Comparison of specificity of <i>in vivo</i> immune responses with <i>in silico</i> predictions using various algorithms.

<p>Individual rabbit (Panel A, n = 8 animals) or mouse (Panel B, n = 10 animals) immune sera were analyzed by ELISA and reported as OD values (bar graph). X-axis indicates the peptide ID and span; Y-axis indicates the OD value of the ELISA after subtracting the background measured for pre-immune sera. Horizontal line indicates threshold OD as described in Materials and Methods. Brackets indicate the segments of the protein predicted to be immunogenic by the <i>KTApred</i> (black), the <i>Bepipred</i> (blue) or the <i>Discotope</i> (green) algorithms. Open circles (grey) above individual bars identify peptides predicted by the <i>ABCpred</i> prediction tool. Asterisks above individual bars indicate positive responses detected by MALDI-TOF MS analysis. The MALDI-TOF data are the mean of three independent experiments using pooled immune sera. Responses from pooled pre-immunes were subtracted.</p

Fine specificity of PfCelTOS-specific T cells.

<p>Reactivity was determined by ELISpot analysis measuring <i>Pf</i>CelTOS-specific IFN-γ responses. Mouse splenocytes from three strains (inbred BALB/c and C57BL/6 and outbred ICR) were tested against a panel of 43 overlapping peptides (AA = amino acid position within the protein). Putative binding to indicated MHC class I and class II (in bold) alleles was determined by Rankpep analysis. Underlined amino acids designate predicted binding motif for indicated MHC allele. Shading and intensity of shading indicates the magnitude of the T cell response after <i>ex vivo</i> stimulation with the peptides.</p

Proteasomal and Cathepsin cleavage maps of CelTOS.

<p>The proteasomal and Cathepsin D, L and S peptide fragments were separated by UFLC, analyzed on an LCMS-IT-TOF mass spectrometer and then identified using the MASCOT data base. The peptides derived from the proteasomal degradation of CelTOS (blue lines) are denoted above the sequence, and Cathepsin D (red lines), Cathepsin L (green lines) and Cathepsin S (purple lines) are denoted below the sequence, represent the identified peptides. The data shown is representative of two separate experiments.</p

B-cell epitope prediction using second generation methods.

a<p>Accuracy measures the percentage of correct epitope classification across all residues.</p>b<p>A_roc is the area under the curve constructed by the Receiver Operational Characteristics (ROC), which is the function of the sensitivity and specificity of the epitope mapping score. A_roc = 1 indicates perfect prediction of epitopes, A_roc = 0.5 indicates completely random predictions.</p>c<p>p-value is the probability that the observed A_roc value was obtained by chance. In the null positive and null negative methods, all and no residues, respectively, were classified as epitopes.</p><p>A_roc standard errors (SE) were estimated using bootstrapping. <i>p</i>-values were calculated using permutation tests <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071610#pone.0071610-Larsen1" target="_blank">[6]</a>.</p

Consolidation of structural properties from <i>in silico</i> predictions and <i>in vivo</i> immune responses.

<p>Computational epitope predictions (gray) are shown for <i>ABCpred</i>, <i>Discotope</i>, and <i>Bepipred</i>. Experimental epitope mapping using antibody peptide scanning (black) from both rabbit and mouse anti-<i>Pf</i>CelTOS serum antibodies are significant above an OD-cutoff of the mean background responses plus three standard deviations from the mean. Computational secondary structure propensities for α-helix (blue), coiled (red) regions and disordered propensity (green) reported in a relative scale −1 to 1. All computational epitope definitions are based on classifications using default score cutoff values for <i>Discotope</i> and <i>Bepipred</i>. Score cutoff for <i>ABCpred</i> was optimized to maximize accuracy. Only results from computational methods with statistically significant predictions (<i>p</i><0.001) are shown.</p

Top-ranked structure predictions of the CelTOS protein using Rosetta (A), i-TASSER (B), and QUARK (C) as backbone traces.

<p>Predicted conformational epitopes by <i>Discotope</i> are shown as colors for epitopes I (yellow), II (orange), and III (red). Regions predicted to be epitopes by <i>Discotope</i> but not found to be antigenic in peptide scans are shown in magenta.</p

Figure 6

<p>Relationship of growth inhibition with time to infection. Time to infection in individuals with upper quartile GIA results (>60% inhibition) tested against 3D7 compared to lower quartiles GIA results (<60% inhibition) controlling for age (Cox regression, p = 0.0438). Kaplan-Meier curves are divided by child and adult to illustrate age effects on growth inhibition. Similar analyses controlling for age were performed with D10 and FVO GIA.</p

Figure 2

<p>GIA of 5 representative Kenyan samples tested in parallel against 3 parasite lines (D10, 3D7 and FVO). Individual A has significant inhibitory activity against the 3 lines tested whereas individual D has essentially no inhibitory activity against all lines tested. Individual B has a growth inhibition pattern that reflects that of the entire population (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003557#pone-0003557-g001" target="_blank">Figure 1</a>). Other individual samples demonstrate variable growth inhibitory activity against the different lines tested.</p

Figure 7

<p>Comparison between different GIA methods. Panel A depicts box plots for D10 GIA using the same 54 dialyzed Kenyan plasma samples to compare one growth cycle (harvest at ring stage) to two growth cycles (harvest at trophozoite stage). Parasitemia was assessed by flow cytometry (10× SYBR Green 1 stain for one cycle, GFP for two cycles). Two growth cycles of D10 GIA had a statistically significant higher median inhibition (18.5% inhibition) compared to one cycle D10 GIA (8.3% inhibition; p = 0.0003; Wilcoxon signed-rank test). Panel B shows the Bland-Altman plot to assess the degree of agreement between paired results after one vs. two growth cycles. The points display the difference in growth inhibition between the two assays (y-axis) against their corresponding average values (x-axis). The horizontal lines correspond to the mean difference (solid line) ±2SD (dashed lines). The mean shows a bias of -11.05, indicating the two assays are producing different results with a trend of increasing differences with increased averages. Panel C depicts box plots for 3D7 GIA using the same 54 Kenyan samples. All samples were dialyzed prior to use. 3D7 GIA using one cycle of parasite growth, harvest at the trophozoite stage with parasitemia measured by pLDH was compared to 3D7 GIA using two growth cycles, harvest at the trophozoite stage with parasitemia measured by flow cytometry (EtBr). No statistically significant difference was noted (Wilcoxon signed-rank test). Panel D shows the Bland-Altman plot to assess the degree of agreement between paired assay results. The points display the difference in growth inhibition between the two assays (y-axis) against their corresponding average values (x-axis). The horizontal lines correspond to the mean difference (solid line) ±2SD (dashed lines). Panel E depicts box plots for D10 GIA using the same 54 Kenyan samples either dialyzed or not dialyzed. GIA was performed using one cycle of parasite growth (harvest at ring stage) followed by flow cytometry to measure parasitemia (SYBR Green 1 stain for dialyzed samples, Hoechst stain for non dialyzed samples). No statistically significant difference was noted (Wilcoxon signed-rank test). Panel F shows the Bland-Altman plot to assess the degree of agreement between paired assay results. The points display the difference in growth inhibition between the two assays (y-axis) against their corresponding average values (x-axis). The horizontal lines correspond to the mean difference (solid line) ±2SD (dashed lines). The mean has minimal bias (−2.8) with a small trend of increasing differences with increased averages.</p

Summary of GIA methodologies performed with 54 Kenyan plasma samples.

<p>HO = Hoechst 33342, Troph = trophozoite, pLDH = parasite lactic dehydrogenase, SYBR = SYBR Green I, GFP = green fluorescent protein, EtBr = Ethidium Bromide. CWRU = Case Western Reserve University, WRAIR = Walter Reed Army Institute of Research, WEHI = Walter and Eliza Hall Institute</p