Despite the importance of vaccinia virus in basic and applied immunology, our knowledge of the human immune response directed against this virus is very limited. CD4+ T cell responses are an important component of immunity induced by current vaccinia-based vaccines, and likely will be required for new subunit vaccine approaches, but to date vaccinia-specific CD4+ T cell responses have been poorly characterized, and CD4+ T cell epitopes have been reported only recently. Classical approaches used to identify T cell epitopes are not practical for large genomes like vaccinia. We developed and validated a highly efficient computational approach that combines prediction of class II MHC-peptide binding activity with prediction of antigen processing and presentation. Using this approach and screening only 36 peptides, we identified 25 epitopes recognized by T cells from vaccinia-immune individuals. Although the predictions were made for HLA-DR1, eight of the peptides were recognized by donors of multiple haplotypes. T cell responses were observed in samples of peripheral blood obtained many years after primary vaccination, and were amplified after booster immunization. Peptides recognized by multiple donors are highly conserved across the poxvirus family, including variola, the causative agent of smallpox, and may be useful in development of a new generation of smallpox vaccines and in the analysis of the immune response elicited to vaccinia virus. Moreover, the epitope identification approach developed here should find application to other large-genome pathogens.

T cells generally recognize peptide antigens bound to MHC proteins through contacts with residues found within or immediately flanking the seven-to nine-residue sequence accommodated in the MHC peptide-binding groove. However, some T cells require peptide residues outside this region for activation, the structural basis for which is unknown. Here, we have investigated a HIV Gagspecific T cell clone that requires an unusually long peptide antigen for activation. The crystal structure of a minimally antigenic 16-mer bound to HLA-DR1 shows that the peptide C-terminal region bends sharply into a hairpin turn as it exits the binding site, orienting peptide residues outside the MHC-binding region in position to interact with a T cell receptor. Peptide truncation and substitution studies show that both the hairpin turn and the extreme C-terminal residues are required for T cell activation. These results demonstrate a previously unrecognized mode of MHC-peptide-T cell receptor interaction.antigen presentation ͉ receptors ͉ antigen ͉ protein conformation C lass II MHC proteins are cell-surface glycoproteins that bind antigens in the form of short peptides and present them for recognition to T cell receptors (TCRs) on the cell surface of CD4 ϩ T cells (1). Naturally processed peptides isolated from class II MHC proteins found in antigen-presenting cells are usually 15-25 residues long (2, 3). The central region of these peptides interacts directly with class II MHC proteins, typically with specific recognition of an approximate nine-residue stretch (4). X-ray crystallography of human and murine class II MHC proteins has revealed that peptides bind to the protein in an extended polyproline type II conformation, with several peptide side chains bound into polymorphic pockets that line the peptide-binding groove (5-12). A hydrogen-bond network between the conserved residues on the class II MHC and the peptide main-chain carbonyl and amide groups, independent of the sequence of the peptide, stabilizes the MHC-peptide complex (13), and enforces the polyproline conformation that directs some of the side chains into the MHC pockets and leaves the others accessible for TCR interactions (14). Generally, pockets accommodate the side chains of peptide residues at the P1, P4, P6, and P9 positions, with smaller pockets or shelves in the binding site accommodating the P3 and P7 residues. Minor variations on this theme have been observed, for example, in some complexes the P9 interactions are weak or absent (5, 10). In the canonical conformation, the side chains of residues at positions P Ϫ1, P2, P5, and P8 are solvent-accessible and point toward the TCR, with portions of other side chains and the peptide main chain also exposed for potential TCR interaction in the central region of the complex.The interaction of TCR with MHC-peptide complexes also is expected to be relatively stereotyped, with complementaritydetermining regions (CDRs) from the V␣ and V␤ domain lying across the peptide-MHC complex, typically with CDR3 loops of both vari...