Deimmunization of flagellin for repeated administration as a vaccine adjuvant.

Flagellin, a protein-based Toll-like receptor agonist, is a versatile adjuvant applicable to wide spectrum of vaccines and immunotherapies. Given reiterated treatments of immunogenic biopharmaceuticals should lead to antibody responses precluding repeated administration, the development of flagellin not inducing specific antibodies would greatly expand the chances of clinical applications. Here we computationally identified immunogenic regions in Vibrio vulnificus flagellin B and deimmunized by simply removing a B cell epitope region. The recombinant deimmunized FlaB (dFlaB) maintains stable TLR5-stimulating activity. Multiple immunization of dFlaB does not induce FlaB-specific B cell responses in mice. Intranasally co-administered dFlaB with influenza vaccine enhanced strong Ag-specific immune responses in both systemic and mucosal compartments devoid of FlaB-specific Ab production. Notably, dFlaB showed better protective immune responses against lethal viral challenge compared with wild type FlaB. The deimmunizing B cell epitope deletion did not compromise stability and adjuvanticity, while suppressing unwanted antibody responses that may negatively affected vaccine antigen-directed immune responses in repeated vaccinations. We explain the underlying mechanism of deimmunization by employing molecular dynamics analysis.

The cytochrome d oxidase complex regulated by fexA is an Achilles' heel in the in vivo survival of vibrio vulnificus.

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.

A stealth adhesion factor contributes to Vibrio vulnificus pathogenicity: Flp pili play roles in host invasion, survival in the blood stream and resistance to complement activation.

The tad operons encode the machinery required for adhesive Flp (fimbrial low-molecular-weight protein) pili biogenesis. Vibrio vulnificus, an opportunistic pathogen, harbors three distinct tad loci. Among them, only tad1 locus was highly upregulated in in vivo growing bacteria compared to in vitro culture condition. To understand the pathogenic roles of the three tad loci during infection, we constructed single, double and triple tad loci deletion mutants. Interestingly, only the Δtad123 triple mutant cells exhibited significantly decreased lethality in mice. Ultrastructural observations revealed short, thin filamentous projections disappeared on the Δtad123 mutant cells. Since the pilin was paradoxically non-immunogenic, a V5 tag was fused to Flp to visualize the pilin protein by using immunogold EM and immunofluorescence microscopy. The Δtad123 mutant cells showed attenuated host cell adhesion, decreased biofilm formation, delayed RtxA1 exotoxin secretion and subsequently impaired translocation across the intestinal epithelium compared to wild type, which could be partially complemented with each wild type operon. The Δtad123 mutant was susceptible to complement-mediated bacteriolysis, predominantly via the alternative pathway, suggesting stealth hiding role of the Tad pili. Complement depletion by treating with anti-C5 antibody rescued the viable count of Δtad123 in infected mouse bloodstream to the level comparable to wild type strain. Taken together, all three tad loci cooperate to confer successful invasion of V. vulnificus into deeper tissue and evasion from host defense mechanisms, ultimately resulting in septicemia.

Vibrio vulnificus RtxA1 cytotoxin targets filamin A to regulate PAK1- and MAPK-dependent cytoskeleton reorganization and cell death.

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.

A built-in adjuvant-engineered mucosal vaccine against dysbiotic periodontal diseases.

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.

More robust gut immune responses induced by combining intranasal and sublingual routes for prime-boost immunization.

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.

Mucosal immunization with a flagellin-adjuvanted Hgp44 vaccine enhances protective immune responses in a murine Porphyromonas gingivalis infection model.

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.

Racial Differences in Expression Levels of miRNA Machinery-Related Genes, Dicer, Drosha, DGCR8, and AGO2, in Asian Korean Papillary Thyroid Carcinoma and Comparative Validation Using the Cancer Genome Atlas.

Aberrant regulation of microRNA (miRNA) machinery components is associated with various human cancers, including papillary thyroid carcinoma (PTC), which is the most common type of thyroid cancer, and a higher prevalent female malignancy. The purpose of this study is to investigate racial differences in mRNA expression levels of four miRNA machinery components, Dicer, Drosha, DGCR8, and AGO2, and their correlations with clinicopathological characteristics. Forty PTC samples from female Asian Korean PTC patients were enrolled. Using qPCR, we examined mRNA expression levels of the components and next validated our results by comparison with results of female white American in the TCGA PTC project. Interestingly, mRNA expression levels of the selected factors were altered in the TCGA PTC samples. However, only Drosha showed a significantly lower expression level in Asian Korean PTC samples. Furthermore, the mRNA expression levels of the four components showed no association with clinicopathological characteristics in both groups. On the other hand, positive correlations were observed between altered mRNA expression levels of Dicer and Drosha and DGCR8 and Drosha in TCGA PTC samples. These findings collectively revealed that altered mRNA expression levels of miRNA machinery components might be responsible for racial differences in the carcinogenesis of PTC.

Caffeic acid, morin hydrate and quercetin partially attenuate sulfur mustard-induced cell death by inhibiting the lipoxygenase pathway.

Sulfur mustard (SM) is an alkylating agent, which has been used as in chemical warfare in a number of conflicts. As the generation of reactive oxygen species (ROS), and adducts in DNA and proteins have been suggested as the mechanism underlying SM­induced cytotoxicity, the present study screened several antioxidant candidates, including tannic acid, deferoxamine mesylate, trolox, vitamin C, ellagic acid and caffeic acid (CA) to assess their potential as therapeutic agents for SM­induced cell death. Among several antioxidants, CA partially alleviated SM­induced cell death in a dose­dependent manner. Although CA treatment decreased the phosphorylation of p38 mitogen­activated protein (MAP) kinase and p53, p38 MAP kinase inhibition by SB203580 did not affect SM­induced cell death. As CA has also been reported as a 15­lipoxygenase (15­LOX) inhibitor, the role of 15­LOX in SM­induced cytotoxicity was also examined. Similar to the results observed with CA, treatment with PD146176, a specific 15­LOX inhibitor, decreased SM­induced cytotoxicity, accompanied by decreases in the production of tumor necrosis factor­α and 15­hydroxyeicosatetraenoic acid. Furthermore, the present study investigated the protective effects of two natural 15­LOX inhibitors, morin hydrate and quercetin, in SM­induced cytotoxicity. As expected, these inhibitors had similar protective effects against SM­induced cytotoxicity. These antioxidants also reduced the generation of ROS and nitrate/nitrite. Therefore, the results of the present study indicated that the natural products, CA, quercetin and morin hydrate, offer potential as adjuvant therapeutic agents for SM­induced toxicity, not only by reducing inflammation mediated by the p38 and LOX signaling pathways, but also by decreasing the generation of ROS and nitrate/nitrite.

Flagellin is a strong vaginal adjuvant of a therapeutic vaccine for genital cancer.

Cervical cancer is a high-incidence female cancer most commonly caused by human papilloma virus (HPV) infection of the genital mucosa. Immunotherapy targeting HPV-derived tumor antigens (TAs) has been widely studied in animal models and in patients. Because the female genital tract is a portal for the entry of HPV and a highly compartmentalized system, the development of topical vaginal immunotherapy in an orthotopic cancer model would provide an ideal therapeutic. Thus, we examined whether flagellin, a potent mucosal immunomodulator, could be used as an adjuvant for a topical therapeutic vaccine for female genital cancer. Intravaginal (IVAG) co-administration of the E6/E7 peptides with flagellin resulted in tumor suppression and long-term survival of tumor-bearing mice. In contrast to IVAG vaccination, intranasal (IN) or subcutaneous (SC) immunization did not induce significant tumor suppression in the same model. The vaginal adjuvant effect of the flagellin was completely abolished in Toll-like receptor-5 (TLR5) knock-out mice. IVAG immunization with the E6/E7 peptides plus flagellin induced the accumulation of CD4+ and CD8+ cells and the expression of T cell activation-related genes in the draining genital lymph nodes (gLNs). The co-administered flagellin elicited antigen-specific IFNγ production in the gLNs and spleen. The intravaginally administered flagellin was found in association with CD11c+ cells in the gLNs. Moreover, after immunization with a flagellin and the E6/E7 peptides, the TLR5 expression in gLN cells was significantly upregulated. These results suggest that flagellin serves as a potent vaginal adjuvant for a therapeutic peptide cancer vaccine through the activation of TLR5 signaling.

All Three TonB Systems Are Required for Vibrio vulnificus CMCP6 Tissue Invasiveness by Controlling Flagellum Expression.

TonB systems actively transport iron-bound substrates across the outer membranes of Gram-negative bacteria. Vibrio vulnificus CMCP6, which causes fatal septicemia and necrotizing wound infections, possesses three active TonB systems. It is not known why V. vulnificus CMCP6 has maintained three TonB systems throughout its evolution. The TonB1 and TonB2 systems are relatively well characterized, while the pathophysiological function of the TonB3 system is still elusive. A reverse transcription-PCR (RT-PCR) study showed that the tonB1 and tonB2 genes are preferentially induced in vivo, whereas tonB3 is persistently transcribed, albeit at low expression levels, under both in vitro and in vivo conditions. The goal of the present study was to elucidate the raison d'être of these three TonB systems. In contrast to previous studies, we constructed in-frame single-, double-, and triple-deletion mutants of the entire structural genes in TonB loci, and the changes in various virulence-related phenotypes were evaluated. Surprisingly, only the tonB123 mutant exhibited a significant delay in killing eukaryotic cells, which was complemented in trans with any TonB operon. Very interestingly, we discovered that flagellum biogenesis was defective in the tonB123 mutant. The loss of flagellation contributed to severe defects in motility and adhesion of the mutant. Because of the difficulty of making contact with host cells, the mutant manifested defective RtxA1 toxin production, which resulted in impaired invasiveness, delayed cytotoxicity, and decreased lethality for mice. Taken together, these results indicate that a series of virulence defects in all three TonB systems of V. vulnificus CMCP6 coordinately complement each other for iron assimilation and full virulence expression by ensuring flagellar biogenesis.

Molecular characterization of vulnibactin biosynthesis in Vibrio vulnificus indicates the existence of an alternative siderophore.

Vibrio vulnificus is a halophilic estuarine bacterium that causes fatal septicemia and necrotizing wound infections in humans. Virulent V. vulnificus isolates produce a catechol siderophore called vulnibactin, made up of one residue of 2, 3-dihydroxybenzoic acid (2, 3-DHBA) and two residues of salicylic acid (SA). Vulnibactin biosynthetic genes (VV2_0828 to VV2_0844) are clustered at one locus of chromosome 2, expression of which is significantly up-regulated in vivo. In the present study, we decipher the biosynthetic network of vulnibactin, focusing specifically on genes around SA and 2, 3-DHBA biosynthetic steps. Deletion mutant of isochorismate pyruvate lyase (VV2_0839) or 2, 3-dihydroxybenzoate-2, 3-dehydrogenase (VV2_0834) showed retarded growth under iron-limited conditions though the latter showed more significant growth defect than the former, suggesting a dominant role of 2, 3-DHBA in the vulnibactin biosynthesis. A double deletion mutant of VV2_0839 and VV2_0834 manifested additional growth defect under iron limitation. Though the growth defect of respective single deletion mutants could be restored by exogenous SA or 2, 3-DHBA, only 2, 3-DHBA could rescue the double mutant when supplied alone. However, double mutant could be rescued with SA only when hydrogen peroxide was supplied exogenously, suggesting a chemical conversion of SA to 2, 3-DHBA. Assembly of two SA and one 2, 3-DHBA into vulnibactin was mediated by two AMP ligase genes (VV2_0836 and VV2_0840). VV2_0836 deletion mutant showed more significant growth defect under iron limitation, suggesting its dominant function. In conclusion, using molecular genetic analytical tools, we confirm that vulnibactin is assembled of both 2, 3-DHBA and SA. However, conversion of SA to 2, 3-DHBA in presence of hydrogen peroxide and growth profile of AMP ligase mutants suggest a plausible existence of yet unidentified alternative siderophore that may be composed solely of 2, 3-DHBA.

Anti-tumoral effect of the mitochondrial target domain of Noxa delivered by an engineered Salmonella typhimurium.

Bacterial cancer therapy relies on the fact that several bacterial species are capable of targeting tumor tissue and that bacteria can be genetically engineered to selectively deliver therapeutic proteins of interest to the targeted tumors. However, the challenge of bacterial cancer therapy is the release of the therapeutic proteins from the bacteria and entry of the proteins into tumor cells. This study employed an attenuated Salmonella typhimurium to selectively deliver the mitochondrial targeting domain of Noxa (MTD) as a potential therapeutic cargo protein, and examined its anti-cancer effect. To release MTD from the bacteria, a novel bacterial lysis system of phage origin was deployed. To facilitate the entry of MTD into the tumor cells, the MTD was fused to DS4.3, a novel cell-penetrating peptide (CPP) derived from a voltage-gated potassium channel (Kv2.1). The gene encoding DS4.3-MTD and the phage lysis genes were placed under the control of PBAD , a promoter activated by L-arabinose. We demonstrated that DS4.3-MTD chimeric molecules expressed by the Salmonellae were anti-tumoral in cultured tumor cells and in mice with CT26 colon carcinoma.

Contribution of six flagellin genes to the flagellum biogenesis of Vibrio vulnificus and in vivo invasion.

Vibrio vulnificus is a halophilic pathogenic bacterium that is motile due to the presence of a single polar flagellum. V. vulnificus possesses a total of six flagellin genes organized into two loci (flaFBA and flaCDE). We proved that all six of the flagellin genes were transcribed, whereas only five (FlaA, -B, -C, -D, and -F) of the six flagellin proteins were detected. To understand roles of the six V. vulnificus flagellins in motility and virulence, mutants with single and multiple flagellin deletions were constructed. Mutations in flaB or flaC or the flaCDE locus resulted in a significant decrease in motility, adhesion, and cytotoxicity, whereas single mutations in the other flagellin genes or the flaFBA locus showed little or no effect. The motility was completely abolished only in the mutant lacking all six flagellin genes (flaFBA flaCDE). Surprisingly, a double mutation of flaB and flaD, a gene sharing 99% identity with the flaB at the amino acid level, resulted in the largest decrease in motility, adhesion, and cytotoxicity except for the mutant in which all six genes were deleted (the hexa mutant). Additionally, the 50% lethal doses (LD50s) of the flaB flaD and the flaFBA flaCDE mutants increased 23- and 91-fold in a mouse model, respectively, and the in vitro and in vivo invasiveness of the mutants was significantly decreased compared to that of the wild type. Taken together, the multiple flagellin subunits differentially contribute to the flagellum biogenesis and the pathogenesis of V. vulnificus, and among the six flagellin genes, flaB, flaD, and flaC were the most influential components.

Flagellin enhances tumor-specific CD8⁺ T cell immune responses through TLR5 stimulation in a therapeutic cancer vaccine model.

Tumor antigen (TA)-specific immunotherapy is an emerging approach for cancer treatment. Potent adjuvants are prerequisites to the immunotherapy for overcoming the low immunogenicity of TAs. We previously demonstrated that a bacterial flagellin, Vibrio vulnificus FlaB, has potent adjuvant activity in various vaccination models. In this study, we investigated whether the FlaB protein could be a potent adjuvant for a human papillomavirus 16 E6 and E7 (E6/E7) peptide-based anticancer immunotherapy. We used an E6/E7-expressing TC-1 carcinoma implantation animal model and tested TA-specific immunomodulation by FlaB. We co-administered the E6/E7 peptide either with or without FlaB into TC-1 tumor-bearing mice and then analyzed the antitumor activity of the peptide. FlaB significantly potentiated specific antitumor immune responses elicited by the peptide immunization, as evidenced by retarded in vivo tumor growth and significantly prolonged survival. We noticed that TC-1 cells do not express Toll-like receptor 5 (TLR5) on their surface and the TLR5 signaling pathway in TC-1 cells was not responsible for the antitumor effect of FlaB. FlaB potentiated the CTL activity and Ag-specific IFN-γ production of CD8(+) T cells from the draining lymph node and spleen. In addition, this antitumor activity was abrogated following the in vivo depletion of CD8(+) T cells and in TLR5 knockout (KO) or MyD88 KO mice. These results suggest that flagellin could enhance TA-specific CD8(+) CTL immune responses through TLR5 stimulation in cancer immunotherapy.

The pyrH gene of Vibrio vulnificus is an essential in vivo survival factor.

We have suggested an important role of the pyrH gene during the infectious process of Vibrio vulnificus. Previously, we have identified 12 genes expressed preferentially during human infections by using in vivo-induced antigen technology. Among the in vivo-expressed genes, pyrH encodes UMP kinase catalyzing UMP phosphorylation. Introduction of a deletion mutation to the pyrH gene was lethal to V. vulnificus, and an insertional mutant showed a high frequency of curing. We constructed a site-directed mutant strain (R62H/D77N) on Arg-62 and Asp-77, both predicted to be involved in UMP binding, and characterized the R62H/D77N strain compared with the previously reported insertional mutant. We further investigated the essential role of the pyrH gene in the establishment of infection using the R62H/D77N strain. Cytotoxicity was decreased in the R62H/D77N strain, and the defect was restored by an in trans complementation. The intraperitoneal 50% lethal dose of the R62H/D77N strain increased by 26- and 238,000-fold in normal and iron-overloaded mice, respectively. The growth of the R62H/D77N strain in 50% HeLa cell lysate, 100% human ascitic fluid, and 50% human serum was significantly retarded compared to that of the isogenic wild-type strain. The R62H/D77N mutant also had a critical defect in the ability to survive and replicate even in iron-overloaded mice. These results demonstrate that pyrH is essential for the in vivo survival and growth of V. vulnificus and should be an attractive new target for the development of antibacterial drugs and replication-controllable live attenuated vaccines.