Sonja Wolfheimer

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.

Epicutaneous immune modulation with Bet v 1 plus R848 suppresses allergic asthma in a murine model

Combining allergen(s) with an adjuvant is a strategy to improve the efficacy and safety of allergen‐specific immunotherapy. Here, we aimed at investigating the adjuvant effects of polyadenylic–polyuridylic acid (poly(A:U)), a TLR3 agonist, and R848 (resiquimod), a TLR7 agonist, in epicutaneous immunotherapy with Bet v 1, the major birch pollen allergen, to intervene in birch pollen allergy.

MPLA shows attenuated pro‐inflammatory properties and diminished capacity to activate mast cells in comparison with LPS

Monophosphoryl lipid A (MPLA), a nontoxic TLR4 ligand derived from lipopolysaccharide (LPS), is used clinically as an adjuvant in cancer, hepatitis, and malaria vaccines and in allergen‐specific immunotherapy. Nevertheless, its cell‐activating effects have not been analyzed in a comprehensive direct comparison including a wide range of different immune cells. Therefore, the objective of this study was the side‐by‐side comparison of the immune‐modulating properties of MPLA and LPS on different immune cells.

Molecular cloning of plane pollen allergen Pla a 3 and its utility as diagnostic marker for peach associated plane pollen allergy.

Non‐specific lipid transfer proteins (nsLTP) are considered to provoke allergic symptoms to plane tree pollen, which are frequently associated with peach allergy.

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.

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.

Monophosphoryl Lipid a as an adjuvant for immune therapy? A detailed in vitro comparison to LPS

Monophosphoryl lipid A (MPL) is a non-toxic TLR4 ligand, derived from Salmonella minnesota R595 (Re) lipopolysaccharide (LPS) by chemical modification. It is clinically used as an adjuvant for cancer treatment (Fendrix®, Ceravix®) and allergen specific immunotherapy (Pollinex® Quattro, ORALVAC®). Nevertheless, reports on the mechanism of adjuvant activity are limited. The aim of this study was to compare the immune modulating capacities of MPL and LPS in vitro. In both human and murine lung epithelial cell lines (LA-4, A549) LPS induced a higher CCL2 secretion than MPL. In murine BM-derived myeloid dendritic cells (mDC), LPS as well as MPL stimulation resulted in the same pattern of cytokine secretion (IL-1β, IL-6, IL-10 and TNF-α). At high concentrations of MPL, IL-1β secretion was 4-fold higher compared to LPS, whereas LPS stimulation resulted in higher secretion of IL-6, IL-10 and TNF-α, respectively. Moreover, mDC stimulation with both adjuvants resulted in a pronounced cell activation pattern characterized by CD40 and CD69 upregulation, at which LPS proved to be more potent than MPL (thresholds for mDC activation: MPL: 100 ng/ml, LPS: 1 ng/ml). In MyD88-/- and Trif-/- mDC, MPL-induced cytokine secretion was absent in MyD88- but only reduced in Trif-deficient mDC. LPS induced cytokine secretion was mostly unchanged in Trif-/- mDC. Furthermore, the co-administration of MPL and Ova resulted in enhanced IFN-γ and IL-5 secretion from OVA-specific DO11.10 CD4+ T cells co-cultured with BALB/c mDC which was not observed for LPS controls. In line with this result, stimulation with a covalent fusion protein of MPL and Ova (MPL:Ova) resulted in enhanced cytokine secretion from both mDC (IL-1β, IL-6, TNF, IL-10, IL-12) and CD4 T cells (IL-5, IL-13, IL-2, IFN-γ, IL-17) compared to equimolar concentrations of MPL and Ova provided individually or as a mixture. Interestingly, Ova induced IL-9 secretion from CD4+ T cells was dose-dependently repressed when fused to MPL. In summary, using in vitro assay systems we observed similar but attenuated immune responses induced by MPL in comparison to LPS. MPL applied together with allergen (either mixed or covalently fused) on CD4+ T cells boosted allergen-specific TH1-, TH2-, and TH17-adaptive responses. Although considered safe in humans, further studies should critically assess the adjuvant capacity of MPL in order to evaluate potential non-desired immunological effects.

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.

LTP cross-reactivity - primary sensitization to mugwort pollen LTP Art v 3, facilitates subsequent sensitisation to peach LTP Pru p 3 in mice

Non-specific lipid transfer proteins (nsLTPs) are important pollen and food allergens in the Mediterranean area. Peach LTP Pru p 3 is considered as primary sensitizer inducing LTP cross-reactive IgE antibodies. Despite the evidence on the importance of Pru p 3, it is unclear whether reactivity to multiple LTPs is due to antibody cross-reactivity or to independent sensitization events. Aim of the study was (1) to compare antigenicity of LTPs from pollen and food, and (2) to investigate whether primary sensitization to LTPs promotes secondary sensitization to LTPs from unrelated sources. IgE sensitization to peach LTP was compared in different mouse strains (C3H/HeJ, BALB/c, CBA/J). IgE high responder mice (CBA/J) were used to compare the antigenicity and IgE cross-reactivity of LTPs from pollen (mugwort/Art v 3, plane tree/Pla a 3, ragweed/Amb a 6) and foods (peach/Pru p 3, cherry/Pru av 3, hazelnut/Cor a 8). To investigate whether an established Th2 immune response to LTPs facilitates a secondary immune response and cross-reactivity to unrelated LTPs, CBA/J mice were immunized with (1) Pru p 3 followed by Art v 3, (2) Art v 3 followed by Pru p 3, and (3) a mixture of Pru p 3 and Art v 3. Induction of Pru p 3 and Art v 3 specific IgE and IgG titers, and cross-reactivity between the two LTPs were determined by ELISA. CBA and C3H mice showed strong IgE-responses to Pru p 3, whereas BALB/c mice did not. Remarkably, a species-specific immune response lacking any IgE- and IgG-cross-reactivity was observed for all tested LTPs except for Pru p 3 and Pru av 3. Although LTPs show high structural similarity, Pru p 3 and Art v 3 displayed different antigenicity. In comparison to Art v 3, Pru p 3 showed much lower IgE-sensitizing capacity. In contrast, IgE response to Pru p 3 was promoted in mice previously sensitized to Art v 3, which was even the case upon concurrent administration of both LTPs. Neither sequential immunization nor immunization with the mixture induced IgE antibodies to unrelated LTPs, e.g. Cor a 8. In summary, the immune response to LTPs in mice varied strongly between strains. The LTPs studied, displayed different immunogenic properties and did not induce cross-reactive IgE antibodies. Primary sensitization to the pollen LTP Art v 3 conditioned the animals for subsequent sensitization to the food LTP Pru p 3. The extent to which the findings are applicable to the manifestation of clinical cross-reactivity to LTPs in humans needs to be investigated.