Carlos Pastor-Vargas

Epitope Mapping of Atlantic Salmon Major Allergen by Peptide Microarray Immunoassay

Background: IgE epitope mapping of allergens reveals important information about antigen elicitors involved in allergic reactions. The peptide-based microarray immunoassay offers an advantage of scale and parallel design over previous methods of epitope mapping. It has been used to map epitopes of some food allergens but has never been used with fish allergens. Objective: We sought to develop a peptide microarray immunoassay to map allergenic fish epitopes of two isoforms of Atlantic salmon (Salmo salar) parvalbumin, Sal s 1 beta 1 and Sal s 1 beta 2. Methods: Sera from 16 fish-allergic patients with specific IgE to salmon parvalbumin were used. Twelve healthy volunteers were used as negative controls. A library of overlapping peptides was synthesized commercially, representing the primary sequence of Sal s 1 beta 1 and Sal s 1 beta 2. Peptides were used to analyze allergen-specific IgE antibodies by immunolabeling with patient sera. Results: Three antigenic regions, not previously described, were identified in Sal s 1 beta 1. Two of them correlated with those previously reported in Gad c 1, parvalbumin from Baltic cod (Gadus callarias). No allergenic regions were found in Sal s 1 beta 2. This could be explained by crucial amino acid substitutions between isoforms. Conclusions: We have identified three antigenic regions in Sal s 1 beta 1 using a peptide microarray immunoassay. These three sequential epitopes formed a unique antigenic determinant in the three-dimensional model of the protein. In addition, we proved that isoforms from the same protein might have a different allergenic behavior.

Identification of sole parvalbumin as a major allergen: study of cross-reactivity between parvalbumins in a Spanish fish-allergic population.

Background Fish allergy is becoming an important health problem in Spain, a country with the third highest level of fish consumption after Japan and Portugal. The most common fish allergens are parvalbumins. In our area, the most widely consumed fish species are lean, such as whiff (Lepidorhombus whiffiagonis) and sole (Solea solea). Adverse reactions to fish are usually related to these species, a fact that is largely unknown to allergists in other countries.

Epitope mapping of the major allergen from Atlantic cod in Spanish population reveals different IgE-binding patterns.

SCOPE IgE-epitope mapping of allergens reveal important information about antigen components involved in allergic reactions. The peptide-based microarray immunoassay has been used to map epitopes of some food allergens. We developed a peptide microarray immunoassay to map allergenic epitopes in parvalbumin from Atlantic cod (Gad m 1), the most consumed cod species in Spain. METHODS AND RESULTS Sera from 13 fish-allergic patients with specific IgE to cod parvalbumin were used. A library of overlapping peptides was synthesized, representing the primary sequence of Gad m 1. Peptides were used to analyze allergen-specific IgE antibodies in patient sera. 100% of the patients recognized one antigenic region of 15 amino acids in length in Gad m 1. This region only partially correlated with one of the three antigenic determinants of Gad c 1 (Allergen M), parvalbumin from Baltic cod (Gadus callarias). In the 3D model of the protein, this region was located on the surface of the protein. CONCLUSION We have identified a relevant antigenic region in Gad m 1. This epitope could be considered as a severity marker and provides additional information to improve fish allergy diagnosis and the design of safe immunotherapeutic tools.

Dehydrated egg white: An allergen source for improving efficacy and safety in the diagnosis and treatment for egg allergy

Raw and cooked eggs are used as allergens in oral food challenge (OFC). Raw egg is the best option, as it keeps proteins intact and retains their allergenicity, albeit microbiologically safe manipulation is difficult. Therefore, the use of dehydrated egg white (DEW) could improve the efficacy and safety profile of OFC. The aim of the study was to compare the allergenicity of DEW, a product that undergoes a double heat treatment (pasteurization and drying), with that of raw egg white (REW) and determine the efficacy of DEW in the diagnosis of egg allergy.

Allergy to goat’s and sheep’s milk in a population of cow’s milk–allergic children treated with oral immunotherapy*

To cite this article: Rodríguez del Río P, Sánchez‐García S, Escudero C, Pastor‐Vargas C, Sánchez Hernández JJ, Pérez‐Rangel I, Ibáñez MD. Allergy to goat’s and sheep’s milk in a population of cow’s milk–allergic children treated with oral immunotherapy. Pediatr Allergy Immunol 2012: 23: 128–132.

Arginine kinase from the cellar spider (Holocnemus pluchei): a new asthma-causing allergen.

Background: We report a 31-year-old farmer whose work consists in handling cereal and vegetables, who consulted our clinic because of asthma symptoms after inhalation of dust during manipulation of the deposited material, usually inside the warehouse. Methods and Results: Skin prick tests and specific immunoglobulin E (IgE) determinations were negative with common aeroallergens. The patient noted the presence of many spiders in the warehouse, which were identified as the cellar spider Holocnemus pluchei and the common house spider, Tegenaria domestica. Extracts of spider bodies brought in by the patient were obtained and used to perform in vivo and in vitro studies. Molecular characterization of IgE-binding bands was performed by mass spectrometry. We obtained positive prick tests to the extracts of the bodies of both spiders. Immunoblotting displayed different bands in both spider extracts, in a range of 20–70 kDa. All were hemocyanins, except for a 17-kDa protein of Holocnemus identified as an arginine kinase (AK). Bronchial challenge was positive with the extract of the cellar spider and with the AK, but was negative with the domestic house spider. Conclusion: We present the first case of respiratory allergy due to sensitization to AK from a common spider, confirmed by bronchial provocation tests.

Involvement of Can f 5 in a Case of Human Seminal Plasma Allergy

Background: The existence of IgE binding to dog dander extract without IgE antibodies against the described dog allergens (Can f 1, 2, 3 and 4) implies the presence of other dog allergens yet to be identified. Recently, an IgE-binding protein was isolated from dog urine and identified as prostatic kallikrein; it has been named Can f 5. Cross-reactivity between a dog dander allergen and human prostate-specific antigen (PSA) has been described. The aim of this study was to identify the dog dander allergen that presents cross-reactivity with PSA and demonstrate its clinical relevance in our patient with human seminal plasma allergy. Methods: SDS-PAGE immunoblotting and inhibition tests were performed. Mass spectrometry was carried out to identify the protein involved in the allergy reactions. Results: SDS-PAGE immunoblotting-inhibition with an IgE-binding protein from dog prostatic secretion showed total IgE binding inhibition to a 28-kDa IgE-reactive band identified as PSA. The electroeluted protein from dog prostatic secretion was identified by mass spectrometry as Can f 5. IgE immunoblotting of human seminal plasma incubated with the serum of the patient revealed two IgE-binding bands (28 and 32.7 kDa). Both SDS-PAGE immunoblotting inhibition assays, with human seminal plasma or purified PSA in solid phase, showed complete IgE binding inhibition when the serum of the anaphylactic patient was preincubated with dog dander extract or recombinant Can f 5. Conclusions: The dog dander allergen that shows cross-reactivity with human PSA has been characterized and turns out to be the recently described Can f 5. We demonstrated the clinical relevance of this cross-reactivity in a patient.

Targeting antigens to an invariant epitope of the MHC Class II DR molecule potentiates the immune response to subunit vaccines

Recombinant subunit and peptidic vaccines in general present a reduced immunogenicity in vaccinated individuals with respect to the whole pathogen from which they derived. The generation of strong immune responses to these vaccines requires the use of potent adjuvants, high antigen doses and repetitive vaccinations. In this report, we document the enhanced antibody response obtained against two recombinant subunit vaccines by means of targeting to antigen-presenting cells by a recombinant single chain antibody. This antibody, named APCH1, recognizes an epitope of MHC Class II DR molecule preserved in different animal species, including humans. We showed that vaccinal antigens translationally fused to APCH1 antibody and produced by recombinant baculoviruses in insect larvae (Trichoplusia ni), elicited an increased antibody response in comparison with the same antigens alone or fused to a carrier molecule. These results suggest that targeting of antigens to this invariant MHC Class II epitope has immunopotentiating effects that could circumvent the reduced potency of peptidic or subunit vaccines, opening the possibility of widespread application of APCH1 as a new adjuvant antibody of general use.

Watermelon Profilin: Characterization of a Major Allergen as a Model for Plant-Derived Food Profilins

Background: Plant profilins have been reported as minor allergens. They are a well-known pan-allergen family responsible for cross-reactivity between plant-derived foods and pollens. Watermelon profilin has been reported to be a major allergen in watermelon (Citrullus lanatus).The aim of this study was to characterize recombinant watermelon profilin, confirming its reactivity for diagnostic purposes and the development of immunotherapy. Methods: Native profilin was purified from watermelon extract by affinity chromatography using poly-L-proline. Recombinant His-tagged profilin was produced in Pichia pastoris yeast using pPICZαA vector and purified by metal chelate affinity chromatography. ELISA and immunoblot were carried out with sera from 17 watermelon-allergic patients. Biological activity was tested by the basophil activation test. Results: Native profilin and recombinant profilin were purified and identified by mass spectrometry. Both show similar IgE reactivity in vitro and are biologically active. Conclusions: Similarities were found in the IgE-binding patterns and biological activity of recombinant profilin and native profilin. Recombinant profilin may be a powerful tool for specific diagnosis.

Allergy to uncommon pets: new allergies but the same allergens.

The prevalence of exotic pet allergies has been increasing over the last decade. Years ago, the main allergy-causing domestic animals were dogs and cats, although nowadays there is an increasing number of allergic diseases related to insects, rodents, amphibians, fish, and birds, among others. The current socio-economic situation, in which more and more people have to live in small apartments, might be related to this tendency. The main allergic symptoms related to exotic pets are the same as those described for dog and cat allergy: respiratory symptoms. Animal allergens are therefore, important sensitizing agents and an important risk factor for asthma. There are three main protein families implicated in these allergies, which are the lipocalin superfamily, serum albumin family, and secretoglobin superfamily. Detailed knowledge of the characteristics of allergens is crucial to improvement treatment of uncommon-pet allergies.