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

Novel antigens combined with CSP provide greater protection than CSP alone.

<p>(A) Each C57Bl/6 mouse (circles) was immunized with a combination of <i>Py</i>CSP DNA (2.5 μg) with novel antigen DNA (2.5 μg) by GG, using the schedule described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159449#pone.0159449.g002" target="_blank">Fig 2</a>. (B) Each BALB/c mouse (triangles) was immunized with a combination of <i>Pb</i>CSP DNA (10 μg) with novel antigen DNA (10 μg) by EP, using the same schedule used for the GG immunizations. Data were compared to the negative control group immunized with a combination of CSP and EV tested in the same immunization study. Significant reduction in LS parasite burden was determined by Kruskal-Wallis test followed by Mann-Whitney test and p<0.05 was considered as significant. Green box indicates p<0.05 and red box indicates p>0.05. A complete statistical analysis is provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159449#pone.0159449.s011" target="_blank">S6 Table</a>.</p

Gene expression profile of <i>Pf</i> genes and selection algorithm used for antigen selection.

<p>(A) Heat map of gene expression by tiling microarray. Red color represents the genes expressing above the 50^th percentile, while green color represents genes expressing below the 50^th. The heat map shows the expression profile of genes across SS, 24h and 48h axenically cultured LS, and BS parasites. (B) Venn diagram shows stage-specific expression of genes by tiling microarray. Color coding of Venn regions matches the color bar provided for the heat map in Panel A. (C) Selection of genes for vaccine evaluation in two rodent models. One hundred and thirty-one genes were selected from among the upregulated genes identified by tiling-microarray, and 124 were confirmed by qPCR to be transcribed at higher levels in LS versus SS and/or BS parasite samples. Twenty-one of these 124 genes were selected for further evaluation as vaccine candidates.</p

Protein features and transcriptional profiles of selected candidate genes.

<p>Protein features and transcriptional profiles of selected candidate genes.</p

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

Pre-erythrocytic antigens selected for vaccine evaluation.

<p>Pre-erythrocytic antigens selected for vaccine evaluation.</p