Gordon Dougan

The Genetics of Salmonella and Vaccine Development

The advent of molecular biological techniques coupled with the availability of in vivo and in vitro models for studying virulence has significantly contributed to our understanding of salmonella pathogenesis. Genes and gene clusters have been identified which are known to be required for in vivo growth and survival but other genes that are necessary for the expression of full virulence by pathogenic salmonellae have still to be identified. Salmonella strains harbouring fully defined mutations have been constructed and are under evaluation in systems such as the murine typhoid model. Some of these attenuated derivatives are excellent candidate vaccine strains, eliciting potent local and systemic responses in the host. Fully genetically defined strains are now being evaluated in humans as candidate oral typhoid vaccines. Because such live vaccine strains are capable of eliciting potent immune responses they have been considered as carriers for delivering heterologous antigens to the mammalian immune system. Problems have been encountered with this approach, a major obstacle being the instability of expression of foreign antigens from recombinant plasmids. We are tackling this by utilising promotors to direct the expression of heterologous sequences that are tightly regulated but switch on in particular environments within host tissue We have demonstrated the utility of such an approach by using the nirB promotor to control the expression of heterologous antigens and have shown that this may be a generally applicable approach to obtaining the stable in vivo expression of heterologous antigens in salmonella vaccine strains.

In vivo and in vitro Characterisation of Attenuated TNphoA Mutants of Salmonella typhimurium C5

Defined mutations in such genes as aroA, purA and ompR have been shown to attenuate Salmonella species, and strains harbouring such mutations make good, single dose oral vaccine strains (O’Callaghan et al 1988, Dorman et al 1989, Chatfield et al 1992). As an approach to identifying other virulence factors associated at or near the bacterial surface TnphoA was used to mutagenise the mouse virulent strain of S. typhimurium, C5. The TnphoA transposon is based on Tn5 but carries the Escherichia coli alkaline phosphatase gene lacking the promoter and signal sequence (Manoil and Beckwith 1985). Functional activity of the alkaline phosphatase enzyme only occurs when secreted, thereby indicating insertion of the transposon into a gene whose product is secreted. Activity of the enzyme can be screened using a chromogenic substance such as X-gal, resulting in white colonies for non-functional activity and blue colonies for a functional fusion. Using this method a bank of mutants was obtained and screened for attenuation in BALB/c mice using the natural oral route of infection (Miller et al 1989). of these, 15 were found to be attenuated, 9 having defects in LPS biosynthesis which were not used in this study. of the remaining 6 mutants 2 have been shown to harbour a mutation in htrA, which encodes a stress protein and is thought to be important in survival within macrophages (Johnson et al 1991). A third mutant has now been shown to harbour a mutation in osmZ, which encodes a histone-like protein which may be involved in DNA supercoiling (Higgins et al 1988). The remaining 3 mutants were screened for their in vivo properties by both the oral and intravenous (i.v.) routes in BALB/c mice (Table 1).

The Development of Genetically Defined Live Bacterial Vaccines

It has long been recognised that vaccination with live organisms can induce more effective protection against infectious diseases than the use of dead vaccines presented in conjunction with currently available adjuvants. Live vaccines offer particular advantages in their ability to induce cell-mediated immune responses which are often critical for the establishment of solid protection against certain infectious agents. The route of vaccine delivery is also of importance. Oral vaccines can induce immune responses at mucosal surfaces and such responses are often lacking in individuals who receive parenteral vaccines. The combination of live organisms with oral delivery thus offers potential advantages for creating practical and efficacious vaccines. Many problems were experienced with early live vaccines because of the frequent occurrence of reversion to virulence and batch to batch variation. These problems were difficult to deal with because the attenuating lesions present in avirulent vaccine strains derived from virulent pathogens were uncharacterised at the genetic level. Recent advances in our understanding of the genetics and the mechanisms employed by pathogens to establish infections in the host has allowed the construction of genetically defined attenuated derivatives of many pathogens.