A PLASMID FAMILY CONTAINING 2 DIFFERENT EXPRESSION CASSETTES SUITABLEFOR IMMUNOMODULATION AND GENETIC IMMUNIZATION

We have developed an improved eukaryotic expression vector that consis ts of two distinct, complete, and differentially regulated transcripti on units. The peculiarities of this prototype vector, named pRC110, ar e represented by two different strong promoter/enhancer sequences, cyt omegalovirus and Rous sarcoma virus, that independently drive transcri ption of two recombinant cDNAs, which may be easily cloned into specif ic rare restriction sites. Moreover, we describe a simple way to intro duce an optimal translational start site context 5' to any peptide to be cloned in our vectors, thus allowing the correct and efficient expr ession of even a single part of a larger gene or a short synthetic pep tide lacking its own AUG and neighboring regions. We demonstrate the i n vivo expression efficacy of pRC110 for use in genetic vaccination th rough direct intramuscular gene transfer: specific antibodies are rais ed against one of the encoded peptides 3 weeks after muscle injection, and efficient transcription of the other syngeneic cDNA, mouse interl eukin-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 immunologica l response studies, when the concomitant local production of an immuno genic peptide and of a syngeneic immunomodulating cytokine is required . (C) 1998 Academic Press.