Inhibition studies to test the co-purification of PCR inhibitors.

<p>Two different concentrations of IC DNA were used as the DNA template in the qPCR reactions to test the co-purification of PCR inhibitors in samples extracted from whole blood using ME or EZ methods; High DNA concentration or Low DNA concentration. Control experiments did not contain genomic DNA in the reaction. Each column shows the method which the genomic DNA present [or not for the controls] was extracted [ME or EZ] and amount of IC plasmid present [High or Low].</p

Analysis of parasite densities in clinical samples using absolute qPCR and microscopy.

<p>Absolute quantitative qPCR was performed using plasmid DNA as the standard to analyze clinical samples. The log₁₀ parasite densities in terms of parasite/µl was determined from qPCR assays and compared to the log₁₀ parasite densities as determined by expert microscopist. The correlation coefficient of parasite densities measured using the two methods was calculated using the nonparametric Spearman correlation coefficient. There was a statistically significant correlation between parasite density measured by microscopy and absolute quantitative qPCR.</p

Analysis of the multiplex real-time PCR assay.

<p>Multiplex qPCR assays were performed containing both primer and probe sets for all four targets or primer and probe set for single target. Analysis 1 shows data from multiplex assay analyzed as multiplex where all four targets were analyzed simultaneously. Analysis 2 shows data from multiplex assay but data was analyzed as a single assay for each target. Analysis 3 shows data from single assays.</p

Primers and probes sequences used for qPCR assays in this study.

<p>Primer and probes for amplification of <i>Plasmodium</i> spp., <i>P. falciparum</i>, <i>P. vivax</i>, RNaseP and internal control (IC) plasmid DNA assays. Sequences for Forward (F), Reverse(R) primers and the Probe (P) are shown.</p

Linear regression plots for absolute qPCR assays.

<p>Real-time PCR assays were performed using plasmid DNAs for each assay. Plasmid DNA was 10-fold serially diluted at each point and ran in 4–8 replicates. A linear regression plot was generated using GraphPad Prism. The slope, the Y-intercept and the r² value were determined. Data shown confirms that these assays perform with high efficiencies.</p

Absolute qPCR CT values obtained vs. the genomic equivalence used.

<p>Data showing the mean C_T values obtained from qPCR assays performed using plasmid DNAs. Plasmid DNAs were 10-fold serially diluted 10-log [times] and ran in 4–8 replicates.</p

Titration of reaction master mix volume in qPCR reaction.

<p>Multiplex qPCR reactions were set-up that contained descending reaction master mix from 10 µl to 1 µl and 1 µl DNA template in each reaction. Experiments were performed in total replicates of 8. All the four targets in the multiplex qPCR assay were analyzed simultaneously. There was a negative correlation between reaction master mix volume and assay sensitivity. As the volume of reaction master mix increased, the sensitivity of the qPCR decreased with 1 µl reaction master mix reaction being the most sensitive for PLU, FAL and VIV assays.</p

VMP001-NP immunization elicits high avidity antibodies capable of agglutinating live sporozoites.

<p>(A) Avidity indices of anti-VMP001 IgG sera obtained from mice immunized as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031472#pone-0031472-g003" target="_blank">Fig. 3</a> with soluble VMP001+MPLA (red circles) or VMP001-NP+MPLA (blue circles) were characterized over 6 months following vaccination. *, <i>p</i><0.01 and **, <i>p</i><0.001, analyzed by two-way ANOVA, followed by a Bonferroni post-test. (B) Anti-VMP001 IgG antibodies elicited with soluble VMP001+MPLA (red circles) or VMP001-NP+MPLA (blue circles) were further examined for their affinities against key fragments of VMP001, including peptides representing the Type I repeat, AGDR motif, Region I, Region II, C-terminus, and scrambled negative peptide control. Sera from non-immunized mice were also included as controls (black squares). (C,D) Sera obtain from mice on day 63 post-immunizations with 2.5 µg VMP001 and 25 µg MPLA in either (C) soluble or (D) VMP001-NP formulations were incubated with live VK210 sporozoites, and immunoflurescence assay was performed to assess recognition of native CSP present on the surface of live sporozoites by anti-VMP001 IgG sera. Mice immunized with VMP001-NP vaccines raised sera that agglutinated live VK210 subtype of <i>P. vivax</i>.</p

VMP001-NP immunization triggers germinal center formation.

<p>(A) C57Bl/6 mice were vaccinated with 0.1 µg VMP001 and 5 µg MPLA in either soluble or VMP001-NP formulations, and on day 21, inguinal dLNs were isolated and analyzed for germinal center formation. The number of isotype-switched germinal center B cells (GL-7⁺PNA⁺), gated on B220⁺IgD^low populations in dLNs was measured with flow cytometric analysis. (B) Inguinal dLNs were cryo-sectioned on day 21 post-immunization and stained with anti-B220, anti-IgD, and anti-GL-7 (markers for B cells, immature B cells, and germinal center, respectively) and examined by confocal microscopy. Scale bars, 50 µm. *, <i>p</i><0.05, analyzed by Student's <i>t</i> test.</p

Synthesis of PLGA NPs with surface-conjugated VMP001 (VMP001-NPs).

<p>(A) Schematic illustration of synthesis of lipid-enveloped PLGA NPs with surface-conjugated VMP001. PLGA NPs were incubated with thiolated VMP001, conjugating the antigen to maleimide-functionalized lipids displayed on the particle membranes. Particles were then PEGylated in a reaction with PEG-thiol. (B) A scanning electron microcopy image of VMP001-NPs. Scale bar, 2 µm. (C) Confocal microscopy image of VMP001-NPs incubated with anti-his-tag and fluorescent secondary antibodies to detect particle surface-conjugated VMP001. Scale bar, 10 µm.</p