identification of the vaccine strain and its di f f er ent i at i on from wild 
type strains. 
2) Auxotrophic Strains 
Another approach by which bacterial pathogens may be suitably 
attenuated to serve as live vaccine strains is to render them auxotrophic 
for substrates that are unavailable in the human or animal tissues or body 
fluids. The best examples of this variety of attenuation are the Aro- 
derivatives of S. t yph i and S. t yphi mur i urn . These mutants have deletions 
of the Ar o A gene rendereing them unable to persist in the mammalian body 
because of the lack of 2,3, dihydroxybenzoate. As a consequence these 
attenuated mutants cannot proliferate to reach high numbers in the 
mammmal i an host and cause disease but they persist sufficiently long to 
stimulate immune responses. Arc- mutants of S. t yph i mur i urn have been 
shown to be safe and protective vaccines in mice and cattle, while the 
safety and immunogenic ity of an Arc-, Pur- S. t yphi vaccine strain (541Ty) 
has recently been demonstrated in Phase 1 clinical studies in man. 
Auxotrophic mutants can be prepared by recombinant DNA technology, as 
well as by the classical genetic techniques (using phages to create the 
deletions) employed to prepare 541Ty. These mutants should possess some 
stable marker allowing them to be clearly discernable from wild type 
or gani sms . 
3) Proven Attenuated Bacteria Acting as "Carrier" Strains to Express 
Cloned Genes of other Organisms 
Attenuated S. typhi strain Ty21a, because of its record of safety and 
its stimulation of both cel 1 -medi ated as well as humoral immune responses, 
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Recombinant DNA Research, Volume 1 1 
