Cellular immune responses by vaccine group and days post immunization.

<p>A. Geometric mean CS-specific CD4⁺ T cells producing IFN-γ by ELISpot. Comparisons between ARR and RRR on days 42, 77, 105,140, and 236 were significant (P < .05) by Student’s <i>t</i>-test on log-transformed data. Error bars represent standard deviation of the mean. B. Geometric mean CS-specific CD4⁺ T cell responses expressing ≥2 cytokine/activation markers among IL2, IFN-γ, TNF-α, and CD40L, per million PBMC. Comparisons between ARR and RRR on days 42, 77, 105, 140 were significant (P < .05) by Student’s <i>t</i>-test on log-transformed data. Error bars represent standard deviation of the mean. C. Pie charts showing the pattern of CS-specific polyfunctional CD4⁺ CD40L⁺ T cells expressing ≥2 cytokine markers at Day 77 (day of challenge). Each slice within the pie chart represents a specific combination of two or three cytokines. The size of the pie chart is proportional to the magnitude of the response.</p

Vaccine efficacy against positive blood slide parasitemia after sporozoite challenge (ATP cohort for vaccine efficacy).

<p>Incremental VE of ARR over RRR, -17.6 (-107.4–33.3); P = .768</p><p>ARR; first dose with Ad35.CS, second and third doses with RTS,S/AS01B</p><p>RRR; three doses of RTS,S/AS01B</p><p>VE (%), Vaccine Efficacy</p><p>CI, 95% Lower and Upper Confidence Intervals</p><p>P-value; Two-sided Fisher Exact Test</p><p>Vaccine efficacy; 100*(1 –attack rate ARR/ attack rate RRR)</p><p>Vaccine efficacy against positive blood slide parasitemia after sporozoite challenge (ATP cohort for vaccine efficacy).</p

Flow diagram.

<p>The number of subjects completing each immunization and controlled human malaria infection. ARR = first dose with Ad35.CS, second and third doses with RTS,S/AS01_B. RRR = three doses of RTS,S/AS01_B</p

Seropositivity rates and Geometric Mean Titer (GMT) for anti-CS antibodies overall and by protection status (ATP cohort for immunogenicity).

<p>ARR = first dose with Ad35.CS, and the second and third doses with RTS,S/AS01_B</p><p>RRR = three doses with RTS,S/AS01_B</p><p>NP = non-protected</p><p>P = protected</p><p>P values calculated using log-transformed antibody levels in Students t-test</p><p>Seropositivity rates and Geometric Mean Titer (GMT) for anti-CS antibodies overall and by protection status (ATP cohort for immunogenicity).</p

Reverse cumulative distribution curves by post challenge protection status for CS anti-repeat antibodies on day 77-Day of challenge for subjects in ARR protected (solid red) and non-protected (dashed red) compared to RRR protected (solid black) and non-protected (dashed black) subjects.

<p><i>Y</i>-axis represents proportion of subjects at each antibody level.</p

Frequency of CS-specific CD4+ T cells expressing at least 2 cytokines/activation markers per million PBMC, by treatment group and protection status (ATP cohort for immunogenicity).

<p>ARR = first dose with Ad35.CS, and the second and third doses with RTS,S/AS01_B</p><p>RRR = three doses with RTS,S/AS01_B</p><p>P values calculated using log-transformed cell frequency per million PBMC in Students t-test</p><p>NP = non-protected; P = protected</p><p>Frequency of CS-specific CD4+ T cells expressing at least 2 cytokines/activation markers per million PBMC, by treatment group and protection status (ATP cohort for immunogenicity).</p

Trial design.

<p>Subjects were immunized week 0, 4, 8 and 24 and challenged week 28 (blue arrows). Samples for measuring cell-mediated immunity (ELISpot assay and flow cytometry) were collected at six time points (black arrows), and for measuring antibody levels (ELISA, IFA and growth inhibition assay) at similar time points plus after the DNA immunizations (gray arrows). See text for details.</p

Development of parasitemia in the immunized and infectivity control subjects.

<p><b>Panel A</b>: Parasitemia-free survival curves (Kaplan-Meier) for immunized volunteers and infectivity controls based on microscopic examination of peripheral blood smears. <b>Panel B</b>: Quantitative(q)-PCR measurements of parasitemia in immunized and challenge controls (error bars show standard deviation) (see reference 28).</p

Rank correlations between pre-existing anti-Ad5 NAb titers and ELISpot, CD4+ T cell and CD8+ T cell IFN-γ activities, ELISA and Sporozoite IFA titers.

<p>Pre-existing Ad5 NAb titers measured just prior to Ad immunization were tested for negative correlations with CSP and AMA1 activities by IFN-γ ELISpot, total IFN-γ CD4+ T cell ICS, total IFN-γ CD8+ T cell ICS, ELISA and sporozoite IFA for all volunteers (n = 15). r = rank correlation coefficient, and p = p value for the null hypothesis that the correlation is zero (two-tailed). Significant correlations are shown in bold.</p

Schematic of DNA and Adenovirus CSP and AMA1 vaccines.

<p>Each panel presents the native protein (top of each panel) and the protein expressed by the DNA or Ad construct (middle and bottom of each panel) for the CSP (Panel <b>A</b>) and AMA1 (Panel <b>B</b>) vaccine antigens. N = amino terminus; C = carboxy terminus; TPA = human tissue plasminogen activator signal sequence; TM = transmembrane domain. See text for explanation. Identical colors indicate identical sequences. Not represented is a single amino acid substitution (G → R) in the AMA DNA construct at position 143.</p