carrier can effectively improve the immune efficacy of vaccines,and Glu-FTH mainly induces Th2 type immune response.This is mainly due to the high uptake rate of DC to Glu-FTH,and the maturation of DC induced by Glu-FTH.