protein Gag-Pol-Nef of HIV-1 from clade B (referred as MVA-B), that in DNA prime/MVA boost protocols in mice induced strong immune responses to HIV-1 antigens [17,18,20]. In macaques, a similar MVA construct expressing Env (gp120 from SHIV89.6P) and Gag-Pol-Nef (from SIVmac239) showed strong specific CD4+ and CD8+ T-cell immune responses with a bias for CD8+, and high protection after challenge with SHIV89.6P [22]. Furthermore, the expression of HIV-1 antigens from MVA-B selectively induced in human dendritic cells (DCs) the expression of different cellular genes that might act as regulators of immune responses to HIV-1 antigens [24] and MVA-B-infected DCs co-cultured with autologous T lymphocytes induced a highly functional HIV-1-specific CD8+ T- cell responses including proliferation, secretion of IFN-c, IL-2, TNF-a, MIP1b, MIP1a, RANTES and IL-6, and strong cytotoxic activity against autologous HIV-1-infected CD4+ T lymphocytes [25]. Based on these previous results, MVA-B has recently entered a phase I clinical trial in healthy volunteers in Spain. However, more efficient poxvirus MVA-B vectors that enhance the magnitude, breath, polyfunctionality and durability of the immune responses to HIV-1 antigens are desirable. This is particularly relevant when a single immunogen is desirable for mass vaccination purposes to simplify the immunization protocols and reduce manufacturing cost. Deletion in the vector backbone of MVA-B of known or suggested immunomodulatory VACV genes, which antagonize host specific immune responses, is a general strategy that could enhance immunogenicity of the vector against HIV-1 antigens. In this study, we have generated a new HIV-1 vaccine candidate, termed MVA-B DC6L, which contains a deletion in the vector backbone of MVA-B of the VACV C6L gene. We have analyzed the expression of C6, the innate immune responses elicited by MVA-B DC6L in human THP-1 cells and monocyte-derived DCs (moDCs) and we have examined if the deletion of C6L gene in the candidate HIV/AIDS vaccine vector MVA-B could improve the humoral and T-cell memory immune responses to HIV-1 antigens in mice. We showed that C6 is expressed early during viral infection and localizes in the cytoplasm of infected cells. MVA-B DC6L replicates in cell culture at the same level as parental MVA-B, indicating that C6 is not essential for virus replication. Furthermore, MVA-B DC6L up-regulated the expression of IFN-b and IFN-a/b-inducible genes (IFIT1 and IFIT2) in human THP-1 cells and moDCs, suggesting that C6 inhibits the IFN-b signalling pathway by blocking some unknown component involved in the induction of IFN-b. In DNA prime/MVA boost immunization protocols in mice comparing MVA-B DC6L and MVA-B, MVA-B DC6L signifi- cantly enhanced the magnitude and polyfuncionality of the HIV-1- specific CD4+ and CD8+ T-cell memory immune responses, which are mostly mediated by CD8+ T cells of effector phenotype in both immunization groups. HIV-1-specific CD4+ T-cell memory responses induced by MVA-B and MVA-B DC6L were preferen- tially Env-specific. However, while MVA-B elicited more Env- and Gag-specific CD8+ T-cell memory responses, MVA-B DC6L induced preferentially Gag-Pol-Nef (GPN)-specific CD8+ T-cell memory responses. Moreover, MVA-B DC6L enhanced the levels of antibodies against HIV-1 Env in comparison to MVA-B. Altogether, our findings revealed that C6 is a new VACV immunomodulatory protein, and that deletion of C6L in MVA represents an attractive alternative to increase the immunogenicity of MVA vaccine candidates. Results Generation and in vitro characterization of MVA-B DC6L The function of the VACV C6L is unknown, although it is predicted that have an immunomodulatory role [3]. To analyze C6L Deletion in MVA-B Improves HIV Immunogenicity the possible immunomodulatory function of C6L, we have constructed an MVA-B deletion mutant lacking VACV gene C6L (MVA-B DC6L, see Materials and Methods) from the previously described recombinant MVA-B (expressing HIV-1 Env, Gag, Pol and Nef antigens from clade B) [18]. The diagram of the MVA-B DC6L deletion mutant is shown in Figure 1A. PCR using primers for the C6L locus confirmed the deletion of the C6L gene in MVA- B DC6L (Figure 1B). Deletion of C6L from MVA-B DC6L was also confirmed by DNA sequencing (data not shown). In addition, analysis by Western blot confirmed that MVA-B DC6L expresses HIV-1 antigens BX08gp120 and IIIBGPN at the same level as their parental virus MVA-B (Figure 1C). C6 is non-essential in cell culture The mere isolation of MVA-B DC6L deletion mutant demon- strated that the C6 protein is not essential for MVA replication. To determine whether deletion of C6L altered virus multiplication, we compared the growth of MVA-B DC6L and MVA-B in DF-1 cells. Kinetics studies revealed that deletion of C6L in the MVA-B genome did not affect virus replication. Hence, C6L is not essential for virus propagation in cultured cells (Figure 1D). Furthermore, similar to the parental virus MVA-B or MVA, MVA-B DC6L is an attenuated virus which does not replicate in mammalian cells (Figure S1). C6 is expressed early in infection Western blot analyses using rabbit polyclonal antibodies raised against C6 (produced as described in Material and Methods) identified a 18.2 kDa protein in DF-1 cells infected with MVA- B, but not with MVA-B DC6L (Figure 2A). We then determined when C6 is expressed in DF-1 cells infected with WR and MVA (Figure 2B). C6 expression was detected at 3 hours post-infection, and increased with time for at least 22 hours. Treatment of DF-1 cells at the onset of infection with cytosine arabinoside (AraC), an inhibitor of viral DNA replication and therefore late protein expression, did not abrogate C6 expression, indicating that C6L is an early gene. The increased amount of early C6 protein at 22 h post-infection compared to what is produced when viral DNA replication is inhibited by AraC (Figure 2B), is probably due to enhanced stability of the protein after virus DNA synthesis and to the contribution of progeny virus. The intracellular localization of C6 was examined by immuno- fluorescence in DF-1 cells infected with different VACV strains (Figure 2C). C6 was detected in the cytoplasm, presumably in viral factories, of DF-1 cells infected with WR, MVA and MVA-B, but not in MVA-B DC6L. The reduced fluorescence intensity of C6 (in green) indicates low levels of protein expression in comparison with the late protein A27 (in red). MVA-B DC6L up-regulates IFN-b expression in human macrophages and dendritic cells As a first step to determine whether C6 impairs the response of innate immune cells to MVA-B, we examined by real time PCR the expression of IFN-b, IFN-b-induced genes (IFIT1, IFIT2) and chemokines by human THP-1 macrophages infected for 1, 3 and 6 hours with MVA, MVA-B and MVA-B DC6L (Figure 3A). Compared to MVA and MVA-B, MVA-B DC6L (5 PFU/cell, Figure 3A and 1 PFU/cell, data not shown) markedly up- regulated IFN-b as well as IFIT1 and IFIT2 expression in THP- 1 cells. MVA-B DC6L also increased the expression of MIP-1a and RANTES, but not that of IL-8 and IP-10 (Figure 3A and data not shown). To confirm that C6 impaired IFN-b and IFN-b-dependent gene expression in innate immune cells, we infected human moDCs PLoS ONE | www.plosone.org 2 August 2011 | Volume 6 | Issue 8 | e24244