Fig 1: Schematic representation of the experimental approach. P. vivax–infected red blood cells are cultured until the majority have matured into schizonts, after which schizont stages are enriched and used for invasion assays; if enough schizonts are available, they are stored for subsequent mRNA sequencing. Three different invasion assay setups are used: invasion into SAO reticulocyte-enriched RBCs (reRBCs), band 3 blocking with a polyclonal antibody (pAb), and band 3 blocking with a PvTRAg38 peptide. The number of newly invaded ring- and young trophozoite-stage parasites is counted by light microscopy, and compared to the paired control (non-SAO reRBCs without antibody/peptide), resulting in a normalized percentage of invasion inhibition. Figure created with BioRender.com.
Fig 2: Visualisation of the differential expression analysis groups and outcome. (A) Plot showing the invasion inhibition level in SAO reRBCs for all nine isolates for which mRNA-seq data were obtained. From strong to weak inhibition, the isolates were split into three groups of three isolates. For four isolates (Pv004, Pv023, Pv024, Pv025) the SAO invasion inhibition level was repeated twice using SAO10 and SAO20 reRBCs. In those cases, the average invasion inhibition level was used and shown in the plot. (B) Heatmap showing, for each isolate, normalized expression values of the 20 genes in the candidate band 3 ligand list with the highest fold change. Isolates are ordered from weak (left) to strong (right) invasion inhibition levels in SAO reRBCs, and genes are ordered from high (top) to lower fold change (bottom). Darker fields indicate higher expression values.
Fig 3: P. vivax reticulocyte invasion efficiency is dependent on band 3 receptor availability. Dot plot showing P. vivax reticulocyte invasion inhibition in invasion assays using (A) two SAO reRBC samples (SAO10 and SAO20), (B) a polyclonal antibody (pAb) against band 3 and (C) a PvTRAg38 peptide (amino acid region 187-208). Percent inhibition for each P. vivax isolate is pairwise-normalized to the invasion observed in the control reRBCs (non-SAO reRBCs, in absence of antibodies or peptides). Dots represent isolates. Black diamonds represent the mean percentage of invasion inhibition, with whiskers showing the standard deviation. Isolates used twice in the presence of different SAO reRBCs or at a different TRAg38 peptide concentration are shown in the same color. Mouse IgG 50 µg/mL and 100 µg/mL = band 3 pAb positive control, BSA = PvTRAg38 peptide positive control. Wilcoxon signed rank test was performed to test for significant differences between the invasion assays and their positive controls. **=p<0.01, *=p<0.05, NS, not significant.
Fig 4: Receptor expression on SAO and non-SAO reticulocytes as measured by flow cytometry. Surface abundance of band 3, DARC, GYPC, and GYPA receptors on SAO and non-SAO reticulocytes (only CD71+ RBCs were measured) is expressed as median fluorescent intensity (MFI). The SAO reticulocytes used were SAO10 and SAO20, which were also used in the invasion assays (only SAO20 for GYPA). The dots indicate reRBC samples from hemochromatosis blood collected in Belgium, and triangles reRBC samples from cord blood collected in Papua New Guinea. reRBCs were incubated with commercial fluorescently tagged antibodies, using primary (band 3, GYPC, GYPA) or secondary (DARC) binding. The anti-band 3 antibody used was reported to specifically target wildtype band 3 (Groves et al., 1993). The non-SAO reRBC sample from Papua New Guinea (triangle) has a homozygous GYPC exon 3 deletion, resulting in a low MFI for GYPC compared with the other reRBC samples. No statistical tests could be performed due to the small number of SAO reRBCs.
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