.Federal Register / Vol. 49, No. 80 / Tuesday, April 24, 1984 / Notices 
17673 
in Appendix K-ll-A through K.-II-F is 
permitted. 
4. A reference to Appendix C would 
be added to the fourth sentence of 
Appendix K-I, Se/ectioh of Physical 
Containment Levels. That sentence 
would read as follows: 
The Pl-LS level of physical containment is 
required for large-scale research or 
production of viable organisms containing 
recombinant DNA molecules which require 
Pi containment at the laboratory scale (See 
Appendix C). 
As a possible substitute, NIH staff 
proposes the following alternate 
modification of Appendix K-I, Selection 
of Physical Containment Levels. The 
following sentence would be added 
following the fourth sentence of 
Appendix K-I, Selection of Physical 
Containment Levels : 
(The Pl-LS level of physical containment is 
recommended for large-scale research or 
production of viable organisms for which Pi 
is recommended at the laboratory scale such 
as those described in Appendix C). 
II. Proposal to Clone Shiga-Like Toxin 
Gene From E. coli 
Dr. Alison O'Brien of the Uniformed 
Services University of the Health 
Sciences (USUHS) in collaboration with 
Dr. Randall Holmes (USUHS) proposes 
to clone in Escherichia coli K-12 the 
structural gene of the Shiga-like toxin of 
E. coli. Shiga-like toxin has activity 
similar to the activity of Shigella 
dysenteriae toxin. 
In a first submission in September 
1982, the investigators proposed to clone 
the Shiga-like toxin gene in E. coli EKl 
host-vector systems using plasmid, 
cosmid, or lambda cloning vectors. In 
support of their proposal, Drs, O’Brien 
and Holmes offered the following 
arguments: 
1. Clinical isolates of E. coli have 
already been demonstrated to elaborate 
large amounts of toxin indistinguishable 
from that produced by Shigella 
dysenteriae 1 (Shiga). Therefore, the 
genes for Shiga-like toxin production are 
present in the E. coli gene pool found in 
nature. 
2. Human volunteers fed large 
numbers of Shigella dysenteriae 1 
organisms that produced Shiga toxin but 
could not colonize the bowel did not 
become ill. Therefore, any accidential 
ingestion of the organism to be 
manufactured, a toxin-producing E. coli 
K-12 strain that cannot colonize the 
human intestinal tract, would pose little 
hazard to man. 
3. Purification of Shiga toxin in 
several laboratories and E. coli Shiga- 
like toxin in the investigators’ laboratory 
has not identified any excessive risk 
from the aerosolization of toxin that 
probably occurs during the process of 
toxin preparation. In one laboratory, 
toxin was isolated from 500 liters of 
culture with only Pi physical 
containment. 
4. Shiga toxin is a potent cytotoxin for 
a subline of HeLa cells (a human 
cervical carcinoma tissue culture cell 
line), but the toxin has no effect on 
many other human, monkey, and rodent 
tissue culture cells. Therefore, the toxin 
is quite cell-type specific, and this 
limited spectrum of activity suggests 
that it would be non-toxic for most cells 
in the human body. 
5. Contrary to the old literature, Shiga 
toxin is not a neurotoxin. By 1955, it was 
established that the paralysis observed 
in rabbits and mice (but not monkeys, 
guinea pigs, hamsters, or rats) when 
toxin is given intravenously is a 
reflection of the effect of toxin on the 
endothelium of small blood vessels, not 
a direct effect on nerve cells. 
The request was summarized in the 
Federal Register of September 22, 1982 
(47 FR 41924). The RAC discussed the 
request submitted by Dr. O’Brien at the 
October 25, 1982, meeting. The 
committee by a vote of twelve in favor, 
none opposed, and one abstention, 
recommended that the Initial 
experiments be performed under P4 -f 
EKl containment conditions. The NIH 
accepted the RAC recommendation that 
P4 + EKl containment is adequate to 
contain safely the experiments proposed 
by Drs. O'Brien and Holmes and 
appropriate language was added to 
Appendix F of the Guidelines. 
In December 1983, Drs. O’Brien and 
Holmes requested reconsideration of 
containment levels in veiw of 
information which had recently become 
available. The requested approval at the 
P2 level of physical containment for the 
following reasons: 
1. Epidemiology studies have been 
performed on over 150 K coli strains 
isolated from human and animal stools. 
These studies have shown that the 
majority (80%) of the strains made 
detectable levels of Shiga-like toxin. 
Moreover, four of four substrains of the 
well-characterized bacterium EL coli K- 
12 were shown to make low levels of the 
toxin. Thus, cloning of the Shiga-like 
toxin gene from clinical isolates of E. 
coli into laboratory strains of E. coli K- 
12 will not involve the introduction of a 
"foreign” toxin Into the organism. 
2. Production of low levels of Shiga- 
like toxin was observed in 2 of 15 
normal human gut flora E coli strains 
from asymptomatic infants. 
3. Strains of Vibrio cholerae and 
Vibrio parahaemolyticus were tested 
and shown to produce Shiga-like toxin. 
Thus, the gene(s) for Shiga-like toxin are 
present in naturally occurring isolates of 
the family Vibrionaceae and not 
restricted to the Enterobactcriaceae. In 
volunteer studies, some of the strains of 
V. cholerae that produce Shiga-lie toxin 
did not cause disease. Therefore, the 
ability to produce Shiga-like toxin is not 
equivalent with virulence in humans 
challenged by the oral route. 
4. Phages from two clinical isolates of 
E. coli have been shown to control high- 
level production of Shiga-like toxin in E. 
coli K-12 host strains by phage 
conversion. Thus, either the structural 
gene(s) for the Shiga-like toxin or 
regulatory genes that control high-level 
production of the toxin are present on 
wild-type phages from clinical isolates 
of E. coli. In this sense, "cloning” of 
genes that affect production of Shiga- 
like toxin onto phage genomes has 
already occurred in nature. 
In additon, the U.S. Cholera Panel of 
the National Institute of Allergy and 
Infectious Diseases (NIAID) 
recommended that NIH reconsider the 
ban: 
* * * on Shiga toxin cloning experiments 
in containment facilities other than P4. This 
strict requirement will prevent most 
laboratories from deleting the Shiga gene 
from candidate V. cholerae and ETEC 
vaccine strains. Shiga toxin is now found in 
many nonpathogenic EL coli, including the 
common vector host, E. coli K-12. 
The proposal was published in the 
January 5, 1984, Federal Register (49 FR 
696). The RAC reviewed the proposal of 
Drs. O’Brien and Holmes at the 
February 6, 1984, meeting. By a vote of 
nine in favor, five opposed and four 
abstentions, the RAC recommended that 
Drs. O'Brien and Holmes and coworkers 
be allowed to proceed with cloning the 
gene for Shiga-like toxin under P2 
physical containment conditions in E. 
coli' K-12, restricted to using EK2 
plasmid vectors, commencing first with 
the use of pBR325 and pBR322, and 
proceeding to other EK2 plasmid vectors 
only if those are unsatisfactory. 
By a vote of eight in favor, four 
opposed, and five abstentions, the RAC 
passed the same motion but with the 
names of the investigators deleted from 
the motion. 
It has been the practice of NIH not to 
accept RAC recommendations that do 
not indicate a clear concensus. 
Accordingly, NIH did not accept the 
RAC recommendations offered at the 
February 8, 1984, meeting. The 
investigators have approval, however, to 
conduct these experiments at the P4 
level of containment under their 
previous permission which appears in 
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