PRODUCTION & CHARACTERIZATION OF Bet v 1 RECOMBINANT ALLERGEN INTENDED FOR IMMUNOTHERAPY

L'allergie respiratoire au pollen de bouleau affecte un nombre important de personnes dans le monde. Il est estimé que 100 millions d'individus sont sensibilisés à l'allergène majeur du pollen de bouleau nommé Bet v 1. L’immunothérapie allergénique, basée sur l'administration répétée de l'allergène incriminé, permet la rééducation du système immunitaire du patient d’un profil TH2 vers un profil TH1/Treg et à terme la diminution des symptômes allergiques. Ces travaux de thèse avaient donc pour finalité de produire et de caractériser l'allergène recombinant Bet v 1, à des fins d’immunothérapie allergénique. Dans ce cadre, différentes méthodes analytiques ont été développées et appliquées afin d'optimiser le procédé de production via l'élimination de différentes impuretés liées au produit ou au procédé de production et de documenter la structure de l’allergène. En particulier, l'utilisation de la spectrométrie de masse a permis la détermination de la masse exacte de l'allergène ainsi que la vérification complète de sa séquence en acides aminés. Les travaux en spectrométrie de masse ont également contribué aux détections et identifications de diverses impuretés et produits de dégradations et ont ainsi conduit à plusieurs optimisations du procédé industriel de production de l'allergène recombinant. Les activités immunologiques de certains produits de dégradations ont également été investiguées. La structure tertiaire (spatiale) de l'allergène a été déterminée par diffraction aux rayons X. Enfin, ces travaux ont permis de documenter la qualité de l'allergène recombinant rBet v 1 afin i) de l'établir comme substance de référence pour la Pharmacopée Européenne et ii) de procéder à une étude clinique d’immunothérapie allergénique de phase II auprès de 483 patients allergiques au pollen de bouleau.Respiratory allergy to birch pollen affects a large number of people in the world. It is estimated that 100 million people are sensitized to the major allergen from birch pollen, namely Bet v 1. Allergen immunotherapy, based on the repeated administration of the allergen of interest, allows the modification of the patient's immune response from a TH2 to a TH1/Treg pattern and thus the reduction of allergic symptoms. This study was therefore aimed to produce and characterize the recombinant Bet v 1 (rBet v 1) allergen, for immunotherapy purpose.In this context, various analytical methods have been developed and applied in order to optimize the production of rBet v 1 via the reduction of process or product-related impurities as well as to document the quality of the purified allergen. In particular, the use of mass spectrometry has allowed the determination of the exact mass of the intact allergen and the complete verification of its amino acid sequence. Mass spectrometry data have also contributed to the detection and identification of impurities and degradation products and have therefore led to several optimizations of the industrial process for the production of the recombinant allergen. Immunological activities of certain degradation products were also investigated and the allergen tertiary structure was determined by X-ray diffraction. Finally, this study was decisive in order i) to establish rBet v 1 as a chemical reference substance for the European Pharmacopoeia as well as ii) to perform a phase II clinical study conducted in 483 patients with birch pollen-induced rhinoconjunctivitis

Specific IgE recognition of pollen allergens from subtropic grasses in patients from the subtropics

Background Pollens of subtropical grasses, Bahia (Paspalum notatum), Johnson (Sorghum halepense), and Bermuda (Cynodon dactylon), are common causes of respiratory allergies in subtropical regions worldwide. Objective To evaluate IgE cross-reactivity of grass pollen (GP) found in subtropical and temperate areas. Methods Case and control serum samples from 83 individuals from the subtropical region of Queensland were tested for IgE reactivity with GP extracts by enzyme-linked immunosorbent assay. A randomly sampled subset of 21 serum samples from patients with subtropical GP allergy were examined by ImmunoCAP and cross-inhibition assays. Results Fifty-four patients with allergic rhinitis and GP allergy had higher IgE reactivity with P notatum and C dactylon than with a mixture of 5 temperate GPs. For 90% of 21 GP allergic serum samples, P notatum, S halepense, or C dactylon specific IgE concentrations were higher than temperate GP specific IgE, and GP specific IgE had higher correlations of subtropical GP (r = 0.771-0.950) than temperate GP (r = 0.317-0.677). In most patients (71%-100%), IgE with P notatum, S halepense, or C dactylon GPs was inhibited better by subtropical GP than temperate GP. When the temperate GP mixture achieved 50% inhibition of IgE with subtropical GP, there was a 39- to 67-fold difference in concentrations giving 50% inhibition and significant differences in maximum inhibition for S halepense and P notatum GP relative to temperate GP. Conclusion Patients living in a subtropical region had species specific IgE recognition of subtropical GP. Most GP allergic patients in Queensland would benefit from allergen specific immunotherapy with a standardized content of subtropical GP allergens

UAV Photogrammetry for archaeological survey: The Theaters area of Pompeii

Aerial surveying is, in the domain of archeology and architecture, an exponential field of application and interest. Beyond the increasing flying capabilities and skills of existing UAV, the operational potentiality of such aircraft oversteps the imagination of their own creators. Initially conceived for robotic civilian and military surveying applications, recent experiments have tested autonomous flight, self decision-making and multi-agent convergent tasks. Based upon 10 years of aerial surveying experience, part of the MAP activity is concerned: firstly, unmanned flight and related developments within a specific institutional partnership and secondly, advanced tool-making workflow for architecture and urban design 3D modeling and image analysis protocols. In this paper we will describe a particular operational pipeline tested on the "Theaters Area" of Pompeii in Italy, starting from aerial data collection to accurate 3D processing with the help of innovative co-operative technologies. Keywords—component; UAV photogrammetry, orthoimages, archaeological sites, monitoring

UAV Photogrammetry for archaeological survey: The Theaters area of Pompeii

Aerial surveying is, in the domain of archeology and architecture, an exponential field of application and interest. Beyond the increasing flying capabilities and skills of existing UAV, the operational potentiality of such aircraft oversteps the imagination of their own creators. Initially conceived for robotic civilian and military surveying applications, recent experiments have tested autonomous flight, self decision-making and multi-agent convergent tasks. Based upon 10 years of aerial surveying experience, part of the MAP activity is concerned: firstly, unmanned flight and related developments within a specific institutional partnership and secondly, advanced tool-making workflow for architecture and urban design 3D modeling and image analysis protocols. In this paper we will describe a particular operational pipeline tested on the "Theaters Area" of Pompeii in Italy, starting from aerial data collection to accurate 3D processing with the help of innovative co-operative technologies. Keywords—component; UAV photogrammetry, orthoimages, archaeological sites, monitoring

UAV photogrammetry for archaeological survey: the Theaters area of Pompeii

Aerial surveying is, in the domain of archeology and architecture, an exponential field of application and interest. Beyond the increasing flying capabilities and skills of existing UAV, the operational potentiality of such aircraft oversteps the imagination of their own creators. Initially conceived for robotic civilian and military surveying applications, recent experiments have tested autonomous flight, self decision-making and multi-agent convergent tasks. Based upon 10 years of aerial surveying experience, part of the MAP activity is concerned: firstly, unmanned flight and related developments within a specific institutional partnership and secondly, advanced tool-making workflow for architecture and urban design 3D modeling and image analysis protocols. In this paper we will describe a particular operational pipeline tested on the "Theaters Area" of Pompeii in Italy, starting from aerial data collection to accurate 3D processing with the help of innovative co-operative technologies

CCR10 + ILC2s with ILC1-like properties exhibit a protective function in severe allergic asthma

International audienceWe previously showed that patients with severe allergic asthma have high numbers of circulating ILC2s expressing CCR10.METHOD:Herein, CCR10+ ILC2s were further analyzed in the blood of healthy individuals or patients with allergic and non-allergic asthma. Characteristics of human CCR10+ and CCR10- ILC2s were assessed by flow cytometry as well as single-cell multiplex RT-qPCR. The role of CCR10+ ILC2s in asthma pathophysiology was studied in allergen-treated mice.RESULTS:When compared to healthy controls, CCR10+ ILC2s are enriched in the blood of both allergic and non-allergic severe asthmatic patients, and these cells are recruited to the lungs. Plasma concentrations of the CCR10 ligand CCL27 are significantly increased in severe asthmatics when compared to non-asthmatic patients. CCR10+ ILC2s secrete little TH 2 cytokines, but exhibit ILC1-like properties, including a capacity to produce IFN-γ. Also, single-cell analysis reveals that the CCR10+ ILC2 subset is enriched in cells expressing amphiregulin. CCR10+ ILC2 depletion, as well as blocking of IFN-γ activity, exacerbates airway hyperreactivity in allergen-challenged mice, providing evidence for a protective role of these cells in allergic inflammation.CONCLUSIONS:Frequencies of circulating CCR10+ ILC2s and CCL27 plasma concentrations represent candidate markers of asthma severity. The characterization of CCR10+ ILC2s in human samples and in mouse asthma models suggests that these cells downregulate allergic inflammation through IFN-γ production