Non-replicating recombinant vaccinia virus expressing CD80 to enhance T-cell stimulation

The following method describes the generation of a recombinant vaccinia virus expressing a costimulatory molecule (human CD80 or B7.1).The procedure first requires the cloning, by classical methods not described here, of the gene of interest, e.g. CD80, into a vaccinia shuttle plasmid under the control of a virus-specific promoter enabling a transcription during the early phase of infection. Flanking the insert, the plasmid contains viral sequences and a selection maker needed for the insertion into the viral genome. The successive plaque isolation of recombinant virus on cell monolayer described here is based on the transient "gpt" selection system which enables other insertions in different loci of the same virus. Finally, after verification amplification and titration of the recombinant vector, replication will be impaired by a psoralen-UV treatment in order to produce a non-replicating virus. Expression and function of inserts, following infection of cells, are verified by specific phenotypic and functional assays.