Allergy / Prevention of allergy by viruslike nanoparticles (VNP) delivering shielded versions of major allergens in a humanized murine allergy model

Background: In highrisk populations, allergenspecific prophylaxis could protect from sensitization and subsequent development of allergic disease. However, such treatment might itself induce sensitization and allergies, thus requiring hypoallergenic vaccine formulations. We here characterized the preventive potential of viruslike nanoparticles (VNP) expressing surfaceexposed or shielded allergens. Methods: Fulllength major mugwort pollen allergen Art v 1 was selectively targeted either to the surface or to the inner side of the lipid bilayer envelope of VNP. Upon biochemical and immunological analysis, their preventive potential was determined in a humanized mouse model of mugwort pollen allergy. Results: Viruslike nanoparticles expressing shielded version of Art v 1, in contrast to those expressing surfaceexposed Art v 1, were hypoallergenic as they hardly induced degranulation of rat basophil leukemia cells sensitized with Art v 1specific mouse or human IgE. Both VNP versions induced proliferation and cytokine production of allergenspecific T cells in vitro. Upon intranasal application in mice, VNP expressing surfaceexposed but not shielded allergen induced allergenspecific antibodies, including IgE. Notably, preventive treatment with VNP expressing shielded allergenprotected mice from subsequent sensitization with mugwort pollen extract. Protection was associated with a Th1/Tregdominated cytokine response, increased Foxp3+ Treg numbers in lungs, and reduced lung resistance when compared to mice treated with empty particles. Conclusion: Viruslike nanoparticles represent a novel and versatile platform for the in vivo delivery of allergens to selectively target T cells and prevent allergies without inducing allergic reactions or allergic sensitization.DKW1248SFB F4605SFB F4609(VLID)313247

Prospective assessment of pre-existing and de novo anti-HLA IgE in kidney, liver, lung and heart transplantation

IntroductionAntibody mediated rejection (ABMR) is a major factor limiting outcome after organ transplantation. Anti-HLA donor-specific antibodies (DSA) of the IgG isotype are mainly responsible for ABMR. Recently DSA of the IgE isotype were demonstrated in murine models as well as in a small cohort of sensitized transplant recipients. In the present study, we aimed to determine the frequency of pre-existing and de novo anti-HLA IgE antibodies in a cohort of 105 solid organ transplant recipients.MethodsWe prospectively measured anti-HLA IgE antibodies in a cohort of kidney (n=60), liver, heart and lung (n=15 each) transplant recipients before and within one-year after transplantation, employing a single-antigen bead assay for HLA class I and class II antigens. Functional activity of anti-HLA IgE antibodies was assessed by an in vitro mediator release assay. Antibodies of the IgG1-4 subclasses and Th1 and Th2 cytokines were measured in anti-HLA IgE positive patients.ResultsPre-existing anti-HLA IgE antibodies were detected in 10% of renal recipients (including 3.3% IgE-DSA) and in 4.4% of non-renal solid organ transplant recipients (heart, liver and lung cohort). Anti-HLA IgE occurred only in patients that were positive for anti-HLA IgG, and most IgE positive patients had had a previous transplant. Only a small fraction of patients developed de novo anti-HLA IgE antibodies (1.7% of kidney recipients and 4.4% of non-renal recipients), whereas no de novo IgE-DSA was detected. IgG subclass antibodies showed a distinct pattern in patients who were positive for anti-HLA IgE. Moreover, patients with anti-HLA IgE showed elevated Th2 and also Th1 cytokine levels. Serum from IgE positive recipients led to degranulation of basophils in vitro, demonstrating functionality of anti-HLA IgE.DiscussionThese data demonstrate that anti-HLA IgE antibodies occur at low frequency in kidney, liver, heart and lung transplant recipients. Anti-HLA IgE development is associated with sensitization at the IgG level, in particular through previous transplants and distinct IgG subclasses. Taken together, HLA specific IgE sensitization is a new phenomenon in solid organ transplant recipients whose potential relevance for allograft injury requires further investigation

N-Glycosylation engineering of plants for the biosynthesis of glycoproteins with bisected and branched complex N-glycans

Glycoengineering is increasingly being recognized as a powerful tool to generate recombinant glycoproteins with a customized N-glycosylation pattern. Here, we demonstrate the modulation of the plant glycosylation pathway toward the formation of human-type bisected and branched complex N-glycans. Glycoengineered Nicotiana benthamiana lacking plant-specific N-glycosylation (i.e. β1,2-xylose and core α1,3-fucose) was used to transiently express human erythropoietin (hEPO) and human transferrin (hTF) together with modified versions of human β1,4-mannosyl-β1,4-N-acetylglucosaminyltransferase (GnTIII), α1,3-mannosyl-β1,4-N-acetylglucosaminyltransferase (GnTIV) and α1,6-mannosyl-β1,6-N-acetylglucosaminyltransferase (GnTV). hEPO was expressed as a fusion to the IgG-Fc domain (EPO-Fc) and purified via protein A affinity chromatography. Recombinant hTF was isolated from the intracellular fluid of infiltrated plant leaves. Mass spectrometry-based N-glycan analysis of hEPO and hTF revealed the quantitative formation of bisected (GnGnbi) and tri- as well as tetraantennary complex N-glycans (Gn[GnGn], [GnGn]Gn and [GnGn][GnGn]). Co-expression of GnTIII together with GnTIV and GnTV resulted in the efficient generation of bisected tetraantennary complex N-glycans. Our results show the generation of recombinant proteins with human-type N-glycosylation at great uniformity. The strategy described here provides a robust and straightforward method for producing mammalian-type N-linked glycans of defined structures on recombinant glycoproteins, which can advance glycoprotein research and accelerate the development of protein-based therapeutics

Towards the role of carbohydrate reactive IgE

Glykosylierte Allergene aus Allergenquellen wie Pflanzen oder Insektengift beinhalten Kohlenhydrat bzw Zucker -Epitope (CCDs) die IgE-Reaktivität in circa 20 % aller allergischen Patienten auslösen können, jedoch geringe allergische Aktivität aufweisen. Sie führen wegen ihres Vorkommens in nicht verwandten Allergenquellen zu hohen Kreuzreaktivitäten und dadurch zu falsch positiven Resultaten in der in vitro Allergiediagnostik. Um die Genauigkeit und Verlässlichkeit von serologischen Allergietests zu verbessern und um den Mechanismus der Interaktion zwischen CCDs und IgE zu verstehen, haben wir es uns als Ziel gesetzt ein Molekül zu entwickeln welches es ermöglicht Zucker-von Peptid-reaktivem IgE zu unterscheiden. Wir fügten N-Glykosylierungsstellen in ein nicht allergenes, strikt monomeres Protein, Myoglobin (aus Pferdeherz) ein und exprimierten es in Insektenzellen um ein authentisches Insekten-Glykosylierungsprofil zu erhalten. Die Charakterisierung der Moleküle erfolgte mit biochemischen und biophysikalischen Methoden. Wir konnten die Expression, Reinigung und Charakterisierung von Glykoproteinen zeigen, welche ausschließlich Zucker-IgE Epitope aufweisen. Damit konnten wir auch CCD spezifisches IgE in Seren von Allergikern quantifizieren, welches mit durchschnittlich 3.6% des Gesamt-IgE eher gering ist. In Inhibitionsexperimenten untersuchten wir die IgE-Kreuzreaktivität der Glykoproteine mit Zuckern aus unterschiedlichen Allergenquellen und konnten diese nicht nur in Pflanzen und Insektengift sondern auch in Milben nachweisen. Zusätzlich zeigte die Verwendung des glykoslierten Proteins im Inhibitionstest, dass HHM 2 die Unterscheidung von peptid-spezifischen und kohlenhydrate-spezifischem IgE ermöglicht. Zusätzlich konnten wir eine Hierarchie der IgE Bindung an glykosylierte Pflanzenallergene, bezüglich der Anzahl der IgE-positiven Seren als auch der Höhe des spezifischen IgEs, feststellen. Dies deutet auf Graspollen als primäre Sensibilisierungsquelle gegen Kohlenhydrate hin.The carbohydrate moieties of glycosylated allergens found in plants and insect venoms can elicit IgE reactivity in about 20% of allergic patients and are responsible for cross-reactivity of sera and are so called cross-reactive carbohydrate determinants (CCD). CCD-specific IgE antibodies show low or no allergenic activity. Therefore, IgE cross-reactivity to carbohydrates in unrelated allergen sources can lead to false positive in vitro diagnostic test results. In order to improve in vitro allergy diagnosis by blocking CCD cross-reactivity upon unrelated allergen sources and to elucidate the mechanism of interactions between IgE and CCDs we aimed to design a molecule which enables to dissect carbohydrate from peptide specific IgE. We designed N-glycosylation sites and grafted them in different numbers onto a non-allergenic protein backbone, horse heart myoglobin (HHM), a strictly monomeric protein with a molecular weight of 17.6 kDa. Recombinant proteins were expressed in an insect cell line to obtain an authentic N-glycosylation profile. The resulting molecules were characterized with biochemical and biophysical approaches and occurred as monomeric folded proteins. Recombinant HHM 1 with one glycosylation site as well as HHM 2 containing two glycosylation sites bound exclusively carbohydrate specific IgE and were used to quantify CCD specific IgE in sera of allergic patients, obtaining an average of 3.6 % of total IgE. In a set of inhibition experiments we investigated the cross-reactivity of glycosylated HHM 2 with carbohydrates from various allergen sources, and showed that glycosylated HHM 2 resembles glyco-epitopes from insects, plants and mites. The usage of HHM 2 as CCD-inhibitor showed remarkably high inhibition rates of IgE reactivity to natural glycosylated allergens on a micro-arrayed allergen chip and allowed to dissect carbohydrate specific IgE from peptide specific IgE. In addition, we could detect a hierarchy of IgE binding to glycans from different plant allergens, regarding frequency of IgE recognition and specific IgE levels. This finding hints to grass pollen as source for primary sensitization to carbohydrates.Abweichender Titel laut Übersetzung der Verfasserin/des VerfassersArbeit an der Bibliothek noch nicht eingelangt - Daten nicht geprüftMedizinische Universität Wien, Diss., 2019(VLID)447786

The culprit insect but not severity of allergic reactions to bee and wasp venom can be determined by molecular diagnosis

Background. Allergy to bee and wasp venom can lead to life-threatening systemic reactions. The identification of the culprit species is important for allergen-specific immunotherapy. Objectives. To determine a panel of recombinant bee and wasp allergens which is suitable for the identification of bee or wasp as culprit allergen sources and to search for molecular surrogates of clinical severity of sting reactions. Methods. Sera from eighty-seven patients with a detailed documentation of their severity of sting reaction (Mueller grade) and who had been subjected to titrated skin testing with bee and wasp venom were analyzed for bee and wasp-specific IgE levels by ImmunoCAPTM. IgE-reactivity testing was performed using a comprehensive panel of recombinant bee and wasp venom allergens (rApi m 1, 2, 3, 4, 5 and 10rVes v 1 and 5) by ISAC chip technology, ImmunoCAP and ELISA. IgG4 antibodies to rApi m 1 and rVes v 5 were determined by ELISA and IgE/ IgG4 ratios were calculated. Results from skin testing, IgE serology and IgE/IgG4 ratios were compared with severity of sting reactions. Results. The panel of rApi m 1, rApi m 10, rVes v 1 and rVes v 5 allowed identification of the culprit venom in all but two of the 87 patients with good agreement to skin testing. Severities of sting reactions were not associated with results obtained by skin testing, venom-specific IgE levels or molecular diagnosis. Severe sting reactions were observed in patients showing < 1 ISU and < 2kUA/L of IgE to Api m 1 and/or Ves v 5. Conclusion. We identified a minimal panel of recombinant bee and wasp allergens for molecular diagnosis which may permit identification of bee and/or wasp as culprit insect in venom-sensitized subjects. The severity of sting reactions was not associated with parameters obtained by molecular diagnosis

Three-dimensional structure of the wheat -amylase Tri a 17, a clinically relevant food allergen

Non peer reviewe

Complex IgE sensitization patterns in ragweed allergic patients: Implications for diagnosis and specific immunotherapy

Abstract Background Ragweed (Ambrosia artemisiifolia) is one of the most important allergen sources, worldwide, causing severe respiratory allergic reactions in late summer and fall, in sensitized patients. Amb a 1 has been considered as the most important allergen in ragweed but 12 ragweed pollen allergens are known. The aim of our study was to investigate IgE reactivity profiles of ragweed allergic patients and to associate them with clinical symptoms. Methods IgE sensitization profiles from clinically well‐characterized ragweed allergic patients (n = 150) were analyzed using immunoblotted ragweed pollen extract. Immunoblot inhibition experiments were performed with two Amb a 1 isoforms and CCD markers and basophil activation experiments were performed with IgE serum before and after depletion of Amb a 1‐specific IgE. Results By IgE‐immunoblotting 19 different IgE reactivity patterns with and without Amb a 1‐sensitization were found. The majority of patients (>95%) suffered from rhino‐conjunctivitis, around 60% reported asthma‐like symptoms and about 25% had skin reactions. Patients with complex IgE sensitization profiles tended to have more clinical symptoms. Serum with and without Amb a 1‐specific IgE induced basophil activation. Conclusions Ragweed pollen allergic patients exhibit complex IgE reactivity profiles to ragweed allergens including Amb a 1 isoforms and cross‐reactive carbohydrates indicating the importance of Amb a 1 isoforms and additional allergens for diagnosis and allergen‐specific immunotherapy of ragweed allergy