Abstract
Poxviruses and Alphaviruses constitute two promising viral vectors that have been used extensively as expression systems, or as vehicles for vaccine purposes. Poxviruses, like vaccinia virus (VV) are well-established vaccine vectors having large insertion capacity, excellent stability, and ease of administration. In turn, replicons derived from Alphaviruses like Semliki Forest virus (SFV) are potent protein expression and immunization vectors but stocks are difficult to produce and maintain. In an attempt to demonstrate the use of a Poxvirus as a means for the delivery of small vaccine vectors, we have constructed and characterized VV/SFV hybrid vectors. A SFV replicon cDNA was inserted in the VV genome and placed under the control of a VV early promoter. The replicon, transcribed from the VV genome as an early transcript, was functional, and thus capable of initiating its own replication and transcription. Further, we constructed a VV recombinant additionally expressing the SFV structural proteins under the control of a vaccinia synthetic early/late promoter. Infection with this recombinant produced concurrent transcription of the replicon and expression of SFV structural proteins, and led to the generation of replicon-containing SFV particles that were released to the medium and were able to infect additional cells. This combined VV/SFV system in a single virus allows the use of VV as a SFV delivery vehicle in vivo. The combination of two vectors, and the possibility of generating in vivo single-cycle, replicon containing alphavirus particles, may open new strategies in vaccine development or in the design of oncolytic viruses.
Highlights
Virus-based expression systems have been derived from members of diverse virus families, including widely different RNA and DNA viruses
Coinfection of Cells with Vaccinia and Semliki Forest virus (SFV) Particles To ascertain whether Vaccinia virus (VV) and SFV replication cycles are compatible, i.e., can take place concurrently in the same cells, we carried out coinfections of cells with vaccinia virus and SFV replicons
We used a vaccinia virus b– Glucuronidase recombinant and single-cycle SFV particles harboring the b–Galactosidase gene, and gene expression mediated by each system was measured
Summary
Virus-based expression systems have been derived from members of diverse virus families, including widely different RNA and DNA viruses. Vaccinia virus (VV), the representative member of the Poxviridae, constitutes an extensively used protein expression and vaccine vector. In addition to many beneficial characteristics for vaccine use, a major advantage of VV vectors is their large DNA genome that provides considerable insertion capacity, allowing the expression of large and/or multiple genes. Alphavirus-based vectors are expression systems which are smaller in size and insertion capacity, but constitute attractive vaccine candidates shown to induce strong immune responses. Alphaviruses are members of the family Togaviridae, whose genome is a positive-sense single-stranded RNA molecule of approximately 12 kb. The 59 two-thirds of the incoming genome is translated, producing the viral replicase nonstructural proteins (nsP1–4). The replicase synthesizes negative-sense copies of the genome, which serve as templates for both progeny genomes and for transcription of an mRNA from the internal subgenomic promoter [5]
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