Activation and Kinetics of Circulating T Follicular Helper Cells, Specific Plasmablast Response, and Development of Neutralizing Antibodies following Yellow Fever Virus Vaccination

Abstract

A single dose of the replication-competent, live-attenuated yellow fever virus (YFV) 17D vaccine provides lifelong immunity against human YFV infection. The magnitude, kinetics, and specificity of B cell responses to YFV 17D are relatively less understood than T cell responses. In this clinical study, we focused on early immune events critical for the development of humoral immunity to YFV 17D vaccination in 24 study subjects. More specifically, we studied the dynamics of several immune cell populations over time and the development of neutralizing Abs. At 7 d following vaccination, YFV RNA in serum as well as several antiviral proteins were detected as a sign of YFV 17D replication. Activation of Th1-polarized circulating T follicular helper cells followed germinal center activity, the latter assessed by the surrogate marker CXCL13 in serum. This coincided with a plasmablast expansion peaking at day 14 before returning to baseline levels at day 28. FluoroSpot-based analysis confirmed that plasmablasts were specific to the YFV-E protein. The frequencies of plasmablasts correlated with the magnitude of neutralizing Ab titers measured at day 90, suggesting that this transient B cell subset could be used as an early marker of induction of protective immunity. Additionally, YFV-specific memory B cells were readily detectable at 28 and 90 d following vaccination, and all study subjects tested developed protective neutralizing Ab titers. Taken together, these studies provide insights into key immune events leading to human B cell immunity following vaccination with the YFV 17D vaccine.

FIGURE 1.

Vaccination strategy and detection of viral RNA and immune response–related proteins in serum following YFV 17D vaccination. (A) Vaccination and sampling schedule for all study subjects (n = 24). (B) YFV NS5 copies in serum as detected by RT-PCR. Dotted line at y = 20 denotes limit of detection. Plots represent median with 95% confidence interval (CI) (n = 14). (C) Fold change day 7 and day 14, in comparison with day 0, of 87 immune response–related proteins (n = 10). Statistical analysis was performed using nonparametric Friedman test and corrected for multiple comparisons with Dunn multiple comparison test. The protein comparison p values were adjusted by false discovery rate using the Benjamini–Hochberg method with Q value set at 5%.*p < 0.05.

FIGURE 2.

Magnitude and kinetics of NK, T, and B cell activation following YFV 17D vaccination. (A) CD38 and Ki67 coexpression on freshly isolated CD56^bright and CD56^dim cells over time in one representative study subject. Plot of CD38 and Ki67 coexpression on (B) CD56^bright and (C) CD56^dim cells at days 0, 7, 14, 28, and 90 following vaccination. (D) CD38 and Ki67 coexpression on freshly isolated CD8⁺ and CD4⁺ T cells over time in one representative study subject. Plots of CD38 and Ki67 coexpression on (E) CD8⁺ T cells and (F) CD4⁺ T cells at days 0, 7, 14, 28, and 90 following vaccination. (G) CD38 and Ki67 coexpression on freshly isolated CD19⁺ B cells over time in one representative study subject. (H) Plots of CD38 and Ki67 coexpression on B cells at days 0, 7, 14, 28, and 90 after vaccination. All plots represent median with 95% confidence interval (CI). For all plots: day 0, n = 24; day 7, n = 15; day 14, n = 22; day 28, n = 18; and day 90, n = 12. Statistical analysis was performed using nonparametric Wilcoxon matched-pairs signed-rank test. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 3.

cTfh cells and germinal center activity following YFV 17D vaccination. (A) Gating strategy of cTfh cells defined as CXCR5⁺ CD4⁺ T cells with Th1-polarized CXCR3⁺ and Th2/17-polarized CXCR3⁻ subsets. (B) Total cTfh, (C) Th1-polarized CXCR3⁺ cTfh, and (D) CXCR3⁻ cTfh cell frequencies of CD4⁺ T cells over time following vaccination. (E) Representative gating of frequencies of Th1-polarized CXCR3⁺ cTfh cells expressing CCR7, Ki67, ICOS, and PD1. (F) Expression of CCR7 and activation of Th1-polarized CXCR3⁺ cTfh cells over time as assessed by dual expression of (G) PD1 and Ki67, (H) ICOS and Ki67, and (I) PD1 and ICOS. Comparison of expression of CCR7, Ki67, ICOS, and PD1 between Th1-polarized CXCR3⁺ (green) and Th2/17-polarized CXCR3⁻ (blue) cTfh cells on (J) day 7 and (K) day 14 following vaccination. (L) Change in germinal center activity over time as defined by serum CXCL13 concentrations determined by ELISA (n = 10). All graphs plotted as median with 95% confidence interval (CI) (n = 8 unless otherwise stated). Statistical analysis was performed using nonparametric Friedman test and corrected for multiple comparisons with Dunn multiple comparison test (B–D, F–I, and L) or nonparametric Wilcoxon matched-pairs signed-rank test. *p < 0.05, **p < 0.01.

FIGURE 4.

Plasmablast responses following YFV 17D vaccination. (A) CD38^high and CD27^high coexpression on CD19⁺CD20^−/low B cells define plasmablasts in freshly isolated PBMCs over time. Displayed is one representative study subject along with IgA, IgG, and Ki67 expression on each subset. (B) Total plasmablast and plasmablast (C) IgA⁺, (D) IgG⁺, and (E) IgA⁻IgG⁻ subset frequencies. Median with 95% confidence interval (CI) are plotted at days 0, 7, 14, 28, and 90 following vaccination. (F) Proportions of Ig expression shift on plasmablasts over time. (G) Ki67 expression in total plasmablasts and (H) IgA⁺, (I) IgG⁺, and (J) IgA⁻IgG⁻ and subsets plotted as median with 95% CI over time. For all plasmablast graphs, excluding IgA⁺, IgA⁺ Ki67⁺, IgA⁻IgG⁻, and IgA⁻IgG⁻ Ki67⁺ subsets, day 0, n = 24; day 7, n = 15; day 14, n = 22; day 28, n = 18; and day 90, n = 12. For IgA⁺ and Ki67⁺ IgA⁺ plasmablasts, day 0, n = 11; day 7, n = 15; day 14, n = 20; day 28, n = 16; and day 90, n = 10. Statistical analysis was performed using nonparametric Wilcoxon matched-pairs signed-rank test. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 5.

Specificity of plasmablasts following YFV 17D vaccination. (A) Total IgG⁺ plasmablasts (yellow), IgA⁺ plasmablasts (green), and YFV-E–specific IgG⁺ plasmablasts (red). Shown are FluoroSpot wells from a representative study subject’s cryopreserved PBMCs over time following vaccination with YFV 17D. (B) Total number of IgA⁺ and (C) IgG⁺ Ab-secreting cells over time following vaccination with YFV 17D. (D) Kinetics of YFV-E–specific IgG⁺ plasmablasts per million PBMCs at days 0, 7, 14, and 28. (B and C) Median is plotted with 95% confidence interval (CI) (n = 8). Statistical analysis was performed using nonparametric Friedman test and corrected for multiple comparisons with Dunn multiple comparison test. *p < 0.05, ***p < 0.001.

FIGURE 6.

Neutralizing Ab titers correlation with IgG⁺ plasmablast frequencies following YFV 17D vaccination. (A) Kinetics of development of neutralizing Ab titers over time (day 0, n = 21; day 28, n = 17; and day 90, n = 12). (B) Correlation between ED₉₀ titers at day 90 and day 14 frequencies of IgG⁺ plasmablasts (n = 10). Statistical analysis was performed using nonparametric Wilcoxon matched-pairs signed-rank test and nonparametric Spearman correlation test. ***p < 0.001.

FIGURE 7.

Kinetics and specificity of memory B cells following YFV 17D vaccination. (A) The B cell compartment was divided into CD27⁺IgD⁻ memory B cells, CD27⁺IgD⁺ unswitched memory B cells, CD27⁻IgD⁻ naive B cells, and CD27⁻IgD⁻ double-negative B cells over time. Memory B cells were further defined by their class-switched IgA or IgG expression. (B) Total memory B cells, (C) IgG⁺ memory B cell, and (D) IgA⁺ memory B cell frequencies following vaccination with YFV 17D. (E) Total unswitched memory B cell, (F) naive B cell, and (G) double-negative B cell frequencies over time. (H) Representative FluoroSpot wells at days 0, 28, and 90 detecting YFV-E⁺ IgG⁺ memory B cells. (I) The number of detectable YFV-E⁺ IgG⁺ memory B cells before and after vaccination at days 28 and 90 (n = 8). All data plotted as median with 95% confidence interval (CI) at days 0, 7, 14, and 28 after vaccination (n = 8). Statistical analysis was performed using nonparametric Friedman test and corrected for multiple comparisons with Dunn multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 8.

Homing characteristics of memory B cells following YFV 17D vaccination. (A) Gating strategy of homing receptor expression on memory B cells and IgG⁺ subsets from a representative study subject. Frequency of CCR7, CXCR3, and CXCR5 expression on memory B cells (B–D) and IgG⁺ memory B cells (E–G) at days 0, 7, 14, and 28 after vaccination with YFV 17D (n = 8). In all graphs, the median is plotted with 95% confidence interval (CI). Statistical analysis was performed using nonparametric Wilcoxon matched-pairs signed-rank test. *p < 0.05, **p < 0.01, ***p < 0.001.