Epitope mapping

Epitope mapping of the CD4 T cell response to Engerix-B vaccine
Conclusion:
After deconvolution of the response to matrix peptide pools, single peptides against which a CD4 T cell response was detected were selected for in vitro expansion and sorting of single peptide-specific CD4 T cells for TCR-beta sequencing. Aim was to track vaccine-specific CD4 T clonotypes within memory CD4 T cell repertoire pre and post-vaccination.
Notes:
A set of 15-mers peptides with an 11-amino acid overlap spanning the 226 amino acids along the small S protein of hepatitis B (HB) surface antigen (HBsAg), The set, composed of 54 single peptides was used in a matrix-based strategy to map epitopes against which the immune response is directed. The matrix layout enables efficient identification of epitopes within the antigen using a minimal number of cells. For this purpose, a matrix of 15 pools, 7 rows and 8 columns, referred to as matrix peptide pool, was designed so that each peptide is in exactly one row-pool and one column-pool, thereby allowing for the identification of positive peptides at the intersection of positive pools. Matrix peptide pools that induced a CD4 T cell response (as determined by CD40L/CD154 assay described below) which meets the threshold criteria for a positive response were considered in the deconvolution process. Top six single peptides were considered for peptide-specific T cell expansion and sorting. A master peptide pool is composed of all of the 54 single peptides and was used to identify and sort total vaccine-specific CD4 T cells. Each peptide was used at a final concentration of 2 µg/ml. Gates for CD40L+ CD4 T cells were set using cells left unstimulated.