Ghanian sample summary.

<p>The frequency of inferred number of strains per sample (left) and and the panmixia coefficient by number of strains (right). MAP estimates used.</p

Parameters and definitions for the model and its description.

<p>Parameters and definitions for the model and its description.</p

Table of simulated parameter values. <i>C</i> is the number of read counts while <i>M</i>, <i>K</i> and <i>α</i> are as in Table 1.

<p>Table of simulated parameter values. <i>C</i> is the number of read counts while <i>M</i>, <i>K</i> and <i>α</i> are as in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004824#pcbi.1004824.t001" target="_blank">Table 1</a>.</p

Performance for parameter inference.

<p>Upper row: mean squared deviation for strain frequencies by number of read counts (left) and by number of SNPs (right). Lower row: absolute normalized deviation for panmixia coefficient by number of read counts (left) and by number of SNPs.</p

Examples of real and model-simulated data.

<p>For three samples (rows), we present the observed data WSAF plotted against the PLAF (first column), a diagram of the inferred model indicating the bands, proportions, and panmixia coefficient (second column), and data simulated under the inferred model. Panmixia coefficient and strain proportions are the MAP values. In the second column, the model’s PLAF-varying mixture densities are shown in grey scale, with black equal to one.</p

Component inference.

<p><i>Maximum a posteriori</i> (MAP) inferred number of components by number of read counts across 10 simulations, with dotted line at the true number of components.</p

Number of strains by F-statistic.

<p>Boxplot of the inbreeding coefficient (1 − <i>F</i>_<i>is</i>) for each sample grouped by the MAP number of inferred strains.</p

Identification of Novel Pre-Erythrocytic Malaria Antigen Candidates for Combination Vaccines with Circumsporozoite Protein

<div><p>Malaria vaccine development has been hampered by the limited availability of antigens identified through conventional discovery approaches, and improvements are needed to enhance the efficacy of the leading vaccine candidate RTS,S that targets the circumsporozoite protein (CSP) of the infective sporozoite. Here we report a transcriptome-based approach to identify novel pre-erythrocytic vaccine antigens that could potentially be used in combination with CSP. We hypothesized that stage-specific upregulated genes would enrich for protective vaccine targets, and used tiling microarray to identify <i>P</i>. <i>falciparum</i> genes transcribed at higher levels during liver stage versus sporozoite or blood stages of development. We prepared DNA vaccines for 21 genes using the predicted orthologues in <i>P</i>. <i>yoelii</i> and <i>P</i>. <i>berghei</i> and tested their efficacy using different delivery methods against pre-erythrocytic malaria in rodent models. In our primary screen using <i>P</i>. <i>yoelii</i> in BALB/c mice, we found that 16 antigens significantly reduced liver stage parasite burden. In our confirmatory screen using <i>P</i>. <i>berghei</i> in C57Bl/6 mice, we confirmed 6 antigens that were protective in both models. Two antigens, when combined with CSP, provided significantly greater protection than CSP alone in both models. Based on the observations reported here, transcriptional patterns of <i>Plasmodium</i> genes can be useful in identifying novel pre-erythrocytic antigens that induce protective immunity alone or in combination with CSP.</p></div

GG DNA immunization and reduction of LS parasite burden post-sporozoite challenge.

<p>(A) Experimental design for the immunization and challenge studies. Mice were immunized 3 times at 3 week intervals with VR1020 plasmid DNA carrying the <i>Pb</i> or <i>Py</i> antigen. Two weeks after the last boost mice were challenged with 10,000 <i>Pb</i> or 20,000 <i>Py</i> sporozoites intravenously and livers were harvested 40h post-challenge. *DNA dose is 5 μg (GG), 25 μg + 35 μg GM-CSF DNA (IM) or 20 μg (EP). (B) Meta-analyses of 7 independent immunization experiments and resulting LS parasite burden reduction in <i>Py</i> in BALB/c model by GG immunizations. (C) Meta-analyses of 10 independent immunization experiments and resulting LS parasite burden reduction in <i>Pb</i> in C57Bl/6 model induced by GG immunizations. Each circle represents one mouse. Green color indicates significant difference as compared to EV immunized groups tested in the same immunization studies (p<0.05). Red color indicates p>0.05 and therefore no significant difference in LS parasite burden reduction as compared to EV immunized group. Purple color indicates LS parasite burden reduction by CSP (positive control). A complete statistical analysis is provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159449#pone.0159449.s010" target="_blank">S5 Table</a>.</p

Expression of novel antigens by <i>Py</i> LS parasites.

<p>(A) Strategy for generation of myc-tagged <i>Py</i>PF3D7_1241500. (B) Immunofluorescence assay using <i>Py</i>17XNL grown 24h in HepG2-CD81 cells, showing expression of <i>Py</i>PF3D7_1241500 protein detected by Alexa-594 conjugated anti-myc antibody (red). UIS4 (green) was used as a PVM marker and DAPI to identify nuclei. Scale bar represents 10 μm.</p