Figure 1, 2, 3, 4, 5, 6 and supplementary figure 1

<b>Figure 1.</b> Dynamics of antibody responses to ProC6C constituent antigens. Antibody reactivity to the ProC6C constituent antigens, Pfs230-Pro and Pfs48/45-6C, in individuals immunized with ProC6C-AlOH/Matrix-M (G2D, geometric mean titer, GMT-blue line) or a control vaccine (G2E, GMT-black line) at each study time point (D0, D14, D42, D70, D140, and D180). GMT is indicated in Table 1. <b>Figure 2.</b> Biological activity of antibodies. Significant TRA denoted by a closed circles and non-significant TRA by open circles. A) The biological activity (functionality) for the ProC6C-AlOH/Matrix-M (G2D) and control group (G2E) is plotted for each volunteer at D70 and D180 from purified IgG at 15 mg/mL in the SMFA (with human complement) as TRA. The median for each group is indicated by a line and p values were determined by Mann Whitney test. Paired aligned dot plots showing the anti-Pfs48/45-6C (B) and anti-Pfs230-Pro (C) antibody levels at D70 and D180. Individuals were categorized into 3 subgroups based on their TRA levels: “a” significant TRA at both time points; “b” significant TRA at D70 only; and “c” insignificant TRA at both timepoints. Pairwise comparisons were performed using the Paired t test (log-transformed ELISA titers were used for all analyses) D) Pfs48/45-6C IgG concentrations TB31F equivalence (μg/mL) at D70 and D180 for each subgroup (“+” significant TRA and “-“ non-significant TRA). GMT indicated by horizontal line. TB31F threshold of 2.1 mg/mL indicated by red hashed line. Unpaired t-tests were utilized. E) Pfs230-Pro IgG arbitrary units (a.u.) at D70 and D180 for each subgroup (“+” significant TRA and “-“ non-significant TRA). GMT indicated by horizontal line. Unpaired t-tests were utilized; **** p &lt; 0.0001, *** p &lt; 0.001, ** p &lt; 0.01, * p &lt; 0.05, ns p &gt; 0.05. <b>Figure 3.</b> Correlation between biological activity and Pfs48/45-6C and Pfs230-Pro specific antibodies. G2D individuals indicated by closed blue circles and G2E individuals indicated by closed black circles. A) Correlation of anti-Pfs48/45-6C serum antibodies with TRA at D70 and D180 in ProC6C-AlOH/Matrix-M vaccinated individuals (G2D). B) Correlation of Pfs230-Pro antibodies with TRA at D70 and D180 in ProC6C-AlOH/Matrix-M vaccinated individuals (G2D). C) Correlation of anti-Pfs48/45-6C serum antibodies with TRA at D70 and D180 in control vaccinated individuals (G2E). D) Correlation of Pfs230-Pro antibodies with TRA at D70 and D180 in control vaccinated individuals. The biological activity is plotted in log of mean oocyst ratio (LMR) between control and test IgGs (y axis) respective to square root (sqrt) of antibody levels of Pfs48/45-6C or Pfs230-Pro (x axis). For ease of comprehension, the y axis shows corresponding percent TRA values instead of LMR. Correlations were assessed using Spearman’s rank correlation. The p value and correlation coefficient (r) in each panel are shown. <b>Figure 4.</b> Correlation between antibodies against epitope I (competition) and total Pfs48/45-6C IgG and TRA. D70 and D180 epitope I antibody reactivity was determined in volunteers vaccinated with ProC6C-AlOH/Matrix-M (G2D) using the TB31F competition ELISA. The square root (sqrt) of D70 antibody levels of Pfs48/45-6C IgG (y axis) is plotted against the sqrt of epitope I IgG. Antibody levels are given as TB31F equivalence (μg/mL) for Pfs48/45-6C IgG. The coefficient of correlation and p value using Spearman’s rank test are provided. The red dotted line shows a y=x. <b>Figure 5.</b> Receiver Operating Characteristic (ROC) curves. Receiver operating characteristic curves were generated for the square root of IgG: anti-Pfs48/45-6C (A) and anti-Epitope I (B) relative to significant TRA. A) Anti-Pfs48/45-6C demonstrated an AUC of 0.8933 and a threshold of 8.7 mg/mL. B) Anti-Epitope I demonstrated an AUC of 0.8267 a threshold of 5.5 mg/mL. In contrast Pfs230-Pro IgG did not demonstrate a high level of specificity and sensitivity (Supplementary Figure S1). Youden’s topleft analysis was utilized to determine the optimal threshold on the ROC curve as indicated by dot and light dotted lines. Black dotted line y=x. <b>Figure 6.</b> Functional activity of ProC6C IgG is independent of complement. The TRA of ProC6C-AlOH/Matrix-M (G2D) sera is plotted for each volunteer at D70 with (+C) and without (-C) human complement. p values determined by Wilcoxon matched-pairs signed rank test; ns, p &gt; 0.05.<b>Supplementary Figure S1. </b>ROC Plot of Square Root Pfs230-Pro antibody IgG concentration and TRA based on significant TRA. Pfs230-Pro demonstrated an AUC of 0.628 which was not significant (<i>p </i>= 0.222<i>) </i>with a threshold of 18.9 mg/mL anti-Pfs230-Pro IgG.