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Abstracts / Human Immunology 74 (2013) 1–49
NEW APPROACHES TO DEFINE BINDING MOTIFS AND SUPERTYPES IN PEPTIDE BINDING ANALYSIS. Rico Buchli 1, Rebecca McAdams 1, Aaron D. Rennels 1, Rodney S. VanGundy 1, William H. Hildebrand 1,2. 1 R & D, Pure Protein LLC, Oklahoma City, OK, USA; 2 Microbiology and Immunology, Oklahoma University Health Sciences Center, Oklahoma City, OK, USA. Aim: Classification of allele overlapping peptide-binding specificities has become an important issue in vaccine design with direct implications concerning population coverage. In this study, we used a combinatoric approach not only to discover epitope/HLA combinations suitable to be utilized in peptide binding assays, but also to evaluate the degree of overlapping peptide binding capacities among the alleles tested. Methods: A set of 20 different FITC-labeled reference peptides was applied in combination with over 50 sHLA alleles to determine their binding capabilities. During the process, each peptide candidate was incubated with activated sHLA, and the peptide/HLA interaction was monitored over time using Fluorescence Polarization (FP). This assay combination allows the direct measurement of the ratio between free and bound labeled ligand in solution without any separation steps. The technique of FP is based on the principal that if a fluorescent-labeled peptide binds to the sHLA molecule of higher molecular weight, polarization values will increase due to the slower molecular rotation of the bound probe. Results: Data analysis showed that the majority of peptide candidates were capable of binding with various degrees of overlap, reflecting the ability of HLA class I alleles to share the binding of identical peptides. The approach resulted in the identification of over 40 new peptide/HLA combinations applicable for epitope discovery and characterization. However, comparison to published binding motifs and supertype classifications showed several inconsistencies indicating the need of more refinement in their definition. Conclusions: Our approach suggests that broadly cross-reactive peptide epitopes can be identified and greatly enhance the effectiveness of future vaccine designs by providing a more extensive population coverage. They will also have a profound benefit in the understanding of antigenic peptide selection, degeneration and discrimination during T-cell mediated immune responses.
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Abstracts / Human Immunology 74 (2013) 1–49
NEW APPROACHES TO DEFINE BINDING MOTIFS AND SUPERTYPES IN PEPTIDE BINDING ANALYSIS. Rico Buchli 1, Rebecca McAdams 1, Aaron D. Rennels 1, Rodney S. VanGundy 1, William H. Hildebrand 1,2. 1 R & D, Pure Protein LLC, Oklahoma City, OK, USA; 2 Microbiology and Immunology, Oklahoma University Health Sciences Center, Oklahoma City, OK, USA. Aim: Classification of allele overlapping peptide-binding specificities has become an important issue in vaccine design with direct implications concerning population coverage. In this study, we used a combinatoric approach not only to discover epitope/HLA combinations suitable to be utilized in peptide binding assays, but also to evaluate the degree of overlapping peptide binding capacities among the alleles tested. Methods: A set of 20 different FITC-labeled reference peptides was applied in combination with over 50 sHLA alleles to determine their binding capabilities. During the process, each peptide candidate was incubated with activated sHLA, and the peptide/HLA interaction was monitored over time using Fluorescence Polarization (FP). This assay combination allows the direct measurement of the ratio between free and bound labeled ligand in solution without any separation steps. The technique of FP is based on the principal that if a fluorescent-labeled peptide binds to the sHLA molecule of higher molecular weight, polarization values will increase due to the slower molecular rotation of the bound probe. Results: Data analysis showed that the majority of peptide candidates were capable of binding with various degrees of overlap, reflecting the ability of HLA class I alleles to share the binding of identical peptides. The approach resulted in the identification of over 40 new peptide/HLA combinations applicable for epitope discovery and characterization. However, comparison to published binding motifs and supertype classifications showed several inconsistencies indicating the need of more refinement in their definition. Conclusions: Our approach suggests that broadly cross-reactive peptide epitopes can be identified and greatly enhance the effectiveness of future vaccine designs by providing a more extensive population coverage. They will also have a profound benefit in the understanding of antigenic peptide selection, degeneration and discrimination during T-cell mediated immune responses.