Cow's milk allergy: from allergens to new forms of diagnosis, therapy and prevention.

The first adverse reactions to cow's milk were already described 2,000 years ago. However, it was only 50 years ago that several groups started with the analysis of cow's milk allergens. Meanwhile the spectrum of allergy eliciting proteins within cow's milk is identified and several cow's milk allergens have been characterized regarding their biochemical properties, fold and IgE binding epitopes. The diagnosis of cow's milk allergy is diverse ranging from fast and cheap in vitro assays to elaborate in vivo assays. Considerable effort was spent to improve the diagnosis from an extract-based into a component resolved concept. There is still no suitable therapy available against cow's milk allergy except avoidance. Therefore research needs to focus on the development of suitable and safe immunotherapies that do not elicit severe side effect.

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.

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.

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.

Cloning, expression, and mapping of allergenic determinants of alphaS1-casein, a major cow's milk allergen.

Milk is one of the first components introduced into human diet. It also represents one of the first allergen sources, which induces IgE-mediated allergies in childhood ranging from gastrointestinal, skin, and respiratory manifestations to severe life-threatening manifestations, such as anaphylaxis. Here we isolated a cDNA coding for a major cow's milk allergen, alphaS1-casein, from a bovine mammary gland cDNA library with allergic patients' IgE Abs. Recombinant alphaS1-casein was expressed in Escherichia coli, purified, and characterized by circular dichroism as a folded protein. IgE epitopes of alphaS1-casein were determined with recombinant fragments and synthetic peptides spanning the alphaS1-casein sequence using microarrayed components and sera from 66 cow's milk-sensitized patients. The allergenic activity of ralphaS1-casein and the alphaS1-casein-derived peptides was determined using rat basophil leukemia cells transfected with human FcepsilonRI, which had been loaded with the patients' serum IgE. Our results demonstrate that ralphaS1-casein as well as alphaS1-casein-derived peptides exhibit IgE reactivity, but mainly the intact ralphaS1-casein induced strong basophil degranulation. These results suggest that primarily intact alphaS1-casein or larger IgE-reactive portions thereof are responsible for IgE-mediated symptoms of food allergy. Recombinant alphaS1-casein as well as alphaS1-casein-derived peptides may be used in clinical studies to further explore pathomechanisms of food allergy as well as for the development of new diagnostic and therapeutic strategies for milk allergy.

Hypoallergenic derivatives of the major birch pollen allergen Bet v 1 obtained by rational sequence reassembly.

BACKGROUND: At least 100 million patients suffer from birch pollen allergy. OBJECTIVE: Rational design of recombinant derivatives of the major birch pollen allergen, Bet v 1, characterized by reduced IgE reactivity, preservation of sequences relevant for the induction of allergen-specific blocking IgG, and maintenance of T-cell epitopes for immunotherapy of birch pollen allergy. METHODS: Three recombinant mosaic proteins derived from Bet v 1 were generated by reassembly of codon-optimized genes coding for Bet v 1 fragments containing the elements for the induction of allergen-specific blocking IgG antibodies and the major T-cell epitopes. The proteins were expressed in Escherichia coli as recombinant mosaic molecules and compared with the Bet v 1 wild-type protein by chemical and structural methods, regarding IgE-binding and IgG-binding capacity, in basophil activation assays and tested for the in vivo induction of IgG responses. RESULTS: Three recombinant Bet v 1 (rBet v 1) mosaic proteins with strongly reduced IgE reactivity and allergenic activity were expressed and purified. Immunization with the recombinant hypoallergens induced IgG antibodies that inhibited IgE reactivity of patients with allergy to Bet v 1 comparable to those induced with the rBet v 1 wild-type allergen. CONCLUSION: We report the generation and preclinical characterization of 3 hypoallergenic rBet v 1 derivatives with suitable properties for immunotherapy of birch 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.

Efficacy of recombinant birch pollen vaccine for the treatment of birch-allergic rhinoconjunctivitis.

BACKGROUND: Recombinant DNA technology has the potential to produce allergen-specific immunotherapy vaccines with defined composition. OBJECTIVE: To evaluate the effectiveness of a new recombinant birch pollen allergen vaccine in patients with birch pollen allergy. METHODS: A multicenter, randomized, double-blind, placebo-controlled trial was undertaken to compare the following 3 vaccines in 134 adults with birch pollen allergy: recombinant birch pollen allergen vaccine (rBet v 1a), licensed birch pollen extract, natural purified birch pollen allergen (nBet v 1), and placebo. Patients received 12 weekly injections followed by monthly injections of the maintenance dose containing 15 microg Bet v 1 for 2 years. RESULTS: Significant reductions (about 50%) in rhinoconjunctivitis symptoms (rBet v 1, P = .0002; nBet v 1, P = .0006; birch extract, P = .0024), rescue medication (rBet v 1, P = .0011; nBet v 1, P = .0025; birch extract, P = .0063), and skin sensitivities (P < .0001) were observed in the 3 actively treated groups compared with placebo during 2 consecutive pollen seasons. Clinical improvement was accompanied by marked increases in Bet v 1-specific IgG levels, which were higher in the rBet v 1-treated group than in the birch and nBet v 1-treated groups. New IgE specificities were induced in 3 of 29 patients treated with birch pollen extract, but in none of the 32 rBet v 1-treated or 29 nBet v 1-treated patients. No severe systemic adverse events were observed in the rBet v 1-treated group. CONCLUSION: The rBet v 1-based vaccine was safe and effective in treating birch pollen allergy, and induced a highly specific immune response.

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.

Mimotopes identify conformational epitopes on parvalbumin, the major fish allergen.

Parvalbumin, the major fish allergen, is recognized by allergen-specific IgE of more than 90% of all fish-allergic patients. A detailed knowledge of allergenic structures is crucial for developing a vaccine inducing blocking antibodies specifically directed towards the IgE binding epitopes. In the present study we aimed to use the phage display technique to generate mimotopes, which mimic epitopes on parvalbumin. Parvalbumin-specific IgE was purified from sera of fish-allergic patients and used for screening of a constrained decamer phage library. After four rounds of biopanning using parvalbumin-specific IgE, five phage clones were selected which were specifically recognized by parvalbumin-specific IgE as well as IgG. DNA sequencing and peptide alignment revealed a high degree of sequence similarities between the mimotopes. Interestingly, on the surface of natural parvalbumin three regions could be defined by computational mimotope matching. In accordance, previously defined allergenic peptides of cod parvalbumin highlighted areas in close proximity or overlapping with the mimotope matching sites. From the presented data we conclude that our approach identified conformational epitopes of parvalbumin relevant for IgE and IgG binding. We suggest that these mimotopes are suitable candidates for an epitope-specific immunotherapy of fish-allergic patients.

Clustering of conformational IgE epitopes on the major dog allergen Can f 1.

Immunoglobulin E (IgE)-associated allergy affects more than 25% of the population. Can f 1 is the major dog allergen associated with respiratory symptoms but the epitopes recognized by allergic patients IgE on Can f 1 are unknown. To characterize IgE epitopes of Can f 1 recognized by dog allergic patients, six overlapping peptides spanning the Can f 1 sequence were synthesized. In direct IgE epitope mapping experiments peptides were analyzed for IgE reactivity by dot blot and Enzyme-linked immunosorbent assay (ELISA) with sera from dog allergic patients. For indirect epitope-mapping, rabbits were immunized with the peptides to generate specific IgG antibodies which were used to inhibit allergic patients' IgE binding to Can f 1. IgE binding sites were visualized on a model of the Can f 1 three-dimensional structure. We found that Can f 1 does not contain any relevant sequential IgE epitopes. However, IgE inhibition experiments with anti-peptide specific IgGs showed that Can f 1 N- and C-terminal portion assembled a major conformational binding site. In conclusion, our study is the first to identify the major IgE epitope-containing area of the dog allergen Can f 1. This finding is important for the development of allergen-specific treatment strategies.

Affinity determinations of purified IgE and IgG antibodies against the major pollen allergens Phl p 5a and Bet v 1a: discrepancy between IgE and IgG binding strength.

Allergen-specific IgE and IgG antibodies coexist in allergic individuals, but only IgE has anaphylactogenic capacity. This study aimed to determine the association, dissociation and equilibrium constants for the interaction of allergen-specific IgE and IgG with the major grass and birch pollen allergens Phl p 5a and Bet v 1a. We isolated specific IgE and IgG antibodies from pollen allergic patients' sera by a two-step affinity chromatography protocol and controlled the high purity in a recombinant allergen chip microarray. Surface plasmon resonance measurements of polyclonal IgE and IgG species revealed that their affinities diverge widely, being in the range of 10(-10) and 10(-11) M for IgE, but only 10(-6)-10(-7) M for IgG. Moreover, murine monoclonal IgG1 antibodies against the allergens showed affinities of 10(-7)-10(-8) M. Thus, we conclude from our data that even stringently affinity matured IgG cannot score the superior affinity of IgE antibodies to allergens.

Microarrayed allergen molecules: diagnostic gatekeepers for allergy treatment.

Type I allergy is an immunoglobulin E (IgE)-mediated hypersensitivity disease affecting more than 25% of the population. Currently, diagnosis of allergy is performed by provocation testing and IgE serology using allergen extracts. This process defines allergen-containing sources but cannot identify the disease-eliciting allergenic molecules. We have applied microarray technology to develop a miniaturized allergy test containing 94 purified allergen molecules that represent the most common allergen sources. The allergen microarray allows the determination and monitoring of allergic patients' IgE reactivity profiles to large numbers of disease-causing allergens by using single measurements and minute amounts of serum. This method may change established practice in allergy diagnosis, prevention, and therapy. In addition, microarrayed antigens may be applied to the diagnosis of autoimmune and infectious diseases.

Effects of nasal corticosteroids on boosts of systemic allergen-specific IgE production induced by nasal allergen exposure.

BACKGROUND: Allergen exposure via the respiratory tract and in particular via the nasal mucosa boosts systemic allergen-specific IgE production. Intranasal corticosteroids (INCS) represent a first line treatment of allergic rhinitis but their effects on this boost of allergen-specific IgE production are unclear. AIM: Here we aimed to determine in a double-blind, placebo-controlled study whether therapeutic doses of an INCS preparation, i.e., nasal fluticasone propionate, have effects on boosts of allergen-specific IgE following nasal allergen exposure. METHODS: Subjects (n = 48) suffering from grass and birch pollen allergy were treated with daily fluticasone propionate or placebo nasal spray for four weeks. After two weeks of treatment, subjects underwent nasal provocation with either birch pollen allergen Bet v 1 or grass pollen allergen Phl p 5. Bet v 1 and Phl p 5-specific IgE, IgG1-4, IgM and IgA levels were measured in serum samples obtained at the time of provocation and one, two, four, six and eight weeks thereafter. RESULTS: Nasal allergen provocation induced a median increase to 141.1% of serum IgE levels to allergens used for provocation but not to control allergens 4 weeks after provocation. There were no significant differences regarding the boosts of allergen-specific IgE between INCS- and placebo-treated subjects. CONCLUSION: In conclusion, the application of fluticasone propionate had no significant effects on the boosts of systemic allergen-specific IgE production following nasal allergen exposure. TRIAL REGISTRATION: http://clinicaltrials.gov/NCT00755066.

Characterization of a novel isoform of alpha-nascent polypeptide-associated complex as IgE-defined autoantigen.

The nascent polypeptide-associated complex is required for intracellular translocation of newly synthesized polypeptides in eukaryotic cells. It may also act as a transcriptional coactivator in humans and various eukaryotic organisms and binds to nucleic acids. Recently, we provided evidence that a component of nascent polypeptide-associated complex, alpha-nascent polypeptide-associated complex, represents an IgE-reactive autoantigen for atopic dermatitis patients. By oligonucleotide screening we isolated a complete cDNA coding for a so far unknown alpha-nascent polypeptide-associated complex isoform from a human epithelial cDNA library. Southern blot hybridization experiments provided further evidence that alpha-nascent polypeptide-associated complex is encoded by a gene family. Recombinant alpha-nascent polypeptide-associated complex was expressed in Escherichia coli as a soluble, His-tagged protein, and purified via nickel affinity chromatography. By circular dichroism analysis it is demonstrated that purified recombinant alpha-nascent polypeptide-associated complex represents a folded protein of mixed alpha-helical and beta-sheet conformation with unusual high thermal stability and remarkable refolding capacity. Complete recombinant alpha-nascent polypeptide-associated complex (215 amino acids) and its 86 amino acid C-terminal fragment specifically bound IgE autoantibodies. Recombinant alpha-nascent polypeptide-associated complex also inhibited IgE binding to natural alpha-nascent polypeptide-associated complex, demonstrating the presence of common IgE epitopes between the recombinant and natural protein. Furthermore, recombinant alpha-nascent polypeptide-associated complex induced specific lymphoproliferative responses in peripheral blood mononuclear cells of a sensitized atopic dermatitis patient. As has been proposed for environmental allergens it is possible that T cell responses to IgE-defined autoantigens may contribute to the chronic skin manifestations in atopic dermatitis.

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.