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 trial.
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 prevaccination, 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 immunized 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 transaminase). 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 transfusions (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 screened.
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 [22
]. The transmission intensity is 22–53 infective bites per year [23
]. Junju is served by a local government dispensary, which has an active ITN distribution programme. Inpatient care is available at Kilifi District Hospital.
The antigen insert used in the vaccine was TRAP, joined to a multiple epitope (ME) string from six P. falciparum
pre-erythrocytic antigens [7
]. 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 produced 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 (5 × 107 plaque-forming units) followed by MVA ME-TRAP vaccination (1.5 × 108 plaque-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 °C, 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 resuscitation 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.
Efficacy (malaria episodes).
The primary endpoint was a clinical episode of malaria, defined as an axillary temperature greater than 37.5 °C, with a P. falciparum parasitaemia greater than 2,500 parasites/μl. The presence of any falciparum parasitaemia with fever above 37.5 °C was a secondary endpoint.
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 [24
]. An endpoint with low specificity will lead to an incorrect estimate of efficacy [25
]. 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 [22
], 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 dihydroartemisinin 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 treatment.
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 °C, 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 mononuclear 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 MabTech antibodies (MabTech, Stockholm, Sweden) according to manufacturer's instructions. Freshly isolated PBMCs were incubated at 4 × 105 cells/well in a volume of 100 μl with 10 μg/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 μg/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 × 106 PBMCs were incubated in 0.5 ml of 10 μg/ml/peptide of pooled TRAP and ME peptides in a 24-well plate. On days 3 and 7, 250 μl of culture supernatant was replaced with 250 μl 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 discolouration 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% 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 conducted.
Intradermal poxvirus vaccinations are associated with skin discolouration and blistering [21
]. 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/μl) 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.