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Array of peptides tested in the <i>F. tularensis</i> CTL screen in C57BL/6 LVS immunized mice.

<p>Summary of the total number of peptides selected, evaluated and responding in the various subsets (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036440#pone-0036440-g001" target="_blank">Figure 1</a>). Data on Subset I and II is from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036440#pone.0036440-Zvi1" target="_blank">[14]</a>. Peptides which were shown to stimulate lymphocytes and induce IFNγ production are referred to as “responders". P-values are provided for evaluation of significance of difference between number of responders in the cluster-based Subsets I and III versus Subset II, and versus Subset IV.</p

Magnitude of T-cell response among identified CTL epitopes (Subsets I, III, V).

<p>Responders identified from clusters with densities of 1.0–1.4 (dark cyan) (determined from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036440#pone.0036440-Zvi1" target="_blank">[14]</a>), from clusters with densities of 0.8–1.0 (light cyan) and from the high affinity-based approach (light blue) are classified according to magnitude of T-cell response, as deduced from number of spots in the EliSpot assay. The bars represent the percent of responders per subset in each of the classes, while the actual number of responders is given in brackets on the top of the corresponding bar. Classification (expressed in number of spots/million cells) is: Low: 5–20; Medium: 20–32; High> = 33).</p

Distribution of clusters of CTL epitopes in a representative non-membranal and membrane-spanning protein and alignment with transmembrane α-helices and loops.

<p>Sample plots of the topological map (generated by TMHMM predictions) of two representative proteins. Red segments represent the probability of having a helical region, while the thin blue and pink lines represent inside and outside loops, respectively. In the lower part of every protein chart, a bar represents a cluster of predicted epitopes. Co-localization of the putative MHC binders with the predicted helix or loop region in the protein is marked by a grey box. (A) A sample of a non-membranal protein (gi|89256893) carrying a cluster at density 0.8, harboring 23 putative epitopes, four of which are a responders. This represents the 18% non-membranal proteins carrying high density clusters (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036440#pone-0036440-g004" target="_blank">Figure 4</a>). (B) A sample of a transmembrane protein (gi|89256946) carrying four clusters (clusters #1–4) within a α-helical transmembrane domain, (marked by “TM"), and an additional cluster (cluster #5) co-localized with an outside loop domain (marked by “loop") between two transmembranal regions. The total number of putative epitopes in clusters #1–4 is 102 and in cluster #5 is 17, which contain 3 and 2 responders, respectively. This sample protein provides an example for the high frequency of clusters in helical regions but also demonstrates the presence of high density clusters in loops (there are examples of other proteins, not shown, where clusters span both transmembrane and loop regions).</p

Comparison of cluster-based and high affinity-based screens conducted in C57BL/6 and BALB/c mouse models.

<p>Comparison of cluster-based and high affinity-based screens conducted in C57BL/6 and BALB/c mouse models.</p

Distribution of parental proteins containing responders according to their membrane topology.

<p>Parental proteins containing responder peptides identified by the cluster-based, high affinity-based and random-based approaches are classified as follows: Membrane-spanning proteins (with 10 or more predicted transmembranal domains (TM) and/or TMs spanning at least a third of the protein) in dark blue; partially membranal proteins (less than 10 predicted TMs) in blue; proteins with no predicted transmembranal domains (except signal peptide) in light blue. . P-values are provided for evaluation of significance of difference between membranal proteins in the cluster-based Subsets I and III versus Subset V and versus Subset II.</p

Flowchart of down-selection of putative MHC binders for experimental evaluation.

<p>The whole-genome immunoinformatic analysis conducted on the 1754 <i>F. tularensis</i> ORF products and the downstream selection of the various subsets of peptides is provided. Boxes shaded in light blue: Subsets I and II were described in a previous study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036440#pone.0036440-Zvi1" target="_blank">[14]</a> and are added for the sake of completeness. Boxes shaded in cyan: Subsets III–V of peptides selected and evaluated in this study. In all down selection steps referred to as : “Selected for evaluation" (in Subset III, IV and V), clusters or peptides are selected at random as an operational filter for reduction of number of peptides to be experimentally tested, unlike Subset II, were selection of peptides was at random with respect to clusters. Subset III contains 401 peptides selected from clusters having density of 0.8 up to 1.0 (1.0 not included); Subset IV is composed of 200 peptides selected from the overall 3016 predicted MHC binders (IC₅₀< = 500 nM) located outside the high-density cluster regions in the 41 source proteins of Subset I (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036440#pone-0036440-g002" target="_blank">Figure 2</a>); Subset V includes 92 predicted epitopes having an IC₅₀ of 10 nM and lower, representing putative high-affinity MHC binders.</p

Induction of T cell response to selected CTL epitopes following LVS or DNA-PolyEp immunization.

<p>^a CTL epitopes were derived from six proteins respectively: FTL_1916_400–410, FTL_0966_126–133, FTL_1708_13–23, FTL_0283_95–104, FTL_1101_64–74, and FTL_1673_308–318.</p><p>^b Stimulating antigens: 10 mM of the individual peptides or 10⁷ CFU/ml of formalin-inactivated LVS.</p><p>^c C57BL/6 mice were immunized with LVS or the DNA-PolyEp vaccine with CTL epitopes numbered (1)-(6) (see Materials and Methods).</p><p>^d Data represent the mean and SD of three experiments (at least two animals per experiment) that were carried out in duplicate.</p><p>^e The pCI vector was used to express a 186 bp DNA fragment encoding for the 1–6 epitopes (see Materials and Methods).</p><p>^f The peptide “ICYVSTNIM”, an identified CTL epitope in LVS not included in the DNA-PolyEp vaccine, was used as a positive control (see Materials and Methods).</p><p>^g Scrambled sequence of peptide (5).</p><p>^h LVS was inactivated by formalin (see Materials and Methods).</p

Survival after lethal inhalational <i>F. tularensis</i> LVS challenge.

<p>Two weeks after the completion of the immunization schedule (see Materials and Methods), 10 mice in each vaccine group were challenged <i>i.n.</i> with 10⁴ CFU LVS (equivalent to 10 LD₅₀) and monitored for survival for 28 days. squares, DNA-PolyEP immunization; circles, pCI immunization; triangles, non-immunized mice.</p

Bacterial burden in vaccinated mice after <i>F. tularensis</i> LVS challenge.

<p>Mice were vaccinated and challenged as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085215#pone-0085215-g002" target="_blank">figure 2</a>. At two, three and five days post-challenge, 3 mice in each group were sacrificed, and their spleens (A) and livers (B) were removed for bacterial load inspection by CFU counts; similar results were obtained by quantitative real time-PCR. The data represent the means ± SD from 2 independent experiments. Black squares, DNA-PolyEp immunization; white circles, pCI immunization; grey triangles, non-immunized mice.</p