The

n e w e ng l a n d j o u r na l

of

m e dic i n e

Persistent Immune Responses after Ebola Virus Infection

To the Editor: Ebola virus is a highly virulent
emerging pathogen and a causative agent of viral
hemorrhagic fever.1 Studies of the pathogenesis
of Ebola virus infection in humans have indicated that recovery is largely dependent on the development of an immune response.1-3 To investigate the persistence of immune response in
humans, we examined levels of cytokine expression after in vitro whole-blood stimulation in
persons 12 years after infection with the Gulu
strain of Sudan Ebola virus (SUDV-gul).
The study was conducted in accordance with
the Declaration of Helsinki and was approved by
the Uganda Ministry of Health, the Uganda National Council for Science and Technology, and
the ethics committee of the Uganda Virus Research Institute. All participants provided written informed consent.
We obtained whole-blood samples from six
survivors of the Ebola virus outbreak that occurred in 2000 in the Gulu district of Uganda,4
as well as from four persons in the local community who were not infected. To obtain data on
fresh whole-blood stimulation assays, despite the
geographically remote nature of this cohort, reaction tubes were preloaded with antigen on site
and incubated with whole blood. Plasma supernatants then were transported to the laboratory
for analysis. Enzyme-linked immunosorbent as-

says (ELISAs) for detection of antiSUDV-gul

IgG antibody levels in serum and a viral plaquereduction neutralization test with the lowest serum dilution inhibiting 80% or more of the
plaques (PRNT80) were performed.5
After samples obtained from survivors of
Ebola virus infection underwent whole-blood
stimulation with inactivated SUDV-gul and purified glycoprotein of SUDV-gul (GP1-649), there was
a significant increase in levels of cytokine expression associated with regulators of the proinflammatory and T-cellderived immune response. Cytokine levels were measured in the
plasma supernatants of samples after wholeblood stimulation in the field. As compared with
the samples obtained from the control group, in
the samples obtained from the survivors of Ebola
virus infection there were significant increases
in levels of interleukins 1, 1, 5, 8, 10, 12p70,
and 15 and tumor necrosis factor and
interferon-. The levels of interleukins 2 and 17
also increased, but as compared with the samples from the control group, these increases
were not significant, perhaps because of the
sample size.
Survivors of Ebola virus infection and persons in the noninfected control group had similar baseline expression of cytokines and similar
increased expression after stimulation with phyto-

Table 1. Results of Enzyme-Linked Immunosorbent Assay IgG and PRNT80 Assay Tests, According to Study Group.*
Test

Survivors of SUDV-gul Infection

1

Noninfected Controls
6

10

IgG titer
SUDV-gul

1:6400

1:3200

1:3200

1:3200

1:6400

1:3200

1:50

1:50

1:50

1:50

GP1-649

>25,600

1:1600

>25,600

>25,600

1:1600

>25,600

1:50

1:50

1:50

1:50

Immunoreactivity
SUDV-gul (S:N)

28.50

69.82

22.60

26.69

12.59

41.56

3.64

0.44

0.77

0.85

GP1-649 (PP)

14.93

5.32

7.33

14.07

3.89

12.97

0.02

0.03

0.02

0.03

PRNT80 titer

1:160

1:40

1:160

1:320

<1:40

1:80

<1:40

<1:40

<1:40

<1:40

* GP1-649 denotes purified recombinant glycoprotein, PP percent positivity, PRNT80 plaque-reduction neutralization test with a cutoff value
of 80% neutralization at a serum dilution of 1:40 or lower, S:N signal-to-noise ratio, and SUDV-gul Gulu strain of Sudan Ebola virus.
An immunoglobulin titer of 1:50 was considered negative for immunoreactivity.
Immunoglobulin immunoreactivity was obtained at a serum dilution of 1:400. Cutoff values for SUDV-gul (5.48 S:N) and GP1-649 (0.43 PP)
immunoreactivity were determined as previously described.5

492

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correction

hemagglutinin leukoagglutinin. There were no

differences in complete blood count, general
medical history, or treatment during infection
among persons who had been infected with
Ebola virus.
Results of ELISA IgG against inactivated
SUDV-gul and GP1-649 revealed that all SUDV-gul
survivors tested positive for both SUDV-gul and
GP1-649. No immunoreactivity was observed in
the noninfected control group. PRNT80 results
showed that in the survivor group, four persons
were positive for neutralization and two persons
were negative. All serum samples obtained from
noninfected persons that were not immuno
reactive were negative in neutralization assays
(Table 1).
Survivors of Ebola virus infection had per
sistent serum-neutralizing activity and IgG im
munoreactivity against the viral protein GP1-649
12years after infection, and this protein stimulated high levels of cytokine expression after
whole-blood stimulation.
Ariel Sobarzo, Ph.D.
David E. Ochayon, M.Sc.
Ben-Gurion University of the Negev
Beer-Sheva, Israel

Julius J. Lutwama, Ph.D.

Steven Balinandi, M.Sc.
Uganda Virus Research Institute
Entebbe, Uganda

Ofer Guttman, M.Sc.

Robert S. Marks, Ph.D.
Ben-Gurion University of the Negev
Beer-Sheva, Israel

Ana I. Kuehne, Ph.D.

John M. Dye, Ph.D.
U.S. Army Medical Research Institute of Infectious Diseases
Fort Detrick, MD

Victoria Yavelsky, Ph.D.

Eli C. Lewis, Ph.D.
Leslie Lobel, M.D., Ph.D.

3. Sobarzo A, Groseth A, Dolnik O, et al. Profile and persis-

tence of the virus-specific neutralizing humoral immune response in human survivors of Sudan ebolavirus (Gulu). J Infect
Dis 2013;208:299-309.
4. Lamunu M, Lutwama JJ, Kamugisha J, et al. Containing a
haemorrhagic fever epidemic: the Ebola experience in Uganda
(October 2000-January 2001). Int J Infect Dis 2004;8:27-37.
5. Sobarzo A, Perelman E, Groseth A, et al. Profiling the native
specific human humoral immune response to Sudan ebolavirus
strain Gulu by chemiluminescence enzyme-linked immunosorbent assay. Clin Vaccine Immunol 2012;19:1844-52.
DOI: 10.1056/NEJMc1300266
Correspondence Copyright 2013 Massachusetts Medical Society.

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correction

Ben-Gurion University of the Negev

Beer-Sheva, Israel
leslie.lobel@gmail.com
Drs. Dye, Yavelsky, Lewis, and Lobel contributed equally to
this letter.
Disclosure forms provided by the authors are available with
the full text of this letter at NEJM.org.
1. Kuhn JH. Filoviruses: a compendium of 40 years of epide-

miological, clinical, and laboratory studies. Arch Virol Suppl

2008;20:13-360.
2. Mohamadzadeh M, Chen L, Schmaljohn AL. How Ebola and
Marburg viruses battle the immune system. Nat Rev Immunol
2007;7:556-67.

Continuing Medical Education: Sofosbuvir for Hepatitis C Genotype 2 or 3 in Patients without Treatment Options (May 16,
2013;368:1951). Question 1 should have begun, Which one of
the following statements summarizes the results in the population of patients with hepatitis C virus (HCV) infection for
whom interferon treatment was not an option and who were
randomly assigned . . . , rather than Which one of the following statements summarizes the results of the trial involving patients with hepatitis C virus (HCV) infection who were
randomly assigned . . . . In Choices C and D, after initiation of treatment should have been . . . after the end of
treatment. The examination is correct at NEJM.org.

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