Plasmid DNA initiates replication of yellow fever vaccine in vitro and elicits virus-specific immune response in mice

Abstract

Yellow fever (YF) causes an acute hemorrhagic fever disease in tropical Africa and Latin America. To develop a novel experimental YF vaccine, we applied iDNA infectious clone technology. The iDNA represents plasmid that encodes the full-length RNA genome of 17D vaccine downstream from a cytomegalovirus (CMV) promoter. The vaccine was designed to transcribe the full-length viral RNA and to launch 17D vaccine virus in vitro and in vivo. Transfection with 10 ng of iDNA plasmid was sufficient to start replication of vaccine virus in vitro. Safety of the parental 17D and iDNA-derived 17D viruses was confirmed in AG129 mice deficient in receptors for IFN-α/β/γ. Finally, direct vaccination of BALB/c mice with a single 20 μg dose of iDNA plasmid resulted in seroconversion and elicitation of virus-specific neutralizing antibodies in animals. We conclude that iDNA immunization approach combines characteristics of DNA and attenuated vaccines and represents a promising vaccination strategy for YF.

Keywords: 17D vaccine; DNA vaccine; Flavivirus; Live attenuated vaccine; YFV; Yellow fever virus.

Fig. 1

Preparation of the YF 17D iDNA plasmids encoding the full-length 17D RNA downstream from the CMV promoter. (a) Schematic depiction of two iDNA plasmids, pYF17D-5 and pYF17D-16. Indicated are CMV promoter (shaded arrow), positions of the 5’ and 3’ ends of the full-length 17D cDNA, as well as intron within pYF17D-16 (shaded box). The 17D nucleotides are indicated according to 17D genome, Genbank X03700 (b) Plasmid yields in E. coli DH5α and Stbl3 cells. Miniprep DNA isolations were performed from each E. coli strain as indicated. The DNA yields were compared by gel densitometry analysis. (c) Infectious center assay (ICA) of Vero cells transfected with 200 ng of pYF17D-5 and pYF17D-16 iDNA. Transfected cells were covered with 1% agarose overlay and incubated for 4 days to form plaques, which were visualized using neutral red. (d) Indirect immunofluorescence assay (IFA) of Vero cells transfected with 200 ng of pYF17D-5 and pYF17D-16 iDNA. After transfection, aliquots of transfected Vero cells were seeded in 8-well chamber slides, fixed at 72 h in cold acetone and processed by IFA using YF-specific mouse polyclonal antiserum and FITCI-conjugated goat antibody to mouse IgG (H+L). Propidium iodide (PI) counterstain was used to visualize cell nuclei. Expression of 17D antigens after transfection of iDNA plasmid is indicated by green fluorescent Vero cells.

Fig. 2

Expression of YF 17D antigens and secretion of viruses in Vero cells transfected with 200 ng of iDNA or infected with 10³ PFU of 17D virus, by western blot and plaque assay. (a) Western blot of Vero cells transfected with indicated iDNA plasmid or infected with 17D virus. Cells were harvested at day 9 posttransfection or postinfection and solubilized in SDS-PAGE sample buffer with no 2-Mercaptoethanol. Lane 1, pYF17D-5 transfected cells; lane 2, pYF17D-16 transfected cells; lane 3, 17D-infected cells; lane 4, untreated control Vero cells; lane 5, SeeBlue Plus2 protein standard. (b) Plaque assay of growth medium from Vero cells transfected with indicated iDNA or infected with 17D virus. Growth medium samples were taken at day 8. Plaque assay was performed on Vero cell monolayers for 4 days and plaques were visualized with neutral red. (c) Growth curves of 17D viruses in iDNA-transfected (solid lines) and 17D virus-infected (dashed lines) Vero cells. Vero cells were transfected by electroporation with 200 ng of indicated iDNA plasmids (Fig. 1a) or infected with 10³ PFU of 17D virus. Designations of the viruses and plasmids are shown. Plaque assay was done in duplicates. Each data point represents average of two measurements. Standard deviations are not shown to improve clarity of the graph.

Fig. 3

Transfections of Vero cells with escalating amounts of iDNA to start replication of YF 17D virus. Vero cells were transfected by electroporation with indicated amounts of pYF17D-16 iDNA or mock-transfected using PBS. Aliquots of medium from transfected cells were taken every 24 h and virus titer in the medium samples was determined by plaque assay. Plaque assay was done in duplicates. Standard deviations are not shown to improve clarity of the graph. The result was reproduced three times with various quantities of iDNA.

Fig. 4

Replication of YF 17D viruses in AG129 mice. AG129KO mice (129/Sv/Ev background) were inoculated with parental biological YF 17D virus or with YF 17D virus recovered from YF17D-16 iDNA. Neurotropic, viscerotropic properties and induction of IFN-γ were assessed. Briefly, three mice from each group were sacrificed at indicated time points and neurotropic and viscerotropic features as well as induction of IFN-γ were measured as described in Materials and Methods. Panels (a) and (b), replication kinetics and viral loads in brain and liver tissue, respectively. Panels (c) and (d), induction of IFN-γ mRNA in brain and liver tissue, respectively.

Fig. 5

Detection of serum antibody in sera of iDNA-vaccinated or 17D virus-vaccinated BALB/c mice, by IFA. (a) BALB/c mice 1–8 were injected i.m. with 20 µg of pYF17D-16 iDNA that encoded the full-length 17D genomic RNA as a cDNA copy downstream from the CMV promoter (Fig. 1a). After iDNA injection, mice were electroporated in order to launch live attenuated 17D vaccine in vivo and to elicit 17D-specific antibody response. (b) Mice 9–15 were vaccinated i.m. by injection with 10⁴ PFU of 17D virus. Sera were taken at day 21 post-vaccination and probed at 1:10 dilution with acetone-fixed monolayers of Vero cell that were previously incubated with 100 PFU of 17D virus. After incubation with mouse antisera, monolayers were incubated with FITC-conjugated goat antimouse IgG antibody to visualize foci of YF 17D-expressing cells. The YF 17D-specific mouse antisera showed green fluorescent cell foci indicating binding to 17D-infected Vero cells.