Alphavirus-Based Vaccines

Lundstrom, Kenneth

Several types of vector systems have been engineered. There are three types of replication-deficient vectors consisting of naked RNA, recombinant particles and layered DNA vectors (Figure 1 ). The application of naked RNA vectors involves the use of in vitro transcribed RNA from an expression vector consisting of the viral nonstructural replicase genes and the foreign gene of interest downstream of the strong subgenomic promoter. The production of recombinant particles requires the co-transfection of in vitro transcribed RNA from an expression vector (as described above) and a helper vector supplying the viral structural genes into mammalian cell lines (for example, baby hamster kidney (BHK) cells). The generated particles are capable of one round of infection of a broad range of host cells, but due to the selective packaging of only expression vector RNA, no further virus production occurs. The layered DNA vector system consists of delivery of a DNA vector providing foreign gene expression from a CMV promoter. Furthermore, the engineering of vectors with an additional subgenomic promoter to the full-length genome allows for the generation of replication-proficient particles, which can provide improved delivery and extended gene expression. All alphavirus vectors described take advantage of the extremely efficient RNA replication, resulting in some 200,000 RNA copies from each RNA molecule. The essential question is: which vector system to use? Obviously, replication-proficient particles can provide efficient delivery, but suffer from potential insufficiency related to safety aspects. Although replication-deficient particles provide a higher level of safety, there is still a marginal risk of the generation of replication-proficient particles through non-homologous recombination. To minimize any unwanted recombination events, a split helper vector system with capsid and envelope genes expressed from separate helper vectors has been engineered [8] .