INFLUENCE OF AMINO-ACIDS OF A CARRIER PROTEIN FLANKING AN INSERTED T-CELL DETERMINANT ON T-CELL STIMULATION

CD4(+) helper T cells recognize antigen in association with MHC class II molecules. To investigate the role of the context of a minimal T cell epitope on presentation and recognition in association with MHC class II molecules, an epitope of the 65 kDa heat shock protein of Mycobacterium tuberculosis was inserted at four different sites in the outer membrane protein PhoE of Escherichia coli. Only some of the constructs could stimulate the epitope-specific T cell clone A2b in vitro. One non-stimulatory construct could be made stimulatory by denaturation, suggesting that the protein conformation prevents correct presentation of the epitope. Other non-stimulatory constructs did not become stimulatory with denaturation. One of these constructs could be made stimulatory by substitution of either one of the two amino acids directly preceding the minimal epitope in the recombinant protein. In synthetic peptides the presence of these upstream residues did not interfere with T cell recognition, suggesting that these residues influence processing of the recombinant protein. Flanking amino acids also influenced induction of a T cell response against the inserted epitope in vivo. These results demonstrate that insertion of minimal T cell epitopes in carrier proteins for the design of vaccines might fail because of the inhibiting effects of flanking residues.