A Fusion Protein of Derp2 Allergen and Flagellin Suppresses Experimental Allergic Asthma

PURPOSE: The house dust mite (HDM) is one of the most important sources of indoor allergens and a significant cause of allergic rhinitis and allergic asthma. Our previous studies demonstrated that Vibrio vulnificus flagellin B (FlaB) plus allergen as a co-treatment mixture improved lung function and inhibited eosinophilic airway inflammation through the Toll-like receptor 5 signaling pathway in an ovalbumin (OVA)- or HDM-induced mouse asthma model. In the present study, we fused the major mite allergen Derp2 to FlaB and compared the therapeutic effects of the Derp2-FlaB fusion protein with those of a mixture of Derp2 and FlaB in a Derp2-induced mouse asthma model. METHODS: BALB/c mice sensitized with Derp2 + HDM were treated with Derp2, a Derp2 plus FlaB (Derp2 + FlaB) mixture, or the Derp2-FlaB fusion protein 3 times at 1-week intervals. Seven days after the final treatment, the mice were challenged intranasally with Derp2, and airway responses and Derp2-specific immune responses were evaluated. RESULTS: The Derp2-FlaB fusion protein was significantly more efficacious in reducing airway hyperresponsiveness, lung eosinophil infiltration, and Derp2-specific IgE than the Derp2 + FlaB mixture. CONCLUSIONS: The Derp2-FlaB fusion protein showed a strong anti-asthma immunomodulatory capacity, leading to the prevention of airway inflammatory responses in a murine disease model through the inhibition of Th2 responses. These findings suggest that the Derp2-FlaB fusion protein would be a promising vaccine candidate for HDM-mediated allergic asthma therapy.

Development of a Novel Subunit Vaccine Targeting Fusobacterium nucleatum FomA Porin Based on In Silico Analysis

Selecting an appropriate antigen with optimal immunogenicity and physicochemical properties is a pivotal factor to develop a protein based subunit vaccine. Despite rapid progress in modern molecular cloning and recombinant protein technology, there remains a huge challenge for purifying and using protein antigens rich in hydrophobic domains, such as membrane associated proteins. To overcome current limitations using hydrophobic proteins as vaccine antigens, we adopted in silico analyses which included bioinformatic prediction and sequence-based protein 3D structure modeling, to develop a novel periodontitis subunit vaccine against the outer membrane protein FomA of Fusobacterium nucleatum. To generate an optimal antigen candidate, we predicted hydrophilicity and B cell epitope parameter by querying to web-based databases, and designed a truncated FomA (tFomA) candidate with better solubility and preserved B cell epitopes. The truncated recombinant protein was engineered to expose epitopes on the surface through simulating amino acid sequence-based 3D folding in aqueous environment. The recombinant tFomA was further expressed and purified, and its immunological properties were evaluated. In the mice intranasal vaccination study, tFomA significantly induced antigen-specific IgG and sIgA responses in both systemic and oral-mucosal compartments, respectively. Our results testify that intelligent in silico designing of antigens provide amenable vaccine epitopes from hard-to-manufacture hydrophobic domain rich microbial antigens.

Flagellin Modulates the Function of Invariant NKT Cells From Patients With Asthma via Dendritic Cells

PURPOSE: Invariant natural killer T (iNKT) cells play a critical role in the pathogenesis of asthma. We previously reported the association between circulating Th2-like iNKT cells and lung function in asthma patients and the suppressive effect of Toll-like receptor 5 ligand flagellin B (FlaB) on asthmatic in a mouse model. Thus, we investigated whether FlaB modulates the function of circulating iNKT cells in asthmatic patients. METHODS: Peripheral blood mononuclear cells (PBMCs) were treated with FlaB, and the secreted and intracellular cytokines of iNKT cells were evaluated by using ELISA and flow cytometry, respectively, following stimulation with alpha-galactosylceramide. Foxp3+ iNKT cells were also measured. To determine the effect of FlaB-treated dendritic cells (DCs) on iNKT cells, we co-cultured CD14+ monocyte-derived DCs and T cells from patients with house dust mite-sensitive asthma and analyzed intracellular cytokines in iNKT cells. RESULTS: A reduction of IL-4 and IL-17 production by iNKT cells in PBMCs after FlaB treatment was alleviated following blocking of IL-10 signaling. A decrease in the frequencies of IL-4+ and IL-17+ iNKT cells by FlaB-treated DCs was reversed after blocking of IL-10 signaling. Simultaneously, an increase in Foxp3+ iNKT cells induced by FlaB treatment disappeared after blocking of IL-10. CONCLUSIONS: FlaB may inhibit Th2- and Th17-like iNKT cells and induce Foxp3+ iNKT cells by DCs via an IL-10-dependent mechanism in asthmatic patients. In patients with a specific asthma phenotype associated with iNKT cells, FlaB may be an effective immunomodulator for iNKT cell-targeted immunotherapy.

Tetanus toxin fragment C fused to flagellin makes a potent mucosal vaccine

PURPOSE: Recombinant subunit vaccines provide safe and targeted protection against microbial infections. However, the protective efficacy of recombinant subunit vaccines tends to be less potent than the whole cell vaccines, especially when they are administered through mucosal routes. We have reported that a bacterial flagellin has strong mucosal adjuvant activity to induce protective immune responses. In this study, we tested whether FlaB could be used as a fusion partner of subunit vaccine for tetanus. MATERIALS AND METHODS: We constructed fusion proteins consisted with tetanus toxin fragment C (TTFC), the nontoxic C-terminal portion of tetanus toxin, and a Toll-like receptor 5 agonist from Vibrio vulnificus (FlaB). Mice were intranasally administered with fusion protein and protective immune responses of the vaccinated mice were analyzed. RESULTS: FlaB-TTFC recombinant protein induced strong tetanus-specific antibody responses in both systemic and mucosal compartments and prolonged the survival of mice after challenge with a supra-lethal dose of tetanus toxin. CONCLUSION: This study establishes FlaB as a successful fusion partner for recombinant subunit tetanus vaccine applicable through mucosal route, and it further endorses our previous observations that FlaB could be a stable adjuvant partner for mucosal vaccines.

Towards Vaccine 3.0: new era opened in vaccine research and industry

No abstract available.

Destructive Intestinal Translocation of Vibrio vulnificus Determines Successful Oral Infection

Vibrio vulnificus causes primary septicemia as a result of the consumption of contaminated seafood. The intestinal epithelial layer is the first host barrier encountered by V. vulnificus upon oral intake; however, epithelial translocation (invasion) of V. vulnificus has not been extensively studied. In this study, we investigated in vivo translocation of V. vulnificus using clinical (CMCP6) and environmental isolates (96-11-17M). And we analyzed physiological changes of intestinal epithelium concurrent with bacterial translocation by using polarized HCA-7 transwell culture system. The efficiency of epithelial translocation of 97-11-17M strains was significantly lower than that of pathogenic clinical isolate CMCP6 in a murine ligated ileal loop model. In an oral infection model, the survival rate was reciprocally related with efficacy of in vivo epithelial translocation. These results indicate that efficient translocation of V. vulnificus through intestinal epithelium is highly correlated with successful oral infection. We determined translocation of the bacteria from upper to lower chamber, changes of transepithelial electric resistance (TER) and cytotoxicity of the polarized HCA-7 cells to understand general features of V. vulnificus invasion. Bacterial translocation was accompanied by big decrease of TER (about 90%) and about 50% cytotoxicity of the epithelial cells. Taken together, these results indicate that V. vulnificus actively translocates the epithelium by destruction of epithelium and the efficiency of intestinal invasion by V. vulnificus is critical for successful oral infection. From this result, it is suggested that integrity of intestinal barrier is an important factor for susceptibility to oral infection of V. vulnificus.

Intranasal immunization with a flagellin-adjuvanted peptide anticancer vaccine prevents tumor development by enhancing specific cytotoxic T lymphocyte response in a mouse model

PURPOSE: Human papillomavirus (HPV) is a significant cause of cervical cancer-related deaths worldwide. Because HPV is a sexually transmitted mucosal pathogen, enhancement of antigen-specific mucosal immune response likely serves good strategy for vaccination. However, mucosal vaccines generally do not induce strong enough immune responses. Previously we proved that a bacterial flagellin, Vibrio vulnificus FlaB, induce strong antigen-specific immune responses by stimulating the Toll-like receptor 5. In this study, we tested whether FlaB could serve as an effective mucosal adjuvant for a peptide-based HPV preventive cancer vaccine. MATERIALS AND METHODS: Mice were intranasally administered with a mixture of FlaB and E6/E7 protective peptides in 5-day interval for a total of two times. Five-days after the last vaccination, cellular immune responses of the vaccinated mice were analyzed. Tumor growth was also observed after a subcutaneous implantation of TC-1 cells bearing E6/E7 antigens. RESULTS: Intranasal administration of the E6/E7 peptide mixture with FlaB elicited a strong antigen-specific cytotoxic T lymphocyte activity and antigen-specific interferon-gamma production from splenocytes and cervical lymph node cells. Furthermore, FlaB, as a mucosal adjuvant, conferred an excellent protection against TC-1 tumor challenge with high survival rates in E6/E7 immunized animals. CONCLUSION: These results indicate that FlaB can be a promising mucosal adjuvant for nasal HPV vaccine development.

Mucosal vaccine adjuvants update

Mucosal vaccination, capable of inducing protective immune responses both in the mucosal and systemic immune compartments, has many advantages and is regarded as a blue ocean in the vaccine industry. Mucosal vaccines can offer lower costs, better accessability, needle-free delivery, and higher capacity of mass immunizations during pandemics. However, only very limited number of mucosal vaccines was approved for human use in the market yet. Generally, induction of immune responses following mucosal immunization requires the co-administration of appropriate adjuvants that can initiate and support the effective collaboration between innate and adaptive immunity. Classically, adjuvant researches were rather empirical than keenly scientific. However, during last several years, fundamental scientific achievements in innate immunity have been translated into the development of new mucosal adjuvants. This review focuses on recent developments in the concepts of adjuvants and innate immunity, mucosal immunity with special interest of vaccine development, and basic and applied researches in mucosal adjuvant.

Toll-Like Receptor Ligands as Cancer Immunotherapeutics

Toll-like receptors (TLRs) are pattern recognition receptors (PRRs) expressed in a wide spectrum of cell types that recognize distinctive ligands and subsequently activate adaptive immune responses. TLR ligands are considered a promising target for development of immunomodulatory agents. Extensive clinical investigations are currently underway to develop TLR ligands-based non-specific immunostimulants and vaccine adjuvants. It has been well accepted that cancer cells develop a strategy to avoid host immune responses by producing inhibitory molecules. In addition, tumor-associated antigens are often not strong enough to induce effective anti-cancer immune responses. In this context, immunostimulants or adjuvants are critically required for more effective cancer immunotherapies. Here, we discuss recent progresses in the field of cancer immunotherapy under special emphasis on the TLR ligands as a component of immunostimulatory agents.

TLR4, 5, and 9 Agonists Inhibit Murine Airway Invariant Natural Killer T Cells in an IL-12-Dependent Manner

PURPOSE: Invariant natural killer T (iNKT) cells may play an important role in the pathogenesis of asthma in mice and humans. Thus, an agent that modulates the function of iNKT cells may have therapeutic potential to control asthma. We hypothesized that lipopolysaccharide (LPS)-, flagellin-, or CpG-induced changes in the cytokine milieu may modify and even inhibit the function of airway iNKT cells in asthma. METHODS: Because increased alpha-galactosylceramide (GalCer)-induced airway hyperreactivity (AHR) reflects the presence of airway iNKT cells, alpha-GalCer-induced AHR, as well as inflammatory cells and cytokines in bronchoalveolar lavage (BAL) fluid, were determined 24 hours after in vivo treatment with LPS, flagellin, or CpG in naive BALB/c mice. Intracellular IL-4 and IFN-gamma were measured in spleen iNKT cells after in vitro treatment with LPS, flagellin, or CpG. A role for IL-12 following the treatments was determined. RESULTS: Intranasal administration of LPS, flagellin, or CpG reduced development of alpha-GalCer-induced AHR, eosinophilic airway inflammation, and Th1 and Th2 cytokine responses in BAL fluid, while producing IL-12 in BAL fluid. Intraperitoneal administration of IL-12 mAb blocked the suppressive effect of LPS, flagellin, or CpG. In vitro treatment with LPS, flagellin, or CpG reduced production of IL-4 and IFN-gamma from alpha-GalCer-stimulated spleen iNKT cells; these effects were ameliorated by addition of anti-IL-12 mAb. CONCLUSIONS: TLR4, 5, and 9 agonists may suppress the function of airway and spleen iNKT cells via IL-12-dependent mechanisms. Anergy of iNKT cells by IL-12 might play a role in suppression by these TLR agonists.

Enhancement of antitumor effect using dendritic cells activated with natural killer cells in the presence of Toll-like receptor agonist

Dendritic cells (DCs) play a role in natural killer (NK) cell activation, while NK cells are also able to activate and mature DCs. Toll-like receptors (TLRs) on the surface of DCs and NK cells induce the maturation and activation of these cells when engaged with their cognate ligand. We investigated to generate potent DCs by maturation with NK cells in the presence of TLR agonist in vitro and tested the efficacy of these DC vaccinations in mouse colon cancer model. The optimal ratios of DCs versus NK cells were 1:1 to 1:2. Immature DCs were mature with NK cells in the presence of lipopolysaccharide, which is TLR4 agonist, and further addition of IL-2 induced phenotypically and functionally mature bone marrow-derived DCs. These potent DCs exhibited not only high expression of several costimulatory molecules and high production of IL-12p40 and IL-12p70, but also high allogeneic T cells stimulatory capacity, and the induction of the high activities to generate tumor-specific CTLs. Consistently, vaccination with these DCs efficiently inhibited CT-26 tumor growth in mouse colon cancer model when compared to other vaccination strategies. Interestingly, combination therapy of these DC-based vaccines and with low-dose cyclophosphamide showed dramatic inhibition effects of tumor growth. These results suggest that the DCs maturated with NK cells in the presence of TLR agonist are potent inducer of antitumor immune responses in mouse model and may provide a new source of DC-based vaccines for the development of immunotherapy against colon cancer.

Protective Effect of Ginsan Against Vibrio vulnificus Infection

Ginsan, a botanic polysaccharide extracted from Panax ginseng, has recently been reported to modulate mucosal immune response. In this study, we investigated the protective effect of Ginsan against fatal Vibrio vulnificus mucosal infection. A lethal dose of V. vulnificus (1.0 x 106 CFU/mouse) was nasally inoculated to mice. The bacterial count in the nasal associated lymphoid tissue (NALT) of the mouse was significantly reduced in the Ginsan-treated group. The Ginsan-treated group showed improved survival compared to the control group (100% vs 18%). To elucidate the effect of Ginsan on modulating host immune response, cytokine mRNA expressions involved in mediating inflammation were determined by semiquantitative RT-PCR in the NALTs of the infected mice. Most of the cytokine mRNAs were similarly expressed as the control group. However, COX-1 mRNA expression level was higher in Ginsan-treated group compared to the control group. The protective effect of Ginsan was antagonized by treating with a specific COX-1 inhibitor, SC-560. Thus, these data suggest that the protective effect of Ginsan against V. vulnificus infection is partly mediated by modulating COX-1 expression.

Roles of Flagellar Hook-Associated Proteins in Vibrio vulnificus Motility and Virulence

The bacterial flagellar structure can be divided into the basal body, the hook and the filament. Three minor components called hook associated proteins (HAP1, HAP2 and HAP3) form a junction between the hook and the filament (HAP1 and HAP3) and a capping structure at the distal end of flagellar filament (HAP2). Vibrio vulnificus is a halophilic pathogenic bacterium that is locomotive by means of a polar flagellum. From a V. vulnificus genome sequencing project, we obtained sequences of V. vulnificus flgK (Vv-flgK), flgL (Vv-flgL), and flaH (Vv-flaH) genes that encode HAP1, HAP3, and HAP2, respectively. To investigate roles of the HAP proteins, deletion mutants of the Vv-flgK, Vv-flgL and Vv-flaH were constructed. Electron microscopic analysis showed that the Vv-flgK or Vv-flgL mutant did not produce an intact polar flagellum while the Vv-flaH mutant produced a fragile flagellar structure. Western blot analysis against a major polar flagellin proposed that the null HAP1 and HAP3 mutations resulted in a failure of normal flagellar assembly since flagellins produced by the mutants were secreted out in the culture supernatants without long flagellar filaments. Motility was completely abolished by a single mutation in HAP1 or HAP3, and the HAP2 mutant showed a decreased motility. Also each of the mutants showed an impaired cytotoxicity and adherence to HeLa cell compared with the isogenic wild type strain. LD(50) increased by 10- and 11-fold in the V. vulnificus HAP3 and HAP2 mutant, respectively. These results suggest that the HAP proteins play important roles in polar flagellation and the virulence of V. vulnificus.

Effect of the Heat Shock Protein 70 on the Adjuvanticity Induced by a Bacterial Flagellin of Vibrio ulnificus

Recently we have shown that a bacterial flagellin, Vibrio vulnifiucs FlaB (Vv-FlaB), has a strong adjuvant activity to induce protective immune response. In order to investigate the adjuvanticity of Vv-FlaB, we prepared highly purified recombinant protein by using an intein fusion protein purification system. However, in the process of the purification, we unexpectedly encountered a contamination with a 70 kDa protein. We proved the 70 kDa protein as the heat shock protein 70 (HSP70) by Western blotting. Unfortunately, it was reported that the HSP70 has a strong adjuvanticity. In this study we investigated the role of contaminating HSP70 on the Vv-FlaB-mediated adjuvanticity. We separated Vv-FlaB and HSP70 by using a high performance protein purification chromatography and compared adjuvant activities of Vv-FlaB, HSP70 and Vv-FlaB/HSP70 mixture. Using an intranasal immunization mouse model, we observed that co-administration of the flagellin with tetanus toxoid (TT) induced significantly enhanced TT-specific antibody (Ig) responses. However contaminating doses of HSP70 did not affect the adjuvanticity of Vv-FlaB and furthermore HSP70 alone did not enhance TT-specific Ig response and protective immunity against lethal challenge with tetanus toxin. These results show that the HSP70 contaminating Vv-FlaB preparations did not affect the adjuvanticity of Vv-FlaB.

A Bacterial Flagellin, Vibrio vulnificus FlaB, Induces Human Dendritic Cell Maturation

The motile marine bacterium, Vibrio vulnificus has a total of six flagellins. Flagellin is a structural component of flagellar filament in various locomotive bacteria and is the ligand of Toll-like receptor 5 (TLR5). TLRs, highly expressed on various types of cells including dendritic cells (DCs), recognize invading microorganisms and finally trigger host immune responses. In this study, we prepared all of six recombinant flagellin proteins and assessed the effect of six flagellins on IL-8 activation through TLR5 recognition. Although showed different activities, five out of the six flagellins stimulated significant IL-8 activation. We also investigated the immunomodulatory roles of Vv-FlaB, the crucial building block of V. vulnificus flagellar filament, on human dendritic cells. Treatment of immature DCs with Vv-FlaB resulted in an increased expression of co-stimulatory molecules and induced strong allo-T cell proliferative activities of the DCs. These results show that the Vv-FlaB may serve an epochal immune adjuvant possessing effective immunomodulatory activities.

A Two Component Signal Transduction System Required for the Virulence of Vibrio vulnificus

Vibrio vulnificus is an estuarine bacterium that opportunistically infects humans with underlying hepatic diseases, heavy alcohol drinking habits, and other immunocompromised conditions. A locus in the V. vulnificus genome was cloned and sequenced, which showed similarities to the bacterial two-component signal transduction system. The locus encoded a putative sensor kinase, designated vvgS, and a divergently transcribed putative response regulator, designated vvgR. VvgS was predicted to be a protein belonging to the tripartite hybrid sensor kinase subfamily such as ArcB and BvgS. VvgR showed similarities to well known response regulators. An insertional mutation of vvgS in a V. vulnificus strain led to a remarkable decrease of cytotoxicity to HeLa cells, while the production of exotoxins, the hemolysin and the protease, slightly increased by the vvgS mutation. Adhesion to HeLa cells only slightly decreased. However, the vvgS mutation had no effect on the motility of V. vulnificus. The vvgS mutation resulted in a significant decrease of lethality to mice. These results indicate that vvgRS two-component system plays a very important role in regulating novel virulence factor(s) of V. vulnificus associated with cytotoxicity other than hemolysin and protease during the infection process.

A Two Component Signal Transduction System Required for the Virulence of Vibrio vulnificus

Vibrio vulnificus is an estuarine bacterium that opportunistically infects humans with underlying hepatic diseases, heavy alcohol drinking habits, and other immunocompromised conditions. A locus in the V. vulnificus genome was cloned and sequenced, which showed similarities to the bacterial two-component signal transduction system. The locus encoded a putative sensor kinase, designated vvgS, and a divergently transcribed putative response regulator, designated vvgR. VvgS was predicted to be a protein belonging to the tripartite hybrid sensor kinase subfamily such as ArcB and BvgS. VvgR showed similarities to well known response regulators. An insertional mutation of vvgS in a V. vulnificus strain led to a remarkable decrease of cytotoxicity to HeLa cells, while the production of exotoxins, the hemolysin and the protease, slightly increased by the vvgS mutation. Adhesion to HeLa cells only slightly decreased. However, the vvgS mutation had no effect on the motility of V. vulnificus. The vvgS mutation resulted in a significant decrease of lethality to mice. These results indicate that vvgRS two-component system plays a very important role in regulating novel virulence factor(s) of V. vulnificus associated with cytotoxicity other than hemolysin and protease during the infection process.

Close Encounter of V. vulnificus with Host Cells is a Prerequisite to the Cytotoxicity of yet Unidentified New Virulence Factor

Live Vibrio vulnificus is highly cytotoxic to host cells in vivo and in vitro. The two most representative cytotoxins, cytolytic hemolysin and elastolytic protease, have been regarded to play major roles in the cytotoxicity of V. vulnificus. To further determine roles of the two cytotoxins in V. vulnificus pathogenesis, we constructed a double mutant of vvhA and vvpE genes, encoding a hemolysin and a protease, respectively. However, the cytotoxicity and the LD50 of a vvhA/vvpE double mutant showed no significant difference from those of the isogenic wild type strain. From these results, we came to speculate that yet unidentified, key cytotoxic factors should play a major role in the cytotoxic activity of V. vulnificus. The HeLa cells encountered with V. vulnificus became rounded, following detachment from the bottom of culture plate, and were killed eventually. However, the bacterial culture supernatant did not show any effect on the morphology and viability of HeLa cells. Also, no cytotoxicity could be noted when V. vulnificus was not allowed to contact with HeLa cells in the TranswellR system. Chloramphenicol, at lower concentration than minimum inhibitory concentration (MIC), decreased the cytotoxicity of a vvhA/vvpE double mutant to HeLa cells in a dose dependent manner. These results suggest that close encounter of V. vulnificus with host cells is a prerequisite to the cytotoxicity and that a yet unidentified virulence factor (s) should play an important role in the contact-dependent cytotoxicity.

Effect of Salinity, Temperature, and Glucose on the Production of Vibrio vulnificus Hemolysin

Among the exotoxins produced by V. vulnificus, hemolysin (HS) has been reported to be the most potent one. To investigate the factors up- or down-regulating HS production in the context of pathogenesis, we observed the effects of salinity or/and temperature shifting, glucose, and acidic pH on the production of HS by V. vulnificus C7184 strain in vitro. Significantly more HS was produced when V. vulnificus was cultured in 0.9% salinity and 37 degrees C than in 2.5% and 25 degrees C. When the culture condition reflecting natural habitat of V. vulnificus (2.5% salinity and 25degrees C) was changed into that reflecting human body (0.9% salinity and 37 degrees C), 2.5 fold or more HS was produced than in the V. vulnificus being cultured continuously in 0.9% NaCl at 37 degrees C. This result suggests that V. vulnificus somehow recognizes the shifting in salinity and temperature and stimulate HS production. Glucose addition in the culture medium resulted in a dose- dependent decrease in the HS production. Glucose itself and acidic pH resulting from its metabolism both appeared to inhibit the HS production. Glucose in itself had more dominant role in suppressing the HS production than the lowered pH accompanying the metabolism of glucose. This result suggests that HS production is down-regulated in the presence of glucose and under environmental acidic pH.

Phenolate Siderophore Stimulates Growth of Vibrio vulnificus: Application of CAS Agar Diffusion Assay - Comparison of Siderophore Production Among Strains

No abstract available.