Levels of anti-AMA1 antibody elicited with the four multi-allele vaccine formulations in rabbits.

<p>Gp 1 rabbits were immunised with seven AMA1 antigens (DiCo mix and AMA1 from FVO, HB3, 3D7 and CAMP parasite strains) in CoVaccine HT™, and the other groups were immunised with DiCo mix in CoVaccine HT™ (Gp 2), Montanide IMS (Gp 3) and Montanide ISA 51 (Gp 4) respectively. Antibody titres were determined by a standardized ELISA with DiCo 1, DiCo 2, DiCo 3, FVO, HB3, 3D7 and CAMP AMA1-coated plates. Data is presented on a Log2 scale as dotplots with a boxplot superpose indicating the lower and upper quartiles as well as the median per immunisation group. Within the same immunisation group, plotting symbols represent the antibody titre of individual rabbits on all coating/capture antigens.</p

Mean % antibody depletion from FVO and 3D7 AMA1-coated plates.

<p>Antibody depletion after competition ELISA, values reported as mean % depletion (95%CI).</p><p>7Ag – vaccine containingDiCo mix + four AMA1 alleles from FVO, HB3, 3D7 and CAMP parasite strains.</p><p>3Ag – vaccine containing DiCo mix.</p><p>NM &DM – competitor antigen mixtures comprising natural AMA1 alleles (FVO, HB3, 3D7, CAMP) and DiCo mix, respectively.</p

Alignment of the protein sequences (aa25-545) of <i>Pf</i>AMA1 antigens used in this study.

<p>DiCo proteins were used to immunise rabbits and as capture antigens in ELISA. Natural allele AMA1 proteins were used to immunize rabbits, and as capture and competitor antigens in (competition) ELISAs. All proteins were produced in <i>Pichia pastoris</i> and are devoid of N-glycosylation sites. These have been replaced with amino acid residues (indicated in red) that occur in AMA1 sequences from other malarial species (N162Q, T288V, S373D, N422D, S423K, N499Q). Residue 162 is unique as it is also a polymorphic residue. Additionally, all sequences contain a point mutation at position 376 (K to R, indicated in orange). This was necessary to prevent protein cleavage by <i>P. pastoris</i> proteases.</p

Growth inhibition of <i>Plasmodium</i> parasites by antibodies elicited with the three DiCo mix vaccines.

<p>Antibodies from all immunisation groups were tested on each of six culture-adapted strains (7G8, CAMP, FCR3, HB3, L32 and NF54) of <i>P. falciparum</i>. Plots represent the mean % inhibition ± SEM for all antibody samples within an immunisation group. Blue filled circles (•) represent Gp 2 (DiCo mix in CoVaccine HT™, n = 8), green open diamonds (◊) represent Gp 3 (DiCo mix in Montanide IMS, n = 8) and red filled squares (▪) represent Gp 4 (DiCo mix in Montanide ISA 51, n = 5).</p

Relationship between ELISA antibody titre and <i>in vitro</i> parasite growth inhibition.

<p>Association of antibody levels with <i>in vitro</i> antibody functionality for three of the four immunisation groups (Gps 2, 3 and 4) is shown for parasite strains whose AMA1 allelic antigens were available for antibody measurement. In order to obtain an optimal estimate of the association, growth inhibition data at all four antibody concentrations tested (6.0, 3.0, 1.5 and 0.75 mg/ml) for each sample were included. Plots are based on a four-parameter logistic function, and each symbol/colour represents individual rabbits in the same immunisation group.</p

Mean % antibody depletion from DiCo 1, 2 and 3-coated plates.

<p>Antibody depletion after competition ELISA, values reported as mean % depletion (95%CI).</p><p>7Ag – vaccine containing DiCo mix + four AMA1 alleles from FVO, HB3, 3D7 and CAMP parasite strains.</p><p>3Ag – vaccine containing DiCo mix.</p><p>NM &DM – competitor antigen mixtures comprising natural AMA1 alleles (FVO, HB3, 3D7, CAMP) and DiCo mix, respectively.</p

Refinement statistics.

<p>Refinement statistics.</p

Germline sequences and somatic mutations of the R31C2 variable domains.

<p>(A) Amino acid and nucleotide sequences of the R31C2 V_L domain compared with the <i>Vκ</i> and <i>Jκ</i> germline genes. (B) Amino acid and nucleotide sequences of the R31C2 V_H domain compared with the <i>VH</i>, <i>D</i> and <i>JH</i> germline genes.</p

Comparison of the D2 loop of PkAMA1 and PfAMA1.

<p>(A) PkAMA1. (B) PfAMA1 (PDB entry 2Z8V). The two structures are viewed from equivalent orientations and are shown in surface representation with Domain 1 in green, Domain 2 in light brown, and the D2 loop in red. The D2 loop conformation of both orthologues is shown in ribbon representation and the N- and C-terminal residues are labelled.</p

Crystallographic data.

<p>* Pearson correlation coefficient of two half data sets [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123567#pone.0123567.ref058" target="_blank">58</a>].</p><p>Crystallographic data.</p