REDD1 is required for optimal cell survival independent of stimulation.

<p>Wildtype (WT) and knockout (KO) mouse lymph node cells were stimulated with 1.5 μg/ml PHA and cell survival was measured by flow cytometry with dead cell staining. <b>(A)</b> Representative flow plots of dead cell staining, gated on CD4 or CD8 T cells. The percentage of WT and KO cells falling within the gate are indicated in the corner of each panel. <b>(B)</b> Average percentages of live CD4 or CD8 T cells. N = 8 WT; N = 8 KO. *p = 0.01 to 0.05; **p = 0.001 to 0.01; ***p < 0.001.</p

REDD1 does not affect upregulation of activation markers CD69 or CD25.

<p>Wildtype (WT) and knockout (KO) mouse lymph node cells were stimulated with 1.5 μg/ml PHA and CD69 and CD25 expression was measured by flow cytometry. <b>(A)</b> Representative flow plots of CD69 and CD25 staining gated on CD4 or CD8 T cells. The percentage of WT and KO cells falling within the gate are indicated in the corner of each panel. <b>(B)</b> Average percentages of CD4 or CD8 T cells expressing CD69 and CD25 after stimulation. N = 4 WT; N = 4 KO.</p

REDD1 does not affect PHA induced apoptosis.

<p>Wildtype (WT) and knockout (KO) mouse lymph node cells were stimulated with 1.5 μg/ml PHA and cell survival was measured by flow cytometry with PI/Annexin V staining. <b>(A)</b> Representative flow plots of Annexin V/PI staining gated on CD4 or CD8 T cells. <b>(B)</b> Average percentages of live (PI-/Annexin V-), dead (PI+AnnexinV+), and apoptotic (Annexin V+) CD4 or CD8 T cells. N = 4 WT; N = 4 KO. *p = 0.01 to 0.05; **p = 0.001 to 0.01; ***p < 0.001.</p

REDD1 mRNA and protein is expressed in lymphoid tissues and is upregulated during T cell activation.

<p>Primary human CD4 T cells were stimulated with 1.5 μg/ml PHA + 100 U/ml IL-2 or 3 CD3/CD28 beads/cell + 100 U/ml IL-2. REDD1 mRNA <b>(A)</b> and protein <b>(B)</b> expression was determined using qRT-PCR and immunoblot, respectively. <b>(C)</b> Mouse splenocytes were stimulated with 1.5 μg/ml PHA + 20 U/ml IL-2 and REDD1 mRNA expression was determined by qRT-PCR. All qRT-PCR data is presented as fold change compared to the unstimulated cells. qRT-PCR and immunoblot data are representative of 3 individual experiments.</p

REDD1 is required for optimal T cell proliferation.

<p>Wildtype (WT) and knockout (KO) mouse lymph node cells were labeled with CFSE and stimulated with 1.5 μg/ml PHA for 72 hours. <b>(A)</b> Representative flow plots of CFSE staining gated on CD4 T cells. <b>(B)</b> Average percentages of CD4 T cells that have undergone the indicated number of divisions in 72 hours. Statistically significant differences between the WT and KO populations for each division are indicated next to the legend. Statistically significant differences between 'few divisions' (divisions 0–2) and 'multiple divisions' (divisions 3–8) are also indicated. N = 8 WT; N = 8 KO. *p = 0.01 to 0.05; **p = 0.001 to 0.01; ***p < 0.001.</p

Life cycle of Chikungunya virus in Africa showing the interconnection between the sylvatic cycle on the left and the urban cycle on the right.

<p>Particularly in Africa, the virus is maintained in a sylvatic cycle comprising non-human primates and different species of forest-dwelling mosquitoes including <i>Aedene</i> mosquitoes (<i>Ae. Africanus</i>, <i>Ae. furcifer-taylori</i>, <i>Ae. dalzieli</i>, etc.,) and non <i>Aedene</i> mosquitoes (Mansonia, Culex, etc.) <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0000623#pntd.0000623-Diallo1" target="_blank">[10]</a>.</p

Comparative properties of DNA vaccines over other vaccine approaches.

<p>Comparative properties of DNA vaccines over other vaccine approaches.</p

Levels of CHIKV-specific IgG in mice immunized with CHIKV vaccines.

<p>Each group of C57BL/6 mice (<i>n</i> = 5) was immunized with 12.5 µg of pVax1 control vector or CHIKV vaccine plasmids as indicated at 0 and 2 wk. Mice were bled 2 wk after each immunization, and each group's serum pool was diluted to 1∶100 and 1∶500 for reaction with specific vaccine constructs. Serum was incubated for 1 h at 37°C on 96-well plates coated with 2 mg/ml of respective CHIKV peptides, and antibody was detected using anti-mouse IgG-HRP and OD was measured at 405 nm.</p

DNA vaccinated mice are capable of producing antibodies against the antigens encoded in the DNA vaccine.

<p>Hela cells transfected with DNA plasmid vaccine encoding the CHIKV Capsid (left) and Envelope (right) genes were examined for protein expression using confocal microscopy. Serum collected from mice immunized with the DNA vaccine was used as the primary antibody for detection of CHIKV proteins. Two days post-transfection, the cells, treated with serum and then with an anti-mouse IgG conjugated with Alexa-Fluor 488, were visualized under the Ziess LSM510 META NLO Laser Scanning Confocal Microscope (×63). Expression of high levels of CHIKV proteins in these cells revealed the presence of CHIKV-specific antibodies, thereby validating the efficacy of the DNA vaccine in inducing antibodies.</p

Life cycle of Chikungunya virus inside infected cells.

<p>Characteristically, there are two rounds of translation: (+) sense genomic RNA (49S′ = 11.7 kb) acts directly as mRNA and is partially translated (5′ end) to produce non-structural proteins (nsp's). These proteins are responsible for replication and formation of a complementary (−) strand, the template for further (+) strand synthesis. Subgenomic mRNA (26 S = 4.1 kb) replication occurs through the synthesis of full-length (−) intermediate RNA, which is regulated by nsp4 and p123 precursor in early infection and later by mature nsp's. Translation of the newly synthesized sub-genomic RNA results in production of structural proteins such as Capsid and protein E2-6k-E1 (from 3′ end of genome). Assembly occurs at the cell surface, and the envelope is acquired as the virus buds from the cell and release and maturation almost simultaneous occurred. Replication occurs in the cytoplasm and is very rapid (∼4 h) <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0000623#pntd.0000623-Edwards1" target="_blank">[28]</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0000623#pntd.0000623-Strauss1" target="_blank">[29]</a>.</p