We previously demonstrated that vaccinia virus (VV)-specific CD4(+) cytolytic T cells can persist for >50 years after immunization against
smallpox in the absence of re-exposure to VV. Nevertheless, there have been few studies focusing on CD4(+) T cell responses to
smallpox vaccination. To ensure successful vaccination, a candidate
vaccine should contain immunodominant CD4(+)
T cell epitopes as well as CD8(+) T and
B cell epitopes. In the present study, we established cytotoxic CD4(+) T cell lines from VV-immune donors, which recognize
epitopes in VV
proteins D1R and A24R in association with
HLA-DR1 Ags. Comparisons of sequences between different members of the poxvirus family show that both
epitopes are completely conserved among VV, variola viruses, and most mammalian poxviruses, including
monkeypox,
cowpox, and
ectromelia. The CD4(+) T cell lines lysed VV-infected, Ag- and
peptide-pulsed targets, and the lysis was inhibited by
concanamycin A. We also detected these
peptide-specific cytolytic and IFN-gamma-producing CD4(+) T cells in short-term bulk cultures of PBMC from each of the three VV-immune donors tested. These are the first VV-specific CD4(+)
T cell epitopes identified in humans restricted by one of the most common
MHC class II molecules,
HLA-DR1, and this information may be useful in analyzing CD4(+) T cell responses to pre-existing or new generation VV
vaccines against
smallpox.