Advances and novel developments in molecular allergology

Abstract The continuous search for new allergens and the design of allergen derivatives improves the understanding of their allergenicity and aids the design of novel diagnostic and immunotherapy approaches. This article discusses the recent developments in allergen and epitope discovery, allergy diagnostics and immunotherapy. Structural information is crucial for the elucidation of cross‐reactivity of marker allergens such as the walnut Jug r 6 or that of nonhomologous allergens, as shown for the peanut allergens Ara h 1 and 2. High‐throughput sequencing, liposomal nanoallergen display, bead‐based assays, and protein chimeras have been used in epitope discovery. The binding of natural ligands by the birch pollen allergen Bet v 1 or the mold allergen Alt a 1 increased the stability of these allergens, which is directly linked to their allergenicity. We also report recent findings on the use of component‐resolved approaches, basophil activation test, and novel technologies for improvement of diagnostics. New strategies in allergen‐specific immunotherapy have also emerged, such as the use of virus‐like particles, biologics or novel adjuvants. The identification of dectin‐1 as a key player in allergy to tropomyosins and the formyl peptide receptor 3 in allergy to lipocalins are outstanding examples of research into the mechanism of allergic sensitization.

Precision medicine in allergology requires the identification of genes and biomarkers for diagnosis or monitoring of treatment efficacy. 4 Such novel discoveries should be discussed, and their merits and demerits be evaluated as the research progresses. As clinical trials for the evaluation of treatment options of allergic diseases are becoming more important, the European Academy of Allergy and Clinical Immunology (EAACI) is actively involved in harmonizing and validating AIT study designs. 5 Besides, the treatment of allergic diseases with biologics 6 or small molecules 7 is gaining importance. Here, we review recent advances in allergen discovery, diagnostic approaches, AIT strategies, biomarkers of allergic diseases, and mechanisms leading to allergic sensitization including an evaluation on their clinical relevance (Box 1).

| ALLERG EN MOLECULE S
Continuous identification and characterization of novel allergens is required for understanding the mechanisms of allergic sensitization, improving diagnostics and developing immunotherapy strategies. 1

| Identification of new allergens and allergenic determinants
Several novel allergens were recently accepted by the WHO/IUIS Allergen Nomenclature Sub-Committee. An allergenic glutathione S-transferase, Per a 5.0101, was extracted from the American cockroach (Periplaneta americana) and its immunodominant IgE epitopes were predicted using in silico approaches. 8 Unlike the German cockroach (Blattella germanica) allergens Bla g 1 and 2, which are predominantly found in fecal extracts, Bla g 6, 9, and 11 are present in the whole body, and are now also recognized as major allergens. 9 Par h 1, a pollen defensin-like protein, was discovered from the feverfew weed (Parthenium hysterophorus), a so far overlooked allergen source that causes pollen-induced allergic rhinitis. 10 Among foods, a novel vicilin, Jug r 6, was identified from the English walnut (Juglans regia). 11 The low reactivity to cartilaginous fish due to their α-parvalbumin content indicated dietary alternatives for patients sensitized to β-parvalbumins, major allergens of bony fish. 12 The European paper wasp (Polistes dominula) is gaining importance in venom allergy research due to its invasive nature. Perez-Riverol et al reported the absence of cross-reactive carbohydrate determinants (CCD), which are a common cause of false-positive results in venom allergy diagnosis of Polistes species. 13 Until recently, only Pol d 5 from P dominula venom (PDV) was commercially available for component-resolved diagnosis (CRD). 14 A dipeptidyl peptidase IV, Pol d 3, was identified as another major allergen in PDV. 15 Resistance to β-lactam antibiotics is increasing worldwide.
Consequently, novel β-lactamase inhibitors like clavulanic acid (CLV) are co-formulated with such antibiotics. Currently, no immunoassay is available for detecting IgE against CLV. Two possible IgE-binding antigenic determinants of CLV were proposed as a result of protein haptenation. 16 Moreover, two benzylpenicillin-haptenated peptides, derived from human serum albumin, were involved in sensitization to penicillin. 17

| New technologies for epitope mapping and the discovery of allergens
Several official databases for searching published epitopes are available. 18 Based on the immune epitope database, 19 25 times more linear than conformational epitopes are currently reported. Besides the in silico approaches, epitopes can be defined by physicochemical and biological assays. Liposomal nanoallergen display was used to analyze the individual and combined immunogenicity of eight linear epitopes from the major peanut allergen Ara h 2. 20 For milk and peanut allergens, bead-based epitope assays were utilized to identify patient-specific IgE epitopes using customized peptide libraries. 21 However, such a Luminex-based microplate setup allows only a single antibody-isotype measurement at a time. Ara h 1-derived peptides displayed on phages were screened using allergic patients' sera combined with high-throughput (HTP) sequencing, which revealed the target-unrelated peptide (TUP) problem. 22 To overcome this issue, comprehensive putative TUP databases should be included for the screening. Immunodominant linear IgE epitopes of the oyster allergen Cra g 1 were also identified by a physicochemical method, the isothermal titration microcalorimetry. 23 On the other hand, in silico approaches enable the design of chimeric proteins, where conformational epitopes of an allergen are grafted on low or nonallergenic homologues. The conformational IgE epitope profile of the Bet v 1-related soy allergen Gly m 4 was identified by grafting its IgE-binding areas on related nonallergenic proteins. 24 Reversely, the olive pollen allergen Ole e 15 was mutated by introducing non-IgE binding patches from its human homologue, which allowed the definition of conformational IgE-binding epitopes. 25 Yet, the studies on grafting technologies were done with small patient numbers, and correctly folded mutated chimeras are difficult to produce. Other technologies to discover epitopes such as amide hydrogen/deuterium exchange coupled with mass spectrometry, heteronuclear single quantum coherence-NMR, monoclonal antibody (mAb) binding tests coupled with mutagenesis, and computational prediction tools, were reviewed elsewhere. 26 These novel approaches for epitope discovery are summarized in Table 1.
Finally, HTP genome and proteome screenings combined with bioinformatics are becoming state-of-the-art. Previously unreported 24 Pacific oyster allergens were identified using genome information, in silico allergen prediction and confirmation by IgE immunoblots. 27

| Clinically relevant cross-reactive allergens
For the design of efficient diagnostic or immunotherapy strategies, it is crucial to understand the cross-reactivities among allergens from various sources, which were the topic of several recent studies. The major tree nut allergens are often highly cross-reactive. While investigating the natural Jug r 2, another walnut vicilin, Jug r 6 was discovered, which proved to be an IgE cross-reactive marker between walnut, hazelnut, pistachio, and sesame. 11 Moreover, examining purified natural allergens is essential for the detection of native posttranslational modifications (PTMs) such as glycosylation that may be important for IgE cross-reactivity. Concomitant sensitization to hazelnut and peanut starts early in life, and their cross-reactivity is possibly dominated by T-cell epitopes of the 7S vicilins Cor a 11 and Ara h 1. 28 Examples of nonhomologous proteins that cross-react on the IgE and T-cell levels are discussed in a recent review. 3 The structure of Can f 6 was resolved, which contributed to the understanding of its cross-reactivity with the cat allergen Fel d 4. 29 However, conformational epitopes that selectively recognize either of these allergens are yet to be discovered to diagnose genuine dog or cat sensitization. Red meat allergy is induced by IgE specific for galactose-α-1,3-galactose (α-Gal), which is structurally similar to the blood group B-antigen. A study of red meat allergic patients showed that blood group B individuals had almost no IgE to α-Gal, indicating that self-tolerance reduces the risk of developing red meat allergy. 30 The data from a multi-center meta-analysis showed that the presence of B-antigen in blood reduces the risk of developing red meat allergy. 31

Approach Outcome and clinical implications
In silico allergen prediction Discovery of 24 previously unreported allergens from Pacific oyster, filling a major gap in the management of shellfish allergic patients 26 Liposomal nanoallergen display Analysis of the contribution of high-and low-affinity IgE binding epitopes of Ara h 2 to the allergic response 20 Bead-based assays Validation of bead-based epitope assays for screening of food allergy 21 Prediction of clinical status of milk allergy, identifying a new phenotype of patients who are tolerant to fermented milk products 50 Mapping of conformational epitopes using chimeras Discovery of Ole e 15 epitopes and identification of two major IgE-binding areas 25 TA B L E 1 Novel approaches in recent discoveries of allergens and epitopes

| Structural stability of allergens
The structural stability of allergens can be an intrinsic characteristic of the proteins themselves such as heat and protease resistance or may be influenced by the presence of ligands or PTMs. 32  Evidence is accumulating on the allergic sensitizing capacity of food matrix-associated lipids, for instance in α-Gal-mediated delayed allergic reactions to red meat. 37 The allergenic glycan α-Gal is present in both protein and lipid extracts from red meat. However, only α-Gal bound to lipids was transported across the intestinal cell monolayer. Food allergen passage through the gastro-intestinal tract depends on its stability to digestion and its absorption rate.
Peanut proteins are quickly absorbed into the circulation, detectable as early as 5 minutes after ingestion, and can stay immunologically intact for up to 2 days, still capable of inducing basophil activation and wheal reaction in some patients. 38 Sensitivity of an allergen to gastro-intestinal digestion also shapes its ability to induce oral tolerance. When the calcium-binding residues of carp parvalbumin Cyp c 1 were mutated, the structural stability of the allergen, hence its resistance to digestion, were drastically affected. 39 Unlike the natural allergen, the mutated form could not induce prophylactic tolerance in a mouse model of fish allergy. 39 Interestingly, proteins with allergenic activity were found to be relatively more abundant and stable than nonallergens in extracts from birch pollen, timothy grass, ragweed pollen, and German cockroach. 40

| D IAG NOS TI C APPROACHE S
Allergies are usually diagnosed by bronchial/nasal provocation test, oral food challenge, skin prick test (SPT), intradermal test, and allergen-specific IgE (sIgE) quantification. Additional approaches such as basophil activation test (BAT), T-cell proliferation, and CRD may help to improve the precision and comprehensiveness of the diagnostics ( Figure 1).

| Improvements in diagnosis
Component-resolved diagnosis provides detailed data to determine sensitization profiles, to predict outcomes for persistent allergies, and to avoid allergen challenges. In a longitudinal study with egg-allergic infants, followed up until 4 years of age, Gal d 1-sIgE significantly increased the risk for a persistent egg allergy further in childhood. 41 The presence of sIgE to all four known egg allergens was associated with a high risk for persistent raw-egg allergy. CRD was also helpful to differentiate true walnut allergy from the walnut-pecan dual allergy, reducing the need for oral food challenges. 42 While sIgE to Jug r 1 and 4 performed best in diagnosing walnut allergy, sIgE to Jug r 2, 4, and 6 indicated a dual allergy. CRD, when performed prior to a nasal provocation test (NPT), was a safe predictive tool to diagnose allergy to dog dander. 43

| New approaches for diagnosis
Among the new diagnostic tools, ALEX 2® (Macroarray Diagnostics), a nano bead-based platform for allergy diagnosis, correlated well with the widely used ImmunoCAP ISAC (Thermo Fisher Scientific). 57 The nanofluidic assay (NFA) abioSCOPE ® allows accurate quantification of IgE at picomolar range within 5 minutes at the point-of-care. 58 Validation of a chip-based microfluidic immuno-affinity BAT (miBAT) was compared to conventional BAT. 59

| NE W S TR ATEG IE S FOR ALLERG EN -S PECIFIC IMMUNOTHER APY
The goal of AIT is clinical desensitization, meaning an increase in the threshold allergen amount needed to induce allergic symptoms. 64 The primary outcome of successful AIT is a subjective measure such  were not surface-exposed. 87 In the context of the hygiene hypothesis, arabinogalactans from cowshed dust extract were shown to bind human dendritic cells (DCs) and downregulate subsequent T-cell stimulation. 88 Such molecules could be added in formulations while designing prophylactic vaccines.
Antigen-specific human mAbs can be produced utilizing humanized mice, phage display, by generating mAbs from immortalized human memory B cells, or by retrieving the sequence of immunoglobulin genes from single B cell clones. 89 A combinatorial phage-displayed single-chain variable fragment (ScFv) library was constructed from the PBMCs of a nonallergic individual, who had been immunized with hypoallergenic Bet v 1 fragments. 90 Bet v 1-specific phage clones were converted into soluble ScFvs, which recognized native Bet v 1 and its homologues from alder pollen, hazelnut and apple.
AIT-induced affinity-selected human monoclonal IgG4 prevented IgE engagement of the major cat allergen Fel d 1 in a clinical study conducted with cat-allergic patients. 91 Moreover, polyclonal anti-Fel d 1 chicken antibodies produced in eggs were able to neutralize the allergen in the cat when these eggs were added to its diet. 92 Outbred llamas are exploited for their immune responses with a wide diversity of Ab variable regions, and were used to produce bispecific antibodies against type 2 cytokines. 93 So far, the best-characterized mAb for allergy treatment is omalizumab. However, its use has certain limitations such as high costs and strict patient exclusion criteria. 94

| EMERG ING B I OMARK ER S IN ALLERGY
Allergy covers a variety of disease manifestations which all have in common the involvement of IgE. Biomarkers are needed to classify these diseases manifestations, as IgE alone is not specific enough.
Precision medicine requires biomarkers for choosing the correct treatment of patients displaying various forms of allergic diseases.
Recently, biomarker discovery has gained momentum.

| Diagnostic biomarkers
Clinically applicable biomarkers for diagnosing asthma were characterized by the "SAVED" model which was reported in an EAACI position paper. 99  Emerging data indicate regulatory innate lymphoid cells (ILCregs) as a candidate biomarker cell type for monitoring the successful tolerance induction following an AIT. 111 Although not validated yet, serum levels of vitamin D may provide an insight for natural tolerance development in egg-allergic infants and cow's milk-allergic children. 103,112 Omalizumab has been approved for the treatment of chronic spontaneous urticaria. 113 Accordingly, total serum IgE levels, serum autoantibody levels and lack of basophil activation can be used to monitor the response. 114 In a post hoc analysis of atopic diseases, absolute eosinophil counts correlated with patients' improvements after therapeutic interventions. 115 A critical evaluation is needed when comorbidities are present as the profile of the analyzed biomarkers may change. Allergic sensitization is also influenced by other components from allergen sources. African green monkeys developed peanut-specific IgG but not IgE when sensitization with defatted peanut extract was performed, 118 possibly due to the lack of peanut lipids.

| NOVEL DISCOVERIE S IN MECHANIS MS OF ALLERG I C S EN S ITIZ ATI ON
Moreover, peanut lipids acted as adjuvants on human keratinocytes, inducing pro-inflammatory response and potentially aiding allergic sensitization. 119 Similarly, low molecular weight components from bony fish extracts induced barrier damage and pro-inflammatory cytokine release by bronchial epithelial cells. 120 When provided the right milieu of cytokines, antibodies undergo class-switch recombination, which decides the quality of the immune response. In peanut allergy, the class switch to IgE occurs already in the gut, 121 supporting the emerging evidence of germinal center-independent class-switching. 122 Nonetheless, the period of allergen exposure can affect the residence of IgE-switched plasma cells and their half-life. 123 Sequence differences of the pattern recognition receptor dectin-1 on respiratory epithelial cells were reported between allergic and healthy individuals. 124 This study showed a critical role of dectin-1 variants in regulating IL-33 homeostasis. Other important players at the epithelial barrier are innate lymphoid 2 cells which may express CD1a and present lipid antigens. 125 In addition, formyl peptide receptors (FPRs) from DCs were studied in the context of antigen presentation. 126 Peptide metabolites from the allergenic lipocalins, but not from nonallergenic homologues, activated FPR3 signaling and abrogated IL-12 production.

| CON CLUS IONS
Molecular allergology has come a long way since its start in the late 1980s. 2 While most, if not all of the important allergen types have been identified, 127 future research will have to elucidate the molecular mechanisms of allergic sensitization and define the signal transduction cascades that ultimately lead to the production of sIgE. This will impact on prevention strategies. Furthermore, recent advances in molecular-based allergy diagnosis should be implemented into routine diagnosis to improve patient management and to decide on personalized approaches for immunotherapy, as recently discussed by Ansotegui et al. 128 Developing novel technologies for use in allergy diagnosis and management is crucial since it (a) aids to an in-depth understanding of the disease, (b) collects information on advantages and disadvantages of each method for each type of allergy, and (c) encourages further research for advancing top-notch techniques that are clinically applicable, effective and affordable. Novel treatments using next-generation adjuvants, biologics, and small molecules are expected to advance treatment options. Allergy vaccines will be improved and fine-tuned with the aim to achieve long-lasting improvements. As shown in Box 2 on future research perspectives, there are many fields where molecular allergology will move forward.

ACK N OWLED G M ENTS
Authors ÖÜ and HB gratefully acknowledge the support of the Austrian Science Fund (FWF) Doctoral Program MCCA W1248-B30.
The authors also wish to thank Anna Głobińska for help in preparing the figure.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.