Co-Expression of miRNA Targeting the Expression of PERK, but Not PKR, Enhances Cellular Immunity from an HIV-1 Env DNA Vaccine

Adam K Wheatley,Damian F J Purcell,Jesse G Toe,Rob J Center,Marina R Alexander,Marit Kramski

doi:10.1371/journal.pone.0018225

Adam K Wheatley, Damian F J Purcell + Show 4 more

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https://doi.org/10.1371/journal.pone.0018225

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Small non-coding micro-RNAs (miRNA) are important post-transcriptional regulators of mammalian gene expression that can be used to direct the knockdown of expression from targeted genes. We examined whether DNA vaccine vectors co-expressing miRNA with HIV-1 envelope (Env) antigens could influence the magnitude or quality of the immune responses to Env in mice. Human miR-155 and flanking regions from the non-protein encoding gene mirhg155 were introduced into an artificial intron within an expression vector for HIV-1 Env gp140. Using the miR-155-expressing intron as a scaffold, we developed novel vectors for miRNA-mediated targeting of the cellular antiviral proteins PKR and PERK, which significantly down-modulated target gene expression and led to increased Env expression in vitro. Finally, vaccinating BALB/c mice with a DNA vaccine vector delivering miRNA targeting PERK, but not PKR, was able to augment the generation of Env-specific T-cell immunity. This study provides proof-of-concept evidence that miRNA effectors incorporated into vaccine constructs can positively influence vaccine immunogenicity. Further testing of vaccine-encoded miRNA will determine if such strategies can enhance protective efficacy from vaccines against HIV-1 for eventual human use.

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Despite intensive research and the development of new generation vectors and delivery modalities, broadly protective vaccines against many common chronic viral infections, such as hepatitis-C virus (HCV) and HIV-1, have met with limited clinical success
PKR can be activated via intracellular signalling in response to Type 1 interferons [3], or by direct binding of double-stranded RNA (dsRNA) [4] and upon activation, PKR mediates multiple functions including the phosphorylation of eukaryotic initiation factor 2-a [5], the activation of transcription factors IkB and NFkB [6] and the induction of apoptosis by interactions with pro-apoptotic mediators such as Fas-associated death domain (FADD) [7] or C/EBP homologous protein (CHOP) [8]. eIF-2a is an essential factor required for the initiation of mammalian mRNA translation [9] and the phosphorylation of eIF-2a prevents recycling back into the ribosomal initiation complex leading to a cell-wide shutoff of protein synthesis [10]
To examine if the PKR response limits the efficient expression of Env from DNA vaccines, we co-transfected HeLa cells with a fluorescent HIV-1 Env reporter plasmid, pNL140.EGFP, encoding a truncated Env gp140 fused to EGFP (Env.EGFP), together with cDNA expression plasmids expressing human wild-type PKR or the N-terminal dsRNA binding domain of PKR (PKR-N), previously shown to inhibit PKR activation in a dominant-negative fashion [42]

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Despite intensive research and the development of new generation vectors and delivery modalities, broadly protective vaccines against many common chronic viral infections, such as HCV and HIV-1, have met with limited clinical success. One potential area for improvement of vaccination strategies using recombinant viral vectors and/or pure nucleic acid for the expression of viral antigens may lie in preventing cellular antiviral responses that limit efficient antigen expression. Multiple and overlapping intracellular antiviral response pathways mediate the detection of viral infection and the induction of early innate immune effectors. PKR can be activated via intracellular signalling in response to Type 1 interferons [3], or by direct binding of dsRNA [4] and upon activation, PKR mediates multiple functions including the phosphorylation of eukaryotic initiation factor 2-a (eIF-2a) [5], the activation of transcription factors IkB and NFkB [6] and the induction of apoptosis by interactions with pro-apoptotic mediators such as Fas-associated death domain (FADD) [7] or C/EBP homologous protein (CHOP) [8]. The activity of PKR can be positively and negatively regulated by interactions with cellular proteins such as PKR-activating protein (PACT, PRKRA) [11,12] or TAR-RNA binding protein (TRBP) [13]

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