D2.186 - In vivo and ex vivo evaluation of the immunological responses induced by a grass depigmented-polymerized allergenic extract in dogs

Modified allergenic extracts constitute an alternative for the treatment of Canine Atopic Dermatitis (CAD), a chronic, inflammatory skin disease in dogs that are often sensitized to environmental allergens. However, the mechanisms of action of modified extracts in dogs remain largely unknown. In particular, depigmented-polymerized extracts—whose safety and efficacy in human allergen immunotherapy are well established—have never been studied in dogs. The objective of this study was to evaluate the immunogenicity and in vitro efficacy of a depigmented-polymerized extract of Phleum pratense in dogs.

Native (NAT) and depigmented-polymerized (POL) extracts from P. pratense pollen were subcutaneously administered in stabled non-atopic beagle dogs (3 animals per type of extract) and blood samples were collected at different days. Levels of IgG, IgG1 and IgG2 were determined in serum samples by immunoblot and/or ELISA. The blocking capacity of dog IgG over human IgE was analysed by ELISA inhibition. Finally, PBMCs from atopic dogs (n=3) were stimulated for 36 and 60 h with both type of extracts. Afterwards, B, T and Treg cell activation was analysed by flow cytometry and cytokine production by Luminex technology.

Immunization with native and depigmented-polymerized extracts induced high levels of specific IgG, IgG1 and IgG2 with respect to sera pre-immunization. Moreover, IgG induced by both types of extracts had the capacity to reduce the binding of IgE from human grass-allergic patients to the allergen in more than 80%.

Regarding cellular studies, higher stimulation indexes (mean±SD) of activated B and T cells were obtained for POL extract (B cells: 4.77±2.62; T cells: 1.72±0.64) compared to NAT extract (B cells: 1.06±0.10; T cells: 0.95±0.09). No relevant differences were found between type of extracts in Treg induction. Finally, both types of extracts induced higher levels (pg/mL; mean±SD) of cytokines IFN-γ (NAT: 136.6±146.4; POL: 45.8±30.9) and IL-10 (NAT: 2784±1795; POL: 3077±2269) with respect to the non-stimulated cells (IFN-γ: 23.7±15.0; IL-10:1768±1367).

Depigmented-polymerized extract of P. pratense induced potent immunogenic and tolerogenic responses in dogs, suggesting its potential in allergen immunotherapy for treating CAD.