Figure. Flow cytometric analysis of T-cell responses to smallpox antigens after recent smallpox vaccination and in long-term vaccinated or not vaccinated persons. Interferon (IFN)-γ synthesis by T cells after an in vitro stimulation with vaccinia antigens was analyzed in eight healthy donors selected as recently vaccinated, long-term vaccinated, and not vaccinated persons. A representative experiment is reported in this figure. Panels A and D refer to an unvaccinated healthy donor (25-year-old white man, current neutralizing antibodies absent). A long-term vaccinated healthy person is reported in panels B and E (29-year-old white man received two doses of vaccine virus by scarification >20 years ago, current vaccinia neutralizing antibody titer of 1:8). Results from a recently vaccinated person are shown in panels C and F (31-year-old white man, single dose of Dryvax vaccine virus [Wyeth Labs, Marietta, PA] by scarification, January, 2002, current vaccinia neutralizing antibody titer of 1:32). Serum was tested for standard neutralization assay. Briefly, 0.1 mL of serial twofold dilutions of each serum was mixed with an equal volume of vaccinia virus suspension containing ~100 TCID₅₀. After incubation, virus-antibody mixtures, medium, and virus controls were inoculated onto monolayers of Vero cells seeded in 96-well plates. Concomitant retitration of virus suspension was run in parallel. After 48-h incubation at 37°C, the cytopathic effect was observed under light microscope, and the microplates were stained with crystal violet. For T-cell assays, peripheral blood mononuclear cells cultures were stimulated with vaccinia virus (panels A–C) or cytomegalovirus antigens (panels D–F), and intracellular IFN-γ synthesis was analyzed in CD3(+) T cells. Percentages in panel quadrants refer to total lymphocytes.