The magnitude and breadth of the vaccine-induced T-cell response were assessed in splenocytes collected 2?weeks after the 2nd vaccination (week 6) by an IFN- ELISPOT assay using two sets of peptide pools: (i) 8 overlapping peptide pools covering the HTI sequence and assembled in separate pools for each protein subunit included in HTI (HTI pool-1 to pool-8) and (ii) 19 additional peptide pools spanning the full-length sequences of Gag (n?=?6 pools), Pol (n?=?8), Nef (n?=?2), Tat (n?=?1), and Vif (n?=?2). HIVACAT T-cell Immunogen (HTI) sequence which is usually 529 amino acids in length, includes more than 50 optimally defined CD4+ and CD8+ T-cell epitopes restricted by a wide range of HLA class I and II molecules and covers viral sites where mutations led to a dramatic reduction in viral replicative fitness. In both, C57BL/6 mice and Indian rhesus macaques immunized with an HTI-expressing DNA plasmid (DNA.HTI) induced broad and balanced T-cell responses to several segments within Gag, Pol, and Vif. DNA.HTI induced robust CD4+ and CD8+ T cell responses that were increased by a booster vaccination using modified computer virus Ankara (MVA.HTI), expanding the DNA.HTI induced response to up to 3.2% IFN- T-cells in macaques. HTI-specific T cells showed a central and effector memory phenotype with a significant fraction of the IFN-+ CD8+ T cells being Granzyme B+ and able to degranulate (CD107a+). Conclusions These data demonstrate the immunogenicity of a novel HIV-1?T cell vaccine concept that induced broadly balanced responses to vulnerable sites of HIV-1 while avoiding the induction of responses to potential decoy targets that may divert effective T-cell responses towards variable and less Isoorientin protective Isoorientin viral determinants. Electronic supplementary material The online version of this article (doi:10.1186/s12967-015-0392-5) contains supplementary material, which is available to authorized users. computer virus control or lack thereof [1-3]. Among these, CD8+ cytotoxic T lymphocytes (CTL) responses to HIV-1 Gag have most consistently been associated with reduced viral loads in both HIV-1 clade B- and C-infected cohorts [2,4]. This is in line with data from post-hoc analyses of the STEP vaccine trial, where individuals in whom vaccine-induced responses targeted 3 different Gag epitopes achieved a lower viral load than subjects without Gag responses [5]. CD4+ T-cell responses to Gag have also been Isoorientin associated with relative HIV-1 control [6,7]. However, it remains unclear whether the relative benefit of Gag is due to high protein expression levels, rapid representation of viral particle-derived CTL epitopes [8], reduced susceptibility of Gag-specific CTL to Nef-mediated immune evasion strategies [9] or particular amino acid composition Rabbit Polyclonal to Collagen V alpha1 and inherently greater immunogenicity [10]. In addition, the elevated level of conservation of Gag across viral isolates [11] and the severe fitness reductions caused by CTL escape variants [12-16] may provide Gag-specific T-cell responses with a particular advantage. At the same time, it is also clear that not all Gag-specific responses exert the same antiviral activity, suggesting that a rational selection of Gag components could help focus vaccine induced responses onto the most protective targets. The same likely applies for all other viral proteins as well, as they may contain some regions that are of particular value for inclusion in a vaccine while other regions or proteins may induce less useful T cell responses. As such, effective vaccine design should probably aim to induce broad and evenly distributed responses to conserved and vulnerable sites of the computer virus while avoiding the induction of responses to regions that can be highly immunogenic but that may act as potential decoy targets and divert responses away from more relevant targets [17-22]. The failure of various T-cell vaccine candidates expressing entire HIV-1 proteins in large human clinical trials and data from post-trial analyses suggesting a sieve effect on the infecting viral strains, indicate the urgent need to improve vaccine immunogen design [23-26]. Here, we describe a rational design and pre-clinical testing of a novel approach to HIV-1?T cell immunogen development and its implication for HIV-1 control. Starting with a comprehensive screening of large cohorts of clade B and C HIV-1-infected individuals, we identified viral targets associated with relative HIV-1 control [27,28]. These earlier analyses in aggregate identified 26 regions in HIV-1 Gag, Pol, Vif and Nef proteins that (i) were.