BRIEF REPORT
Safety and Immunogenicity of DNA/
Modified Vaccinia Virus Ankara
Malaria Vaccination in African Adults
Vasee S. Moorthy,1,2 Margaret Pinder,1 William H. H. Reece,2
Kate Watkins,2 Sowsan Atabani,1 Carolyn Hannan,2 Kalifa Bojang,1
Keith P. W. J. McAdam,1 Joerg Schneider,2 Sarah Gilbert,2
Adrian V. S. Hill2
1
Medical Research Council Laboratories, Banjul, The Gambia; 2Nuffield
Department of Medicine, University of Oxford, John Radcliffe Hospital,
Oxford, United Kingdom
The present study is an investigation of the safety and immunogenicity of DNA and modified vaccinia virus Ankara
(MVA) candidate vaccines, each encoding the malaria DNA
sequence multiple epitope–thrombospondin related adhesion protein (ME-TRAP), against Plasmodium falciparum.
DNA ME-TRAP and MVA ME-TRAP are safe and immunogenic for effector and memory T cell induction. MVA METRAP, with or without prior DNA ME-TRAP immunization,
was more immunogenic and more cross-reactive in malariaexposed individuals than in malaria-naive individuals, a finding suggesting that recombinant MVA vaccines are particularly promising for the development of a malaria vaccine for
exposed populations. Both CD4+ and CD8+ T cells were induced by these vaccines.
On average, a child dies every 30 s of malaria in sub-Saharan
Africa [1]. An effective malaria vaccine is urgently needed. In
several animal models of malaria, cytotoxic T cells are critical
for protection against infection [2], and there is indirect evidence that cytotoxic T cells play a similar role in human Plasmodium falciparum malaria [3]. If a so-called priming immunization with a plasmid DNA encoding a preerythrocytic
Received 10 February 2003; accepted 7 May 2003; electronically published 30 September
2003.
Presented in part: Keystone conference, “Malaria’s challenge: from infants to genomics to
vaccines,” Keystone, Colorado, February 2002; American Society of Tropical Medicine and
Hygiene Annual Meeting, Atlanta, Georgia, November 2001; World Health Organization
meeting, “Safety of DNA vaccines,” Geneva, Switzerland, February 2001.
Potential conflict of interest: A.V.S.H. is a cofounder of and an equity holder in Oxxon
Pharmaccines, a company developing prime-boost therapeutic vaccines.
Financial support: Wellcome Trust Training Fellowship in Clinical Tropical Medicine (award
060147 to V.S.M.); Wellcome Trust Principal Fellowship (to A.V.S.H.).
Reprints or correspondence: Dr. Adrian V. S. Hill, Nuffield Dept. of Clinical Medicine, Level
7, John Radcliffe Hospital, Oxford, OX3 9DU, UK ([email protected]).
The Journal of Infectious Diseases 2003; 188:1239–44
2003 by the Infectious Diseases Society of America. All rights reserved.
0022-1899/2003/188080-0020$15.00
malaria antigen is followed by a boosting immunization with
a recombinant, nonreplicating viral vector encoding the same
antigen, there results a 10-fold amplification of CD8+ T cell
immunogenicity, compared with that produced by DNA vaccination alone and, in 2 mouse models of malaria, complete
protection [4]. In these experiments, protection correlated with
secretion of interferon-g by splenocytes in an ex vivo ELISPOT
assay. Similar results have been published for human immunodeficeincy virus (HIV) [5], tuberculosis (TB) [6], and Ebola
virus [7] (DNA/adenovirus) models. A particularly immunogenic viral vector for boosting immunization is modified vaccinia virus Ankara (MVA), a vector suitable for human use.
MVA is a highly attenuated vaccinia virus with an exceptionally
good safety profile [8].
We have developed 2 novel vaccine candidates, DNA multiple epitope (ME)–thrombospondin related adhesion protein
(TRAP) and MVA ME-TRAP. The ME-TRAP construct consists
of the ME string [9] of 18 T cell epitopes and 2 B cell epitopes,
from 6 preerythrocytic P. falciparum antigens and the T9/96
strain of an entire, well-characterized preerythrocytic antigen,
TRAP [10]. Since August 1999, we have investigated, in the
first prime-boost trials of DNA-based vaccines in humans, their
safety, immunogenicity, and efficacy in phase 1/2a trials in Oxford. In this report, we describe the first African phase 1 trial
to assess the potential of this new vaccine technology.
Volunteers and methods. Volunteers were recruited from
the periurban community of Bakau, which is on the coast of
The Gambia. Approval was obtained from the Joint Gambian
Government/Medical Research Council Ethics Committee and
the Central Oxford Research Ethics Committee. Written, informed consent was obtained from all volunteers, after obtaining
initial community consent, having discussion with volunteers in
the local languages, and disseminating information sheets and
consent forms translated into local languages in Arabic script.
The trial was conducted in accordance with Medical Research
Council, UK, guidelines for the conduct of clinical trials.
Potential volunteers underwent thorough clinical evaluation and were screened for hematological (full blood count),
renal (plasma creatinine and urinalysis), and hepatic (plasma
alanine aminotransferase [ALT]) dysfunction. A total of 20
semi-immune, healthy adults, 18–45 years old, were enrolled.
An independent safety monitor based in The Gambia monitored the study.
For comparison between malaria-exposed and -naive individuals, ELISPOT data from assays performed on volunteers
from the United Kingdom are included in the analysis. The
BRIEF REPORT • JID 2003:188 (15 October) • 1239
UK study is reported in full elsewhere [11]. The UK DNA/
MVA group consisted of 9 volunteers (3 volunteers in each
group), who received either 1, 2, or 3 1-mg immunizations of
DNA ME-TRAP followed by 2 immunizations of 3 ⫻ 107 pfu
of MVA ME-TRAP [12]. In these 3 groups, effector T cell
frequencies were equivalent, and, therefore, the vaccination regimen received by UK DNA/MVA vaccinees were comparable
to those received by Gambian DNA/MVA vaccinees. The UK
MVA-alone group consisted of 5 volunteers who received 3
immunizations 3 ⫻ 107 pfu of MVA ME-TRAP (identical to the
immunizations given to the Gambian MVA-alone group).
The 2 study vaccines were DNA ME-TRAP and MVA METRAP. The individual epitopes that constitute the ME string
are described in detail elsewhere [9]. The clinical vaccines were
manufactured by contract manufacturers (DNA ME-TRAP by
Qiagen and MVA ME-TRAP by IDT). Regulatory approval for
prior UK phase 1 studies of these vaccines was obtained from
the UK Medicines Control Agency (MCA). The Gambian government accepts MCA approval in the absence of a Gambian
regulatory authority.
The present study was a phase 1 open-label trial. All vaccinations in both the UK group and the Gambian group were
administered at 3-week intervals. A total of 12 volunteers received 2 1-mg immunizations of DNA ME-TRAP intramuscularly, followed by 2 immunizations of 3 ⫻ 107 pfu of MVA
ME-TRAP intradermally; 8 volunteers received 3 immunizations of 3 ⫻ 107 pfu of MVA ME-TRAP intradermally. Eligible
volunteers were allocated to each of these 2 groups alternately,
in order of enrollment. Each volunteer was observed for at least
1 h after vaccination. Study visits were scheduled for 1, 2, 3,
7, and 28 days after each vaccination and for 8–10 weeks after
the final vaccination. A total of 6 screened, healthy, unvaccinated volunteers were bled 3 times on days 0, 56–84, and 140–
156 to assess, in the Gambian population, background variation
in effector T cell responses over the study period.
Each Gambian volunteer had 30 mL of venous blood drawn
from an antecubital vein, on the following 5 occasions: screening (day ⫺28 to day ⫺7); 1 week after the first vaccination (in
the MVA group only; day 7); 1 week after the second vaccination
(in both groups; day 28); 1 week after the third vaccination (in
both groups; day 49); 1 week after the fourth vaccination (in
the DNA/MVA group only; day 70); and 8–10 weeks after the
final vaccination (day 142–156). Complete blood counts and
ALT and creatinine assays were performed according to the
standard operating procedures of Medical Research Council
Laboratories, The Gambia.
ELISPOTs were performed on Millipore MAIP S45 plates
with MabTech antibodies, in accordance with the manufacturer’s instructions. Quantities of 4 ⫻ 10 5 freshly isolated peripheral blood mononuclear cells (PBMCs) were incubated for
18–20 h on the ELISPOT plates, in the presence of 25 mg/mL
1240 • JID 2003:188 (15 October) • BRIEF REPORT
of peptides, before being developed. The number of spot-forming cells (SFCs) were counted by the AutoImmun Diagnostika
system. Individual 8–17-mer epitopes were used for epitopes
from the ME string, whereas 20-mer epitopes overlapping by
10 were used to span TRAP, with both T9/96 and 3D7 strains
of TRAP spanned in their entirety. Because of cell-number
limitations, peptides were assayed in pools, and cells were assayed in duplicate for each pool. Cell separations were performed on frozen cells by use of Miltenyi Biotech magnetic cell
sorting beads and were checked by costaining and fluorescence
activated–cell sorter analysis.
Recombinant T9/96 TRAP and 3D7 TRAP were coated overnight at 4C on Nunc immunoplates, at a concentration of 4
mg/mL. Standard ELISAs were performed, with incubations for
2 h at 37C.
Peptide pools 5–9 spanned 3D7 TRAP, and 10–13 spanned
T9/96 TRAP (figure 1). Spots were summed, across relevant
Figure 1. Breadth of effector T cell responses, by ex vivo ELISPOT.
The figure represents a time course for 1 Gambian volunteer. A total of
13 peptide pools are shown along the X-axis. Pool 1 is the negative
control (cells and no peptide); pools 2–4 span the multiple epitope (ME)
string; pools 5–9 span the 3D7 thrombospondin-related adhesion protein
(TRAP); and pools 10–13 span the T9/96 TRAP. The ME string pools
included all individual Plasmodium falciparum epitopes from the epitope
string as 1 pool of CD4 epitopes and 2 pools of CD8 epitopes. Pools 5–
9 each consisted of 10 20-mer 3D7 TRAP peptides; pools 10 and 12, 10
20-mer T9/96 TRAP peptides; pool 11, 30 20-mer T9/96 TRAP peptides;
and pool 13, 7 20-mer peptides, which cover the N-terminal region completely conserved between T9/96 and 3D7 TRAP strains. Post-DNA, 7
days after the second DNA immunization; Post-DNA/MVA, 7 days after
the first modified vaccinia virus Ankara immunization. PBMCs, peripheral
blood mononuclear cells; SFCs, spot-forming cells.
pools, and the “no peptide” negative-control spot counts were
subtracted from the requisite number of times (figure 2A–2D).
Arithmetic means, geometric means, medians, and interquartile
ranges (IQRs) were derived; all 4 are provided for initial immunogenicity values presented in the text, to allow for evaluation of the distribution of these summed-response data.
Groups of individual summed responses were compared by the
Mann-Whitney test. This test may not have power to detect
some true differences but was used because variance between
groups was often markedly dissimilar. It was not always possible
to obtain normality via conventional transformations.
Results. A total of 18 of 20 vaccinees completed the study
protocol: 2 withdrew their consent after the second DNA immunization; neither had experienced any adverse events. After
a total of 24 DNA ME-TRAP immunizations, there were no
local or systemic adverse events. The MVA vaccine was well
tolerated (table 1). All the tabulated adverse events were mild
(no interference with activities of daily living). The 1 moderate
adverse event, transient limitation of arm abduction, occurred
after MVA vaccination. MVA causes a characteristic local reaction after intradermal administration, with redness and induration peaking at 48–72 h. In 5 of 18 subjects who received
first immunizations of MVA ME-TRAP, there was a blister of
!2 mm in diameter at the center of the indurated lesion, which,
in all cases, healed without complications over 1–3 weeks.
Analysis of the hematology and biochemistry safety assays revealed no adverse events.
The 6 unvaccinated volunteers had geometric mean effector
T cell frequencies of 5.3, 7.2, and 6.6 SFCs/106 PBMCs, at the
beginning, middle, and end of the study, respectively. Effector
T cells were induced in vaccinees with specificity for all peptide
pools; TRAP-specific frequencies were higher than ME-specific
frequencies (figure 1). After DNA ME-TRAP immunization,
effector cell frequency showed a small but non–statistically significant increase. MVA ME-TRAP immunization induced much
greater effector T cell frequencies. Immunogenicity of MVA
ME-TRAP after prior DNA ME-TRAP (in terms of vaccineinduced T9/96 TRAP–specific effectors) was 13-fold higher in
Gambian volunteers than in UK volunteers (arithmetic mean,
175.4 vs. 51.4; geometric mean, 69.8 vs. 19.8; median, 172.0
vs. 31.2; IQR, 23.4–239.4 vs. 15.6–131.0 SFCs/106 PBMCs;
P p .03) (figure 2A and 2C). Immunogenicity was also higher
in Gambians who received MVA ME-TRAP immunization
without prior DNA ME-TRAP than in UK adults who received
the same regimen (arithmetic mean, 55.4 vs. 17.2; geometric
mean, 23.0 vs. 11.0; median, 28.8 vs. 15.2; IQR, 10.6–105.6 vs.
3.4–40.7 SFCs/106 PBMCs; P p .10) (figure 2B and 2D). T9/
96 TRAP effector T cell frequency after MVA boosting was
higher in Gambians who received prior DNA ME-TRAP than
in Gambians who received MVA without prior DNA (geometric
Figure 2. Magnitude, duration, and cross-reactivity of effector T cell responses, by ex vivo ELISPOT. Prevaccination responses are set to zero.
Arithmetic mean (SE) time courses for Gambian DNA/modified vaccinia virus Ankara (MVA) group (A), Gambian MVA group (B), UK DNA/MVA group
(C), and UK MVA group (D). Final, 8–10 weeks after the final vaccination. PBMCs, peripheral blood mononuclear cells; SFCs, spot-forming cells; VAC,
vaccination.
BRIEF REPORT • JID 2003:188 (15 October) • 1241
Table 1. Reactogenicity after each dose of modified vaccinia
virus Ankara multiple epitope–thrombospondin related adhesion
protein.
Dose 2
(n p 18)
Dose 1
(n p 18)
Adverse Event
Total
PB
Total
Dose 3
(n p 7)
PB
Total
PB
Local
Discoloration, median
(range), mm
9 (6–18)
7 (4–11)
7 (5–9)
Itching
7
3
1
Pain
5
0
0
Blisters
5
0
0
Systemic
Temperature ⭓37.5C
1
1
0
0
0
0
Headache
2
1
1
1
2
1
Malaise
1
1
0
0
1
0
Myalgia
0
0
1
0
0
0
Arthalgia
1
1
0
0
0
0
Nausea
0
0
0
0
0
0
NOTE. Data for discoloration were recorded on day 2. All other data are
no. of volunteers who experienced adverse event during 3-day follow-up. n,
number of volunteers who received each dose and for whom diary cards were
completed; PB, probably related.
mean, 69.8 vs 23.0 SFCs/106 PBMCs; P p .07) (figure 2A and
2B). After the second MVA immunization in the Gambian
DNA/MVA group, the geometric mean effector T cell frequency
was 63.3 SFCs/106 PBMCs, compared with 69.8 SFCs/106
PBMCs after the first MVA immunization. In both the UK
DNA/MVA group (data not shown) and the Gambian MVAalone group (figure 2B), effector T cell frequency kinetics
showed a decay from 7 days after the first MVA immunization
to 7 days after the second MVA immunization. In the group
with the strongest responses (DNA/MVA-vaccinated Gambian
adults), geometric mean effector frequencies at the 8–10 week
follow-up were 70.6% of the peak frequencies.
To assess the ability of a malaria vaccine to induce a crossreactive T cell response, we evaluated the effector response to
the 3D7 strain of TRAP, a heterologous strain with 6% sequence
variance at the amino-acid level, compared with T9/96. Crossrecognition of responses induced in Gambians was far in excess
of that seen in UK volunteers (vaccine-induced response to
3D7 after DNA-then-MVA immunization in Gambian adults
vs. that in UK adults; geometric mean, 65.1 vs 5.5 SFCs/ 106
PBMCs; P p .01) (figure 2A and 2C). Before vaccination, T
cell responses in the group of Gambians who received MVA
without prior DNA were higher to 3D7 than to T9/96. In this
group, immunogenicity was correspondingly greater for 3D7
than for T9/96 (vaccine-induced responses; geometric mean,
25.9 vs 15.0 SFCs/106 PBMCs; P p .16) (figure 2B).
The induced effectors are of both CD4+ and CD8+ T cell
subsets (figure 3). Most responses were either CD4+ or mixed
1242 • JID 2003:188 (15 October) • BRIEF REPORT
CD4+ and CD8+ responses, but pure CD8+ responses were
also seen.
A total of 12 of 20 Gambian volunteers had titers of antiTRAP antibodies (to both or either of T9/96 and/or 3D7 strains)
that were statistically significantly above titers in malaria-naive
individuals. There was no statistically significant increase or decrease of antibody titers after vaccination (data not shown).
Discussion. Most vaccines in widespread use have been
developed and formulated for optimal antibody induction. The
prime-boost approach outlined in the present report is an example of a new approach that targets the maximization of T
cell immunogenicity. Potent T cell induction is likely to be
necessary to vaccinate effectively against intracellular organisms
such as HIV, Mycobacterium tuberculosis, and liver-stage P. falciparum and for cancer immunotherapy.
The ability of MVA vaccines to amplify preexisting T cell
responses induced by priming with DNA vaccines in animal
models suggests that the vaccines may be more immunogenic
in African individuals who have been primed by natural exposure to malaria. Elsewhere, in a mouse model, it has been
shown that immunogenicity of a single dose of a recombinant
vaccinia vaccine is not protective but that prior exposure to
Figure 3. Subset distribution of effector T cell responses. The figure
represents T cell subsets for DNA/modified vaccinia virus Ankara (MVA)–
vaccinated volunteers (A) and MVA-vaccinated volunteers (B); each set
of 3 bars represents ELISPOT response to a single pool of peptides (various
pools, from 5–13 in figure 1A) at the maximal time point (usually 7 days
after the first MVA immunization). Assays were performed on frozen/
thawed cells, on unseparated CD4-depleted and CD8-depleted cell populations. The nos. along the X-axis are volunteer nos.; 2 different pooled
responses were assayed from both volunteers 95 and 84. PBMCs, peripheral blood mononuclear cells; SFCs, spot-forming cells.
malaria sporozoites boosts this immunogenicity to protective
levels [13], resulting in protection that is T cell mediated. Our
findings confirm this prediction. The immunogenicity of an
MVA malaria vaccine (with or without DNA priming) was of
greater magnitude in previously exposed Gambian individuals
than in malaria-naive individuals from the United Kingdom.
There are genetic and environmental differences between
malaria-naive and -exposed individuals in this study, other than
their status with regard to previous exposure to malaria, but
we feel that these differences are unlikely to account for the
reported altered immunogenicity. Genetic and, in particular,
HLA differences between the populations might have been most
evident in responses to the HLA class I restricted peptides of
the epitope string, but these contributed very little to the overall
response level (figure 1). Interestingly, even with sporozoite
priming, further priming by DNA immunization is still necessary for optimal T cell induction by MVA immunization.
The greatly increased cross-recognition demonstrated in Africans in this study provides encouragement for further work
with T cell–inducing DNA and recombinant viral vaccines. Lack
of strain transcendence has long been viewed as a potential
obstacle to malaria vaccination. The Gambian volunteers who
received MVA without prior DNA vaccination (figure 2B) had
higher prevaccination effector T cell frequencies to the 3D7
strain of TRAP than to the T9/96 strain. After immunization,
frequencies of T cells specific for 3D7 TRAP increased more
than did those specific for T9/96, which is consistent with the
well-recognized immunological phenomenon termed “original
antigenic sin” [14].
Not only CD8+ T cells, but also high frequencies of CD4+ T
cells, were induced by DNA/MVA and MVA immunizations.
Although CD4+ T cells have traditionally been considered to
be helper T cells for either antibody production or CD8+ T cell
cytotoxicity, it is now clear that many CD4+ T cells have effector
activity. Directly cytotoxic CD4+ T cell clones confer protection
in murine adoptive-transfer experiments [15], and such cytotoxic CD4+ T cell clones are present in attenuated sporozoite
immunization–protected humans [16].
In mouse models, contraction of ∼90%–95% of the effector
T cell pool by apoptosis occurs after infectious challenge over
the course of 2 weeks (for CD8+ T cells) or 7 weeks (for CD4+
T cells), in 1 model [17]. We examined the kinetics of such
contraction in humans. We demonstrated the persistence of a
residual memory pool with rapid effector function 8–10 weeks
after final vaccination, with frequencies at this time 150% of
the peak frequencies.
In all groups studied, the second MVA immunizations, which
were performed 3 weeks after the first MVA immunizations,
show no increase in immunogenicity. In fact, effector T cell
frequencies show a reduction that is consistent with natural
decay kinetics, from the peak frequency 7 days after the first
MVA immunization. This may be because, at this interval, a
host immune response to the highly immunogenic MVA vector
prevents infection of host cells and recombinant protein expression after the second MVA immunization. A study is underway to evaluate immunogenicity of the second MVA immunization after a 12-month interval.
It has been difficult to induce strong effector T cell immune
responses through vaccination. We present a safe strategy that
is immunogenic for effector T cell induction in malaria and is
likely to be applicable to other fields. Prime-boost approaches
with viral-vector boosts have been seen as an excellent option
for improving the efficacy of prophylactic DNA vaccines in
diseases in which T cell responses are protective. The confirmation of the ability of MVA to boost preexisting T cell responses in humans implies that MVA and the DNA/MVA combination may also be effective for immunotherapy of chronic
infections and tumors. Examples that may merit evaluation are
TB infection before onset of disease, hepatitis B virus–infected
individuals at risk of disease progression, and immunotherapy
of HIV-positive and melanoma patients. The first field-efficacy
study of prime-boost immunization against malaria in Africans
is underway.
Acknowledgments
We gratefully acknowledge the generous contribution of the
volunteers who participated in this study, without which the
study would not have been possible. S. Baldeh was the field
supervisor; S. Allen was the safety monitor; T. Corrah provided
clinical support; Matron P. Collier Njai, Sister V. Thomas, and
the nursing staff on the Medical Research Council ward in
Fajara provided nursing assistance; H. Whittle provided scientific support and advice; I. Sambou, C. E. M. Allsop, and S.
Correa provided laboratory assistance; and David Jeffries provided statistical advice.
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