Purpose: Allergy is a hypersensitivity disease IgE-mediated, affecting more than 25% of the population. The
symptoms of IgE-mediated allergies reactions can be transiently ameliorated pharmacologically, but the only
curative treatment of allergies is Allergen-Specific Immunotherapy (SIT). Recombinant hypoallergenic
allergen 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 Lipid Tranfer Proteins
(Par j 1 and Par j 2). By means of DNA recombinant technology, a hybrid hypoallergenic (PjEDcys),
expressing disulphide bond variants of Par j 1 and Par j 2, was generated. The aim of this research project is
to study the immunological mechanisms activated by the major allergens of Parietaria judaica, Par j 1 and
Par j 2, and hypoallergenic hybrid rPjEDcys. Moreover, the project I am involved is trying to address the
question whether this engineered hypoallergenic derivative can be a potential products for safer Allergen
Specific Immunotherapy (SIT).
Methods: Par j 1, Par j 2 and PjEDcys were produced as recombinant proteins. Human Peripheral Blood
Mononuclear Cell (PBMC) from P. judaica allergic patients were stimulated in vitro with wild-type
recombinant allergens and hybrid. PBMC proliferation assay, cytokine secretion assay, magnetic cell sorting
of different subset of regulatory T cells, multiparametric flow cytometric analysis and molecular
characterization using Real Time-PCR on sorted cells allow to study the biological properties of wild-type
recombinant allergens and hybrid hypoallergenic derivate.
Results: In vitro analysis suggested that PjEDcys have a reduced allergenity and maintained T cells
reactivity. PBMC of P. judaica allergic patients stimulated in vitro with the hybrid and the wild-type
recombinant allergens scored a percentage of proliferating CD4+ and CD56+ cell higher than unstimulated
sample. Consistent with these data, cytokine secretion assay on CD4+ cells demonstrated that PBMC
stimulation with rPjEDcys showed a percentage of IL-5 and IL-13 secreting T CD4+ cells lower than the
wild-type allergens. Both rPjEDcys and wild-type stimulation promote the secretion of IFN- \u3b3 and IL-10 by
T CD4+ cells. Finally whit the aim to study which subset of regulatory cells respond to wild-tipe allergens
and hypoallergenic hybrid new experiment are setting.
Discussion: In this experimental setting, the use of the major allergens of Pj and the hybrid polypeptides,
rPjEDcys allows me to study the immunological mechanisms activated by the two different antigen
stimulation and to investigate differences between the wild-type allergen and the hypoallergenic mutant
rPjEDcys. Our data showed that CD4+ cells are clearly the predominant cell population proliferating in
response to mixture of Par j 1 and Par j 2 allergens. The hypoallergenic derivate rPjEDcys retain the ability
to stimulate CD4+ cells proliferation like the mixture of allergens (rPar j 1 and rPar j 2). Moreover these
results highlighted a particular interesting datum; the mixture of allergens and the rPjEDcys hybrid showed
the ability to stimulate an innate immune response, inducing CD56+ cells proliferative response. Cytokine
secretion assay demonstrate that rPjEDcys reduce the secretion of IL-5 and IL-13, Th2 cytokines with a
critical role in the development of allergy, compared to wild-type allergens. This may reflect the different
biological function exerted by rPjEDcys.
Conclusion: Collectivelly, our findings demonstrate that PjEDcys show a reduced allergenicity but
maintained its immunogenicity and maybe it is also capable to regulate and redirect the immune response.
These results suggest that PjEDcys represent a useful approach for immunotherapy of allergic disease