N-terminal Domain of the Spike Protein of Porcine Epidemic Diarrhea Virus as a New Candidate Molecule for a Mucosal Vaccine.

Porcine epidemic diarrhea virus (PEDV) is a contagious coronavirus infecting pigs that leads to significant economic losses in the swine industry. Given that PEDV infection occurs in gut epithelial cells mainly via the fecal-oral route, induction of PEDV-specific immune responses in the mucosal compartment is required for protective immunity against viral infection. However, an effective mucosal vaccine against the currently prevalent PEDV strain is not available. In this study, we demonstrated that the N-terminal domain (NTD) of the spike (S) protein of PEDV represents a new vaccine candidate molecule to be applied via the mucosal route. We first established an Escherichia coli expression system producing the partial NTD (NTD231-501) of the PEDV S protein. Orally administered NTD231-501 protein specifically interacted with the apical area of M cells in the follicle-associated epithelium of Peyer's patch. Additionally, the NTD protein induced antigen-specific immune responses in both the systemic and mucosal immune compartments when administered orally. Collectively, we propose the NTD of the PEDV S protein to be a candidate mucosal vaccine molecule.

Antimicrobial activity of the chloroform fraction of Drynaria fortunei against oral pathogens.

Drynaria fortunei (D. fortunei), widely used in traditional Korean medicine, is reportedly effective in treating inflammation, hyperlipidemia, bone fractures, oxidative damage, arteriosclerosis, rheumatism, and gynecological diseases. The objective of this study was to evaluate the antibacterial effects of the chloroform fraction of D. fortunei (DFCF) and assess the synergistic effects of DFCF with antibiotics against bacterial pathogens. This was carried out by calculating the minimal inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) and performing checkerboard dilution test and time-kill assays. The MICs/MBCs for DFCF, ampicillin, and gentamicin against all oral strains were >39-2,500/5,000 µg/mL, 0.25-64/0.25-64 µg/mL, and 0.5-256/1-512 µg/mL, respectively. DFCF exhibited the highest activity against the periodontic pathogens Prevotella intermedia and Porphylomonas gingivalis. DFCF in combination with ampicillin showed a strong synergistic effect against oral bacteria (fractional inhibitory concentration (FIC) index ≤0.5), whereas on combining with gentamicin, it reduced the on half-eighth times than used alone (FICI ≤ 0.5). DFCF combined with ampicillin or gentamicin killed 100% of most tested bacteria within 3-4 h. The results of this study demonstrate the antimicrobial and synergistic activity of DFCF and antibiotics against oral pathogens.

Antibacterial and synergistic effects of the n-BuOH fraction of Sophora flavescens root against oral bacteria.

The antibacterial activity of an extract and several fractions of Sophora flavescens (S. flavescens) root alone and in combination with antibiotics against oral bacteria was investigated by checkerboard assay and time-kill assay. The minimum inhibitory concentration/minimum bactericidal concentration (MIC/MBC) values for all examined bacteria were 0.313-2.5/0.625-2.5 µg/mL for the n-BuOH fraction, 0.625-5/1.25-10 µg/mL for the EtOAc fraction, 0.25-8/0.25-16 µg/mL for ampicillin, 0.5-256/1-512 µg/mL for gentamicin, 0.008-32/0.016-64 µg/mL for erythromycin, and 0.25-64/0.5-128 µg/mL for vancomycin. The n-butanol (n-BuOH) and ethyl acetate (EtOAc) fractions exhibited stronger antibacterial activity against oral bacteria than other fractions and extracts. The MICs and MBCs were reduced to between one half and one quarter when the n-BuOH and EtOAc fractions were combined with antibiotics. After 24 h of incubation, combination of 1/2 MIC of the n-BuOH fraction with antibiotics increased the degree of bactericidal activity. The present results suggest that n-BuOH and EtOAc extracts of S. flavescens root might be applicable as new natural antimicrobial agents against oral pathogens.

Fucoidan inhibits LPS-induced inflammation in vitro and during the acute response in vivo.

Studies have been focused on natural products with antibacterial and anti-inflammatory activities, such as fucoidan. Many in vivo studies have evaluated the effect of fucoidan on tumor growth, diabetes, obesity, ischemia reperfusion, and oxidative stress. However, the effects of fucoidan on bacteria-induced gingival inflammation and periodontitis have not been reported. We previously characterized the anti-inflammatory effect of fucoidan in vitro. Here, we confirmed the anti-inflammatory activity of fucoidan in a macrophage cell line in terms of its inhibition of the expression of inflammatory mediators and pro-inflammatory cytokines. Additionally, we confirmed the ability of fucoidan to inhibit gingival inflammation, expression of pro-inflammatory cytokines, and neutrophil recruitment in the gingival tissue of mice injected with LPS prepared from P. gingivalis. Interestingly, however, fucoidan did not inhibit the expression of pro-inflammatory cytokines in a P. gingivalis-infected mouse model of periodontitis. Additionally, fucoidan treatment did not lead to clearance of P. gingivalis or improvement of P. gingivalis infection-mediated bone loss in the periodontitis model. We conclude that fucoidan exerts anti-inflammatory effects in vitro and in vivo, together with a limited antibacterial effect in vivo.

Sophoraflavanone G prevents Streptococcus mutans surface antigen I/II-induced production of NO and PGE2 by inhibiting MAPK-mediated pathways in RAW 264.7 macrophages.

BACKGROUND: Sophora flavescens AITON (Leguminosae) is a typical traditional Korean medical herb considered to exhibit antibacterial, anti-inflammatory, and antipyretic effects, and is also used for the treatment of skin and mucosal ulcers, sores, diarrhea, gastrointestinal hemorrhage, arrhythmia, and eczema. OBJECTIVE AND DESIGN: This study examined the inhibitory effects of sophoraflavanone G (SF) of S. flavescens on the bacterial fibrillar protein, Antigen I/II (AgI/II)-N recombinant protein isolated from Streptococcus mutans(rAg I/II)-induced production of nitric oxide (NO) and prostaglandin E2 (PGE2). The investigation was focused on whether SF could inhibit the production of proinflammatory mediators such as nitric oxide (NO) and prostaglandin (PG) E2 as well as the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, tumor necrosis factor (TNF)-a, interleukin (IL)-6, nuclear factor (NF)-κB and mitogen-activated protein kinases (MAPKs) in rAgI/II-stimulated RAW 264.7 cells using Griess reagent, Enzyme linked immunosorbent assay (ELISA), and Western blotting analysis. RESULTS: SG significantly inhibited the production of NO and PGE2 and pro-inflammatory cytokines, such as interleukin (IL)-1ß, IL-6, and tumor necrosis factor α in Ag I/II-N-stimulated RAW264.7 cells, which were mediated by the down-regulation of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) expression. The SF inhibited the phosphorylation of IκB-α, nuclear translocation of p65, and subsequent activation of NF- κB in the rAgI/II-stimulated cells. In addition, the SF suppressed the rAgI/II-stimulated activation of ERK MAPK as well as the MAPK inhibitor significantly reduced the rAgI/II-induced production of NO and PGE2. CONCLUSION: Collectively, we suggest that the SF inhibits the expression and production of inflammatory mediators by blocking the ERK MAPK mediated pathway and inhibiting the activation of NF-κB.

Intestinal anti-inflammatory activity of the seeds of Raphanus sativus L. in experimental ulcerative colitis models.

ETHNOPHARMACOLOGICAL RELEVANCE: Water extract of Raphanus sativus L. (RSL) seeds was traditionally used to treat digestive inflammatory complaints in Korean culture. RSL seeds exerted antioxidant, anti-inflammatory, and anti-septic functions, suggesting their pharmacological potential for the treatment of inflammatory pathologies associated with oxidative stress such as inflammatory bowel disease. AIM OF THIS STUDY: We evaluated the intestinal anti-inflammatory effects of RSL seed water extract (RWE) in experimental rat models of trinitrobenzenesulphonic acid (TNBS)- or dextran sodium sulfate (DSS)-induced colitis. MATERIALS AND METHODS: RWE was characterized by determining the content of sinapic acid as a reference material and then assayed in the DSS and TNBS models of rat colitis. Male Sprague-Dawley rats were divided into 10 groups (n=7/group): non-colitic control, DSS or TNBS control, DSS colitis groups treated with RWE (100mg/kg) or mesalazine (25mg/kg), and TNBS colitis groups treated with various doses (10, 40, 70, and 100mg/kg) of RWE or mesalazine (25mg/kg). RWE or mesalazine treatment started the same day of colitis induction and rats were sacrificed 24h after the last treatment followed by histological and biochemical analyses. RESULTS: Oral administration with RWE suppressed intestinal inflammatory damages in both DSS- and TNBS-induced colitic rats. The treatment with 100mg/kg RWE recovered intestinal damages caused by TNBS or DSS to levels similar to that of mesalazine, decreasing the activity of myeloperoxidase activity and the secretion of tumor necrosis factor (TNF)-α and interleukin (IL)-1ß. RWE treatment inhibited malondialdehyde production and glutathione reduction in colon of colitis rats. The administration of RWE at dose of 100mg/kg also suppressed the TNBS- or DSS-stimulated expression of TNF-α, IL-1ß, monocyte chemotactic protein-1, inducible nitric oxide, and intercellular adhesion molecule-1. Furthermore, RWE inhibited p38 kinase and DNA-nuclear factor-κB binding activities, both of which were stimulated in the colitic rats. CONCLUSIONS: The current findings show that RWE ameliorates intestinal oxidative and inflammatory damages in DSS and TNBS models of rat colitis, suggesting its beneficial use for the treatment of intestinal inflammatory disorders.

N-acetyl cysteine inhibits H2O2-mediated reduction in the mineralization of MC3T3-E1 cells by down-regulating Nrf2/HO-1 pathway.

There are controversial findings regarding the roles of nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway on bone metabolism under oxidative stress. We investigated how Nrf2/HO-1 pathway affects osteoblast differentiation of MC3T3-E1 cells in response to hydrogen peroxide (H2O2), N-acetyl cysteine (NAC), or both. Exposing the cells to H2O2 decreased the alkaline phosphatase activity, calcium accumulation, and expression of osteoblast markers, such as osteocalcin and runt-related transcription factor-2. In contrast, H2O2 treatment increased the expression of Nrf2 and HO-1 in the cells. Treatment with hemin, a chemical HO-1 inducer, mimicked the inhibitory effect of H2O2 on osteoblast differentiation by increasing the HO-1 expression and decreasing the osteogenic marker genes. Pretreatment with NAC restored all changes induced by H2O2 to near normal levels in the cells. Collectively, our findings suggest that H2O2-mediated activation of Nrf2/HO-1 pathway negatively regulates the osteoblast differentiation, which is inhibited by NAC.

Antimicrobial peptide LL-37 promotes antigen-specific immune responses in mice by enhancing Th17-skewed mucosal and systemic immunities.

The human antimicrobial peptide LL-37 is known to have chemotactic and modulatory activities on various cells including monocytes, T cells, and epithelial cells. Given that LL-37 enhances chemotactic attraction and modulates the activity of DCs, it is conceivable that it might play a role as an immune adjuvant by skewing the immune environment toward immunostimulatory conditions. In this study, we characterized the mucosal adjuvant activity of LL-37 using model and pathogenic Ags. When LL-37-conjugated Ag was administered orally to mice, a tolerogenic Peyer's patch environment was altered to cell populations containing IL-6-secreting CD11c(+), CD11c(+) CD70(+), and Th17 cells capable of evoking a subsequent LL-37-conjugated Ag-specific immune response in both systemic and mucosal immune compartments. In addition, we showed presentation of formyl peptide receptor, an LL-37 receptor, on M cells, which may aid the initiation of an LL-37-mediated enhanced immune response through targeting and transcytosis of the conjugated Ag. Based on our findings, we conclude that LL-37 has potential as an oral mucosal adjuvant, not only by enhancing the delivery of LL-37-conjugated Ag to M cells, but also by triggering T-cell-mediated Ag-specific immune responses through modulation of the mucosal immune environment.

COMP-angiopoietin 1 increases proliferation, differentiation, and migration of stem-like cells through Tie-2-mediated activation of p38 MAPK and PI3K/Akt signal transduction pathways.

Recombinant COMP-Ang1, a chimera of angiopoietin-1 (Ang1) and a short coiled-coil domain of cartilage oligomeric matrix protein (COMP), is under consideration as a therapeutic agent capable of inducing the homing of cells with increased angiogenesis. However, the potentials of COMP-Ang1 to stimulate migration of mesenchymal stem cells (MSCs) and the associated mechanisms are not completely understood. We examined the potential of COMP-Ang1 on bone marrow (BM)-MSCs, human periodontal ligament stem cells (PDLSCs), and calvarial osteoblasts. COMP-Ang1 augmented Tie-2 induction at protein and mRNA levels and increased proliferation and expression of runt-related transcription factor 2 (Runx2), osterix, and CXCR4 in BMMSCs, but not in osteoblasts. The COMP-Ang1-mediated increases were inhibited by Tie-2 knockdown and by treating inhibitors of phosphoinositide 3-kinase (PI3K), LY294002, or p38 mitogen-activated protein kinase (MAPK), SB203580. Phosphorylation of p38 MAPK and Akt was prevented by siRNA-mediated silencing of Tie-2. COMP-Ang1 also induced in vitro migration of BMMSCs and PDLSCs. The induced migration was suppressed by Tie-2 knockdown and by CXCR4-specific peptide antagonist or LY294002, but not by SB203580. Furthermore, COMP-Ang1 stimulated the migration of PDLSCs into calvarial defect site of rats. Collectively, our results demonstrate that COMP-Ang1-stimulated proliferation, differentiation, and migration of progenitor cells may involve the Tie-2-mediated activation of p38 MAPK and PI3K/Akt pathways.

Monoclonal antibodies specific to Streptococcus mutans GS-5 glucosyltransferase-C inhibit bacterial glucosyltransferase.

Glucosyltransferase-C (GTFC) is a virulence factor of Streptococcus mutans. Additionally, GTFC represents an essential element required for bacterial cell coherence, allowing for the formation of dental plaque, which leads to dental caries. As such, monoclonal antibodies (MAbs) against S. mutans are believed to offer some protection against dental caries. In the current study, we amplified an approximately 1.5 kb fragment of the N-terminal half of the S. mutans gtfC gene by PCR, then induced expression of this gene. This protein was designated GTFCN. After the expressed protein was purified, it was used as an immunogen and injected into BALB/c mice. We selected and established two MAbs by producing hybridomas (HCN17 and HCN37). The anti-GTFCN antibody isotype was confirmed as IgG2a for HCN17 and IgG2b for HCN37. The anti-GTFCN antibody was found to specifically react with the GTFCN protein. The enzymatic activity of the crude glucosyltransferase of S. mutans GS-5 was significantly inhibited at a concentration of 350 ng of MAb/mL. These results suggest that the monoclonal anti-GTFCN antibodies could represent an alternative modality for passive immunization to prevent S. mutans aggregation and dental plaque.

Rapid detection of S. mutans surface antigen I/II using a sensitive monoclonal anti-Ag I/II antibody by ELISA.

The cell-surface protein antigen I/II (Ag I/II) is expressed in oral streptococci, which are known as the causative agent of a number of diseases including dental caries, endocarditis, gingivitis, and periodontal disease. Consequently, monoclonal antibodies (MAb) capable of recognizing the streptococcal Ag I/II protein could be a useful tool for the diagnosis and cure of these diseases. In this study, a previously generated monoclonal anti-Ag I/II antibody, ckAg I/II, was used to detect a small amount of Streptococcus mutans (S. mutans) surface antigen Ag I/II. The ckAg I/II was proved to be very sensitive and able to detect as little as 1 ng of recombinant Ag I/II protein within 5 min and Ag I/II in saliva within 10 min, as well as native Ag I/II in 20 µL of culture supernatant by ELISA. These results suggest that ckAg I/II can be used as a fast and efficient diagnostic tool to detect Ag I/II.

Application of an M-cell-targeting ligand for oral vaccination induces efficient systemic and mucosal immune responses against a viral antigen.

Oral mucosal vaccination is an alternative method to overcome the pitfalls of current injection-based vaccines, such as pain and high cost of vaccination. It is a feasible and economic vaccine application, especially in developing countries. However, achieving effective antigen delivery into mucosal lymphoid organs and efficient immune stimulation are prerequisites to successful oral mucosal vaccination. One promising approach for oral mucosal vaccine development is exploring the potential of M cells via M-cell-targeting ligands that have the potential to deliver ligand-conjugated antigens into mucosal lymphoid organs and evoke conjugated-antigen-specific systemic and mucosal immune responses. Here, we investigated the M-cell-targeting ligand, Co1, in inducing specific immune responses against a pathogenic viral antigen, envelope domain III (EDIII) of dengue virus, to provide the foundation for oral mucosal vaccine development against the pathogen. After oral administration of Co1-conjugated EDIII antigens, we observed efficient antigen delivery into Peyer's patches. We also report the elicitation of EDIII-specific immunity in systemic and mucosal compartments by Co1 ligand (located in the C-terminus of EDIII). Furthermore, the antibodies induced by the ligand-conjugated EDIII antigen showed effective virus-neutralizing activity. The results of this study suggest that the M-cell-targeting strategy using Co1 ligand as a mucosal adjuvant may be applicable for developing oral vaccine candidates against pathogenic viral antigen.

C5a receptor-targeting ligand-mediated delivery of dengue virus antigen to M cells evokes antigen-specific systemic and mucosal immune responses in oral immunization.

Oral mucosal immunization is a feasible and economic vaccination strategy. In order to achieve a successful oral mucosal vaccination, antigen delivery to gut immune inductive site and avoidance of oral tolerance induction should be secured. One promising approach is exploring the specific molecules expressed on the apical surfaces of M cells that have potential for antigen uptake and immune stimulation. We previously identified complement 5a receptor (C5aR) expression on human M-like cells and mouse M cells and confirmed its non-redundant role as a target receptor for antigen delivery to M cells using a model antigen. Here, we applied the OmpH ligand, which is capable of targeting the ligand-conjugated antigen to M cells to induce specific mucosal and systemic immunities against the EDIII of dengue virus (DENV). Oral immunization with the EDIII-OmpH efficiently targeted the EDIII to M cells and induced EDIII-specific immune responses comparable to those induced by co-administration of EDIII with cholera toxin (CT). Also, the enhanced responses by OmpH were characterized as Th2-skewed responses. Moreover, oral immunization using EDIII-OmpH did not induce systemic tolerance against EDIII. Collectively, we suggest that OmpH-mediated targeting of antigens to M cells could be used for an efficient oral vaccination against DENV infection.

Synergistic effect of fucoidan with antibiotics against oral pathogenic bacteria.

BACKGROUND: Fucoidan is a sulphated polysaccharide that is primarily extracted from brown seaweeds; it has been broadly studied in recent years due to its numerous biological properties, including anticoagulant, antithrombotic, antitumour and antiviral activities. OBJECTIVE AND DESIGN: In this study, fucoidan was evaluated against oral bacteria, either alone or with antibiotics, via the broth dilution method and chequerboard and time-kill assay. RESULTS: Minimum inhibitory concentration/minimum bactericidal concentration (MIC/MBC) values for the fucoidan against all the tested bacteria ranged between 0.125 and 0.50/0.25 and 1.00mgml(-1), for ampicillin 0.125 and 64/0.5 and 64µgml(-1) and for gentamicin 2 and 256/4 and 512µgml(-1), respectively. Furthermore, the MIC and MBC were reduced to one half-eighth as a result of the combination of the fucoidan with antibiotics. One to 3h of treatment with MIC50 of fucoidan with MIC50 of antibiotics resulted from an increase of the rate of killing in colony forming units (CFUs) ml(-1) to a greater degree than was observed with alone. CONCLUSION: These results suggest that fucoidan is important in the antibacterial actions of the agents.

Nasal immunization with major epitope-containing ApxIIA toxin fragment induces protective immunity against challenge infection with Actinobacillus pleuropneumoniae in a murine model.

Actinobacillus pleuropneumoniae is an infective agent that leads to porcine pleuropneumonia, a disease that causes severe economic losses in the swine industry. Based on the fact that the respiratory tract is the primary site for bacterial infection, it has been suggested that bacterial exclusion in the respiratory tract through mucosal immune induction is the most effective disease prevention strategy. ApxIIA is a vaccine candidate against A. pleuropneumoniae infection, and fragment #5 (aa. 439-801) of ApxIIA contains the major epitopes for effective vaccination. In this study, we used mice to verify the efficacy of intranasal immunization with fragment #5 in the induction of protective immunity against nasal challenge with A. pleuropneumoniae and compared its efficacy with that of subcutaneous immunization. Intranasal immunization of the fragment induced significantly higher systemic and mucosal immune responses measured at the levels of antigen-specific antibodies, cytokine-secreting cells after antigen exposure, and antigen-specific lymphocyte proliferation. Intranasal immunization not only efficiently inhibited the bacterial colonization in respiratory organs, but also prevented alveolar tissue damage in infectious condition similar to that of a contaminated pig. Moreover, intranasal immunization with fragment #5 provided acquired protective immunity against intranasal challenge with A. pleuropneumoniae serotype 2. In addition, it conferred cross-protection against serotype 5, a heterologous pathogen that causes severe disease by ApxI and ApxII secretion. Collectively, intranasal immunization with fragment #5 of ApxIIA can be considered an efficient protective immunization procedure against A. pleuropneumoniae infection.

A monoclonal antibody specific to glucosyltransferase B of Streptococcus mutans GS-5 and its glucosyltransferase inhibitory efficiency.

Glucosyltransferase-B (GTFB) of Streptococcus mutans is considered a virulence factor because of its activity in the production of insoluble glucan, which is key to the bacterial attachment onto dental surfaces, leading to the formation of dental caries. Local passive immunization with monoclonal antibodies against GTFB is considered to be an effective way to prevent dental caries. Here we amplified a 1.3 kb fragment of the N-terminal half of the gtfB gene (193-1530) of S. mutans by PCR and expressed the truncated protein (GTFBN). The expressed, purified protein was used as an immunogen in BALB/c mice. We selected and established one hybridoma (HBN8) that was capable of producing anti-GTFBN using ELISA, dot blot, and Western blot analyses. The monoclonal anti-GTFBN antibody was purified by affinity chromatography and its isotype was confirmed as IgG2a. The anti-GTFBN antibody inhibited the enzymatic activity of crude glucosyltransferase of S. mutans GS-5 in a dose-dependent manner. These data suggest that the anti-GTFBN antibody could be used as a vaccine to prevent the aggregation of S. mutans on tooth surfaces, and thus prevent the formation of dental caries.

Mucosal Immune System and M Cell-targeting Strategies for Oral Mucosal Vaccination.

Vaccination is one of the most effective methods available to prevent infectious diseases. Mucosa, which are exposed to heavy loads of commensal and pathogenic microorganisms, are one of the first areas where infections are established, and therefore have frontline status in immunity, making mucosa ideal sites for vaccine application. Moreover, vaccination through the mucosal immune system could induce effective systemic immune responses together with mucosal immunity in contrast to parenteral vaccination, which is a poor inducer of effective immunity at mucosal surfaces. Among mucosal vaccines, oral mucosal vaccines have the advantages of ease and low cost of vaccine administration. The oral mucosal immune system, however, is generally recognized as poorly immunogenic due to the frequent induction of tolerance against orally-introduced antigens. Consequently, a prerequisite for successful mucosal vaccination is that the orally introduced antigen should be transported across the mucosal surface into the mucosa-associated lymphoid tissue (MALT). In particular, M cells are responsible for antigen uptake into MALT, and the rapid and effective transcytotic activity of M cells makes them an attractive target for mucosal vaccine delivery, although simple transport of the antigen into M cells does not guarantee the induction of specific immune responses. Consequently, development of mucosal vaccine adjuvants based on an understanding of the biology of M cells has attracted much research interest. Here, we review the characteristics of the oral mucosal immune system and delineate strategies to design effective oral mucosal vaccines with an emphasis on mucosal vaccine adjuvants.

M cells expressing the complement C5a receptor are efficient targets for mucosal vaccine delivery.

In the mucosal immune system, M cells are known as specialized epithelial cells that take up luminal antigens, although the receptors on M cells and the mechanism of antigen uptake into M cells are not well-understood. Here, we report the expression of the complement C5a receptor (C5aR) on the apical surface of M cells. C5ar mRNA expression in co-cultured Caco-2 human M-like cells was six-fold higher than in mono-cultured cells. C5aR expression was detected together with glycoprotein 2, an M-cell-specific protein, on the apical surface of M-like cells and mouse Peyer's patch M cells. Interestingly, after oral administration of Yersinia enterocolitica which expresses outer membrane protein H (OmpH) that is homologous to the Skp α1 domain of Escherichia coli, a ligand of C5aR, dense clustering and phosphorylation of C5aR were detected in M cells. Finally, targeted antigen delivery to M cells using C5aR as a receptor was achieved using the OmpH α1 of Y. enterocolitica such that the induction of ligand-conjugated antigen-specific immune responses was confirmed in mice after oral immunization of the OmpH ß1α1-conjugated antigen. Collectively, we identified C5aR expression on M cells and suggest that C5aR could be used as a target receptor for mucosal antigen delivery.

Development of a monoclonal antibody against glucosyltransferase D of Streptococcus mutans GS 5.

Glucosyltransferases GtfB, GtfC, and GtfD of Streptococcus mutans are virulent factors involved in dental caries. Consequently, they are considered to be target molecules in the development of vaccines against dental caries. Among them, GtfD plays a significant role in the sucrose-dependent cellular adhesion of S. mutans, and a number of studies have suggested that the N-terminus of GtfD is an important part of its role in enzymatic activity. In this study, we generated monoclonal antibodies against the N-terminus of GtfD (anti-GtfDN antibody) in an initial attempt to investigate its preventive efficacy against dental caries. To obtain anti-GtfDN monoclonal antibodies, the gene for the N-terminus of gtfD (2 kb) was cloned into an Escherichia coli expression vector, pQE30; then the expressed protein (about 75 kDa) was purified. The purified GtfDN protein was injected into BALB/c mice, and hybridoma clones were established. We obtained three hybridoma clones (HDN9, HDN11, and HDN28) capable of producing anti-GtfDN antibodies. Their binding specificity was characterized by ELISA, dot blot, and Western blot analysis after purification using affinity column chromatography. The isotype of the monoclonal antibodies was confirmed to be IgG2a.

Characterization of antigenic determinants in ApxIIA exotoxin capable of inducing protective immunity to Actinobacillus pleuropneumoniae challenge.

Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia. Among the virulence factors of the pathogen, ApxIIA, a bacterial exotoxin, is expressed by many serotypes and presents a plausible target for vaccine development. We characterized the region within ApxIIA that induces a protective immune response against bacterial infection using mouse challenge model. Recombinant proteins spanning the length of ApxIIA were produced and antiserum to the full-length ApxIIA was induced in mice. This antiserum recognized fragments #2, #3 and #5 with high binding specificity, but showed poor recognition for fragments #1 and #4. Of the antisera induced in mice by injection of each fragments, only the antiserum to fragment #4 failed to efficiently recognize the full-length antigen, although the individual antisera recognized their cognate antigens with almost equal efficiency. The protective potency of the immunogenic proteins against a challenge injection of bacteria in vivo correlated well with the antibody titer. Fragment #5 induced the highest level of protective activity, comparable to that by the full-length protein. These results support the use of fragment #5 to produce a vaccine against A. pleuropneumoniae challenge, since the small antigen peptide is easier to handle than is the full-length protein and can be expressed efficiently in heterologous expression systems.