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The differentiation of naive CD8+ T cells into effector cells is important for establishing immunity. However, the effect of heterogeneous naive CD8+ T cell populations is not fully understood. Here, we demonstrate that steady-state naive CD8+ T cells are composed of functionally heterogeneous subpopulations that differ in their ability to differentiate into type 17 cytotoxic effector cells (Tc17) in inflammatory disease models. The differential ability of Tc17 differentiation was not related to T-cell receptor (TCR) diversity and antigen specificity but was inversely correlated with self-reactivity acquired during development. Mechanistically, this phenomenon was linked to differential levels of intrinsic TCR sensitivity and basal SMAD3 expression, generating a wide spectrum of Tc17 differentiation potential within naive CD8+ T cell populations. These findings suggest that developmental self-reactivity can determine the fate of naive CD8+ T cells to generate functionally distinct effector populations and achieve immense diversity and complexity in antigen-specific T-cell immune responses.

Therapeutic cancer vaccines, which induce or amplify tumor-specific T cell responses, are a critical component of multiple combination cancer immunotherapy regimens. Innovative neoantigen identification continually prompts the development of vaccine platforms. However, vaccine monotherapy is not sufficient to eradicate tumors. Thus, therapeutic strategies combining cancer vaccines and treatment with other immune modulators have been expl, ored. Previously, we showed that flagellin has an excellent adjuvant activity to induce effective immune responses to co-administered peptide epitopes through TLR5 stimulation in mouse TC-1 tumor models and flagellin-expressing bacteria modulate the tumor microenvironment (TME) toward enhanced immunogenicity.Given that short- and long-peptides undergo different fates of internalization, processing, and MHC-restricted presentation by professional antigen-presenting cells (APCs), we compared the antitumor activity of flagellin-adjuvanted peptide vaccines by employing the E7 CD8 epitope short peptide (E7-SPFlagellin adjuvanted E7-LP35 vaccine (FlaB-LP35Vax) showed significantly higher antitumor activity than flagellin adjuvanted E7-SP vaccine (FlaB-SPVax) and flagellin adjuvanted E7-LP20 vaccine (FlaB-LP20Vax) in a mouse TC-1 tumor model. Coadministration of flagellin was essential for E7-mediated tumor suppression. PD-1 blockade enhanced the therapeutic efficacy of FlaB-LP35Vax but not FlaB-SPVax. Taken together, E7-LP35 is an optimal tumor antigen for flagellin-adjuvanted E7 cancer vaccines, and the combination of FlaB-LP35Vax with anti-PD-1 antibody treatment induced long-term antitumor immune responses.This result suggests that cooperation between CD4

Toll-like receptors (TLRs) make a crucial contribution to the innate immune response. TLR5 was expressed in embryoid body derived from mouse embryonic stem cells (mESCs) and βIII-tubulin-positive cells under all-trans retinoic acid-treated condition. TLR5 was upregulated during neural differentiation from mESCs and augmented the neural differentiation of mESCs via nuclear factor-κB and interleukin 6/CREB pathways. Besides, TLR5 was expressed in SOX2- or doublecortin-positive cells in the subgranular zone of the hippocampal dentate gyrus where adult neurogenesis occurs. TLR5 inhibited the proliferation of adult hippocampal neural stem cells (NSCs) by regulating the cell cycle and facilitated the neural differentiation from the adult hippocampal NSCs via JNK pathway. Also, TLR5 deficiency impaired fear memory performance in mice. Our data suggest that TLR5 is a crucial modulator of neurogenesis from mESCs and adult hippocampal NSCs in mice and represents a new therapeutic target in neurological disorders related to cognitive function.

Background Glioblastoma (GBM) is the most aggressive type of brain tumor with heterogeneity and strong invasive ability. Treatment of GBM has not improved significantly despite the progress of immunotherapy and classical therapy. Epidermal growth factor receptor variant III (EGFRvIII), one of GBM-associated mutants, is regarded as an ideal therapeutic target in EGFRvIII-expressed GBM patients because it is a tumor-specific receptor expressed only in tumors. Flagellin B (FlaB) originated from Vibrio vulnificus, is known as a strong adjuvant that enhances innate and adaptive immunity in various vaccine models. This study investigated whether FlaB synergistically could enhance the anti-tumor effect of EGFRvIII peptide (PEGFRvIII). Methods EGFRvIII-GL261/Fluc cells were used for glioblastoma-bearing mouse brain model. Cell-bearing mice were inoculated with PBS, FlaB alone, PEGFRvIII alone, and PEGFRvIII plus FlaB. Tumor growth based on MRI and the survival rate was investigated. T cell population was examined by flow cytometry analysis. Both cleaved caspase-3 and CD8 + lymphocytes were shown by immunohistochemistry (IHC) staining. Results The PEGFRvIII plus FlaB group showed delayed tumor growth and increased survival rate when compared to other treatment groups. As evidence of apoptosis, cleaved caspase-3 expression and DNA disruption were more increased in the PEGFRvIII plus FlaB group than in other groups. In addition, the PEGFRvIII plus FlaB group showed more increased CD8 + T cells and decreased Treg cells than other treatment groups in the brain. Conclusions FlaB can enhance the anti-tumor effect of PEGFRvIII by increasing CD8 + T cell response in a mouse brain GBM model.

[Purpose] Heterogeneity and the tumor microenvironment are major obstacles to overcoming incurable advanced gastric cancer (AGC). Although Epstein-Barr virus-positive and microsatellite instability subtypes can be detected early and properly treated, they are rare in AGC. In this regard, recent combinational therapy of immune checkpoint inhibitors (ICIs) such as PD-1/PD-L1 inhibitors and other various therapeutics has been tried, but there are clinically limited doses and drug resistance. We hypothesized that CAR-T targeting PD-L1, the common tumor-associated antigen (TAA), which expresses in most advanced cancer cells, would make a significant breakthrough in curing a solid tumor. This study aimed to evaluate the killing effects of anti-PD-L1 CAR-T against gastric cancer cell lines. Removal of PD-L1-expressing cells in the AGC TME will improve the clinical outcomes of current therapies. [Results] We constructed a second-generation PD-L1 CAR lentiviral vector targeting PD-L1, which has an intermediate affinity with PD-L1 in solid tumor TME. As the secondary signaling motif, CD28 was chosen and tested along with the CD3 zeta chain. The effector CAR-T, named Vax-CAR-T, specifically recognized the PD-L1-expressing AGC cell line, SNU638, and exhibited rapid and robust cytotoxic activity in vitro. When compared to untransduced T cells, the level of the CD25 activation marker was higher in Vax-CAR-T cells. Besides, the inhibitory markers of Tim3 and LAG3 were expressed even higher than the untransduced. Despite high Tim3 and LAG3 expression in the final manufacturing process, Vax-CAR-T cells significantly suppressed tumor growth in both bigger (>95mm3) and smaller (≤50mm3) AGC xenograft NSG mouse models. More importantly, Vax-CAR-T-treated mice have well tolerated the CAR-T therapy with no significant adverse events during the CRS monitoring period, 2 weeks after Vax-CAR-T injection and have gradually recovered weight gains 5 days after CAR-T cell injection. [Conclusion] We believe that a new autologous CAR-T targeting PD-L1 could be a promising new tool for removing heterogeneous solid tumors in TME, which would be a significant step forward in improving current conventional and immunotherapeutic strategies. Citation Format: Hye-Seong Park, Eun-Ji Choi, Jae-In Yu, Kyung-Sik Lee, Yeo-Jin Lim, Se-Ryeong Choi, Yoonjoo Choi, Bum Chan Park, Jae Eun Park, Mihwa Kim, Je-Jung Lee, Joon Haeng Rhee. The efficacy and safety of PD-L1-specific CAR-T in advanced gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 889.

Objectives: This study aimed to evaluate potential therapeutic effect of Metagonimus yokogawai on the OVA-induced allergic rhinitis model. Methods: OVA-sensitized mice were used to assess potential therapeutic effect of the extract protein of M. yokogawai (My-TP). My-TP was administrated via the intralymphatic route to cervical lymph nodes. The frequencies of sneezing or nasal rubbing were recorded. Histopathologic evaluation was performed for eosinophil infiltrations in the tissues of the nasal mucosa and skin. The mRNA relative expressions of the cytokine profiles including Th1, Th2, Th17, and Treg subsets in the nasal mucosa, cervical lymph nodes, and spleen were analyzed by quantitative real-time reverse-transcriptase polymerase chain reaction. The potential underlying mechanism was investigated by examining cytokine profiles including IL-4 and Treg subsets from lymphocytes of the spleen by flow cytometry. Results: Intralymphatic injection of My-TP reduced allergic symptoms and eosinophil infiltration in the nasal mucosa. My-TP-treated group showed markedly decreased levels of OVA-specific IgE and WBC counts in nasal lavage. My-TP-treated group showed the decreased expression levels of IL-4, while those of IL-10 were increased in both the nasal mucosa. The levels of IFN-γ and IL-17 were also decreased in the nasal mucosa and cervical lymph nodes. The immunological mechanism may involve the downregulation of Th2 response and upregulation of Tregs in the nasal mucosa and cervical lymph nodes. Conclusions: Our results provide the first evidence of potential therapeutic effect of M. yokogawai in OVA-sensitized allergic rhinitis mice, suggesting that a Treg/Th2 reorganization may play a role in clinical course of allergic rhinitis.

Purpose We investigated whether adoptive cell therapy with ex vivo–activated natural killer (NK) cells enhances the therapeutic efficacy of local tumor radiation therapy (RT) using a human triple-negative breast cancer xenograft model. Methods and Materials NK cells from healthy donors were expanded ex vivo. MDA-MB-231/Luc-GFP cells were subcutaneously implanted into the thighs of NSG mice. The animals were divided into 4 experimental groups: control, RT, NK, and RT + NK. On day 17 after tumor implantation, tumors from the RT groups were irradiated. The ex vivo–expanded NK cells were intravenously administered twice, on days 17 and 19. Primary and secondary tumors were evaluated using long-term bioluminescence imaging, and histopathology was performed on resected tumor tissue specimens. Results The luciferase signals of the primary tumors in the RT + NK group were significantly lower than those of comparably sized primary tumors in the RT group. The long-term migration and infiltration of NK cells into the primary tumor sites were significantly higher in RT + NK than in NK mice. Moreover, lymphatic metastasis to the axillary lymph nodes and liver and lung metastases were highly suppressed in the RT + NK group, as demonstrated by BLI and p53 immunohistochemistry. The long-term survival of the RT + NK group was significantly higher than that of the RT or NK groups. Conclusions Reduction in tumor burden by combining RT and systemic NK cell therapy improved the suppression of primary tumor growth, with efficient NK cell migration and penetration into the primary tumor site. Administered NK cells were maintained in the primary tissue for a significantly longer time in RT + NK group compared with NK group. Both lymphatic spread and distant metastasis to the lungs and liver were effectively suppressed by the combined therapy.

Purpose Glioblastoma (GBM) is the most aggressive type of brain tumor with heterogeneity and strong invasive ability. Treatment of GBM has not improved significantly despite the progress of immunotherapy and classical therapy. Epidermal growth factor receptor variant III (EGFRvIII), one of GBM-associated mutants, is regarded as an ideal therapeutic target in EGFRvIII-expressed GBM patients because it is a tumor-specific receptor expressed only in tumors. Flagellin B (FlaB) originated from Vibrio vulnificus is known as a strong adjuvant that enhance innate and adaptive immunity in various vaccine models. This study performed whether FlaB synergistically could enhance the anti-tumor effect of EGFRvIII peptide (PEGFRvIII). Methods EGFRvIII-GL261/Fluc cells were used for glioblastoma-bearing mouse brain model and cell-bearing mice were inoculated with PBS, FlaB alone, PEGFRvIII alone, and PEGFRvIII plus FlaB. Survival rate through tumor growth was investigated by MRI. T cell population was examined by flow cytometry analysis. Both cleaved caspase-3 and CD8+ lymphocytes were shown by immunohistochemistry (IHC) staining. Results The PEGFRvIII plus FlaB group showed delayed tumor growth and increased survival rate when compared to other treatment groups. As an evidence of apoptosis, cleaved caspase-3 expression and DNA disruption were more increased in the PEGFRvIII plus FlaB group than in other groups. In addition, the PEGFRvIII plus FlaB group showed more increased CD8+ T cells and decreased Treg cells than other treatment groups in the brain. Conclusion FlaB can enhance the anti-tumor effect of PEGFRvIII by increasing CD8+ T cell response in a mouse brain GBM model.

Vibrio vulnificus is a halophilic estuarine bacterium causing severe opportunistic infections. To successfully establish an infection, V. vulnificus must adapt to redox fluctuations in vivo. In the...

RtxA1 is a major cytotoxin of Vibrio vulnificus (V. vulnificus) causing fatal septicemia and necrotic wound infections. Our previous work has shown that RpoS regulates the expression and secretion of V. vulnificus RtxA1 toxin. This study was conducted to further investigate the potential mechanisms of RpoS on RtxA1 secretion. First, V. vulnificus TolCV1 and TolCV2 proteins, two Escherichia coli TolC homologs, were measured at various time points by Western blotting. The expression of TolCV1 was increased time-dependently, whereas that of TolCV2 was decreased. Expression of both TolCV1 and TolCV2 was significantly downregulated in an rpoS deletion mutation. Subsequently, we explored the roles of TolCV1 and TolCV2 in V. vulnificus pathogenesis. Western blot analysis showed that RtxA1 toxin was exported by TolCV1, not TolCV2, which was consistent with the cytotoxicity results. Furthermore, the expression of TolCV1 and TolCV2 was increased after treatment of the host signal bile salt and the growth of tolCV1 mutant was totally abolished in the presence of bile salt. A tolCV1 mutation resulted in significant reduction of V. vulnificus induced-virulence in mice. Taken together, TolCV1 plays key roles in RtxA1 secretion, bile salt resistance, and mice lethality of V. vulnificus, suggesting that TolCV1 could be an attractive target for the design of new medicines to treat V. vulnificus infections.

[Purpose] Immunosuppressive tumor microenvironment (TME) is the major hurdle to cancer immunotherapy. Advanced hepatocellular carcinoma (aHCC) is one of the representative cold tumors with immunosuppressive TME and presents obvious correlation between immune check point expression, such as programmed cell death ligand 1 (PD-L1), and poorer prognosis. In aHCC patients, cancer cells and stellate cells in TME appeared to express high level of PD-L1, which should suppress activities of antitumor immune cells. In this context, the combination of PD-1/PD-L1 inhibitors and other therapeutic modalities are tried to treat aHCC. The chimeric antigen receptor (CAR) T cell therapy is a potent way of eradicating cells expressing targetable antigens. Removal of PD-L1 expressing cells in the aHCC TME will improve the clinical outcomes of current therapies against aHCC. In the present study, we developed an autologous anti-PD-L1 CAR T strategy and show high efficacy of tumor suppression using an HCC xenograft model. [Result] We constructed second generation CAR lentiviral vector expressing an unique anti-PD-L1 scFv manifesting moderated affinity. The scFv was specifically generated by phage display technique using human B cell-derived naïve cDNA library, which targets PD-L1 with intermediate affinity in solid tumor TME. As the secondary signaling motif, CD28 was chosen and tested along with CD3 zeta chain. The effector CAR T, named VPC1, cells specifically recognized PD-L1 expressing HCC cells and exhibited rapid and robust cytotoxic activity in 2D and 3D culture experiments. In an HCC xenograft NSG mouse model, VPC1 CAR T cells significantly suppressed the tumor growth. More importantly, VPC-1-treated animals have well tolerated the CAR T therapy with no significant adverse manifestations during one month observation period and exhibited gradual weight gains since 3 days after CAR T cell injection. [Conclusion] We hereby report that a new autologous PD-L1 CAR-T therapeutic is capable of eradicating solid tumors expressing PD-L1, which would significantly potentiate efficacies of current conventional and immunotherapeutic strategies. Citation Format: Kyung-Sik Lee, Hye-Seong Park, Guk-Yeol Park, Gi-Chan Lee, Eun-Ji Choi, Jae-in Yu, Seung-Joo Yang, Mihwa Kim, Yoonjoo Choi, Je-Jung Lee, Joon Haeng Rhee, Bum Chan Park, Jae Eun Park. Development of an anti hepatocellular carcinoma CAR T strategy targeting PDL1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6189.

This study aimed to evaluate potential therapeutic effect of Metagonimus yokogawai on the OVA-induced allergic rhinitis model.OVA-sensitized mice were used to assess potential therapeutic effect of the extract protein of M. yokogawai (My-TP). My-TP was administrated via the intralymphatic route to cervical lymph nodes. The frequencies of sneezing or nasal rubbing were recorded. Histopathologic evaluation was performed for eosinophil infiltrations in the tissues of the nasal mucosa and skin. The mRNA relative expressions of the cytokine profiles including Th1, Th2, Th17, and Treg subsets in the nasal mucosa, cervical lymph nodes, and spleen were analyzed by quantitative real-time reverse-transcriptase polymerase chain reaction. The potential underlying mechanism was investigated by examining cytokine profiles including IL-4 and Treg subsets from lymphocytes of the spleen by flow cytometry.Intralymphatic injection of My-TP reduced allergic symptoms and eosinophil infiltration in the nasal mucosa. My-TP-treated group showed markedly decreased levels of OVA-specific IgE and WBC counts in nasal lavage. My-TP-treated group showed the decreased expression levels of IL-4, while those of IL-10 were increased in both the nasal mucosa. The levels of IFN-γ and IL-17 were also decreased in the nasal mucosa and cervical lymph nodes. The immunological mechanism may involve the downregulation of Th2 response and upregulation of Tregs in the nasal mucosa and cervical lymph nodes.Our results provide the first evidence of potential therapeutic effect of M. yokogawai in OVA-sensitized allergic rhinitis mice, suggesting that a Treg/Th2 reorganization may play a role in clinical course of allergic rhinitis.

Vibrio vulnificus (V. vulnificus) infection, frequently resulting in fatal septicemia, has become a growing health concern worldwide. The present study aimed to explore the potential agents that could protect against V. vulnificus cytotoxicity, and to analyze the possible underlying mechanisms. First, we observed that 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt hydrate (DIDS) significantly suppressed V. vulnificus cytotoxicity to host cells by using a lactate dehydrogenase (LDH) assay. DIDS did not exhibit any effect on host cell viability, bacterial growth, microbial adhesion and swarming motility. DIDS effectively lowered V. vulnificus RtxA1 toxin-induced calcium influx into host mitochondria and RtxA1 binding to host cells. To further elucidate the underlying mechanism, the synthesis and secretion of RtxA1 toxin were investigated by Western blotting. Intriguingly, DIDS selectively inhibited the secretion of RtxA1 toxin, but did not influence its synthesis. Consequently, the outer membrane portal TolC, a key conduit for RtxA1 export coupled with tripartite efflux pumps, was examined by RT-PCR and Western blotting. We found that DIDS significantly reduced the expression of TolCV1 protein at the transcriptional level. Taken together, these results suggest that DIDS is a promising new paradigm as an antimicrobial drug that targets TolC-mediated toxin.

Neutropenic sepsis is a fatal consequence of chemotherapy, and septic complications are the principal cause of mortality. Chemotherapy-induced neutropenia leads to the formation of microscopic ulcers in the gastrointestinal epithelium that function as a portal of entry for intraluminal bacteria, which translocate across the intestinal mucosal barrier and gain access to systemic sites, causing septicemia. A cyclophosphamide-induced mouse model was developed to mimic the pathophysiologic sequence of events that occurs in patients with neutropenic sepsis. The TLR5 agonist bacterial flagellin derived from Vibrio vulnificus extended the survival of cyclophosphamide-treated mice by reducing the bacterial load in internal organs. The protective effect of flagellin was mediated by the antimicrobial protein lipocalin 2 (Lcn2), which is induced by TLR5-NF-&kappa, B activation in hepatocytes. Lcn2 sequestered iron from infecting bacteria, particularly siderophore enterobactin-dependent members of the Enterobacteriaceae family, thereby limiting their proliferation. Lcn2 should be considered for the treatment of neutropenic sepsis and gastrointestinal damage during chemotherapy to prevent or minimize the adverse effects of cancer chemotherapy.

Mucosal surfaces are the interface between the host’s internal milieu and the external environment, and they have dual functions, serving as physical barriers to foreign antigens and as accepting sites for vital materials. Mucosal vaccines are more favored to prevent mucosal infections from the portal of entry. Although mucosal vaccination has many advantages, licensed mucosal vaccines are scarce. The most widely studied mucosal routes are oral and intranasal. Licensed oral and intranasal vaccines are composed mostly of whole cell killed or live attenuated microorganisms serving as both delivery systems and built-in adjuvants. Future mucosal vaccines should be made with more purified antigen components, which will be relatively less immunogenic. To induce robust protective immune responses against well-purified vaccine antigens, an effective mucosal delivery system is an essential requisite. Recent developments in biomaterials and nanotechnology have enabled many innovative mucosal vaccine trials. For oral vaccination, the vaccine delivery system should be able to stably carry antigens and adjuvants and resist harsh physicochemical conditions in the stomach and intestinal tract. Besides many nano/microcarrier tools generated by using natural and chemical materials, the development of oral vaccine delivery systems using food materials should be more robustly researched to expand vaccine coverage of gastrointestinal infections in developing countries. For intranasal vaccination, the vaccine delivery system should survive the very active mucociliary clearance mechanisms and prove safety because of the anatomical location of nasal cavity separated by a thin barrier. Future mucosal vaccine carriers, regardless of administration routes, should have certain common characteristics. They should maintain stability in given environments, be mucoadhesive, and have the ability to target specific tissues and cells.

Bacterial cancer therapy relies on the properties of certain bacterial species capable of targeting and proliferating within solid malignancies. If these bacteria could be loaded with antitumor proteins, the efficacy of this approach could be greatly increased. However, because most antitumor proteins are also toxic to normal tissue, they must be expressed by bacteria that specifically target and exclusively localize to tumor tissue. As a strategy for treating solid malignancies, we recently evaluated L-asparaginase (L-ASNase) delivered by tumor-targeted Salmonella. In this system, L-ASNase was expressed under the control of the araBAD promoter (PBAD) of the E. coli arabinose operon, which is induced by injection of L-arabinose. Here, we further improved the performance of recombinant Salmonella in cancer therapy by exploiting the quorum-sensing (QS) system, which uses cell mass-dependent auto-induction logic. This approach obviates the necessity of monitoring intratumoral bacterial status and inducing cargo protein expression by administration of an exogenous compound. Recombinant Salmonella in tumors expressed and secreted active L-ASNase in a cell mass-dependent manner, yielding significant anticancer effects. These results suggest that expression of a therapeutic protein under the control of the QS system represents a promising engineering platform for the production of recombinant proteins in vivo.

Breast cancer (BC) is the second most common solid malignant tumor that metastasizes to the brain. Despite emerging therapies such as immunotherapy, whether the tumor microenvironment (TME) in breast cancer brain metastasis (BCBM) has potential as a target of new treatments is unclear. Expression profiling of 770 genes in 12 pairs of primary BC and matched brain metastasis (BM) samples was performed using the NanoString nCounter PanCancer IO360TM Panel. Immune cell profiles were validated by immunohistochemistry (IHC) in samples from 50 patients with BCBM. Pathway analysis revealed that immune-related pathways were downregulated. Immune cell profiling showed that CD8+ T cells and M1 macrophages were significantly decreased, and M2 macrophages were significantly increased, in BM compared to primary BC samples (p = 0.001, p = 0.021 and p = 0.007, respectively). CCL19 and CCL21, the top differentially expressed genes, were decreased significantly in BM compared to primary BC (p &lt, 0.001, both). IHC showed that the CD8+ count was significantly lower (p = 0.027), and the CD163+ and CD206+ counts were higher, in BM than primary BC (p &lt, 0.001, both). A low CD8+ T cell count, low CD86+ M1 macrophage count, and high M2/M1 macrophage ratio were related to unfavorable clinical outcomes. BC exhibits an immunosuppressive characteristic after metastasis to the brain. These findings will facilitate establishment of a treatment strategy for BCBM based on the TME of metastatic cancer.

Chronic periodontitis is caused by interactions between the oral polymicrobial community and host factors. Periodontal diseases are associated with dysbiotic shift in oral microbiota. Vaccination against periodontopathic bacteria could be a fundamental therapeutic to modulate polymicrobial biofilms. Because oral cavity is the site of periodontopathic bacterial colonization, mucosal vaccines should provide better protection than vaccines administered systemically. We previously reported that bacterial flagellin is an excellent mucosal adjuvant. In this study, we investigated whether mucosal immunization with a flagellin-adjuvanted polypeptide vaccine induces protective immune responses using a Porphyromonas gingivalis infection model. We used the Hgp44 domain polypeptide of Arg-gingipain A (RgpA) as a mucosal antigen. Intranasal (IN) immunization induced a significantly higher Hgp44-specific IgG titer in the serum of mice than sublingual (SL) administration. The co-administration of flagellin potentiated serum IgG responses for both the IN and SL vaccinations. On the other hand, the anti-Hgp44-specific IgA titer in the saliva was comparable between IN and SL vaccinations, suggesting SL administration as more compliant vaccination route for periodontal vaccines. The co-administration of flagellin significantly potentiated the secretory IgA response in saliva also. Furthermore, mice administered a mixture of Hgp44 and flagellin via the IN and SL routes exhibited significant reductions in alveolar bone loss induced by live P. gingivalis infections. An intranasally administered Hgp44-flagellin fusion protein induced a comparable level of Hgp44-specific antibody responses to the mixture of Hgp44 and flagellin. Overall, a flagellin-adjuvanted Hgp44 antigen would serve an important component for a multivalent mucosal vaccine against polymicrobial periodontitis.

The mortality rate associated withVibrio vulnificussepsis remains high. Anin vitrotime-kill assay revealed synergism between tigecycline and ciprofloxacin. The survival rate was significantly higher in mice treated with tigecycline plus ciprofloxacin than in mice treated with cefotaxime plus minocycline. Thus, combination treatment with tigecycline-ciprofloxacin may be an effective novel antibiotic regimen forV. vulnificussepsis.

Vibrio vulnificus is a halophilic estuarine bacterium causing severe opportunistic infections. To successfully establish an infection, V. vulnificus must adapt to redox fluctuations in vivo. In the present study, we show that deletion of V. vulnificus fexA gene caused hypersensitivity to acid and reactive oxygen species. The ΔfexA mutant exhibited severe in vivo survival defects. For deeper understanding the role of fexA gene on the successful V. vulnificus infection, we analyzed differentially expressed genes in ΔfexA mutant in comparison with wild type under aerobic, anaerobic or in vivo culture conditions by genome-scale DNA microarray analyses. Twenty-two genes were downregulated in the ΔfexA mutant under all three culture conditions. Among them, cydAB appeared to dominantly contribute to the defective phenotypes of the ΔfexA mutant. The fexA deletion induced compensatory point mutations in the cydAB promoter region over subcultures, suggesting essentiality. Those point mutations (PcydSMs) restored bacterial growth, motility, cytotoxicity ATP production and mouse lethality in the ΔfexA mutant. These results indicate that the cydAB operon, being regulated by FexA, plays a crucial role in V. vulnificus survival under redox-fluctuating in vivo conditions. The FexA-CydAB axis should serve an Achilles heel in the development of therapeutic regimens against V. vulnificus infection.

The specific recognition between a monoclonal antibody (mAb) and its epitope can be used in a tag system that has proved valuable in a wide range of biological applications. Herein, we describe a novel tag called RA-tag that is composed of a seven amino acid sequence (DIDLSRI) and recognized by a highly specific mAb, 47RA, against the bacterial toxin Vibrio vulnificus RtxA1/MARTXVv. By using recombinant proteins with the RA-tag at the N-terminal, C-terminal, or an internal site, we demonstrated that the tag system could be an excellent biological system for both protein purification and protein detection in enzyme-linked immunosorbent, Western blot, flow cytometry, and immunofluorescence staining analyses in Escherichia coli, mammalian cell lines, yeast, and plant. In addition, our RA-tag/47RA mAb combination showed high sensitivity and reliable affinity (KD = 5.90 × 10−8 M) when compared with conventional tags. Overall, our results suggest that the RA-tag system could facilitate the development of a broadly applicable tag system for biological research.

Background & Aims Dysregulation of the microbiome has been associated with development of complex diseases, such as obesity and diabetes. However, no method has been developed to control disease-associated commensal microbes. We investigated whether immunization with microbial antigens, using CpG oligodeoxynucleotides and/or curdlan as adjuvants, induces systemic antigen-specific IgA and IgG production and affects development of diseases in mice. Methods C57BL/6 mice were given intramuscular injections of antigens (ovalbumin, cholera toxin B-subunit, or pneumococcal surface protein A) combined with CpG oligodeoxynucleotides and/or curdlan. Blood and fecal samples were collected weekly and antigen-specific IgG and IgA titers were measured. Lymph nodes and spleens were collected and analyzed by enzyme-linked immunosorbent assay for antigen-specific splenic T-helper 1 cells, T-helper 17 cells, and memory B cells. Six weeks after primary immunization, mice were given a oral, nasal, or vaginal boost of ovalbumin; intestinal lamina propria, bronchial lavage, and vaginal swab samples were collected and antibodies and cytokines were measured. Some mice were also given oral cholera toxin or intranasal Streptococcus pneumoniae and the severity of diarrhea or pneumonia was analyzed. Gnotobiotic mice were gavaged with fecal material from obese individuals, which had a high abundance of Clostridium ramosum (a commensal microbe associated with obesity and diabetes), and were placed on a high-fat diet 2 weeks after immunization with C ramosum. Intestinal tissues were collected and analyzed by quantitative real-time polymerase chain reaction. Results Serum and fecal samples from mice given injections of antigens in combination with CpG oligodeoxynucleotides and curdlan for 3 weeks contained antigen-specific IgA and IgG, and splenocytes produced interferon-gamma and interleukin 17A. Lamina propria, bronchial, and vaginal samples contained antigen-specific IgA after the ovalbumin boost. This immunization regimen prevented development of diarrhea after injection of cholera toxin, and inhibited lung colonization by S pneumoniae. In gnotobiotic mice colonized with C ramosum and placed on a high-fat diet, the mice that had been immunized with C ramosum became less obese than the nonimmunized mice. Conclusions Injection of mice with microbial antigens and adjuvant induces antigen-specific mucosal and systemic immune responses. Immunization with S pneumoniae antigen prevented lung infection by this bacteria, and immunization with C ramosum reduced obesity in mice colonized with this microbe and placed on a high-fat diet. This immunization approach might be used to protect against microbe-associated disorders of intestine.

Cytoskeletal rearrangement and acute cytotoxicity occur in Vibrio vulnificus-infected host cells. RtxA1 toxin, a multifunctional autoprocessing repeats-in-toxin (MARTX), is essential for the pathogenesis of V. vulnificus and the programmed necrotic cell death. In this study, HeLa cells expressing RtxA1 amino acids 1491–1971 fused to GFP were observed to be rounded. Through yeast two-hybrid screening and subsequent immunoprecipitation validation assays, we confirmed the specific binding of a RtxA11491–1971 fragment with host-cell filamin A, an actin cross-linking scaffold protein. Downregulation of filamin A expression decreased the cytotoxicity of RtxA1 toward host cells. Furthermore, the phosphorylation of JNK and p38 MAPKs was induced by the RtxA1-filamin A interaction during the toxin-mediated cell death. However, the phosphorylation of these MAPKs was not observed during the RtxA1 intoxication of filamin A-deficient M2 cells. In addition, the depletion of pak1, which appeared to be activated by the RtxA1-filamin A interaction, inhibited RtxA1-induced phosphorylation of JNK and p38, and the cells treated with a pak1 inhibitor exhibited decreased RtxA1-mediated cytoskeletal rearrangement and cytotoxicity. Thus, the binding of filamin A by the RtxA11491–1971 domain appears to be a requisite to pak1-mediated MAPK activation, which contributes to the cytoskeletal reorganization and host cell death.

Gram-negative Vibrio species secrete multifunctional autoprocessing repeats-in-toxin (MARTX) toxins associated with bacterial pathogenesis. Here, the cross-reactivity and cross-protectivity of mAbs against V. vulnificus RtxA1/MARTXVv was evaluated. Passive administration of any of these mAbs (21RA, 24RA, 46RA, 47RA and 50RA) provided strong protection against lethal V. cholerae infection. Interestingly, 24RA and 46RA, which map to the cysteine protease domain of V. cholerae MARTXVc , inhibited CPD autocleavage in vitro; this process is involved in V. cholerae pathogenesis. These results generate new insight into the development of broadly protective mAbs and/or vaccines against Vibrio species with MARTX toxins.

Background Although the hygiene hypothesis suggests that microbial infections could subvert asthma and thus a microbial product might serve as a therapeutic adjuvant for asthma, the relationship between bacterial components and asthma is complex. Recently, low levels of flagellin, the Toll-like receptor (TLR) 5 ligand, have been reported to promote asthma. Objective We show that a therapeutic dose of flagellin suppresses asthma and that the effect occurs through generating regulatory dendritic cells (rDCs) and regulatory T (Treg) cells. Methods Ovalbumin (OVA)–induced wild-type and TLR5 knockout asthmatic mice were treated intranasally with a mixture of OVA and 10 μg of a flagellin B (FlaB; of Vibrio vulnificus ). OVA/FlaB-treated rDCs were adoptively transferred to mice with OVA-induced asthma. Anti-CD25 mAb was used to deplete Treg cells. A mixture of house dust mite (HDM) and FlaB was used to treat mice with HDM-induced asthma. Blood CD14 + monocyte-derived dendritic cells from HDM-sensitive asthmatic patients were treated with FlaB and incubated with autologous CD4 + T cells. Results An OVA/FlaB mixture ameliorated OVA-induced asthma by inhibiting T H 1/T H 2/T H 17 responses in a TLR5-dependent manner through generating rDCs and Treg cells. The adoptive transfer of OVA/FlaB-treated dendritic cells inhibited OVA-induced asthma, whereas the depletion of CD25 + cells eliminated the inhibitory effect. A similar effect of FlaB was observed in mice with HDM-induced asthma. In patients with HDM-sensitive asthma, FlaB-treated rDCs inhibited HDM-stimulated T H 1/T H 2 responses while enhancing Treg cells in an IL-10–dependent manner. Conclusion These findings collectively suggest that flagellin could be used as a tolerogenic adjuvant to treat allergic asthma.

Immune checkpoint inhibitors have now become a standard therapy for malignant melanoma. However, immune checkpoint inhibitor monotherapy has shown effective in only a limited number of patients. Photodynamic (PDT) and immune combined therapy is a promising modality for the treatment of metastatic melanoma. Herein, we report the combination therapy with PDT and flagellin adjuvanted- Tyrp1/Trp2/gp100 peptides based vaccination (Fla-vax) in mouse models of B16-F10 melanoma cancer. We used the bilateral tumor model to determine whether the Fla-vax could induce systemic anti-tumor effects. The combination therapy with PDT and Fla-vax sufficiently induced systemic anti-tumor immune responses for local and abscopal tumor control,, with a significant increase in the systemic IFNγ/TNFα secretion from cytotoxic T lymphocytes (CTLs) and tumor-infiltrated effector memory CD8+ T cells. It was also found that the combined therapy greatly induced the number of migratory CD103+ DCs which is secreting CXCL10. The therapeutic benefit of PDT and Fla-vax proved to be CD8+ T-cell dependent, and the antitumor activity was additive with that provided by programmed death-1 (PD-1)-targeted immunotherapy. These results suggest the combination of active immunotherapy with tumor ablation by PDT as a feasible novel treatment strategy for advanced melanoma cancer.

Norovirus causes acute and debilitating gastroenteritis, characterized by vomiting and diarrhea. We recently reported a recombinant GII. 4 P domain particle (Pd) vaccine adjuvanted with a flagellin, Vibrio vulnificus FlaB, effectively promoting both humoral and cell-mediated immune responses. In the previous study, we found that sublingual (SL) immunization induced higher fecal secretory IgA (SIgA) responses while intranasal (IN) route provided higher amplitude of humoral and cellular immune responses in the systemic compartment. We hypothesized that the combination of IN and SL routes should induce more potent and sustained SIgA responses in the gut. In this study, we have tried combinatorial prime-boost immunization employing both IN and SL routes. The IN priming and SL boosting with the Pd+FlaB vaccine enhanced highest SIgA responses in feces, accompanying increased Pd-specific memory B cells and plasma cells in spleen and bone marrow, respectively. Notably, the strongest long-lasting SIgA response in feces was induced by combined IN prime and SL boost vaccination, which was sustained for more than 3 months. Significantly enhanced gut-homing B cell and follicular helper T cell responses in mesenteric lymph nodes (mLNs) were observed in the IN prime and SL boost combination. IN priming was a requisite for the robust induction of Pd-specific IFNγ, IL-2, IL-4 and IL-5 cytokine responses in the systemic immune compartment. Collectively, the IN prime and SL boost combination was the best option for inducing balanced long-lasting immune responses against the norovirus antigen in both enteric and systemic compartments. These results suggest that immune responses in specific mucosal compartments may be programmed by employing different prime-boost immunization routes.

Periodontitis is associated with a dysbiotic shift in the oral microbiome. Vaccine approaches to prevent microbial shifts from healthy to diseased state in oral biofilms would provide a fundamental therapeutic strategy against periodontitis. Since dental plaque formation is a polymicrobial and multilayered process, vaccines targeting single bacterial species would have limited efficacy in clinical applications. In this study, we developed a divalent mucosal vaccine consisting of a mixture of FlaB-tFomA and Hgp44-FlaB fusion proteins targeting virulence factors of inflammophilic bacteria Fusobacterium nucleatum and Porphyromonas gingivalis, respectively. Introduction of peptide linkers between FlaB and antigen improved the stability and immunogenicity of engineered vaccine antigens. The intranasal immunization of divalent vaccine induced protective immune responses inhibiting alveolar bone loss elicited by F. nucleatum and P. gingivalis infection. The built-in flagellin adjuvant fused to protective antigens enhanced antigen-specific antibody responses and class switch recombination. The divalent vaccine antisera recognized natural forms of surface antigens and reacted with diverse clinical isolates of Fusobacterium subspecies and P. gingivalis. The antisera inhibited F. nucleatum-mediated biofilm formation, co-aggregation of P. gingivalis and Treponema denticola, and P. gingivalis-host cell interactions. Taken together, the built-in adjuvant-engineered mucosal vaccine provides a technological platform for multivalent periodontitis vaccines targeting dysbiotic microbiome.

Dendritic cell (DC)-based vaccines are considered useful in cancer immunotherapy, and the interaction of DC and adjuvants is important in the design of the next generation vaccines. In this study, whether DC combined with Rv2299c derived from mycobacteria could improve anti-tumor immune responses in a colon cancer mouse model was evaluated. MC38 cell lines were injected subcutaneously to establish colon-cancer-bearing mice and the following four groups were evaluated: PBS control, tumor antigen (TA) loaded-DC, Rv2299c, and a combination of TA-loaded-DC and Rv2299c. The combination treatment with TA-loaded-DC and Rv2299c exhibited greater inhibition of tumor growth compared to other groups. These effects were associated with the reduction of suppressor cells, such as myeloid-derived suppressor cells and regulatory T cells, and the induction of effector cells, such as CD4+ T cells and CD8+ T cells, in spleen, and with the activation of cytotoxic T Lymphocytes and NK cells. These results suggest that TA-loaded-DC vaccination with Rv2299c derived from mycobacteria enhanced anti-tumor immunity in a mouse colon cancer model by inhibiting the generation of immune-suppressive cells and recovering numbers of effector cells, and demonstrated superior polarization of the Th1/Th2 balance in favor of the Th1 immune response.

RtxA1 toxin, which results in cytoskeletal rearrangement, contact cytotoxicity, hemolysis, tissue invasion, and lethality in mice, is the most potent cytotoxic virulence factor of Vibrio vulnificus. Bioinformatics analysis of rtxA1 predicted 4 functional domains that presumably performed discrete functions during host cell killing. V. vulnificus RtxA1 has a unique domain designated as RtxA1-D2, corresponding to amino acids 1951-2574, which is absent in Vibrio cholerae multifunctional-autoprocessing repeats-in-toxin, suggesting that this domain confers specific biological functions to V. vulnificus RtxA1. HeLa cells expressing green fluorescent protein-RtxA1-D2 became round and lost their viability. A yeast 2-hybrid system identified prohibitin (PHB) 1 as the host partner of RtxA1-D2. The specific interaction of RtxA1-D2 with PHB1 was confirmed by performing immunoprecipitation. Interestingly, V. vulnificus RtxA1 up-regulated PHB1 expression on the cytoplasmic membrane of host cells. Extracellular signal-regulated kinase and p38 mitogen-activated protein kinase pathways were confirmed as being important in the up-regulation of PHB1 by using inhibitors. Down-regulation of PHB1 by small interfering RNAs decreased the cytotoxicity of RtxA1-D2 against HeLa cells. The pretreatment of an anti-PHB1 antibody impaired the cytotoxicity of V. vulnificus RtxA1. These results suggest that the involvement PHB1 in the RtxA1 cytotoxicity has significant implications for the pathogenesis of V. vulnificus infections.

We report a method of cancer immunotherapy using an attenuated Salmonella typhimurium strain engineered to secrete Vibrio vulnificus flagellin B (FlaB) in tumor tissues. Engineered FlaB-secreting bacteria effectively suppressed tumor growth and metastasis in mouse models and prolonged survival. By using Toll-like receptor 5 (TLR5)-negative colon cancer cell lines, we provided evidence that the FlaB-mediated tumor suppression upon bacterial colonization is associated with TLR5-mediated host reactions in the tumor microenvironment. These therapeutic effects were completely abrogated in TLR4 and MyD88 knockout mice, and partly in TLR5 knockout mice, indicating that TLR4 signaling is a requisite for tumor suppression mediated by FlaB-secreting bacteria, whereas TLR5 signaling augmented tumor-suppressive host reactions. Tumor microenvironment colonization by engineered Salmonella appeared to induce the infiltration of abundant immune cells such as monocytes/macrophages and neutrophils via TLR4 signaling. Subsequent secretion of FlaB from colonizing Salmonella resulted in phenotypic and functional activation of intratumoral macrophages with M1 phenotypes and a reciprocal reduction in M2-like suppressive activities. Together, these findings provide evidence that nonvirulent tumor-targeting bacteria releasing multiple TLR ligands can be used as cancer immunotherapeutics.

Vibrio vulnificus causes rapidly progressing septicemia with an extremely high mortality rate (≥50%), even with aggressive antibiotic treatment. The bacteria secrete multifunctional autoprocessing repeats-in-toxin (MARTX) toxins, which are involved in the pathogenesis of Gram-negative Vibrio species. Recently, we reported that immunization with the C-terminal region of V. vulnificus RtxA1/MARTX Vv , RtxA1-C, elicits a protective immune response against V. vulnificus through a poorly defined mechanism. In this study, we generated a panel of new monoclonal antibodies (MAbs) against V. vulnificus RtxA1-C and investigated their protective efficacies and mechanisms in a mouse model of infection. Prophylactic administration of seven MAbs strongly protected mice against lethal V. vulnificus infection (more than 90% survival). Moreover, three of these MAbs (21RA, 24RA, and 47RA) demonstrated marked efficacy as postexposure therapy. Notably, 21RA was therapeutically effective against lethal V. vulnificus infection by a variety of routes. Using Fab fragments and a neutropenic mouse model, we showed that 21RA and 24RA mediate protection from V. vulnificus infection through an Fc-independent and/or neutrophil-independent pathway. In contrast, 47RA-mediated protection was dependent on its Fc region and was reduced to 50% in neutropenic mice compared with 21RA-mediated and 24RA-mediated protection. Bacteriological study indicated that 21RA appears to enhance the clearance of V. vulnificus from the blood. Overall, these studies suggest that humoral immunity controls V. vulnificus infection through at least two different mechanisms. Furthermore, our panel of MAbs could provide attractive candidates for the further development of immunoprophylaxis/therapeutics and other therapies against V. vulnificus that target the MARTX toxin.

Vibrio vulnificus is a foodborne pathogen that is prevalent in coastal waters worldwide. Infection with V. vulnificus causes septicemia with fatality rates exceeding 50% even with aggressive antibiotic therapy. Several vaccine studies to prevent V. vulnificus infection have been performed but have had limited success. In this study, we identified the C-terminal region (amino acids 3491 to 4701) of the V. vulnificus multifunctional autoprocessing RTX (MARTXVv or RtxA1) protein, RtxA1-C, as a promising antigen that induces protective immune responses against V. vulnificus. Vaccination of mice with recombinant RtxA1-C protein with adjuvant elicited a robust antibody response and a dramatic reduction in blood bacterial load in mice infected intraperitoneally. Vaccination resulted in significant protection against lethal challenge with V. vulnificus. Furthermore, intraperitoneal passive immunization with serum raised against the recombinant RtxA1-C protein demonstrated marked efficacy in both prophylaxis and therapy. These results suggest that active and passive immunization against the C-terminal region of the RtxA1 protein may be an effective approach in the prevention and therapy of V. vulnificus infections.

Flagellin, the cognate ligand for toll-like receptor 5, has potent adjuvant activity in various vaccines. However, its efficacy in generating dendritic cells (DCs) remains contentious. This study assessed how efficaciously Vibrio vulnificus FlaB (v-FlaB) could be used in generating a potent DC to induce antigen-specific cytotoxic T lymphocytes (CTLs). Mature DCs (mDCs) induced by the combination of v-FlaB/TNFα/IFNα were significantly more potent in inducing specific anticancer immune responses compared with the standard DCs that were maturated by the conventional cytokine cocktail of TNFα/IL-1β/IL-6/PGE(2). The potent mDCs produced a higher level of interleukin (IL)-12p70 and polarized naive CD4(+) T cells more towards Th1-type cells, markedly increased antigen-specific CD8(+) T-cell number and significantly enhanced the induction of lytic enzymes in antigen-specific CD8(+) CTLs and sensitized CD3(+) T cells to produce higher number of interferon (IFN)γ-secreting cells. As a result, the mDCs produced more potent antigen-specific CTLs against the MART-1 and expressed higher levels of homing receptors CCR5 and CXCR3. More importantly, the v-FlaB/TNFα/IFNα-DCs generated from melanoma patients produced strong autologous CTLs with efficient cytotoxic activities. In conclusion, v-FlaB combined with tumor necrosis factor (TNF)α and IFNα can generate potent DCs which produce functionally active CTLs and that may have potential as a potent cancer vaccine.

Department of Microbiology, Chonnam National University Medical School, Kwangju 501-746, KoreaReceived May 3, 2013, Accepted May 20, 2013Guanosine-5'-diphosphate 3'-diphosphate (ppGpp) serves as alarmone in bacterial stringent responses. In thisstudy, an affinity column was constructed by immobilizing ppGpp to NHS-Sepharose for isolating ppGpp-binding proteins. A novel ppGpp-binding protein, YjgA, was isolated and characterized by MALDI-TOF MS(matrix-assisted laser desorption ionization-time-of-flight mass spectrometry) coupled with two-dimensionalgel electrophoresis. YjgA and truncated forms of YjgA were cloned and over-expressed in BL21 (DE3). Thebinding affinity of YjgA to ppGpp was determined by equilibrium dialysis. The interaction of YjgA withppGpp was very specific, considering that the dissociation constant of YjgA with ppGpp was measured as 5.2± 2.0 µM, while the affinities to GTP and GDP were about 60 and 30 times weaker than ppGpp. Expression ofyjgA gene in Escherichia coli K-12 MG1655 was examined by reverse transcription polymerase chain reaction(RT-PCR). RT-PCR results revealed that yjgA was expressed from early to late stationary phase. The yjgAdeletion mutant exhibited decreased cell number at stationary phase compared to parent strain and the over-expression of YjgA increased the cell number. These results suggested that YjgA might stimulate cell divisionunder stationary phase. In most prokaryotic genome, about half of the protein candidates are hypothetical, thatare expected to be expressed but there is no experimental report on their functions. The approach utilized in thisstudy may serve as an effective mean to probe the functions of hypothetical proteins. Key Words : YjgA, ppGpp, Stringent responses, Stationary phase, Hypothetical proteinIntroductionProkaryotes show stringent response to various nutritionalchanges, including amino acids (Haseltine and Block, 1973),carbon (Flardh et al., 1994), nitrogen (Silberbach et al.,2005) and phosphate (Bougdour and Gottesman, 2007)limitation. Cashel and Gallant first discovered guanosine-5'-diphosphate 3'-diphosphate (ppGpp) accumulation in bacteriaduring nutrient starvation (Cashel and Gallant, 1969), andRelA and SpoT serve as ppGpp synthetases in E. coli(Cashel et al., 1996). ppGpp binds to the β and β' subunits ofcore RNA polymerase, inhibits stable RNA biosynthesis(rRNA and tRNA) during growth inhibition (Ryals et al.,1982; Toulokhonov et al., 2001), and arrests cell cycles(Ferullo and Lovett, 2008). A complete absence of ppGppcreates its own phenotypic features including multiple aminoacid requirements, poor survival of aged culture, aberrantcell division, morphology, and immotility (Potrykus andCashel, 2008). Importantly, ppGpp influences the pattern ofoverall gene expression that requires for survival in stressconditions (Song et al., 2004). In E. coli, ppGpp acts as aglobal regulatory affects on gene expression through itsinfluence on the levels of global regulators such as IHF,RpoS and RpoH (Lange et al., 2005). The absence of ppGppimpairs or severely delays the accumulation of RpoS, astress response sigma factor (Gentry et al., 1993). In addi-tion to transcription, ppGpp regulates translation by inter-acting with EF-G and IF2 (Mitkevich et al., 2010). ppGpp isinvolved in the acid stress response via lysine decarboxylaseLdc1 (Kanjee et al., 2011), phosphate metabolism viapolynucleotide phosphorylase (Gatewood and Jones, 2010),and replication via DnaG primase (Maciag et al., 2010).With such pleotropic eects, ppGpp seems to be a majorregulator of bacterial growth rate (Potrykus et al., 2011). Inspite of the diverse functions connected to ppGpp, the mech-anism of ppGpp regulation remains controversial (Vrentas etal., 2008). The first genome structure was announced in 1995 andmore than 1,000 genome sequences are currently availablein public domain (Karin et al., 2010). Although the wholegenome provides rich information on metabolic pathwaysand their regulations, about a half of proteins in mostgenomes are annotated as hypothetical proteins (Bhatia etal., 1997). Hypothetical proteins, also referred as ‘putative’,‘uncharacterized’ or ‘unknown’ proteins, are those whoseexistence can be predicted but no experimental reports ontheir functions are available. Even for Escherichia coli K-12,the most studied organism, there are still about 1,500 genesthat have not been experimentally categorized (Koonin andGalperin, 2003). The functions of hypothetic proteins are

Vibrio vulnificus causes fatal septicemia in susceptible subjects after the ingestion of raw seafood. In the present study, the roles of cyclic adenosine monophosphate (cAMP) receptor protein (CRP) in V. vulnificus pathogenesis were investigated. A mutation in the V. vulnificus crp gene resulted in a significant down-regulation of various virulence phenotypes, except for RtxA1-mediated cytoskeletal rearrangement. Bacterial growth was impeded by the crp mutation. In addition, colony morphology was converted from opaque to translucent type by this mutation, which implies a decrease in capsule production. The crp mutant also showed significant decrease in motility and adhesion to host cells. V. vulnificus CRP positively regulated production of hemolysin and protease at transcriptional level. All these changes in the crp mutant were fully complemented in trans by a plasmid harboring the wild-type gene. In contrast, CRP negatively regulated the expression of RtxA1. The crp mutant caused the cytoskeletal rearrangement in HeLa cells, which is a hallmark activity of RtxA1 toxin. Taken together, CRP seems to play a dual regulatory role in various virulence traits of V. vulnificus.

We report a method of cancer immunotherapy using an attenuated

Background Noroviruses (NoVs) are a major cause of childhood gastroenteritis and foodborne diseases worldwide. Lack of appropriate animal models or cell-based culture systems makes the development and evaluation of NoV-specific vaccines a daunting task. VP1 is the major capsid protein of the NoVs that acts as a binding motif to human histo-blood group antigens (HBGAs) through its protruding 2 (P2) domain and can serve as a protective antigen candidate for vaccine development. Methods Recombinantly produced NoV specific P domain (Pd) vaccine was inoculated into groups of mice either alone or in conjugation with mucosal adjuvant FlaB, the flagellar protein from Vibrio vulnificus. Antigen specific humoral and cell mediated immune responses were assessed by enzyme linked immunosorbent assay (ELISA) or fluorescent activated cell sorting (FACS). A comparative analysis of various routes of vaccination viz. intranasal, sublingual and subcutaneous, was also done. Results In this study, we show that a recombinant Pd-vaccine administered through intranasal route induced a robust TH2-dependent humoral immune response and that the combination of vaccine with FlaB significantly enhanced the antibody response. Interestingly, FlaB induced a mixed TH1/TH2 type of immune response with a significant induction of IgG1 as well as IgG2a antibodies. FlaB also induced strong IgA responses in serum and feces. FlaB mediated antibody responses were toll like receptor 5 (TLR5) dependent, since the FlaB adjuvanticity was lost in TLR5−/− mice. Further, though the Pd-vaccine by itself failed to induce a cell mediated immune response, the Pd-FlaB combination stimulated a robust CD4+IFNγ+ and CD8+IFNγ+ T cell response in spleen and mesenteric lymph nodes. We also compared the adjuvant effects of FlaB with that of alum and complete Freund’s adjuvant (CFA). We found that subcutaneously inoculated FlaB induced more significant levels of IgG and IgA in both serum and feces compared to alum or CFA in respective samples. Conclusion We validate the use of TLR5 agonist as a strong mucosal adjuvant that would facilitate the development of NoV specific vaccines for humans and veterinary use. This study also highlights the importance of route of immunization in inducing the appropriate immune responses in mucosal compartments.

In the modern concept of gene regulation, 'DNA looping' is the most common underlying mechanism in the interaction between RNA polymerase (RNAP) and transcription factors acting at a distance. This study demonstrates an additional mechanism by which DNA-bound proteins communicate with each other, by analysing the bacterial histone-like nucleoid-structuring protein (H-NS), a general transcriptional silencer. The LEE5 promoter (LEE5p) of enteropathogenic Escherichia coli was used as a model system to investigate the mechanism of H-NS-mediated transcription repression. We found that H-NS represses LEE5p by binding to a cluster of A tracks upstream of -114, followed by spreading to a site at the promoter through the oligomerization of H-NS molecules. At the promoter, the H-NS makes a specific contact with the carboxy terminal domain of the α subunit of RNAP, which prevents the processing of RNAP-promoter complexes into initiation-competent open promoter complexes, thereby regulating LEE5p from distance.

Background & Aims Foxp3 + T-regulatory cells (Tregs) maintain intestinal homeostasis under conditions of continuous challenge with inflammatory microbes. However, plasticity of the Treg population under certain conditions has been reported; Foxp3 + Tregs can be converted to Foxp3 − CD4 + T cells. Methods We used mice with a T cell–induced colitis model to study the regulatory role of type I interferons (IFNs) in adaptive immunity. We transferred CD4 + CD45RB hi (RB hi ) T cells, with or without CD4 + CD45RB lo CD25 + T cells, from wild-type or IFN-αβR −/− mice into Rag1 −/− recipients. We analyzed induction of colitis by flow cytometry, confocal microscopy, and enzyme-linked immunosorbent assay and reverse-transcription polymerase chain reaction analyses. IFN-αβR −/− Rag −/− mice were given injections of recombinant IFN-α following transfer of IFN-αβR −/− RB hi T cells and CD4 + Foxp3 + cells from Foxp3-eGFP mice. Results Signaling by type I IFNs was required for maintenance of Foxp3 expression and the suppressive activity of Tregs in mice. Transfer of CD4 + CD45RB lo CD25 + Tregs from IFN-αβR −/− mice did not prevent T-cell induction of colitis in mice. Foxp3 expression by Tregs transferred from IFN-αβR −/− mice was significantly lower than that of Tregs from wild-type mice. Administration of recombinant IFN-α reduced T cell–mediated colitis by increasing the number of Foxp3 + Tregs and their suppressive functions. Conclusions Type I IFNs regulate intestinal homeostasis by maintaining Foxp3 expression on Tregs in colons of mice under inflammatory conditions.

Transcription start points in bacteria are influenced by the nature of the RNA polymerase·promoter interaction. For Escherichia coli RNA polymerase holoenzyme containing σ70, it is presumed that specific sequence in one or more of the -10, extended -10 and -35 elements of the promoter guides the RNAP to select the cognate start point. Here, we investigated the promoter driving expression of the LEE1 operon in enteropathogenic E. coli and found two promoters separated by 10 bp, LEE1 P1A (+1) and LEE1 P1B (+10) using various in vitro biochemical tools. A unique feature of P1B was the presence of multiple transcription starts from five neighbouring As at the initial transcribed region. The multiple products did not arise from stuttering synthesis. Analytical software based on information theory was employed to determine promoter elements. The concentration of the NTP pool altered the preferred transcription start points, albeit the underlying mechanism is elusive. Under in vivo conditions, dominant P1B, but not P1A, was subject to regulation by IHF.

Cellular immunotherapy is emerging as a potential immunotherapeutic modality in multiple myeloma (MM). We have developed potent immunotherapeutic agent (VAX-DC/MM) generated by dendritic cells (DCs) loaded with autologous myeloma cells irradiated with ultraviolet B. In this study, we evaluated the safety and efficacy of VAX-DC/MM in patients with relapsed or refractory MM. This trial enrolled relapsed or refractory MM patients who had received both thalidomide- and bortezomib-based therapies. Patients received the intradermal VAX-DC/MM injection every week for 4 weeks. Patients were treated with 5 × 106 or 10 × 106 cells, with nine patients treated at a higher dose. The median time from diagnosis to VAX-DC/MM therapy was 56.6 months (range, 28.5-130.5). Patients had received a median of five prior treatments, and 75% had received autologous stem cell transplantation. VAX-DC therapy was well-tolerated, and the most frequent adverse events were local reactions at the injection site and infusion-related reactions. In seven of nine patients who received 10×106 cells, an immunological response (77.8%) was observed by interferon-gamma ELISPOT assay or a mixed lymphocyte reaction assay for T-cell proliferation. The clinical benefit rate was 66.7% including one (11.1%) with minor response and five (55.6%) with stable disease; three (33.3%) patients showed disease progression. In conclusion, VAX-DC/MM therapy was well-tolerated, and had disease-stabilizing activity in heavily pretreated MM cases. Further studies are needed to increase the efficacy of VAX-DC/MM in patients with MM.

Curcumin, a natural polyphenolic flavonoid extracted from the rhizome of Curcuma longa L., has many beneficial biological activities. However, there are relatively few reports of the effects of curcumin on pathogen infections. This study examined the effect of curcumin on a Vibrio vulnificus infection. The cytotoxicity of V. vulnificus to HeLa cells was significantly inhibited by curcumin (at 10 or 30 μM). To further examine the inhibitory mechanism of curcumin against V. vulnificus-mediated cytotoxicity, the level of bacterial growth, bacterial motility, cell adhesion, RTX toxin expression and host cell reactions were evaluated. Curcumin inhibited V. vulnificus growth in HI broth. Curcumin inhibited both bacterial adhesion and RTX toxin binding to the host cells, which can be considered the major protective mechanisms for the decrease in V. vulnificus cytotoxicity. Curcumin also inhibited host cell rounding and actin aggregation, which are the early features of cell death caused by V. vulnificus. In addition, curcumin decreased the V. vulnificus-induced NF-κB translocation in HeLa cells. Finally, curcumin protected mice from V. vulnificus-induced septicemia. In conclusion, curcumin may be an alternative antimicrobial agent against fatal bacterial infections.

Streptococcus pneumoniae is a major respiratory pathogen that causes high levels of mortality and morbidity in infants and the elderly. Despite the use of antibiotics and vaccines, fatal pneumococcal disease remains prevalent. Pneumococcal surface protein A (PspA), a highly immunogenic surface protein produced by all strains of S. pneumoniae, can elicit protective immunity against fatal pneumococcal infection. We have previously demonstrated that the Vibrio vulnificus FlaB, a bacterial flagellin protein and agonist of TLR5, has strong mucosal adjuvant activity and induces protective immunity upon co-administration with tetanus toxoid. In this study, we have tested whether intranasal immunization with recombinant fusion proteins consisted of PspA and FlaB (PspA-FlaB and FlaB-PspA) is able to elicit more efficient protective mucosal immune responses against pneumococcal infection than immunization with PspA alone or with a stoichiometric mixture of PspA and FlaB. When mice were intranasally immunized with fusion proteins, significantly higher levels of anti-PspA IgG and IgA were induced in serum and mucosal secretions. The mice immunized intranasally with the FlaB-PspA fusion protein were the most protected from a lethal challenge with live S. pneumoniae, as compared to the mice immunized with PspA only, a mixture of PspA and FlaB, or the PspA-FlaB fusion protein. FlaB-PspA also induced a cross protection against heterologous capsular types. These results suggest that a FlaB-PspA fusion protein alone could be used as an anti-pneumococcal mucosal vaccine or as an effective partner protein for multivalent capsular polysaccharide conjugate vaccines.

Vibrio vulnificus is a lethal pathogen that is responsible for most seafood-related deaths. The mortality rate for susceptible individuals is higher than 50% within 1–2 days after contact with the bacteria. Recent studies suggest that RtxA1, a secreted bacterial toxin, plays a key role in the cytotoxicity and pathogenesis of V. vulnificus. Despite the important role of RtxA1 in the virulence of V. vulnificus, few monoclonal antibodies (MAbs) against RtxA1 have been generated or characterized. In this study, we purified a recombinant RtxA1 protein with a mass of approximately 130 kDa that contains GD-rich repeats proposed to play an important role in host cell killing, and generated a panel of 10 new MAbs against the recombinant RtxA1 protein. By performing competition binding assays and expressing RtxA1 fragments via nested deletions of the gene, we spatially mapped our new MAbs to three fragments of the recombinant RtxA1 protein. Western blot analysis and immunostaining studies showed that the MAbs efficiently reacted with secreted and host cell-targeted RtxA1 from V. vulnificus. These data suggest that the RtxA1 MAbs might be effectively used as important biomaterials for manufacturing toxin-neutralizing therapeutic MAbs targeting the bacterial RtxA1 toxin and could be relevant for developing rapid and specific diagnostic tools against lethal V. vulnificus infections.

Optimization of the specific affinity of cardiac delivery vector could significantly improve the efficiency of gene/protein delivery, yet no cardiac vectors to date have sufficient target specificity for myocardial infarction (MI). In this study, we explored bacterial tropism for infarcted myocardium based on our previous observations that certain bacteria are capable of targeting the hypoxic regions in solid tumors. Out of several Escherichia coli or Salmonella typhimurium strains, the S. typhimurium defective in the synthesis of ppGpp (ΔppGpp S. typhimurium) revealed accumulation and selective proliferation in the infarcted myocardium without spillover to noncardiac tissue. The Salmonellae that were engineered to express a variant of Renilla luciferase gene (RLuc8), under the control of the E. coli arabinose operon promoter (P(BAD)), selectively targeted and delivered RLuc8 in the infarcted myocardium only upon injection of L-arabinose. An examination of the infarct size before and after infection, and estimations of C-reactive protein (CRP) and procalcitonin indicated that intravenous injection of ΔppGpp S. typhimurium did not induce serious local or systemic immune reactions. This current proof-of-principle study demonstrates for the first time the capacity of Salmonellae to target infarcted myocardium and to serve as a vehicle for the selective delivery of therapeutic agents in MI.