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

Construction and characterization of CHIKV DNA vaccine.

<p>(A) Schematic representation of pMCE321 construct. The flanking enzyme sites used for cloning, Kozak expression element, CMV promoter, human IgE-leader, CHIKV fusion gene (E3-E2-E1), and cleavage sites (CS) are indicated and were cloned into the pVax1 vector. (B) Expression of pMCE321 constructs was confirmed <i>in vitro</i> using Envelope-E1 antiserum for the Western blot of CHIKV envelope antigens expressed in Vero and BHK-21 cells by Western blotting. Arrows indicate the positions of E1 protein expression. (C) Immunofluorescent assay showing staining of Vero cells transfected with pCHIKV-E1, pCHIKV-E2, or pMCE321 constructs and transient expression of the envelope proteins. (D). FACS analysis of envelope expression in transfected cells (0.5×10⁶ cells). Vero cells were transfected with indicated constructs and stained with anti-Env sera raised in mice, followed by staining with secondary PE-conjugated anti-mouse IgG antibody as indicated. Two representative FACS histograms are shown.</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 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

Clinical observation in Chikungunya patients.

<p>Clinical observation in Chikungunya patients.</p

Immunogenicity of CHIKV DNA vaccine in nonhuman primates.

<p>(A) IFN-γ ELISpot assay results presented are from individual macaques 2 weeks after the fifth immunization against pMCE321 administered vaccine. PBMCs harvested from animals immunized with pMCE321 were used in the IFN-γ assay. We also tested PBMCs that were depleted of CD8⁺ T cells by magnetic bead separation before <i>in vitro</i> stimulation. The PBMCs were incubated in the presence of the following stimulators and controls: R10 medium (negative control), Con A (5 µg/ml positive control), and 10 µg/ml CHIKV peptide mix. Data are presented as the SI (experimental counts/spontaneous counts), where the spontaneous count wells are from the R10-negative control wells as described in <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 of triplicate cultures and are representative of three independent experiments. (B) nAb titers from sera of DNA vaccinated monkeys are shown. The pMCE321 DNA vaccine construct induced nAb responses ranging from 80-1,280 titers and mimicked those induced in convalescent patient sera.</p