Figure 6. Immunization with MVA-B DC6L enhances the humoral immune responses elicited against HIV-1 gp160 protein. Serum was collected from individual mouse immunized with DNA-B/MVA-B, DNA-B/MVA-B DC6L or DNA-w/MVA (n = 4 in DNA-B/ MVA-B and DNA-w/MVA; n = 3 in DNA-B/MVA-B DC6L), 53 days after the last immunization. Anti-gp120 antibody titers were determined by ELISA as described in Materials and Methods. Titers represent the last dilution of the serum that signals 3-fold higher than signals obtained with the serum of na¨ıve mice. The dotted line represents the limit of detection of the ELISA. The horizontal bar represents the mean value. Each dot represents one mouse. * p,0.05. doi:10.1371/journal.pone.0024244.g006 post-infection and at low virus doses, probably because long-term viral exposure and high viral doses induce extensive apoptosis [26]. Previous experiments revealed in macrophages infected with MVA a critical role for TLR2-TLR6-MyD88 in the production of IFN-b-independent chemokines and of MDA-5-IPS-1 in the production of IFN-b-dependent chemokines [40]. Phosphorylation of IRF3, IRF7 and STAT-1, which are essential for the transcription of the IFN-b gene (IRF3) and critical targets of the IFN-b signalling pathway required for the transcriptional activation of IFN-b-dependent genes (IRF7 and STAT-1), were detected in THP-1 cells infected with MVA and associated with the induction of IFN-b [40]. Whether C6 blocks directly or indirectly IRF3, IRF7 or STAT-1 expression or activation by phosphorylation is under investigation. Since our goal is to develop modified MVA-B with enhanced immunogenicity to HIV-1 antigens, we carried out a detail characterization of the HIV-1-specific memory immune responses induced in mice using DNA prime/MVA boost approach, and compared parental MVA-B with the deletion mutant MVA-B DC6L. We used ICS, which allows a more extensive character- ization of T-cell effector functions at the single-cell level [41]. Our findings revealed that at 53 days post-boost, DNA-B/MVA-B DC6L triggered higher magnitude and polyfunctionality of total HIV-1-specific CD4+ and CD8+ T-cell memory immune respons- es (specific for Env, Gag and GPN) than DNA-B/MVA-B. The vaccine-induced T-cell memory responses were predominantly mediated by CD8+ T cells in both immunization groups, with most of the response mediated by CD8+ EM and TEMRA T cells, which have been described to have a powerful and direct antiviral capacity [30,31,32,33] and have been associated with HIV-1 viral control in early and chronic infection [42,43,44]. HIV-1-specific CD4+ T-cell memory responses were preferentially Env-specific in both immunization groups, similar to what it is obtained with other MVA HIV-1 vaccines as MVA-CMDR [45] or other C6L Deletion in MVA-B Improves HIV Immunogenicity NYVAC HIV-1 vaccines [21,46,47]. Furthermore, immunization with DNA-B/MVA-B DC6L induced preferentially GPN-specific CD8+ T-cell memory responses compared with DNA-B/MVA-B. The shift towards GPN-response triggered by MVA-B DC6L might result from the activation of the intrinsic pathway of antigen presentation by the Gag-Pol-Nef intracellular polyprotein due to the increased IFN-b production promoted by the deletion of C6L. However, it is not clear yet whether the enhanced CD8+ T-cell response to GPN is due to a greater breadth of response or could reflect an enhanced response to single epitopes. The functional relevance of Env-specific CD4+ T-cell responses or GPN-specific CD8+ T-cell responses in setting of vaccination for prevention of HIV-1 infection needs to be further explored. Further experiments need to be done in other animal models such as non-human primates to determine the possible benefits of the response elicited by MVA-B DC6L. Interestingly, enhanced Gag response has been associated with better control of virus in macaques infected with SIV and in HIV-1-infected individuals [48,49]. How significant are our in vivo findings with regard to immune requirements for HIV-1 protection? While definition of correlates of protection to HIV-1 remains to be firmly established, there are a number of markers that can be used as potential indicators for an effective HIV-1 vaccine, such as: 1) activation of HIV-1-specific CD4+ and CD8+ T cells; 2) triggering polyfunctional responses; 3) enhanced magnitude and breath of the immune response; 4) induction of long-term memory cells of effector phenotype; 5) production of neutralizing antibodies with broad specificities. Several features of the T-cell response to HIV-1 are correlated with control of viral replication [50,51], and a correlation of the CD8+ T-cell response with a lowering of peak viremia in acute HIV-1 infection has been described [52,53]. Also, in non-human primates there is a good correlation between vaccine-induced HIV-1-specific cellular immunogenicity and protection after a challenge with a pathogenic SHIV [22,27,28], where CD8+ T cells play an important role in immunity to HIV-1 [54]. Several studies have demonstrated that T-cell polyfunctionality is associated with protective antiviral immunity [[50,51,55,56,57], for a review [33]]. In HIV-1-infected patients that are nonprogressors, HIV-1- specific CD8+ T cells were polyfunctional [50]. Furthermore, the generation of memory CD8+ T cells of EM and TEMRA phe- notypes have been associated with the control of HIV-1 infection in patients [42,43,44] and in non-human primates [58]. Moreover, it was described recently that effector memory T-cell responses elicited after vaccination in non-human primates could control highly pathogenic SIVmac239 infection early after mucosal chal- lenge, showing the important role of memory T-cell responses [59]. These observations suggest that polyfunctional CD8+ T cells and effector memory T cells are important components of a protective immune response [33]. Importantly, both MVA-B and MVA-B DC6L triggered immune responses that fulfill several of the characteristics of a promising candidate HIV-1 vaccine. Indeed immunization with DNA-B/MVA-B DC6L induced activation of HIV-1-specific CD4+ and CD8+ T cells, enhanced magnitude and polyfunctionality of the immune response, triggered long-term memory T cells of effector phenotype, and increased the levels of antibodies directed against Env. However, the immunological parameters required for protec- tion against HIV-1 infection in humans remain unknown. The phase III Thai clinical trial reported a modest protection of about 31% against HIV-1 infection in vaccinees with the combination of recombinant vaccines canarypox and gp120, in spite of poor neutralizing antibodies and of reduced T-cell responses against HIV-1 [60]. The phase III Thai trial has pointed out that further developments of poxvirus vectors is needed. Among MVA vectors, PLoS ONE | www.plosone.org 9 August 2011 | Volume 6 | Issue 8 | e24244