Viral Vectored Blood Stage Malaria Vaccine Figure 9. Multi-functionality of CD4+ T cell response induced by vaccination. The multi-functional composition of the CD4+ T cell response (measured in Figure 8) is shown. The pie charts summarize the fractions of AMA1-specific CD4+ cells that are positive for a given number of functions (IFN-c, TNF-a and IL-2). Individual data points and median percentage of the parent CD4+ T cell response (open bars) are shown for each of the functional populations indicated on the x-axis. doi:10.1371/journal.pone.0020977.g009 the inclusion of the tPA leader sequence as demonstrated by the capture ELISA assay. Either or both of these mechanisms could account for the increased immunogenicity of the AMA1 vaccine constructs in which the native signal peptide was replaced with tPA. The construct that included the PfAMA1 C-terminal domains (transmembrane and cytoplasmic) allowed cell surface localization, but this was found to improve humoral immunoge- nicity only in combination with the native signal peptide, and did not significantly further improve the tPA containing construct. In agreement with these data, it has been previously reported that antigens expressed at the cell surface were better than intracellular antigens at inducing antibody responses [30]. Polymorphism in AMA1 has been one of the main hurdles in vaccine development targeting this antigen. AMA1 polymor- phisms are under balancing selection, especially within domains I and II that are the most polymorphic. It has been speculated that these polymorphisms are maintained due to selective pressure by the host immune system [60,61,62,63]. In an attempt to overcome the allele-specific functionality of the IgG induced by the first generation vaccines, bi-allelic PfAMA1 vectored vaccines were developed that also incorporated the tPA leader and C-terminal region of one allele of PfAMA1. Multi-allele AMA1 protein vaccines have been developed by several groups. Approaches have included a 50:50 mixture of recombinant 3D7 and FVO AMA1 antigen formulated in Alhydrogel 6 CpG adjuvant (AMA1-C1) [64], as well as a diversity covering (DiCo) approach that covers 97% of the amino acid variability in PfAMA1 [21]. These multi- allelic vaccines show better cross-reactivity in terms of GIA than mono-allelic vaccines, and interestingly in one study have been shown to focus antibody responses on conserved B cell epitopes [18]. Data on human antibody responses to PfAMA1 show that affinity purified antibodies from individuals living in malaria endemic areas can inhibit P. falciparum growth in vitro [49], and Cortes et al. have also examined the allele specificities of plasma samples of adults from Papua New Guinea and found that antibodies against conserved regions of the molecule were much more common than allele-specific antibodies [14]. In this study in mice, the AdHu5-MVA and ChAd63-MVA bi-allelic PfAMA1 vaccination regimes induced antibodies that bound both the 3D7 and FVO recombinant proteins by ELISA. Although slightly weaker responses were observed at the day 14 time-point when using ChAd63, these responses equalized by day 55 and were PLoS ONE | www.plosone.org 12 June 2011 | Volume 6 | Issue 6 | e20977