Viral Vectored Blood Stage Malaria Vaccine Figure 1. Murine antibody responses generated by AdHu5-MVA PfAMA1 (3D7) vaccination. 5–6 week old female BALB/c mice (n = 7/ group) were immunized via the intradermal route with 561010 vp of AdHu5-PfAMA1 and boosted 8 weeks later with 16107 pfu MVA-PfAMA1. 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 mice 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). (C) The correlation between the 3D7 and FVO antibodies at day 70 was analyzed using Pearson’s rank correlation (R2 = 0.49, P #0.05). (D) IgG isotypes were assessed at day 70 in the serum against recombinant FVO PfAMA1. doi:10.1371/journal.pone.0020977.g001 capture ELISA was performed to quantify the amount of PfAMA1 protein secreted/expressed from the two vectors: i) AdHu5-tPA- PfAMA1 and ii) AdHu5-N-PfAMA1. In agreement with the hypothesis that the mammalian leader sequence could enhance transgene secretion and/or protein expression, there was consis- tently more PfAMA1 protein in both the supernatant and the lysate of the cells infected with the vector containing the tPA signal peptide as compared to those infected with the vector containing the native parasite AMA1 signal peptide (Figure 5A). Comparative immunogenicity of modified PfAMA1 transgene inserts Four groups of BALB/c mice were subsequently immunized with the four different AdHu5 constructs: i) AdHu5-tPA-PfAMA1, ii) AdHu5-tPA-PfAMA1-C, iii) AdHu5-N-PfAMA1 and iv) AdHu5- N-PfAMA1-C. The total IgG response to the ectodomain of PfAMA1 was measured by ELISA in the serum 12 days post vaccination against recombinant PfAMA1 (3D7) protein (Figure 5B). Inclusion of the human tPA leader sequence in the vaccine led to significantly higher (P#0.05) antibody titers than when the native parasite N- terminus was used, presumably due to increased efficiency of antigen secretion and/or expression (Figure 5A). Interestingly, the antibody response was maintained when the native C-terminus was included in the vaccine construct in conjunction with the tPA leader, and in fact responses tended to be stronger when the C-terminus was used in conjunction with the native N-terminal signal sequence. However, the addition of the C-terminus did not further enhance the antibody response when the tPA leader sequence was used in place of the native N-terminus. These data indicate that although cell surface localization may in some cases enhance the antibody immunogenicity of AdHu5 recombinants, the efficiency of antigen secretion and/or level of expression may be of greater importance. Generation and immunogenicity of a bi-allelic PfAMA1 construct A bi-allelic PfAMA1 vaccine construct was designed in an attempt to address the issue of antigenic polymorphism in PfAMA1. The 3D7 and FVO alleles utilized in other candidate protein vaccines [17] were included and are two of the most divergent in terms of amino acid (aa) polymorphism differing by 24 aa [20]. The construct contained the ectodomains of the two alleles fused in tandem and linked by a glycine-proline linker, as well as the tPA leader sequence (at the N-terminus) and the TM region and C- terminus of the FVO allele [44]. For pre-clinical studies this construct was cloned into MVA (referred to as MVA-bi-allelic), AdHu5 (AdHu5-bi-allelic) and the chimpanzee adenovirus ChAd63 PLoS ONE | www.plosone.org 4 June 2011 | Volume 6 | Issue 6 | e20977