Next generation sequencing of DNA-launched Chikungunya vaccine virus

Abstract

Chikungunya virus (CHIKV) represents a pandemic threat with no approved vaccine available. Recently, we described a novel vaccination strategy based on iDNA® infectious clone designed to launch a live-attenuated CHIKV vaccine from plasmid DNA in vitro or in vivo. As a proof of concept, we prepared iDNA plasmid pCHIKV-7 encoding the full-length cDNA of the 181/25 vaccine. The DNA-launched CHIKV-7 virus was prepared and compared to the 181/25 virus. Illumina HiSeq2000 sequencing revealed that with the exception of the 3' untranslated region, CHIKV-7 viral RNA consistently showed a lower frequency of single-nucleotide polymorphisms than the 181/25 RNA including at the E2-12 and E2-82 residues previously identified as attenuating mutations. In the CHIKV-7, frequencies of reversions at E2-12 and E2-82 were 0.064% and 0.086%, while in the 181/25, frequencies were 0.179% and 0.133%, respectively. We conclude that the DNA-launched virus has a reduced probability of reversion mutations, thereby enhancing vaccine safety.

Keywords: Alphavirus; CHIKV; Chikungunya fever; Chikungunya virus; DNA vaccine; Live attenuated vaccine.

Figure 1

Preparation and characterization of iDNA-launched CHIKV-7 vaccine virus. (a) Schematic depiction of the CHIKV-7 virus production in Vero cells transfected with iDNA plasmid. The full-length cDNA copy of CHIKV genome (solid line) was placed downstream from the CMV promoter (open arrow) to transcribe the full-length viral RNA. Within the RNA, indicated are the non-structural genes (open box), 26S promoter (solid arrow), and structural genes (filled box). (b) Infectious center assay (ICA) of transfected Vero cells. Vero cells were transfected by electroporation using 1μg of iDNA plasmid pCHIKV-7. Aliquot of transfected cells was seeded into 6-well plates for ICA as described in Materials and Methods. (c) Expression of CHIKV antigens in iDNA-transfected Vero cells by immunofluorescence assay (IFA) at 18, 30 and 48 h post-transfection. Sham-transfected cells are shown as negative control (NC). Aliquots of transfected cells were seeded in 8-well chamber slides, fixed at indicated times in cold acetone and processed by IFA using mouse CHIKV-specific antibody VR-64, followed by FITC-conjugated secondary antibody to mouse IgG (H+L). Propidium iodide (PI) nuclear counterstain was used to visualize cell nuclei (Jones and Kniss, 1987). Red fluorescence indicates nuclear staining with the PI. (d) Expression of CHIKV antigens in Vero cells transfected with pCHIKV-7 DNA or infected with 181/25 P1 virus, by western blot. Cells were harvested at 60 h. Proteins in cells lysates were separated by SDS-PAGE. Western blot was done using mouse antibody VR-64 followed by AP-conjugated secondary antibody to mouse IgG (H+L). Lane 1, pCHIKV-7 transfected Vero cells; lane 2, medium from untreated Vero cells; lane 3, 181/25-infected cells; lane 4, untreated Vero cells; M, See BluePlus2 protein molecular weight marker (Thermo). Arrows indicate bands resulting from the CHIKV structural protein expression.

Figure 2

Characterization of the CHIKV-7 vaccine virus and 181/25 virus by growth curve and next generation sequencing. (a) Growth curves of CHIKV-7 virus in iDNA-transfected Vero cells (solid line) and of 181/25 virus in infected Vero cells (dashed line). Vero cells were transfected with 1 μg of pCHIKV-7 iDNA plasmid or infected with 10³ PFU of 181/25 virus. Aliquots of growth medium supernatants were collected and the virus was quantitated by direct plaque assay. Virus presence in the growth medium was determined by plaque assay in duplicates. Each data point represents average of three measurements. Standard deviations are indicated. Data at -1 h indicate PFU of 181/25 virus at the start of infection and IC number corresponding to 1 μg iDNA at the start of transfection, respectively. Data from 0 h show PFU/ml. (b) Plaque morphology of CHIKV-7 virus from transfected Vero cells. Average size of total 51 plaques is 4.30±0.93 mm, as measured using magnified image; with five largest plaques 1.37 times larger than the average size. (c) Plaque morphology of 181/25 P1 virus from transfected Vero cells. Average size of total 48 plaques is 5.01±1.78 mm, as measured using magnified image, with five largest plaques 1.78 times larger than the average size. (d) Comparison of sequence coverage and depth of the virus genomes. RNA was isolated from each virus by Trizol LS and sequenced using Illumina HiSeq2000. Sequencing products were assembled to a reference CHIKV 181/25 sequence. The locations of the nonstructural and structural polyproteins are indicated.