Fine characterization of immunological mechanisms mediated by the major allergens of Parietaria judaica and hypoallergenic hybrid, rPjEDcys.

Allergy is a hypersensitivity disease IgE-mediated, affecting more than 30% of the population living in the industrialized countries. The symptoms of allergic reactions can be transiently ameliorated pharmacologically, but the only curative treatment of allergies is Allergen-Specific Immunotherapy (SIT). Recombinant hypoallergenic derivatives with reduced allergenic activity have been engineered to reduce side effects during SIT. Parietaria judaica (Pj) pollen contains two major allergens belonging to the family of non specific-Lipid Transfer Proteins (ns-LTP): Par j 1 and Par j 2. The allergens Par j 1 and Par j 2 are recognized in approximately 95% of allergic patients. By means of DNA recombinant technology, a hybrid hypoallergenic (PjEDcys), expressing disulphide bond variants of Par j 1 and Par j 2, has been generated. The aim of this research project is to study the immunological mechanisms activated by the major allergens of Parietaria judaica, rPar j 1 and rPar j 2, and the hypoallergenic hybrid rPjEDcys. Moreover, the project I am involved in is trying to address the question whether this engineered hypoallergenic derivative can be a potential product for safer Allergen Specific Immunotherapy. In vitro analysis suggested that rPjEDcys has a reduced allergenity and maintains T cells reactivity. In particular we showed that PBMC of Pj allergic patients stimulated in vitro with the hybrid and the wild-type recombinant allergens scored a percentage of proliferating CD4+ cells higher than unstimulated samples. We also demonstrate that components of innate immune system (CD56+ cells) proliferate in response to wild-type allergens and rPjEDcys. Furthermore, cytokine secretion assays on CD4+ cells demonstrated that rPjEDcys induces a lower the secretion of two Th2 cytokines that are critical in the development of allergy such as IL-5 and IL-13 compared to wild-type allergens. Furthermore we observed the induction of a Treg cell subset (defined as CD4+ CD25++ CD127-) in response to rPjEDcys and the wild-type allergens. However, the number of these Treg cells and the intensity of CD25 expression is higher in response to hypoallergen hybrid than Parietaria major allergens. We also characterized these cells at molecular level by REAL-TIME PCR. Moreover, we addressed the kinetic of functional surface marker expression, such as GARP (Glycoprotein A Repetitions Predominant), LAP (Latency-Associated Peptide) CD39 and PD1 on CD4+ cells. Our analyses demonstrated that rPjEDcys induces a number of GARP-LAP-CD39 co-expressing cells and CD4+CD25++PD1+ higher than wild-type recombinant allergens. These results suggest that rPjEDcys represents a useful approach for immunotherapy of allergic disease.