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ISIS Press Release 09/07/07
Antibodies from Hybrid GM Tobacco Plants
Proposed release has not considered risks of antibodies to wild life and human
beings, nor the horizontal transfer of antibiotic resistance marker genes to
disease-causing bacteria. Prof.
Joe Cummins and Dr. Mae-Wan
Ho
This report has been submitted to the USDA, please circulate widely
to your policy-makers
Antibody against tooth decay
The Animal and Plant Health Inspection Service
(APHIS) of the United States Department of Agriculture (USDA) has prepared
an environmental assessment in response to a request for a permit submitted
by Planet Biotechnology for environmental release of a genetically engineered Nicotiana interspecies hybrid [1], and it is open
for public comment by 13 July 2007 at http://www.regulations.gov/fdmspublic/component/main.
The interspecific
hybrid line 06PBCarHG1 (Nicotiana tabacum
x N. glauca) was obtained by
crossing a transgenic N. tabacum
(producing an antimicrobial monoclonal antibody that binds to the bacterium
Streptococcus mutans associated
with tooth decay in humans) with N. glauca.
The application, submitted in 2005, requested permission to release the transgenic
hybrid on 100 acres in Daviess County, Kentucky, beginning June 2007 and ending
fall of 2007. Following harvest of the Nicotiana hybrid leaves, the company will
extract and purify the CaroRx™ antibody intended to prevent tooth decay. In
the United States, CaroRx™ is an Investigational New Drug
(BB-IND # 7526) and in the European Union, it is a registered Medical Device
[1].
Caro
RX antibody used for passive immunization should not be presumed safe
The Nicotiana hybrid line 06PBCarHG1 produces
the constituent parts of the secretory
CaroRxTM antibody - light chain, heavy chain and J chain from
mouse, and the secretory component from rabbit - all driven by the cauliflower
mosaic virus (CaMV) promoter. These constituent parts were cloned and expressed
in tobacco by independent transformation events. The events were combined
into a single line by classical breeding methods. The monoclonal
antibody produced in genetically modified (GM) tobacco is a synthetic mixture
of gene sequences of mouse and
rabbit. The application of the synthetic antibody leads to passive immunity
to the bacterium Streptococcus mutans
in humans treated with the antibody.
The production
of monoclonal antibodies in plants has been described in a number of patents
[2, 3]. This involves the assembly of monoclonal antibodies in transgenic
tobacco plants [4] and the generation of secretory antibodies in plants [5].
Recombinant plant monoclonal antibodies were tested in human subjects for
preventive immunotherapy against dental caries [6].
That small clinical study proved the concept, but was not sufficient to establish
that the treatment was safe for a large heterogeneous population.
The APHIS environmental assessment for Caro RX antibody
in transgenic hybrid tobacco presumes that the recombinant antibody will be
contained on the 100-acre test-site because there will be little or no pollen
produced by the hybrid tobacco plants. There is a good chance that the dust
and debris from decaying plant parts will lead to surface and ground water
being contaminated with the transgenic antibody, but no provisions were made
to monitor for such contamination. As the genes and proteins produced in the
transgenic hybrid tobacco are synthetic approximations of natural antibodies,
the product should not be presumed safe for wild and protected animals before
appropriate laboratory studies are conducted.
It is worth
mentioning that the transgenic antibody may not be required for passive
immunization to prevent dental caries. A mouth rinse of egg yolk from chickens
immunized against Streptococcus mutans effectively prevented
the bacterium from colonizing the human subjects [7].
In contrast, using transgenic probiotic bacteria for
immunisation is riskier than transgenic tobacco antibodies. Lactobacilli modified to contain single chain
antibodies against Streptococcus mutans
can provide continuous release of recombinant antibody into the oral cavity,
and is considered therapeutically superior to the mouth wash procedure [8].
However, such transgenic probiotic bacteria may turn them into serious pathogens
preadapted for invading the human gut, and we have recommended that Genetically
Modified Probiotics Should Be Banned [9] (ISIS scientific publication).
DNA
vaccines for active immunization
Passive immunity is not the only approach to controlling Streptococcus mutans.
Antigens from the bacterium are delivered using oral or nasal applications.
The preferred delivery of the antigen is through an anti-caries DNA plasmid
carrying the gene that codes for the bacterial antigen. Intra-nasal application
of the plasmid significantly reduced dental caries in rats [10]. Genetic adjuvants,
such as gene for interleukin-5 or the cholera toxin gene, enhanced protection
against caries in mice [11]. Intranasal immunization of rabbits and monkeys
with an anti-caries vaccine proved effective in preventing dental caries [12].
An anti-caries DNA found to produce extended immunity in mice was approved for
human trials by the United States Food and Drug Administration [13]. Active
immunization may be inherently superior to passive immunization and the use
of DNA vaccines will avoid potential environmental pollution associated with
the production of passive vaccines in crop plants. However, large doubts remain
over the safety of DNA vaccines [14] (How
to Stop Bird Flu Instead, SiS 35), as acknowledged by the researchers
themselves [15], pointing to risks that include autoimmune disease, contamination
with bacterial toxins, immune cross reactions with human proteins, immune tolerance,
integration of the nucleic acid into the genome of cells including germ cells
and transmission to the next generations, recombination with host viruses and
bacteria to create new pathogens, and transfer of antibiotic resistance marker
genes to bacteria. The strong promoters from viruses may trigger cancer, and
lastly, liver toxicity from small interference RNAs. Do these risks apply to
the DNA vaccines used? It is important that post release safety monitoring should
be put in place.
Risks from horizontal transfer of antibiotic resistance markers
The hybrid line 06PBCarHG1
contains two additional protein products expressed under the control of a
plant recognized nos promoter
(one of very few bacterial promoters known to be active in plants). These
proteins are NPTII (from E.coli),
an enzyme conferring resistance to kanamycin, used as a selectable marker,
and NOS (from A. tumefaciens),
an enzyme that forms nopaline from the amino acid arginine and alpha-ketoglutaric
acid, but was not used as a selectable marker in the construction of 06PBCarHG1.
Line 06PBCarHG1 also contains trfA
(from E. coli) that encodes
a DNA-binding protein important for plasmid DNA replication and add3 (from E. coli) that codes
for resistance to the antibiotic streptomycin/spectinomycin. These genes are
driven by bacterial promoters not recognized by plants, and are therefore
not expressed in 06PBCarHG1. Additional non-coding sequences contained in
the transformed plant, but not converted into protein products in the transgenic
hybrid 06PBCarHG1 are colEI
and rk2 origin of replication, both from E. coli and the nos terminator from A. tumefaciens
[1]. The antibiotic resistance genes pose dangers
all the same to humans, plants and animals when transferred horizontally to
pathogenic bacteria, whether they are active in the transgenic plants or not.
The sequences trfA and rk2 increase the chance of replicating transgenic
DNA and hence the likelihood for unintended horizontal gene transfer. The
possibility of horizontal transfer of transgenic DNA appears not to have been
addressed
Conclusion
The large field test release
of transgenic tobacco modified with a mouse-rabbit hybrid antibody intended
for passive immunization against dental caries entails environmental risks
that outweigh the benefits. It has not been demonstrated that passive immunization
offered by the transgenic tobacco is superior to using the egg yolk from chickens
immunized with the bacteria. Furthermore, active immunization using DNA vaccines
may provide long-term protection
against the disease without the
environmental risks, though the potential risks of the DNA vaccines also need
to be addressed.
References
1. U.S.
Department of Agriculture Animal and Plant Health Inspection Service Biotechnology
Regulatory Services USDA/APHIS Environmental Assessment In response to the
Planet Biotechnology permit application 05-354-03r for an environmental release
to produce antibodies in genetically engineered N. tabacum X N. glauca hybrid
plants 2007 http://www.regulations.gov/fdmspublic/component/main
2. Hiatt
A, Ma J, Lehner T and Mostov K. Method for producing imunoglobulins containing
protection proteins in plants and their use 2004 United States Patent 6,303,341
3. Hein
M, Hiatt A and Ma J. Transgenic crops expressing assembled secretory antibodies
2006 Units States Patent 6,995,014
4. Ma
JK, Lehner T, Stabila P, Fux CI and Hiatt A. Assembly of monoclonal antibodies
with IgG1 and IgA heavy chain domains in transgenic tobacco plants. Eur J Immunol. 1994 Jan;24(1):131-8.
5. Ma
JK, Hiatt A, Hein M, Vine ND, Wang F, Stabila P, van Dolleweerd C, Mostov
K and Lehner T. Generation and assembly of secretory antibodies in plants.
Science 1995, 268(5211), 716-9.
6. Ma
JK, Hikmat BY, Wycoff K, Vine ND, Chargelegue D, Yu L, Hein MB and Lehner
T. Characterization of a recombinant plant monoclonal secretory antibody and
preventive immunotherapy in humans. Nat
Med. 1998, 4(5), 601-6.
7. Hatta
H, Tsuda K, Ozeki M, Kim M, Yamamoto T, Otake S, Hirasawa M, Katz J, Childers
NK and Michalek SM. Passive immunization against dental plaque formation
in humans: effect of a mouth rinse containing egg yolk antibodies (IgY) specific
to Streptococcus mutans. Caries Res.
1997, 31(4), 268-74.
8. Kruger
C, Hultberg A, van Dollenweerd C, Marcotte H and Hammarstrom L. Passive immunization
by lactobacilli expressing single-chain antibodies against Streptococcus
mutans. Mol Biotechnol. 2005, 31(3), 221-31.
9. Cummins
J and Ho MW. Genetically modified probiotics should be banned. Microbial Ecology in Health and Disease 2005,
17, 66-68.
10. Xu
QA, Yu F, Fan MW, Bian Z, Chen Z, Peng B, Jia R and Guo JH. Protective efficacy
of a targeted anti-caries DNA plasmid against cariogenic bacteria infections.
Vaccine 2007 25(7), 1191-5.
11. Han TK and Dao ML. Enhancement of salivary IgA
response to a DNA vaccine against Streptococcus mutans wall-associated protein
A in mice by plasmid-based adjuvants. J Med Microbiol. 2007, 56(5), 675-80.
12. Jia
R, Guo JH, Fan MW, Bian Z, Chen Z, Fan B, Yu F and Xu QA.Immunogenicity of
CTLA4 fusion anti-caries DNA vaccine in rabbits and monkeys. Vaccine 2006, 24(24):5192-200.
13. Xu QA, Yu F, Fan MW, Bian Z, Chen Z, Fan B, Jia
R and Guo JH. Immunogenicity and persistence of a targeted anti-caries DNA
vaccine. J Dent Res. 2006, 85(10), 915-8.
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