New Zealand white rabbits were used for all rabbit experiments. The vaccines were shipped from Oxford and the immunization of rabbits and collection of sera was performed by Biogenes GmbH, Berlin, Germany (for the mono-allelic AMA1 rabbit experiment) or Agro-bio, France (for comparison of mono-allelic and bi-allelic vaccines). Rabbits were immunized i.d. or intramuscularly (i.m.) with 561010 vp of AdHu5 or ChAd63 expressing AMA1 and 56108 pfu of MVA AMA1. Total IgG ELISA The antibodies induced by vaccination were measured by ELISA as previously described [28]. Briefly, recombinant PfAMA1 protein was adsorbed to 96 well Nunc-Immuno Maxisorp plates at a concentration of 2 mg/ml in PBS. 3D7 AMA1 was a kind gift from Dr Chetan Chitnis (ICGEB, New Delhi, India) and FVO AMA1 was a kind gift from Dr Mike Blackman (NIMR, London, UK). After blocking, the serum was incubated for 2 h and the bound antibodies were detected using alkaline phosphatase-conjugated goat anti-mouse (Sigma A3562) or anti-rabbit (Sigma A8025) IgG (whole molecule) diluted 1:5000. Serum antibody endpoint titers were taken as the x-axis intercept of the dilution curve at an absorbance value 36 standard deviations greater than the OD405 for naive mouse serum (typical cut-off OD405 for positive sera = 0.15). Na¨ıve mice sera were pooled and used as controls for all the ELISAs and they were negative against both recombinant 3D7 and FVO PfAMA1 protein. Standardized ELISAs for PfAMA1 were performed as described elsewhere [11]. Isotype ELISA Mouse IgG isotype ELISA assays were performed exactly as for total IgG, except antibodies were detected by incubation for 1 h at room temperature (RT) with biotinylated rat anti-mouse IgG1 or IgG2a mAbs diluted to 1 mg/ml in Phosphate Buffered Saline +0.05% Tween. Plates were washed 66 in PBS/T and then incubated for 30 min at RT with ExtrAvidin conjugated to alkaline phosphatase (Sigma) diluted 1:5000 in PBS/T. After the incubation the plates were developed as for total IgG. In vitro Growth Inhibitory Activity (GIA) Assay Sera from immunized animals were used to test their ability to inhibit parasite growth. The purified IgG was mixed with P. falciparum infected human red blood cells and the growth inhibition activity of the serum was determined by measuring parasite lactate dehydrogenase (pLDH) release after a 40–48 hour incubation period. Purified IgG was tested against 3D7, FVO, or FCR3 strains of P. falciparum at 5 or 10 mg/ml. The mouse and rabbit GIA assays were performed using a standardized method [10] at the GIA Reference Centre (Laboratory of Malaria and Vector Research, NIH) against both 3D7 and FVO strains of P. falciparum parasites. The rabbit GIA assay (Figure 3B) against the 3D7 and FCR3 strains of P. falciparum was undertaken at the Biomedical Primate Research Centre, The Netherlands against the 3D7 and FCR3 parasite strains following a similar protocol [21]. Multiparameter Flow Cytometry Cytokine secretion by peripheral blood mononuclear cells (PBMCs) and splenocytes were assayed by intracellular cytokine staining (ICS). PBMCs and Splenocytes were prepared as previously described and added to a 96-well U-bottom plate. 50 ml of Brefeldin A (GolgiPlug) at 1 mg/ml and 50 ml of peptide pools [44] diluted in complete medium at a final concentration of 1 mg/ml were added to the test wells. In the control wells Viral Vectored Blood Stage Malaria Vaccine GolgiPlug and medium alone were added. Cells were incubated at 37uC 5% CO2 for 5 h. After incubation the cells were left at 4uC overnight. The next day cells were surface stained for 30 min at 4uC with PerCP-Cy5.5-conjugated anti-mouse CD8a (clone 53- 6.7) and Pacific blue (PB)-labelled anti-mouse CD4 (clone LT34) diluted in PBS with 0.1% BSA at a dilution individually assessed by titration for each antibody. Cells were then permeabilized in 100 ml 16 Cytofix/Cytoperm solution for 20 min at 4uC and then intracellularly stained for 30 min at 4uC with APC-conjugated anti-mouse IFN-c (clone XMG1.2), FITC-conjugated anti-mouse TNFa (clone 145-2C11) and PE-conjugated anti-mouse IL-2 (clone JES6-5H4). After staining, cells were resuspended in 200 ml PBS containing 1% formalin. Stained cells were acquired using a LSRII flow cytometer (BD Biosciences) and data analyzed using FlowJo v9 (Tree Star Inc, USA), Pestle and SPICE (Mario Roederer, VRC, NIAID, NIH) software. Background responses in unstimulated cells were subtracted from the stimulated responses prior to analysis. Immunofluorescence Assay (IFA) Hela cells (obtained from European Collection of Cell Cultures, UK) were grown on 22 mm coverslips in 6-well plates to about 50% confluency. 20 ml Lipofectamine 2 K and 500 ml Optimem (Invitrogen) (for each well) were mixed and incubated for 5 min at RT. 4 mg plasmid DNA of the individual constructs corresponding to the transgenic insert in recombinant adenoviruses and comprising a human CMV promoter and bovine growth hormone polyadenylation signal were gently mixed with 500 ml of Optimem (per well). The DNA-Optimem and the Lipofectamine-Optimem solutions were combined and incubated for 20 min at RT. These were then used to transfect the cells grown on the coverslips by adding 1 ml of the solution to each well. These were incubated for 3–4 h at 37uC and 5% CO2 in a humidified incubator and then 1 ml of Optimem was added to each well and left overnight. The plates were kept on ice and the cells were gently washed twice with ice-cold PBS. The cells were then fixed with 4% paraformalde- hyde in PBS for 10 min on ice and then 20 min at RT. The cells were then washed with PBS three times over 10 min and quenched with 50 mM ammonium chloride for 5 min. After this the cells were permeabilized (if required) with 0.2% Tween-100 in PBS for 5 min and washed 3 times with PBS. If the cells were not permeabilized only the cell surface protein is accessible for immunolabelling. The cells were then blocked with 0.5% BSA in PBS for 30 min and then incubated with polyclonal mouse serum from mouse immunized with AdHu5-PfAMA1 and MVA- PfAMA1 diluted 1:100 in PBS. After 30 min Alexa 548- conjugated anti-mouse IgG (1:200 dilution) was added (Invitrogen, UK) and incubated for another 30 min. After the incubation the cells were washed with PBS and the cover slip was mounted using 30 ml Mowiol-DAPI (49,69-diamino2-phenylindole, 500 ng/ml). Slides were imaged using a Leica DMI3000B epifluorescence microscope and a Qicam (Qimaging). The parasite immunofluorescence assay was performed accord- ing to a published protocol [66]. Cold methanol-fixed smears prepared from a synchronized culture of P. falciparum with fully mature schizonts were used for the assay. The slides were fixed with acetone and incubated for 30 min with a serial dilution of the post immunization serum and control serum in PBS containing 1% BSA and 0.01% sodium azide. After incubation these were washed and incubated with a 1:80 dilution of FITC-conjugated goat anti-rabbit IgG (Dako, UK) for 30 min. The slides were immersed in a solution of 0.1% Evans blue and 0.001% DAPI and mounted on coverslips and examined microscopically as above. PLoS ONE | www.plosone.org 14 June 2011 | Volume 6 | Issue 6 | e20977