Safety aspects related to recombinant protein expression from Semliki Forest virus vectors

Semliki Forest virus vectors (SFV) have been developed for efficient transgene expression to result in high receptor yields (50-200 pmol receptor/mg protein) in a variety of mammalian host cells. Transfer of the SFV technology to mammalian cells growing in suspension cultures has made it feasible to produce hundreds of milligrams of receptor proteins in a short time. Large-scale production, however, raises the questions of the safety of handling virally infected cells for down-stream processing. Analysis of cell culture medium and SFV-infected cells revealed that some infectious particles were still present. Replacement of virus-containing medium at 2 h post-infection efficiently removed the majority of infectious replication-deficient SFV particles. Washes with PBS further reduced the number of infectious particles significantly both in the medium and associated with cells to levels that allowed safe handling of SFV-infected cells outside the cell culture facility for biochemical, pharmacological, or electrophysiological assays or down-stream processes in connection to receptor purification. Furthermore, engineering of novel temperature-sensitive mutant SFV vectors resulted in temperature-controlled transgene expression, which completely eliminates the risk of contaminating laboratory personnel.