Apple Allergy Research Articles

Immunotherapy (oral and sublingual) for food allergy to fruits.

Food allergy is an abnormal immunological response following exposure (usually ingestion) to a food. Elimination of the allergen is the principle treatment for food allergy, including allergy to fruit. Accidental ingestion of allergenic foods can result in severe anaphylactic reactions. Allergen-specific immunotherapy (SIT) is a specific treatment, when the avoidance of allergenic foods is problematic. Recently, studies have been conducted on different types of immunotherapy for the treatment of food allergy, including oral (OIT) and sublingual immunotherapy (SLIT). To determine the efficacy and safety of oral and sublingual immunotherapy in children and adults with food allergy to fruits, when compared with placebo or an elimination strategy. The Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, EMBASE, CINAHL, and AMED were searched for published results along with trial registries and the Journal of Negative Results in BioMedicine for grey literature. The date of the most recent search was July 2015. Randomised controlled trials (RCTs) comparing OIT or SLIT with placebo or an elimination diet were included. Participants were children or adults diagnosed with food allergy who presented immediate fruit reactions. We used standard methodological procedures expected by the Cochrane Collaboration. We assessed treatment effect through risk ratios (RRs) for dichotomous outcomes. We identified two RCTs (N=89) eligible for inclusion. These RCTs addressed oral or sublingual immunotherapy, both in adults, with an allergy to apple or peach respectively. Both studies enrolled a small number of participants and used different methods to provide these differing types of immunotherapy. Both studies were judged to be at high risk of bias in at least one domain. Overall, the quality of evidence was judged to be very low due to the small number of studies and participants and possible bias. The studies were clinically heterogeneous and hence we did not pool the results. A study comparing SLIT with placebo for allergy to peach did not detect a significant difference between the number of patients desensitised at six months following a double-blind placebo-controlled food challenge (RR 1.16, 95% confidence interval (CI) 0.49 to 2.74). The second study, comparing OIT versus no treatment for apple allergy, found an effect on desensitisation in favour of the intervention using an oral provocation test at eight months, but results were imprecise (RR 17.50, 95% CI 1.13 to 270.19). Neither study reported data on evidence of immunologic tolerance. In both studies, the incidence of mild and moderate adverse events was higher in the intervention groups than in the controls. In the study comparing SLIT with placebo, patients in the intervention group experienced significantly more local adverse reactions than participants in the control group (RR 3.21, 95% CI 1.51 to 6.82), though there was not a significant difference in the number of participants experiencing systemic adverse reactions (RR 0.81, 95% CI 0.22 to 3.02). In the study of OIT, two of the 25 participants in the intervention group reported relevant side effects, whereas no participants in the control group reported relevant side effects. There is insufficient evidence for using OIT or SLIT to treat allergy to fruit, specifically related to peach and apple. Mild or moderate adverse reactions were reported more frequently in people receiving OIT or SLIT. However, these reactions could be treated successfully with medications.

Recombinant Mal d 1 facilitates sublingual challenge tests of birch pollen-allergic patients with apple allergy.

It is still unclear whether allergen‐specific immunotherapy (AIT) with birch pollen improves birch pollen‐related food allergy. One reason for this may be the lack of standardized tests to assess clinical reactions to birch pollen‐related foods, for example apple. We tested the applicability of recombinant (r) Mal d 1, the Bet v 1‐homolog in apple, for oral challenge tests. Increasing concentrations of rMal d 1 in 0.9% NaCl were sublingually administered to 72 birch pollen‐allergic patients with apple allergy. The dose of 1.6 μg induced oral allergy syndromes in 26.4%, 3.2 μg in 15.3%, 6.3 μg in 27.8%, 12.5 μg in 8.3%, 25 μg in 11.1%, and 50 μg in 4.2% of the patients. No severe reactions occurred. None of the patients reacted to 0.9% NaCl alone. Sublingual administration of 50 μg of rMal d 1 induced no reactions in three nonallergic individuals. Our approach allows straight forward, dose‐defined sublingual challenge tests in a high number of birch pollen‐allergic patients that inter alia can be applied to evaluate the therapeutic efficacy of birch pollen AIT on birch pollen‐related food allergy.

High prevalence of lipid transfer protein sensitization in apple allergic patients with systemic symptoms.

BackgroundApple allergy manifests as two main groups of clinical entities reflecting different patterns of allergen sensitization: oral allergy syndrome (OAS) and generalized symptoms (GS).ObjectiveWe analysed the sensitization profile to a wide panel of different components of food allergens (rMal d 1, Mal d 2, rMal d 3, rMal d 4, rPru p 3, rBet v 1 and Pho d 2) for a population of Mediterranean patients with OAS and GS to apple.MethodsPatients (N = 81) with a history of apple allergy that could be confirmed by positive prick-prick test and/or double-blind-placebo-controlled food challenge (DBPCFC), were included. Skin prick test (SPT) and ELISA were performed using a panel of inhalant, fruit and nut allergens. ELISA and ELISA inhibition studies were performed in order to analyse the sensitization patterns.ResultsThirty-five cases (43.2%) had OAS and 46 (56.8%) GS. SPT showed a significantly higher number of positive results with peach, cherry and hazelnut in those with GS. ELISA showed a significantly high percentage of positive cases to rMal d 3, rMal d 4, rPru p 3 and Pho d 2 in patients with OAS and GS compared to controls, and to rBet v 1 in patients with OAS vs controls and between OAS and GS patients. Three different patterns of recognition were detected: positive to LTP (rMal d 3 or rPru p 3), positive to profilin (rMal d 4 and Pho d 2), or positive to both. There were also patients with rMal d 1 recognition who showed cross-reactivity to rBet v 1.ConclusionIn an apple allergy population with a high incidence of pollinosis different patterns of sensitization may occur. LTP is most often involved in those with GS. Profilin, though more prevalent in patients with OAS, has been shown to sensitise patients with both types of symptoms.

Placental immune response to apple allergen in allergic mothers

The immunological milieu in the placenta may be crucial for priming the developing foetal immune system. Early imbalances may promote the establishment of immune-mediated diseases in later life, including allergies. The initial exposure to allergens seems to occur in utero, but little is known about allergen-induced placental cytokine and chemokine release. The release of several cytokines and chemokines from placenta tissue after exposure to mast cell degranulator compound 48/80 or apple allergen in placentas from allergic and healthy mothers was to be analysed. Four placentas from women with apple allergy and three controls were applied in a placental perfusion model with two separate cotyledons simultaneously perfused with and without apple allergen (Mal d 1). Two control placentas were perfused with compound 48/80. In outflow, histamine was quantified spectrophotofluorometrically, IL-2, IL-4, IL-6, IL-10, TNF and IFN-γ by a cytometric multiplex bead array and IL-13 and CXCL10, CXCL11, CCL17 and CCL22 with an in-house multiplex Luminex assay. Compound 48/80 induced a rapid release of histamine, CXCL10, CXCL11, CCL17 and CCL22, but not of the other factors. Apple allergen induced a time-dependent release of IL-6 and TNF, but not of histamine, in placentas of women with apple allergy compared with the unstimulated cotyledon. CCL17 levels were slightly increased after allergen stimulation in control placentas. Allergens can induce placental cytokines and chemokines distinctly in allergic and healthy mothers. These mediators may affect the prenatal development of the immune system and modify the risk of diseases related to immune disorders in childhood such as allergies.

Recombinant Mal d 1 is a reliable diagnostic tool for birch pollen allergen–associated apple allergy

From the Department of Clinical and Experimental Medicine, Faculty of Health Sciences, and CIMed, Centre for Individualised Medicine, Faculty of Health Sciences, Linkoping University, Linkoping, Sweden; the State Key Laboratory of Reproductive Medicine, Central Laboratory of Nanjing First Hospital, Nanjing Medical University, Nanjing, China; the Department of Pathophysiology and Allergy Research, Medical University of Vienna, Vienna, Austria; and the Department of Pediatrics, University of Gothenburg, Gothenburg, Sweden. E-mail: mikael.benson@liu.se. Or: hui.wang@gu.se. *These authors contributed equally to this work. These authors share senior authorship of this work. Supported by the European Commission under the 7th Framework Programme, grant agreement no. 223367; MultiMod; the Swedish Medical Research Council; and the National Natural Science Foundation of China (grant no. 81201598). Disclosure of potential conflict of interest: S. Zhao has received grants from the National Natural Science Foundation of China. B. Bohle has received grants from the Austrian Science Fund and Christian Doppler Research Foundation, has consultant arrangements with the Food Allergy Resource and Research Program, and has grants/grants pending with the Austrian Science Fund. M. Benson has received grants and has grants/grants pending from the European Union and the Swedish Research Council. The rest of the authors declare that they have no relevant conflicts of interest.

Characterization of Apple Allergy in A Mediterranean Population

Characterization of Apple Allergy in A Mediterranean Population

Oral exposure to Mal d 1 affects the immune response in patients with birch pollen allergy

Antibodies and T cells specific for the major birch pollen allergen Bet v 1 cross-react with structurally related food allergens, such as Mal d 1 in apple. We sought to evaluate the effects of oral uptake of Mal d 1 on the allergen-specific immune response in patients with birch pollen allergy. Patients received 50 μg of rBet v 1 sublingually on 2 consecutive days outside of the birch pollen season. One year later, equal amounts of rMal d 1 were administered. Blood samples were collected before and after oral exposure, as well as before and after the intermediate birch pollen season. Allergen-specific IgE levels were determined by using ImmunoCAP. Proliferation of allergen-stimulated PBMCs was assessed, as well as the expression of IL-5, IL-13, IL-10, IFN-γ, and forkhead box protein 3 (Foxp3) in isolated T cells (real-time PCR). Allergen-specific T-cell lines were analyzed for epitope recognition. Orally administered Bet v 1 transiently reduced Bet v 1-specific serum IgE levels, as well as Bet v 1- and Mal d 1-induced T-cell proliferation, and enhanced the expression of IL-5, IL-10, and Foxp3. Orally applied Mal d 1 significantly decreased Bet v 1- and Mal d 1-specific IgE levels and induced IL-5 and IL-10 but no Foxp3 expression. In contrast to Bet v 1, Mal d 1 triggered IFN-γ production and T cells with a different epitope repertoire. Inhalation of birch pollen significantly enhanced allergen-specific IgE levels, T-cell proliferation, and IL-5, IL-10, IL-13, and Foxp3 expression. Two sublingual administrations of 50 μg of Mal d 1 were well tolerated and induced transient immune responses seen during peripheral tolerance development. Thus recombinant Mal d 1 might be suitable and relevant for sublingual treatment of birch pollen-related apple allergy.

Continuous apple consumption induces oral tolerance in birch‐pollen‐associated apple allergy

Patients with birch pollen allergy (major allergen: Bet v 1) have often an associated oral allergy syndrome (OAS) to apple, which contains the cross-reactive allergen Mal d 1. As successful birch pollen immunotherapy does not consistently improve apple related OAS symptoms, we evaluated whether regular apple consumption has an effect on OAS and immune parameters of Mal d 1 or Bet v 1 allergy. A total of 40 patients with a clear history of birch pollen rhinoconjunctivitis and associated OAS to apple were included in an open, randomized, controlled clinical trial: 27 patients consumed daily defined amount of apple (1-128 g), doubling the amount every two to three weeks, while 13 patients remained untreated. Primary endpoint was the proportion of patients that achieved tolerance to at least 128 g of apple at the end of the study after 8 months. Exploratory endpoints were questionnaire about cross-reactive food and pollen allergy symptoms, conjunctival provocation test with birch pollen and Bet v 1, and in vitro tests (tIgE, sIgE, and IgG4 to Mal d 1 and Bet v 1; basophil activation test with both allergens). Seventeen of 27 patients in active group and none of 13 patients in control group (P = 0.0001) could tolerate a whole apple after the intervention. However, differences in endpoints reflecting systemic immune reactivity did not reach statistical significance. In patients with OAS to apple, tolerance can be safely induced with slowly, gradually increasing consumption of apple. However, the observation of a relapse after discounting of apple consumption and absence of immunologic changes suggest that induced tolerance is only transient.

Peach allergy: different clinical profiles across Europe

Rosaceae fruit allergy is the most common food allergy in Europe in patients over 5 years of age. The two fruits most frequently involved are peach and apple. Different clinical phenotypes of peach allergy are observed across Europe in relation to different allergen sensitisation patterns to the peach allergens Pru p1 (Bet v 1 homologue), Pru p 3 (peach lipid transfer protein, LTP), and Pru p 4 (peach profilin). In areas rich in birch trees of Central and Northern Europe peach allergy is linked to birch pollinosis and apple allergy, and the major allergen involved is Pru p 1. These patients present mild oropharyngeal symptoms (oral allergy syndrome, OAS) upon peach ingestion. Profilin co-sensitisation is also found in a minority of patients, but the clinical presentation is similarly OAS. In contrast, in the Mediterranean areas free of birch trees peach allergy may result from a primary sensitisation to Pru p 3, or be linked to pollen allergy with sensitisation to pollen profilin and secondary to Pru p 4, or a combination of both. In patients monosensitised to Pru p 3 the phenotype is severe and the great majority experience systemic reactions including anaphylaxis. In subjects monosensitised to Pru p 4, peach allergy is linked to a pollen allergy, generally to grass pollen, and the phenotype is mild with OAS in all the patients. When sensitisation to Pru p 3 and Pru p 4 are combined systemic reactions are less frequently observed than in patients monosensitised to Pru p3, and OAS is more frequently reported. Sensitisation to Pru p 2, the thaumatin like protein in peach, may be found in combination with Pru p 3 and 4, but its clinical relevance has not yet been established. Peach allergy across Europe is an interesting model food allergy that illustrates how different allergen sensitisation profiles determine the clinical phenotypes of fruit allergy. The reason for the (almost) lack of sensitisation to Pru p 3 in Central and Northern Europe still remains unclear.

Birch-Apple Syndrome Treated with Birch Pollen Immunotherapy

Background: The most common pollen-fruit cross-reaction is the birch-apple syndrome. Allergen immunotherapy (IT) is clearly effective for birch allergy, but its efficacy on apple allergy is controversial. We performed a randomized study on patients with birch-apple syndrome to evaluate the outcome of subcutaneous immunotherapy (SCIT) and sublingual immunotherapy (SLIT). Methods: Forty patients underwent IT with a birch extract (Staloral; Stallergenes, Antony, France), 20 by SCIT and 20 by SLIT. After 1 year of treatment, 15 patients (8 for SCIT and 7 for SLIT) accepted to undergo an oral apple challenge. Measurements of specific IgE to Bet v 1 and Mal d 1 and related allergens Api g 1 and Dau c 1 were obtained in 10 patients, at baseline and after IT. Results: Two of 8 SCIT-treated patients (25%) and 1 of 7 SLIT-treated patients (14.2%) developed complete tolerance to apple. In the remaining patients, an increase in the provocative dose was found in 3 of the SCIT-treated (37.5%) and 2 of the SLIT-treated patients (28.6%). Changes in the levels of specific IgE to Mal d 1 were unrelated to clinical results. Conclusions: These findings suggest that different doses of birch extract may be needed in different patients to improve the associated apple allergy and that a finer diagnostic work-up in selecting patients with birch-apple syndrome who are candidates to respond to birch pollen IT also concerning apple allergy is required.

Consumer attitudes towards hypoallergenic apples that alleviate mild apple allergy

The development of genetically modified (GM) foods with benefits for consumers may be more acceptable than GM foods with benefits that accrue to industry or producers. The Santana apple is a novel hypoallergenic product suitable for many apple allergic consumers with mild symptomology. The Santana also needs fewer pesticides to be applied in production. A survey was conducted among consumers who bought the Santana in a large-scale “sales pilot”. The Santana was perceived to be beneficial by many apple allergic consumers. Non-allergic consumers were less positive about genetically modified hypoallergenic apples. Overall, traditional breeding was the preferred production strategy, although acceptance of genetic modification as a process did increase with increasing perceived personal benefit associated with products, in particular those which were “medically-related”, or perceived to reduce allergic reactions. Consumer preferences for reduced pesticide usage were also found, although this was more contingent on type of production processes applied.

Allergenicity of different apple cultivars assessed by means of skin prick test and sensitisation to recombinant allergens Mal d 1 and Mal d 3 in a group of Italian apple-allergic patients

To evaluate differences in skin prick test response to apple cultivars in patients with apple allergy, 19 patients (10 adults and 9 children) underwent prick-to-prick skin prick test with eleven commercial and noncommercial apple cultivars, and evaluation of specific IgE to apple and recombinant apple allergens Mal d 1 and Mal d 3. The results show that different reactions might be evoked in a single patient by different apple cultivars and also separately for the peel and the pulp of a single cultivar. The cultivars were ranked according to their allergenicity level, being Jonathan, Jonagold, Golden Delicious and Fiesta the most allergenic when considering the pulp and Gala, Fiesta and Golden Delicious for the peel. Further investigations are needed to clarify if a single patient can be allergic only to well-defined apple cultivars and which allergometric tests are necessary to ascertain this.

Application of multiplexed immunoglobulin E determination on a chip in component‐resolved diagnostics in allergy

Cite this as: E. F. Knol and A. C. Knulst, Clinical & Experimental Allergy, 2010 (40) 190–192.

Identification of European Allergy Patterns to the Allergen Families PR-10, LTP, and Profilin from Rosaceae Fruits

High fruit intakes are associated with significant health benefits but fruit allergy sufferers may be discouraged from eating fruit due to the symptoms they experience. Knowledge about allergens involved in fruit allergy and the frequent cross-reactions to other allergens is essential to (a) design the best strategy for fruit allergy testing (b) prescribe optimal avoidance diets, and (c) design technological solutions for development of hypoallergenic fruits. The objective of this review was to investigate whether some characteristic disease entities could be identified in Europe for allergy to Rosaceae fruits. Five allergy patterns were found involving the allergen families PR-10, LTP, and profilin. In the Western Mediterranean area allergies to Rosaceae fruits are caused by monosensitization to LTP, monosensitization to profilin, or co-sensitization to both these allergens. On the contrary, monosensitization to PR-10 and, to a lesser degree, co-sensitization to profilin and PR-10 is dominant in Northern and Central Europe. LTP sensitization is present both in pollinosis and non-pollinosis patients and is associated with peach allergy in particular. The disease pattern for patients sensitized to profilin is characterized by several concomitant allergies including grass and other pollens, Rosaceae and non-Rosaceae fruits. Finally, PR-10 sensitization is primarily associated to concomitant birch pollen and apple allergy.

DEVELOPMENT OF A PROTOCOL THAT ALLOWS SAFE CONSUMPTION OF THE HYPOALLERGENIC APPLE CULTIVAR SANTANA

The apple (Malus ×domestica) cultivar Santana was shown to have a low allergenicity in a previously reported clinical trial. A protocol was developed to validate these results outside a clinical setting and to market the Santana as a hypoallergenic apple for large-scale consumption. This protocol was designed to ensure safe consumption of the Santana by consumers suffering from apple allergy. We tested the protocol in a pilot in 100 supermarket stores and evaluated in an internet-based survey with 295 respondents. Forty-one percent of the consumers with an apple allergy did not have an allergic reaction to the Santana. Only five percent of the allergic consumers that tried the Santana reported a strong allergic reaction. In concordance with this, the majority (78%) of the mild apple allergic consumers appreciated the Santana. The developed protocol is considered suitable for safe consumption of the Santana providing that information about the possibility of having an allergic reaction among consumers with a mild apple allergy is added to the label.

Lipid transfer protein in diagnosis of birch–apple syndrome in children

Lipid transfer protein (LTP) has been reported as an important allergen inducing severe systemic reactions in allergic subjects. The aim of the present study was to estimate the role of LTP in diagnosis of apple allergy in children allergic to birch pollen. The prospective study was carried out on a group of 21 children sensitized to birch pollen and apple allergens, confirmed by presence of specific IgE in the sera of patients. Allergens separated by electrophoresis were probed by Western blotting using hypersensitive patients’ sera. Food hypersensitivity to apple was verified by oral food challenge test. Six patients’ sera were hypersensitive to birch pollen and apple proteins. Almost all sera recognized specifically the main allergen of apple peal Mal d 3 with molecular weight <10 kDa (LTP). Positive oral challenge to apple was found in 52.4% of investigated children. Children allergic to Mal d 1 presented different clinical symptoms. Sensitization to Mal d 3 is very common in children with apple allergy. No correlation between clinical symptoms and sensitization to recombinant apple allergens has been found, LTP may be useful in diagnosis of apple allergy.

Successful sublingual immunotherapy with birch pollen has limited effects on concomitant food allergy to apple and the immune response to the Bet v 1 homolog Mal d 1

Cross-reactivity between the major birch pollen allergen, Bet v 1, and the apple protein, Mal d 1, frequently causes food allergy. To investigate the effects of successful sublingual immunotherapy (SLIT) with birch pollen extract on apple allergy and the immune response to Bet v 1 and Mal d 1. Before and after 1 year of SLIT, Bet v 1-sensitized patients with oral allergy syndrome to apple underwent nasal challenges with birch pollen and double-blind placebo-controlled food challenges with apple. Bet v 1-specific and Mal d 1-specific serum antibody levels and proliferation in PBMCs and allergen-specific T-cell lines (TCLs) were determined. Bet v 1-specific TCLs were mapped for T-cell epitopes. In 9 patients with improved nasal provocation scores to birch pollen, apple-induced oral allergy syndrome was not significantly reduced. Bet v 1-specific IgE and IgG(4) levels significantly increased. Bet v 1-specific T-cell responses to all epitopes and those cross-reactive with Mal d 1 significantly decreased. However, neither Mal d 1-specific IgE and IgG(4) levels nor Mal d 1-induced T-cell proliferation changed significantly. In contrast, Mal d 1-specific TCLs showed increased responses to Mal d 1 after 1 year of SLIT. This longitudinal study indicates that pollen SLIT does not efficiently alter the immune response to pollen-related food allergens, which may explain why pollen-associated food allergy is frequently not ameliorated by pollen immunotherapy even if respiratory symptoms significantly improve. SLIT with birch pollen may have no clinical effect on associated apple allergy.

Characterization of recombinant Mal d 4 and its application for component‐resolved diagnosis of apple allergy

Profilins are ubiquitous panallergens that have been extensively characterized; yet, their clinical relevance is still unclear. The aim of the present study was to produce recombinant apple profilin (rMal d 4) and to evaluate its allergenic activity and its potency for component-resolved allergy diagnosis. Complementary DNA-derived Mal d 4 was cloned, expressed in Escherichia coli and subsequently purified via poly (l-proline) sepharose. A total of 28 sera from apple-allergic patients were used for IgE-ELISA, immunoblot, RAST and basophil histamine release (BHR) test. In addition, skin prick tests (SPTs) were performed in five patients. Four different complementary DNA coding for apple profilin, Mal d 4, each with an open reading frame of 393 nucleotides, were identified. One isoform Mal d 4.0101 was expressed in Escherichia coli and subsequently purified. Mass spectroscopy revealed the expected mass of 13.826 for rMal d 4.0101, and circular dichroism analysis data were typical for a folded protein and small-angle X-ray scattering measurement identified the protein as a monomer. All the serum samples displayed IgE binding to rMal d 4.0101 in IgE ELISA, immunoblot and RAST. In immunoblotting, IgE binding to natural Mal d 4 was partially/completely inhibited by preincubation with rMal d 4.0101, and RAST values to apple extract were significantly reduced upon serum pretreatment with rMal d 4.0101. SPTs and BHR assays using purified rMal d 4.0101 were positive. Purified rMal d 4.0101 was destroyed within seconds when subjected to pepsin digestion. Apple profilin complementary DNAs were identified. The physicochemical and allergenic properties of purified recombinant Mal d 4.0101 were evaluated showing that the recombinant protein was equal to the natural protein as shown by inhibition assays. Thus, Mal d 4 represents another example suitable for component-resolved diagnosis of food allergy.

Apple allergy across Europe: How allergen sensitization profiles determine the clinical expression of allergies to plant foods

Allergy to a plant food can either result from direct sensitization to that food or from primary sensitization to pollen, latex, or another food. We sought to investigate the primary sensitizers in apple allergy across Europe, the individual allergens involved, and whether these differences determine the clinical presentation. Patients (n = 389) with positive case histories and skin prick test responses to fresh apple were selected in the Netherlands, Austria, Italy, and Spain. Skin prick tests and RASTs to a panel of pollens and plant foods were performed, as well as RASTs to Bet v 1 and the apple allergens Mal d 1, 2, 3, and 4. In the Netherlands, Austria, and Italy apple allergy is mild (>90% isolated oral symptoms) and related to birch pollinosis and sensitization to Bet v 1 and its apple homologue, Mal d 1, which has an odds ratio of local reactions of 2.85 (95% CI, 1.47-5.55). In Spain apple allergy is severe (>35% systemic reactions) and related to peach allergy and sensitization to Mal d 3 (nonspecific lipid transfer protein), which has an odds ratio of systemic reactions of 7.76 (95% CI, 3.87-15.56). The analysis of individual apple allergens in a clinical context has provided insight into the sensitization pathway and into the intrinsic risk an allergen bears to induce mild or severe food allergy. Information on the sensitization pathway is essential to develop preventive strategies in food allergy. The application of individual food allergens with a known intrinsic risk will improve the prognostic value of diagnostic tests.

A mutant of the major apple allergen, Mal d 1, demonstrating hypo‐allergenicity in the target organ by double‐blind placebo‐controlled food challenge

Allergen-specific immunotherapy for food allergy has been hindered by severe side-effects in the past. Well-characterized hypo-allergenic recombinant food allergens potentially offer a safe solution. To demonstrate hypo-allergenicity of a mutated major food allergen from apple, Mal d 1, in vitro and in vivo. A mutant of the major apple allergen, Mal d 1, was obtained by site-directed mutagenesis exchanging five amino acid residues. Fourteen patients with combined birch pollen-related apple allergy were included in the study. Hypo-allergenicity of the mutant rMal d 1 (rMal d 1mut) compared with rMal d 1 was assessed by in vitro methods, i.e. RAST (inhibition), immunoblotting and basophil histamine release (BHR) and in vivo by skin prick test and double-blind placebo-controlled food challenge (DBPCFC). RAST analysis (n = 14) revealed that IgE reactivity to rMal d 1mut was twofold lower than that of the wild-type molecule (95% confidence interval (CI): 1.7-2.4). RAST inhibition (n = 6) showed a 7.8-fold decrease in IgE-binding potency (95% CI: 3.0-12.6). In contrast to this moderate decrease in IgE-binding potency, the biological activity of rMal d 1mut assessed by SPT and BHR decreased 10-200-fold. Hypo-allergenicity was confirmed by DBPCFC (n = 2) with both recombinant molecules. A moderate decrease in IgE-binding potency translates into a potent inhibition of biological activity. This is the first study that confirms by DBPCFC that a mutated recombinant major food allergen is clinically hypo-allergenic. This paves the way towards safer immunotherapy for the treatment of food-allergic patients.