A DNA Vaccine against Chikungunya Virus Is Protective in Mice and Induces Neutralizing Antibodies in Mice and Nonhuman Primates

Chikungunya virus (CHIKV) is an emerging mosquito-borne alphavirus indigenous to tropical Africa and Asia. Acute illness is characterized by fever, arthralgias, conjunctivitis, rash, and sometimes arthritis. Relatively little is known about the antigenic targets for immunity, and no licensed vaccines or therapeutics are currently available for the pathogen. While the Aedes aegypti mosquito is its primary vector, recent evidence suggests that other carriers can transmit CHIKV thus raising concerns about its spread outside of natural endemic areas to new countries including the U.S. and Europe. Considering the potential for pandemic spread, understanding the development of immunity is paramount to the development of effective counter measures against CHIKV. In this study, we isolated a new CHIKV virus from an acutely infected human patient and developed a defined viral challenge stock in mice that allowed us to study viral pathogenesis and develop a viral neutralization assay. We then constructed a synthetic DNA vaccine delivered by in vivo electroporation (EP) that expresses a component of the CHIKV envelope glycoprotein and used this model to evaluate its efficacy. Vaccination induced robust antigen-specific cellular and humoral immune responses, which individually were capable of providing protection against CHIKV challenge in mice. Furthermore, vaccine studies in rhesus macaques demonstrated induction of nAb responses, which mimicked those induced in convalescent human patient sera. These data suggest a protective role for nAb against CHIKV disease and support further study of envelope-based CHIKV DNA vaccines

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

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

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

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

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