Viral Vectored Blood Stage Malaria Vaccine Figure 3. Growth inhibitory activity of sera from immunized mice and rabbits. 5–6 week old female BALB/c mice (n = 7) were immunized as in Figure 1. Sera were obtained two weeks post boost (day 70) by terminal bleed and the IgG was purified from the pooled sera. The GIA activity of the serum was assessed by incubating the IgG at a final concentration of 5 mg/ml with P. falciparum cultures. The ELISA titers and the % growth inhibition of the pooled serum are shown against both the homologous 3D7 and heterologous FVO strains of PfAMA1 (A). New Zealand white rabbits (n = 8) were immunized as in Figure 2. Two weeks after the final vaccination the sera were obtained. The effect of the purified IgG on parasite growth from each individual rabbit was evaluated by incubating the IgG at a concentration of 6 mg/ml with mature P. falciparum schizonts. The % GIA values for individual rabbits and the median are shown against both the 3D7 and FCR3 strains at 6 mg/ml IgG concentration (B). There was no correlation between the 3D7 and FCR3 GIA (C). There was a significant positive correlation between the 3D7 ELISA titers and 3D7 GIA (R2 = 0.68, *P #0.05) and with FCR3 GIA (R2 = 0.51, *P #0.05) using Pearson’s rank correlation (D). doi:10.1371/journal.pone.0020977.g003 vaccination. However, the immunogenicity of the mono-allelic and bi-allelic AMA1 vaccine constructs was indistinguishable based on the absolute IgG titers. Comparison of antibody responses and serum GIA generated in rabbits after vaccination with mono-allelic or bi-allelic PfAMA1 vaccines New Zealand white rabbits (n = 4) were primed with 1) ChAd63-bi-allelic, 2) AdHu5-bi-allelic or 3) the mono-allelic AdHu5-PfAMA1 (3D7) vaccines. Groups 1) and 2) were boosted with MVA-bi-allelic and group 3) was boosted with the mono- allelic MVA-PfAMA1 (3D7) vaccine. Antibody levels were measured two weeks after the final vaccination against 3D7 and FVO PfAMA1 (Figure 7A,B). As seen in mice, there was no significant difference in the antibody response generated after the three vaccination regimes either against 3D7 or FVO PfAMA1. Cross-reactive antibodies were measured against the FVO recombinant protein after immunization with the AdHu5- MVA PfAMA1 (3D7) vaccination regime as seen previously (Figure 2B). However, when antibody functionality was assessed using the in vitro assay of GIA, the mono-allelic vaccine once again induced strain-specific activity: 87.5% GIA against the 3D7 parasite but only 18.5% GIA against FVO (Figure 7C,D), in agreement with the previous study (Figure 3B). However the ChAd63-MVA bi-allelic and AdHu5_MVA bi-allelic vaccina- tion regimes induced antibodies that showed comparable GIA against both the 3D7 (58.5% and 79.5% respectively) and FVO (54% and 57% respectively) parasites. GIA against 3D7 and FVO parasite strains following bi-allelic immunization also correlated significantly (Figure 7E). These data confirm that even though mono- and bi-allelic PfAMA1 vaccine constructs induce cross-reactive IgG that are indistinguishable by ELISA assay, the IgG induced by the bi-allelic vaccines was capable of reducing the growth of both parasite strains encoded by the vaccine in vitro. T cell immunogenicity and multi-functionality of the mono-allelic and bi-allelic PfAMA1 vaccines in mice T cell responses were also analyzed in BALB/c mice by ICS following re-stimulation of splenocytes with one of four pools of overlapping peptides (15 mers, overlapping by 10 aa) corre- sponding to the 3D7 and FVO coding sequence in the bi-allelic vaccine [44]. Pool 1 contained 64 peptides common to both alleles of PfAMA1. Pools 2 and 3 (47 peptides in each) contained allele-specific peptides unique to 3D7 and FVO respectively (each peptide pair differ by 1 or 2 aa). Pool 4 had only 12 PLoS ONE | www.plosone.org 6 June 2011 | Volume 6 | Issue 6 | e20977