significant proportion of this immunity is directed to antigens expressed on the blood-stage parasites or infected erythrocytes. This immunity is mostly antibody mediated [4], although more recently the importance of cellular immunity has begun to be recognized in mice [5,6] and humans [7,8]. Blood-stage malaria subunit vaccine development has thus mainly focused on antibody-inducing protein-in-adjuvant formu- lations targeting well-studied merozoite antigens, including merozoite surface proteins (MSPs) and antigens secreted from apical organelles of the parasite during erythrocyte invasion, such as apical membrane antigen 1 (AMA1). To date there have been over 30 phase I/II clinical trials with these blood-stage antigens reported [3], but the limited success of such candidates, combined with reports of vaccine allele-specific efficacy in field trials [9] and in in vitro assays of purified IgG growth inhibitory activity (GIA) [10,11], indicates that blood-stage vaccines may need to include multiple alleles of the same antigen to achieve significant efficacy against the many strains of P. falciparum in the field. PfAMA1 has been one of the leading blood-stage malaria vaccine candidate antigens for a considerable time, and there have been numerous pre-clinical and clinical AMA1 vaccine studies (reviewed in Ref [12]). Field studies have primarily addressed the importance of antibodies to PfAMA1 to clinical immunity, showing that in naturally exposed individuals the prevalence of PfAMA1-specific IgG increases with age and that this is associated with reduced risk of clinical malaria [13,14,15]. However, the PfAMA1 antigen is polymorphic, probably as a result of immune selection operating on this important target of naturally occurring immunity, and antibodies raised against individual naturally- occurring alleles of this antigen inhibit growth of P. falciparum strains in vitro in a strain-specific manner. A clinical trial of an PfAMA1 3D7 allele protein vaccine (FMP2.1) showed that sera from vaccinees, although capable of inhibiting growth of 3D7 strain P. falciparum parasites in vitro, afforded no inhibition of the heterologous FVO strain [16]. Several different approaches have thus been developed to address the issue of polymorphism [3]. The AMA1-C1 recombi- nant protein vaccine in clinical development is composed of a mixture of the 3D7 and FVO alleles [17] – two of the most divergent in terms of polymorphism and which differ by 24 amino acids out of 622 amino acids. It has been shown that vaccination with multivalent or chimeric PfAMA1 vaccines increases the levels of antibodies to common allele epitopes and/or broadens inhibition of malaria strains [18,19,20]. Immunization with diversity-covering (DiCo) PfAMA1 sequences, that on average incorporate 97% of the amino acid variability observed in PfAMA1, has also been shown to induce broader functional immunity than immunization with mixtures of naturally-occurring alleles [21]. Conversely, studies of T cell-mediated immunity against PfAMA1 and/or blood-stage malaria infection in humans have been more limited. In one epidemiological study, seven T cell epitopes in PfAMA1 were reported, with some localized to conserved areas of PfAMA1 – an important observation in terms of vaccination strategy against this polymorphic antigen [22]. Individuals showing a proliferative response to one PfAMA1 peptide corresponding to aa 259–271 were found to be less likely to be parasitemic on follow up [23]. Another study has shown that repeated ultra-low dose exposure of malaria-na¨ıve human volunteers to blood-stage parasites and drug cure can induce sterile protection against a blood-stage challenge. In this study there was no detectable antibody response against the parasite. A cell-mediated immune response was measured – mainly charac- terized by a proliferative CD4+ and CD8+ T cell response and Viral Vectored Blood Stage Malaria Vaccine Th1-type cytokine induction [7]. In another study, volunteers were protected against malaria challenge with infected mosqui- toes after sporozoite inoculation and chloroquine drug cure, and parasite-specific pluripotent effector memory T cells before challenge were found to be associated with protection [8]. In rodent malaria models, there is strong evidence for the role of CD4+ T cells, interferon-gamma (IFN-c) and nitric oxide contributing to the control of blood-stage parasitaemia [6,24], including those cells against the AMA1 antigen [5]. Recently a study in the P. chabaudi AS model showed that the failure to maintain long-term protective responses was due to a gradual decline in the parasite-specific memory CD4+ T cell response, despite persistent B cell memory and circulating antibodies [25]. This study provides an important insight into T and B cell memory to malaria and encourages vaccination strategies that induce memory T cells to ensure long-term efficacy. With increasing evidence of the role for T cells, as well as antibodies, in blood-stage malaria immunity, vaccine development strategies should focus on vaccine platforms capable of generating both humoral and cellular immunity [26]. This strategy could induce a broader repertoire of immune responses to target such polymor- phic malarial proteins. Recently, replication-deficient recombinant viral vectored vaccination regimens have been described that are capable of inducing potent T cell and antibody responses against encoded transgenes [27]. When targeting the blood-stage malaria antigen MSP1, high level antibody-mediated protection could be achieved in the P. yoelii mouse model of blood-stage malaria infection by using a priming immunization with a recombinant human adenovirus serotype 5 (AdHu5) vector followed by a booster immunization with the poxvirus vector modified vaccinia virus Ankara (MVA) [28]. The same regime induced effector CD8+ T cells that could reduce P. yoelii parasite burden during the preceding liver-stage infection [29]. AdHu5 and poxvirus vaccines encoding P. falciparum MSP1 and AMA1 have also been reported [30,31,32]. The advantages of using recombinant adenovirus vectors as vaccine carriers are numer- ous and certain serotypes, such as AdHu5, are highly immunogenic [27]. However, the host generates an immune response not only to the transgene but to the vector as well [33,34]. AdHu5 vectors have been developed for vaccine delivery for several diseases and tested in rodents, primates and recently in humans as a vectored vaccine against HIV-1 [35] and malaria . Anti-AdHu5 immunity has been shown in pre-clinical and clinical studies to hamper the immunogenicity of recombinant AdHu5 vaccines [36,37,38]. Due to the need to overcome this problem, simian adenovirus vaccine vectors, such as chimpanzee ChAd63 (previously known as AdCh63), have been developed for which there is less pre-existing immunity in human populations [39,40]. We have recently reported that this vector exhibits comparable immunogenicity to AdHu5 when recombinant for the P. falciparum liver-stage antigen TRAP and blood-stage antigen MSP1 [32,41,42]. Here we report the iterative development of human and simian adeno- and pox- viral vectored vaccines targeting two alleles of PfAMA1. These vectors were tested in mice and rabbits to assess the cellular and humoral immunogenicity of these vaccines in a prime-boost vaccination regime. PfAMA1-specific antibody induction by these viral vectors was optimized by using mammalian secretory signal sequences and inclusion of the native transmembrane domain for cell surface antigen localization. These data support the rational design of vectored vaccine transgene inserts for optimization of immune responses against multiple alleles of the same antigen. PLoS ONE | www.plosone.org 2 June 2011 | Volume 6 | Issue 6 | e20977