Cellular and humoral immune responses of convalescent <i>Salmonella</i>-infected mice to recombinant <i>Salmonella</i> antigens.

<p><b>A</b>) Antigen-specific CD4 T cell frequencies as detected by CD154 upregulation (red) and IFNγ (green) or IL-17 (blue) secretion. The data represent means ± SE of three mice. Responses to <i>Salmonella</i> antigens in non-infected control mice were subtracted (see also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002966#ppat.1002966.s001" target="_blank">Fig. S1</a>). <b>B</b>) Serum antibody responses to <i>Salmonella</i> antigens. The data represent means ± SE of 11 convalescent mice (filled circles) and means ± SE for ten non-infected control mice (open circles).</p

<i>Salmonella</i> antigen protectivity does not correlate with in vivo antigen abundance but depends on antigen localization within the <i>Salmonella</i> cell.

<p><b>A</b>) Relationship between antigen protectivity and in vivo abundance as determined by quantitative proteome analysis of ex vivo purified <i>Salmonella</i> (means ± SD for three independently infected mice; b.d., below detection threshold). <b>B</b>) Relationship between antigen protectivity and antigen localization within <i>Salmonella</i> (C, cytosol; IM, inner membrane; P, periplasm; LP, outer-membrane associated lipoprotein; OMP, outer membrane protein; S, surface). Statistical significance of differences between internal and outer membrane/surface antigens was tested using the non-parametric Mann-Whitney U test.</p

Schematic model for cellular immunity to <i>Salmonella</i>.

<p><i>Salmonella</i> (yellow) reside in intracellular vacuoles in infected host cells. <i>Salmonella</i> possesses internal (green) and surface-associated (red) antigens. <b>Left</b>) Live <i>Salmonella</i> shield internal antigens, but some of their surface-associated antigens are accessible for processing and presentation. As a consequence, T cells specific for <i>Salmonella</i> surface antigens can recognize these infected cells and initiate antibacterial immune effector mechanisms. In contrast, T cells specific for internal <i>Salmonella</i> antigens fail to detect host cells that contain exclusively intact <i>Salmonella</i>. <b>Right</b>) Dead <i>Salmonella</i> release internal antigens. As a consequence, both surface-exposed and internal antigens can be processed, presented, and recognized by cognate T cells. However, this recognition is unproductive for infection control since it targets <i>Salmonella</i> that are already dead.</p

CD4 T cell responses to <i>Salmonella</i> expressing an ovalbumin model antigen in various compartments.

<p><b>A</b>) Schematic overview of fusion proteins that target an immunodominant ovalbumin epitope (OVA) to various <i>Salmonella</i> cell compartments. <b>B</b>) Flow cytometric analysis of ovalbumin-specific CD4 T cell activation in a T cell receptor-transgenic adoptive transfer model. Mice were infected with control <i>Salmonella</i> expressing GFP (left) or <i>Salmonella</i> expressing LPP_OVA (right). Ovalbumin-specific transgenic CD4 T cells were detected with a clonotypic monoclonal antibody and analyzed for forward scatter and expression of the very early activation marker CD69. The dashed line was used to count CD4 T cell blasts. Similar observations were made for more than hundred mice in several independent experiments. <b>C</b>) Relationship between <i>Salmonella</i> Peyer's patches colonization and OVA-specific CD4 T cell induction in mice infected with <i>Salmonella</i> expressing high levels of LPP_OVA (filled circles) or low levels of GFP_OVA (open circles). Data represent means ± SEM's for groups of five to six animals from three independent experiments. CD4 T cell blasts correlated with <i>Salmonella</i> Peyer's patches colonization for both strains (Spearman test, <i>P</i><0.05 in both cases). The slopes of the two curves differed (ANCOVA; <i>P</i><0.05). <b>D</b>) OVA-specific CD4 T cell induction in mice infected with <i>Salmonella</i> expressing OVA at various levels (open circles, low abundance; filled circles, high abundance) in four different compartments. The dashed line represents CD4 T cell responses to saturating levels of cytosolic OVA. The star represents data for <i>Salmonella</i> expressing moderate levels of cytosolic OVA together with cholera toxin B and AIDA. Data represent means ± SEM's for groups of ten to twenty mice. Statistical significance of differences to <i>Salmonella</i> expressing saturating levels of cytosolic OVA were tested using Mann-Whitney U test (*, <i>P</i><0.05; **, <i>P</i><0.01).</p

Structural models and exposed immune epitopes of various <i>Salmonella</i> outer membrane proteins.

<p>The outer membrane is shown as a grey area, predicted CD4 T cell epitopes in exposed loops are shown in red, potential antibody binding sites are shown in blue, and overlapping T and B cell epitopes are shown in magenta. Partially exposed epitopes are shown in pale colors. Amino acid residues that differ between <i>Salmonella enterica</i> serovars Typhimurium and Typhi are shown in green. For TolC only the outer membrane-associated part is shown. LPS structures as observed in FhuA-LPS crystals <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002966#ppat.1002966-Ferguson1" target="_blank">[93]</a> are also shown.</p

Properties of <i>Salmonella</i> antigens.

1<p>, t-test vs. non-infected control mice;</p>2<p>, t-test of log-transformed values vs. non-infected control mice;</p>3<p>, log-rank test vs. control mice immunized with GFP;</p><p>b.t.; below quantification threshold; n.d., not determined; n.a., not applicable for secreted proteins SseB and SseD.</p

Detection of intact and damaged <i>Salmonella</i> cells in infected mouse tissues.

<p><b>A</b>) Flow cytometry of a spleen homogenate infected with <i>Salmonella sifB::gfp</i> using 488 nm excitation. Gate 1 contains GFP-positive <i>Salmonella</i>. The inset shows the relationship between flow cytometry data and plate counts for individual mice, the dashed line represents a 1∶1 ratio. <b>B</b>) Confocal micrographs of liver cryosections infected with <i>Salmonella sifB::gfp</i> that were stained with antibodies to <i>Salmonella</i> lipopolysaccharide (red) and macrophage marker CD68 (blue). Individual color channels are shown with inverted grey scale for better visualization of weak staining. Micrographs represent typical observations for four independently infected mice. <b>C</b>) Confocal micrographs of lipopolysaccharide-positive particles that lack detectable GFP (even when contrast was increased compared to B). Such particles were absent in non-infected control sections.</p