Food and Drug Reactions and Anaphylaxis
Mutational analysis of major, sequential IgE-binding epitopes in αs1-casein, a major cow's milk allergen,☆☆

https://doi.org/10.1067/mai.2003.1617Get rights and content

Abstract

Background: Allergy to cow's milk is common in early childhood, and no therapy other than avoidance exists. In murine models of peanut allergy, immunotherapy with mutated, engineered, proteins appears promising. Objective: We sought to identify the critical amino acids (AAs) for immunoglobulin E (IgE) binding within the major B-cell epitopes of αs1-casein, a major cow's milk allergen. This will provide the necessary information to alter the cDNA to encode a protein capable of activating milk-specific T cells, but with reduced IgE-binding capacity. Methods: For mutational analysis of the IgE-binding epitopes, peptides of 10-14 AAs in length were synthesized on a derivatized cellulose membrane with single or multiple AA substitutions. Membranes were immunolabeled with pooled sera from 15 cow's-milk-allergic patients and with 8 individual sera. Results: With the pooled sera, substitution of a single AA led to complete abrogation of IgE binding to 2 of 8 peptides and diminished binding in the remainder. Substitution of multiple AAs led to an abrogation of binding in the remaining peptides. In 4 of the 8 peptides, the critical AA identified with pooled sera did not result in significant reduction of IgE binding with 1 or more individual patients. For these patients, other critical AAs were identified, indicating a more heterogeneous pattern in IgE recognition. Conclusion: This study indicates that single or multiple AA substitutions within IgE-binding epitopes result in reduced binding of milk-specific IgE antibodies by patients' sera. However, for future immunotherapeutic interventions with mutated peptides, critical AAs should be evaluated with individual patient sera to determine B-cell-epitope heterogeneity. (J Allergy Clin Immunol 2003;112:433-7.)

Section snippets

Patient population

Sera from 15 patients with documented IgE-mediated cow's milk hypersensitivity (mean age, 8.5 years; range, 2-18 years) were used to identify the AAs essential for IgE binding. Milk-specific IgE antibodies in these sera ranged from 25 to >100 kU/L (median, >100 kU/L), as measured by the CAP system FEIA (Pharmacia Diagnostics, Uppsala, Sweden). In 11 children, CMA was confirmed by open (n = 3) or double-blind, placebo-controlled food challenges (n = 8). The remaining 4 children had a history of

Results

Pooled sera from 15 patients and individual sera from 8 randomly selected patients whose serum was included in the pool were used to identify AAs essential for epitope-specific recognition by IgE antibodies. In the first phase of experiments, each residue within an epitope was substituted with alanine (or, when alanine was present, with glycine) 1 at a time. Fig 1 shows an example of the immunolabeling with pooled sera for peptide AA 109-120.

. Selected part of the SPOT membrane, showing 1 of the

Discussion

Within the last few years, the primary AA sequences of a number of food allergens and their IgE-binding regions have been identified. To develop safe immunotherapeutic agents for food allergy, modified, recombinant proteins need to be engineered that will not bind serum and mast cell IgE. Therefore, critical AAs within the IgE-binding regions need to be identified to provide the necessary information to alter the cDNAs that encode proteins capable of activating milk-specific T cells, but not

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    Supported by grants AI 44236 and AI 24439 from the National Institute of Allergy and Infectious Diseases, grant M01 RR00071 from the Division of Research Resources, National Institutes of Health, and the Bunning Family Fund.

    ☆☆

    Reprint requests: Kirsten Beyer, MD, Department of Pediatrics, Charite-CVK, Augustenburgerplatz 1, 13353 Berlin, Germany.

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