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Background House dust mites (HDMs) are among the most important allergen sources containing many different allergenic molecules. Analysis of patients from a double-blind, placebo-controlled allergen-specific immunotherapy (AIT) study indicated that patients may benefit from AIT to different extents depending on their molecular sensitization profiles. Objective Our aim was to investigate in a real-life setting whether stratification of patients with HDM allergy according to molecular analysis may enhance AIT success. Methods Serum and nasal secretion samples from patients with HDM allergy (n = 24) (at baseline, 7, 15, 33, and 52 weeks) who had received 1 year of treatment with a well-defined subcutaneous AIT form (Alutard SQ 510) were tested for IgE and IgG reactivity to 15 microarrayed HDM allergen molecules with ImmunoCAP Immuno-solid-phase Allergen Chip technology. IgG subclass levels to allergens and peptides were determined by ELISA, and IgG blocking was assessed by basophil activation. In vitro parameters were related to reduction of symptoms determined by combined symptom medication score and visual analog scale score. Results Alutard SQ 510 induced protective IgG mainly against Dermatophagoides pteronyssinus (Der p) 1 and Der p 2 and to a lesser extent to Der p 23, but not to the other important allergens such as Der p 5, Der p 7, and Der p 21, showing better clinical efficacy in patients sensitized only to Der p 1 and/or Der p 2 as compared with patients having additional IgE specificities. Conclusion Stratification of patients with HDM allergy according to molecular sensitization profiles and molecular monitoring of AIT-induced IgG responses may enhance the success of AIT.

Purpose of review More than 30 years ago, the first molecular structures of allergens were elucidated and defined recombinant allergens became available. We review the state of the art regarding molecular AIT with the goal to understand why progress in this field has been slow, although there is huge potential for treatment and allergen-specific prevention. Recent findings On the basis of allergen structures, several AIT strategies have been developed and were advanced into clinical evaluation. In clinical AIT trials, promising results were obtained with recombinant and synthetic allergen derivatives inducing allergen-specific IgG antibodies, which interfered with allergen recognition by IgE whereas clinical efficacy could not yet be demonstrated for approaches targeting only allergen-specific T-cell responses. Available data suggest that molecular AIT strategies have many advantages over allergen extract-based AIT. Summary Clinical studies indicate that recombinant allergen-based AIT vaccines, which are superior to existing allergen extract-based AIT can be developed for respiratory, food and venom allergy. Allergen-specific preventive strategies based on recombinant allergen-based vaccine approaches and induction of T-cell tolerance are on the horizon and hold promise that allergy can be prevented. However, progress is limited by lack of resources needed for clinical studies, which are necessary for the development of these innovative strategies.

MeDALL (Mechanisms of the Development of ALLergy; EU FP7-CP-IP; Project No: 261357; 2010-2015) has proposed an innovative approach to develop early indicators for the prediction, diagnosis, prevention and targets for therapy. MeDALL has linked epidemiological, clinical and basic research using a stepwise, large-scale and integrative approach: MeDALL data of precisely phenotyped children followed in 14 birth cohorts spread across Europe were combined with systems biology (omics, IgE measurement using microarrays) and environmental data. Multimorbidity in the same child is more common than expected by chance alone, suggesting that these diseases share causal mechanisms irrespective of IgE sensitization. IgE sensitization should be considered differently in monosensitized and polysensitized individuals. Allergic multimorbidities and IgE polysensitization are often associated with the persistence or severity of allergic diseases. Environmental exposures are relevant for the development of allergy-related diseases. To complement the population-based studies in children, MeDALL included mechanistic experimental animal studies and in vitro studies in humans. The integration of multimorbidities and polysensitization has resulted in a new classification framework of allergic diseases that could help to improve the understanding of genetic and epigenetic mechanisms of allergy as well as to better manage allergic diseases. Ethics and gender were considered. MeDALL has deployed translational activities within the EU agenda.

A large number of allergens have been discovered but we know little about their potential to induce inflammation (allergenic activity) and symptoms. Nowadays, the clinical importance of allergens is determined by the frequency and intensity of their IgE antibody binding (allergenicity). This is a rather limited parameter considering the development of experimental allergology in the last 20 years and the criteria that support personalized medicine. Now it is known that some allergens, in addition to their IgE antibody binding properties, can induce inflammation through non IgE mediated pathways, which can increase their allergenic activity. There are several ways to evaluate the allergenic activity, among them the provocation tests, the demonstration of non-IgE mediated pathways of inflammation, case control studies of IgE-binding frequencies, and animal models of respiratory allergy. In this review we have explored the current status of basic and clinical research on allergenic activity of indoor allergens and confirm that, for most of them, this important property has not been investigated. However, during recent years important advances have been made in the field, and we conclude that for at least the following, allergenic activity has been demonstrated: Der p 1, Der p 2, Der p 5 and Blo t 5 from HDMs; Per a 10 from P. americana; Asp f 1, Asp f 2, Asp f 3, Asp f 4 and Asp f 6 from A. fumigatus; Mala s 8 and Mala s 13 from M. sympodialis; Alt a 1 from A. alternata; Pen c 13 from P. chrysogenum; Fel d 1 from cats; Can f 1, Can f 2, Can f 3, Can f 4 and Can f 5 from dogs; Mus m 1 from mice and Bos d 2 from cows. Defining the allergenic activity of other indoor IgE antibody binding molecules is necessary for a precision-medicine-oriented management of allergic diseases.

Purpose of Review The aim of this article is to discuss how allergen-specific immunotherapy (AIT) can be improved through molecular approaches. We provide a summary of next-generation molecular AIT approaches and of their clinical evaluation. Furthermore, we discuss the potential of next generation molecular AIT forms for the treatment of severe manifestations of allergy and mention possible future molecular strategies for the secondary and primary prevention of allergy. Recent Findings AIT has important advantages over symptomatic forms of allergy treatment but its further development is limited by the quality of the therapeutic antigen preparations which are derived from natural allergen sources. The field of allergy diagnosis is currently undergoing a dramatic improvement through the use of molecular testing with defined, mainly recombinant allergens which allows high-resolution diagnosis. Several studies demonstrate that molecular testing in early childhood can predict the development of symptomatic allergy later on in life. Summary Clinical studies indicate that molecular AIT approaches have the potential to improve therapy of allergic diseases and may be used as allergen-specific forms of secondary and eventually primary prevention for allergy.

PURPOSE OF REVIEW: Molecular allergology uses pure, mainly recombinant and structurally defined allergen molecules and allergen-derived epitopes to study mechanisms of IgE-associated allergy, to diagnose, and even predict the development of allergic manifestations and to treat and prevent IgE-associated allergies. Atopic dermatitis, a chronic inflammatory skin disease is almost always associated with IgE sensitization to allergens. However, also non-IgE-mediated pathomechanisms seem to be operative in atopic dermatitis and it is often difficult to identify the disease-causing allergens. Here we review recent work showing the usefulness of molecular allergology to study mechanisms of atopic dermatitis, for diagnosis and eventually for treatment and prevention of atopic dermatitis. RECENT FINDINGS: IgE sensitization to airborne, food-derived, microbial allergens, and autoallergens has been found to be associated with atopic dermatitis. Using defined allergen molecules and non-IgE-reactive allergen derivatives, evidence could be provided for the existence of IgE- and non-IgE-mediated mechanisms of inflammation in atopic dermatitis. Furthermore, effects of epicutaneous allergen administration on systemic allergen-specific immune responses have been studied. Multi-allergen tests containing micro-arrayed allergen molecules have been shown to be useful for the identification of culprit allergens in atopic dermatitis and may improve the management of atopic dermatitis by allergen-specific immunotherapy, allergen avoidance, and IgE-targeting therapies in a personalized medicine approach. SUMMARY: Molecular allergology allows for dissection of the pathomechanisms of atopic dermatitis, provides new forms of allergy diagnosis for identification of disease-causing allergens, and opens the door to new forms of management by allergen-specific and T cells-targeting or IgE-targeting interventions in a personalized medicine approach.

Allergic reactions to fungi were described 300 years ago, but the importance of allergy to fungi has been underestimated for a long time. Allergens from fungi mainly cause respiratory and skin symptoms in sensitized patients. In this review, we will focus on fungi and fungal allergens involved in respiratory forms of allergy, such as allergic rhinitis and asthma. Fungi can act as indoor and outdoor respiratory allergen sources, and depending on climate conditions, the rates of sensitization in individuals attending allergy clinics range from 5% to 20%. Due to the poor quality of natural fungal allergen extracts, diagnosis of fungal allergy is hampered, and allergen-specific immunotherapy is rarely given. Several factors are responsible for the poor quality of natural fungal extracts, among which the influence of culture conditions on allergen contents. However, molecular cloning techniques have allowed us to isolate DNAs coding for fungal allergens and to produce a continuously growing panel of recombinant allergens for the diagnosis of fungal allergy. Moreover, technologies are now available for the preparation of recombinant and synthetic fungal allergen derivatives which can be used to develop safe vaccines for the treatment of fungal allergy.

Background Sensitization to individual cat and dog allergen molecules can contribute differently to development of allergy to these animals. Objective We sought to investigate the association between sensitization patterns to cat and dog allergen molecules during childhood and symptoms to these furry animals up to age 16 years. Methods Data from 779 randomly collected children from the Barn/Children Allergy/Asthma Milieu Stockholm Epidemiologic birth cohort at 4, 8, and 16 years were used. IgE levels to cat and dog were determined by using ImmunoCAP, and levels to allergen molecules were determined by using an allergen chip based on ISAC technology (Mechanisms for the Development of Allergy chip). Allergy was defined as reported rhinitis, conjunctivitis, or asthma at exposure to cat or dog. Results Cross-sectionally, IgE to Fel d 1 and cat extract had similar positive predictive values for cat allergy. IgE to Can f 1 showed a higher positive predictive value for dog allergy than dog extract IgE. Sensitizations to Fel d 1 and Can f 1 in childhood were significantly associated with symptoms to cat or dog at age 16 years. Polysensitization to 3 or more allergen molecules from cat or dog was a better longitudinal predictor of cat or dog symptoms than results of IgE tests with cat or dog allergen extract, respectively. Cross-sectionally, cat/dog-polysensitized children had higher IgE levels and more frequent symptoms to cat and dog than monosensitized children. Conclusions Sensitization to Fel d 1 and Can f 1 in childhood and polysensitization to either cat or dog allergen molecules predict cat and dog allergy cross-sectionally and longitudinally significantly better than IgE to cat or dog extract.

Background: Commercial skin prick test (SPT) extracts used for the diagnosis of dog allergy are prepared by extracting allergens from natural sources, e.g. dog hair and dander. Due to different starting material and extraction methods used, it is likely that extracts differ regarding their allergen contents. Methods: The total protein content and composition of dog SPT extracts from 5 European manufacturers were compared by silver-stained SDS-PAGE. Specific antibody probes were generated to detect major and minor allergens in each extract by immunoblotting. Additionally, sera of patients suffering from dog allergy were used to detect dog allergens in SPT extracts. Results: SPT extracts showed a 20-fold variation regarding the total protein content. The contents of the major dog allergen Can f 1 and of Can f 2 varied considerably between the extracts. In one of the extracts, neither Can f 1 nor Can f 2 could be detected by immunoblotting. The contents of the minor dog allergen Can f 3, albumin, also showed great variability. In one of the dog SPT extracts, the presence of human serum albumin (HSA) was detected with HSA-specific antibodies. Conclusion: The observed variability of commercial dog SPT extracts regarding their allergen contents likely has a negative influence on the accuracy of diagnosis of dog allergy.

Asthma, rhinitis, and eczema are complex diseases with multiple genetic and environmental factors interlinked through IgE-associated and non–IgE-associated mechanisms. Mechanisms of the Development of ALLergy (MeDALL; EU FP7-CP-IP; project no: 261357; 2010-2015) studied the complex links of allergic diseases at the clinical and mechanistic levels by linking epidemiologic, clinical, and mechanistic research, including invivo and invitro models. MeDALL integrated 14 European birth cohorts, including 44,010 participants and 160 cohort follow-ups between pregnancy and age 20years. Thirteen thousand children were prospectively followed after puberty by using a newly standardized MeDALL Core Questionnaire. Amicroarray developed for allergen molecules with increased IgE sensitivity was obtained for 3,292 children. Estimates of air pollution exposure from previous studies were available for 10,000 children. Omics data included those from historical genome-wide association studies (23,000 children) and DNA methylation (2,173), targeted multiplex biomarker (1,427), andtranscriptomic (723) studies. Using classical epidemiology and machine-learning methods in 16,147 children aged 4years and 11,080 children aged 8years, MeDALL showed the multimorbidity of eczema, rhinitis, and asthma and estimated that only 38% of multimorbidity was attributable to IgE sensitization. MeDALL has proposed a new vision of multimorbidity independent of IgE sensitization, and has shown that monosensitization and polysensitization represent 2 distinct phenotypes. The translational component of MeDALL is shown by the identification of a novel allergic phenotype characterized by polysensitization and multimorbidity, which is associated with the frequency, persistence, and severity of allergic symptoms. The results of MeDALL will help integrate personalized, predictive, preventative, and participatory approaches in allergic diseases.

The major turnip (Brassica rapa) pollen allergen, belongs to a family of calcium-binding proteins (i.e., two EF-hand proteins), which occur as highly cross-reactive allergens in pollen of weeds, grasses and trees. In this study, the IgE binding capacity and allergenic activity of three recombinant allergen variants containing mutations in their calcium-binding sites were analyzed in sensitized patients with the aim to identify the most suitable hypoallergenic molecule for specific immunotherapy. Analysis of the wildtype allergen and the mutants regarding IgE reactivity and activation of basophils in allergic patients indicated that the allergen derivative mutated in both calcium-binding domains had the lowest allergenic activity. Gel filtration and circular dichroism experiments showed that both, the wildtype and the double mutant, occurred as dimers in solution and assumed alpha-helical fold, respectively. However, both fold and thermal stability were considerably reduced in the double mutant. The use of bioinformatic tools for evaluation of the solvent accessibility and charge distribution suggested that the reduced IgE reactivity and different structural properties of the double mutant may be due to a loss of negatively charged amino acids on the surface. Interestingly, immunization of rabbits showed that only the double mutant but not the wildtype allergen induced IgG antibodies which recognized the allergen and blocked binding of allergic patients IgE. Due to the extensive structural similarity and cross-reactivity between calcium-binding pollen allergens the hypoallergenic double mutant may be useful not only for immunotherapy of turnip pollen allergy, but also for the treatment of allergies to other two EF-hand pollen allergens.

Allergy diagnosis based on purified allergen molecules provides detailed information regarding the individual sensitization profile of allergic patients, allows monitoring of the development of allergic disease and of the effect of therapies on the immune response to individual allergen molecules. Allergen microarrays contain a large variety of allergen molecules and thus allow the simultaneous detection of allergic patients' antibody reactivity profiles towards each of the allergen molecules with only minute amounts of serum. In this article we summarize recent progress in the field of allergen microarray technology and introduce the MeDALL allergen-chip which has been developed for the specific and sensitive monitoring of IgE and IgG reactivity profiles towards more than 170 allergen molecules in sera collected in European birth cohorts. MeDALL is a European research program in which allergen microarray technology is used for the monitoring of the development of allergic disease in childhood, to draw a geographic map of the recognition of clinically relevant allergens in different populations and to establish reactivity profiles which are associated with and predict certain disease manifestations. We describe technical advances of the MeDALL allergen-chip regarding specificity, sensitivity and its ability to deliver test results which are close to in vivo reactivity. In addition, the usefulness and numerous advantages of allergen microarrays for allergy research, refined allergy diagnosis, monitoring of disease, of the effects of therapies, for improving the prescription of specific immunotherapy and for prevention are discussed. This work was supported by the European Commission’s Seventh Framework Programme under grant agreement No. 261357 and in part by project F4605 of the Austrian Science Fund (FWF) and the Christian Doppler Research Association, Austria