Probing immunogenicity of a T cell epitope by L-alanine and D-amino acid scanning.
All residues of the I-Ed restricted fragment 24-36 of a snake toxin were individually changed into L-alanine and the corresponding D-enantiomer. Four analogs substituted with L-Ala at positions 25;30, 31 and 33, and nine analogs substituted with a D-residue along the stretch 25-33 lost most (position 28) or all their capacity to stimulate a toxin-specific T hybridoma. None of these analogs stimulated splenocytes from mice immunized with the peptide 24-36. Only the L-A31 and D-W29 modified analogs could prime a T cell response which, however, showed no cross-reactivity with the native peptide, demonstrating that T cell response selectivity can be deeply modified by mutation or configuration inversion of a single residue. Our data suggest that (i) the region 25-33 is the core of the T epitope that binds to I-Ed, and (ii) Y25 R30 and R33 contribute to the peptide binding by anchoring into pockets of I-Ed. In agreement with T cell priming observations, only the L-A31 and D-W29 modified analogs elicited strong antibody responses, just like the peptide 24-36, whereas nearly all other analogs were less immunogenic. All but the L-Ala30 and L-Ala33 modified analogs were recognized by a 24-36 specific antiserum as well as the native peptide. Altogether, our results show that substitution by D-amino acid in a peptide could be particularly well-suited for either minimizing the risk of hypersensitivity or designing peptidic vaccines.
Maillère
B
B
CEA, Département d'Ingénierie et d'Etudes des Protéines, Gif-sur-Yvette, France.
Mourier
G
G
Cotton
J
J
Hervé
M
M
Leroy
S
S
Ménez
A
A
D000409:Alanine / Q000235:genetics* / Q000276:immunology*; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis; D000917:Antibody Formation; D000939:Epitopes / Q000235:genetics* / Q000276:immunology*; D006825:Hybridomas; D008213:Lymphocyte Activation; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010455:Peptides / Q000235:genetics / Q000276:immunology; D013237:Stereoisomerism; D013601:T-Lymphocytes / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010455:Peptides; D000409:Alanine