Enhancing Allergen-Presentation Platforms for Sublingual Immunotherapy.

Sublingual immunotherapy (SLIT) relies on high doses of allergens to treat patients with type I allergies. Although SLIT is commonly performed without any adjuvant or delivery system, allergen(s) could be further formulated with allergen-presentation platforms to better target oral dendritic cells eliciting regulatory immune responses. Improving the availability of allergens to the immune system should enhance SLIT efficacy, while allowing to decrease allergen dosing. Herein, we present an overview of adjuvants and vector systems that have been, or could be, considered as candidate allergen-presentation platforms for the sublingual route. Such platforms encompass adjuvants capable of stimulating allergen-specific TH1 and/or regulatory CD4+ T-cell responses, including 1,25-dihydroxy vitamin D3, glucocorticoids, Toll-like receptor ligands as well as selected bacterial probiotic strains. A limiting factor for SLIT efficacy is the number of dendritic cells capturing the allergens in the upper layers of oral tissues. Thus, adsorption or encapsulation of the allergen(s) within mucoadhesive particulate vector (or delivery) systems also has the potential to significantly enhance SLIT efficacy due to a facilitated allergen uptake by tolerogenic oral dendritic cells.

The regulatory dendritic cell marker C1q is a potent inhibitor of allergic inflammation.

The complement subunit C1q was recently identified as a marker for monocyte-derived regulatory dendritic cells supporting the differentiation of interleukin (IL)-10-secreting CD4+ T cells with a suppressive activity. Furthermore, C1q expression is upregulated in peripheral blood mononuclear cells of allergic patients in the course of successful allergen immunotherapy. Herein, we investigated a potential direct role of C1q in downregulating allergic inflammation. In mice with ovalbumin (OVA) or birch pollen (BP)-induced allergic asthma, C1q is as efficacious as dexamethasone to reduce both airway hyperresponsiveness (AHR), eosinophil, and ILC2 infiltrates in bronchoalveolar lavages, as well as allergen-specific T helper 2 cells in the lungs. Administration of C1q does not expand IL-10+/Foxp3+ regulatory T cells in the lungs, spleen, or in the blood. Depletion of plasmacytoid dendritic cells (pDCs) abrogates the capacity of C1q to reduce AHR and eosinophilic infiltrates in OVA-sensitized mice. Also C1q treatment inhibits the activation of human and mouse pDCs by CpGs, thereby demonstrating a critical role for pDCs in the anti-inflammatory activity of C1q. We conclude that regulatory dendritic cells can mediate a potent direct anti-inflammatory activity via the expression and/or secretion of molecules such as C1q, independently of their capacity to expand the pool of regulatory T cells.

Development and evaluation of a sublingual tablet based on recombinant Bet v 1 in birch pollen-allergic patients.

BACKGROUND: Sublingual immunotherapy (SLIT) applied to type I respiratory allergies is commonly performed with natural allergen extracts. Herein, we developed a sublingual tablet made of pharmaceutical-grade recombinant Bet v 1.0101 (rBet v 1) and investigated its clinical safety and efficacy in birch pollen (BP)-allergic patients. METHODS: Following expression in Escherichia coli and purification, rBet v 1 was characterized using chromatography, capillary electrophoresis, circular dichroism, mass spectrometry and crystallography. Safety and efficacy of rBet v 1 formulated as a sublingual tablet were assessed in a multicentre, double-blind, placebo-controlled study conducted in 483 patients with BP-induced rhinoconjunctivitis. RESULTS: In-depth characterization confirmed the intact product structure and high purity of GMP-grade rBet v 1. The crystal structure resolved at 1.2 Å documented the natural conformation of the molecule. Native or oxidized forms of rBet v 1 did not induce the production of any proinflammatory cytokine by blood dendritic cells or mononuclear cells. Bet v 1 tablets were well tolerated by patients, consistent with the known safety profile of SLIT. The average adjusted symptom scores were significantly decreased relative to placebo in patients receiving once daily for 5 months rBet v 1 tablets, with a mean difference of 17.0-17.7% relative to the group treated with placebo (P < 0.025), without any influence of the dose in the range (12.5-50 µg) tested. CONCLUSION: Recombinant Bet v 1 has been produced as a well-characterized pharmaceutical-grade biological drug. Sublingual administration of rBet v 1 tablets is safe and efficacious in patients with BP allergic rhinoconjunctivitis.

Characterization of oral immune cells in birch pollen-allergic patients: impact of the oral allergy syndrome and sublingual allergen immunotherapy on antigen-presenting cells.

BACKGROUND: A detailed characterization of human oral immune cells is needed to better understand local mechanisms associated with allergen capture following oral exposure. METHODS: Oral immune cells were characterized by immunohistology and immunofluorescence in biopsies obtained from three healthy individuals and 23 birch pollen-allergic patients with/without oral allergy syndrome (OAS), at baseline and after 5 months of sublingual allergen immunotherapy (AIT). RESULTS: Similar cell subsets (i.e., dendritic cells, mast cells, and T lymphocytes) were detected in oral tissues from healthy and birch pollen-allergic individuals. CD207+ Langerhans cells (LCs) and CD11c+ myeloid dendritic cells (DCs) were found in both the epithelium and the papillary layer of the Lamina propria (LP), whereas CD68+ macrophages, CD117+ mast cells, and CD4+ /CD8+ T cells were rather located in both the papillary and reticular layers of the LP. Patterns of oral immune cells were identical in patients with/without OAS, except lower numbers of CD207+ LCs found in oral tissues from patients with OAS, when compared to OAS- patients (P < 0.05). A 5-month sublingual AIT had a limited impact on oral immune cells, with only a significant increase in IgE+ cells in patients from the active group. Colocalization experiments confirmed that such IgE-expressing cells mostly encompass CD68+ macrophages located in the LP, and to a lesser extent CD207+ LCs in the epithelium. CONCLUSION: Two cell subsets contribute to antigen/allergen uptake in human oral tissues, including (i) CD207+ LCs possibly involved in the physiopathology of OAS and (ii) CD68+ macrophages likely critical in allergen capture via IgE-facilitated mechanisms during sublingual AIT.

Respiratory allergen from house dust mite is present in human milk and primes for allergic sensitization in a mouse model of asthma.

There is an urgent need to identify environmental risk and protective factors in early life for the prevention of allergy. Our study demonstrates the presence of respiratory allergen from house dust mite, Der p 1, in human breast milk. Der p 1 in milk is immunoreactive, present in similar amounts as dietary egg antigen, and can be found in breast milk from diverse regions of the world. In a mouse model of asthma, oral exposure to Der p through breast milk strongly promotes sensitization rather than protect the progeny as we reported with egg antigen. These data highlight that antigen administration to the neonate through the oral route may contribute to child allergic sensitization and have important implications for the design of studies assessing early oral antigen exposure for allergic disease prevention. The up-to-now unknown worldwide presence of respiratory allergen in maternal milk allows new interpretation and design of environmental control epidemiological studies for allergic disease prevention.

Heterogeneity of antibody responses among clinical responders during grass pollen sublingual immunotherapy.

BACKGROUND: During allergen-specific sublingual immunotherapy (SLIT), the relevance of changes in specific IgE and IgG antibody titres to treatment efficacy remains to be evaluated at an individual patient level. OBJECTIVE: To investigate whether antibody responses can be used as biomarkers for SLIT efficacy. METHODS: Comprehensive quantitative, qualitative and functional analyses of allergen-specific IgA, IgE, IgG1-4 and IgM responses were performed using purified Phl p 1 to 12 allergens in sera, saliva and nasal secretions from 82 grass pollen allergic patients. These patients were enrolled in a randomized, double-blind placebo-controlled study and assessed in an allergen challenge chamber (ClinicalTrials.gov NCT00619827). Antibody responses were monitored in parallel to clinical responses before and after daily sublingual treatment for 4 months with either a grass pollen or a placebo tablet. RESULTS: A significant mean improvement (i.e. 33-40.6%) in rhinoconjunctivitis total symptom scores was observed in SLIT recipients, irrespective of their baseline patterns of IgE sensitization (i.e. narrow, intermediate, broad) to grass pollen allergens. SLIT did not induce any de novo IgE sensitization. Clinical responders encompassed both immunoreactive patients who exhibited strong increases in titres, affinity and/or blocking activity of grass-pollen-specific IgGs (representing 17% of treated patients), as well as patients with no detectable antibody responses distinguishing them from the placebo group. No significant changes were detected in antibody titres in saliva and nasal washes, even in clinical responders. CONCLUSIONS AND CLINICAL RELEVANCE: Sublingual immunotherapy with a grass pollen tablet is efficacious irrespective of the patients' baseline sensitization to either single or multiple grass pollen allergens. Seric IgG responses may contribute to SLIT-induced clinical tolerance in a fraction (i.e. 17%) of patients, but additional immune mechanisms are involved in most patients. Consequently, antibody responses cannot be used as a marker of SLIT efficacy at an individual patient level.

Allergen-specific CD4+ T cell responses in peripheral blood do not predict the early onset of clinical efficacy during grass pollen sublingual immunotherapy.

BACKGROUND: Surrogate biomarkers of efficacy are needed in support of allergen-specific immunotherapy. OBJECTIVE: The aim of this study was to relate changes in peripheral CD4(+) T cell responses to clinical efficacy during sublingual immunotherapy (SLIT). METHODS: Allergen-specific CD4(+) T cell responses were assessed in peripheral blood mononuclear cells (PBMCs) from 89 grass pollen-allergic individuals enrolled in a double-blind placebo-controlled SLIT study conducted in an allergen exposure chamber (ClinicalTrials.gov NCT00619827). Surface phenotype, proliferative responses, cytokine production and gene expression were analysed in coded samples at baseline, and after 2 and 4 months of SLIT, in PBMCs after in vitro allergen stimulation or among MHC class II/peptide (pMHCII)-tetramer-positive CD4(+) T cells. RESULTS: SLIT induced a 29.3% improvement of the average rhinoconjunctivitis total symptom score in the active group, when compared to the placebo group. In parallel, only minor changes in proportions of CD4(+) T cells expressing Th1 (CCR5(+), CXCR3(+)), Th2 (CRTh2(+), CCR4(+)) and Treg (CD25(+), CD127(-), Foxp3(+)) markers were detected. A down-regulation of IL-4 and IL-10 gene expression and IL-10 secretion (P < 0.001) were observed, as well as a decrease in the frequency of potential "pro-allergic" CD27(-) Th2 cells from patients receiving active tablets (P < 0.001), but without any correlation with clinical benefit. pMHCII-tetramer analyses failed to document any major impact in both numbers and polarization of circulating Phl p 1- and Phl p 5-specific CD4(+) T cells, confirming that early clinical improvement during SLIT is not associated with dramatic alterations in T lymphocyte responses. CONCLUSION & CLINICAL RELEVANCE: Changes in patterns of peripheral CD4(+) T cells are not markers for the early onset of efficacy during SLIT.

Sublingual allergen immunotherapy: mode of action and its relationship with the safety profile.

Allergen immunotherapy reorients inappropriate immune responses in allergic patients. Sublingual allergen immunotherapy (SLIT) has been approved, notably in the European Union, as an effective alternative to subcutaneous allergen immunotherapy (SCIT) for allergic rhinitis patients. Compared with SCIT, SLIT has a better safety profile. This is possibly because oral antigen-presenting cells (mostly Langerhans and myeloid dendritic cells) exhibit a tolerogenic phenotype, despite constant exposure to danger signals from food and microbes. This reduces the induction of pro-inflammatory immune responses leading to systemic allergic reactions. Oral tissues contain relatively few mast cells and eosinophils (mostly located in submucosal areas) and, in comparison with subcutaneous tissue, are less likely to give rise to anaphylactic reactions. SLIT-associated immune responses include the induction of circulating, allergen-specific Th1 and regulatory CD4+ T cells, leading to clinical tolerance. Although 40-75% of patients receiving SLIT experience mild, transient local reactions in the oral mucosa, these primary reactions rarely necessitate dose reduction or treatment interruption. We discuss 11 published case reports of anaphylaxis (all nonfatal) diagnosed according to the World Allergy Organization criteria and relate this figure to the approximately 1 billion SLIT doses administered worldwide since 2000. Anaphylaxis risk factors associated with SCIT and/or SLIT should be characterized further.

Establishment of recombinant major allergens Bet v 1 and Phl p 5a as Ph. Eur. reference standards and validation of ELISA methods for their measurement. Results from feasibility studies.

The potency of allergen extracts is determined as total allergenic activity without consideration of their composition and the units differ from one manufacturer to another, making it very difficult to compare the different products. Recently, purified major allergens have been obtained by recombinant DNA technology and produced under Good Manufacturing Practice (GMP) conditions. In principle, such recombinant allergens could be established as reference standards and could help for the standardisation of the major allergen content of allergen extracts. Two recombinant major allergens, one from birch pollen, rBet v 1, and one from Timothy grass pollen, Phl p 5a, have been selected at the end of the CREATE programme as a potential starting point for the establishment as European Pharmacopoeia (Ph. Eur.) Reference Standards through a project run by the Biological Standardisation Programme (BSP) of the European Directorate for the Quality of Medicines & HealthCare (EDQM). To this end, bulk candidate recombinant materials, produced under GMP conditions, were procured from two European manufacturers and subsequently formulated and lyophilised. Four ELISA systems from three different manufacturers were included in the project, two for Bet v 1 and two for Phl p 5a with the aim of establishing reference methods for determination of the respective major antigens both in natural allergen extracts as well as in recombinant allergen products. The project was run in 3 phases: a preparatory and preliminary testing phase (feasibility phase or Phase 1), an extended feasibility phase carried out in 3 laboratories (Phase 2) to confirm the transferability of the methods and an international collaborative study with a large number of participating laboratories (Phase 3). This article describes the work done in Phase 1 and Phase 2, i.e. the physico-chemical and biological characterisation of the recombinant candidate reference standards, the assessment of their suitability for the intended purpose as well as the evaluation of the candidate ELISA systems. The results show that both candidate reference standards are suitable for the intended purpose. In addition, three out of the four ELISA systems that were included in the preliminary phase were found to be appropriate for further evaluation in the collaborative study which was organised in 2011. The results of the collaborative study will be published separately.

Changes in basophil activation during grass-pollen sublingual immunotherapy do not correlate with clinical efficacy.

BACKGROUND: Biomarkers predicting the safety and efficacy of sublingual immunotherapy (SLIT) remain to be established. METHODS: Eighty-nine patients with allergic rhinoconjunctivitis to grass pollen received either a placebo or five-grass-pollen daily tablet sublingually for 4 months. Following exposure in an allergen challenge chamber, clinical responders and nonresponders were identified individually by evaluating their rhinoconjunctivitis total symptom score (RTSS). Activation of peripheral blood basophils was measured by cytofluorometry before and after 2 or 4 months of immunotherapy, based on CD203c surface expression following allergen stimulation. RESULTS: Patients receiving the grass-pollen tablet had a relative mean improvement of 29.3% vs placebo in the average RTSS after 4 months of SLIT (P < 0.0003). No significant changes in basophil activation were noticed after 2 or 4 months of SLIT despite induction of specific IgGs. Among individual clinical responders, basophil activation was either decreased, increased, or unmodified during SLIT. Levels of basophil activation prior to immunotherapy were not predictive of local adverse reactions associated with immunotherapy. A moderate association was found between basophil activation and allergen-specific IgE levels, skin reactivity, or RTSS, suggesting that the former is, to some extent, indicative of disease severity. As such, patients with the highest level of basophil activation before treatment were more likely to benefit clinically from SLIT. CONCLUSIONS: Allergen reactivity of peripheral blood basophils is not a biomarker for adverse events or early onset of clinical responses to SLIT.

Molecular variability of group 1 and 5 grass pollen allergens between Pooideae species: implications for immunotherapy.

BACKGROUND: Differences between major allergens from distinct grass species remain to be investigated, both in terms of structure and antigenicity. METHODS: Group 1 and 5 allergens purified from five common Pooideae species were analysed by mass spectrometry (MS). Major histocompatibility complex (MHC) class II-restricted T cell epitopes were identified using predictive algorithms and human leucocyte antigen (HLA)-binding assays. CD4+ T cell reactivity and IgE binding were assessed based on the induction of CD154 expression in peripheral blood mononuclear cells and using competitive ELISA assays, respectively. RESULTS: MS analysis of group 5 pollen allergens reveals considerable intra- and inter-species variability in amino acid sequence, with 30-50 predominant isoforms found for each species. Differences in the amino acid sequence as well as N- and O-glycosylation contribute to the variability of group 1 allergens, yielding 5-10 main isoforms, depending on the species. Out of 14 MHC class II-restricted T cell epitopes identified within group 1, only one is conserved among the five grass species. Significant differences in binding affinities for HLA-DR molecules result in variable CD4+ T cell recognition of group 1 and 5 allergens purified from the various species. Up to 38% and 85% of patients exhibit seric IgE responses to species-restricted (or semi-restricted) epitopes associated with group 1 or 5 allergens, respectively. CONCLUSION: Major pollen allergens from distinct grass species bear both shared and species-restricted T and B cell immune epitopes. When compared with single extracts, a five grass pollen extract is thus more suitable for specific immunotherapy, as it contains a broader repertoire of the IgE epitopes to which patients are sensitized.