Dietrich Kraft

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.

Identification of allergens in fruits and vegetables: IgE cross-reactivities with the important birch pollen allergens Bet v 1 and Bet v 2 (birch profilin)

BACKGROUND In this study serum samples collected from 20 patients with birch pollen allergy were investigated. All patients had experienced allergic symptoms after contact with or ingestion of particular fresh fruits and vegetables known as birch pollen-related foods. METHODS Serum samples were tested by means of immunoblotting for IgE reactivities with proteins in extracts of birch pollen, apple, pear, celery, carrot, and potato. Anti-Bet v 1 and anti-Bet v 2 antibodies were used to investigate cross-reactivity. Inhibition studies were performed by preincubation of sera with recombinant Bet v 1 and Bet v 2. RESULTS IgE binding to proteins, corresponding to the major birch pollen allergen Bet v 1 and to Bet v 2 (birch pollen profilin) could be observed. An allergen homologous to Bet v 1 could be detected in apple, pear, and celery when a Bet v 1-specific monoclonal antibody was used. Testing a polyclonal rabbit anti-Bet v 2 antibody with extracts of the respective plants revealed the presence of profilins in every source tested. Inhibition with recombinant Bet v 1 and Bet v 2 led to complete blocking or marked reduction of IgE binding to proteins of comparable molecular weights in the respective food extracts, indicating IgE cross-reactivity. CONCLUSION Our results indicate that many plant-derived food agents contain proteins with high homology to the birch pollen allergens Bet v 1 and Bet v 2 and must therefore be considered as potentially threatening for patients with tree pollen allergy.

Modulation of IgE reactivity of allergens by site-directed mutagenesis: potential use of hypoallergenic variants for immunotherapy

Specific immunotherapy is an efficient treatment for patients suffering from type I allergy. The mechanisms underlying successful immunotherapy are assumed to operate at the level of T helper cells, leading to a modulation of the immune response to allergens. During immunotherapy, increasing doses of allergens are given on a regular basis, and the beneficial effects for the patient depend on the concentration of allergen used. On the other hand, the risk of IgE‐mediated anaphylactic side effects also increase with the amount of allergen applied per injection. Therefore, we have proposed the use of hypoallergenic (low IgE binding activity) forms of allergens for immunotherapy. We evaluated by site‐directed mutagenesis the contributions of individual amino acid residues/positions for IgE binding to Bet v 1, the major allergen of birch pollen. We found that IgE binding to Bet v 1 depended on at least six amino acid residues/positions. Immunoblot analyses and inhibition experiments showed that the multiple‐point Bet v 1 mutant exhibited extremely low reactivity with serum IgE from birch pollen‐allergic patients. In vivo (skin prick) tests showed that the potency of the multiple‐point mutant to induce typical urticarial type I reactions in pollen‐allergic patients was significantly lower than for wild‐type Bet v 1. Proliferation assays of allergen‐specific T cell clones demonstrated that these six amino acid exchanges in the Bet v 1 sequence did not influence T cell recognition. Thus, the Bet v 1 six‐point mutant displayed significantly reduced IgE binding activity, but conserved T cell activating capacity, which is necessary for immunomodulation. The approach described here may be generally applied to produce allergen variants to be used in a safe therapy form of immediate‐type allergies.—Ferreira, F., Ebner, C., Kramer, B., Casari, G., Briza, P., Kungl, A. J., Grimm, R., Jahn‐Schmid, B., Breiteneder, H., Kraft, D., Breitenbach, M., Rheinberger, H.‐J., Scheiner, O. Modulation of IgE reactivity of allergens by site‐directed mutagenesis: potential use of hypoallergenic variants for immunotherapy. FASEB J. 12, 231–242 (1998)

Common epitopes of birch pollen and apples--studies by western and northern blot.

Eighty-three sera from patients with birch-pollen allergy were investigated for IgE antibodies against apple allergens by means of immunoblotting. In immunoblots, 81 patients (97.6%) exhibited IgE directed against the major allergen of birch, Bet v I (17 kd), and these patients also demonstrated IgE binding to apple allergens in the molecular weight range 17 to 18 kd. Inhibition studies by preincubation of sera with birch-pollen extract led to complete blocking of IgE binding to this 17 to 18 kd protein, whereas preincubation with apple extract could not diminish IgE binding to Bet V I. Furthermore, a 17 kd protein in apple extract could be detected by immunoblotting with a Bet v I-specific monoclonal antibody. Northern blotting with a Bet v I cDNA clone as a probe revealed cross-hybridization of birch and apple allergen coding nucleic acids under conditions of high stringency, suggesting significant homology of the nucleic acid level. Our results support the concept that antigens in birch pollen and apples share allergenic epitopes leading to IgE cross-reactivities that may cause clinical manifestations when a special threshold level of specific IgE antibodies is reached.

Allergen-specific immunotherapy with a monophosphoryl lipid A-adjuvanted vaccine: reduced seasonally boosted immunoglobulin E production and inhibition of basophil histamine release by therapy-induced blocking antibodies.

Background Allergen‐specific immunotherapy represents a causal form of treatment for IgE‐mediated allergies. The allergen extract‐based analyses of immunotherapy‐induced effects yielded highly controversial results regarding a beneficial role of therapy‐induced IgG antibodies.

Conversion of the major birch pollen allergen, Bet v 1, into two nonanaphylactic T cell epitope-containing fragments: candidates for a novel form of specific immunotherapy.

A novel approach to reduce the anaphylactic activity of allergens is suggested. The strategy makes use of the presence of conformational immunoglobulin E (IgE) epitopes on one of the most common allergens. The three dimensional structure of the major birch pollen allergen, Bet v 1, was disrupted by expressing two parts of the Bet v 1 cDNA representing amino acids 1-74 and 75-160 in Escherichia coli. In contrast to the complete recombinant Bet v 1, the fragments showed almost no allergenicity and exhibited random coil conformation as analyzed by circular dichroism. Both nonanaphylactic fragments induced proliferation of human Bet v 1-specific T cell clones, indicating that they harbored all dominant T cell epitopes and therefore may be considered as a basis for the development of a safe and specific T cell immunotherapy.

Skin testing with recombinant allergens rBet v 1 and birch profilin, rBet v 2: Diagnostic value for birch pollen and associated allergies

OBJECTIVE This study assesses the value of two recombinant birch allergens for diagnosis of patients sensitized to birch pollen with or without associated food allergy. METHODS Fifty-one patients with positive skin test responses to Betulaceae and seven nonallergic control subjects were investigated; specific IgE antibodies were evaluated by specific immunoassay and blot immunodetection. RESULTS Among 51 patients, 47 reacted to rBet v 1 and 10 to rBet v 2. Seven patients reacted to both recombinant allergens. In skin prick tests we found a correlation between the wheal produced by the commercial birch extract and the wheal produced by rBet v 1. Among 47 patients with positive test responses to rBet v 1, 83% had IgE binding to the Bet v 1 protein as determined by immunoblotting. Among 10 patients sensitized to rBet v 2, six had IgE binding to Bet v 2. Eleven patients with negative results, as determined by immunoblotting, had low levels of birch IgE in the sera (less than 10 kU/L) and low concentrations of IgE to rBet v 1 or rBet v 2 in ELISA. The nonallergic control subjects (n = 7) did not react to rBet v 1 or rBet v 2 in skin prick tests, nor did they have detectable amounts of specific IgE to rBet v 1 or rBet v 2. Histamine release tests confirmed sensitization to Bet v 1 in two patients with discordant results; for Bet v 2, one patient had positive results only at a high concentration, and one had results that remained negative. Thirty-four patients had birch pollinosis, and all reacted to rBet v 1. Patients who were monosensitized to birch never reacted to rBet v 2. Sensitization to rBet v 2 was only found in patients who reacted to other pollens (mainly grass). Twenty-nine patients demonstrated allergy to apples, cherries, or hazelnuts; and all reacted to rBet v 1. Among 11 patients with allergy to Umbelliferae, only three reacted to rBet v 2. CONCLUSIONS Use of the two recombinant allergens (rBet v 1 and rBet v 2) always permits the diagnosis of birch sensitization. Sensitization to rBet v 1 is specific for birch and Rosaceae allergies, whereas sensitization to birch profilin, Bet v 2, is encountered in multisensitized subjects and is not always related to Umbelliferae allergy.

OLIGODEOXYNUCLEOTIDES CONTAINING CPG MOTIFS INDUCE IL-12, IL-18 AND IFN-GAMMA PRODUCTION IN CELLS FROM ALLERGIC INDIVIDUALS AND INHIBIT IGE SYNTHESIS IN VITRO

The effects of phosphorothioate oligonucleotides containing CpG motifs (CpG‐ODN) on cultured cells from allergic patients and non‐atopic individuals were investigated. In peripheral blood mononuclear cells (PBMC) CpG‐ODN led to a significant increase of IFN‐γ. By intracellular cytokine staining, IFN‐γ production could be attributed to NK cells and inhibition experiments indicated an IL‐12‐dependent mechanism. Moreover, CpG‐ODN increased mRNA expression of IL‐12 and IL‐18 in PBMC. In this respect, no significant difference between allergic and non‐atopic individuals was observed. Monocyte‐derived dendritic cells were identified as one IL‐12‐ and IL‐18‐producing source. In addition, stimulation of PBMC derived from atopic patients with CpG‐ODN led to a considerable increase of polyclonal IgG and IgM synthesis. In contrast, the production of total IgE was suppressed. CpG‐ODN induced a significant rise of IgG and IgM specific for allergens to which the patients were sensitized, whereas allergen‐specific IgE levels remained unchanged. Our data suggest that CpG‐ODN display a strong influence on the ongoing immune response and might represent potential adjuvants for specific immunotherapy of type I allergy.

Recombinant birch pollen allergens (rBet v 1 and rBet v 2) contain most of the IgE epitopes present in birch, alder, hornbeam, hazel, and oak pollen: A quantitative IgE inhibition study with sera from different populations ☆ ☆☆ ★ ★★

BACKGROUND Pollen from trees of the order Fagales are important allergen sources in most parts of the world. Clinical, immunochemical, and molecular biology studies indicate that they contain cross-reactive allergens. The major birch pollen allergen, Bet v 1, and birch profilin, Bet v 2, a highly cross-reactive allergen, have been cloned and expressed in Escherichia coli. OBJECTIVE The purpose of this study was to demonstrate the presence of allergens in Fagales pollens that share IgE epitopes with recombinant Bet v 1 and Bet v 2 and to determine the percentage of birch, alder, hornbeam, hazel, and oak pollen-specific IgE that can be preabsorbed with rBet v 1 and rBet v 2 from 102 sera of different populations of subjects allergic to Fagales tree pollen. METHODS The presence of rBet v 1- and rBet v 2-homologous allergens in tree pollen extracts was investigated by IgE immunoblot inhibition experiments, and the percentage of tree (birch, alder, hornbeam, hazel, and oak) pollen-specific IgE that was bound by a mixture of rBet v 1 and rBet v 2 was determined by RAST-based quantitative IgE inhibition experiments. The clinical significance of IgE antibody cross-reactivity was studied by skin prick testing with rBet v 1, rBet v 2, and Fagales pollen extracts. RESULTS Natural birch, alder, hornbeam, hazel, and oak pollen contain allergens that share IgE epitopes with rBet v 1 and rBet v 2. A combination of rBet v 1 and rBet v 2 accounted for 82% of tree pollen-specific IgE on average. Most of the tree pollen-specific IgE was directed against rBet v 1. CONCLUSION rBet v 1 and rBet v 2 contain most of the Fagales pollen-specific IgE epitopes and may therefore substitute natural tree pollen extracts not only for diagnosis but also for patient-tailored immunotherapy of tree pollen allergy.

Quantitative IgE inhibition experiments with purified recombinant allergens indicate pollen-derived allergens as the sensitizing agents responsible for many forms of plant food allergy

BACKGROUND Type I allergic symptoms in the oropharyngeal mucosa upon contact with plant-derived food in patients with pollen allergies have been termed oral allergy syndrome (OAS). IgE cross-reactivity between pollen and food allergens represents the molecular basis for this phenomenon. The sensitizing allergen source (pollen or plant food) in OAS is a controversial issue. OBJECTIVE We sought to determine the primary sensitizing molecules in patients with OAS. METHODS We used recombinant birch pollen (rBet v 1 and rBet v 2) and plant food allergens (apple, rMal d 1; celery, rApi g 1; and carrot, rDau c 1), as well as natural pollen (birch and timothy grass) and plant food (apple, peach, kiwi, hazelnut, celery, and carrot) allergens, to identify cross-reactive allergens by using qualitative immunoblot inhibitions. In addition, we determined the percentage of plant food-specific IgE that can be preadsorbed with recombinant and natural pollen allergens by quantitative RAST inhibitions by using sera from 71 patients with OAS. RESULTS Preincubation of sera with recombinant and natural pollen allergens led to an almost complete inhibition of IgE binding to plant food allergens in Western blots, as well as in RAST inhibition experiments. In contrast, recombinant plant food allergens poorly inhibited IgE binding to Bet v 1. CONCLUSION Most IgE epitopes in plant food recognized by patients with OAS are resembled by pollen allergens. Thus pollen allergens may be responsible for the elicitation and maintenance of OAS.