FLANKING RESIDUES ALTER ANTIGENICITY AND IMMUNOGENICITY OF MULTIUNIT CTL EPITOPES

CD8(+) CTL responses constitute a critical component for vaccines deve loped to eliminate intracellular pathogens, One approach to achieve br oad CTL diversity is based on genetically linking immunogenic peptides from multiple proteins to form poly-epitope Ags, To address the influ ence of flanking residues on class I Ag presentation, H-2(d)-restricte d HIV-1 and mouse hepatitis virus CTL epitopes were linked via various spacer residues, The resulting 20 to 31 amino acid peptides were expr essed using recombinant vaccinia viruses to monitor both CTL. recognit ion and induction, Our data indicate that recognition is profoundly in fluenced by the nature of intervening residues forming carboxyl-termin al flanks for one and amino-terminal flanks for the other epitope, Fla nking amino acids with aromatic (tyrosine), basic (lysine), and small aliphatic side chains (alanine) supported efficient CTL recognition of both epitopes, By contrast, acidic and helix breaking residues (glyci ne, proline) specifically inhibited recognition of the adjacent amino- terminal epitope, Flanking residues inhibitory for recognition were al so detrimental for CTL induction, suggesting similar processing mechan isms in vitro and in vivo, The ratios of peptide-specific CTL precurso rs primed by the tandem epitopes varied up to 50-fold depending on mol ecular context, These data demonstrate a substantial role of carboxyl- flanking residues in governing the efficiency of class I Ag presentati on both in vitro and in vivo, The dramatic influence of flanking resid ues on the hierarchy of CTL responses indicates that CTL induction by poly-epitope Ags can be optimized by strategically linking epitopes vi a selection of appropriate spacer residues.