An Allergomic Study Reveals Two Novel Venom Allergens, Phospholipase <scp>A1</scp> and Antigen 5, From the Social Wasp <i>Apoica pallens</i>

Hymenoptera venoms are prominent elicitors of systemic IgE-mediated allergic reactions and sometimes fatal anaphylaxis [1]. In Latin America, Hymenoptera stings are one of the major causes of hypersensitive reactions, along with drugs and foods [2]. Despite hosting a wide diversity of clinically relevant insects (bees, ants, and wasps), allergy diagnostics in Brazil is commonly based on non-standardised venom extracts [3]. The use of this type of raw material in clinical practice often compromises identification of the venom responsible for the primary sensitization [4] and the outcome of the immunotherapy. Currently, no venom allergens from Brazilian native species are available for the rational design of component-resolved diagnostics (CRD) [5]. In contrast to European wasps, no recombinant venom allergens from Brazilian Hymenoptera are commercially available as surrogates for venom extracts-based diagnostics, which is often affected by detection of clinically irrelevant cross-reactive IgE, in addition to low sensitivity. Apoica pallens (Hymenoptera, Vespidae) is a clinically relevant Neotropical social wasp commonly found in rural areas, occurring from Mexico to Argentina. Despite causing a high number of sting accidents and IgE-mediated anaphylaxis, A. pallens venom allergy is a neglected human health problem in most Latin American countries, including Brazil [6]. A. pallens nests were collected at São Paulo State University and in surrounding areas, in Rio Claro, São Paulo State, southeast Brazil (SISGEN protocol A0C2FB3). After collection, the insects were immediately frozen, dissected, and the crude venom was extracted. The venom was fractionated thorough a cation exchange chromatography, and the proteins were sequenced and identified by using proteomic shotgun approach, through digestion in solution with trypsin and chymotrypsin. This strategy permitted the unambiguous sequencing of PLA1 and antigen 5, which will appear in the UniProt Knowledgebase under accession codes C0HMC9 and C0HMD0, respectively. Multiple alignment analysis showed high to moderate levels of primary sequence similarity of the PLA1 and antigen 5 with homologous venom proteins from other clinically relevant social wasps. The specific IgE (sIgE) reactivities to these proteins were detected by immunoblotting, using a pool of sera from A. pallens venom allergic patients. The IgE immunoreactivities of purified native PLA1 and antigen 5 were assessed by ELISA. The allergens were incubated with individual sera from patients (n = 22) with a clinical history of allergic reaction to A. pallens venom and sensitization confirmed by UniCAP250 (cutoff i3, sIgE ≤ 0.35 kU/L). Only peptide epitope-based sIgE reactivity was detected, as previously reported for allergens of venoms from Neotropical wasps including A. pallens, which lack cross-reactive carbohydrate determinants (CCD) [7]. Both purified allergens were recognised by the sera of all the sensitised patients (n = 22). All the sera also presented reactivity to A. pallens crude venom (Figure 1A). PLA1 and antigen 5 are unique marker allergens with no homologues in HBV. As expected, there was no detected cross-reactivity of PLA1 and antigen 5 with sera from HBV mono-sensitised patients (Figure 1B), suggesting that they could be valuable novel diagnostic tools for distinguishing wasp venom from Apid sensitizations during routine diagnostics in Brazil [5]. These results strongly suggested that the combined use of the novel allergens would provide high diagnostic sensitivity. Finally, investigation was made of the peptide-based cross-reactivity among these allergens and their homologues in Polistes dominula, the European paper wasp. Consistent with results obtained previously for venom PLA1 of the Brazilian paper wasp P. paulista (Pol p 1) [8], extensive peptide-based cross-reactivity was detected in ELISA for PLA1 and antigen 5 with sera from Polistes sensitised patients (Figure 1C). Due to the features reported above, the WHO/IUIS Allergen Nomenclature Sub-Committee considered both proteins as novel allergens from the venom of the social wasp A. pallens. Hence, as allergens, PLA1 was officially named Apo p 1 (https://www.allergen.org/viewallergen.php?aid=1160), while antigen 5 was named Apo p 5 (https://www.allergen.org/viewallergen.php?aid=1161). Venom allergens from clinically relevant Brazilian native wasps remain largely unexplored. To date, only the major components from P. paulista wasp venom (Pol p 1, Pol p 2, and Pol p 5) have been officially annotated as wasp venom allergens [9]. Therefore, given the lack of data on A. pallens allergenic venom compounds, a comprehensive biochemical and immunological characterisation was performed for the Apo p 1 and Apo p 5 as novel venom allergens. To our knowledge, this represents the first conclusive evidence of extensive cross-reaction among homologous allergens from major elicitors of wasp venom allergy in Europe and Latin America. The high levels of similarity of the primary sequences and the 3-D structural models of Apo p 1 and Apo p 5 with their counterparts in PDV and P. paulista (Hymenoptera: Vespidae), suggest the common linear and conformational peptide epitopes as the structural basis for the cross-reactivity detected among members of the Polistinae subfamily. The high levels of diagnostic sensitivity of Pol d 1 and Pol d 5 for the detection of A. pallens sensitization indicates that they may potentially be used as surrogates for venom extracts in routine IgE testing. Hence, Apo p 1 and Apo p 5 are novel candidates for the development of molecular diagnostics of A. pallens venom allergy, which could enable better rationale for therapeutic decisions regarding Brazilian allergic patients. A detailed description of the methods and results is available at: https://osf.io/fmsyc/?view_only=3a1a76cf7b614e07b8dc1cf74dcb0f7d. Amilcar Perez-Riverol: Formal analysis, investigation, methodology, writing – review and editing. Gabriel Hideki Izuka Moraes: Investigation, methodology. José Roberto Aparecido dos Santos-Pinto: Supervision, validation, writing – review and editing. Luis Gustavo Romani Fernandes: Formal analysis, investigation, methodology, writing – review and editing. Alexis Musacchio Lasa: Investigation and Methodology. Brita Dorn: Formal analysis, investigation, methodology, writing – review and editing. Maria Beatrice Biló: Resources, validation, writing – review and editing. Ricardo de Lima Zollner: Resources, supervision, validation, writing – review and editing. Thilo Jakob: Resources, supervision, validation, writing – review and editing. Mario Sergio Palma: Resources, supervision, validation, writing – review and editing. The authors declare no conflicts of interest. The data that supports the findings of this study are available in the supporting information of this article.