Sample inclusion.

<p>Malaria treatments with amodiaquine+sulfadoxine-pyrimethamine (AQ+SP) and available samples for inclusion in the study. Further details on children not randomized have been previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052571#pone.0052571-Dorsey1" target="_blank">[17]</a>.</p

Relationship between anti-apical membrane antigen 1 (AMA-1) antibody levels and risk of treatment failure.

<p>A consistent relationship was seen across the range of values observed. Anti-AMA-1 levels (log-transformed) were divided into five equal intervals, each representing a 10-fold increase in level.</p

Antibody responses to <i>Plasmodium falciparum</i> antigens.

a<p>Positive cutoff = mean of 20 <i>P. falciparum</i> unexposed controls +3 standard deviations.</p>b<p>AU = arbitrary units, where an AU of 100 is equivalent to the antibody level of pooled serum from 50 African adults.</p><p>Note: Generalized estimating equations (GEE) were used to estimate antibody prevalence, geometric mean antibody level, and fold change.</p><p>Circumsporozoite protein (CSP); liver stage-antigen (LSA); apical membrane antigen 1 (AMA-1); merozoite surface protein 1, 2, or 3 (MSP-1, MSP-2, MSP-3); amino- or carboxy-terminal region of glutamine rich protein (GLURP-R0, GLURP-R2).</p

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

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

Sera or Cell Transfer Studies.

<p>(<b>A</b>) Pooled sera isolated from mice immunized with <i>Pf</i>CSP-SAPN or <i>Pf</i>CSP-KMY-SAPN but not sera from <i>Pv</i>CSP-SAPN or <i>Pv</i>CSP-KMY-SAPN immunized mice transferred to naïve mice conferred protection from challenge with Tg-<i>Pb/PfCSP</i> sporozoites. (<b>B</b>) In a separate experiment sera or washed splenocytes from mice were transferred. Whereas sera from <i>Pf</i>CSP-SAPN or <i>Pf</i>CSP-KMY-SAPN transferred protection sera from <i>Pv</i>CSP-SAPN or <i>Pv</i>CSP-KMY-SAPN immunized mice did not. On the contrary, total splenocytes from <i>Pf</i>CSP-KMY-SAPN or <i>Pv</i>CSP-KMY-SAPN transferred protection while splenocytes from <i>Pf</i>CSP-SAPN or <i>Pv</i>CSP-SAPN did not. (<b>C</b>) Two wks post final immunization with <i>Pf</i>CSP-KMY-SAPN 1.33×10⁶ enriched CD8⁺ T-cells were adoptively transferred to naïve animals which were then challenged 72 hrs post transfer. *P<0.05. Mice were challenged with 5,000 Tg-<i>Pb/PfCSP</i> sporozoites. n = 10.</p

Antibody responses and protective efficacy induced by SAPN vaccinations in Mice.

<p>C57BL/6 and Balb/C mice make high titer antibodies when immunized with the <i>Pf</i>CSP SAPN. (A) Mice were given vaccine intraperitoneally (i.p.) or intramuscularly (i.m.) at wk 0, 2 and 4. Titers were determined 2 wks after each dose. n = 10 per group per experiment; data are representative of one of three independent experiments with similar results. (B) Two wks post 3^rd dose of <i>Pf</i>CSP-SAPN, C57BL/6 mice were challenged with 5,000 Tg-<i>Pb/Pf</i>CSP, Balb/C with 10,000, Tg-<i>Pb/Pf</i>CSP sporozoites. Shown are three separate experiments with C57BL/6 mice and two experiments with Balb/C mice. n = 10. Infectivity sham control mice were given PBS. Mice were considered protected if they had no detectable parasitemia by day 15 following challenge. (C) Titer of anti-<i>Pf</i>CSP repeat antibody of the serum from individual mice in each SAPN vaccinated group two days before challenge. The line represents the mean titers of the individual mice in that group. n = 6 or 7 depending if serum was available. Those SAPN immunized mice that were protected against malaria are represented by (•); those that developed parasitemia and died by (▴). (D) Protection following challenge. At the predetermined time point, from wk 6 to wk 52 of the study, C57BL/6 mice in a select immunized group (n = 6 or 7) and a time matched PBS-sham vaccinated group were challenged with 5,000 sporozoites. Mice receiving <i>Pf</i>CSP-SAPN immunization (black bars) and matched sham PBS vaccinated mice (white bars). ***P<0.0001.</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