Construction of recombinant fowlpox viruses carrying multiple vaccine antigens and immunomodulatory molecules

Here we describe plasmid vectors and selection protocols developed to allow the construction of recombinant fowlpox viruses (rFPVs) with up to three insertions of foreign DNA in the viral genome. Transient dominant selection allows the construction of recombinant viruses that do not retain the selection markers and can therefore be used for the insertion of additional genes at other sites in the viral genome. A SYBR Green real-time PCR sequence detection assay was applied to the identification of recombinant viruses from individual plaques, eliminating the need for amplification and hybridization from the transient dominant protocol and resulting in significant savings in time at each round of plaque purification. Dominant selection techniques allow more rapid recombinant virus construction; however, as the markers are retained along with the gene of interest, they can only be used to generate the final recombinant. rFPV vaccines constructed using these techniques have reached preclinical nonhuman primate and phase I human clinical trials in prime/boost vaccination studies as human immunodeficiency virus (HIV) therapeutic and prophylactic vaccines.