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

Comparative properties of DNA vaccines over other vaccine approaches.

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

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

Survival and weight loss following H5N1 ferret challenge.

<p>(a) Kaplan-Meier curve of ferrets challenged with an H5N1 virus (A/Vietnam/1203/04). Ferrets were immunized and electroporated three times with the indicated constructs. One month following the final immunization, ferrets were challenged intranasally with a lethal dose of an H5N1 influenza virus. (b) Maximum weight loss in each group. Error bars represent 1 standard deviation from the mean, and are shown only in the positive direction for clarity.</p

Induction of antigen-specific immune responses in BALB/C mice.

<p>(a) Quantification of IFN-γ secreting CD8+ T cells at one month following the final immunization. Splenocytes were stimulated with pools of overlapping peptides spanning the length of the antigen, separated into multiple pools. Also included are CD8-depleted controls. (b) Endpoint antibody ELISA from serum collected from mice immunized with pH5HA. All error bars represent ±1 standard deviation from the mean, and are representative of three independent experiments.</p

Kaplan-Meier survival curve in mice challenged with (a) H1N1 influenza (A/PR/8/34) and (b) H5N1 influenza (A/Vietnam/1203/04).

<p>All mice were immunized with pNP (except for naïve) and depleted of CD4+ T cells, CD8+ T cells, both, or neither.</p

Hemagglutination inhibition activity in rhesus macaques following two immunizations.

<p>HI activity is shown for clade 1 (A/Vietnam/1203/04), clade 2.1 (A/Anhui/01/05), and a more divergent clade 2.1 (A/Indonesia/05/05). Naïve monkeys showed no detectable HI activity against any of the indicated strains.</p

Results following an H5N1 challenge of immunized mice.

<p>Mice were challenged with 100LD₅₀ of an H5N1 virus (A/Hanoi/05/2005). (a) Kaplan-Meier curve showing the percent survival in each group. (b) Average weight loss among the surviving members of each group (and the weight loss seen in naïve mice before death). Error bars represent 1 standard deviation from the mean, and are shown only in the positive direction for clarity.</p