Monofunctional and polyfunctional CD8+ T cell activities to CSP and AMA1 after DNA/Ad (Panels A and B) and AdCA (Panels C and D) immunization are shown as color-coded filled circles that represent the percent of CD8+ T cells containing cytokine(s). Black horizontal bar denotes highest activities in DNA/Ad compared with AdCA activities. Activities to individual CSP and AMA1 peptide pools are shown in Figures S1 and S2. Panel A: DNA/Ad CSP: IFN-γ monofunctional activities of protected v11 and v18 were higher (v11, 0.14%; v18, 0.1%) than the two positive non-protected volunteers (v03, 0.07%; v15, 0.09%). Non-protected v15 developed the highest IFN-γ/IL2 polyfunctional (0.20%) and IL2 monofunctional (0.5%) activities. Protected v11 developed positive IFN-γ/IL2 polyfunctional (0.07%) and IL2 monofunctional (0.06%) activities. Panel B: DNA/Ad AMA1: IFN-γ monofunctional activities of protected v10, v11 and v18 (v10, 0.16%; v11, 0.58%, v18, 0.49%) were higher than the eight non-protected volunteers (range 0.05–0.14%). Non-protected v17 and protected v10 also developed lower TNF-α monofunctional (0.07%, 0.09%) activity; v11 also developed IFN-γ/TNF-α polyfunctional (0.31%) activity and lower IFN-γ/IL2 polyfunctional (0.04%), and IFN-γ/IL2/TNF-α polyfunctional (0.05%) activities. Panel C: AdCA CSP: 11 volunteers developed IFN-γ monofunctional activities (0.05%–0.48%), of whom one volunteer developed IFN-γ/IL2 polyfunctional, three developed IL2 monofunctional and two additional volunteers also developed IL2 monofunctional activities. Four volunteers had higher IFN-γ and two volunteers had higher IL2 monofunctional activities than protected volunteers in DNA/Ad. Panel D: AdCA AMA1: all 16 volunteers developed positive IFN-γ monofunctional – boxed volunteers are above scale (1.60% and 1.79% respectively), of whom nine developed lower IFN-γ/IL2 polyfunctional or IFN-γ/TNF-α polyfunctional activities; three volunteers developed IL2 monofunctional and TNF-α monofunctional activities. Four volunteers had higher IFN-γ monofunctional and eight volunteers had higher IFN-γ/IL2 polyfunctional activities than protected volunteers in the DNA/Ad trial.