Beda M. Stadler

Allergenicity prediction by protein sequence

Potential allergenicity of transgenic proteins for consumption must be investigated before their introduction into the food chain. A prerequisite is sequence analysis. We have critically reviewed the performance of the current guidelines proposed by the Food and Agriculture Organization (FAO) and the World Health Organization (WHO) for allergenicity prediction based on protein sequence and show that its precision is very low. To improve prediction, we propose a new strategy based on sequence motifs identified from a new allergen database. If tested on random test sequences and known allergens, both methods are apparently very sensitive. However, the precision of our motif‐based prediction (95.5%) is superior to the current method (36.6%). We conclude that the proposed motif‐based prediction is a superior alternative to the current method for use in the decision‐tree approach for allergenicity assessment.

Cultures of purified human natural killer cells: Growth in the presence of interleukin 2

Abstract We have isolated highly enriched populations of LGL, which are virtually devoid of mature typical lymphocytes (as enumerated by morphological and surface antigen analysis using monoclonal antibodies e.g., OKT3) in comparison to T cells which contain greater than 95% sheep erythrocyte-forming cells and are devoid of LGL and NK K activities. Both types of cells grew in the presence of crude or partially purified IL-2. Cultures of LGL could be initiated consistently even in the absence of lectins and the cultured LGL retained their characteristic morphology and cytotoxic activity. However, within 7–10 days after initiation, the cultured LGL changed in surface phenotype to become antigenically indistinguishable from cultured T cells.

Recombinant Lactobacillus johnsonii as a mucosal vaccine delivery vehicle

Lactobacilli are considered to be safe organisms making them attractive as vehicles for oral vaccination. We report that Lactobacillus johnsonii (Lj) partially survived simulated gastric conditions in vitro, suggesting that it could be used as an oral vaccine delivery vehicle. In order to test this approach, we used the cell wall anchored proteinase PrtB, isolated from Lactobacillus delbrueckii subsp. bulgaricus as a model antigen. Using a new vector system, we demonstrated expression of both proteinase PrtB alone and a mimotope peptide derived from tetanus toxin integrated in the sequence of proteinase PrtB (TTmim-PrtB fusion protein) on the surface of Lj. Oral immunisation of mice with recombinant Lj, expressing the TTmim-PrtB fusion protein induced a systemic IgG response against Lj and recombinantly expressed proteinase PrtB but no antibody response against the tetanus toxin mimotope suggesting that the mimotope was not sufficiently immunogenic to induce an immune response. Interestingly, a proteinase PrtB specific fecal IgA response was also induced, indicating that the proteinase PrtB fusion protein expressed as a cell surface protein on Lj can induce both systemic and local mucosal immune responses.

Allergen mimotopes in food enhance type I allergic reactions in mice

BIP1 is a murine IgG antibody capable of enhancing the IgE binding to Bet v 1, the major birch pollen allergen. We have previously generated a mimotope of BIP1, designated Bet mim 1, from a constrained phage display peptide library. We demonstrated that oral immunization of BALB/c mice with the Bet mim 1 mimotope resulted in the induction of Bet v 1‐specific IgG. The aim of this study was to test the influence of such an oral immunization with Bet mim 1 on a subsequent type I allergic response to Bet v 1. Phages displaying Bet mim 1 or control mimotopes, or PBS alone, were delivered to BALB/c mice by intragastric gavages prior to systemic sensitization with recombinant Bet v 1 and Al(OH)3, an adjuvant inducing preferentially IgE antibody responses. Only mice fed with Bet mim 1‐phages displayed substantially enhanced type I allergic skin reactivity to Bet v 1, as compared to mice pretreated with control mimotopes or PBS. A gastric digestion assay indicated that Bet v 1 and its homologue from apple, Mal d 1, were degraded within seconds under physiological conditions. In contrast, phage‐displayed mimotopes were resistant to digestion. Our data indicate that allergen mimics in the diet that resist digestion, can induce allergen specific IgG able to enhance an allergic response. We therefore conclude that sensitization via the oral route may represent a mechanism for aggravating type I allergic reactions, probably leading to an earlier onset of symptoms even at lower allergen dosage.—Jensen‐Jarolim, E., Wiedermann, U., Ganglberger, E., Zürcher, A., Stadler, B. M., Boltz‐Nitulescu, G., Scheiner, O., Breiteneder, H. Allergen mimotopes in food enhance type I allergic reactions in mice. FASEB J. 13, 1586–1592 (1999)

Human anti-FcεRIα autoantibodies isolated from healthy donors cross-react with tetanus toxoid

Natural antibodies (Ab) reacting with self antigens have been shown to be present in all individuals. These autoantibodies (auto‐Ab) can be either pathogenic or non‐pathogenic. Auto‐Ab reacting with the α‐subunit of the high‐affinity receptor for IgE (FcϵRIα) have been implicated in the pathogenesis of a subset of patients with chronic idiopathic urticaria (CIU). Intravenous immunoglobulin (IVIg) preparations have been used with variable clinical benefit in the treatment of these patients. Here we show that anti‐FcϵRIα auto‐Ab are present in a therapeutic IVIg preparation as well as in atopic and chronic urticaria patients and healthy individuals. We affinity‐purified the anti‐FcϵRIα Ab from an IVIg preparation using recombinant FcϵRIα. Interestingly, these anti‐FcϵRIα auto‐Ab showed no evidence of histamine release but strongly cross‐reacted with an external antigen, tetanus toxoid (TTd) with a higher affinity for TTd than for the FcϵRIα. Since the cross‐reacting Ab are non‐anaphylactogenic, there is no evidence that TTd immunization may contribute to the pathogenesis of CIU. However, our results may indicate that the anti‐FcϵRIα auto‐Ab belong to the natural Ab and serve as the parental Ab for some anti‐TTd Ab.

Conditional autoimmunity mediated by human natural anti-Fc(epsilon)RIalpha autoantibodies?

Natural antibodies provide an early defense mechanism against pathogens, show a frequent self‐reactivity, and are present throughout life. Two questions concern the physiological control of self‐reactivity and the pathogenetic link to autoimmune disease. Here we propose a concept of conditional autoimmunity involving natural antibodies against the α chain of the high‐affinity receptor for IgE (FcεRIα). Like other natural antibodies, anti‐FcεRIα antibodies are found in sera of healthy donors. We now report the first human recombinant anti‐FcεRIα autoantibodies isolated by repertoire cloning from a human tonsillar IgM library. These high‐affinity antibodies recognize FcεRIα on cells and trigger histamine release from freshly isolated blood basophils. However, the latter effect requires IgE removal from the FcεRI. The same conditional histamine release is seen when using sera from individual normal donors and affinity‐purified anti‐FcεRIα antibodies isolated from multidonor therapeutic IgG preparations. We propose that such anti‐FcεRIα antibodies can become pathogenic and that this is dependent on the state of occupancy of the FcεRIα by its natural ligand IgE. We suggest that an imbalance between FcεRIα occupancy and natural anti‐FcεRIα antibodies may be implicated in the pathogenesis of autoimmune urticaria.—Horn, M. P., Pachlopnik, J. M., Vogel, M., Dahinden, M., Wurm, F., Stadler, B. M., Miescher, S. M. Conditional autoimmunity mediated by human natural anti‐FcɛRIα autoantibodies? FASEB J. 15, 2268–2274 (2001)

Accelerated dissociation of IgE:FcεRI complexes by disruptive inhibitors actively desensitizes allergic effector cells

BACKGROUND The remarkably stable interaction of IgE with its high-affinity receptor FcεRI on basophils and mast cells is critical for the induction of allergic hypersensitivity reactions. Because of the exceptionally slow dissociation rate of IgE-FcεRI complexes, such allergic effector cells permanently display allergen-specific IgE on their surface and immediately respond to allergen challenge by releasing inflammatory mediators. We have recently described a novel macromolecular inhibitor that actively promotes the dissociation of IgE from FcεRI through a molecular mechanism termed facilitated dissociation. OBJECTIVE Here we assessed the therapeutic potential of this non-immunoglobulin-based IgE inhibitor E2_79, a designed ankyrin repeat protein (DARPin), as well as a novel engineered biparatopic DARPin bi53_79, and directly compared them with the established anti-IgE antibody omalizumab. METHODS IgE-FcεRI complex dissociation was analyzed in vitro by using recombinant proteins in ELISA and surface plasmon resonance, ex vivo by using human primary basophils with flow cytometry, and in vivo by using human FcεRI α-chain transgenic mice in a functional passive cutaneous anaphylaxis test. RESULTS We show that E2_79-mediated removal of IgE from primary human basophils fully abrogates IgE-dependent cell activation and release of proinflammatory mediators ex vivo. Furthermore, we report that omalizumab also accelerates the dissociation of IgE from FcεRI, although much less efficiently than E2_79. Using the biparatopic IgE targeting approach, we further improved the disruptive potency of E2_79 by approximately 100-fold and show that disruptive IgE inhibitors efficiently prevent passive cutaneous anaphylaxis in mice expressing the human FcεRI α-chain. CONCLUSION Our findings highlight the potential of such novel IgE inhibitors as important diagnostic and therapeutic tools for management of allergic diseases.

DARPins as Bispecific Receptor Antagonists Analyzed for Immunoglobulin E Receptor Blockage

The concept of multispecific antibodies is of high therapeutic interest but has failed to produce pharmaceutical products due to the poor biophysical properties of such molecules. Here, we propose an alternative and simple way to generate bispecific binding molecules using designed ankyrin repeat proteins (DARPins). For this purpose, monovalent DARPins with different epitope specificities were selected against the alpha chain of the high-affinity receptor for human immunoglobulin E (IgE) (FcepsilonRIalpha). Two of the isolated binders interfering with IgE binding to the receptor were joined to each other or to themselves via a flexible protein linker. The resulting bivalent and bispecific DARPins were tested for their ability to prevent allergen-induced cell degranulation using rat basophilic leukemia cells stably transfected with human FcepsilonRIalpha. The bispecific DARPin construct was the most potent one, efficiently blocking the IgE-FcepsilonRI interaction and preventing the release of proinflammatory mediators. Noteworthy, the multivalent and multispecific DARPin construct did not show any alteration of the beneficial biophysical properties of the monovalent parental DARPins. Hence, bispecific DARPins may be used to generate receptor antagonists simultaneously targeting different epitopes on the same molecule. Moreover, they easily overcome the limiting immunoglobulin binding paradigm (one binding molecule=one epitope) and thereby represent an alternative to monoclonal antibodies in cases where the immunoglobulin scaffold is unsuitable.

Oral anti‐IgE immunization with epitope‐displaying phage

An essential requirement for oral vaccines is the ability to survive the harsh environment of the stomach in an antigenically intact form. As bacteriophages are adapted to this environment we used epitope‐displaying M13 bacteriophages as carriers for an experimental oral anti‐IgE vaccine. The feasibility of this approach was tested in a simulated gastric fluid using two different mimotopes as well as an anti‐idiotypic Fab of the non‐anaphylactogenic monoclonal anti‐IgE antibody BSW17. All phage clones remained infective after this treatment. However, only epitopes displayed on the pVIII protein were still recognized by BSW17 whereas pIII‐expressed epitopes were rapidly inactivated. Surprisingly, when used for oral immunization of mice all phage clones induced anti‐IgE antibodies. In contrast, oral immunization with the purified, pVIII protein displaying the mimotope induced anti‐phage but no anti‐IgE antibodies. After feeding a single dose of mimotope‐displaying bacteriophage, phage DNA could be detected in mouse feces for 10 days. Our results show that epitope‐displaying bacteriophages can be used to induce an epitope‐specific antibody response via the oral route.

A mimotope defined by phage display inhibits IgE binding to the plant panallergen profilin

Birch pollen and mugwort pollen allergies are often associated with hypersensitivity to plant foods. This clinical and serological cross‐reactivity is mediated by IgE antibodies reacting with homologous proteins in pollen and food. Cross‐reacting homologs of the important birch pollen allergen Bet v  2 (profilin) could be detected in other pollen, fruits, nuts, and vegetables, such as celery tuber. We purified IgG/IgE antibodies from the serum of an exclusively profilin‐allergic patient using affinity columns either coupled with protein extracts from mugwort pollen, birch pollen, or celery tuber. Constrained and unconstrained random nonapeptide libraries were pooled and screened with the anti‐profilin antibody preparations to define cross‐reactive ligands. Specific ligands were enriched by successive panning rounds using the profilin‐specific antibodies in series. After the last panning round enriched phage clones were screened with purified profilin‐specific antibodies and IgE‐binding clones were sequenced. Five out of eight positive clones (62.5 %) displayed the same circular peptide CAISGGYPVC. This peptide was synthesized and examined for its ability to inhibit IgE binding to blotted mugwort pollen, birch pollen, or celery tuber profilin. Inhibition studies showed reduction of IgE binding to profilins in all three protein extracts. As the sequence of the mimotope did not show any homology to the known birch profilin sequence this peptide is considered to mimic a common conformational IgE epitope for these examined profilins.