<i>In vitro</i> characterization of MVA-B deletion mutants.

<p>(A) Scheme of MVA-B deletion mutant's genome map, adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066894#pone.0066894-Antoine1" target="_blank">[25]</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066894#pone.0066894-Najera2" target="_blank">[69]</a>. The different regions are indicated by capital letters. The right and left terminal regions are shown. Below the map, the deleted or fragmented genes are depicted as black boxes. The deleted <i>C6L</i> and <i>K7R</i> genes are indicated. The HIV-1 Gag-Pol-Nef (from isolate IIIB) and gp120 (from isolate BX08) clade B sequences driven by the synthetic early/late (sE/L) virus promoter inserted within the TK viral locus (J2R) are indicated (adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066894#pone.0066894-Gomez1" target="_blank">[16]</a>). (B) PCR analysis of <i>C6L</i> and <i>K7R</i> loci. Viral DNA was extracted from DF-1 cells mock-infected or infected at 1 PFU/cell with MVA-WT, MVA-B, MVA-B ΔC6L or MVA-B ΔC6L/K7R. Primers spanning <i>C6L- or K7R-</i>flanking regions were used for PCR analysis of the <i>C6L</i> or <i>K7R</i> loci, respectively. The DNA products corresponding to the parental virus or to the deletion are indicated by an arrow on the right. Molecular size marker (1 Kb ladder) with the corresponding sizes (base pairs) is indicated on the left. Lane Mock, cells not infected. (C) Expression of HIV-1 _BX08gp120 and _IIIBGPN proteins. DF-1 cells were mock-infected or infected at 1 PFU/cell with MVA-WT, MVA-B, MVA-B ΔC6L or MVA-B ΔC6L/K7R. At 24 hours post-infection, cells were lysed in Laemmli buffer, fractionated by 8% SDS-PAGE and analyzed by Western-blot using rabbit polyclonal anti-gp120 antibody or polyclonal anti-gag p24 serum. Arrows on the right indicate the position of HIV-1 _BX08gp120 and _IIIBGPN proteins. (D) Viral growth kinetics in DF-1 cells. DF-1 cells were infected at 0.01 PFU/cell with MVA-B, MVA-B ΔC6L or MVA-B ΔC6L/K7R. At different times post-infection (0, 24, 48, and 72 hours), cells were harvested and virus titers in cell lysates were determined by plaque immunostaining assay with anti-WR antibodies. The mean of two independent experiments is shown.</p

Immunization with MVA-B deletion mutants enhances the magnitude and polyfunctionality of HIV-1-specific CD4⁺ and CD8⁺ T cell memory immune responses.

<p>Splenocytes were collected from mice (n = 4 per group) immunized with DNA-φ/MVA-WT, DNA-B/MVA-B, DNA-B/MVA-B ΔC6L or DNA-B/MVA-B ΔC6L/K7R 52 days after the last immunization and HIV-1-specific CD4⁺ and CD8⁺ T cell memory immune responses were analyzed by flow cytometry as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066894#pone-0066894-g003" target="_blank">Figure 3</a>. Values from unstimulated controls were subtracted in all cases. <i>p</i> values indicate significantly higher responses compared to DNA-B/MVA-B immunization group, and also between MVA-B deletion mutants. Data are from one experiment representative of three experiments. (A) Magnitude of HIV-1-specific CD4⁺ and CD8⁺ T cells. The values represent the sum of the percentages of T cells secreting IFN-γ and/or TNF-α and/or IL-2 against Env+Gag+GPN peptide pools. *** <i>p</i><0.001. (B) Percentage of Env, Gag and GPN HIV-1-specific CD4⁺ and CD8⁺ T cell memory immune responses. Frequencies were calculated by reporting the number of T cells producing IFN-γ and/or TNF-α and/or IL-2 to the total number of CD4⁺ and CD8⁺ T cells in the different immunization groups. (C) Polyfunctionality of HIV-1-specific CD4⁺ and CD8⁺ T cells. Functional profile of HIV-1-specific CD4⁺ (left panel) and CD8⁺ (right panel) T cell memory immune responses in the different immunization groups. Responses are grouped and color-coded on the basis of the number of functions. All the possible combinations of the responses are shown on the X axis. The percentages of T cells secreting IFN-γ and/or TNF-α and/or IL-2 against Env+Gag+GPN peptide pools are shown on the Y axis. The pie charts summarize the data. Each slice corresponds to the proportion of HIV-1-specific CD4⁺ or CD8⁺ T cells producing one, two or three cytokines (IFN-γ and/or TNF-α and/or IL-2) within the total HIV-1-specific CD4⁺ or CD8⁺ T cells. ** <i>p</i><0.005, *** <i>p</i><0.001.</p

Complex antigen presentation pathway for an HLA-A*0201-restricted epitope from Chikungunya 6K protein

<div><p>Background</p><p>The adaptive cytotoxic T lymphocyte (CTL)-mediated immune response is critical for clearance of many viral infections. These CTL recognize naturally processed short viral antigenic peptides bound to human leukocyte antigen (HLA) class I molecules on the surface of infected cells. This specific recognition allows the killing of virus-infected cells. The T cell immune T cell response to Chikungunya virus (CHIKV), a mosquito-borne <i>Alphavirus</i> of the <i>Togaviridae</i> family responsible for severe musculoskeletal disorders, has not been fully defined; nonetheless, the importance of HLA class I-restricted immune response in this virus has been hypothesized.</p><p>Methodology/Principal findings</p><p>By infection of HLA-A*0201-transgenic mice with a recombinant vaccinia virus that encodes the CHIKV structural polyprotein (rVACV-CHIKV), we identified the first human T cell epitopes from CHIKV. These three novel 6K transmembrane protein-derived epitopes are presented by the common HLA class I molecule, HLA-A*0201. One of these epitopes is processed and presented via a complex pathway that involves proteases from different subcellular locations. Specific chemical inhibitors blocked these events in rVACV-CHIKV-infected cells.</p><p>Conclusions/Significance</p><p>Our data have implications not only for the identification of novel <i>Alphavirus</i> and <i>Togaviridae</i> antiviral CTL responses, but also for analyzing presentation of antigen from viruses of different families and orders that use host proteinases to generate their mature envelope proteins.</p></div

MVA-B deletion mutants induces the production of IFN-β, type I IFN inducible genes, TNF-α and MIP-1α in macrophages and dendritic cells.

<p>Human THP-1 macrophages (A) and moDCs (B, C) were mock-infected or infected with MVA-WT, MVA-B, MVA-B ΔC6L or MVA-B ΔC6L/K7R (5 PFU/cell in A, and 1 PFU/cell in B and C). At different time post-infection (3 h and 6 h in A, 6 h in B), RNA was extracted and the mRNA levels of IFN-β, TNF-α, MIP-1α, type I IFN inducible genes (IFIT1 and IFIT2), and HPRT were analyzed by RT-PCR. Results were expressed as the ratio of gene to HPRT mRNA levels. A.U: arbitrary units. <i>p</i> values indicate significantly higher responses compared to DNA-B/MVA-B immunization group. * <i>p</i><0.05, ** <i>p</i><0.005, *** <i>p</i><0.001. Data are means ± SD of duplicate samples and are representative of three independent experiments. (C) Human moDCs were mock-infected or infected with 1 PFU/cell of MVA-WT, MVA-B, MVA-B ΔC6L or MVA-B ΔC6L/K7R. Six hours later, cell-free supernatants were collected to quantify the concentration of IFN-β by ELISA and the concentration of TNF-α and MIP-1α by Luminex. Data are means ± SD of duplicates and are representative of three independent experiments.</p

Recognition of infected HLA-A*0201⁺ DC by CHIKV 6K_51-59- or VACV D12I_251-259-specific CD8⁺ T cells in the presence of various protease inhibitors.

<p>Mouse HLA-A*0201⁺ DC infected as in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006036#pntd.0006036.g004" target="_blank">Fig 4</a> were treated before ICS assay with protease inhibitors including DANLME (aspartic protease A1 inhibitor), dec-RVKR (furin and other members of the propotein convertase family), STBI (trypsin, chymotrypsin and plasmin), Z-LL₂ (signal peptide peptidase), or zVAD (caspase). The percentage of specific inhibition was calculated as in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006036#pntd.0006036.g005" target="_blank">Fig 5</a>. Data shown as mean ± SD of four independent experiments (*** P <0.001 of CHIKV 6K_51-59-specific CD8⁺ T cells vs. no inhibitor (white asterisks) or VACV D12I_251-259 ligand with the inhibitor (black asterisks)).</p

Humoral immune responses elicited by MVA-B and MVA-B deletion mutants against HIV-1 gp120 protein.

<p>Serum was collected from individual mouse immunized with DNA-B/MVA-B, DNA-B/MVA-B ΔC6L or DNA-B/MVA-B ΔC6L/K7R or DNA-φ/MVA-WT (n = 4), 10 days (left panel) or 52 days (right panel) after the last immunization. Levels of gp120-specific IgG binding antibodies were measured by ELISA. Titers represent the last dilution of the serum that signals 3-fold higher than signals obtained with the serum of naïve mice. The horizontal bar represents the mean antibodies titer for each group. Each dot represents data obtained from one mouse. Data are from one experiment representative of three experiments.</p

Diversity of proteases and processing pathway involved in CHIKV 6K_51-59 epitope presentation.

<p>The model shows the components of the antigen presentation pathway proposed for the CHIKV 6K_51-59 epitope. Stop-transfer signals are indicated by rectangular blocks, and signal sequences by dashed cylinders. Subcellular organelles are shown as colored boxes: cytosol (yellow), ER (blue), trans-Golgi network (mauve) and lysosomes (green). CHIKV proteins are capsid (CP, maroon), p62 (yellow), 6K (green), E1 (peach), E2 (blue), and E3 (brown). The CHIKV 6K_51-59 epitope is depicted in red. The role of the distinct proteases is deduced from CD8⁺ T cell sensitivity to the various inhibitors, except for the signal peptidase, whose role was described in the generation of the Alphavirus 6K protein [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006036#pntd.0006036.ref059" target="_blank">59</a>].</p

Effect of LC and Leu-SH inhibitors on recognition of CHIKV 6K_51-59- or VACV D12I_251-259 viral epitopes.

<p>Mouse HLA-A*0201⁺ DC infected as in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006036#pntd.0006036.g004" target="_blank">Fig 4</a> were treated before ICS assay with LC (proteasome inhibitor) or Leu-SH (ERAP and other metallo-aminopeptidases). The percentage of specific inhibition was calculated as in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006036#pntd.0006036.g005" target="_blank">Fig 5</a>. Data shown as mean ± SD of four independent experiments (*** P <0.001; ** P <0.01, as in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006036#pntd.0006036.g007" target="_blank">Fig 7</a>).</p

Phenotypic profile of memory HIV-1-specific CD4⁺ and CD8⁺ T cells.

<p>FACS plots of HIV-1 Env-, Gag- and GPN-specific CD4⁺ and CD8⁺ T memory cells. CD127 and CD62L expression was used to identify central memory (Tcm: CD127⁺/CD62L⁺), effector memory (Tem CD127⁺/CD62L⁻) and effector (Te: CD127⁻/CD62L⁻) sub-populations. The T cell memory sub-populations are depicted as density plots. Blue dots represent T cells producing IFN-γ and/or TNF-α and/or IL-2. Data are from one experiment representative of three experiments.</p

CHIKV 6K peptide specificity of HLA-A*0201-restricted CD8⁺ T cell lines.

<p>Mouse HLA-A*0201⁺ DC pre-pulsed with 10⁻⁶ M of indicated CHIKV 6K synthetic peptide were analyzed by ICS for CD8⁺ T cell activation with CHIKV peptide-specific CD8⁺ T cells from HLA-A*0201-transgenic mice immunized with rVACV-CHIKV and restimulated <i>in vitro</i> with the appropriate CHIKV 6K synthetic peptide. Graph data shown as mean ± SD of four independent experiments (*** P <0.001). Representative ICS panels with non-specific or CHIKV peptide-specific CD8⁺ T cell lines are depicted beneath the graphs. The percentages of IFNγ-expressing CD8⁺ T cells are indicated in each dot plot.</p