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

Abstract

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 P. falciparum 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 P. yoelii and P. berghei and tested their efficacy using different delivery methods against pre-erythrocytic malaria in rodent models. In our primary screen using P. yoelii in BALB/c mice, we found that 16 antigens significantly reduced liver stage parasite burden. In our confirmatory screen using P. berghei 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 Plasmodium genes can be useful in identifying novel pre-erythrocytic antigens that induce protective immunity alone or in combination with CSP.

Fig 1. Gene expression profile of Pf genes and selection algorithm used for antigen selection.

(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.

Fig 2. GG DNA immunization and reduction of LS parasite burden post-sporozoite challenge.

(A) Experimental design for the immunization and challenge studies. Mice were immunized 3 times at 3 week intervals with VR1020 plasmid DNA carrying the Pb or Py antigen. Two weeks after the last boost mice were challenged with 10,000 Pb or 20,000 Py 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 Py in BALB/c model by GG immunizations. (C) Meta-analyses of 10 independent immunization experiments and resulting LS parasite burden reduction in Pb 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 S5 Table.

Fig 3. Novel antigens combined with CSP provide greater protection than CSP alone.

(A) Each C57Bl/6 mouse (circles) was immunized with a combination of PyCSP DNA (2.5 μg) with novel antigen DNA (2.5 μg) by GG, using the schedule described in Fig 2. (B) Each BALB/c mouse (triangles) was immunized with a combination of PbCSP 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 S6 Table.

Fig 4. Expression of novel antigens by Py LS parasites.

(A) Strategy for generation of myc-tagged PyPF3D7_1241500. (B) Immunofluorescence assay using Py17XNL grown 24h in HepG2-CD81 cells, showing expression of PyPF3D7_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.