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

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

CXCR5⁺ follicular cytotoxic T cells control viral infection in B cell follicles

During unresolved infections, some viruses escape immunological control and establish a persistant reservoir in certain cell types, such as human immunodeficiency virus (HIV), which persists in follicular helper T cells (TFH cells), and Epstein-Barr virus (EBV), which persists in B cells. Here we identified a specialized group of cytotoxic T cells (TC cells) that expressed the chemokine receptor CXCR5, selectively entered B cell follicles and eradicated infected TFH cells and B cells. The differentiation of these cells, which we have called 'follicular cytotoxic T cells' (TFC cells), required the transcription factors Bcl6, E2A and TCF-1 but was inhibited by the transcriptional regulators Blimp1, Id2 and Id3. Blimp1 and E2A directly regulated Cxcr5 expression and, together with Bcl6 and TCF-1, formed a transcriptional circuit that guided TFC cell development. The identification of TFC cells has far-reaching implications for the development of strategies to control infections that target B cells and TFH cells and to treat B cell–derived malignancies

Engineered miRNA sequences.

<p>Engineered miRNA sequences.</p

Expression of mature miR-155 does not inhibit efficient Env expression.

<p>(<b>A</b>) Un-transfected HeLa cells (lane 1) or HeLa cells transfected with 2 µg of either pCMV-Empty vector backbone (lane 2), pMIR155HG (lane 3) or Env expression vectors pAD8-140 miR-155S (lane 4), pAD8-140 miR-155 (lane 5) or pAD8-140 (lane 6). Forty-eight hours post-transfection, total RNA was isolated and resolved using denaturing urea PAGE. Membranes were subsequently probed with an [³²P]-ATP radio-labelled oligonucleotide complementary to the mature guide strand of miR-155. Equal loading of RNA was confirmed by probing with a [³²P]-CTP labelled, DNA probe specific for U6 snRNA. (<b>B</b>) Protein lystates were isolated from similarly transfected HeLa and resolved using SDS-PAGE. Expression levels of Env were analysed by immunoblotting with anti-gp120 D7324 and anti-β-actin antibodies.</p

Design of Env expression plasmids for the co-expression of miR-155.

<p>(<b>A</b>) Diagram of the pMIR155HG construct. A CMV immediate early promoter drives transcription through an artificial intron consisting of the CMV intron A splice donor (SD) and C57Bl/6 IgG heavy chain splice acceptor (SA). The transcript encodes the 3^rd exon of the human <i>mir155hg</i> gene and terminates at a bovine growth hormone poly-adenylation signal (pA). (<b>B</b>) The HIV-1 Env expression plasmid pAD8-140, expresses a truncated and cleavage site modified Env protein (gp140) and is based on a native <i>env</i> cDNA derived after splicing of exons 1 and 4bE from the full genomic mRNA. The HIV-1 5′ UTR, <i>rev</i> and 3′ UTR regions are derived from strain NL4.3, whilst the area bounded by KpnI and BamHI encode a heterologous <i>env</i> gp140 gene from strain ADA. A CMV promoter drives transcription through the artificial intron described above. The 4 kb transcript encodes for Vpu, gp140 and a truncated Nef protein. The HIV-1 intron is bounded by SD4 and SA7 and Rev is expressed from the spliced 2 kb mRNA. (<b>C</b>) Overlap extension PCR was used to incorporate the entire MIR155HG exon 3 sequence, or a shortened 122 bp variant into the artificial intron of pAD8-140 to create the vectors pAD8-140 miR-155 and pAD8-140 miR-155S respectively.</p

miRNA targeting murine PKR and PERK but not PACT augment Env expression <i>in vitro</i>.

<p>(<b>A</b>) Green fluorescence as a marker of Env expression from the pNL-140.EGFP reporter constructs. HeLa cells were transfected with 1 µg of pCMV-Empty (lane 1), pNL-140.EGFP (lane 2), or pNL-140.EGFP co-expressing miR-NS (lane 3), miR-huPKR (lane 4), miR-huPERK (lane 5) or miR-huPACT (lane 6). Forty-eight hours post-transfection, the relative fluorescence intensity of Env.EGFP was determined and is presented relative to cells transfected with reporter alone as the Mean +/− SEM of four independent transfections. Results were compared to the miR-NS control using a Mann-Whitney U test. (* indicates p<0.05). (<b>B</b>) LTA cells were similarly transfected with 1 µg of pCMV-Empty (lane 1), pNL-140.EGFP (lane 2), or pNL-140.EGFP co-expressing miR-NS (lane 3), miR-muPKR (lane 4), miR-muPERK (lane 5) or miR-muPACT (lane 6) and analysed for green fluorescence by flow cytometry 48 h post-transfection. The relative fluorescence intensity was determined relative to cells transfected with reporter alone and is presented as the Mean +/− SEM of four independent transfections. Results were compared to the miR-NS control using a Mann-Whitney U test. (* indicates p<0.05). Secretion of Env.EGFP from the pNL-140.EGFP reporter constructs was measured by ELISA. Half-log-2 serial dilutions of culture supernatants from (<b>C</b>) HeLa or (<b>D</b>) LTA transfected with pNL-140.EGFP alone or co-expressing miRNA were incubated in a 96-well plate coated with anti-gp120 D7324 antibodies. Bound Env.EGFP was detected using anti-HIV human sera, HRP-conjugated anti-human IgG and colorimetric reaction of the TMB substrate was measured and is shown as the Mean +/− SEM of two separate transfections.</p

Oligonucleotides.

<p>Oligonucleotides.</p