Western and adenylation analyses of glycerol gradient fractionated editosomes from KREX2 null and parental cell lines.

<p>Gradient fractions from BF 427wt (<b>A.</b>), BF-KREX2-null (<b>B.</b>), PF 29.13 (<b>C.</b>) and PF-KREX2-null (<b>D.</b>) were probed using antibodies recognizing editosome proteins KREPA1, KREPA2, KREL1, and KREPA3 (top panel) or by adenylation of ligases KREL1 and KREL2 (bottom panel). Editosome sedimentation of KREX2 null samples is shifted toward upper part of the gradient (i.e. smaller in size) relative to parental controls. KREL1 particularly shifts up in the gradient.</p

Real-time PCR analysis of KREX2 null cells.

<p>RNA abundance for nuclear mRNAs KREX1 and KREX2 (black bars), never-edited mRNAs COI and ND4 (light grey bars), pre-edited mRNAs (white bars), and edited mRNAs (dark grey bars) is calculated relative to parental cell line for both BF (427wt) and PF (29.13). Analysis was performed in triplicate. For each target amplicon, the relative change in RNA abundance was determined by using either 18S rRNA (left bar in each pair) or β-tubulin (right bar in each pair) as an internal control. Asterisks denote mRNAs that were not detected in KREX2 null cells. <b>A.</b> A significant loss of CYb editing is shown in BF KREX2 null cells. <b>B.</b> PF KREX2 null cells have predominant decreases in CR3 and ND3 edited mRNAs.</p

PF KREX2 null cells grow slower than parental cells in vitro, but BF KREX2 null cells grow indistinguishably from parental cells both in vitro and in vivo.

<p><b>A.</b> Cumulative growth of BF 427wt (solid squares) and derived KREX2 null cells (open squares) in vitro. <b>B.</b> Increasing parasitemia in 2 mice infected with either BF 427wt (squares) or derived KREX2 null cells (triangles) in vivo. <b>C.</b> Cumulative growth of PF 29.13 (solid symbols) and derived KREX2 null cells (open symbols) in vitro. In normal SDM-79 media (squares), KREX2 null cells grow more slowly than parental 29.13 cells. In media lacking glucose (triangles) KREX2 null cells grow indistinguishably from parental 29.13 cells. Addition of 6 mM glucose to glucose-free media re-establishes growth defect of KREX2 null cells (circles).</p

Cleavage activity maintained in KREX2 null cells.

<p>Glycerol gradient fractionated editosomes from KREX2 null and parental cell lines were examined for insertion (<b>A.</b>) or deletion (<b>B.</b>) cleavage activity. Hydroxyl (OH) and T1 nuclease (T1) ladders were used as references. Positive control reaction using 20S mitochondrial fraction (20S+) requires gRNA (+g) for cleavage, which is absent without gRNA (−g). Arrows denote cleavage product.</p

Pre-cleaved editing assays of glycerol gradient fractionated editosomes from KREX2 null and parental cell lines.

<p>Editing activities are maintained in KREX2 null cells. 20S glycerol gradient fraction from purified mitochondria is used as a positive control. Asterisks in schematics denote location of radiolabel. Pre-cleaved insertion assays for BF (<b>A.</b>) and PF (<b>B.</b>) demonstrate KREX2 null cells maintain both TUTase and ligase activity. Pre-cleaved deletion assays for BF (<b>C.</b>) and PF (<b>D.</b>) demonstrate KREX2 null cells maintain both exoUase and ligase activity.</p

KREN1 editosomes isolated from KREX2 null cells have exoUase activity, while KREN2 editosomes do not.

<p><b>A.</b> Western analysis of KREN1 (N1) or KREN2 (N2) TAP purified editosomes from either PF 29.13, BF-KREX2-null, or PF-KREX2-null cells using antibodies recognizing editosome proteins KREPA1, KREPA2, KREL1, and KREPA3 (top panel) or KREX1 (bottom panel). Note KREX1 signal is restricted to N1 editosomes. <b>B.</b> Expected cleavage specificity observed with KREN1 and KREN2 editosomes isolated from BF-KREX2-null cells. Insertion (top panel) and deletion (bottom panel) cleavage labeled as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033405#pone-0033405-g005" target="_blank">Figure 5</a>. <b>C.</b> Modified pre-cleaved deletion substrate assays U-specific exonuclease activity. Schematic depicts base change from U to A within the unpaired string of Us typically removed in pre-cleaved deletion assays. Left panel shows that only KREN1 editosomes from BF-KREX2-null cells have U-specific exonuclease activity, while it is possessed by both KREN1 and KREN2 editosomes from PF 29.13. Right panel shows similar restriction of U-specific exonuclease activity to KREN1 editosomes from PF-KREX2-null cells. Open triangles indicate increasing amount of extract used in assays. Arrow denotes fully edited product. Note also the lack of -2U product (indicated by black wedges) with KREN2 editosomes isolated from KREX2 null cells.</p

Volunteer gene expression trends visualized as heatmaps.

<p>Gene expression trends are shown for two differentially expressed BTMs and one gene set linked to parasitemia. Gene-wise expression levels of the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199392#pone.0199392.g004" target="_blank">Fig 4</a> DE modules are visualized as heatmap. Column color bars indicate grouping of subjects into early (red), average (green) and late (blue) group, based on time point of parasitemia detection. Each row corresponds to one gene.</p

DE genes determined by limma pairwise visit comparison.

<p>DE was pronounced at a BH-adjusted p-value < 0.05 and >1.5 fold expression change. (a) DE genes (red: up-regulated genes, blue: down-regulated genes) identified for each tested contrast are visualized as bars. The number of DE genes per contrast is indicated and additionally emphasized by the length of the bars. The bar width / x-axis indicates the log2 fold expression change of each DE gene. The Venn diagrams display the overlaps between (b) all DE genes of contrasts 5/0 and 9/5, (c) the 5/0 up-regulated and 9/5 down-regulated DE genes and (d) the 5/0 down-regulated and 9/5 up-regulated DE genes.</p

Image_2_Longitudinal immune profiling after radiation-attenuated sporozoite vaccination reveals coordinated immune processes correlated with malaria protection.pdf

BackgroundIdentifying immune processes required for liver-stage sterilizing immunity to malaria remains an open problem. The IMRAS trial comprised 5x immunizations with radiation-attenuated sporozoites resulting in 55% protection from subsequent challenge.MethodsTo identify correlates of vaccination and protection, we performed detailed systems immunology longitudinal profiling of the entire trial time course including whole blood transcriptomics, detailed PBMC cell phenotyping and serum antigen array profiling of 11 IMRAS radiation-attenuated sporozoite (RAS) vaccinees at up to 21 timepoints each.ResultsRAS vaccination induced serum antibody responses to CSP, TRAP, and AMA1 in all vaccinees. We observed large numbers of differentially expressed genes associated with vaccination response and protection, with distinctly differing transcriptome responses elicited after each immunization. These included inflammatory and proliferative responses, as well as increased abundance of monocyte and DC subsets after each immunization. Increases in Vδ2 γδ; T cells and MAIT cells were observed in response to immunization over the course of study, and CD1c+ CD40+ DC abundance was significantly associated with protection. Interferon responses strongly differed between protected and non-protected individuals with high interferon responses after the 1st immunization, but not the 2nd-5th. Blood transcriptional interferon responses were correlated with abundances of different circulating classical and non-classical monocyte populations.ConclusionsThis study has revealed multiple coordinated immunological processes induced by vaccination and associated with protection. Our work represents the most detailed immunological profiling of a RAS vaccine trial performed to date and will guide the design and interpretation of future malaria vaccine trials.</p

Volunteer gene expression trends visualized as boxplots.

<p>Gene expression trends are shown for two differentially expressed BTMs and one gene set linked to parasitemia. Boxplots with gene-wise baseline-subtracted expression values are shown separately for subjects with early (red), average (green) and late (blue) detection of blood stage parasitemia.</p