A Candidate HIV/AIDS Vaccine (MVA-B) Lacking Vaccinia Virus Gene C6L Enhances Memory HIV-1-Specific T-Cell Responses Juan Garcı´a-Arriaza1, Jose´ Luis Na´ jera1, Carmen E. Go´ mez1, Nolawit Tewabe1€, Carlos Oscar S. Sorzano 2 , Thierry Calandra3, Thierry Roger3, Mariano Esteban 1 * 1 Department of Molecular and Cellular Biology, Centro Nacional de Biotecnolog´ıa, Consejo Superior de Investigaciones Cient´ıficas (CSIC), Madrid, Spain, 2 Biocomputing Unit, Centro Nacional de Biotecnolog´ıa, Consejo Superior de Investigaciones Cient´ıficas (CSIC), Madrid, Spain, 3 Infectious Diseases Service, Department of Medicine, Centre Hospitalier Universitaire Vaudois and University of Lausanne, Lausanne, Switzerland Abstract The vaccinia virus (VACV) C6 protein has sequence similarities with the poxvirus family Pox_A46, involved in regulation of host immune responses, but its role is unknown. Here, we have characterized the C6 protein and its effects in virus replication, innate immune sensing and immunogenicity in vivo. C6 is a 18.2 kDa protein, which is expressed early during virus infection and localizes to the cytoplasm of infected cells. Deletion of the C6L gene from the poxvirus vector MVA-B expressing HIV-1 Env, Gag, Pol and Nef antigens from clade B (MVA-B DC6L) had no effect on virus growth kinetics; therefore C6 protein is not essential for virus replication. The innate immune signals elicited by MVA-B DC6L in human macrophages and monocyte-derived dendritic cells (moDCs) are characterized by the up-regulation of the expression of IFN-b and IFN-a/b-inducible genes. In a DNA prime/MVA boost immunization protocol in mice, flow cytometry analysis revealed that MVA-B DC6L enhanced the magnitude and polyfunctionality of the HIV-1-specific CD4+ and CD8+ T-cell memory immune responses, with most of the HIV-1 responses mediated by the CD8+ T-cell compartment with an effector phenotype. Significantly, while MVA-B induced preferentially Env- and Gag-specific CD8+ T-cell responses, MVA-B DC6L induced more Gag-Pol-Nef-specific CD8+ T-cell responses. Furthermore, MVA-B DC6L enhanced the levels of antibodies against Env in comparison with MVA-B. These findings revealed that C6 can be considered as an immunomodulator and that deleting C6L gene in MVA-B confers an immunological benefit by enhancing IFN-b-dependent responses and increasing the magnitude and quality of the T-cell memory immune responses to HIV-1 antigens. Our observations are relevant for the improvement of MVA vectors as HIV-1 vaccines. Citation: Garc´ıa-Arriaza J, Na´jera JL, Go´ mez CE, Tewabe N, Sorzano COS, et al. (2011) A Candidate HIV/AIDS Vaccine (MVA-B) Lacking Vaccinia Virus Gene C6L Enhances Memory HIV-1-Specific T-Cell Responses. PLoS ONE 6(8): e24244. doi:10.1371/journal.pone.0024244 Editor: Maciej S. Lesniak, The University of Chicago, United States of America Received April 1, 2011; Accepted August 4, 2011; Published August 31, 2011 Copyright: 2011 Garc´ıa-Arriaza et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This investigation was supported by grants from FIPSE-360731/09, the Ministry of Science and Innovation (SAF2008-02036), Foundation Bot´ın of Spain, the Swiss National Science Foundation (310000-114073/1 and 310030-118266), and the Santos Suarez Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: mesteban@cnb.csic.es € Current address: Takeda Pharmaceutical Company, London, United Kingdom Introduction Poxvirus vectors express numerous genes encoding for immu- nomodulatory proteins that interfere with host anti-viral response [1]. The VACV C6L gene is present in the genome of VACV strains Western Reserve (WR) (VACV-WR_022), Copenhagen (C6L) and MVA (MVA 019L), but absent in New York Vaccinia Virus strain (NYVAC). C6L is presumably an immediate-early gene based on the analysis of the C6L promoter (www.poxvirus. org) and a genome-wide transcriptome analysis that detected C6 mRNA 30 minutes post-infection [2]. C6L encodes a 157 amino acid protein with a predicted molecular weight of 18.2 kDa (www. poxvirus.org). Bioinformatic analyses clustered C6L to the poxvirus BCL-2-like gene family that includes A46R, A52R, B15R (named B14R in WR) and K7R [3], a family of proteins that inhibit the Toll-like receptor (TLR) signalling pathway at different levels [4,5,6,7,8,9,10,11,12]. C6 protein is present at low levels in VACV intracellular mature virions (IMV) [13], and binds to KRT4 (keratin 4), PDCD6IP (programmed cell death 6 interacting protein) and TNNI2 (troponin I) [14]. Moreover, a C6 epitope (amino acids 74–82, GFIRSLQTI in WR and SFIRSLQNI in MVA) is highly immunogenic in BALB/c mice, and WR elicited high levels of C6L-specific IFN-c secreting cells in mice, similarly to VACV E3L, F2L and A52R peptides [15]. All these charac- teristics suggest that C6 may have an important immunomodu- latory function by antagonizing with the TLR signalling pathway. The highly attenuated VACV strain MVA is one of the most promising vectors to be used as an effective vaccine against HIV-1 [16]. MVA has an excellent safety profile, and MVA recombinants expressing HIV-1 antigens induce protection after simian/human immunodeficiency virus (SHIV) challenge, and elicit strong, broad, polyfunctional and durable immune responses to HIV-1 antigens in different animal models and humans trials [[17,18, 19,20,21,22], for a review [23]]. We have previously constructed a recombinant MVA express- ing codon-optimized Env as monomeric gp120 and the poly- PLoS ONE | www.plosone.org 1 August 2011 | Volume 6 | Issue 8 | e24244