DNA coding for bacteriophage T7 RNA polymerase was ligated to a vaccinia virus transcriptional promoter and integrated within the vaccinia virus genome. The recombinant vaccinia virus retained infectivity and stably expressed T7 RNA polymerase in mammalian cells. Target genes were constructed by inserting DNA segments that code for (3-galactosidase or chloramphenicol acetyltransferase into a plasmid with bacteriophage T7 promoter and terminator regions. When cells were infected with the recombinant vaccinia virus and transfected with plasmids containing the target genes, the latter were expressed at high levels. Chloramphenicol acetyltransferase activity was 400-600 times greater than that observed with conventional mammalian transient-expression systems regulated either by the enhancer and promoter regions of the Rous sarcoma virus long terminal repeat or by the simian virus 40 early region. The vaccinia/T7 hybrid virus forms the basis of a simple, rapid, widely applicable, and efficient mammalian expression system. Recombinant DNA technology has made it possible to develop molecular cloning vectors that allow expression of heterologous genes in prokaryotic and eukaryotic cells.

BCG, a live attenuated tubercle bacillus, is the most widely used vaccine in the world and is also a useful vaccine vehicle for delivering protective antigens of multiple pathogens. Extrachromosomal and integrative expression vectors carrying the regulatory sequences for major BCG heat-shock proteins have been developed to allow expression of foreign antigens in BCG. These recombinant BCG strains can elicit long-lasting humoral and cellular immune responses to foreign antigens in mice.

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A novel expression system based on coinfection of cells with two recombinant vaccinia viruses has been developed. One recombinant vaccinia virus contained the bacteriophage T7 RNA polymerase gene under control of a vaccinia virus promoter. The second recombinant vaccinia virus contained a target gene of choice flanked by bacteriophage T7 promoter and termination sequences. Maximum expression of the target gene occurred when cells were infected with 10 PFU of each recombinant virus. Although T7 RNA polymerase synthesis began shortly after infection, the target gene was not expressed until late times and was largely inhibited when DNA replication was blocked. Target gene transcripts were analyzed by agarose gel electrophoresis and had the predicted size. With this system, Escherichia coli (-galactosidase, hepatitis B virus surface antigen, and human immunodeficiency virus envelope proteins were made. In each case, the level of synthesis was greater than had previously been obtained with the more conventional recombinant vaccinia virus expression system. Molecular cloning vectors have been developed that allow expression of heterologous genes in procaryotic and eucaryotic cells. Genetic elements carried by these vectors typically confer drug resistance, ability to replicate autonomously, and regulatory controlling elements juxtaposed to the target gene of interest. While this approach has been extensively used in bacterial and mammalian cell expression systems, certain features of one system may be advantageous over another. Bacteria are easy to use and high expression is often attainable, but synthesis of native eucaryotic proteins is frequently not achieved. Mammalian cells can faithfully express eucaryotic proteins, but usually at relatively low levels. An expression system that used procaryotic transcriptional elements in mammalian cells might have important advantages since the catalytic activity, induciblity, and promoter specificity are well characterized.For these reasons, a chimeric expression system that used favorable transcriptional components from bacteria in a eucaryotic milieu may offer a highly specific and efficient method for the synthesis of ecuaryotic proteins.A eucaryotic transient-expression system based on a recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase in the cytoplasm of infected cells was recently described (5). Plasmids containing the target genes flanked by T7 promoter and termination sequences were introduced into infected cells by transfection procedures. The efficiency of the vaccinia virus-T7 transient system relative to that of more conventional mammalian transient expression systems was attributed to the high catalytic activity of T7 RNA polymerase and stringent T7 promoter specificity. Since it is possible to infect tissue culture cells synchronously with vaccinia virus, the absolute level of expression was probably limited by transfection of the target gene. It would seem that the efficiency of the system would be enhanced if vaccinia virus were used...

A recombinant vaccinia virus that directs the synthesis of bacteriophage T7 RNA polymerase provides the basis for the expression of genes that are regulated by T7 promoters in mammalian cells. The T7 transcripts, which account for as much as 30% of the total cytoplasmic RNA at 24 hr after infection, are largely uncapped. To improve the translatability of the uncapped RNA, the encephalomyocarditis virus (EMCV) untranslated region (UTR) was inserted between the T7 promoter and the chloramphenicol acetyltransferase (CAT) gene. Experiments with a reticulocyte extract demonstrated that the EMCV UTR conferred efficient and cap-independent translatability to CAT RNA synthesized in vitro by T7 RNA polymerase. In cells infected with recombinant vaccinia viruses containing the T7 promoter-regulated CAT gene, the EMCV UTR increased the amount of CAT RNA on polyribosomes. The polyribosome-derived CAT RNA, which contained the EMCV UTR, was translated in vitro in a capindependent fashion as well. Use of the EMCV UTR significantly enhanced the vaccinia/T7 hybrid expression system as it resulted in a 4-to 7-fold increase in total CAT activity. A further =2-fold improvement was achieved by incubating the cells in hypertonic medium, which favors the translation of uncapped picornavirus RNA over cellular mRNAs. With this newly modified expression system, CAT was the predominant protein synthesized by infected cells and within 24 hr accounted for >10% of the total cell protein.A eukaryotic expression system based on a recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase in the cytoplasm of infected mammalian cells was described previously (1-3). The target gene for the bacteriophage RNA polymerase, flanked by T7 promoter and termination sequences, was introduced into infected cells either by transfection of a recombinant plasmid or by infection with a second recombinant vaccinia virus. Through the use of transfection or infection protocols, it was found that T7 lacZ transcripts comprise 10% or 30%6, respectively, of the total cytoplasmic RNA at 24 hr after infection. The T7 transcripts were initiated correctly, but only 5-10% contained 5'-terminal cap structures, providing an explanation for the discrepancy between the major amount ofRNA made and the moderate amount of protein expressed. The more efficient capping of vaccinia virus mRNA, compared to T7 RNA, may be due to association of the viral RNA guanylyltransferase with the viral RNA polymerase (4). In addition, the 5' end stem-loop structure of the 17 transcripts, which was found to be crucial for its stability (3), might interfere with capping as well as with ribosome binding and scanning.To improve the translatability of the uncapped 17 tran-