Engineered oncolytic bacteria HCS1 exerts high immune stimulation and safety profiles for cancer therapy.

Background and rationale: Attenuated Salmonella typhimurium VNP20009 has been used to treat tumor-bearing mice and entered phase I clinical trials. However, its mild anticancer effect in clinical trials may be related to insufficient bacterial colonization and notable adverse effects with increasing dosages. Guanosine 5'-diphosphate-3'-diphosphate (ppGpp) synthesis-deficient Salmonella is an attenuated strain with good biosafety and anticancer efficacy that has been widely investigated in various solid cancers in preclinical studies. Integration of the advantages of these two strains may provide a new solution for oncolytic bacterial therapy. Methods: We incorporated the features of ΔppGpp into VNP20009 and obtained the HCS1 strain by deleting relA and spoT, and then assessed its cytotoxicity in vitro and antitumor activities in vivo. Results: In vitro experiments revealed that the invasiveness and cytotoxicity of HCS1 to cancer cells were significantly lower than those of the VNP20009. Additionally, tumor-bearing mice showed robust cancer suppression when treated with different doses of HCS1 intravenously, and the survival time and cured mice were dramatically increased. Furthermore, HCS1 can increase the levels of pro-inflammatory cytokines in tumor tissues and relieve the immunosuppression in the tumor microenvironments. It can also recruit abundant immune cells into tumor tissues, thereby increasing immune activation responses. Conclusion: The newly engineered Salmonella HCS1 strain manifests high prospects for cancer therapeutics and is a promising option for future clinical cancer immunotherapy.

Synergistic cancer immunotherapy utilizing programmed Salmonella typhimurium secreting heterologous flagellin B conjugated to interleukin-15 proteins.

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.

Photodynamic therapy-improved oncolytic bacterial immunotherapy with FAP-encoding S. typhimurium.

Genetically engineered bacterial cancer therapy presents several advantages over conventional therapies. However, the anticancer effects of bacterium-based therapies remain insufficient, and serious side effects may be incurred with the increase in therapeutic dosages. Photodynamic therapy (PDT) suppresses tumor growth by producing reactive oxygen species (ROS) through specific laser-activated photosensitizers (PSs). Tumor-specific PS delivery and activatable ROS generation are two critical aspects for PDT advancement. Here, we propose PDT-enhanced oncolytic bacterial immunotherapy (OBI) by using genetically engineered avirulent Salmonella expressing a fluorogen-activating protein (FAP). Upon binding to a fluorogen, FAP could be activated and generate fluorescence and ROS. The instant activation of persistent fluorescence was detected in tumors through bacterium-based imaging. In a colon cancer model, PDT-OBI showed an enhanced tumor inhibition effect and prolonged animal survival. Mechanically, PDT generated ROS, resulting in the killing of cancer cells and over-accumulated bacteria. The pathogen-associated molecular patterns and damage-associated molecular patterns released from the destroyed bacteria and cancer cells recruited and activated immune cells (macrophages, neutrophils, and dendritic cells), which released additional proinflammatory cytokines (TNF-α and IL-1ß); reduced anti-inflammatory cytokines (IL-10); and further enhanced immune cell infiltration in a positive-feedback manner, thus reducing bacterium-induced side effects and improving anticancer activities. This synergistic therapy has promising potential for cancer immunotherapy.

Circ_0002111 modulates the growth process of papillary thyroid carcinoma cells by targeting the miR-363-3p/HMGB1 axis.

Previous studies have suggested that circular RNAs (circRNAs) are engaged in the progression of papillary thyroid carcinoma (PTC). However, the mechanism of circ_0002111 in PTC is still unclear. In this study, quantitative real-time PCR was carried out to measure the expressions of circ_0002111, microRNAs (miRNAs) and high-mobility group box 1 (HMGB1). Immunohistochemistry assay and western blot were applied for the determination of protein levels. The assays of 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide and thymidine analog 5-ethynyl-2'-deoxyuridine were deployed to assess PTC cell viability and proliferation, respectively. Besides, the capacities of cell apoptosis, invasion and angiogenesis were determined by flow cytometry, transwell and tube formation assays, respectively. Moreover, the interaction between miR-363-3p and circ_0002111 or HMGB1 was confirmed using a dual-luciferase reporter assay. Lastly, we established a xenograft model for the examination of the function of circ_0002111 in vivo. It was found that the expression of circ_0002111 was enhanced in PTC tissues and cells. Silencing circ_0002111 apparently retarded the viability, proliferation, invasion and tube formation, as well as expedited the apoptosis of PTC cells. Besides, circ_0002111 knockdown impeded the growth of the tumor in vivo. For mechanism analysis, circ_0002111 adjusted the expression of HMGB1 by sponge adsorption of miR-363-3p. Moreover, miR-363-3p inhibitor regained the influence of cellular malignant phenotype caused by circ_0002111 knockdown. Additionally, miR-363-3p overexpression impacted the cell functions by targeting HMGB1 in PTC. Thus, silencing circ_0002111 constrained the progression of PTC by the miR-363-3p/HMGB1 axis, which perhaps provided a novel idea of the therapeutic in PTC.

Comparison of Anticancer Activities and Biosafety Between Salmonella enterica Serovar Typhimurium ΔppGpp and VNP20009 in a Murine Cancer Model.

Salmonella Typhimurium defective in guanosine 5'-diphosphate-3'-diphosphate (ppGpp) synthesis (ΔppGpp) is an attenuated strain with good biosafety and excellent anticancer efficacy. It has been widely applied in preclinical studies of anticancer therapy for various types of solid cancer. VNP20009 is another genetically modified auxotrophic strain with 108-kb deletion, purI- , msbB- , and many single nucleotide polymorphisms (SNPs); it has shown promising therapeutic efficacy in various preclinical tumor models and entered phase I clinical trials. Here, the invasion activities and virulence of ΔppGpp were obviously lower than those of the VNP20009 strain when tested with cancer cells in vitro. In addition, the MC38 tumor-bearing mice showed comparable cancer suppression when treated with ΔppGpp or VNP20009 intravenously. However, the ΔppGpp-treated mice showed 16.7% of complete cancer eradication and prolonged survival in mice, whereas VNP20009 showed higher toxicity to animals, even with equal tumor size individually. Moreover, we found decreased levels of inflammatory cytokines in circulation but strengthened immune boost in tumor microenvironments of ΔppGpp-treated mice. Therefore, the engineered ΔppGpp has high potential for cancer therapeutics, and it is a promising option for future clinical cancer therapy.

An all-in-one adjuvanted therapeutic cancer vaccine targeting dendritic cell cytosol induces long-lived tumor suppression through NLRC4 inflammasome activation.

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.

Targeting of pancreatic cancer cells and stromal cells using engineered oncolytic Salmonella typhimurium.

Pancreatic cancer is resistant to conventional therapeutic interventions, mainly due to abundant cancer stromal cells and poor immune cell infiltration. Here, we used a targeted cancer therapy approach based on attenuated Salmonella typhimurium engineered to express cytolysin A (ClyA) to target cancer stromal cells and cancer cells and treat pancreatic cancer in mice. Nude mice bearing subcutaneous or orthotopic human pancreatic cancers were treated with engineered S. typhimurium expressing ClyA. The tumor microenvironment was monitored to analyze stromal cell numbers, stromal cell marker expression, and immune cell infiltration. The attenuated bacteria accumulated and proliferated specifically in tumor tissues after intravenous injection. The bacteria secreted ClyA into the tumor microenvironment. A single dose of ClyA-expressing Salmonella markedly inhibited growth of pancreatic cancer both in subcutaneous xenograft- and orthotopic tumor-bearing nude mice. Histological analysis revealed a marked decrease in expression of stromal cell markers and increased immune cell (neutrophils and macrophages) infiltration into tumors after colonization by ClyA-expressing bacteria. ClyA-expressing S. typhimurium destroyed cancer stromal cells and cancer cells in mouse models of human pancreatic cancer. This approach provides a novel strategy for combining anticancer and anti-stromal therapy to treat pancreatic cancer.

Genetically engineered oncolytic bacteria as drug delivery systems for targeted cancer theranostics.

Drug delivery systems based on genetically engineered oncolytic bacteria have properties that cannot be achieved by traditional therapeutic interventions. Thus, they have attracted considerable attention in cancer therapies. Attenuated bacteria can specifically target and actively penetrate tumor tissues and play an important role in cancer suppression as the "factories" of diverse anticancer drugs. Over the past decades, several bacterial strains including Salmonella and Clostridium have been shown to effectively retard tumor growth and metastasis, and thus improve survival in preclinical models or clinical cases. In this review, we summarize the unique properties of oncolytic bacteria and their anticancer mechanisms and highlight the particular advantages compared with traditional strategies. With the current research progress, we demonstrate the potential value of oncolytic bacteria-based drug delivery systems for clinical applications. In addition, we discuss novel strategies of cancer therapies integrating oncolytic bacteria, which will provide hope to further improve and standardize the current regimens in the near future.

Targeted cancer immunotherapy with genetically engineered oncolytic Salmonella typhimurium.

Conventional chemotherapies have some limitations, including the lack of selectivity, high toxicity to normal tissues, multidrug resistance, and tumor relapse. Recently, great progress was made in immunotherapies for anticancer research, with bacteria-mediated cancer therapy one of the most promising approaches among them. Attenuated Salmonella have very specific targeting to various solid cancers, making them ideal vectors for the delivery and expression of immunostimulators. They have native bacterial immunogenicity and induce strong anticancer immunity in vivo. In this review, the recent advances in Salmonella-mediated cancer immunotherapies and the related mechanisms of Salmonella-based cancer therapies are summarized.

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.

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

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

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.

Inhibition of the ubiquitination of HSF1 by FBXW7 protects the intestine against ischemia-reperfusion injury.

Epithelial apoptosis is an important factor in intestinal ischemia-reperfusion (I/R) injury. Heat shock factor 1 (HSF1) is a classical stress response factor that directly regulates the transcription of heat shock proteins (HSPs) under stress conditions. Although HSPs are involved in protecting the intestine against I/R, the mechanism whereby HSF1 is regulated in I/R is poorly understood. Here, we show that the ubiquitin ligase FBXW7 targets HSF1 for ubiquitination and degradation in intestinal I/R. In this study, we found that FBXW7 expression was upregulated at the transcriptional level in intestinal mucosae subjected to I/R. In Caco-2 and IEC-6 cells subjected to hypoxia/reoxygenation (H/R), a high FBXW7 level led to excessive HSF1 ubiquitination and degradation. FBXW7 knockdown attenuated HSF1 ubiquitination and downregulation and accelerated HSPB1 and HSP70 expression. In addition, FBXW7 deletion alleviated the apoptosis of intestinal epithelial cells, as evidenced by decreased activation of caspase-3 and caspase-9. The results suggest that FBXW7 suppression protects against intestinal I/R, at least partly through the HSF1/HSP pathway. These findings indicate that FBXW7 may be a potential therapeutic target for inhibiting intestinal mucosa apoptosis during intestinal I/R.

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.

PKCζ phosphorylates TRAF2 to protect against intestinal ischemia-reperfusion-induced injury.

Intestinal ischemia-reperfusion (I/R) is a common clinical problem that occurs during various clinical pathological processes. Excessive apoptosis has an indispensable role in intestinal I/R injury. Tumor necrosis factor receptor-associated factor 2 (TRAF2) and PKCζ have an essential role in apoptosis. Here, we aimed to investigate the effects of PKCζ and TRAF2 and to explore the correlation between PKCζ and TRAF2 in intestinal I/R injury. Mice were subjected to intestinal I/R injury in vivo. In vitro experiments were conducted by treating Caco-2 cells with hypoxia/reoxygenation (H/R) stimulation to simulate intestinal I/R. Intestinal tissue samples and Caco-2 cells were examined using various approaches. Intestinal I/R induced the membrane translocation and phosphorylation of PKCζ. Pretreatment with the PKCζ activator phosphatidylcholine remarkably attenuated gut injury by suppressing apoptosis. H/R induced PKCζ to combine with TRAF2, which was phosphorylated by PKCζ at Ser55, but not at Ser11, under intestinal I/R or H/R conditions. In addition, TRAF2 Ser55 phosphorylation increased cell survival by inhibiting cell apoptosis in the H/R model. Mechanistically, TRAF2 Ser55 phosphorylation promoted NF-κB activation but suppressed c-Jun activation in Caco-2 cells under H/R conditions. The results of this study demonstrate that the PKCζ/TRAF2 pathway represents a novel protective mechanism against intestinal I/R injury. Therefore, the PKCζ/TRAF2 pathway is a novel target for potential treatments of intestinal I/R injury-related diseases.

Two-step enhanced cancer immunotherapy with engineered Salmonella typhimurium secreting heterologous flagellin.

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.

Norovirus (NoV) specific protective immune responses induced by recombinant P dimer vaccine are enhanced by the mucosal adjuvant FlaB.

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.

Antifungal drug itraconazole targets VDAC1 to modulate the AMPK/mTOR signaling axis in endothelial cells.

Itraconazole, a clinically used antifungal drug, was found to possess potent antiangiogenic and anticancer activity that is unique among the azole antifungals. Previous mechanistic studies have shown that itraconazole inhibits the mechanistic target of rapamycin (mTOR) signaling pathway, which is known to be a critical regulator of endothelial cell function and angiogenesis. However, the molecular target of itraconazole that mediates this activity has remained unknown. Here we identify the major target of itraconazole in endothelial cells as the mitochondrial protein voltage-dependent anion channel 1 (VDAC1), which regulates mitochondrial metabolism by controlling the passage of ions and small metabolites through the outer mitochondrial membrane. VDAC1 knockdown profoundly inhibits mTOR activity and cell proliferation in human umbilical vein cells (HUVEC), uncovering a previously unknown connection between VDAC1 and mTOR. Inhibition of VDAC1 by itraconazole disrupts mitochondrial metabolism, leading to an increase in the cellular AMP:ATP ratio and activation of the AMP-activated protein kinase (AMPK), an upstream regulator of mTOR. VDAC1-knockout cells are resistant to AMPK activation and mTOR inhibition by itraconazole, demonstrating that VDAC1 is the mediator of this activity. In addition, another known VDAC-targeting compound, erastin, also activates AMPK and inhibits mTOR and proliferation in HUVEC. VDAC1 thus represents a novel upstream regulator of mTOR signaling in endothelial cells and a promising target for the development of angiogenesis inhibitors.

Antisense proline-arginine RAN dipeptides linked to C9ORF72-ALS/FTD form toxic nuclear aggregates that initiate in vitro and in vivo neuronal death.

Expanded GGGGCC (G4C2) nucleotide repeats within the C9ORF72 gene are the most common genetic mutation associated with both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Sense and antisense transcripts of these expansions are translated to form five dipeptide repeat proteins (DRPs). We employed primary cortical and motor neuron cultures, live-cell imaging, and transgenic fly models and found that the arginine-rich dipeptides, in particular Proline-Arginine (PR), are potently neurotoxic. Factors that anticipated their neurotoxicity included aggregation in nucleoli, decreased number of processing bodies, and stress granule formation, implying global translational dysregulation as path accountable for toxicity. Nuclear PR aggregates were also found in human induced motor neurons and postmortem spinal cord tissues from C9ORF72 ALS and ALS/FTD patients. Intronic G4C2 transcripts, but not loss of C9ORF72 protein, are also toxic to motor and cortical neurons. Interestingly, G4C2 transcript-mediated neurotoxicity synergizes with that of PR aggregates, suggesting convergence of mechanisms.