M. Focke

Heterogeneity of commercial timothy grass pollen extracts

Background The diagnosis and specific immunotherapy of allergy is currently performed with allergen extracts prepared from natural allergen sources.

Developments in allergen-specific immunotherapy: from allergen extracts to allergy vaccines bypassing allergen-specific immunoglobulin E and T cell reactivity

Allergen‐specific immunotherapy (SIT) is the only specific and disease‐modifying approach for the treatment of allergy but several disadvantages have limited its broad applicability. We argue that the majority of the possible disadvantages of SIT such as unwanted effects, poor efficacy and specificity as well as inconvenient application are related to the poor quality of natural allergen extracts, which are the active ingredients of all currently available allergy vaccines. Because of the progress made in the field of molecular allergen characterization, new allergy vaccines based on recombinant allergens, recombinant hypoallergenic allergen derivatives and allergen‐derived T cell peptides have entered clinical testing and hold promise to reduce the side‐effects and to increase the specificity as well as the efficacy of SIT. Here, we present a refined immunotherapy concept, which is based on the use of peptides derived from allergen surfaces that exhibit reduced, allergen‐specific IgE as well as T cell reactivity. These peptides when fused to non‐allergenic carriers give rise to allergen‐specific protective IgG responses with T cell help from a non‐allergenic carrier molecule. We summarize the experimental data demonstrating that such peptide vaccines can bypass allergen‐specific IgE as well as T cell activation and may be administered at high doses without IgE‐ and T cell‐mediated side‐effects. Should these peptide vaccines prove efficacious and safe in clinical trials, it may become possible to develop convenient, safe and broadly applicable forms of SIT as true alternatives to symptomatic, drug‐based allergy treatment.

Non-anaphylactic surface-exposed peptides of the major birch pollen allergen, Bet v 1, for preventive vaccination

Background Almost 100 million allergic patients are sensitized to the major birch pollen allergen, Bet v 1, a 17 kDa protein containing most of the IgE epitopes present in pollens of trees belonging to the Fagales order and plant‐derived food.

Molecular composition and biological activity of commercial birch pollen allergen extracts

Background  Commercial extracts used for diagnosis and treatment of allergy are currently prepared from natural allergen sources. The aim of this study was to analyse birch pollen allergen extracts produced for in vivo diagnosis of birch pollen allergy regarding their contents of individual birch pollen allergens (Bet v 1, Bet v 2 and Bet v 4).

Skin prick test extracts for dog allergy diagnosis show considerable variations regarding the content of major and minor dog allergens.

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.

Analysis of Epitope-Specific Immune Responses Induced by Vaccination with Structurally Folded and Unfolded Recombinant Bet v 1 Allergen Derivatives in Man

Previously, we have constructed recombinant derivatives of the major birch pollen allergen, Bet v 1, with a more than 100-fold reduced ability to induce IgE-mediated allergic reactions. These derivatives differed from each other because the two recombinant Bet v 1 fragments represented unfolded molecules whereas the recombinant trimer resembled most of the structural fold of the Bet v 1 allergen. In this study, we analyzed the Ab (IgE, IgG subclass, IgA, IgM) response to Bet v 1, recombinant and synthetic Bet v 1-derived peptides in birch pollen allergic patients who had been vaccinated with the derivatives or adjuvant alone. Furthermore, we studied the induction of IgE-mediated skin responses in these patients using Bet v 1 and Bet v 1 fragments. Both types of vaccines induced a comparable IgG1 and IgG4 response against new sequential epitopes which overlap with the conformational IgE epitopes of Bet v 1. This response was 4- to 5-fold higher than that induced by immunotherapy with birch pollen extract. Trimer more than fragments induced also IgE responses against new epitopes and a transient increase in skin sensitivity to the fragments at the beginning of therapy. However, skin reactions to Bet v 1 tended to decrease one year after treatment in both actively treated groups. We demonstrate that vaccination with folded and unfolded recombinant allergen derivatives induces IgG Abs against new epitopes. These data may be important for the development of therapeutic as well as prophylactic vaccines based on recombinant allergens.

Carrier‐bound Alt a 1 peptides without allergenic activity for vaccination against Alternaria alternata allergy

The mould Alternaria alternata is a major elicitor of allergic asthma. Diagnosis and specific immunotherapy (SIT) of Alternaria allergy are often limited by the insufficient quality of natural mould extracts.

Identification of Allergens in Oilseed Rape (Brassica napus) Pollen

Background: Pollen from oilseed rape (OSR), Brassica napus, an increasingly cultivated oilplant from the Brassicaceae, has been recognized as a potential cause of allergic sensitization. Allergens have been hardly investigated. Methods: We characterized IgE binding proteins in OSR pollen by immunoblot, immunoblot inhibition and specific monoclonal antibodies using sera from 89 patients sensitized to OSR. Results: Two low–molecular–weight allergens of 6/8 kD and 14 kD as well as a high molecular–weight cluster (27–69 kD) comprising six cross–reactive peptides could be identified. The three allergens were recognized by 50, 34 and 80% of patients, respectively. Immunoblot IgE binding to OSR could be totally inhibited by rye pollen and moderately by birch pollen (6/8 and 14 kD) while mugwort had little effect. An anti–profilin–specific monoclonal antibody bound specifically to a 14–kD protein in OSR. Binding to the 6/8–kD rape allergen could be effectively inhibited by rAln g 2, a calcium–binding protein from alder. Periodate treatment led to a significant reduction in IgE binding to the 27 to 69–kD OSR allergens indicating that carbohydrate determinants are involved in IgE binding. OSR proteins were capable to quench IgE binding to timothy grass pollen proteins of ≥60 kD suggesting that grass pollen group 4 allergens cross–react with the 27 to 69–kD cluster in OSR. Conclusions: The data demonstrate that OSR pollen is allergenic and indicate that the identified allergens represent cross–reacting homologues of well–known pollen allergens, i.e. calcium–binding proteins, profilins, and high–molecular–weight glycoproteins. Via cross–reactivity, exposure to OSR pollen may be a prolonging and aggravating factor in underlying birch and grass pollen allergy.

Cross-reactivity between Ficus benjamina latex and fig fruit in patients with clinical fig allergy.

Background  Anaphylactic reactions to fig fruits (Ficus carica) have been reported from subjects sensitized to Ficus benjamina (FB) latex allergens. Figs may also be involved in the latex–fruit syndrome.

Disruption of Allergenic Activity of the Major Grass Pollen Allergen Phl p 2 by Reassembly as a Mosaic Protein

The recognition of conformational epitopes on respiratory allergens by IgE Abs is a key event in allergic inflammation. We report a molecular strategy for the conversion of allergens into vaccines with reduced allergenic activity, which is based on the reassembly of non-IgE-reactive fragments in the form of mosaic proteins. This evolution process is exemplified for timothy grass pollen-derived Phl p 2, a major allergen for more than 200 million allergic patients. In a first step, the allergen was disrupted into peptide fragments lacking IgE reactivity. cDNAs coding for these peptides were reassembled in altered order and expressed as a recombinant mosaic molecule. The mosaic molecule had lost the three-dimensional structure, the IgE reactivity, and allergenic activity of the wild-type allergen, but it induced high levels of allergen-specific IgG Abs upon immunization. These IgG Abs crossreacted with group 2 allergens from other grass species and inhibited allergic patients’ IgE binding to the wild-type allergen. The mosaic strategy is a general strategy for the reduction of allergenic activity of protein allergens and can be used to convert harmful allergens into safe vaccines.