Novel vaccine against Venezuelan equine encephalitis combines advantages of DNA immunization and a live attenuated vaccine

Abstract

DNA vaccines combine remarkable genetic and chemical stability with proven safety and efficacy in animal models, while remaining less immunogenic in humans. In contrast, live-attenuated vaccines have the advantage of inducing rapid, robust, long-term immunity after a single-dose vaccination. Here we describe novel iDNA vaccine technology that is based on an infectious DNA platform and combines advantages of DNA and live attenuated vaccines. We applied this technology for vaccination against infection with Venezuelan equine encephalitis virus (VEEV), an alphavirus from the Togaviridae family. The iDNA vaccine is based on transcription of the full-length genomic RNA of the TC-83 live-attenuated virus from plasmid DNA in vivo. The in vivo-generated viral RNA initiates limited replication of the vaccine virus, which in turn leads to efficient immunization. This technology allows the plasmid DNA to launch a live-attenuated vaccine in vitro or in vivo. Less than 10 ng of pTC83 iDNA encoding the full-length genomic RNA of the TC-83 vaccine strain initiated replication of the vaccine virus in vitro. In order to evaluate this approach in vivo, BALB/c mice were vaccinated with a single dose of pTC83 iDNA. After vaccination, all mice seroconverted with no adverse reactions. Four weeks after immunization, animals were challenged with the lethal epidemic strain of VEEV. All iDNA-vaccinated mice were protected from fatal disease, while all unvaccinated controls succumbed to infection and died. To our knowledge, this is the first example of launching a clinical live-attenuated vaccine from recombinant plasmid DNA in vivo.

Fig. 1. Preparation of pTC83 iDNA containing the full-length TC-83 cloned genome and generation of TC-83 virus in transfected CHO cells

(a) Schematic representation of pTC83 plasmid. Restriction sites used for preparation of the full-length TC-83 clone are indicated. (b) Indirect immunofluorescence assay (IFA) of CHO cells transfected with pTC83 iDNA. IFA was performed at 24 hr (left panel) and 48 hr post electroporation. In order to visualize nuclei in transfected cells, the 4',6-diamidino-2-phenylindole (DAPI) stain was used. (c) Western blot of CHO cells transfected with pTC83 iDNA (left panel) and plaque assay of the supernatant from CHO cells transfected with pTC83 iDNA (right panel). Western blot was performed at 24 hr post electroporation using ATCC antiserum against VEEV. Plaque assay was carried out in Vero cell monolayers. (d) Replication of iDNA-derived TC-83 virus in infected Vero cells. Vero cells were infected with 100 PFU of iDNA-derived TC-83 virus. Plaque titer was determined in duplicates, error bars are not visible at the log scale shown.

Fig. 2. Replication kinetics of TC-83 virus in virus-infected and iDNA-transfected CHO cells

(a) IFA of CHO cells transfected with 5 µg or 1 µg of iDNA (upper panel) or with 10⁵ PFU of TC-83 virus (lower panel). (b) Growth curves of TC-83 viruses in virus-infected and in iDNA-transfected CHO cells. Left panel, CHO cells were either infected with 10⁴–10⁵ PFU of virus, or transfected with 0.2–5 µg of pTC83 iDNA. Right panel, CHO cells were transfected with 8 ng to 1 µg of pTC83 iDNA. Plaque titer was determined in duplicates, error bars are not visible at the scale shown.

Fig. 3. Detection of TC-83 virus and antibody in vivo

(a) Detection of viremia in plasma samples collected from pTC83 iDNA-vaccinated BALB/c mice. BALB/c mice were injected intravenously (i.v.) with mixture of pTC83 iDNA and TransIT transfection reagent (Mirus, Madison, WI). Plasma samples were collected on day 1 post inoculation and incubated with Vero cells for virus amplification. At day 3, culture medium was collected and assayed by plaque titration using Vero cell monolayers. Left panel, TC83 virus recovered from plasma of iDNA-inoculated mice. Right panel, virus recovered from plasma after immunization with prototype VEEV TC-83 vaccine. (b) Detection of serum antibody in the plasma samples 1–10 from pTC83 iDNA-vaccinated BALB/c mice, by IFA. Ten BALB/c mice were vaccinated by electroporation with pTC83 iDNA. Plasma samples from individual mice were collected on day 21 post vaccination and probed with monolayers of CHO cells infected with TC-83-virus at MOI=0.1. Plasma samples were used at 1:20 dilution.

Fig. 4

Survival of BALB/c mice after challenge with VEEV. Mice were vaccinated by electroporation with 50 µg of pTC83 iDNA. On day 28 after vaccination, mice were transferred into BSL3 facility and challenged s.c. with 10⁵ PFU of VEEV virus. Mice were daily monitored for signs of illness and deaths. Uniform lethality was observed for control mice while all iDNA-vaccinated mice survived challenge.