Structure of the Metal–Support Interface and Oxidation State of Gold Nanoparticles Supported on Ceria

An Au/CeO₂ model catalyst was prepared by deposition–precipitation, with the aim of obtaining a sample suitable for a detailed X-ray absorption fine structure (XAFS) analysis of the gold–ceria interface structure. The results demonstrate the existence of a large interface between the gold particle and the support oxide, characterized by well-defined Au–O and Au–Ce interactions extending up to ∼6.4 Å. The complex interface structure is retained after CO treatment up to 250 °C and subsequent reoxidation at 400 °C. The analysis of the XANES spectra, and the Au–O distance of 2.21 Å, longer than Au–O bond lengths previously reported for Au/ceria catalysts, suggest a low oxidation state for the gold atoms placed at the interface between Au and ceria

Deletion of <i>A46R</i> gene from NYVAC-C enhances innate immune responses.

<p>Human macrophages were mock-infected (0) or infected with NYVAC-WT, NYVAC-C or NYVAC-C-Δ46R (1 or 5 PFU/cell). 24 hours later, cell-free supernatants were collected to quantify the concentrations of TNF and IL-6 by bioassay and of IL-8 by ELISA. Data are means ± SD of duplicates and are representative of three independent experiments. * <i>p</i><0.05, ** p<0.005.</p

Adaptive HIV-specific T cell immune responses elicited by <i>A46R</i> deletion mutant in the spleen of BALB/c mice in heterologous prime/boost immunization protocol.

<p>(A) Magnitude of the vaccine-specific CD4 or CD8 T cell response. The HIV-specific CD4 or CD8 T cells were measured 10 days after the last immunization by ICS assay following stimulation of splenocytes derived from immunized animals (n=4) with the different HIV peptide pools. The total value in each group represents the sum of the percentages of CD4⁺ or CD8⁺ T cells secreting IFN-γ and/or IL-2 and/or TNF-α (CD4) or CD107a and/or IFN-γ and/or IL-2 and/or TNF-α (CD8) against all HIV peptide pools. All data are background-subtracted. *** <i>p</i><0.001. <i>p</i> value indicates significantly higher responses compared to parental group or between DNA-C/NYVAC-C-ΔA46R and DNA-C/NYVAC-C immunization groups. (B) Flow cytometry profiles of vaccine-induced CD4 or CD8 T cell responses against Env pool. (C) Functional profile of the adaptive HIV-specific CD4 or CD8 T cell response in the different immunization groups. The possible combinations of the responses are shown on the <i>x</i> axis, whereas the percentages of the functionally distinct cell populations within the total CD4 or CD8 T cell population are shown on the <i>y</i> axis. Combinations that did not contribute significantly to the functional profile are not shown. Responses are grouped and colour-coded on the basis of the number of functions. The non-specific responses obtained in the control group DNA-ϕ/NYVAC-WT were subtracted in all populations. ** p<0.005, *** <i>p</i><0.001. <i>p</i> values indicate significantly higher responses compared to DNA-C/NYVAC-C immunization group.</p

Memory HIV-specific T cell immune responses elicited by <i>A46R</i> deletion mutant in the spleen of BALB/c mice after prime/boost immunization.

<p>(A) Magnitude of the vaccine-specific CD4 or CD8 T cell responses. The HIV-specific CD4 or CD8 T cells were measured 53 days after the last immunization by ICS assay following stimulation of splenocytes derived from immunized animals (n=4) with the different HIV peptide pools. The total value in each group represents the sum of the percentages of CD4⁺ or CD8⁺ T cells secreting IFN-γ and/or IL-2 and/or TNF-α (CD4) or CD107a and/or IFN-γ and/or IL-2 and/or TNF-α (CD8) against all HIV peptide pools. All data are background-subtracted. *** <i>p</i><0.001. <i>p</i> value indicates significantly higher responses compared to parental group or between DNA-C/NYVAC-C and DNA-C/NYVAC-C-ΔA46R immunization groups. (B) Functional profile of the memory HIV-specific CD8 T cell response in the different immunization groups. The possible combinations of the responses are shown on the <i>x</i> axis, whereas the percentages of the functionally distinct cell populations within the total CD8 T cell population are shown on the <i>y</i> axis. Combinations that did not contribute significantly to the functional profile are not shown. Responses are grouped and colour-coded on the basis of the number of functions. *** <i>p</i><0.001. <i>p</i> values indicate significantly higher responses compared to DNA-C/NYVAC-C immunization group. (C) Phenotypic profile of memory HIV-specific CD8 T cells. Representative FACS plots showing the percentage of Env-specific CD8 T cells with central memory (TCM; CD127⁺CD62L⁺), effector memory (TEM; CD127⁺CD62L^-) or effector (TE; CD127^-CD62L^-) phenotype.</p

Analysis of apoptosis induced by MVA-C-ΔF1L deletion mutant.

<p>(A) PARP cleavage was analyzed by Western-blot in HeLa cells mock-infected or infected with MVA wt, MVA-C or MVA-C-ΔF1L at 5 PFU/cell for 16 hours. Detection of cellular β-actin protein was used as an internal loading control. (B) Annexin V binding assay of HeLa, 3T3 cells or murine DCs infected with MVA wt, MVA-C or MVA-C-ΔF1L at 5 PFU/cell. At 16 hours post-infection, the infected cells were stained with Annexin V and propidium iodide as described under Materials and Methods and the percentages of early apoptotic cells (Annexin V positive, PI negative) were determined by flow cytometry. All data are mock-infected cells-subtracted. HeLa or 3T3 cells treated with staurosporine (0.5 µM) were used as positive control (not shown). *** <i>p</i><0.001. <i>p</i> value indicates significantly higher response compared to MVA wt or MVA-C-infected cells.</p

MVA-C-ΔF1L deletion mutant enhances the production of type I IFNs and cytokines in murine macrophages (A) and dendritic cells (B).

<p>BMDMs or BMDCs were mock-infected (0) or infected with MVA-C or MVA-C-ΔF1L at 1 or 5 PFU/cell for 6 hours (upper panels) or 24 hours (lower panels). IFN-α, IFN-β, IL-1β, TNF, IL-6, IL-12p40 and MIP-1α mRNA (upper panels) and protein (lower panels) levels were quantified by RT-PCR and ELISA, respectively. mRNA results are expressed as the ratio of the gene of interest to <i>Hprt</i> mRNA levels. A.U: arbitrary units. ELISA results are means ± SD of duplicate samples from one experiment and are representative of two experiments. * <i>p</i><0.05 for all conditions comparing MVA-C-ΔF1L to MVA-C at the same MOI.</p

Generation and <i>in vitro</i> characterization of MVA-C-ΔF1L deletion mutant.

<p>(A) Scheme of construction of the plasmid transfer vector pGem-RG-F1L. The plasmid transfer vector pGem-RG-F1L was obtained by sequential cloning of markers dsRed2 and rsGFP and <i>F1L</i> recombination flanking sequences into the plasmid pGem-7Zf. The dsRed2 gene under the control of the synthetic early/late (E/L) promoter was excised from plasmid pG-dsRed2 and inserted into pGem-7Zf to generate pGem-Red. The rsGFP gene under the control of the synthetic E/L promoter was amplified by PCR from plasmid pG-dsRed2, digested and inserted into the plasmid pGem-Red to generate pGem-Red-GFP. MVA genome was used as template to amplify the left flanking sequence of <i>F1L</i> gene. The PCR product was digested and inserted into pGem-Red-GFP previously digested and dephosphorylated by incubation with Calf intestinal Alkaline Phosphatase (CIP) to generate the plasmid pGem-RG-fiF1L. The right flanking sequence of <i>F1L</i> gene was amplified by PCR from MVA genome. The PCR product was digested and inserted into plasmid pGem-RG-fiF1L to generate the plasmid transfer vector pGem-RG-F1L. (B) Confirmation of <i>F1L</i> gene deletion by PCR analysis. Viral DNA was extracted from DF-1 cells infected with MVA wt or MVA-C-ΔF1L at 5 PFU/cell. Primers fdF1L-S and fiF1L-BR spanning <i>F1L</i> flanking sequences were used for PCR analysis of <i>F1L</i> locus. In parental MVA, a 1380 bp-product is obtained while in deletion mutant a unique 1529 bp-product is observed. (C) Analysis of virus growth of MVA-C-ΔF1L in CEF cells. Monolayers of CEF cells were infected with MVA-C or MVA-C-ΔF1L at 0.01 PFU/cell. At different times post-infection (0, 24, 48 and 72 hours), cells were collected and infectious viruses were quantified by immunostaining assay.</p

Analysis of the stability of gp120 and GPN proteins expressed by MVA-C-ΔF1L.

<p>(A) Thirty individual plaques from MVA-C-ΔF1L were grown in DF-1 cells, lysed, proteins fractionated by SDS-PAGE and analyzed by Western-blot with specific antibodies. The expression of gp120 or GPN proteins in mock-infected cells or cells infected with MVA wt, MVA-C or with individual plaques from MVA-C-ΔF1L (1–30) is shown. Arrows indicate the correct size of gp120 and GPN proteins. (B) Schematic representation of the HIV-1 inserts within the TK viral locus of MVA-C. The positions and sequences of the different sets of primers used for PCR analysis and sequencing of GPN polyprotein and the expected sizes of PCR products are represented. (C) Identification of mutations of GPN polyprotein. Viral DNA extracted from DF-1 cells infected with plaques 19 or 26 of MVA-C-ΔF1L was used as template to amplify and sequence different regions of GPN polyprotein. In plaque 19, one cytosine insertion was identified at position 1355–1362 producing a frame-shift and a premature stop codon at position 3216. In plaque 26, one cytosine insertion was identified at position 3136–3141 producing a frame-shift and a premature stop codon at position 3216.</p

Functional profile of the adaptive HIV-1-specific CD8 T cell response in the different immunization groups.

<p>All the possible combinations of the responses are shown on the <i>x</i> axis, whereas the percentages of the functionally distinct cell populations within the total CD8 T cell population are shown on the <i>y</i> axis. Responses are grouped and color-coded on the basis of the number of functions. The non-specific responses obtained in the control group DNA-φ/MVA wt were subtracted in all populations. * <i>p</i><0.05; *** <i>p</i><0.001. <i>p</i> values indicate significantly higher responses compared to DNA-C/MVA-C immunization group.</p

Functional profile and expression of co-stimulatory molecules and of co-inhibitory receptors of HIV-specific CD8 T-cell responses during acute and chronic HIV infections.

<p>Analysis of the functional profile (<b>A</b>), of the expression of CD27 and CD28 (<b>B</b>) and of the expression of 2B4, CD160 and PD-1 (<b>C</b>) in HIV-specific CD8 T cells in patients with acute (PHI-B), untreated chronic progressive (CP-B) or non-progressive (LTNP) HIV infection. Representative examples of the distinct flow cytometry panels are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003423#ppat.1003423.s001" target="_blank">Fig. S1B</a>-D. Regarding the functional profile (<b>A</b>), although TNF-α was detected, analyses are restricted to the expression of IFN-γ, IL-2 and perforin for clarity. All possible combinations of the distinct markers are shown on the <i>x</i> axis, whereas the percentages of the distinct cell subsets within virus-specific CD8 T cells are shown on the <i>y</i> axis. The pie charts summarize the data, and each slice corresponds to a certain combination of molecules. Colors in the pie charts are based on the colored boxes at the bottom of the panel.</p