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ISIS Report 07/01/09
Dangers of HPV Vaccine Production in Plants, Microbes, and
Viruses
Widespread
releases of hazardous transgenes and vaccines have the potential to create
viruses more deadly than the ones the vaccines protect against
Prof. Joe Cummins and Dr.
Mae-Wan Ho
Human papilloma virus (HPV) vaccines are already commercialised and promoted
worldwide in a bid to protect young girls and women from cervical cancer [1,
2] (Recombinant Cervical Cancer Vaccines,
SiS 29; The
HPV Vaccine Controversy, SiS 41), while there is still major uncertainty
over their efficacy and safety, especially in the long term. One obstacle to
the adoption of the vaccines by developing countries is that the two available
are very costly. There appears to have been a rush to create cheap oral HPV
vaccines in transgenic plants, microbes and viruses that do not require refrigeration
and can be distributed relatively inexpensively, but would involve widespread
releases of hazardous transgenes and products into the open environment. Some
of these are near commercialization, and regulators must be warned against the
approval of such production methods unless and until strict containment and
safeguards are put in place.
HPV
vaccines in crop plants
The main concern over the
vaccines produced in crop plants is that transgenes
from tests sites or production farms can
readily spread by pollen or by mechanical dispersal of seeds. Debris
from transgenic crops can also spread transgenes and vaccine proteins through
contaminating surface and groundwater. Debris in the form of dust in the air
can impact on the respiratory mucosa directly, with the potential of triggering
acute and delayed immune reactions in humans and animals exposed. HPV vaccines
have already been associated with various adverse acute immune reactions some
of which resulted in death [2]. People subject to persistent exposure to the crop vaccine are likely to develop oral
tolerance rendering them susceptible to virus infection [3] (Pharm Crops for Vaccines and Therapeutic
Antibodies, SiS 24)..
There have been reports since 2006 that HPV virus and L1 proteins were produced
in plants including transgenic potato, tobacco and a wild tobacco
N. benthamiana [4]. HPV L1 virus-like particles were expressed in
transgenic potatoes and these particles were found to immunize animals fed
the potatoes. The gene for the particle protein L1 had been optimized for
activity in potato by codon alterations. The full length message had a C-terminal
signal sequence for nuclear localization of the
protein and production of the L1 protein is enhanced by removal of the signal
sequence for nuclear localization. The oral immunization using transgenic
potato had to be enhanced by ingesting LI protein produced from insect
cell (baculovirus) cultures.[5]. Comparing the production of HPV19 L1 in cytoplasm
or chloroplast of Nicotiana benthamiana showed that the vaccine was
produced most effectively in the chloroplasts. Adjustments in codon preferences
showed that the human codon preference was most effective in enhancing
production of the vaccine. The optimally engineered gene configuration produced
up to 11 percent of the plant’s soluble protein
as L1 vaccine protein [6]. The HPV 16 L1 protein produced in N. benthamiana
proved very immunogenic following injection in mice [7]. Another N.
benthamiana chloroplast transformation produced up to 1.5 percent total
leaf protein as HPV L1 whose half life in the leaf was at least 8 hours [8].
The plant chloroplast
system not only produces satisfactorily high levels of HPV L1 protein but
avoided the spread of the transgene in pollen for the most part. But the spread of L1 protein in plant debris polluting surface
and ground water and in dust to the respiratory tracts of humans and other
animals cannot be avoided unless the transgenic plants are carefully confined
in a secure greenhouse facility
Transgenic microbes
as oral Vaccines
The transgenic yeast, Schizosaccharomyces pombe,
modified to produce HPV 16 L1 [1]. Currently, a lyophilized preparation of
S. pombe containing HPV 16 L1 as an
oral vaccine was the subject of a patent application [9]. S. pombe
is a native of Africa and has been
used there to make beer. The potential pollution
of the African environment with transgenic pombe yeasts requires fuller consideration.
A bacterial
system has been developed for both a prophylactic and a therapeutic treatment
for cervical cancer. Bacterial expression vectors are designed to produce
coat protein ( L1) or tumour associated proteins of HPV. These proteins are
displayed on the surface of the modified bacterium. The bacteria-based vaccine
is potentially capable of preventing viral infection and of targeting cancer
cells. Gram positive bacteria such as Lactobacilli, or gram negative
bacteria such as Salmonella, may both serve as display vectors [10].
Vaccine production from viruses
A rabbit papilloma virus
similar to the human virus served as a model for producing vaccine using tobacco
mosaic virus (TMV). The DNA codes for epitopes (protein amino acid sequences
that are recognized by elicited antibodies) were identified and used to modify
coat proteins from TMV. The modified TMV proteins were capable of eliciting
antibodies that were active against the rabbit papilloma virus. The modified
TMV coat proteins served as a vaccine to prevent rabbit paillomavirus infection.
The modified vaccine was produced rapidly and in quantity by infecting N.
benthamiana with modified TMV [11]. Using modified TMV to produce recombinant
vaccines is convenient, but inherently hazardous, as the recombinant virus may give rise to new pathogens.
A potyvirus
(potato virus A) coat protein gene was modified by fusing an epitope from the HPV L2 minor protein to its N terminus, and an epitope
from E7 oncooprotein (cancer gene) to its C terminus. That construct was
cloned into a potato virus X vector, and used to transform N. benthamiana
and the food crop Brassica rapa variety Rapa
(turnip
tops). Both transformed crops produced edible vaccine believed
to be capable of both preventing and treating HPV cancers [12]. The
purified HPV vaccine was most stable as freeze dried material stored at minus
20 degrees C [13]. N. benthamiana is not a food crop nor is it used
to produce tobacco. B. rapa is both a food crop and a weed known to
spread transgenic pollen great distances, and is
almost certain to cross pollinate Brassica food crops. No transgenic
crops producing vaccines and drugs should be allowed in open fields for reasons
stated earlier.
The Cervarix
vaccine available commercially [2] is produced by GlaxoSmithKline using a
baculovirus vector propagated in an insect cell line. A number of other vaccines
are also being produced using baculovirus vectors. Baculoviruses are soil
inhabiting viruses that infect insects. Baculovirus expression vectors propagated
in insect cells were originally hampered by the
appearance of many interfering baculovirusese with chromosomal deletions,
which arise as an intrinsic property of the
native baculovirus [14,15]. The intrinsic deletions in the viral chromosome
may provide a source of diversity as the virus faces environmental challenges.
Such instability is undesirable in producing vaccines. Some progress has
been achieved in making more stable baculovirus expression vector lines [16].
Nevertheless, regulators and the vaccine producer have not made public comment
about the genetic stability of the baculovirus lines producing Cervarix vaccine,
nor the fact that baculovirus is capable of
infecting mammalian cells and tissues.
If the GM baculovirus infects mammalian cells and tissues in vivo, they would
also transfer transgenes to those infected cells as gene therapy experiments
have demonstrated since 2001 [17]. Baculovirus can also serve as a gene delivery
vector for stem cell and bone tissue engineering [18].
The use of GM
viruses to produce HPV vaccines in yeast, insect cells, crop plants and bacteria
has proceeded without much warning. And the
pharmaceutical corporations commercializing such products appear to have scant
regard over the safety of their products.
Hazards
of horizontal transfer of transgenes
A safety issue
that has been persistently ignored by regulators is horizontal transfer of
transgenes to unrelated species. GM microbes and viruses have the strongest
potential to transfer transgenes horizontally and contribute to creating new
pathogenic bacteria and viral strains Recent evidence confirms that transgenic
DNA does jump species to bacteria and even plants and
animals [19] (Horizontal Gene Transfer
from GMOs Does Happen, SiS 39), as some
of us had predicted. The widespread use of eukaryotic cell cultures and crops
plants to produce vaccines in conjunction with viruses creates abundant opportunities
for horizontal gene transfer and recombination to generate potentially more
deadly viruses than the vaccines are meant to protect against.
References
1. Cummins
J. Recombinant cervical cancer vaccines Science in Society 29, 20-21,
2006.
2. Cummins J and Ho MW. The HPV vaccine controversy. Science
in Society 41 (to appear).
3. Cummins
J. Pharm crops for vaccines and therapeutic antibodies. Science in Society 24, 22-23,
2004.
4. Santia
L, Huanga Z, Mason H. Virus-like particles production in green plants. Particle-based
Vaccines Methods 2006, 40, 66-76.
5. Warzecha
H, Mason HS, Lane C, Tryggvesson A, Rybicki E, Williamson AL, Clements JD,
Rose RC.Oral immunogenicity of human papillomavirus-like particles expressed
in potato. J Virol. 2003; 77(16),:8702-11.
6. Maclean
J, Koekemoer M, Olivier AJ, Stewart D, Hitzeroth II, Rademacher T, Fischer
R, Williamson AL, Rybicki EP.Optimization of human papillomavirus type 16
(HPV-16) L1 expression in plants: comparison of the suitability of different
HPV-16 L1 gene variants and different cell-compartment localization. J
Gen Virol. 2007; 88(Pt 5), 1460-9.
7. Fernández-San
Millán A, Ortigosa SM, Hervás-Stubbs S, Corral-Martínez P, Seguí-Simarro JM,
Gaétan J, Coursaget P, Veramendi J.Human papillomavirus L1 protein expressed
in tobacco chloroplasts self-assembles into virus-like particles that are
highly immunogenic. Plant Biotechnol J. 2008; 6(5), 427-41.
8. Lenzi
P, Scotti N, Alagna F, Tornesello ML, Pompa A, Vitale A, De Stradis A, Monti
L, Grillo S, Buonaguro FM, Maliga P, Cardi T.Translational fusion of chloroplast-expressed
human papillomavirus type 16 L1 capsid protein enhances antigen accumulation
in transplastomic tobacco. Transgenic Res. 2008, 17(6), 1091-102.
9. Sasagawa
T, Tohda H, Hama Y. Edible vaccine United States Patent Application 2007
20070154491
10.
Sung
M, Poo H, Lee J, Jung C, Hong S, Kim C, Park S, Pyo H. United States
Patent 2009, 7,425,438
11.
Palmer
KE, Benko A, Doucette SA, Cameron TI, Foster T, Hanley KM, McCormick AA, McCulloch
M, Pogue GP, Smith ML, Christensen ND. Protection of rabbits against cutaneous
papillomavirus infection using recombinant tobacco mosaic virus containing
L2 capsid epitopes. Vaccine 2006, 24(26), :5516-25
12.
Hoffmeisterová
H, Čeřovská N, Moravec T, Plchová H, Folwarczna J, Velemínský J.
Transient expression of fusion gene coding for the HPV-16 epitopes fused to
the sequence of potyvirus coat protein using different means of inoculation
of Nicotiana benthamiana and Brassica rapa , cv. Rapa plants. Plant
Cell, Tissue and Organ Culture 2008, 94, 261-7.
13.
Čeřovská
N, Hoffmeisterová H, Moravec T, Plchová H, Folwarczna J, Hadámková R. Optimum
storage conditions for product of transiently expressed epitopes of Human
papillomavirus using Potato virus X-based vector. Biologia Plantarum
2008, 52, 184-6.
14.
Pijlman
GP, van den Born E, Martens DE, Vlak JM. Autographa californica baculoviruses
with large genomic deletions are rapidly generated in infected insect cells.
Virology. 2001, 283(1),132-8.
15.
Pijlman
GP, van Schijndel JE, Vlak JM. Spontaneous excision of BAC vector sequences
from bacmid-derived baculovirus expression vectors upon passage in insect
cells.J Gen Virol. 2003; 84(Pt 10), 2669-78.
16.
Pijlman
GP, de Vrij J, van den End FJ, Vlak JM, Martens DE.Evaluation of baculovirus
expression vectors with enhanced stability in continuous cascaded insect-cell
bioreactors. Biotechnol Bioeng. 2004; 87(6),743-53.
17.
Pieroni
L, Maione D, La Monica N. In vivo gene transfer in mouse skeletal muscle mediated
by baculovirus vectors. Hum Gene Ther. 2001,12(8), 871-81.
18.
Chuang
CK, Sung LY, Hwang SM, Lo WH, Chen HC, Hu YC. Baculovirus as a new gene delivery
vector for stem cell engineering and bone tissue engineering. Gene Ther.
2007t;14(19), 1417-24.
19.
Ho
MW and Cummins J. Horizontal gene transfer from GMOs does happen. Science in Society 39, 22-24,
2008.
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