Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Wed, 05 Dec 2018 12:55:25 Journal of General Virology (1999), 80, 345–353. Printed in Great Britain ................................................................................................................................................................................................................................................................................... Development and use of a 293 cell line expressing lac repressor for the rescue of recombinant adenoviruses expressing high levels of rabies virus glycoprotein D. A. Matthews, 1 D. Cummings, 1 C. Evelegh, 1 F. L. Graham 1, 2 and L. Prevec 1, 2 Departments of Biology 1 and Pathology 2 , McMaster University, 1280 Main St W., Hamilton, Ontario, Canada L8S 4K1 An expression cassette designed for high-level production of rabies virus glycoprotein (RG) could not be rescued into a replication-defective, adenovirus-based vector using standard procedures. To overcome this difficulty, a 293-based cell line, designated 293LAP13, was constructed that contained and expressed a derivative of the lac repressor protein. The lac operator sequence, to which the repressor binds, was incorporated into an expression cassette, containing a promoter and intron, designed for high-level production of RG. Insertion of a single operator sequence immediately downstream of the transcription start site and the use of the 293LAP13 cell line allowed recombinant viruses that could not be isolated with 293 cells to be rescued efficiently. The operator-containing virus reached higher titres in 293LAP13 than in parental 293 cells and also produced plaques more efficiently in 293LAP13 cells. Moreover, in non-complementing human and canine cell lines, adenovirus vectors with a promoter–intron expression cassette expressed RG at much higher levels than vectors lacking the intron. These observations, together with the demonstration that expression of RG by operator-containing vectors was repressed markedly in 293LAP13 cells and that this inhibition was relieved at least partly by IPTG, suggest that the 293LAP13 cell line may be useful for the rescue and propagation of many vectors in which high expression of the desired protein prevents vector rescue in 293 cells. Introduction Human adenoviruses (Ad) have become one of the vectors of choice for the delivery and expression of foreign proteins for gene therapy, immunotherapy and vaccination (for a recent review see Hitt et al., 1997). In particular, Ad vectors have been shown to be effective vaccines for the delivery of rabies virus glycoprotein (RG) antigens to non-human hosts. Protective immune responses have been induced in animals immunized with infectious (Prevec et al., 1990 ; Charlton et al., 1992; Yarosh et al., 1996) and defective (Xiang et al., 1996) Ad vectors delivered by parenteral and oral routes. Using vectors containing the RG gene in E3, we showed that the level of protective immunity induced in treated animals correlated with the amount of antigen produced in cell culture (Yarosh et al., 1996). Because high-level antigen expression by recombinant virus vaccines allows for the use of lower doses and gives Author for correspondence : Frank L. Graham (at the Department of Biology). Fax 1 905 521 2955. e-mail grahammcmaster.ca greater efficacy, and because one of our principal objectives is to use Ad-based vectors expressing RG as oral vaccines for wildlife, it is clearly important to obtain the highest possible level of production in infected cells. In general, replication-defective Ad vectors have the early transcription regions E1 and E3 deleted and cassettes inserted containing an appropriate exogenous promoter and the gene(s) to be expressed. Such viruses, often called ‘ first-generation vectors ’, are produced and replicated in E1-complementing cell lines such as the human 293 cell line (Graham et al., 1977). We have described procedures employing bacterial plasmids and in vivo recombination to generate E1 - , E3 - vectors with inserts in E1 or E3 (Graham & Prevec, 1995). Basically, the foreign gene (or promoter–gene cassette) is cloned into one of a series of shuttle plasmids and co-transfected into 293 cells with a complementing plasmid encoding the majority of the Ad genome, in order to rescue recombinant Ad vectors containing the desired transgene. This procedure has been used suc- cessfully to produce many recombinant viruses expressing proteins from a wide variety of sources. While the ease with which particular vectors are generated 0001-5866  1999 SGM DEF