| Reference | ||
|---|---|---|
| Reference Type | Submission | IEDB_Reference:1000399 |
| Title | HLA DRB1*01:01 binding capacity of selected SARS-derived peptides | |
| Authors | John Sidney; Jason Botten; Benjamin Neuman; Michael Buchmeier; Alessandro Sette | |
| Affiliations | La Jolla Institute for Allergy and Immunology, San Diego, CA, USA, and The Scripps Research Institute, La Jolla, CA, USA | |
| Submitter | John Sidney | IEDB_Submission:1000049 |
| Year | 2006 | |
| Abstract | To identify potential SARS-derived HLA class II epitopes we scanned the SARS sequence (see PMIDs: 12730500 and 12730501) to identify a set of 752 15-mer peptides (from a set of about 10,000 possible 15-mers) that contain the previously described HLA DR-supermotif (Southwood, et al., PMID 9531296), associated with the majority of HLA DR promiscuous binding peptides. Each peptide was tested for binding to HLA DRB1*01:01, the prototype molecule for the DR supertype. Previous data has shown that peptides binding DRB1*01:01 with high affinity also tend to bind other DRB1 alleles and, similarly, that the vast majority of HLA class II promiscuous epitopes also bind DRB1*01:01 with high affinity. Thus, while HLA DRB1*01:01 has an inherently high rate of binding compared to other class II specificities, DRB1*01:01 binding affinity has predictive value for identifying and ranking the most promising prospective promiscuous T cell epitope candidates. The pre-selection proved successful, and 694 (92%) of the peptides bound DRB1*01:01 with an affinity of 1000 nM, or better, including 116 (15%) with affinities <2 nM. These data confirm the efficacy of the DR-supermotif for identifying high affinity binding peptides, and provide a set of candidates for further studies examining the immune response to SARS. Subsequent binding studies could examine the capacity of the top 116 peptides to bind additional common HLA DR molecules, including DRB1*0301, DRB1*0401, DRB1*0701, DRB1*1101, DRB1*1501 and DRB5*0101. HLA DRB1*01:01 binding was determined in quantitative competition assays based on the inhibition of binding of a radiolabeled standard peptide to purified MHC molecules. Assays were performed essentially as previously described (PMIDs 18432745, 15078927 and 9531296). Briefly, 0.1-1 nM of radiolabeled peptide was incubated at room temperature with 1 nM to 1 µM purified MHC class II molecule in the presence of a mixture of protease inhibitors. After a 2-day incubation, binding of the radiolabeled peptide to the given MHC class II molecule was determined by capturing MHC/peptide complexes on Greiner Lumitrac 600 microplates (Greiner Bio-One, Monroe, NC) coated with the LB3.1, L243, SPVL3, HB180 or B7/21 monoclonal antibodies, and measuring bound cpm using the TopCount microscintillation counter (PerkinElmer, Waltham, MA). The concentration of peptide yielding IC50 of the binding of the radiolabeled peptide was calculated. Peptides were typically tested at six different concentrations covering a 100,000-fold dose range, and in three or more independent assays. Under the conditions used, where [label] < [MHC] and IC50 ≥ [MHC], the measured IC50 values are reasonable approximations of the true Kd values. This work has been supported by funds provided through NIH NIAID contract N01-AI-40023. | |
| Curation Last Updated | 2024-08-19 20:01:32 | |
| Related Information | |
|---|---|
| Epitopes | |
| Bcell Assays | 0 |
| Tcell Assays | 0 |
| MHC Ligand Assays | |