Results Antibody responses induced by AdHu5-MVA PfAMA1 (3D7) prime boost vaccination in mice and rabbits PfAMA1-specific IgG antibody responses were measured by ELISA in the serum of BALB/c mice and New Zealand white rabbits after an AdHu5-MVA PfAMA1 (3D7) heterologous prime- boost vaccination regimen using an eight week interval [28]. Both vectors encoded a first generation PfAMA1 (3D7) vaccine construct (aa 25–546) including the tPA leader sequence and with mutations in potential N-linked glycosylation sites (as described in Methods). In mice the kinetics of the total IgG response was measured by ELISA against both the homologous 3D7 allele (vaccine strain) (Figure 1A) and the heterologous FVO allele (Figure 1B) to assess the induction of cross-reactive antibodies. All mice seroconverted after the AdHu5-PfAMA1 (3D7) prime (assessed at day 14) and these responses significantly increased (P#0.05) by day 55 (pre-boost), in agreement with murine data for similar AdHu5 vectors encoding the P. yoelii and P. falciparum MSP1 antigens [28,32]. The MVA boost immunization on day 56 significantly increased (P#0.05) the PfAMA1-specific IgG anti- body response as assessed on day 70 (two weeks post boost). Cross- reactive antibodies were measured against the heterologous FVO allele and there was a significant correlation (R2 = 0.49, P = 0.03) between the antibodies to 3D7 and FVO PfAMA1 at the peak of the response, day 70 (Figure 1C). The IgG isotypes (IgG1 and IgG2a) were also measured in the sera and there was a balanced Th1-type IgG2a and Th2-type IgG1 response in mice at the day 70 time-point (Figure 1D). The kinetic of the IgG antibody response in rabbits after the same immunization regime was different to that seen in mice. After immunization with AdHu5-PfAMA1 (3D7) all the rabbits serocon- verted, but the antibody response tended to be lower at day 55 post prime in comparison to day 14 (P#0.05) (Figure 2A,B), unlike in mice where there was a significant increase in the antibody response over time after the AdHu5-PfAMA1 (3D7) prime. Administration of the MVA boost on day 56 significantly increased the antibody response by day 70. Measurement of cross-reactive antibodies against FVO AMA1 showed there was a stronger correlation between the 3D7 and FVO antibodies at day 70 in rabbits (R2 = 0.98, P#0.0001) than in mice (Figure 2C). The antibodies induced by vaccination recognized native AMA1 protein as shown by parasite immunofluorescence assay (IFA) (Figure 2D). IFA was performed using slides of P. falciparum schizont infected red blood cells (RBCs) which were incubated with day 70 sera from immunized rabbits. The sera bound to the P. falciparum schizont stages as shown by the punctate apical staining of merozoites that is typical of that seen for AMA1 (Figure 2E). Functional activity of antibodies generated in mice and rabbits The functional activity of antibodies induced by the AdHu5- MVA PfAMA1 (3D7) vaccination regime was determined using a standardized assay of growth inhibitory activity (GIA) [43], which measures the growth inhibition of blood-stage P. falciparum parasites by purified IgG. Mouse sera obtained at the day 70 time-point were pooled and the GIA was determined using a final concentration of 5 mg/mL purified IgG. The samples showed a GIA of 98% against 3D7 parasites (vaccine strain), but lower levels against the heterologous FVO parasites (54%) (Figure 3A). Purified control mouse IgG shows no significant biological activity against either parasite strain in this assay [28]. The antibody response (measured using a standardized ELISA [11]) against the 3D7 allele was higher than FVO (Figure 3A). Viral Vectored Blood Stage Malaria Vaccine Similar assays were performed for individual rabbit serum samples taken two weeks after the final immunization, and GIA was assessed against the homologous 3D7 and heterologous FCR3 strains using purified IgG at a final concentration of 6 mg/mL. The AMA1 sequence of FCR3 differs from that of the FVO strain by only a single amino acid [18]. In rabbits there was moderate growth inhibition against the homologous 3D7 strain (medi- an = 44%, n = 8) but little or no GIA against the heterologous FCR3 strain (median = 7%, n = 8) (Figure 3B). This strain-specific GIA of purified rabbit IgG has been reported in numerous other studies using recombinant protein AMA1 vaccines [12,19]. There was no correlation between 3D7 and FCR3% GIA (Figure 3C). However, as seen in other studies [32] there was a positive correlation between the 3D7 AMA1 ELISA titers and 3D7 GIA (R2 = 0.68, P#0.05) and also with FCR3 GIA (R2 = 0.51, P#0.05) (Figure 3D). Generation of modified mono-allelic PfAMA1 vaccine constructs These preliminary data in mice and rabbits highlighted the need to further optimize the PfAMA1 vaccine constructs in terms of inducing antibodies capable of inhibiting the growth of divergent P. falciparum strains. One recent study has demonstrated the ability of co-immunization with two alleles of AMA1 to focus the induced antibodies on conserved epitopes [18] and another demonstrated enhanced antibody-inducing capability of an AdHu5 vaccine expressing PfAMA1 (3D7) when localized to the cell surface in comparison to intracellular expression [30]. The first generation mono-allelic PfAMA1 vaccine described here utilized the signal peptide from the human tissue plasminogen activator (tPA) as a leader sequence in place of the native parasite N-terminal (N) PfAMA1 signal sequence. However, the transmembrane domain (TM) and the cytoplasmic tail (C) of PfAMA1 were not included in this construct. As part of the process to develop a second generation vaccine construct based on PfAMA1, the potential effects on antibody immunogenicity of the addition of the native N and/or C termini were tested. Three new recombinant AdHu5 vectors were thus generated (second generation mono-allelic PfAMA1 (3D7) vaccines) encoding the ectodomain of PfAMA1 (aa 25–546) with either the native N terminal signal peptide or tPA leader and either with or without the native C-terminal transmembrane and cytoplasmic domains (aa 547–623) (Figure 4A), in order to comparatively assess antibody immunogenicity. Effect of the addition of the C-terminus on cellular localization of PfAMA1 In order to confirm differences in the subcellular localization of the PfAMA1 antigen following inclusion of the C-terminus in the vaccine construct, an in vitro immunofluorescence assay was performed. Transfection of HeLa cells with plasmid construct tPA-PfAMA1-C containing the AMA1 C-terminus showed a cell surface localization of PfAMA1 in both the permeabilized and non-permeabilized cells (Figure 4D,E). In contrast, when using the construct tPA-PfAMA1 that expressed the construct without the C-terminal membrane anchor, PfAMA1 expression was only detectable in the permeabilized cells, presumably localized in the endoplasmic reticulum (Figure 4B,C). These data confirmed that the addition of the C- terminus led to the cell surface expression of PfAMA1. Effect of replacing the native parasite signal peptide with tPA as a leader sequence The immunoflourescence assay does not quantify the effect of replacing the N-terminal parasite signal sequence with tPA, so a PLoS ONE | www.plosone.org 3 June 2011 | Volume 6 | Issue 6 | e20977