A novel allergen-adjuvant conjugate suitable for allergen-specific Th2 redirection

Background: In a previous paper we described a substituted 8-hydroxyadenine named SA-2 as able to in vitro revert the TH2 phenotype of human allergen specific T cells by triggering TLR7. The conjugation of the adjuvant to the allergen is a novel and efficacious strategy of allergen immunotherapy because it enhances the immunogenicity and reduces the allergenicity of the vaccine. In this work we synthetized another adenine derivative named SA-26E, which was chemically conjugated to the purified natural allergen Derp2 (nDer p2-Conj) from Dermatophagoides pteronyssinus and we assessed this conjugate for its immunomodulatory activity on human cells. Method: nDer p2-Conj was characterized by MALDI-TOF analysis and evaluated for induction of NF-jB in CD14+ cells, TLR-triggering in transfected HEK293 cell lines, cytokine production in BDCA4+ and CD14+ cells, Th-skewing effects in allergen- specific human T cell lines and T cell clones and allergenicity in BASOTEST assays. Results: nDer p2-Conj induced nuclear translocation of p50 and trigger TLR7, induced innate cells to produce IFN-a and IL-12 at similar levels than dispersible TLR ligand R-848. As a consequence, the conjugate reverted TH2-prone allergen T cell lines into IFN-c-producing cells (TH1/ TH0 phenotype) as assessed by cytokine production in the supernatants, intracellular cytokine expression and TH-related transcription factor expression (GATA-3 and T-bet). nDer p2-Conj was also able to revert the phenotype of TH2 established cells, as demonstrated by culturing CRTH2 cells with this conjugate. We further demonstrated that the TH1 differentiation induced by allergen conjugate can be reverted by the addition of neutralizing antibodies against TH1 citokines. In addition, Derp2 specific T cell clones derived from the T cell lines cultured in the presence of Derp2 conjugate showed a TH1/ TH0 profiling. Finally, the nDer p2-Conj reduced basophils activation in comparison with uncoupled allergen protein. Conclusion: The system of chemical conjugation to relevant proteins as allergens confers the ability to induce the production of regulatory cytokines in innate immunity cells by TLR7 triggering and to down-regulate TH2 allergen-specific T cells, suggesting a relevant role in the development of novel immunotherapeutic strategies in allergic disorders.