Methods: Mimotopes specific to the epitopes of the major shellfish allergen tropomyosin were identified by screening the one-bead-one-compound (OBOC) peptide library. The OBOC library is a chemical synthetic library allowing high throughput screening of mimotopes with quantitative estimation on binding affinity. This method is advantageous over the conventional phage-displayed libraries by allowing the use of polyclonal antibodies or even untreated serum samples. The mimicry potential of the mimotopes was validated by both in silico and in vivo analysis. To investigate the therapeutic potential of mimotopes for allergy diseases, we used a mouse model of shrimp allergy through intragastric gavage of tropomyosin with cholera toxin as adjuvant followed by oral challenge. Splenocyte proliferation and local cytokine expression in the jejunum were analyzed to elucidate possible mechanisms of therapeutic effects of the mimotopes.
Results: Twenty-five mimotopes specific to shrimp tropomyosin were identified by screening OBOC peptide library. In silico analysis revealed six clusters of mimotopes, with the mimotopes in each cluster sharing at least three or more identical amino acid residues at the same position. With the automated epitope mapping tool EpiSearch, the six clusters of mimotopes could be mapped to six epitope regions of shrimp tropomyosin, of which five were identical to the previous reported epitopes. One mimotope from each cluster were synthesized and conjugated to the carrier protein keyhole limpet hemocyanin (KLH) for in vivo analysis. BALB/c mice immunized with mimotope-KLH conjugate were found to have an elevated level of tropomyosin-specific IgG but not in mice immunized with KLH alone or an irrelevant mimotope. The therapeutic potential of these mimotopes were further investigated with the use of the BALB/c mouse model of shrimp allergy. Sensitized mice were injected with a mixture of six mimotope-KLH conjugates, one from each cluster, before receiving a subsequent oral challenge. Compared to the control mice receiving KLH alone, the mimotopes-treated mice demonstrated a suppressed splenocyte proliferation response to tropomyosin and a reduced expression of cytokines in the jejunum.
Conclusion: The OBOC peptide library is a useful tool in identifying mimotopes for allergens with multiple epitopes. Mimotopes specific to the tropomyosin were identified by screening OBOC library and validated by in silico and in vivo experiments. The mimotopes could be potential therapeutic candidates for allergy diseases.
[The present work was supported by grants from the Research Grants Council (CUHK 463911) and the Health and Medical Research Fund (02130206), HKSAR Government and from the Food Allergy and Anaphylaxis Network.]