Edzard Spillner

Hymenoptera allergens: from venom to "venome"

In Western Europe, Hymenoptera venom allergy (HVA) primarily relates to venoms of the honeybee and the common yellow jacket. In contrast to other allergen sources, only a few major components of Hymenoptera venoms had been characterized until recently. Improved expression systems and proteomic detection strategies have allowed the identification and characterization of a wide range of additional allergens. The field of HVA research has moved rapidly from focusing on venom extract and single major allergens to a molecular understanding of the entire “venome” as a system of unique and characteristic components. An increasing number of such components has been identified, characterized regarding function, and assessed for allergenic potential. Moreover, advanced expression strategies for recombinant production of venom allergens allow selective modification of molecules and provide insight into different types of immunoglobulin E reactivities and sensitization patterns. The obtained information contributes to an increased diagnostic precision in HVA and may serve for monitoring, re-evaluation, and improvement of current therapeutic strategies.

Predominant Api m 10 sensitization as risk factor for treatment failure in honey bee venom immunotherapy

BACKGROUNDnComponent 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.nnnOBJECTIVEnWe performed a retrospective analysis of component-resolved sensitization profiles in HBV-allergic patients and association with treatment outcome.nnnMETHODSnHBV-allergic patients who had undergone controlled honey bee sting challenge after at least 6xa0months of HBV immunotherapy (nxa0=xa0115) were included and classified as responder (nxa0=xa079) or treatment failure (nxa0=xa036) 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.nnnRESULTSnNo 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; Pxa0=xa0.0013) 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 sIgG4 induction was observed only in those patients who were treated with HBV in which Api m 10 was detectable.nnnCONCLUSIONSnComponent-resolved sensitization profiles in HBV allergy suggest predominant IgE sensitization to Api m 10 as a risk factor for treatment failure in HBV immunotherapy.

Basophil Activation Test Using Recombinant Allergens: Highly Specific Diagnostic Method Complementing Routine Tests in Wasp Venom Allergy

Background Skin testing can expose allergic subjects to potential systemic reactions, sensitization against unrelated proteins, and increased risk of future sting reactions. Therefore the continuous improvement of in vitro diagnostic methods is desirable. Recombinant allergens have been shown to improve the sensitivity of specific IgE (sIgE) detection in vitro whilst no data is available regarding their application and reliability in basophil activation test (BAT). Here we aimed to compare the specificity and sensitivity of recombinant allergens Ves v 1, Ves v 2, Ves v 3 and Ves v 5 in both specific IgE (sIgE) detection in vitro and basophil activation test. Methods sIgE detection by ELISA or ImmunoCAP and BAT towards the panel of recombinant allergens Ves v 1, Ves v 2, Ves v 3 and Ves v 5 were performed in 43 wasp venom allergic patients with a history of anaphylactic reaction and sIgE seropositivity, as well as 17 controls defined as subjects with a history of repetitive wasp stings but absence of any allergic symptom. Results The BAT performed with the recombinant allergens Ves v 1, Ves v 2, Ves v 3 and Ves v 5 markedly improved the specificity of diagnosis in wasp venom allergic subjects when compared to the respective sIgE detection in serum. Conclusions BAT performed with the recombinant allergens Ves v 5, Ves v 3 and Ves v 1 provides an emerging highly specific in vitro method for the detection of wasp venom allergy, compared to the sIgE detection. Recombinant allergens applied to BAT represent a step forward in developing reliable in vitro tests for specific diagnosis of allergy.

rApi m 3 and rApi m 10 improve detection of honey bee sensitization in Hymenoptera venom allergic patients with double sensitization to honey bee and yellow jacket venom

Recombinant allergens improve the diagnostic precision in Hymenoptera venom allergy (HVA), in particular in patients with double sensitization to both honey bee (HBV) and yellow jacket venom (YJV). While currently available vespid allergens allow the detection of >95% of YJV‐allergic patients, the sensitization frequency to the only available HBV marker allergen rApi m 1 in HBV‐allergic patients is lower. Here, we demonstrate that sIgE to additional HBV marker allergens rApi m 3 and rApi m 10 allows the detection of genuine HBV sensitization in 46–65% of Api m 1 negative sera. This is of particular relevance in patients with double sensitization to HBV and YJV that did not identify the culprit insect. Addition of sIgE to rApi m 3 and rApi m 10 provides evidence of HBV sensitization in a large proportion of rApi m 1‐negative patients and thus provides a diagnostic marker and rationale for VIT treatment with HBV, which otherwise would have been missing.

IgE recognition of chimeric isoforms of the honeybee (Apis mellifera) venom allergen Api m 10 evaluated by protein array technology

Api m 10 has recently been established as novel major allergen that is recognized by more than 60% of honeybee venom (HBV) allergic patients. Previous studies suggest Api m 10 protein heterogeneity which may have implications for diagnosis and immunotherapy of HBV allergy. In the present study, RT-PCR revealed the expression of at least nine additional Api m 10 transcript isoforms by the venom glands. Two distinct mechanisms are responsible for the generation of these isoforms: while the previously known variant 2 is produced by an alternative splicing event, novel identified isoforms are intragenic chimeric transcripts. To the best of our knowledge, this is the first report of the identification of chimeric transcripts generated by the honeybee. By a retrospective proteomic analysis we found evidence for the presence of several of these isoforms in the venom proteome. Additionally, we analyzed IgE reactivity to different isoforms by protein array technology using sera from HBV allergic patients, which revealed that IgE recognition of Api m 10 is both isoform- and patient-specific. While it was previously demonstrated that the majority of HBV allergic patients display IgE reactivity to variant 2, our study also shows that some patients lacking IgE antibodies for variant 2 display IgE reactivity to two of the novel identified Api m 10 variants, i.e. variants 3 and 4.

Diagnostics in Hymenoptera venom allergy: current concepts and developments with special focus on molecular allergy diagnostics

BackgroundThe high rate of asymptomatic sensitization to Hymenoptera venom, difficulty in correctly identifying Hymenoptera and loss of sensitization over time make an accurate diagnosis of Hymenoptera venom allergy challenging. Although routine diagnostic tests encompassing skin tests and the detection of venom-specific IgE antibodies with whole venom preparations are reliable, they offer insufficient precision in the case of double sensitized patients or in those with axa0history of sting anaphylaxis, in whom sensitization cannot be proven or only to the presumably wrong venom.MethodsSystematic literature research and review of current concepts of diagnostic testing in Hymenoptera venom allergy.Resultsxa0and discussionImprovements in diagnostic accuracy over recent years have mainly been due to the increasing use of molecular allergy diagnostics. Detection of specific IgE antibodies to marker and cross-reactive venom allergens improves the discrimination between genuine sensitization and cross-reactivity, and this provides axa0better rationale for prescribing venom immunotherapy. The basophil activation test has also increased diagnostic accuracy by reducing the number of Hymenoptera venom sensitizations overlooked with routine tests. This paper reviews current concepts of diagnostic testing in Hymenoptera venom allergy and suggests fields for further development.

AllergoOncology – the impact of allergy in oncology: EAACI position paper

Th2 immunity and allergic immune surveillance play critical roles in host responses to pathogens, parasites and allergens. Numerous studies have reported significant links between Th2 responses and cancer, including insights into the functions of IgE antibodies and associated effector cells in both antitumour immune surveillance and therapy. The interdisciplinary field of AllergoOncology was given Task Force status by the European Academy of Allergy and Clinical Immunology in 2014. Affiliated expert groups focus on the interface between allergic responses and cancer, applied to immune surveillance, immunomodulation and the functions of IgE‐mediated immune responses against cancer, to derive novel insights into more effective treatments. Coincident with rapid expansion in clinical application of cancer immunotherapies, here we review the current state‐of‐the‐art and future translational opportunities, as well as challenges in this relatively new field. Recent developments include improved understanding of Th2 antibodies, intratumoral innate allergy effector cells and mediators, IgE‐mediated tumour antigen cross‐presentation by dendritic cells, as well as immunotherapeutic strategies such as vaccines and recombinant antibodies, and finally, the management of allergy in daily clinical oncology. Shedding light on the crosstalk between allergic response and cancer is paving the way for new avenues of treatment.

Application of recombinant antigen 5 allergens from seven allergy-relevant Hymenoptera species in diagnostics

Hymenoptera stings can cause severe anaphylaxis in untreated venom‐allergic patients. A correct diagnosis regarding the relevant species for immunotherapy is often hampered by clinically irrelevant cross‐reactivity. In vespid venom allergy, cross‐reactivity between venoms of different species can be a diagnostic challenge. To address immunological IgE cross‐reactivity on molecular level, seven recombinant antigens 5 of the most important Vespoidea groups were assessed by different diagnostic setups.

Decline of Ves v 5‐specific blocking capacity in wasp venom‐allergic patients after stopping allergen immunotherapy

While allergen‐specific immunotherapy (AIT) is very efficient in hymenoptera venom (HV)‐allergic patients, long‐term outcome after finishing AIT is not well investigated, especially regarding mechanisms that are suggested to contribute to allergen‐specific tolerance. Here, we analyse the Ves v 5‐inhibitory activity of sera from wasp venom‐allergic patients using the novel cell‐free enzyme‐linked immunosorbent facilitated antigen binding (ELIFAB) assay. Compared to pre‐AIT, sera from patients undergoing AIT displayed an increased ability to inhibit Ves v 5 binding by IgE antibodies. In contrast, this inhibitory activity was reduced in patients having finished AIT 5–12 years ago. Allergen‐blocking capacity correlated with serum concentrations of Ves v 5‐specific IgG4 which rose during AIT but almost reached pretreatment levels in patients who had stopped AIT more than 5 years ago. These data raise questions about how long allergen tolerance is maintained in AIT‐treated HV‐allergic patients and suggest that the ELIFAB assay might be an easy‐to‐use tool assessing long‐term tolerance in patients treated with HV‐AIT.

Structure of the omalizumab Fab

Omalizumab is a humanized anti-IgE antibody that inhibits the binding of IgE to its receptors on mast cells and basophils, thus blocking the IgE-mediated release of inflammatory mediators from these cells. Omalizumab binds to the Fc domains of IgE in proximity to the binding site of the high-affinity IgE receptor FcℇRI, but the epitope and the mechanisms and conformations governing the recognition remain unknown. In order to elucidate the molecular mechanism of its anti-IgE activity, the aim was to analyse the interaction of omalizumab with human IgE. Therefore, IgE Fc Cℇ2-4 was recombinantly produced in mammalian HEK-293 cells. Functionality of the IgE Fc was proven by ELISA and mediator-release assays. Omalizumab IgG was cleaved with papain and the resulting Fab was purified by ion-exchange chromatography. The complex of IgE Fc with omalizumab was prepared by size-exclusion chromatography. However, crystals containing the complex were not obtained, suggesting that the process of crystallization favoured the dissociation of the two proteins. Instead, two structures of the omalizumab Fab with maximum resolutions of 1.9 and 3.0u2005Å were obtained. The structures reveal the arrangement of the CDRs and the position of omalizumab residues known from prior functional studies to be involved in IgE binding. Thus, the structure of omalizumab provides the structural basis for understanding the function of omalizumab, allows optimization of the procedure for complex crystallization and poses questions about the conformational requirements for anti-IgE activity.