Viral Vectored Blood Stage Malaria Vaccine Figure 5. Use of a mammalian secretory signal sequence enhances antigen expression and antibody immunogenicity of an AdHu5 vectored PfAMA1 vaccine. (A) A549 cells were infected either with the same number of infectious units of AdHu5-tPA-PfAMA1 or AdHu5-N- PfAMA1. The quantity of PfAMA1 protein was assessed in the supernatant and lysate by a PfAMA1 capture ELISA. The figure shows the OD405 values for both the supernatant and the lysate for the two constructs. (B) Four groups of 5–6 week old female BALB/c mice were immunized with one of the four constructs i) AdHu5-tPA-PfAMA1, ii) AdHu5-tPA-PfAMA1-C, iii) AdHu5-N-PfAMA1, or iv) AdHu5-N-PfAMA1-C at a dose of 161010 vp. The total IgG response was measured by ELISA in the serum 12 days after immunization against P. falciparum 3D7 recombinant protein. The median and endpoint titers (log 10) for individual mice are shown. The asterisk indicates a significant difference in endpoint titer between the groups by ANOVA (* P #0.05, ** P #0.01). doi:10.1371/journal.pone.0020977.g005 Figure 6. Antibody responses in mice induced by the mono-allelic and bi-allelic PfAMA1 vaccine constructs. 6 week old female BALB/c mice (n = 6) were primed with 16109 vp of each of the adenovirus constructs i) ChAd63-biallelic, ii) AdHu5-biallelic and iii) AdHu5-PfAMA1 (3D7). Groups i) and ii) were boosted at day 56 with 16107 pfu of MVA-biallelic and group iii) was boosted with the mono-allelic MVA-PfAMA1 (3D7). The total IgG response was measured by ELISA in the serum at day 14, 55 and 70 against both the 3D7 (A) and FVO (B) alleles. The median and the range of the endpoint titer (log 10) are shown in the figure. There were significant differences between the ELISA titres at day 14 by one-way ANOVA: 3D7 PfAMA1 ChAd63-biallelic versus AdHu5-monoallelic P ,0.001; FVO PfAMA1 ChAd63-biallelic versus AdHu5-biallelic P ,0.05. The MVA boost significantly increased IgG titres against both alleles in all groups (* P ,0.05 by Wilcoxon signed rank test). doi:10.1371/journal.pone.0020977.g006 PLoS ONE | www.plosone.org 8 June 2011 | Volume 6 | Issue 6 | e20977