© INS~TrUT PASTEuR/ELsEVI~.R Res. Microbiol. Paris ~1990 ~1990, 141, 769-773 USE OF LIVE ATTENUATED BACTERIA TO STIMULATE IMMUNITY N.F. Fairweatber, S.N. Chatfield, I.G. Charles, M. Roberts, M. Lipscombe, L. Jing Li, D. Strugnell, S. Comerford, J. Tite and G. Dongan (') Department of Molecular Biology, Welicome Research Langley Court, Beckenham, Kent BR3 3BS, (UK) Introduction. Many bacteria, including Salmonella, Listeria, and Yersinia species, can cause severe systemic infections of mammals. These invasive bacteria appear to have deve- loped sophisticated mechanisms for entry into, and survival within, eukaryotic cells. Genetic manipulation of these organisms has led to the isolation of mutants which have an impaired ability to survive in vivo, and some of these mutants have been used as live oral vaccines to immunize against virulent infection. In this review, we shall outline some of the work carried out in our laboratory on the analysis of these bacteria, including the construction of auxotrophic mutants and their use in carry- ing foreign antigens to the immune system. In addition, the use of secreted proteins as immunogens and as carriers of epitopes will be discussed. Attenuation of Salmonella. The early work of Hoiseth and Stacker (1981) demonstrated that aroA mutants of Salmonella typhimurium were avirulent in mice and cattle, and could be used as live oral vaccines. We have extended this approach to the construction of mutants defective in other genes of the aromatic biosynthetic pathway, including aroA, aroC and aroD. Double aroA aroD and aroA aroC mutants of S. typhimurium are at- tenuated to the same extent as single aroA mutants and confer solid immunity in mice after a single oral dose (Dougan et al., 1988). Based on the results of these studies, we have constructed aroA aroC mutants of S. typhi. These mutants are defined at the molecular level and contain precise deletions within the aroA and aroC genes. The deletions have been checked by polymerase chain reaction (PCR) amplification of the flanking DNA followed by cloning and sequencing through the deletion. Such mutants are thus candidates for a live oral typhoid vaccine and perhaps for the carri- age of foreign antigens to the immune system. It is now clear that many mutations in many different genes may give rise to reduced virulence of Salmonella species. The ompR gene encodes a positive regula- tor which controls the expression of the outer membrane proteins ompC and ompF. In contrast to individual mutants of ompC or ompF, OmpR mutants of S. typhimu- rium are highly attenuated in mice and confer protection ag~;,nst challenge with viru- lent bacteria (Dorman et al., 1989). (*) To whom correspondence should be addressed.