Dose-dependent T-cell Dynamics and Cytokine Cascade Following rVSV-ZEBOV Immunization

Abstract

Background: The recent West African Ebola epidemic led to accelerated efforts to test Ebola vaccine candidates. As part of the World Health Organisation-led VSV Ebola Consortium (VEBCON), we performed a phase I clinical trial investigating rVSV-ZEBOV (a recombinant vesicular stomatitis virus-vectored Ebola vaccine), which has recently demonstrated protection from Ebola virus disease (EVD) in phase III clinical trials and is currently in advanced stages of licensing. So far, correlates of immune protection are incompletely understood and the role of cell-mediated immune responses has not been comprehensively investigated to date.

Methods: We recruited 30 healthy subjects aged 18-55 into an open-label, dose-escalation phase I trial testing three doses of rVSV-ZEBOV (3×10⁵ plaque-forming units (PFU), 3×10⁶ PFU, 2×10⁷ PFU) (ClinicalTrials.gov; NCT02283099). Main study objectives were safety and immunogenicity, while exploratory objectives included lymphocyte dynamics, cell-mediated immunity and cytokine networks, which were assessed using flow cytometry, ELISpot and LUMINEX assay.

Findings: Immunization with rVSV-ZEBOV was well tolerated without serious vaccine-related adverse events. Ebola virus-specific neutralizing antibodies were induced in nearly all individuals. Additionally, vaccinees, particularly within the highest dose cohort, generated Ebola glycoprotein (GP)-specific T cells and initiated a cascade of signaling molecules following stimulation of peripheral blood mononuclear cells with Ebola GP peptides.

Interpretation: In addition to a benign safety and robust humoral immunogenicity profile, subjects immunized with 2×10⁷ PFU elicited higher cellular immune responses and stronger interlocked cytokine networks compared to lower dose groups. To our knowledge these data represent the first detailed cell-mediated immuneprofile of a clinical trial testing rVSV-ZEBOV, which is of particular interest in light of its potential upcoming licensure as the first Ebola vaccine. VEBCON trial Hamburg, Germany (NCT02283099).

Keywords: Cytokines; Ebola vaccine; Humoral and cell-mediated immune responses; Phase I study; T-cell responses; rVSV-ZEBOV.

Graphical abstract

Fig. 1

Trial profile. Flow diagram of screening, enrollment and vaccination in the three cohort groups. For each cohort group, 4 to 5 eligible individuals withdrew from the study. One participant aborted the study after day 28 due to moving abroad (3 × 10⁶ PFU). One subject missed visit day 56 (2 × 10⁷ PFU).

Fig. 2

Local and systemic adverse events. a) Recorded adverse events of vaccinees during 180 days post vaccination. Events are depicted in frequency (%) of participants among respective cohort presenting at least one adverse event of the category. Adverse events with the severity of mild, moderate and severe are grouped into grade 1, 2 and 3, respectively. Cohort 1: 3 × 10⁵ PFU, Cohort 2: 3 × 10⁶ PFU, Cohort 3: 2 × 10⁷ PFU. b) Number of related solicited and unsolicited adverse events reported over time in the 3 × 10⁵ PFU (cohort 1 (green)), 3 × 10⁶ PFU (cohort 2 (blue)), and 2 × 10⁷ PFU (cohort 3 (red)) dose group.

Fig. 3

Ebola virus antibody titers increase following administration of rVSV-ZEBOV. a) Neutralizing antibodies against infectious EBOV isolate Mayinga were analyzed. Analysis was started with a 1:8 dilution. Seropositivity is defined by a GMT > 8. b) Pseudovirion neutralization assay assessing the 50% serum neutralization capacity (PsVNA50) complemented by homologous glycoprotein. a) and b): The results are expressed in neutralization titers. Statistical analysis was performed using Mann-Whitney-Wilcoxon test (*p < 0.05; **p < 0.01; ***p < 0.005) (Graphs: Box and Whiskers, Min to Max, Line: Median).

Fig. 4

Kinetics of plasmablasts and T-cell activation in response to rVSV-ZEBOV immunization. a) Plasmablasts peaked at day 7 post vaccination in all dose groups. B cells were stained using antibodies against CD19, CD24 and CD38. Representative contour plots depict B-cell subsets of naïve (CD19⁺ CD24^low/intermCD38^interm), memory (CD19⁺ CD24^{−/interm/high}CD38^low/interm), transitional (CD19⁺ CD24⁺ CD38^high) and plasmablasts (CD19⁺ CD24⁻ CD38^high). Numbers in contour plots represent the percentage of plasmablasts of CD19⁺ B cells. b) Percentages of CD19⁺ CD24⁻ CD38^high over time in immunized subjects. Each dot and line depicts one subject (3 × 10⁵ PFU: n = 10; 3 × 10⁶ PFU: n = 8; 2 × 10⁷ PFU: n = 9). c) Graph highlights the fold induction of plasmablasts at day 7 compared to day 0. Each dot represents one subject (bar graph, line: median value). d) T-cell activation in subjects early after vaccination. Activation was analyzed by HLA DR and CD38 staining. Representative contour plots depict the gating strategy for activation of CD4⁺ (upper plot) and CD8⁺ T cells (lower plot). e) Percentages of HLA DR⁺ CD38⁺ of CD4⁺ (upper row) and CD8⁺ T cells (lower row) are represented in the graphs. Each dot and line represents one subject. (3 × 10⁵ PFU: n = 10; 3 × 10⁶ PFU: n = 6; 2 × 10⁷ PFU: n = 8). Statistical analysis was performed using Mann-Whitney-Wilcoxon test (*p < 0.05; **p < 0.01; ***p < 0.005).

Fig. 5

Ebola GP-specific T-cell responses. Kinetics of antigen-specific CD8⁺ T-cell responses. a) Four different peptide pools (GP1a, GP1b, GP2, SP) that cover the whole Ebola GP protein (Kikwit) were used for PBMC stimulation. Graphs represent frequencies (%) of total cytokine responses of CD8⁺ T cells (IFNγ, TNFα, IL2, MIP1β). Each dot represents one subject. (3 × 10⁵ PFU: n = 10; 3 × 10⁶ PFU: n = 7; 2 × 10⁷ PFU: n = 8) (Box and Whiskers, Min to Max, Line: median) b) Fold induction of total cytokines of CD8⁺ T cells from day 0 to day 56 (Bar graph, line: median value). c) Piecharts represent composition of cytokines induced by all four peptide pools. They show the average values of CD8⁺ T-cell responses of day 56 of the 2 × 10⁷ PFU dose cohort.

Fig. 6

CTL-responses following rVSV-ZEBOV vaccination. a) Degranulation of CD8⁺ T cells was measured by CD107a staining. Graphs depict the sum of frequency of CD107a expression induced by all four peptide pools (GP1a, GP1b, GP2, SP) (Box and Whiskers, Min to Max, Line: median). b) The breadth of peptide pool responses increases by vaccine dose. An assay response was determined by 3-fold over the background. The darker the grey, the more peptide pool responses, the higher the breadth of response. Statistical analysis was performed using Mann-Whitney-Wilcoxon test (*p < 0.05; **p < 0.01; ***p < 0.005).

Fig. 7

PBMCs induce broad cytokine profile following stimulation with Ebola GP peptide pools. a) PBMCs were stimulated with two peptide pools (MP1, MP2 (Appendix Table 5)) that cover the whole Ebola GP (Kikwit). Following 16 h stimulation, supernatants of both stimulations were pooled together and a 27-plex LUMINEX assay was performed (blue: high expression, green: low expression). b) TH1-belonging cytokines are induced in subjects immunized with 2 × 10⁷ PFU. Graphs depict the cytokines IL2 (upper left graph), IL7 (upper right graph), IL15 (middle left graph), IFNγ (middle right graph) and TNFα (lower left graph) (Box and Whiskers, Min to Max, Line: median). Statistical analysis was performed using Mann-Whitney-Wilcoxon test (*p < 0.05; **p < 0.01; ***p < 0.005).

Fig. 8

Cytokines show an interlocked network following peptide stimulation. Correlogram of correlations among 27 cytokines for all dose cohorts for day 14 following vaccination. The highest median correlation coefficient (mcc) was observed in the 2 × 10⁷ PFU cohort with 0.52 in contrast to 0.39 and 0.29 in the middle and lowest dose group, respectively. Red indicates a negative and blue a positive correlation. The more intense the colour, the greater the correlation magnitude. Correlogram was plotted using R software.