Molecular Microbiology (1990) 4(12), 2111-2118 Generation of a cytotoxic T-lymphocyte response using a Salmonella antigen-delivery system J. L. Flynn,^* W. R. Weiss,^ K. A. H. S. Seifert,^ S. Kumar,* and M. So' ^Department of Moleeular Biology. Research Institute of Scripps Clinic, La Jolla. California 92037, USA. ^Infectious Disease Department, Naval Medical Research Institute, Bethesda, Maryland 20814, USA. ^Department of Microbiology and Immunology. Northwestern University School of Medicine. Chicago. Illinois 60611, USA. ''Laboratory of Parasitic Diseases, Nationai Institutes of Health, Bethesda. Maryland 20892, USA. Summary We have constructed a general-use vector for the cloning and stable expression of foreign genes in the chromosome of attenuated Salmonella typhimurium. Using this chromosomal expression vector (CEV), we expressed the circumsporozoite (CS) gene of the mouse malaria Plasmodium yoelil in an aroA S. typhi- murium strain. Mice immunized with CS-expressing Salmonella recombinants mount a CS-specific cyto- toxic T-lymphocyte (CTL) response. This is the first demonstration that attenuated Salmonella can elicit a specific CTL response to a foreign protein in mice. The ability to easily and stably express foreign genes from the Salmonella chromosome and the generation of specific CTL greatly expands the potential of Sal- monella as an antigen-delivery system. Introduction Salmonella typhimurium is a pathogen that causes a typhoid fever-like disease in mice. During infection, Sal- monella elicits both humoral and cellular immune re- sponses, both of which are necessary for protection against subsequent infection. Heat-killed Salmonella does not protect against a virulent challenge although strong antibody responses are generated. Salmonella strains with lesions in galE{Moseretai, 1980), aroA (Hoiseth and Stocker, 1981), and cya (Curtiss et al., 1987) genes. Received 16 July, 1990. Preseni addresses: "Department of Microbiology and Immunology, Albert Einstein College of Medicine. Bronx, New York 10461, USA (for correspondence); ^Department of Molecular Genetics and Biochemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15261 USA- among others, have greatly increased lethal dose (50%) (LD50) values for inbred mouse strains. Mice immunized with these attenuated strains are protected against a lethal challenge with virulent Salmonella. Therefore live strains appear to elicit the appropriate (B- and T-cell) immune responses for protection. Genes from a variety of path- ogens have been expressed in attenuated S. typhimurium (Poirier et ai. 1988; Nevrton et ai, 1989; Sadoff et ai, 1988) and immunization of mice with recombinant strains generated antibody (Poirier et ai, 1988; Newton et ai, 1989; Brown et ai, 1987) or delayed-type hypersensitivity (DTH) (Sadoff etai, 1988) responses against the foreign antigen. For these reasons, there is a great deal of interest in attenuated Salmonella antigen-delivery systems as a means of examining the immune response to various antigens (Dougan et ai, 1987). Plasmids have been the primary method used to express foreign genes in Salmonella. The use of plasmids in antigen-delivery systems may have several drawbacks. The expression of many foreign antigens can result in plasmid instability, leading to loss of the plasmid (C'Call- aghan et ai. 1988); alternatively, the cloned sequences may undergo deletion or other rearrangements. High copy-number plasmids or a strong promoter can result in the overexpression of foreign proteins, which can be lethal to the bacterial cell (Shatzman etai, 1983). A plasmid can be integrated into the bacterial chromosome by a single recombinational event (Hone etai, 1988), but in the case of a protein which is deleterious to the cell, the plasmid could recircularize and be lost. Although a number of prokaryotic genes may be stably expressed and the proteins maintained in Salmonella, eukaryotic, including parasite, genes and gene products may be more difficult to stabilize. Antibiotic selection can maintain a reasonable level of an unstable plasmid in vitro, but it is not feasible to continue antibiotic selection in mice. One method for stabilizing foreign expressed DNA in Salmonella is to integrate the gene into the chromosome. There have been two reports of methods used to insert genes into the chromosome of Salmonella (Hone ef a/., 1988; Strugnall et ai, 1990). We have also developed a general system for stably inserting foreign genes into the chromosome of S. typhimurium. This system uses a defective transposable element which stably inserts into the bacterial chromo- some to express a gene from any organism of interest in S. typhimurium. We have termed this type of system