A novel allergen-adjuvant conjugate suitable for specific immunotherapy of respiratory allergy

Abstract

Background: Several approaches to find a better adjuvant, focus immunomodulation, and reduce allergenicity are under investigation to improve the efficacy and safety of specific immunotherapy. Objective: We performed an investigation of the in vitro and in vivo effects of a purified allergen chemically conjugated to a novel 8-OH modified adenine as an adjuvant. Methods: Purified group 2 major allergen from house dust mite chemically conjugated to 4-(6-amino-9-benzyl-8-hydroxy-9H-purin-2-ylsulfanyl)-butyric acid succinimidyl ester was analyzed by using mass spectrometry. The adduct (nDer p 2-Conj) was assayed for Toll-like receptor activation on transfected HEK293 cells, stimulation of innate cells, and effects on the functional phenotype of specific T-cell lines and clones by means of flow cytometry, real-time PCR, and expression of T H -related transcription factors. Lung cells and sera of nDer p 2-Conjsensitized C57Bl/6 mice were studied by means of cytology, histology, real-time PCR, and ELISA. Results: nDer p 2-Conj stimulated IL-12 and IFN-a production from monocytes and plasmacytoid dendritic cells, respectively, retaining the ability to trigger Toll-like receptor 7 exclusively, and expanded human allergen-specific lymphocytes with reduced ability to produce T H 2-related cytokines and increased IFN-g levels, as based on GATA-3/T-bet expression. In vivo adduct-sensitized mice exhibited reduced eosinophil infiltration and IL-13 expression in the airways, IFN-g upregulation together with IgE downregulation, and an increase in allergenspecific IgG 2a levels in sera. The conjugate exhibited reduced ability to activate human FcεRI 1 cells without inducing T H 17 cells or autoantibodies.

Dermatophagoides pteronyssinus group 2 allergen bound to 8-OH modified adenine reduces the Th2-mediated airway inflammation without inducing a Th17 response and autoimmunity

“…Furthermore, significantly higher levels of the IL-1␤, IL-6 and IL-23 were also induced by the nDer p2-Conj than nDer p2 when cultured with purified CD11c+ spleen cells from wt C57BL/6 mice (data not shown). We also confirmed the induction of higher levels of proinflammatory (IL-27, CXCL10) and regulatory (IL-10) cytokines by the nDer p2-Conj than the corresponding free allergen (Filì et al, 2013;Nencini et al, 2015).…”

“…SA26E synthesis and its binding to nDer p2 or OVA were performed as previously described (Filì et al, 2013;Nencini et al, 2015). Briefly, SA26E was dissolved in 130 l DMSO (Sigma Aldrich, Milan, Italy) and chemically conjugated with 3 mg of purified OVA or 1.5 mg of nDer p2 in phosphate buffer (to final volumes of 1.5 and 3 ml, respectively) by overnight incubation at 4 • C with continuous rotation.…”

“…Lung sections were stained with haematoxylin and eosin (H&E) and periodic acid Schiff (PAS) to evaluate lung inflammation and goblet cell hyperplasia according to scores previously described (Filì et al, 2013;Vultaggio et al, 2011) 2.7. Generation of BMDC BMDC were prepared according to well-defined protocols (Vultaggio et al, 2009) and 1 × 10 6 cells were cultured in vitro at d8 for 6 h (mRNA detection) and 72 h (protein detection) with nDer p2 (10 g/ml) or nDer p2-Conj (10 g/ml), or medium alone.…”

“…Total lung RNA from snap-frozen mouse lungs were extracted using TRIzol reagent (RNAwiz, Invitrogen, Milan, Italy), whereas total RNA from MNC were extracted using the RNeasy mini kit (Qiagen, Milan, Italy) as previously described (Filì et al, 2013). Real-time quantitative PCR was performed on an ABI PRISM 7700 Sequence Detector (Applied Biosystems, Warrington, UK) with Applied Biosystems predesigned TaqMan Gene Expression assays and reagents according to the manufacturer's instructions.…”

“…The conjugate (nDer p2-Conj) between the modified adenine SA26E, signaling through human and murine TLR7, and the natural Dermatophagoides Pteronyssinus 2 (nDer p2) allergen, was able to redirect nDer p2-specific Th2 responses in vitro and to prevent the development of airway inflammation in vivo in a short term protocol (Filì et al, 2013). Moreover, when administered in allergen-sensitized mice in a therapeutic setting, both nDer p2-and ovalbumin (OVA)-Conj improved airway inflammation and redirected Th2 effector cells into IL-10-and IFN-␥-producing T cells associated with reduced IgE and increased IgG2a antibodies (Nencini et al, 2015).…”