Peter Briza

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)

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.

Art v 1, the major allergen of mugwort pollen, is a modular glycoprotein with a defensin-like and a hydroxyproline-rich domain

In late summer, pollen grains originating from Compositae weeds (e.g., mugwort, ragweed) are a major source of allergens worldwide. Here, we report the isolation of a cDNA clone coding for Art v 1, the major allergen of mugwort pollen. Sequence analysis showed that Art v 1 is a secreted allergen with an N‐terminal cysteine‐rich domain homologous to plant defensins and a C‐terminal proline‐rich region containing several (Ser/Ala)(Pro)2–4 repeats. Structural analysis showed that some of the proline residues in the C‐terminal domain of Art v 1 are posttranslationally modified by hydroxylation and O‐glycosylation. The O‐glycans are composed of 3 galactoses and 9–16 arabinoses linked to a hydroxyproline and represent a new type of plant O‐glycan. A 3‐D structural model of Art v 1 was generated showing a characteristic “head and tail” structure. Evaluation of the antibody binding properties of natural and recombinant Art v 1 produced in Escherichia coli revealed the involvement of the defensin fold and posttranslational modifications in the formation of epitopes recognized by IgE antibodies from allergic patients. However, posttranslational modifications did not influence T‐cell recognition. Thus, recombinant nonglycosylated Art v 1 is a good starting template for engineering hypoallergenic vaccines for weed‐pollen therapy.

Distinct Roles of Secreted HtrA Proteases from Gram-negative Pathogens in Cleaving the Junctional Protein and Tumor Suppressor E-cadherin

Background: The function of HtrA proteases in bacterial infections is widely unknown. Results: Secreted HtrA from various bacterial pathogens exhibits a conserved specificity for cleavage of E-cadherin. Conclusion: HtrA-mediated E-cadherin cleavage is a prevalent novel mechanism in bacterial pathogenesis. Significance: HtrA activity plays a direct role in the pathogenesis of different bacteria. The periplasmic chaperone and serine protease HtrA is important for bacterial stress responses and protein quality control. Recently, we discovered that HtrA from Helicobacter pylori is secreted and cleaves E-cadherin to disrupt the epithelial barrier, but it remained unknown whether this maybe a general virulence mechanism. Here, we show that important other pathogens including enteropathogenic Escherichia coli, Shigella flexneri, and Campylobacter jejuni, but not Neisseria gonorrhoeae, cleaved E-cadherin on host cells. HtrA deletion in C. jejuni led to severe defects in E-cadherin cleavage, loss of cell adherence, paracellular transmigration, and basolateral invasion. Computational modeling of HtrAs revealed a conserved pocket in the active center exhibiting pronounced proteolytic activity. Differential E-cadherin cleavage was determined by an alanine-to-glutamine exchange in the active center of neisserial HtrA. These data suggest that HtrA-mediated E-cadherin cleavage is a prevalent pathogenic mechanism of multiple Gram-negative bacteria representing an attractive novel target for therapeutic intervention to combat bacterial infections.

Morphogenetic Pathway of Spore Wall Assembly in Saccharomyces cerevisiae

ABSTRACT The Saccharomyces cerevisiae spore is protected from environmental damage by a multilaminar extracellular matrix, the spore wall, which is assembled de novo during spore formation. A set of mutants defective in spore wall assembly were identified in a screen for mutations causing sensitivity of spores to ether vapor. The spore wall defects in 10 of these mutants have been characterized in a variety of cytological and biochemical assays. Many of the individual mutants are defective in the assembly of specific layers within the spore wall, leading to arrests at discrete stages of assembly. The localization of several of these gene products has been determined and distinguishes between proteins that likely are involved directly in spore wall assembly and probable regulatory proteins. The results demonstrate that spore wall construction involves a series of dependent steps and provide the outline of a morphogenetic pathway for assembly of a complex extracellular structure.

IgE cross-reactivity between Ascaris and domestic mite allergens: the role of tropomyosin and the nematode polyprotein ABA-1

Background:  Analysis of cross‐reactivity between the nematode Ascaris ssp. and dust mites, two important allergen sources in the tropics, will contribute in understanding their influence on asthma and atopy. The objective of this study was to investigate immunoglobulin E (IgE) cross‐reactivity between Ascaris and two domestic mites in the tropics.

Expression levels of parvalbumins determine allergenicity of fish species

To cite this article: Griesmeier U, Vázquez‐Cortés S, Bublin M, Radauer C, Ma Y, Briza P, Fernández‐Rivas M, Breiteneder H. Expression levels of parvalbumins determine allergenicity of fish species. Allergy 2010; 65: 191–198.

Yeast ascospore wall assembly requires two chitin deacetylase isozymes.

Chitin deacetylases are required for spore wall rigidity in Saccharomyces cerevisiae. Two chitin deacetylase genes (CDA1 and CDA2) have been identified in yeast. In this report we studied the biochemical properties of the chitin deacetylases encoded by CDA1 and CDA2 and we show how their elimination directly affects the ascospore wall assembly.

Enolases are highly conserved fungal allergens.

BACKGROUND Lack of knowledge of the identity of fungal allergens still is a major obstacle for improvement of diagnosis and therapy of allergies to moulds. We have therefore further analyzed the allergens of the two moulds, Alternaria alternata and Cladosporium herbarum and found that enolases (EC 4.2.1.11) are major allergens, at least of the two fungal species just mentioned. METHODS The enolases of Alternaria and Cladosporium were cloned from cDNA libraries constructed from vegetative cells of the two moulds by immunological screening with sera from selected patients allergic to the moulds. The two enolases were expressed as recombinant nonfusion proteins and used for determination of the incidence of allergy to enolase among a cohort of patients. RESULTS Sequencing of the two enolases showed very close relationships with other known fungal enolase sequences. Competition experiments using immunoblots of the recombinant nonfusion proteins showed nearly complete identity of the epitopes on both enolases. Serum from a patient reactive to Cladosporium enolase reacted equally well with the enolases of Alternaria, Saccharomyces and Candida. About 50% each of the sera from patients reactive to Cladosporium and Alternaria were strongly reactive to the recombinant enolases. CONCLUSIONS Enolases are therefore considered to be highly conserved major fungal allergens.

Mutational Analysis of Amino Acid Positions Crucial for IgE-Binding Epitopes of the Major Apple (Malus domestica) Allergen, Mal d 1

Background: Individual amino acid residues of the major birch pollen allergen, Bet v 1, have been identified to be crucial for IgE recognition. The objective of the present study was to evaluate whether this concept was applicable for the Bet v 1-homologous apple allergen, Mal d 1. Methods: A Mal d 1 five-point mutant was produced by PCR techniques, cloned into pMW 172 and expressed in Escherichia coli BL21(DE3) cells. To evaluate the allergenic properties of the engineered protein compared to Mal d 1 wild-type IgE immunoblotting, ELISA, peripheral blood monocytes proliferation assays, and skin prick tests were performed. Results: The Mal d 1 mutant showed reduced capacity to bind specific IgE as compared to wild-ype Mal d 1 in in vitro assays in the majority of the sera tested. In ELISA, 10 out of 14 serum samples displayed an 88–30% decrease in IgE binding to Mal d 1 mutant compared to wild-type Mal d 1. Skin prick tests in apple-allergic patients (n = 2) confirmed the markedly decreased ability of the Mal d 1 mutant to induce allergic reactions in vivo. However, the relevant T cell epitopes were present in the mutated molecule according to peripheral blood mononuclear cell proliferation assays. Conclusions: Our findings suggest that it is possible to modulate the IgE-binding properties of allergens by single amino acid substitutions at crucial positions which might be useful for future immunotherapy of birch-pollen-associated food allergies which are not ameliorated by birch pollen immunotherapy.