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Background: Whereas sublingual allergen immunotherapy (AIT) is routinely performed without any adjuvant or delivery system, there is a strong scientific rationale to better target the allergen(s) to oral dendritic cells known to support regulatory immune responses by using appropriate presentation platforms., Objective: To identify a safe presentation platform able to enhance allergen-specific tolerance induction., Methods: Virosomes with membrane-integrated contiguous overlapping peptides (COPs) of Bet v 1 and TLR4 or TLR2/TLR7 agonists were assessed for induction of Bet v 1-specific IgG1, IgG2a and IgE antibodies, hypersensitivity reactions and body temperature drop following subcutaneous injection in naive CD-1 mice. The most promising candidate, Bet v 1 COPs anchored to virosomes with membrane-incorporated TLR4 agonist (Vir.A-Bet v 1 COPs), was further evaluated by the sublingual route in a therapeutic setting in BALB/c mice with birch pollen-induced allergic asthma. Airway hyperresponsiveness, pro-inflammatory cells in bronchoalveolar lavages and polarization of Th cells in the lungs and spleen were then assessed., Results: Both types of adjuvanted virosomes coupled to Bet v 1 COPs triggered a boosted Th1 immunity. Given a more favourable safety profile, Vir.A-Bet v 1 COPs were further evaluated and shown to able to fully reverse asthma symptoms and lung inflammation in a sublingual therapeutic model of birch pollen allergy., Conclusions and Clinical Relevance: We report herein for the first time on the capacity of a novel and safe presentation platform, that is virosomes with membrane-integrated TLR4 agonist, to improve dramatically sublingual AIT efficacy in a murine model due to its intrinsic dual properties of targeting and stimulating to further promote anti-allergic immune responses. As such, our study paves the ground for further clinical development of this allergen presentation platform for patients suffering from respiratory allergies., (© 2020 John Wiley & Sons Ltd.)

Background: Eligibility to immunotherapy is based on the determination of IgE reactivity to a specific allergen by means of skin prick or in vitro testing. Biomarkers predicting the likelihood of clinical improvement during immunotherapy would significantly improve patient selection., Methods: Proteins were differentially assessed by using 2-dimensional differential gel electrophoresis and label-free mass spectrometry in pretreatment sera obtained from clinical responders and nonresponders within a cohort of 82 patients with grass pollen allergy receiving sublingual immunotherapy or placebo. Functional studies of Fetuin-A (FetA) were conducted by using gene silencing in a mouse asthma model, human dendritic cell in vitro stimulation assays, and surface plasmon resonance., Results: Analysis by using quantitative proteomics of pretreatment sera from patients with grass pollen allergy reveals that high levels of O-glycosylated sialylated FetA isoforms are found in patients exhibiting a strong decrease in rhinoconjunctivitis symptoms after sublingual immunotherapy. Although FetA is involved in numerous inflammatory conditions, its potential role in allergy is unknown. In vivo silencing of the FETUA gene in BALB/c mice results in a dramatic upregulation of airway hyperresponsiveness, lung resistance, and T H 2 responses after allergic sensitization to ovalbumin. Both sialylated and nonsialytated FetA bind to LPS, but only the former synergizes with LPS and grass pollen or mite allergens to enhance the Toll-like receptor 4-mediated proallergic properties of human dendritic cells., Conclusions: As a reflection of the patient's inflammatory status, pretreatment levels of sialylated FetA in the blood are indicative of the likelihood of clinical responses during grass pollen immunotherapy., (Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Allergy to the short ragweed (Ambrosia artemisiifolia) pollen is a major health problem. The ragweed allergen repertoire has been recently expanded with the identification of Amb a 11, a new major allergen belonging to the cysteine protease family. To better characterize Amb a 11, a recombinant proform of the molecule with a preserved active site was produced in Escherichia coli, refolded, and processed in vitro into a mature enzyme. The enzymatic activity is revealed by maturation following an autocatalytic processing resulting in the cleavage of both N- and C-terminal propeptides. The 2.05-Å resolution crystal structure of pro-Amb a 11 shows an overall typical C1A cysteine protease fold with a network of molecular interactions between the N-terminal propeptide and the catalytic triad of the enzyme. The allergenicity of Amb a 11 was confirmed in a murine sensitization model, resulting in airway inflammation, production of serum IgEs, and induction of Th2 immune responses. Of note, inflammatory responses were higher with the mature form, demonstrating that the cysteine protease activity critically contributes to the allergenicity of the molecule. Collectively, our results clearly demonstrate that Amb a 11 is a bona fide cysteine protease exhibiting a strong allergenicity. As such, it should be considered as an important molecule for diagnosis and immunotherapy of ragweed pollen allergy., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

As of today, allergen immunotherapy is performed with aqueous natural allergen extracts. Recombinant allergen vaccines are not yet commercially available, although they could provide patients with well-defined and highly consistent drug substances. As Bet v 1 is the major allergen involved in birch pollen allergy, with more than 95% of patients sensitized to this allergen, pharmaceutical-grade recombinant Bet v 1-based vaccines were produced and clinically tested. Herein, we compare the clinical results and modes of action of treatments based on either a birch pollen extract or recombinant Bet v 1 expressed as hypoallergenic or natural-like molecules. We also discuss the future of allergen immunotherapy with improved drugs intended for birch pollen-allergic patients suffering from rhinoconjunctivitis.

Background: Allergy to pollen from short ragweed (Ambrosia artemisiifolia) is a serious and expanding health problem in the United States and in Europe., Objective: We sought to investigate the presence of undescribed allergens in ragweed pollen., Methods: Ragweed pollen proteins were submitted to high-resolution gel electrophoresis and tested for IgE reactivity by using sera from 92 American or European donors with ragweed allergy. Pollen transcriptome sequencing, mass spectrometry (MS), and recombinant DNA technologies were applied to characterize new IgE-binding proteins., Results: High-resolution IgE immunoblotting experiments revealed that 50 (54%) of 92 patients with ragweed allergy were sensitized to a 37-kDa allergen distinct from Amb a 1. The full-length cDNA sequence for this molecule was obtained by means of PCR cloning after MS sequencing of the protein combined with ragweed pollen RNA sequencing. The purified allergen, termed Amb a 11, was fully characterized by MS and confirmed to react with IgEs from 66% of patients. This molecule is a 262-amino-acid thiol protease of the papain family expressed as a combination of isoforms and glycoforms after proteolytic removal of N- and C-terminal propeptides from a proform. Three-dimensional modeling revealed a high structural homology with known cysteine proteases, including the mite Der p 1 allergen. The protease activity of Amb a 11, as well as its capacity to activate basophils from patients with ragweed allergy, were confirmed. The production of a nonglycosylated recombinant form of Amb a 11 in Escherichia coli established that glycosylation is not required for IgE binding., Conclusion: We identified the cysteine protease Amb a 11 as a new major allergen from ragweed pollen. Given the similar physicochemical properties shared by the 2 major allergens, we hypothesize that part of the allergenic activity previously ascribed to Amb a 1 is rather borne by Amb a 11., (Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Background: Allergy to short ragweed (Ambrosia artemisiifolia) pollen is a serious and expanding health problem in North America and Europe. Whereas only 10 short ragweed pollen allergens are officially recorded, patterns of IgE reactivity observed in ragweed allergic patients suggest that other allergens contribute to allergenicity. The objective of the present study was to identify novel allergens following extensive characterization of the transcriptome and proteome of short ragweed pollen., Methods: Following a Proteomics-Informed-by-Transcriptomics approach, a comprehensive transcriptomic data set was built up from RNA-seq analysis of short ragweed pollen. Mass spectrometry-based proteomic analyses and IgE reactivity profiling after high resolution 2D-gel electrophoresis were then combined to identify novel allergens., Results: Short ragweed pollen transcripts were assembled after deep RNA sequencing and used to inform proteomic analyses, thus leading to the identification of 573 proteins in the short ragweed pollen. Patterns of IgE reactivity of individual sera from 22 allergic patients were assessed using an aqueous short ragweed pollen extract resolved over 2D-gels. Combined with information derived from the annotated pollen proteome, those analyses revealed the presence of multiple unreported IgE reactive proteins, including new Amb a 1 and Amb a 3 isoallergens as well as 7 novel candidate allergens reacting with IgEs from 20-70% of patients. The latter encompass members of the carbonic anhydrase, enolase, galactose oxidase, GDP dissociation inhibitor, pathogenesis related-17, polygalacturonase and UDP-glucose pyrophosphorylase families., Conclusions: We extended the list of allergens identified in short ragweed pollen. These findings have implications for both diagnosis and allergen immunotherapy purposes.

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., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Second generation sublingual allergy vaccines based upon recombinant allergens combined with vector systems are being developed as an alternative to conventional allergen extracts. Herein, we evaluated the efficacy of a recombinant form of the major allergen Bet v 1a (rBet v 1a) formulated as a mucoadhesive particle in a preclinical model of birch pollen (BP) respiratory allergy., Materials and Methods: BALB/c mice were sensitized to BP extracts by intraperitoneal injections followed by aerosol exposures. Sensitized mice underwent sublingual immunotherapy (SLIT) twice a week for eight weeks with either a BP extract or rBet v 1a formulated in amylopectin-based microparticles (MPA). SLIT efficacy was assessed using whole body plethysmography, lung histology and cell counts in broncho-alveolar lavages (BAL) as read outs. BP and/or rBet v 1a-specific T cell and antibody responses were monitored in lung and serum, respectively. IgA levels were measured in saliva., Results: Mice sensitized to BP exhibit chronic airway hyperresponsiveness (AHR), lung inflammation (documented by compliance and resistance measurements), eosinophil infiltrates in BAL, as well as Bet v 1-specific Th2 biased responses. Both SLIT with soluble rBet v 1a (50μg/dose) and BP extract (equivalent to 50μg rBet v 1 per dose) lead to a significant reduction in AHR, lung eosinophilia and Th2 responses. A sub-optimal dose of 5μg of rBet v 1a displays a similar level of efficacy with a significant decrease of Th2 responses when formulated with MPA microparticles. In addition, allergen vectorization with mucoadhesive particles allows a faster reduction in AHR in sensitized animals., Conclusion: We demonstrate in a murine model of chronic BP respiratory allergy the efficacy of SLIT with vectorized rBet v 1a. Thus, combining recombinant allergens with mucoadhesive vector systems paves the ground for improved second generation sublingual allergy vaccines., (Copyright © 2013 Elsevier Ltd. All rights reserved.)