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

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

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

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

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

The effects of FTY720 on accumulation of RTDC and LVS in the MdLN.

<p>Mice infected with 10⁵ LVS were treated with FTY720 diluted in carrier solution, or with carrier solution alone, as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000211#s4" target="_blank">Materials and Methods</a>. To examine DC trafficking, MdLNs were collected 48 hrs post infection and analyzed for RTDC representation (CD11b^high/autofluorescence^low) as described in the legend to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000211#ppat-1000211-g003" target="_blank">Figure 3</a>. An experiment was conducted on MdLN pools, each consisting of 6 organs. MdLNs were derived from infected mice treated with either carrier alone (A) or with FTY720 (B). Non-infected, FTY720-treated mice (C) served as controls. Bacterial colonization of the MdLN (D) was examined 48 hrs post infection in each one of the 6 individual FTY720- treated and 6 mock-treated mice, <i>P</i><0.01. Three independent experiments, each conducted with groups of six mice, exhibited similar correlation between RTDC and LVS recruitment.</p

Cell recruitment to MdLN following airway infection with LVS.

<p>MdLNs were isolated at various time points after intra-nasal infection of mice. Single cell suspensions were analyzed by flow cytometry as indicated on the legend to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000211#ppat-1000211-g003" target="_blank">Figure 3</a>. Number of AMΦ and RTDC at different time points post infection are presented as bar diagrams.</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

Interaction of BMDC and LVS in vitro Alter.

<p>(A) Propagation of LVS in J774A.1 cells and BMDC: Infection was performed as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000211#s4" target="_blank">Materials and Methods</a> section, and gentamicin was added 1 hr post pulsing with bacteria. Cells were harvested 2 or 24 hrs post infection, washed, and lysed with DOC. Lysates were diluted and plated for CFU enumeration. Each bar represents the average CFUs in three infection wells. The entire experiment represents one of three repetitions. For visualization of cell-associated bacteria by fluorescence microscopy, infected J774A.1 or BMDC cells were stained with anti-LVS antibodies 24 hrs post LVS infection. (B) Expression of CCR7 by BMDC: Cells were analyzed for CCR7 expression 24 hours post infection with the live or formaldehyde-killed LVS. Non-pulsed cells and cells pulsed with 1 µg/ml <i>E. coli</i> LPS served as negative and positive controls, respectively. Gray lines denote isotype-matched immunoglobulin controls. Fractions of CCR7^positive cells are indicated. (C) DC were pulsed as indicated above, and expression of co-stimulatory molecules was examined as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000211#s4" target="_blank">Materials and Methods</a>. Geomeans of fluorescence intensities are presented as relative values, using the values obtained for each marker by pulsing with 1 µg/ml <i>E. coli</i> LPS as 100. Results are presented by bar diagrams as the averages of three independent experiments. (D) Migration of BMDC: Cells were pulsed as indicated above, 24 hrs later cells were examined for migration in Transwell chambers towards CCL19 or control medium. Non-pulsed cells and cells pulsed with LPS served as controls. Results are presented as percent cells migrating from the upper compartments to the lower compartment. Each bar represents the average migration in 3 Transwells. The experiment was repeated 3 times. Data presented in this figure were generated by using BALB/c BMDC. Results were confirmed with DC derived from C57BL6 mice.</p