Introduction

Over the past few decades, innovations in molecular biology, biotechnology, and health technology have substantially advanced the diagnosis and treatment of allergic diseases. The availability of well-defined molecules for allergy diagnosis enabled a better endotyping of patients, resulting in personalized care. The large number of characterized allergen molecules created a need to compile existing knowledge into a handbook dedicated both to clinicians and to scientists. The first edition of the European Academy of Allergy and Clinical Immunology (EAACI) Molecular Allergology User’s Guide (MAUG), edited by Paolo Matricardi, Jörg Kleine-Tebbe, Hans-Jürgen Hoffmann, Rudolf Valenta, and Markus Ollert, was launched in 2016 and quickly became one of the most cited articles in the journal Pediatric Allergy and Immunology [1]. The rapid progress in discovering new allergenic molecules, the availability of new diagnostic methods, and new clinical studies emphasizing the importance of specific immunoglobulin E (IgE) profiles for prediction of clinical cross-reactivity, prognosis, and symptom severity made an update mandatory. A new dedicated task force was proposed by a team formed by Karin Hoffmann-Sommergruber, Christiane Hilger, Stephanie Dramburg, Alexandra Santos, and Leticia de las Vecillas and approved by the EAACI Executive Committee in 2021. All of the authors of the previous edition were invited to contribute with an update of their chapters, but also expert juniors were included as co-authors for most chapters (Fig. 1). In line with the first edition, MAUG 2.0 is divided into four sections:

  • Section A: General concepts

  • Section B: Using molecular allergology in clinical practice

  • Section C: Cross-reactive molecules

  • Section D: Important molecules (this section is only included in the book [2], but not in the Pediatric Allergy and Immunology supplement [3])

Fig. 1
figure 1

First and updated editions of the Molecular Allergology User’s Guide

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Great efforts were dedicated to the homogenization of the existing illustrations and tables, as well as to the creation of new artwork for a better and more intuitive reading. This article gives an overview of the most important updates of the new edition of the Molecular Allergology User’s Guide, MAUG 2.0. All content is freely available at the EAACI Knowledge Hub (https://hub.eaaci.org/resources_documents/molecular-allergology-users-guide-2-0/) and in the journal Pediatric Allergy and Immunology (https://doi.org/10.1111/pai.13854).

Section A—General concepts

A new chapter included in MAUG 2.0 discusses the role of molecular allergology for allergen immunotherapy (AIT). Clinical studies, where molecular allergology has been applied to the development of immunotherapy are revised, including emerging strategies such as the use of allergen fragments, AIT based on wild-type recombinant allergens, hypoallergens, and the ligation of allergens to adjuvants.

Several advances have been made to standardize extracts in order to increase the quality and enable the comparison between products, under the EU-funded project CREATE. The development of specific monoclonal antibodies now makes it possible to monitor the content of major allergens in allergen extracts. Current practices and current market documentation requirements to use allergen extracts in AIT and in in vivo tests are improving the quality of therapeutical products. Biomarkers assessing immunotherapy effectiveness are emerging. The chapter revises the evidence on using the basophil activation test (BAT), specific IgG4, or the detection of molecular allergen-specific IgE related to high clinical reactivity in order to elucidate AIT efficacy, as well as the identification and relevance of protein epitopes recognized by allergen-specific antibodies.

The BAT uses flow cytometry to measure the expression of degranulation markers (e.g., CD63, CD203c) on the surface of basophils following stimulation with allergen or controls. The BAT can be useful to confirm the diagnosis of allergy to food, insect venom, or respiratory allergies. Allergen extracts or individual allergen molecules can be used for basophil stimulation. Individual allergen molecules can add specificity to the BAT, particularly in some cases, for instance, in food allergy: BAT to Pru p 3 was more specific than BAT to peach in one study and BAT to Ara h 2 was more accurate than BAT to peanut in another study. Api m 1 and Ves v 5 in cases of suspected bee and wasp venom allergies can also be more helpful than the crude extracts, especially in the case of cross-reactivity or cross-reactive carbohydrate determinant (CCD) sensitization. The new chapter provides an extensive overview of the literature and the use and relevance of the BAT in food, insect venom, and respiratory allergy.

To date, no unique intrinsic properties making a molecule an allergen have been identified. The authors theorize that any molecule can be an allergen if it comes into contact with the immune system of a susceptible genotype leading to the induction of a Th2 response. Within this complex immune reaction, allergen-specific IgE antibodies are produced and act as important mediators for cell degranulation. Therefore, allergens are often classified as “major” and “minor,” according to their ability to induce specific IgE in a population. However, this concept is theoretically incomplete, as the clinical relevance of an allergen is also connected to its capacity to induce inflammation. The mechanisms of atopy are still somewhat poorly defined, but the evolution of IgE responses has been described in several large cohorts, particularly throughout childhood. The observations range from a broadening of the IgE profile over time (“molecular spreading”) and a distinct role of “initiator molecules,” over a co-occurrence of allergen-specific IgG and IgE, to specific clinical phenotypes associated with distinct sensitization patterns. Particularly the observed broadening of the immune response and associated clinical severity led to the hypothesis that a prophylactic AIT should ideally be anticipated to prevent the development of a broad IgE response.

The discovery of immunogenic and widely cross-reactive carbohydrate determinants (CCDs) in the early 1980s was accompanied by great enthusiasm, which was dampened as increasing evidence led to the conclusion that they are of low to no clinical relevance. In the diagnostic work-up, CCDs are, however, important as patients with IgE to these cross-reactive determinants usually present with a multitude of positive results in extract-based assays that are not related to positive skin prick test results or an associated history of symptoms. Immunoglobulin E to CCDs is most frequently produced by patients sensitized to grass pollen and/or insect venom, but also helminth exposure may cause reactivity to “classic” CCD epitopes. Chapter A10 delivers comprehensive, yet easily understandable information on the biochemistry, immunological role (protective or aggressive?), and confounding potential of CCD for in vitro diagnostics, completed with illustrative clinical cases.

Many allergen molecules have been well characterized regarding their structure and function, but for most of them, these characteristics do not explain their allergenicity. A new area of research is thus focusing on protein–ligand interactions as well as on matrix components that are required to induce Th2-type immune responses. Ligand binding can affect protein conformation and enhance IgE binding; it can increase protein stability against gastric, thermal, or lysosomal degradation; or even play an immunomodulatory role. To acknowledge substantial advances in the field, a new chapter is dedicated to small molecules as immunomodulators and allergen ligands. The chapter discusses recent findings on allergens belonging to major protein families, such as (a) lipocalins, a highly diverse family of mammalian and arthropod allergens with a transport function for small hydrophobic molecules; (b) pathogenesis-related proteins (PR-10), a highly conserved group of pollen allergens with a hydrophobic ligand binding cavity; (c) serum albumins, highly conserved mammalian plasma proteins with a multitude of transport functions; (d) Niemann–Pick protein C2 (NCP2) family, group 2 mite allergens containing a large hydrophobic cavity for lipid ligands; and (e) the nonspecific lipid transfer proteins (nsLTPs), allergens involved in the defense of plants against abiotic and biotic stress and accommodating a huge number of hydrophobic ligands. In addition, lipids not binding to allergens, but associated with specific allergens such as nsLTPs, are able to modulate the immune response through the activation of innate pathways by binding to the CD1d receptor and interacting with invariant NKT cells.

In the 1990s, immunoassays containing purified batches of marker allergens were developed to assess the environmental exposure on a molecular level. Since then, a number of studies have provided reliable data to improve allergenic risk assessment. A new chapter on molecular exposure has been added, summarizing the current sampling methods and the immunoassays available. These assays allow us to measure the environmental allergen content in certain geographic areas, help to investigate the occupational exposures in relation to allergenic compounds, and are applied for food allergen detection. Also, indoor allergen exposure can be followed by sampling dust. Furthermore, this approach is also used for efficacy testing of therapeutic products and enables monitoring of allergen exposure during clinical trials. A number of different test formats are available including ELISA assays, bead-based assays using a multiplex format, and lateral flow devices. Alternative methods are quantitative polymerase chain reaction (PCR) tests, DNA-based biosensors, and mass spectrometry methods. In summary, relevant tests are established and available. However, there is a need for harmonization of allergenic exposure assessments including multicenter studies and commonly agreed reference material. This will pave the way for consensus documents regarding allergen levels relevant for allergen labeling of products and exposure risks.

Section B—Using molecular allergology in clinical practice

The chapter on cat, dog, and horse allergy has been expanded to cover all furry animals. New allergens have been characterized for cats and dogs, but also for small mammals (Fig. 2). Recent clinical studies emphasize the importance of molecular diagnosis for dog allergy. Whereas monosensitization to Can f 5 may point to tolerance of female dogs, polysensitization is associated with clinical allergy to dog. The section on clinical management now includes diagnostic algorithms for allergy to cat, dog, and horse, as well as background information on specific markers and markers of cross-reactivity.

Fig. 2
figure 2

Known allergens of dog, cat, and horse, as listed in the database of the World Health Organization/IUIS Allergen Nomenclature Sub-Committee (www.allergen.org). Color codes: proteins belonging to the lipocalin family are depicted in blue, serum albumins are shown in orange, latherins in dark green, immunoglobulins in light gray, cystatin in purple, secretoglobin in dark red, NPC2 in yellow, kallikrein in dark gray, and lysozyme in light green

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Although insects are consumed in many regions worldwide, they have only recently been introduced in Europe. They are very much appreciated as a promising alternative source of protein and can be consumed as a whole dried insect or in the form of powder added to various products such as pasta or burgers. The new chapter on edible insects gives background knowledge on the main insect species consumed, the cross-reactive pan-allergens such as tropomyosin and arginine kinase, and their relevance in clinical cross-reactivity to shrimp and shellfish (Fig. 3). Primary sensitization was confirmed in an occupational setting in employees working in a facility rearing or processing mealworms. Overall, data on sensitization to edible insects are limited and diagnostic tests are not yet available. A diagnostic algorithm and the description of case reports give further insight into this new emerging allergy in Europe and North America.

Fig. 3
figure 3

Cross-reactivity of tropomyosin (TM) and arginine kinase (AK) between different allergenic sources. Solid line tropomyosin, dashed line arginine kinase

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The first MAUG edition contained two chapters on specific occupational allergies, allergy to latex, and allergy to laboratory animals. To take into account the many challenges in occupational allergy, a whole new chapter is now dedicated to the topic. The new chapter includes sections of the previous chapters on latex and animal allergy and adds new content for wheat allergy in baker’s asthma as well as other examples on plant- and animal-derived occupational allergies occurring in workers exposed to these sources. An example of a new allergen source is the industrial culturing and handling of Cannabis sativa and its derived products. Other examples of inhalant respiratory allergen sources are soybean, green coffee bean, or wood dust. Relevant sources of animal respiratory allergy are laboratory animals, cattle, and seafood. Microbial-derived allergens affect workers in waste collection and composting, but also workers employed in industries producing microbial-derived enzymes or in companies using those products during the manufacturing process. Diagnostic algorithms and tables of relevant allergens support the clinical diagnosis and management.

Allergy to mammalian meat is a relatively rare form of food allergy, but the diagnosis can be complex due to the existence of different forms of meat allergy. Besides a primary meat allergy, for which allergens are still not well characterized, secondary meat allergies related to milk or animal dander are more frequent. Another form of meat allergy, characterized by the presence of specific IgE antibodies to the carbohydrate galactose-alpha‑1,3‑galactose (α-gal) and a delayed onset of allergic symptoms upon food ingestion, is associated with tick bites. The term “alpha-gal syndrome” (AGS) is the preferred term to describe allergic reactions to α‑gal, which can occur via ingestion of mammalian food or via parenteral administration of drugs derived from mammals. The chapter on mammalian meat allergy has been substantially updated, acknowledging recent studies on the α‑gal syndrome and providing a clinical algorithm for the diagnosis of the different forms of mammalian meat allergy (Fig. 4).

Fig. 4
figure 4

Diagnostic algorithm for patients with allergic reactions to meat

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As part of the section on allergy to fish, the new MAUG edition has included a dedicated chapter to Anisakis simplex (AS) allergy. In the past few decades, it has become increasingly recognized as an allergenic component found mainly in raw or uncooked fish. Its allergenicity decreases when the larvae are dead, after freezing fish at −20 °C for 24 h, or after cooking it for more than 5 min at 60 °C.

The AS seroprevalence ranges between 0.2 and 15% in the general population when detecting specific IgE to the whole extract, which is not always related to symptomatic sensitization. Molecular diagnosis has increased the accuracy to diagnose allergy to Anisakis simplex, mainly with the detection of specific IgE to the major allergen Ani s 1. Another 13 allergens of this parasite have been described so far in the literature. The diagnosis of Anisakis simplex allergy diagnosis also includes IgE testing for other invertebrates such as house dust mites, crustaceans, and nematodes to rule out a potential cross-reactivity. Furthermore, calculating the ratio between sensitization to Anisakis and Ascaris-IgE, a potentially cross-reactive nematode, has been shown to increase the diagnostic specificity (Anisakis-IgE/Ascaris-IgE ≥ 4.4, specificity > 95%).

Although buckwheat consumption has increased exponentially, mainly by patients with gluten-related disorders and as a hidden food allergen, buckwheat allergy is still a rare condition. However, it can induce anaphylactic reactions.

The diagnosis of buckwheat allergy is primarily based on clinical findings. A buckwheat allergy is more likely if the individual is sensitized to the buckwheat extract and to Fag e 2, which is the only commercially available allergen associated with buckwheat. Despite their high sensitivity, the prick test and the detection of specific IgE to the whole extract have a low specificity. Fag e 2 is a 2S albumin, associated with severe reactions. However, even in the absence of sensitization to this allergen, clinical reactivity to this cereal remains possible. Other buckwheat allergens (e.g., Fag e 1, 3, and 5) have been involved in allergic reactions in Fag e 2-negative patients without a history of a severe reaction, and an oral food challenge may be needed to rule out allergy or confirm the diagnosis. A part on buckwheat allergy has been included in the wheat allergy chapter.

In the recent past, the use of Cannabis sativa for various medical and non-medical purposes has significantly increased, which is reflected by numerous reports about allergic reactions to related products. Therefore, a part on cannabis-food syndrome was added to the chapter on fruit and vegetable allergy and specific aspects of cannabis allergy in the occupational setting are summarized in the chapter on occupational allergy. At present the following allergens are described: nsLTP, a Bet v 1 homologue, profilin, and oxygen-evolving enhancer protein. Can s 3, the nsLTP, is regarded as a major allergen and the cross-reactive component accounting for the cannabis-food syndrome. Symptoms of cannabis allergy range from cutaneous contact urticaria to anaphylaxis. In occupational allergy due to contact with C. sativa during production and processing, workers developed both skin and respiratory symptoms.

Section C—Cross-reactive molecules

Due to its clinical relevance, a full chapter on seed storage proteins has been added to the new edition of MAUG. Seed storage proteins are molecular allergens related to clinically relevant sensitizations to nuts, seeds, and legumes. They include 2S albumins, 7S globulins, and 11S globulins. Immunoglobulin E cross-reactivity can occur between allergens from different families of seed storage proteins but mainly happens between allergens from related plants with a high protein sequence identity. However, the clinical impact of cross-reactivity is still unknown. 2S albumins are major and clinically relevant allergens in peanut and tree nuts (e.g., hazelnut, walnut, and cashew nuts). A high risk of cross-reactivity between walnut and pecan nut or cashew nut and pistachio has been described. 7S globulins (vicilins) are major and clinically relevant allergens for legumes. Risks of cross-reactivity between peanuts and peas or lupine, and between peas and lentils, have been previously reported (Fig. 5). Related to 11S globulins (legumins), they have been described as clinically relevant allergens to almonds, hazelnuts, and peanuts. Despite their high clinical impact, there is still a lack of commercial availability of all relevant allergenic seed storage proteins for routine diagnosis.

Fig. 5
figure 5

In vitro cross-reactivity between 7S globulins from tree nuts, seeds, and legumes. Strong cross-reactivity has been shown for cashew and pistachio (black arrow). Limited cross-reactivity is indicated with a black arrow; cross-reactivity only confirmed in vitro and limited knowledge regarding clinical relevance is indicated with a gray arrow

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Gibberellin-regulated proteins (GRP) are plant defense proteins present in the pulp and peel of fruits as well as in pollen grains (Fig. 6). The relatively small, antimicrobial proteins are resistant to heat and proteolysis, and their three-dimensional structure displays a cleft, likely to bind a ligand that has not yet been identified. The main elicitors involved in a GRP-mediated pollen food allergy syndrome are peach, citrus fruits, apricot, cherry, and pomegranate. To date, Cupressaceae is the only tree family known to express allergenic pollen GRP whose clinical relevance has been shown among European patients exposed to Mediterranean cypress as well as Japanese patients with reactions to Japanese cedar. In addition, many plant foods are exposed to an exogenous gibberellin treatment to improve the yield and quality of foods. It is likely, though not yet proven, that this addition of synthetic gibberellin might affect the expression of GRP and therefore the allergenicity of plant foods and pollen. Detailed information on the clinical relevance and management of allergies to GRP can be found in the respective MAUG 2.0 chapter and are completed by four clinical case reports.

Fig. 6
figure 6

Evolutionary relationships of taxa (phylogenetic tree) and 3D modeling of nine allergenic gibberellin-regulated proteins (GRP) and the prototype reference GRP snakin‑1 from potato. Three conformational epitopic regions were predicted using the software Disco Tope 2.0. They are shaded in yellow and orange

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Oleosins are important allergens in nuts and seeds. They are lipophilic proteins with a unique structure composed of a central hydrophobic domain surrounded by hydrophilic domains. Since oleosins are lipophilic, they are under-represented in aqueous allergen extracts, which are made of defatted nuts or seeds, typically used in allergy tests, such as the skin prick test and specific IgE testing. Oleosins are resistant to heat and enzymatic processes and thus they can behave as primary food allergens, responsible for sensitization and potentially severe allergic reactions. An increase in allergenicity has been described with certain forms of food processing such as roasting. The combination of being able to cause severe allergic reactions and being absent or under-represented in allergen extracts means that oleosins are placed in the middle of clinical conundrums such as that of patients who report severe allergic reactions to nuts or seeds but test negative. This is why it is important to test for oleosins specifically, but technical challenges of isolating and solubilizing these proteins have limited their availability thus far.

Conclusion

The second edition of the Molecular Allergology User’s Guide represents the most up-to-date knowledge on molecular allergology, compiled by almost 100 authors who are renowned experts in their field. The new edition reviews the current literature, pinpoints missing diagnostic tools, and gives perspectives for further research. Most importantly, however, it aims at being a valuable tool for daily clinical practice.