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

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

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

Env-A244 peptides generated by CAT and proteasomal cleavage of CAT degradation products.

a<p>The number of Env-A244 peptides identified by mass spectrometry.</p>b<p>As reported in the Los Alamos database.</p

Env-A244 peptides derived by CAT D, K, and L cleavage induce IFN-γ from HIV-1 Env-specific CD4⁺ T-cells.

<p>CAT D, K, and L digests of Env-A244 were incubated with Env-specific CD4⁺ T-cell lines generated from six RV144 phase III trial volunteers. The Env-specific T cell lines were incubated with Env-CMDR peptide pool, Gag-CMDR peptide pool, media containing the buffers used for CAT degradation, and CAT D, K, and L degraded ovalbumin (OVA) as a non-HIV antigen control. The CD4+ T cell lines were then analyzed for the induction of IFN-γ by ELISPOT. No IFN-γ was detected in the CAT buffer controls or from the Gag-CMDR peptide pool (panels A–F); Four of the six cell lines tested with CAT degraded OVA also did not induce IFN-γ (panels A–C and E). Five out of the six cell lines (panels A–E) incubated with the Env-CMDR peptide pool induced IFN-γ. These same cell lines (A–E) also induced IFN-γ when stimulated with Env-A244 peptides derived from CAT D and K degradations. The cell line that did not induce IFN-γ with either Env-CMDR peptide pool or Env-A244 peptides derived from CAT D, K and L degradations, is a cell line derived from a placebo volunteer (panel F). Only one out of the 6 cell lines (panel A) incubated with Env-A244 peptides derived from CAT L induced IFN-γ. CD4⁺ T-cell lines derived from 144193 and 144620 (vaccinees) and 144432 (placebo) were tested for their response to SEB (panel G). All three of the cell lines showed a response to SEB demonstrating that the cells are functional. The data represent the mean ± S.D. of triplicate wells. *denotes p≤0.05 and **denotes p≤0.01. N/D: Not determined.</p

A schematic representation of the differential processing of the components of the RV144 vaccine.

<p>The RV144 vaccine consists of canarypox vector ALVAC (vCP1521), carrying HIV clade B and circulating recombinant form (CRF)01_AE <i>gag</i>, <i>pro</i> and <i>env</i> genes, and AIDSVAX®B/E genetically engineered gp120 protein from viruses of subtypes B and CRF01_AE. After entry into the cell, the ALVAC canary pox vector will generate protein antigens in the cytosol after transcription and translation. These proteins are then processed through the classical MHC class I pathway involving the proteasomal complex. The phagocytosed AIDSVAX®B/E protein antigens will enter the endosome/lysosome compartment and will be cleaved by the CAT. Some of the peptides generated within the endosome/lysosome compartment can be retrotranslocated into the cytosol and enter the classical MHC class I pathway. The peptides containing the MHC class II epiotpes enter the MIIC compartment from the endosome/lysosome where they are then loaded onto class II molecules and transported to the cell surface.</p

Env-A244 peptides derived by CAT D, K, and L cleavage induce a poly-functional cytokine response from antigen-specific CD4⁺ T-cell lines.

<p>HIV-1 Env specific CD4⁺ T cell lines derived from two volunteers from the RV144 phase III trial (144620 and 144193) were stimulated with either the CMDR Env peptides, SEB, peptides generated from Env-A244 cleaved by CAT D, CAT K, or CAT L, or cultured in media only. The CD4+ T cells were analyzed for the generation of CD107a (red), IFN-γ (brown), IL-2 (light green), MIP1β (light blue) and TNF-α (purple) by flow cytometry. The pie charts (panels A and B) demonstrate the fraction of cells with one (green), two (red), three (purple), four (orange), or five (light blue) cytokines generated from the same cell, and the circular lines arching the pie chart represent the five cytokines/chemokines examined. The data is denoted in a graphical format in panels C and D as a percentage of CD4⁺ T-cells capable of generating each of the 31 possible cytokine combinations. The color designations for each of the experimental conditions shown in panels C and D are as follows: unstimulated (blue bar); SEB (yellow bar); CMDR peptides (red bar); CAT-D Env-A244 derived peptides (green bar); CAT-K Env-A244 derived peptides (orange bar); and CAT-L Env-A244 derived peptides (purple bar).</p