GPIs of RH and PTG strains induce TNF-α and IL-12p40 secretion by macrophages.

<p>(<b>A</b>) Macrophages were incubated for 24 h with medium alone, or with individual GPIs of the PTG strain extracted from 1×10⁸ parasites and assayed for TNF-α cytokine production. (<b>B</b>) Macrophages were incubated for 24 h with medium alone, or with individual GPIs of the RH (left panel) and the PTG strain (right panel) extracted from 2×10⁸ parasites, respectively, and assayed for IL-12p40 cytokine production. (<b>C</b>) Macrophages were incubated for 24 h with medium alone, or with GPIa (3 mM), a chemically synthesized structure of RH strain GPI III core glycan and assayed for IL-12p40 cytokine production. ^***P<0.0005 PTG GPI II compared to medium control. ND, not determined.</p

Protein-free Glc-GalNAc-substituted GPIs are clustered on extracellular parasites.

<p>Staining after permeabilization of HFF cells infected (72 h p.i.) with RH (upper panel) and PTG (lower panel) strains was performed using mAb T54 E10, recognizing both the EtN-PO₄ and the Glc-GalNAc side-branch epitopes of protein-free GPIs. Filled arrowheads point to intracellular parasites residing inside parasitophorous vacuoles. Unfilled arrowheads point to extracellular parasites. DIC, differential interference contrast.</p

Comparison of AHM and inositol quantifications of protein-free GPIs and GPI-anchored proteins of RH and PTG strains.

a<p>Copies per cell. In parentheses are percentages of PTG values compared to RH values. Values are means ± SD of three replicates.</p

GPIs of the PTG strain activate TLR4/NF-κB signaling.

<p>(<b>A</b>) CHO cells expressing TLR2 (TLR2⁺), TLR4 (TLR4⁺), or neither (TLR2⁻/TLR4⁻) were either untreated (black line) or exposed to the four different GPIs (II, III, V and VI) extracted from 1×10⁹ parasites (gray line). CD25 expression was measured by FACS analysis 18 h after stimulation. The figure is representative of three independent experiments. Percentage = [percentage of CD25 expression (M2) on GPI-stimulated cells] minus [percentage of CD25 expression (M2) on medium stimulated cells]. (<b>B</b>) Macrophages were incubated for 15, 30, or 60 min with GPI VI extracted from 4×10⁸ PTG strain parasites. Total nuclear protein content was tested using a NF-κB assay as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085386#s2" target="_blank">Materials and Methods</a>. The figure is a representative experiment with PTG GPI VI and similar results were obtained with GPIs II, III and V of the PTG strain. ^**P<0.005 compared to medium control.</p

RH and PTG strains have different GPI profiles.

<p>(<b>A</b>) Metabolically labeled ([³H]-glucosamine) parasite glycolipids were extracted and separated by TLC as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085386#s2" target="_blank">Materials and Methods</a>. TLC chromatograms were scanned for radioactivity using a Berthold LB 2842 linear analyzer. Six different peaks representing individual GPIs are detected in the RH strain <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085386#pone.0085386-Striepen1" target="_blank">[35]</a>, whereas only four major peaks are present in the PTG strain. (<b>B</b>) Structures of <i>T. gondii</i> RH GPIs. GPI VI (Man-Man-[GalNAc]Man-GlcN-PI) is neither substituted with an EtN-PO₄ nor with the Glc linked to the GalNAc residue. GPIs I, II and III contain an EtN-PO₄ residue (dashed frame line). GPIs I, II, IV and V are substituted with a Glc that is linked to the GalNAc side-branch in α1–4 linkage (thickened frame line). GPIs I, II and IV, V, respectively, have identical carbohydrate moieties and differ in their fatty acid composition.</p

GPI core glycans and PIs of RH and PTG strains have similar structures.

<p>(<b>A</b>) HPAEC analysis of the core glycans generated from <i>T. gondii</i> PTG strain protein-free GPIs II, III, V and VI. TLC-purified D-[³H]-glucosamine labeled GPIs were dephosphorylated, deaminated and reduced. The resulting neutral glycans were analysed before (untreated) and after Jack bean α-mannosidase or hexosaminidase treatments. The elution positions of the co-injected glucose oligomer standards are indicated at the top of each profile (bold digits) and given as DU. (<b>B</b>) Predicted Man-Man-(Glc-GalNAc)Man-anhydromannitol core glycan structure derived from protein-free GPIs II and V (right panel), and predicted Man-Man-(GalNAc)Man-anhydromannitol core glycan structure derived from protein-free GPIs III and VI (left panel) of the PTG strain. (<b>C</b>) ES-MS spectra of the PI moieties released by deamination from purified protein-free GPIs from RH (upper panel) and PTG (lower panel) strains.</p

Carbohydrate composition analysis of protein-free GPIs and GPI-anchored proteins of RH and PTG strains.

a<p>Glucose is a common contaminant. Values are means of molar ratios ± SD of three replicates.</p><p>Values are means of molar ratios ± SD of three replicates.</p>**<p>P<0.005,</p>*<p>P<0.05 RH protein-free GPI and GPI-anchored protein samples compared to PTG protein-free GPI and GPI-anchored protein samples, respectively.</p

Activation of antigen-specific IFN-γ-producing CD8+ T cell precursors in mice immunized with pIRES I or pIRES II.

<p>(A-B) Spleen cells from BALB/c mice spleen cells were stimulated with the MHC-I-restricted p24-specific peptide, and the p24-specific IFN-γ-producing CD8⁺ T cells were detected by intracellular cytokine staining (A) or ELISPOT assay (B). (C–D) Spleen cells from C57BL/6 mice were stimulated with the MHC-I-restricted E7-specific peptide, and the E7-specific IFN-γ-producing CD8⁺ T cells were detected by IFN-γ intracellular staining (C) or ELISPOT assay (D). Mice were i.m. immunized with three doses of the DNA vaccines with one week intervals between doses (100 µg/dose). The CD8⁺ T-cell responses were analyzed two weeks after the last dose. *p<0.05. Data represent the compilation of two independent experiments with four mice per immunization group (n = 8) and results expressed by each animal analyzed. pIRES is the empty vector used as immunization control.</p

Construction of bicistronic DNA vaccines encoding HPV, HIV and HSV antigens for expression in mammalian cells.

<p>(A) Schematic linear representation of the trivalent DNA vaccines. pIRES I and pIRES II contain gDp24 and gDE7 chimeric gene fusions, which are inverted with regard to the CMV promoter and IRES sequence. The empty vector pIRES Ø was used as a control. The nucleotide numbers corresponding to the IRES sequence and the cloned chimeric genes are indicated. (B) In vitro expression of the chimeric proteins encoded by pIRES I (left panels) and pIRES II (right panels). Non-permeabilized pIRES I- or pIRES II-transfected COS-7 cells were labeled with antigen-specific antibodies for the simultaneous detection of the HSV-1 protein gD and the HIV-1 protein p24 or the HPV-16 oncoprotein E7. Green, gD; red, p24 or E7; yellow, co-localization of gD with p24 or E7; blue, DAPI nuclear staining.</p

Induction of <i>in vivo</i> E7- and p24-specific cytolytic CD8⁺ T cell responses in immunized mice.

<p>(A–B) <i>In vivo</i> antigen-specific CD8⁺ T cell-dependent cytotoxic responses in the vaccinated mice was measured two weeks after the last immunization dose. Spleen cells from BALB/c or C57BL/6 mice were labeled with CFSE and pulsed with synthetic peptides representing the immunodominant MHC-I-restricted epitopes of p24 (A) or E7 (B). Data shown in A and B represent the compilation of two independent experiments, encompassing four and five mice per group (n = 9) with results based on the response of each animal. (C) The protective immunity elicited in BALB/c mice immunized with pIRES I or pIRES II was measured after challenge with a recombinant vaccinia virus expressing the HIV-1 protein Gag. Female BALB/c mice were challenged with 2×10⁶ P.F.U. of rVV-Gag, and 5 days later, the level of viable vaccinia virus in the ovaries was determined after titration in Vero cells. Data shown in C represent the compilation of two independent experiments carried out with pooled samples from five mice per group. *p<i><</i>0.05. (D) Prophylactic and (E) therapeutic anti-tumor immunity in C57BL/6 mice immunized with pIRES I or PIRES II. The prophylactic anti-tumor effects were determined in five vaccinated female mice after the s.c. transplantation of 7.5×10⁴TC-1 cells two weeks after the last vaccination. The therapeutic anti-tumor effects induced by the vaccines were determined after transplantation of 7.5×10⁴TC-1 cells one day before the administration of the first vaccine dose. Data shown in D and E represent the compilation of two independent experiments, with five mice per group. The survival curves D and E raised <i>p</i> values of 0.0001 and 0.0006, respectively, in the Logrank test for trend. pIRES is the empty vector used as immunization control.</p