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Unique Human Immune Signature of Ebola Virus Disease in Guinea

Paula Ruibal  1   2   3   4 Lisa Oestereich  2   3   4 Anja Lüdtke  1   2   3   4 Beate Becker-Ziaja  2   3   4 David M Wozniak  2   3   4 Romy Kerber  2   3   4 Miša Korva  4   5 Mar Cabeza-Cabrerizo  2   4 Joseph A Bore  4 Fara Raymond Koundouno  4 Sophie Duraffour  2   4 Romy Weller  4   6 Anja Thorenz  4   7 Eleonora Cimini  4   8 Domenico Viola  4   8 Chiara Agrati  4   8 Johanna Repits  4 Babak Afrough  4   9 Lauren A Cowley  4   10 Didier Ngabo  4   9 Julia Hinzmann  4   11 Marc Mertens  4   12 Inês Vitoriano  4   9 Christopher H Logue  4   9 Jan Peter Boettcher  4   11 Elisa Pallasch  2   3   4 Andreas Sachse  4   11 Amadou Bah  4   13 Katja Nitzsche  3   4 Eeva Kuisma  4   9 Janine Michel  4   11 Tobias Holm  2   3   4 Elsa-Gayle Zekeng  4 Isabel García-Dorival  4   14 Roman Wölfel  3   4   15 Kilian Stoecker  3   4   15 Erna Fleischmann  3   4   15 Thomas Strecker  3   4   16 Antonino Di Caro  4   8 Tatjana Avšič-Županc  4   5 Andreas Kurth  4   11 Silvia Meschi  4   8 Stephane Mély  4   17 Edmund Newman  4   9 Anne Bocquin  4   17 Zoltan Kis  4   18   19 Anne Kelterbaum  3   4   16 Peter Molkenthin  3   4   15 Fabrizio Carletti  4   8 Jasmine Portmann  4   20 Svenja Wolff  3   4   16 Concetta Castilletti  4   8 Gordian Schudt  3   4   16 Alexandra Fizet  4   21 Lisa J Ottowell  4   9 Eva Herker  1 Thomas Jacobs  2 Birte Kretschmer  22 Ettore Severi  19 Nobila Ouedraogo  11 Mar Lago  23 Anabel Negredo  24 Leticia Franco  24 Pedro Anda  24 Stefan Schmiedel  25 Benno Kreuels  2   3   25 Dominic Wichmann  3   25 Marylyn M Addo  3   25 Ansgar W Lohse  3   25 Hilde De Clerck  26 Carolina Nanclares  26 Sylvie Jonckheere  26 Michel Van Herp  26 Armand Sprecher  26 Gao Xiaojiang  27   28 Mary Carrington  27   28 Osvaldo Miranda  29 Carlos M Castro  29 Martin Gabriel  2   3   4 Patrick Drury  30 Pierre Formenty  30 Boubacar Diallo  30 Lamine Koivogui  31 N'Faly Magassouba  32 Miles W Carroll  4   9 Stephan Günther  2   3   4 César Muñoz-Fontela  1   2   3   4
Affiliations

Unique Human Immune Signature of Ebola Virus Disease in Guinea

Paula Ruibal et al. Nature.

Abstract

Despite the magnitude of the Ebola virus disease (EVD) outbreak in West Africa, there is still a fundamental lack of knowledge about the pathophysiology of EVD. In particular, very little is known about human immune responses to Ebola virus. Here we evaluate the physiology of the human T cell immune response in EVD patients at the time of admission to the Ebola Treatment Center in Guinea, and longitudinally until discharge or death. Through the use of multiparametric flow cytometry established by the European Mobile Laboratory in the field, we identify an immune signature that is unique in EVD fatalities. Fatal EVD was characterized by a high percentage of CD4(+) and CD8(+) T cells expressing the inhibitory molecules CTLA-4 and PD-1, which correlated with elevated inflammatory markers and high virus load. Conversely, surviving individuals showed significantly lower expression of CTLA-4 and PD-1 as well as lower inflammation, despite comparable overall T cell activation. Concomitant with virus clearance, survivors mounted a robust Ebola-virus-specific T cell response. Our findings suggest that dysregulation of the T cell response is a key component of EVD pathophysiology.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Extended Data Figure 1
Extended Data Figure 1. Initial immunophenotyping data from Guéckédou
a, Graph depicting the number of samples tested by the EMLab unit in Guéckédou by function of time since the beginning of the outbreak. The blue square indicates the period in which leftover whole blood samples from the diagnostic activities were shipped to the BSL-4 laboratory in Hamburg for initial immunophenotyping. b, Demographic data of the Guéckédou EVD patient cohort. Adults were ≥ 18 years of age and pediatric patients were < 18 years of age. c, Comparison of the expression of CTLA-4 assessed by Median Fluorescence Intensity Ratio (MFIR) in CD4 and CD8 T cells of EVD patients (POS, black boxes) and non-EVD controls (NEG, green boxes). MFIR represents the ratio between the CTLA-4-specific signal divided by the Fluorescence Minus One (FMO) signal of the same cell population. d, Comparison between CTLA-4 MFIR values in CD8 T cells from fatal (black) vs. surviving (blue) EVD cases. In all panels the ends of the whiskers in the box-and-whisker plots represent the 10th and 90th percentile, respectively. Statistical analysis was performed by non-parametric Mann-Whitney test. The level of significance is indicated as follows: ns, not significant; * p≤0.05; *** p≤0.0001.
Extended Data Figure 2
Extended Data Figure 2. Epidemiological data of patients tested by EMLab unit in Coyah
a and b, Demographic data of the Coyah EVD patient cohort. Adults were ≥ 18 years of age and pediatric patients were < 18 years of age. The median age of the 157 patients in the study was 26 years (interquartile range (IQR) 20–38 years). Percentages of males and females were comparable within all groups, with adults accounting for 79% of patients. c, Box and whisker plots depicting statistical association between Ct values and outcome. The case-fatality ratio (CFR) was 51.6%. Fatalities and survivors were compared via non-parametric Mann-Whitney test; *** p<0.001. d, Correlation between Ct value and age of the patients. The Ct value did not correlate with age. However, survivors clustered in a group characterized by Ct value higher than 18 and by age less than 40 years (cluster encircled in blue). Statistical significance was tested by non-parametric Spearman correlation analysis. e, Ct values correlated negatively with symptom scoring, so that low Ct values were associated with severe disease symptoms. Symptom score was calculated as the summation of individual symptoms (bleeding, liver dysfunction, respiratory distress, kidney failure, neurological symptoms and anorexia) from ‘0’ (no symptoms) to ‘6’ (all symptoms present). In the box and whisker plot the ends of the whiskers represent the 10th and 90th percentile, respectively. Statistical analysis was performed by non-parametric Mann-Whitney test. The level of significance is indicated as follows: ns, not significant; * p≤0.05; *** p≤0.0001.
Extended Data Figure 3
Extended Data Figure 3. Gating strategy for flow cytometry studies in Guinea
All samples evaluated in the field were aliquoted for four panels. All panels had the following common gating: G1: Lymphocyte gate; G2: Live cells; G3: Singlets; G4: T cells; G5: CD4 T cells; G6: CD8 T cells. Panel 1 Panel 1, 2, 3, and 4 evaluated expression of HLA-DR, Ki-67, CTLA-4, and PD-1, respectively, in either CD4 or CD8 T cells.
Extended Data Figure 4
Extended Data Figure 4. Gating strategy for flow cytometry in Hamburg
Cryopreserved PBMC samples from Coyah were thawed as indicated in the Methods section. The following gates were used for sample analysis: G1: Lymphocyte gate; G2: Live cells; G3: Singlets, G4: T cells; G5: CD4 T cells; G6: CD8 T cells. In G5 or G6, samples were evaluated for co-expression of the indicated cell markers. Dextramer staining was evaluated in G6 following protocols described in the Methods section.
Extended Data Figure 5
Extended Data Figure 5. Correlation of double positive PD-1+/CTLA-4+ CD8 T cells with Ct values and lymphopenia
a, Graph showing correlation between the frequency of CD8 T cells co-expressing PD-1 and CTLA-4 and the Ct value. Correlation analysis was done via non-parametric Spearman correlation test. b, Box and whisker plots depicting the concentration of CD4 and CD8 T cells in blood of fatal and surviving EVD patients. The ends of the whiskers in the box-and-whisker plots represent the 10th and 90th percentile, respectively.
Extended Data Figure 6
Extended Data Figure 6. In silico peptide analysis and dextramer design
a, Selection of peptides consisting of 9 amino acid residues corresponding to the EBOV NP sequence predicted to bind the indicated HLA alleles. IC50 values for peptide binding to HLA were predicted by the artificial neural network (ANN) at the Immune Epitope Database and Analysis Resource (IEDB) (www.iedb.org). b, Dextramer background was determined by staining of HLA-matched healthy donor peripheral blood leukocyte samples. T cells were gated as indicated in Extended Data Fig. 4. Plots in the upper row represent stainig of a FMO (fluorescent minus one) sample in which the APC channel was left empty. Lower rows show backgroud dextramer staining as indicated. The mean background staining plus minus standard deviation is indicated for each dextramer and the FMO.
Extended Data Figure 7
Extended Data Figure 7. Longitudinal analysis of CTLA-4 expression in the CD8 T cell compartment during the course of EVD in two patients
a, Graph depicts the levels of expression of CTLA-4 in CD8 T cells of a fatal vs. a surviving EVD case over the course of disease. Both patients were treated in Europe. Samples were taken at consecutive days starting immediately after patient admission as indicated. MFIR represents the ratio between the CTLA-4-specific signal divided by the Fluorescence Minus One (FMO) signal of the same cell population. b, Longitudinal analysis of CTLA-4 expression in CD8 T cells and Ct values in survivors from Coyah.
Figure 1
Figure 1. Immune signature of EVD at the time of admission
a, Day of symptom onset at admission. b, Frequencies of CD8 T cells (left) and CD4 T cells (right) positive for the indicated markers. Black boxes represent fatal cases and blue boxes represent survivors. c, Plots of four survivors and four fatalities showing frequencies of CTLA-4+ CD8 T cells. d, Levels of plasma cytokines in fatal vs surviving EVD cases. Statistical analysis was performed by Mann-Whitney test: ns, not significant; * p≤0.05; ** p≤0.01; *** p≤0.001.
Figure 2
Figure 2. Functional properties of T cells in EVD patients
a, Frequencies of CD8 T cells co-expressing the indicated markers. b, Plots of two surviving and two fatal EVD patients depicting CD8 T cell populations co-expressing the indicated markers. Overlays indicate activation status of CTLA-4+ CD8 T cells (red). Total CD8 T cells are represented by black contours. c, CD4 T cells co-expressing the indicated markers. d, Plots of two surviving and two fatal patients depicting CD4 T cell populations co-expressing the indicated markers. Overlays indicate activation status of CTLA-4+ CD4 T cells (red). Total CD4 T cells are represented by black contours. e, Correlation between frequency of CD8 T cells (upper graph) and CD4 T cells (lower graph) expressing CTLA-4 and Ct values. f, Correlation between the frequency of CD8 T cells (upper graph) and CD4 T cells (lower graph) positive for PD-1 and Ct values. Statistics was performed by Mann-Whitney test. ns, not significant; * p≤0.05; ** p≤0.01.
Figure 3
Figure 3. Longitudinal evaluation of EBOV-specific T cell immunity
a, Frequency of EBOV-specific CD8 T cells in EVD patients at admission. Statistics was performed by Mann-Whitney test: ns, not significant. b, Frequency of CD8 T cells specific for the HLA-A*02:01-restricted peptide FLSFASLFL. Two fatal patients (F1 and F2, see panel d) are shown and day of sample evaluation post-onset is indicated between brackets. A surviving patient (S1) and fatal patient (F3) are shown for positive HLA-A*02:01 dextramer staining. c, Frequencies of CD8 T cells specific for the HLA-B*35:01-restricted FPQLSAIAL peptide are shown in survivors (S3, S4 and S5) with serial sampling. d, Plots of one survivor (S2) showing staining of EBOV-specific CD8 T cells restricted for HLA-A*23:01 AYQGDYKLF. e, Longitudinal patient data at the indicated days after symptom onset. Left axis represents the frequency of CD8 T cells co-expressing CD38 and HLA-DR (black), CTLA-4 and PD-1 (red) and dextramer positive cells (blue). Dextramer background was assessed in HLA-matched healthy donors (Extended Data Fig. 6b), and background dextramer staining is shown as dashed blue lines. The corresponding flow cytometry plots are shown in panels a-d. Ct values are represented for each patient with dashed lines and values depicted in the right axis.

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