Allergen hybrids - next generation vaccines for Fagales pollen immunotherapy.

BACKGROUND: 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. OBJECTIVES: T cells are considered key players in the modification of an allergic immune response during specific immunotherapy (SIT), therefore we thought to combine linear T cell epitope-containing stretches of the five most important Fagales allergens from birch, hazel, alder, oak and hornbeam resulting in a Fagales pollen hybrid (FPH) molecule applicable for SIT. METHODS: A Fagales pollen hybrid was generated by PCR-based recombination of low IgE-binding allergen epitopes. Moreover, a structural-variant FPH4 was calculated by in silico mutagenesis, rendering the protein unable to adopt the Bet v 1-like fold. Both molecules were produced in Escherichia coli, characterized physico-chemically as well as immunologically, and tested in mouse models of allergic sensitization as well as allergy prophylaxis. RESULTS: Using spectroscopic analyses, both proteins were monomeric, and the secondary structure elements of FPH resemble the ones typical for Bet v 1-like proteins, whereas FPH4 showed increased amounts of unordered structure. Both molecules displayed reduced binding capacities of Bet v 1-specific IgE antibodies. However, in a mouse model, the proteins were able to induce high IgG titres cross-reactive with all parental allergens. Moreover, prophylactic treatment with the hybrid proteins prevented pollen extract-induced allergic lung inflammation in vivo. CONCLUSION: The hybrid molecules showed a more efficient uptake and processing by dendritic cells resulting in a modified T cell response. The proteins had a lower IgE-binding capacity compared with the parental allergens, thus the high safety profile and increased efficacy emphasize clinical application for the treatment of Fagales multi-sensitization.

A hypoallergenic variant of the major birch pollen allergen shows distinct characteristics in antigen processing and T-cell activation.

BACKGROUND: BM4 is a novel genetically engineered variant of the major birch pollen allergen Bet v 1 that lacks the typical Bet v 1-like fold and displays negligible IgE-binding but strong T cell-activating capacity. The aim of this study was to elucidate possible differences between BM4 and Bet v 1 in internalization, antigen processing, and presentation. METHODS: Proliferative responses to BM4 and Bet v 1 of peripheral blood mononuclear cells and Bet v 1-specific T-cell clones were compared. Fluorescently labeled BM4 and Bet v 1 were used to study surface binding, endocytosis, and intracellular degradation by monocyte-derived DC (mdDC). Both proteins were digested by endolysosomal extracts of mdDC. BM4- and Bet v 1-pulsed mdDC were employed to assess the kinetics of activation of Bet v 1-specific T-cell clones and the polarization of naïve T cells. RESULTS: BM4 displayed a significantly stronger T cell-activating capacity than Bet v 1. Furthermore, BM4 showed increased surface binding and internalization as well as faster endolysosomal degradation compared with Bet v 1. BM4-pulsed mdDC induced enhanced proliferative responses at earlier time-points in Bet v 1-specific T-cell clones and promoted less IL-5 production in T cells than Bet v 1-pulsed mdDC. CONCLUSION: The loss of the Bet v 1-fold changes the protein's interaction with the human immune system at the level of antigen-presenting cells resulting in altered T-cell responses. By combining low IgE-binding with strong and modulating T cell-activating capacity, BM4 represents a highly interesting candidate for specific immunotherapy of birch pollen allergy.

Genetic allergen modification in the development of novel approaches to specific immunotherapy.

In the recent past, multiple allergens from relevant allergen sources have been cloned, sequenced and produced as recombinant proteins. The availability of recombinant allergens with immunological characteristics similar to their natural counterparts has improved the diagnosis of allergic disorders and increased our knowledge of the biochemical, structural and immunological characteristics of proteins with allergenic potential. Moreover, the use of defined recombinant proteins as vaccines substituting currently used total protein extracts from allergen sources may improve specific immunotherapy (SIT) of Type I allergy. In addition to producing well-defined batches of wild-type allergens, the recombinant technology offers the possibility to easily and selectively modify their properties or functions. Diverse modifications of allergens can be genetically engineered, e.g. variants with reduced IgE-binding capacity, multi-mers of single allergens or hybrids consisting of different allergens. Furthermore, allergens can be genetically fused with proteins that promote immune responses, which counterregulate the disease-eliciting T-helper type 2-dominated immune response in allergic individuals and may therefore, improve the efficacy of SIT. This review will introduce different concepts of allergen modification using genetic engineering to improve vaccines for SIT of Type I allergy.

Pollen-food syndromes associated with weed pollinosis: an update from the molecular point of view.

Pollinosis patients often display adverse reactions upon the ingestion of plant-derived foods as a result of immunoglobulin E (IgE) cross-reactive structures shared by pollen and food allergen sources. The symptoms of such pollen-food syndromes (PFS) or class 2 food allergies range from local oral allergy syndrome to severe systemic anaphylaxis. Two clinical syndromes, the celery-mugwort-spice syndrome and the mugwort-mustard-allergy syndrome have been described in association with weed pollinosis. However, other associations between weed pollinosis and hypersensitivity to certain kinds of food have also been observed, like the mugwort-peach, the ragweed-melon-banana, the plantain-melon, the pellitory-pistachio, the goosefoot-fruit, the Russian thistle-saffron, and the hop-celery association. The number of allergen sources involved, the allergens, and influencing factors including geography, diet, and food preparation contribute to the high clinical complexity of PFS. So far, known causative cross-reactive allergens include profilins, lipid transfer proteins, and high-molecular weight allergens and/or glycoallergens. The current usage of nonstandardized allergen extracts poses additional problems for both diagnosis and therapy of PFS patients. Further identification and characterization of involved allergens is inescapable for better understanding of PFS and vaccine development. Panels of recombinant allergens and/or hypo-allergens are promising tools to improve both PFS diagnostics and therapy.