Domain-specificities of AS02_A and ISA720 induced anti-AMA1 were similar.

<p>End-point titer was determined against chimeric proteins displaying <i>P. falciparum</i> domains 1, 2, 3, 1+2 or 2+3 on <i>P. berghei</i> AMA1 scaffold (D1, D2, D3, D1+2, D2+3 chimeras respectively). Domain-specific titer was calculated by expressing the domain-specific end-point titer as a percentage of titer against the full-length 3D7 AMA1 protein. Mean and standard error for 6 animals per group was plotted.</p

Parasitemia profiles of FCH/4 and FVO challenged groups.

<p>Daily parasitemia of animals plotted against days-post-challenge (days). Solid lines represent a virulent parasitemia profile that required drug treatment for high parasitemia (>200,000/µL) within 15 days of challenge. Broken lines represent a slower progressing and self-limiting infection profile. The challenge strains are indicated in parentheses (FCH/4 or FVO). Two monkeys in the AMA+ISA_FCH/4 group (AI3181, AI-3176) were re-challenged on day 16.</p

3D7 AMA1 vaccination slowed growth rate of FCH/4 but not FVO strain.

<p>Group-wise mean cumulative parasitemia of the FCH/4 (left panel) and FVO (right) challenged animals plotted against days post challenge. Solid line, PBS control group; broken line, AMA+ISA; dotted line, AMA+AS02_A.</p

The FCH/4 strain AMA1 is more homologous to 3D7 AMA1 as compared to FVO strain AMA1.

<p>Top panel shows the amino acid differences between 3D7, FVO and FCH/4 AMA1. Polymorphisms within the grey cells are included in the sequence boundary of 3D7 AMA1 vaccine, amino acid 83–531. The disulphide bonded domains (D1, D2 and D3) are marked by thick lines. D1 (amino acid 95–300), D2 (308–404) and D3 (439–584). Lower panel shows the crystal structure of AMA1 (amino acids 97–531) with the location of the 3D7-FVO (left) and 3D7-FCH/4 (right) amino acid differences shown as solid balls (D1 polymorphisms - red; D2 - green and D3 polymorphisms - blue). The residues of the C1 cluster (187, 190, 196, 197, 200, 204, 206 and 225) are circled in green.</p

Immunogenicity and efficacy data of individual animals.

<p>Vaccine and challenge groups, animal ID, ELISA endpoint titer against 3D7 or FVO AMA1 coated plates, IFA titer against 3D7 and FVO schizonts, GIA activity against <i>P. falciparum</i> 3D7 or FVO target parasite and parasite burden (mean, peak and cumulative counts) between days 4 and 11 post challenge are shown. ** Two animals in the PBS+ISA_FVO group died due to unrelated causes during the vaccination phase. GIA was not done (nd). The ELISA titer values are 1000X.</p

3D7 AMA1 vaccination reduced the parasite burden of the FCH/4 strain.

<p>Mean, peak and cumulative parasitemia (parasites/µL) of individual animals and their mean (line) between days 4–11 post challenge.</p

Recombinant 3D7 AMA1 vaccine was pure and folded correctly.

<p>Left panel shows a coomassie blue stained SDS-PAGE analysis of the 3D7 AMA1 vaccine under non-reducing (NR) and reducing conditions (Red). Right panel shows positive reactivity of only the non-reduced 3D7 AMA1 protein with a conformational monoclonal antibody 4G2dc1.</p

Domain1+2 end-point titer of protected animals exceeded 100,000.

<p>FCH/4 challenged AMA+AS02_A and AMA+ISA group titers against full-length 3D7 AMA1 (3D7) or chimeras D1, D2, D3, D1+2, D2+3 that display the corresponding <i>P. falciparum</i> 3D7 strain AMA1 domains on a <i>P. berghei</i> AMA1 scaffold (Pb). End-point titers against the <i>P. berghei</i> AMA1 scaffold protein are also plotted. Animals protected in the AMA+ISA group (AI-3176, AI-3179 and AI-3181) had the highest D1+2 end-point titer (red dotted line).</p

Monoclonal antibodies against AMA1.

<p> GIA values are mean of 3 or more experiments against 3D7 strain.</p><p>^# 1F9 tested at 0.6 mg/ml in GIA.</p><p> Strain reactivity out of 7 allelic proteins by dot blot.</p><p> Binding location assigned by dot blot or western blot against linear and crystal domain chimeras.</p><p><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003840#ppat.1003840-Coley2" target="_blank">[29]</a><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003840#ppat.1003840-Kocken1" target="_blank">[30]</a>. previously described AMA1 mAbs </p

Biological activity of monoclonal antibodies.

<p>(<b>A</b>) Binding of 3D7 AMA1 (OD₄₅₀) to immobilized RON2 peptide inhibited by serial dilutions of the mAbs. Negative control mAb 5G8 binds to the N-terminal prosequence. (<b>B</b>) Western blot of a 3D7 parasite processing inhibition assay using 200 µg/ml mAbs. Top panel shows the merozoite bound full-length (83 kDa) 3D7 parasite AMA1 and the product of N-terminal processing (66 kDa). Bottom panel shows the co-migrating products of normal shedding (48+44 kDa) and the product of anomalous AMA1 processing (52 kDa). These fragments were captured from the culture supernatant using a sub-inhibitory concentration of polyclonal anti-3D7 AMA1 sera (1∶2500) in the processing assay <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003840#ppat.1003840-Dutta4" target="_blank">[34]</a>. (<b>C</b>) GIA against 3D7 target strain, using 1×IC₃₀ dose of individual mAbs (black), 2×IC₃₀ dose of individual mAbs (gray), 1×IC₃₀+1×IC₃₀ mixture of two 1e-loop mAbs (green) or 1e-loop+domain2-loop mAb (blue) or 1e-loop+domain-3 mAb (orange) or domain2 loop+domain-3 mAb (red). Mean+s.e.m. of 3 experiments; (*) p<0.05 comparing the mean of each group to the mean of 2×IC₃₀ dose of individual mAbs (gray bars). (<b>D</b>) GIA against the 3D7 parasite strain using increasing concentrations of mAb 1E10, with (red line) or without (blue line) the addition of 1×IC₃₀ concentration of mAb 4G2 (1.8 mg/ml, expected 30% GIA in green). Predicted inhibition for additive interaction (black line) was calculated according to “Bliss independence” as has been applied to determine synergy by Williams <i>et al. </i><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003840#ppat.1003840-Williams1" target="_blank">[55] </a><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003840#ppat.1003840-Bliss1" target="_blank">[56]</a>; data are mean+s.e.m. of triplicate wells. (<b>E</b>) Inhibition of 7 parasite strains using 2 mg/ml of the RON2 inhibitory mAb or a mixture of 1 mg/ml each of the RON2 inhibitory mAbs and processing inhibitory mAb 1E10; a representative of two experiments is shown.</p