Assembly, structures and mass spectrum analyses of prototype multivalent B, CD4+ and CD8+ epitopes HER-2 glyco-lipopeptide molecules.

<p>(<b>A</b>) The RAFT moiety <b>1</b> was assembled from an orthogonally protected linear decapeptide following the standard Fmoc/tBu strategy, as we previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011216#pone.0011216-Renaudet1" target="_blank">[21]</a>. The amino acid sequences of CD4⁺ and CD8⁺ epitopes and cyclic template are given using one letter code. Unusual amino acids are designated as dA (L-alanine), Cha (cyclohexyl alanine) and Ahx (L-2-aminohexanoic acid). Each compound displays clustered Tn analog on the upper domain of the cyclodecapeptide RAFT template. HER-GP and HER-GLP contain respectively either HER_420–429-PADRE chimeric peptide or PAM- HER_420–429-PADRE chimeric lipopeptide on the lower domain. (<b>B</b>) Mass spectrum (MS) analysis was obtained by electron spray ionization (ESI-MS) in the positive mode. The multi-charged ions observed for HER-GP (<i>m/z</i>: 841.7 [M+6H]⁶⁺, 1009.7 [M+5H]⁵⁺, 1261.7 [M+4H]⁴⁺, 1682.2 [M+3H]³⁺), HER-GLP-1 (<i>m/z</i>: 882.5 [M+6H]⁶⁺, 1057.5 [M+5H]⁵⁺, 1321.4 [M+4H]⁴⁺, 1761.6 [M+3H]³⁺) and HER-GLP-2 (<i>m/z</i>: 881.3 [M+6H]⁶⁺, 1057.6 [M+5H]⁵⁺, 1321.4 [M+4H]⁴⁺, 1761.7 [M+3H]³⁺) correspond to the expected deconvoluated masses calculated for [M+H]⁺ (5044.3 for HER-GP, 5282.8 for HER-GLP-1 and 5282.9 for HER-GLP-2).</p

Immunotherapeutic efficacy of linear HER-GLP-1 and branched HER-GLP-2 vaccine constructs.

<p>NT2 cells (1×10⁵/mouse) were injected s.c. in the mammary fat pad of 40 female B10.D1 mice (5 wk old). Eight days later, when tumor diameter reached 3–4 mm, mice were divided into 4 groups of 10 mice each. Group 1 was immunized s.c. four times at seven day intervals with the self-adjuvanting linear HER-GLP-1 (<i>GLP-1</i>), group 2 was immunized with the branched HER-GLP-2 (<i>GLP-2</i>), group 3 was immunized with both HER-GLP-1 and HER-GLP-2 (<i>both</i>), and group 4 was injected with PBS alone as control (<i>Mock</i>). (<b>A</b>) <b>Tumor progression.</b> Local tumor dimensions were measured with calipers as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011216#s4" target="_blank"><i>Materials and Methods</i></a>. The average of tumor diameters (in millimeters) in the course of 50 days is presented. (<b>B</b>) <b>Survival.</b> Mice from the same experiment were monitored daily for 90 days and were sacrificed when moribund, which corresponded to a tumor diameter of 18 mm. The results are presented as mean+SEM. Both <b>A</b> and <b>B</b> present <i>p</i> values calculated to compare the two groups of HER-GLP-1 and HER-GLP-2 immunized mice (i.e. group 1 and group 2). Data are representative of two independent experiments.</p

Cross-presentation pathways of linear HER-GLP-1 and branched HER-GLP-2 vaccine constructs in dendritic cells.

<p>(<b>A</b>) Dendritic cells were pre-incubated with brefeldin A for 1 h, followed by the addition of linear HER-GLP-1, branched HER-GLP-2 or non-lipidated HER-GP construct (<i>open bars</i>). As a positive control, DCs were left untreated with antigen-processing inhibitors but were pulsed with linear HER-GLP-1, branched HER-GLP-2 or parent non-lipidated HER-GP (<i>open bars</i>). After overnight incubation, DCs were then washed and added to the HER_420–429-specific CD8⁺ T cells for additional 5 hrs. IFN-γ produced by HER_420–429-specific CD8⁺ T cells was tested by ELISpot assay. Panel (<b>B</b>) and (<b>C</b>) represents identical experiments conducted in the presence of epoxomycin and monensin inhibitors, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011216#s4" target="_blank"><i>Materials and Methods</i></a>. Results are representative of three independent experiments.</p

Immunization with branched HER-GLP-2 induces stronger RAFT-specific IgGs than linear HER-GLP-1.

<p>Three groups of B10.D1 mice (<i>n</i> = 10) were immunized subcutaneously two times with an interval of 14 days, with linear HER-GLP-1 (50 µM/mouse) or branched HER-GLP-2 (50 µM/mouse) or non-lipidated HER-GP (50 µM/mouse) as shown in (panel <b>A</b>) or injected with PBS alone. Ten days after the 2^nd immunization, serum was collected from each mouse and the level of RAFT-specific IgG (panel <b>B</b>) was measured by ELISA. MCF7 cells (4×10⁵ cells) were incubated for 30 min with 10 µl of mice sera from HER-GLP-1, HER-GLP-2, HER-GP immunized or PBS-injected control mice (Mock) at 1∶250 dilutions and analyzed by flow cytometry using FITC labeled goat anti mouse IgG antibody. The binding efficiency was calculated in terms of mean fluorescent intensity (MFI) after subtracting the background of serum binding from mock-immunized mice (panel <b>C</b>). A representative data showing the binding of serum from HER-GLP-1 and HER-GLP-2 immunized mice (solid lines) and serum from mock-immunized mice (broken lines) with MCF7 cells (panel <b>D</b>). The results are representative of three experiments.</p

Strength measurements and histological stainings of quadriceps muscle of WT and VCP^R155H/+ mice on normal and different lipid-enriched diets.

<p>(A,B) Grip strength and Rotarod measurements analyses at 15-months of age, (C,D) H&E and (E,F) Oil Red O staining of quadriceps muscle from WT and VCP^{<b>R155H/+</b>} animals placed on the 9%, 12%, 30%, and 48% LED from birth. The number of mice analyzed per experiment is 8–10. Insets show magnified pathology, including rimmed vacuoles (orange arrows), atrophy (blue arrows), centralized nuclei (green arrows), and increased lipid droplets (white arrows).</p

Phenotypic and functional maturation of dendritic cells induced by linear HER-GLP-1, branched HER-GLP-2 and HER-GP molecules.

<p>(<b>A</b>) Immature DCs were derived from mouse bone marrow and either left untreated (none) or incubated in vitro for 48 hrs with equimolar amount of linear HER-GLP-1, branched HER-GLP-2, or non-lipidated HER-GP analog. Phenotypic markers for DC maturation (major histocompatibility complex (MHC) class II, CD80, CD86) were analyzed by FACS and plotted in terms of calculated MFI. (<b>B</b>) IL-12p35 and TNF-α released by linear HER-GLP-1, branched HER-GLP-2 and HER-GP induced matured DCs were measured by cytokine assay, as described in <i>Materials and Method</i>. Panel (<b>C</b>) shows the Inhibition of HER_420–429-Specific IFN-γ spot-forming CD8+ T cells by anti TLR2 antibody as described in <i>Materials and Method</i>. The results are representative of three experiments.</p

Survival curves of WT, VCP^R155H/+, and VCP^R155H/R155H mice on normal and varying lipid-enriched diets.

<p>Kaplan-Meier survival probability (%) analysis for the WT, VCP^{<b>R155H/+</b>}, and VCP^{<b>R155H/R155H</b>} pregnant dams placed on the (A) 6% normal diet, (B) 9% LED, (C) 12% LED, (D) 30% LED, and (E) 48% LED. (F) Combined survival curve of all the LED regimens for the WT, VCP^{<b>R155H/+</b>}, and VCP^{<b>R155H/R155H</b>}. The number of mice analyzed per experiment is 35–40.</p

Immunization with linear HER-GLP-1 induces stronger and long-lasting HER_420–429-specific IFN-γ producing CD8+ T cell responses than branched HER-GLP-2.

<p>Three groups of B10.D1 mice (<i>n</i> = 10) were immunized subcutaneously three times with HER-GLP-1 (50 µM/mouse), HER-GLP-2 (50 µM/mouse) or HER-GP (50 µM/mouse) as shown in panel <b>A</b>) or injected with PBS alone (Mock) with an interval of 14 days. Ten days after the 3^rd immunization, spleen cells were isolated and stimulated <i>in vitro</i> for 4 days with HER_420–429 peptide and assayed for IFN-γ producing CD8+ T cells by ELISpot. Mean values (± SD) of IFN-γ spot-forming CD8+ T cells were plotted against each group of mice and are shown in (<b>B</b>). Kinetics of HER_420–429-specific IFN-γ producing CD8+ T cells were measured in mice immunized with HER-GLP-1, HER-GLP-2 and HER-GP from 0 to 60 days of post immunization and is shown in (<b>C</b>). The results are representative of three experiments.</p

Relative uptake of linear HER-GLP-1; branched HER-GLP-2 and non-lipidated HER-GP molecules by bone marrow derived immature dendritic cells.

<p>(<b>A</b>) Primary cultures of bone marrow derived DC populations were incubated for 30 min at 37°C with Alexa Fluor 488-labeled HER-GLP-1, HER-GLP-2 or HER-GP at an equimolar concentration of 1 uM each. Left panel shows the dot plot representation of loaded HER-GLP and HER-GP constructs on CD11b/c⁺ cells and right panel shows the subsequent cytoplasmic localization of HER-GLP and HER-GP constructs by confocal microscopy. (<b>B</b>) Shows the uptake kinetics of HER-GLP and HER-GP constructs by CD11b/c⁺ cells following incubation at different time intervals as measured by the endocytosis assay uptake. Endocytosis assay uptake of Alexa Fluor 488-labeld HER-GP and HER-GLPs by DCs was determined for 120 min both at 4C° and 37C°. Subsequently, DCs were washed three times with phosphate-buffered saline/bovine serum albumin 1% and total uptake of Alexa Fluor 488-labeld was measured by FACS analyses and expressed by the difference in geometric mean that resulted from subtracting the values obtained at 4C° from the values obtained at 37C°. This formula determines the amount of HER-GP and HER-GLPs that is actively internalized. The results are representative of five experiments.</p

HIV-1 Tat induces IL-6 and IL-8 production in monocytes/macrophages.

<p>(<b>A-B</b>) and <b>(C-D)</b> Human monocytes (0.5x10⁶) or <b>(E-F)</b> Wt peritoneal mice macrophages (0.5x10⁶) were incubated with increasing amounts of recombinant GST-Tat 1–101 protein (Tat), a deleted mutant GST-Tat 1–45 protein carrying the first 45 amino acid (Tat 1–45) or an equal amount of GST protein alone (GST). Untreated cells were used as negative controls. Tat heat-inactivated for 20 min at 95°C or Tat previously incubated with mAb anti-Tat for 60 min at 37°C, or GST were used for the control of the specificity. <b>(C-D)</b> Monocytes were isolated from PBMC using either adherence protocol as described in Material and Methods, or positive selection using CD14 MicroBead according to the manufacturer instruction (Miltenyi Biotec), CD14 negative fraction of PBMC was used as control. Cells were either kept untreated or treated with Synthetic Tat. After 24 h of treatment, human or mouse IL-6 and human IL-8 and mouse CXCL1/KC cytokines were measured in the culture supernatants by ELISA. Cytokine production is expressed in ng/ml. The data represent means and standard deviation (SD) of three independent experiments. Asterisks represent <i>P</i> values comparing "untreated" group versus "Treated (as indicated)" group * for p < 0.05, ** p < 0.01, *** p < 0.001, ns non significant. Statistical significance comparing different group linked with a black line above the compared bar and are denoted with # for p < 0.05, ## p < 0.01, ### p < 0.001, ns non significant.</p