Schematic description of the construction of a recombinant vaccinia virus that expresses a synthetic DNA encoding for a polyepitope protein that contains 6 HLA A2–restricted LMP1 epitopes.

See the box in this figure and Table 1 for details about the LMP1 epitopes. Each of the alternate epitope sequences is underlined. The DNA sequence encoding this polyepitope protein was constructed using epitope sequence–specific primers (referred to as LMP-A, LMP-B, LMP-C, and LMP-D) and a technique based on mutual priming and overlap extension as described in “Materials and methods.” The nucleic acid sequence of the fragment coded (from the 5′ end)BamHI and PstI restriction sites, a Kozak sequence, a methionine start codon, 6 contiguous minimal LMP1 CTL epitopes, a stop codon, and XbaI and HindIII restriction sites at the 3′ end. This DNA insert was subcloned behind the vaccinia p7.5 promoter in the plasmid shuttle vector pTK-7.5A using specific restriction enzymes for the construction of recombinant vaccinia virus.