A universal approach to eliminate antigenic properties of alpha-gliadin peptides in celiac disease

Abstract

Celiac disease is caused by an uncontrolled immune response to gluten, a heterogeneous mixture of wheat storage proteins, including the α-gliadins. It has been shown that α-gliadins harbor several major epitopes involved in the disease pathogenesis. A major step towards elimination of gluten toxicity for celiac disease patients would thus be the elimination of such epitopes from α-gliadins. We have analyzed over 3,000 expressed α-gliadin sequences from 11 bread wheat cultivars to determine whether they encode for peptides potentially involved in celiac disease. All identified epitope variants were synthesized as peptides and tested for binding to the disease-associated HLA-DQ2 and HLA-DQ8 molecules and for recognition by patient-derived α-gliadin specific T cell clones. Several specific naturally occurring amino acid substitutions were identified for each of the α-gliadin derived peptides involved in celiac disease that eliminate the antigenic properties of the epitope variants. Finally, we provide proof of principle at the peptide level that through the systematic introduction of such naturally occurring variations α-gliadins genes can be generated that no longer encode antigenic peptides. This forms a crucial step in the development of strategies to modify gluten genes in wheat so that it becomes safe for celiac disease patients. It also provides the information to design and introduce safe gluten genes in other cereals, which would exhibit improved quality while remaining safe for consumption by celiac disease patients.

Figure 1. Presence of DQ2-Glia-α1 and DQ2-Glia-α2 epitopes in diploid wheat.

Pepsin-trypsin digests of 29 diploid wheat accessions were prepared and tested in a competition ELISA with a mAb specific for a sequence partially overlapping with the DQ2-Glia-α1 and DQ2-Glia-α2 epitopes and after deamidation with T cell clones specific for the DQ2-Glia-α1 and DQ2-Glia-α2 epitopes. A: results of the competiton ELISA. B: T cell proliferation assay with a DQ2-Glia-α1 specific T cell clone. C: T cell proliferation assay with a DQ2-Glia-α2 specific T cell clone. IL-2: proliferation of the T cell clone under the influence of interleukin-2. Background: proliferation of the T cells in the presence of antigen presenting cells but no antigen. Positive control: proliferation of the T cell clone in the presence of a synthetic peptide encoding the specific α-gliadin epitope and antigen presenting cells. cpm: counts per minute. AA: diploid accessions with an A genome; SS: diploid accessions with an S genome; DD: diploid accessions with a D genome.

Figure 2. Amino acid substitution eliminates toxicity of known α-gliadin epitopes.

The DQ2-Glia-α3 epitope and the known 33-mer were synthesized in deamidated form either as the original sequence or after substitution in each epitope of the prolines at position 8 with serine. These peptides were tested in T cell proliferation assays. A: T cell proliferation assay using a DQ2-Glia-α3 T cell clone. B: T cell proliferation assay using a DQ2-Glia-α1 T cell clone. Positive control: synthetic peptide encoding the specific minimal T cell epitope. Stim. index: stimulation index defined as the specific proliferation of a sample divided by the background proliferation.

Figure 3. Response of DQ2-Glia-α1 epitope specific T cell clones against single and multiple P to S substituted peptides.

Five T cell clones derived from 3 CD patients were tested against the deamidated form of the DQ2-Glia-α1 peptide (sequence PFPQPELPY) and variants thereof in which prolines at position 3, 5, 8 and 10 were systematically substituted for serine, both as single substitutions and in all possible combinations. Shown is the response to the substituted peptides relative to unsubstituted DQ2-Glia-α1 epitope. The introduced substitutions are underlined. The most C-terminal substituted proline at position 10 lies outside the 9 amino acid core of the T cell stimulatory peptide.