CLINICAL AND VACCINE IMMUNOLOGY, Nov. 2006, p. 1267–1277
Vol. 13, No. 11
A DNA Vaccine for Ebola Virus Is Safe and Immunogenic in a Phase
I Clinical Trial?†
Julie E. Martin,1Nancy J. Sullivan,1Mary E. Enama,1Ingelise J. Gordon,1Mario Roederer,1
Richard A. Koup,1Robert T. Bailer,1Bimal K. Chakrabarti,1Michael A. Bailey,1
Phillip L. Gomez,1Charla A. Andrews,1Zoe Moodie,2Lin Gu,2
Judith A. Stein,1Gary J. Nabel,1Barney S. Graham,1*
and the VRC 204 Study Team1‡
Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 40 Convent Drive,
Bethesda, Maryland 20892-3017,1and Statistical Center for HIV/AIDS Research and Prevention,
Fred Hutchinson Cancer Research Center, Seattle, Washington 98109-10242
Received 2 May 2006/Returned for modification 9 June 2006/Accepted 20 August 2006
Ebola viruses represent a class of filoviruses that causes severe hemorrhagic fever with high mortality.
Recognized first in 1976 in the Democratic Republic of Congo, outbreaks continue to occur in equatorial Africa.
A safe and effective Ebola virus vaccine is needed because of its continued emergence and its potential for use
for biodefense. We report the safety and immunogenicity of an Ebola virus vaccine in its first phase I human
study. A three-plasmid DNA vaccine encoding the envelope glycoproteins (GP) from the Zaire and Sudan/Gulu
species as well as the nucleoprotein was evaluated in a randomized, placebo-controlled, double-blinded, dose
escalation study. Healthy adults, ages 18 to 44 years, were randomized to receive three injections of vaccine at
2 mg (n ? 5), 4 mg (n ? 8), or 8 mg (n ? 8) or placebo (n ? 6). Immunogenicity was assessed by enzyme-linked
immunosorbent assay (ELISA), immunoprecipitation-Western blotting, intracellular cytokine staining (ICS),
and enzyme-linked immunospot assay. The vaccine was well-tolerated, with no significant adverse events or
coagulation abnormalities. Specific antibody responses to at least one of the three antigens encoded by the vaccine
as assessed by ELISA and CD4?T-cell GP-specific responses as assessed by ICS were detected in 20/20 vaccinees.
CD8?T-cell GP-specific responses were detected by ICS assay in 6/20 vaccinees. This Ebola virus DNA vaccine was
safe and immunogenic in humans. Further assessment of the DNA platform alone and in combination with
replication-defective adenoviral vector vaccines, in concert with challenge and immune data from nonhuman
primates, will facilitate evaluation and potential licensure of an Ebola virus vaccine under the Animal Rule.
Outbreaks of infection with Ebola virus result in a rapid and
severe disease with high mortality, for which there is currently
no licensed antiviral treatment or vaccine. While outbreaks
remain unpredictable, they have occurred with increasing fre-
quency in equatorial Africa, west of the Rift Valley, where they
have infected both humans and nonhuman primates and have
significantly depleted chimpanzee and gorilla populations in
Central Africa. Although a potential reservoir has been sug-
gested (15), the risk of zoonotic transmission remains high and
unpredictable (1). Announcements of Ebola virus outbreaks
cause widespread fear and have socioeconomic consequences
beyond the direct impact on infected persons. Outbreaks of
Ebola virus infection have become more frequent since its
discovery in 1976 and reemergence in 1995, and there are now
areas in which the infection appears to be endemic. Although
there have been no human outbreaks of Ebola virus in the
United States, the virus caused an outbreak in imported lab-
oratory nonhuman primates in Reston, Va., in 1989. It is also
considered to be a potential bioweapon. For these reasons,
vaccine development for Ebola virus and other filoviruses has
become a priority.
Outbreaks of hemorrhagic fever caused by the Ebola virus
are associated with high mortality rates. The highest lethality is
associated with the Zaire subtype, one of four species identi-
fied to date (9, 21). An outbreak of hemorrhagic fever reported
in October 2000 in the Gulu district of Uganda was confirmed
as Ebola virus and resulted in the deaths of dozens of people
(6). Another outbreak in Gabon and the Republic of Congo
likely involved several independent introductions. It continued
from 2001 through 2003 and resulted in more than 100 deaths
(3, 28). The triggers for such outbreaks are not understood,
although there may be a correlation with climatic changes (31).
These periodic but devastating outbreaks underscore the dif-
ficulty in controlling this virus that emerges periodically via
uncertain primary transmission routes and then disappears
into an unclearly defined natural reservoir.
Infection with Ebola virus initially results in an influenza-like
syndrome that progresses to severe illness manifested by co-
* Corresponding author. Mailing address: Vaccine Research Center,
NIAID/NIH, 40 Convent Drive, MSC-3017, Room 2502, Bethesda,
MD 20892-3017. Phone: (301) 594-8468. Fax: (301) 480-2771. E-mail:
† Supplemental material for this article may be found at http://cvi
‡ The VRC 204 Study Team includes Margaret M. McCluskey,
Brenda Larkin, Sarah Hubka, Lasonji Holman, Laura Novik, Pamela
Edmonds, Steve Rucker, Michael Scott, Colleen Thomas, LaChonne
Stanford, Ed Tramont, Woody Dubois, Tiffany Alley, Erica Eaton,
Sandra Sitar, Ericka Thompson, Andrew Catanzaro, Joseph Casazza,
Janie Parrino, Laurence Lemiale, Rebecca Sheets, Ellen Turk, Laurie
Lamoreaux, Jennifer Fischer, Mara Abashian, John Rathmann, and
?Published ahead of print on 20 September 2006.
agulation abnormalities, disseminated intravascular coagula-
tion, multi-organ system involvement, and an exaggerated but
nonprotective inflammatory response (1). Fatality rates range
from 50 to 90%, and death is frequently due to bleeding and
hypotensive shock (22). The rapid advancement of severe dis-
ease following Ebola virus infection allows little opportunity to
develop natural immunity, and there is no effective antiviral
therapy currently available. Vaccination offers a propitious in-
tervention to prevent infection and limit spread as well as an
important public health benefit for health care workers in-
volved in care of patients and the containment of outbreaks.
Because of the potential safety concerns associated with
using conventional vaccination strategies, such as attenuated
or inactivated Ebola virus as an immunogen, the vaccination
strategies that have been evaluated in published preclinical
studies to date for Ebola virus have focused on the use of live
and replication-defective vectors and virus-like particles. Pub-
lished studies have included expression of Ebola virus protein
subunits in live vaccinia virus vectors, selected DNA plasmids,
Ebola virus-like particles, replication-competent vesicular sto-
matitis virus, Venezuela equine encephalitis virus replicons,
recombinant parainfluenza virus type 3, replication-defective
recombinant adenovirus (rAd), and DNA combined with rAd
prime-boost strategies (5, 12, 14, 19, 22, 24, 26, 31). While
many of these approaches have been evaluated in a nonhuman
primate model (10), only DNA/rAd-, rAd-, or vesicular stoma-
titis virus-based vaccines have shown efficacy in primates.
DNA vaccination conferred an Ebola virus-specific immune
response in guinea pigs and mice (4, 25, 32) that protected
against a challenge with Ebola virus adapted to produce lethal
infection in rodents (1, 4, 8). Humoral and T-cell-mediated
immunity were elicited in these animal models, but antibody
titers appeared to correlate better with protection following
immunization with plasmids carrying genes for Ebola virus
Zaire proteins. DNA vaccination followed by a boost with
recombinant adenoviral vectors encoding Ebola viral proteins
uniformly protected nonhuman primates from an otherwise-
lethal dose of Ebola virus (25). Protection correlated with the
development of Ebola virus-specific CD8?T-cell and antibody
production. The vaccinated animals remained protected and
asymptomatic following challenge with a lethal dose of the
highly pathogenic, wild-type, 1976 Mayinga strain of Ebola
virus Zaire (22, 24, 25).
Ideally, an effective vaccine would provide immunity to the
multiple Ebola virus species that have been isolated in human
infections and may require multiple antigenic specificities.
There is a concern that combining multiple expression vectors
in the same vaccine may result in interference of expression of
some of the constructs (23). However, a multigene vaccine
containing antigens for Ebola virus glycoproteins from the
Zaire, Ivory Coast, and Sudan viruses induced specific immune
responses to all three subtypes without evidence of interfer-
ence in an animal model (24, 25). Additionally, a series of
studies showed that both GP and sGP (a soluble form of the
glycoprotein) conferred optimal protection in the guinea pig
model (25, 32). We report the results of the first human clinical
trial of a candidate Ebola virus DNA vaccine and show that
plasmids expressing Ebola virus GP (Zaire [Z]), GP (Sudan/
Gulu [S/G]), and NP (Z) are safe and well-tolerated and in-
duce Ebola virus-specific antibody and T-cell responses in
MATERIALS AND METHODS
Study design. Protocol VRC 204 was a single-site, phase I, randomized, pla-
cebo-controlled, double-blinded, dose escalation study to examine safety and
tolerability, dose, and immune response to an investigational Ebola virus plasmid
DNA vaccine. Healthy adult volunteers 18 to 44 years of age were recruited at
the NIAID Vaccine Research Center Clinic, National Institutes of Health (Be-
thesda, Md.). Human experimental guidelines of the U.S. Department of Health
and Human Services were followed in the conduct of clinical research, and the
protocol was reviewed and approved by the National Institute of Allergy and
Infectious Diseases (NIAID) Institutional Review Board. Three sequential
groups of volunteers were enrolled between November 2003 and July 2004 to
receive placebo or vaccine at doses of 2.0 mg, 4.0 mg, and 8.0 mg, respectively.
Group 1 subjects (n ? 7) were randomized in a ratio of 5 vaccine/2 placebo;
group 2 (n ? 10) and group 3 (n ? 10) subjects were randomized in a ratio of 8
vaccine/2 placebo. Total enrollment was 27 volunteers (21 vaccine, 6 placebo). In
all groups, the vaccine was administered on study days 0, 28 ? 7, and 56 ? 7 (with
at least 21 days between injection days).
Safety through at least 2 weeks after the second injection at each dose level
was reviewed by a data and safety monitoring board prior to enrolling volunteers
into the next dose level group. For the 2.0-mg and 4.0-mg immunizations, a single
dose of vaccine or placebo required a volume of 1.0 ml, administered by intra-
muscular injection in the lateral deltoid muscle. The maximum concentration of
vaccine formulation is 4 mg/ml; therefore, two 1.0-ml injections (one intramus-
cular injection in each deltoid muscle) of this formulation were necessary to
deliver the 8.0-mg dose. Placebo was administered the same way as vaccine for
each dosage group. All intramuscular injections were administered with the
Biojector 2000 needle-free injection management system. Adverse reactions
were evaluated by laboratory and clinical evaluations at scheduled study visits,
coded using the Medical Dictionary for Regulatory Activities, and severity graded
using a scale of 0 to 5. Solicited reactogenicity was collected by study subject
report on 7-day diary cards. Subjects were followed for a total of 12 months, and
the study was completed in August 2005.
Vaccine. This multigene plasmid DNA vaccine is a mixture of three plasmids
in equal concentrations that were constructed to produce three Ebola virus
proteins designed to elicit broad immune responses to multiple Ebola virus
subtypes. These proteins include the nucleoprotein (NP) derived from the Zaire
strain of Ebola virus, which exhibits highly conserved domains, as well as two
glycoproteins (GP), which mediate viral entry, from the Zaire strain (homolo-
gous to the Ivory Coast strain) and the Sudan/Gulu species (associated with
recent outbreaks of hemorrhagic fever in Africa). The Ebola virus GP genes
expressed by plasmid DNA constructs in this vaccine contain deletions in the
transmembrane region of GP that were intended to eliminate potential cellular
toxicity observed in the in vitro experiments using plasmids expressing the full-
length wild-type GPs (33). In addition, the Ebola virus GP inserts have been mod-
ified to optimize expression in human cells. The three plasmids in this vaccine are
incapable of replication in animal cells and would not permit the generation of an
infectious virion even if recombination or gene duplication were to occur.
The vaccine plasmids were prepared by cloning the Ebola virus gene sequences
into the VR-1012 expression vector produced by Vical, Inc. (San Diego, CA)
(13). The VR-1012 expression vector is very similar to the vector backbone used
in a plasmid DNA-based malaria vaccine (29) and a multiclade human immu-
nodeficiency virus (HIV) DNA vaccine that has been tested in humans (11). To
generate the vaccine (EBODNA012-00-VP) tested in this clinical trial, Ebola
virus GP gene sequences were subcloned into a slightly modified VR-1012
plasmid backbone containing the human T-cell leukemia virus 1 R region trans-
lational enhancer for improved expression (2). The CMV/R expression vector
has been tested in a clinical trial of a multiclade HIV DNA vaccine (11) and in
other candidate vaccines currently undergoing evaluation in clinical studies by
the Vaccine Research Center and the Division of AIDS, NIAID, National
Institutes of Health.
The DNA plasmids were produced in bacterial cell cultures containing a
kanamycin selection medium. The process involved Escherichia coli fermenta-
tion, purification, and formulation as a sterile liquid injectable dosage form for
intramuscular injection. Following growth of bacterial cells harboring the plas-
mid, the plasmid DNA was purified from cellular components.
The vaccine was produced by Vical, Inc. (San Diego, CA), under current Good
Manufacturing Practices conditions and met lot release specifications prior to
administration. This naked DNA product involves no lipid, viral, or cellular
vector components. A phosphate-buffered saline placebo control, pH 7.2, was
1268 MARTIN ET AL.CLIN. VACCINE IMMUNOL.
produced under current Good Manufacturing Practices conditions by Bell-More
Labs, Inc. (Hampstead, MD).
Measurement of antibody responses: enzyme-linked immunosorbent assay
(ELISA). Endpoint titers of antibodies directed against Ebola virus antigens NP
(Z), GP (S/G), and GP (Z) were determined using 96-well Immulon 2 plates
(Dynex Technologies) coated with a preparation of purified recombinant pro-
teins according to methods adapted from those described previously (11). Biotin-
labeled anti-human immunoglobulin G (IgG), IgA, or IgM and streptavidin
conjugated with horseradish peroxidase and 3,5?,5,5?-tetramethylbenzidine sub-
strate was used to develop the reaction, which was detected on a Spectramax
microplate spectrophotometer (Molecular Devices, Sunnyvale, CA). The end-
point titer was calculated as the most dilute serum concentration that gave an
optical density reading of ?0.2 above background.
Measurement of antibody responses by immunoprecipitation and Western
blot analysis. Antibody responses were measured in a semiquantitative assay
combining immunoprecipitation (IP) of crude cell-free supernatants containing
GP (Z) or GP (S/G) or cell lysates containing NP protein with volunteer sera,
followed by Western blotting for GP or NP as previously described (7). Briefly,
sera (10 ?l) from immunized individuals were used to immunoprecipitate Ebola
virus proteins either from 100 ?l of cell-free supernatant or from cell lysates of
293 cells (100 ?l of cell lysate is equivalent to 300 to 400 ?g of total protein)
transfected with vectors encoding transmembrane-deleted Ebola virus GP (Z) or
transmembrane-deleted Ebola virus GP (S/G) or Ebola virus NP (Z). Immune
complexes were separated by sodium dodecyl sulfate–7.5% polyacrylamide gel
electrophoresis (SDS-PAGE) and analyzed by immunoblotting using the follow-
ing Ebola virus protein-specific antibodies: mouse monoclonal 12B5 (generous
gift from Mary Kate Hart, USAMRIID) against GP (Z), rabbit polyclonal B83
(generous gift from Barton Haynes, Duke University) against GP (S/G), or
mouse monoclonal 1C9 (generous gift from Barton Haynes, Duke University)
against NP. Preimmune sera (10 ?l) from those individuals were used as controls.
The gels were scanned, and the intensity of each band was quantified by densi-
tometry using the program ImageQuant; results are presented graphically to
facilitate comparisons among groups.
Measurement of T-cell responses and cell preparation. Peripheral blood
mononuclear cells (PBMC) were prepared by standard Ficoll-Hypaque density
gradient centrifugation (Pharmacia, Uppsala, Sweden). PBMC were frozen in
heat-inactivated fetal calf serum containing 10% dimethyl sulfoxide in a Forma
CryoMed cell freezer (Marietta, OH). Cells were stored at ?140°C. All immu-
nogenicity assays were performed on thawed specimens; average viability was
Antibodies. Unconjugated mouse anti-human CD28, unconjugated mouse anti-
human CD49d, allophycocyanin-conjugated mouse anti-human CD3, fluorescein
isothiocyanate-conjugated mouse anti-human CD8, peridinin chlorophyll pro-
tein-conjugated mouse anti-human CD4, and a mixture of phycoerythrin-conju-
gated mouse anti-human gamma interferon (IFN-?) and interleukin 2 (IL-2)
monoclonal antibodies were obtained from Becton Dickinson Immunocytometry
Systems (BDIS; San Jose, CA). All reagents were independently titrated to
determine the optimum concentrations for staining.
Peptides and cell stimulation. Peptides 15 amino acids in length, overlapping
by 11, and corresponding to the vaccine inserts were synthesized at ?85% purity
as confirmed by high-performance liquid chromatography (24). Peptides were
pooled for each protein, NP (Z), GP (S/G), and GP (Z), and used at a final
TABLE 1. Demographic characteristics at enrollment
Characteristic and subcategory
No. (%) of subjects with characteristic in dose group
2 mg (n ? 5) 4 mg (n ? 8)8 mg (n ? 8)Placebo (n ? 6)Overall (n ? 27)
Black or African American
American Indian/Alaskan Native
Native Hawaiian or other Pacific Islander
30.0 or over
Less than high school graduate
High school graduate/GED
aAge at enrollment day.
bHeight and weight (used for BMI) were from the screening evaluation.
VOL. 13, 2006 PHASE I CLINICAL TRIAL OF EBOLA DNA VACCINE1269
concentration of 500 ng per stimulation. Cell stimulation was performed as
described previously (11). Briefly, one million PBMC in 200 ?l R-10 medium
(RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, 100
U/ml penicillin G, 100 ?g/ml streptomycin sulfate, and 1.7 mM sodium gluta-
mate) were incubated with 1 ?g/ml each of costimulatory anti-CD28 and -CD49d
monoclonal antibodies and 2.5 ?g/ml of each peptide in wells of 96-well V-
bottom plates. Cells incubated with only costimulatory antibodies were included
in every experiment to control for spontaneous production of cytokine and
activation of cells prior to addition of peptides. Staphylococcal enterotoxin B (10
?g/ml; Sigma-Aldrich) was used as a positive control for lymphocyte activation.
Cultures were incubated at 37°C in a 5% CO2incubator for 6 h in the presence
of brefeldin A (10 ?g/ml; Sigma, St. Louis, MO).
Intracellular cytokine and immunofluorescence staining. Cells were perme-
abilized for 7 min in 200 ?l of a solution containing 67 ?l Tween 20 (Sigma), 106
?l deionized water, and 27 ?l of 10? FACS-Lyse solution (BDIS) at room
temperature, washed twice in cold Dulbecco’s phosphate-buffered saline con-
taining 1% fetal bovine serum and 0.02% sodium azide (FACS [fluorescence-
activated cell sorting] buffer), and stained directly with conjugated anti-human
CD3, anti-human CD4, anti-human CD8, and anti-human IFN-? and IL-2 anti-
bodies for 15 min on ice. Stained cells were then immediately washed twice with
cold FACS buffer. The cells were resuspended in Dulbecco’s phosphate-buffered
saline containing 1% paraformaldehyde (Electron Microscopy Systems, Fort
Washington, PA) and stored at 4°C until analysis. Four-parameter flow cytomet-
ric analysis was performed on a FACSCalibur flow cytometer (BDIS). Following
intracellular cytokine staining (ICS), between 50,000 and 250,000 events were
acquired, gated on small lymphocytes, and assessed for CD3, CD8, CD4, and
IFN-?/IL-2 expression. Results were analyzed using FlowJo software (Tree Star
Software, Ashland, OR). The same cytokine, CD4, and CD8 gates were used for
the entire trial.
ELISPOT. Vaccine-induced T-cell responses were also detected by enzyme-
linked immunospot assays (ELISPOT) according to a modification of previously
published methods (11) using a commercially available ELISPOT kit (BD Bio-
sciences). PBMC were stimulated overnight at 37°C in triplicate wells at a density
of 2 ? 105cells/well for all stimulations other than staphylococcal enterotoxin B,
which was conducted at 5 ? 104cells/well. Following incubation, cells were lysed,
and the wells were washed and incubated for 2 h at room temperature in the
presence of biotinylated IFN-? detection antibodies. Subsequently, the wells
were incubated with an avidin-horseradish peroxidase solution for 1 h at room
temperature, followed by a 20-min incubation with the AEC substrate solution.
The plate was air dried for a minimum of 2 hours prior to spot quantitation on
a CTL ELISPOT image analyzer (Cellular Technology Ltd., Cleveland, OH).
Results were expressed as mean spot-forming cells per million PBMC.
Statistical methods. Positive response rates to any antigen (GP [S/G], GP [Z],
or NP [Z]) and to each individual antigen were used to summarize the T-cell
response data; exact two-sided 95% confidence intervals (29) are reported. The
positivity criteria for the ICS data consisted of a statistical hypothesis test for a
difference in the stimulated and unstimulated wells followed by the requirement
of a minimal level of response. For an individual’s response to be categorized as
positive, it had to be statistically significant and had to exceed the threshold for
positivity. Positivity thresholds were based on an ICS validation study of HIV
peptides completed at the VRC. The thresholds were selected to give a 1%
false-positive rate across PBMC from 34 HIV type 1-seronegative individuals
stimulated with eight HIV peptide pools in the validation data set. Only 2 of the
272 samples (0.007) had responses exceeding the thresholds. The validation study
results using HIV peptides are expected to be relevant for the Ebola virus
peptides; hence, in addition to the statistical hypothesis test for positivity, the
same thresholds were used. For the ICS responses, Fisher’s exact test was applied
to each antigen-specific response versus the negative control response, with a
Holm adjustment for the multiple comparisons. The nominal significance level
was ? ? 0.01, and the minimum threshold for background-corrected percent
positive response was 0.0241 for CD4?and 0.0445 for CD8?. To determine
positivity of the ELISPOT responses, a permutation test was applied to each
antigen-specific response versus negative control responses using the Westfall-
Young approach to adjust for the multiple comparisons. The nominal signifi-
cance level was ? ? 0.05. In addition, for the sample to be categorized as positive,
the result had to achieve a statistically significant difference and be above a
predetermined cutoff set at a false-positive rate of ?1% (i.e., the mean difference
in the antigen-stimulated wells and the negative control wells had to be greater
than or equal to 10 spot-forming cells per 2 ? 105PBMC). A variance filter for
the antigen-specific responses was also used: samples with a ratio of antigen-well
variance (median, ?1) greater than or equal to 100 were discarded from the
analysis; no such samples were found in the data set. SAS (version 9.1; SAS
Institute) and Splus (version 6.0; Insightful) were used for all analyses.
TABLE 2. Local and systemic reactogenicitya
Symptom and intensity
No. (%) of patients with reaction in dose group
(n ? 5)
(n ? 8)
(n ? 8)
(n ? 6)
Pain or tenderness
Any local symptom
Any systemic symptom
aThe local injection site reactions were recorded by clinicians at 30 to 45 min
postinjection and were then recorded as self-assessments at home by subjects on
a 7-day diary card. Systemic reactions were recorded as self-assessments at home
by subjects on a 7-day diary card following each injection.
bA single severe systemic symptom (malaise) was related to a foot fracture
which occurred 6 days following vaccination.
1270 MARTIN ET AL.CLIN. VACCINE IMMUNOL.
Study population demographics. A total of 27 healthy adult
volunteers were enrolled, with 5 in the 2-mg dose group, 8 each
in the 4-mg and 8-mg dose groups, and 6 in the placebo group.
Table 1 includes demographic data regarding subject gender,
age, race/ethnicity, body mass index (BMI), and educational
level at the time of enrollment. The subject population was
66.7% male and 33.3% female with a mean age of 33.1 years
(range of 18 to 44 years). Subjects were predominantly white
(96.3%) and non-Hispanic/Latino (96.3%). The mean BMI
was 25.6 (range, 19.5 to 42.2). All subjects had an educational
level of high school or higher, with 48.1% having college level
degrees and 33.3% holding advanced degrees.
Vaccine safety. Due to a theoretical concern over GP-medi-
ated cytopathicity (22), coagulation parameters of study sub-
jects were closely monitored. At enrollment and throughout
the study, D-dimer, prothrombin time, partial thromboplastin
time, fibrinogen, complete blood count, and red blood cell
smears were evaluated. There were no reportable coagulation
Two subjects were withdrawn from the vaccination schedule
due to serious adverse events that were assessed as “possibly”
related to vaccination: a grade 4 creatine phosphokinase ele-
vation 2 weeks after first vaccination and a grade 2 herpes
zoster thoracic dermatome eruption 3 weeks after the second
vaccination, both in 8-mg recipients. Of note, the grade 4
creatine phosphokinase elevation was associated with vigorous
exercise. These events resolved without sequelae, and these
subjects continued to participate in the study and attended all
study visits. Although only six of eight subjects in the 8-mg dose
group received all three injections, the immunogenicity and
safety laboratory values for all subjects are included in the
FIG. 1. Specific antibody responses to all vaccine components by IP-Western blot analysis. Sera from three representative subjects from each
vaccine dose group are shown for each antigen (A). Sera were drawn at week 12, 4 weeks following the third vaccination. Antibody responses were
specific and not cross-reactive to other vaccine antigens based on immunoblotting with monoclonal antibodies (B).
VOL. 13, 2006 PHASE I CLINICAL TRIAL OF EBOLA DNA VACCINE1271
analyses. One subject in the 2-mg dose group chose to with-
draw after the second vaccination; another subject (in the
placebo group) withdrew after the third injection. Neither of
these subjects returned for further visits and, therefore, they
were not included in the immunogenicity analysis due to a lack
of samples at time points following their withdrawal. As a
result, 20 of 21 vaccinees had immune responses assessed. All
subjects are represented in the safety data through the time
The diary cards showed that 90.5% (19/21) of subjects who
received vaccine (at any dose level) experienced at least one
local injection site symptom (mild to moderate pain/tender-
ness, mild induration, or mild skin discoloration) following a
vaccination. The systemic symptoms recorded on diary cards
included malaise, myalgia, headache, nausea, and fever, as well
as local injection site symptoms (Table 2). The study vaccina-
tions were well-tolerated and safe in healthy subjects, ages 18
to 44 years.
Antibody responses. Ebola virus-specific humoral responses
were detected in all vaccinees. GP- and NP-specific antibody
responses were detected by IP-Western blot analysis (Fig. 1).
Initial analysis of three representative subjects at different vac-
cine doses, 4 weeks following the third dose of vaccine (week
12), revealed antibodies specific for GP (Zaire) or GP (Sudan/
Gulu) (Fig. 1A, left and middle panels) and to NP (Fig. 1A,
right panel). These data demonstrate that specific antibodies to
each antigen can be induced by the vaccine independently and
are not cross-reactive (Fig. 1B). All (100%) of the 2-mg and
4-mg recipients and 75% (6/8) of the 8-mg recipients made GP
antibodies. Three-fourths (75%) of the 2-mg and 87.5% of the
4-mg and 8-mg recipients produced an NP-specific antibody
response (Fig. 2A). All (100%) vaccinees made a specific an-
tibody response detected by ELISA to at least one of the three
antigens encoded by the vaccine, with 19 of 20 vaccinees pro-
ducing a GP (Z)- and GP (S/G)-specific antibody response at
one or more time points (data not shown). This antibody re-
sponse was detected after the second dose of vaccine in some
subjects, peaked after the third dose (week 12), and waned
over the course of 1 year (Fig. 2B). Antibody titers (reciprocal
dilution) at week 12 ranged from undetectable to 4,000 for
either GP antigen (see Tables S1 and S2 in the supplemental
material). Ebola virus-specific neutralizing antibody, measured
by a pseudotyped virus neutralization assay (23), was not detected
in any study subject (data not shown), as might be expected with
DNA vaccination in the absence of rAd boosting.
T-cell responses. CD4?and CD8?T-cell responses were
assessed by ICS for all three antigens encoded by the vaccine
for all study subjects. GP (S/G) was the stronger T-cell im-
munogen of the two GP antigens encoded by the vaccine, and
NP induced the weakest response of the three antigens but was
still measurable in the majority of vaccinees. An Ebola virus-
specific CD4?T-cell response was demonstrated in all vaccin-
ees by ICS, and many of these responses occurred by week 4,
following just one dose of vaccine. CD4?T-cell responses for
GP (S/G) were detected in 100% of vaccinees by week 10. By
week 12, 100% of 2-mg (4/4) and 88% of 4-mg (7/8) and 8-mg
(7/8) recipients produced a CD4?T-cell GP (Z)-specific re-
sponse; by week 52, 100% of 2-mg (4/4) and 4-mg (8/8) recip-
FIG. 2. Kinetics and frequency of antibody responses. (A) Percentages of responders following the third vaccination by the IP-Western assay
for all subjects are shown. The y axis represents the percentage of responders with a positive assay, and the x axis represents the vaccine dose group.
White bars, GP (Z); black bars, GP (S/G); gray bars, NP (Z). (B) Kinetics of the antibody response for all subjects is shown over the 52 weeks
of the study. The geometric mean titer of the log10reciprocal dilution and standard deviation of the antibody response to GP (S/G) are plotted
against the number of weeks after initial vaccination for each of the three dose levels. Vaccinations were given at 0, 4, and 8 weeks. The threshold
for positivity in this assay was a reciprocal dilution of 30 and is shown as a dashed line. Of note, only six of eight subjects in the 8-mg dose group
received all three vaccinations in the series, yet all vaccinees are included in the immunogenicity analysis.
1272 MARTIN ET AL.CLIN. VACCINE IMMUNOL.
ients and 88% (7/8) of 8-mg recipients produced a CD4?T-cell
response to the GP (Z) antigen. By week 10, 75% (3/4) of 2-mg
and 4-mg recipients (6/8) and 100% of 8-mg (8/8) recipients
developed a CD4?T-cell response to NP (Fig. 3).
CD8?T-cell Ebola-specific responses were detected less
frequently than CD4?T-cell responses but were present in
30% (6/20) of all vaccinees by ICS. By week 10, 25% (1/4) of
2-mg, none of the 4-mg, and 13% (1/8) of 8-mg recipients
produced a CD8?T-cell response to GP (S/G). By week 12,
none of the 2-mg and 25% (4/16) of the 4-mg and 8-mg recip-
ients generated a CD8?T-cell response to GP (Z). None of
the 2-mg or 4-mg vaccinees and only one of the 8-mg vaccinees
produced a measurable CD8?T-cell response to the NP anti-
gen by week 10 as assessed by ICS (Fig. 3).
Analyses were also performed on all study subjects for all
three antigens by ELISPOT. Consistent with the ICS results,
the dominant antigen was GP (S/G). By week 12, 50% (2/4) of
2-mg, 63% (5/8) of 4-mg, and 63% (5/8) of 8-mg recipients
developed a positive ELISPOT response to GP (S/G). By week
12, 50% of 2-mg (2/4), 50% of 4-mg (4/8), and 38% of 8-mg
(3/8) recipients had a positive ELISPOT response to GP (Z) as
well. By week 24, 25% (1/4) of 2-mg, 13% (1/8) of 4-mg, and
63% (5/8) of 8-mg recipients displayed positive ELISPOT re-
sponses to the NP antigen (Fig. 3).
The magnitude of the CD8?T-cell response was slightly less
than that seen in the CD4?T-cell analysis as assessed by ICS.
The GP (S/G) immunogen induced slightly higher-magnitude
CD4?T-cell responses compared to the other immunogens in
the vaccine. The magnitude of the GP (Z)-specific CD4?re-
sponse was 70% of the GP (S/G) response, while the magni-
tude of the NP-specific CD4?response was 16% of the GP
(S/G) response. A correlation was not seen in the low number
of positive responses as assessed by ICS for CD8?T cells (Fig.
4). Analysis of the kinetics of the T-cell responses revealed that
the responses peaked between weeks 10 and 12 and, in general,
detectable responses were not sustained, although there was a
trend in the higher dose group toward a slightly greater dura-
tion of detectable responses (Fig. 5). Consistent with the ICS
and antibody responses, the ELISPOT response was of great-
est magnitude for the GP (S/G) antigen (see Table S3 in the
The rapid progression of severe disease after Ebola virus
infection allows little opportunity to develop protective immu-
nity, and there is currently no effective antiviral therapy.
Therefore, vaccination offers a promising intervention to pre-
FIG. 3. Frequency of CD4?and CD8?T-cell responses by ICS and ELISPOT analysis. Frequency (percent responders) is represented on the
left y axis. The week of analysis is shown on the lower x axis, the antigen assessed is shown on the upper x axis, and vaccine dose group is shown
on the right y axis. The frequency of CD4?ICS responses is shown by red bars, the frequency of CD8?ICS responses is shown by green bars, and
the frequency of positive ELISPOT responses is shown with blue bars. The schedule of the three DNA vaccinations is represented by arrows along
the lower x axis.
VOL. 13, 2006 PHASE I CLINICAL TRIAL OF EBOLA DNA VACCINE1273
vent infection or severe disease and limit spread to contacts
and would be an important public health benefit for health
care workers involved in the care of patients and containment
of outbreaks. Another compelling reason for accelerated de-
velopment of an Ebola virus vaccine relates to its contribution
to biodefense (17). The Centers for Disease Control and Pre-
vention (6) Category A agents are highly contagious and
largely lack effective vaccines or treatments (18) and include
the filoviruses (Ebola and Marburg viruses).
Gene-based vaccine technology provides a safe avenue for
producing candidate vaccines for select agents without the
need for extreme biocontainment. Gene-based vectors for filo-
viruses are particularly attractive vaccine approaches because
of their capacity to induce both humoral and cell-mediated
immune responses, both of which may be important for pro-
tection. The concept of using bacterium-derived plasmid DNA
to deliver vaccine antigens has many attractive features, includ-
ing (i) ease and flexibility of construction, (ii) scalable manu-
facturing capacity, (iii) stability, (iv) intracellular production of
vaccine antigen, (v) transient expression, (vi) no induction of
antivector immunity, (vii) induction of both CD4?and CD8?
T-cell responses as well as antibody, and (viii) lack of local or
systemic reactogenicity. However, DNA vaccines have not per-
formed well enough to be considered as a vaccine platform in
humans until recently. A hepatitis B virus DNA vaccine ad-
ministered by a needle-free particle-mediated delivery was
shown to be safe and immunogenic in a phase I clinical trial
(20). Additionally, DNA vaccination against malaria was
shown to be safe and immunogenic, especially as a priming
vaccination in a prime-boost regimen (16, 30). Recently, a
multiclade HIV DNA vaccine based on a similar design to the
Ebola virus DNA vaccine described here was shown to be safe
and immunogenic in healthy adults (11).
The broad immunogenicity of this Ebola virus DNA vaccine
suggests that immunization by plasmid DNA delivery is a via-
ble platform and merits further development. The consistent
immunogenicity of the Ebola virus DNA vaccine described
here likely reflects a combination of factors, including optimi-
zation of vector design, manufacturing methods, delivery, sam-
ple processing, and immunological assays. Additional work is
needed to further improve the efficiency and consistency of
Nonhuman primate studies have shown that an rAd5 vaccine
effectively prevents disease, and DNA vaccination prior to
boosting with rAd5 also confers protection and markedly in-
creases the magnitude of the immune response (22, 24, 25).
Further vector and construct optimization may further in-
crease protective immunity of this DNA vaccine. Recently, the
importance of the GP transmembrane region in the design of
the immunogen has been described. Reduced protection with
FIG. 4. Magnitude of antigen-specific T-cell responses to vaccine components. GP (S/G) (x axes) are shown in relation to each of the other two
antigens, GP (Z) and NP (Z) (y axes). All antigen-specific CD4?and CD8?T-cell responses for all subjects (vaccine and placebo recipients) as
assessed by ICS are shown. CD4?and CD8?T-cell responses are shown as a percentage of total CD4?or CD8?T cells on the x and y axes. CD4
responses are shown in the upper graphs, and CD8?responses are shown in the lower graphs. The red dashed line represents the threshold of
positivity (0.0445% for CD8?T cells and 0.0241% for CD4?T cells).
1274 MARTIN ET AL.CLIN. VACCINE IMMUNOL.
FIG. 5. Kinetics of CD4?and CD8?T-cell responses to GP (S/G). Responses were assessed by ICS and are shown over the course of the study. Results are presented
by dose group, with a blue line indicating the mean response over time for all subjects in a given group. The median of the response is represented in the box and whisker plots at each time point. The red dashed lines represent the positivity threshold for the ICS assay.
VOL. 13, 2006 PHASE I CLINICAL TRIAL OF EBOLA DNA VACCINE1275
an Ebola virus rAd immunogen containing a GP transmem-
brane region deletion compared to a point mutation in this
region or wild-type GP constructs has been found (22). Addi-
tionally, it was found that the NP gene is dispensable for
immune protection, and the addition of NP in a candidate
vaccine may diminish the immune response to Ebola virus GP
(23). Therefore, future formulations of this DNA product will
include multiple GP constructs encoding GP in either its wild-
type form or a modified form to optimize vaccine potency.
Because Ebola virus from Ivory Coast has been observed in
only one limited outbreak and is closely related to Ebola virus
Zaire, it is not included in vaccine formulations.
This is the first report of an evaluation of a candidate Ebola
virus vaccine in humans. This three-plasmid DNA candidate
Ebola virus vaccine was safe and well-tolerated in 21 healthy
adults. Importantly, DNA immunization induced both Ebola
virus-specific antibody and T-cell responses to the GP and NP
antigens. While Ebola virus-specific neutralizing antibody
could not be detected in vaccinees, the range of antibody titers
measured by ELISA was similar to those seen in nonhuman
primates following vaccination with similar vaccine constructs
(24). Recently, in a series of nonhuman primate studies dem-
onstrating protection from Ebola virus with vaccine constructs
expressing similar antigens as used in this clinic trial, IgG as
measured by ELISA correlated with survival. In functional
assays, serum antibodies were neither neutralizing nor enhanc-
ing, suggesting that IgG levels may reflect the overall level of
immune stimulation. Although antibody-dependent enhance-
ment of Ebola virus replication has been observed in tissue
culture, there is no evidence of antibody-dependent enhance-
ment in humans or in animal studies, and only protection,
rather than enhancement, has been observed in animal studies
evaluating DNA or rAd-based vaccine strategies (23, 27). In
the clinical trial described here, the vaccine-induced antibody
and T-cell-mediated immune responses were greatest to the
GP immunogens, with a less frequent response to the NP
immunogen, and Ebola virus-specific CD4?T-cell responses
were more frequent than CD8?T-cell responses. While the
presence of Ebola virus GP-specific IgG seems to predict sur-
vival in nonhuman primates, the definite correlate(s) of pro-
tection from Ebola virus infection is not known, and it is
possible that T-cell responses also contribute to protection.
Therefore, we believe it is important that a candidate Ebola
virus vaccine be capable of eliciting both Ebola virus-specific
antibody and T-cell responses.
Further studies are needed to determine the optimal pre-
ventive gene-based Ebola virus vaccine strategy. Our develop-
ment plan includes evaluation of DNA vaccination alone, rAd5
vaccination alone, and a heterologous prime-boost strategy of
DNA priming followed by rAd boosting. Even if the optimal
strategy were determined to be heterologous prime-boost, the
potential vaccines would need to be independently demon-
strated as safe and immunogenic. Since the prophylactic effi-
cacy of an Ebola virus vaccine cannot feasibly or ethically be
demonstrated in a human trial, the combination of safety and
immunogenicity data from phase I, II, and III human trials and
efficacy data from nonhuman primate studies will ultimately
need to be utilized to obtain licensure of an Ebola virus vaccine
under the Animal Rule.
The successful evaluation of a DNA vaccine to multiple
Ebola virus subtypes reported here provides the opportunity
for further clinical evaluation of candidate Ebola virus DNA
vaccines alone or in combination with Ebola virus rAd vaccines
as a heterologous prime-boost strategy. Evaluation of gene-
based candidate vaccines in humans will continue in parallel
with efforts to define immunological correlates of vaccine-in-
duced protection in nonhuman primate models of Ebola virus
infection. Together, these studies will provide the scientific
basis for identifying a vaccine strategy for the prevention of
Ebola virus and other filovirus infections in humans.
We thank the study volunteers who graciously gave their time and
understand the importance of finding a safe and effective Ebola virus
vaccine. We also thank NIH Clinical Center staff, NIAID staff, PRPL
and OCPL staff, the members of the Intramural NIAID DSMB,
EMMES Corporation (Phyllis Zaia, Lihan Yan, and others), Vical
Incorporated (David Kaslow and others), Biojector, Inc. (Richard
Stout and others), and other supporting staff (Richard Jones, Kathy
Rhone, Katina Bryan, Theodora White, Ariella Blejer, and Monique
Young) who made this work possible. We are grateful as well for the
advice and important preclinical contributions of VRC investigators
and key staff, including Daniel Douek, Yue Huang, Wing-Pui Kong,
Peter Kwong, Norman Letvin, Abraham Mittelman, Steve Perfetto,
Srini Rao, Robert Seder, Jessica Wegman, Richard Wyatt, Ling Xu,
and Zhi-yong Yang.
This work was funded by the National Institute of Allergy and
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