CMV-specific memory phenotypes and MVA vector-specific responses. (A) Memory phenotype of CMV-specific T cells following Triplex vaccination. The top panel shows representative FACS plots (from UPN 13; gray boxes in the subsequent panels indicate the selected time point) relative to the gating hierarchy used for the memory phenotype analysis of IFN-γ producing T cells. From left to right, lymphocytes gated by use of forward and side scatter; histogram plots indicating the CD3 (peridinin chlorophyll protein Cychrome 5 [PerCP Cy5.5] conjugated) and the CD8 (violet laser excited coumarin dye [V450] conjugated) gating; subsequent gating of CD8⁺ IFN-γ⁺ (APC conjugated) T cells unstimulated or stimulated with the pp65 library (as specified on the plot title), and respective percentages shown in the upper right gate of each contour plot. By gating pp65-specific CD8⁺ IFN-γ⁺ T cells, 4 subpopulations were identified according to the expression of CD28 and CD45RA (far right dot plot). CD45RA⁺ CD28⁺ cells were classified as naive (upper right quadrant); CD45RA⁻ CD28⁺ cells were classified as central memory (T_CM, lower right quadrant), and CD28⁻ cells were classified as effector. Within the effector T-cell group, 2 subpopulations were identified: CD45RA⁻ CD28⁻ (TEM, T effector memory, lower left quadrant) and CD45RA⁺CD28⁻ (TEMRA, revertant T-effector memory re-expressing CD45RA, upper left quadrant) T cells. The middle panel of line graphs shows the longitudinal profiles of levels of CMV-specific CD8⁺ and CD4⁺ T cells producing IFN-γ in CMV-seronegative UPN 13, UPN 14, and UPN 18 following stimulation with the peptide library indicated in the legend. The lower panel of line graphs shows the percentages of the respective memory phenotypes indicated in the legend for CMV-specific CD8⁺ IFN-γ⁺ T cells. Memory phenotypes were analyzed when CMV-specific IFN-γ production by T cells in response to a CMV-specific library was ≥0.2%. UPN 13 had been vaccinated against smallpox. Arrows show time of vaccinations (days 0 and 28). The star symbol indicates that UPN 14 and UPN 18 seroconverted and were tested to be CMV seropositive on day 360. (B) T-cell response to the MVA vector. Box plots show levels of vaccinia-specific CD8 T cells on a square-root scale. Colored lines indicate DL and the fitted means are shown as detailed in Figure 2. (C) MVA-specific neutralizing antibodies. The right plot shows MVA specific NT for DL2 and DL3 cohorts, expressed as inhibition dilution (ID₅₀), which is the serum dilution that caused 50% reduction in fluorescence. Box plots cover central 50% of observations, and the central bars show median; whiskers extend to at most 1.5 times box length. All patients are individually shown by a line, as specified in the legend; “pre72” indicates that the subject received smallpox vaccination being born before 1972, during the compulsory smallpox vaccination campaign. The percentage of infection neutralization (ID inhibition dilution) was calculated as follows: (1 − [percentage of fluorescence in Epstein-Barr virus transformed lymphoblastoid cell line cells incubated with serum from vaccinated subject/[percentage of fluorescence in untreated Epstein-Barr virus transformed lymphoblastoid cell line controls]) × 100. Dilutions for each sample ranged from 1:20 to 1:1000. The ID₅₀ is the serum dilution that caused 50% reduction in fluorescence and was calculated by determining the linear slope of the graph plotting ID versus serum dilution by using the next higher and lower ID values that were closest to 50% neutralization.