A Monovalent Chimpanzee Adenovirus Ebola Vaccine

The
n e w e ng l a n d j o u r na l
of
m e dic i n e
original article
A Monovalent Chimpanzee Adenovirus
Ebola Vaccine — Preliminary Report
Tommy Rampling, M.R.C.P., Katie Ewer, Ph.D., Georgina Bowyer, B.A.,
Danny Wright, M.Sc., Egeruan B. Imoukhuede, M.D., Ruth Payne, M.R.C.P.,
Felicity Hartnell, M.B., B.S., Malick Gibani, M.R.C.P., Carly Bliss, B.A.,
Alice Minhinnick, M.B., Ch.B., Morven Wilkie, M.R.C.P., Navin Venkatraman, M.R.C.P.,
Ian Poulton, Dip.H.E., Natalie Lella, B.A., Rachel Roberts, M.Sc.,
Kailan Sierra-Davidson, B.A., Verena Krähling, Ph.D., Eleanor Berrie, Ph.D.,
Francois Roman, M.D., Iris De Ryck, Ph.D., Alfredo Nicosia, Ph.D., Nancy J. Sullivan, Ph.D.,
Daphne A. Stanley, M.S., Julie E. Ledgerwood, D.O., Richard M. Schwartz, Ph.D.,
Loredana Siani, Ph.D., Stefano Colloca, Ph.D., Antonella Folgori, Ph.D.,
Stefania Di Marco, Ph.D., Riccardo Cortese, M.D., Stephan Becker, Ph.D.,
Barney S. Graham, M.D., Richard A. Koup, M.D., Myron M. Levine, M.D.,
Vasee Moorthy, D.Phil., Andrew J. Pollard, Ph.D., Simon J. Draper, D.Phil.,
W. Ripley Ballou, M.D., Alison Lawrie, Ph.D., Sarah C. Gilbert, Ph.D.,
and Adrian V.S. Hill, D.M.
A BS T R AC T
Background
The West African outbreak of Ebola virus disease has caused more than 8500 deaths.
A vaccine could contribute to outbreak control in the region. We assessed a monovalent formulation of a chimpanzee adenovirus 3 (ChAd3)–vectored vaccine encoding
the surface glycoprotein of Zaire ebolavirus (EBOV), matched to the outbreak strain.
Methods
After expedited regulatory and ethics approvals, 60 healthy adult volunteers in Oxford,
United Kingdom, received a single dose of the ChAd3 vaccine at one of three dose
levels: 1×1010 viral particles, 2.5×1010 viral particles, and 5×1010 viral particles
(with 20 participants per group). Safety was assessed over the next 4 weeks. Antibodies were measured on enzyme-linked immunosorbent assay (ELISA) and T-cell responses on enzyme-linked immunospot (ELISpot) and flow-cytometry assays.
The authors’ affiliations are listed in the
Appendix. Address reprint requests to
Dr. Hill at the Jenner Institute, University
of Oxford, Old Road Campus Research
Bldg., Headington, Oxford OX3 7DQ,
United Kingdom, or at [email protected]
.ox.ac.uk.
Drs. Rampling and Ewer contributed
equally to this article.
This article was published on January 28,
2015, at NEJM.org.
DOI: 10.1056/NEJMoa1411627
Copyright © 2015 Massachusetts Medical Society.
Results
No safety concerns were identified at any of the dose levels studied. Fever developed
in 2 of the 59 participants who were evaluated. Prolonged activated partial-thromboplastin times and transient hyperbilirubinemia were observed in 4 and 8 participants, respectively. Geometric mean antibody responses on ELISA were highest
(469 units; range, 58 to 4051; 68% response rate) at 4 weeks in the high-dose group,
which had a 100% response rate for T cells on ELISpot, peaking at day 14 (median,
693 spot-forming cells per million peripheral-blood mononuclear cells). Flow cytometry revealed more CD4+ than CD8+ T-cell responses. At the vaccine doses
tested, both antibody and T-cell responses were detected but at levels lower than
those induced in macaques protected by the same vaccine.
Conclusions
The ChAd3 monovalent vaccine against EBOV was immunogenic at the doses tested.
(Funded by the Wellcome Trust and others; ClinicalTrials.gov number, NCT02240875.)
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1
The
T
n e w e ng l a n d j o u r na l
he current outbreak of Ebola virus disease (EVD) in West Africa has led to
more than 8500 deaths.1 An effective vaccine may be necessary to contain this international public health emergency. No new vaccine
has been first tested in humans and rapidly developed within months in an attempt to control a
major infectious disease outbreak. However,
both the chimpanzee adenovirus (ChAd) and
modified vaccinia virus Ankara (MVA) vectors,2
which were developed by the Vaccine Research
Center of the National Institute of Allergy and
Infectious Diseases in collaboration with Okairos, had already been manufactured to clinical
grade at the time of the acceleration of the EVD
outbreak in early August 2014. These events provided the opportunity to design a rapid clinicaldevelopment program that could lead to deployment of the vaccine in 2015.
ChAd vectors make up a new-generation vaccine technology that first reached clinical assessment in 20073-5 and are generally used clinically with an MVA booster dose.6-14 A single
dose of 1010 or 1011 virus particles of chimpanzee
adenovirus type 3 (ChAd3, also called cAd3)
encoding the Zaire ebolavirus (EBOV) wild-type
surface glycoprotein had shown efficacy in cynomolgus macaques, which encouraged the assessment of a single-dose vaccine in this phase 1
trial, called EBL01.2 The Guinea outbreak strain
of ebolavirus is 97% identical in amino acid sequence to the well-characterized Zaire strain.15
Although the original clinical-development plan
for this Ebola vaccine included the use of a bivalent vaccine formulation of Zaire and Sudan
strains16 that would use both ChAd3 and MVA
primarily for biodefense, the ChAd3 vaccine encoding just the Zaire strain appeared to be a
potentially advantageous monovalent formulation for outbreak control on the basis of efficacy
data in macaques and was thus selected for testing in this study.
Biomanufacturing of large amounts of the
ChAd3 vaccine was a limiting factor in the development of an accelerated plan to undertake
large-scale efficacy trials and deployment. The
use of the monovalent formulation halves the
manufacturing challenge, as compared with use
of the bivalent vaccine, which includes a second
vector encoding the Sudan strain glycoprotein16
with only 60% identity to the Guinea outbreak
strain. Moreover, previous preclinical and clini2
of
m e dic i n e
cal assessments of viral vectors encoding multiple antigens or mixtures of vectors encoding
different antigens have sometimes shown reduced immunogenicity when more than a single
antigen vector was used.17,18 A further consideration in the trial design was the possibility that
a lower dose of ChAd might be sufficiently immunogenic with this insert, as suggested by
previous studies of clinical-dose response with
ChAd vectors,3 a factor that could thus allow the
deployment of more vaccine doses from each
manufacturing run.
With input from the World Health Organization (WHO),19 we therefore developed a plan for
rapid clinical assessment of the safety and immunogenicity of a monovalent formulation of
the ChAd3 vaccine against EBOV in August 2014
at three clinical sites (Oxford, United Kingdom;
Lausanne, Switzerland; and Bamako, Mali), with
an aim to immunize and evaluate 240 participants by late November. We report here on the
safety and immunogenicity of this monovalent
vaccine — now prioritized for use in a phase 3
trial and, potentially, for outbreak control — in
its first trial in Oxford, United Kingdom.
Me thods
Study Participants
The study was conducted at the Centre for Clinical Vaccinology and Tropical Medicine at the
University of Oxford. Participants were healthy
adults between the ages of 18 and 50 years who
provided written informed consent (Table 1).
Ethics and Regulatory Approval
The study was reviewed and approved by the U.K.
National Research Ethics Service, Committee
South Central–Oxford A, the Medicines and
Healthcare Products Regulatory Agency, and the
Oxford University Clinical Trials and Research
Governance team, who monitored compliance
with Good Clinical Practice guidelines. Safety
oversight was provided by an independent data
and safety monitoring board. The ChAd3 vaccine
was provided by the Vaccine Research Center of
the National Institute of Allergy and Infectious
Diseases and GlaxoSmithKline.
Study Design
EBL01 was a phase 1, dose-escalation, open-label
study assessing the safety and immunogenicity
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A Monovalent Chimpanzee Adenovirus Ebola Vaccine — Preliminary Report
of the experimental monovalent ChAd3 vaccine
against EBOV. Eligible participants were assigned
to receive the ChAd3 vaccine as a single intramuscular injection in one of three dose groups:
group 1 (1×1010 viral particles), group 2 (2.5×1010
viral particles), and group 3 (5×1010 viral particles). The first nine vaccinations occurred in a
stepwise dose-escalation manner, with three
participants in the low-dose group being vaccinated and followed for a minimum of 48 hours
before proceeding to immunize volunteers in
the next dose group. Full details regarding the
study conduct are provided in the protocol,
which is available with the full text of this article at NEJM.org.
Study Vaccine
The ChAd3 drug substance was manufactured at
Advent, a subsidiary of Okairos (now Glaxo­
SmithKline), and the drug product was manufactured at the Vaccine Research Center Vaccine Pilot Plant, under contract with the Vaccine Clinical
Materials Program, Leidos Biomedical Research.
The vaccine is a sterile, aqueous, buffered solution that includes the ChAd3-vectored vaccine
encoding the surface glycoprotein of EBOV in
single-dose vials of 9.1×1010 particle units per
milliliter, as determined on high-performance
liquid chromatography. Different dose levels
were achieved by adjusting the volume of vaccine
injected from 110 μl (in group 1) to 275 μl (in
group 2) and 550 μl in group 3.
Assessment of Safety
Participants were observed for 60 minutes after
vaccination. Follow-up visits were scheduled for
days 1, 7, 14, 28, 90, and 180 after vaccination,
with an additional visit at day 10 for participants
in group 3. All participants were given access to
an electronic diary card on which to record all
solicited symptoms for 7 days after vaccination
and unsolicited symptoms for 28 days after vaccination. A review of symptoms occurred at
each follow-up visit, in addition to testing that
included a full blood count and the measurement of urea and electrolytes, liver enzymes,
activated partial-thromboplastin time, prothrombin time, and fibrinogen. Severity grading of adverse events and the assignment of a
causal relationship for unsolicited adverse
events were conducted according to predefined
criteria.
Table 1. Characteristics of the Participants at Baseline.*
Characteristic
Group 1
(N = 20)
Group 2
(N = 20)
Group 3
(N = 20)
All
Participants
(N = 60)
6 (30)
13 (65)
13 (65)
32 (53)
14 (70)
7 (35)
7 (35)
28 (47)
1 (5)
1 (2)
Sex — no. (%)
Male
Female
Age
Group — no. (%)
18–20 yr
0
0
21–30 yr
9 (45)
8 (40)
11 (55)
28 (47)
31–40 yr
6 (30)
8 (40)
3 (15)
17 (28)
41–50 yr
5 (25)
4 (20)
5 (25)
14 (23)
Mean — yr
31.7±9.0
33.5±8.0
31.3±9.3
32.2±8.7
Range — yr
21–48
22–49
18–48
18–49
19 (95)
19 (95)
18 (90)
55 (92)
Race — no. (%)†
White
Black
0
0
0
0
Asian
0
1 (5)
1 (5)
2 (3)
Mixed
1 (5)
0
1 (5)
3 (5)
0
1 (5)
1 (5)
2 (3)
Body-mass index‡
Value — no. (%)
<18.5
11 (55)
11 (55)
16 (80)
37 (62)
25–29.9
18.5–24.9
8 (40)
6 (30)
3 (15)
18 (30)
≥30
1 (5)
2 (10)
0
3 (5)
Mean
24.5±3.3
24.6±3.4
22.7±2.3
24.0±3.1
Range
18.5–30.5
17.4–33.0
17.1–27.4
17.1–33.0
*Plus–minus values are means ±SD. There were no significant differences between the study groups.
†Race was self-reported.
‡The body-mass index is the weight in kilograms divided by the square of the
height in meters.
ELISA Analyses
We assessed anti–glycoprotein IgG responses to
EBOV using enzyme-linked immunosorbent assay (ELISA) by means of a commercial kit (AE
320620-1, Alpha Diagnostic International [ADI]),
according to the manufacturer’s instructions,
with serum diluted at 1:100 and 1:500. Optical
density was read at 450 nm on an EL800 microplate reader (BioTek). Values are reported with or
without the subtraction of the prevaccination optical density and were converted to micrograms
per milliliter with the use of the reference serum
provided by the manufacturer. We also used end-
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3
The
n e w e ng l a n d j o u r na l
Table 2. Adverse Events.*
Symptom
and Intensity
Group 1
(N = 19)
Group 2
(N = 20)
Group 3
(N = 20)
All Participants
(N = 59)
number (percent)
Local
Pain
Mild
10 (53)
14 (70)
7 (35)
31 (53)
Moderate
2 (11)
2 (10)
3 (15)
7 (12)
Mild swelling
3 (16)
1 (5)
2 (10)
6 (10)
Mild redness
3 (16)
7 (35)
3 (15)
13 (22)
Mild warmth
10 (53)
7 (35)
4 (20)
21 (36)
2 (11)
4 (20)
0
6 (10)
0
1 (5)
1 (5)
2 (3)
Mild
3 (16)
5 (25)
6 (30)
14 (24)
Moderate
0
2 (10)
1 (5)
3 (5)
Severe
0
1 (5)
0
1 (2)
Mild itch
m e dic i n e
point titration to perform assays to compare the
responses directly with titers that were associated with protection in macaque efficacy trials2
and with a method used in a trial of a bivalent
ChAd3 vaccine in humans conducted by the National Institutes of Health (NIH).16 The end-point
dilution ELISA for the NIH EC90 (the concentration at which there is a 90% decrease in antigen
binding) assay was performed as reported previously,2 and values are also presented with or
without the subtraction of the prevaccination
optical density to maintain consistency with previous studies in which this assay was used.
T-Cell Assays
Systemic
Moderate fever
Feverishness
Myalgia
Mild
7 (37)
6 (30)
Moderate
0
3 (15)
11 (55)
0
3 (5)
Mild
1 (5)
3 (15)
3 (15)
7 (12)
Moderate
0
1 (5)
1 (5)
2 (3)
Mild
7 (37)
11 (55)
8 (40)
26 (44)
Moderate
2 (11)
3 (15)
1 (5)
6 (10)
11 (58)
8 (40)
10 (50)
29 (49)
2 (11)
2 (10)
3 (15)
7 (12)
Mild
3 (16)
1 (5)
4 (20)
8 (14)
Moderate
1 (5)
2 (10)
1 (5)
4 (7)
Severe
0
1 (5)
0
1 (2)
Mild
6 (32)
5 (25)
7 (35)
18 (31)
Moderate
2 (11)
1 (5)
2 (10)
5 (8)
Headache
Fatigue
Mild
Moderate
Nausea
Malaise
Severe
Use of acetaminophen,
NSAID, or aspirin
for symptoms
0
1 (5)
0
8 (42)
8 (40)
7 (35)
Ex vivo enzyme-linked immunospot (ELISpot)
and intracellular cytokine staining assays were
performed largely as described previously7,11
with the use of overlapping peptide pools. (Additional details are provided in the Methods section in the Supplementary Appendix, available at
NEJM.org.)
24 (41)
Arthralgia
1 (2)
23 (39)
*Shown are the maximum solicited local and systemic reactogenicity symptoms collected for 7 days after vaccination. Frequency is calculated as the
number of participants counted once at the time of the worst severity of the
event. Intensity categories in which all the values were zero are not shown.
NSAID denotes nonsteroidal antiinflammatory drug. The case definitions for
these adverse events can be found in the protocol.
4
of
R e sult s
Study Population
From September 17, 2014, to November 18, 2014,
a total of 60 of the 88 participants who were
screened for eligibility were vaccinated (Fig. S1 in
the Supplementary Appendix). One participant in
group 1 withdrew on day 1 after vaccination owing to an aversion to venipuncture. The participant had reported no symptoms at the time of
withdrawal but declined to attend additional
follow-up visits. E-mail correspondence on day
10 after vaccination confirmed that the participant remained well, with no symptoms to report.
All the remaining 59 participants completed at
least 28 days of follow-up.
Safety
A full listing of the frequency and maximum severity of solicited, unsolicited, and laboratory adverse events according to dose group are provided
in Table 2, and in Tables S1 and S2 in the Supplementary Appendix. A majority of the adverse
events that were reported in all dose groups were
mild in severity, with no unexpected serious adverse reactions or serious adverse events (Table 2).
Two participants (one in group 2 and one in group
3) had an episode of moderate fever (temperature,
38.1°C and 38.9°C, respectively). Both events oc-
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A Monovalent Chimpanzee Adenovirus Ebola Vaccine — Preliminary Report
curred within the first 24 hours after vaccination,
and neither persisted for more than 24 hours.
A prolonged activated partial-thromboplastin
time was observed in four participants during
the first 2 weeks after vaccination: in three participants in group 2 (two with a grade 1 elevation and one with a grade 2 elevation) and in one
participant in group 3 with a grade 1 elevation.
None of the prolongations were associated with
symptoms or clinical features of coagulopathy.
The elevation had fully resolved in all participants by 10 weeks after vaccination. No further
abnormality was found in any of these participants on extended hematologic and coagulation
evaluation.
Transient induction of an antiphospholipid
antibody causing an in vitro artifact on the laboratory assay for activated partial-thromboplastin
time was reported previously after the administration of adenovirus vectors.20,21 Transient mild
lymphocytopenia was noted on day 1 after vaccination in five participants in group 1, four in
group 2, and eight in group 3, and moderate
lymphocytopenia was noted in two participants
each in group 2 and group 3 on day 1. Transient
mild-to-moderate elevations in bilirubin were
recorded in six participants in group 2 and group
3 combined. Transient hyperbilirubinemia in the
severe range was recorded in two participants
(one in group 2 and one in group 3) who had a
prevaccination diagnosis of Gilbert’s syndrome.
Antibody Responses
We measured optical density to assess IgG responses against the surface glycoprotein of EBOV
with a single serum dilution before immunization and at days 14 and 28 (Fig. 1A and 1B). Antibody responses were highest at 28 days after
vaccination, with no significant difference in response at any time point among the three dose
groups. Vaccination induced a significant increase in the antibody titer in all groups (P<0.001
by the Wilcoxon matched-pairs test) (Fig. 1, and
Fig. S2A in the Supplementary Appendix).
We also used the EC90 end-point titration
method described in Methods to compare the
responses directly with titers that were associated with protection in macaque efficacy trials2
and in a trial of bivalent ChAd3 vaccine in humans conducted by the NIH16 (Fig. 1C and 1E,
and Fig. S2B in the Supplementary Appendix). In
the analyses of samples obtained from 58 par-
ticipants on day 28, the geometric mean titer
after the subtraction of prevaccination responses
was 235 units in group 1, 402 in group 2, and
469 in group 3 (Fig. 1E).
The results of the two above-mentioned assays, the ELISA with the ADI kit and the endpoint dilution by means of the NIH method,2,16
were highly correlated with each other. We used
linear regression to determine a conversion factor of 1 optical-density unit on the ADI scale to
a dilution factor of 1202 on the NIH EC90 assay
(Fig. 1C), which yielded geometric mean values
of 481 units in group 1, 514 in group 2, and 681
in group 3. We also used a heat-killed virus antigen ELISA to assess the induction of antibody
titers against the Guinea outbreak strain of
Ebola virus, using the Guinea strain whole virus
rather than the surface glycoprotein from the
Zaire strain as the antigen. In these analyses, 6
of 35 participants (17%) who were tested had a
positive response (2 of 10 in group 1, 1 of 12 in
group 2, and 3 of 13 in group 3) (Fig. 1D).
ELISpot Responses
T-cell responses to the 10 peptide pools were assessed by means of interferon-γ ELISpot assays
on days 0, 7, 14, and 28 (Fig. 2A). Before vaccination, responses to the EBOV glycoprotein were
below the level of detection of the assay in 72%
of the participants (median, 50 spot-forming
cells [SFCs] per million peripheral-blood mononuclear cells; 95% confidence interval, 53 to 63)
(Fig. 2B). ELISpot responses peaked at day 14
(Fig. 2C), with median responses of 431 SFCs (interquartile range, 203 to 783) in group 1, 387
SFCs (interquartile range, 281 to 834) in group 2,
and 693 SFCs (interquartile range, 348 to 866) in
group 3. There was no significant difference in
the magnitude of the immune response at any
time point among the different dose groups on
the basis of the Kruskal–Wallis test.
Flow Cytometry with Intracellular Cytokine
Staining
At 28 days after vaccination, we assessed the
CD4+ and CD8+ T-cell responses to vaccination
according to the secretion of interferon-γ, interleukin-2, or tumor necrosis factor α. Cytokines
were predominantly detected from CD4+ T cells,
and cytokine expression tended to be higher in
group 3 than in group 1 (P = 0.06 by the Kruskal–
Wallis test with Dunn’s correction for multiple
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5
The
n e w e ng l a n d j o u r na l
A
of
m e dic i n e
B
P=0.001
4
63%
70%
90%
ADI EBOV Glycoprotein
ADI EBOV Glycoprotein
2.0
1.5
1.0
Group 1
Group 2
Group 3
0.5
0.0
0
7
14
21
28
3
2
1
0
Day 0
Day
Day 14 Day 28
Day 0
Group 1
C
Day 14 Day 28
Day 0
Group 2
Day 14 Day 28
Group 3
D
100
EBOV Antigen ELISA (Day 28)
NIH EC90 (Day 28)
104
103
102
r=0.77
P<0.001
101
0.1
1.0
10−1
r=0.51
P=0.003
10−2
0.1
10.0
1.0
ADI EBOV Glycoprotein (Day 28)
10.0
ADI EBOV Glycoprotein (Day 28)
E
NIH EC90 (Day 0 subtracted)
104
103
102
101
100
Day 14
Day 28
Group 1
Day 14
Day 28
comparisons) (Fig. 3A). Expression of the degranulation marker CD107a was also detected
from CD8+ T cells (Fig. 3B). Both CD4+ and
CD8+ T cells showed polyfunctional and monofunctional phenotypes, with dual positive cells
predominating (Fig. 3C and 3D).
6
Day 14
Group 2
Day 28
Group 3
Discussion
The safety and immunogenicity profile of this
monovalent chimpanzee adenovirus–vectored
vaccine supported its assessment either alone or
as part of a heterologous prime-boost vaccination
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A Monovalent Chimpanzee Adenovirus Ebola Vaccine — Preliminary Report
Figure 1 (facing page). Antibody Responses to the Zaire
ebolavirus Glycoprotein.
Panel A shows anti–glycoprotein IgG levels, as measured with the use of an enzyme-linked immunosorbent assay (ELISA) (Alpha Diagnostic International
[ADI]), at baseline and on days 14 and 28 after vaccination with increasing doses of a chimpanzee adenovirus
3 (ChAd3)–vectored vaccine against Zaire ebolavirus
(EBOV). The antibody levels in the three dose groups
are shown in optical-density (OD) units without subtraction of background levels on the y axis. The analyses are for 19 patients in group 1, 20 in group 2, and
19 in group 3. No significant differences in responses
among doses at any time point were detected. Lines
represent geometric mean IgG responses for each
group. Panel B shows individual responses at days 0,
14, and 28 for each group in geometric means and
95% confidence intervals. The percentage of participants with positive results at the peak time points on
day 28 are indicated. The dotted line represents the
positive threshold (optical density, 1.024), as calculated from the mean +3 SD of the day 0 responses for all
participants. Panel C shows Spearman’s correlation for
the results on the ADI ELISA and the NIH EC90 endpoint dilution ELISA for 58 samples tested with the two
assays for IgG antibodies against EBOV. Linear regression of these data indicates that 1 OD unit with the
ADI assay is equivalent to 1202 units on the NIH EC90
scale for serum diluted at 1:500. On the basis of this
conversion, the geometric mean responses were 481
units for group 1, 514 for group 2, and 681 for group 3
on the NIH EC90 scale, representing reciprocal serum
dilutions. Panel D shows Spearman’s correlation for
the ADI ELISA and the EBOV antigen ELISA using heatkilled Guinea strain virus in samples obtained from 35
participants. Panel E shows titers for 58 participants
with the use of the NIH EC90 ELISA reported previously16 (with 19 participants in group 1, 20 in group 2, and
19 in group 3). The geometric mean titers in these
groups were 235, 402, and 469, respectively, after the
subtraction of prevaccination responses at baseline.
regimen for the prevention of infection and disease by the Guinea outbreak strain of EBOV. A
considerable effort by many groups and agencies
allowed for a successful application for funding,
the filling of the monovalent vaccine, submission
and approval of regulatory and ethical applications, completion of contractual arrangements,
and initiation of this clinical trial in approximately 1 month. The prioritization of this trial by
several funders, regulators, and reviewers in response to the declaration of an international public health emergency was key to rapid progress.
No safety concerns were identified for this
ChAd vector, with the majority of the recorded
local and systemic adverse events being mild and
short-lived. These findings are similar to the
safety profile of other simian adenoviral vectors
that have been assessed clinically.3-7,11-14,18 As of
mid-December 2014, approximately 250 participants had received this monovalent vaccine, with
no reports of serious vaccine-related adverse
events.22,23
Accrual of safety data from this trial allowed
for initiation of immunization with the same
monovalent vaccine in Bamako, in early October
2014,23 and to date 91 participants have been
immunized at that center.22 In previous trials of
some viral-vectored vaccines, reduced immunogenicity has been observed among African populations, as compared with the responses among
northern Europeans,11,24 which highlights the
importance of immunogenicity assessments in
West Africa for a vaccine that is intended for use
in the control of the current Ebola outbreak.
In our study, immunogenicity assessments
included measurements of glycoprotein-specific
antibodies against EBOV by means of an ELISA
analysis for which the manufacturer provided a
conversion factor and control serum, which allowed for an estimated readout in micrograms
per milliliter, as well as both interferon-γ
ELISpot and flow-cytometry assays performed
on fresh cells. These ex vivo T-cell assays provide
a sensitive measure of T-cell immunogenicity
that is not subject to diminution by cryopreservation of peripheral-blood mononuclear cells.
Antibody responses, as measured on ELISA,
showed a weak dose–response relationship, with
a maximal geometric mean response at day 28
after immunization for the dose groups tested in
this study. The range of ELISA values that were
measured by means of end-point titration in the
high-dose group (group 3) was 58 to 4051, with
a geometric mean titer of 469, which was generally lower than the titers of 967 to 6600 that
were reported in macaques protected by 2×1010
viral particles of the bivalent ChAd3 vector,2 although some overlap is evident.
T-cell responses that were measured on
ELISpot assays were as expected for this viral
vector, with responses peaking at day 14 at
about 700 SFCs in the high-dose group.6 Flow
cytometry on day 28 showed a predominantly
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7
The
n e w e ng l a n d j o u r na l
A T-Cell Response
800 57
55
58
58
Group 1
Group 2
SFC/106 PBMC
600
Group 3
400
200
0
0
7
14
21
28
Day
B Day 0
SFC/106 PBMC
103
102
101
1
2
3
Dose Group
C Day 14
SFC/106 PBMC
104
89.5%
80.0%
100%
1
2
3
103
102
101
Dose Group
CD4+ T-cell response, in contrast to the predominant CD8+ T-cell responses induced by
chimpanzee adenoviral vectors in many animal
models. Although direct comparison is difficult
because responses in the macaque study were
reported as a percentage of the subpopulation of
memory cells rather than as a percentage of all
CD4+ and CD8+ lymphocytes,2 the magnitude
8
of
m e dic i n e
Figure 2. Responses to Peptides Spanning the Glycoprotein at Increasing Doses of Vaccine.
Panel A shows median responses on interferon-γ
­enzyme-linked immunospot (ELISpot) assays to 10
summed glycoprotein peptide pools at increasing doses of the ChAd3 vaccine. Numbers indicate the number of data points included at each time point. Responses are measured in the number of spot-forming
cells (SFCs) per million peripheral-blood mononuclear
cells (PBMCs). Panel B shows responses before immunization at baseline. Panel C shows responses at the
peak of the cell-mediated immune response at 14 days
after vaccination, with the percentages of participants
who had a positive response (as indicated by a significant increase over the background prevaccination level)
shown for each group. In Panels B and C, the horizontal lines represent medians. The analyses are for 19 patients in group 1, 20 in group 2, and 19 in group 3.
of responses that we observed, about 0.07% of
CD8+ T cells, appears to be lower than that reported for macaques protected by this vaccine.2
As expected, there was substantial polyfunctionality in the observed T-cell responses, but the
role of T-cell quality in protection against ebolavirus remains unclear.
If greater immunogenicity is required, one
option is to use a higher dose of vaccine, as suggested by the improved immunogenicity of a
bivalent formulation of this vaccine16 encoding
the glycoproteins of both the Sudan and Zaire
strains at a dose of 2×1011 viral particles, and
two trials of higher-dose monovalent vaccines
are in progress. Another possible option for inducing a higher response is to boost the immunogenicity of ChAd-primed antibody and T-cell
responses by means of an MVA vector encoding
the same antigen.3-5 Reported antibody and T-cell
responses in humans with the use of a booster
dose of an MVA vector are increased by a factor
of approximately 30 for antibodies and by a factor of 5 to 10 for T-cell responses.3-5,7 In macaques, the activation of antibodies that was
observed with an MVA vector encoding the EBOV
glycoprotein was increased by a factor of 30, as
compared with ChAd3 priming alone, and there
was an increase in the frequency of antigenspecific T cells by a factor of approximately 5,
with improved durability of vaccine efficacy.2
Phase 1 assessment of a booster study of some
of these vaccinees with an MVA vector began in
late November 2014. Future phase 3 efficacy trials could consider the potential need for such a
booster dose.
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A Monovalent Chimpanzee Adenovirus Ebola Vaccine — Preliminary Report
A Day 28
B Day 28
CD4+
CD8+
P=0.001
100
CD107a Expresssion
(% of CD8+ T-cell subset)
Total Cytokine Response
(% of subset)
100
10−1
10−2
10−3
1
2
3
1
2
3
10−1
10−2
10−3
Dose Group
1
2
3
Dose Group
C CD4+ T Cells
Group 1
Group 2
Group 3
Interferon-γ Interleukin-2 TNF-α
+
+
+
+
+
−
+
−
+
+
−
−
−
+
+
−
+
−
−
−
+
D CD8+ T Cells
Group 1
Group 2
Group 3
Interferon-γ Interleukin-2 TNF-α
+
+
+
+
+
−
+
−
+
+
−
−
−
+
+
−
+
−
−
−
+
Figure 3. Flow Cytometry with Intracellular Cytokine Staining at 28 Days after Vaccination.
Panel A shows the percentages of cells secreting interferon-γ, interleukin-2, and tumor necrosis factor α (TNF-α) from CD4+ and CD8+
T cells, as quantified for each dose group and shown as the percentage of cells expressing any one of the three cytokines. Positive glycoprotein-specific CD4+ T-cell responses were detected in 50% of samples in group 1, 71% of samples in group 2, and 92% of samples in
group 3. For CD8+ T cells, the corresponding positivity rates were 20%, 64%, and 54%, respectively. For CD4+ T cells, there was a trend
toward higher cytokine expression in group 3 than in group 1 (P = 0.06 by the Kruskal–Wallis test with Dunn’s correction for multiple
comparisons). Panel B shows the expression of CD107a cells (a functional marker for the identification of activity of lytic cells) at each
dose. The horizontal lines indicate medians, and I bars indicate interquartile ranges. Panels C and D show the proportions of CD4+ and
CD8+ T cells producing any combination of interferon-γ, interleukin-2, and TNF-α. The analyses are for 10 patients in group 1, 14 in
group 2, and 13 in group 3.
In conclusion, the safety and immunogenicity
profile of the ChAd3 vaccine in our study encourage further assessment of this vector alone
and in heterologous prime-boost regimens in
future phase 1 and phase 2 trials, as well as
consideration of the inclusion of this vaccine
vector in proposed phase 3 trials in countries in
which Ebola is endemic in early 2015.
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9
The
n e w e ng l a n d j o u r na l
The views expressed in this article are those of the authors
and do not necessarily represent the position or policies of the
World Health Organization.
Supported by the Wellcome Trust, the United Kingdom Department for International Development, the Medical Research
Council (grant 106325/Z/14/Z), and the Oxford Biomedical Research Centre of the U.K. National Institute for Health Research.
The ChAd3 vaccine was provided by the Vaccine Research Center
of the National Institute of Allergy and Infectious Diseases and
GlaxoSmithKline.
Disclosure forms provided by the authors are available with
the full text of this article at NEJM.org.
of
m e dic i n e
We thank the Wellcome Trust award scientific advisory board
members (Brian Greenwood, Peter Piot, Pontiano Kaleebu, Allan Saul, Paul Kaye, Charlie Weller, and Morven Roberts), John
Mascola (NIH), Marie-Paul Kieny (WHO), Samba Sow (Center
for Vaccine Development, Mali), and Umberto Dalessandro
(Medical Research Council, Gambia) for their advice; Carly Banner and Geoff Lees for arranging contracts; Mary E. Enama
(NIH) and Nina M. Berkowitz (NIH) for their contributions to
the protocol; Zonghui Hu (NIH) for statistical analysis; and the
Medicines and Healthcare Products Regulatory Agency and the
Oxfordshire research ethics committee for their exceptionally
rapid review.
Appendix
The authors’ affiliations are as follows: the Jenner Institute and Centre for Clinical Vaccinology and Tropical Medicine, University of
Oxford, and the National Institute for Health Research Oxford Biomedical Research Centre, Oxford, United Kingdom (T.R., K.E., G.B.,
D.W., E.B.I., R.P., F.H., M.G., C.B., A.M., M.W., N.V., I.P., N.L., R.R., K.S.-D., E.B., A.J.P., S.J.D., A.L., S.C.G., A.V.S.H.); Institute of
Virology, Philipps University Marburg, Marburg, Germany (V.K., S.B.); GlaxoSmithKline Biologicals, Rixensart, Belgium (F.R., I.D.R.,
W.R.B.); ReiThera, Rome (A.N., L.S., S.C., A.F., S.D.M.), and CEINGE and the Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Naples (A.N.) — both in Italy; Vaccine Research Center, National Institute of Allergy and
Infectious Diseases, National Institutes of Health, Bethesda (N.J.S., D.A.S., J.E.L., R.M.S., B.S.G., R.A.K.), and Center for Vaccine Development, University of Maryland School of Medicine, Baltimore (M.M.L.) — both in Maryland; and Keires, Basel (R.C.), and the World
Health Organization, Geneva (V.M.) — both in Switzerland.
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