boosted comparably by MVA. These antibody data are in agreement with those for similar vectors encoding P. falciparum MSP1 [32], and confirm that, at least in mice, ChAd63 can be used to replace AdHu5 in this adenovirus-MVA prime-boost vaccination regime without compromising the humoral immuno- genicity of the transgene product. Since seropositivity against AdHu5 is too prevalent to permit its widespread deployment in humans, these data support its replacement with ChAd63 as a vaccine vector. Notably in these mice, overall IgG titers were comparable whether using mono- or bi-allelic vaccines. Rabbits were thus immunized with both the vaccine constructs in order to purify IgG and assay for functional GIA against both the 3D7 and FVO strains of PfAMA1 to provide a comparison of the efficacy of the two vaccine constructs. Encouragingly the bi-allelic vaccines induced antibodies in rabbits that showed significant GIA against both the 3D7 and FVO strains unlike the mono-allelic vaccine. These data have also been confirmed in a recent macaque study, showing the ChAd63-MVA bi-allelic PfAMA1 regime induces significant GIA against both the 3D7 and FCR3 strains [44]. However, it has also been reported that the allele specificity of antibodies to PfAMA1 depends on the species immunized [19]. Interestingly, unlike for mice and rabbits discussed earlier, immunization of rhesus monkeys with recombinant PfAMA1 showed a completely different strain specificity pattern in terms of GIA. Even when monkeys were immunized with single-allele PfAMA1 vaccines, the elicited antibodies could bind indistin- guishably to both homologous and heterologous PfAMA1 proteins on ELISA plates, and the antibodies showed cross-reactive biological activities, as judged by GIA and antigen reversal GIA assays [19]. Thus despite the evidence in our recent macaque study, these rabbit data confirm the value, at least in this species, of developing multi-allelic PfAMA1 vectored vaccines. Splenic T cell responses in mice to both common and unique pools of peptides were also measured here by flow cytometry, and CD4+ T cells to the common pool displayed a multi-functional phenotype in terms of IFN-c TNFa and IL-2 production. This is encouraging because if CD4+ or CD8+ T cells recognizing common epitopes in humans are important for protection, then they could help to confer efficacy against heterologous strains. In agreement with this hypothesis, there have been suggestions based on analysis of sequence polymorphism frequencies that the selection pressure on AMA1 domain III is mediated by T-cell immunity [13]. In another recent study, pluripotent effector memory T cells against infected erythrocytes have been associated with protection in humans after a vaccination with sporozoites and treatment with choloroquine [8]. In the P. chabaudi model, in agreement with another study [5], we have shown a protective role of vaccine induced CD4+ T cells against blood-stage infection by in vivo depletion and adoptive transfer studies [Biswas et al., submitted]. We have also shown that MSP1-specific CD8+ T cells can reduce the liver-stage parasite burden in the P. yoelii model [29], and a similar mechanism may be relevant to AMA1 – an antigen also expressed by sporozoites [65] and most likely also late in the liver-stage of infection. Although we have shown here that the ChAd63 vector induced comparable antibody immunogenicity to AdHu5, there were some differences in relation to other studies using this vector for the pre- erythrocytic ME-TRAP antigen [41] and the blood-stage antigen MSP1 in mice [32]. Unlike for ME-TRAP and MSP1, the AMA1- specific CD4+ and CD8+ T cell responses were lower following ChAd63_MVA immunization in comparison to those induced by AdHu5_MVA expressing the same vaccine construct. It is possible these differences may be antigen- and/or insert-specific and, in Viral Vectored Blood Stage Malaria Vaccine agreement with these murine data, weak AMA1-specific T cell responses were also induced in rhesus macaques following immunization with this vector, although they boosted to high levels following MVA [44]. Here we have described the rational design of bi-allelic PfAMA1 vectored vaccines that induce functional antibodies against two parasite strains. The addition of the mammalian tPA leader sequence to the adenoviral vectored vaccine insert significantly increases the antibody response, and this adenovirus-MVA prime- boost vaccination regime also induces T cell responses to conserved epitopes. The responses to conserved B and T cell epitopes following bi-allelic AMA1 immunization may improve the strain-transcending efficacy of PfAMA1 vaccines in humans. Overall, these data support the rational design of vectored vaccine transgene inserts for optimization of immune responses against multiple alleles of the same antigen, as well as the continued development of these vectors towards phase I/IIa clinical testing. Materials and Methods Generation of viral vectors expressing AMA1 The first generation mono-allelic AMA1 construct encoded the ectodomain of P. falciparum 3D7 AMA1 (amino acid 25–546), and included a number of amino acid substitutions to remove potential sites of N-linked glycosylation and was codon optimized for expression in humans (GeneArt, Regensburg, Germany), as described elsewhere [17]. The native predicted N-terminal signal sequence (N), transmembrane domain (TM) and the C-terminal cytoplasmic domain (C) were not included in the construct. The human tissue plasminogen activator (tPA) leader sequence replaced the native AMA1 signal sequence as previously described [28]. This construct was used to make recombinant E1 and E3 deleted human adenovirus serotype 5 (AdHu5) and the attenuated orthopoxvirus modified vaccinia virus Ankara (MVA) expressing AMA1. The methods used for the generation of the vectors have been previously described [32,42]. The second generation mono-allelic AMA1 vaccines are described in Figure 4A. A final construct was designed to contain a fusion of two divergent alleles of AMA1 (3D7 and FVO) linked by a Glycine-Proline (GGGPGGG) linker. It also had the human tPA leader sequence and the TM and C terminus from the FVO strain and was human codon optimized. This construct (3483 bp in length) is described elsewhere [44] and was cloned into AdHu5 and MVA as before, as well as E1/E3 deleted chimpanzee adenovirus 63 (ChAd63, previously known as AdCh63) as described [32]. Animals and Immunizations Female BALB/c (H-2d) mice 5–6 weeks old (John Radcliffe Hospital, Oxford, UK or Harlan, UK) were used in all experiments. All procedures were performed according to the terms of the UK Animals (Scientific Procedures) Act Project License (PPL 30/2414) and Personal License (PIL 30/7792) and were approved by the University of Oxford Animal Care and Ethical Review Committee. The vaccines were diluted in endotoxin-free Dulbecco’s PBS prior to immunization. Vaccines were administered intradermally (i.d.) bilaterally into the ears in a total volume of 50 ml for both recombinant adenovirus and MVA. The doses of recombinant adenovirus vaccines used were 561010 viral particles (vp), 161010 vp or 16109 vp and the dose of recombinant MVA vaccine used was always 16107 plaque forming units (pfu) [29,44]. The interval between the prime immunization with AdHu5-AMA1 or ChAd63-AMA1 and boosting with MVA-AMA1 was 8 weeks for all the regimens. PLoS ONE | www.plosone.org 13 June 2011 | Volume 6 | Issue 6 | e20977