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Metilases de Modificação do DNA , Enzimas Reparadoras do DNA , Resistencia a Medicamentos Antineoplásicos , Glioblastoma , Células-Tronco Neoplásicas , Temozolomida , Regulação para Cima , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Temozolomida/uso terapêutico , Temozolomida/farmacologia , Humanos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Metilases de Modificação do DNA/metabolismo , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Regulação para Cima/efeitos dos fármacos , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Antineoplásicos Alquilantes/uso terapêutico , Antineoplásicos Alquilantes/farmacologiaRESUMO
Periodontal disease (PD) is caused by microbial dysbiosis and accompanying adverse inflammatory responses. Due to its high incidence and association with various systemic diseases, disease-modifying treatments that modulate dysbiosis serve as promising therapeutic approaches. In this study, to simulate the pathophysiological situation, we established a "temporary ligature plus oral infection model" that incorporates a temporary silk ligature and oral infection with a cocktail of live Tannerella forsythia (Tf), Pophyromonas gingivalis (Pg), and Fusobacterium nucleatum (Fn) in mice and tested the efficacy of a new trivalent mucosal vaccine. It has been reported that Tf, a red complex pathogen, amplifies periodontitis severity by interacting with periodontopathic bacteria such as Pg and Fn. Here, we developed a recombinant mucosal vaccine targeting a surface-associated protein, BspA, of Tf by genetically combining truncated BspA with built-in adjuvant flagellin (FlaB). To simultaneously induce Tf-, Pg-, and Fn-specific immune responses, it was formulated as a trivalent mucosal vaccine containing Tf-FlaB-tBspA (BtB), Pg-Hgp44-FlaB (HB), and Fn-FlaB-tFomA (BtA). Intranasal immunization with the trivalent mucosal vaccine (BtB + HB + BtA) prevented alveolar bone loss and gingival proinflammatory cytokine production. Vaccinated mice exhibited significant induction of Tf-tBspA-, Pg-Hgp44-, and Fn-tFomA-specific IgG and IgA responses in the serum and saliva, respectively. The anti-sera and anti-saliva efficiently inhibited epithelial cell invasion by Tf and Pg and interfered with biofilm formation by Fn. The flagellin-adjuvanted trivalent mucosal vaccine offers a novel method for modulating dysbiotic bacteria associated with periodontitis. This approach leverages the adjuvant properties of flagellin to enhance the immune response, aiming to restore a balanced microbial environment and improve periodontal health.
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Alzheimer's disease (AD) and related tauopathies are associated with pathological tau protein aggregation, which plays an important role in neurofibrillary degeneration and dementia. Targeted immunotherapy to eliminate pathological tau aggregates is known to improve cognitive deficits in AD animal models. The tau repeat domain (TauRD) plays a pivotal role in tau-microtubule interactions and is critically involved in the aggregation of hyperphosphorylated tau proteins. Because TauRD forms the structural core of tau aggregates, the development of immunotherapies that selectively target TauRD-induced pathological aggregates holds great promise for the modulation of tauopathies. In this study, we generated recombinant TauRD polypeptide that form neurofibrillary tangle-like structures and evaluated TauRD-specific immune responses following intranasal immunization in combination with the mucosal adjuvant FlaB. In BALB/C mice, repeated immunizations at one-week intervals induced robust TauRD-specific antibody responses in a TLR5-dependent manner. Notably, the resulting antiserum recognized only the aggregated form of TauRD, while ignoring monomeric TauRD. The antiserum effectively inhibited TauRD filament formation and promoted the phagocytic degradation of TauRD aggregate fragments by microglia. The antiserum also specifically recognized pathological tau conformers in the human AD brain. Based on these results, we engineered a built-in flagellin-adjuvanted TauRD (FlaB-TauRD) vaccine and tested its efficacy in a P301S transgenic mouse model. Mucosal immunization with FlaB-TauRD improved quality of life, as indicated by the amelioration of memory deficits, and alleviated tauopathy progression. Notably, the survival of the vaccinated mice was dramatically extended. In conclusion, we developed a mucosal vaccine that exclusively targets pathological tau conformers and prevents disease progression.
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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 a context of murine inflammatory disease models, such as inflammatory bowel disease and graft-versus-host disease. The differential ability of Tc17 differentiation is not related to T-cell receptor (TCR) diversity and antigen specificity but is inversely correlated with self-reactivity acquired during development. Mechanistically, this phenomenon is linked to differential levels of intrinsic TCR sensitivity and basal Suppressor of Mothers Against Decapentaplegic 3 (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.
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Linfócitos T CD8-Positivos , Inflamação , Camundongos , Animais , Modelos Animais de Doenças , Diferenciação Celular , Inflamação/patologia , Receptores de Antígenos de Linfócitos T/metabolismoRESUMO
Innate immune cells generate a multifaceted antitumor immune response, including the conservation of essential nutrients such as iron. These cells can be modulated by commensal bacteria; however, identifying and understanding how this occurs is a challenge. Here we show that the food commensal Lactiplantibacillus plantarum IMB19 augments antitumor immunity in syngeneic and xenograft mouse tumor models. Its capsular heteropolysaccharide is the major effector molecule, functioning as a ligand for TLR2. In a two-pronged manner, it skews tumor-associated macrophages to a classically active phenotype, leading to generation of a sustained CD8+ T cell response, and triggers macrophage 'nutritional immunity' to deploy the high-affinity iron transporter lipocalin-2 for capturing and sequestering iron in the tumor microenvironment. This process induces a cycle of tumor cell death, epitope expansion and subsequent tumor clearance. Together these data indicate that food commensals might be identified and developed into 'oncobiotics' for a multi-layered approach to cancer therapy.
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Ferro , Microambiente Tumoral , Animais , Ferro/metabolismo , Camundongos , Microambiente Tumoral/imunologia , Humanos , Macrófagos Associados a Tumor/imunologia , Macrófagos Associados a Tumor/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Receptor 2 Toll-Like/metabolismo , Receptor 2 Toll-Like/imunologia , Camundongos Endogâmicos C57BL , Lipocalina-2/metabolismo , Lipocalina-2/imunologia , Feminino , Simbiose/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Ativação de Macrófagos/imunologia , Camundongos KnockoutRESUMO
Cancer immunotherapy has made tremendous advancements in treating various malignancies. The biggest hurdle to successful immunotherapy would be the immunosuppressive tumor microenvironment (TME) and low immunogenicity of cancer cells. To make immunotherapy successful, the 'cold' TME must be converted to 'hot' immunostimulatory status to activate residual host immune responses. To this end, the immunosuppressive equilibrium in TME should be broken, and immunogenic cancer cell death ought to be induced to stimulate tumor-killing immune cells appropriately. Photodynamic therapy (PDT) is an efficient way of inducing immunogenic cell death (ICD) of cancer cells and disrupting immune-restrictive tumor tissues. PDT would trigger a chain reaction that would make the TME 'hot' and have ICD-induced tumor antigens presented to immune cells. In principle, the strategic combination of PDT and immunotherapy would synergize to enhance therapeutic outcomes in many intractable tumors. Novel technologies employing nanocarriers were developed to deliver photosensitizers and immunotherapeutic to TME efficiently. New-generation nanomedicines have been developed for PDT immunotherapy in recent years, which will accelerate clinical applications.
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Imunoterapia , Nanopartículas , Neoplasias , Fotoquimioterapia , Fármacos Fotossensibilizantes , Microambiente Tumoral , Fotoquimioterapia/métodos , Microambiente Tumoral/imunologia , Microambiente Tumoral/efeitos dos fármacos , Humanos , Neoplasias/terapia , Neoplasias/imunologia , Imunoterapia/métodos , Animais , Fármacos Fotossensibilizantes/uso terapêutico , Terapia Combinada , Nanomedicina/métodosRESUMO
Addressing age-related immunological defects through therapeutic interventions is essential for healthy aging, as the immune system plays a crucial role in controlling infections, malignancies, and in supporting tissue homeostasis and repair. In our study, we show that stimulating toll-like receptor 5 (TLR5) via mucosal delivery of a flagellin-containing fusion protein effectively extends the lifespan and enhances the healthspan of mice of both sexes. This enhancement in healthspan is evidenced by diminished hair loss and ocular lens opacity, increased bone mineral density, improved stem cell activity, delayed thymic involution, heightened cognitive capacity, and the prevention of pulmonary lung fibrosis. Additionally, this fusion protein boosts intestinal mucosal integrity by augmenting the surface expression of TLR5 in a certain subset of dendritic cells and increasing interleukin-22 (IL-22) secretion. In this work, we present observations that underscore the benefits of TLR5-dependent stimulation in the mucosal compartment, suggesting a viable strategy for enhancing longevity and healthspan.
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Longevidade , Receptor 5 Toll-Like , Animais , Camundongos , Flagelina/metabolismo , Mucosa Intestinal/metabolismo , Longevidade/genética , Pulmão/metabolismoRESUMO
The E6 and E7 proteins of specific subtypes of human papillomavirus (HPV), including HPV 16 and 18, are highly associated with cervical cancer as they modulate cell cycle regulation. The aim of this study was to investigate the potential antitumor effects of a messenger RNA-HPV therapeutic vaccine (mHTV) containing nononcogenic E6 and E7 proteins. To achieve this, C57BL/6j mice were injected with the vaccine via both intramuscular and subcutaneous routes, and the resulting effects were evaluated. mHTV immunization markedly induced robust T cell-mediated immune responses and significantly suppressed tumor growth in both subcutaneous and orthotopic tumor-implanted mouse model, with a significant infiltration of immune cells into tumor tissues. Tumor retransplantation at day 62 postprimary vaccination completely halted progression in all mHTV-treated mice. Furthermore, tumor expansion was significantly reduced upon TC-1 transplantation 160 days after the last immunization. Immunization of rhesus monkeys with mHTV elicited promising immune responses. The immunogenicity of mHTV in nonhuman primates provides strong evidence for clinical application against HPV-related cancers in humans. All data suggest that mHTV can be used as both a therapeutic and prophylactic vaccine.
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Proteínas Oncogênicas Virais , Infecções por Papillomavirus , Vacinas contra Papillomavirus , Neoplasias do Colo do Útero , Humanos , Feminino , Animais , Camundongos , Papillomavirus Humano , Proteínas Oncogênicas Virais/genética , Infecções por Papillomavirus/prevenção & controle , RNA Mensageiro/genética , Proteínas E7 de Papillomavirus/genética , Camundongos Endogâmicos C57BL , Vacinação/métodos , Imunização , Neoplasias do Colo do Útero/prevenção & controleRESUMO
Flagellin, the TLR5 agonist, shows potent adjuvant activities in diverse vaccines and immunotherapies. Vibrio vulnificus flagellin B expressed in eukaryotic cells (eFlaB) could not stimulate TLR5 signaling. Enzymatic deglycosylation restored eFlaB's TLR5 stimulating functionality, suggesting that glycosylation interferes with eFlaB binding to TLR5. Site-directed mutagenesis of N-glycosylation residues restored TLR5 stimulation and adjuvanticity. Collectively, deglycosylated eFlaB may provide a built-in adjuvant platform for eukaryotic-expressed antigens and nucleic acid vaccines.
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The use of appropriately designed immunotherapeutic bacteria is an appealing approach to tumor therapy because the bacteria specifically target tumor tissue and deliver therapeutic payloads. The present study describes the engineering of an attenuated strain of Salmonella typhimurium deficient in ppGpp biosynthesis (SAM) that could secrete Vibrio vulnificus flagellin B (FlaB) conjugated to human (hIL15/FlaB) and mouse (mIL15/FlaB) interleukin-15 proteins in the presence of L-arabinose (L-ara). These strains, named SAMphIF and SAMpmIF, respectively, secreted fusion proteins that retained bioactivity of both FlaB and IL15. SAMphIF and SAMpmIF inhibited the growth of MC38 and CT26 subcutaneous (sc) tumors in mice and increased mouse survival rate more efficiently than SAM expressing FlaB alone (SAMpFlaB) or IL15 alone (SAMpmIL15 and SAMphIL15), although SAMpmIF had slightly greater antitumor activity than SAMphIF. The mice treated with these bacteria showed enhanced macrophage phenotype shift, from M2-like to M1-like, as well as greater proliferation and activation of CD4+ T, CD8+ T, NK, and NKT cells in tumor tissues. After tumor eradication by these bacteria, ≥50% of the mice show no evidence of tumor recurrence upon rechallenge with the same tumor cells, indicating that they had acquired long-term immune memory. Treatment of mice of 4T1 and B16F10 highly malignant sc tumors with a combination of these bacteria and an immune checkpoint inhibitor, anti-PD-L1 antibody, significantly suppressed tumor metastasis and increased mouse survival rate. Taken together, these findings suggest that SAM secreting IL15/FlaB is a novel therapeutic candidate for bacterial-mediated cancer immunotherapy and that its antitumor activity is enhanced by combination with anti-PD-L1 antibody.
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Interleucina-15 , Neoplasias , Humanos , Animais , Camundongos , Interleucina-15/genética , Salmonella typhimurium , Neoplasias/terapia , Proteínas , Imunoterapia , Linhagem Celular TumoralRESUMO
Flagellin is the cognate ligand for host pattern recognition receptors, toll-like receptor 5 (TLR5) in the cell surface, and NAIP5/NLRC4 inflammasome in the cytosol. TLR5-binding domain is located in D1 domain, where crucial amino acid sequences are conserved among diverse bacteria. The highly conserved C-terminal 35 amino acids of flagellin were proved to be responsible for the inflammasome activation by binding to NAIP5. D2/D3 domains, located in the central region and exposed to the outside surface of flagellar filament, are heterogeneous across bacterial species and highly immunogenic. Taking advantage of TLR5- and NLRC4-stimulating activities, flagellin has been actively developed as a vaccine adjuvant and immunotherapeutic. Because of its immunogenicity, there exist worries concerning diminished efficacy and possible reactogenicity after repeated administration. Deimmunization of flagellin derivatives while preserving the TLR5/NLRC4-mediated immunomodulatory activity should be the most reasonable option for clinical application. This review describes strategies and current achievements in flagellin deimmunization.
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Inflamassomos , Receptor 5 Toll-Like , Receptor 5 Toll-Like/metabolismo , Imunidade Inata , Flagelina/genética , Flagelina/química , Bactérias/metabolismoRESUMO
Primary and adaptive resistance to immune checkpoint therapies (ICT) represent a considerable obstacle to achieving enhanced overall survival. Innate immune activators have been actively pursued for their antitumor potential. Herein we report that a syngeneic 4T1 mammary carcinoma murine model for established highly-refractory triple negative breast cancer showed enhanced survival when treated intra-tumorally with either the TLR5 agonist flagellin or CBLB502, a flagellin derivative, in combination with antibodies targeting CTLA-4 and PD-1. Long-term survivor mice showed immunologic memory upon tumor re-challenge and a distinctive immune activating cytokine profile that engaged both innate and adaptive immunity. Low serum levels of G-CSF and CXCL5 (as well as high IL-15) were candidate predictive biomarkers correlating with enhanced survival. CBLB502-induced enhancement of ICT was also observed in poorly immunogenic B16-F10 melanoma tumors. Combination immune checkpoint therapy plus TLR5 agonists may offer a new therapeutic strategy to treat ICT-refractory solid tumors.
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Melanoma Experimental , Receptor 5 Toll-Like , Animais , Camundongos , Imunidade Adaptativa , Citocinas , Flagelina/farmacologia , Melanoma Experimental/tratamento farmacológico , Receptor 5 Toll-Like/agonistasRESUMO
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.
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Neoplasias Encefálicas , Glioblastoma , Animais , Neoplasias Encefálicas/tratamento farmacológico , Caspase 3 , Modelos Animais de Doenças , Receptores ErbB/genética , Flagelina , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Camundongos , PeptídeosRESUMO
Background: 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. Methods: 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-SP49-57) and E7 long peptides (E7-LP2043-62 and E7-LP3543-77). Because combinations take center stage in immune checkpoint inhibitor (ICI) therapy, we evaluated the best E7 peptide vaccine component for combination with anti-PD-1 in the mouse TC-1 model. Results: Flagellin 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. Conclusions: This result suggests that cooperation between CD4+ and CD8+ cell-mediated immune responses is essential for the success of combination therapy with cancer vaccines and ICIs.
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Therapeutic cancer vaccines (TCVs) should induce robust tumor-specific T cell responses. To achieve this, TCVs incorporate T cell epitopes and strong adjuvants. Here, we report an all-in-one adjuvanted cancer vaccine platform that targets the intracellular compartment of dentritic cells and subsequently induces effective cytotoxic T cell responses. We screened a novel peptide (DCpep6) that specifically binds and transmits into CD11c+ cells through a novel in vivo phage biopanning. We then engineered a protein-based TCV (DEF) consisting of DCpep6 (D), an optimized HPV E7 tumor antigen (E), and a built-in flagellin adjuvant (F) as a single molecule. DEF was stably expressed, and each component was functional. In vivo-administered DEF rapidly biodistributed in draining LNs and internalized into CD11c+ cells. DEF immunization elicited strong antitumor T cell responses and provided long-term survival of TC-1 tumor-implanted mice. The DEF-mediated antitumor effect was abolished in NLRC4-/- mice. Taken together, we propose a protein-based all-in-one TCV platform that intracellularly codelivers tumor antigen and inflammasome activator to DCs to induce long-lasting antitumor T cell responses.
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Vacinas Anticâncer , Neoplasias , Adjuvantes Imunológicos/farmacologia , Animais , Antígenos de Neoplasias/metabolismo , Linfócitos T CD8-Positivos , Citosol , Células Dendríticas , Inflamassomos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/metabolismoRESUMO
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 ßIII-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.
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Células-Tronco Neurais , Receptor 5 Toll-Like , Animais , Proliferação de Células , Células-Tronco Embrionárias/metabolismo , Hipocampo , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Neurais/metabolismo , Neurogênese/fisiologia , Receptor 5 Toll-Like/metabolismoRESUMO
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 < 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 < 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.
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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.
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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.