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Human Papillomaviruses (HPV) are the causative agents for cervical, skin,
head and neck tumors. In developing countries, cervical cancer is one of the
main causes of cancer-related deaths. However, Italian researchers have now
developed an immunologically active, cost-efficient vaccine against HPV,
March 2006 by Katharina Schoebi, Checkbiotech.

Currently, more than 150 different types of Human Papillomaviruses are
known. The most commonly found HPVs in cervical carcinomas are HPV 16 and
18. The cancer causing factors of these viruses are the proteins E6 and E7,
which are known as onco-proteins. Onco-proteins represent a promising target
for the development of a therapeutic vaccine against HPV-associated tumors.
This has been affirmed by an experiment showing that mice eating crude plant
extracts containing the onco-protein E7 are partially protected against
HPV-induced cancers.

Unfortunately, attempts to produce large amounts of E7 protein have
practically failed until now, because E7 is a short-lived protein that is
rapidly degraded. Nevertheless, some E7-based HPV vaccines are currently
being explored and the first promising results have been disclosed, but they
still need further improvement to ensure protection.

One method to produce high amounts of E7 protein in a relatively short time
is by genetically engineering plants. An advantage of transgenic plant
systems is that they generally lack human pathogens, oncogenic DNA sequences
and endotoxins, explained Dr. Rosella Franconi from ENEA (Ente per le nuove
Tecnologie, Energia e Ambiente). ?This minimizes health risks and lowers the
production costs.?

Plants can be genetically modified by either stable or transient
transformation. With stable transformation, the introduced DNA integrates in
the genome of the plant, whereas with transient transformation, the
introduced gene sequences do not integrate into the genome of the plant.

Ideal tools for transient transformation are viruses, whose genomes contain
the new gene sequences of interest. When the plant is infected with the
virus, the virus spreads and replicates and the genes introduced in its
genome are proliferated in high amounts. The fact that viruses are usually
not transmitted by pollen ? which ensures genetic contamination of wild
growing plants is prevented - is a further advantage of viral systems.

In 2002, Dr. Franconi and her research teams from the IRE (the Cancer
Institute ?Regine Elena?) and the ISS (Istituto Superiore di Sanit?
Laboratory of Virology), in Rome, produced the HPV16 E7 protein in the
cytoplasm of tobacco (Nicotiana benthamiana) plants. To do so, they used the
potato virus X (PVX). PVX is a safe tool for genetic engineering, because it
does not infect animals, but does effectively transfer genetic material to a
variety of plant species.

Mice fed with crude extracts of transgenic tobacco plants showed strong
immune responses and about 40 percent of the animals were protected from
HPV-induced tumors. The researchers hypothesized that the low percentage of
responders could be due to the low amount of E7 antigen in the plant extract
vaccine dose. Only 3 to 4 microgram per gram fresh leaf of E7 protein were
produced and the quantity administered to the mice was 20-fold lower than
that known to prevent tumor growth.

Dr. Franconi and her team therefore wondered if they could enhance the
production levels of E7 by targeting it to the plant secretory pathway,
which is used by cells to transport material to the outside. The researchers
targeted the protein to the secretory pathway by adding a so-called signal
sequence to the protein. Signal sequences guide proteins to specific areas
for accumulation, for example to the endoplasmic reticulum, where the
proteins are prepared and modified. This offered a natural way to
pre-concentrate the E7 protein.

After examining their results, Dr. Franconi?s team found that the E7
production level was five-fold higher (15 microgram protein per gram fresh
leaf) compared to the production level in the cytoplasm, Dr. Franconi
previously observed (The International Journal of Immunopathology and
Pharmacology, in press).

?This result may be related to a positive effect on protein stability,? Dr.
Franconi told Checkbiotech. In the endoplasmic reticulum, there are few
enzymes that degrade proteins, but many so called chaperons that help
proteins fold correctly. An additional advantage of targeting E7 proteins to
the secretory pathway is that the protein purification is further
simplified, because the proteins might be naturally released from the roots
or leaves of the plants.

Since the E7 proteins accumulated in the spaces between the cells, referred
to as the apoplast, their concentration was much higher and the efficacy of
the plant vaccines was improved. Mice vaccinated with these plant extracts
showed a stronger immune response that more effectively protected them from
tumors than mice vaccinated with plant extracts producing the E7 protein in
the cytoplasm. Dr. Franconi said, ?We believe that a further enhancement of
this anti-tumor immunity can be achieved by a combination of E7-containing
extracts with immunostimulatory genes, or by application of two different
antigens.?

The fact that freeze-dried N. benthamiana leaf tissues containing high
amounts of E7 antigen are stable for at least one year at room temperature,
further improves production and storage of the vaccine. In addition, it
offers the possibility to use such plant extracts as edible vaccines.
?Edible plant based vaccines offer a palatable oral delivery system without
the costly purification processes required for injectable vaccines,? Dr.
Franconi explained.

However, a great disadvantage of oral vaccine delivery is that the digestive
system could degrade the antigens. This might limit the use of oral vaccines
against pathogens that can survive in the harsh environment of the digestive
system.

By using a plant system for antigen production, however, the vaccine
antigens inside the plant cells are naturally protected by the cell wall.
Dr. Franconi affirmed, ?Edible vaccines are now considered to be more stable
and practical in the harsh environment of the gastrointestinal tract than
purified vaccine antigens.?

Oral immunization with tobacco plants, however, could raise the problem of
toxicity, since many tobacco cultivars produce high levels of toxic
alkaloids. Varieties with only low alkaloid amounts, however, have been
shown to be safe production and delivery systems for therapeutic vaccine.s
Mice fed with such tobacco leaves showed no symptoms of toxicity.

Dr. Franconi?s work represents a significant step toward the development of
a vaccine against cancers caused by the Human Papillomavirus type 16. Since
HPV16 is an enormous problem - particularly in the developing world - a
vaccine against this virus needs to be highly effective, temperature-stabile
and easy to administer. Furthermore, it should be possible to produce the
vaccine on a large scale and cost-efficiently. Plants, as both a production
and delivery system for vaccines, meet these requirements.

?Their simple demand for solar light, water and minerals make plants an
economic system for the production of proteins.?

Katharina Schoebi is a biologist and Chief Science Writer for Checkbiotech.
Contact her at katharina.schoebi@bluewin.ch.

Giorgi C. et al. Plant derived Human Papillomavirus 16 E7 oncoprotein
induces immune response and specific tumor protection. Cancer Research.
(2002) 62, pp. 3654-3658

Franconi R. & Venuti A. HPV Vaccines in Plants: an appetising solution to
Control Infection and Associated Cancers in Papillomavirus. Editor ? S.
Campo, Horizon Scientific Press - Caister Academic Press, Norfolk, NR18 0JA,
U.K. (in press).

Franconi R. et al. Exploiting the plant secretory pathway to improve the
anti-cancer activity of a plant-based HPV16 E7 vaccine. The International
Journal of Immunopathology and Pharmacology (in press).

www.checkbiotech.org

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