A Phase 2b Randomised Trial of the Candidate
Malaria Vaccines FP9 ME-TRAP and MVA ME-TRAP
among Children in Kenya
Philip Bejon1*, Jedidah Mwacharo1, Oscar Kai1, Tabitha Mwangi1,2, Paul Milligan3, Stephen Todryk2,
Sheila Keating2, Trudie Lang2, Brett Lowe1, Caroline Gikonyo1, Catherine Molyneux1, Greg Fegan1,3,
Sarah C. Gilbert4, Norbert Peshu1, Kevin Marsh1,5, Adrian V. S. Hill2,4
1 Kenya Medical Research Institute, Centre for Geographical Medicine Research (Coast), Kilifi, Kenya, 2 Centre for Clinical Vaccinology and Tropical
Medicine, University of Oxford, Oxford, United Kingdom, 3 London School of Hygiene and Tropical Medicine, London, United Kingdom, 4 Wellcome
Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom, 5 Nuffield Department of Clinical Medicine, Oxford University, John
Radcliffe Hospital, Oxford, United Kingdom
Trial Registration: ISRCTN:
Funding: PB holds a Wellcome Trust
training fellowship (073597), KM is
supportedbytheWellcome Trust, and
AVSH is a Wellcome Trust Principal
by the Wellcome Trust and the Gates
Malaria Partnership at the London
School of Hygiene and Tropical
Medicine. The sponsors had no role in
trial design, conduct, analysis,
presentation, or decision to publish.
Competing Interests: AVSH is
cofounder of and an equity holder in
Oxxon Therapeutics, a company
developing prime-boost therapeutic
vaccines. No other authors have any
conflicts of interest.
Citation: Bejon P, Mwacharo J, Kai O,
Mwangi T, Milligan P, et al. (2006) A
phase 2b randomised trial of the
candidate malaria vaccines FP9 ME-
TRAP and MVA ME-TRAP among
children in Kenya. PLoS Clin Trials 1(6):
Received: June 19, 2006
Accepted: September 1, 2006
Published: October 20, 2006
Copyright: ? 2006 Bejon et al. This
is an open-access article distributed
under the terms of the Creative
Commons Attribution License, which
permits unrestricted use,
distribution, and reproduction in any
medium, provided the original
author and source are credited.
Abbreviations: ATP, according to
protocol; CI, confidence interval; EIR,
entomological inoculation rate;
immunosorbent spot; ITN,
insecticide-treated net; ITT, intention
to treat; MVA, modified vaccinia
virus Ankara; PBMC, peripheral blood
* To whom correspondence should
be addressed. E-mail: pbejon@well.
Objective: The objective was to measure the efficacy of the vaccination regimen FFM ME-
TRAP in preventing episodes of clinical malaria among children in a malaria endemic area. FFM
ME-TRAP is sequential immunisation with two attenuated poxvirus vectors (FP9 and modified
vaccinia virus Ankara), which both deliver the pre-erythrocytic malaria antigen construct
multiple epitope–thrombospondin-related adhesion protein (ME-TRAP).
Design: The trial was randomised and double-blinded.
Setting: The setting was a rural, malaria-endemic area of coastal Kenya.
Participants: We vaccinated 405 healthy 1- to 6-year-old children.
Interventions: Participants were randomised to vaccination with either FFM ME-TRAP or
control (rabies vaccine).
Outcome Measures: Following antimalarial drug treatment children were seen weekly and
whenever they were unwell during nine months of monitoring. The axillary temperature was
measured, and blood films taken when febrile. The primary analysis was time to a parasitaemia
of over 2,500 parasites/ll.
Results: The regime was moderately immunogenic, but the magnitude of T cell responses was
lower than in previous studies. In intention to treat (ITT) analysis, time to first episode was
shorter in the FFM ME-TRAP group. The cumulative incidence of febrile malaria was 52/190
(27%) for FFM ME-TRAP and 40/197 (20%) among controls (hazard ratio ¼ 1.52). This was not
statistically significant (95% confidence interval [CI] 1.0–2.3; p ¼ 0.14 by log-rank). A group of
346 children were vaccinated according to protocol (ATP). Among these children, the hazard
ratio was 1.3 (95% CI 0.8–2.1; p ¼ 0.55 by log-rank). When multiple malaria episodes were
included in the analyses, the incidence rate ratios were 1.6 (95% CI 1.1–2.3); p ¼ 0.017 for ITT,
and 1.4 (95% CI 0.9–2.1); p ¼ 0.16 for ATP. Haemoglobin and parasitaemia in cross-sectional
surveys at 3 and 9 mo did not differ by treatment group. Among children vaccinated with FFM
ME-TRAP, there was no correlation between immunogenicity and malaria incidence.
Conclusions: No protection was induced against febrile malaria by this vaccine regimen.
Future field studies will require vaccinations with stronger immunogenicity in children living in
www.plosclinicaltrials.org October | 2006 | e290001
P PL Lo oS S CLINICAL TRIALS
More than 1 million people die each year from malaria, and
this number is likely to increase . A vaccine is urgently
needed. There is evidence that T cells are protective against
malaria in animal models , field studies , and following
immunization with irradiated sporozoites . This evidence
has prompted the development of a heterologous prime-
boost strategy to induce T cell responses against pre-
erythrocytic stages of parasite development . The prime-
boost strategy uses two different vectors to deliver a common
antigen construct, which achieves an expansion of T cells
reactive to the common antigen, rather than to the vectors
Prime-boost vaccination with FP9 (an attenuated fowlpox
virus) then with modified vaccinia virus Ankara (MVA), both
recombinant for the pre-erythrocytic antigen construct (the
multiple-epitope string and thombospondin-related adhesion
protein, ME-TRAP ) was safe, immunogenic, and partially
protective in malaria-naı ¨ve adults exposed to experimental
challenge . After controlled bites from Plasmodium falcipa-
rum-infected mosquitoes, some individuals were fully pro-
tected and vaccinees showed mean delays in time to
parasitaemia, corresponding to a mean 92% reduction in
malaria parasites completing pre-erythrocytic development
. However, prime-boost vaccination with DNA priming and
MVA (delivering the ME-TRAP insert) was not protective
against parasitaemia in semi-immune adults .
However, incidence studies in semi-immune adults are
complicated by the fact that adults have greater naturally
acquired immunity than do children; this natural immunity
increases with age . Reinfection rates are similar despite
different entomological inoculation rates (EIRs) [11,12], and
so such studies might then not identify vaccine-induced
reductions in malaria parasites completing pre-erythrocytic
development. Incidental insecticide-treated net (ITN) use was
not associated with a reduced risk of parasitaemia in
longitudinal follow-up studies of semi-immune adults
[10,13]. Thus, a partially effective pre-erythrocytic vaccine
might be more effective against febrile disease in children
than against asymptomatic parasitaemia in semi-immune
adults. Furthermore, FP9 priming was significantly more
immunogenic and protective than DNA priming in mice ,
and more protective in nonimmune volunteers, although not
significantly so (p ¼ 0.3) in the small sample studied .
The response to vaccination was measured by two different
enzyme-linked immunosorbent spot (ELISPOT) assays. An ex
vivo ELISPOT assay was used to count IFN-c-producing
effector T cells after overnight incubation with antigen, and a
cultured ELISPOT assay was used to measure resting memory
cells . These assays appear to measure different cell
populations. A study of naturally acquired immunity to
circumsporozoite protein demonstrated no association be-
tween cultured ELISPOT and ex vivo ELISPOT results .
Furthermore, the T cells induced by vaccination that are
identified by cultured ELISPOT persist for at least six months
after vaccination of naı ¨ve participants, despite waning
numbers of cells detected by ex vivo ELISPOT . The
cultured ELISPOT response to TRAP after prime boost and
to the circumsporozoite protein after the malaria vaccine
RTS,S in various adjuvants was associated with protection
following sporozoite challenge [17,18].
www.plosclinicaltrials.orgOctober | 2006 | e290002
Efficacy of FFM ME-TRAP in Children
Background: Malaria kills over a million people a year worldwide, and
young children in sub-Saharan are particularly at risk. Cheap, safe, and
effective vaccines are needed. One strategy involves a double-
vaccination process. This approach (termed ‘‘prime-boost’’) uses two
different delivery methods to transmit the same antigen (part of a
protein from the malaria parasite that can trigger an immune response).
The two-step vaccination is designed to achieve a greater immune
response than with just one vaccination. One research group, based in
Oxford in the UK, is using an antigen called ‘‘ME-TRAP,’’ which is
delivered using first a strain of modified fowlpox virus (called FP9), then a
weakened vaccinia virus (called MVA). Previous studies done in adult UK
volunteers have been promising, achieving an immune response and
some protection against malaria when volunteers were deliberately
infected. However, this approach has not been tested in the group most
in need of a vaccine—young African children. Therefore a field trial was
conducted among 405 healthy children aged 1–6 years, in a region of
Kenya with year-round malaria transmission. Children were randomized
to receive either the sequence of vaccines delivering ME-TRAP or to
receive a rabies vaccine (as control, but which still gives the children
some benefit for taking part in the trial). The children were followed up
for nine months, and the primary aim of the trial was to compare the
occurrence of clinical malaria (fever combined with malaria parasites in
the blood) in the two groups.
What this trial shows: In the 387 children receiving vaccine and having
at least one follow-up visit the vaccine did produce an immune response;
however, this immune response did not seem to be protective, as the
occurrence of malaria was slightly higher in the group receiving the
candidate vaccine—although this difference was not statistically
significant. Safety data were also collected; the number and severity of
adverse events were similar between volunteers receiving the rabies
vaccine and those receiving the candidate malaria vaccine, and any
serious events were not judged to be linked to the vaccines by the trial’s
data safety monitoring board.
Strengths and limitations: The methods used in the trial were robust,
using appropriate randomization procedures and blinding of partic-
ipants and researchers. Outcome measures (clinical malaria, defined as
fever together with parasites in the blood over 2,500/microliter) were
clinically relevant. In order to detect cases of malaria in vaccinated
children, health workers visited children weekly, and children with a
temperature over 37.5 8C were tested for parasites in the blood. (In
between the weekly visits, self-report and referral for assessment also
allowed detection of cases.) This process of active detection of malaria
cases (as opposed to obtaining data on clinical malaria only from self-
report or referral) enables a smaller sample size to be used in the trial,
but it is not clear whether this approach is more or less specific at picking
up malaria cases than are passive methods. The researchers aimed to
ensure that their case detection methods were specific; for children with
normal temperature, but reported by their parents as feverish, parasite
tests were done only if subsequent temperature readings were high.
Contribution to the evidence: Previous studies of ME-TRAP using the
FP9 and MVA vectors have shown the candidate vaccine is safe and
induces a strong immune response. The safety result was also supported
by the findings of the current trial, conducted in young Kenyan children,
but a 5-fold lower immune response was found compared to previous
studies. The trial showed that this weak immune response was not
effective at preventing clinical cases of malaria in this group of children,
although it is not clear why the immune response was lower than
The Editorial Commentary is written by PLoS staff, based on the reports of the
academic editors and peer reviewers.
Immunogenicity and safety data were acquired for adults
and then children in Phase 1 studies in Kenya before Phase 2b
studies were undertaken, including the FFM ME-TRAP
regimen (i.e., two sequential FP9 vaccinations followed by
MVA, where both vectors deliver ME-TRAP) that is used the
present study [19–21]. Local and systemic reactogenicity was
mild. Immunogenicity was lower than, but comparable to,
that seen among partially protected volunteers in sporozoite
The primary aim of this Phase 2b trial was to evaluate
safety, immunogenicity, and efficacy of the vaccination in the
target population (i.e., children in a malaria-endemic area),
using mild febrile malaria as an endpoint. To detect cases, we
used active weekly monitoring for fever, and made blood
films from febrile children. Field workers lived in the
community, and were easily accessed by parents when fever
occurred between regular visits.
The study was randomised, controlled, and double blind.
Ethical approval was obtained from the Kenyan Medical
Research Institute National Ethics Committee, the Central
Oxford Research Ethics Committee, and the London School
of Hygiene and Tropical Medicine Ethics Committee. An
independent Data Safety Monitoring Board and a Local
Safety Monitor were appointed. The Data Safety Monitoring
Board reviewed all serious adverse events as they occurred,
approved the selection of vaccine dose based on previous
Phase 1 studies, and reviewed the analysis plan. Research was
conducted in accordance with the Helsinki Declaration of
1975 (revised 1983). The trial was conducted according to
Good Clinical Practice. Oxford University, as trial sponsor,
arranged external monitoring and oversaw the conduct of the
At least one dose of vaccine was given to 405 children.
Blood tests for immunology, safety, and cross-sectional
assessments of malaria parasitaemia were conducted prevac-
cination, at screening, 1 wk after the third vaccination, then
at 3 mo and at 9 mo. Monitoring for solicited adverse events
was conducted for 1 wk after each vaccination. Unsolicited
adverse events and episodes of malaria were monitored
throughout the 1 y study duration. Children were screened in
February 2005, immunised between March 2005 and May
2005, and followed up until February 2006. Monitoring is
continuing in a further study.
The participating children were aged 1–6 y (inclusive),
healthy, and resident in the study area. After a series of
public meetings and individual discussions, a screening date
was set at which study information was repeated, and consent
was sought before proceeding. Participants were not immu-
nized until at least one week after their parents signed
consent, to allow the parents time to consider their decision.
Children were screened by history, examination, and blood
tests (complete blood count, creatinine, and alanine trans-
aminase). Those with clinically significant illness were
excluded. Clinically evident immunosuppression was one of
the criteria for exclusion, but no children were excluded on
this basis. HIV testing was not conducted. Abnormal alanine
transaminase or creatinine levels were exclusion criteria, but
anaemia without clinically significant signs and symptoms was
not (although iron supplementation was given to the 27
children with haemoglobin . 8 g/dl). Recent blood trans-
fusions (within the previous 2 mo), current participation in
another clinical trial, or receipt of another experimental
vaccine were also exclusion criteria. Eligible children were
invited to attend vaccination in the order in which they were
The study was carried out in Junju sublocation in Kilifi
District, on the Kenyan coast. Junju contains a group of five
closely related villages within the Chonyi area of Kilifi
district. Junju lies between Kilifi and Mombasa, 14 km inland
from the coastal road. Kilifi is malaria-endemic. There are
two high-transmission seasons, but low-level transmission
continues all year-round . The transmission intensity is
22–53 infective bites per year . Junju is served by a local
government dispensary, which has an active ITN distribution
programme. Inpatient care is available at Kilifi District
The antigen insert used in the vaccine was TRAP, joined to a
multiple epitope (ME) string from six P. falciparum pre-
erythrocytic antigens . The ME string contains 14 pre-
erythrocytic MHC class I epitopes from six P. falciparum pre-
erythrocytic antigens, three class II epitopes, two pre-
erythrocytic B cell epitopes and pb9 (a P. berghei T-cell
epitope that allows pre-clinical potency and stability testing).
The vectors used were an attenuated fowlpox virus, FP9,
and MVA. Recombinant vaccine stock was supplied to
contract manufacturer IDT (Rosslau, Germany), who pro-
duced clinical lots under Good Manufacturing Practice
conditions. Single batches of FP9 ME-TRAP and MVA ME-
TRAP were used throughout (both batch #051204).
The trial vaccination regimen was two sequential FP9 ME-
TRAP vaccinations (53107plaque-forming units) followed by
MVA ME-TRAP vaccination (1.53108plaque-forming units),
given intradermally. The control was rabies vaccine (Aventis
Pasteur, WISTAR strain), administered according to the same
timings. Rabies was also given intradermally, at 0.25 IU.
Vaccinations were spaced four weeks apart (acceptable range
three to five weeks).
Vaccines were shipped to Kenya on dry ice with logged
temperature monitoring, stored at?80 8C, and transported to
the field in cool boxes for use within six hours. Vaccines were
given intradermally over the deltoid area of the nondominant
arm, using a 27-gauge needle to raise a visible intradermal
bleb. Volunteers were observed for one hour with resuscita-
tion facilities available for advanced life support. Children
received vitamin A supplements with each vaccination, as per
Government of Kenya guidelines, and parents were supplied
with two doses of paracetamol syrup for use if the child
developed fever at night.
The objectives of this Phase 2b trial were to evaluate safety,
immunogenicity, and efficacy of the vaccination in the target
population (i.e., children in a malaria-endemic area), using
mild febrile malaria as an endpoint.
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Efficacy of FFM ME-TRAP in Children
Efficacy (malaria episodes). The primary endpoint was a
clinical episode of malaria, defined as an axillary temperature
greater than 37.5 8C, with a P. falciparum parasitaemia greater
than 2,500 parasites/ll. The presence of any falciparum
parasitaemia with fever above 37.5 8C was a secondary
Asymptomatic parasitaemia is common in children under
endemic conditions (70% of children in this study). If a child
with asymptomatic parasitaemia is evaluated during an
unrelated viral illness, they will be wrongly ascertained as a
case of febrile malaria . An endpoint with low specificity
will lead to an incorrect estimate of efficacy . Since active
surveillance was used, the specificity of the endpoint was
maximized in three ways. A previously defined threshold
parasitaemia was used for the diagnosis of malaria , all
children received curative treatment for malaria before
monitoring began, and only children in whom fever was
confirmed by measurement (rather than simply by verbal
report) had blood films made.
The curative treatment was given one week after the final
vaccination, using seven days of directly observed dihydroar-
temisinin monotherapy (2 mg/kg on the first day, followed by
1 mg/kg for 6 d). Blood films were taken to confirm that
children were parasite-negative one week after the end of
Children were then visited every week by field workers. The
mother was asked whether she thought the child was hot, and
the axillary temperature was also measured. When the
temperature was greater than 37.5 8C, a blood film was made
and a rapid near-patient test for malaria conducted. The
blood film was reviewed within 24–48 h, but treatment
decisions were based on the rapid test result.
When the mother reported that the child was hot, but an
objectively elevated temperature was not identified, blood
films and rapid testing was not performed, but the field
worker returned to the child three times in the next 24 h.
Rapid testing and blood films were performed if the child’s
temperature was elevated on any of these visits. Parents could
bring their children for assessment between regular weekly
visits if they thought the child had developed fever, and the
child was assessed as above. Field workers were recruited
from the villages in which the study was conducted, and so
were readily accessible to the parents. Treatment for episodes
of malaria was with the Government of Kenya-recommended
first-line treatment, artemether-lumefantrine.
During blood tests for safety and T cell response, blood
films were made on all children. The results of this testing was
not available for several weeks, during which monitoring of
febrile illnesses continued. Asymptomatic parasitaemia was
therefore not treated unless the child developed a fever. All
blood films (from well children and febrile children) were
counted in duplicate by two microscopists, and a third count
was conducted if they were discrepant.
Immunogenicity by ELISPOT. Peripheral blood mononu-
clear cells (PBMCs) were separated at screening, one week
after the last vaccination, then at three months and at nine
months. PBMCs were incubated in RPMI media (Sigma-
Aldrich, St. Louis, Missouri, United States) with 10% human
AB serum. ELISPOTs used Millipore MAIP S45 plates
(Millipore, Billerica, Massachusetts, United States) and Mab-
Tech antibodies (MabTech, Stockholm, Sweden) according to
manufacturer’s instructions. Freshly isolated PBMCs were
incubated at 4 3 105cells/well in a volume of 100 ll with 10
lg/ml peptides for 18–20 h before the ELISPOT was
developed. Individual 8- to 17-residue epitopes were pooled
for the ME string. Peptides of 20 residues overlapped by ten
residues were used for TRAP. TRAP peptides were pooled
according to region. Phytohaemagglutinin (Sigma-Aldrich)
was used at 20 lg/ml as positive control, and PBMCs were
cultured in medium alone as negative control. Spot-forming
cell numbers were counted by an ELISPOT plate reader
(version 3.0; Autoimmun Diagnostika, Strassberg, Germany).
For cultured ELISPOTs, 1 3 106PBMCs were incubated in
0.5 ml of 10 lg/ml/peptide of pooled TRAP and ME peptides
in a 24-well plate. On days 3 and 7, 250 ll of culture
supernatant was replaced with 250 ll of culture medium
containing 20 IU/ml of recombinant IL-2. On day 9 the cells
were washed three times and left overnight before the
standard ELISPOT assay.
Field workers visited patients daily for the first three days
after vaccination and at one week after vaccination. Solicited
adverse events were recorded, the diameters of skin dis-
colouration were noted, and blistering was measured. Loss of
the epidermis or upper part of the dermis was described as a
deroofed blister. If unsolicited adverse events were reported,
these were assessed and documented by a medically qualified
investigator. Blood tests for routine biochemistry (plasma
alanine transaminase and creatinine) and haematology
(complete blood counts) were conducted 7 d after the final
vaccination, then at 3 mo and 9 mo.
We expected the incidence of febrile malaria to be 50%. The
study was designed to detect 35% efficacy with 80% power by
enrolling 400 children. In fact malaria transmission was lower
than expected (as in most of East Africa in 2005); only a 25%
Table 1. Baseline Covariates
Parasitaemia at screening visit
Age category (y)
Numbers of children within vaccination group for each covariate are shown. The
percentage of all children in the vaccination group with each covariate is given in
aITN use was defined as sleeping every night under an ITN with fewer than three holes
that could comfortably admit a finger. ITN data are missing for four children at
baseline and six at the end. Data on parasitaemia at screening were missing for one
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Efficacy of FFM ME-TRAP in Children
incidence was recorded, so the actual trial was only powered
to detect a 50% efficacy.
The randomization sequence was generated by the trial
statistician in the UK, who had no involvement in enrolment,
follow-up, or assessment of participants. Eligible children
were assigned a randomization number, and vaccine group
was allocated using restricted randomization in blocks of ten
after sorting the list of eligible individuals by age and village.
Opaque randomization envelopes were prepared and sealed
in the UK without the involvement of any investigators
participating in enrolling or assessing children at the study
site. Each child was assigned the envelope bearing his or her
study number; the envelope was opened when the child
attended for the first vaccination.
The investigators in Kenya enrolled children. Study numbers
were applied sequentially. A list of eligible children was then
ordered according to age and village, and matched to the list
of randomization card numbers. If children did not attend
for the first vaccination, the card was then assigned to a child
of similar age from the same village.
The nurses who administered vaccinations did not take part
in any other trial-related procedure, and were subsequently
based in Kilifi District Hospital rather than the trial site. They
drew up vaccinations according to the instructions in the
randomisation envelope, and documented the vaccination in
notes that were not available to the investigators until after
unblinding, after follow-up was completed for all children.
The randomisation envelopes were resealed after each
vaccination and stored in a locked office for reuse at
subsequent vaccinations. Neither parents nor investigators
were told of the vaccination allocation, and the investigators
did not enter the vaccination room while vaccinations were
Intradermal poxvirus vaccinations are associated with skin
discolouration and blistering . These effects were unlikely
to have compromised blinding, since children were routinely
assessed by field workers who were unaware of an expected
difference in local reactogenicity. Furthermore, 16% of
intradermal rabies vaccinations in the trial were associated
with skin discolouration.
No interim analyses were planned or conducted, and the
analysis plan was approved by the Data Safety Monitoring
Board before unblinding. The primary analysis was a log-rank
test comparing the time to the first or only episode of malaria
(defined as fever with parasitaemia above 2,500/ll) between
the vaccination groups, stratified by age group, ITN use, and
village, for both according-to-protocol (ATP) and intention-
to-treat (ITT) participants. The hazard ratio and 95%
confidence interval (CI) were estimated by Cox’s regression
adjusted for the same covariates. Age group was a categorical
variable with three levels (ages 1–2 y, 2–5 y, and 5–6 y). Village
had five levels. ITN use was defined as sleeping under a
treated net every night, which had fewer than three holes into
which a finger could comfortably fit.
Poisson regression was used to estimate the incidence rate
ratio taking into account all malaria episodes, adjusted for
the same covariates. A period of 28 days after each malaria
episode was deducted from the person time at risk, since
individuals were assumed not to be at risk of malaria during
this period. In secondary analyses, malaria was defined as
fever with parasitaemia at any density. ELISPOT results were
log-transformed, substituting one spot per million for
negative results (half the lower limit of detection), and then
Student’s t-tests used to compare results. To examine the
effect of ELISPOT results on episodes of malaria, the
responses were divided into tertiles, and the tertile was then
used as a categorical variable.
The analysis plan specified that monitoring of malaria
episodes would continue for a further nine months, after
which a second analysis of episodes would be conducted.
A total of 530 children were screened. Eligible children were
invited to attend for vaccination in the order in which they
were screened. Vaccination continued until 400 children
Children were recruited over a five-week period, and all
immunizations took place from March to May 2006.
The trial participants were balanced between treatment
allocation groups in both demographic characteristics at
baseline and progress through the trial (Table 1). Of 386
children, 275 (71%) were parasitaemic at baseline. Antima-
larial treatment was given to 73 children for intercurrent
Figure 1. Trial Profile
After screening for eligibility, parents were invited to bring their children back
to the dispensary for immunisation. Children were randomised on attending
for vaccination. Of the 17 children who attended for the first, but not the final,
vaccination, two had moved out of the area, and parents of the remaining 15
chose not to reattend. No severe adverse events were identified in these
children. Before 9 mo monitoring was complete, eight children had moved
out of the area.
www.plosclinicaltrials.orgOctober | 2006 | e290005
Efficacy of FFM ME-TRAP in Children
febrile malaria during vaccinations. Blood films were not
taken on the days of vaccination, but 179 (52%) of 374
children were parasitaemic one week following final vacci-
nation, before receiving antimalarials at the start of monitor-
In total 346 children were vaccinated ATP, and an additional
41 children were included only in the ITT analyses. The
distribution of ATP and ITT groups by vaccination is given in
Outcomes and Estimation
Efficacy (primary analysis). The time to first episode was
shorter in the malaria vaccine group (Figure 2). The
cumulative incidence of malaria (fever with parasitaemia
over 2,500/ll) in the ITT group was 52/190 children (27%) in
the malaria vaccine group and 40/197 in the control group
(20%), (stratified log-rank test v2¼2.2, p ¼0.14 for log-rank),
with a hazard ratio of 1.5 (95% CI 1.0–2.3). ATP vaccinations
were given to 346 children and followed up. Of these, 40/175
(23%) had malaria in the malaria vaccine group compared to
36/171 (21%) in the control group (stratified log-rank test v2¼
0.36; p ¼ 0.55; hazard ratio 1.3; 95% CI 0.83–2.1).
Ancillary analyses. In a secondary analysis, as specified in
the Report and Analysis Plan (Text S1), episodes were defined
as fever with parasitaemia at any density. In the malaria
vaccine group 69/190 children (36%) had at least one episode,
and 54/197 (27%) in the controls (stratified log-rank test v2¼
2.79; p¼0.095; hazard ratio 1.5 [95% CI 1.0–2.1]), and among
those vaccinated according to protocol, 58/175 (33%) and 48/
171 (28%), respectively (log-rank test v2¼ 1.26, p ¼ 0.26).
Multiple episodes were modelled by Poisson regression,
adjusted for the same covariates (Figure 3). There were 0.46
episodes of malaria (fever with parasitaemia over 2500/ll) per
person-year among the FFM ME-TRAP vaccinees, and 0.36
episodes per person-year among the control group (incident
rate ratio 1.6 [95% CI 1.1–2.3], p ¼ 0.017). Among those
vaccinated according to protocol, the incidence rate ratio was
1.4 (95% CI 0.89–2.1; p ¼ 0.16). When these analyses were
repeated using malaria definition of fever with any para-
sitaemia, the incidence rate ratios were 1.5 (95% CI 1.1–2.0), p
¼ 0.013 for ITT and 1.4 (95% CI 0.96–1.9), p ¼ 0.08 for ATP.
Only two episodes of malaria required hospital admission
(one in each vaccination group), and neither of these met
accepted criteria for severe malaria.
Secondary analysis by subgroup. The Report and Analysis
Plan had considered the possibility that susceptibility might
vary by T cell response to vaccination (either by ex vivo or
cultured ELISPOT). Vaccinees were divided into tertiles
according to the ex vivo or cultured ELISPOT responses
measured one week after vaccination, and each tertile was
compared with the control group (Figure 3). No clear trend
for differential susceptibility according to immunogenicity
was seen, and a log likelihood ratio test suggested that the two
models using immunogenicity (ex vivo and cultured) were not
significantly better than using covariates alone (log likelihood
testing gave p ¼ 0.4 for ex vivo and p ¼ 0.24 for cultured).
There was no indication that immune response predicted
susceptibility when multiple episodes were analyzed (p¼0.4, p
¼ 0.29, respectively, for log likelihood).
However, the arithmetic mean was only 228 spots per
million on ex vivo ELISPOT in the highest tertile. Explor-
atory analysis (not specified in the Report and Analysis Plan)
divided vaccinees into seven groups of 28 children per group
Figure 2. Primary Analysis of Efficacy
The probability of remaining free of clinical malaria is plotted over the 9 mo of monitoring (the primary analysis). Numbers of children at risk are given below the
Kaplan-Meier plots for ITT (top; p ¼ 0.55) and ATP (bottom; p ¼ 0.14). Both plots use an endpoint of over 2,500 parasites/ll and fever.
www.plosclinicaltrials.orgOctober | 2006 | e290006
Efficacy of FFM ME-TRAP in Children
(on average), according to ex vivo T cell response. The highest
responders were at a mean of 325 spots per million, but still
had more frequent episodes of malaria than the control
group (hazard ratio 2.39 [95% CI 1.1–5.9]). The 27 highest
responders by cultured ELISPOT were at mean response of
1,130 spots per million, and the hazard ratio was 1.76 (95% CI
0.78–4.0) compared with controls.
Other endpoints were haemoglobin and parasitaemia
detected at cross-sectional surveys at 3 and 9 mo. These
variables were not different between vaccination groups. The
prevalences of parasitaemia for FFM ME-TRAP and the
control group were, respectively, 29% and 26% at 3 mo (n ¼
346) and 33% and 33% at 9 mo (n¼306). Mean haemoglobin
was 11 g/dl (95% CI 10.8–11.1) before monitoring began (n ¼
362), and 10.1 at 3 mo (95% CI 9.8–10.5) in both the FFM ME-
TRAP and control group (n ¼ 354).
Immunogenicity. The vaccine was moderately immuno-
genic (Figure 4). T cell responses were measured using both
the ex vivo and the cultured ELISPOT, and both assays
detected a response to vaccination. This response was
significantly greater than that measured prevaccination (p
, 0.001), and greater in FFM ME-TRAP vaccinees than in
children receiving control vaccinations (p , 0.001). Ex vivo T
cell responses rose from 30 spots per million before
vaccination (95% CI 21–40) to 107 spots per million (95%
CI 88–127) after vaccination. Cultured responses rose from
123 spots at baseline (95% CI 104–142) to 407 (95% CI 349–
464). Vaccine-induced responses for both ex vivo and
cultured ELISPOT remained above the control group at 9
Figure 3. Secondary Analyses for Efficacy
The results from secondary analyses of malaria episodes are shown. These multiple analyses were generated by varying the case definition and the statistical
methodology, and are shown for both ATP and ITT. The more rigorous case definition (over 2,500 parasites/ll and fever) is the first in each group of comparisons.
Cox regression was used to estimate hazard ratios for time to first episode (A) and Poisson regression was used to estimate incidence rate ratios for the frequency
of episodes (B). Models were adjusted for age, village, and ITN use. The two groups of three points to the right of each panel show the hazard ratios and
incidence rate ratios for subgroups FFM ME-TRAP-vaccinated children. Participants were divided into tertiles based on either ex vivo or cultured ELISPOT
responses. Hazard ratios and incidence rate ratios for each tertile relative to the control group are shown, using a case definition of parasitaemia over 2,500/ll
Figure 4. Immunogenicity
T cell responses to vaccination identified by both ex vivo and cultured
ELISPOT are displayed over time. Median, 25th, and 75th quartile, 5th and
95th quartile, and outlying results are given by box and whisker plots. Data
were available for 400 children at screening (i.e., prevaccination), for 379
children 7 d after the last vaccination, for 345 at 3 mo, and for 304 at 9 mo. T
cell numbers were similar at baseline for ex vivo (p ¼ 0.4) and cultured (p ¼
0.91) responses. Ex vivo responses were higher among FFM vaccinees at 1 wk
(p , 0.001) and 9 mo (p ¼ 0.015), as were cultured responses. Ex vivo
responses did not significantly differ at 3 mo (p ¼ 0.27), but cultured
responses did (p , 0.001).
www.plosclinicaltrials.orgOctober | 2006 | e29 0007
Efficacy of FFM ME-TRAP in Children
mo (the final time point) (p ¼ 0.015 for ex vivo, p , 0.001 for
The vaccine was well tolerated. Ten serious adverse events
occurred. Four occurred after rabies vaccination (severe
malaria, gastroenteritis, trauma, and asthma) and six after
FFM ME-TRAP (severe malaria, an abdominal skin infection
distant to the vaccination site, trauma, gastroenteritis, and
multiple seizures). The serious adverse events identified were
not unexpected events given the study population, were not
closely related to vaccination in timing, and were judged
unlikely to be linked to vaccination by the investigators and
the Data Safety Monitoring Board. Local cutaneous discolou-
ration and blistering was frequent, but blistering with a
diameter greater than 0.5 cm occurred at a rate of 1%–4%
(Table 2). No blisters were greater than 1 cm in diameter or
associated with reports of marked pain. Keloid formation or
hypertrophic scars were not seen. The ranges of routine
haematology and biochemistry results, and frequency of
abnormal results, were similar for the two groups. No
abnormal laboratory results were attributed to vaccination.
FFM ME-TRAP was safe when given to 1- to 6-year-old
children in a malaria-endemic area. It was moderately
immunogenic, but not as immunogenic as in malaria-naı ¨ve
 or semi-immune adults , and it was not protective
against episodes of clinical malaria. Although more malaria
episodes were observed in the malaria vaccine group, the
difference was not significant by primary analysis (ITT).
Although the formal possibility exists that altered peptide
ligand effects , transforming growth factor b production
 and T cell anti-inflammatory responses  induced by
vaccination might increase susceptibility to malaria, there
was no indication in our study that higher T cell responses
predicted greater susceptibility to malaria within the vacci-
nation group (either cultured or ex vivo). Since naturally
acquired responses were not associated with protection ,
it seems unlikely that vaccine-induced responses could lead to
greater susceptibility by suppressing naturally acquired
responses. The DNA-MVA regime tested in Gambia induced
stronger T cell responses to TRAP than seen here, without
evidence of enhanced susceptibility . There is therefore
no obvious hypothesis to link vaccine-induced responses with
enhanced susceptibility, and the significance testing by
primary analysis indicates that this was a chance finding.
FFM ME-TRAP was only moderately immunogenic in this
population, despite the strong immunogenicity observed
previously. Stronger immunogenicity was seen in previous
Phase 1 studies in adults and children in Kilifi. Arithmetic
mean responses by ex vivo ELISPOT were 610 spots per
million PBMCs in malaria-naı ¨ve adults  but 350 spots per
million in semi-immune adults in Gambia and 360 spots per
million in Kenya . The arithmetic mean T cell response
during Phase 1 trials in children in Vipingo, Kilifi (where the
EIR is 1 ) was 200 spots per million. In the Phase 2b study
in Junju reported here (where the EIR is 22–53 ),
arithmetic mean responses were 110 spots per million.
Immunogenicity was moderately reduced for semi-immune
adults compared with malaria naı ¨ve adults, and was reduced
further among children in a malaria-endemic area. Several
studies have shown dendritic cell function to be impaired by
malaria and other chronic infections , and placental
Table 2. Solicited Adverse Events: Frequencies of Children Experiencing the Named Adverse Events at Any Time during the First
Week of Monitoring
Adverse EventVaccination Frequency Percentage (95% CI)
Any blister 1st FP9 ME-TRAP
2nd FP9 ME-TRAP
1st FP9 ME-TRAP
2nd FP9 ME-TRAP
1st FP9 ME-TRAP
2nd FP9 ME-TRAP
1st FP9 ME-TRAP
2nd FP9 ME-TRAP
1st FP9 ME-TRAP
2nd FP9 ME-TRAP
19.0% (13%–to 24%)
Blister .0.5 cm diameter8
Marked local pain and reduced activities
Marked local pain without impact on activities
Temperature .39.0 8C
Frequency refers to the number of children experiencing the adverse event listed. ‘‘Reduced activities’’ refers to children whose parents stated that they were not eating or not playing
after receiving the vaccine. ‘‘Marked pain’’ refers to the report given by parents who were offered the options ‘‘none,’’ ‘‘a little,’’ or ‘‘a lot’’ of pain experienced by the child after
vaccination (excluding the immunisation itself).
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Efficacy of FFM ME-TRAP in Children
malaria induces T cell regulatory responses at birth .
There may therefore be a causal association between reduced
immunogenicity and more frequent malaria and other
Different batches of FP9 ME-TRAP can be associated with
different immunogenicity , and work to assess factors that
may underlie this variability is in progress. However, studies
on semi-immune and naı ¨ve adults used the same batch of
vaccine, and preliminary studies in the same group of semi-
immune adults suggested that the batches used for Phase 1
and Phase 2b trials in children had similar immunogenicity. It
is therefore unlikely that most of the observed variation in
immunogenicity is accounted for by batch variation.
Active case detection might produce a less specific endpoint
by detecting milder and more self-limiting illness than would
passive case detection. This problem can be countered by
using a parasitaemia threshold to define malaria cases . It
is conventional to perform blood film examination on history
of fever alone, since relying on a single measurement of fever
can miss cases . However, subjective impressions of fever
are extremely nonspecific . In order to improve the
specificity of cases identified by active case detection, we did
not perform blood films from children with a history of a
fever without an objective fever, but after the initial visit
returned on three occasions in the next 24 hours to measure
the temperature again. This approach appeared to be safe in
Is it nevertheless possible that a case definition with poor
specificity obscured vaccine efficacy? This seems unlikely.
Febrile malaria was identified in only five (6%) of 74 5- to 6-
year-olds, compared with 19 (29%) of 65 1- to 2-year-olds,
although fever and asymptomatic parasitaemia were common
in both age groups. A study using passive case detection
would probably require a 4-fold increase in sample size for
similar power .
It is unclear whether reductions in liver parasites similar to
those seen in sporozoite challenge  might have occurred in
this trial without a reduction in clinical episodes, but these
reductions might be expected among children with the
highest T cell responses. These data, together with the Phase
2a and 2b trial data for RTS,S/AS02 [35,36] suggest that
greater than 90% reductions in liver parasites are required
for protection in the field. This trial also suggests that similar
pre-erythrocytic vaccines should achieve T cell responses
considerably higher than those seen here in order to provide
measurable efficacy in children, and raises the possibility that
T cell-inducing vaccines may show lower immunogenicity in
higher-transmission settings. Further development of potent
T cell-inducing vaccinations against malaria will examine
different vector combinations and antigen inserts, in order to
improve both immunogenicity and the breadth of responses
Found at DOI: 10.1371/journal.pctr.0010029.sd001 (48 KB DOC).
Found at DOI: 10.1371/journal.pctr.0010029.sd002 (135 KB PDF).
Alternative Language Abstract S1.
Translation of the Abstract into Kiswahili
Found at DOI: 10.1371/journal.pctr.0010029.sd003 (23 KB DOC).
Report and Analysis Plan
Found at DOI: 10.1371/journal.pctr.0010029.sd004 (37 KB DOC).
The participants and their parents are thanked. The Junju dispensary
committee facilitated the study. The study was performed with the
permission of Kenyan Medical Research Institute National Ethics
Committees, and Central Oxford Research Ethics Committee, the
National Health Service Central Office for Research Ethics Commit-
tees. The study was published with the permission of the Director of
Kenyan Medical Research Institute. The Data Safety Monitoring
Board members were David Mabey (chair), Tim Peto, Lucy Dorrell,
and Kathryn Maitland.
PB, PM, TL, CM, GF, NP, KM, and AVSH designed the study. PB, GF,
and NP analyzed the data. PB enrolled patients. PB, OK, TM, PM, ST,
SK, TL, SCG, NP, KM, and AVSH wrote the paper. JM, OK, TM, ST,
SK, BL, CM, and SCG collected data or did experiments. JM, OK, ST,
and SK were involved with the immunological laboratory studies. TM
helped with data collection and fieldwork. TL was the project manger
for this trial, and contributed to the trial design and management of
this study. BL assisted with the experimental design and ensured
accuracy of all the laboratory assays. CG was responsible for fieldwork
to ensure that standards of informed consent were met and were
continuously monitored to maintain those standards. CM assisted in
designing the community engagement elements of the trial, which
included helping to write the consent forms, to assess community
understanding of the information, and systems to respond to issues
raised. SCG designed the vaccines used in the study, produced seed
stocks, carried out quality control, and assisted with regulatory
submissions for the vaccines. KM was Dr. Bejon’s on-site PhD
supervisor and had overall responsibility for the conduct of the trial
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