1,721,005 research outputs found
Analysis of apoptosis induced by MVA-C-ΔF1L deletion mutant.
No full text<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
Significant genes associated with specific immune function in MVA-C-infected mDCs and pDCs.
No full text<p>A) Gene expression heatmap using selected set of immune related genes resulting from comparing myeloid DCs infected with MVA-C (MDC_MVC) and mock-infected (MDC_Mock) groups. B) Gene expression heatmap using selected set of IFN-induced genes resulting from comparing myeloid DCs infected with MVA-C (MDC_MVC) and mock-infected (MDC_Mock) groups. C) Gene expression heatmap using selected set of IFN signaling genes resulting from comparing plasmacytoid DCs infected with MVA-C (PDC_MVC) and mock-infected (PDC_Mock) groups. D) Gene expression heatmap using selected set of inflammation associated genes resulting from comparing myeloid DCs infected with MVA-C (MDC_MVC) and mock-infected (MDC_Mock) groups. E) Gene expression heatmap using selected set of inflammation associated genes resulting from comparing plasmacytoid DCs infected with MVA-C (PDC_MVC) and mock-infected (PDC_Mock) groups. The genes selected are differentially expressed (FC>1.3 and adjusted p-value<0.05). Red and Blue correspond to up- and down-regulation respectively.</p
Genes induced by MVA-C in human mDCs and pDCs.
No full text<p>A) Gene expression heatmap using the top 50 differentially expressed genes resulting from comparing myeloid DCs infected with MVA-C (MDC_MVC) and mock-infected (MDC_Mock) groups. B) Gene expression heatmap using the top 50 differentially expressed genes resulting from comparing plasmacytoid DCs infected with MVA-C (PDC_MVC) and mock-infected (PDC_Mock) groups. Genes selected as differentially expressed based on the following criteria: adjusted p-value<0.05 and |FC|>1.3. The scale shows the level of gene expression where Red and Blue correspond to up- and down-regulation respectively.</p
Analysis of the stability of gp120 and GPN proteins expressed by MVA-C-ΔF1L.
No full text<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
Signaling pathways induced upon MVA-C infection of mDCs and pDCs.
No full text<p>Selected significant top 10 pathways and their genes members resulting from comparing myeloid DCs infected with MVA-C (MDC_MVC) and mock-infected (MDC_Mock) groups (A) or plasmacytoid DCs infected with MVA-C (PDC_MVC) and mock-infected (PDC_Mock) groups (B). Each row is an upregulated canonical pathway for innate immunity (Ingenuity software); each column represents an up- (red) or down- (blue) regulated gene (p<0.05 and |FC|>2) included in 1 or more regulated pathway(s). The over-representation test was performed using Fisher Exact Test and the significance, displayed on the right side, is achieved for p<0.05 (-log(p)>1.3).</p
MVA-C induces cytokine and chemokine production in human primary macrophages.
No full text<p>A) Human primary macrophages were infected at 5 pfu/cell with MVA-WT or MVA-C. At 3 h.p.i., cells were collected, RNA extracted and levels of different mRNAs were defined by RT-PCR. B) Human primary macrophages were infected (1 or 5 pfu/cell) with MVA-WT or MVA-C. Supernatants were collected at 24 h and used to quantify IL-1β, IL-8, IFNβ, MIP-1α, RANTES, IP-10 and IFNα by ELISA. Experiments were performed in triplicate samples from two human donors. Data are means and standard deviation from one experiment representative of at least two experiments.</p
Systems analysis of MVA-C induced immune response reveals its significance as a vaccine candidate against HIV/AIDS of clade C.
Get PDFBased on the partial efficacy of the HIV/AIDS Thai trial (RV144) with a canarypox vector prime and protein boost, attenuated poxvirus recombinants expressing HIV-1 antigens are increasingly sought as vaccine candidates against HIV/AIDS. Here we describe using systems analysis the biological and immunological characteristics of the attenuated vaccinia virus Ankara strain expressing the HIV-1 antigens Env/Gag-Pol-Nef of HIV-1 of clade C (referred as MVA-C). MVA-C infection of human monocyte derived dendritic cells (moDCs) induced the expression of HIV-1 antigens at high levels from 2 to 8 hpi and triggered moDCs maturation as revealed by enhanced expression of HLA-DR, CD86, CD40, HLA-A2, and CD80 molecules. Infection ex vivo of purified mDC and pDC with MVA-C induced the expression of immunoregulatory pathways associated with antiviral responses, antigen presentation, T cell and B cell responses. Similarly, human whole blood or primary macrophages infected with MVA-C express high levels of proinflammatory cytokines and chemokines involved with T cell activation. The vector MVA-C has the ability to cross-present antigens to HIV-specific CD8 T cells in vitro and to increase CD8 T cell proliferation in a dose-dependent manner. The immunogenic profiling in mice after DNA-C prime/MVA-C boost combination revealed activation of HIV-1-specific CD4 and CD8 T cell memory responses that are polyfunctional and with effector memory phenotype. Env-specific IgG binding antibodies were also produced in animals receiving DNA-C prime/MVA-C boost. Our systems analysis of profiling immune response to MVA-C infection highlights the potential benefit of MVA-C as vaccine candidate against HIV/AIDS for clade C, the prevalent subtype virus in the most affected areas of the world
Systems analysis of MVA-C induced immune response reveals its significance as a vaccine candidate against HIV/AIDS of clade C
Get PDFBased on the partial efficacy of the HIV/AIDS Thai trial (RV144) with a canarypox vector prime and protein boost, attenuated poxvirus recombinants expressing HIV-1 antigens are increasingly sought as vaccine candidates against HIV/AIDS. Here we describe using systems analysis the biological and immunological characteristics of the attenuated vaccinia virus Ankara strain expressing the HIV-1 antigens Env/Gag-Pol-Nef of HIV-1 of clade C (referred as MVA-C). MVA-C infection of human monocyte derived dendritic cells (moDCs) induced the expression of HIV-1 antigens at high levels from 2 to 8 hpi and triggered moDCs maturation as revealed by enhanced expression of HLA-DR, CD86, CD40, HLA-A2, and CD80 molecules. Infection ex vivo of purified mDC and pDC with MVA-C induced the expression of immunoregulatory pathways associated with antiviral responses, antigen presentation, T cell and B cell responses. Similarly, human whole blood or primary macrophages infected with MVA-C express high levels of proinflammatory cytokines and chemokines involved with T cell activation. The vector MVA-C has the ability to cross-present antigens to HIV-specific CD8 T cells in vitro and to increase CD8 T cell proliferation in a dose-dependent manner. The immunogenic profiling in mice after DNA-C prime/MVA-C boost combination revealed activation of HIV-1-specific CD4 and CD8 T cell memory responses that are polyfunctional and with effector memory phenotype. Env-specific IgG binding antibodies were also produced in animals receiving DNA-C prime/MVA-C boost. Our systems analysis of profiling immune response to MVA-C infection highlights the potential benefit of MVA-C as vaccine candidate against HIV/AIDS for clade C, the prevalent subtype virus in the most affected areas of the world
Generation and <i>in vitro</i> characterization of MVA-C-ΔF1L deletion mutant.
No full text<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
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