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Federal Register / Vol. 50, No. 226 / Friday, November 22. 1985 / Notices 
vaccine strain which produces as much 
Shiga toxin as the parent pathogenic 
strain but lacks invasiveness factors did 
not cause disease when fed to human 
volunteers. In the single case in which 
the strain reverted to invasive, the 
human volunteer became ill. 
"Dr. Formal said he had constructed a 
strain by introducing half of the 
Sliige/la flexneri chromosome and an 
SLT converting phage into E. co/i K-12. 
The introduced Shigella flexneri genes 
were thought to include all of the genes 
necessary for virulence. The constructed 
strain had no effect on monkeys in 
feeding experiments although it 
produced 10 7 CDs per mg protein of SLT 
under optimal laboratory conditions. 
"Dr. Gill asked whether concern 
exists about transfer to other organisms 
of a high copy number plasmid carrying 
the Shiga toxin gene and its control 
elements. 
“Dr. Newland said the probability of 
transfer of the SLT or Shiga toxin gene 
by phage in nature is higher than the 
probability of transfer from K-12 by 
poorly mobilizable plasmid vectors. 
"Dr. Gottesman asked what 
laboratory conditions are necessary to 
obtain optimal expression of the SLT 
gene in the E. coli K-12 host-vector 
system. Dr. Newland replied that 
antibiotic pressure is necessary. In 
addition, several other procedures such 
as using specially treated medium are 
also required. If an SLT clone is simply 
cultured in the conditions usually used 
to culture the host-vector system, toxin 
production is generally a factor of 10* 
lower than the highest level of 
expression obtained under optimal 
conditions with that clone. 
“Dr. Newland said he had observed 
that the recombinant plasmid carrying 
the SLT gene appears to be rapidly lost 
from the E. coli host-vector system in 
the absence of selective antibiotic 
pressure. The plasmid without the SLT 
gene is not so rapidly lost in the absence 
of selective pressure. He and Dr. O'Brien 
hypothesized that high levels of toxin 
may be toxic to bacteria. The rapid loss 
of the recombinant plasmid in the 
absence of selective pressure may be an 
additional safety factor. 
“Dr. Gottesman asked whether the 
working group would consider 
modifying Appendix F-1V-H by raising 
the upper level of toxin expression to 10 8 
CDs per mg protein in cell lysates in 
exchange for an assurance that the 
plasmid will be rapidly lost in the 
absence of antibiotic pressure. 
Alternatively, an upper level of 
expression need not be fixed if the 
plasmid will be rapidly lost since rapid 
plasmid loss will provide a measure of 
safety. 
“Dr. Levine said one problem with Dr. 
Gottesman's proposal is that language 
gerferic for experiments involving E. coli 
may not apply to experiments involving 
Vibrio or Shigella. A second concern is 
the variability of the SLT assay; without 
an internal standard it is difficult to 
know how much toxin is produced. He 
asked Dr. O'Brien how great a difference 
is observed using the same procedure 
from experiment to experiment. Dr. 
O'Brien replied that a 100 fold difference 
in the amount of CDs produced may be 
observed from experiment to 
experiment. 
“Dr. Gill moved that Appendix F-IV- 
H be amended to read in part: 
‘E. coli host-vector systems expressing 
the Shiga-like toxin gene product may 
be moved from BL3 + EK1 to BL2 + EK1 
containment conditions provided that: 
(1) 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 
0157:H7, grown and measured under 
optimal conditions, or ten times this 
level if the maintenance of the plasmid 
carrying the gene is dependent upon 
growth in the presence of an 
antibiotic . . . .’ 
“Dr. Levine suggested the motion be 
amended to read in part: 
'E. coli host-vector systems expressing 
the Shiga-like toxin gene product may 
be moved from BL3-(-EKl to BL2 + EK1 
containment conditions provided that; 
(1) The amount of toxin produced by the 
modified host vector strain be no greater 
than 10* CDs per mg protein. . . .’ 
“Dr. Formal suggested the motion 
should name a toxin producing strain as 
the standard rather than citing a specific 
number. He suggested Shigella 
dysenteriae 1 strain 60R be used for this 
purpose. Shigella dysenteriae 1 strain 
60R, a rough strain not capable of 
colonizing the human gut, produces 
about ten times more toxin than 933 E. 
coli 0157:H7. Language specifying a 
specific strain w r ould provide an internal 
standard and permit comparison from 
laboratory to laboratory. Dr. Kopecko 
agreed. 
“Dr. Levine amended his motion to 
require use of Shigella dysenteriae 
strain G0R as the standard strain. Dr. 
Formal seconded this motion.. 
“Dr. Gottesman seconded Dr. Gill's 
original motion in order to permit a vote 
on this motion. By a vote of two in favor, 
four opposed, and no abstentions, Dr. 
Gill's motion was refused by the 
working group. . . . 
“The working group then voted on Dr. 
Levine's motion. By a vote of five in 
favor, one opposed, and no abstentions, 
the working group accepted this 
motion. . . . 
“Dr. Kopecko then offered the 
following motion: 
'Campylobacter species have long 
been recognized as members of the 
family Vibrionaceae. Recently 
Campylobacter species have beer! 
separated taxonomically. into an orphan 
genus. Because of the similarities 
between Campylobacter and 
Vibrionaceae. the Working Group on 
Toxins recommends that for purposes of 
cloning SLT Campylobacter can be 
considered as members of the 
Vibrionaceae ' 
“Dr. Levine seconded this motion. 
“Dr. Gill said the most important 
consideration in evaluating a proposal is 
not the species from which the gene was 
originally isolated but the 
characteristics of the gene's product and 
the host-vector system. He said he 
would support Dr. O'Brien's request to 
permit cloning of the SLT gene from 
Campylobacter. He thought Dr. 
Kopecko's motion did not convey the 
idea that the gene, and the 
characteristics of its product are the 
most important considerations. He 
suggested a substitute motion which 
would not refer to the source of the gene 
he developed. . 
“Dr. Formal said although he agreed 
with Dr. Gill, at the present time it 
would be difficult to precisely define tne 
characteristics of SLT toxin. A 
substitute motion would have to contain 
such a definition. 
“Dr. Milewski said Dr. Kopecko’s 
motion was preferable because of the 
way the August 19, 1985, Federal 
Register announcement had been 
written. She said the minutes of the 
working group meeting would show the 
group strongly supports the concept that 
the gene is the most important 
consideration. 
“Dr. Levine called the vote on Dr. 
Kopecko's motion. By a vote of six in 
favor, none opposed, and no 
abstentions, the working group 
approved of the motion. . . .” 
The RAC discussed Dr. O'Brien's 
proposal at the September 23, 1985, 
meeting. 
By a vote of seventeen in favor, none 
opposed, and no abstentions the RAC 
recommended NIH accept the working 
group recommendation to approve Dr. 
O'Brien's request as it appeared in the 
August 19, 1985, Federal Register with 
Campylobacter species considered to b*. 
members of the Vibrionaceae 
By a vote of seventeen in favor, noiu. 
opposed, and no abstentions the RAC 
recommended the NIH accept the 
working group recommendation that 
Appendix F-IV-H be amended to read 
in part: 
[528] 
