MVA-B-induced HIV-1-specific T cell responses across the study. (A) Chronological diagram showing the vaccination schedule followed in the RISVAC02 study and the immunogenicity endpoints. W6, week 6. (B) Percentage of responders at the different time points. The percentage of responders was calculated on the basis of volunteers with a positive IFN-γ ICS. (C) Magnitudes of vaccine-specific CD4⁺ and CD8⁺ T cells at the different time points. The mean values for the total responses (Env, Gag, and GPN) in each T cell population are shown. The box plots show the distribution of responses in positive responders only. The boxes indicate the median (solid line), mean (dashed line), and interquartile range (IQR). P values for significant differences were determined using the Mann-Whitney U test and are represented. (D) Breadth of CD4⁺ and CD8⁺ T cell responses at the different time points. Percentages of responders that recognized 1, 2, or 3 HIV-1 peptide pools in both T cell subsets are shown. (E) Percentages of CD4⁺ and CD8⁺ T cells producing IFN-γ in response to Env, Gag, or GPN peptide pools as measured by ICS at the different time points. The box plots show the distribution of responses in positive responders only. The boxes indicates the median (solid line), mean (dash line), and IQR. P values for significant differences were determined using the Wilcoxon rank sum test with continuity correction and are represented. All data are background subtracted.

Vaccine-induced T cell responses at primary immunogenicity endpoints (weeks 6 and 18). (A) Magnitude of HIV-1-specific CD4⁺ and CD8⁺ T cells after two and three doses of MVA-B. The mean values for the total responses (Env, Gag, and GPN) in each T cell population are shown. The box plots show the distribution of responses in positive responders only. The boxes indicate the median (solid line), mean (dashed line), and interquartile range (IQR). (B) Percentages of HIV-1-specific T cells secreting cytokines in the CD4 and CD8 T cell subsets. The box plots show the distribution of responses in positive responders only. The boxes indicate the median (solid line), mean (dashed line), and IQR. Data points represent the sum of the frequencies obtained against the Env, Gag, and GPN peptide pools. All data are background subtracted. **, P values of <0.005 determined using the Wilcoxon rank sum test with continuity correction, comparing the secretions of the different cytokines at the same time points.

Functional profile of vaccine-induced CD4 and CD8 T cells. The results shown are generated from the determinations in responders at weeks 6 and 18. All the possible combinations of the responses are shown on the x axis, whereas the percentage of the functionally distinct cell populations within the total CD4 and CD8 T cell populations are shown on the y axis. Responses are grouped and color coded on the basis of the number of functions. The bars correspond to the individual data points and interquartile ranges (IQR) after 2 (W6) or 3 (W18) doses of MVA-B. The pie charts showed the average proportion of the CD4 or CD8 vaccine-specific T cell responses according to the functions.

Phenotype of long-lived memory vaccine-induced T cell responses. (A) Distribution of HIV-1 antigen-specific T cells at week 48 based on CCR7 expression in combination with CD45RA. The bars correspond to the individual data points and interquartile ranges (IQR) of the CD4⁺ and CD8⁺ T cell responses against Env, Gag, and GPN with phenotype central memory (TCM; CD45RA⁻ CCR7⁺), effector memory (TEM; CD45RA⁻ CCR7⁻), or terminally differentiated effector memory (TEMRA; CD45RA⁺ CCR7⁻). The pie charts showed the average proportion of the CD4⁺ or CD8⁺ vaccine-specific T cell responses according to the memory phenotype. *, distributions that are different from the CD4 T cell subset at P values of <0.05 (Student's t test). All data are background subtracted. (B) Representative phenotypic profiles of long-lived memory HIV-1-specific CD4 and CD8 T cells. Fresh PBMCs obtained from the responder volunteers at week 48 were stimulated with Env, Gag, or GPN peptide pools. The red dots indicate antigen-specific (IL-2 and IFN-γ) vaccine-induced CD4⁺ T cells, and blue dots indicate antigen-specific (IL-2 and IFN-γ) vaccine-induced CD8⁺ T cells, both overlaid on the total T cell subsets (gray). Neg, background values in unstimulated cells.

Antivector-induced T cell responses across the study. (A) Percentages of CD4⁺ and CD8⁺ T cells producing IFN-γ against MVA-infected cells as measured by ICS at the different time points. The box plots show the distribution of responses in positive responders at weeks 6 and 18. The boxes indicate the median (solid line), mean (dashed line), and interquartile range (IQR). All data are background subtracted. P values for significant differences were determined using the Wilcoxon rank sum test with continuity correction and are represented. (B) Functional profile of MVA-specific CD8 T cells. The results shown are generated from the determinations in all the responders. All the possible combinations of the responses are shown on the x axis, whereas the percentages of the functionally distinct cell populations within the total CD8 T cell populations are shown on the y axis. Responses are grouped and color coded on the basis of the number of functions. The bars correspond to the individual data points and IQR after 2 (week 6 [W6]) or 3 (W18) doses of MVA-B. The pie charts show the average proportion of the MVA-specific CD8⁺ T cell responses according to the functions. (C) Phenotype of long-lived memory MVA-specific T cell responses. Distribution of MVA-specific T cells at week 48 based on CCR7 expression in combination with CD45RA. The bars correspond to the individual data points and IQR of the CD4⁺ and CD8⁺ T cell responses against MVA-infected cells with phenotype central memory (TCM; CD45RA⁻ CCR7⁺), effector memory (TEM; CD45RA⁻ CCR7⁻), or terminal effector memory (TEMRA; CD45RA⁺ CCR7⁻). The pie charts show the average proportions of the CD4⁺ or CD8⁺ MVA-specific T cell responses according to the memory phenotype. *, distributions that are different from the CD4 T cell subset at P values of <0.05 (Student's t test). All data are background subtracted.