A plasmid family containing two different expression cassettes suitable for immunomodulation and genetic immunization.

We have developed an improved eukaryotic expression vector that consists of two distinct, complete, and differentially regulated transcription units. The peculiarities of this prototype vector, named pRC110, are represented by two different strong promoter/enhancer sequences, cytomegalovirus and Rous sarcoma virus, that independently drive transcription of two recombinant cDNAs, which may be easily cloned into specific rare restriction sites. Moreover, we describe a simple way to introduce an optimal translational start site context 5' to any peptide to be cloned in our vectors, thus allowing the correct and efficient expression of even a single part of a larger gene or a short synthetic peptide lacking its own AUG and neighboring regions. We demonstrate the in vivo expression efficacy of pRC110 for use in genetic vaccination through direct intramuscular gene transfer: specific antibodies are raised against one of the encoded peptides 3 weeks after muscle injection, and efficient transcription of the other syngeneic cDNA, mouse interleukin-2, is shown. The development of a "family" of vectors directly deriving from pRC110 is also described, with the common property that one of the encoded proteins may modulate the effects of the other. We recommend the use of pRC110 for genetic immunization and immunological response studies, when the concomitant local production of an immunogenic peptide and of a syngeneic immunomodulating cytokine is required.