Ulrike Schulmeister

A Recombinant Hypoallergenic Parvalbumin Mutant for Immunotherapy of IgE-Mediated Fish Allergy

IgE-mediated allergy to fish is a frequent cause of severe anaphylactic reactions. Parvalbumin, a small calcium-binding protein, is the major fish allergen. We have recently isolated a cDNA coding for carp parvalbumin, Cyp c 1, and expressed in Escherichia coli a recombinant Cyp c 1 molecule, which contained most IgE epitopes of saltwater and freshwater fish. In this study, we introduced mutations into the calcium-binding domains of carp parvalbumin by site-directed mutagenesis and produced in E. coli three parvalbumin mutants containing amino acid exchanges either in one (single mutants; Mut-CD and Mut-EF) or in both of the calcium-binding sites (double mutant; Mut-CD/EF). Circular dichroism analyses of the purified derivatives and the wild-type allergen showed that Mut-CD/EF exhibited the greatest reduction of overall protein fold. Dot blot assays and immunoblot inhibition experiments performed with sera from 21 fish-allergic patients showed that Mut-CD/EF had a 95% reduced IgE reactivity and represented the derivative with the least allergenic activity. The latter was confirmed by in vitro basophil histamine release assays and in vivo skin prick testing. The potential applicability for immunotherapy of Mut-CD/EF was demonstrated by the fact that mouse IgG Abs could be raised by immunization with the mutated molecule, which cross-reacted with parvalbumins from various fish species and inhibited the binding of fish-allergic patients’ IgE to the wild-type allergen. Using the hypoallergenic carp parvalbumin mutant Mut-CD/EF, it may be possible to treat fish allergy by immunotherapy.

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 2000 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.

Cloning, Expression, and Mapping of Allergenic Determinants of αS1-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, αS1-casein, from a bovine mammary gland cDNA library with allergic patients’ IgE Abs. Recombinant αS1-casein was expressed in Escherichia coli, purified, and characterized by circular dichroism as a folded protein. IgE epitopes of αS1-casein were determined with recombinant fragments and synthetic peptides spanning the αS1-casein sequence using microarrayed components and sera from 66 cow’s milk-sensitized patients. The allergenic activity of rαS1-casein and the αS1-casein-derived peptides was determined using rat basophil leukemia cells transfected with human FcεRI, which had been loaded with the patients’ serum IgE. Our results demonstrate that rαS1-casein as well as αS1-casein-derived peptides exhibit IgE reactivity, but mainly the intact rαS1-casein induced strong basophil degranulation. These results suggest that primarily intact αS1-casein or larger IgE-reactive portions thereof are responsible for IgE-mediated symptoms of food allergy. Recombinant αS1-casein as well as αS1-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.

Microarray and allergenic activity assessment of milk allergens

Background Cows milk is one of the most common causes of food allergy affecting approximately 2.5% of infants in the first years of their life. However, only limited information regarding the allergenic activity of individual cows milk allergens is available.

Patients suffering from non-IgE-mediated cow’s milk protein intolerance cannot be diagnosed based on IgG subclass or IgA responses to milk allergens

To cite this article: Hochwallner H, Schulmeister U, Swoboda I, Twaroch TE, Vogelsang H, Kazemi‐Shirazi L, Kundi M, Balic N, Quirce S, Rumpold H, Fröschl R, Horak F, Tichatschek B, Stéfanescu CL, Szépfalusi Z, Papadopoulos NG, Mari A, Ebner C, Pauli G, Valenta R, Spitzauer S. Patients suffering from non‐IgE‐mediated cow’s milk protein intolerance cannot be diagnosed based on IgG subclass or IgA responses to milk allergens. Allergy 2011; 66: 1201–1207.

Visualization of clustered IgE epitopes on α-lactalbumin

BACKGROUND alpha-Lactalbumin (alpha-La) is a major cows 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.

Infant milk formulas differ regarding their allergenic activity and induction of T-cell and cytokine responses

Several hydrolyzed cows milk (CM) formulas are available for avoidance of allergic reactions in CM‐allergic children and for prevention of allergy development in high‐risk infants. Our aim was to compare CM formulas regarding the presence of immunoreactive CM components, IgE reactivity, allergenic activity, ability to induce T‐cell proliferation, and cytokine secretion.

Allergenic activity and ability to induce T cell and cytokine responses of different infant milk formulas

Background Many hydrolyzed cow’s milk (CM) formulas are available for avoidance of allergic reactions in cow’s milk allergic children and for prevention of allergy development in high risk infants. CM formulas were compared regarding the presence of immunoreactive CM components, IgE reactivity, allergenic activity, ability to induce T cell proliferation and allergic or pro-inflammatory cytokine secretion.