ABSTRACT Background IgE is the central driver of allergic responses. Prior studies have defined the conformation of the IgE Fc fragment bound to the FcεRIα ectodomain and the dynamic properties of the IgE Fc. It remains unknown, how these prior studies translate to the complex of a full antibody including the Fab arms with the receptor. Methods For structural analysis, crystallography, cryo‐EM and negative stain EM (ns‐EM) were combined. IgE variants were analyzed by mediator release and CD23 binding assays. Results An ensemble of 10 cryo‐EM structures of the full‐size IgE FcεRIα complex was obtained revealing that the receptor bound IgE adopts a pronounced T‐like conformation. Either Fab arm may rotate up to 40°. Two additional conformations with different arrangements of the Fab arms were captured in ns‐EM. The introduction of additional flexibility into the Fab‐Fc hinge does not compromise the biological activity of IgE, suggesting that the observed conformations of the IgE Fab‐Fc hinge exhibit equivalent biological function. Comparison of the full IgE receptor complex with recent cryo‐EM structures of the intact receptor reveals that FcεRI conformations differ markedly by the orientation of the ectodomain. Hence, our ensemble of IgE FcεRIα structures including the Fab arms enabled critical evaluation of FcεRI conformations. Conclusion Our data reveal the architecture of a full‐size IgE antibody bound to its receptor and a new layer of dynamics in FcεRIα bound IgE on top of the well‐established IgE Fc conformations. Development of novel anti‐IgE therapeutics may take into account these properties of FcεRIα bound IgE.

The presence of allergen-specific IgE in serum is a biomarker for allergic disease. Specific IgE antibodies for research and diagnostics, however, remain scarce. In contrast to prototypic antibodies, camelid species have evolved single domains as moiety for antigen recognition. These so-called nanobodies represent a versatile platform for the development of diagnostic and therapeutic approaches. In this study, we aimed for generating nanobodies and derived IgE formats from an extract-shaped immune repertoire. Timothy grass pollen represents a complex, but well-defined mixture of individual allergens. Therefore, a repertoire library from a timothy grass pollen extract immunised llama was established. The selection by phage display yielded 3 nanobodies with immunoreactivity to the extract. IgE-like nanobody-based human IgE (nb-hIgE) antibodies were produced in mammalian cells and assessed in different immunoassays and commercial platforms. Immunoblotting and diagnostic ImmunoCap analysis of single timothy grass pollen allergens identified the major allergens Phl p 6 and Phl p 4 as targets. Assessment of immunoreactivity further documented significant molecular cross-reactivity with pollen extract of different grass species and variant presence of allergens within extracts of Pooideae grasses. In summary, our study shows that extract-based immunisation enables the generation of allergen-specific nanobodies and derived nb-hIgE formats linking nanobody technologies with allergological applications.

Immunoglobulin E is a mammal specific antibody isotype supporting the immune response against parasites and venoms, but also a driver of allergic responses. Prior studies have defined the conformation of the IgE Fc fragment bound to the cell surface receptor FcεRIα and the dynamic properties of the IgE Fc. It remains unknown, how these prior studies translate to the complex of a full antibody including the Fab arms with the receptor. Here we show that in a cryo-EM structure of the IgE FcεRIα complex, IgE adopts a T-like conformation where the antigen binding Fab arms may be parallel to the cell membrane. Two additional conformations are captured in negative stain EM (ns-EM) where the arrangements of the Fab arms differ from the cryo-EM conformation. Small angle scattering data favors the FcεRIα bound IgE conformation observed by cryo-EM, but the major IgE conformation observed by ns-EM possibly may also occur. In all observed conformations of FcεRIα bound IgE, one Fab arm is fixed relative to the IgE Fc moiety whereas the second Fab may alternate its position. Introduction of flexibility in the Fab-Fc hinge diminishes the biological activity of IgE demonstrating a functional role for the observed defined Fab-Fc hinge conformations. Our data show the organization of a full size antibody on its receptor and reveal a new layer of dynamics in FcεRIα bound IgE on top of the well established spectrum of IgE Fc conformations. Development of novel anti-IgE therapeutics may take into account these distinct FcεRIα bound IgE conformations. Significance statement IgE represents a canonical antibody isotype and is a key molecule for the allergic immune response to environmental triggers driven by mast cells and basophils. The requirements for efficient mediation of IgE’s effects are not fully understood. Here we elucidate the structure of the entire IgE in complex with its high affinity receptor and identify two clearly distinct and dominant conformations, in which one of the Fab arms is fixed relative to the Fc domains. Enforcing IgE flexibility impacts the biological function with potential consequences for the allergic response. This unique behavior makes IgE different from all other isotypes and its understanding sheds light on the allergenic activation of the immune response.

Breast cancer (BC) treatment has traditionally been challenging due to tumor heterogeneity. Bispecific antibodies (bsAbs) offer a promising approach for overcoming these challenges by targeting multiple specific epitopes. In the current study, we designed a new bsAb against the most common BC cell surface proteins (SPs). To achieve this, we analyzed RNA-sequencing data to identify differentially expressed genes, which were further evaluated using Gene Ontology enrichment, Hidden Markov Models, clinical trial data, and survival analysis to identify druggable gene-encoding cell SPs. Based on these analyses, we constructed and expressed a bsAb targeting the mucin 1 (MUC1) and epidermal growth factor receptor (EGFR) proteins, which are the dominant druggable gene-encoding cell SPs in BC. The recombinant anti-MUC1×EGFR bsAb demonstrated efficient production and high specificity for MUC1 and EGFR + cell lines and BC tissue. Furthermore, the bsAb significantly reduced the proliferation and migration of BC cells. Our results suggested that simultaneous targeting with bsAbs could be a promising targeted therapy for improving the overall efficacy of BC treatment.

Immune responses to N-glycan structures from allergens and parasites are often associated with pronounced, high affinity IgE reactivities. Cross-reactive carbohydrate determinants (CCDs) are constituted by modified N-glycan core structures and represent the most frequently recognized epitopes in allergic immune responses. Although recently accepted as potentially allergenic epitopes, the biological and clinical relevance as well as structural and functional characteristics of CCD-specific antibodies remain elusive. In order to gain structural insights into the recognition of CCDs, two specific antibody fragments were isolated from a leporid immune repertoire library and converted into human/leporid IgE and IgG formats. The antibody formats were assessed by ELISA and surface plasmon resonance, structural and functional analyses were performed by X-ray crystallography, mediator release, and ELIFAB assays. The recombinant IgE exhibited highly specific interactions with different types of CCDs on numerous CCD-carrying glycoproteins. Crystal structures of two CCD-specific antibodies, one of which in complex with a CCD-derived disaccharide emphasize that mechanisms of core glycan epitope recognition are as specific as those governing protein epitope recognition. The rIgE triggered immediate cellular responses via FcεRI cross-linking and mediated facilitated antigen presentation by binding of IgE/antigen complexes to CD23, a process that also could be blocked by IgG of allergic patients. Our study provides evidence for the relevance of N-glycan recognition in T H 2 responses and corroborates that IgE and IgG antibodies to ubiquitous carbohydrate epitopes can be equivalent to those directed against proteinaceous epitopes with implications for diagnostic and immunotherapeutic concepts.

For the canonical antibody isotypes, antigen recognition is driven by the variable regions of both heavy and light chains. In contrast, single-domain antibodies or nanobodies (nb) are the antigen-binding moiety of heavy chain only antibodies occurring in camelid species and cartilaginous fish.1, 2 Their small-size, high-yield production, and stability render nanobodies versatile building blocks for the development of binders and unorthodox activatory or inhibitory multi-domain derivatives.3 In the allergological context, single nanobodies were used as anti-IgE molecules and as binding moiety for allergens.4, 5 Our aim was to generate allergen-specific nanobodies and establish a nanobody-based IgE format in the context of hymenoptera venom allergy (Figure 1A,B). In venom-allergic patients, the IgE response is typically directed to a set of major and minor allergens.6 The best-characterized and most abundant honeybee venom (HBV) allergens are phospholipase A2 (Api m 1) and hyaluronidase (Api m 2) (Figure S1A). Therefore, native Api m 1 and recombinant Api m 2 were used for llama immunization (Figure S1B,C). Immune libraries were generated from peripheral blood mononuclear cells. Enrichment by phage display and immunoreactivity of individual phage clones was shown by ELISA (Figure S2). After expression and purification from bacterial supernatant, the nanobodies showed the expected molecular masses in SDS-PAGE and immunoreactivity to their target in ELISA (Figure S3A–C). Exemplary nanobodies against Api m 1 (AM1-1 and AM1-3) and against Api m 2 (AM2-A1 and AM2-C2) were then converted into homodimeric IgE formats by fusion to IgE CH2-4 domains. After purification from supernatant of stably transfected HEK293 cells, SDS-PAGE and immunoblotting analyses corroborated proper dimerization of the nb-IgE (Figure 1C, Figure S4). Expression yields of 10–20 mg/L pointed at a favorable performance of the nb-IgE in mammalian hosts as compared to the limited yields often observed for entire IgE antibodies.7 The nb-IgEs remained reactive to both their particular target allergen and the FcεRIα in ELISA (Figure 1D). The antibodies exhibited sIgE reactivity in Euroline assays to HBV and individual allergens without cross-reactivity to other HBV or yellow jacket venom allergens (Figure 2A,B). Furthermore, individual nb-IgE clones were applied on the ImmunoCAP test system. Concentration-dependent sIgE reactivity was detected for Api m 1, Api m 2, and HBV (Figure 2C) corresponding well to the total IgE levels. The sIgE reactivity against Api m 1 was found highly comparable to that of HBV. In contrast, a clearly reduced IgE reactivity to HBV compared with the component Api m 2 pointed toward a lower sensitivity of HBV for patients with sensitization to Api m 2 possibly due to a limited accessibility of immunoreactive Api m 2 in the test. Furthermore, an oligoclonal artificial human serum was established by combining the 4 nb-IgE against Api m 1 and Api m 2 in equimolar concentration. Assessment of sIgE reactivity verified the added sIgE levels for HBV, Api m 1, and Api m 2 (Figure 2D). Notably, the reduced sIgE level for HBV reflected the reduced reactivity of the individual Api m 2-specific nb-IgE to HBV. Hence, artificial sera comprising molecularly defined IgE surrogates of adjustable concentration might replace human sera in applications such as assessment of diagnostic test systems, round-robin tests, and diagnostic and therapeutic extracts, but also mechanistical analyses in basic research. In summary, we have established nanobodies as a toolbox for the generation of important downstream formats like the novel nb-IgE. This platform also paves the way for the generation of other formats including blocking IgG formats, and hence, has the potential to provide advanced tools for diagnostics, functional analyses, and interventional studies. Gratefully acknowledged is the excellent technical assistance by Nanna Breum Nielsen, Britta Dorn, and Manuel Schulze-Dasbeck. Figure 1 A and B was created with BioRender.com. This study was supported by the Novo Nordisc Foundation, grant NNF19OC0058484, and the Independent Research Foundation Denmark, grant 9041-00291A. A patent application covering part of the manuscript has been filed by JBA, CS, MM, and ES. The other authors have no financial conflict of interest and nothing to disclose. Appendix S1 Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Minibodies (single-chain Fv-CH3) are fusion proteins of a single-chain variable fragment (scFv) to the human IgG1 CH3 domain. They exhibit superior properties as compared to whole antibodies due to their smaller size and less complex composition, and also as compared to scFvs due to the two antigen-binding domains, for immunotherapy and imaging of various carcinomas including breast cancer. In the current study, efficient production of the recombinant anti-MUC-1 minibody for its dominant format (VH-VL) was obtained in the periplasmic space of the Escherichia coliBL21 (DE3) expression system. The active recombinant protein was successfully purified from soluble fraction. Functional assays presented the in vitro targeting properties and specificity of the expressed anti-MUC-1 HL minibody in the MUC-1 positive cell lines compared to normal cell.

Matricellular proteins are involved in the crosstalk between cells and their environment and thus play an important role in allergic and inflammatory reactions. Periostin, a matricellular protein, has several documented and multi-faceted roles in health and disease. It is differentially expressed, usually upregulated, in allergic conditions, a variety of inflammatory diseases as well as in cancer and contributes to the development and progression of these diseases. Periostin has also been shown to influence tissue remodelling, fibrosis, regeneration and repair. In allergic reactions periostin is involved in type 2 immunity and can be induced by IL-4 and IL-13 in bronchial cells. A variety of different allergic diseases, among them bronchial asthma and atopic dermatitis (AD), have been shown to be connected to periostin expression. Periostin is commonly expressed in fibroblasts and acts on epithelial cells as well as fibroblasts involving integrin and NF-κB signalling. Also direct signalling between periostin and immune cells has been reported. The deposition of periostin in inflamed, often fibrotic, tissues is further fuelling the inflammatory process. There is increasing evidence that periostin is also expressed by epithelial cells in several of the above-mentioned conditions as well as in cancer. Augmented periostin expression has also been associated with chronic inflammation such as in inflammatory bowel disease (IBD). Periostin can be expressed in a variety of different isoforms, whose functions have not been elucidated yet. This review will discuss potential functions of periostin and its different isoforms in allergy and inflammation.

BACKGROUND:IgE is the central antibody isotype in TH2-biased immunity and allergic diseases. The structure of intact IgE and the impact of IgE-targeting molecules on IgE however remain elusive. In order to obtain insights into IgE biology and the clinical impact, we aimed for structure determination of IgE and the complex of IgE with the anti-IgE antibody ligelizumab.METHODS:Structures of two distinct intact IgE with specificity for cross-reactive carbohydrate determinants and Der p 2 as well as complexes of ligelizumab-Fab with IgE and IgE Fc were assessed by negative stain electron microscopy and solution scattering. Inhibition of IgE binding and displacement of receptor-bound IgE were assessed using cellular assays, basophil activation testing and ELIFAB assays.RESULTS:Our data reveal that the investigated IgE molecules share an overall rigid conformation. In contrast to the IgE Fc fragment, the IgE Fc in intact IgE is significantly less asymmetrically bent. The proximal and the distal Fabs are rigidly tethered to the Fc. Binding of ligelizumab to IgE in a 2:1 stoichiometry induces an extended and twofold symmetrical conformation of IgE, which retains a rigid Fab-Fc architecture. Analyses of effector cell activation revealed that ligelizumab inhibits IgE binding without displacing receptor-bound IgE. Together with an interference of CD23 binding, the data underline a functional activity similar to omalizumab.CONCLUSIONS:Our data reveal the first structures of intact IgE suggesting that the IgE Fab is fixed relative to the Fc. Furthermore, we provide a structural rationale for the inhibitory mechanism of ligelizumab.

Yellow jacket and honey bee stings are among the most common elicitors of severe anaphylactic reactions in Europe.1 The only causal therapy to prevent future anaphylactic reactions is allergen immunotherapy (AIT). AIT is well established for patients allergic to honey bee (HBV) and yellow jacket venom (YJV).2 However, the efficacy of AIT with HBV is lower than with YJV.3 So far, the reason for this difference is not clear. Recently, we have identified Api m 10 as a major allergen in HBV4 and described that a dominant Api m 10 sensitization is associated with an increased risk for treatment failure of AIT in HBV-allergic patients.5 In addition, we and others reported that Api m 10 is absent or underrepresented in several therapeutic HBV preparations.5-7 To characterize the IgE response to Api m 10 in detail, we now analyzed linear IgE epitopes recognized by patients allergic to HBV. Synthetic 15-mer peptides with 12 amino acids overlay were generated (>95% purity, CASLO ApS) spanning the whole amino acid sequence of Api m 10 (isoform variant 1; 204 amino acids) and coupled to macroarrays (Macro Array Diagnostics). Recombinant (r) HBV allergens (rApi m 1, 2, 3, and 10) served as positive controls (for more details see this article's online supporting information). IgE binding was evaluated in sera obtained from HBV-allergic patients prior to initiation of AIT that displayed specific (s) IgE to Api m 10 (>0.35kUA/L) as determined by ImmunoCAP (ThermoFisher Scientific) (Api m 10-sIgE median [range]: 3.79 [0.59-66.6] kUA/L; n = 40) and (for more details see online supporting information) in sera from healthy controls without a history of HBV allergy and without Api m 10 sensitization (n = 10). All patients' sera displayed positive IgE reactivity for rApi m 10 in the macroarray analysis (5.37 [0.17-24] kUA/L) that showed a reasonable correlation with the values obtained by ImmunoCAP (r = .7041, P < .0001). Individual sera displayed IgE reactivity to up to 29 Api m 10 peptides, and collectively 43 peptides of the 64 tested were recognized by IgE of HBV-allergic patients. Three Api m 10 regions were identified that were recognized by more than 40% of tested sera. Higher numbers of recognized peptides correlated with higher Api m 10-sIgE concentrations (r = .6500, P < .0001; data not shown). Healthy control sera did not show IgE binding to any of the Api m 10 peptides or to any of the HBV allergens tested (data not shown). Interestingly, one peptide, P54 (Api m 10160-174; amino acid sequence: ADSDVTTLPTLIGKN), was recognized by 100% of the Api m 10-positive sera and hence represents the dominant linear IgE epitope of Api m 10 (Figure 1A). IgE reactivity to P54 was higher than to any other Api m 10 peptide (Figure 1A) and represented on average 67% of the total Api m 10 peptide-sIgE for the tested patient cohort (data not shown). The correlation of Api m 10-sIgE and P54-sIgE (as determined by macroarray) showed a positive association (Figure 1C; Spearman's rank correlation coefficient; r = .7783, P < .0001). To verify the significance of P54 in HBV allergy, ImmunoCAP inhibition experiments were performed. Preincubation of sIgE Api m 10-positive sera (n = 7) with increasing concentrations of P54 (1-100 µg/mL) resulted in a dose-dependent inhibition of IgE reactivity to rApi m 10 as determined by ImmunoCAP (Figure 2A), while no inhibition was observed with the control peptide P12 (n = 3). Vice versa, preincubation of serum with rApi m 10 inhibited binding of IgE to the Api m 10 peptides on the macroarray (Figure 2B), while binding of sIgE to other HBV allergens on the macroarray (such as rApi m 2, 3, or 4) was not inhibited (for more details see online supporting information). Api m 10 is a major allergen in HBV and a marker allergen to discriminate HBV from YJV sensitization.8 In addition, a dominant Api m 10 sensitization has been associated with an increased risk for AIT treatment failure in HBV allergy.5 Although Api m 10 is a major allergen in HBV allergy, the quantity of Api m 10 in the venom is of low abundance (approx. 1%).9 In addition, Api m 10 is easily degraded in reconstituted venom preparation6 and has been reported to be underrepresented or absent in therapeutic venom preparations,5-7 suggesting a potential link with treatment failure in dominantly Api m 10 sensitized HBV-allergic patients. However, the lack of results from prospective clinical studies makes it difficult to exclude other predominant sensitizations as risk factors for treatment failure and to conclude that the therapeutic failure of HBV VIT is really due to the lack of Api m 10 in therapeutic preparations. Indeed, currently we do not even know whether and how much Api m 10 is required for effective HBV AIT in Api m 10 sensitized patients. Provided that a link between lack of Api m 10 and lower therapeutic efficacy can be confirmed in prospective studies, strategies to overcome this lack may be of particular interest. In theory, this lack could be compensated by spiking therapeutic HBV preparations with rApi m 10. However, due to the intrinsic instability of Api m 10, alternative strategies may be needed. In this context, the identification of a major Api m 10 IgE epitope, such as P54, that is recognized by all tested sera may become highly relevant. A short immunodominant peptide is easier to produce and thus may have relevant advantages over the use of full-length rApi m 10. Mapping of corresponding T-cell epitopes of Api m 10 is currently underway to address the relevance of P54 for the induction of protective T-cell responses. Finally, due to its dominant IgE reactivity, P54 may substitute rApi m 10 in diagnostic approaches, for example, in a peptide-based sIgE assay. Larger patient samples are needed to address the feasibility and assay performance of this approach. In summary, we identified a major IgE epitope of the HBV allergen Api m 10 that is recognized by all sera from HBV-allergic patients sensitized to Api m 10. This may be of use for diagnostic purposes and potentially also for augmentation of therapeutic HBV preparations that lack Api m 10 immunoreactivity. The assistance of Andrea Komann, Heike Neumann, Medical Centre—University of Freiburg, Britta Dorn, Justus-Liebig University Gießen, Germany, and Peter Forstenlechner, Macro Array Diagnostics, Vienna, Austria, is gratefully acknowledged. MMR, CM, AJ, MM, ES, SM and AR declare that they have no potential conflict of interest. WP has given lectures for ALK-Abelló and Thermo Fisher Scientific, served on advisory boards for ALK-Abelló and received research funding from ALK-Abelló. TJ has given lectures for ALK-Abelló, Allergy therapeutics/ Bencard, Novartis, and Thermo Fisher Scientific, served on advisory boards for ALK-Abelló, Allergopharma, Allergy therapeutics/ Bencard, and Novartis and received research funding by ALK-Abelló, Novartis, and Thermo Fisher Scientific. This study is funded by a grant (NR66-0004) of the von-Behring-Röntgen-Stiftung and by a research grant (7/2017GI) of the University Medical Center Giessen and Marburg (UKGM) to TJ. Patent pending, registration number EP19199738. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Core fucosylation of N‐glycans is catalyzed by fucosyltransferase 8 and is associated with various types of cancer. Most reported fucosyltransferase inhibitors contain non‐drug‐like features, such as charged groups. New starting points for the development of inhibitors of fucosyltransferase 8 using a fragment‐based strategy are presented. Firstly, we discuss the potential of a new putative binding site of fucosyltransferase 8 that, according to a molecular dynamics (MD) simulation, is made accessible by a significant motion of the SH3 domain. This might enable the design of completely new inhibitor types for fucosyltransferase 8. Secondly, we have performed a docking study targeting the donor binding site of fucosyltransferase 8, and this yielded two fragments that were linked and trimmed in silico. The resulting ligand was synthesized. Saturation transfer difference (STD) NMR confirmed binding of the ligand featuring a pyrazole core that mimics the guanine moiety. This ligand represents the first low‐molecular‐weight compound for the development of inhibitors of fucosyltransferase 8 with drug‐like properties.

Molecular cross-reactivity caused by allergen homology or cross-reactive carbohydrate determinants (CCDs) is a major challenge for diagnosis and immunotherapy of insect venom allergy. Venom phospholipases A1 (PLA1s) are classical, mostly non-glycosylated wasp and ant allergens that provide diagnostic benefit for differentiation of genuine sensitizations from cross-reactivity. As CCD-free molecules, venom PLA1s are not causative for CCD-based cross-reactivity. Little is known however about the protein-based cross-reactivity of PLA1 within vespid species. Here, we address PLA1-based cross-reactivity among ten clinically relevant Hymenoptera venoms from Neotropical and temperate regions including Polybia paulista (paulistinha) venom and Vespula vulgaris (yellow jacket) venom. In order to evaluate cross-reactivity, sera of mice sensitized with recombinant PLA1 (rPoly p 1) from P. paulista wasp venom were used. Pronounced IgE and IgG based cross-reactivity was detected for wasp venoms regardless the geographical region of origin. The cross-reactivity correlated well with the identity of the primary sequence and 3-D models of PLA1 proteins. In contrast, these mice sera showed no reaction with honeybee (HBV) and fire ant venom. Furthermore, sera from patients monosensitized to HBV and fire ants did not recognize the rPoly p 1 in immunoblotting. Our findings reveal the presence of conserved epitopes in the PLA1s from several clinically relevant wasps as major cause of PLA1-based in vitro cross-reactivity. These findings emphasize the limitations but also the potential of PLA1-based HVA diagnostics.

Background: Although venom immunotherapy (VIT) with partially purified venom (Pharmalgen®) is highly effective in preventing systemic reactions in yellow jacket (YJ) venom allergic patients, its cost-effectiveness has recently been questioned. Up-dosing is particularly expensive while the reductive effect on adverse reactions to VIT has never been demonstrated. Objective: Initiation of YJ-VIT with 100 μg Pharmalgen® was compared to up-dosing with a modified rush regimen in terms of adverse reactions, clinical efficacy, immunological effects and costs. Methods: In this randomized clinical trial, eighteen patients received YJ-VIT initiation by a single injection of 100 μg Pharmalgen® followed by 3 booster injections every 4 weeks, or, alternatively, 12 updose injections in 7 weeks followed by one booster injection 5 weeks later. Adverse reactions were registered. Clinical efficacy was assessed by a sting challenge and compared to sting challenge outcomes in twenty untreated patients. Allergen-blocking IgG capacity was assessed with the ELIFAB assay. Direct medical costs and indirect costs were estimated as actual costs from a societal perspective. Results: No systemic adverse reactions were observed to either VIT regimen and both regimens offered complete protection against systemic reactions to subsequent sting challenges. In contrast, 20% of untreated patients reacted systemically to a sting challenge. Allergen blocking IgG capacity was increased equally by the two VIT regimens. The 100-μg regimen and modified rush regimen cost $323.76 and $856.52, respectively. Conclusions and clinical relevance: This is the first indication that YJ-VIT may be initiated at the maintenance dose in selected patients, thereby avoiding time-consuming regimens and reducing costs. Further studies on the safety of this approach are warranted.

Insect venom hypersensitivity represents an important health problem in Neotropical regions. In Latin America, Hymenoptera stinging is reported to be 1 of the 3 major causes (14%) of severe allergic reactions.1Sole D. Ivancevich J.C. Borges M.S. Coelho M.A. Rosario N.A. Ardusso L.R. et al.Anaphylaxis in Latin America: a report of the online Latin American survey on anaphylaxis (OLASA).Clinics (Sao Paulo). 2011; 66: 943-947Crossref PubMed Scopus (60) Google Scholar In Europe and Northern America, venoms and allergens of clinically relevant species, honeybee and yellow jacket, have been extensively characterized. Allergy testing using venoms and recombinant components can yield precise information on sensitization profiles and even potential risk factors for venom immunotherapy.2Frick M. Fischer J. Helbling A. Rueff F. Wieczorek D. Ollert M. et al.Predominant Api m 10 sensitization as risk factor for treatment failure in honey bee venom immunotherapy.J Allergy Clin Immunol. 2016; 138: 1663-1671.e9Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar In contrast, little is known about the venoms from Neotropical species. Clinically relevant Hymenoptera species in South America include honeybee, fire ants, and importantly various wasps endemic in different regions of the continent. Systematic studies showed that in contrast to Europe and Northern America, the most commonly found wasp species in Neotropical regions belong to the Epiponini tribe of the Polistinae subfamily including, for example, the genera Polybia, Apoica, and Agelaia.3Locher G.D.A. Togni O.C. Silveira O.T. Giannotti E. The social wasp fauna of a Riparian forest in Southeastern Brazil (Hymenoptera, Vespidae).Sociobiology. 2014; 61: 225-233Crossref Scopus (25) Google Scholar Despite the high incidence, the severity of sting events, and the broad diversity of clinically relevant Hymenoptera identified in this geographical zone, venom allergy diagnosis is essentially based on either commercial venom extracts from European and North American species, or noncommercial venoms from endemic species. The use of extracts from other than the relevant species and the potentially limited cross-reactivity might result in lower sensitivity. Although the use of components in Hymenoptera venom allergy is now state-of-the-art, important molecular allergens from endemic species are not available for molecular diagnosis.4Perez-Riverol A. Justo-Jacomini D.L. Zollner Rde L. Brochetto-Braga M.R. Facing Hymenoptera venom allergy: from natural to recombinant allergens.Toxins (Basel). 2015; 7: 2551-2570Crossref PubMed Scopus (24) Google Scholar Extract-based diagnostics however are often prone to high levels of cross-reactivity due to molecular similarity of the venoms and the presence of the cross-reactive carbohydrate determinants (CCDs).2Frick M. Fischer J. Helbling A. Rueff F. Wieczorek D. Ollert M. et al.Predominant Api m 10 sensitization as risk factor for treatment failure in honey bee venom immunotherapy.J Allergy Clin Immunol. 2016; 138: 1663-1671.e9Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar Insect venom CCDs are defined by an alpha-1,3-linked fucose residue at the innermost N-acetylglucosamine of the N-glycan of mostly oligomannosidic type.5Homann A. Schramm G. Jappe U. Glycans and glycan-specific IgE in clinical and molecular allergology: sensitization, diagnostics, and clinical symptoms.J Allergy Clin Immunol. 2017; 140: 356-368Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar Extensive CCD-based cross-reactivity of honeybee venom (HBV) and yellow jacket venom (YJV), and protein-based cross-reactivity between allergens from Polistes and YJV are the most relevant problems in Hymenoptera venom allergy diagnostics in Europe and Northern America, respectively.6Golden D.B. New directions in diagnostic evaluation of insect allergy.Curr Opin Allergy Clin Immunol. 2014; 14: 334-339Crossref PubMed Scopus (30) Google Scholar Irrespective of a potential clinical relevance of CCD, carbohydrate-related cross-reactivity causes up to 69% to 75% of double-positive test results to HBV and YJV.7Seismann H. Blank S. Braren I. Greunke K. Cifuentes L. Grunwald T. et al.Dissecting cross-reactivity in hymenoptera venom allergy by circumvention of alpha-1,3-core fucosylation.Mol Immunol. 2010; 47: 799-808Crossref PubMed Scopus (64) Google Scholar Failure in the identification of the culprit venom due to CCD-related cross-reactivity often leads to the inclusion of multiple venoms in the immunotherapy, which results in increasing risks of de novo sensitizations. Recently, we reported that the venoms of European and North American Polistes species are devoid of CCDs.8Blank S. Neu C. Hasche D. Bantleon F.I. Jakob T. Spillner E. Polistes species venom is devoid of carbohydrate-based cross-reactivity and allows interference-free diagnostics.J Allergy Clin Immunol. 2013; 131: 1239-1242Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar So far, we interpreted the lack of alpha-1,3 core fucosylation primarily as a biological peculiarity because CCDs are found within various species ranging from plants to helmiths and in other Hymenoptera such as honeybee and yellow jackets. The presence of CCDs and therefore the molecular basis for cross-reactivity among venoms of Neotropical insects remained largely unexplored. Here, we addressed the CCD-based IgE reactivity of 6 venoms of Neotropical wasps as compared with HBV, and 2 venoms of common European wasps: Vespula vulgaris (yellow jacket) and Polistes dominula (Mediterranean wasp). After harvesting of the nests, venoms were collected manually using 1000 venom glands of each species, and the quality and composition were analyzed by SDS-PAGE (Fig 1; see the Methods section in this article's Online Repository at www.jacionline.org). Notably, the proteins within one genus appear similar although pronounced variations in size and relative intensity were found for different genera. The general presence of oligomannosidic N-linked glycans was evaluated using biotinylated Concanavalin A, a lectin that recognizes α-linked mannose residues in the predominantly high mannose-type glycans found on insect venom allergens. Presence of α1,3-core fucosylation and therefore CCDs was detected using an anti–horseradish peroxidase (HRP) serum. HBV (i1), YJV (i3), P dominula venom (i4), and CCD-positive rVes v 3 were used as controls. Immunoblotting analyses revealed the presence of several glycoproteins in all the venoms tested (Fig 2, A; see also data in this article's Online Repository). Presence of CCDs was detected for HBV, YJV, and rVes v 3. In contrast and similar to P dominula venom, none of the venom extracts of Neotropical wasps was recognized by the anti-HRP antibodies. ELISA analyses under nondenaturing conditions (Fig 2, B; see also data in this article's Online Repository) corroborated the immunoblotting results. These data suggest that the venom components of all the Neotropical wasps analyzed here lack α1,3-core fucosylation and therefore presence of CCDs (Fig 2, A). To verify the lack of CCD-reactivity, we selected sera of allergic patients with elevated specific IgE (sIgE) level to CCDs. As shown in Fig 2, C, none of the Neotropical wasp venoms was recognized by the patients' sera in ELISA while pronounced signals were obtained for the CCD-positive HBV, YJV, and rVes v 3. Our finding supports previous results showing that venoms of 6 Polistes species from Europe and Northern America are devoid of CCDs.8Blank S. Neu C. Hasche D. Bantleon F.I. Jakob T. Spillner E. Polistes species venom is devoid of carbohydrate-based cross-reactivity and allows interference-free diagnostics.J Allergy Clin Immunol. 2013; 131: 1239-1242Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar Taxonomically, the Neotropical wasps assessed here belong to a different tribe of the Polistinae subfamily. Together the absence of CCDs found for all members of this subfamily suggests a general absence in all Polistinae and provides a new perspective on the biology of CCDs. Recent findings suggest developmental implications of the α1,3-core fucosylation in plants, supporting also the neural relevance of the epitope in insects.9Pedersen C.T. Loke I. Lorentzen A. Wolf S. Kamble M. Kristensen S.K. et al.N-glycan maturation mutants in Lotus japonicus for basic and applied glycoprotein research.Plant J. 2017; 91: 394-407Crossref PubMed Scopus (21) Google Scholar Against this background not the absence of CCDs but their presence on HBV and YJV might represent the biological peculiarity. The lack of CCDs in all the clinically relevant wasps from Southern America has major implications for the procedures currently used for allergy diagnosis. While allergen components remain unavailable, crude venoms from these species allow for reliable determination of CCD-independent sensitization. Therefore, the differentiation of sensitization to HBV or wasp venom is significantly facilitated in particular in those patients having sIgE to CCDs. Because of frequent lack of information, positive sIgE to these venoms now can be interpreted as a purely venom-based sensitization in patients with and without sIgE to CCDs. Moreover, the quantitative determination of sIgE levels to venoms of wasps from different tribes might allow the identification of culprit insects even within taxonomically related species. The rational design of strategies for differential allergy diagnosis involving carefully selected venoms and recombinant components will contribute to dissect the complex picture of wasp venom allergy in Neotropical regions worldwide. We acknowledge the financial support received from FAPESP (Sao Paulo Research Foundation) (grant no. 2014/13936-7), CAPES-PDSE (grant no. 88881.132448/2016-01), and CAPES-DS and Postgraduate Program of Biological Sciences (Cellular and Molecular Biology) at UNESP, Rio Claro (for providing doctoral scholarships to A.P.R.). Nests from Neotropical wasps were captured around or within the campus of Universidade Estadual Paulista “Júlio de Mesquita Filho” (UNESP), Rio Claro, SP, Brazil (licensed by Sisbio, No. 58500). The wasps were immediately anesthetized at low temperature (−80°C). The venom glands of 1000 insects were extracted using sterile tweezers, washed with sterile ultrapure water, and suspended in a protease inhibitor mix (2 mM leupeptine and 1 mM phenylmethylsulfonyl fluoride; Sigma-Aldrich, St Louis, Mo). Then, the gland suspensions were macerated, washed 3 times with the protease inhibitor mix, and centrifuged at 10,000g for 15 minutes at 4°C. The resulting supernatants were collected and stored at −20°C until use. HBV (i1), YJV (i3), and P dominula venom (i4) were obtained from Euroimmun (Lübeck, Germany). Recombinant Ves v 3 was produced in HighFive insect cells, which are known to establish CCDs. SDS-PAGE was performed using standard protocols according to Laemmli,E1Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature. 1970; 227: 680-685Crossref PubMed Scopus (207012) Google Scholar using a Mini-Protean Tetra Cell System (BioRad, Sao Paulo, Brazil). After running, the gels were stained with Coomassie Brilliant Blue R-250 (CBB) (Sigma Aldrich, Sao Paulo, Brazil). For immunoblotting analysis for detection of glycosylation and CCDs, the venoms were subjected to (12%) SDS-PAGE and then transferred to a 0.22-μm nitrocellulose membrane using a semi-dry system (Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell, Bio-Rad). For HRP detection the membranes were blocked for 30 minutes at room temperature with 4% nonfat dry milk powder (AppliChem, Darmstadt, Germany) in TBS buffer (20 mM Tris-HCl and 150 mM NaCl; pH 7.4). The membrane was then incubated overnight (4°C, 150 rpm) with rabbit anti-HRP serum (1:5000) diluted in TBS buffer with 2% nonfat dried milk powder. After washing 3 times with TBS buffer containing 0.5% Tween, the membrane was incubated for 1 hour at room temperature with goat anti-rabbit IgG (Sigma-Aldrich, Copenhagen, Denmark) (1:20,000) conjugated with alkaline phosphatase. Bound antibodies were visualized using 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitro blue tetrazolium (NBT) as substrate (Sigma, Copenhagen, Denmark). For the detection of glycosylation, the membranes containing the venoms were blocked using a Blocking Solution (Vector Labs, Peterborough, United Kingdom) (1 hour at room temperature). After washing with TBS-T, 2 μg/mL of biotinylated Concanavalin A (Vector Labs), a jack bean lectin that binds internal and nonreducing terminal α-D-mannosyl and α-D-glucosyl groups in glycans, was added. After incubation for 30 minutes at room temperature, the membranes were washed and then incubated with streptavidin conjugated to alkaline phosphatase (1:70,000) (Sigma) for 30 minutes at room temperature. Bound lectin then was visualized as above. The patient sera were selected for a high level of sIgE to CCDs (class 5) and negative sIgE to HBV and YJV components as determined by Euroline assay measurements. For detection of CCDs by the rabbit anti-HRP serum and sIgE in patient sera, 384-well microtiter plates (Nunc, Thermo Fisher Scientific, Ulm, Germany) were coated with 20 ng of the venoms (4°C, overnight). Coated plates were then blocked with 4% nonfat dry milk powder (AppliChem, Darmstadt, Germany) in TBS at room temperature for 1 hour. After washing with TBS, either rabbit anti-HRP serum (1:5000) or patient sera (1:2) (diluted in TBS and 2% nonfat dry milk powder) were added to the plates and incubated overnight at 4°C. A goat anti-rabbit IgG (1:20,000) (Sigma-Aldrich) and a goat anti-human IgE (1:20,000) (Biozol, Eching, Germany), both conjugated to alkaline phosphatase, were added for detection of sIgE to HRP, or insect CCDs. After incubation for 1 hour with gentle shaking at room temperature, the detection was performed by adding 50 μL of the substrate solution per well (5 mg/mL 4-nitrophenylphosphate; AppliChem, Darmstadt, Germany). The absorbance was read at 405 nm. A signal that duplicates the means of the negative control was interpreted as positive result. Three independent experiments for each type of antibodies were conducted.E2Seismann H. Blank S. Braren I. Greunke K. Cifuentes L. Grunwald T. et al.Dissecting cross-reactivity in hymenoptera venom allergy by circumvention of alpha-1,3-core fucosylation.Mol Immunol. 2010; 47: 799-808Crossref PubMed Scopus (91) Google Scholar

Anti-IgE therapeutics interfere with the ability of IgE to bind to its receptors on effector cells. Here we report the crystal structure of an anti-IgE single-domain antibody in complex with an IgE Fc fragment, revealing how the antibody inhibits interactions between IgE and the two receptors FcεRI and CD23. The epitope overlaps only slightly with the FcεRI-binding site but significantly with the CD23-binding site. Solution scattering studies of the IgE Fc reveal that antibody binding induces a half-bent conformation in between the well-known bent and extended IgE Fc conformations. The antibody acts as functional homolog of CD23 and induces a closed conformation of IgE Fc incompatible with FcεRI binding. Notably the antibody displaces IgE from both CD23 and FcεRI, and abrogates allergen-mediated basophil activation and facilitated allergen binding. The inhibitory mechanism might facilitate strategies for the future development of anti-IgE therapeutics for treatment of allergic diseases.

TH2-biased immunity to parasites and allergens is often associated with increased levels of antigen-specific and high affinity IgE. The role in reacting against minute amounts of target structures and to provoke severe anaphylactic reactions renders IgE a mechanistically outstanding isotype. IgE represents the least abundant serum antibody isotype and exhibits a variety of peculiarities including structure, extensive glycosylation and effector functions. Despite large progress in antibody technologies, however, the recombinant access to isotypes beyond IgG such as IgE still is scarce. The capacity of expression systems has to meet the complex structural conformations and the extensive posttranslational modifications that are indispensable for biological activity. In order to provide alternatives to mammalian expression systems with often low yield and a more complex glycosylation pattern we established the recombinant production of the highly complex IgE isotype in insect cells. Recombinant IgE (rIgE) was efficiently assembled and secreted into the supernatant in yields of >30 mg/L. Purification from serum free medium using different downstream processing methods provided large amounts of rIgE. This exhibited a highly specific interaction with its antigen, therapeutic anti-IgE and its high affinity receptor, the FcεRI. Lectins and glyco-proteomic analyses proved the presence of prototypic insect type N-glycans on the epsilon heavy chain. Mediator release assays demonstrated a biological activity of the rIgE comparable to IgE derived from mammalian cells. In summary the expression in insect cells provides rIgE with variant glycosylation pattern, but retained characteristics and biological activity. Therefore our data contribute to the understanding of functional and structural aspects and potential use of the IgE isotype.

Background: Component resolution recently identified distinct sensitization profiles in honey bee venom (HBV) allergy, some of which were dominated by specific IgE to Api m 3 and/or Api m 10, which have been reported to be underrepresented in therapeutic HBV preparations.Objective: We performed a retrospective analysis of component-resolved sensitization profiles in HBV-allergic patients and association with treatment outcome.Methods: HBV-allergic patients who had undergone controlled honey bee sting challenge after at least 6 months of HBV immunotherapy (n = 115) were included and classified as responder (n = 79) or treatment failure (n = 36) on the basis of absence or presence of systemic allergic reactions upon sting challenge. IgE reactivity to a panel of HBV allergens was analyzed in sera obtained before immunotherapy and before sting challenge.Results: No differences were observed between responders and nonresponders regarding levels of IgE sensitization to Api m 1, Api m 2, Api m 3, and Api m 5. In contrast, Api m 10 specific IgE was moderately but significantly increased in nonresponders. Predominant Api m 10 sensitization (>50% of specific IgE to HBV) was the best discriminator (specificity, 95%; sensitivity, 25%) with an odds ratio of 8.444 (2.127-33.53; P = .0013) for treatment failure. Some but not all therapeutic HBV preparations displayed a lack of Api m 10, whereas Api m 1 and Api m 3 immunoreactivity was comparable to that of crude HBV. In line with this, significant Api m 10 sIgG(4) induction was observed only in those patients who were treated with HBV in which Api m 10 was detectable.Conclusions: Component-resolved sensitization profiles in HBV allergy suggest predominant IgE sensitization to Api m 10 as a risk factor for treatment failure in HBV immunotherapy.

Table of contents ORAL ABSTRACTS Symposium 1: Biochemistry, structure and environment of the allergen: what makes a protein an allergen? O1 Two cell-membrane peptidases carrying galactose-alpha-1,3-galactose are implicated in delayed anaphylactic reactions upon pork kidney ingestion in patients with IgE-antibodies to alpha-Gal Christiane Hilger, Kyra Swiontek, Jörg Fischer, François Hentges, Christiane Lehners, Martine Morisset, Bernadette Eberlein, Tilo Biedermann, Markus Ollert O2 Structure solution of Pla l 1 suggests similar folding of Ole e 1-like family members but distinct immunological properties Sabrina Wildner, Teresa Stemeseder, Regina Freier, Peter Briza, Roland Lang, Eva Batanero, Mayte Villalba, Jonas Lidholm, Thomas Hawranek, Fatima Ferreira, Hans Brandstetter, Gabriele Gadermaier Symposium 2: New allergen molecules in the spotlight O3 Identification of the cysteine protease Amb a 11 as a novel major allergen from short ragweed (Ambrosia artemisiifolia) Philippe Moingeon, Rachel Groeme, Julien Bouley, Véronique Bordas, Maxime Le Mignon, Laetitia Bussières, Aurélie Lautrette, Laurent Mascarell, Vincent Lombardi, Véronique Baron-Bodo, Henri Chabre, Thierry Batard, Emmanuel Nony O4 Production and characterization of polybia paulista recombinant antigen 5: a valuable diagnostic tool Karine Marafigo De Amicis, Alexandra Sayuri Watanabe, Daniele Danella Figo, José Roberto Aparecido Dos Santos-Pinto, Mario Sergio Palma, Fabio Fernandes Morato Castro, Jorge Kalil, Therese Wohlschlager, Peter Briza, Sabrina Wildner, Fatima Ferreira-Briza, Gabriele Gadermaier, Keity Souza Santos Symposium 3: Progress in molecular and cellular diagnosis O5 Basophil activation test with recombinant Pru p 3; identifying genuine peach allergic patients Margaretha Faber, Athina Van Gasse, Vito Sabato, Margo M. Hagendorens, Chris H. Bridts, Luc S. De Clerck, Araceli Diaz Perales, Didier Ebo O6 Nanofluidic technology enables rapid, near-patient quantification of allergen-specific IgE Petra Zavadakova, Aurélie Buchwalder, Fabien Rebeaud, Iwan Märki Symposium 4: Relevance of molecular diagnostics for intervention and treatment O7 Longitudinal analysis of Bet v 1-specific epitope repertoires during birch pollen immunotherapy Barbara Gepp, Nina Lengger, Christian Möbs, Wolfgang Pfützner, Christian Radauer, Barbara Bohle O8 A natural CCD-free tool: is polistes sp. venom suitable for polybia paulista diagnosis and therapy? Karine Marafigo De Amicis, Alexandra Sayuri Watanabe, Clovis Eduardo Galvao, Daniele Danella Figo, Jose Roberto Aparecido Santos-Pinto, Mario Sergio Palma, Fabio Fernandes Morato Castro, Jorge Kalil, Fatima Ferreira, Gabriele Gadermaier, Keity Souza Santos Symposium 5: The advent of molecular allergology in epidemiology O9 Peanut oleosins: from identification to diagnostic testing Christian Schwager, Skadi Kull, Frauke Schocker, Jochen Behrends, Wolf-Meinhard Becker, Uta Jappe O10 Endotypes of oral allergy syndrome in childhood: a molecular diagnostic approach Carla Mastrorilli, Salvatore Tripodi, Carlo Caffarelli, Riccardo Asero, Arianna Dondi, Giampaolo Ricci, Carlotta Povesi Dascola, Elisabetta Calamelli, Andrea Di Rienzo Businco, Annamaria Bianchi, Tullio Frediani, Carmen Verga, Iride Dello Iacono, Diego Peroni, Giuseppe Pingitore, Roberto Bernardini, Paolo Maria Matricardi Symposium 6: Molecular AIT: which approaches will make it to market? O11 Mbc4: an innovative molecule to tackle birch pollen and concomitant food allergies Heidi Hofer, Claudia Asam, Michael Hauser, Peter Briza, Martin Himly, Christof Ebner, Fatima Ferreira O12 Challenges and solutions associated with the production of recombinant Bet v 1 allergen as a therapeutic protein Emmanuel Nony, Maxime Le Mignon, Pierrick Lemoine, Karine Jain, Kathy Abiteboul, Monica Arvidsson, Sabina Rak, Philippe Moingeon Clinical Cases: Breakthroughs and headaches from CRD: interactive session CC1 Anaphylaxis caused by lipid transfer proteins: a complex clinical pattern syndrome Inês Mota, Filipe Benito Garcia, Angela Gaspar, Cristina Arêde, Susana Piedade, Graça Sampaio, Graça Pires, Luís Miguel Borrego, Cristina Santa-Marta, Mário Morais-Almeida CC2 IgE sensitization profile in a patient with asteraceae pollen-exotic fruits association Florin-Dan Popescu, Mariana Vieru, Florin-Adrian Secureanu CC3 Food-dependent: exercise induced anaphylaxis. Which component to blame? Rosa Anita Rodrigues Fernandes, Isabel Carrapatoso, Raquel Gomes, Celso Pereira, Ana Todo-Bom CC4 Anaphylaxis to intravenous iron preparations in a patient that tolerates oral administration María Cecilia Martín Fernández De Basoa, Javier Barrios Regio, Juan De Castro Cordova, Antón Fernández Ferreiro CC5 IgE sensitization pattern in an adult patient with oral allergy syndrome to peanuts and pollinosis from southern Romania Florin-Dan Popescu, Mariana Vieru, Florin-Adrian Secureanu CC6 Evidence of specific IgE to plant-derived cross-reactive carbohydrate determinant in a patient with delayed anaphylaxis to red meat Mariana Vieru, Florin-Dan Popescu, Florin-Adrian Secureanu POSTER PRESENTATIONS Poster Session 1: Molecular allergology and epidemiology P1 Atopic children produce stronger and more frequent IgG responses than non-atopic children: longitudinal data from the German MAS birth cohort Olympia Tsilochristou, Serena Perna, Alina Schwarz, Alexander Rohrbach, Antonio Cappella, Laura Hatzler, Carl-Peter Bauer, Ute Hoffmann, Johannes Forster, Fred Zepp, Antje Schuster, Raffael D’amelio, Ulrich Wahn, Thomas Keil, Susanne Lau, Paolo Maria Matricardi P2 The IgG sensitization profiles against 112 allergenic components support the absence of a protective role of IgG in allergic individuals, outside of the context of SIT Pol André Apoil, Claire Mailhol, Anne Broué-Chabbert, Agnès Juchet, Alain Didier, Elodie Carrer, Thomas Lanot, Antoine Blancher P3 The immune response against the timothy grass pollen allergen Phl p 5 in non-allergic humans Almedina Kurtaj, Christoph Hillebrand, Gerda Fichtinger, Martin Danzer, Christian Gabriel, Theresa Thalhamer, Sandra Scheiblhofer, Josef Thalhamer, Richard Weiss P4 Analyzing the cross-reactivity profile of the major ragweed allergen Amb a 1 Martin Wolf, Michael Hauser, Ulrike Pichler, Teresa Twaroch, Gabriele Gadermaier, Christof Ebner, Hidenori Yokoi, Toshiro Takai, Alain Didierlaurent, Adriano Mari, Peter Briza, Heidrun Behrendt, Angela Neubauer, Frank Stolz, Fátima Ferreira, Michael Wallner P5 LTP (Pru p 3) sensitisation in skin prick test: which means in clinical practice? Sara Carvalho, Tatiana Lourenço, Joana Cosme, Fátima Cabral Duarte, Amélia Spínola Santos, Ana Célia Costa, Manuel Pereira Barbosa P6 IgE profiles, allergen exposure and lifestyle of 501 Austrian pupils: investigation of influences on the development of allergic sensitizations Teresa Stemeseder, Eva Klinglmayr, Bettina Schweidler, Lisa Lueftenegger, Stephanie Moser, Patrick Doppler, Roland Lang, Martin Himly, Gertie J. Oostingh, Arne Bathke, Joerg Zumbach, Thomas Hawranek, Gabriele Gadermaier P7 Molecular profiles of sensitization to perennial inhalant allergens in a middle European region Petr Panzner, Martina Vachova, Tomas Vlas, Marek Maly P8 Evolution of the IgE response to house dust mite allergen molecules in childhood Daniela Posa, Serena Perna, Stephanie Hofmaier, Laura Hatzler, Alexander Rohrbach, Carl-Peter Bauer, Ute Hoffmann, Johannes Forster, Fred Zepp, Antje Schuster, Philippe Stock, Ulrich Wahn, Linus Grabenhenrich, Thomas Keil, Susanne Lau, Kuan-Wei Chen, Yvonne Resch, Susanne Vrtala, Rudolf Valenta, Paolo Maria Matricardi P9 Tropomyosin (Pen a1): to include or not to include in skin prick testing? Joana Cosme, Sara Carvalho, Tatiana Lourenço, Amélia Spínola Santos, Manuel Pereira Barbosa Immunoallergy Department - Hospital de Santa Maria – Centro Hospitalar Lisboa Norte, Lisbon, Portugal, Lisbon, Portugal; Immunoallergy Department - Hospital de Santa Maria – Centro Hospitalar Lisboa Norte, Lisbon, Portugal; Faculdade de Medicina de Lisboa, Lisbon, Portugal P10 Component-resolved IgE profiles in Georgian patients Tamar Abramidze, Nino Lomidze, Maia Gotua P11 Cross reactivity between food and pollen allergens in Lithuania according to spIgE evaluation Austeja Dapkeviciute, Ruta Einikyte, Jolita Norkuniene, Laima Skrickiene, Asta Miskiniene, Violeta Kvedariene P12 Distribution of inhalant allergy in the population of Lithuania Ruta Einikyte, Austeja Dapkeviciute, Jolita Norkuniene, Laima Skrickiene, Asta Miskiniene, Violeta Kvedariene Poster Session 2: Allergen molecules: identification, characterization, structure and function P13 Interference of antigen 5-based cross-reactivity in the diagnosis of hymenoptera venom allergy Maximilian Schiener, Bernadette Eberlein, Carmen Moreno-Aguilar, Gunilla Pietsch, Mareike Mc Intyre, Lea Schwarze, Dennis Rußkamp, Tilo Biedermann, Edzard Spillner, Ulf Darsow, Carsten Schmidt-Weber, Markus Ollert, Simon Blank P14 IgE cross-reactivity between European Hymenoptera and Asian hornet (Vespa velutina) venom allergens Cyril Longé, Andrea Brazdova, Jean-Louis Brunet, Claire Schwartz, Bruno Girodet, François Lavaud, Joelle Birnbaum, Nhân Pham Thi, Magalie Duchateau, Julia Chamot-Rooke, Laurence Guilloux, Marie-Ange Selva, Rémy Couderc, Hélène Sénéchal, Jean-Pierre Sutra, Pascal Poncet P15 Carbohydrate composition of house dust mite extracts and major group 1 and group 2 allergens Steffen Augustin, Linda Pump, Martin Wald, Thomas Eichhorn, Frank Fischer, Christoph Willers P16 Specificity of monoclonal antibodies against cross-reactive carbohydrate determinants Michaela Miehe, Melanie Plum, Sara Wolf, Frederic Jabs, Tim Raiber, Frank Bantleon, Henning Seismann, Thilo Jakob, Edzard Spillner P17 Red meat allergic patients have a selective IgE response to the a-Gal glycan Danijela Apostolovic, Anh Thu Tran, Sara Sanchez-Vidaurre, Tanja Cirkovic Velickovic, Maria Starkhammar, Carl Hamsten, Marianne Van Hage P18 Specificity of non-specific lipid transfer proteins and influence of the ligands on their three-dimensional structure Pawel Dubiela, Piotr Humeniuk, Sabine Pfeifer, Merima Bublin, Tomasz Borowski, Karin Hoffmann-Sommergruber P19 Real-time PCR analysis of Pru av 1 and Pru av 3 allergens Martie C.M. Verschuren, Shanna Bastiaan-Net, Defien Depoortere, Kay Foetisch, Stephan Scheurer, Harry J Wichers, Theo Noij P20 Specificity of anti-Pru av 1 antibodies for the detection of Pru av 1 isoallergens Martie C.M. Verschuren, Shanna Bastiaan-Net, Nikki M.E. Van Uden, Karel Vandenberghe, Kay Foetisch, Stephan Scheurer, Harry J. Wichers H.J., Theo H.M. Noij P21 Enhancing recombinant production yield of Bet v 1 through codon usage harmonization Anargyros Roulias, Maria Alejandra Parigiani, Heidi Hofer, Claudia Asam, Christof Ebner, Fátima Ferreira P22 Structural and dynamic insights into the world of PR-10 allergens Linda Ahammer, Sarina Grutsch, Martin Tollinger Poster Session 3: Allergen molecules: identification, characterization, structure and function P23 Purification of polcalcin from different pollen allergenic sources by antibody-affinity chromatography Raquel Moya, Mª Angeles López-Matas, Raquel Reyes, Jerónimo Carnés P24 Variations of wheat allergens in cultivars measured through a targeted quantitative mass spectrometry approach Colette Larré, Hélène Rogniaux, Roberta Lupi, Sandra Denery-Papini P25 Art v 1, Amb a 4 and Par h 1 defensin-like proteins share similar structural features but distinct immunological and allergenic properties Isabel Maria Pablos, Stephanie Eichhorn, Yoan Machado, Peter Briza, Christof Ebner, Jung-Won Park, Alain Didierlaurent, Naveen Arora, Stefan Vieths, Gabriele Gadermaier, Fatima Ferreira P26 Homogeneity or diversity of IgE-binding proteins in wheat dependant exercise induced anaphylaxis? Sandra Denery-Papini, Charlene Tanaka, Florence Pineau, Roberta Lupi, Martine Drouet, Etienne Beaudouin, Martine Morisset, Susan Altenbach P27 Deciphering the role of disulfide bonds and of repetitive epitopes in immunoglobulin E binding to wheat gliadins Sandra Denery-Papini, Hamza Mameri, Chantal Brossard, Roberta Lupi, Florence Pineau, Jean Charles Gaudin, Denise Anne Moneret-Vautrin, Etienne Beaudouin, Evelyne Paty, Martine Drouet, Olivier Tranquet, Colette Larré P28 Assessment of the allergenicity of soluble fractions from bread and durum wheats genotypes Roberta Lupi, Stefania Masci, Olivier Tranquet, Denise-Anne Moneret-Vautrin, Sandra Denery-Papini, Colette Larré P29 Isolation and characterization of Ara h 12 and Ara h 13: defensins, a novel class of peanut allergens Skadi Kull, Arnd Petersen, Marisa Böttger, Sandra Rennert, Wolf-Meinhard Becker, Susanne Krause, Martin Ernst, Thomas Gutsmann, Johann Bauer, Buko Lindner, Uta Jappe P30 Allergenicity attributes of different peanut market types Stef Koppelman, Shyamali Jayasena, Dion Luykx, Erik Schepens, Danijela Apostolovic, Govardus De Jong, Tom Isleib, Julie Nordlee, Joe Baumert, Steve Taylor, Soheila Maleki P31 The impact of peanut lipids on Ara h 1-induced immune responses in monocytes-derived dendritic cells Chiara Palladino, Barbara Gepp, Sofía Sirvent, Alba Angelina, Merima Bublin, Christian Radauer, Nina Lengger, Thomas Eiwegger, Oscar Palomares, Heimo Breiteneder P32 Compared allergenicity of native and thermally aggregated ovalbumin as large agglomerated particles Mathilde Claude, Roberta Lupi, Grégory Bouchaud, Marie Bodinier, Chantal Brossard, Sandra Denery-Papini P33 Simulation of the gastrointestinal digestion of the hazelnut allergens Cor a 9 and Cor a 11 by an in-vitro model and characterisation of peptidic products including epitopes by HPLC-MS/MS Robin Korte, Julia Bräcker, Jens Brockmeyer P34 Analysis of distribution of rice allergens in brown rice grain and allergenicity of the products containing rice bran Rie Satoh, Reiko Teshima Poster Session 4: Molecular approaches in AIT P35 Production of a recombinant hypoallergenic variant of the major peanut allergen Ara h 2 for allergen-specific immunotherapy Angelika Tscheppe, Dieter Palmberger, Merima Bublin, Christian Radauer, Chiara Palladino, Barbara Gepp, Nina Lengger, Reingard Grabherr, Heimo Breiteneder P36 Mutagenesis of amino acids critical for calcium-binding leads to the generation of a hypoallergenic Phl p 7 variant Marianne Raith, Linda Sonnleitner, Doris Zach, Konrad Woroszylo, Margit Focke-Tejkl, Herbert Wank, Thorsten Graf, Annette Kuehn, Ines Swoboda P37 Are birch pollen allergen immunotherapy induced blocking antibodies protective for cross-reactive allergens? Claudia Asam, Sara Huber, Heidi Hofer, Roland Lang, Thomas Hawranek, Fátima Ferreira, Michael Wallner P38 High success of 58 subcutaneous immunotherapy for pets allergy in a polyallergic cohort of patients: a component resolved individually adapted treatment (CRIAT) Fabienne Gay-Crosier P39 Neutrophils are potential antigen presenting cells in IgE- mediated allergy Dominika Polak, Birgit Nagl, Claudia Kitzmüller, Barbara Bohle P40 Characterization of allergen-specific CD8+ T cells in type I allergy Nazanin Samadi, Claudia Kitzmüller, Rene Geyeregger, Barbara Bohle, Beatrice Jahn-Schmid Poster Session 5: Molecular and cellular diagnostic tests P41 Nanofluidic-based biosensors allow quantification of total circulating IgE from a drop of blood in 5 minutes Aurélie Buchwalder, Ariel Gomez, Fabien Rebeaud, Iwan Märki P42 Allergen microarray for the analysis of serum IgE binding profile and allergenic activity Jaana Haka, Liisa Hattara, Marika Heikkinen, Merja H Niemi, Juha Rouvinen, Petri Saviranta, Pekka Mattila, Kristiina Takkinen, Marja-Leena Laukkanen P43 Generation of a well-characterized panel of periplaneta americana allergens for component resolved diagnosis Stephanie Eichhorn, Isabel Pablos, Bianca Kastner, Bettina Schweidler, Sabrina Wildner, Peter Briza, Jung-Won Park, Naveen Arora, Stefan Vieths, Gabriele Gadermaier, Fatima Ferreira P44 Improved diagnostic sensitivity of recombinant Api m 1 and Ves v 5 in diagnosis of Hymenoptera venom allergy Mira Silar, Julij Selb, Rok Kogovsek, Mitja Kosnik, Peter Korosec P45 Added value of biomarkers of primary sensitization and cross-reactivity in patients with hymenoptera venom allergy Leticia Pestana, Alcinda Campos Melo, Ana Mendes, Maria Elisa Pedro, Manuel Pereira Barbosa, Maria Conceição Pereira Santos P46 Cosensitization to Alt a 1 and Act d 2: more than a fortuitous association? Françoise Bienvenu, Claire Goursaud, Lorna Garnier, Sandrine Jacquenet, Michaël Degaud, Sébastien Viel, Annick Barre, Pierre Rougé, Jacques Bienvenu, Joana Vitte P47 Molecular diagnosis for peanut allergy: ALFA method performs as well as established methods for Ara h 1, Ara h 2, Ara h 6, Ara h 9 and CCD Amel Bensalah, Isabelle Cleach, Laurent Mousseau, Chantal Agabriel, Valérie Liabeuf, Joëlle Birnbaum, Jean-Louis Mège, Joana Vitte P48 Evaluation of a food challenge service in relation to specific IgE to molecular components in children with suspected peanut allergy James Gardner, Minal Gandhi, Harsha Kariyawasam, Giuseppina Rotiroti P49 Component resolved diagnosis in cereal allergy Isabel Carrapatoso, Celso Pereira, Frederico Regateiro, Emília Faria, Ana Todo-Bom Poster Session 6: Molecular diagnosis in prevention and therapy P50 Pretreatment molecular sensitizations determine the sIgG4 induction during the updosing of SCIT and may be useful to identify clinically relevant additional sensitizations Johannes Martin Schmid, Ronald Dahl, Hans Juergen Hoffmann P51 Usefulness of recombinant latex allergens in immunotherapy’s decision and follow-up Inês Mota, Filipe Benito Garcia, Angela Gaspar, Mário Morais-Almeida P52 Omega-5-gliadin in the diagnosis of wheat-dependent anaphylaxis induced by ibuprofen but not by exercise Joana Cosme, Letícia Pestana, Amélia Spínola Santos, Manuel Pereira Barbosa P53 Food dependent exercise-induced anaphylaxis: a component-resolved and in vitro depletion approach to access IgE cross-reactivity Diana Silva, Teresa Vieira, Ana Maria Pereira, André Moreira, Luís Delgado P54 Olive pollen allergens: what are we missing? Sara Prates, Cátia Alves, Elena Finelli, Paula Leiria Pinto P55 Purified Alt a 1 extract in Alternaria alternata allergy diagnosis Bárbara Kong Cardoso, Cíntia Cruz, Filipa Semedo, Elza Tomaz, Filipe Inácio P56 Use of specific IgE Bos d8 (casein) to aid early introduction of dietary baked milk in children with cows’ milk allergy James Gardner, Santanu Maity, Giuseppina Rotiroti, Minal Gandhi P57 Molecular characterisation and immunoreactivity of a peanut ingredient for use in oral food challenges Ivona Baricevic-Jones, Justin T. Marsh, Phil E. Johnson, Anuradha Balasundaram, Anya-May Hope, Aafke Taekema, Angela Simpson, Aida Semic-Jusufagic, E.N. Clare Mills P58 Specific IgE to recombinant allergens of hazelnut and oral food challenge in children Gourdon Dubois Nelly, Sellam Laetitia, Pereira Bruno, Michaud Elodie, Messaoudi Khaled, Evrard Bertrand, Fauquert Jean-Luc Poster session 7/8: miscellaneous P59 What defines a protein as an allergen? A discussion of sources and sufficiency Richard E. Goodman P60 Cat allergy: relationship between clinical and molecular diagnostic María Cecilia Martín Fernández De Basoa, Antón Fernández Ferreiro, Elena Rodríguez Plata P61 Anaphylaxis to rabbit: the cat came in last Luis Amaral, Borja Bartolomé, Alice Coimbra, Jose L Placido P62 Dog allergy: relationship between clinical and molecular diagnostic María Cecilia Martín Fernández De Basoa, Antón Fernández Ferreiro, Elena Rodríguez Plata P63 Correlation of serum timothy grass-pollen specific IgE levels determined by two immunoblot test systems Mariana Vieru, Florin-Dan Popescu, Florin-Adrian Secureanu, Carmen Saviana Ganea P64 Development of oral food challenge formulations for diagnosis of fish allergy using powdered fish ingredients Carol Ann Costello, Ivona Baricevic-Jones, Martin Sorensen, Clare Mills, Adrian Rogers, Aage Otherhals P65 Fish and peanut allergens interact with plasma membranes of intestinal and bronchial epithelial cells and induce differential gene expression of cytokines and chemokines Tanja Kalic, Isabella Ellinger, Chiara Palladino, Barbara Gepp, Eva Waltl, Verena Niederberger-Leppin, Heimo Breiteneder P66 Interleukin 4 affects fat tissue metabolism and expression of pro-inflammatory factors in isolated rat adipocytes Dawid Szczepankiewicz, Ewa Pruszynska-Oszmalek, Marek Skrzypski, Krzysztof W. Nowak, Aleksandra Szczepankiewicz P67 Ozone induced airway hyperreactivity in PD-L2−/− mice model Gwang-Cheon Jang P68 Thymic stromal lymphopoietin (TSLP) and its receptor as targets for the development of anti-inflammatory inhibitory agents Iva Markovic, Andreas Borowski, Tina Vetter, Andreas Wohlmann, Michael Kuepper, Karlheinz Friedrich P69 The mononuclear phagocyte system in experimentally-induced allergic rhinitis Ibon Eguiluz Gracia, Anthony Bosco, Ralph Dollner, Guro Reinholt Melum, Anya C Jones, Maria Lexberg, Patrick G Holt, Espen Sønderaal Bækkevold, Frode Lars Jahnsen P70 Expression of histamine metabolizing enzymes is increased in allergic children Aleksandra Szczepankiewicz, Paulina Sobkowiak, Marta Rachel, Beata Narozna, Dorota Jenerowicz, Witold Swiatowy, Anna Breborowicz P71 Modifying the glycosylation of human IgE towards oligomannosidic structures does not affect its biological activity Melanie Plum, Sara Wolf, Frank Bantleon, Henning Seismann, Frederic Jabs, Michaela Miehe, Thilo Jakob, Edzard Spillner P72 Flying Labs: an educational initiative to transfer allergy research into high-school settings Michael Wallner, Heidi Hofer, Fatima Ferreira, Reinhard Nestelbacher P73 Clinical significance of antihistamines and Kujin, an anti-allergic Kampo medicine Hiroyuki Fukui

Neuregulin ligands and their erbB receptors are important for the development of Schwann cells (SCs), the glial cells of the peripheral nervous system (PNS). ErbB3 deficiency is characterized by a complete loss of SCs along axons of the peripheral nerves, impaired fasciculation and neuronal cell death. We performed comparative gene expression analysis of dorsal root ganglia (DRG) explant cultures from erbB3 deficient and wild type mice to identify genes that are involved in SC development and migration. The extracellular matrix (ECM) gene periostin was found to exhibit the most prominent down regulation in erbB3 deficient DRG. Expression analysis revealed that the periostin expressing cell population in the PNS corresponds to SC precursors and SCs and is particularly high in migratory SCs. Furthermore; stimulation of SCs with neuregulin‑1 (NRG1) or transforming growth factor β (TGFβ) resulted in an upregulation of periostin expression. Interestingly, DRG explant cultures of periostin deficient mice revealed a significant reduction of the number of migrating SCs. These data demonstrate that the expression of periostin is stimulated by erbB ligand NRG1 and influences the migration of SC precursors.

Monoclonal antibodies have become a mainstay for the targeted treatment of cancer today. Some of the most successful targets of monoclonal antibodies are constituted by the epidermal growth factor receptor family spearheaded by the epidermal growth factor receptor (EGFR). Prompted by studies indicating that IgE compared to IgG may harness alternate effector functions to eradicate malignant cells, we addressed the establishment, engineering, and the potential tumoricidal effects of recombinant anti-EGFR IgE. Therefore, two different therapeutic EGFR-specific antibodies, 225 and 425, were chosen for re-cloning into different chimeric IgE and IgG formats and produced in human cells. Simultaneous antibody binding to the sEGFR demonstrated accessibility of both epitopes for recombinant IgE. Proliferation and cytotoxicity assays demonstrated signal blocking and effector mediating capability of IgE isotypes. Pronounced degranulation in the presence of sEGFR upon activation exclusively with two IgE antibodies verified the epitope proximity and provides evidence that tumor-targeting by anti-EGFR IgE is safe with regard to soluble target structures. Degranulation mediated by tumor cells expressing EGFR could be demonstrated for singular and combined IgE antibodies; however, use of two IgE specificities was not superior to use of one IgE alone. The data suggest that the surface distribution of EGFR is optimally suited to mount a robust effector cell trigger and corroborate the potential and specificity of the IgE/IgE receptor network to react to xenobiotic or pathogenic patterns for targeting malignancies.