Viral Vectored Blood Stage Malaria Vaccine Figure 2. P. falciparum AMA1 specific total IgG responses induced by immunization in rabbits. New Zealand rabbits (n = 8) were immunized via the intradermal route with 561010 vp of AdHu5-PfAMA1 (3D7) and boosted 8 weeks later with 56107 pfu of MVA-PfAMA1 (3D7). The AMA1-specific antibody response against both homologous 3D7 (A) and heterologous FVO (B) PfAMA1 recombinant protein was measured in the serum at day 14 (post prime), 55 (pre boost) and 70 (post boost). Data show the responses from individual rabbits and the lines represent the median. The asterisk indicates a significant difference between the different time-points as analyzed by Wilcoxon signed rank test (* P #0.05, ** P #0.01). (C) The correlation between the 3D7 and FVO antibodies at day 70 was analysed using Pearson’s rank correlation. There was a highly significant positive correlation (R2 = 0.96, P #0.0001). (D) IFA titers were measured using sera taken two weeks following the boost immunization. Individual IFA titers against 3D7 and FCR3 P. falciparum strains are shown with the median response (FCR3 AMA1 differs from FVO AMA1 by only a single amino acid substitution [18]). (E) When serum from immunized rabbits was used to label P. falciparum schizonts, typical AMA1-specific punctate apical staining of merozoites was seen. doi:10.1371/journal.pone.0020977.g002 (ChAd63-bi-allelic). This simian vector has also been used in the development of other P. falciparum candidate malaria vaccines encoding the ME-TRAP [41] and MSP1 [32,42] antigens, and was used in order to circumvent issues surrounding pre-existing anti- vector immunity to AdHu5 in humans. Mice were primed with either i) ChAd63-bi-allelic, ii) AdHu5- bi-allelic or iii) the mono-allelic AdHu5-PfAMA1 (3D7) vectors. Groups i) and ii) were boosted with MVA-bi-allelic and group iii) was boosted with the mono-allelic MVA-PfAMA1 (3D7) vaccine. Total IgG was measured in the serum on days 14, 55 and 70 by ELISA against recombinant 3D7 and FVO PfAMA1. AMA1 specific IgG responses were detected in all the groups to both 3D7 (Figure 6A) and FVO (Figure 6B) recombinant proteins. Although minor, but statistically significant differences were noted between groups by one-way ANOVA analysis at day 14, by day 55 these differences in IgG antibody responses had equalized between the different groups, suggesting that after priming with ChAd63 rather than AdHu5, the antibodies take slightly longer to reach maximal levels. The MVA immunization significantly increased the antibody response against both alleles in all the groups to similar levels by day 70 (P#0.05). Thus, in agreement with data for the MSP1 antigen [32], the ChAd63 and AdHu5 vectors expressing the bi-allelic PfAMA1 construct primed similar IgG responses, which remained equivalent after a heterologous MVA boost PLoS ONE | www.plosone.org 5 June 2011 | Volume 6 | Issue 6 | e20977