Antibody-Dependent Natural Killer Cell Activation After Ebola Vaccination

Abstract

Background: Antibody Fc-mediated functions, such as antibody-dependent cellular cytotoxicity, contribute to vaccine-induced protection against viral infections. Fc-mediated function of anti-Ebola glycoprotein (GP) antibodies suggest that Fc-dependent activation of effector cells, including natural killer (NK) cells, could play a role in vaccination against Ebola virus disease.

Methods: We analyzed the effect on primary human NK cell activation of anti-Ebola GP antibody in the serum of United Kingdom-based volunteers vaccinated with the novel 2-dose heterologous adenovirus type 26.ZEBOV, modified vaccinia Ankara-BN-Filo vaccine regimen.

Results: We demonstrate primary human NK cell CD107a and interferon γ expression, combined with down-regulation of CD16, in response to recombinant Ebola virus GP and post-vaccine dose 1 and dose 2 serum samples. These responses varied significantly with vaccine regimen, and NK cell activation was found to correlate with anti-GP antibody concentration. We also reveal an impact of NK cell differentiation phenotype on antibody-dependent NK cell activation, with highly differentiated CD56dimCD57+ NK cells being the most responsive.

Conclusions: These findings highlight the dual importance of vaccine-induced antibody concentration and NK cell differentiation status in promoting Fc-mediated activation of NK cells after vaccination, raising a potential role for antibody-mediated NK cell activation in vaccine-induced immune responses.

Keywords: Ebola; antibody; natural killer cell; vaccine.

Figure 1.

Flow cytometry gating strategy for natural killer (NK) cell CD107a, CD16, and interferon (IFN) γ expression. Flow cytometry plots show CD3⁻CD56⁺ NK cell (gating strategy shown in Supplementary Figure 1A) CD107a (A), CD16 (B) and IFN-γ (C) expression in response to 5% prevaccination (visit 0), post–dose 1 (visit 1), and post–dose 2 (visit 2) vaccination serum and plate-bound Ebola virus glycoprotein antigen. Whole human peripheral blood mononuclear cells from 1 nonvaccinated single donor were used for NK cell activation assays in Figures 1–4, and the NK cell differentiation phenotype of the donor is shown in Supplementary Figure 1D.

Figure 2.

Antibody-dependent natural killer (NK) cell responses to plate-bound Ebola virus glycoprotein after adenovirus type 26.ZEBOV, modified vaccinia Ankara (MVA)–BN-Filo vaccination. The median and 95% confidence interval of NK cell CD107a, CD56^dimCD16 mean fluorescence intensity (MFI), and interferon (IFN) γ responses to prevaccination (visit 0), post–dose 1 (visit 1) and post–dose 2 (visit 2) vaccination serum sample are shown. A, All vaccine arms combined (n = 72). NK cell CD107a, CD16, and IFN-γ responses were analyzed according to NK cell differentiation subset, defined by CD56, CD57, and NKG2C expression (gating strategy shown in Supplementary Figure 1C). B, Each individual serum donor is represented by a dot with a line at the median. The proportion of total NK cell CD107a and IFN-γ expression (after dose 2) attributed to each subset is shown as a pie graph, with each slice representing the median. C, Comparisons across vaccination visits and between subsets were performed using 1-way analysis of variance with Holm-Sidak test for multiple comparisons. ‡P < .001.

Figure 3.

Antibody-dependent natural killer (NK) cell activation varies with vaccine regimen. NK cell CD107a (A), CD56^dimCD16 mean fluorescence intensity (MFI) (B), and interferon (IFN) γ (C) expression was plotted according to vaccine regimen (groups 1–5) for prevaccination (visit 0), post–dose 1 (visit 1), and post–dose 2 (visit 2) time points; graphs show median only. Comparisons between visits within each group were performed using 1-way analysis of variance with Dunn correction for multiple comparisons and summarized in Supplementary Table 1.

Figure 4.

Natural killer (NK) cell activation correlates with anti–glycoprotein (GP) antibody concentration and pseudovirion virus neutralizing antibody (psVNA) titers. Post–dose 2 anti-GP antibody concentrations (A) or Ebola GP–specific pseudovirion virus neutralizing antibody (psVNA) titers (B) (determined previously by Milligan et al [21]) were correlated with post–dose 2 NK cell CD107a, CD56^dimCD16 mean fluorescence intensity (MFI) and interferon (IFN) γ expression, all vaccination groups combined. A 2-phase nonlinear regression model was fitted in prism, and R² goodness-of-fit analysis is shown; P values were determined by Pearson correlation, with significance defined as P < .05. Abbreviations: ELISA, enzyme-linked immunosorbent assay; IC₅₀, median inhibitory concentration; IgG, immunoglobulin G.

Figure 5.

Natural killer (NK) cell activation varies with NK cell donor. A, NK cell CD107a, CD56^dimCD16 mean fluorescence intensity (MFI), and interferon (IFN) γ expression (multiple nonvaccinated donors; n = 16) in response to 5% pooled prevaccination (Pre) and post–dose 2 (Post) vaccination serum (group 2) and plate-bound Ebola virus glycoprotein. B, NK cell subset frequency distribution is shown for each donor. C, D, NK cell CD107a, CD16, and IFN-γ responses were also analyzed according to NK cell differentiation subset (C) and the proportion of total NK cell IFN-γ expression (after dose 2) attributed to each subset is shown as a pie graph, with each slice representing the median (D). Graphs show before-and-after plots with a line connecting each donor or 1 dot per donor with a line representing the median. Comparisons between pre- and postvaccination serum responses were performed using Wilcoxon signed rank test and comparison between subsets using 1-way analysis of variance with Dunn correction for multiple comparisons. *P < .05; †P < .01; ‡P < .001.