Fig 1.
Clinical trial layout for study participants and vaccination strategy with sampling timeline.
(A) The flowchart depicts the screening and enrollment of healthy volunteers in the study. Enrolled study participants were divided into seven vaccine cohorts. Study participant dropouts and total missed visits are depicted. (B) Schematic of vaccination strategy and sampling schedule for enrolled study participants. Peripheral blood and serum samples were taken at the time-points indicated by blood drop symbol. See Methods for vaccination details.
Table 1.
Cohort characteristics.
Table 2.
Summary of registered adverse events over the entire study period.
Fig 2.
Clinical laboratory assessment following concomitant flavivirus vaccination.
(A) Total number of YFV RNA copies/mL serum in cohorts receiving YFV vaccine. (B) Comparison of day 7 YFV RNA copies/mL between cohorts (plot depicts median with IQR). (C) Median clinical chemistry data-points during first four time-points of the study. Grey backgrounds denote the normal range in healthy adults. (D) Total numbers of CD45+, CD4+, and CD8+ cells over time following vaccination (left panels) and comparison of day 14 median absolute cell numbers in the respective vaccine cohorts (right panels, plots depict median with IQR). (E) Heat-map of fold change of serum TNF and IL-18 levels following the first vaccine dose. The legend denotes color-coding of the seven cohorts. Statistical analyses in (A), (C), and (E) were performed using nonparametric Friedman test with Dunn’s multiple comparison tests, in (B) and (D day 14 plots), using nonparametric Kruskal-Wallis test with Dunn’s multiple comparison tests, and in (D timeline) using nonparametric Wilcoxon matched-pairs signed-rank tests. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 3.
Seroconversion and development of neutralizing antibody following concomitant flavivirus vaccination.
(A) TBEV specific IgG levels before and after concomitant vaccination with TBEV and YFV or only TBEV. The dotted line denotes manufacturer’s positive threshold (120 Vienna units) and samples ≥1000 Vienna units (upper limit of quantification) were plotted at 1000 x 2 Vienna units. (B) Comparisons of day 30 and final TBEV-specific IgG levels between cohorts A1, A2 and C. (C) JEV specific IgG levels before and after concomitant vaccination with JEV and YFV or only JEV. The dotted line denotes manufacturer’s positive threshold (20 RU/ml) and samples ≥200 RU/mL were plotted at 200 RU/mL. (D) Comparisons of day 30 and final JEV-specific IgG levels between cohorts B1, B2 and D. (E) TBEV neutralizing antibody titers before and at days 30 and 210 after concomitant vaccination with TBEV and YFV or only TBEV. Study participants with nAbs detected at day 0 (A1, n = 1; A2, n = 1) excluded from analyses. The dotted line denotes positive threshold (≥ 5). (F) Comparisons of final nAb titers against TBEV between cohorts (percent seropositive individuals as denoted above). (G) JEV neutralizing antibody titers before and at days 30 and 60 after concomitant vaccination against JEV and YFV or only JEV. Dotted line denotes positive threshold (≥ 5). (H) Comparisons of final nAbs titers against JEV between cohorts (percent seropositive individuals as denoted above). (I) YFV neutralizing antibody titers before and at days 30 and 60 after concomitant vaccination with TBEV or JEV and YFV, or only YFV. The dotted line denotes positive threshold (≥ 5). (J) Comparisons of final nAbs titers against YFV between respective cohorts. (K) Comparison of virus-specific nAb-based seroconversion rates between same or different arms of YFV and TBEV or JEV vaccinated cohorts. The legend denotes color coding of the seven cohorts. All plots are depicted with geometric mean values. Statistical analyses in (A), (C), (E), (G), and (I) were performed using nonparametric Friedman test with Dunn’s multiple comparison tests, in (B), (D), (F), (H), and (J) using nonparametric Kruskal-Wallis test with Dunn’s multiple comparison tests and in (K) Fisher’s exact test. ns ≥ 0.05, *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 4.
B cell, T cell and NK cell activation following concomitant flavivirus vaccination.
(A) Representative gating strategy identifying B cells, CD4+ and CD8+ T cells as well as CD56dim NK cells. Representative FACS plot of CD38+ Ki67+ (B) B cells, (C) CD4+ T cells, (D) CD8+ T cells, and (E) CD56dim NK cells (left panels) with corresponding plot of the respective cohorts’ frequency of activation medians before and following vaccination program (middle panels). Plots comparing peak activation at day 14 between the vaccination cohorts (right panels depicting median values with IQR). The legend denotes color-coding of the seven cohorts. Statistical analyses were performed using nonparametric Kruskal-Wallis test with Dunn’s multiple comparison tests. *p < 0.05 and ***p < 0.001.
Fig 5.
Germinal center activity and plasmablast expansion following concomitant flavivirus vaccination.
(A) Median CXCL13 serum fold changes over time following vaccination program (left panel), and comparison of peak levels between cohorts at days 7 and 14 (right panel). (B) Representative gating strategy for identifying plasmablasts. (C) Median plasmablast expansion over time following vaccination and (D) comparison of peak expansion between cohorts at day 14. The legend denotes color-coding of the seven cohorts. All plots are depicted with median values with IQR. Statistical analyses were performed using nonparametric Kruskal-Wallis test with Dunn’s multiple comparison tests. *p < 0.05, **p < 0.01, ***p < 0.001.