The general idea of this study was to develop candidate specific IgY antibodies for an oral therapy targeting the ESBL-producing gram negative bacteria. As the family of ESBLs constantly grows and there is lack of their clear classification in the literature, the specific aim was to build a proof of concept study based on the parental enzyme ß-lactamase TEM-1 to investigate different specific IgYs strategies to inhibit the growth of TEM-1 producing E.coli. This research included a bioinformatic analysis of the TEM-1 structure in the context of TEM-derivative ESBLs. Then, two IgY strategies were designed to target the ß-lactamase TEM-1-producing E.coli (BW25113 ΔbamBΔtolC) with IgYs: as a complement to antibiotics (IgYs against the enzyme TEM-1 used in combination with ampicillin) and as an alternative to antibiotics (IgYs against the bacteria TEM-1-producing E.coli without the addition of ampicillin). A good inhibitory effect of (a)TIgY, (a)p2IgY (in the presence of ampicillin) and eIgY, hIgY (without the ampicillin) on TEM-1-producing E.coli was observed in vitro. Moreover, they had the typical configuration of avian antibodies and were highly specific to their antigens. This study presents a model system to develop specific IgYs against a therapeutic target of interest. The activity of these IgYs complementary or alternatively to antibiotics should be further investigated in vivo in an animal infectious model. IgYs developed in this study might also be good candidates for further investigation as a broad-spectrum treatment against a variety of ESBL-producing E.coli. The aTIgY which was developed against the whole TEM-1 might also target its derivatives, as they have similar 3D structure with single amino acids mutations in the sequence. The ap2IgY was generated against catalytic and conservative residues, characteristic for the whole class A of ß-lactamases, thus it might target also the active site of ESBL-s from this class. The strategy used to generate eIgY and hIgY was efficient and IgYs could be generated directly against ESBL-producing bacteria.

In this investigation of immunotherapy (IT) children 6-16 years old with rhinoconjunctivitis due to birch polli­nosis were included. I. Methodological studies. To monitor IT a reliable provocation test is desirable. The conjunctival provocation test (CPT) was evaluated in 20 children with four repeated challenges. The test was found to have a good preci­sion, it was simple and appeared to be clinically safe. After repeated tests the levels of IgE antibodies against birch increased considerably in three children, indicating an immunological response. A pollen peak affects the symptoms of an atopic individual for several days. Thus pollen counts for previous days must be taken into account when relating symptom scores with the counts. A dynamic time series model was therefore developed by which groups of atopic patients could be compared when exposed to different amounts of pollens. II: Cross-reactivity between deciduous trees during IT. Immunotherapy with pollen allergen preparations made from either birch (B) or a mixture of birch, alder and hazel (M) were compared. As measured with symptom scores the children in the M group improved at least as much as those in the B group. In the B group but not in the M group the improvement correlated with immunochemical findings before IT or early during the treatment, probably an unsignificant finding. Otherwise there was little difference between the two groups. Analysis of sera with crossed radioimmunoelectrophoresis in 20 children revealed that 60% of the children below 13 years had de­veloped IgE antibodies during IT against allergens against which they had not been allergic before IT. This had no appearent clinical implications. III: Oral immunotherapy (OIT). A pilot study of 18 children treated with high doses of a birch pollen allergen preparation in enteric coated capsules and 8 untreated controls indicated that OIT was effective as shown by lower symptom scores, less conjuctival sensitivity and increased levels of IgE antibodies against birch. However, the gastrointestinal side-effects were pronounced. Therefore a second double-blind study, in 30 children, was performed reducing the side-effects through a different dose schedule. Compared with the placebo group, the ac­tively treated children had lower symptom scores (p = 0.04), reduced skin sensitivity (p = 0.01), increasing levels of IgE (p = 0.001) and IgG (p = 0.007) antibodies against birch before the birch pollen season and a suppression of the seasonal increase in levels of IgE antibodies against birch (p <0.001). After three months of OIT but not after ten months they also had a lower sensitivity in CPT than the controls (p = 0.01). The intestinal permeability as assessed by the urinary recovery of differently-sized polyethyleneglycols was studied in 24 of the children during IT. No changes were seen in the group of actively treated children. In two ad­ditional children openly treated with OIT small bowel biopsies were taken with normal morphological findings. Thus OIT did not result in a generalized inflammation of the small bowel. / digitalisering@umu

La prévalence des allergies alimentaires IgE-médiées aurait triplé au cours de la dernière décennie avec des études Nord-Américaines atteignant les 8% chez les enfants. Quoiqu’il n’y ait à ce jour aucun traitement curatif pour les allergies alimentaires, l’immunothérapie oral (OIT) constitue une nouvelle approche expérimentale prometteuse. Cette dernière consiste en l’administration de doses progressive d’allergènes par voie orale sur une période prolongée dans le but d’instaurer un état de désensibilisation et possiblement une tolérance orale soutenue. Cette approche a été démontrée sécuritaire et permettrait la désensibilisation à haute dose de plus de 80% des participants allergiques aux arachides, lait ou œufs. Dans cette thèse, nous présentons 2 études de phase 1 portant sur des protocoles d’OIT, destinés à optimiser l’efficience du traitement chez les sujets avec allergies alimentaires multiples. Près de 30% des enfants avec allergie alimentaire sont allergiques à plus d’un aliment, une proportion qui augmente à 70% lorsqu’on considère les cas les plus sévères. Ces enfants sont à risque augmenté de réactions accidentelles et souffrent d’un impact plus grand sur leur qualité de vie. Dans la première étude, en créant un mélange individualisé avec un ratio stochiométrique 1:1 entre les protéines des aliments allergiques de l’enfant, nous démontrons qu’il est possible de désensibiliser jusqu’à 5 aliments simultanément avec un profil d’innocuité similaire à une monothérapie. Dans la seconde étude, nous utilisons un traitement à l’omalizumab, un anticorps monoclonal anti-IgE, pour permettre une désensibilisation orale multi-allergénique fortement accélérée. Lorsque comparé à l’approche sans omalizumab, ce protocole s’associe à une nette diminution du temps requis pour atteindre les doses d’entretien, passant d’une médiane de 21 à 4 mois, sans affecter le profil d’innocuité. Alors que ces études fournissent des approches cliniques raisonnables pour désensibiliser la population multi-allergique, plusieurs questions persistent, notamment en ce qui a trait à l’induction de tolérance permanente. Une barrière majeure à cet égard réside dans notre piètre compréhension des mécanismes sous-jacents à l’immunothérapie. Prenant avantage d’échantillons cliniques bien caractérisés provenant des essais cliniques ci-haut mentionnés, nous utilisons les nouvelles technologies de séquençage TCR pour suivre la distribution clonale des lymphocytes T spécifiques aux arachides durant une immunothérapie orale. Nous démontrons que l’OIT s’associe à des changements significatifs dans les fréquences des clones spécifiques, suggérant un processus d’épuisement clonal et de remplacement. Nous démontrons par ailleurs que le test de prolifération lymphocytaire, traditionnellement utilisé pour évaluer la réponse cellulaire allergique, est dominé par une distribution polyclonale hautement non-spécifique. Cette observation a des implications majeures considérant que la plupart de la littérature actuelle sur la réponse T se base sur cette technique. En somme, cette thèse jette les bases pour des programmes de recherche translationnelle pour optimiser et personnaliser les protocoles cliniques actuels et développer de nouvelles avenues d’investigation et de traitement pour améliorer la prise en charge des sujets avec allergies alimentaires. / The prevalence of IgE-mediated food allergies has tripled over the last decade with prospective studies indicating that up to 8% of children may be affected in North America. There is currently no cure for food allergy but oral immunotherapy (OIT) is an experimental approach to treat food allergies. It consists in the progressive administration from minute to large amounts of the allergenic food by the mouth over a prolonged period of time to induce a state of desensitization and possibly sustained tolerance. This approach has been shown to be safe and to allow desensitization to high doses in over 80% of participants allergic to peanuts, milk or egg. In this thesis, we present two phase 1 trials on OIT protocols designed to efficiently treat multiple foods allergies. About 30% of children with food allergy are allergic to more than one food. This proportion increases to 70% when considering the most severe cases. Children with multiple food allergies are at higher risk of accidental reactions and suffer from greater impact on quality of life than those with single food allergies. By creating a customized treatment mix with a 1:1 stoichiometric ratio for the child’s relevant food proteins, we were first able to safely desensitize up to 5 foods simultaneously with a safety profile similar to single allergen therapy and a minimal increase in time to maintenance. Then, taking advantage of recent evidence showing that omalizumab, an anti-IgE receptor monoclonal antibody, can significantly raise reaction thresholds in food allergic subjects, we used short courses of omalizumab to allow very rapid oral desensitization to various foods in a second phase 1 study. When compared to “standard” multi-OIT, the omalizumab-enabled rush protocol resulted in a decreased time to maintenance from a median of 21 to 4 months. While these studies provide reasonable clinical approaches to this population, many questions remain, especially with regards to long term tolerance. A major limit to our progress in improving these protocols stems from our lack of understanding of the underlying immune mechanisms of oral immunotherapy. Taking advantage of well phenotyped samples from the afore-mentioned trials, we used next-generation high-throughput TCR sequencing to follow clonal distribution of peanut specific T cells during oral immunotherapy. We found that OIT is associated with significant changes in food-specific clonal frequencies, suggesting clonal exhaustion and replacement as an underlying mechanism of OIT. In addition, we show that the proliferation assay which is traditionally used to assess the cellular response is dominated by a highly non-specific polyclonal distribution. This observation has important implications considering most of the current literature on T cell response to immunotherapy is based on this assay. This highlights the need for the development of new tools to assess the cellular allergic response. Overall this thesis lays the ground for further comprehensive translational research programs on the treatment of food allergy.

Problématique: En immunothérapie orale (ITO), le manque de variété et l’aversion envers les doses d’allergènes peuvent compromettre l’adhérence au traitement, toutefois essentielle pour maintenir la désensibilisation. Le but de l’étude était de développer et de valider une nouvelle intervention nutritionnelle pour l’utilisation d’options d’équivalences à domicile. Méthodes: L’intervention a été développée selon les besoins de familles déjà en ITO, exprimés lors d’entrevues préliminaires. De nouveaux patients débutant l’ITO ont ensuite été invités dans un essai contrôlé randomisé pour évaluer l’impact de l’intervention. Les participants (n = 30) ont été randomisés en 3 groupes : A) Consultation nutritionnelle avec outils d’options d’équivalences (intervention complète); B) Consultation nutritionnelle sans les outils (intervention partielle) et C) Groupe contrôle avec l’intervention complète retardée de 4 semaines. La compétence des parents pour le calcul de doses d’équivalences était suivie de façon longitudinale par une série d’exercices pratiques. Résultats: Les résultats aux exercices étaient en moyenne supérieurs avec l’intervention complète (93,3% ± 3,1), comparés au groupe contrôle sans intervention (1,7% ± 1,7, p<0,001). La compétence était maintenue 12 semaines plus tard (résultats de 88,9% ± 4,7). Sans les outils, l'acquisition initiale (résultats de 46,7% ± 7,3) et la rétention après 4 semaines (résultats de 26,7% ± 5,1) étaient inférieures, mais augmentaient après l’ajout des outils (résultats de 83,3% ± 7,5). La satisfaction et la diversité des doses ont également augmenté avec l’intervention complète. Conclusion: Cette étude démontre l'efficacité d'un programme d'intervention nutritionnelle pour accompagner la gestion des doses d'allergènes à domicile. L'utilisation de documents écrits est essentielle pour en obtenir tout le bénéfice. / Background: During oral immunotherapy (OIT), lack of palatability or diversity in daily allergen doses can compromise treatment adherence, which is essential to maintain benefit. The aim of the study was to develop and validate a nutritional intervention program on the use of whole food alternatives for allergen daily dosing during OIT. Methods: The program was initially developed based on preliminary interviews with families already on OIT. Patients beginning OIT were then invited to participate to an open-label randomized controlled trial to assess the impact of the intervention. Participants (n=30) were randomized into 3 arms when they transferred to whole foods: A) Dietitian counselling with supporting documents (full intervention); B) Dietitian counselling without document; C) Control group where full intervention was delayed by 4 weeks. Parent competency was followed longitudinally using a series of practical food dose calculation exercises. Results: Results of exercises at week 4 were in average higher in the full intervention group (93.3% ± 3.1) compared to reference group without intervention (1.7% ± 1.7, p<0.0001). Competency was maintained 12 weeks after intervention (results of 88.9% ±4.7). Without written documents, the initial acquisition (results of 46.7% ±7.3) and retention of competency at 4 weeks (results of 26.7% ±5.1) were lower, but competency was rescued by adding written documents (results of 83.3% ±7.5). Patient satisfaction and food diversity also increased with full intervention. Conclusion: This study demonstrates the efficacy of a nutritional intervention program to help patients and their parents manage their OIT allergen doses. The use of written documents is essential to achieve the full benefit.