Method for generating recombinant poxviruses

A method of generating recombinant poxvirus, wherein the poxvirus is vaccinia virus (VACV), and wherein a recombinant vaccina virus (rVACV) is generated expressing a gene of interest (GOI) the method comprising

• homologously recombining a replication-inducible VACV (vIND) parental virus genome and a DNA shuttle vector to generate the rVACVs with the GOI,
• wherein expression of one or more essential genes in the parental vIND is controlled by an inducible mechanism, wherein the one or more essential genes is required for viral replication of the vIND,
• wherein the DNA shuttle vector comprises an expression cassette comprising the gene of interest operably linked to a promoter, the cassette flanked by right and left regions that are homologous to the parental vIND virus genome, wherein the right flanking region is homologous to a region in the parental vIND genome that is upstream of the inducible mechanism and the left flanking region is homologous to a region in the parental vIND genome that is downstream of the inducible gene, such that homologous recombination between vIND and the DNA shuttle vector leads to insertion of the expression cassette into the parental vIND virus genome between the homologous regions and results in loss of the inducible mechanism control on the expression of the one or more essential genes in the rVACV,
• transfecting the DNA shuttle vector into parental vIND-infected cells, and
• purifying the rVACVs from the parental vIND by serial passage in the absence of the parental inducer.