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.

Cor a 1-reactive T cells and IgE are predominantly cross-reactive to Bet v 1 in patients with birch pollen-associated food allergy to hazelnut.

BACKGROUND: IgE- and T-cell cross-reactivity contribute to the birch pollen-food syndrome. OBJECTIVES: We performed a comprehensive analysis of T-cell cross-reactivity in primary cell cultures, facilitating the identification of allergen-specific T-cell subpopulations from individual patients. METHODS: Patients with birch pollen allergy and associated food allergy to hazelnuts, carrots, or both were analyzed for IgE cross-reactivity, T-cell responses, and T-cell cross-reactivity to recombinant Bet v 1.0101 (Bet v 1; birch), Cor a 1.0401 (Cor a 1; hazelnut), and Dau c 1.0104 (Dau c 1; carrot). A novel flow cytometry-based method using a 2-step staining process with fluorescent dyes was established to identify subpopulations of cross-reactive T cells. RESULTS: IgE-binding inhibition tests of individual sera revealed that the vast majority of Cor a 1-reactive IgE was cross-reactive to Bet v 1, whereas Bet v 1-reactive IgE was only partially inhibited by preincubation with Cor a 1. Primary stimulation of T cells with Bet v 1 or Cor a 1 resulted in a significant increase in specific responses to Cor a 1 or Bet v 1 after secondary stimulation, respectively, indicating T-cell cross-reactivity between birch and hazelnut allergens in all patients of the study cohort. Preactivation with Dau c 1 induced less pronounced effects. A novel flow cytometry-based proliferation assay identified a predominant Cor a 1/Bet v 1-cross-reactive T-cell subpopulation within highly Bet v 1/Cor a 1-responsive T cells. CONCLUSION: Analysis of primary allergen-specific T cells combined with flow cytometry-based proliferation assays facilitates investigation of allergen-specific T-cell subpopulations in subjects and might be helpful to evaluate the effect of birch-specific immunotherapy on pollen-associated food allergies.

Allergic Reaction to a Commercially Available Insect Snack Caused by House Cricket (Acheta domesticus) Tropomyosin.

SCOPE: Edible insects contain allergens with potential cross-reactivity to other invertebrates. Here, this study examines IgE-reactive proteins in a house cricket snack (Acheta domesticus) leading to an allergic reaction in a 27-year old man followed by a similar reaction days later after eating shrimps. METHODS AND RESULTS: Prick to prick tests verify the IgE-mediated allergy to crickets and skin prick testing confirms a type I sensitization to house dust mite without any clinical relevance for the patient, and to shrimp extracts, but is negative for several other foods. Serological testing reveals a sensitization to shrimps, shrimp tropomyosin, and house dust mite tropomyosin. IgE-immunodetection shows that the cricket allergic patient is sensitized to two proteins of 45 and >97 kDa using aqueous control cricket extract, but to only one protein at around 45 kDa when using the causative, seasoned insect snack extract. Mass spectrometry data and IgE-inhibition experiments clearly identify this protein belonging to the tropomyosin allergen family. CONCLUSION: This case report suggests that cricket tropomyosin may be an elicitor of allergic reactions even in previously not allergic patients, although it cannot be excluded the patient reacted additionally to other ingredients of the snack.

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.

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.

Component-Resolved Diagnosis of American Cockroach (Periplaneta americana) Allergy in Patients From Different Geographical Areas.

Background: Manifestation of respiratory allergy to American cockroach (Periplaneta americana) is prominent in the subtropical and tropical areas. However, co-existing perennial indoor inhalant allergies frequently compromise clinical diagnosis of cockroach allergy, and the analysis of sensitization pattern is limited by the lack of Periplaneta allergens widely available for component-resolved diagnostics (CRD). Objective: To evaluate a collection of previously described recombinant Periplaneta allergens for CRD in cockroach allergy. Methods: A panel of nine recombinant Periplaneta allergens (Per a 1-5, 7-10) was generated, purified, and subjected to physicochemical characterization by applying circular dichroism (CD) spectroscopy, dynamic light scattering (DLS), amino acid (AA) analysis, and mass spectrometry (MS). Patients (n = 117) from India, Korea, Venezuela, and Iran, reporting perennial respiratory indoor allergies with IgE sensitization to cockroach (P. americana and/or Blattella germanica), were included. The sensitization profile was monitored by the experimental ImmunoCAP testing. Results: ImmunoCAP testing confirmed IgE sensitization to Periplaneta and/or Blattella extract in 98 of 117 patients (r = 0.95). Five out of 117 patients were sensitized to only one of the two cockroach species. Within the whole study group, the prevalence of sensitization to individual allergens varied from 4% (Per a 2) to 50% (Per a 9), with the highest IgE values to Per a 9. Patients from four countries displayed different sensitization profiles at which Per a 3 and Per a 9 were identified as major allergens in India and Korea. Periplaneta-derived lipocalin and myosin light chain were characterized as new minor allergens, designated as Per a 4 and Per a 8. Periplaneta extract showed higher diagnostic sensitivity than all individual components combined, suggesting the existence of allergens yet to be discovered. Conclusion: Utilization of a panel of purified Periplaneta allergens revealed highly heterogeneous sensitization patterns and allowed the classification of lipocalin and myosin light chain from Periplaneta as new minor allergens.

Identification and Characterization of IgE-Reactive Proteins and a New Allergen (Cic a 1.01) from Chickpea (Cicer arietinum).

SCOPE: Chickpea (Cicer arietinum) allergy has frequently been reported particularly in Spain and India. Nevertheless, chickpea allergens are poorly characterized. The authors aim to identify and characterize potential allergens from chickpea. METHODS AND RESULTS: Candidate proteins are selected by an in silico approach or immunoglobuline E (IgE)-testing. Potential allergens are prepared as recombinant or natural proteins and characterized for structural integrity by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), circular dichroism (CD)-spectroscopy, and mass spectrometry (MS) analysis. IgE-sensitization pattern of Spanish chickpea allergic and German peanut and birch pollen sensitized patients are investigated using chickpea extracts and purified proteins. Chickpea allergic patients show individual and heterogeneous IgE-sensitization profiles with extracts from raw and boiled chickpeas. Chickpea proteins pathogenesis related protein family 10 (PR-10), a late embryogenesis abundant protein (LEA/DC-8), and a vicilin-containing fraction, but not 2S albumin, shows IgE reactivity with sera from chickpea, birch pollen, and peanut sensitized patients. Remarkably, allergenic vicilin, DC-8, and PR-10 are detected in the extract of boiled chickpeas. CONCLUSION: Several IgE-reactive chickpea allergens are identified. For the first time a yet not classified DC-8 protein is characterized as minor allergen (Cic a 1). Finally, the data suggest a potential risk for peanut allergic patients by IgE cross-reactivity with homologous chickpea proteins.

Identification and implication of an allergenic PR-10 protein from walnut in birch pollen associated walnut allergy.

SCOPE: English walnut (Juglans regia) belongs to the most important allergenic tree nuts. Co-sensitization with birch (Betula verrucosa) pollen has been reported. We aimed to identify a walnut allergen homologous to the major birch pollen allergen Bet v 1. METHODS AND RESULTS: A cDNA encoding a Bet v 1-homologous allergen (Jug r 5) in walnut kernels was cloned by RT-PCR. Jug r 5 was expressed in Escherichia coli, purified by column chromatography and characterized by circular dichroism spectroscopy. Specific IgE levels to walnut, Bet v 1, and Jug r 5 in birch pollen allergics (n = 16) with concomitant walnut allergy were measured by ImmunoCAP: 44% of the patients were tested positive to walnut while 94% were reactive to Jug r 5, and 100% to Bet v 1. Jug r 5 and Bet v 1 allergens showed bidirectional IgE cross-reactivity by competitive ELISA and were capable of inducing histamine release from effector cells. Immunoblot competition experiments demonstrated the presence of IgE-reactive Jug r 5 in walnut extract, but at low levels. CONCLUSION: A Bet v 1-like allergen was identified in walnut. Diagnostic use of Jug r 5 will compensate for the low sensitivity of walnut extract for patients with birch pollen associated walnut allergy.

Identification of Sola l 4 as Bet v 1 homologous pathogenesis related-10 allergen in tomato fruits.

SCOPE: The aim was to investigate the potential contribution of a major birch pollen Bet v 1-homologous allergen to birch pollen-associated tomato fruit allergy. METHODS AND RESULTS: Two isoforms of a Bet v 1-homologous protein (designated Sola l 4) from tomato fruit were identified by cDNA-cloning and produced as recombinant proteins. Allergen-specific IgE levels to tomato, birch pollen, Bet v 1, and Sola l 4 were determined in birch pollen allergic patients with allergy or tolerance to tomato. Sola l 4 was recognized in 76% of birch/tomato allergic patients, while tomato- and Bet v 1-specific IgE was detectable in 64% and 81% of sera. Almost all patients sensitized to Bet v 1 reacted with Sola l 4. Both Sola l 4 isoforms displayed allergenic potency and IgE-cross-reactivity with Bet v 1 as investigated by competitive ELISA and in vitro mediator release assay. Nevertheless, the reactivity pattern of patients' sera was diverse. CONCLUSION: Sola l 4, a novel pathogenesis related-10 protein, qualifies as major allergen in tomato fruits. Data suggest Sola l 4 as class II allergen. IgE-testing using Sola l 4 showed low clinical specificity, but high sensitivity in tomato allergic patients and will further improve component-resolved allergy diagnosis.