The Journal of Infectious Diseases
BRIEF REPORT
Comprehensive Characterization of
Cellular Immune Responses Following
Ebola Virus Infection
Christine Dahlke,1,3,a Sebastian Lunemann,2,a Rahel Kasonta,1,3 Benno Kreuels,1
Stefan Schmiedel,1 My L. Ly,1,3 Sarah K. Fehling,4,5 Thomas Strecker,4,5
Stephan Becker,4,5 Marcus Altfeld,2,3 Abdourahmane Sow,6 Ansgar W. Lohse,1,3
César Muñoz-Fontela,2 and Marylyn M. Addo1,3
1 EBOV can hide from the immune system and about the conse-
1st Department of Medicine, University Medical Center Hamburg-Eppendorf, 2Leibniz Institute
for Experimental Virology, Heinrich-Pette-Institute, Hamburg, 3German Center for Infection
Research (DZIF), partner site Hamburg-Lübeck-Borstel-Riems, 4German Center for Infection
Research (DZIF), partner site Giessen-Marburg-Langen, Braunschweig, and 5Institute for
Virology, Philipps University Marburg, Germany; and 6West African Health Organisation, Bobo-
Dioulasso, Burkina Faso
The West African Ebola virus disease (EVD) outbreak was the
largest EVD outbreak in history. However, data on lymphocyte
dynamics and the antigen specificity of T cells in Ebola survi-
vors are scarce, and our understanding of EVD pathophysiology
is limited. A case of EVD survival in which the patient cleared
Ebola virus (EBOV) infection without experimental drugs al-
lowed for the detailed examination of lymphocyte dynamics.
We demonstrate the persistence of T-cell activation well beyond
viral clearance and detect EBOV-specific T cells. Our study pro-
vides significant insights into lymphocyte specificity during the
recovery phase of EVD and may inform novel strategies to treat
EVD.
Keywords. EVD; EVD survivor; convalescent phase; cellu-
lar immune response; lymphocyte dynamics; T-cell activation;
Ebola GP–specific T-cell responses; polyfunctional T-cell
responses.
The Ebola outbreak in West Africa was the most dramatic Ebola
virus disease (EVD) outbreak in history. During this outbreak,
27 patients with EVD were medically evacuated from Africa to
Europe and the United States [1], providing novel opportunities
to fill critical gaps in our understanding of the mechanisms un-
derlying the immunopathophysiology of EVD.
Data on immunity to Ebola virus (EBOV) infection in Ebola
survivors are scarce, owing to factors including unpredictability,
limited numbers of cases in previous outbreaks, and limited
Received 1 July 2016; accepted 17 October 2016; published online 31 October 2016.
Presented in part: 13th Kongress für Infektionskrankheiten und Tropenmedizin, Würzburg,
Germany, June 2016; 46th Annual Meeting of the German Society for Immunology, Hamburg,
Germany, September 2016.
a
C. D. and S. L. contributed equally to this study.
Correspondence: M. M. Addo, University Medical Center Hamburg Eppendorf, 1st Depart-
ment of Medicine, Division of Tropical Medicine, Martinistr. 52, 20246 Hamburg, Germany
(m.addo@uke.de).
The Journal of Infectious Diseases® 2017;215:287–92
© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of
America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.
DOI: 10.1093/infdis/jiw508
state-of-the-art laboratory resources available in EVD epicen-
ters. Consequently, T- and B-cell dynamics remain incomplete-
ly understood. Human data are so far limited to very few reports
describing longitudinal T- and B-cell dynamics in the context of
EVD [2–5].
Reports about EBOV persistence in sanctuary sites have
emerged recently [6], yielding questions about how and where
quences that persistence has on both human hosts and their
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contacts. A case of EVD survival at the University Medical Cen-
ter Hamburg-Eppendorf (UKE) provided the unique opportu-
nity to perform a comprehensive examination of lymphocyte
dynamics during the convalescent phase at days 37 and 46
after first appearance of EVD symptoms [7]. Importantly, our
results represent the first immunity data from an EVD survivor
during the convalescent phase who cleared the infection
without experimental drugs [7]. Beyond the assessment of neu-
tralizing antibodies, our approach involved a detailed analysis of
T- and B-cell dynamics and the investigation of antigen-specific
T cells via intracellular staining (ICS) and enzyme-linked im-
munospot (ELISPOT) analysis.
Despite the patient’s full clinical recovery from EVD symp-
toms, a high fraction of cytotoxic T cells and persistent immune
activation were detectable well beyond clearance of plasma vire-
mia. EBOV glycoprotein (GP)–specific T cells were detectable
but of low magnitude and were predominantly found in the
CD8+ T-cell population with the largest fraction of cells produc-
ing tumor necrosis factor α (TNF-α) but not interferon γ (IFN-
γ), interleukin 2 (IL-2), or macrophage inflammatory protein
(MIP-1β; hereafter, “TNF-α single producers”), as well as
among CD107a+ cells. Our data provide detailed insight into
the understanding of lymphocyte dynamics during the recovery
phase of EVD, which may contribute to future design of thera-
peutics and vaccines.
METHODS
Peptides
Overlapping peptides (OLPs) cover the Zaire EBOV GP. Se-
quences and pools are shown in Supplementary Table 1.
Patient and Control Specimens
Blood specimens from the patient and the healthy controls
were collected at the UKE. Donors provided written informed
consent. Peripheral blood mononuclear cells (PBMCs) were
isolated via Ficoll density gradient centrifugation from
blood collected in ethylenediaminetetraacetic acid (EDTA)–
lined tubes and cryopreserved using standard operating
procedures.
BRIEF REPORT • JID 2017:215 (15 January) • 287
Phenotyping and ICS
Phenotyping and ICS were performed using cryopreserved
PBMCs. Staining panels are depicted in Supplementary Table 2.
For ICS, PBMCs were incubated for 6 hours at 37°C with EBOV
GP peptide pools in the presence of CD28/CD49d, GolgiStop,
and GolgiPlug. Negative controls were unstimulated but treated
with dimethyl sulfoxide. Positive controls were stimulated with
PMA and CEF (a pool consisting of peptides from cytomegalo-
virus, Epstein-Barr virus, and influenza virus). Cells were ana-
lyzed on a BD LSRFortessa and evaluated with FlowJo 10.
ELISPOT
EBOV GP–specific T-cell responses were assessed using over-
lapping peptides by IFN-γ ELISPOT (MabtechELISpotPLUS)
as described by Streeck et al [8].
Neutralizing Antibody Assay
The neutralizing antibody assay was performed with plasma
samples, as previously described [9].
RESULTS
The interaction of EBOV with the human immune system re-
mains incompletely understood. We here report new insight
into the long-lasting immune responses of an EVD survivor fol-
lowing recovery.
Ten days after first symptoms, the patient was transferred to
Hamburg, Germany, and treated in an isolation unit for indi-
viduals with highly contagious diseases. The initial clinical
course has been previously described [7]. He experienced a se-
vere EBOV infection with the associated complications of sep-
ticemia, encephalopathy, and respiratory failure [7]. After
recovery and EBOV-negative results of polymerase chain reac-
tion analysis of plasma, he was transferred to the infectious dis-
ease unit (Supplementary Figure 1). Blood specimens were
collected in EDTA-lined tubes on days 37 and 46 after illness.
Lymphocyte Dynamics
Initial analysis of CD4+, CD8+, and CD19+ lymphocyte dynam-
ics revealed minor differences in their respective frequencies as
compared to findings for healthy controls (SF2). Subsequently,
the expression of CCR7 and CD45RA was evaluated (Figure 1A).
While antigen-experienced effector memory (EM) T cells
(CCR7−CD45RA−) were only slightly increased in the CD4+
compartment (21% on days 37 and 46, compared with a
mean [±SD] of 13% ± 1.5% in controls), the proportion of
EM CD8+ T cells expanded, compared with controls (39% on
day 37 and 36% on day 46, compared with a mean [±SD] of
19% ± 7% in controls).
T-cell activation is a critical element of the antiviral immune
defense. We therefore assessed activation via HLA-DR and
CD38 staining (Figure 1B). Analysis of CD8+ T cells revealed
a high frequency of activated cells, composing 68% (on day
37) and 62% (on day 46) of the main CD8+ T-cell population,
compared with a mean (±SD) of 8% ± 4% among controls, and
288 • JID 2017:215 (15 January) • BRIEF REPORT
81% (on day 37) and 80% (on day 46) of the EM subpopulation,
compared with a mean (±SD) of 14% ± 9%. Analysis of CD4+ T
cells and their corresponding EM subset revealed increased T-
cell activation too, but to a lesser extent.
Cytotoxicity was assessed via GrzB staining. The CD4+ and
CD8+ T-cell populations showed a high proportion of GrzB+
cells (Figure 1C).
Regulatory lymphocytes play an important role in immune
homeostasis. Whereas the regulatory T-cell (Treg) population
(CD4+CD25 hiCD127low) was quantitatively comparable to
that in healthy donors (SF2), the 2 B-cell subsets
(CD19+CD24hiCD38hi and CD24hiCD27+), in which regulatory
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B-cells are enriched [10], showed slightly lower proportions
than in controls (Figure 1D).
The slight expansion of plasmablasts (CD19+CD24−CD38high)
in the patient with EVD indicates activation of the humoral im-
mune response and may mirror generation of antibodies. Indeed,
we detected EBOV-specific neutralizing antibodies with geomet-
ric mean titers of 91 (on day 37) and 76 (on day 46; SF3).
EBOV GP–Specific T-Cell Responses
To define EBOV-specific cellular immune responses, we investi-
gated the induction of 4 cytokines (IFN-γ, IL-2, TNF-α, and
MIP-1β) in response to EBOV GP–specific OLP stimulation,
via ICS (Figure 2). Antigen-specific T cells were predominantly
found in the CD8+ compartment and not in the CD4+ compart-
ment. Whereas TNF-α/IFN-γ double producers were predomi-
nantly observed upon SP and GP2 stimulation, GP1a
stimulation resulted in TNF-α induction (Figure 2A and 2B).
Only limited expression was observed for MIP-1β, and no induc-
tion was detectable for IL-2. Limited polyfunctionality (maximal-
ly 3 functions) was observed in CD8+ T cells in response to GP2
and SP peptide pools (Figure 2C), and none were observed in
CD4+ T cells (data not shown). Antigen-specific cytotoxic lym-
phocytes were characterized by staining with CD107a, with the
strongest induction observed in the GP1a pool (Figure 2B).
To increase sensitivity, EBOV-specific T-cell responses were
also assessed by IFN-γ ELISPOT (Figure 2D). Significant
responses were observed for peptide pools GP2.2 (376 spot-
forming cells [SFCs] per million peripheral blood mononuclear
cells [PBMCs] on day 37) and SP (202 SFCs per million PBMCs
on day 37), confirming the ICS data described above. A higher
response was observed for the master pool MP1.
In summary, both ICS and ELISPOT revealed EBOV-specific
antiviral T-cell activity, albeit with a low magnitude.
DISCUSSION
EBOV represents one of the most deadly human pathogens, and
to date little is understood about the human immunity to EBOV
infection. Immunopathogenesis, factors that influence survival,
and correlates of protection have not been completely
elucidated.
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Figure 1. Lymphocyte dynamics during the convalescent phase of Ebola virus (EBOV) disease (EVD). A, CD4+ (left) and CD8+ (right) T-cell subsets were stained by
CCR7 and CD45RA to distinguish naive (CCR7+CD45RA+), effector memory (EM; CCR7−CD45RA−), central memory (CM; CCR7+CD45RA−), and effector memory RA
(CCR7−CD45RA+) T cells. In the upper row, data are represented in contour plots. The control plot depicts a representative experiment. The lower row shows the
distribution of T-cell subsets. The control pie chart represents the median value of measured results for 5 controls. B, T-cell activation was analyzed by HLA-DR and
CD38 staining to investigate CD4+, CD8+, and the respective EM T cells. At left, contour plots show the gating strategy. At right, the graph depicts the results (control
values are for 5 individuals). C, Cytotoxic T cells were assessed via GrzB staining. At left, contour plots show results for the patient with EVD on day 37 after symptom
onset for CD4+ (upper plot) and CD8+ (lower plot) T cells. Results are highlighted in the graph (red triangle, EVD survivor; circle, 2 healthy controls). D, Regulatory B
cells are enriched in transitional B cells (CD24+CD38high; blue gate; upper plot) and the CD19+CD24hiCD27+ population (lower plot). At left, contour plots depict rep-
resentative plots for the patient with EVD (on day 37) and 1 healthy control. At right, results are highlighted in the graph (data are for 5 controls). Values from controls
are shown as median values with SDs.

BRIEF REPORT • JID 2017:215 (15 January) • 289

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Figure 2. Ebola virus (EBOV) glycoprotein (GP)–specific T-cell responses found in CD8+ T cells. A, EBOV GP–specific interferon γ (IFN-γ)/tumor necrosis factor α
(TNF-α) T-cell responses. At left, graphs depict the observed IFN-γ/TNF-α responses in CD8+ (upper graph) and CD4+ (lower graph) compartments specific for the 4
EBOV GP peptide pools (control values are for 2 individuals). At right, representative dot plots show EBOV GP–specific IFN-γ/TNF-α T-cell responses for both CD8+
(upper panel) and CD4+ (lower panel) T cells (data are for patients with EVD on day 37 after first appearance of EVD symptoms). B, Cytokine and cytotoxic T-
lymphocyte responses upon stimulation with EBOV GP peptide pools (control values are for 2 individuals). C, The distribution of the functionality of CD8+ T-cell
responses to the 4 EBOV GP peptide pools on days 37 and 46 after first appearance of EVD symptoms. D, IFN-γ enzyme-linked immunospot assay of responses
against EBOV GP peptide pools. A CEF pool (consisting of peptides from cytomegalovirus, Epstein-Barr virus, and influenza virus) was used as positive control. The
assay was performed in duplicate. The graph shows median values and SDs (control values are for 2 individuals). Abbreviations: PBMC, peripheral blood mono-
nuclear cell; SFC, spot-forming cell.

290 • JID 2017:215 (15 January) • BRIEF REPORT

 The medical management of the EVD survivor at the UKE
provided the unique opportunity to examine lymphocyte dy-
namics during the early convalescent phase of EVD in compre-
hensive detail. This report is the first to show T- and B-cell
dynamics and antigen-specific T cells in an EVD survivor,
who received only supportive therapy and no experimental
drugs.
There is no consensus about the correlates of protection
against EBOV, but vaccine-induced antigen-specific antibodies
were reported in surviving nonhuman primates [11]. Plasma
from the Hamburg patient effectively inhibited replication of
EBOV (isolate Mayinga) with titers similar to levels observed
in the EVD survivor treated in Frankfurt, who was analyzed
at similar time points [12]. Furthermore, neutralizing antibodies
were detected in 3 patients treated in Spain during their conva-
lescent phase [13]. These data reveal robust EBOV-specific
humoral response following infection, with generation of
neutralizing antibodies that persisted for at least 5 weeks
after illness.
Antigen-specific cellular immunity may contribute impor-
tantly to protection against EBOV infection. But so far, immu-
nodominant EBOV-specific T-cell responses in humans remain
largely unknown. Our approach involved the analysis of anti-
gen-experienced and polyfunctional T cells following EBOV in-
fection. The increased number of EM and TEMRA cells in the
CD8+ compartment imply antigen experience, but ELISPOT
analysis of the reactivity of EBOV GP–specific T cells revealed
an overall low IFN-γ response upon stimulation with GP
peptide pools. To increase the breadth of cytokine analysis,
we performed ICS for 3 additional readouts and observed pre-
dominantly TNF-α single-producing T cells and a low frequen-
cy of IFN-γ/TNF-α double-producing T cells. But taking into
consideration that immune responses were analyzed at days
37 and 46 after illness onset, some antiviral functions may al-
ready have played their part. One recent study in nonhuman
primates revealed a peak in IFN-γ and IL-2 expression by
CD8+ T cells early, at day 14 after infection [14]. However,
the significance of virus-specific CD8+ T cells, monofunctional-
ity or polyfunctionality, and the combination of cytokines fol-
lowing EBOV infection in humans remains to be identified.
More human data are required to identify cellular key factors
contributing to survival or protection.
Lymphocyte activation has been suggested as a marker for
survival following EBOV infection. This was first described by
Baize et al, based on their observation of increased messenger
RNA levels for several activation markers in PBMCs [15].
Viral infections are typically associated with massive expansion
of activated T cells, which generally declines following pathogen
clearance. Although viable EBOV was not detected for around
20 days in analyzed body fluids, T cells remained highly activat-
ed with a strong cytotoxic profile, specifically in the CD8+ T-cell
compartment. There is a question of whether the observed
ongoing T-cell activation reflects the previously experienced
septicemia, nonspecific bystander activation, or the persistence
of virus or viral antigens in sanctuary sites. Support for possible
virus persistence is provided by 2 recent publications describing
a similar phenotype of long-lasting activated cytotoxic T cells
[2, 3] and the emerging reports on post-EVD syndrome
(PEVDS) and EBOV persistence in compartments other than
the bloodstream [6]. It is therefore conceivable that antigen
may have persisted at a low level, contributing to T-cell activa-
tion and cytotoxicity.
In light of EBOV persistence and PEVDS, the convalescent
phase of EVD survivors represents an important state to an-
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alyze. However, thus far human data are scarce. To this date,
only a few recent publications specifically investigated T- and
B-cell dynamics, of which 2 focused on the convalescent
phase. A comparison of these data with results associated
with the Hamburg patient may contribute to our understand-
ing of immune responses following EBOV infection. Long-
lasting lymphocyte dynamics and immunity following
EBOV infection were analyzed in 6 patients. Of note, all of
these analyzed patients received experimental drugs, in con-
trast to the Hamburg patient. Further, it needs to be consid-
ered that analysis time points in all these patients were highly
heterogeneous, with different rates of disease progression, dis-
similar therapies, distinct viral loads, and differences in pro-
tocols and assays.
A clear difference was observed in the type of cytokines.
Ahmed et al investigated EBOV-specific T-cell specificity in
3 EVD survivors and observed consistent IFN-γ responses in
single-producing cells to nucleoprotein and variable responses
to GP. Only 1 survivor showed a strong IFN-γ response to GP
[2]. We detected predominantly TNF-α (and CD107a) induc-
tion. High T-cell activation was consistent in all 3 studies,
and in the context of the observed cytotoxic profile, these
data indicate a robust immune response following EBOV infec-
tion and may suggest possible antigen persistency. In contrast to
previous reports, recent data from us and others do not show
immune suppression but prolonged immune activation with
high cytotoxicity, elements that may have contributed to viral
control. Further, these similarities were detected irrespective
of the patient treatment strategy, suggesting that these experi-
mental treatments had little impact on T-cell activation and fur-
ther assuming delayed clearance of EBOV in humans, which
needs to be further investigated.
In sum, detailed analysis of immune responses and dynamics
following EBOV infection may help to broaden our understand-
ing of possible mechanisms leading to EBOV persistence and
the development of PEVDS. Identification of immune respons-
es and of antigens and epitopes targeted in patients with natural
immunity to EBOV infection may also be critical for our under-
standing of immunopathogenesis and may inform strategic
Ebola vaccine design and monitoring.
BRIEF REPORT • JID 2017:215 (15 January) • 291
Supplementary Data
Supplementary materials are available at http://jid.oxfordjournals.org.
Consisting of data provided by the author to benefit the reader, the posted
materials are not copyedited and are the sole responsibility of the author, so
questions or comments should be addressed to the author.
Notes
Acknowledgments. We thank Claudia Beisel, Till Bornscheuer, Jakob
Cramer, Claudia Frey, Johannes Jochum, Stefan Lüth, Hanna Matthews,
Claudia Röder, Thierry Rolling, Camilla Rothe, Helmut Salzer, Guido Schä-
fer, Christoph Schramm, Julian Schulze zur Wiesch, Christof Vinnemeier,
Tobias Werner, Dominic Wichmann, Dorothea Wiemer, and all of the
other physicians and nurses involved in the clinical treatment of the patient;
Florian Harder, Tom Hildebrandt, Hannes Kalb, and Kristina Scholz, for
intensive support in setting up, maintaining, and running the unit for the
treatment of highly contagious infections at the University Medical Center
Hamburg–Eppendorf (UKE); Nadine Biedenkopf, for organizing sample
shipment; Joseph Poetsch and Madeleine Zinser, for critical review of the
manuscript; and Gotthard Ludwig and Michael Schmidt (Philipps Univer-
sity Marburg), for their technical assistance with biosafety level 4
procedures.
Financial support. This work was supported by the German Center for
Infection Research (DZIF).
Potential conflicts of interest. All authors: No reported conflicts. All au-
thors have submitted the ICMJE Form for Disclosure of Potential Conflicts
of Interest. Conflicts that the editors consider relevant to the content of the
manuscript have been disclosed.
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