B-cell linear epitopes mapping of antigen-5 allergen from Polybia paulista wasp venom José Roberto Aparecido dos Santos-Pinto, PhD, Lucilene Delazari.

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Presentation transcript:

B-cell linear epitopes mapping of antigen-5 allergen from Polybia paulista wasp venom José Roberto Aparecido dos Santos-Pinto, PhD, Lucilene Delazari dos Santos, PhD, Helen Andrade Arcuri, PhD, Anally Ribeiro da Silva Menegasso, MSc, Paloma Napoleão Pêgo, MSc, Keity Souza Santos, PhD, Fábio Morato Castro, MD, PhD, Jorge Elias Kalil, MD, PhD, Salvatore Giovanni De-Simone, PhD, Mario Sergio Palma, PhD Journal of Allergy and Clinical Immunology Volume 135, Issue 1, Pages 264-267.e8 (January 2015) DOI: 10.1016/j.jaci.2014.07.006 Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 B-cell linear epitopes mapping along the primary sequence of the P paulista venom antigen-5 detected in the SPOT synthesis assay with the sera of patients who were sensitive to the venom of the wasp P paulista. IgG-reactive peptides (A); IgE-reactive peptides (B); the smallest peptide sequence corresponding to the B-cell linear epitope of antigen-5 that is immunoreactive to human IgE (C); P paulista venom antigen-5 primary sequence (D) showing regions of IgG reactivity (black bars), IgE reactivity (grey bars), and the identification of the smallest peptide sequence corresponding to the B-cell linear epitope that was immunoreactive to human IgE. Journal of Allergy and Clinical Immunology 2015 135, 264-267.e8DOI: (10.1016/j.jaci.2014.07.006) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Three-dimensional mapping of B-cell linear epitopes of antigen-5 that were reactive to human IgG shown in yellow: epitope 1 (A), epitope 2 (B), epitope 3 (C), epitope 4 (D), epitope 5 (E), epitope 6 (F), epitope 7 (G), epitope 8 (H), and epitope 9 (I); epitope 7 is also reactive to human IgE. The elements of secondary structures shown in blue correspond to helices; those shown in red and green correspond to beta-sheets and loops, respectively; meanwhile, the regions represented in yellow correspond to the spatial location of the linear epitopes for human IgG and/or IgE. Journal of Allergy and Clinical Immunology 2015 135, 264-267.e8DOI: (10.1016/j.jaci.2014.07.006) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 Intensity analysis of the reactivity of peptide sequences corresponding to B-cell linear epitope 7 of P paulista antigen-5 reactive to human IgE. C5.1-VGHYTQVVWAKTKE, C5.2-VGHYTQVV, C5.3-TQVVWAKTKE, C5.4-WAKTKE and Control (-). Journal of Allergy and Clinical Immunology 2015 135, 264-267.e8DOI: (10.1016/j.jaci.2014.07.006) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E2 Intensity analysis of the reactivity of peptide sequences corresponding to B-cell linear epitopes of antigen-5 immunoreactive to human IgG. Journal of Allergy and Clinical Immunology 2015 135, 264-267.e8DOI: (10.1016/j.jaci.2014.07.006) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E3 Evaluating the potential epitopes of antigen-5 by indirect ELISA with a pool of sera from patients allergic to P paulista venom. Peptide reactivity with human IgG (A); peptide reactivity with human IgE (B). Journal of Allergy and Clinical Immunology 2015 135, 264-267.e8DOI: (10.1016/j.jaci.2014.07.006) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E4 Alignment of the sequences of known antigen-5 homologs found in the venoms of different species of social wasps: POLPI-Polybia paulista; POLEX-Polistes exclamans; POLAN-Polistes annularis; POLFU-Polistes fuscatus; POLGA-Polistes gallicus; POLDO-Polistes dominula; VESVU-Vespula vulgaris; VESGE-Vespula germanica; VESMC-Vespula maculifrons; VESPE-Vespula pensylvanica; VESSQ-Vespula squamosa. The conserved B-cell epitopes identified in P paulista antigen-5 are conserved in all these sequences and are shown inside the rectangles indicating epitopes 1 to 9. Journal of Allergy and Clinical Immunology 2015 135, 264-267.e8DOI: (10.1016/j.jaci.2014.07.006) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E5 Biological activities assay for the peptide sequences corresponding to B-cell linear epitopes of antigen-5 immunoreactive to human IgE (epitope 7) and immunoreactive to human IgG (epitopes 1 to 9). Degranulation activity in rat peritoneal mast cells (A); the activity was determined by measuring the release of the granule marker, β-D-glucosaminidase, which co-localizes with histamine. The values for β-D-glucosaminidase released in the medium were expressed in the percentage of total enzyme activity. Hemolytic activity in washed rat red blood cells (B); the absorbance measured at 540 nm from lysed washed rat red blood cells in presence of 1% (v/v) Triton X-100 (Sigma, St Louis, Mo) was considered as 100%. Chemotaxis of polymorphonucleated leukocytes (C). The results were compared with the activities measured for the standard mast cell degranulating and hemolytic activity peptide melittin and compared with the activities measured for the standard chemotactic activity peptide protonectin 1-6. Data are represented as means ± SDs (n = 5). Journal of Allergy and Clinical Immunology 2015 135, 264-267.e8DOI: (10.1016/j.jaci.2014.07.006) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions