Alphaviruses are unique in allowing PKR activation and eIF2α phosphorylation in infected cells.

<p><i>A</i>) Susceptibility of MEFs to viruses used in this study. Cells were infected with the indicated viruses to a moi of 25 pfu/cell and 6 h later pulsed with [³⁵S]-Met for 30 min. Labeled proteins were resolved by SDS-PAGE followed of autoradiography. <i>B</i>) PKR activation and eIF2α phosphorylation in wild type or <i>PKR</i>^o/o MEFs infected with the indicated viruses at 4 and 6 hpi. Note the mobility shift of PKR band upon activation in SINV and SFV-infected cells (upper panel). eIF2α phosphorylation in wild type (+/+) and <i>PKR</i> knock-out cells (o/o) infected with the indicated viruses. Only Alphavirus-infected cultures showed a strong eIF2α phosphorylation. For VSV-infected cells, a slight increase in eIF2α phosphorylation was also observed.</p

eIF2α phosphorylation and translational resistance of SINV virus also operates in infected animals.

<p><i>A</i>) IF analysis of brains from wild type and <i>PKR</i> knock-out mice infected with SINV at 4 dpi. Adjacent sections were incubated with anti-SINV and anti-phosphoeIF2α antibodies as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016711#s4" target="_blank">Materials and Methods</a>. <i>B</i>) IF analysis of spleens from mice infected with VV-Luc at 1dpi. Sections were incubated with anti-VVp14 (reactive against the envelope protein A27) and anti-phosphoeIF2α antibodies. Note that spleen cells expressing viral antigens did not react with anti- phosphoeIF2α antibodies. <i>C</i>) Expression of <i>PKR</i> in different mouse organs from uninfected animals. Equivalent amounts of protein extracts were analyzed by immunoblot against PKR, total eIF2α and β-actin. <i>D</i>) Attenuation of ΔDLP mutant virus in wild type, but not in <i>PKR</i>^o/o mice. Animals were inoculated with 10⁷ of WT and 2×10⁷ of ΔDLP mutant viruses. Viral yields in mouse brains at 4 dpi were titrated by plaque assay. Results are the mean from 10 animals inoculated for each group in three independent experiments. SD from each group is also showed.</p

Alphavirus Replicon DNA Expressing HIV Antigens Is an Excellent Prime for Boosting with Recombinant Modified Vaccinia Ankara (MVA) or with HIV gp140 Protein Antigen

<div><p>Vaccination with DNA is an attractive strategy for induction of pathogen-specific T cells and antibodies. Studies in humans have shown that DNA vaccines are safe, but their immunogenicity needs further improvement. As a step towards this goal, we have previously demonstrated that immunogenicity is increased with the use of an alphavirus DNA-launched replicon (DREP) vector compared to conventional DNA vaccines. In this study, we investigated the effect of varying the dose and number of administrations of DREP when given as a prime prior to a heterologous boost with poxvirus vector (MVA) and/or HIV gp140 protein formulated in glucopyranosyl lipid A (GLA-AF) adjuvant. The DREP and MVA vaccine constructs encoded Env and a Gag-Pol-Nef fusion protein from HIV clade C. One to three administrations of 0.2 μg DREP induced lower HIV-specific T cell and IgG responses than the equivalent number of immunizations with 10 μg DREP. However, the two doses were equally efficient as a priming component in a heterologous prime-boost regimen. The magnitude of immune responses depended on the number of priming immunizations rather than the dose. A single low dose of DREP prior to a heterologous boost resulted in greatly increased immune responses compared to MVA or protein antigen alone, demonstrating that a mere 0.2 μg DREP was sufficient for priming immune responses. Following a DREP prime, T cell responses were expanded greatly by an MVA boost, and IgG responses were also expanded when boosted with protein antigen. When MVA and protein were administered simultaneously following multiple DREP primes, responses were slightly compromised compared to administering them sequentially. In conclusion, we have demonstrated efficient priming of HIV-specific T cell and IgG responses with a low dose of DREP, and shown that the priming effect depends on number of primes administered rather than dose.</p></div

<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

T cell responses induced by tested vaccine candidates.

<p>(A) Mice (<i>n</i> = 3 per group) were immunized once with 5 μg of DREP-C-ENV or 5 μg of DREP-C-GPN alone, or both constructs either mixed or given at separate sites. DREP constructs were given i.d. followed by EP. Splenocytes were assayed 10 days post-immunization with IFN-γ Elispot using peptides PADPNQEM (Env), VGPTPVNI (Pol1) and YYDPSKDLI (Pol2). (B-D) DREP-C-ENV and DREP-C-GPN induce T cells and antibodies that are boosted by multiple administrations. Mice (<i>n</i> = 5 per group) were immunized by i.d. EP one to three times with 5 or 0.1 μg of each DREP-C-ENV and DREP-C-GPN given at separate sites. One group was given DNA-C (5 μg of each construct) 3 times i.m. Boost immunizations were given with a 3 week interval between each administration. (B) Serum was assayed with ELISA for anti-gp140 IgG antibodies 3 weeks after the last immunization. Responses are shown as box plots, with whiskers representing 5–95 percentiles. (C) Splenocytes were assayed with IFN-γ Elispot 10 days and 3 weeks after last immunization using the peptides described above. (D) ICS for CD107a, IFN-γ, IL-2 and TNF-α was performed on splenocytes 10 days after the last immunization. The percentage of CD8+ T cells expressing a certain number of cytokines is shown. Specific markers expressed are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117042#pone.0117042.s002" target="_blank">S2 Fig.</a> Statistical analyses were performed to compare mice given a different number of administrations of the same dose of DREP-C. In addition, mice given the same number of administrations but with different doses/regimens were compared in separate statistical comparisons. Abbreviations: D-ENV, DREP-C-CN54ENV; D-GPN, DREP-C-ZM96GPN. * <i>P</i> < 0.05; ** <i>P</i> < 0.01</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

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

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

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

Antibody responses following DREP-C prime and boost with MVA-C and/or gp140/GLA.

<p>Mice (<i>n</i> = 5 per group) were primed 1 or 3 times with DREP-C, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117042#pone.0117042.g001" target="_blank">Fig. 1B-D</a>. Six weeks after the last administration of DREP-C, mice were boosted with one of the following: (A, E) MVA in one side, (B, F) CN54gp140/GLA in one side, (C, G) MVA-C in one side followed by CN54gp140/GLA in the other side three weeks after MVA-C administration, or (D, H) MVA-C in one side and gp140/GLA in the other side simultaneously. The DREP-C doses are stated in the figure. The booster doses given were 5×10⁶ TCID50 of MVA, 10 μg of gp140 mixed with 0.7 μg of GLA. Three weeks after the last immunization, serum was assayed with ELISA for anti-gp140 (A-D) IgG, (E-H) IgG1 (red) and IgG2a (blue). Responses are shown as box plots, with whiskers representing 5–95 percentiles. (I-L) IgG2a:IgG1 ratio means, with error bars representing standard error of the mean. Groups that were given the same booster following a DREP-C prime were compared statistically. * <i>P</i> < 0.05.</p