Modification of <i>var</i> transcription by mosquito transmission.

<p>(A) The rate difference of the median expression for each individual <i>var</i> variant and the housekeeping controls shows the overexpression of the entire <i>var</i> gene family <i>in vivo</i> with exception of the <i>var2csa</i> gene PFL0030c in comparison to the parental Master Cell Bank (MCB) parasite line. Each point reflects the median for the volunteer samples at the day of patent infection (n = 18) divided by the median observed for the parasite generations 6, 8 and 21 from two vials of the MCB cell line (n = 6). Housekeeping genes used as controls, <i>var</i> gene names and groups are indicated. (B) Differences in gene expression on group level between the pre-mosquito MCB parasite lines and parasites recovered from the infected volunteers (VOL) at the day of patent infection determined by thick blood smear are displayed as group median with interquartile range (IQR). Significant differences in distributions between MCB and VOL series were tested via a Wilcoxon rank-sum test using a Bonferroni corrected significance level. The graph contains a scale brake at the y-axis to account for the huge variability in the gene expressions. Group affiliations are indicated above the graph. Red (A), orange (A, subfamily <i>var3</i>), dark red (A, subfamily <i>var1</i>), purple (B/A), blue (B), turquoise (B/C), green (C) and yellow (E).</p

<i>Var</i> transcription profiles of parasites from infected volunteers 1–2 days before parasites were microscopically detectable.

<p>(A) Heat map showing the individual <i>var</i> gene expression profiles for samples obtained from volunteers one or two days before parasites were present in the thick blood smear. To correct for individual differences in the overall <i>var</i> expression levels, the expression for each <i>var</i> gene was normalized against the total <i>var</i> expression in each sample. Expression is ranked by mean expression obtained from “day of patent infection” samples (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005538#ppat.1005538.g001" target="_blank">Fig 1A</a>). The color scale indicates the relative expression levels with red representing values above the median, blue representing values below the median, and white representing median. Grey means not detected. (B) Relative gene expression is shown in a dot plot for the volunteer samples one time point before thick blood smear positivity (n = 8). Gene IDs of <i>var</i> genes and controls are indicated on the x-axis, <i>var</i> gene groups are indicated above the graph. (C) The distribution of <i>var</i> transcripts according to <i>var</i> group affiliation in the volunteer samples 1–2 days before parasites were detected in the thick blood smear is displayed by summarization of the total <i>var</i> gene expression and calculation of the proportion for each <i>var</i> group. The genomic proportion of each <i>var</i> gene group is indicated after the color code. (D) The pairwise Spearman’s rank correlation heat map demonstrates a positive correlation between the expression profiles on the day of first microscopically detectable parasitemia and 1–2 days before in the same volunteer and between volunteer samples.</p

<i>Var</i> transcription profiles of parental parasite lines used for volunteer infection.

<p>(A) The gene expression of each <i>var</i> gene and control genes relative to <i>sbp1</i> expression is shown in dot plots for the pre-mosquito Master Cell Bank (MCB) parasite line. Each point represents values observed per test generation for the MCB samples taken from two independently thawed parasite stocks after 6, 8 and 21 parasite generations, respectively (n = 6). The median expression per <i>var</i> gene is shown, housekeeping genes used as controls, <i>var</i> gene names and groups are indicated. (B) The MCB cell line exclusively expresses the group E <i>var2csa</i> gene as shown by the proportion of <i>var</i> transcripts according to <i>var</i> group affiliation. The genomic proportion of each <i>var</i> gene group is indicated after the color code.</p

<i>Var</i> transcription profiles of parasites recovered from infected volunteers at the day of first microscopically detectable parasitemia.

<p>(A) Heat map showing the individual <i>var</i> gene expression for all volunteer samples taken when parasites were present in the thick blood smear ranked by mean expression. To correct for individual differences in the overall <i>var</i> expression levels, the expression for each <i>var</i> gene was normalized against the total <i>var</i> expression in each sample. The color scale indicates the relative expression levels with red representing values above the median, blue representing values below the median, and white representing median. Grey means not detected. The number of sporozoites (200, 800, 2500 and 3200) and mode of injection (iv = intravenous, id = intradermal) used for each volunteer are indicated below. (B) The distribution of the relative gene expression per <i>var</i> gene and control genes is shown in a dot plot for all volunteer samples at the day of patent infection defined as parasites present in the thick blood smear (n = 18). Each point represents a <i>var</i> gene expression value relative to the normalizing gene <i>sbp1</i> observed per volunteer sample and the median expression per <i>var</i> gene is marked. Housekeeping genes used as controls, <i>var</i> gene names and groups are indicated. (C) Proportion of <i>var</i> gene expression by group across all volunteers at the day of patent infection. For comparison, genomic proportion of each <i>var</i> gene group is indicated after the color code. (D) Comparison of the expression levels between subtelomeric and centromeric <i>var</i> gene variants. The box plot shows the distribution of transcript levels for each individual <i>var</i> gene relative to <i>sbp1</i> according to the chromosomal localization of the genes for all 18 volunteer samples at the day of patent infection. Gene expression varied significantly between both gene sets (Wilcoxon rank-sum test, p<0.0001) with median expression of 232 (IQR: 87–510) for telomeric <i>var</i> genes and 40 (IQR: 18–94) for centromeric <i>var</i> genes. (E) The heat map of pairwise Spearman’s rank correlation coefficients (R) between expression profiles illustrates the positive correlation between all 18 volunteer samples at the day of patent infection. Volunteer samples were ranked by the sum of their correlation coefficients. The color scale indicates the correlation coefficient in the range from 0 to 1. With exception of isolate 25.1 versus the isolates 02.1 (p = 0.0015), 25.3 (p = 0.0029) and 32.4 (p = 0.0030) all expressions correlated at a significance level below 0.001.</p

Overview of volunteer characteristics infected with PfSPZ Challenge.

<p>Overview of volunteer characteristics infected with PfSPZ Challenge.</p