9 
no immune response is stimulated, can cause severe disease and sometimes 
death. The two groups disagreed, however, over the appropriate conditions 
for cloning the gene for Shiga toxin. Shiga toxin is comparably powerful 
pharmacologically to botulimm and tetanus toxin. However, Shiga toxin 
differs from these toxins in that a few molecules of Shiga toxin have not 
been shown to cause disease. To illustrate this argument, Dr. Levine 
offered the example of an attenuated Shiga bacillus vaccine strain which 
produced as much Shiga toxin as the parent pathogenic strain tut did not 
cause disease when fed to human volunteers; the attenuated strain lacked 
invasive potential . The production of Shiga toxin without concord tan t 
invasive potential does not confer upon the organism the ability to cause 
disease in man. He surmised that many individuals in the assembly had 
Shiga toxin-producing EL ooli in their guts. 
Dr. Levine said the two working group factions could not agree upon appro- 
priate containment for cloning the gene coding for Shiga toxin. As there 
was no pressing need to fix containment for Shiga toxin at that time, Shiga 
toxin was classified in Appendix F-l with botulinum and tetanus toxin. 
Dr. Levine said members of the Working Group on Toxins at the meeting in 
the spring of 1981 had been worried that recombinant DNA laboratory proce- 
dures could introduce the Shiga toxin gene into EL_ coli . Today we knew 
that the Shiga toxin gene already exists and is expressed in many EL_ ooli 
strains in nature. Thus, a "new" organism would not be created by cloning 
the Shiga toxin gene in EL ooli K-12. He also pointed out that since 
investigators have shotgun cloned the EL ooli genome into EL_ coli K-12, 
the Shiga-like toxin gene has almost certainly inadvertently been cloned. 
Dr. Levine said Shiga toxin is also produced by the Vibrionaceae . He 
emphasized the Importance of research on the Shiga toxin gene to develop- 
ment of a cholera vaccine. Dr. Levine explained that several laboratories 
are attempting to create a genetically engineered live attenuated cholera 
vaccine. These vaccines stimulate potent antibody responses and produce 
immunity tut do not produce cholera toxin. However, about one-quarter of 
the human volunteers testing this vaccine develop mild diarrhea . Upon 
exantination, it vets found that these vaccine strains produce enterotoxins 
such as Shiga toxin. In order to understand whether the mild diarrhea 
is caused by the Shiga toxin or whether proliferation in the gut of the 
attenuated bacteria alone causes illness, the Shiga toxin gene must be 
identified and eliminated from the genome of the attenuated V. cholera 
vaccine strain. ' 
Dr. Levine said development of a single-dose potent inexpensive cholera 
vaccine is important to world health, and it is critical that the Shiga 
toxin gene be identified, studied, and eliminated frcm the cholera vaccine. 
Dr. Levine suggested that Dr. O'Brien's proposed dealing of the gene for 
Shiga-like toxin be allcwad to proceed under P2 containment conditions 
^- n coli K-12 using EX2 vectors such as the plasmids pBR322 or pBR325. 
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