A flagellin-conjugate protein induces dual NLRC4- and NLRP3-inflammasome activation which modulates inflammatory cytokine secretion from macrophages.

Background: A recombinant fusion protein combining the adjuvant and TLR5-ligand flagellin with the major birch pollen allergen Bet v 1 (rFlaA:Betv1) has been suggested to prevent the manifestation of birch allergy. Noteworthy, rFlaA:Betv1 induced both pro- and anti-inflammatory responses which were differentially regulated. However, the mechanism by which flagellin fusion proteins modulate allergen-specific immune responses, especially the mechanisms underlying IL-1ß secretion and their contribution to the overall immune responses remains elusive. Objective: To investigate the mechanisms underlying the production of IL-1ß from rFlaA:Betv1 stimulated macrophages. Methods: Macrophages were derived from mouse peritoneal-, human buffy-coat-, and PMA-differentiated THP-1 (wild type or lacking either ASC, NLRP3, or NLRC4) cells. Macrophages were stimulated with non-modified rFlaA:Betv1, mutant variants lacking either the flagellin DC0 domain or a sequence motif formerly described to mediate TLR5-activation, and respective controls in the presence or absence of inhibitors interfering with MAPK- and NFκB-signaling. Cytokine secretion was analyzed by ELISA and intracellular signaling by Western Blot. To study the contribution of IL-1ß to the overall immune responses, IL1R-deficient mouse peritoneal macrophages were used. Results: rFlaA:Betv1 consistently activated all types of investigated macrophages, inducing higher IL-1ß secretion compared with the equimolar mixture of both proteins. rFlaA:Betv1-induced activation of THP-1 macrophages was shown to be independent of either the TLR5-activating sequence motif or the flagellin DC0 domain but depended on both NLRP3- and NLRC4-inflammasomes. In addition, NFκB and SAP/JNK MAP kinases regulated rFlaA:Betv1-induced inflammasome activation and cytokine secretion by modulating pro-Caspase-1- and pro-IL-1ß-expression in THP-1 macrophages. Finally, lack of IL-1ß positive feedback via the IL1R strongly diminished the rFlaA:Betv1-induced secretion of IL-1ß, IL-6, and TNF-α from peritoneal macrophages. Conclusion: The mechanisms contributing to rFlaA:Betv1-induced IL-1ß secretion from macrophages were shown to be complex, involving both NLRC4- and NLRP3-inflammsomes, as well as NFκB- and SAP/JNK MAP kinase-signaling. Better understanding the mechanisms regulating the activation of immune cells by novel therapeutic candidates like the rFlaA:Betv1 fusion protein will allow us to further improve and develop new treatment strategies when using flagellin as an adjuvant.

Stimulation of naïve B cells with a fusion protein consisting of FlaA and Bet v 1 induces regulatory B cells ex vivo.

BACKGROUND: The experimental fusion protein rFlaA:Betv1 was shown to efficiently suppress allergen-specific sensitization in mice. However, the detailed mechanism of rFlaA:Betv1-mediated immune modulation is not fully understood. In this study, we investigated the effect of rFlaA:Betv1 on naïve murine B cells. METHODS: Immune modulating capacity of rFlaA:Betv1 was screened in IL-10 reporter mice. B cells were isolated from spleens of naïve C57Bl/6, TLR5-/- , or MyD88-/- mice, stimulated with rFlaA:Betv1 and controls, and monitored for the expression of the regulatory B cell markers CD1d, CD24, CD38, and surface IgM by flow cytometry. Secreted cytokines, antibodies, and reactivity of the induced antibodies were investigated by ELISA and intracellular flow cytometry. Suppressive capacity of rFlaA:Betv1-stimulated B cells was tested in mDC:CD4+ T cell:B cell triple cultures. RESULTS: Upon in vivo application of rFlaA:Betv1 into IL-10-GFP reporter mice, CD19+ B cells were shown to produce anti-inflammatory IL-10, suggesting B cells to contribute to the immune-modulatory properties of rFlaA:Betv1. rFlaA:Betv1-induced IL-10 secretion was confirmed in human B cells isolated from buffy coats. In vitro stimulation of naïve murine B cells with rFlaA:Betv1 resulted in an mTOR- and MyD88-dependent production of IL-10 and rFlaA:Betv1 induced Bet v 1-reactive IgG production, which was not observed for IgA. rFlaA:Betv1-stimulated B cells formed a CD19+ CD24+ CD1d+ IgM+ CD38+ Breg subpopulation capable of suppressing Bet v 1-induced TH2 cytokine secretion in vitro. CONCLUSION: rFlaA:Betv1 can act as a thymus-independent B cell antigen, stimulating the mTOR- and MyD88-dependent differentiation of B cells displaying a regulatory phenotype, IL-10 secretion, antigen-binding antibody production, and a suppressive capacity in vitro.

Role of Glycolysis and Fatty Acid Synthesis in the Activation and T Cell-Modulating Potential of Dendritic Cells Stimulated with a TLR5-Ligand Allergen Fusion Protein.

Trained immune responses, based on metabolic and epigenetic changes in innate immune cells, are de facto innate immune memory and, therefore, are of great interest in vaccine development. In previous studies, the recombinant fusion protein rFlaA:Betv1, combining the adjuvant and toll-like receptor (TLR)5-ligand flagellin (FlaA) and the major birch pollen allergen Bet v 1 into a single molecule, significantly suppressed allergic sensitization in vivo while also changing the metabolism of myeloid dendritic cells (mDCs). Within this study, the immune-metabolic effects of rFlaA:Betv1 during mDC activation were elucidated. In line with results for other well-characterized TLR-ligands, rFlaA:Betv1 increased glycolysis while suppressing oxidative phosphorylation to different extents, making rFlaA:Betv1 a suitable model to study the immune-metabolic effects of TLR-adjuvanted vaccines. In vitro pretreatment of mDCs with cerulenin (inhibitor of fatty acid biosynthesis) led to a decrease in both rFlaA:Betv1-induced anti-inflammatory cytokine Interleukin (IL) 10 and T helper cell type (TH) 1-related cytokine IL-12p70, while the pro-inflammatory cytokine IL 1ß was unaffected. Interestingly, pretreatment with the glutaminase inhibitor BPTES resulted in an increase in IL-1ß, but decreased IL-12p70 secretion while leaving IL-10 unchanged. Inhibition of the glycolytic enzyme hexokinase-2 by 2-deoxyglucose led to a decrease in all investigated cytokines (IL-10, IL-12p70, and IL-1ß). Inhibitors of mitochondrial respiration had no effect on rFlaA:Betv1-induced IL-10 level, but either enhanced the secretion of IL-1ß (oligomycin) or decreased IL-12p70 (antimycin A). In extracellular flux measurements, mDCs showed a strongly enhanced glycolysis after rFlaA:Betv1 stimulation, which was slightly increased after respiratory shutdown using antimycin A. rFlaA:Betv1-stimulated mDCs secreted directly antimicrobial substances in a mTOR- and fatty acid metabolism-dependent manner. In co-cultures of rFlaA:Betv1-stimulated mDCs with CD4+ T cells, the suppression of Bet v 1-specific TH2 responses was shown to depend on fatty acid synthesis. The effector function of rFlaA:Betv1-activated mDCs mainly relies on glycolysis, with fatty acid synthesis also significantly contributing to rFlaA:Betv1-mediated cytokine secretion, the production of antimicrobial molecules, and the modulation of T cell responses.

Modulation of dendritic cell metabolism by an MPLA-adjuvanted allergen product for specific immunotherapy.

Background: Recently, bacterial components were shown to enhance immune responses by shifting immune cell metabolism towards glycolysis and lactic acid production, also known as the Warburg Effect. Currently, the effect of allergen products for immunotherapy (AIT) and commercial vaccines on immune cell metabolism is mostly unknown. Objective: To investigate the effect of AIT products (adjuvanted with either MPLA or Alum) on myeloid dendritic cell (mDC) metabolism and activation. Methods: Bone marrow-derived mDCs were stimulated with five allergoid-based AIT products (one adjuvanted with MPLA, four adjuvanted with Alum) and two MPLA-adjuvanted vaccines and analyzed for their metabolic activation, expression of cell surface markers, and cytokine secretion by ELISA. mDCs were pre-incubated with either immunological or metabolic inhibitors or cultured in glucose- or glutamine-free culture media and subsequently stimulated with the MPLA-containing AIT product (AIT product 1). mDCs were co-cultured with allergen-specific CD4+ T cells to investigate the contribution of metabolic pathways to the T cell priming capacity of mDCs stimulated with AIT product 1. Results: Both the MPLA-containing AIT product 1 and commercial vaccines, but not the Alum-adjuvanted AIT products, activated Warburg metabolism and TNF-α secretion in mDCs. Further experiments focused on AIT product 1. Metabolic analysis showed that AIT product 1 increased glycolytic activity while also inducing the secretion of IL-1ß, IL-10, IL-12, and TNF-α. Both rapamycin (mTOR-inhibitor) and SP600125 (SAP/JNK MAPK-inhibitor) dose-dependently suppressed the AIT product 1-induced Warburg Effect, glucose consumption, IL-10-, and TNF-α secretion. Moreover, both glucose- and glutamine deficiency suppressed secretion of all investigated cytokines (IL-1ß, IL-10, and TNF-α). Glucose metabolism in mDCs was also critical for the (Th1-biased) T cell priming capacity of AIT product 1-stimulated mDCs, as inhibition of mTOR signaling abrogated their ability to induce Th1-responses. Conclusion: The AIT product and commercial vaccines containing the adjuvant MPLA were shown to modulate the induction of immune responses by changing the metabolic state of mDCs. Better understanding the mechanisms underlying the interactions between cell metabolism and immune responses will allow us to further improve vaccine development and AIT.

The Fusion Protein rFlaA:Betv1 Modulates DC Responses by a p38-MAPK and COX2-Dependent Secretion of PGE2 from Epithelial Cells.

Developing new adjuvants/vaccines and better understanding their mode-of-action is an important task. To specifically improve birch pollen allergy treatment, we designed a fusion protein consisting of major birch pollen allergen Betv1 conjugated to the TLR5-ligand flagellin (rFlaA:Betv1). This study investigates the immune-modulatory effects of rFlaA:Betv1 on airway epithelial cells. LA-4 mouse lung epithelial cells were stimulated with rFlaA:Betv1 in the presence/absence of various inhibitors with cytokine- and chemokine secretion quantified by ELISA and activation of intracellular signaling cascades demonstrated by Western blot (WB). Either LA-4 cells or LA-4-derived supernatants were co-cultured with BALB/c bone marrow-derived myeloid dendritic cells (mDCs). Compared to equimolar amounts of flagellin and Betv1 provided as a mixture, rFlaA:Betv1 induced higher secretion of IL-6 and the chemokines CCL2 and CCL20 from LA-4 cells and a pronounced MAPK- and NFκB-activation. Mechanistically, rFlaA:Betv1 was taken up more strongly and the induced cytokine production was inhibited by NFκB-inhibitors, while ERK- and p38-MAPK-inhibitors only suppressed IL-6 and CCL2 secretion. In co-cultures of LA-4 cells with mDCs, rFlaA:Betv1-stimulated LA-4 cells p38-MAPK- and COX2-dependently secreted PGE2, which modulated DC responses by suppressing pro-inflammatory IL-12 and TNF-α secretion. Taken together, these results contribute to our understanding of the mechanisms underlying the strong immune-modulatory effects of flagellin-containing fusion proteins.

The Flagellin:Allergen Fusion Protein rFlaA:Betv1 Induces a MyD88- and MAPK-Dependent Activation of Glucose Metabolism in Macrophages.

TLR5 ligand flagellin-containing fusion proteins are potential vaccine candidates for many diseases. A recombinant fusion protein of flagellin A and the major birch pollen allergen Bet v 1 (rFlaA:Betv1) modulates immune responses in vitro and in vivo. We studied the effects of rFlaA:Betv1 on bone marrow-derived macrophages (BMDMs). BMDMs differentiated from BALB/c, C57BL/6, TLR5-/-, or MyD88-/- mice were pre-treated with inhibitors, stimulated with rFlaA:Betv1 or respective controls, and analyzed for activation, cytokine secretion, metabolic state, RNA transcriptome, and modulation of allergen-specific Th2 responses. Stimulation of BMDMs with rFlaA:Betv1 resulted in MyD88-dependent production of IL-1ß, IL-6, TNF-α, IL-10, CD69 upregulation, and a pronounced shift towards glycolysis paralleled by activation of MAPK, NFκB, and mTOR signaling. Inhibition of either mTOR (rapamycin) or SAP/JNK-MAPK signaling (SP600125) resulted in dose-dependent metabolic suppression. In BMDM and T cell co-cultures, rFlaA:Betv1 stimulation suppressed rBet v 1-induced IL-5 and IL-13 secretion while inducing IFN-γ production. mRNA-Seq analyses showed HIF-1a, JAK, STAT, phagosome, NLR, NFκB, TNF, TLR, and chemokine signaling to participate in the interplay of cell activation, glycolysis, and immune response. rFlaA:Betv1 strongly activated BMDMs, resulting in MyD88-, MAPK-, and mTOR-dependent enhancement of glucose metabolism. Our results suggest macrophages are important target cells to consider during restauration of allergen tolerance during AIT.

Organ-Specific Expression of IL-1 Receptor Results in Severe Liver Injury in Type I Interferon Receptor Deficient Mice.

Upon treatment with polyinosinic:polycytidylic acid [poly(I:C)], an artificial double-stranded RNA, type I interferon receptor-deficient (IFNAR-/-) mice develop severe liver injury seen by enhanced alanine aminotransferase (ALT) activity in the serum that is not observed in their wildtype (WT) counterparts. Recently, we showed that liver injury is mediated by an imbalanced expression of interleukin (IL)-1ß and its receptor antagonist (IL1-RA) in the absence of type I IFN. Here we show that despite comparable expression levels of IL-1ß in livers and spleens, spleens of poly(I:C)-treated IFNAR-/- mice show no signs of injury. In vitro analyses of hepatocytes and splenocytes revealed that poly(I:C) had no direct toxic effect on hepatocytes. Furthermore, expression levels of cytokines involved in other models for liver damage or protection such as interferon (IFN)-γ, transforming growth factor (TGF)-ß, IL-6, IL-10, IL-17, and IL-22 were comparable for both organs in WT and IFNAR-/- mice upon treatment. Moreover, flow cytometric analyses showed that the composition of different immune cells in livers and spleens were not altered upon injection of poly(I:C). Finally, we demonstrated that the receptor binding IL-1ß, IL1R1, is specifically expressed in livers but not spleens of WT and IFNAR-/- mice. Accordingly, mice double-deficient for IFNAR and IL1R1 developed no liver injury upon poly(I:C) treatment and showed ALT activities comparable to those of WT mice. Collectively, liver injury is mediated by the organ-specific expression of IL1R1 in the liver.

NFκB- and MAP-Kinase Signaling Contribute to the Activation of Murine Myeloid Dendritic Cells by a Flagellin A:Allergen Fusion Protein.

Fusion proteins incorporating the TLR5-ligand flagellin are currently undergoing clinical trials as vaccine candidates for many diseases. We recently reported a flagellin:allergen fusion protein containing the TLR5-ligand flagellin A (FlaA) from Listeriamonocytogenes and the major birch pollen allergen Bet v 1 (rFlaA:Betv1) to prevent allergic sensitization in an experimental mouse model. This study analyzes the signaling pathways contributing to rFlaA:Betv1-mediated pro- and anti-inflammatory cytokine secretion and cell metabolism in myeloid dendritic cells (mDCs) in vitro. The influence of mammalian target of rapamycin (mTOR)-, NF?B-, and MAP kinase (MAPK)-signaling on cytokine secretion and metabolic activity of bone marrow (BM)-derived mDCs stimulated with rFlaA:Betv1 were investigated by pre-treatment with either mTOR- (rapamycin), NF?B- (dexamethason, BMS-345541, TPCA-1, triptolide, or BAY-11) or MAPK- (SP600125, U0126, or SB202190) inhibitors, respectively. rFlaA:Betv1-mediated IL-10 secretion as well as activation of mDC metabolism, rather than pro-inflammatory cytokine secretion, were inhibited by rapamycin. Inhibition of NFκB-signaling suppressed rFlaA:Betv1-induced IL-12, while inhibition of MAPK-signaling dose-dependently suppressed rFlaA:Betv1-induced IL-10 as well as pro-inflammatory IL-6 and TNF-α production. Notably, with the exception of a partial JNK-dependency, rFlaA:Betv1-mediated effects on mDC metabolism were mostly NF?B- and MAPK-independent. Therefore, MAPK-mediated activation of both NFκB- and mTOR-signaling likely is a key pathway for the production of pro- and anti-inflammatory cytokines by flagellin fusion protein vaccines.

The vaccine adjuvant MPLA activates glycolytic metabolism in mouse mDC by a JNK-dependent activation of mTOR-signaling.

INTRODUCTION: The detoxified TLR4-ligand MPLA is a successfully used adjuvant in clinically approved vaccines. However, its capacity to activate glycolytic metabolism in mDC and the influence of MPLA-induced metabolic changes on cytokine secretion are unknown. AIM: To analyze the capacity of MPLA to activate mDC metabolism and the mechanisms contributing to MPLA-induced metabolism activation and cytokine secretion. METHODS: C57BL/6 bone-marrow-derived myeloid dendritic cells (mDCs) were stimulated with LPS or MPLA and analyzed for intracellular signaling, cytokine secretion, and metabolic state. mDC were pre-treated with rapamycin (mTOR-inhibitor), U0126, SP600125, SB202190 (MAPK kinase inhibitors), as well as dexamethasone (MAPK- and NFκB-inhibitor) and analyzed for MPLA-induced cytokine secretion and cell metabolic state. RESULTS: Stimulation of mDCs with either LPS or MPLA resulted in a pronounced, mTOR-dependent activation of glucose metabolism characterized by induction of the Warburg Effect, increased glucose consumption from the culture medium, as well as release of LDH. Compared to LPS, MPLA induced significantly lower cytokine secretion. The activation of mDC metabolism was comparable between LPS- and MPLA-stimulated mDCs. The MPLA-induced cytokine secretion could be partially inhibited using mTOR-, MAP kinase-, and NFκB-inhibitors, whereas the activation of glucose metabolism was shown to depend on both mTOR- and JNK-signaling. SUMMARY: The MPLA-induced activation of glycolytic metabolism in mouse mDC was shown to depend on a JNK-mediated activation of mTOR-signaling, while both MAPK- and NFB-signaling contributed to pro-inflammatory cytokine secretion. Understanding the mechanisms by which MPLA activates dendritic cells will both improve our understanding of its adjuvant properties and contribute to the future development and safe application of this promising adjuvant.

Author Correction: Conjugation of wildtype and hypoallergenic mugwort allergen Art v 1 to flagellin induces IL-10-DC and suppresses allergen-specific TH2-responses in vivo.

A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.

Critical role of mammalian target of rapamycin for IL-10 dendritic cell induction by a flagellin A conjugate in preventing allergic sensitization.

BACKGROUND: Fusion proteins incorporating the Toll-like receptor 5 ligand flagellin are currently undergoing clinical trials as vaccine candidates for many diseases. OBJECTIVE: We studied the mechanisms of immune modulation by a flagellin:allergen fusion protein containing the Toll-like receptor 5 ligand flagellin A from Listeria monocytogenes and the birch pollen allergen Bet v 1 (recombinant flagellin A [rFlaA]:Betv1). METHODS: BALB/c mice were vaccinated with rFlaA:Betv1 in an experimental Bet v 1 sensitization model. Myeloid dendritic cells (mDCs) were differentiated from mouse bone marrow, and PBMCs were isolated from subjects with birch pollen allergy. Cells were stimulated with equimolar amounts of rFlaA, rBet v 1, rFlaA plus rBet v 1, or the rFlaA:Betv1 conjugate and analyzed for cell activation, cytokine secretion, and metabolic state. RESULTS: rFlaA:Betv1 displayed strong immune-modulating properties both in vivo and in vitro, as characterized by secretion of both proinflammatory and anti-inflammatory cytokines from murine mDCs and PBMCs from patients with birch allergy. rFlaA:Betv1 suppressed TH2 responses from Bet v 1-specific CD4+ T cells and prevented allergic sensitization in a mouse allergy model. Aggregation of rFlaA:Betv1 resulted in stronger protein uptake accompanied by an increased resistance to microsomal digestion. Remarkably, rFlaA:Betv1 induced activation of mammalian target of rapamycin, which increased the metabolic activity of the stimulated mDCs. rFlaA:Betv1-mediated IL-10 secretion, but not proinflammatory cytokine secretion, was inhibited by rapamycin in mDCs. CONCLUSION: These results provide evidence that mammalian target of rapamycin is a key player involved in prevention of TH2 responses by flagellin A conjugate vaccines.

Targeting of Immune Cells by Dual TLR2/7 Ligands Suppresses Features of Allergic Th2 Immune Responses in Mice.

BACKGROUND: TLR ligands can promote Th1-biased immune responses, mimicking potent stimuli of viruses and bacteria. AIM: To investigate the adjuvant properties of dual TLR2/7 ligands compared to those of the mixture of both single ligands. METHODS: Dual TLR2/7 ligands: CL401, CL413, and CL531, including CL264 (TLR7-ligand) and Pam2CysK4 (TLR2-ligand), were used. Immune-modulatory capacity of the dual ligands with the individual ligands alone or as a mixture in mouse BMmDCs, BMmDC:TC cocultures, or BMCMCs was compared and assessed in naïve mice and in a mouse model of OVA-induced intestinal allergy. RESULTS: CL413 and CL531 induced BMmDC-derived IL-10 secretion, suppressed rOVA-induced IL-5 secretion from OVA-specific DO11.10 CD4+ TCs, and induced proinflammatory cytokine secretion in vivo. In contrast, CL401 induced considerably less IL-10 secretion and led to IL-17A production in BMmDC:TC cocultures, but not BMCMC IL-6 secretion, or IL-6 or TNF-α production in vivo. No immune-modulating effects were observed with single ligands. All dual TLR2/7 ligands suppressed DNP-induced IgE-and-Ag-specific mast cell degranulation. Compared to vaccination with OVA, vaccination with the mixture CL531 and OVA, significantly suppressed OVA-specific IgE production in the intestinal allergy model. CONCLUSIONS: Based on beneficial immune-modulating properties, CL413 and CL531 may have utility as potential adjuvants for allergy treatment.

Conjugation of wildtype and hypoallergenic mugwort allergen Art v 1 to flagellin induces IL-10-DC and suppresses allergen-specific TH2-responses in vivo.

Allergies to weed pollen including members of the Compositae family, such as mugwort, ragweed, and feverfew are spreading worldwide. To efficiently treat these newly arising allergies, allergen specific immunotherapy needs to be improved. Therefore, we generated novel vaccine candidates consisting of the TLR5-ligand Flagellin A from Listeria and the major mugwort allergen Art v 1 including either the wild type Art v 1 sequence (rFlaA:Artv1) or a hypoallergenic variant (rFlaA:Artv1hyp) with reduced IgE-binding capacity. Immune modulating capacity of these constructs and respective controls was evaluated in vitro and in vivo. Incorporation of hypoallergenic Art v 1 derivative did not interfere with the resulting fusion proteins' immune stimulatory capacity. Both rFlaA:Artv1 and rFlaA:Artv1hyp induced a prominent, mTOR-dependent, IL-10 secretion from murine dendritic cells, and suppressed allergen-specific TH2-cytokine secretion in vitro and in vivo. Both conjugates retained the capacity to induce rFlaA-specific antibody responses while efficiently inducing production of Art v 1-specific IgG1 and IgG2a antibodies in mice. Interestingly, only the suppression of TH2-cytokine secretion by rFlaA:Artv1 (but not rFlaA:Artv1hyp) was paralleled by a strong secretion of IFN-γ. In summary, we provided evidence that incorporating hypoallergens into flagellin:allergen fusion proteins is a suitable strategy to further improve these promising vaccine candidates.

Prevention of intestinal allergy in mice by rflaA:Ova is associated with enforced antigen processing and TLR5-dependent IL-10 secretion by mDC.

Conjugated vaccines consisting of flagellin and antigen activate TLR5 and induce strong innate and adaptive immune responses. Objective of the present study was to gain further insight into the mechanisms by which flagellin fusion proteins mediate their immune modulating effects. In a mouse model of Ova-induced intestinal allergy a fusion protein of flagellin and Ova (rflaA:Ova) was used for intranasal and intraperitoneal vaccination. Aggregation status of flaA, Ova and flaA:Ova were compared by light scattering, uptake of fluorescence labeled proteins into mDC was analyzed, processing was investigated by microsomal digestion experiments. Mechanism of DC-activation was investigated using proteasome and inflammasome inhibitors. Immune responses of wildtype, IL-10(-/-), TLR5(-/-) mDCs and Ova-transgenic T cells were investigated. Mucosal and i.p.-application of rflaA:Ova were able to prevent allergic sensitization, suppress disease-related symptoms, prevent body weight loss and reduction in food uptake. Intranasal vaccination resulted in strongest suppression of Ova-specific IgE production. These protective effects were associated with increased aggregation of rflaA:Ova and accompanied by tenfold higher uptake rates into mDC compared to the mixture of both proteins. Microsomal digestion showed that stimulation with rflaA:Ova resulted in faster degradation and the generation of different peptides compared to rOva. rflaA:Ova-mediated activation of mDC could be suppressed in a dose-dependent manner by the application of both inflammasome and proteasome inhibitors. Using TLR5(-/-) mDC the rflaA:Ova induced IL-10 secretion was shown to be TLR5 dependent. In co-cultures of IL-10(-/-) mDC with DO11.10 T cells the lack of rflaA:Ova-mediated IL-10 secretion resulted in enhanced levels of both TH2 (IL-4, IL-5) and TH1 (IL-2 and IFN-y) cytokines. In summary, mucosal vaccination with flaA:Ova showed strongest preventive effect. Stimulation with rflaA:Ova results in strong immune modulation mediated by enhanced uptake of the aggregated fusion protein, likely resulting in a different processing by DC as well as stronger TLR5 mediated cell activation.

A fusion protein of flagellin and ovalbumin suppresses the TH2 response and prevents murine intestinal allergy.

BACKGROUND: The Toll-like receptor (TLR) 5 agonist flagellin is associated with immunomodulatory functions. OBJECTIVE: We sought to investigate whether Listeria monocytogenes-derived flagellin A (flaA) can modulate ovalbumin (OVA)-specific T-cell responses and prevent OVA-induced intestinal allergy. METHODS: Bone marrow-derived myeloid dendritic cells from BALB/c, C57BL/6, or TLR signaling-deficient (MyD88(-/-)) mice were stimulated with rOVA, rflaA, rflaA plus rOVA, or a recombinant fusion protein consisting of rflaA and rOVA (rflaA:OVA). The immunomodulating properties of rflaA plus rOVA and rflaA:OVA were investigated by means of DC-T-cell coculture with CD4(+) T cells from OVA-T-cell receptor transgenic or OVA/alum-immunized mice. rflaA:OVA was applied as a prophylactic and therapeutic vaccine in a murine model of intestinal allergy. RESULTS: rflaA:OVA induced upregulation of TLR5 and dose-dependent IL-6 and IL-10 secretion by myeloid dendritic cells. IL-10 contributed to repressing IL-4 and IFN-γ secretion by OVA-T-cell receptor transgenic CD4(+) T cells. Moreover, rflaA:OVA suppressed CD4(+) T cells derived from T(H)2-biased mice on OVA/alum immunization. In a murine model of intestinal allergy, prophylactic vaccination with rflaA:OVA reduced T-cell activation. Protection from intestinal allergy included suppression of OVA-specific IgE while inducing OVA-specific IgG(2a). Equimolar amounts of rflaA or rOVA provided alone or as a mixture did not have comparable effects. Moreover, therapeutic vaccination was shown to reduce allergic symptoms and T-cell activation in the spleen. CONCLUSION: The rflaA:OVA fusion protein showed strong TLR-mediated immunomodulating capacities probably attributed by the proximity of adjuvant and allergen, leading to the prevention of intestinal allergy in a murine disease model. Therefore recombinant flaA:allergen fusion proteins are promising vaccine candidates for intervention in patients with IgE-mediated allergy.

Protein unfolding strongly modulates the allergenicity and immunogenicity of Pru p 3, the major peach allergen.

BACKGROUND: Allergen-specific immunotherapy for food allergies, including peach allergy, has not been established. Use of allergens with reduced allergenic potential and preserved immunogenicity could improve the safety and efficacy of allergen-specific immunotherapy. OBJECTIVE: We sought to create a hypoallergenic derivative of the major peach allergen Pru p 3 and to characterize its biochemical and immunologic properties. METHODS: A Pru p 3 folding variant generated by means of reduction and alkylation was investigated for structural integrity and stability to gastrointestinal enzymes. IgE reactivity and allergenic potency were determined by means of immunoblotting, ELISA, and in vitro mediator release assay with sera from patients with peach allergy. T-cell immunogenicity was investigated by using human allergen-specific T cells and CBA/J mice immunized with either native Pru p 3 (nPru p 3) or reduced and alkylated (R/A) Pru p 3. Pru p 3 processing by endolysosomal fractions of dendritic cells and antigenicity was examined in mice. RESULTS: Unfolding of Pru p 3 reduced its high resistance to gastrointestinal proteolysis and almost completely abrogated its IgE reactivity and allergenic potency. However, R/A Pru p 3 was capable of stimulating human and murine T cells. Endolysosomal degradation of R/A Pru p 3 was accelerated in comparison with nPru p 3, but similar peptides were generated. IgG and IgE antibodies raised against nPru p 3 showed almost no cross-reactivity with R/A Pru p 3. Moreover, the antigenicity of R/A Pru p 3 was strongly reduced. CONCLUSION: Unfolded Pru p 3 showed reduced allergenicity and antigenicity and preserved T-cell immunogenicity. The hypoallergenic variant of Pru p 3 could be a promising vaccine candidate for specific immunotherapy of peach allergy.