P < 0.05 was considered statistically significant. == Disclosure of potential conflicts of interest == The authors report no potential conflicts of interest. == Acknowledgment == The authors would like to thank Enago (www.enago.cn) for the English language review. == Funding == This study was funded by grants from National Natural Science Foundation of China (81570972) to Prof. of GLU and poly(I:C). Immunization with GLU-FTH achieved lower caries scores compared with those of the other vaccines. Administration of GLU-FTH nanoparticles enhanced phagocytosis by DCs and their maturation. Thus, self-assembling GLU-FTH is usually a highly effective anticaries mucosal vaccine that enhanced antibody production and inhibitedS. mutansinfection in rodents. KEYWORDS:adjuvants, dental caries, dendritic cells, mucosal immunity, nanoparticle, salivary IgA == Introduction == Streptococcus mutansis the primary pathogen that causes dental caries. Antigen I/II (PAc) and glucosyltransferase (GTF) are the 2 major virulence factors associated with the adherence ofS. mutansto substrates.1GTFs play a major role in the sucrose-dependent accumulation ofS. mutanson tooth surfaces through glucan synthesis. The putative catalytic and glucan-binding regions (CAT and GLU, respectively) of S. mutans GTF-I are major antigens that induce the production GnRH Associated Peptide (GAP) (1-13), human of salivary immunoglobulin A (IgA) antibodies that inhibit bacterial adherence and colonization. Therefore, these antigens were chosen as immunogens for developing an anticaries vaccine.2The findings of numerous studies of DNA-based or protein-based anticaries vaccines3,4,5,6have not been translated to the clinic, primarily because of their low immunogenicity, in terms of the unsatisfactory immune responses induced through mucosal administration, which are characterized by the production of IgA with transient, variable, and insufficient titers.7 Proteins that form GnRH Associated Peptide (GAP) (1-13), human nanoparticles are suitable for antigen presentation and immune activation.8,9For example, ferritin forms self-assembling synthetic nanoparticles that increase the ability to induce broadly neutralizing antibodies against influenza computer virus.10Further, a ferritin heavy chain (FTH) was used as a nanoplatform for antigen delivery to develop a dendritic cell (DC)-based vaccine.11Moreover, certain self-assembling peptides act as effective adjuvants that stimulate the induction of potent local IgA antibody responses.12Therefore, we considered GnRH Associated Peptide (GAP) (1-13), human it important to determine whether multifunctional ferritin cage nanostructures can be used to enhance the induction of salivary IgA antibody to protect against caries. To answer this question, here we incorporated GLU and a linker sequence (GGGGSGGGGSGGGGS) at the N-terminus of FTH to form a fusion protein designated GLU-FTH. We then conducted anin vivostudy to evaluate the ability of GLU-FTH to induce a GLU-specific salivary IgA antibody response that guarded against colonization byS. mutans. Further, we evaluated the phagocytosis of nanoparticles by DCsin vitroas well as the ability of nanoparticles to enhance DC maturation. We believe that our findings will inspire a strategy for developing specific mucosal vaccines for other infectious diseases. == Results GnRH Associated Peptide (GAP) (1-13), human == == Construction, purification, and characterization of GLU-FTH == To construct multifunctional nanoparticles, recombinant GLU, ferritin (FTH), and GLU-ferritin (GLU-FTH) were Rabbit Polyclonal to SCFD1 expressed inE. coli. The proteins were purified using size-exclusion chromatography and analyzed using SDS-PAGE (Fig. 1A) and immunoblot (Fig. 1B). The estimated relative molecular masses of the 3 purified proteins were identical to their theoretical values (33.4, 21, and 57.7 kDa, respectively). These results indicate that GLU was fused to the N-terminus of the ferritin subunit. Transmission electron micrographic images of the purified proteins revealed a hollow spherical architecture with a thin size distribution. These results indicate that fusion of the GLU to the N-terminus of the ferritin subunit did not interfere with self-assembly of the subunits to form the characteristic 24-subunit ferritin cage (Fig. 1C). The diameters of the FTH and GLU-FTH cages were narrowly distributed (14.1 1.1 nm and 15.9 1.4 nm, respectively) (Fig. 1D). == Physique 1. == Generation and characterization of ferritin cages displaying FTH and GLU-FTH. (A) SDS-PAGE analysis of FTH, FTH-GLU, and GLU. (B) Immunoblot analysis of fusion protein using an anti-His tag monoclonal antibody as the probe. M, protein marker; Lane 1, FTH; Lane 2, GLU-FTH; and Lane 3, GLU. (C) TEM images of FTH and GLU-FTH protein cages. The upper panels show the protein cages at a higher magnification. (D) TEM analysis of the size distributions of FTH and GLU-FTH. == Nasal immunization of GLU-FTH induces GLU-specific immune responses == To determine the immunogenicity of GLU-FTH, we intranasally immunized mice with purified proteins and boosted them on days 14 and 28. FTH alone did not induce a detectable titer of specific IgG or IgA. These results show that intranasal immunization using FTH was not immunogenic. Significantly enhanced levels of GLU-specific serum IgG, serum IgA, and.