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[SANET-MG] Antibodies from Hybrid GM Tobacco Plants comments due by friday
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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