C6L Deletion in MVA-B Improves HIV Immunogenicity Figure 3. MVA-B DC6L induces the production of IFN-b and type I IFN inducible genes in macrophages and dendritic cells. Human THP-1 macrophages (A) and moDCs (B, C, D) were infected with MVA, MVA-B and MVA-B DC6L (5 PFU/cell in A, and 0.2, 0.02 and 0.002 PFU/cell in B, C and D). At different time post-infection (1 h, 3 h and 6 h in A, 6 h in B), RNA was extracted and the mRNA levels of IFN-b, type I IFN inducible genes (IFIT1 and IFIT2), chemokines and HPRT were analyzed by RT-PCR. Results were expressed as the ratio of gene to HPRT mRNA levels. A.U: arbitrary units. Data are means 6 SD of duplicate samples. * p,0.05, ** p,0.005. (C, D) Human moDCs were infected with 0.2, 0.02 and 0.002 PFU/cell of MVA, MVA-B and MVA-B DC6L. Six hours later, cell-free supernatants were collected to quantify the concentration of IFN-b by ELISA (C) and the concentration of type I IFN using the HL116 reporter cell line (D). Results were expressed in absorbance values at 450 nm (C), and in luciferase units (D). Data are means 6 SD of duplicates and are representative of two independent experiments. * p,0.05. doi:10.1371/journal.pone.0024244.g003 To have a detailed assessment of the quality of T-cell memory responses, we next evaluated the production of IFN-c and/or IL-2 by HIV-1-specific CD4+ and CD8+ T-cell memory cells (Figure 5). DNA-B/MVA-B DC6L increased the polyfunctionality of HIV-1- specific CD4+ and CD8+ T memory cells consisting of cells producing both IFN-c and IL-2 [CD4+ T cells: 34% in DNA-B/ MVA-B DC6L vs. 16% in DNA-B/MVA-B, (p,0.005); CD8+ T cells: 29% in DNA-B/MVA-B DC6L vs. 16% in DNA-B/MVA-B, (p,0.005)] (Figure 5). Altogether, these findings established that immunization with DNA-B/MVA-B DC6L significantly increased the magnitude and polyfunctionality of HIV-1-specific CD4+ and CD8+ T-cell memory responses, with most of the response mediated by EM and TEMRA T cells. HIV-1-specific CD4+ T-cell memory responses were preferentially Env-specific following DNA-B/ MVA-B and DNA-B/MVA-B DC6L vaccination. Yet, DNA-B/ MVA-B DC6L induced an immunodominance towards CD8+ GPN-specific T-cell memory responses, while DNA-B/MVA-B induced preferentially CD8+ Env- and Gag-specific T-cell memory responses. MVA-B DC6L enhances the levels of antibodies against HIV-1 gp120 Since cells infected with MVA-B release monomeric gp120 [18], we evaluated whether DNA-B/MVA-B and DNA-B/MVA-B DC6L immunization stimulated the production of antibodies against HIV-1 Env. Anti-gp120 antibodies in serum from individual mouse collected 53 days post-boost were quantified by ELISA, measuring the levels of specific antibodies reactive against gp160 protein from the HIV-1 clone LAV (clade B). Compared to DNA-B/MVA-B, DNA-B/MVA-B DC6L immunization in- creased 44-fold the levels of antibodies reactive against gp160 protein (Figure 6). Therefore, MVA-B DC6L increases the humoral immune responses against HIV-1 Env. Discussion The MVA vector, despite of its attenuated phenotype, still contains genes that encode proteins that can interfere with host immune responses to viral infection [34], and it is described that deletion of immunomodulatory proteins in orthopoxviruses can enhance immune responses ([17,34,35,36,37,38,39]). The function of some of these genes, like the VACV gene C6L, is unknown. We report here the immunomodulatory role of C6L, showing the effects of the C6 protein on virus replication, innate immune sensing and immunogenicity in vivo. MVA-B, the attenuated VACV vector MVA expressing the clade B HIV-1 antigens Env, as monomeric gp120, and Gag, Pol and Nef, as a polyprotein of about 160 kDa is considered a vaccine candidate against HIV/AIDS [18] based on preclinical studies in different animal models [17,18,20,22] and on gene signatures triggered in human DCs infected with MVA-B, where the expression of HIV-1 proteins induced the expression of immuno- modulatory molecules such as cytokines, cytokine receptors, chemokines, chemokine receptors and molecules involved in antigen uptake and processing [24]. Moreover, human DCs exposed to MVA-B induced highly functional HIV-1-specific CD8+ T-cell responses in HIV-1 infected individuals [25]. Thus, due to the good immunogenicity behavior of MVA-B, a prophylactic phase I clinical trial was initiated in Spain. To improve the immunogenicity elicited by MVA-B and to investigate the possible immunomodulatory role of C6L we have removed from the MVA-B viral genome the C6L gene, generating the deletion mutant termed MVA-B DC6L. First, we showed in cultured cells that MVA-B DC6L does not express the C6 protein, but efficiently produced the four HIV-1 antigens (Env, Gag, Pol and Nef) in a stable manner and at the same level as MVA-B during the course of virus infection. Also, MVA-B DC6L replicates similarly to MVA-B in cultured cells, indicating that deletion of C6L has no effect on virus propagation. Therefore, C6L is not essential for viral replication in cell culture. Moreover, similar to MVA-B, MVA-B DC6L maintains an attenuated phenotype and does not replicate in mammalian cells. Western blot analyses demonstrated that C6 is expressed early in cells infected with the VACV strains WR and MVA. This early expression profile is consistent with genome-wide transcriptome analyses that detected C6 mRNA 30 minutes post-infection [2]. Most VACV immuno- modulatory proteins are expressed early during infection, and the early expression pattern of C6 suggests that it is involved in immune evasion as we confirmed in experiments using human macrophages and DCs. In addition, C6 localizes to the cytoplasm of infected cells, opening the possibility that C6 modulates, directly or indirectly, intracellular signalling pathways controlling immune responses. Yeast two-hybrid and pull-down assays revealed that VACV C6 protein binds to three host human cell proteins [14]. However, none of these proteins seems to be directly related with the host immune response. One of the C6 binding partners is programmed cell death 6 interacting protein (PDCD6IP/ALIX), which has been involved in the regulation of apoptosis, cytokinesis and HIV- 1 budding. VACV C6 also interacts with keratin 4 (KRT4), present in intermediate filaments, and which also binds IMV surface protein A27. C6 protein has also been detected in a low proportion in intracellular mature virions [13], similar to other proteins of the poxvirus family Pox_A46 (A46). One possible reason for presence of C6 in the virion could be that C6 is necessary for viral cycle early after virus entry or that C6 have a function in IMV-cell attachment, fusion, and/or microtubule transport through their interaction with KRT4. Finally, C6 also binds to troponin I, skeletal, fast (TNNI2), a co-activator of estrogen receptor-related receptor a (ERRa), suggesting that C6 could have a role in ERRa-mediated transcriptional activity. Additional experiments will be required to decipher the relation- ship between the C6 interaction with binding partners and C6 immunomodulatory function. A bioinformatic analysis indicated that C6L has sequence similarities with the poxvirus family Pox_A46, a poxvirus Bcl-2- like gene family, which includes A46R, A52R, K7R and B15R PLoS ONE | www.plosone.org 6 August 2011 | Volume 6 | Issue 8 | e24244