Although antigen specific CD4+ T-cell responses were detected in all experimental groups vaccinated with Env, increased LY2835219 distributor immunogenicity mediated by miR-PERK expression was limited to CD8+ T-cells. The observation that the knockdown of PERK failed to augment the secretion of Env proteins in vitro may explain the lack of augmentation of the primary CD4+ T-cell response, which is largely driven by the uptake and processing of extracellular antigens by dendritic cells, a class of professional antigen presenting cells. When taken together, our findings describe a model whereby APC are being directly transfected following DNA vaccination and are efficiently expressing both Env and miRmuPERK in vivo. Reductions in intracellular PERK expression leads to increased intracellular accumulation of HIV-1 Env antigens, a proportion of which upon degradation by the proteasome and ERresident transporter associated with antigen processing complex, may facilitate increased incorporation of Env peptides into the MHC Class I presentation pathway. In contrast with PERK, the ability of miR-PKR to increase Env expression in vitro did not translate into increased immunogenicity, presumably reflecting a failure to augment antigen expression or presentation in vivo. One possibility may be that interactions between TLR-9 and unmethylated CpG-dinucleotides within E.coli-derived DNA plasmids, and not HIV-1 Env expression, activates PKR responses in eukaryotic cells during transfection. Notably, the DNA vectors utilised in this study contain 15 primate-optimised CpG motifs within the backbone. The stimulation of TLR-9 signalling with CpG-containing oligonucleotides induces the secretion of type-I interferons from plasmacytoid dendritic cells and monocytes. Furthermore, TLR-9 is highly expressed in many tumour-derived cell lines, including HeLa cells, and the treatment of HeLa with CpGODN stimulates the secretion of chemokine monocyte chemoattractant protein-1, indicating that TLR-9-dependent signalling pathways are functional in this cell line. Alternatively, the intracellular concentrations of Env mRNA produced in vitro after lipid-based transfection that are available as substrate for PKR activation may be significantly higher than that obtained after plasmid uptake in vivo following vaccination with naked DNA plasmids. In either case, the knockdown of PKR as a molecular Trichostatin A side effects adjuvant may be of limited value for DNA vaccines and may have more application in recombinant viral vectors dependent upon infection and/or active replication, where intracellular concentrations of viral mRNAs may be significant. This study provides proof-of-principle evidence that RNAi effectors incorporated into vaccine constructs can positively influence vaccine immunogenicity. Furthermore, the co-expression of engineered miRNA, or multiple miRNA, has the potential to improve the effectiveness of current vaccines that rely upon the de novo expression of antigens, such as DNA vaccines and recombinant viral vectors, by ameliorating confounding factors that act to limit maximal antigen expression such as the activation of cellular antiviral pathways or the induction of cellular apoptosis.