Characterization of mutants of a highly cross-reactive calcium-binding protein from Brassica pollen for allergen-specific immunotherapy.

The major turnip (Brassica rapa) pollen allergen, belongs to a family of calcium-binding proteins (i.e., two EF-hand proteins), which occur as highly cross-reactive allergens in pollen of weeds, grasses and trees. In this study, the IgE binding capacity and allergenic activity of three recombinant allergen variants containing mutations in their calcium-binding sites were analyzed in sensitized patients with the aim to identify the most suitable hypoallergenic molecule for specific immunotherapy. Analysis of the wildtype allergen and the mutants regarding IgE reactivity and activation of basophils in allergic patients indicated that the allergen derivative mutated in both calcium-binding domains had the lowest allergenic activity. Gel filtration and circular dichroism experiments showed that both, the wildtype and the double mutant, occurred as dimers in solution and assumed alpha-helical fold, respectively. However, both fold and thermal stability were considerably reduced in the double mutant. The use of bioinformatic tools for evaluation of the solvent accessibility and charge distribution suggested that the reduced IgE reactivity and different structural properties of the double mutant may be due to a loss of negatively charged amino acids on the surface. Interestingly, immunization of rabbits showed that only the double mutant but not the wildtype allergen induced IgG antibodies which recognized the allergen and blocked binding of allergic patients IgE. Due to the extensive structural similarity and cross-reactivity between calcium-binding pollen allergens the hypoallergenic double mutant may be useful not only for immunotherapy of turnip pollen allergy, but also for the treatment of allergies to other two EF-hand pollen allergens.

Staphylococcus aureus fibronectin-binding protein specifically binds IgE from patients with atopic dermatitis and requires antigen presentation for cellular immune responses.

BACKGROUND: Staphylococcus aureus superinfections occur in more than 90% of patients with atopic dermatitis (AD) and aggravate skin inflammation. S aureus toxins lead to tissue damage and augment T-cell-mediated skin inflammation by a superantigen effect. OBJECTIVE: To characterize IgE-reactive proteins from S aureus. METHODS: A genomic S aureus library was screened with IgE from patients with AD for DNA clones coding for IgE-reactive antigens. One was identified as fibronectin-binding protein (FBP). Recombinant FBP was expressed in Escherichia coli, purified, and tested for specific IgE reactivity in patients with AD. Its allergenic activity was studied in basophil activation experiments and T-cell cultures. The in vivo allergenic activity was investigated by sensitizing mice. RESULTS: Using IgE from patients with AD for screening of a genomic S aureus library, an IgE-reactive DNA clone was isolated that coded for FBP. Recombinant FBP was expressed in E coli and purified. It reacted specifically with IgE from patients with AD and exhibited allergenic activity in basophil degranulation assays. FBP showed specific T-cell reactivity requiring antigen presentation and induced the secretion of proinflammatory cytokines from PBMCs. Mice sensitized with FBP mounted FBP-specific IgE responses, showed FBP-specific basophil degranulation as well as FBP-specific T-cell proliferation, and mixed T(h)2/T(h)1 cytokine secretion. CONCLUSION: Evidence is provided that specific humoral and cellular immune responses to S aureus antigens dependent on antigen presentation represent a novel mechanism for S aureus-induced skin inflammation in AD. Furthermore, FBP may be used for the development of novel diagnostic and therapeutic strategies for S aureus infections.

Carrier-bound, nonallergenic Ole e 1 peptides for vaccination against olive pollen allergy.

BACKGROUND: Trees of the family Oleaceae (olive and ash) are important allergen sources in Mediterranean countries, Northern and Central Europe, and North America. The major olive pollen allergen Ole e 1 represents the majority of allergenic epitopes in olive pollen and cross-reacts with Fra e 1, the major ash pollen allergen. OBJECTIVE: We sought to develop a safe vaccine for the treatment of Oleaceae pollen allergy. METHODS: We synthesized 5 peptides ranging from 32 to 36 amino acids, which covered the whole sequence of Ole e 1. The IgE and T-cell reactivity of the peptides was compared with that of Ole e 1 by means of dot blot experiments, as well as ELISA, and in proliferation assays. Rabbits were immunized with non-IgE-reactive, keyhole limpet hemocyanin-coupled peptides or Ole e 1. The reactivity of the IgG antibodies with Ole e 1 and their ability to inhibit IgE binding to nOle e 1 was evaluated by means of ELISA. RESULTS: Only the C-terminal Ole e 1 peptide showed IgE binding, whereas the other peptides were nonallergenic. Immunization of rabbits with Ole e 1-derived peptides bound to the carrier molecule keyhole limpet hemocyanin induced in rabbits the production of Ole e 1-specific IgG antibodies, which cross-reacted with Fra e 1, and inhibited olive and ash pollen-sensitized patients' IgE binding to Ole e 1. CONCLUSION: Two non-IgE-binding peptides with low T-cell reactivity from the N-terminus of Ole e 1 were identified that might represent safe vaccine candidates for immunotherapy of Oleaceae pollen allergy.

Visualization of clustered IgE epitopes on alpha-lactalbumin.

BACKGROUND: alpha-Lactalbumin (alpha-La) is a major cow's milk (CM) allergen responsible for allergic reactions in infants. OBJECTIVE: We performed molecular, structural, and immunologic characterization of alpha-La. METHODS: Recombinant alpha-lactalbumin (ralpha-La) was expressed in Escherichia coli, purified to homogeneity, and characterized by means of mass spectrometry and circular dichroism, and its allergenic activity was studied by using microarray technology, as well as in a basophil histamine release assay. IgE epitope mapping was performed with synthetic peptides. RESULTS: According to circular dichroism analysis, ralpha-La represented a folded protein with a high thermal stability and refolding capacity. ralpha-La reacted with IgE antibodies from 57.6% of patients with CM allergy (n = 66) and induced the strongest basophil degranulation with sera from patients with CM allergy who had exhibited gastrointestinal symptoms or severe systemic reactions on CM exposure. ralpha-La contained sequential and conformational IgE epitopes. Superposition of IgE-reactive peptides onto the 3-dimensional structure of alpha-La revealed a close vicinity of the N- and C-terminal peptides within a surface-exposed patch. CONCLUSIONS: ralpha-La can be used for the diagnosis of patients with severe allergic reactions to CM and serves as a paradigmatic tool for the development of therapeutic strategies for CM allergy.

Mast cell-derived proteases control allergic inflammation through cleavage of IgE.

BACKGROUND: Cross-linking of mast cell-bound IgE releases proinflammatory mediators, cytokines, and proteolytic enzymes and is a key event in allergic inflammation. OBJECTIVE: We sought to study the effect of proteases released on effector cell activation on receptor-bound IgE and their possible role in the regulation of allergic inflammation. METHODS: Using molar ratios of purified recombinant tryptase and human IgE, we studied whether tryptase can cleave IgE. Similar experiments were performed with mast cell lysates in the presence or absence of protease inhibitors. IgE cleavage products were detected in supernatants of allergen cross-linked, cultivated mast cells and in tissue fluids collected from patients' skin after IgE-mediated degranulation. The effects of protamine, an inhibitor of heparin-dependent proteases on IgE-mediated allergic in vivo skin inflammation in human subjects were studied. RESULTS: We show that beta-tryptase, a major protease released during mast cell activation, cleaves IgE. IgE degradation products were detected in tryptase-containing tissue fluids collected from sites of allergic inflammation. The biologic significance of this mechanism is demonstrated by in vivo experiments showing that protease inhibition enhances allergic skin inflammation. CONCLUSION: We suggest that IgE cleavage by effector cell proteases is a natural mechanism for controlling allergic inflammation.

Exposure to a farming environment has allergen-specific protective effects on TH2-dependent isotype switching in response to common inhalants.

BACKGROUND: IgE synthesis by human B cells results from allergen-dependent, T(H)2-mediated isotype switching. Exposure to a farming environment protects against IgE responses. OBJECTIVE: We reconstructed allergen-dependent switching patterns in vivo to identify the level or levels at which farm exposure acts to protect against atopy. METHODS: Serum IgG1 to IgG4 and IgE to grass (rPhl p 1 and rPhl p 5), cat (rFel d 1), and mite (rDer p 2) were assessed by means of ELISA in the Allergy and Endotoxin study population (812 children). Farm exposure was defined as currently living on a farm, exposure to stables/farm milk in the first year of life, or both. RESULTS: Farm exposure did not affect allergen-specific IgG2 and IgG3 levels but had complex allergen-specific effects on IgG1, IgG4, and IgE levels. Exposure protected against grass-specific responses at every step along the IgG1/IgG4/IgE switching pathway but had no significant effect on mite responses. Protection from cat responses was concentrated at the IgG1 level. For all allergens, failure to express IgG1 was associated with low prevalence of IgG4 or IgE responses. Notably, coexpression of IgG1, IgG4, and IgE to grass was associated with increased risk of allergic disease and higher IgE levels compared with production of IgG1 and IgE without IgG4, suggesting IgG4 coexpression marks stronger activation of T(H)2-dependent events. CONCLUSION: The protective effects of farm exposure were confined to T(H)2-dependent IgG1, IgG4, and IgE expression and were allergen and switch stage specific, suggesting that distinct mechanisms regulate individual steps within allergen-induced class switching in vivo. CLINICAL IMPLICATIONS: Environmental interventions to prevent IgE expression might need to be tailored to specific allergens.

Allergen cleavage by effector cell-derived proteases regulates allergic inflammation.

The key event of allergic inflammation, allergen-induced crosslinking of mast cell-bound IgE antibodies, is accompanied by release of inflammatory mediators, cytokines, and proteases, in particular beta-tryptase. We provide evidence that protease-mediated cleavage of allergens represents a mechanism that regulates allergen-induced mast cell activation. When used in molar ratios as they occur in vivo, purified beta-tryptase cleaved major grass and birch pollen allergens, resulting in defined peptide fragments as mapped by mass spectrometry. Tryptase-cleaved allergens showed reduced IgE reactivity and allergenic activity. The biological relevance is demonstrated by the fact that lysates from activated human mast cells containing tryptase levels as they occur in vivo cleaved allergens. Additionally, protamine, an inhibitor of heparin-dependent effector cell proteases, augmented allergen-induced release of mediators from effector cells. Protease-mediated allergen cleavage may represent an important mechanism for terminating allergen-induced effector cell activation.

Molecular characterization of polygalacturonases as grass pollen-specific marker allergens: expulsion from pollen via submicronic respirable particles.

Grass pollen belong to the most important allergen sources involved in the elicitation of allergic asthma. We have isolated cDNAs coding for Bermuda grass (Cynodon dactylon) and timothy grass (Phleum pratense) pollen allergens, belonging to a family of pectin-degrading enzymes (i.e., polygalacturonases). The corresponding allergens, termed Cyn d 13 and Phl p 13, represent glycoproteins of approximately 42 kDa and isoelectric points of 7.5. rPhl p 13 was expressed in Escherichia coli and purified to homogeneity. Immunogold electron microscopy using rabbit anti-rPhl p 13 Abs demonstrated that in dry pollen group 13, allergens represent primarily intracellular proteins, whereas exposure of pollen to rainwater caused a massive release of cytoplasmic material containing submicronic particles of respirable size, which were coated with group 13 allergens. The latter may explain respiratory sensitization to group 13 allergens and represents a possible pathomechanism in the induction of asthma attacks after heavy rainfalls. rPhl p 13 was recognized by 36% of grass pollen allergic patients, showed IgE binding capacity comparable to natural Phl p 13, and induced specific and dose-dependent basophil histamine release. Epitope mapping studies localized major IgE epitopes to the C terminus of the molecule outside the highly conserved functional polygalacturonase domains. The latter result explains why rPhl p 13 contains grass pollen-specific IgE epitopes and may be used to diagnose genuine sensitization to grass pollen. Our finding that rabbit anti-rPhl p 13 Abs blocked patients' IgE binding to the allergen suggests that rPhl p 13 may be used for immunotherapy of sensitized patients.

Microarrayed allergens for IgE profiling.

Diagnosis of type I allergy is based on anamnesis, provocation testing, and serological determination of total and specific IgE. Currently, in vivo and in vitro diagnostic tests employ allergen extracts prepared from various allergen sources (e.g., pollen, mites, animal dander, moulds, foods, venoms, etc.). The application of recombinant DNA technology to the field of allergen characterization has allowed to reveal the molecular nature of the most common allergens. To date a continuously increasing number of allergen sequences has become available and panels of recombinant allergens-assembling the epitope complexity of natural allergens sources-can be produced. The use of recombinant allergens instead of crude, natural extracts for allergy diagnosis allows us to determine the individual IgE reactivity profile of each patient. To enable a comprehensive analysis of the patient's IgE binding pattern to a large number of individual allergens, a new type of serological test is required. In this paper, we applied microarray technology to create a multi-allergen test system, based on microarrayed recombinant allergens.