Circulating follicular T helper cells and cytokine profile in humans following vaccination with the rVSV-ZEBOV Ebola vaccine

Abstract

The most recent Zaire Ebolavirus (ZEBOV) outbreak was the largest and most widespread in recorded history, emphasizing the need for an effective vaccine. Here, we analyzed human cellular immune responses induced by a single dose of the rVSV-ZEBOV vaccine candidate, which showed significant protective efficacy in endemic populations in Guinea. This is the first in-depth characterization of ZEBOV-GP specific, circulating follicular T cells (cTfh). Since antibody titers correlated with protection in preclinical models of ZEBOV infection, Tfh were predicted to correlate with protection. Indeed, the ZEBOV-specific cTfh data correlated with antibody titers in human vaccines and unexpectedly with the Tfh17 subset. The combination of two cutting edge technologies allowed the immuno-profiling of rare cell populations and may help elucidate correlates of protection for a variety of vaccines.

Figure 1. Clustering of net cytokine signatures in response to rVSV-ZEBOV vaccination.

Correlogram depicts the relationships between cytokines produced by PBMC from subjects in Cohort 1 (Panel a), Cohort 2 (Panel b) and Cohort 3 (Panel c) in response to stimulation with ZEBOV-GP-peptides. Colors indicate the level of correlation (blue – positive, red – negative, white – no correlation). See Table 1 for actual cytokine data.

Figure 2. Frequency of ZEBOV-GP-specific cTfh increases after vaccination in peripheral blood.

Cohorts 1, 2 and 3 received a single IM inoculation of 3 × 10⁶, 2 × 10⁷ or 1 × 10⁸ pfu, respectively. Lymphocytes from Day 0, Day 28 and Day 56 were stimulated with EBOV peptides and stained for the expression of CD3, CD4, CD154, CXCR5. Box plot represent n = 10 subjects per vaccine cohort and time point. Frequencies reported here are the percentage CD154⁺ (ZEBOV-specific) CD4⁺ CXCR5⁺ cells within viable CD3⁺ population of PBMC. Statistical differences between vaccine cohorts were found on Day 28 (p = 0.004, ANOVA) and Day 56 (p = 0.013, ANOVA).

Figure 3. Kinetic of ZEBOV-GP-specific cTfh as a function of vaccine dose and time.

Cohorts 1, 2 and 3 received a single IM inoculation of 3 × 10⁶, 2 × 10⁷ or 1 × 10⁸ pfu, respectively. Lymphocytes from Day 0, Day 28 and Day 56 were stimulated with ZEBOV-GP- peptides and enriched based on the expression of activation marker CD154. Box plot represent n = 10 subjects per vaccine cohort and time point. See Supplementary Fig. S1 for gating strategy. The frequency reported is percentage of CXCR5⁺ cells within enriched CD4⁺CD154⁺ T cells. The frequencies were statistically different between the cohorts on Day 28 (p < 0.001, ANOVA) and Day 56 (p = 0.009), but not for Day 0 (p = 0.78). Brackets with asterisks indicate statistical differences between time points. See Supplementary Table S1 for detailed statistical analysis.

Figure 4. Changes in ZEBOV-GP-specific Tfh subpopulations as a function of vaccine dose and time.

PBMC enriched based on the expression of activation marker CD154 were subsequently analyzed by flow cytometry for the expression of CD3, CD4, CXCR5 and the subset specific markers CCR6 and CXCR3. Responses in the various cohorts (cohort 1 (Panel a,b), cohort 2 (Panel c,d), cohort 3 (Panel e,f)) are shown for Day 28 (Panel a,c,e) and Day 56 (Panel b,d,f) in box plots. The bold line next to each box plot represents the median frequency of each cTfh population at Day 0. Data expressed as absolute numbers of CD3⁺CD4⁺CXCR5⁺CCR6⁻CXCR3⁻ (Tfh2), CD3⁺CD4⁺CXCR5⁺CCR6⁻CXCR3⁺(Tfh1), and CD3⁺CD4⁺CXCR5⁺CCR6⁺CXCR3⁻(Tfh17) within CD4⁺CD154⁺ T cells (see Supplementary Fig. S1 for gating strategy). See Supplementary Table S2 for detailed statistical analysis.

Figure 5. Correlation between frequency of ZEBOV-GP-specific cTfh cells and antibody titers.

Scatterplots comparing the ELISA titer (measured as ELISA units/mL) and frequency of CD154⁺CD4⁺CXCR5⁺ (cTfh, top row) or frequency of CD154⁺CD4⁺CXCR5⁺CCCR6⁺CXCR3⁻ (cTfh17, bottom row) at Day 28 (left column) and Day 56 (right column). Pearson correlation coefficient R² and p-values are shown. Data from all cohorts were pooled to achieve a large sample size. No correlations between antibody titer and cTfh1 or cTfh2 were observed (Supplementary Fig. 2).