Fig 1.
Overview of the vaccine cohorts.
(A) Blood sampling schedule and (B) administration sites for all vaccine cohorts included in the study. The number (n) of study participants in each vaccine cohort is written and applies to all subsequent Figs. (C) Out of the three structural and seven non-structural proteins encoded by the flavivirus genome, capsid (C), envelope (E), and non-structural protein-5 (NS5), were selected as targets for overlapping peptide pool-generation. Peptides, 15-mers with 11 amino acid (aa) overlap, were pooled for the respective protein and the number of peptides for each pool indicated.
Fig 2.
Kinetics of the antigen-specific CD4
+ and CD8+ T cell response in study participants vaccinated against TBEV, JEV, and YFV. (A) Representative flow cytometry gating for the AIM assay used to identify antigen-specific CD4+ T cells (CD69+CD40L+) and antigen-specific CD8+ T cells (CD69+41BB+) after PBMC stimulation with DMSO (negative control), SEB (positive control), and peptide pool (E, C or NS5). (B-D) Frequency of TBEV-, JEV- and YFV-specific E, C, and NS5 CD4+ T cells in the (B) TBEV (cohort C), (C) JEV (cohort D) and (D) YFV vaccinated (cohort E), respectively. (E-G) Frequency of TBEV-, JEV- and YFV-specific E, C, and NS5 CD8+ T cells in the (E) TBEV (cohort C), (F) JEV (cohort D) and (G) YFV vaccinated (cohort E), respectively. (D-G) Statistical analysis assessed by Friedman test, with Siegel and Castellan’s All-Pairs Comparisons post hoc tests with Bonferroni correction. Significant results are shown, where: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.
Fig 3.
Influence of co-vaccination on antigen-specific CD4
+ and CD8+ T cell responses. (A) Frequency of TBEV E-specific CD4+ T cells between study participants from cohort C (TBEV) and cohort A (TBEV+YFV) and (B) frequency of JEV E-specific CD4+ T cells between cohort D (JEV) and cohort B (JEV+YFV). (C) Frequency of TBEV E-specific CD8+ T cells between cohort C and cohort A and (D) frequency of JEV E-specific CD8+ T cells between cohort D and cohort B. (A-D) Statistical analysis assessed by Mann-Whitney rank test at each time point. Distribution of data points is determined by boxplot with median and 25th-75th percentiles; whiskers are drawn from 1.5 times the IQR. Significant results are shown, where: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.
Fig 4.
Influence of co-vaccination on antigen-specific T cell responses upon vaccination in the same or different upper arms.
(A) Frequency of TBEV E-specific CD4+ T cells compared between cohort A1 and A2. (B) Frequency of JEV E-specific CD4+ T cells compared between cohort B1 and B2. (C) Frequency of TBEV E-specific CD8+ T cells compared between cohort A1 and A2. (D) Frequency of JEV E-specific CD8+ T cells compared between cohort B1 and B2. (A-D) Statistical analysis assessed by Mann-Whitney rank test at each time point. Distribution of data points is determined by boxplot with median and 25th-75th percentiles; whiskers are drawn from 1.5 times the IQR. Significant results are shown, where: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.
Fig 5.
Kinetics of the antigen-specific CD4+ memory effector T cell (TEM) responses.
(A) Representative flow cytometry gating of memory CD4+ T cells based on CD45RA and CCR7 expression within bulk CD4+ T cells (left panel) and within CD69+CD40L+ of CD4+ T cells after peptide stimulation (right panel). (B) Frequency of TBEV E-, C- and NS5-specific CD4+ TEM cells in TBEV vaccinated (cohort C). (C) Frequency of TBEV E-, C- and NS5-specific CD4+ TEM cells in YFV+TBEV vaccinated (cohort A). (D) Frequency of JEV E-, C- and NS5-specific CD4+ TEM cells in JEV vaccinated (cohort D). (E) Frequency of JEV E-, C- and NS5-specific CD4+ TEM cells in YFV+JEV vaccinated (cohort B). (F) Frequency of YFV E-, C- and NS5-specific CD4+ TEM cells in YFV vaccinated (cohort E). (B-F) Statistical analysis assessed by Friedman test, with Siegel and Castellan’s All-Pairs Comparisons post hoc tests with Bonferroni correction. Significant results are shown, where: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.
Fig 6.
Cross-reactive E-specific T cell responses in the vaccine cohorts.
(A) Sequence alignments between TBEV, JEV, and YFV. Heatmaps are annotated according to percent of amino acid sequence similarity. (B-C) Frequency of TBEV, JEV, and YFV E-specific CD4+ T cells, and (D-E) TBEV, JEV, and YFV E-specific CD8+ T cells, in study participants from all vaccine cohorts expressed as fold change relative to day 0. Line plots show median and 95% confidence interval (shaded area).