Additional file 1 of Cellular and antibody response in GMZ2-vaccinated Gabonese volunteers in a controlled human malaria infection trial

Additional file 1: Figure S1a. Gating strategies for cytokine producing CD4+ T cell identification. Figure S1b. Gating strategy for GMZ2-reactive memory B cell identification. Figure S2. Estimated number of CD4+ T cells producing cytokines on unstimulated, vaccine antigen GMZ2 and Staphylococcal endoterotoxin B (SEB) stimulated cells following immunization. Symbols represent individual samples in unstimulated, GMZ2 stimulated and SEB-stimulated conditions. All time points per volunteer were measured in a single experiment after several optimization tests, and individual volunteers were measured in separate experiments. Red lines represent median values with interquartile range. p value lower than 0.05 is considered significant. Figure S3. Estimated number of B cells with or without GMZ2-reactivity following immunization. Symbols represent individual samples. All time points per volunteer were measured in a single experiment after several optimization tests, and individual volunteers were measured in separate experiments. Red lines represent the median values with interquartile range. p value lower than 0.05 is considered significant. Figure S4. Association between pre/post-immunization GMZ2-specific immune cells and trial outcome. Dot plot graphs show the relation between the estimated number of pre/post-immunization GMZ2 stimulated cytokine producing CD4+ T cells (upper side), the number of and B cells subsets (bottom side) regarding clinical malaria status after CHMI. Monotone increase of parasitemia with symptoms (Malaria) is represented by black spots. Low oscillating parasitemia with no symptoms (Control) plus individuals with neither parasitemia nor symptoms (Protected) are represented by open circles. Comparison of the cell number of GMZ2 stimulated CD4+ T cells, of CD20+ B cells and the GMZ2-specific B subsets was performed using Mann-Whitney (for T cells) or unpaired t-tests (for B cells). Data are from a single experiment after several optimization tests, and individual volunteers were measured in separate experiments. Symbols represent individual samples. Figure S5. Post-immunization cell frequencies and the time to treatment after CHMI. Graphs show the time to first malaria treatment regarding the fraction of specific triple and double positive CD4+ T cells, total B cells and the CD27+/- cluster subsets of GMZ2-specific within CD20+IgG+ B cells (a), or the number of GMZ2-stimulated triple and double positive CD4+ T cells, and the number of total B cells and different GMZ2+B cells (b) at D84. Values above the median are represented in red whereas data below the median are shown in blue. The Log-rank test was used to compare the two curves. p value lower than 0.05 is considered significant. Figure S6. Correlation between the estimated number of B cell phenotypes and the anti-GMZ2 IgG concentration at baseline. The association between the estimated number of CD20+IgG+ B cells, the estimated number of GMZ2-specific B cells and the anti-GMZ2 IgG concentration, was performed on D0 data using Pearson’s correlation after log transformation. Data are from a single experiment after several optimization tests, and individual volunteers were measured in separate experiments. Symbols represent individual samples. A p-value less than 0.05 is considered as statistically significant. Figure S7. B cell phenotypes frequency following immunization regarding vaccine intervention. Frequencies of CD20+IgG positive, CD20+IgG+GMZ2-specific and GMZ2-specific CD27+/- B cells between D0 and D84 are compared for all volunteers regarding vaccine intervention. Vaccinated subjects with Rabies control vaccine are represented with opened squares. GMZ2 vaccinated discriminate vaccinees receiving 100µg GMZ2-Alhydrogel (opened dots), 30µg GMZ2-CAF01 (grey dots), and 100µg GMZ2-CAF01 (black dots). Wilcoxon test following by Bonferroni correction for multiple comparison is performed to test statistical significance. p value below 0.05 is considered statistically significant. Data are from a single experiment after several optimization tests, and individual volunteers were measured in separate experiments. Symbols represent individual samples. Red lines represent the median values with interquartile range. p value lower than 0.05 is considered significant. Table S1. Cox proportional analysis post-immunization