Qing-an Xu

TLR2-dependent modulation of osteoclastogenesis by Porphyromonas gingivalis through differential induction of NFATc1 and NF-κB

Osteolytic diseases, including rheumatoid arthritis, osteomyelitis, and periodontitis, are usually associated with bacterial infections. However, the precise mechanisms by which bacteria induce bone loss still remain unclear. Evidence exists that Toll-like receptor (TLR) signaling regulates both inflammation and bone metabolism and that the receptor activator of NF-κB ligand (RANKL) and its receptor RANK are the key regulators for bone remodeling and for the activation of osteoclasts. Here, we investigate the direct effects of the periodontal pathogen Porphyromonas gingivalis on osteoclast differentiation and show that P. gingivalis differentially modulates RANKL-induced osteoclast formation contingent on the state of differentiation of osteoclast precursors. In addition, although an optimal induction of cytokines by P. gingivalis is dependent on TLR2 and TLR4, as well as myeloid differentiation factor 88 and Toll/IL-1R domain-containing adaptor-inducing IFN-β, P. gingivalis utilizes TLR2/ myeloid differentiation factor 88 in modulating osteoclast differentiation. P. gingivalis modulates RANKL-induced osteoclast formation by differential induction of NFATc1 and c-Fos. More importantly, RANKL-mediated lineage commitment also has an impact on P. gingivalis-induced cytokine production. RANKL inhibits P. gingivalis-induced cytokine production by down-regulation of TLR/NF-κB and up-regulation of NFATc1. Our findings reveal novel aspects of the interactions between TLR and RANK signaling and provide a new model for understanding the mechanism underlying the pathogenesis of bacteria-mediated bone loss.

Immunogenicity and Persistence of a Targeted Anti-caries DNA Vaccine

We have previously reported that a targeted anti-caries DNA vaccine, pGJA-P, induced accelerated and increased antibody responses compared with a non-targeted anti-caries DNA vaccine. Recently, pGJA-P/VAX, a new targeted anti-caries DNA vaccine for human trials, was constructed by replacing the pCI vector used in the construction of pGJA-P with pVAX1, the only vector authorized by the US Food and Drug Administration in clinical trials. Here, we report on our exploration of the kinetics of the antibody responses generated following pGJA-P/VAX immunization and the persistence of pGJA-P/VAX at both the inoculation site and the draining lymph nodes. Intranasal vaccination of mice with pGJA-P/VAX induced strong antibody responses that lasted for more than 6 months. Furthermore, pGJA-P/VAX could still be detected at both the inoculation site and the draining cervical lymph nodes 6 months after immunization. Thus, the persistent immune responses are likely due to the DNA depot in the host, which acts as a booster immunization.

Extensive Description and Comparison of Human Supra-Gingival Microbiome in Root Caries and Health

Knowledge of the polymicrobial etiology of root caries is limited. To conduct a comprehensive research study on root caries, we utilized 454-pyrosequencing of 16S rRNA gene libraries and quantitative PCR to compare supra-gingival bacterial communities from healthy sites and carious sites of 21 patients with root caries (Patient-controls and Patient-cases) and the sites of 21 healthy individuals (Healthy-controls) from two nursing homes. Healthy-controls and Patient-cases showed no significant differences in terms of biomass, species richness, and species diversity. However, as for beta diversity based on either community membership metric (unweighted UniFrac) or community structure metric (weighted UniFrac), Healthy-controls and Patient-cases were clearly distinguished from each other, appearing more variable in the community membership and structure in root caries microbiome but relatively conserved in the health microbiome. The Patient-controls group was at an intermediate stage between Healthy-controls and Patient-cases, but was more inclined to the former. Demonstrated in both relative abundance and prevalence of species in health and root caries, Propionibacterium acidifaciens, Streptococcus mutans, Olsenella profusa, Prevotella multisaccharivorax, and Lactobacillus crispatus were found to be most associated with root caries, whereas Delftia acidovorans, Bacteroidetes[G-2] sp., Lachnospiraceae[G-3] sp., and Prevotella intermedia are most associated with health. Our study provides a basis for further elucidating the microbial etiology of root caries in the elderly.

Construction of a New Fusion Anti-caries DNA Vaccine

Mutans streptococci (MS) are generally considered to be the principal etiological agent of dental caries. MS have two important virulence factors: cell- surface protein PAc and glucosyltransferases (GTFs). GTFs have two functional domains: an N-terminal catalytic sucrose-binding domain (CAT) and a C-terminal glucan-binding domain (GLU). A fusion anti-caries DNA vaccine, pGJA-P/VAX, encoding two important antigenic domains, PAc and GLU, of S. mutans, was successful in reducing the levels of dental caries caused by S. mutans in gnotobiotic animals. However, its protective effect against S. sobrinus infection proved to be weak. Does the DNA vaccine need an antigen of S. sobrinus to enhance its ability to inhibit infection? To answer this question, in this study, we cloned the catalytic (cat) fragment of S. sobrinus gtf-I, which demonstrated its ability to inhibit water-insoluble glucan synthesis by S. sobrinus, into pGJA-P/VAX to produce a new anti-caries DNA vaccine.

A Mucosal Subunit Vaccine Protects against Lethal Respiratory Infection with Francisella tularensis LVS

Francisella tularensis (FT) is a highly virulent pathogen for humans and other mammals. Severe morbidity and mortality is associated with respiratory FT infection and there are concerns about intentional dissemination of this organism. Therefore, FT has been designated a category A biothreat agent and there is a growing interest in the development of a protective vaccine. In the present study, we determine the protective potential of a subunit vaccine comprised of the FT heat shock protein DnaK and surface lipoprotein Tul4 against respiratory infection with the live vaccine strain (LVS) of FT in mice. First, we establish an optimal intranasal immunization regimen in C57BL/6 mice using recombinant DnaK or Tul4 together with the adjuvant GPI-0100. The individual immunization regimens induced robust salivary IgA, and vaginal and bronchoalveolar IgA and IgG antigen-specific antibodies. Serum IgG1 and IgG2c antibody responses were also induced, indicative of a mixed type 2 and type 1 response, respectively. Next, we show that immunization with DnaK and Tul4 induces mucosal and systemic antibody responses that are comparable to that seen following immunization with each antigen alone. This immunization regimen also induced IFN-γ, IL-10 and IL-17A production by splenic CD4+ T cells in an antigen-specific manner. Importantly, over 80% of the mice immunized with DnaK and Tul4, but not with each antigen alone, were protected against a lethal respiratory challenge with FT LVS. Protection correlated with reduced bacterial burden in the lung, liver and spleen of mice. This study demonstrates the potential of DnaK and Tul4 as protective antigens and lends support to the notion of combining distinct, immunodominant antigens into an effective multivalent tularemia vaccine.

Mucosal and systemic immunization with targeted fusion anti-caries DNA plasmid in young rats

Early life vaccination is necessary to protect young children from dental caries. Our group had previously reported that a plasmid DNA vaccine pGJA-P/VAX against the glucosyltransferase (GTF) enzyme and cell surface antigen AgI/II (PAc) of Streptococcus mutans (S. mutans) elicited a specific and protective immunity in adult experimental animal models. In this report, early life immunization with the same plasmid was studied following intranasal (i.n.) and intramuscular (i.m.) delivery in murine models. The potential of inducing mucosal and systemic immune responses to special antigens was measured by ELISA. In addition, cytokine production and protection effectiveness against dental caries formation were also investigated. In the i.n. route, rats were primed when they were 5 days old, and boosted after 10 and 20 days with either plasmid pGJA-P/VAX-bupivacaine complexes, or pGJA-P/VAX alone, or empty vector. The pGJA-P/VAX-bupivacaine combination was able to mount the immune responses characterized by increased antibody levels of specific salivary IgA and serum IgG, preferential IFN-gamma production and significant reduction in the dental caries lesions. In the i.m. route, rats were vaccinated with either pGJA-P/VAX alone or empty vector with the same immunization schedule as the i.n. route. Plasmid pGJA-P/VAX alone induced a significant increase in the serum IgG and IFN-gamma production. However, it was not effective in eliciting specific salivary IgA and in decreasing the dental caries formation. All these findings indicate the feasibility of immunity with a targeted fusion DNA vaccine to a young immune system.

Enhanced efficacy of CTLA-4 fusion anti-caries DNA vaccines in gnotobiotic hamsters

AbstractAim:To evaluate the comparative immunogenicity and protective efficacy of the cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) fusion anti-caries DNA vaccines pGJA-P/VAX1, pGJA-P, and non-fusion anti-caries DNA construct pGLUA-P in hamsters. In addition, the ability of CTLA-4 to target pGJA-P/VAX1-encoding antigen to dendritic cells was tested in vitro.Methods:All DNA constructs contain genes encoding the A-P regions of a cell surface protein (PAc) and the glucan binding (GLU) domain of glucosyltransferases (GTFs) of cariogenic organism Streptococcus mutans. Human dendritic cells were mixed with the CTLA-4-Ig-GLU-A-P protein expressed by pGJA-P/VAX1-transfected cells and analyzed by flow cytometry. Gnotobiotic hamsters were immunized with anti-caries DNA vaccines by intramuscular injection or intranasal administration. Antibody responses to a representative antigen PAc were assayed by ELISA, and caries protection was evaluated by Keyes caries scores.Results:A flow cytometric analysis demonstrated that CTLA-4-Ig-GLU-A-P protein was capable of binding to human dendritic cells. pGJA-P/VAX1 and pGJA-P induced significantly higher specific salivary and serum anti-PAc antibody responses than pGLUA-P. Significantly fewer caries lesions were also observed in hamsters immunized with pGJA-P/VAX1 and pGJA-P. There was no significant difference in the anti-PAc antibody level or caries scores between pGJA-P/VAX1 and pGJA-P-immunized groups.Conclusion:Antigen encoded by CTLA-4 fusion anti-caries DNA vaccine pGJA-P/VAX1 could specifically bind to human dendritic cells through the interaction of CTLA-4 and B7 molecules. Fusing antigen to CTLA-4 has been proven to greatly enhance the immunogenicity and protective efficacy of anti-caries DNA vaccines.

Immunogenicity and Protective Efficacy of a Targeted Fusion DNA Construct against Dental Caries

Targeting antigens to antigen-presenting cells by fusion to cytotoxic T lymphocyte-associated antigen 4 (CTLA4) has been shown to be a highly efficient method to enhance the efficacy of DNA vaccines. The purpose of this study was to determine the immunogenicity and protective efficacy of the targeted fusion DNA construct pGJA-P, which contains the signal peptide and extracellular regions of human CTLA4 gene, the hinge and Fc regions of human Igγ1 gene, the glucan-binding domain of the Streptococcus mutans gtfB gene and the A-P fragment of the S. mutans pac gene, compared with the fusion DNA construct pGLUA-P, which contains only the glucan-binding domain of the S. mutansgtfB gene and the A-P fragment of the S. mutans pac gene. BALB/c mice were immunized with pGJA-P, pGLUA-P, or pCI (vector) by the intramuscular or intranasal route. Specific anti-PAc and anti-GTF-I serum IgG and salivary IgA antibody responses were assessed by an enzyme-linked immunosorbent assay. Wistar rats were orally challenged with S. mutans and immunized with pGJA-P, pGLUA-P, or pCI intramuscularly or intranasally, and caries activity was evaluated by the Keyes method. pGJA-P induced accelerated and increased serum and salivary antibody responses in mice compared with pGLUA-P. Rats immunized with pGJA-P had significantly fewer caries lesions than rats immunized with pGLUA-P (p < 0.01). Thus, this study demonstrates that the targeted DNA construct pGJA-P can enhance both systemic and mucosal immunity and may be a useful strategy for improving the protective efficacy of anticaries DNA vaccines.

Intranasal co-delivery of IL-6 gene enhances the immunogenicity of anti-caries DNA vaccine

Aim:To investigate the effects of co-delivering IL-6 expressing plasmid pCI-IL-6 on the immunogenicity of the anti-caries DNA vaccine pCIA-P, which encodes the surface protein antigen PAc of Streptococcus mutans.Methods:Plasmid pCI-IL-6 was constructed by inserting the murine IL-6 gene into the pCI vector. Expression of IL-6 in vitro was assessed using Western blot analysis. BALB/c mice were intranasally co-immunized with pCIA-P plus pCI-IL-6 on d 0 and 14. Anti-PAc IgG and secretory IgA (sIgA) were assessed by ELISA. Splenocytes from the mice were re-stimulated with the PAc protein, and IFN-γ and IL-4 production was measured using ELISA. Splenocyte proliferation was analyzed with flow cytometry. Rats were similarly immunized, and dental caries scores were determined using the Keyes method.Results:Marked expression of IL-6 was found in COS-7 cells transfected with pCI-IL-6. In the pCI-IL-6 co-immunized mice, the specific IgG antibodies in serum and sIgA antibodies in saliva were significantly higher than those in the control mice at weeks 4 and 8. Moreover, the secretion of IFN-γ from splenocytes in response to re-stimulation with PAc protein was significantly higher in the pCI-IL-6 co-immunized mice than that in the control mice, whereas the secretion of IL-4 had no significant difference. The proliferation of splenocytes from the pCI-IL-6 co-immunized mice was significantly higher than that from the mice immunized with pCIA-P and pCI vector. In the rat caries model, the pCI-IL-6 co-immunization rats displayed lower caries scores than the control rats.Conclusion:Intranasal co-delivery of IL-6 gene significantly enhances the immunogenicity of the anti-caries DNA vaccine.

Anti-caries DNA vaccine-induced secretory immunoglobulin A antibodies inhibit formation of Streptococcus mutans biofilms in vitro

Aim:To investigate the effects of anti-caries DNA vaccine-induced salivary secretory immunoglobulin A (S-IgA) antibodies on Streptococcus mutans (S. mutans) adherence and biofilms formation in vitro.Methods:Adult female Wistar rats were intranasally immunized with the anti-caries DNA vaccine pGJA-P/VAX. Their saliva samples were collected at different times after the immunization, and S-IgA antibody level in the saliva and its inhibition on S. mutans adherence were examined. The effects of S-IgA in the saliva with the strongest inhibitory effects were examined at 3 different stages, ie acquired pellicles, biofilm formation and production of mature biofilms. The number of viable bacteria and depth of the biofilm at 16 h in each stage were determined using counting colony forming units and using a confocal laser scanning microscopy (CLSM). The participation of S-IgA in acquired pellicles and its aggregation with S. mutans were also observed under CLSM.Results:The S-IgA titer in saliva reached its peak and exhibited the strongest inhibition on S. mutans adhesion at 10 weeks after the immunization. The colonies and depth of the biofilm in the saliva-pretreated group were 41.79% and 41.02%, respectively, less than the control group. The colonies and depth of the biofilm in the co-culture group were 27.4% and 22.81% less than the control group. The assembly of S. mutans and S-IgA was observed under CLSM after co-cultivation. In the mature-stage biofilm, no differences were observed between the different groups.Conclusion:These results demonstrate that the anti-caries DNA vaccine induces the production of specific S-IgA antibodies that may prevent dental caries by inhibiting the initial adherence of S. mutans onto tooth surfaces, thereby reducing the accumulation of S. mutans on the acquired pellicles.