is being used to carry and express cloned genes of critical, putatively 
protective antigens of other organisms. For example, modified Ty21a 
expressing the plasmid-encoded 0 antigen of S. sonnei and Ty21a expressing 
the cloned genes for Vibrio cholerae 01 serotype Inaba have been 
prepared. Known attenuated strains, such as Ty21a, carrying cloned genes 
from other organisms should be excluded from the guidelines, as long as 
the introduced genes do not encode a potent holotoxin. 
4) Strains with Deletions of Chromosomal Genes Encoding Critical 
Virulence Properties 
For some bacterial pathogens, a chromosomal gene product is an 
absolute necessity for full expression of pathogenicity. One such example 
is V. chol er ae 01. The severe diarrheal purging characteristic of cholera 
gravis is the consequence of the effects of cholera enterotoxin which 
consists of five B (binding) subunits and one A (biologically active, 
ADF'-r i bosyl at i ng ) subunit. Ingestion of minute amounts (5 meg) of 
purified cholera enterotoxin can result in severe purging. Similarly, 
deletion of the genes encoding the A subunit renders the mutant unable to 
cause cholera gravis. An example of such a vaccine strain is CVD 103, a 
genetically-engineered A-B+ mutant of a V. cholerae classical Inaba 
strain. CVD 103 does not cause severe diarrhea, is highly immunogenic and 
is highly protective. 
Live vaccines attenuated by the deletion of critical virulence 
properties should also be exempt, as long as they have a stable marker to 
di f f er en t i at e them from wild type strains and particularly if they have a 
further mutation in the r ec A gene. The latter defect virtually assures 
that DNA introduced by conjugation will not be incorporated into the 
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