Clinical trial design

We conducted the study at Centre for Clinical Vaccinology and Tropical Medicine and Vaccinology, University of Oxford, with the CHMI procedure performed at Imperial College, London. We recruited healthy malaria naïve adults aged 18–50 years old from the Oxford area during Feb 2009 to Jan 2010 (inclusion and exclusion criteria are described in Supplementary Methods). All volunteers gave written informed consent prior to participation and the study was conducted according to the principles of the Declaration of Helsinki and in accordance with Good Clinical Practice (GCP). Approvals were granted by the Oxfordshire Research Ethics Committee (OXREC A 09/HO604/9) and the Medicines and Healthcare products Regulatory Agency (EudraCT 2008-006804-46). The trial was registered with ClinicalTrials.gov number NCT00890760. The Clinical Biomanufacturing Facility, University of Oxford (ChAd63) and IDT Biologika, Rosslau, Germany (MVA), manufactured study vaccines under Good Manufacturing Practice conditions. A Local Safety Monitor provided safety oversight and GCP compliance was externally monitored.

We vaccinated 15 volunteers with ChAd63 ME-TRAP 5 × 1010 v.p. intramuscularly followed by MVA ME-TRAP 2 × 108 p.f.u. intradermally 56 days later. We vaccinated another group of 10 volunteers with a single dose of ChAd63 ME-TRAP 5 × 1010 v.p. intramuscularly. Group allocation was non-randomized. Volunteers attended clinical follow-up at days 2, 14 and 28 following ChAd63 ME-TRAP, and at days 56, 63 and 76 following MVA ME-TRAP. Safety assessment including blood sampling for safety was conducted as previously described17,18,23 (adverse event assessment criteria are shown in Supplementary Tables S4–S7). One volunteer in the prime-boost group in CHMI A was unable to proceed to challenge having moved from the study area.

Five Anopheles stephensi mosquitoes, each with 102–104 sporozoites per salivary gland, were allowed to bite each subject, thus delivering 3D7 strain P. falciparum sporozoites, 14–21 days after the final vaccination. This procedure took place over 2 days with the same number of vaccinees and controls exposed each day. Monitoring took place twice daily by using Giemsa-stained thick blood films, which were considered positive if a single morphologically correct parasite was seen, and by quantitative PCR starting on day 6.5 until day 14 and then once daily until the end of the study period at day 21. Subjects were treated with Riamet after the first confirmed positive blood film or at day 21 if no parasitemia was detected. In addition to vaccinated subjects, six unvaccinated subjects were infected with malaria as infectivity controls. We reviewed volunteers at 35, 90 and 150 days following challenge for safety and immunology assessment.

Sporozoite CHMI group sizes are typically small but power to detect differences between the vaccinees and the controls is improved by the use of Kaplan–Meier analysis of time to patent parasitaemia. Partially effective vaccines should delay average time to parasitaemia in non-sterilely protected individuals if they protect any number of volunteers fully. The primary endpoint analysis was time to patent parasitaemia for the vaccine groups (prime-boost (n=14) and ChAd63-alone (n=10)) compared with unvaccinated controls (n=12) by Kaplan–Meier analysis (two-tailed). Significance testing used the log-rank test.

Immunological and molecular assays

Ex vivo (18-h stimulation) and cultured ELISPOT (10-day stimulation) assays were performed using Multiscreen IP ELISPOT plates (Millipore), human IFNγ SA-ALP antibody kits (Mabtech) and BCIP NBT-plus chromogenic substrate (Moss Inc). Cells were cultured in RPMI (Sigma) containing 10% heat-inactivated, sterile-filtered fetal calf serum, previously screened for low reactivity (Labtech International). Antigens were tested in duplicate with 250,000 PBMC added to each well of the ex vivo ELISPOT plate18 and 100,000 cultured T cells in the cultured ELISPOT assay. TRAP peptides were 20 amino acids in length, overlapping by 10 amino acids (Neopeptide), assayed in 6 pools of 7–10 peptides at 10 μg ml−1. Responses were averaged across duplicates, responses in unstimulated (negative control) wells were subtracted and then responses in individual pools were summed for each strain of the TRAP antigen. ME responses were assayed in a single pool and peptide pool configurations are shown in Supplementary Tables S2 and S3. Staphylococcal enzyme B (0.04 μg ml−1) and phytohaemmagglutinin-L (20 μg ml−1) were used as a positive control. Epitope mapping was performed using individual 20mer peptides spanning the length of the T9/96 TRAP protein in single ELISPOT wells, each containing 100,000 PBMC. Plates were counted using an AID automated ELISPOT counter (AID Diagnostika GmbH, algorithm C), using identical settings for all plates and counts were adjusted only to remove artefacts. Responses to the negative control were always <80 SFC per million PBMC. Responses of >50 SFC per million after subtraction of background were considered positive.

For flow cytometry, responses were assessed by either a 7-colour (part A) or 12-colour (part B) staining panel, which was performed on freshly isolated PBMC, in parallel with ELISPOT assays, aliquots of 1 × 106 cells in 1 ml of medium containing anti-CD28 and anti-CD49d at 1 μg ml−1 (Becton Dickinson) and 62.5 μg ml−1 of CD107a-PeCy5 (eBioscience)) in part B were stimulated with either no antigen, a pool of all 56 peptides spanning the T9/96 strain of the TRAP antigen (1 μg ml−1) or a positive control, Staphylococcal enterotoxin B (Sigma, 1 μg ml−1) in 5 ml polystyrene FACS tubes for 18 h. Brefeldin A and monensin, both at 1 μg ml−1, were added for the last 16 h. Cells were incubated with a dead cell discrimination dye (VIVID, 1/80, Invitrogen), and then surface-stained at 4°C with CD4-APC (1/20, eBioscience) or CD4-Qdot 625 (1/50, Invitrogen), CD14- and CD19-Pacific Blue (both 1/50, Becton Dickinson). After permeabilisation, intracellular staining was performed at room temperature with CD3-PeCy5 (part A, 1/20, eBioscience) or CD3-Alexa Fluor 700 (part B, 1/100, eBioscience) plus CD8-APC-Alexa Fluor 780 (1/50) and IFN-γ-FITC (1/50), IL-2-PE (1/100) and TNFα-Pe-Cy7 (1/50, all eBioscience) and fixed in 4% paraformaldehyde. Acquisition was performed on the day of staining on a BD LSRII; at least 500,000 events were collected per sample. Data was prepared and analysis performed using FlowJo v8.8.6 (Treestar Inc,), Pestle v1.6 and Spice v5.05 (Mario Roederer, Vaccine Research Centre, NIAID, NIH). Dead cells (Vivid+), monocytes (CD14+) and B cells (CD19+) were excluded from the analysis. A time gate was first evaluated, and then cells were gated on lymphocytes, singlets, live CD3+, CD8+ or CD4+ (excluding double-positives), and then IFNγ and combinations of markers. A sample gating strategy is provided in Supplementary Fig. S2. Responses to peptide were determined after subtraction of the response in the unstimulated control for each sample. Pie charts were created using absolute measures with a threshold of 0.001%. Mean fluorescence integrity (MFI) was calculated using the geometric mean of the cytokine-positive population and integrated MFI (iMFI) represents the integration of the frequency with the geometric mean of the cytokine-secreting population, giving a measure of total amount of cytokine production.

nAb titres to ChAd63 vector were determined as follows. One day prior to assay, GripTite 293 cells (Invitrogen) were seeded in 96-well plates (3 × 104 cells/well). Heat-inactivated test sera were diluted fourfold from 1:9 to 1:2,304 in 10% fetal bovine sera (FBS) in DMEM and incubated 1:1 with ChAd63 expressing the secreted alkaline phosphatase (SEAP) gene (8 × 107 vp ml−1) for 1 h at 37 °C. Serum and virus were then added to 293 cells in a volume of 200 μl in duplicate for 1 h, after which serum and virus were aspirated and replaced with fresh 10% FBS in DMEM. A virus-only control was included. After 22–26 h at 37 °C, 50 μl of medium was assayed for SEAP activity using a Phospha-Light TROPIX phosphatase assay (Applied Biosystems) in black assay plates and luminescence was measured after 45 min on a Thermo-Fisher Varioskan Flash Luminometer. Anti-vector neutralization titres were defined as the dilution of serum showing 50% reduction in SEAP activity, based on observed % inhibition values relative to SEAP activity from virus alone.

Anti-TRAP IgG ELISAs were performed in Nunc-Immuno Maxisorp 96-well plates (Thermo Scientific) coated with 1 μg ml−1 of TRAP protein in carbonate–bicarbonate coating buffer (Sigma) overnight at 4 °C. Plates were washed with PBS Tween and blocked with 1% BSA in PBS. Sera were diluted at a starting concentration of 1:100, added in duplicate and serially diluted threefold. Plates were incubated for 2 h at room temperature and then washed as before. Goat anti-human whole IgG conjugated to alkaline phosphatase (Sigma) was added for 1 h at room temperature. Following a final wash, plates were developed by adding p-nitrophenylphosphate at 1 mg ml−1 in diethanolamine buffer (Pierce). Optical density (OD) was read at 405 nm on an ELx800 microplate reader. Serum antibody endpoint titres were taken as the x axis intercept of the dilution curve at an absorbance value three s.d greater than the OD405 for serum before vaccination. A standard positive serum sample was included in each assay as a reference control.

PCR for blood parasite density after CHMI used blood samples filtered on custom 24-well Whatman VFE plates and washed with PBS to reduce white blood cell numbers. DNA extraction was performed on 0.5 ml of the filtered blood using Qiagen QIAamp Blood mini kit using a modified vacuum manifold protocol as follows: volumes of protease, lysis buffer and ethanol were 40 μl, 400 μl and 400 μl, respectively, and wash buffer volumes increased to 750 μl. The second wash buffer allowed to soak on the DNA purification columns for 2 min before the vacuum was applied. Columns were transferred to collection tubes and centrifuged at 13,000 r.p.m. for 4 min to dry. DNA was eluted with 50 μl sterile 10 mM Tris (pH 8.0), incubating for 1 min at room temperature before centrifugation (1 min, 8,000 r.p.m.) to collect the DNA sample. Quantitative PCR was performed on extracted DNA, amplifying a 133-bp product from the 18S ribosomal gene of P. falciparum in a TaqMan probe-based QPCR using the following primers: forward- 5′-GTAATTGGAATGATAGGAATTTACAAGGT-3′, reverse 5′-TCAACTACGAACGTTTTAACTGCAAC-3′ and a TaqMan probe 5′-FAM-AACAATTGGAGGGCAAG-NFQ-MGB-3′ (Applied Biosystems)45,46. Triplicate reactions of 25 μl were set up with 5 μl DNA template, 12.5 μl Universal PCR Master Mix, no Amperase UNG (Applied Biosystems), 1 μl forward primer, 1 μl reverse primer, 0.25 μl TaqMan Probe (all at 10 pmol μl−1) and 5.25 μl nuclease-free water. Reactions were run on an Applied Biosystems Step One Plus PCR System with an initial 10 min hot start activation at 95 °C, followed by 15 s denaturation at 95 °C and a 60 s annealing and extension at 60 °C for 45 cycles. Each plate contained a no template control and a standard curve made by serial dilutions of a plasmid containing the 133-bp PCR product that had been adjusted to give a parasites per ml equivalent (based on copies of 18S per parasite genome, blood sample volume extracted from and volume of DNA template used per reaction) of 105, 104, 103, 3 × 102, 102, 4 × 101 and 2 × 101. Automatic quantitation was performed with Applied Biosystems Step One plus software v2.1. Mean parasite equivalent values of <20 or with only 1 positive replicate were classed as negative. Laboratory staff performing blood film microscopy and qPCR were blinded to the group allocation of volunteers.

Data analysis and statistics

Time to parasitaemia as measured by thick-film microscopy was the primary efficacy endpoint and this was analysed using Kaplan–Meier log-rank test. Assessments of efficacy compared unvaccinated controls with groups of immunized volunteers. Immunological analysis compared markers of T-cell immunogenicity (summed ex vivo and cultured ELISPOT responses, and percentage of antigen-specific CD4+ and CD8+ cells by intracellular cytokine staining) and antibody responses between groups. Group averages (mean or median) were described. Significance testing of differences between group means (for normally distributed data as assessed by the D’Agostino and Pearson omnibus normality test) used the two-tailed Student’s t-test, or medians used the two-tailed Mann–Whitney U-test. Immunological correlations with time to patent parasitaemia or other variables were pre-specified in the trial protocol as secondary endpoints and prioritized analysis of T-cell subsets (particularly CD8+ subpopulations), based on observations from pre-clinical studies with these vaccines. Analyses were performed using the two-tailed Spearman’s rank correlation co-efficient (r s ) as data were either non-normally distributed or the number of observations was too small to allow an accurate estimation of the underlying distribution. Correction of P-values for multiple comparisons was made using the Bonferroni method, which multiplies the P value by the number of comparisons assessed. For log-transformed PCR data, an arbitrary value of 1 was added to each PCR value to allow log transformation of 0.0 values (y=log (1+PCR)). The area under curves was calculated using the trapezoid rule and compared between groups as described above. Regression models were fitted to log-transformed flow cytometry data with frequency of given T-cell populations as the independent variable and time to parasitaemia as the dependent variable, and then Akaike’s information criterion was used as an aid for choosing between competing models. Lower values of the index indicate the preferred model, that is, the one with the fewest parameters that still provides an adequate fit to the data. An alpha level of 0.05 was considered statistically significant. Statistical analysis was performed using Prism 5.0, Graph Pad Software.

Additional clinical information

Over 300 individuals had previously participated in controlled human malaria infections by mosquito bite at the Oxford centre and over a 1,000 further volunteers worldwide, all as part of vaccine studies. The clinical trial methodologies have been found to be safe and have been reviewed recently47,48. In this trial volunteers received as compensation sums ranging from £1,500 to £1,800, varying according to the number of clinic visits made.