Food Allergy and Anaphylaxis Meeting (FAAM 2013

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Clinical and Translational Allergy 2013, Volume 3 Suppl 3 http://www.ctajournal.com/supplements/3/S3

MEETING ABSTRACTS

Open Access

Food Allergy and Anaphylaxis Meeting (FAAM 2013): Abstracts Nice, France. 7-9 February 2013 Edited by Karin Hoffmann-Sommergruber, Antonella Muraro, Berber Vlieg-Boerstra, Carsten Bindslev-Jensen, Anthony Dubois, Phillippe Eigenmann, Susanne Halken, Giovanni Pajno, Lars K Poulsen, Graham Roberts, Ronald van Ree, Valerie Verhasselt and Thomas Werfel Published: 25 July 2013 These abstracts are available online at http://www.ctajournal.com/supplements/3/S3

ORAL PRESENTATIONS O1 Emergency treatment of food anaphylaxis: a report of 152 cases registered by the Allergy Vigilance Network A Moneret-Vautrin Nancy University, Vandoeuvre les Nancy, France Clinical and Translational Allergy 2013, 3(Suppl 3):O1 Background: International guidelines are in place for the management of severe food anaphylaxis. Their implementation in emergency services is examined to identify the points needing improvement. Methods: One hundred and fifty-two cases of severe anaphylaxis were reported to the Allergy Vigilance Network in France and Belgium in 2011. Information was recorded on a standardised form. A thorough analysis was then undertaken. Results: 78 paediatric and 74 adult cases were reported. Two deaths occurred. Personnel and emergency centres involved in the management were known in 147 cases, and treatment in 136 cases. First-aid was provided at home by patients themselves on 43 occasions or by the family physician 12 times. 49 called for an ambulance. 89 required hospitalisation in an Emergency Department. Epinephrine auto-injectors were used by 4 patients only. Medicalized ambulances treated 23 patients, and used epinephrine in 11/23 cases. Emergency departments treated 58 patients and used epinephrine in 19 cases. When epinephrine had been injected before arriving at the ED, no further injection in hospital was necessary. In total, epinephrine was given to 37 patients (27.2%). The observation period was much shorter than recommended in the Directives. Patients were discharged without care summaries. Allergists implemented school management plans for all children. 68 to 80% of patients were prescribed self-injectable epinephrine. Conclusion: Personal care plans including patient education on the use of epinephrine should be more widely used. The frequent prescription of selfinjectable epinephrine to the patients is almost at no use. As patients fail to use them when necessary, paramedics should be equipped with autoinjectors and trained to identify severe symptoms. We recommend improved coordination between emergency doctors, paediatricians and allergists. Disclosure of interest: None declared. References 1. Moneret-Vautrin DA, Morisset M, et al: Allergy 2005, 60:443-51. 2. Muraro A, Roberts G, et al: Allergy 2007, 62(8):857-71. 3. Worm M, Timmermans F, et al: Allergy 2010, 65:671-80. 4. Capps JA, Sharmab V, et al: Resuscitation 2010, 81:653-7. 5. Arroabarren E, Lasa EN, et al: Pediatr Allergy Immunol 2011, 22:708-714.

O2 Drug anaphylaxis in children – data of the German-speaking anaphylaxis registry S Gernert1*, M Worm2, H Ott3, S Hompes2, L Lange1 1 Department of Pediatrics, St. Marien-Hospital Bonn, Bonn, Germany; 2 Department of Dermatology and Allergy, Charité – Universitätsmedizin Berlin, Berlin, Germany; 3Department of Pediatric Dermatology, Catholic Children’s Hospital Wilhelmstift Hamburg, Hamburg, Germany Clinical and Translational Allergy 2013, 3(Suppl 3):O2 Background: Anaphylaxis caused by drugs seems to be less common in children compared with adults. The anaphylaxis-registry collects data of patients with severe allergic reactions from 89 allergy centres in Germany, Austria and Switzerland. Aim of this study was to investigate the circumstances and relevance of drug-induced anaphylaxis in children. Methods: The analysed data included anaphylactic reactions registered from July 2006 until February 2012. The data are delivered by a password-controlled internet-basedquestionnaire. Only severe reactions with pulmonary and/or cardiovascular symptoms are accepted. 704 of the 2926 anaphylactic reactions occurred in children. They were caused by foods (406 cases), insects (180 cases), drugs (50 cases) and others (68 cases). Results: 19 of 50 cases of drug induced anaphylaxis were induced by subcutaneous immunotherapy (SCIT). In the 31 remaining cases most frequent triggers were analgetics (n=14) and antibiotics (n=10) followed by a small number of other drugs such as fentanyl and lidocaine. Nearly all of the children suffered from respiratory symptoms (86%) in addition to skin symptoms, 50% had cardiovascular symptoms and only a small number (22%) had gastrointestinal symptoms. Most of the anaphylactic reactions after SCIT started at the location (medical center, hospital) where the SCIT was administered; only 3 of the SCIT-induced reactions started after the patients left the medical center. Most of the anaphylactic reactions triggered by other drugs took place at home. Only 35% occurred in a medical center or hospital. Although only severe reactions were included, only a small number of the children (12%, 42% of the reactions related to SCIT) received intramuscular, intravenous or inhalative adrenaline. Most of the children (67%, 84%) were treated with antihistamines and/or corticosteroids. Inhaled beta-2-agonists were part of the anaphylaxis therapy in only 19% (SCIT 42%). 38% of the children received an emergency kit after the anaphylactic reaction. This kit included self-injectable adrenaline in 20%, antihistamine in 32%, inhaled beta-2-agonists in 14% and corticosteroids in 36%.

© 2013 various authors, licensee BioMed Central Ltd. All articles published in this supplement are distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Table 1(abstract O1) Clinical features of severe food anaphylaxis treated by epinephrine Clinical features

% paediatric population 78 cases

% adult population 74 cases

% total population 152 cases

% treatment by epinephrine 37 cases

Anaphylactic shock

8.9

24.2

16.4

54.2

Systemic reaction Acute asthma

66,6 2.6

51.3 5.4

59.2 3.9

16.3 66.7

Laryngeal angioedema

11.5

12.2

11.8

16.7

Conclusion: Drug-induced anaphylaxis in children is mainly triggered by analgetics, antibiotics and SCIT. Most of the reactions take place at the children’s home and not in hospitals or medical centers. Disclosure of interest: None declared.

O3 Food allergens in mattress dust in Norwegian homes: a significant source of allergen exposure? RJ Bertelsen1, CK Faeste2, B Granum1, E Egaas2, SJ London3, K-H Carlsen4, KL Carlsen4, M Lovik1* 1 Dept. Food, Water and Cosmetics, Norwegian Institute of Public Health, Oslo, Norway; 2Dept. Chemistry and Toxicology, Norwegian Veterinary Institute, Oslo, Norway; 3National Institute of Environmental Health Sciences, National Institutes of Health, Dept. of Health and Human Services, Research Triangle Park, Durham, NC, USA; 4Dept. Pediatrics, Oslo University Hospital, Oslo, Norway Clinical and Translational Allergy 2013, 3(Suppl 3):O3 Background: Sensitization to food allergens is supposed mostly to be caused by ingestion of the allergen. However, sensitization may also be initiated in the respiratory tract or through the skin. Little is known about sources and prevalence of food allergens in environmental samples. We aimed to describe the presence of food allergens in mattress dust from the homes of 13 year old Norwegian adolescents in relation to home characteristics. Methods: Food allergens from egg, peanut, fish, and milk were measured by dot-blot analysis in mattress dust from 143 homes of 13-year olds in Oslo, Norway. The results of the dot-blot analyses were semi-quantifiable and dichotomized into no detection and confirmed detection. Associations between food allergen detection and home characteristics (collected by study investigators and by parental questionnaires) were assessed by chisquare tests and by multivariate logistic regression models. Results: Fish allergen was found in 46%, peanut in 41%, milk in 39%, and egg allergen in 22% of the mattresses, and only three dust samples contained none of the four food allergens. Milk allergen was more likely to be found in mattresses from beds that are usually made (covered) during the day (53%) than in beds that are not covered during the day (31%), p=0.01. All four food allergens were more frequently detected in small dwellings (0.35 to ovalbumin (OVA) or ovomucoid (OVM). Egg allergy or tolerance at inclusion was assessed through a double-blind placebo-controlled egg challenge. Allergic children were divided in 2 groups: G1 underwent OIT with raw egg white, whereas G2 continued avoiding egg. Tolerant children constituted group G3. Additionally, 2 groups of atopic (nut-allergic, G4) and non atopic children (G5) were included. Serum sIgE, sIgG4 and sIgA to OVA and OVM were determined at baseline (T0) in all groups, and repeated 9 months after in G1 and G2. A second DBPCFC was performed in G2 in T1. Comparisons among groups were performed through non parametrical tests using SPSS 19.0. 2-tailed p < 0.05 was considered significant. Results: Number of children recruited: G1 (28); G2 (24); G3 (21); G4 (10); G5 (10). sIgE decreased and sIgG4 increased in desensitized children (G1) 9 months after OIT-start, but not in egg-allergic controls (G2) (p > 0.001). 4 children in G2 developed spontaneous tolerance over follow-up. In them, sIgE decreased and sIgG4 increased in T1 versus T0. Similarly, sIgE was lower and OVA-sIgG4 was higher in transient egg-allergic (G3) than in persistent egg-allergic individuals (G1 plus G2 in T0) (p < 0.05). Atopic eggtolerant children (G4) had higher sIgG4 than persistent egg-allergic, transient egg-allergic and non atopic children (p < 0.05). No changes in sIgA were detected in desensitized children or in those 4 developing natural tolerance over follow-up. No differences were found in sIgA among the 5 groups. Conclusion: The humoral immune mechanisms involved in natural and induced tolerance to egg allergens seem to be similar. Serum sIgG4, but not sIgA, is involved in induced tolerance in egg-allergic children, as well as with natural tolerance in transient egg-allergic and atopic individuals. Disclosure of interest: None declared. References 1. Savilahti E: Pediatr Allergy Immunol , doi: 10.1111/pai.12004. 2. Tay SS: Clin Exp Allergy 2007, 37(10):1512-1518. 3. Savilahti E: J Allergy Clin Immunol 2010, 125(6):1315-1321.

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O7 Benefits of using heated egg (HE) in the management of egg allergy A Claver*, E Botey, B Navarro, D Nathalie, A Cistero-Bahima Allergy, Instituto Universitario Dexeus. UAB, Barcelona, Spain Clinical and Translational Allergy 2013, 3(Suppl 3):O7 Background: Previous studies suggest that extensive heating and food matrix diminish the allergenicity of egg white proteins, making it possible to be tolerated by some children with egg allergy. Methods: 50 patients (Table 1) underwent an OFC with HE performed in two different days. The first day began with gradual doses of cookies (a brand containing egg) and later home-breaded chicken. Subsequently, tolerant patients incorporated HE into their diet (cookies or food products containing egg daily and home-breaded foods 2-3 days/week). A second challenge with gradual doses of a serving size of a home-made cake containing 4 eggs was performed a week later. Regular consumption was advised for tolerant subjects. All children were periodically controlled. Factors including SPT and sIgE levels, psychological and others were used to determine a subsequent challenge with less-heated-egg (hard-boiled egg, omelette and raw consecutively). Results: 100% of patients tolerated cookies (2-4 units). 90% (45/50) tolerated breaded chicken in the first OFC; 2 patients presented mild anaphylaxis (1 adrenalin dose was required) and 3 mild abdominal pain treated with oral antihistamines. They were instructed a regular cookie intake and 3 were successfully re-challenged a month later. 91.6% (44/48) tolerated cake. 4 patients presented mild abdominal pain. In the next 612 months 72% (36/50) underwent an OFC with hard-boiled egg (negative in 29/5 gastrointestinal symptoms/1 cough/1 angioedema). 30% were challenged with omelette (14 passed/1 vomiting) and 12% with raw egg (all 6 passed). During the home dosing, 20% presented mild abdominal pain with excellent response to oral antihistaminic (50% of which later achieved hard-boiled egg tolerance). SPT wheal diameter to OVM and raw egg white and OVM sIgE levels decreased significantly from the baseline in patients tested. All patients, including the 2 that only tolerated cookies, reported a substantial improvement in quality of life. Conclusion: HE is well tolerated and safe. Thus, strict dietary avoidance may not be necessary at any stage of the protocol, including at the start. Disclosure of interest: None declared.

O8 Treatment of life-threatening food allergy C Nilsson1,2*, A Nopp3, M Wickman1,4, G Johansson3, G Lilja1,2 1 Allergy Department, Sachs’ Children’s Hospital, Stockholm, Sweden; 2 Dept of Clinical Science and Education, Södersjukhuset, Stockholm, Sweden; 3 Dept of Medicine, Clinical Immunology and Allergy, Karolinska Institutet, Stockholm, Sweden; 4Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden Clinical and Translational Allergy 2013, 3(Suppl 3):O8 Background: Food allergy may be life-threatening and affected individuals have impairment in quality of life. Children with multiple and severe food allergies usually have other allergic manifestations like allergy to furry animals and pollen, causing eczema and asthma. Treatment with the anti-IgE (omalizumab) is indicated for severe allergic asthma. Oral immunotherapy (OIT) has been performed in studies, but has shown to be associated with allergic side-effects. Basophil allergen threshold sensitivity (CD-sens) is suggested to mirror the patient’s allergen sensitivity. Methods: Two girls, 16 (N) and 10 (L) years, with repeated life-threatening anaphylaxis from cow’s milk, severe asthma, severe eczema and allergy also

Table 1(abstract O7) Baseline clinical characteristics Gender

32 male/18 female

Mean age (y;range)

5.7 (0.5-16)

History of immediate reaction

50 (28 anaphylaxys, 13 cutaneous, 9 gastrointestinal)

Mean SPT to ovomucoid (OVM) and raw egg white (EW) (mm; range)

OVM: 7.39 (0-18.5) EW: 10.7 (5.5-18.5)

Mean OVM and EW sIgE levels (KU/L; range)

OVM: 8.86 (0.1-100) EW: 11.53 (0.1-100)

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to egg, nuts, peanuts, furry animals and pollens, have been treated with omalizumab followed by OIT during tapering down of the omalizumab treatment. The dose of omalizumab was assessed according to the manufacturer recommendations of asthma treatment. CD-sens was performed, before and during the treatment period. The OIT started with 1-5 ml of fresh milk with increase of another 5 ml every week until a normal serving portion of 300 ml was reached. Results: CD-sens to milk, high at starting point, was zero three months after omalizumab treatment. A milk challenge was performed and the girls passed the challenge without any symptoms and had 300 ml (L) and 450 ml (N) of cow’s milk, respectively. The girls went through the OIT treatment during protection of omalizumab without any side effects. Milk is now daily consumed without restrictions. After some months with omalizumab both girls experienced an improvement of their asthma, needed lower amount of inhalation steroids, had a normal sleeping pattern and improved concentration at school. L did no longer need extra supported teaching to keep up with schoolwork. Both girls experienced a better quality of their hair and nails. Conclusion: Children/teenagers with a “systemic” allergic disease as indicated by multiple allergic manifestations including severe food allergy, asthma, and eczema seem to benefit in general by treatment combining omalizumab and OIT. Disclosure of interest: None declared. O9 Why allergen detection by ELISA is insufficient to protect allergic consumers B Popping Scientific Development, Eurofins, Hamburg, Germany Clinical and Translational Allergy 2013, 3(Suppl 3):O9 Background: In 2010, a multi-allergen detection method based on mass spectrometric (LC-MS/MS) analysis was developed as described in JCA1218. This method was able to demonstrate that seven allergens could be detected in a single analysis. Later, it could be shown that the same method is capable to detect allergens in processed materials while conventional methods like ELSIA and PCR failed in the same matrix, even when amounts as high as 1000mg/kg were present. This was reported in JAOAC 94/4. In mid-2012 a case of a finished product was reported, which caused allergic reactions in children. The product was analysed by ELISA and LC-MS/MS. The ELISA failed to detect the allergens while mass spectrometric analysis clearly demonstrated the presence. This is the first case where evidence suggests that conventional methods like ELISA and PCR are not sensitive enough in some processed materials to protect allergic individuals, while LC-MS/MS is still able to detect these allergens. Methods: To protect the allergic consumer, analytical methods need to be capable of detecting allergens in finished products that typically contain multiple allergens. An LC/MS/MS method for simultaneous detection of seven allergens was developed and compared with commercially available ELISA kits. The detection capabilities of this novel method were demon-strated by analyzing incurred material containing milk, egg, soy, peanut, hazelnut, walnut, and almond. Bread was chosen as a model matrix. To assess the influence of baking on the method’s performance, analysis was done before and after baking. Results: It was demonstrated that in the model matrix bread allergens in concentrations of 1000 mg/kg for egg, soya and milk remained undetected by ELISA while the developed mass spectrometric method was well capable of detecting the allergens. Conclusion: A review of applicable methodology for the detection of allergens in different matrices is required to improve safety for affected consumers and to allow industry to make a meaningful risk assessment. Disclosure of interest: None declared. References 1. Heick J, Fischer M, Pöpping B: Journal of Chromatography A 2011, 1218:938-943. 2. Heick, Fischer, Tamm, Pöpping: J AOAC 2011, 94(4):1060-8.

O10 Abstract not submitted for publication

Clinical and Translational Allergy 2013, 3(Suppl 3):O10

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O11 Mutational analysis of immunodominant epitopes of caprine b-casein recognized by IgE antibodies from patients allergic to goat’s milk and tolerant to cow’s milk S Hazebrouck1*, S Ah-Leung1, E Bidat2, E Paty3, M-F Drumare1, S Tilleul1, K Adel-Patient1, J-M Wal1, H Bernard1 1 Laboratoire d’Immuno-Allergie Alimentaire, INRA, Gif-sur-Yvette, Paris, France; 2Service de Pédiatrie, AP-HP, Hôpital Ambroise Paré, BoulogneBillancourt, France; 3Université Paris Descartes, AP-HP, Hôpital Necker Enfants Malades, Paris, France Clinical and Translational Allergy 2013, 3(Suppl 3):O11 Background: Several cases of allergy to goat’s milk (GM) without allergy to cow’s milk (CM) have been reported. GM-allergy has also been reported in CM-allergic children successfully treated with oral immunotherapy. We previously demonstrated that IgE antibodies from GM-allergic/CM-tolerant patients recognize the caprine b-casein (bcap) without cross-reacting with the bovine b-casein (bbov) despite a high sequence identity (91%). We aimed in the present work to identify the critical amino acids in the noncross-reactive IgE-binding epitopes of bcap. Methods: Using site-directed mutagenesis, recombinant bcap was modified by performing residue substitutions with the corresponding amino acids found in bbov. The IgE-binding capacity of the different modified bcap was then evaluated with sera from 9 GM-allergic/CM-tolerant patients and 9 CMallergic patients. The specificity of murine monoclonal antibodies (mAb) raised against caprine caseins was also analyzed in order to further characterize non-cross-reactive epitopes. The allergenic activity of recombinant bcap was finally assessed by degranulation tests of RBL cells passively sensitized with human IgE antibodies. Results: The substitutions A55T/T63P/L75P in the N-terminal part and P148H/S152P in the C-terminal part of bcap induced the greatest decrease of IgE-reactivity of GM-allergic/CM-tolerant patients toward the caprine allergen. The threonine 63 was found to be particularly critical, as confirmed by the specificity of mAb SCB1D, whose ability to bind bcap was abolished by the substitution T63P. The recombinant bcap containing the five substitutions was unable to induce the degranulation of RBL cells passively sensitized with IgE from GM-allergic/CM-tolerant patients but was still fully allergenic when testing sera from CM-allergic patients. Conclusion: Most of the critical substitutions supporting the restricted IgE specificity of GM-allergic/CM-tolerant patients toward bcap involved proline residues. This probably affects both the primary and secondary structures of non-cross-reactive epitopes since proline are frequently found in turns in protein structures. The drastic influence of substitution T63P on the binding of mAB SCB1D to bcap confirmed the immunodominant role of the epitope encompassing threonine 63, as initially observed with GM-allergic/CMtolerant patients. Disclosure of interest: None declared.

O12 Molecule-based diagnosis of Apium graveolens allergy: is there a need to increase the current allergen panel? G Gadermaier1*, E Vejvar1, P Briza1, K Hoffmann-Sommergruber2, W Hemmer3, F Ferreira1 1 Christian Doppler Laboratory for Allergy Diagnosis and Therapy, University of Salzburg, Salzburg, Austria; 2Department of Pathophysiology and Allergy Research, Medical University of Vienna, Vienna, Austria; 3FAZ-Floridsdorf Allergy Center, Vienna, Austria Clinical and Translational Allergy 2013, 3(Suppl 3):O12 Background: Consumption of celery (Apium graveolens) has the potential to trigger severe allergic reactions in sensitized patients and thus requires labeling on food products. The aim of the study was to determine the sensitization profile to celery allergens and to identify novel IgE binding proteins in this source. Methods: Immunoblots were performed with extracts of celeriac and celery stalks using sera of A. graveolens sensitized patients from Austria (n=23). Purified allergens were obtained from natural source (celeriac) or produced as recombinant proteins in E. coli and IgE reactivity to rApi g 1, rApi g 2, nApi g 5, and nApi g 6 was tested in ELISA. IgE cross-inhibition assays were performed with purified lipid transfer proteins (LTP). Celeriac

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extract was separated in 2D gel electrophoresis and IgE reactive spots were subjected to mass spectrometry-based analysis. Results: Immunoblots showed distinct IgE reactive bands at 10 and 16 kDa while broad reactivity was observed in the range between 25 and 100 kDa. IgE reactivity to Api g 1, which is solely present in celery tuber and absent in stalks was 60% in A. graveolens sensitized patients. Fifty-six percent reacted to Api g 5 while 26-34% of patients were sensitized to Api g 6 (LTP2) and Api g 2 (LTP1) present in celery tuber and stalks, respectively. Interestingly, Api g 6 and Api g 2 sensitization pattern did not correlate and limited IgE cross-reactivity was observed. Eighteen IgE reactive spots from celeriac extract were obtained in 2D gel electrophoresis. Mass analysis identified them as members of the fructose bisphosphate aldolase, malate dehydrogenase, GAPDH, triosephosphate isomerase, chitinase, and manganese superoxide dismutase families. Conclusion: In vitro diagnosis using purified A. graveolens allergens is able to substitute extracts that might give rise to unspecific reactivity. In order to further enhance sensitivity and specificity, newly identified allergens should be produced and included in molecule-based diagnosis of celery allergy. Disclosure of interest: None declared.

O13 Role of carbohydrate moiety in immunoreactivity of Ovomucoid S Benedé, I López Expósito, E Molina, R López-Fandiñ* Bioactivity and Food Analysis, Institute for Food Science Research (CIAL) (CSIC-UAM), Madrid, Spain Clinical and Translational Allergy 2013, 3(Suppl 3):O13 Background: An important question pertaining to glycoproteins is whether the covalently-bound carbohydrate moieties contribute to its immunogenic activity. Ovomucoid (OM) is, for these kinds of studies, a model compound in view of its relatively high carbohydrate content but the role of the carbohydrate moieties on its immunogenic activity still remains controversial. The aim of this study was to establish the effect of glycosylation on the IgEbinding properties and biological activity of OM. Methods: Carbohydrate moieties of OM were removed by enzymatic deglycosylation with PNGase F and the degree of deglycosylation of the protein was evaluated by SDS-PAGE with PAS staining and RP-HPLC. Changes in secondary and tertiary structure of OM due to deglycosylation were evaluated by circular dichroism. Immunoreactivity of native and deglycosylated OM was evaluated by inhibition ELISA and western blot using the sera from ten egg-allergic patients with IgE levels from 37 to more than 100 kU/L. Using PBMCs from egg-allergic patients, the cellular proliferative potency and the capacity to stimulate the secretion of interleukins of native and deglycosylated OM were assessed. Basophil activation by OM, with and without carbohydrates, was assessed using human basophils passively sensitized with serum from children with egg allergy. Results: PNGase F effectively released carbohydrates attached to OM after five days at 37° without apparent structural changes. Immunoassays showed that, in eight out of ten sera of egg-allergic patients, the IgE binding to deglycosylated OM was less effective than to the native protein. The results of immunoblotting showed that six out of ten sera, previously incubated with the deglycosylated protein, kept their IgE binding capacity to the glycosylated protein, supporting the idea that carbohydrates may play a role in antigen recognition. In spite of these results, no influence of glycosylation on T cell activation and proliferation was found. Conclusion: The carbohydrate chains attached to OM play a role in the reactivity against IgE, but their clinical relevance might be low. Disclosure of interest: None declared.

O14 Sensitization to Cor a 9 and Cor a 14 is highly specific for a severe hazelnut allergy in Dutch children and adults L Masthoff1*, L Mattsson2, L Zuidmeer-Jongejan3, J Lidholm2, K Andersson2, JH Akkerdaas3, SA Versteeg3, C Garino4, Y Meijer5, P Kentie5, A Versluis1, CF den Hartog Jager1, CA Bruijnzeel-Koomen1, AC Knulst1, R van Ree3, E van Hoffen1, SG Pasmans1 1 Dermatology/Allergology, University Medical Center Utrecht, Utrecht, the Netherlands; 2Thermo Fisher Scientific, Uppsala, Sweden; 3Experimental Immunology, Academic Medical Center, Amsterdam, the Netherlands;

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4

Pharmaceutical Sciences, Drug & Food Biotechnology Center, University of Piemonte Orientale “A. Avogadro”, Novara, Italy; 5Pediatric Pulmonology/ Allergology, University Medical Center Utrecht, Utrecht, the Netherlands Clinical and Translational Allergy 2013, 3(Suppl 3):O14

Background: Component-resolved diagnosis has been shown to improve diagnosis of food allergy. The aim of this study was to evaluate whether component-resolved diagnosis may help to identify patients at risk of severe allergic reactions to hazelnut. Methods: A total of 161 hazelnut-sensitized patients were included: 40 children and 15 adults with objective symptoms in DBPCFC and 24 adults with a convincing severe history were compared to 41 children and 41 adults with no or subjective symptoms in DBPCFC (grouped together). IgE levels to hazelnut extract and single components were analyzed with ImmunoCAP. Results: IgE to hazelnut extract was significantly higher in children with severe than with no or mild symptoms. Sensitization to rCor a 1.04 was common among both children and adults, while sensitization to rCor a 8 was rare. In 13% of children and 49% of adults with a severe hazelnut allergy, only sensitization to rCor a 1.04 was observed and not to other water-soluble allergens. Sensitization to nCor a 9 and/or rCor a 14 was strongly associated with a severe hazelnut allergy. Using adapted cut-off levels, a diagnostic discrimination between severity groups was obtained. IgE to either nCor a 9 ≥ 1 kUA/L or rCor a 14 ≥ 5 kUA/L (children) and IgE to either nCor a 9 ≥ 1 kUA/L or rCor a 14 ≥ 1 kUA/L (adults) had a specificity of >90% and accounted for 83% of children and 44% of adults with a severe hazelnut allergy. Conclusion: Sensitization to Cor a 9 and Cor a 14 is highly specific for patients with objective symptoms in DBPCFC as marker for a more severe hazelnut allergic phenotype. Disclosure of interest: None declared.

O15 Immunoglobulin E and G4 epitopes of the major allergen of birch pollen Bet v 1 share residues critical for antibody binding N Groh1, B Subbarayal2, L Vogel1, C Möbs3, NW de Jong4, W Pfützner3, RG van Wijk4, J Lidholm5, L Meisel1, S Randow1, T Holzhauser1, B Bohle2, S Vieths1, D Schiller1* 1 Division of Allergology, Paul-Ehrlich-Institut, Langen, Germany; 2 Pathophysiology and Allergy Research and Christian Doppler Laboratory for Immunomodulation, Medical University of Vienna, Vienna, Austria; 3 Department of Dermatology and Allergology, Philipps University Marburg, Marburg, Germany; 4Department of Allergology, Erasmus MC-University Medical Center, Rotterdam, the Netherlands; 5Thermo Fisher Scientific, Uppsala, Sweden Clinical and Translational Allergy 2013, 3(Suppl 3):O15 Background: Millions of patients with allergy to birch pollen develop clinically cross-reactive IgE against Bet v 1-like proteins in plant foods. Specific immunotherapy (SIT) with birch pollen extracts induces the biosynthesis of Bet v 1-specific immunoglobulin (Ig)G4. IgG4 is believed to act as a blocking antibody preventing IgE binding to Bet v 1, thus alleviating allergic symptoms. Only little information on the location and relationship of IgE and IgG4 binding sites of Bet v 1 is available. In this study we seek to identify epitopes of IgE and IgG4 antibodies on Bet v 1. Methods: A competitive immunoscreening of phage-displayed peptides was applied to predict Bet v 1 epitopes of allergen-specific IgE and IgG4 antibodies by bioinformatic means. Predicted epitope residues potentially critical for antibody binding were substituted by site-directed mutagenesis. Recombinant Bet v 1 (rBet v 1) and rBet v 1 variants were purified from Escherichia coli. The proteins were physicochemically characterized using circular dichroism (CD) and dynamic light scattering. To test the IgE and IgG4 interactions with rBet v 1 variants, western blot analyses, ELISA, and cellular mediator release assays were performed. Results: Several rBet v 1 variants were expressed in E. coli. Circular dichroism and structural modeling of the variants revealed Bet v 1-like theoretical secondary structure topology. The rBet v 1 variants showed reduced IgE and IgG4 binding with sera of birch pollen allergic subjects in western blot analyses and competitive ELISAs. The rBet v 1 variants showed decreased IgE-mediated mediator release in humanized rat basophil leukemia cells sensitized with sera of birch pollen allergic subjects.

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Conclusion: We identified critical residues in IgE and IgG4epitopes of Bet v 1. Although patient-specific variability was observed, the antibody interactions of the respective rBet v 1 variants were compromised for both IgE and IgG4, respectively. We conclude that epitopes for IgE and IgG4 share common residues critical for antibody interaction, suggesting an overlap of IgE and IgG4-binding sites on the molecular surface of Bet v 1. The knowledge of clinically relevant immunoglobulin-allergen interactions on the molecular level enables new strategies in the diagnosis, prognosis, and therapy of both birch pollen allergies and birch pollen-related food allergies. Disclosure of interest: None declared.

O16 Identification of allergens and epitopes involved in allergy to deamidated gluten S Denery1*, C Brossard1, C Larré1, M Bodinier1, F Pineau1, M Pietri1, DA Moneret-Vautrin2, E Paty3 1 UR 1268 BIA Nantes, INRA, Nantes, France; 2Service d’allergologie, Centre Hospitalier Jean Monnet, Epinal, France; 3Service de Pneumologie et Allergologie Pédiatriques, Groupe Hospitalier Necker, Paris, France Clinical and Translational Allergy 2013, 3(Suppl 3):O16 Background: Gluten proteins can be modified by deamidation to enhance their solubility and technological applications. However severe allergic reactions have been reported after the consumption of food products containing deamidated gluten in subjects tolerant to wheat. This work aimed to characterize allergen profiles for these patients in comparison with those of patients allergic to wheat and identify IgE-binding epitopes. Methods: Sera were obtained from 15 patients allergic to deamidated gluten (DG) and from 9 patients allergic to wheat proteins (WP). IgE-binding profiles were characterized both in ELISA and in a humanized rat basophilic leukemia (RBL) cell model. Epitopes were mapped on g and ω2-gliadin sequences by Pepscan and effect of glutamine/glutamic acid substitutions were studied. Results: Compared to the heterogeneous pattern of allergens detected by IgE from patients allergic to WP, responses of patients allergic to DG were homogeneous. In ELISA, all the sera displayed IgE-binding to deamidated g and ω2-gliadins and deamidated total gliadins, frequently with high concentrations. These modified proteins induced RBL degranulation with most of the sera from DG-allergic patients. A consensus epitope was found on native g and ω2-gliadins (QPQQPFPQ); it was repeated several times in their sequences. The substitution of two or three glutamines of this epitope into glutamic acid at positions Q3 or Q4 and Q8 (QPEEPFPE) increased its recognition the best. Conclusion: Allergy to DG is a separate entity from wheat allergy characterized by a homogeneous IgE response. Deamidated ω2-gliadins or the dominant IgE-binding epitope QPEEPFPE could be used as tools for the diagnosis of this new allergy. Disclosure of interest: None declared. References 1. Gourbeyre P, Denery-Papini S, Larré C, Gaudin J-C, Brossard C, Bodinier M: Wheat gliadins modified by deamidation are more efficient than native gliadins in inducing a th2 response in balb/c mice experimentally sensitized to wheat allergens. Mol Nutr Food Res 2012, 56(2):336-344. 2. Denery-Papini S, Bodinier M, Larré C, Brossard C, Pineau F, Triballeau S, Pietri M, Battais F, Mothes T, Paty E, Moneret-Vautrin D-A: Allergy to deamidated gluten in patients tolerant to wheat: specific epitopes linked to deamidation. Allergy 2012, 67:1023-1032. O17 Enzymatic hydrolysis of gluten proteins changes the dose-response of oral sensitization, but not the gluten epitopes S Kroghsbo1, NB Andersen1, TF Rasmussen1, S Jacobsen2, CB Madsen1* 1 Division of Toxicology and Risk Assessment, DTU National Food Institute, Soborg, Denmark; 2Enzyme and Protein Chemistry, DTU Systems Biology, Lyngby, Denmark Clinical and Translational Allergy 2013, 3(Suppl 3):O17 Background: Wheat gluten proteins are very complex proteins containing hundreds of components present as monomers, oligomers and polymers that by definition are not soluble in water. To improve utilization, gluten proteins can be hydrolyzed by enzymes or acid. This increases solubility and provide proteins with new functional properties. During the last decade,

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cases of severe allergic reaction to hydrolyzed wheat proteins have been reported in subjects tolerant to wheat. Methods: Brown Norway rats bred and kept on a gluten-free diet for at least three generations were sensitized either orally or intraperitoneally (i.p.) with two different commercial gluten products; unmodified (U-gluten) and enzymatic hydrolyzed gluten (EH-gluten). No adjuvant was used for sensitization. For oral sensitization rats were dosed by daily gavage for 35 days with 0.2, 2 or 20 mg of gluten protein suspended in PBS. For i.p. sensitization rats were immunized three times two weeks apart with 200 µg of gluten protein in PBS. Sera obtained at sacrifice were analyzed by ELISA for levels of specific IgG1 and IgE and for IgG1-binding capacity by inhibition ELISA. Biological functionality of specific IgE was examined by rat basophilic leukemia cell (RBL) assay. Results: Enzymatic hydrolysis significantly increased solubility of gluten proteins. Regardless of the route of dosing, U-gluten and EH-gluten were found to have sensitizing capacity. Sensitization by the i.p. route induced a strong specific IgG1 and IgE response in all animals. No difference in levels or functionality of specific IgG1 and IgE were found between i.p. immunized groups. A clear dose-response in levels of specific IgG1 and IgE was found for rats dosed orally with EH-gluten while U-gluten induced comparable antibody levels and number of responders irrespective of the dose used for oral sensitization. All doses of U-gluten gave a higher response than low dose EH-gluten and a lower response than high dose EH-gluten. As for the i.p. studies, IgG1-binding was not influenced by the gluten product used for oral sensitization. Conclusion: Our results indicate that epitopes are not lost and new epitopes are not formed or disclosed during enzymatic hydrolysis. While enzymatic hydrolysis does not change sensitization by the i.p. route, doseresponse was clearly changed when dosed orally. As EH-gluten was a less potent sensitizer at low dose our results indicate that enzymatic hydrolysis reduces sensitizing capacity despite the fact that epitopes seem unharmed. Disclosure of interest: None declared.

O18 Dietary long chain n-3 polyunsaturated fatty acid induced regulatory T-cells contribute to the prevention of oral sensitization to cow’s milk protein in mice L Van Den Elsen1*, B van Esch1,2, G Hofman1, B van de Heijning2, J Garssen1,2, L Willemsen3 1 Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, the Netherlands; 2Danone Research, Wageningen, the Netherlands; 3Utrecht University, Utrecht, the Netherlands Clinical and Translational Allergy 2013, 3(Suppl 3):O18 Background: Cow’s milk allergy is one of the most common food allergies in children. Dietary lipid composition may affect the susceptibility to develop allergic diseases. Aim of this study was to assess whether and how dietary supplementation with long chain n-3 polyunsaturated fatty acids (n-3 LCPUFA) prevents the establishment of food allergy. Methods: Donor mice were fed a control or fish oil diet before and during oral sensitization with whey. Acute allergic skin response, serum immunoglobulins as well as dendritic cell (DC) and T-cell subsets in mesenteric lymph nodes (MLN), spleen and/or small intestine were assessed. Besides serum transfer experiments, adoptive transfer experiments with splenocytes of whey-sensitized donor mice fed a control or fish oil diet were performed. For this purpose naïve recipient mice received splenocytes either or not ex vivo depleted of CD25+ cells, or MACS isolated CD4+CD25+ regulatory T-cells (Treg). Recipient mice were sham- or whey-sensitized and fed control diet. Results: The acute skin response as well as whey-IgE and -IgG1 levels were reduced in sensitized donor mice fed the fish oil diet as compared to the control diet. Serum transfer confirmed the Th2 type humoral response to be suppressed since sera of fish oil fed sensitized mice had a diminished capacity to induce an allergic response in naïve recipient mice compared to control sera. Furthermore, the acute skin response was diminished upon passive sensitization with hyperimmune serum in fish oil fed naïve recipient mice indicating suppression of the effector response by n-3 LCPUFA. Fish oil fed whey-sensitized donor mice showed an increased % Treg inducing CD11b+CD103+CD8a- DC in MLN in association with enhanced FoxP3+ Treg in spleen and intestine compared to sham mice. In whey-sensitized recipient mice transferred with splenocytes from whey-sensitized fish oil fed donor mice, the acute allergic skin response was similar to sham-sensitized recipients. Ex vivo depletion of Treg prevented this transfer of tolerance.

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Transfer of CD4+CD25+ Treg (85% was FoxP3+) from fish oil fed whey sensitized donors prevented the acute allergic skin response most pronounced. Conclusion: Dietary n-3 LCPUFA largely prevent allergic sensitization in a mouse model for cow’s milk allergy by suppressing the humoral response, enhancing local and systemic Treg and reducing acute allergic symptoms. FoxP3+ Treg play an important role in whey allergy prevention by n-3 LCPUFA. Disclosure of interest: L Van Den Elsen: None declared, B van Esch: Employee of Danone Research, G Hofman: None declared, B van de Heijning: Employee of Danone Research, J Garssen: Employee of Danone Research, L Willemsen: None declared.

O19 Does maternal diet during pregnancy and lactation affect allergy outcomes in their offspring? A systematic review of food based approaches M Netting1*, P Middleton2, M Makrides1 1 Child Nutrition Research Centre, Women’s and Children’s Health Research Institute, North Adelaide, Australia; 2ARCH: Australian Research Centre for Health of Women and Babies, The Robinson Institute, Discipline of Obstetrics & Gynaecology School of Paediatrics and Reproductive Health, The University of Adelaide, North Adelaide, Australia Clinical and Translational Allergy 2013, 3(Suppl 3):O19 Background: Dietary strategies may prevent childhood allergies and reduce the burden of disease. We undertook this systematic review to evaluate the relationship between maternal diet and childhood allergies. Methods: We included studies published up to August 2011 which compared a food-based maternal dietary intervention (intake during pregnancy &/or lactation) with another intervention or no intervention, as well as studies examining associations between maternal dietary intake during pregnancy &/or lactation and allergic outcomes in their children from the index pregnancy. Primary outcomes included child eczema, asthma, hayfever and sensitization (to food or environmental allergens). Studies assessing dietary supplements were not eligible for inclusion, nor were studies where dietary intakes were expressed only in terms of nutrients. Studies exclusively investigating maternal nut and peanut consumption during pregnancy &/or lactation were excluded. Results: 43 studies were included in this systematic review: 11 intervention studies, 27 prospective cohort studies, four retrospective cohort studies and one case-control study. In the RCTs no significant difference was noted overall in the prevalence of eczema and asthma in the offspring of women on diets free from common food allergens during pregnancy. One study (with multiple interventions) reported a lower rate of sensitisation in the intervention group at all ages followed up. Of the prospective cohorts only a few associations were reported between maternal dietary intake and development of allergy considering the overall number that were investigated. Of the results that were consistent, maternal dietary patterns were associated with less risk of allergic disease in offspring included ‘Mediterranean’ dietary patterns and diets rich in fruits and vegetables, fish and vitamin D containing foods. Foods associated with higher risk include vegetable oils and margarine, nuts and fast food. Studies differed in terms of the atopic potential of the participants and the final outcomes were not standardised in terms of age at follow up or diagnosis. Conclusion: The development of atopic disease is complex and multifactorial, depending on genetic potential along with many environmental influences. Maternal diet during pregnancy and lactation is one factor that may have the potential to influence the development of allergy in the child. Disclosure of interest: None declared.

O20 Changes in food allergy and sensitisation over a decade: two birth cohorts from the same geographical area VK Patil1,2*, C Venter1,3, J Grundy1, R Kurukulaaratchy1,2, T Dean1,3, SH Arshad1,2 1 David Hide Asthma & Allergy Research Centre, Newport, UK; 2CES, University of Southampton, Southampton, UK; 3SHSSW, University of Portsmouth, Portsmouth, UK Clinical and Translational Allergy 2013, 3(Suppl 3):O20 Background: Prevalence of food allergy is considered to be increasing with some even suggesting a food allergy epidemic. We aimed to look at

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the changes in reported symptoms of food allergy and food allergen sensitisation over a 10 year period in the same geographical area. We compared the prevalence of reported food allergy and food allergen sensitisation in 9-10 year old children between the Isle of Wight (IoW) birth cohort and Food Allergy and Intolerance Research (FAIR) cohort. Methods: Subjects from the 1989 IOW birth cohort (n=1456) were followed up at 1, 2, 4, 10 and 18 years; the 10 year follow up was completed in 1999-2001. The FAIR cohort was established in 2001 (n=988) and children were followed up at 1, 3 and 9 years; the 9 year follow up was completed in 2010-2011. Reported symptoms were based on the questionnaire data and sensitisation to food allergens was defined as ≥3mm response on skin prick test (SPT). Results: Reported food allergy information was available for 1368 and 827 children and SPT was available for 1034 and 588 children in the IoW and FAIR cohort respectively. Reported allergy to Milk (1.2% to 2.1%), Egg (0.7% to 1.6%), Peanut (1.1% to 1.7%) showed an increase but not statistically significant. Reported allergy to Fish (0.4% to 0.1%) decreased but this was not significant. Statistically significant increase was seen for wheat (0.3% to 1.9%, p0.05). Maternal avoidance of milk products during pregnancy and lactation, as well as use in elevated amounts was not related to an early sensitization to milk allergens (p>0.05). Conclusion: The prevalence of sensitization to cow‘s milk in EuroPrevall Lithuanian birth cohort was 3.4%. In children less than 6 months the sensitization rate was significantly higher in boys, and for older than 18 months – in girls. In children, sensitized to more than one food allergen, SCORAD index was significally higher comparing to sensitized only to milk. There were no statistically significant relationships between maternal disease, use of antibiotic, maternal diet during pregnancy and lactation, parental allergy and sensitization to milk. Disclosure of interest: None declared.

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O22 Antibodies induced by immunization with a hypoallergenic mutant of the major fish allergen Cyp c 1 inhibit allergic symptoms in a mouse model of fish allergy AC Gstoettner1*, U Baranyi2, M Focke-Tejkl1, I Swoboda1, F Stolz3, T Wekerle2, B Linhart1, R Valenta1 1 Pathophysiology and Allergy Research, Medical University of Vienna, Vienna, Austria; 2Department of Surgery, Medical University of Vienna, Vienna, Austria; 3Biomay, Vienna, Austria Clinical and Translational Allergy 2013, 3(Suppl 3):O22 Background: Fish allergy is one of the most frequently occurring allergies to animal-derived food and may cause severe anaphylactic reactions. Currently allergen-specific immunotherapy for fish allergy is not available as it may induce severe side-effects. A hypoallergenic mutant of the major carp allergen Cyp c 1 (mCyp c 1) has recently been developed for the treatment of IgE-mediated fish allergy. We analysed the effect of antibodies induced by immunization with mCyp c 1 on allergic symptoms in a mouse model of fish allergy. Methods: C3H/HeJ mice were sensitized to Cyp c 1 by intragastric gavage or subcutaneous immunization using Cholera toxin or aluminium hydroxide as adjuvans, respectively. Rabbits were immunized with the recombinant mCyp c 1. Antibody responses to mCyp c 1 and wild-type Cyp c 1 were investigated by ELISA using 7 overlapping Cyp c 1-derived synthetic peptides. In order to study if mCyp c 1-specific antibodies can protect, Cyp c 1-sensitized mice received mCyp c 1-specific or control antibodies before they were challenged orally with Cyp c 1. Results: Intragastric sensitization of C3H/HeJ mice induced Cyp c 1-specific IgE antibodies, which did not bind to Cyp c 1-derived peptides, indicating sensitization to conformational epitopes. Rabbit anti-mCyp c 1-specific IgG antibodies recognized the wildtype Cyp c 1 allergen and also reacted with peptide epitopes. They were able to block human and mouse IgE-binding to rCyp c 1 in vitro. In vivo administration of mCyp c 1-specific IgG antibodies reduced allergic symptoms after oral allergen challenge demonstrating that allergen-specific IgG antibodies can protect against food allergy. Conclusion: Vaccination with mCyp c 1 induces IgG antibody responses, which can protect against fish allergy in a murine model. This work was supported by the FAST project of the FP7 of the European Union. Disclosure of interest: None declared.

O23 Involvement of PAF metabolism in anaphylaxis and prevention by Rush-SOTI Z Guo1,2*, NM Tsuji2,3 1 Laboratory for Epithelial Immunobiology, RIKEN Yokohama Institute, Yokohama City, Japan; 2Allergy and Mucosal Immune Tolerance Open Laboratory, RIKEN Yokohama Institute, Yokohama City, Japan; 3Biomedical Research Institute, The National Institute of Advanced Industrial Science and Technology, Tsukuba City, Japan Clinical and Translational Allergy 2013, 3(Suppl 3):O23 Background: Allergy is a critical problem of public health in developed countries. Anaphylaxis is a rapid allergic response with symptoms such as urticarial, itching, diarrhea, and even death. Since allergy is caused by the failure of tolerance induction to exogenous antigens, its establishment of tolerance is a fundamental therapy for allergic diseases. Oral tolerance is a physiological way to actively induce unresponsiveness to food antigens. However, oral immunotherapy using antigens occasionally fails to induce complete tolerance in patients that hinder the development of the therapy. Methods: To clarify the mechanism of the induction of allergy and oral tolerance, we have established a protocol with rush administration of oral antigen and evaluated how anaphylaxis is suppressed. Results: Using this rush specific oral tolerance induction (Rush-SOTI) protocol, we found that the drop of body temperature was partially blocked and recovery was accelerated. Antigen-specific IgE was not reduced by Rush-SOTI. Antagonists of both histamine and platelet-activating factor (PAF) completely prevented the drop of body temperature in anaphylactic mice. From the kinetics, it is suggested that histamine acts in the early phase

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while PAF functions in the late phase.We further analyze the cell population involved in the temperature drop. Depletion of Gr-1+ neutrophils but not mast cells partially prevent body temperature drop. Significant reduction of inflammatory cytokines was correlated to this effect. Conclusion: Together with the observation that he kinetics in both mice with Gr-1 + Cell-depletion and Rush-SOTI treatment were similar, it is suggested that Rush-SOTI prevents anaphylaxis by controlling PAF metabolism, and Gr-1+ neutrophils could be a potential therapeutic target. Disclosure of interest: None declared.

O24 Vaccination with recombinant modified vaccinia virus Ankara prevents the onset of intestinal allergy in mice C Bohnen1, A Wangorsch1*, S Schülke1, M Burggraf2, Y Suezer3, A Schwantes3, G Sutter4, Z Waibler5, G Reese1, M Toda2, S Scheurer1, S Vieths1 1 Division of Allergology, Paul-Ehrlich-Institut, Langen, Germany; 2Junior Research Group “Experimental Allergy Models”, Paul-Ehrlich-Institut, Langen, Germany; 3Pr Research Group “Recombinant Measles Virus and Vaccines”, Paul-Ehrlich-Institut, Langen, Germany; 4Institute for Infectious Diseases and Zoonosis, Ludwig-Maximilians-University, Munich, Germany; 5Junior Research Group “Novel vaccination strategies and early immune responses”, PaulEhrlich-Institut, Langen, Germany Clinical and Translational Allergy 2013, 3(Suppl 3):O24 Background: Modified vaccinia virus Ankara (MVA) encoding antigens are considered as safe vaccine candidates for various infectious diseases in humans. Here, we investigated the immune modulating properties of MVA encoding ovalbumin (MVA-OVA) on the allergen-specific immune response. Methods: The immune modulating properties of MVA-OVA were investigated using GM-CSF-derived BMDCs from C57BL/6 mice. OVA expression upon MVA-OVA infection of BMDCs was monitored by immunoblotting and ELISA. Activation and maturation markers on viable MVA-OVA infected mDCs were analyzed by flow cytometry. INF-g, IL-2, and IL-10 secretion was determined in a co-culture of BMDCs infected with wtMVA or MVA-OVA and MACS-sorted OVA-specific OT-I CD8 + and OT-II CD4+T cells. BALB/c mice were vaccinated with wtMVA, MVA-OVA or PBS, sensitized to OVA/alum and challenged with a diet containing chicken egg white. OVA-specific IgE, IgG1, and IgG2a and cytokine secretion from mesenteric lymph node (MLN)-cells were analyzed. Body weight, body temperature, food uptake and health condition of mice were monitored. Results: Infection with wtMVA and MVA-OVA induced comparable activation of mDCs. MVA-OVA infected BMDCs expressed OVA and induced enhanced IFN-g and IL-2 secretion from OVA-specific CD8 + T cells in comparison to OVA, wtMVA or OVA plus wtMVA. Prophylactic vaccination with MVA-OVA significantly repressed OVA-specific IgE, whereas OVAspecific IgG2a was induced. MVA-OVA vaccination suppressed TH2 cytokine production in MLN cells and prevented the onset of allergic symptoms in a mouse model of OVA-induced intestinal allergy. Conclusion: MVA-OVA vaccination induces a strong OVA-specific TH1immune response, likely mediated by the induction of IFN-g and IgG2a. MVA-based vaccines might be suitable for the treatment of allergic diseases. Disclosure of interest: None declared.

O25 The safety of multiple oral immunotherapy in phase one studies at a single center TLR Dominguez1*, A Mehrotra1, S Wilson1, L Winterroth1, A Blakemore1, K Sampson2, K Nadeau1 1 PEDS Allergy & Immunology, Stanford University, Stanford, CA, USA; 2 Stanford University, Stanford, CA, USA Clinical and Translational Allergy 2013, 3(Suppl 3):O25 Background: There are over 15 million people with food allergies, 38% of which suffer from more than 1 food allergy. With increasing interest in oral immunotherapy (OIT) as a potential treatment, the safety of ingesting known allergens is a major concern. Little to no data are available for OIT with multiple simultaneous allergens. Also, the effect of omalizumab as adjunctive therapy in rush multi OIT had not been investigated.

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Methods: After obtaining IRB approval, subjects (pediatric and adults) were consented and enrolled in an ongoing multiple allergen OIT clinical studies: one phase 1 study “standard multi OIT” (n=27), and a second noted as “rush multi OIT” in which food allergen doses were 100x higher than “standard multi OIT” due to adjunct therapy with omalizumab (n=11). Food allergens included were milk, egg, peanut, walnut, cashew, almond, pecan, hazelnut, wheat, soy, and/or sesame. Up to 5 foods were allowed to be given in the OIT regimen simultaneously depending on the subjects’ allergic reactions to screening food challenges. Entry criteria for both studies were the same; they included a positive DBPCFC for each food allergen with positive skin prick test and/or positive food specific IgE >7kU/L for each food allergen. Subjects receiving omalizumab had injections between weeks 1 and 15. Subjects taking omalizumab and OIT were increased on their initial dose by up to 100 fold more than the initial dose given to subjects taking OIT without omalizumab. Safety was the primary outcome. The secondary goal of both studies was to reach 4000mg of each allergen. Results: These studies are still ongoing. At an average of 28 weeks into studies, there have been an estimated 7471 doses in the standard multiple OIT group, with 371 documented clinical reactions (4.9%). There were 150 documented clinical reactions occurring in an estimated 1076 doses (13.9%) in the rush multiple OIT group. Reactions were mostly mild in both groups (508 mild reactions out of 520 total reactions) and mainly consisted of abdominal pain, pruritus, and rash. No subjects withdrew due to adverse side effects. Six reactions (out of 8547 doses) required epinephrine, two in the rush multi OIT study and four in the standard multi OIT study. Conclusion: Multiple OIT should be performed in a carefully monitored hospital environment with well trained staff. More studies are needed to obtain data on short term and long term efficacy and safety of multi OIT to address the high unmet need of multisensitized food allergy patients. Disclosure of interest: None declared. O26 Safety and predictors of adverse events during oral immunotherapy with raw egg white M Vazquez-Ortiz*, M Alvaro, O Dominguez, R Jimenez-Feijoo, MT Giner, MA Martin-Mateos, AM Plaza Paediatric Allergy and Clinical Immunology, Hospital Sant Joan de Deu, Barcelona, Spain Clinical and Translational Allergy 2013, 3(Suppl 3):O26 Background: Oral immunotherapy (OIT) for food allergy has shown promising efficacy results. However, safety is still concerning. Objectives. To evaluate safety of egg-OIT. To identify clinical/immunological predictors of adverse events. Methods: Prospective longitudinal epidemiological intervention study. Egg-allergic children aged 5–18 underwent a Spanish-approved egg-OIT protocol without premedication. Clinical data, skin prick test (SPT) and specific IgE (sIgE) at baseline and 9 months after OIT were registered. All dose-related reactions, treatments needed and cofactors involved were recorded. Through survival analysis, we studied the cumulative probability of reactions resolution over time and clinical/immunological risk factors of reactions persistence. Results: 51 children were recruited. Mean follow-up was 16 months. 74% reached desensitization to one raw egg. 90% of children suffered reactions, 88% of which affected a single organ. Reactions occurred in 7% of doses, being mainly grade 1 (30%) or 2 (32%). Gastrointestinal (37%) and cutaneous (35%) were the most frequent symptoms. Reactions were heterogeneously distributed: (a) 23 children (45%) had occasional symptoms which ceased over time; (b) 28 (65%) children had more frequent and intense symptoms (71% of total reactions). 11 of them, the most difficult cases, had to interrupt the treatment early, whereas in 17 children reactions decreased in frequency but persisted over-time. Reactions persistence was associated with a higher frequency and severity. Kaplan–Meier estimate revealed a cumulative probability of reactions resolution of 25% at 6 months (95% CI: 2.95-9.05) and 50% (95% CI: 7.2-18.8) at 13 months. Cox proportional hazards multivariate regression model identified 2 variables (egg white-sIgE and Sampson’s severity grades 3 or 4 at baseline egg challenge) as independent risk factors of reactions persistence, with hazard ratios of 1.15 (95% CI: 1.05-1.27; p=0.002) and 4.39 (95% CI: 1.78–10.87; p=0.01), respectively. Early withdrawal was associated to pre-existing asthma and higher sIgE levels (p15 eosinophils/ hpf in esophageal biopsies) was retrospectively analyzed. All children followed the so-called modified six-food elimination diet (SFED), excluding 6 main offending foods retrieved together with those eliciting positive SPT and APT, supplemented with a nutritional support by an amino acid formula. Patients had the following treatment sequence: endoscopy/clinical/biological assessment, 3 months elimination diet and second endoscopy/clinical/ biological assessment. Results: Analysis included 49 patients. At enrolment, BMI was significantly lower in females than in males (p