Comparison of Antibody Responses against Two Molecules from Ascaris lumbricoides: The Allergen Asc l 5 and the Immunomodulatory Protein Al-CPI.

Immunity to Ascaris lumbricoides influences the pathogenesis of allergic diseases. Antibody responses to its proteins have been found to be associated with asthma presentation; however, helminth products that induce immunosuppression have been reported, which also raise specific antibodies. We aimed to evaluate antibody responses (IgE, IgG4 and IgG) to two A. lumbricoides molecules, Asc l 5 and Al-CPI (an anti-inflammatory Cysteine Protease Inhibitor), in an endemic population, exploring their relationships with the infection and asthma. The two molecules were produced as recombinant proteins in E. coli expression systems. Specific antibodies were detected by ELISA. Lower human IgE, but higher IgG4 and IgG antibody levels were observed for Al-CPI than for rAsc l 5. The IgE/IgG4 isotype ratio was significantly higher for Asc l 5 than for Al-CPI. In humans Al-CPI did not induce basophil activation as has been previously described for Asc l 5. In mice, Al-CPI induced fewer IgE responses, but more IgG2a antibody titers than rAsc l 5. Our results suggest that these molecules elicit different patterns of immune response to A. lumbricoides.

Biological activity of human IgE monoclonal antibodies targeting Der p 2, Fel d 1, Ara h 2 in basophil mediator release assays.

Background: Human Immunoglobulin E monoclonal antibodies (hIgE mAb) are unique tools for investigating IgE responses. Here, the biological activity of hIgE mAb, derived from immortalized B cells harvested from the blood of allergic donors, targeting three allergens (Der p 2, Fel d 1 and Ara h 2) was investigated. Methods: Three Der p 2-, three Fel d 1- and five Ara h 2-specific hIgE mAb produced by human B cell hybridomas, were combined in pairs and used to passively sensitize humanized rat basophilic leukemia cells and compared with sensitization using serum pools. Sensitized cells were stimulated with corresponding allergens (recombinant or purified), allergen extracts or structural homologs, having 40-88% sequence similarity, and compared for mediator (ß-hexosaminidase) release. Results: One, two and eight pairs of Der p 2-, Fel d 1- and Ara h 2-specific hIgE mAb, respectively, produced significant mediator release (>50%). A minimum hIgE mAb concentration of 15-30 kU/L and a minimum antigen concentration between 0.01-0.1 µg/mL were sufficient to induce a pronounced mediator release. Individual sensitization with one Ara h 2-specific hIgE mAb was able to induce crosslinking independently of a second specific hIgE mAb. Der p 2- and Ara h 2-specific mAb showed a high allergen specificity when compared to homologs. Mediator release from cells sensitized with hIgE mAb was comparable to serum sensitization. Conclusion: The biological activity of hIgE mAb reported here provides the foundation for novel methods of standardization and quality control of allergen products and for mechanistic studies of IgE-mediated allergic diseases, using hIgE mAb.

Bet v 1-independent sensitization to major allergens in Fagales pollen: Evidence at the T-cell level.

BACKGROUND: In birch-dominated areas, allergies to pollen from trees of the order Fagales are considered to be initiated by the major birch pollen allergen Bet v 1. However, the sensitizing activity of Bet v 1-homologs in Fagales pollen might be underestimated. Allergen-specific T-cells are crucial in the sensitization process. The T-cell response to major allergens from alder, hazel, oak, hornbeam, chestnut, beech, and chestnut pollen has not yet been analyzed. Here, we characterized the cellular cross-reactivity of major allergens in Fagales pollen with Bet v 1. METHODS: T-cell-lines (TCL) were established from allergic individuals with Aln g 1, Car b 1, Ost c 1, Cor a 1, Fag s 1, Cas s 1, and Que a 1, and tested for reactivity with Bet v 1 and synthetic overlapping 12-mer peptides representing its primary sequence. Aln g 1-specific TCL was additionally tested with Aln g 1-derived peptides and all allergens. IgE-competition experiments with Aln g 1 and Bet v 1 were performed. RESULTS: T-cell-lines initiated with Fagales pollen allergens varied strongly in their reactivity with Bet v 1 and by the majority responded stronger to the original stimulus. Cross-reactivity was mostly restricted to the epitope Bet v 1142-153 . No distinct cross-reactivity of Aln g 1-specific T-cells with Bet v 1 was detected. Among 22 T-cell epitopes, Aln g 1 contained two immunodominant epitopes. Bet v 1 inhibited IgE-binding to Aln g 1 less potently than Aln g 1 itself. CONCLUSION: The cellular cross-reactivity of major Fagales pollen allergens with Bet v 1 was unincisive, particularly for Aln g 1, most akin to Bet v 1. Our results indicate that humoral and cellular responses to these allergens are not predominantly based on cross-reactivity with the major birch pollen allergen but suggest a Bet v 1-independent sensitization in individuals from birch tree-dominated areas.

Bet v 1-displaying elastin-like polypeptide nanoparticles induce a strong humoral and weak CD4+ T-cell response against Bet v 1 in a murine immunogenicity model.

There is growing concern about the toxicity of colloidal aluminum salts used as adjuvants in subcutaneous allergen immunotherapy (SCIT). Therefore, alternative adjuvants and delivery systems are being explored to replace alum in SCIT. We applied micellar elastin-like polypeptides (ELPs), a type of self-assembling protein, to replace alum as vaccine adjuvant in birch pollen SCIT. ELP and an ELP-Bet v 1 fusion protein were expressed in E. coli and purified by immuno-affinity chromatography and inverse-transition cycling (ITC). Nanoparticles self-assembled from ELP and a 9:1 ELP/ELP-Bet v 1 mixture were characterized by using dynamic light scattering and atomic force microscopy. Allergenicity was assessed by measuring mediator release from rat basophilic leukemia cells transformed with the human FcϵR1 and sensitized with sera derived from human birch pollen allergic patients. Humoral and T-cell immunity were investigated by immunizing naïve mice with the ELP/ELP-Bet v 1 nanoparticles or alum-adsorbed Bet v 1, both containing 36 µg Bet v 1. ELP and ELP/ELP-Bet v 1 self-assembled at 37°C into spherically shaped micelles with a diameter of ~45 nm. ELP conjugation made Bet v 1 hypo-allergenic (10-fold). Compared to alum-adsorbed Bet v 1, ELP/ELP-Bet v 1 nanoparticles induced stronger IgG responses with an earlier onset. Additionally, ELP/ELP-Bet v 1 did not induce Th2 skewing cytokines and IgE. The hypoallergenic character and strong humoral immune response in the absence of a Th2-skewing T-cell response make ELP-based nanoparticles a promising candidate to replace alum in SCIT.

Comprehensive training load monitoring with biomarkers, performance testing, local positioning data, and questionnaires - first results from elite youth soccer.

Load management, i.e., prescribing, monitoring, and adjusting training load, is primarily aimed at preventing injury and maximizing performance. The search for objective monitoring tools to assess the external and internal load of athletes is of great interest for sports science research. In this 4-week pilot study, we assessed the feasibility and acceptance of an extensive monitoring approach using biomarkers, neuromuscular performance, and questionnaires in an elite youth soccer setting. Eight male players (mean ± SD: age: 17.0 ± 0.6 years, weight: 69.6 ± 8.2 kg, height: 177 ± 7 cm, VO2max: 62.2 ± 3.8 ml/min/kg) were monitored with a local positioning system (e.g., distance covered, sprints), biomarkers (cell-free DNA, creatine kinase), questionnaires, neuromuscular performance testing (counter-movement jump) and further strength testing (Nordic hamstring exercise, hip abduction and adduction). Feasibility was high with no substantial impact on the training routine and no adverse events such as injuries during monitoring. Adherence to the performance tests was high, but adherence to the daily questionnaires was low, and decreased across the study period. Occasional significant correlations were observed between questionnaire scores and training load data, as well as between questionnaire scores and neuromuscular performance. However, due to the small sample size, these findings should be treated with caution. These preliminary results highlight the feasibility of the approach in elite soccer, but also indicate that modifications are needed in further large-scale studies, particularly in relation to the length of the questionnaire.

Cationic liposomes bearing Bet v 1 by coiled coil-formation are hypo-allergenic and induce strong immunogenicity in mice.

Although aluminum hydroxide (alum) is widely accepted and used as safe vaccine adjuvant, there is some concern about possible toxicity upon long-lasting repeated exposure during subcutaneous allergen immunotherapy (SCIT). Our objective was to evaluate allergen-bearing liposomes as possible alternative for alum-adsorption in SCIT. A self-assembling, coiled-coil forming peptide pair was used to anchor the major birch pollen allergen Bet v 1 to the surface of cationic liposomes. The resulting nanoparticulate liposomes were characterized with respect to their physicochemical, allergenic and immunological properties. Allergenicity was studied by ImmunoCAP inhibition and rat basophil leukemia (RBL) cell assays. Immunogenicity (immunoglobulin responses) and immune skewing (cytokine responses) were evaluated upon immunization of naïve mice, and compared to alum-adsorbed Bet v 1. Bet v 1-bearing cationic liposomes with a diameter of ∼200 nm showed a positive zeta potential. The coiled-coil conjugation of Bet v 1 to the surface of liposomes resulted in about a 15-fold lower allergenicity than soluble Bet v 1 as judged by RBL assays. Moreover, the nanoparticles induced Bet v 1-specific IgG1/IgG2a responses in mice that were several orders of magnitude higher than those induced by alum-adsorbed Bet v 1. This strong humoral response was accompanied by a relatively strong IL-10 induction upon PBMC stimulation with Bet v 1. In conclusion, their hypo-allergenic properties, combined with their capacity to induce a strong humoral immune response and a relatively strong IL-10 production, makes these allergen-covered cationic liposomes a promising alternative for aluminum salt-adsorption of allergen currently used in SCIT.

Humanized Mediator Release Assay as a Read-Out for Allergen Potency.

Mediator release assays analyze in vitro immunoglobulin E (IgE)-mediated degranulation and secretion of mediators by effector cells, such as mast cells and basophils, upon stimulation with serial dilutions of putative allergens. Therefore, these assays represent an essential tool that mimics the in vivo degranulation process, which occurs upon allergen exposure in sensitized patients or in skin prick tests. Additionally, these assays are usually employed to investigate the allergenic potential of proteins and the reactivity of patients' sera's reactivity. Herein, we describe a simple 2-day protocol using an immortalized rat basophil leukemia cell line transfected and humanized with the human high-affinity IgE plasma-membrane receptor (FcεRI). This variant of the mediator release assay is a robust, sensitive, and reproducible in vitro cell-based system without the need to immobilize the antigen to solid matrices. The protocol consists of the following steps: (1) complement inactivation of human sera, (2) harvesting, seeding, and passive sensitization of the cells, (3) stimulation with antigen to cause mediator release, and (4) measuring of ß-hexosaminidase activity as a surrogate for the released inflammatory mediators, such as histamine. The assay represents a useful tool to assess the capacity of the allergen-IgE cross-linking to trigger cell degranulation and can be implemented to standardize allergen extracts, to compare patients' reactivity to minor or major allergens and to allergenic extracts (pollen, cat dander, etc.), to investigate the potency of allergen homologs, isoforms, and fold-variants (e.g., hypoallergenicity), as well as the effects of ligands on the allergenic activity. A more recent application includes the use of the assay to monitor the treatment efficacy in the course of allergen immunotherapy.

TGFß1 mimetic peptide modulates immune response to grass pollen allergens in mice.

BACKGROUND: Transforming growth factor ß1 (TGFß1) is a cytokine that exerts immunosuppressive functions, as reflected by its ability to induce regulatory T (Treg) cell differentiation and inhibit Th1 and Th2 responses. Hence, peptides that mimic the active core domain of TGFß1 may be promising candidates for modulation of the allergic response. This study aimed to investigate a synthetic TGFß1 mimetic peptide (TGFß1-mim) for its ability to modulate the immune response during allergic sensitization to grass pollen allergens. METHODS: The in vitro action of TGFß1-mim was evaluated in human lung epithelial cells, Jurkat cells, and rat basophilic leukemia cells. The in vivo action was evaluated in a murine model of Phl p 5 allergic sensitization. Additionally, the Th2 modulatory response was evaluated in IL-4 reporter mice. RESULTS: In vitro, TGFß1-mim downregulated TNF-α production, IL-8 gene expression, and cytokine secretion, upregulated IL-10 secretion, and inhibited Phl p 5-induced basophil degranulation. During Phl p 5 sensitization in mice, TGFß1-mim downregulated IL-2, IL-4, IL-5, IL-13, and IFN-γ, upregulated IL-10, and induced Treg cell production. Furthermore, mice treated with TGFß1-mim had lower levels of IgE, IgG1, IgG2a and higher levels of IgA antibodies than control mice. In a reporter mouse, the mimetic inhibited Th2 polarization. CONCLUSION: The TGFß1-mim efficiently modulated various important events that exacerbate the allergic microenvironment, including the production of main cytokines that promote Th1 and Th2 differentiation, and the induction of allergen-specific regulatory T cells, highlighting its potential use in therapeutic approaches to modulate the immune response toward environmental allergens.

Multiple roles of Bet v 1 ligands in allergen stabilization and modulation of endosomal protease activity.

BACKGROUND: Over 100 million people worldwide suffer from birch pollen allergy. Bet v 1 has been identified as the major birch pollen allergen. However, the molecular mechanisms of birch allergic sensitization, including the roles of Bet v 1 and other components of the birch pollen extract, remain incompletely understood. Here, we examined how known birch pollen-derived molecules influence the endolysosomal processing of Bet v 1, thereby shaping its allergenicity. METHODS: We analyzed the biochemical and immunological interaction of ligands with Bet v 1. We then investigated the proteolytic processing of Bet v 1 by endosomal extracts in the presence and absence of ligands, followed by a detailed kinetic analysis of Bet v 1 processing by individual endolysosomal proteases as well as the T-cell epitope presentation in BMDCs. RESULTS: We identified E1 phytoprostanes as novel Bet v 1 ligands. Pollen-derived ligands enhanced the proteolytic resistance of Bet v 1, affecting degradation kinetics and preferential cleavage sites of the endolysosomal proteases cathepsin S and legumain. E1 phytoprostanes exhibited a dual role by stabilizing Bet v 1 and inhibiting cathepsin protease activity. CONCLUSION: Bet v 1 can serve as a transporter of pollen-derived, bioactive compounds. When carried to the endolysosome, such compounds can modulate the proteolytic activity, including its processing by cysteine cathepsins. We unveil a paradigm shift from an allergen-centered view to a more systemic view that includes the host endolysosomal enzymes.

Two Distinct Conformations in Bet v 2 Determine Its Proteolytic Resistance to Cathepsin S.

Birch pollen allergy affects more than 20% of the European allergic population. On a molecular level, birch pollen allergy can be linked to the two dominant allergens Bet v 1 and Bet v 2. Bet v 2 belongs to the profilin family, which is abundant in the plant kingdom. Importantly, the homologous plant profilins have a conserved cysteine motif with a currently unknown functional relevance. In particular, it is unknown whether the motif is relevant for disulfide formation and to what extent it would affect the profilins' structural, functional and immunological properties. Here we present crystal structures of Bet v 2 in the reduced and the oxidized state, i.e., without and with a disulfide bridge. Despite overall structural similarity, the two structures distinctly differ at their termini which are stabilized to each other in the oxidized, i.e., disulfide-linked state. These structural differences translate into differences in their proteolytic resistance. Whereas the oxidized Bet v 2 is rather resistant towards the endolysosomal protease cathepsin S, it is rapidly degraded in the reduced form. By contrast, both Bet v 2 forms exhibit similar immunological properties as evidenced by their binding to IgE antibodies from birch pollen allergic patients and by their ability to trigger histamine release in a humanized rat basophilic leukemia cells (RBL) assay, independent of the presence or absence of the disulfide bridge. Taken together our findings suggest that the oxidized Bet v 2 conformation should be the relevant species, with a much longer retention time to trigger immune responses.