C6L Deletion in MVA-B Improves HIV Immunogenicity Figure 2. Characterization of C6 expression and localization. DF-1 cells were infected with 5 PFU/cell of MVA-B and MVA-B DC6L (A) or WR and MVA (B) in the presence or absence of AraC (B). Cells extracts collected 24 hours post-infection (A) or at the indicated time (B) were analyzed by SDS-PAGE. VACV C6 protein was detected by Western blot using rabbit polyclonal sera against C6. (C) DF-1 cells were infected with WR, MVA, MVA-B or MVA-B DC6L or mock-infected for 18 hours. The localization of C6 was analyzed by immunofluorescence, as described in Materials and Methods. Cells were staining with DAPI (blue, staining cellular nucleus), purified rabbit polyclonal anti-C6 (green) and anti-14K (red). doi:10.1371/journal.pone.0024244.g002 with increasing doses (0.002, 0.02 and 0.2 PFU/cell) of MVA, MVA-B and MVA-B DC6L and measured IFN-b, IFIT1 and IFIT2 mRNA levels 6 h post-infection (Figure 3B). We used low virus doses, since MVA induces apoptosis of human moDCs [26]. Similarly to the results obtained with human THP-1 cells, MVA-B DC6L strongly increased IFN-b expression compared to MVA and MVA-B in moDCs. Whereas the three viruses used at 0.2 PFU/ml similarly stimulated IFIT1 and IFIT2 mRNA expression in moDCs, MVA-B DC6L was a much more potent inducer than MVA and MVA-B at lower infective doses (0.002 PFU/ml, Figure 3B). Furthermore, MVA-B DC6L stimulated the release by moDCs of much higher levels of IFN-b (Figure 3C) and bioactive type I IFNs than MVA and MVA-B (Figure 3D). Thus, deletion of C6L in the MVA-B genome promotes IFN-b production, suggesting that C6 interferes with the signalling pathway controlling IFN-b gene expression in innate immune cells. MVA-B DC6L enhances the magnitude and polyfunctionality of long-lived memory HIV-1-specific T-cell responses Given the immunomodulatory properties of C6, we tested whether deletion of C6 in MVA-B DC6L could enhance its immunogenic properties by analyzing HIV-1-specific T-cell responses in BALB/c mice immunized with MVA-B or MVA-B DC6L using a DNA prime (100 mg of DNA-B, i.m.)/MVA boost (16107 PFU, i.p.) immunization protocol [17,18,22,27,28,29]. Animals primed with sham DNA (DNA-w) and boosted with non-recombinant MVA were used as controls. Considering that memory T-cell responses might be critical for protection against HIV-1 infection [30,31,32,33], we assessed by IFN-c ELISPOT and IFN-c and IL-2 intracellular cytokine staining (ICS) the long- term immunogenicity profile (i.e. 53 days after the boost) elicited by DNA-B/MVA-B and DNA-B/MVA-B DC6L vaccination in splenocytes. IFN-c ELISPOT revealed that, compared to MVA-B, MVA-B DC6L enhanced 2.1-fold (p,0.005) the T-cell memory response against HIV-1 peptide Gag-B (an HIV-1 peptide representative of Gag antigen) (Figure 4A). Non-recombinant MVA, used as a control, did not induce HIV-1-specific memory responses. The phenotype of the HIV-1-specific memory T cells elicited upon immunization with DNA-B/MVA-B and DNA-B/MVA-B DC6L was characterized by polychromatic flow cytometry using ICS. Splenic CD4+ and CD8+ T cells were co-stained for CD44 and CD62L surface markers to define the na¨ıve (CD442/ CD62L+), central memory (CM: CD44+/CD62L+), effector memory (EM: CD44+/CD62L2) and effector memory terminally differentiated (TEMRA: CD442/CD62L 2 ) sub-populations. We also evaluated IFN-c and IL-2 production after in vitro stimulation with different HIV-1 peptide pools (Env-pool, Gag-pool and GPN- pool) that covered the entire HIV-1 sequences present in the poxvirus vector (Figure 4B). The overall HIV-1-specific immune response at 53 days post- boost was mainly mediated by CD8+ T cells (70%–85%) (Figure 4C) of EM and TEMRA phenotypes (Figure 4D), in both immunization groups. However, long-term post-boost immunization with DNA- B/MVA-B DC6L induced a higher magnitude of HIV-1-specific CD4+ and CD8+ T-cell memory responses producing IFN-c and/or IL-2 than DNA-B/MVA-B [CD4+ T cells: 1.91% in DNA-B/ MVA-B DC6L vs. 1.30% in DNA-B/MVA-B, (p,0.005); CD8+ T cells: 10.95% in DNA-B/MVA-B DC6L vs. 3.06% in DNA-B/ MVA-B (p,0.005)] (Figure 4C). Both vectors induced a similar pattern of HIV-1-specific CD4+ T-cell memory responses (with preference towards Env) (Figure 4B and 4C). Interestingly, the pattern of CD8+ T-cell memory responses was different between the two vectors: DNA-B/MVA-B DC6L induced a higher percentage of GPN-specific CD8+ T-cell responses, while DNA-B/MVA-B induced preferentially Env- and Gag-specific CD8+ T-cell responses (Figure 4B and 4C). In both immunization groups, HIV-1-specific CD8+ T cells were mainly of the EM (60.5–63%) and TEMRA (37%–39.5%) phenotypes (Figure 4D). All HIV-1-specific CD4+ T cells were of the EM phenotype in the DNA-B/MVA-B group. Although most of HIV-1-specific CD4+ T cells (82.8%) were of the EM phenotype in the DNA-B/MVA-B DC6L group, a substantial proportion (17.2%) of cells expressed the TEMRA phenotype (Figure 4D). No CM T cells producing IFN-c and/or IL-2 were detected in both immunization groups (Figure 4D). PLoS ONE | www.plosone.org 4 August 2011 | Volume 6 | Issue 8 | e24244