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Federal Register / Vol. 49, No. 179 / Thursday, September 13, 1984 / Notices 
investigators with USUHs constitutes a 
reason perse for requiring an ACIS. 
Dr Holmes suggested the issue as he saw it 
! s not whether an ACIS is necessary for this 
particular experiment but whether any ACIS 
might be needed for toxin related 
recombinant DNA experiments in general. 
Dr. Levine pointed out that when the 
Working Group on Toxins was constituted in 
the spring of 1981 to evaluate the cloning of 
toxin genes, it was clear that experiments 
involving the cloning of the gene encoding 
botulinum toxin pesented a real concern. 
Botulinum toxin is an exoxinosis; i.e., the 
pure toxin if imbibed or ingested orally 
causes illness. Tetanus toxin also presents a 
real concern. Shiga toxin, on the other hand, 
is a very potent toxin when administered 
parenterally; however, there is no evidence 
epidemiologically or pathophysiologically 
that Shiga toxin is an exotoxinosis. In 1981 In 
discussing the appropriate category for 
experiments involving cloning of the Shiga 
toxin gene, the Working Group on Toxins 
was divided. Some individuals said these 
experiments should be in the same category 
as experiments involving the gene for tetanus 
toxin; this position was based on 
consideration of Shiga toxin’s 
pharmacological potency. Others felt Shiga 
toxin should be in a separate category on the 
basis of epidemiological evidence. As the 
hour was late, Shiga toxin was assigned to 
the same category as botulinum and tetanus 
toxin pending further information. Dr Levine 
said most of the Working Group on Toxin 
members who participated in the May 11, 
1984, meeting were members of the working 
group which in the spring of 1981 drew up 
Appendix F to the Guidelines. These 
individuals, thus, had the opportunity at the 
May 11, 1984, meeting to review additional 
data concerning Shiga toxin and to offer 
recommendations. Dr. Levine, pointed out 
that Swiss and West German committees of 
experts have suggested experiments 
involving cloning of the Shiga toxin gene be 
permitted at no higher than P2 + EK1 
containment. He said the recommendations 
of the RAC Working Group on Toxins in 
contrast represent a very conservative 
attitude towards the cloning of the Shiga 
toxin gene. He urged the RAC to accept the 
working group recommendations. 
In response to Dr. King Holmes' stated 
concerns, Dr. Levine said the Working Group 
on Toxins, in devising its guidelines for 
Appendix F, had considered toxicity to 
primates to be of paramount importance and 
more relevant than data generated with 40 
guinea pigs or 40 mice. He emphasized that 
the primate data of Braham, Dack, and Riggs 
show that 20,000 monkey parenteral lethal 
doses will not cause adverse effect when 
administered by means of an intestinal 
pouch. 
Dr. Levine said he did not believe E. coli 
would present a problem by colonizing the 
skin or peritoneal areas; if E. coli is going to 
present a problem, it will present a problem 
in the gut as the numers of E. coli in the gut 
are orders of magnitude greater than in other 
areas of the body. 
Dr. Levine said that E. coli strains which 
cause hemorrhagic colitis, such as 933 E. coli 
0157H7, are smooth E coli strains capable of 
colonizing the human gut. These strains also 
have other virulence factors. Nevertheless, 
these strains are not widespread pathogens. 
He argued that if strains such as 0157H7 
which possess' so many virulence 
characteristics are not widespread 
pathogens, it is inconceivable that a rough E. 
coli strain, such as E. coli K-12 which does 
not colonize or possess virulence factors, 
would become a widespread pathogen. 
Dr. Levine said the infinitesimal risks 
perceived to be associated with cloning the 
Shiga toxin gene in E. coli K-12 must be 
weighed against the actual benefits. He said 
research with the Shiga toxin gene is very 
important to the development of a cholera 
vaccine. He explained that live attenuated 
cholera vaccines which lack cholera toxin are 
a major step forward in controlling cholera 
by immunoprophylaxis. These vaccines, 
however, still cause a mild diarrhea in 
perhaps a third of the recipients. Thus, this 
vaccine is not sufficiently attenuated for 
public health use. Dr. Levine said the mild 
diarrhea may be explained in two ways: (1) 
The diarrhea is a response to the intestine to 
colonization by the live bacterial strain, or (2) 
other diarrhea-causing toxins may be 
produced by the live attenuated strain. Dr. 
O’Brien and her coworkers have shown that 
some cholera vaccine strains do produce 
Shiga toxin. Shiga toxin thus may play a role 
in causing the mild diarrhea associated with 
the live attenauted cholera vaccine strains. 
This possibility must be tested by cloning the 
Shiga toxin gene and deleting it from the 
vaccine strains. Delaying this research will 
adversely affect public health. 
Dr. Holmes asked Dr. Levine to explain 
why if non-invasive V. cholerae vaccine 
strains may cause Shiga, toxin induced 
diarrhea, would there not be similar concerns 
about an E. coli strain producing Shiga toxin? 
Dr. Levine replied that to be a concern the 
bacterium must possess accessory virulence 
properties. These virulence properties need 
not include invasiveness; the organisms must, 
however, possess characteristics that 
maintain the bacteria in a special proximity 
to the intestinal cells. Dr. Levine said the V. 
cholerae vaccine strains colonize the small 
bowel in contrast to E. coli K-12 strains 
which will not colonize the small bowel. 
Dr. Clowes said at the February 0 RAC 
meeting he had supported the motion to 
lower containment requirements to P2 
because: (1) E. coli and Shigella exchange 
genetic information in nature, and (2) other 
virulence factors in addition to toxin 
production are necessary for pathogenicity. 
He said he had abstained during the vote, 
however, because he felt the language of the 
motion was vague. Dr. Clowes said he 
supported the current recommendations of 
the Working Group on Toxins. However, as 
E. coli K-12 probably possesses a 
chromosomal Shiga toxin gene, he would like 
to suggest that the working group 
recommendation on item three of Dr. 
O'Brien’s April 4 request be modified to 
require P2 containment conditions. 
Dr. Fedoroff felt P2 containment was not 
necessary. She pointed out that two 
recombinational events would have to occur 
to generate a plasmid vector carrying the full 
structural gene for Shiga toxin: one 
recombinational event to integrate the 
plasmid into the chromosome, and a second 
to return the plasmid to the 
extrachromosomal state. 
Dr. McKinney said Dr. Clowes' suggestion 
satisfied Dr. Holmes' concern regarding 
inhalation exposure to Shiga toxin-producing 
E. coli. since P2 reduces the probability of 
exposure by aerosol. He supported Dr. 
Clowes' suggestion. 
Dr. Gottesman said she wished to respond 
to certain of Dr. Holmes' concerns. She 
reminded the committee the proposed 
research with the Shiga toxin structural gene 
is to be performed under P3 containment with 
E. coli K-12 host vector systems. P3 
containment conditions severely limit the 
possibility of the organism escaping. In 
addition, the host in this case would be E. 
coli K-12 which is a debilitated strain. In 
addition, Dr. Gottesman argued that Shiga 
toxin exists in E. coli strains in nature; thus, 
the only way in which a novel organism 
might be produced by recombinant -DNA 
techniques is if the plasmid construct 
produces higher levels of toxin than strains in 
nature. Dr. Gottesman felt these 
considerations and the primate data 
indicating that Shiga toxin is not toxic when 
delivered in the gut, address most of the 
concerns. 
Dr. Gottesman moved that RAC 
recommend experiments Involving the 
cloning in E. coli K-12 of the intact structural 
gene(s) of Shigalike toxin of £. coli be 
permitted at P3 + EK1 containment. This is 
the first request in Dr. O'Brien's April 4 
proposal. Dr, Federoff seconded the motion. 
Dr. King Holmes noted that he would support 
the motion as he felt the benefits greatly 
outweigh the risks. By a vote of twenty-one In 
favor, none opposed, and one abstention, the 
RAC recommended the motion. 
Dr. Gottesman then moved RAC approve 
the working group recommendation that E 
coli host-vector systems expressing the Shiga 
toxin gene may be removed from P3 to P2 
containment under the following conditions: 
a. That the amount of toxin produced by 
the modified host-vector systems be no 
greater than that produced by the positive 
control strain, 933 E. coli 0157H7, grown and 
measured under optimal conditions; and 
b. The cloning vehicle Is to be an EKI 
vector, preferably belonging to the class of 
poorly mobilized plasmids, such as pBR322, 
pBR328, and pBR325. 
Dr. Fedoroff seconded the motion. 
Mr. Jeremy Rifkin was recognized and said 
he felt that a critical turning point has been 
reached with this technology. He thought this 
turning point similar to the running point in 
the nuclear technology discussions where it 
became very obvious there was a 
convertibility between the peaceful use of 
nuclear technology and its possible military 
applications. Mr. Rifkin felt this convertibility 
was especially obvious in relation to the use 
of plutonium in the nuclear energy industry 
and its use in military weapons. 
Mr. Rifkin said that in the last few months 
several disturbing events occurred: (1) The 
Wall Street Journal published a seven part 
series on possible military applications of 
genetic engineering in the Soviet Union; (2) 
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