Coupling of a Major Allergen to the Surface of Immune Cells for Use in Prophylactic Cell Therapy for the Prevention of IgE-Mediated Allergy.

Up to a third of the world's population suffers from allergies, yet the effectiveness of available preventative measures remains, at large, poor. Consequently, the development of successful prophylactic strategies for the induction of tolerance against allergens is crucial. In proof-of-concept studies, our laboratory has previously shown that the transfer of autologous hematopoietic stem cells (HSC) or autologous B cells expressing a major grass pollen allergen, Phl p 5, induces robust tolerance in mice. However, eventual clinical translation would require safe allergen expression without the need for retroviral transduction. Therefore, we aimed to chemically couple Phl p 5 to the surface of leukocytes and tested their ability to induce tolerance. Phl p 5 was coupled by two separate techniques, either by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) or by linkage via a lipophilic anchor, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol)-maleimide (DSPE-PEG-Mal). The effectiveness was assessed in fresh and cultured Phl p 5-coupled cells by flow cytometry, image cytometry, and immunofluorescence microscopy. Chemical coupling of Phl p 5 using EDC was robust but was followed by rapid apoptosis. DSPE-PEG-Mal-mediated linkage was also strong, but antigen levels declined due to antigen internalization. Cells coupled with Phl p 5 by either method were transferred into autologous mice. While administration of EDC-coupled splenocytes together with short course immunosuppression initially reduced Phl p 5-specific antibody levels to a moderate degree, both methods did not induce sustained tolerance towards Phl p 5 upon several subcutaneous immunizations with the allergen. Overall, our results demonstrate the successful chemical linkage of an allergen to leukocytes using two separate techniques, eliminating the risks of genetic modifications. More durable surface expression still needs to be achieved for use in prophylactic cell therapy protocols.

Easy assessment of the avidity of polyclonal allergen-specific serum antibodies.

INTRODUCTION: Allergen-specific IgE-blocking IgG antibodies contribute to successful allergen immunotherapy (AIT), however, not much is known about their affinity. Since affinity measurements of polyclonal antibodies in serum are technically challenging we evaluated the applicability of acidic disruption of antibody-allergen complexes by a modified ELISA protocol with monoclonal antibodies (mAbs) specific for the relevant major allergens Betv1 and Mald1. Then, AIT-induced blocking and non-blocking Mald1-specific antibodies in sera from individuals with or without reduced apple allergy were compared. METHODS: After testing their pH stability coated recombinant allergens were incubated with (i) mAbs diluted in PBS or human serum and (ii) sera from individuals after sublingual administration of Mald1 or Betv1 for 16 weeks. Immune complexes were exposed to buffers in the pH range of 6.4-3.4 and residual antibodies were measured. Avidity indexes (AI), defined as the pH removing 50% of antibodies, were compared to the dissociation constants (KD) of mAbs determined by surface plasmon resonance. RESULTS: The selected pH range was applicable to disrupt allergen complexes with highly affine mAbs without compromising allergen integrity. AIs of mAbs accorded with KD values and were unaffected by epitope specificity or the presence of serum proteins. The inter-assay variability was <4% CV. Protective Mald1-specific IgG antibodies from individuals with reduced apple allergy showed a higher collective binding strength than that of the non-protective antibodies of individuals without reduced apple allergy. CONCLUSION: Acidic disruption of allergen-antibody complexes may be used to estimate the net-binding force of polyclonal serum antibodies and eases the investigation of affinity-related research questions in AIT.

Adoptive transfer of allergen-expressing B cells prevents IgE-mediated allergy.

Introduction: Prophylactic strategies to prevent the development of allergies by establishing tolerance remain an unmet medical need. We previously reported that the transfer of autologous hematopoietic stem cells (HSC) expressing the major timothy grass pollen allergen, Phl p 5, on their cell surface induced allergen-specific tolerance in mice. In this study, we investigated the ability of allergen-expressing immune cells (dendritic cells, CD4+ T cells, CD8+ T cells, and CD19+ B cells) to induce allergen-specific tolerance in naive mice and identified CD19+ B cells as promising candidates for allergen-specific cell therapy. Methods: For this purpose, CD19+ B cells were isolated from Phl p 5-transgenic BALB/c mice and transferred to naive BALB/c mice, pre-treated with a short course of rapamycin and an anti-CD40L antibody. Subsequently, the mice were subcutaneously sensitized three times at 4-week intervals to Phl p 5 and Bet v 1 as an unrelated control allergen. Allergen-expressing cells were followed in the blood to monitor molecular chimerism, and sera were analyzed for Phl p 5- and Bet v 1-specific IgE and IgG1 levels by RBL assay and ELISA, respectively. In vivo allergen-induced lung inflammation was measured by whole-body plethysmography, and mast cell degranulation was determined by skin testing. Results: The transfer of purified Phl p 5-expressing CD19+ B cells to naive BALB/c mice induced B cell chimerism for up to three months and prevented the development of Phl p 5-specific IgE and IgG1 antibody responses for a follow-up period of 26 weeks. Since Bet v 1 but not Phl p 5-specific antibodies were detected, the induction of tolerance was specific for Phl p 5. Whole-body plethysmography revealed preserved lung function in CD19+ B cell-treated mice in contrast to sensitized mice, and there was no Phl p 5-induced mast cell degranulation in treated mice. Discussion: Thus, we demonstrated that the transfer of Phl p 5-expressing CD19+ B cells induces allergen-specific tolerance in a mouse model of grass pollen allergy. This approach could be further translated into a prophylactic regimen for the prevention of IgE-mediated allergy in humans.

T-cell subset changes during the first year of pre-seasonal allergoid allergen-specific immunotherapy.

Allergen-specific immunotherapy (AIT) is the only treatment for type I allergy, which achieves long-lasting effects. Repeated subcutaneous applications of allergen extracts cause a protective antibody response and an immune deviation of T cells. In AIT with allergoids, chemically modified allergen extracts are injected. During a so-called special pre-seasonal application scheme, after the initial phase of applying increased doses of allergoids is followed by natural allergen exposure as a maintenance phase. The effectiveness of allergoid vaccines has been described regarding the improvement of clinical symptoms and the development of protective humoral responses. In this longitudinal observational study, we sought to investigate changes at the T cell level in pre-seasonal AIT with allergoid. Different subsets within CD4+ and CD8+ T cells were monitored by flow cytometry in PBMC of patients known to possess protective antibody responses. Compared to before treatment, a small early boost among allergenic Th cells was observed after 4 months of AIT. In line, a slight Th2 bias was observed after 4 months within circulating T follicular T cells, Tfh and Tfc, representing pre-existing memory Th2 cells. Furthermore, it was demonstrated that responsiveness of CD8+ T cells to allergen stimulation decreased during the course of treatment. Apart from that, we found an influence of the meteorological season on the activation profile of Tfh and Tfc over the course of the treatment. Together, this is the first study investigating changes of different T cell subsets over the course of an allergoid AIT against airborne allergens. Our findings match previous reports on conventional AIT, especially the initial increase of Th2 responses. However, the observed changes were less pronounced which may be either due to the modification of allergens or to the reduced maintenance dose provided by natural allergen exposure compared to a perennial protocol.

Food-sensitized pediatric patients show colonic cow's milk protein-specific Th2 cells.

Food allergies have become a health concern worldwide. Around 6% to 10% of children are allergic to cow's milk proteins. We have previously characterized colorectal polyps in patients sensitized to food allergens. These polyps are classified as inflammatory and present a type 2 environment, with elevated interleukin (IL)-13 and IL-4, and are a site of immunoglobulin E synthesis. In this study, we characterized and isolated cow's milk protein-specific T cell lines and T cell clones from the lamina propria of polyps from patients sensitized to these proteins. Isolated T cells responded to cow's milk proteins similarly to peripheral blood T cells, showing antigen-specific cell proliferation and Th2 cytokines release in vitro. T cell clones obtained were all CD4+ T cells and expressed the membrane TCRαß receptor and secreted higher IL-4, IL-5, and IL-13 amounts than unstimulated cells, whereas interferon γ secretion remained unchanged. Remarkably, the gut homing chemokine receptor CCR9 was augmented in cow's milk-specific peripheral and lamina propria T cells, and CCL25 was found to be expressed in the inflammatory polyp tissue and not in the adjacent mucosa. In conclusion, we isolated and characterized cow's milk-specific lamina propria CD4+ Th2 cells from colonic inflammatory polyps. CCR9 expression on these cells, along with increase secretion of CCL25 in the polyp, favors recruitment and cow's milk-specific allergic response within the inflammatory polyp tissue. Our findings may be critical to understand the underlying mechanism that promotes immunoglobulin E synthesis in the colon of cow's milk proteins allergic patients, contributing to the development of novel T cell-targeted immunotherapies.

The role of IgG1 and IgG4 as dominant IgE-blocking antibodies shifts during allergen immunotherapy.

BACKGROUND: The induction of allergen-specific IgE-blocking antibodies is a hallmark of allergen immunotherapy (AIT). The inhibitory bioactivity has largely been attributed to IgG4; however, our recent studies indicated the dominance of IgG1 early in AIT. OBJECTIVES: Here, the IgE-blocking activity and avidity of allergen-specific IgG1 and IgG4 antibodies were monitored throughout 3 years of treatment. METHODS: Serum samples from 24 patients were collected before and regularly during AIT with birch pollen. Bet v 1-specific IgG1 and IgG4 levels were determined by ELISA and ImmunoCAP, respectively. Unmodified and IgG1- or IgG4-depleted samples were compared for their inhibition of Bet v 1-induced basophil activation. The stability of Bet v 1-antibody complexes was compared by ELISA and by surface plasmon resonance. RESULTS: Bet v 1-specific IgG1 and IgG4 levels peaked at 12 and 24 months of AIT, respectively. Serological IgE-blocking peaked at 6 months and remained high thereafter. In the first year of therapy, depletion of IgG1 clearly diminished the inhibition of basophil activation while the absence of IgG4 hardly reduced IgE-blocking. Then, IgG4 became the main inhibitory isotype in most individuals. Both isotypes displayed high avidity to Bet v 1 ab initio of AIT, which did not increase during treatment. Bet v 1-IgG1 complexes were enduringly more stable than Bet v 1-IgG4 complexes were. CONCLUSIONS: In spite of the constant avidity of AIT-induced allergen-specific IgG1 and IgG4 antibodies, their dominance in IgE-blocking shifted in the course of treatment. The blocking activity of allergen-specific IgG1 should not be underestimated, particularly early in AIT.

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.

Unilateral acute lung injury in pig: a promising animal model.

BACKGROUND: Acute lung injury (ALI) occurs in 23% unilateral. Models of unilateral ALI were developed and used previously without clearly demonstrating the strictly unilateral nature and severity of lung injury by the key parameters characterizing ALI as defined by the American Thoracic Society (ATS). Thus, the use of unilateral ALI remained rare despite the innovative approach. Therefore, we developed a unilateral model of ALI and focused on the crucial parameters characterizing ALI. This model can serve for direct comparisons between the injured and intact lungs within single animals, thus, reducing the number of animals required for valid experimental conclusions. METHODS: We established the model in nine pigs, followed by an evaluation of key parameters in six pigs (main study). Pigs were ventilated using an adapted left double-lumen tube for lung separation and two ventilators. ALI was induced in the left lung with cyclic rinsing (NaCl 0.9% + Triton® X-100), after which pigs were ventilated for different time spans to test for the timing of ALI onset. Ventilatory and metabolic parameters were evaluated, and bronchoalveolar lavage (BAL) was performed for measurements of inflammatory mediators. Finally, histopathological specimens were collected and examined in respect of characteristics defining the lung injury score (LIS) as suggested by the ATS. RESULTS: After adjustments of the model (n = 9) we were able to induce strictly left unilateral ALI in all six pigs of the evaluation study. The median lung injury score was 0.72 (IQR 0.62-0.79) in the left lung vs 0.14 (IQR 0.14-0.16; p < 0.05) in the right lung, confirming unilateral ALI. A significant and sustained drop in pulmonary compliance (Cdyn) of the left lung occurred immediately, whereas Cdyn of the right lung remained unchanged (p < 0.05). BAL fluid concentrations of interleukin-6 and -8 were increased in both lungs. CONCLUSIONS: We established a model of unilateral ALI in pigs, confirmed by histopathology, and typical changes in respiratory mechanics and an inflammatory response. This thoroughly evaluated model could serve as a basis for future studies and for comparing pathophysiological and pharmacological changes in the uninjured and injured lung within the same animal.

Identification of apple cultivars hypoallergenic for birch pollen-allergic individuals by a multidisciplinary in vitro and in vivo approach.

BACKGROUND: Birch pollen-related apple allergy is the most frequent IgE-mediated food allergy in Central-Northern Europe with Mal d 1 as major allergen. Its concentration in apples varies with the cultivar and storage time. Year-round appealing, hypoallergenic cultivars still are needed to satisfy the nutritional needs of affected individuals. We characterized three promising cultivars by multidisciplinary in vitro assays including long-term storage and by clinical challenges of allergic individuals before and after the birch pollen season. METHODS: Proteins were extracted from fruits of 'Santana', 'Golden Delicious' (GD), and three genuine cultivars in November 2018 and April 2019. Mal d 1-levels were analysed by mass spectrometry, SDS-PAGE, immunoblotting, competitive ELISA, and basophil activation tests. Twenty-eight allergic individuals underwent single-blinded open food challenges and skin testing with the cultivars and birch pollen in November 2018 and May 2019. Allergen-specific IgE-levels were determined. RESULTS: After storage all cultivars except 'Santana' were of appealing appearance and taste. Their Mal d 1 content had increased, also reflected by significantly amplified basophil activation and stronger reactions in clinical challenges. Besides, individuals showed boosted reactivity after pollen exposure indicated by enhanced allergen-specific IgE-levels and skin reactions to birch pollen. Still, all cultivars remained significantly less allergenic than GD and comparable to Santana in November 2018 in all assessments except for skin testing. CONCLUSIONS: Combined expertise in pomology and allergology identified promising new cultivars for allergic consumers. The evaluation of hypoallergenic apples should incorporate long-term storage and birch pollen exposure. Basophil activation tests may be suitable in the selection of promising cultivars for oral challenges.

How Do Pollen Allergens Sensitize?

Plant pollen is one of the main sources of allergens causing allergic diseases such as allergic rhinitis and asthma. Several allergens in plant pollen are panallergens which are also present in other allergen sources. As a result, sensitized individuals may also experience food allergies. The mechanism of sensitization and development of allergic inflammation is a consequence of the interaction of allergens with a large number of molecular factors that often are acting in a complex with other compounds, for example low-molecular-mass ligands, which contribute to the induction a type 2-driven response of immune system. In this review, special attention is paid not only to properties of allergens but also to an important role of their interaction with lipids and other hydrophobic molecules in pollen sensitization. The reactions of epithelial cells lining the nasal and bronchial mucosa and of other immunocompetent cells will also be considered, in particular the mechanisms of the activation of B and T lymphocytes and the formation of allergen-specific antibody responses.

The secretome of irradiated peripheral blood mononuclear cells attenuates activation of mast cells and basophils.

BACKGROUND: IgE-mediated hypersensitivity is becoming increasingly prevalent and activation of mast cells and basophils represent key events in the pathophysiology of allergy. We have previously reported that the secretome of γ-irradiated peripheral blood mononuclear cells (PBMCsec) exerts beneficial anti-inflammatory effects. Yet, its ability to alleviate allergic symptoms has not been investigated so far. METHODS: Several experimental in vitro and in vivo models have been used in this basic research study. A murine ear swelling model was used to study the effects of PBMCsec on 48/80-induced mast cell degranulation in vivo. The transcriptional profile of murine mast cells was analysed by single cell RNA sequencing (scRNAseq). Mast cell activation was studied in vitro using primary skin mast cells. Basophils from individuals allergic to birch pollens were used to investigate basophile activation by allergens. Transcriptomic and lipidomic analyses were used to identify mRNA expression and lipid species present in PBMCsec, respectively. FINDINGS: Topical application of PBMCsec on mouse ears (C57BL/6) significantly reduced tissue swelling following intradermal injection of compound 48/80, an inducer of mast cell degranulation. Single cell RNA sequencing of PBMCsec-treated murine dermal mast cells (Balb/c) revealed a downregulation of genes involved in immune cell degranulation and Fc-receptor signalling. In addition, treatment of primary human dermal mast cells with PBMCsec strongly inhibited compound 48/80- and α-IgE-induced mediator release in vitro. Furthermore, PBMCsec remarkably attenuated allergen driven activation of basophils from allergic individuals. Transcriptomic analysis of these basophils showed that PBMCsec downregulated a distinct gene battery involved in immune cell degranulation and Fc-receptor signalling, corroborating results obtained from dermal mast cells. Finally, we identified the lipid fraction of PBMCsec as the major active ingredient involved in effector cell inhibition. INTERPRETATION: Collectively, our data demonstrate that PBMCsec is able to reduce activation of mast cells and basophils, encouraging further studies on the potential use of PBMCsec for treating allergy. FUNDING: Austrian Research Promotion Agency (852748 and 862068, 2015-2019), Vienna Business Agency (2343727, 2018-2020), Aposcience AG, Austrian Federal Ministry of Education, Science and Research (SPA06/055), Danube Allergy Research Cluster, Austrian Science Fund (I4437 and P32953).

Development of a Sequence Searchable Database of Celiac Disease-Associated Peptides and Proteins for Risk Assessment of Novel Food Proteins.

Celiac disease (CeD) is an autoimmune enteropathy induced by prolamin and glutelin proteins in wheat, barley, rye, and triticale recognized by genetically restricted major histocompatibility (MHC) receptors. Patients with CeD must avoid consuming these proteins. Regulators in Europe and the United States expect an evaluation of CeD risks from proteins in genetically modified (GM) crops or novel foods for wheat-related proteins. Our database includes evidence-based causative peptides and proteins and two amino acid sequence comparison tools for CeD risk assessment. Sequence entries are based on the review of published studies of specific gluten-reactive T cell activation or intestinal epithelial toxicity. The initial database in 2012 was updated in 2018 and 2022. The current database holds 1,041 causative peptides and 76 representative proteins. The FASTA sequence comparison of 76 representative CeD proteins provides an insurance for possible unreported epitopes. Validation was conducted using protein homologs from Pooideae and non-Pooideae monocots, dicots, and non-plant proteins. Criteria for minimum percent identity and maximum E-scores are guidelines. Exact matches to any of the 1,041 peptides suggest risks, while FASTA alignment to the 76 CeD proteins suggests possible risks. Matched proteins should be tested further by CeD-specific CD4/8+ T cell assays or in vivo challenges before their use in foods.

A dynamic single cell-based framework for digital twins to prioritize disease genes and drug targets.

BACKGROUND: Medical digital twins are computational disease models for drug discovery and treatment. Unresolved problems include how to organize and prioritize between disease-associated changes in digital twins, on cellulome- and genome-wide scales. We present a dynamic framework that can be used to model such changes and thereby prioritize upstream regulators (URs) for biomarker- and drug discovery. METHODS: We started with seasonal allergic rhinitis (SAR) as a disease model, by analyses of in vitro allergen-stimulated peripheral blood mononuclear cells (PBMC) from SAR patients. Time-series a single-cell RNA-sequencing (scRNA-seq) data of these cells were used to construct multicellular network models (MNMs) at each time point of molecular interactions between cell types. We hypothesized that predicted molecular interactions between cell types in the MNMs could be traced to find an UR gene, at an early time point. We performed bioinformatic and functional studies of the MNMs to develop a scalable framework to prioritize UR genes. This framework was tested on a single-cell and bulk-profiling data from SAR and other inflammatory diseases. RESULTS: Our scRNA-seq-based time-series MNMs of SAR showed thousands of differentially expressed genes (DEGs) across multiple cell types, which varied between time points. Instead of a single-UR gene in each MNM, we found multiple URs dispersed across the cell types. Thus, at each time point, the MNMs formed multi-directional networks. The absence of linear hierarchies and time-dependent variations in MNMs complicated the prioritization of URs. For example, the expression and functions of Th2 cytokines, which are approved drug targets in allergies, varied across cell types, and time points. Our analyses of bulk- and single-cell data from other inflammatory diseases also revealed multi-directional networks that showed stage-dependent variations. We therefore developed a quantitative approach to prioritize URs: we ranked the URs based on their predicted effects on downstream target cells. Experimental and bioinformatic analyses supported that this kind of ranking is a tractable approach for prioritizing URs. CONCLUSIONS: We present a scalable framework for modeling dynamic changes in digital twins, on cellulome- and genome-wide scales, to prioritize UR genes for biomarker and drug discovery.

Neutrophils-typical atypical antigen presenting cells?

Neutrophils are the most abundant cells of the immune system and key in combating infections through phagocytosis, reactive oxygen species, neutrophil extracellular traps, and secretion of cytokines and antimicrobial peptides. Beyond this, they may influence the adaptive immune response by modulating CD4+ T cell responses. In response to cytokines, mainly GM-CSF, but also IFN-γ and TNF-α, neutrophils express major histocompatibility complex class II molecules on their surface. However, to function as antigen-presenting cells for CD4+ T cells, more requirements need to be fulfilled, like antigen internalization, processing into fragments containing T cell epitopes, and their presentation on the cell surface together with costimulatory molecules. Here, studies investigating the key features of antigen-presentation by neutrophils are summarized and discussed. Together, they provide evidence for a potential of neutrophils to specifically activate antigen-experienced CD4+ T cells.

Isotype-specific binding patterns of serum antibodies to multiple conformational epitopes of Bet v 1.

BACKGROUND: Birch pollen is an important elicitor of respiratory allergy. The major allergen, Bet v 1, binds IgE exclusively via conformational epitopes. OBJECTIVE: We identified Bet v 1-specific epitope repertoires of IgE and IgG from birch pollen-allergic and nonallergic subjects. METHODS: Chimeric proteins were created by grafting individual epitope-sized, contiguous surface patches of Bet v 1 onto a nonallergenic structural homolog and expressed in Escherichia coli. Binding of IgE, IgG1, and IgG4 from sera of 30 birch pollen-allergic and 11 nonallergic subjects to Bet v 1, 13 chimeric proteins, and 4 bacterial Bet v 1 homologs were measured by ELISA. The proportion of epitope-specific in-total Bet v 1-specific IgE and the cross-reactivity of Bet v 1-specific IgE with bacterial homologs were determined by competitive ELISA. RESULTS: Thirteen soluble, correctly folded chimeric proteins were produced. IgE from 27 of 30 birch pollen-allergic patients bound to 1 to 12 chimeric proteins (median, 4.0), with patient-specific patterns evident. Three chimeras binding IgE from the majority of sera were identified, the grafted patches of which overlapped with previously published epitopes. Patterns of IgG1 and IgG4 binding to the chimeric proteins did not correspond to the binding patterns of IgE. Sera of 19 of 30 birch pollen-allergic patients contained low amounts of IgE to bacterial homologs. Bacterial proteins were able to partially inhibit IgE binding to Bet v 1. CONCLUSION: Epitopes recognized by Bet v 1-specific antibodies from birch pollen-allergic patients are specific to each patient and differ between IgE, IgG1, and IgG4.