Heidi Hofer

MAESTRO - multi agent stability prediction upon point mutations

BackgroundPoint mutations can have a strong impact on protein stability. A change in stability may subsequently lead to dysfunction and finally cause diseases. Moreover, protein engineering approaches aim to deliberately modify protein properties, where stability is a major constraint. In order to support basic research and protein design tasks, several computational tools for predicting the change in stability upon mutations have been developed. Comparative studies have shown the usefulness but also limitations of such programs.ResultsWe aim to contribute a novel method for predicting changes in stability upon point mutation in proteins called MAESTRO. MAESTRO is structure based and distinguishes itself from similar approaches in the following points: (i) MAESTRO implements a multi-agent machine learning system. (ii) It also provides predicted free energy change (ΔΔG) values and a corresponding prediction confidence estimation. (iii) It provides high throughput scanning for multi-point mutations where sites and types of mutation can be comprehensively controlled. (iv) Finally, the software provides a specific mode for the prediction of stabilizing disulfide bonds. The predictive power of MAESTRO for single point mutations and stabilizing disulfide bonds is comparable to similar methods.ConclusionsMAESTRO is a versatile tool in the field of stability change prediction upon point mutations. Executables for the Linux and Windows operating systems are freely available to non-commercial users from http://biwww.che.sbg.ac.at/MAESTRO.

Tree pollen allergens—an update from a molecular perspective

It is estimated that pollen allergies affect approximately 40% of allergic individuals. In general, tree pollen allergies are mainly elicited by allergenic trees belonging to the orders Fagales, Lamiales, Proteales, and Pinales. Over 25 years ago, the gene encoding the major birch pollen allergen Bet v 1 was the first such gene to be cloned and its product characterized. Since that time, 53 tree pollen allergens have been identified and acknowledged by the WHO/IUIS allergen nomenclature subcommittee. Molecule‐based profiling of allergic sensitization has helped to elucidate the immunological connections of allergen cross‐reactivity, whereas advances in biochemistry have revealed structural and functional aspects of allergenic proteins. In this review, we provide a comprehensive overview of the present knowledge of the molecular aspects of tree pollen allergens. We analyze the geographic distribution of allergenic trees, discuss factors pivotal for allergic sensitization, and describe the role of tree pollen panallergens. Novel allergenic tree species as well as tree pollen allergens are continually being identified, making research in this field highly competitive and instrumental for clinical applications.

Differences in the intrinsic immunogenicity and allergenicity of Bet v 1 and related food allergens revealed by site‐directed mutagenesis

Birch pollen allergies are frequently associated with adverse reactions to various fruits, nuts, or vegetables, described as pollen–food syndrome (PFS) and caused by cross‐reactive IgE antibodies primarily directed against Bet v 1. Specific immunotherapy (SIT) represents an effective treatment for inhalant allergies; however, successful birch pollen SIT does not correlate well with the amelioration of concomitant food allergies.

Allergen hybrids - next generation vaccines for Fagales pollen immunotherapy.

Trees belonging to the order of Fagales show a distinct geographical distribution. While alder and birch are endemic in the temperate zones of the Northern Hemisphere, hazel, hornbeam and oak prefer a warmer climate. However, specific immunotherapy of Fagales pollen‐allergic patients is mainly performed using birch pollen extracts, thus limiting the success of this intervention in birch‐free areas.

Tackling Bet v 1 and associated food allergies with a single hybrid protein

Background Allergy vaccines should be easily applicable, safe, and efficacious. For Bet v 1–mediated birch pollen and associated food allergies, a single wild‐type allergen does not provide a complete solution. Objective We aimed to combine immunologically relevant epitopes of Bet v 1 and the 2 clinically most important related food allergens from apple and hazelnut to a single hybrid protein, termed MBC4. Methods After identification of T cell epitope–containing parts on each of the 3 parental allergens, the hybrid molecule was designed to cover relevant epitopes and evaluated in silico. Thereby a mutation was introduced into the hybrid sequence, which should alter the secondary structure without compromising the immunogenic properties of the molecule. Results MBC4 and the parental allergens were purified to homogeneity. Analyses of secondary structure elements revealed substantial changes rendering the hybrid de facto nonreactive with patients’ serum IgE. Nevertheless, the protein was monomeric in solution. MBC4 was able to activate T‐cell lines from donors with birch pollen allergy and from mice immunized with the parental allergens. Moreover, on immunization of mice and rabbits, MBC4 induced cross‐reactive IgG antibodies, which were able to block the binding of human serum IgE. Conclusion Directed epitope rearrangements combined with a knowledge‐based structural modification resulted in a protein unable to bind IgE from allergic patients. Still, properties to activate specific T cells or induce blocking antibodies were conserved. This suggests that MBC4 is a suitable vaccine candidate for the simultaneous treatment of Bet v 1 and associated food allergies. Graphical abstract Figure. No Caption available.

Amb a 1 isoforms: unequal siblings with distinct immunological features

Ragweed pollen represents a major allergy risk factor. Ragweed extracts contain five different isoforms of the major allergen Amb a 1. However, the immunological characteristics of Amb a 1 isoforms are not fully investigated. Here, we compared the physicochemical and immunological properties of three most important Amb a 1 isoforms.

Allergens of Blomia tropicalis: An Overview of Recombinant Molecules

Allergic diseases are considered a major problem for healthcare systems in both developed and developing countries. House dust mites are well-known triggers of allergic manifestations. While the Dermatophagoides genus is widely distributed globally, Blomia tropicalis is the most prominent mite species in the tropical and subtropical regions of the world. Over the last decades, an increase in sensitization rates to B. tropicalis has been reported, leading to increased research efforts on Blomia allergens. In fact, 8 new allergens have been identified and characterized to different degrees. Here, we provide an overview of recent developments concerning the identification and production of recombinant Blomia allergens, as well as their structural and immunological characterization. Although considerable progress has been achieved, detailed molecule-based studies are still needed to better define the clinical relevance of Blomia allergens. Thus, the establishment of a well-standardized and fully characterized panel of allergens remains a challenge for the development of better diagnosis and therapy of allergic diseases induced by B. tropicalis.

Birch pollen immunotherapy inhibits anaphylaxis to the cross‐reactive apple allergen Mal d 1 in mice

Cross‐reactive apple allergy is a common co‐morbidity of birch pollen allergy, caused by the presence of a Bet v 1 homologue allergen in apple, Mal d 1. Treatment of tree pollen hay fever by immunotherapy is well established, but its effect on the accompanying apple allergy is debated.

Identification of Proteases and Protease Inhibitors in Allergenic and Non-Allergenic Pollen

Pollen is one of the most common causes of allergy worldwide, making the study of their molecular composition crucial for the advancement of allergy research. Despite substantial efforts in this field, it is not yet clear why some plant pollens strongly provoke allergies while others do not. However, proteases and protease inhibitors from allergen sources are known to play an important role in the development of pollen allergies. In this study, we aim to uncover differences in the transcriptional pattern of proteases and protease inhibitors in Betula verrucosa and Pinus sylvestris pollen as models for high and low allergenic potential, respectively. We applied RNA sequencing to Betula verrucosa and Pinus sylvestris pollen. After de-novo assembly we derived general functional profiles of the protein coding transcripts. By utilization of domain based functional annotation we identified potential proteases and protease inhibitors and compared their expression in the two types of pollen. Functional profiles are highly similar between Betula verrucosa and Pinus sylvestris pollen. Both pollen contain proteases and inhibitors from 53 and 7 Pfam families, respectively. Some of the members comprised within those families are implicated in facilitating allergen entry, while others are known allergens themselves. Our work revealed several candidate proteins which, with further investigation, represent exciting new leads in elucidating the process behind allergic sensitization.

The Concept of Pollen Panallergens: Profilins and Polcalcins

Because of their ubiquitous distribution and high level of cross-reactivity, profilins and polcalcins are classified as panallergens. To date, 43 profilin and 15 polcalcin allergens have been identified. Panallergens frequently cause a pattern of multiple sensitizations in skin prick tests. Moreover, IgE specific for a number of biologically unrelated allergen sources exist. These sensitizations and cross-reactivities are usually irrelevant and, only sporadically, do panallergens represent clinically relevant major allergens. On rare occasions, severe allergic reactions to food have been observed among profilin-allergic patients. Pronounced sensitization to polcalcins may possibly be linked to an elevated risk of asthma. Moreover, panallergens affect the analytical specificity of pollen and food allergen extracts in skin prick tests and in IgE-based diagnosis. Thus, sensitization toward panallergens resembles an important indication to complement allergy diagnosis with source-related, species-specific marker major allergens. These marker allergens contribute significantly to the analytical specificity necessary for identifying the disease-eliciting allergen sources for allergen-specific immunotherapy.