Design and characterization of the rAd-sh viruses.

<p>(A) The linear genome of the rAd-sh virus constructed for this study. In constructing the rAd-sh virus, the E1 region (dashed line) is replaced by the sh DNA expression cassette (<i>sh DNA EC</i>), consisting of the U6 promoter (rightward arrow), the shDNA insert with the sense (s) and antisense (as) arms, of 21 base pairs each, followed by of the U6 terminator (empty box). The shaded box between the ‘s’ and ‘as’ arms is a 6-base pair loop sequence. The dotted lines flanking the expression cassette represent plasmid vector sequences. Other elements of the rAd-sh genome include a ∼2.7 Kb deletion in the E3 region (ΔE3), the left (L) and right (R) inverted terminal repeats, and the packaging signal (ψ). The nt sequences of the ‘s’ strands of the sh-5b and sh-scr constructs are shown below. (B) PCR analysis of wild type AdV5 (lanes 1 & 5), rAdsh-E (lanes 4 & 8), rAdsh-5b (lanes 2 & 6) and rAdsh-scr (lanes 3 & 7) using insert-specific (lanes 1–4) and AdV5 E1-specific (lanes 5–8) primers. DNA size markers (sizes in kb shown to the left) were analyzed in lanes ‘M’. The arrows to the right denote the positions of the predicted insert-specific (upper) and AdV5 E1 region-specific (lower) amplicons. (C) RNase protection assay to detect anti-sense strand of sh-5b siRNA. A radiolabeled sense probe was digested with RNases A and T1, either before (lanes 2 & 5) or after hybridization with total RNA isolated from rAdsh-5b-infected Vero cells, harvested on either day 3 (lane 3) or day 8 (lane 6) post-infection. Protected fragments (lower arrow) were analysed on 8 M urea gel and visualized using a phosphoimager. The un-hybridized probe (upper arrow) without any RNase treatment was analysed in parallel (lanes 1 & 4). It is to be noted that in lanes 3 and 6, the cells used for total RNA preparation were challenged with DENV-2 and DENV-4, respectively, at 24 hours post rAdsh-5b infection.</p

The effect of rAd mediated shRNA expression on DENV secretion.

<p>Vero cells were pre-infected either with rAdsh-5b (red curves) or rAdsh-scr (green curves) followed 24 hours later by infection with DENV-1 (A), DENV-2 (B), DENV-3 (C) and DENV-4 (D). Culture supernatants were drawn at daily intervals up to 7 days post DENV infection and analyzed for the presence infectious DENV using a standard plaque assay. Data shown are mean values (n = 6). The vertical bars represent SD.</p

siRNA targets in the DENV NTRs.

<p>A ClustalW2 multiple alignment of 5′ (A) and 3′ (B) NTR sequences of the prototypic representatives of DENV-1, -2, -3 and -4 (described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001735#s2" target="_blank">Methods</a>) showing the sites conserved in two or more serotypes targeted for RNAi in this study. NTR sequences that were utilized to design the sense strand of the sh constructs are shown in red fonts. The names of the sh constructs are shown in italics above the sequences in red fonts. The DENV-4 5′NTR seed sequence identical to the sh-5b target site is underlined.</p

Adenovirus Delivered Short Hairpin RNA Targeting a Conserved Site in the 5′ Non-Translated Region Inhibits All Four Serotypes of Dengue Viruses

<div><h3>Background</h3><p>Dengue is a mosquito-borne viral disease caused by four closely related serotypes of Dengue viruses (DENVs). This disease whose symptoms range from mild fever to potentially fatal haemorrhagic fever and hypovolemic shock, threatens nearly half the global population. There is neither a preventive vaccine nor an effective antiviral therapy against dengue disease. The difference between severe and mild disease appears to be dependent on the viral load. Early diagnosis may enable timely therapeutic intervention to blunt disease severity by reducing the viral load. Harnessing the therapeutic potential of RNA interference (RNAi) to attenuate DENV replication may offer one approach to dengue therapy.</p> <h3>Methodology/Principal Findings</h3><p>We screened the non-translated regions (NTRs) of the RNA genomes of representative members of the four DENV serotypes for putative siRNA targets mapping to known transcription/translation regulatory elements. We identified a target site in the 5′ NTR that maps to the 5′ upstream AUG region, a highly conserved <em>cis</em>-acting element essential for viral replication. We used a replication-defective human adenovirus type 5 (AdV5) vector to deliver a short-hairpin RNA (shRNA) targeting this site into cells. We show that this shRNA matures to the cognate siRNA and is able to inhibit effectively antigen secretion, viral RNA replication and infectious virus production by all four DENV serotypes.</p> <h3>Conclusion/Significance</h3><p>The data demonstrate the feasibility of using AdV5-mediated delivery of shRNAs targeting conserved sites in the viral genome to achieve inhibition of all four DENV serotypes. This paves the way towards exploration of RNAi as a possible therapeutic strategy to curtail DENV infection.</p> </div

The effect of rAd-mediated shRNA expression on DENV RNA accumulation.

<p>Vero cells were pre-infected either with rAdsh-5b (red bars) or rAdsh-scr (green bars) followed 24 hours later by infection with DENV-1, DENV-2, DENV-3 and DENV-4. Total RNA was isolated on days 2 (panels A and B) and 7 (panels C and D) post-DENV infection and analyzed for DENV ’minus’ (panels A and C) and ‘plus’ (panels B and d D) sense viral genomic RNAs by strand-specific real time PCR analyses. DENV RNA was normalized to GAP RNA in each sample analyzed. The data depict DENV RNA levels in rAdsh-5b treated cells relative to those in the corresponding rAdsh-scr treated cells. Each experiment was carried out in triplicate wells and the entire experiment repeated twice.</p

The effect of rAd mediated shRNA expression on on-going DENV infection.

<p>(A) Vero cells in 12-well plates were sequentially infected with DENV-2 (∼25 PFU/well) and 24 hours later, with rAdsh-scr or rAdsh-5b, each at a m.o.i of 5 (top row) or 10 (bottom row). Cells were overlaid with methyl cellulose and plaques visualized as explained in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001735#pntd-0001735-g003" target="_blank">Figure 3</a> legend. Two wells, of four assayed for each sequential infection experiment, are shown. (B) Vero cells in 24-well plates were sequentially infected with DENV-2 (1000 PFUs/well), followed 24 hours later with rAdsh-5b (red curves) or rAdsh-scr (green curve), each at m.o.i. of 5 (solid curve) or 10 (dashed curve). Culture supernatants were drawn at 48 hour intervals up to 7 days post DENV infection and analyzed for the presence of NS1 antigen using BioRad's Platelia Dengue NS1ELISA kit. The data represent plots of NS1 ELISA absorbance as a function of time after DENV infection. Data shown are mean values (n = 4). The vertical bars represent SD. (C) Culture supernatants in (B) were analyzed for the presence infectious DENV using a standard plaque assay. Data shown are mean values (n = 4). The vertical bars represent SD.</p

The effect of NTR-specific shRNAs on DENV replication^a.

a<p>The production of NS1 antigen, measured using BioRad's Platelia assay kit, served as a marker of DENV replication; data shown are from one representative screening experiment.</p>b<p>This indicates the location of the 21 nts corresponding to the sense strand of the sh construct-encoded siRNA, on the DENV genome; numbers indicated correspond to DENV-2 NGC strain (Accession number AF038403).</p>c<p>SLA: stem-loop A; 5′ UAR: 5′ upstream AUG region; 3′ conserved sequence; 3′ UAR: 3′ upstream AUG region; 3′ SL: 3′ stem-loop; nr: not reported.</p>d<p>The percent inhibition was calculated with reference to DENV infectivity (in the presence of transfected sh-scr construct), which was taken as 100%. D-1, D-2, D-3 and D-4 denote DENV-1, DENV-2, DENV-3 and DENV-4, respectively.</p

Additional file 1: Figure S1. of Virus-like particles derived from Pichia pastoris-expressed dengue virus type 1 glycoprotein elicit homotypic virus-neutralizing envelope domain III-directed antibodies

Cloning and expression of DENV-1 E gene into shuttle vector pPICZA. Figure S2. Purification and characterization of recombinant DENV-1 E antigen. Figure S3. Sequence alignment of P. pastoris optimized DENV-1, 2, 3, 4 E showing similarities and differences in the amino acid sequences between four dengue serotypes. (DOC 1608 kb

Design of the DENV-2 E antigen.

<p>(A) Schematic representation of the DENV-2 polyprotein, showing the parts of prM and E included in designing the E antigen for expression in <i>P. pastoris</i>. (B) Design of the DENV-2 E antigen consisting of the 395 aa residue E ectodomain, preceded by the C-terminal 34 aa residues of prM. The grey box denotes the pentaglycine linker peptide joining the C-terminus of E ectodomain to the polyhistidine tag (6×H). (C) The predicted aa sequence of the DENV-2 E antigen shown in ‘B’. The color scheme corresponds to that shown in ‘B’. The first two aa residues (MV) were introduced due to the insertion of the initiator codon in a Kozak consensus context. The downward arrows in ‘B’ and ‘C’ denote the signal cleavage site.</p

<i>Pichia pastoris</i>-Expressed Dengue 2 Envelope Forms Virus-Like Particles without Pre-Membrane Protein and Induces High Titer Neutralizing Antibodies

<div><p>Dengue is a mosquito-borne viral disease with a global prevalence. It is caused by four closely-related dengue viruses (DENVs 1–4). A dengue vaccine that can protect against all four viruses is an unmet public health need. Live attenuated vaccine development efforts have encountered unexpected interactions between the vaccine viruses, raising safety concerns. This has emphasized the need to explore non-replicating dengue vaccine options. Virus-like particles (VLPs) which can elicit robust immunity in the absence of infection offer potential promise for the development of non-replicating dengue vaccine alternatives. We have used the methylotrophic yeast <i>Pichia pastoris</i> to develop DENV envelope (E) protein-based VLPs. We designed a synthetic codon-optimized gene, encoding the N-terminal 395 amino acid residues of the DENV-2 E protein. It also included 5’ pre-membrane-derived signal peptide-encoding sequences to ensure proper translational processing, and 3’ 6× His tag-encoding sequences to facilitate purification of the expressed protein. This gene was integrated into the genome of <i>P. pastoris</i> host and expressed under the alcohol oxidase 1 promoter by methanol induction. Recombinant DENV-2 protein, which was present in the insoluble membrane fraction, was extracted and purified using Ni²⁺-affinity chromatography under denaturing conditions. Amino terminal sequencing and detection of glycosylation indicated that DENV-2 E had undergone proper post-translational processing. Electron microscopy revealed the presence of discrete VLPs in the purified protein preparation after dialysis. The E protein present in these VLPs was recognized by two different conformation-sensitive monoclonal antibodies. Low doses of DENV-2 E VLPs formulated in alum were immunogenic in inbred and outbred mice eliciting virus neutralizing titers >1∶1200 in flow cytometry based assays and protected AG129 mice against lethal challenge (<i>p</i><0.05). The formation of immunogenic DENV-2 E VLPs in the absence of pre-membrane protein highlights the potential of <i>P. pastoris</i> in developing non-replicating, safe, efficacious and affordable dengue vaccine.</p></div