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

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

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

Antibody-mediated neutralization and Hemagglutination Inhibition from CHIKV-infected patient serum.

<p>nAb titers (A) and Hemagglutination Inhibition (HI) antibody responses (B) in patient sera (SRMC-1 to SRMC-30) to CHIKV. Similar results were observed in 2 independent experiments. There is a positive correlation exists between nAb and HI on CHIKV infected patients (C). These relationships were evaluated using the Spearman correlation test using the Prism 5 Graph Pad software. Neutralization of CHIKV infectivity with patient serum (D). The IC50 is defined as the reciprocal of the antiserum dilution at which CHIKV virus entry is 50% inhibited (dashed line). Similar results were observed in 2 independent experiments.</p

CHIKV DNA vaccination and infection.

<p>(A&B) Analysis of proinflammatory cytokines (TNF-α and IL-6) in CHIKV vaccinated and infected mice. Cytokine levels (pg/ml) were assayed by ELISA from the cell free sera from 10 days post infection. These data represent the average 3 wells/mouse and standard deviations of 4 mice. (C) Percent survival in CHIKV-challenged mice. Similar results were observed in 2 independent experiments with at least <i>n</i> = 10 per group for each experiment. (D) The viremia, 5 day after challenge, as measured by a plaque assay. Mice immunized with pVax1 (control) or immunized pMCE321 (vaccine) were challenged with the PC-08 CHIKV strain at a dose of 7 log10 PFU by the intranasal route. Data are mean ± SEM of 5 animals.</p

CHIKV DNA vaccination induces strong immunity in mice.

<p>BALB/c mice were immunized three times, each 2 weeks apart, with 25 µg pVax1 vector or pMCE321-Env and sacrificed 1 week after the 3^rd immunization. (A) Splenocytes from immunized animals were harvested and cultured overnight in the presence of peptide pool matrix spanning the Envelope protein (pool-1 & pool-2) and the IFN-γ response to each pool was measured by ELISpot as described in the <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0000928#s2" target="_blank">Materials and Methods</a>. Values represent the mean and standard deviation of triplicate wells and are representative of three independent experiments. (B) Systemic anti-Env IgG levels after DNA immunization. Each group of inbred BALB/c mice (<i>n = 4</i>) was immunized with indicated vaccines. Mice were bled 1 week after each immunization, and then sera were diluted to 1/100 for reaction with CHIKV-Env. OD was measured at 450 nm. Values and bars represent mean (<i>n = 4</i>) and the SEM. (C and D) Quantification of CHIKV specific neutralizing and HI titer in sera from DNA immunized mice (pVax1/pCHIKV-E1/pCHIKV-E2 and pMCE321) to CHIKV. The nAb titers are plotted as the highest dilution of serum that resulted in at least 50% inhibition of CPE. The highest dilution of the serum that inhibited hemagglutination was recorded as the HI titer. Similar results were observed in three independent experiments with at least <i>n</i> = 4 per group for each experiment.</p

Isolation and identification of CHIKV.

<p>The microphotographs show normal uninfected Vero cells (A) and the Vero cells infected with CHIKV virus isolate. CHIKV infection in Vero cells causes characteristic foamy cytopathic effect (CPE) 48 hours p.i. as seen with the isolate. (B) RT-PCR analysis of CHIKV viral isolates. Agarose gel photograph showing the RT-PCR amplified product (305 bp) of the CHIKV positive patient isolates (Lane 1&2). The uninfected negative control (Lane 3) shows no amplification. (C) Electron micrographs of CHIKV viral isolates (D) Phylogenetic Tree generated with E2 amplicon from CHIKV Isolate. * Indicates the PC-08 CHIKV strain.</p

Histopathology analysis of CHIKV challenged mice.

<p>(A) H&E stained sections of Brain. pMCE321 vaccinated mice showed no severe pathological changes and showed only minimal microglial formation. Capsid immunized mice group showed severe hemorrhage and microglia formation similar to the naïve group. (B) H&E stained sections of Heart, Liver, Kidney and Lungs. Envelope vaccinated group showed minimal or no pathological changes in the organs. The naïve group showed severe pathological changes indicative of virus infection and the Capsid DNA immunized group showed similar pathological changes to the naïve group. Representative data are shown from 2 mice/group.</p