INTRODUCTION: In the doctoral thesis we identified epitopes of the allergen Ves v 5, the major allergen contained in wasp venom, meaning that it is responsible for the majority of allergies caused by a wasp sting.
Allergen epitopes play a crucial role in the process of allergic reaction as they represent the area of IgE antibody binding, consequently leading to effector cell degranulation and manifestation of allergic reaction symptoms. Allergens display epitopes on their surface, and while the presence of monovalent binding of IgE antibody to the allergen is sufficient for the IgE detection in the sera for degranulation of effector cells and the consequent manifestation of allergic symptoms, at least two receptor-bound IgE antibodies need to be cross-linked.
Identification of allergen epitopes is thus a crucial factor in the development of new diagnostic tests and new approaches for immunotherapy treatment. Epitope-based immunotherapy represents a potential alternative to currently used specific immunotherapy, since the synthetic peptides possess certain advantages compared to whole allergen extracts, such as production under standardised conditions, relatively uncomplicated isolation and stability in lyophilised form.
Due to the advancements in the field of next-generation sequencing (NGS) and the development of new bioinformatic tools for analysing phage-display data and protein-peptide interactions, epitope identification reached a new peak as new technologies allowed us to obtain more reliable data.
METHODS: For B-cell epitope identification we utilised phage display technology, using three bacteriophage libraries with a peptide length of 7 or 12 amino acids. Peptides were selected based on the binding to polyclonal anti-Ves v 5 IgG antibodies, which was followed by NGS and bioinformatic analysis of the obtained data (using PuLSE, R, SAROTUP, MEME, Hammock and WebLogo tools). Results were compared to previously identified epitope-like peptides submitted to the online database IEDB. Visualisation of epitope regions on the allergen was performed using Pepitope and Pymol.
Using the peptide microarray, we evaluated the binding of IgE antibodies from 36 wasp sting symptomatic and 36 wasp sting asymptomatic individuals to the selected epitope-like peptides, with the aim of analysing whether antibodies of both groups bind different epitopes. We used linear and cyclic forms of epitope-like peptides.
RESULTS: In the first part we identified epitope-like peptides and, consequently, the major epitope regions of the allergen. Prominent amino acid motifs of the three identified epitope regions are composed of amino acids TKQE, GKI, or KPN. In the online database of previously identified epitopes, we found 42 submissions for the allergen Ves v 5, from which all of them are T-cell epitopes. While the submissions containing TKQ(E) or KPN motifs appear several times, no GKI motif submission has yet been published.
Analysis of the peptide microarray revealed that in 7/12 cases antibodies bind significantly better to linear peptides and in 2/12 to cyclic peptides (in three cases, no difference was observed). Additionally, IgE antibodies bind significantly better to the peptides containing TKQE (P < 0.001) or KPN (P < 0.001) compared to the GKI motif. Sera incubation of symptomatic and asymptomatic individuals revealed that both groups recognise the same epitope regions. However, several identified epitope-like peptides differ among symptomatic and asymptomatic individuals. A more significant difference could be observed for binding affinity, as asymptomatics bind the epitope-like peptides significantly better, both in linear and cyclic conformation (P = 0.001).
CONCLUSION: In the presented doctoral thesis, we introduced a new protocol of epitope identification, implementing NGS technology and bioinformatic tools, which consequently allowed us to identify three epitope regions on the allergen Ves v 5. The utilisation of identified epitopes to distinguish between symptomatic and asymptomatic sensitisation showed that symptomatics and asymptomatics recognise the same epitope regions on the allergen; however, they differ in recognition of side amino acids (amino acids that contribute a smaller share of binding energy) and in the total binding affinity. Further research is needed to clarify the meaning of the obtained results.
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