Stable Conjugates Between A Novel Toll-Like Receptor 7 Ligand and Protein Allergens As Modulators Of Th2 Responses In Vitro and In Vivo

Abstract

U E S D A Y 1002 Stable Conjugates Between A Novel Toll-Like Receptor 7 Ligand and Protein Allergens As Modulators Of Th2 Responses In Vitro and In Vivo Dr. Lucia Fil i, Dr. Alessandra Vultaggio, Dr. Elisa Cardilicchia, Dr. Cinzia Manuelli, Dr. Andrea Casini, Dr. Francesca Nencini, Dr. Laura Maggi, Dr. Sara Pratesi, Dr. Giulia Petroni, Dr. Francesca Boscaro, Prof. Ernesto Giovanni Occhiato, Prof. Sergio Romagnani, Prof. Enrico Maggi, Prof. Paola Parronchi; University of Florence, DENOTHE Center, Florence, Italy, University of Florence, Dept. of Chemistry ‘‘U. Shiff’’, Florence, Italy, University of Florence, Mass Spectrometry Center (CISM), Florence, Italy. RATIONALE: The allergen-specific immunotherapy represents a relevant approach to treat respiratory allergy. A strategy for novel vaccine formulations is to stably link adjuvants with the allergen. The in vitro and in vivo effects of natural Dermatophagoides pteronyssinus 2 and Ovalbumin chemically conjugated to a 8-OH modified adenine (nDer p2-Conj and OVA-Conj) in allergic inflammation were investigated. METHODS: In human setting Toll-like receptor activation on transfected HEK293 cells, stimulation of innate cells, assessment of the functional phenotype of specific T-cell lines and clones by means of flow cytometry, real-time PCR, expression of Th-related transcription factors, variation of allergenicity by BASOTEST. In murine models pre-priming and therapeutic protocols with assessment of TH2-mediated airway inflammation by means of cytology, histology, real-time PCR, and humoral responses by ELISAs. RESULTS: nDer p2-Conj induced human innate cells to produce IFN-a and IL-12 via TLR7 triggering. As a consequence, the conjugate reverted TH2-prone allergen T cell lines into IFN-g-producing cells (TH1/TH0 phenotype). nDer p2and OVA-Conj-primed mice exhibited reduced airway hyperreactivity, eosinophil infiltration and airway IL-13 expression, increased IFN-g and IL-10 levels in allergen-stimulated T cells, up-regulation of allergen-specific IgG2a and reduction of IgE levels. Into therapeutic protocol, OVA-conj redirected established allergen-specific TH2 cell responses into protective phenotypes. No evidence of increased autoantibodies, autoimmunity manifestations or B cell expansion was found. CONCLUSIONS: The co-delivery of a protein with allergenic properties with a modified adenine as a stable conjugate induce modulatory cytokines from innate cells and re-directs in vitro and in vivo pathogenic TH2 responses without eliciting harmful effects.