Alphaviruses are positive-stranded RNA viruses that have a broad host range and therefore are capable of replicating in many vertebrate and invertebrate cells. The single-stranded alphavirus genome is divided into two ORFs. The first ORF encodes the nonstructural proteins that are translated upon entry of the virus into the cytoplasm and are responsible for transcription and replication of viral RNA. The second ORF is under the control of a subgenomic promoter and normally encodes the structural proteins, which are responsible for encapsidation of viral RNA and final assembly into enveloped particles. Expression vectors have been engineered from at least three alphaviruses in which the structural protein gene region has been replaced by heterologous genes and have been shown to express high levels of the heterologous protein in cultured cells. These RNA vectors, known as replicons, are capable of replicating on their own but are not packaged into virus-like particles unless the structural proteins are provided in trans. Thus, replicons are single cycle vectors incapable of spreading from infected to noninfected cells. Because of these features, alphavirus replicon vectors are being developed as a platform vaccine technology for numerous viral, bacterial, protozoan and tumour antigens where they have been shown to be efficient inducers of both humoral and T cell responses. In addition, as the alphavirus structural proteins are not expressed in vaccine recipients, antivector immune responses are generally minimal, allowing for multiple effective immunisations of the same individual.

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Seoul virus (SEOV) is one of four known hantaviruses causing hemorrhagic fever with renal syndrome (HFRS). Candidate naked DNA vaccines for HFRS were constructed by subcloning cDNA representing the medium (M; encoding the G1 and G2 glycoproteins) or small (S; encoding the nucleocapsid protein) genome segment of SEOV into the DNA expression vector pWRG7077. We vaccinated BALB/c mice with three doses of the M or S DNA vaccine at 4-week intervals by either gene gun inoculation of the epidermis or needle inoculation into the gastrocnemius muscle. Both routes of vaccination resulted in antibody responses as measured by ELISA; however, gene gun inoculation elicited a higher frequency of seroconversion and higher levels of antibodies in individual mice. We vaccinated Syrian hamsters with the M or S construct using the gene gun and found hantavirus-specific antibodies in five of five and four of five hamsters, respectively. Animals vaccinated with the M construct developed a neutralizing antibody response that was greatly enhanced in the presence of guinea pig complement. Immunized hamsters were challenged with SEOV and, after 28 days, were monitored for evidence of infection. Hamsters vaccinated with M were protected from infection, but hamsters vaccinated with S were not protected.

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Here we describe a system for promoterless analysis of putative internal ribosome entry site (IRES) elements using an alphavirus (family Togaviridae) replicon vector. The system uses the alphavirus subgenomic promoter to produce transcripts that, when modified to contain a spacer region upstream of an IRES element, allow analysis of cap-independent translation of genes of interest (GOI). If the IRES element is removed, translation of the subgenomic transcript can be reduced >95% compared to the same transcript containing a functional IRES element. Alphavirus replicons, used in this manner, offer an alternative to standard dicistronic DNA vectors or in vitro translation systems currently used to analyze putative IRES elements. In addition, protein expression levels varied depending on the spacer element located upstream of each IRES. The ability to modulate the level of expression from alphavirus vectors should extend the utility of these vectors in vaccine development.