reporter cell line HL116 which stably expresses the luciferase gene under the control of the IFN-a/b-inducible 6–16 promoter [67,68,69]. HL116 cells were grown in DMEM supplemented with 10% FCS and 50 mM hypoxanthine-aminopterin-thymidine. HL116 cells (105 cells in a 96-well plate) were incubated for 6 h with cell culture supernatants or recombinant human IFN-b (Cell Sciences, Canton, MA) as a standard. Luciferase activity in HL116 lysates was quantified using the Luciferase assay reagent (Promega) and a luminometer (EG&E Berthold, Germany). Peptides HIV-1 peptide pools were provided by the EuroVacc Founda- tion. They spanned the entire Env, Gag, Pol and Nef regions from clade B HIV-1 as consecutive 15-mers overlapped by 11 amino acids. The HIV-1BX08 gp120 protein was spanned by the Env-1 and Env-2 pools. The HIV-1IIIB Gag-Pol-Nef fusion protein was spanned by the following pools: Gag-1, Gag-2, GPN-1, GPN-2, GPN-3 and GPN-4. The size and number of peptides included in each pool was previously described [17]. For immunological analysis we grouped the peptides in three main pools: Env, Gag and GPN. The Env-pool comprises Env-1+Env-2; Gag-pool comprises Gag-1+Gag-2; and GPN-pool comprises GPN-1+GPN- 2+GPN-3+GPN-4. HIV-1 peptide Gag-B (AMQMLKETI), from clade B, was produced at Centro Nacional de Biotecnolog´ıa. Mice immunization schedule BALB/c mice (6–8 weeks old) were purchased from Harlan. A DNA prime/MVA boost immunization protocol was performed as previously described [17,18]. Groups of animals (n = 4) received 100 mg of DNA-B (50 mg of pCMV-BX08gp120+50 mg of pCDNA- IIIBGPN) by intramuscular route (i.m.) and two weeks later received an intraperitoneal (i.p.) inoculation of 16107 PFU of the corre- sponding recombinant vaccinia viruses (MVA-B or MVA-B DC6L) in 200 ml of PBS. Mice immunized with sham DNA (DNA-w) followed by MVA booster were used as control group. At 53 days after the last immunization, mice were sacrificed and spleens processed for IFN-c ELISPOT and intracellular cytokine staining (ICS) assays. Two independent experiments have been performed. IFN-c ELISPOT assay IFN-c ELISPOT assay was performed as previously described [17,70]. Intracellular Cytokine Staining assay (ICS) The phenotypes of responding T memory cells were analyzed by ICS and fluorescence-activated cell sorting analysis as described elsewhere [22]. After an overnight rest, 56106 splenocytes (depleted of red blood cells) were resuspended in RPMI 1640 supplemented with 10% FCS and containing 1 ml/ml Golgiplug (BD Biosciences) to inhibit cytokine secretion. Cells were seeded on M96 plates and stimulated with Env-, Gag- or GPN-pools of peptides (5 mg/ml). After 6 h of stimulation, cells were washed, References 1. Alcami A (2003) Viral mimicry of cytokines, chemokines and their receptors. Nat Rev Immunol 3: 36–50. 2. Assarsson E, Greenbaum JA, Sundstrom M, Schaffer L, Hammond JA, et al. (2008) Kinetic analysis of a complete poxvirus transcriptome reveals an immediate-early class of genes. Proc Natl Acad Sci U S A 105: 2140–2145. 3. Gonzalez JM, Esteban M (2010) A poxvirus Bcl-2-like gene family involved in regulation of host immune response: sequence similarity and evolutionary history. Virol J 7: 59. 4. Bowie A, Kiss-Toth E, Symons JA, Smith GL, Dower SK, et al. (2000) A46R and A52R from vaccinia virus are antagonists of host IL-1 and toll-like receptor signaling. Proc Natl Acad Sci U S A 97: 10162–10167. C6L Deletion in MVA-B Improves HIV Immunogenicity stained with anti-CD4-Alexa 700, -CD8-FITC, -CD44-PECy5 and -CD62L-PE antibodies, fixed, permeabilized using the BD Cytofix/CytopermTM Kit (Becton Dickinson) and stained with anti-FN-c-PECY-7 and anti-IL-2-Alexa-647 antibodies (all anti- bodies were from BD Biosciences). Dead cells were excluded using the violet LIVE/DEAD stain kit (Invitrogen). Acquisition and analyses were performed using a LSRII flow cytometer (Becton Dickinson) and FlowJo version 8.5.3 (Tree Star, Ashland, OR). The production of IFN-c and IL-2 was analyzed in each of the T- cell memory populations: central memory (CM: CD44+/CD62L+), effector memory (EM: CD44+/CD62L 2 ) and effector memory terminally differentiated (TEMRA: CD442/CD62L2) sub-popu- lations. Antibody measurements by ELISA Antibodies anti-HIV-1 gp160LAV envelope protein were measured by ELISA as previously described [17,18]. Statistical procedures The statistical analysis of ELISPOT and ICS assays was realized as previously described [17,71]. We have developed a novel approach that corrects measurements for the medium response (RPMI), calculating confidence intervals and p-values. Only antigen responses values significantly larger than the correspond- ing RPMI are represented. Supporting Information Figure S1 Virus growth of MVA-B and MVA-B DC6L in HeLa cells. Monolayers of HeLa cells were infected at 0.01 PFU/ cell with WR, MVA-B or MVA-B DC6L for 0, 24, 48, and 72 h. For comparative purposes, we used the replication-competent WR strain. Cells were collected by centrifugation and infectious viruses associated with the cells (intracellular) and released to the medium (extracellular) during the course of the infection were measured by a plaque immunostaining assay with anti-WR antibodies. Data are from one experiment representative of two experiments. (TIF) Acknowledgments We are grateful to EuroVacc Foundation for peptide pools of HIV-1 antigens from clade B and to NIBSC (UK) for Gag-antibodies. Special thanks to Victoria Jime´nez for excellent technical assistance with the preparation of cells and viruses. We are indebted to Matthieu Perreau for his valuable help with the experiments using moDCs and to Alex Harari and Matthieu Perreau for critical reading of the manuscript. Author Contributions Conceived and designed the experiments: JG-A ME. Performed the experiments: JG-A JLN NT. Analyzed the data: JG-A COSS. Contributed reagents/materials/analysis tools: JG-A CEG TC TR ME. Wrote the paper: JG-A ME. 5. Chen RA, Jacobs N, Smith GL (2006) Vaccinia virus strain Western Reserve protein B14 is an intracellular virulence factor. J Gen Virol 87: 1451–1458. 6. Chen RA, Ryzhakov G, Cooray S, Randow F, Smith GL (2008) Inhibition of IkappaB kinase by vaccinia virus virulence factor B14. PLoS Pathog 4: e22. 7. Graham SC, Bahar MW, Cooray S, Chen RA, Whalen DM, et al. (2008) Vaccinia virus proteins A52 and B14 Share a Bcl-2-like fold but have evolved to inhibit NF-kappaB rather than apoptosis. PLoS Pathog 4: e1000128. 8. Harte MT, Haga IR, Maloney G, Gray P, Reading PC, et al. (2003) The poxvirus protein A52R targets Toll-like receptor signaling complexes to suppress host defense. J Exp Med 197: 343–351. PLoS ONE | www.plosone.org 12 August 2011 | Volume 6 | Issue 8 | e24244