Recombinant phospholipase A1 (Ves v 1) from yellow jacket venom for improved diagnosis of hymenoptera venom hypersensitivity

<p>Abstract</p> <p>Background</p> <p>Hymenoptera venoms are known to cause life-threatening IgE-mediated anaphylactic reactions in allergic individuals. Proper diagnosis of hymenoptera venom allergy using venom extracts is severely affected by molecular cross-reactivities. Although non-glycosylated marker allergens would facilitate the identification of the culprit venom, the major allergen phospholipase A1 (Ves v 1) from yellow jacket venom (YJV) remained unavailable so far.</p> <p>Methods</p> <p>Expression of Ves v 1 as wild type and enzymatically inactivated mutant and Ves v 5 in insect cells yielded soluble proteins that were purified via affinity chromatography. Functionality of the recombinant allergens was assessed by enzymatic and biophysical analyses as well as basophil activation tests. Diagnostic relevance was addressed by ELISA-based analyses of sera of YJV-sensitized patients.</p> <p>Results</p> <p>Both major allergens Ves v 1 and Ves v 5 could be produced in insect cells in secreted soluble form. The recombinant proteins exhibited their particular biochemical and functional characteristics and were capable for activation of human basophils. Assessment of IgE reactivity of sera of YJV-sensitized and double-sensitized patients emphasised the relevance of Ves v 1 in hymenoptera venom allergy. In contrast to the use of singular molecules the combined use of both molecules enabled a reliable assignment of sensitisation to YJV for more than 90% of double-sensitised patients.</p> <p>Conclusions</p> <p>The recombinant availability of Ves v 1 from yellow jacket venom will contribute to a more detailed understanding of the molecular and allergological mechanisms of insect venoms and may provide a valuable tool for diagnostic and therapeutic approaches in hymenoptera venom allergy.</p

Phospholipases A1

Phospholipase A1 (PLA1) is an enzyme that hydrolyzes phospholipids and produces 2-acyl-lysophospholipids and fatty acids. This lipolytic activity is conserved in a wide range of organisms but is carried out by a diverse set of PLA1 enzymes. Where their function is known, PLA1s have been shown to act as digestive enzymes, possess central roles in membrane maintenance and remodeling, or regulate important cellular mechanisms by the production of various lysophospholipid mediators, such as lysophosphatidylserine and lysophosphatidic acid, which in turn have multiple biological functions

Recombinant phospholipase A1 (Ves v 1) from yellow jacket venom for improved diagnosis of hymenoptera venom hypersensitivity.

ABSTRACT:Hymenoptera venoms are known to cause life-threatening IgE-mediated anaphylactic reactions in allergic individuals. Proper diagnosis of hymenoptera venom allergy using venom extracts is severely affected by molecular cross-reactivities. Although non-glycosylated marker allergens would facilitate the identification of the culprit venom, the major allergen phospholipase A1 (Ves v 1) from yellow jacket venom (YJV) remained unavailable so far.Expression of Ves v 1 as wild type and enzymatically inactivated mutant and Ves v 5 in insect cells yielded soluble proteins that were purified via affinity chromatography. Functionality of the recombinant allergens was assessed by enzymatic and biophysical analyses as well as basophil activation tests. Diagnostic relevance was addressed by ELISA-based analyses of sera of YJV-sensitized patients.Both major allergens Ves v 1 and Ves v 5 could be produced in insect cells in secreted soluble form. The recombinant proteins exhibited their particular biochemical and functional characteristics and were capable for activation of human basophils. Assessment of IgE reactivity of sera of YJV-sensitized and double-sensitized patients emphasised the relevance of Ves v 1 in hymenoptera venom allergy. In contrast to the use of singular molecules the combined use of both molecules enabled a reliable assignment of sensitisation to YJV for more than 90% of double-sensitised patients.The recombinant availability of Ves v 1 from yellow jacket venom will contribute to a more detailed understanding of the molecular and allergological mechanisms of insect venoms and may provide a valuable tool for diagnostic and therapeutic approaches in hymenoptera venom allergy

Recombinant phospholipase A1 (Ves v 1) from yellow jacket venom for improved diagnosis of hymenoptera venom hypersensitivity.

ABSTRACT:Hymenoptera venoms are known to cause life-threatening IgE-mediated anaphylactic reactions in allergic individuals. Proper diagnosis of hymenoptera venom allergy using venom extracts is severely affected by molecular cross-reactivities. Although non-glycosylated marker allergens would facilitate the identification of the culprit venom, the major allergen phospholipase A1 (Ves v 1) from yellow jacket venom (YJV) remained unavailable so far.Expression of Ves v 1 as wild type and enzymatically inactivated mutant and Ves v 5 in insect cells yielded soluble proteins that were purified via affinity chromatography. Functionality of the recombinant allergens was assessed by enzymatic and biophysical analyses as well as basophil activation tests. Diagnostic relevance was addressed by ELISA-based analyses of sera of YJV-sensitized patients.Both major allergens Ves v 1 and Ves v 5 could be produced in insect cells in secreted soluble form. The recombinant proteins exhibited their particular biochemical and functional characteristics and were capable for activation of human basophils. Assessment of IgE reactivity of sera of YJV-sensitized and double-sensitized patients emphasised the relevance of Ves v 1 in hymenoptera venom allergy. In contrast to the use of singular molecules the combined use of both molecules enabled a reliable assignment of sensitisation to YJV for more than 90% of double-sensitised patients.The recombinant availability of Ves v 1 from yellow jacket venom will contribute to a more detailed understanding of the molecular and allergological mechanisms of insect venoms and may provide a valuable tool for diagnostic and therapeutic approaches in hymenoptera venom allergy

Recombinant phospholipase A1 (Ves v 1) from yellow jacket venom for improved diagnosis of hymenoptera venom hypersensitivity.

ABSTRACT:Hymenoptera venoms are known to cause life-threatening IgE-mediated anaphylactic reactions in allergic individuals. Proper diagnosis of hymenoptera venom allergy using venom extracts is severely affected by molecular cross-reactivities. Although non-glycosylated marker allergens would facilitate the identification of the culprit venom, the major allergen phospholipase A1 (Ves v 1) from yellow jacket venom (YJV) remained unavailable so far.Expression of Ves v 1 as wild type and enzymatically inactivated mutant and Ves v 5 in insect cells yielded soluble proteins that were purified via affinity chromatography. Functionality of the recombinant allergens was assessed by enzymatic and biophysical analyses as well as basophil activation tests. Diagnostic relevance was addressed by ELISA-based analyses of sera of YJV-sensitized patients.Both major allergens Ves v 1 and Ves v 5 could be produced in insect cells in secreted soluble form. The recombinant proteins exhibited their particular biochemical and functional characteristics and were capable for activation of human basophils. Assessment of IgE reactivity of sera of YJV-sensitized and double-sensitized patients emphasised the relevance of Ves v 1 in hymenoptera venom allergy. In contrast to the use of singular molecules the combined use of both molecules enabled a reliable assignment of sensitisation to YJV for more than 90% of double-sensitised patients.The recombinant availability of Ves v 1 from yellow jacket venom will contribute to a more detailed understanding of the molecular and allergological mechanisms of insect venoms and may provide a valuable tool for diagnostic and therapeutic approaches in hymenoptera venom allergy

Dissecting cross-reactivity in hymenoptera venom allergy by circumvention of alpha-1,3-core fucosylation.

Hymenoptera venom allergy is known to cause life-threatening and sometimes fatal IgE-mediated anaphylactic reactions in allergic individuals. About 30-50% of patients with insect venom allergy have IgE antibodies that react with both honeybee and yellow jacket venom. Apart from true double sensitisation, IgE against cross-reactive carbohydrate determinants (CCD) are the most frequent cause of multiple reactivities severely hampering the diagnosis and design of therapeutic strategies by clinically irrelevant test results. In this study we addressed allergenic cross-reactivity using a recombinant approach by employing cell lines with variant capacities of alpha-1,3-core fucosylation. The venom hyaluronidases, supposed major allergens implicated in cross-reactivity phenomena, from honeybee (Api m 2) and yellow jacket (Ves v 2a and its putative isoform Ves v 2b) as well as the human alpha-2HS-glycoprotein as control, were produced in different insect cell lines. In stark contrast to production in Trichoplusia ni (HighFive) cells, alpha-1,3-core fucosylation was absent or immunologically negligible after production in Spodoptera frugiperda (Sf9) cells. Consistently, co-expression of honeybee alpha-1,3-fucosyltransferase in Sf9 cells resulted in the reconstitution of CCD reactivity. Re-evaluation of differentially fucosylated hyaluronidases by screening of individual venom-sensitised sera emphasised the allergenic relevance of Api m 2 beyond its carbohydrate epitopes. In contrast, the vespid hyaluronidases, for which a predominance of Ves v 2b could be shown, exhibited pronounced and primary carbohydrate reactivity rendering their relevance in the context of allergy questionable. These findings show that the use of recombinant molecules devoid of CCDs represents a novel strategy with major implications for diagnostic and therapeutic approaches

Recombinant phospholipase A1 (Ves v 1) from yellow jacket venom for improved diagnosis of hymenoptera venom hypersensitivity.

ABSTRACT:Hymenoptera venoms are known to cause life-threatening IgE-mediated anaphylactic reactions in allergic individuals. Proper diagnosis of hymenoptera venom allergy using venom extracts is severely affected by molecular cross-reactivities. Although non-glycosylated marker allergens would facilitate the identification of the culprit venom, the major allergen phospholipase A1 (Ves v 1) from yellow jacket venom (YJV) remained unavailable so far.Expression of Ves v 1 as wild type and enzymatically inactivated mutant and Ves v 5 in insect cells yielded soluble proteins that were purified via affinity chromatography. Functionality of the recombinant allergens was assessed by enzymatic and biophysical analyses as well as basophil activation tests. Diagnostic relevance was addressed by ELISA-based analyses of sera of YJV-sensitized patients.Both major allergens Ves v 1 and Ves v 5 could be produced in insect cells in secreted soluble form. The recombinant proteins exhibited their particular biochemical and functional characteristics and were capable for activation of human basophils. Assessment of IgE reactivity of sera of YJV-sensitized and double-sensitized patients emphasised the relevance of Ves v 1 in hymenoptera venom allergy. In contrast to the use of singular molecules the combined use of both molecules enabled a reliable assignment of sensitisation to YJV for more than 90% of double-sensitised patients.The recombinant availability of Ves v 1 from yellow jacket venom will contribute to a more detailed understanding of the molecular and allergological mechanisms of insect venoms and may provide a valuable tool for diagnostic and therapeutic approaches in hymenoptera venom allergy

Recombinant phospholipase A1 (Ves v 1) from yellow jacket venom for improved diagnosis of hymenoptera venom hypersensitivity.

ABSTRACT:Hymenoptera venoms are known to cause life-threatening IgE-mediated anaphylactic reactions in allergic individuals. Proper diagnosis of hymenoptera venom allergy using venom extracts is severely affected by molecular cross-reactivities. Although non-glycosylated marker allergens would facilitate the identification of the culprit venom, the major allergen phospholipase A1 (Ves v 1) from yellow jacket venom (YJV) remained unavailable so far.Expression of Ves v 1 as wild type and enzymatically inactivated mutant and Ves v 5 in insect cells yielded soluble proteins that were purified via affinity chromatography. Functionality of the recombinant allergens was assessed by enzymatic and biophysical analyses as well as basophil activation tests. Diagnostic relevance was addressed by ELISA-based analyses of sera of YJV-sensitized patients.Both major allergens Ves v 1 and Ves v 5 could be produced in insect cells in secreted soluble form. The recombinant proteins exhibited their particular biochemical and functional characteristics and were capable for activation of human basophils. Assessment of IgE reactivity of sera of YJV-sensitized and double-sensitized patients emphasised the relevance of Ves v 1 in hymenoptera venom allergy. In contrast to the use of singular molecules the combined use of both molecules enabled a reliable assignment of sensitisation to YJV for more than 90% of double-sensitised patients.The recombinant availability of Ves v 1 from yellow jacket venom will contribute to a more detailed understanding of the molecular and allergological mechanisms of insect venoms and may provide a valuable tool for diagnostic and therapeutic approaches in hymenoptera venom allergy

Rekombinante Strategien in der Hymenopterengiftallergie und darüber hinaus

Hymenoptera venom allergies affect around one fourth of the population and 1-5% show systemic and sometimes life threatening symptoms. Even if the incidence for fatal events is low, it remains one of the main causes for fatal allergic reactions and the quality of live of affected individuals is significantly reduced. In Europe the main threat is emanating from the western honeybee Apis mellifera and the common wasp Vespula vulgaris. Actually, the only causative therapeutic intervention in hymenoptera venom allergy is the specific immunotherapy (SIT), whereby 80-95% of the treated patients are protected from severe reactions. A prerequisite for a successful therapy is a proper diagnosis, which mainly depends on the correct determination of the culprit venom. However, a considerable number of patients show reactivity to honeybee as well as wasp venom and are therefore diagnosed as double-positive, whereas in up to 75% of these the diagnosis is affected by molecular cross-reactivities. Reasons for this phenomenon are either specific IgE moieties directed against homologous proteins in the venoms, or antibody binding to similar carbohydrate structures on glycoproteins of both venoms, called cross-reactive carbohydrate determinants (CCDs), which cannot be differentiated by the current diagnostic tools. An improvement mainly depends on a thorough knowledge on the allergens involved in hymenoptera venom allergy. Therefore, in the present PhD thesis a new allergen from wasp venom was identified and characterised. Furthermore, already established components were re-evaluated in terms of their allergenic potential and with special regard to CCDs. As a basis for thorough characterisation, all proteins were recombinantly expressed in the baculovirus expression systems in functional form and analysed by various in vitro immunological and cellular assays. Moreover, recombinant antibody technologies were utilised for the generation of IgE, IgG and IgY antibodies as diagnostic tools in allergological and immunological assays in general and with special regard to molecular cross-reactivities. In summary, the presented data allow a revision of the current view on hymenoptera venom allergens and their particular significance, especially concerning the role of CCDs. Moreover, together with the recombinant antibodies, a broad panel of new valuable diagnostic tools for the improvement of allergy diagnosis and beyond were generated which will contribute to a molecular understanding of pathophysiological and allergological mechanisms in hymenoptera venom allergy.Rund ein Viertel der Bevölkerung leidet an Insektengiftallergien, wobei 1-5% systemische bis lebensbedrohliche Reaktionen zeigen können. Auch wenn die Häufigkeit solch fataler Fälle gering ist, stellen sie doch eine der häufigsten Ursachen für fatale allergische Reaktionen dar und die Lebensqualität der Betroffenen ist signifikant reduziert. In Europa geht die Hauptgefährdung von der westlichen Honigbiene Apis mellifera und der gewöhnlichen Wespe Vespula vulgaris aus. Zurzeit stellt die spezifische Immuntherapie (SIT) die einzige kausale therapeutische Intervention dar, wobei 80-95% der behandelten Patienten vor schweren allergischen Reaktionen geschützt sind. Eine Grundvoraussetzung für eine erfolgreiche Therapie ist eine entsprechende Diagnose, die hauptsächlich auf der Bestimmung des verantwortlichen Giftes beruht. Eine Vielzahl von Patienten zeigt jedoch Reaktionen auf Bienen- und Wespengift und wird daher als doppelt positiv eingeschätzt, wobei 75% dieser Diagnosen von molekularen Kreuzreaktionen beeinflusst sind. Gründe für dieses Phänomen sind zum einen IgE Antikörper mit Spezifität für homologe Proteine beider Gifte, oder Antikörper die sich gegen Carbohydratstrukturen auf Glykoproteinen beider Gifte, die als kreuzreaktive Carbohydrat Determinanten (cross-reactive carbohydrate determinants: CCD) bezeichnet werden und nicht in den aktuellen diagnostischen Verfahren differenziert werden können. Um diese diagnostischen Methoden verbessern zu können, bedarf es jedoch eingehender Kenntnis über die beteiligten Allergene in der Insektengiftallergie. Dazu wurde in der vorliegenden Doktorarbeit ein neues Allergen des Wespengifts identifiziert und charakterisiert und zudem bereits bekannte Komponenten im Hinblick auf ihre allergenes Potenzial und die Rolle von CCDs neu bewertet. Dafür wurden alle Proteine rekombinant im Baculovirus-System exprimiert und in verschiedenen immunologischen und zellulären in vitro Untersuchungen analysiert. Weiterführend wurden unterschiedliche rekombinante Antikörperformate für den Einsatz in allergologischen und immunologischen Diagnostikanwendungen hergestellt, wobei auch hier ein Focus auf molekularen Kreuzreaktionen lag. Zusammenfassend erlauben die präsentierten Daten eine Neubewertung der Signifikanz einzelner Insektengiftallergene, besonders im Hinblick auf die Bedeutung von CCDs. Darüber hinaus, wurden zusammen mit den rekombinanten Antikörpern eine Reihe wertvoller diagnostischer Werkzeuge für den Einsatz in der Allergiediagnostik und darüber hinaus zur Verfügung gestellt, die wesentlich zu weiteren Erkenntnissen über die pathophysiologischen und allergologischen Mechanismen der Insektengiftallergie beitragen können