several phase I clinical studies for HIV/AIDS have been performed with DNA prime/MVA boost protocols or with MVA administered alone [61,62,63,64,65] and revealed promis- ing findings. These studies proved the safety and immunogenicity of the MVA vectors and reported an important proportion of responders with multigenic responses that persisted up to one year post-vaccination. In light of our results, one might postulate that MVA-B with deletion of the C6L gene could improve the immunogenicity of the vaccines by enhancing the magnitude, polyfunctional and memory responses T cell responses. In conclusion, we show that deletion of C6L on MVA-B up- regulates IFN-b expression in human macrophages and DCs, and improves MVA-B immunogenicity in vivo, increasing the magni- tude, polyfunctionality and memory T-cell responses against HIV- 1 and the generation of Env-specific antibodies. Thus, VACV C6 protein interferes with host immune responses by at least in part, blocking some component(s) of the IFN-b signalling pathway. Understanding the mechanism of action of C6 will provide new insights in virus-host cell interactions and viral immunomodula- tion. Further work should be devoted to explore the relevance of our findings in a non-human primate model, as MVA-B DC6L represents a promising vector for developing HIV-1 vaccines. Materials and Methods Ethics Statement The animal studies were approved by the Ethical Committee of Animal Experimentation (CEEA-CNB) of Centro Nacional de Biotecnologia (CNB-CSIC, Madrid, Spain) in accordance with national and international guidelines and with the Royal Decree (RD 1201/2005). Permit numbers: 152/07 and 080030. Studies with peripheral blood mononuclear cells (PBMCs) from healthy blood donors recruited by the Blood Center of Lausanne (Switzerland) were approved by the ethics commission for clinical research from the Faculty of Biology and Medicine of Lausanne. Written informed consent was obtained from donors. All infor- mation were kept confidential by the Blood Center. Cells and viruses Primary chicken embryo fibroblast cells (CEF) [18] and DF-1 cells (a spontaneously immortalized chicken embryo fibroblast cell line. ATCC, Manassas, VA) were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS). The human monocytic THP-1 cell line (ATCC, Manassas, VA) was cultured in complete RPMI 1640 medium containing 2 mM L-glutamine, 50 mM 2-mercaptoethanol, 100 IU/ml pen- icillin, 100 mg/ml streptomycin (all from Invitrogen, San Diego, CA) and 10% heat-inactivated FCS (Sigma-Aldrich, St. Louis, MO), as previously described [40]. THP-1 cells were differentiated into macrophages by treatment with 0.5 mM phorbol 12- myristate 13-acetate (PMA, Sigma-Aldrich) for 24 h before usage. Adult peripheral blood mononuclear cells (PBMCs) from healthy donors (recruited by the Blood Center, Lausanne, Switzerland) were seeded in 6-well tissue culture plates (36106 cells/well) in complete RPMI medium supplemented with 10% heat-inactivated FCS and incubated at 37uC for 3 h. Non-adherent cells were removed and medium replaced by fresh complete RPMI medium containing 50 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF) and 20 ng/ml IL-4 (R&D Systems, Minneapolis, MN). moDCs were collected after 7 days of incubation. Cell cultures were performed at 37uC (CEF, THP-1 cells and moDCs) or 39uC (DF-1) in a humidified incubator containing 5% CO2. The poxvirus strains used in this work included: Western Reserve (WR), modified vaccinia virus Ankara (MVA) and the C6L Deletion in MVA-B Improves HIV Immunogenicity recombinant MVA-B expressing the HIV-1BX08 gp120 and HIV- 1IIIB Gag-Pol-Nef proteins [18]. Viruses were grown in CEF cells, purified through two 36% (w/v) sucrose cushions, and titrated by plaque immunostaining assay [66]. Cell lines were infected with viruses as previously described [18,40]. Construction of plasmid transfer vector pGem-RG-C6L wm The plasmid transfer vector pGem-RG-C6L wm was used for the construction of the recombinant virus MVA-B DC6L, with C6L (C6L in Copenhagen strain of VACV is equivalent to MVA 019L in MVA) gene deleted (for simplicity, we used throughout the work the ORF nomenclature of Copenhagen strain to refer the MVA genes). pGem-RG-C6L wm was obtained by sequential cloning of five DNA fragments containing dsRed2 and rsGFP genes and C6L recombination flanking sequences into the plasmid pGem-7Zf(2) (Promega). The construction of the plasmid pGem- Red-GFP wm (4540 bp), containing dsRed2 and rsGFP genes under the control of the synthetic early/late (E/L) promoter was previously described [17]. MVA-B genome was used as the template to amplify the right flank of C6L gene (391 bp) with oligonucleotides RFC6L-AatII-F (59-CTCGTCGACGTCCGAC- CAATCTGGGC-39) (AatII site underlined) and RFC6L-XbaI-R (59-TTCCTATCTAGATTTCTCTGTTTAAA-39) (XbaI site underlined). This right flank was digested with AatII and XbaI and cloned into plasmid pGem-Red-GFP wm previously digested with the same restriction enzymes to generate pGem-RG-RFsC6L wm (4898 bp). The repeated right flank of C6L gene (391 bp) was amplified by PCR from MVA-B genome with oligonucleotides RF9C6L-XmaI-F (59-CTCGTCCCCGGGCGACCAATCTGG- GC-39) (XmaI site underlined) and RF9C6L-ClaI-R (59-TT- CCTAATCGATTTTCTCTGTTTAAA-39) (ClaI site under- lined), digested with XmaI and ClaI and inserted into the XmaI/ClaI-digested pGem-RG-RFsC6L wm to generate pGem- RG-RFdC6L wm (5259 bp). The left flank of C6L gene (413 bp) was amplified by PCR from MVA-B genome with oligonucleotides LFC6L-ClaI-F (59-ATACGCATCGATGATAAACTTAATGA- 39) (ClaI site underlined) and LFC6L-BamHI-R (59-GTTG- TTGGATCCATTGGTAGATGACG-39) (BamHI site under- lined), digested with ClaI and BamHI and inserted into the ClaI/Bam HI-digested pGem-RG-RFdC6L wm. The resulting plasmid pGem-RG-C6L wm (5642 bp) was confirmed by DNA sequence analysis and directs the deletion of C6L gene from MVA- B genome. Construction of MVA-B DC6L deletion mutant MVA-B DC6L deletion mutant was constructed by screening for transient Red2/GFP co-expression using dsRed2 and rsGFP genes as the transiently selectable markers, as previously described [17]. Briefly, 36106 DF-1 cells were infected with MVA-B at a multiplicity of 0.05 PFU/cell and then transfected 1 h later with 6 mg of DNA from plasmid pGem-RG-C6L wm using Lipofecta- mine (Invitrogen) according to the manufacturer’s recommenda- tions. After 72 hours, the cells were harvested, lysed by freeze- thaw cycling and sonicated. Following 6 consecutive rounds of plaque purification in DF-1 cells, MVA-B DC6L was obtained and the deletion of C6L gene was confirmed by PCR amplifying the C6L locus. MVA-B DC6L was grown in CEF cells, purified by centrifugation through two 36% (w/v) sucrose cushions in 10 mM Tris-HCl pH 9, and titrated in DF-1 cells by plaque immuno- staining assay, using rabbit polyclonal antibody against VACV strain WR (Centro Nacional de Biotecnolog´ıa; 1:1000) followed by anti-rabbit-HRP (Sigma; 1:1000), as previously described [66]. PLoS ONE | www.plosone.org 10 August 2011 | Volume 6 | Issue 8 | e24244