groups A and B, P#0.01). Interestingly, there were cross-reactive and comparable CD8+ T cell responses detected to the FVO pool when mice were immunized with the AdHu5-MVA AMA1 (3D7) mono-allelic regime (comparing groups B and C), indicating the amino acid substitutions between the two allelic forms were either not present in the epitope(s) recognized, or did not affect class I MHC presentation or TCR recognition. The dominant CD8+ T cell response in BALB/c mice was to an epitope(s) in the 3D7 pool. There was no response detected to the C-terminal region. CD4+ T cell responses were detected to all four peptide pools, although the overall total magnitude of the response was lower than the total CD8+ T cell response. Consistent with the CD8+ T cell data, the ChAd63-MVA bi-allelic PfAMA1 regime induced an overall weaker CD4+ T cell response in comparison to the equivalent AdHu5-MVA regime (comparing groups A and B, P#0.05 for 3D7 and P#0.01 for FVO and C-term). The bi-allelic AdHu5 vaccine construct tended to induce stronger responses against the common, 3D7 and FVO pools in comparison to the equivalent AdHu5 mono-allelic regime (comparing groups B and C) but this did not reach statistical significance. Also as expected, there was no response detected to the C-terminus when mice were immunised with the mono-allelic 3D7 construct (which does not contain the C-terminus), however the AdHu5 bi-allelic construct which does contain this sequence induced detectable CD4+ T cell responses to this peptide pool. Cross-reactive CD4+ T cell responses were again detected to the FVO pool of peptides when mice were immunized with the mono- allelic 3D7 vaccine, possibly for similar reasons as for the CD8+ T cell responses. The induction of CD4+ T cells against common epitopes could be relevant to the issue of AMA1 polymorphism if these epitopes are important for protection, as indicated by studies of AMA1 in the P. chabaudi rodent malaria model [5,45]. The multi-functionality of the CD4+ T cell response to the common pool was also assessed, given CD4+ T cells producing IFN-c, TNFa and IL-2 simultaneously have been shown to correlate with protection against Leishmania major in mice [46]. All three vaccination regimes induced multi-functional CD4+ T cells, and there were no significant differences in the quantity or quality of multi-functional cells among the different vaccination regimes tested (Figure 9). Discussion This paper describes the development, immunogenicity and in vitro efficacy of a viral vectored vaccination strategy targeting one of the leading blood-stage malaria vaccine candidate antigens PfAMA1. This adenovirus-MVA prime-boost regime induces antibody responses in mice and rabbits, and these antibodies inhibit growth of P. falciparum parasite strains in vitro according to the alleles of PfAMA1 encoded by the recombinant viruses. The same vaccination strategy encoding another blood-stage vaccine candidate antigen (P. falciparum MSP1) has been shown to be similarly effective [32]. In addition to antibodies, this regimen also induces substantial antigen-specific CD8+ and CD4+ T cells in mice. The viral vectored vaccine platform is now offering a safe and reliably immunogenic alternative to recombinant protein-in- adjuvant vaccines not only for malaria vaccine development [47], but also for a wider range of difficult infectious diseases [27]. The first generation AdHu5-MVA mono-allelic AMA1 (3D7) vaccines induced cross-reactive antibodies to the heterologous FVO allele of AMA1 both in mice and rabbits as measured by ELISA, however there was a better correlation in rabbits than in mice between the 3D7 and FVO antibodies at the peak day 70 time-point. This has been seen before with a P. falciparum MSP1 Viral Vectored Blood Stage Malaria Vaccine based vaccine [32], suggesting there are differences in antibody cross-reactivity and B cell epitopes recognized in different mammalian species. These observations may be reflected in differing concentrations of inter-species IgG required to elicit equivalent functional activity in vitro – an observation now reported for both blood-stage antigens MSP1 and AMA1 [19], and also the transmission-blocking vaccine candidate antigen Pfs25 [48]. In another study, IgG isolated from rabbits immunized with a reduced AMA1 ectodomain were reported to not have any growth inhibitory activity in vitro [49]. Importantly, the first generation vaccines described here induced antibodies capable of recognizing antigen in its native conformation in schizont-infected erythro- cytes, an observation also reflected in the measurement of functional GIA. In this study in mice there was 94% GIA against the homologous 3D7 strain but only 54% against the heterologous strain. Similarly in rabbits there was a median GIA of 44% against 3D7 but there was no cross-reactive GIA against the FVO or FCR3 strains of P. falciparum in vitro afforded by purified IgG after immunization with the mono-allelic PfAMA1 (3D7) vaccine. Purified IgG from immunized mice thus shows better cross-strain in vitro inhibition than IgG from rabbits, though conversely in rabbits the correlation between 3D7 and FVO antibodies as measured by ELISA is stronger. These data are not in agreement with a report where the pattern of strain specificity was found to be similar in mice and rabbits [19], but do agree with other AMA1 vaccine studies, whereby the cross-reactivity of antibodies seen by ELISA was not reflected in functional GIA assays [17,49,50,51]. This study also confirms observations made by others that antibodies measured by ELISA against heterologous alleles of AMA1 do not have equal effect on parasite growth in vitro [49]. However, reports describe a clear sigmoidal relationship between in vitro GIA and ELISA titer for homologous alleles of both AMA1 and MSP1 [11]. The assay of GIA remains the gold-standard for pre-clinical screening of blood-stage malaria candidate vaccines. However, a recent Phase IIa clinical trial with a recombinant AMA1 protein vaccine failed to induce significant clinical efficacy or any delay in time to clinical diagnosis even though IgG purified from the vaccinated serum showed very high levels of GIA in vitro [52]. More recent epidemiological studies in the field have shown a weak contribution of GIA towards reduced malaria risk [53], and other studies have shown conflicting results [54,55]. An alternative in vitro assay, measuring antibody-dependent respiratory burst (ADRB) activity from neutrophils has recently been associated with clinical protection in a study from Senegal [56], and may provide a complementary assay for screening vaccine-induced IgG functional activity. In the second generation 3D7 PfAMA1 vaccines, the substitu- tion of the native parasite AMA1 leader sequence with the mammalian tPA leader was shown to lead to significantly improved antibody responses in mice immunized with the recombinant AdHu5 vector. The tPA signal sequence is a specific targeting signal that targets the expressed antigen to the endoplasmic reticulum (ER) [57] and influenza virus nucleopro- tein when fused to tPA was shown to be expressed at higher levels and was found to be in a partially secreted form in vitro [58]. When fused to the tPA signal sequence, viral envelope protein E of Japanese encephalitis virus was expressed at higher levels in transfected cells than the wild-type construct and was mainly localized in the cytosolic and membrane fraction. These tPA fusion constructs elicited stronger humoral and cellular response and better protection against viral challenge in animals [59]. The increased antibody response seen here with this construct can thus be due to better secretion or enhanced PfAMA1 expression due to PLoS ONE | www.plosone.org 10 June 2011 | Volume 6 | Issue 6 | e20977