C6L Deletion in MVA-B Improves HIV Immunogenicity Figure 4. Immunization with MVA-B DC6L enhances the magnitude of HIV-1-specific CD4+ and CD8+ T-cell memory immune responses. Splenocytes were collected from mice (n = 4 per group) immunized with DNA-B/MVA-B, DNA-B/MVA-B DC6L or DNA-w/MVA, 53 days after the last immunization. (A) Gag-B-specific IFN-c secreting splenocytes were quantified by ELISPOT assay. Data are means 6 SD of triplicate cultures. ** p,0.005. (B–D) Flow cytometry phenotypic analysis of Env, Gag and GPN HIV-1 specific CD4+ and CD8+ T-cells. CD44 and CD62L expression was used to identify central memory (CM: CD44+/CD62L+), effector memory (EM: CD44+/CD62L2) and effector memory terminally differentiated (TEMRA: CD442/CD62L2) sub-populations. IFN-y and IL-2 production was analyzed by ICS. (B) A representative flow cytometry is shown. The T-cell memory sub-populations are depicted as density plots. Blue and green dots represent T-cells producing IFN-y and IL-2, respectively. (C) Percentage of splenic Env, Gag and GPN HIV-1-specific CD4+ and CD8+ memory T-cells. Frequencies were calculated by reporting the number of memory T-cells producing IFN-c and/or IL-2 to the total number of CD4+ and CD8+ splenocytes. Values from unstimulated controls were subtracted in all cases. ** p,0.005. (D) Pie charts representing the proportion of CM, EM and TEMRA within the Env, Gag and GPN HIV-1-specific CD4+ and CD8+ memory T-cells. (A–D) Data are from one experiment representative of two experiments. doi:10.1371/journal.pone.0024244.g004 (named B14R in VACV strain WR) [3]. A46 [4,12], A52 [4,7,8], K7 [9,10,11] and B15 [5,6] are intracellular proteins expressed by VACV that inhibit TLR signalling at different levels. A46 contains a Toll/IL-1 receptor (TIR) domain and targets several TIR adaptor proteins (MyD88, TIRAP, TRIF and TRAM) [4,12], blocking MAP kinase activation and TRIF-mediated IRF3 activation. A52 and K7 targets IRAK2 and TRAF6 inhibiting TLR-dependent NF-kB activation [11]. K7 also interacts with DDX3, which is part of the complex that activates transcription factor IRF3, thus inhibiting IRF3 mediated IFN-b gene transcription [9,10]. B15 inhibits IKKb phosphorylation thereby impairing NF-kB activation [5,6]. Considering the sequence similarities of C6 with A46, A52, K7 and B15, we speculate that C6 may interfere with host immune responses through inhibition of TLR signalling pathways. To examine the immunomodulatory role of C6L, we charac- terized the profile of the innate immune sensing induced by MVA- B DC6L, MVA-B and MVA in human macrophages and moDCs. MVA-B DC6L significantly up-regulated IFN-b and IFN-a/b- inducible genes (IFIT1 and IFIT2) mRNA levels and increased IFN-b secretion, suggesting that C6 blocks some component of the IFN-b signalling pathway. The effect was mainly observed 6 hours Figure 5. Immunization with MVA-B DC6L enhances the polyfunctionality of HIV-1-specific CD4+ and CD8+ T-cell memory immune responses. Splenocytes were collected from mice (n = 4 per group) immunized with DNA-B/MVA-B, DNA-B/MVA-B DC6L or DNA-w/MVA, 53 days after the last immunization and analyzed by flow cytometry as described in Figure 4. The polyfunctionality of Env+Gag+GPN HIV-1-specific CD4+ (left part) and CD8+ (right part) memory T cells was defined based on IFN-c and/or IL-2 production. All the possible combinations of the responses are shown on the X axis. The percentages of IFN-c and/or IL-2 producing memory T-cells among total CD4+ and CD8+ T cells are shown on the Y axis. ** p,0.005. The pie charts summarize the data. Each slice corresponds to the proportion of CD4+ or CD8+ T cells producing single (IFN+ or IL-2+) or double (IFN+/IL-2+) responses within the total HIV-1-specific CD4+ or CD8+ T-cell memory populations. The size of the pie chart represents the magnitude of the specific HIV-1 memory immune response induced. doi:10.1371/journal.pone.0024244.g005 PLoS ONE | www.plosone.org 8 August 2011 | Volume 6 | Issue 8 | e24244