United States Patent: 5,914,123
( 1 of 16 )
United States Patent 5,914,123
Arntzen , et al. June 22, 1999
Abstract
The anti-viral vaccine of the present invention is produced in
transgenic plants and then administered through standard vaccine
introduction method or through the consumption of the edible portion
of those plants. A DNA sequence encoding for the expression of a
surface antigen of a viral pathogen is isolated and ligated to a
promoter which can regulate the production of the surface antigen
in a transgenic plant. This gene is then transferred to plant cells
using a procedure that results in its integration into the plant
genome, such as through the use of an Agrobacterium tumefaciens
plasmid vector system. Preferably, the foreign gene is expressed
in an portion of the plant that is edible by humans or animals.
In a preferred procedure, the vaccine is administered through the
consumption of the edible plant as food, preferably in the form
of a fruit or vegetable juice.
Inventors: Arntzen; Charles Joel (The Woodlands, TX); Lam; Dominic
Man-Kit (The Woodlands, TX)
Assignee: Prodigene, Inc. (College Station, TX)
Appl. No.: 479742
Filed: June 7, 1995
U.S. Class:424/439; 424/442; 424/225.1; 424/223.1; 424/93.1; 800/205;
426/615; 426/637
Field of Search: 426/615,637 800/205
424/439,225.1,226.1,227.1,228.1,442,223.1,93.1
References Cited [Referenced By]
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4940835Jul., 1990Shah et al.800/205.
4956282Sep., 1990Goodman et al.435/69.
5484719Jan., 1996Lam et al.435/172.
5654184Aug., 1997Curtiss, III et al..
Foreign Patent Documents
A-0 278 541Jan., 1988EP.
A-0 510 773Apr., 1992EP.
WO 90/02484Mar., 1990WO.
WO 90/10076Sep., 1990WO.
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Primary Examiner: Mosher; Mary E.
Attorney, Agent or Firm: Zarley, McKee, Thomte, Voorhees, &
Sease
Parent Case Text
This is a divisional of U.S. application Ser. No. 08/026,393 filed
Mar. 4, 1993 (now U.S. Pat. No. 5,612,487) and a continuation-in-part
of PCT application PCT/US94/02332 filed Mar. 4, 1994 (designating
the U.S.) which is a continuation-in-part of U.S. application Ser.
No. 07/750,049 filed Aug. 26, 1991 (abandoned). This application
is also a continuation-in-part of U.S. application Ser. No. 08/156,508
filed Nov. 23, 1993 (now U.S. Pat. No. 5,484,719).
Claims
A food comprising transgenic plant material capable of being ingested
for its nutritional value, said transgenic plant expressing a recombinant
immunogen derived from Hepatitis virus.
The food of claim 1 wherein said immunogen is Hepatitis B surface
antigen.
The food of claim 1 wherein said plant is selected from the group
consisting of: tomato and potato.
A food comprising transgenic plant material capable of being ingested
for its nutritional value, said transgenic plant expressing a recombinant
immunogen derived from Transmissible Gastroenteritis Virus.
The food of claim 3 wherein said immunogen is Transmissible Gastroenteritis
Virus S.
The food of claim 4 wherein said plant is selected from the group
consisting of: tomato and potato.
The food of claim 1 or 3 wherein said transgenic plant material
is selected from the group consisting of: edible fruit, leaves,
juices, roots, and seed of said plant.
Description
BACKGROUND OF THE INVENTION
This invention relates generally to vaccines and more particularly
to the production of oral vaccines in edible transgenic plants and
the administration of the oral vaccines such as through the consumption
of the edible transgenic plants by humans and animals.
Diseases have been a plague on civilization for thousands of years,
affecting not only man but animals. In economically advanced countries
of the world, diseases are 1) temporarily disabling; 2) permanently
disabling or crippling; or 3) fatal. In the lesser developed countries,
diseases tend to fall into the latter two categories, permanently
disabling or crippling and fatal, due to many factors, including
a lack of preventative immunization and curative medicine.
Vaccines are administered to humans and animals to induce their
immune systems to produce antibodies against viruses, bacteria,
and other types of pathogenic organisms. In the economically advanced
countries of the world, vaccines have brought many diseases under
control. In particular, many viral diseases are now prevented due
to the development of immunization programs. The virtual disappearance
of smallpox, certainly, is an example of the effectiveness of a
vaccine worldwide. But many vaccines for such diseases as poliomyelitis,
measles, mumps, rabies, foot and mouth, and hepatitis B are still
too expensive for the lesser developed countries to provide to their
large human and animal populations. Lack of these preventative measures
for animal populations can worsen the human condition by creating
food shortages.
The lesser developed countries do not have the monetary funds to
immunize their populations with currently available vaccines. There
is not only the cost of producing the vaccine but the further cost
of the professional administration of the vaccine. Also, some vaccines
require multiple doses to maintain immunity. Therefore, often, the
countries that need the vaccines the most can afford them the least.
Underlying the development of any vaccine is the ability to grow
the disease causing agent in large quantities. At the present, vaccines
are usually produced from killed or live attenuated pathogens. If
the pathogen is a virus, large amounts of the virus must be grown
in an animal host or cultured animal cells. If a live attenuated
virus is utilized, it must be clearly proven to lack virulence while
retaining the ability to establish infection and induce humoral
and cellular immunity. If a killed virus is utilized, the vaccine
must demonstrate the capacity of surviving antigens to induce immunization.
Additionally, surface antigens, the major viral particles which
induce immunity, may be isolated and administered to proffer immunity
in lieu of utilizing live attenuated or killed viruses.
Vaccine manufacturers often employ complex technology entailing
high costs for both the development and production of the vaccine.
Concentration and purification of the vaccine is required, whether
it is made from the whole bacteria, virus, other pathogenic organism
or a sub-unit thereof. The high cost of purifying a vaccine in accordance
with Food and Drug Administration (FDA) regulations makes oral vaccines
prohibitively expensive to produce because they require ten to fifty
times more than the regular quantity of vaccine per dose than a
vaccine which is parenterally administered. Of all the viral vaccines
being produced today only a few are being produced as oral vaccines.
According to FDA guidelines, efficacy of vaccines for humans must
be demonstrated in animals by antibody development and by resistance
to infection and disease upon challenge with the pathogen. When
the safety and immunogenicity levels are satisfactory, FDA clinical
studies are then conducted in humans. A small carefully controlled
group of volunteers are enlisted from the general population to
begin human trials. This begins the long and expensive process of
testing which takes years before it can be determined whether the
vaccine can be given to the general population. If the trials are
successful, the vaccine may then be mass produced and sold to the
public.
Even after these precautions are taken, problems can arise. With
the killed virus vaccines, there is always a chance that one of
the live viruses has survived and vaccination may lead to isolated
cases of the disease. Moreover, since both the killed and live attenuated
types of virus vaccines are made from viruses grown in animal host
cells, the vaccines are sometimes contaminated with cellular material
from the animal host which can cause adverse, sometimes fatal, reactions
in the vaccine recipient. Legal liability of the vaccine manufacturer
for those who are harmed by a rare adverse reaction to a new or
improved vaccine necessitates expensive insurance which ultimately
adds to the cost of the vaccine.
Some vaccines have other disadvantages. Vaccines prepared from whole
killed virus generally stimulate the development of circulating
antibodies (IgM, IgG) thereby conferring a limited degree of immunity
which usually requires boosting through the administration of additional
doses of vaccine at specific time intervals. Live attenuated viral
vaccines, while much more effective, have limited shelf-life and
storage problems requiring maintaining vaccine refrigeration during
delivery to the field..sup.1
Efforts today are being made to produce less expensive vaccines
which can be administered in a less costly manner. Recombinants
or mutants can be produced that serve in place of live virus vaccines.
The development of specific deletion mutants that alter the virus,
but do not inactivate it, yield vaccines that can replicate but
cannot revert to virulence.
Recombinant DNA techniques are being developed to insert the gene
coding for the immunizing protein of one virus into the genome of
a second, a virulent virus type that can be administered as the
vaccine. Recombinant vaccines may be prepared by means of a vector
virus such as vaccines virus or by other methods of gene splicing.
Vectors may include not only a virulent viruses but bacteria as
well. A live recombinant hepatitis A vaccine has been constructed
using attenuated Salmonella typhimurium as the delivery vector via
oral administration..sup.1
Various a virulent viruses have been used as vectors. The gene
for hepatitis B surface antigen (HMsAg) has been introduced into
a gene non-essential for vaccines replication. The resulting recombinant
virus has elicited an immune response to the hepatitis B virus in
test animals. Additionally, researchers have used attenuated bacterial
cells for expressing hepatitis B antigen for oral immunization.
Importantly, when whole cell attenuated Salmonella expressing recombinant
hepatitis antigen were fed to mice, anti-viral T and B cell immune
responses were observed. These responses were generated after a
single oral immunization with the bacterial cells resulting in high-titers
of the antibody. See, e.g., "Expression of hepatitis B virus
antigens in attenuated Salmonella for oral immunization," F.
Schodel and H. Will, Res. Microbiol., 141:831-837 (1990). Others
have had similar success with oral administration routes for recombinant
hepatitis antigens. See, e.g., M. D. Lubeck et al., "Immunogenicity
and efficiacy testing in chimpanzees of an oral hepatitis B vaccine
based on live recombinant adenovirus," Proc. Natl. Acad. Sci.
86:6763-6767 (1989); S. Kuriyama, et al., "Enhancing effects
of oral adjuvants on anti-HBs responses induced by hepatitis B vaccine,"
Clin. Exp. Immunol. 72:383-389 (1988).
Other virus vectors may possess large genomes, e.g. the herpesvirus.
The oral adenovirus vaccine has been modified so that it carries the
HBsAg immunizing gene of the hepatitis B virus. Chimeric polio virus
vaccines have been constructed of which the completely a virulent
type 1 virus acts as a vector for the gene carrying the immunizing
VP1 gene of type 3..sup.1
Immunity to a pathogenic infection is based on the development
of an immune response to specific antigens located on the surface
of a pathogenic organism. For enveloped viruses, the important antigens
are the surface glycoproteins. Glycosylation of viral surface glycoproteins
is not always essential for antigenicity..sup.1 Unglycosylated herpesvirus
proteins synthesized in bacteria have been shown to produce neutralizing
antibodies in test animals..sup.1 However, where recombinant antigens
such as HBsAg are produced in organisms requiring complex fermentative
processes and machinery, the costs and access can be prohibitive.
Viral genes which code for a specific surface antigen that produces
immunity in humans or animals, can be cloned into plasmids. The
cloned DNA can then be expressed in prokaryotic or eukaryotic cells
if appropriately engineered constructions are used. The immunizing
antigens of hepatitis B virus,.sup.2 foot and mouth,.sup.3 rabies
virus, herpes simplex virus, and the influenza virus have been successfully
synthesized in bacteria or yeast cells..sup.1
Animal and human subjects infected by a pathogen present an immune
response when overcoming the invading microorganism. They do so
by initiating at least one of three branches of the immune system
mucosal, humoral or cellular. Mucosal immunity results from the
production of secretory IgA antibodies in the secretions that bathe
mucosal surfaces in the respiratory tract, the gastrointestinal
tract, the genitourinary tact and the secretory glands. McGhee,
J. R. et al. Annals NY Acad. Sci.409:409 (1983). Mucosal antibodies
act to prevent colonization of the pathogen on mucosal surfaces
thus establishing a first line of defense against invasion. The
production of mucosal antibodies can be initiated by either local
immunization of the secretory gland or tissue or by presentation
of the antigen to either the gut-associated lymphoid tissues (GALT;
Peyer's Patches) or the bronchial-associated lymphoid tissue (BALT).
Cebra, J. J. et al. Cold Spring Harbor Symp. Quant. Biol. 41:210
(1976); Bienenstock, J. M., Adv. Exp. Med. Biol. 107:53 (1978);
Weisz-Carrington, P. et al., J. Immunol. 123:1705 (1979); McCaughai,
G. et al., Internal Rev. Physiol. 28:131 (1983). Humoral immunity,
on the other hand, results from the production of IgG and IgM antibodies
in the serun, precipitating phagocytosis of invading pathogens,
neutralization of viruses, or complement-mediated cytotoxicity against
the pathogen. See, Hood et al. supra.
Others have noted that the induction of serum or mucosal antibody
responses to orally administered antigens, however, may be problematic.
Generally, such oral administration requires relatively large quantities
of antigen since the amount of the antigen that is actually absorbed
and capable of eliciting an immune response is usually low. Thus,
the amount of antigen required for oral administration generally far
exceeds that required for parenteral administration. de Aizpurua and
Russell-Jones, J. Exp. Med. 167:440-451 (1988). However, it has been
found that the systemic and mucosal immune systems may be stimulated
by feeding low doses of certain classes of proteins. In particular,
this may be achieved with proteins which share the property of being
able to bind specifically to various glycolipids and glycoproteins
located on the surface of the cells on the mucosal membrane. Such
proteins, called "mucosal immunogens" have been found to
include viral antigens such as viral hemagglutinin. Moreover, dose-response
experiments comparing oral with intramuscular administration revealed
that oral presentation of mucosal immunogens was remarkably efficient
in eliciting a serum antibody response to the extent that the response
elicited by oral presentation was only slightly lower than that elicited
by intramuscular injection of the mucosal immunogen. de Aizpurua and
Russell-Jones, supra.
The hypothesis proposed by these workers that such mucosal immunogens
shared a common ability to bind glycosylated surface proteins on
the mucosal membrane was at least partially confirmed by the inhibition
of mucosal uptake of these mucosal immunogens by certain high levels
of three specific sugars (galactose, lactose or sorbitol). Other
sugars, fructose (the principal sugar found in many plant fruits)
mannose and melibiose, did not inhibit mucosal immunogens from eliciting
antibodies. de Aizpurua and Russell-Jones, supra. Others have found
that certain sugars may, in fact, boost mucosal responses in the
intestine. See, e.g., "Boosted Mucosal Immune Responsiveness
in the Intestine by Actively Transported Hexose," S. Zhang
and G. A. Castro, Gastroenterol., accepted for publication).
Recent advances in genetic engineering have provided the requisite
tools to transform plants to contain foreign genes. Plants that
contain the transgene in all cells can then be regenerated and can
transfer the transgene to their offspring in a Mendelian fashion..sup.4
Both monocotyledenous and dicotyledenous plants have been stably
transformed. For example, tobacco, potato and tomato plants are
but a few of the dicotyledenous plants which have been transformed
by cloning a gene which encodes the expression of 5-enolpyruvyl-shikimate-3-phosphate
syntase..sup.5
Plant transformation and regeneration in dicotyledons by Agrobacterium
tumefaciens (A. tumefaciens) is well documented. The application
of the Agrobacterium tumefaciens system with the leaf disc transformation
method.sup.6 permits efficient gene transfer, selection and regeneration.
Monocotyledons have also been found to be capable of genetic transformation
by Agrobacterium tumefaciens as well as by other methods such as
direct DNA uptake mediated by PEG (polyethylene glycol), or electroporation.
Successful transfer of foreign genes into corn.sup.7 and rice,.sup.8,9
as well as wheat and sorghum protoplasts has been demonstrated.
Rice plants have been regenerated from untransformed and transformed
protoplasts. New methods such as microinjection and particle bombardment
may offer simpler and even more efficient means of transformation
and regeneration of monocotyledons..sup.10
Attempts to produce tnansgenic plants expressing bacterial antigens
of Escherichia coli and of Streptococcus mutans have been made (Curtiss
and Ihnen, WO 90/0248, Mar. 22, 1990). However, until the work of
the present inventors, no transgenic plants had been constructed
expressing viral antigens such as HBsAg..sup.72 In particular, until
the work of the present inventors no such plants had been obtained
which were capable of expressing viral antigens capable of eliciting
an immune response as a mucosal immunogen. Moreover, until the work
reported above no such plants had been obtained capable of producing
particles which were antigenically and physically similar to the
commercially available HBsAg viral antigens derived from human serum
or recombinant yeast. However, none of these references provided
the possibility of testing truly edible vaccines since all such
studies were carried out in the classical tobacco test systems which
plant tissues are not routinely digested by man or animal.
Thus, while prior approaches to obtaining less expensive and more
accessible vaccines have been attempted, there remains a need to
provide alternative sources of such vaccines for new antigens. Particularly,
there remains need to provide alternative sources of vaccines which
are incorporated by plants which are routinely included in human
and animal diets. For instance, while vaccines such as HBsAg have
been produced using antigen particles derived from human serum and
recombinant yeast cells, both sources require greater expense and
provide lower accessibility to technically underdeveloped nations.
Furthermore, while certain bacterial antigens may be expressed in
transgenic plants, until the work of the present inventors it was
unknown whether antigens associated with human or animal viruses
could be expressed in a form physically and antigenically similar
to antigens used in commercial vaccines derived from human serum
or recombinant yeasts. Similarly, while it is now possible to produce
such recombinant antigens in tobacco plants by virtue of the present
inventors work no such antigens have been produced in plants routinely
included in human and animal diets. In particular, prior art approaches
have filed to provide such commercially viable antigen from plants
made to express transgenic hepatitis B viral antigens. Viral antigens,
anti-viral vaccines and transgenic plants expressing the same as
well as methods of making and using such compositions of matter
are needed which provide inexpensive and highly accessible sources
of such medicines in common diet plants of man and animal.
SUMMARY OF THE INVENTION
Recombinant viral antigens, anti-viral vaccines and transgenic
plants expressing the same are provided by the present invention.
These compositions of matter are demonstrated by the present invention
to be made and used by the methods of the invention in a manner
which is potentially less expensive as well as more accessible to
lower technological societies which rely chiefly on agricultural
methods to provide essential raw materials.
More particularly, the present invention overcomes at least some
of the disadvantages of the prior art by providing antigens produced
in edible transgenic plants which antigens are antigenically and
physically similar to those currently used in the manufacture of
anti-viral vaccines derived from human serum or recombinant yeasts.
In a preferred embodiment, these compositions of matter and methods
provide transgenic plants, recombinant viral antigens and anti-viral
vaccines related to the causative agent of human and animal viral
diseases. The diseases of particular interest are those diseases
in which the virus possesses an antigen capable, in at least the
native state of the virus, of eliciting immune responses, particularly
mucosal immune responses. In an embodiment of preference, the pathogen
from which the antigen is derived is the hepatitis pathogen, and
in plants which are routinely included in human and animal diets.
In one embodiment, the compositions of matter and methods of the
invention relate to oral vaccines introduced by consumption of a
transgenic plant-derived antiviral vaccine. Such a plant derived
vaccine may take various forms including purified and partially
purified plant derived viral antigen as well as whole plant, whole
plant parts such as fruits, leaves, stems, tubers as well as crude
extracts of the plant or plant parts. In general, the preferred
state of the composition of matter which is used to induce an immune
response (i.e., whole plant, plant part, crude plant exact, partially
purified antigen or extensively purified antigen) will depend upon
the ability of the immunogen to elicit a mucosal response, the dosage
level of the plant derived antigen required to elicit a mucosal
response, and the need to overcome interference of mucosal immunity
by other substances in the chosen composition of matter (i.e., sugars,
pyrogens, toxins).
The present invention overcomes the deficiencies of the prior art
by producing oral vaccines in one or more tissues of a transgenic
plant, thereby availing large human and animal populations of an
inexpensive means of vaccine production and administration. In a
preferred embodiment the edible fruit, juice, grain, leaves, tubers,
stems, seeds, roots or other plant parts of the vaccine producing
transgenic plant is ingested by a human or an animal thus providing
a very inexpensive means of immunization against disease. In a preferred
embodiment, such plants will be plants routinely included in human
and animal diets. Purification expense and adverse reactions inherent
in existent vaccine production are thereby avoided. The invention
also provides a novel and inexpensive source of antigen for more
traditional vaccine delivery modes. These and other aspects of the
present invention will become apparent from the following description
and drawings.
In one embodiment, the oral vaccine of the present invention is produced
in edible transgenic plants and then administered through the consumption
of a part of those edible plants. A DNA sequence encoding the expression
of a surface antigen of a pathogen is isolated and ligated into
a plasmid vector containing selection markers. A promoter which
regulates the production of the surface antigen in the transgenic
plant is included in the same plasmid vector upstream from the surface
antigen gene to ensure that the surface antigen is expressed in
desired tissues of the plant. Preferably, the foreign gene is expressed
in a portion of the plant that is edible by humans or animals. For
some uses, such as with human infants, it is preferred that the
edible food be a juice from the transgenic plant which can be taken
orally.
In another embodiment, the vaccines (oral and otherwise) are provided
by deriving recombinant viral antigens from the transgenic plants
of the invention in at least a semi-purified form prior to inclusion
into a vaccine. The present invention produces vaccines inexpensively.
Further, vaccines from transgenic plants can not only be produced
in the increased quantity required for oral vaccines but can be
administered orally, thereby also reducing cost. The production
of an oral vaccine in edible transgenic plants may avoid much of
the time and expense required for FDA approval and regulation relating
to the purification of the vaccine.
A principal advantage of the present invention is the humanitarian
good which can be achieved through the production of inexpensive
oral vaccines which can be used to vaccinate the populations of
lesser developed countries who otherwise could not afford expensive
oral vaccines manufactured under present methods or vaccines which
require parenteral administration.
Thus, the invention provides for a recombinant mammalian viral
protein expressed in a plant cell, which protein is known to elicit
an antigenic response in a mammal in at least the native state of
the virus. Preferably, the recombinant viral protein of the invention
will also be one which is known to function as an antigen or immunogen
(used interchangeably herein) as a recombinant protein when expressed
in standard pharmaceutical expression systems such as yeasts or
bacteria or where the viral protein is recovered from mammalian
sera and shown to be antigenic. More preferably still, the antigenic/immunogenic
protein of the invention will be a protein known to be antigenic/immunogenic
when the protein as derived from the native virus, mammalian sera
or from standard pharmaceutical expression systems, is used to induce
the immune response through an oral mode of introduction. In its
most preferred embodiment, the recombinant mammalian viral protein,
known to be antigenic in its native state, will be a protein which
upon expression in the plant cells of the invention, retains at
least some portion of the antigenicity it possesses in the native
state or as recombinantly expressed in standard pharmaceutical expression
systems.
The immunogen of the invention is one derived from a mammalian virus
and which is then expressed in a plant. In certain preferred embodiments,
the mammalian virus from which the antigen is derived will be a
pathogenic virus of the mammal. Thus, it is anticipated that some
of the most useful plant-expressed viral immunogens will be those
derived from a pathogenic virus of a mammal such as a human.
The immunogens of the invention are preferably produced in plants
where at least a portion of the plant is edible. For the purposes
of this invention, an edible plant or portion thereof is one which
is not toxic when ingested by the mammal to be treated with the
vaccine produced in the plant. Thus, for instance, many of the common
food plants will be of particular utility when used in the compositions
and methods of the invention. However, no nutritive value need be
obtained when ingesting the plants of the invention in order for
such a plant to be included within the types of the plants covered
by the claimed invention. Moreover, in some cases, for instance
in the domestic potato, a plant may still be considered edible as
used herein, although some tissues of the plant, but not the entire
plant, may be toxic when ingested (i.e., while potato tubers are
not toxic and thus falling within the definitions of the claimed
invention, the fruit of the potato is toxic when ingested). In such
cases, such plants are still included within the definition of the
claimed invention.
The immunogen of the invention, in a preferred embodiment, is a
mucosal immunogen. For the purposes of the invention, a mucosal
immunogen is an immunogen which has the ability to specifically
prime the mucosal immune system. In a more highly preferred embodiment,
the mucosal immunogens of the invention are those mucosal immunogens
which prime the mucosal immune system and/or stimulate the humoral
immune response in a dose-dependent manner, without inducing systemic
tolerance and without the need for excessive doses of antigen. Systemic
tolerance is defined herein as a phenomenon occurring with certain
antigens which are repeatedly fed to a mammal resulting in a specifically
diminished subsequent anti-antigen response. Of course, while the
immunogens of the invention when used to induce a mucosal response
may also induce a systemic tolerance, the same immunogen when introduced
parenterally will typically retain its immunogenicity without developing
tolerance.
A mucosal response to the immunogens of the invention is understood
to include any response generated when the immunogen interacts with
a mammalian mucosal membrane. Typically, such membranes will be
contacted with the immunogens of the invention through feeding of
the immunogen orally to a subject mammal. Using this route of introduction
of the immunogen to the mucosal membranes provides access to the
small intestine M cells which overlie the Peyer's Patches and other
lymphoid clusters of the gut-associated lymphoid tissue (GALT).
However, any mucosal membrane accessible for contact with the immunogens
of the invention is specifically included within the definition
of such membranes (e.g., mucosal membranes of the air passages accessible
by inhaling, mucosal membranes of the terminal portions of the large
intestine accessible by suppository, etc.).
Thus, the immunogens of the invention may be used to induce both
mucosal as well as humoral responses. Where the immunogens of the
invention are subjected to adequate levels of purification as further
described herein, these immunogens may be introduced parenterally
such as by muscular injection. Similarly, while preferred embodiments
of the invention include feeding of relatively unpurified immunogen
preparations (e.g., portions of edible plants, purees of such portions
of plants, etc.), the introduction of the immunogen to stimulate
the mucosal response may equally well occur through first subjecting
the plant source of the immunogen to various purification procedures
detailed herein or incorporated specifically by reference herein
followed by introduction of such a purified immunogen through any
of the modes discussed above for accessing the mucosal membranes.
The recombinant immunogens of the invention may represent the entire
amino acid sequence of the native immunogen of the virus from which
it is derived. However, in certain embodiments of the invention,
the recombinant immunogen may represent only a portion of the native
molecule's sequence. In either case, the immunogen may be fused
to another peptide, polypeptide or protein to form a chimeric protein.
The fusion of the molecules is accomplished either post-translationally
through covalent bonding of one to another (e.g., covalent bonding
of plant produced hepatitis B viral immunogen with whole hen egg
lysozyme) or pre-translationally using recombinant DNA techniques
(see e.g., supra discussion of poli virus vaccines), both of which
methods are known well to those of skill in the art.
In certain embodiments, the immunogen of the invention will be
an immunogen derived from a hepatitis virus. In particular embodiments,
the hepatitis B virus surface antigen will be selected. Thus, in
a highly preferred embodiment, a viral mucosal immunogen derived
from a hepatitis virus is recombinantly expressed in a plant and
is capable, in the native state of the virus or as a recombinant
protein expressed in any standard pharmaceutical expression system,
of eliciting an immune response, particularly a mucosal immune response.
In other embodiments of the invention, a transgenic plant comprising
a plant expressing a recombinant viral immunogen derived from a
mammalian virus is provided. For purposes of the invention, a transgenic
plant is a plant expressing in at least some of the cells of the
plant a recombinant viral immunogen. The transgenic plant of the
invention, in preferred embodiments, is an edible plant, where the
immunogen is a mucosal immunogen, or more preferably where a mucosal
immunogen capable of binding a glycosylated molecule on the surface
of a membrane of a mucosal cell, and in some embodiments where the
immunogen is a chimeric protein.
In other preferred embodiments, the transgenic plant of the invention
will be a transgenic plant expressing a recombinant viral mucosal
immunogen of hepatitis virus, where the mucosal immunogen is capable
of eliciting an immune response, particularly a mucosal immune response,
in the native state of the virus or as derived from standard pharmaceutical
expression systems.
Also claimed herein are compositions of matter known as vaccines,
where such vaccines are vaccines comprising a recombinant viral
immunogen expressed in a plant. For the purposes of the invention,
a vaccine is a composition of matter which, when contacted with
a mammal, is capable of eliciting an immune response. As described
above, certain preferred vaccines of the invention will be those
vaccines useful against mammalian viruses as a mucosal immunogen,
and more preferably as vaccines wherein the mucosal immunogen is
capable of binding a glycosylated molecule on the surface of a membrane
of a mucosal cell. In some embodiments, the vaccine may comprise
a chimeric protein immunogen. In other embodiments, the vaccine
of the invention will comprise an immunogen derived from a hepatitis
virus. In still other preferred embodiments, the vaccine of the
invention will comprise a mucosal immunogen of hepatitis virus expressed
in a plant, where the mucosal immunogen is capable of eliciting
an immune response, particularly a mucosal immune response, in the
native state of the virus or as derived from standard pharmaceutical
expression systems.
A food composition is also provided by the invention which comprises
at least a portion of a transgenic plant capable of being ingested
for its nutritional value, said plant comprising a plant expressing
a recombinant viral immunogen. For the purposes of the invention,
a plant or portion thereof is considered to have nutritional value
when it provides a source of metabolizable energy, supplementary
or necessary vitamins or co-factors, roughage or otherwise beneficial
effect upon ingestion by the subject mammal. Thus, where the mammal
to be treated with the food is an herbivore capable of bacterial-aided
digestion of cellulose, such a food might be represented by a transgenic
monocot grass. Similarly, although transgenic lettuce plants do
not substantially contribute energy sources, building block molecules
such as proteins, carbohydrates or fats, nor other necessary or
supplemental vitamins or cofactors, a lettuce plant transgenic for
the viral immunogen of a mammalian virus used as a food for that
mammal would fall under the definition of a food as used herein
if the ingestion of the lettuce contributed roughage to the benefit
of the mammal, even if the mammal could not digest the cellulosic
content of lettuce.
As described in the compositions of matter recited above, certain
preferred foods of the invention will include foods where the immunogen
is a mucosal immunogen, or where mucosal immunogen is capable of
binding a glycosylated molecule on the surface of a membrane of
a mucosal cell, or where the immunogen is a chimeric protein or
where, the immunogen is an immunogen derived from a hepatitis virus.
Thus, in a highly preferred embodiment, the food of the claimed
invention will comprise at least a portion of a transgenic plant
capable of being ingested for its nutritional value, where the plant
expresses a recombinant viral mucosal immunogen of hepatitis virus,
and where the mucosal immunogen is capable of binding a glycosylated
molecule on a surface of a membrane of a mucosal cell. In any case,
the foods of the invention may be those portions of a plant including
the fruit, leaves, stems, roots, or seeds of said plant.
Of particular importance to the compositions and methods of the claimed
invention are certain plasmid constructions useful in obtaining
the plants, immunogens, vaccines, and foods of the invention. Thus,
plasmid vectors for transforming a plant are claimed comprising
a DNA sequence encoding a mammalian viral immunogen and a plant-functional
promoter operably linked to the DNA sequence capable of directing
the expression of the immunogen in said plant. In certain embodiments,
the plasmid vector further comprises a selectable or scorable marker
gene to facilitate the detection of the transformed cell or plant.
In certain embodiments, plasmid vector of the invention will comprise
the plant promoter of cauliflower mosaic virus, CaMV35S. As with
other compositions of matter described above, certain preferred
embodiments of the plasmid vector of the invention will be those
where the plant transformed by the plasmid vector is edible, or
where the immunogen encoded by the plasmid vector is a mucosal immunogen,
or more preferably where the immunogen encoded by the plasmid vector
is capable of eliciting an immune response, particularly a mucosal
immune response, in the native state of the virus or as derived
from standard pharmaceutical expression systems, or where the encoded
immunogen is a chimeric protein, or where the encoded immunogen
is an immunogen derived from a hepatitis virus. Thus, in a highly
preferred embodiment, the plasmid vector of the invention useful
for transforming a plant comprises a DNA sequence encoding a mucosal
immunogen of hepatitis virus, where the mucosal immunogen is capable
of eliciting an immune response, particularly a mucosal immune response,
in the native state of the virus or as derived from standard pharmaceutical
expression systems and where a plant-functional promoter is operably
linked to the DNA sequence capable of directing the expression of
the immunogen in the plant. In a very similar embodiment, the invention
provides for DNA fragments useful for microparticle bombardment
transformation of a plant.
Methods for constructing tnansgenic plant cells are also provided
by the invention comprising the steps of constructing a plasmid
vector or a DNA fragment by operably linking a DNA sequence encoding
a viral immunogen to a plant-functional promoter capable of directing
the expression of the immunogen in the plant and then transforming
a plant cell with the plasmid vector or DNA fragment. Where preferred,
the method may be extended to produce transgenic plants from the
transformed cells by including a step of regenerating a transgenic
plant from the transgenic plant cell.
A method for producing a vaccine is also provided by the claimed
invention, comprising the steps of constructing a plasmid vector
or a DNA fragment by operably linking a DNA sequence encoding a
viral immunogen to a plant-functional promoter capable of directing
the expression of the immunogen in the plant, transforming a plant
cell with the plasmid vector or DNA fragment, and then recovering
the immunogen expressed in the plant cell for use as a vaccine.
Again, where preferred, the method provides for an additional step
prior to recovering the immunogen for use as a vaccine, of regenerating
a transgenic plant from the transgenic plant cell.
The recovery of the immunogen from the plant cell or whole plant
may take several embodiments. In one such embodiment, the method
of recovering the immunogen of the invention is accomplished by
obtaining an extract of the plant cell or whole plant or portions
thereof. In embodiments where whole plants are regenerated by the
methods of the invention, the recovery step may comprise merely
harvesting at least a portion of the transgenic plant.
The methods of the invention provide for any of a number of transformation
protocols in order to transform the plant cells and plants of the
invention. While certain preferred embodiments described below utilize
particular transformation protocols, it will be understood by those
of skill in the art that any transformation method may be utilized
with in the definitions and scope of the invention. Such methods
include microinjection, polyethylene glycol mediated uptake, and
electroporation. Thus, certain preferred methods will utilize an
Agrobacterium transformation system, in particular, where the Agrobacterium
system is an Agrobacterium tumefaciens-Ti plasmid system. In other
preferred methods, the plant cell is transformed utilizing a microparticle
bombardment transformation system.
Plants of particular interest in the methods of the invention include
tomato plants and tobacco plants as will be described in more detail
in the examples to follow. However, it will be understood by those
of skill in the art of plant transformation that a wide variety
of plant species are amenable to the methods of the invention. All
such species are included within the definitions of the claimed
invention including both dicotyledon as well as monocotyledon plants.
As will be described in greater detail in the examples to follow,
the methods of the invention by which plants are transformed may
utilize plasmid vectors which are binary vectors. In other embodiments,
the methods of the invention may utilize plasmids which are integrative
vectors. In a highly preferred embodiment, the methods of the invention
will utilize the plasmid vector pB121.
Methods of administering any of the vaccines of the invention are
also provided. In certain general embodiments, such methods comprise
administering a therapeutic amount of the vaccine to a mammal. In
more specific embodiments, these methods entail introduction of
the vaccine either parenterally or non-parenterally into a mammalian
subject. Where a non-parenteral introduction mode is selected, certain
preferred embodiments will comprise oral introduction of the vaccine
into said mammal. Whichever mode of introduction of the vaccine
to the mammalian subject is selected, it will be understood by those
skilled in the art of vaccination that the selected mode must achieve
vaccination at the lowest dose possible in a dose-dependent manner
and by so doing elicit serun and/or secretory antibodies against
the immunogen of the vaccine with minimal induction of systemic
tolerance. Where a mucosal route of vaccination is selected, care
should be taken to introduce the vaccine into the gut lumen of the
mammal at low dosages and in forms which minimize the simultaneous
introduction of interfering compounds such as galactose and galactose-like
saccharides.
In preferred embodiments, methods are provided by the invention
of administering an edible portion of a transgenic plant, which
transgenic plant expresses a recombinant viral immunogen, to a mammal
as an oral vaccine against a virus from which said immunogen is
derived. These methods comprise harvesting at least an edible portion
of the transgenic plant, and feeding the harvested plant or portion
thereof to a mammal in a suitable amount to be therapeutically effective
as an oral vaccine in the mammal.
Similarly, the invention provides for methods of producing and
administering an oral vaccine, comprising the steps of constructing
a plasmid vector or DNA fragment by operably linking a DNA sequence
encoding a viral immunogen to a plant-functional promoter capable
of directing the expression of the immunogen in a plant, transferring
the plasmid vector into a plant cell, regenerating a transgenic
plant from the cell, harvesting an edible portion of the regenerated
transgenic plants, and feeding the edible portion of the plant to
a mammal in a suitable amount to be therapeutically effective as
an oral vaccine. It is this embodiment that will be of particular
utility in underdeveloped countries committed to agricultural raw
products as a main source of most necessities.
Other objects and advantages of the invention will appear from
the following description.
BRIEF DESCRIPTION OF THE DRAWINGS
For a detailed description of the preferred embodiment of the invention,
reference will now be made to the accompanying drawings wherein:
FIG. 1 is a diagrammatic plasmid construct illustrating the construction
of the plasmid vector pHVA-1 containing the HBsAg gene for producing
the HBsAg antigen in a plant; and
FIG. 2 is a map of the coding sequence for two structural genes
and their regulatory elements in the plasmid pHVA-1; and
FIG. 3 is a diagrammatic plasmid construct illustrating the construction
of the plasmid vector pHB101 containing the HBsAg gene for producing
the HBsAg antigen in a plant; and
FIG. 4 is a diagrammatic plasmid construct illustrating the construction
of the plasmid vector pHB102 containing the HBsAg gene for producing
the HBsAg antigen in a plant; and
FIG. 5 is a map of the coding sequence for three structural genes
and their regulatory elements in the plasmids pHB101 and pHB102;
and
FIG. 6A indicates the HBsAg mRNA levels in transgenic tobacco plants;
and FIG. 6B indicates the HBsAg protein levels in transgenic tobacco
plants; and
FIGS. 7A-7B is a micrograph of immunoaffinity purified rHBsAg with
a corresponding histogram; and
FIG. 8 is a sucrose density gradient sedimentation of HBsAg from
transgenic tobacco; and
FIG. 9 is a buoyant density gradient sedimentation of HBsAg from
transgenic tobacco.
FIGS. 10A-10B is an RNA blot of transformed tomato leaf.
FIG. 11 is a tissue blot of tomato leaves.
DESCRIPTION OF THE PREFERRED EMBODIMENT
The present invention has several components which include: using
recombinant DNA techniques to create a plasmid vector which contains
a DNA segment encoding one or more antigenic proteins which confer
immunity in a human or an animal to a particular disease and for
the expression of antigenic protein(s) in desired tissues of a plant;
selecting an appropriate host plant to receive the DNA segment encoding
antigenic protein(s) and subsequently produce the antigenic protein(s);
transferring the DNA segment encoding the antigenic protein(s) from
the plasmid vector into the selected host plant; regenerating the
transgenic plant thereby producing plants expressing the antigenic
protein(s) which functions as a vaccine(s); and administering an
edible part of the transgenic plant containing the antigenic protein(s)
as an oral vaccine to either a human or an animal by the consumption
of a tnansgenic plant part. The present invention thereby provides
for the production of a transgenic plant which when consumed as
food, at least in part, by a human or an animal causes an immune
response. This response is characterized by resistance to a particular
disease or diseases. The response is the result of the production
in the transgenic plant of antigenic protein(s). The production
of the antigenic protein(s) is the result of stable genetic integration
into the transgenic plant of DNA regions designed to cause regulated
expression of antigenic protein(s) in the transgenic plants.
Vaccine(s) and Their Administration
The present invention may be used to produce any type vaccine effective
in immunizing humans and animals against diseases. Viruses, bacteria,
fungi, and parasites that cause disease in humans and animals can
contain antigenic protein(s) which can confer immunity in a human
or an animal to the causative pathogen. A DNA sequence encoding
any of these viral, bacterial, fungal or parasitic antigenic proteins
may be used in the present invention.
Mutant and variant forms of the DNA sequences encoding a antigenic
protein which confers immunity to a particular virus, bacteria,
fungus or parasite in an animal (including humans) may also be utilized
in this invention. For example, expression vectors may contain DNA
coding sequences which are altered so as to change one or more amino
acid residues in the antigenic protein expressed in the plant, thereby
altering the antigenicity of the expressed protein. Expression vectors
containing a DNA sequence encoding only a portion of an antigenic
protein as either a smaller peptide or as a component of a new chimeric
fusion protein are also included in this invention.
The present invention is advantageously used to produce viral vaccines
for humans and animals. The following table sets forth a list of
vaccines now used for the prevention of viral diseases in humans.
Disease Source of Vaccine |
Condition of Virus |
Route of Administration |
Poliomyelitis |
Tissue culture Live attenuated (human diploid cell Killed
line, monkey kidney) |
Oral Subcutaneous |
Measles |
Tissue culture Live attenuated (chick embryo) |
Subcutaneous |
Mumps |
Tissue culture Live attenuated (chick embryo) |
Subcutaneous |
Rubella |
Tissue culture Live attenuated (duck embryo, rabbit, or human
diploid) |
Subcutaneous |
Smallpox |
ymph from calf or Live vaccinia sheep |
Intradermal |
Yellow |
Tissue cultures and eggs Live attenuated |
Subcutaneous |
Fever Viral |
Purified HBsAg from Live attenuated |
Subcutaneous |
hepatitis B |
"health" carriers Recombinant HBsAg Subunit |
Subcutaneous |
Influenza |
Highly purified Killed or subviral forms (chick embryo) |
Subcutaneous |
Rabies |
Human diploid Killed cell cultures |
Subcutaneous |
Adenoviral |
Human diploid Live attenuated infections cell cultures |
Oral |
Japanese B |
Tissue culture Killed encephalitis (hamster kidney) |
Subcutaneous |
Varicella |
Human diploid Live attenuated cell cultures |
Subcutaneous |
The present invention is also advantageously used to produce vaccines
for animals. Vaccines are available to immunize pets and production
animals. Diseases such as: canine distemper, rabies, canine hepatitis,
parvovirus, and feline leukemia may be controlled with proper immunization
of pets. Viral vaccines for diseases such as: Newcastle, Rinderpest,
hog cholera, blue tongue and foot-mouth can control disease outbreaks
in production animal populations, thereby avoiding large economic
losses from disease deaths. Prevention of bacterial diseases in
production animals such as: brucellosis, fowl cholera, anthrax and
black leg through the use of vaccines has existed for many years.
Today new recombinant DNA vaccines, e.g. rabies and foot and mouth,
have been successfully produced in bacteria and yeast cells and
can facilitate the production of a purified vaccine containing only
the immunizing antigen. Veterinary vaccines utilizng cloned antigens
for protozoans and helminths promise relief from parasitic infections
which cripple and kill.
The oral vaccine produced by the present invention is administered
by the consumption of the foodstuff which has been produced from
the transgenic plant producing the antigenic protein as the vaccine.
The edible part of the plant is used as a dietary component while
the vaccine is administered in the process.
The present invention allows for the production of not only a single
vaccine in an edible plant but for a plurality of vaccines into
one foodstuff. DNA sequences of multiple antigenic proteins can
be included in the expression vector used for plant transformation,
thereby causing the expression of multiple antigenic amino acid
sequences in one transgenic plant. Alternatively, a plant may be
sequentially or simultaneously transformed with a series of expression
vectors, each of which contains DNA segments encoding one or more
antigenic proteins. For example, there are five or six different
types of influenza, each requiring a different vaccine. A transgenic
plant expressing multiple antigenic protein sequences can simultaneously
elicit an immune response to more than one of these strains, thereby
giving disease immunity even though the most prevalent strain is
not known in advance.
Vaccines produced in accordance with the present invention may
also be incorporated into the feed of animals. This represents an
important means to produce lower cost disease prevention for pets,
production animals, and wild species.
While the vaccines of the present invention be preferably utilized
directly as oral vaccines of the transgenic plant material, immunogenic
compositions derived from the transgenic plant materials suitable
for use as more traditional immune vaccines may be readily prepared
from the transgenic plant materials described herein. Preferably,
such immune compositions will comprise a material purified from
the transgenic plant. Purification of the antigen may take many
forms known well to those of skill in the art, in particular such
purifications will likely track closely the purification techniques
used successfully in obtaining viral antigen particles from recombinant
yeasts (i.e., those containing HBsAg). In one embodiment, detailed
in the examples to follow, HBsAg viral protein-containing particles,
similar in many respects to those obtained from recombinant yeasts,
were purified from transformed tobacco plants using a particular
purification procedure. Whatever initial purification scheme is
utilized, the purified material will also be extensively dialyzed
to remove undesired small molecular weight molecules (i.e., sugars,
pyrogens) and/or lyophilization of the thus purified material for
more ready formulation into a desired vehicle.
The preparation of vaccines is generally well understood in the
art (e.g., those derived from fermentative yeast cells known well
in the art of vaccine manufacture cite to Valenzuela et al Nature
298, 347-350 (1982), as exemplified by U.S. Pat. Nos.4,608,251;
4,601,903; 4,599,231; 4,599,230; 4,596,792; and 4,578,770, all incorporated
herein by reference. Typically, such vaccines are prepared as injectables,
either as liquid solutions or suspensions. Solid forms suitable
for solution in, or suspension in, liquid prior to injection may
also be prepared.
The preparation may also be emulsified. The active immunogenic ingredient
is often mixed with excipients which are pharmaceutically acceptable
and compatible with the active ingredient. Suitable excipients are,
for example, water, saline, dextrose, glycerol, ethanol, or the like
and combinations thereof. In addition, if desired, the vaccine may
contain minor amounts of auxiliary substances such as wetting or emulsifying
agents, pH buffering agents, or adjuvants which enhance the effectiveness
of the vaccines.
The vaccines are conventionally administered parenterally, by injection,
for example, either subcutaneously or intramuscularly. Additional
formulations which are suitable for other modes of administration
include suppositories and, in some cases, oral formulations or aerosols.
For suppositories, traditional binders and carriers may include,
for example, polyalkalene glycols or triglycerides: such suppositories
may be formed from mixtures containing the active ingredient in
the range of 0.5% to 10%, preferably 1-2%. Oral formulations other
than edible plant portions described in detail herein include such
normally employed excipients as, for example, pharmaceutical grades
of mannitol, lactose, starch, magnesium stearate, sodium saccharine,
cellulose, magnesium carbonate and the like. These compositions
take the form of solutions, suspensions, tablets, pills, capsules,
sustained release formulations or powders and contain 10-95% of
active ingredient, preferably 25-70%.
In many instances, it will be desirable to have multiple administrations
of the vaccine, usually not exceeding six vaccinations, more usually
not exceeding four vaccinations and preferably one or more, usually
at least about three vaccinations. The vaccinations will normally
be at from two to twelve week intervals, more usually from three
to five week intervals. Periodic boosters at intervals of 1-5 years,
usually three years, will be desirable to maintain protective levels
of the antibodies.
The course of the immunization may be followed by assays for antibodies
for the supernatant antigens. The assays may be performed by labeling
with conventional labels, such as radionuclides, enzymes, fluorescers,
and the like. These techniques are well known and may be found in
a wide variety of patents, such as U.S. Pat. Nos. 3,791,932; 4,174,384
and 3,949,064, as illustrative of these types of assays.
Host Plant Selection
A variety of plant species have been genetically transformed with
foreign DNA, using several different gene insertive techniques..sup.10,22-27,29-32
Since important progress is being made to clone DNA coding regions
for vaccine antigens for parasitic tropical diseases and veterinary
parasitic diseases.sup.11-21 the present invention, will have important
means of low cost production of vaccines in a form easily used for
animal treatment.
Since many edible plants used by humans for food or as components
of animal feed are dicotyledenous plants, it is preferred to employ
dicotyledons in the present invention, although monocotyledon transformation
is also applicable especially in the production of certain grains
useful for animal feed.
The host plant selected for genetic transformation preferably has
edible tissue in which the antigenic protein, a proteinaceous substance,
can be expressed. Thus, the antigenic protein is expressed in a
part of the plant, such as the fruit, leaves, stems, seeds, or roots,
which may be consumed by a human or an animal for which the vaccine
is intended. Although not preferred, a vaccine may be produced in
a nonedible plant and administered by one of various other known
methods of administering vaccines.
Various other considerations are made in selecting the host plant.
It is sometimes preferred that the edible tissue of the host plant
not require heating prior to consumption since the heating may reduce
the effectiveness of the vaccine for animal or human use. Also,
since certain vaccines are most effective when administered in the
human or animal infancy period, it is sometimes preferred that the
host plant express the antigenic protein which will function as
a vaccine in the form of a drinkable liquid.
Plants which are suitable for the practice of the present invention
include any dicotyledon and monocotyledon which is edible in part
or in whole by a human or an animal such as, but not limited to,
carrot, potato, apple, soybean, rice, corn, berries such as strawberries
and raspberries, banana and other such edible varieties. It is particularly
advantageous in certain disease prevention for human infants to
produce a vaccine in a juice for ease of administration to humans
such as tomato juice, soy bean milk carrot juice, or a juice made
from a variety of berry types. Other foodstuffs for easy consumption
might include dried fruit.
Methods of Gene Transfer into Plants
There are various methods of introducing foreign genes into both
monocotyledenous and dicotyledenous plants..sup.33,34 The principle
methods of causing stable integration of exogenous DNA into plant
genomic DNA include the following approaches: 1) Agrobacterium--mediated
gene transfer,.sup.35,36,37,53 2) direct DNA uptake,.sup.38 including
methods for direct uptake of DNA into protoplasts,.sup.8 DNA uptake
induced by brief electric shock of plant cells,.sup.41,42 DNA injection
into plant cells or tissues by particle bombardment,.sup.39,44-45
by the use of micropipette systems,.sup.43,47,48 or by the direct
incubation of DNA with germinating pollen;.sup.40,49 or 3) the use
of plant virus as gene vectors..sup.33,51
The Agrobacterium system includes the use of plasmid vectors that
contain defined DNA segments that integrate into the plant genomic
DNA. Methods of inoculation of the plant tissue vary depending upon
the plant species and the Agrobacterium delivery system. A widely
used approach is the leaf disc procedure which can be performed
with any tissue explant that provides a good source for initiation
of whole plant differentiation..sup.6 The Agrobacterium system is
especially viable in the creation of transgenic dicotyledenous plants.
As listed above there are various methods of direct DNA transfer
into plant cells. In electroporation, the protoplasts are briefly
exposed to a strong electric field. In microinjection, the DNA is
mechanically injected directly into the cells using very small micropipettes.
In microparticle bombardment, the DNA is adsorbed on microprojectiles
such as magnesium sulfate crystals or tungsten particles, and the
microprojectiles are physically accelerated into cells or plant
tissues.
The last principle method of vector transfer is the transmission
of genetic material using modified plant viruses. DNA of interest
is integrated into DNA viruses, and these viruses are used to infect
plants at wound sites.
In the preferred embodiment of the present invention, the Agrobacterium-Ti
plasmid system is utilized..sup.53 The tumor-inducing (Ti) plasmids
of A. tumefaciens contain a segment of plasmid DNA called transforming
DNA (T-DNA) which is transferred to plant cells where it integrates
into the plant host genome. The construction of the transformation
vector system has two elements. First, a plasmid vector is constructed
which replicates in Escherichia coli (E. coli). This plasmid contains
the DNA encoding the protein of interest (an antigenic protein in
this invention); this DNA is flanked by T-DNA border sequences that
define the points at which the DNA integrates into the plant genome.
Usually a gene encoding a selectable marker (such as a gene encoding
resistance to an antibiotic such as Kanamycin) is also inserted
between the left border (LB) and right border (RB) sequences; the
expression of this gene in transformed plant cells gives a positive
selection method to identify those plants or plant cells which have
an integrated T-DNA region..sup.52,53 The second element of the
process is to transfer the plasmid from E. coli to Agrobacterium.
This can be accomplished via a conjugation mating system, or by
direct uptake of plasmid DNA by Agrobacterium. For subsequent transfer
of the T-DNA to plants, the Agrobacterium strain utilize must contain
a set of inducible virulence (vir) genes which are essential for
T-DNA transfer to plant cells..sup.53,54
Those skilled in the art should recognize that there are multiple
choices of Agrobacterium strains and plasmid construction strategies
that can be used to optimize genetic transformation of plants. They
will also recognize that A. tumefaciens may not be the only Agrobacterium
strain used. Other Agrobacterium strains such as A. rhizogenes might
be more suitable in some applications.
Methods of inoculation of the plant tissue vary depending upon
the plant species and the Agrobacterium delivery system. A very
convenient approach is the leaf disc procedure which can be performed
with any tissue explant that provides a good source for initiation
of whole plant differentiation. The addition of nurse tissue may
be desirable under certain conditions. Other procedures such as
the in vitro transformation of regenerating protoplasts with A.
tumefaciens may be followed to obtain transformed plant cells as
well..sup.33,53
This invention is not limited to the Agrobacterium-Ti plasmid system
but should include any direct physical method of introducing foreign
DNA into the plant cells, transmission of genetic material by modified
plant viruses, and any other method which would accomplish foreign
DNA transfer into the desired plant cells.
Promoters
Once the host plant has been selected and the method of gene transfer
into the plant determined, a constitutive, a developmentally regulated,
or a tissue specific promoter for the host plant is selected so
that the foreign protein is expressed in the desired part(s) of
the plant.
Promoters which are known or found to cause transcription of a
foreign gene in plant cells can be used in the present invention.
Such promoters may be obtained from plants or viruses and include,
but are not necessarily limited to: the 35S promoter of cauliflower
mosaic virus (CaMV) (as used herein, the phrase "CaMV 35S"
promoter includes variations of CaMV 35S promoter, e.g. promoters
derived by means of ligations with operator regions, random or controlled
mutagenesis, etc.); promoters of seed storage protein genes such
as Zma10Kz or Zmag12 (maize zein and glutelin genes, respectively),
light-inducible genes such as ribulose bisphosphate carboxylase
small subunit (rbcS), stress induced genes such as alcohol dehydrogenase
(Adhl), or "housekeeping genes" that express in all cells
(such as Zmaact, a maize actin gene)..sup.4,55 This invention can
utilize promoters for genes which are known to give high expression
in edible plant parts, such as the patatin gene promoter from potato..sup.56
The plasmid constructed for plant transformation also usually contains
a selectable or scorable marker gene. Numerous genes for this purpose
have been identified..sup.54,57
The following are examples of the production of a vaccine for hepatitis
B in a host transgenic tomato and tobacco plant and are presented
to describe a preferred embodiment and the utility of the present
invention but should not be construed as limiting the claims thereof.
The DNA coding sequence for the hepatitis B surface antigen was
selected for expression in a transgenic plant as Hepatitis B virus
is one of the most widespread viral infections of humans which causes
acute and chronic hepatitis and heptocellular carcinoma..sup.71
Tomato and tobacco plants were selected as the host plants to produce
the hepatitis B recombinant surface antigen as examples of antigenic
protein production in different plant parts. Expression of HbsAg
in tobacco and tomato plants was accomplished by the method of Mason,
H. S. Lam, and Arntzen, C. J., Proceedings of the National Academy
of Sciences, U.S.A. Vol. 89, 11745-11749(1992), herein incorporated
by reference.
EXAMPLE I
A. Construction of Hepatitis B Surface Antigen Expression Vector
pHVA-1
Referring initially to the diagrammatic plasmid construct illustrated
in FIG. 1, the DNA sequence encoding for HBsAg contained within
restriction endonuclease sites Pst I-Hind III on plasmid pWR/HBs-3
was excised and subsequently ligated into the unique Bam H-Sst I
site of the excised beta-glucuronidase (GUS) gene on plasmid pB121
to construct the binary vector plasmid pHVA-1.
Plasmid pB 121, obtained from Clonetech Laboratories, Inc., Palo Alto,
Calif., has cleavage sites for the restriction endonucleases Bam HI
and Sst I located between the CaMV 35S promoter and the GUS structural
gene initiation sequence and between the GUS gene termination sequence
and the NOS polyadenylation signals, respectively. Plasmid pB121 was
selected since the GUS structural gene can be excised from the plasmid
using Bam HI and Sst I, another structural gene encoding an antigenic
protein can be inserted, and the new gene will be functionally active
in plant gene expression. Plasmid pB121 also contains a NPT II gene
encoding neomycin phosphotransferase II; this is an enzyme that confers
Kanamycin resistance when expressed in transformed plant cells, thereby
allowing the selection of cells and tissues with integrated T-DNA.
The NPT II gene is flanked by promoter and polyadenylation sequences
from a Nopaline synthase (NOS) gene.
The HBsAg DNA coding sequence.sup.64,65 was isolated from the plasmid
pWR/BBs-3 (constructed at the Institute of Cell Biology in China)
as a Pst I--Hind III fragment. This fragment was digested with Klenow
enzyme to create blunt ends; the resultant fragment was ligated
at the 5' end with Bam H1 linkers and at the 3' end with Sst 1 linkers,
and then inserted into the pB 121 plasmid at the site where the
GUS coding sequence had been excised, thereby creating plasmid pHVA-1
as shown in FIG. 1.
The plasmid vector pHVA-1 then contains 1) a neomycin phosphotransferase
II (NPT II) gene which provides the selectable marker for kanamycin
resistance; 2) a HBsAg gene regulated by a cauliflower mosaic virus
(CaMV 35S) promoter sequence; and 3) right and left T-DNA border
sequences which effectively cause the DNA sequences for the NOS
and HBsAg genes to be transferred to plant cells and integrated
into the plant genome. The diagrammatic structure of pHVA-1 is shown
in FIG. 2.
B. Transfer of Binary Vector, pHVA-1, to A. tumefaciens
Plasmid pHVA-1, containing the HBsAg gene, was transferred to A.
tumefaciens strain LBA4404 obtained from Clontech Laboratories,
Inc. This strain is widely used since it is "disarmed";
that is, it has intact vir genes, but the T-DNA region has been
removed by in vivo deletion techniques. The vir genes work in trans
to mediate T-DNA transfer to plants from the plasmid pHVA-1.
A. tumefaciens was cultured in AB medium.sup.58 containing two-tenths
milligrams per milliliter (0.2 mg/ml) streptomycin until the optical
density (O.D.) at six hundred nanometers (600 nm) of the culture
reaches about five tenths (0.5). The cells are then centrifuged
at 2000 times gravity (2000.times.G) to obtain a bacterial cell
pellet. The Agrobacterium pellet was resuspended in one milliliter
of ice cold twenty millimolar calcium chloride (20 mM CaCI.sub.2).
Five tenths microgram (0.5 .quadrature.g) of plasmid pHVA-1 DNA
was added to two tenths milliliters (0.2 ml) of the calcium chloride
suspension of A. tumefaciens cells in a one and five tenths milliliter
(1.5 ml) microcentrifuge tube and incubated on ice for sixty minutes.
The plasmid pHVA-1 DNA and A. tumefaciens cells mixture was frozen
in liquid nitrogen for one minute, thawed in a twenty-five degree
Celsius (25.quadrature.C) water bath, and then mixed with five volumes
or one milliliter (1 ml) of rich MGL medium..sup.58 The plasmid
pHVA-1 and A. tumefaciens mixture was then incubated at twenty-five
degrees Celsius (25.quadrature.C) for four hours with gentle shaking.
The mixture was plated on LB, luria broth,.sup.58 agar medium containing
fifty micrograms per milliliter (50 .quadrature.g/ml) kanamycin.
Optimum drug concentration may differ depending upon the Agrobacterium
strain in other experiments. The plates were incubated for three
days at twenty-five degrees Celsius (25.quadrature.C) before selection
of resultant colonies which contained the transformed Agrobacterium
harboring the pHAV-1 plasmids.
The presence of pHVA-1 DNA in the transformed Aerobacterium culture
was verified by restriction mapping of the plasmid DNA purified
by alkaline lysis of the bacterial cells..sup.59
Plant Transformation by A. tumefaciens Containing the UBsAg Gene
as Part of the Ti Vector System
The technique for in vitro transformation of plants by the Agrobacterium-Ti
plasmid system is based on cocultivation of plant tissues or cells
and the transformed Agrobacterium for about two days with subsequent
transfer of plant materials to an appropriate selective medium.
The material can be either protoplast, callus or organ tissue, depending
upon the plant species. Organ cocultivation with leaf pieces is
a convenient method.
Leaf disc transformation was performed in accordance with the procedure
of Horsch et al.sup.6. Tomato and tobacco seedlings were grown in
flats under moderate light and temperature and low humidity to produce
uniform, healthy plants of ten to forty centimeters in height. New
flats were started weekly and older plants were discarded. The healthy,
unblemished leaves from the young plants were harvested and sterilized
in bleach solution containing ten per cent (10%) household bleach
(diluted one to ten from the bottle) and one tenth per cent (0.1%)
Tween 20 or other surfactant for fifteen to twenty minutes with
gentle agitation. The leaves were then rinsed three times with sterile
water. The leaf discs were then punched with a sterile paper punch
or cork borer, or cut into small strips or squares to produce a
wounded edge.
Leaf discs were precultured for one to two days upside down on
MS 104.sup.6 medium to allow initial growth and to eliminate those
discs that were damaged during sterilization or handling. Only the
leaf discs which showed viability as evidenced by swelling were
used for subsequent inoculation. The A. tumefaciens containing pHVA-1
which had been grown in AB medium were diluted one to twenty with
MSO.sup.6 for tomato inoculation and one to ten for tobacco discs.
Leaf discs were inoculated by immersion in the diluted transformed
A. tumefaciens culture and cocultured on regeneration medium MS
104.sup.6 medium for three days. Leaf discs were then washed with
sterile water to remove the free A. tumefaciens cells and placed
on fresh MS selection medium which contained three hundred micrograms
per milliliter (300 .quadrature.g/ml) of kanamycin to select for
transformed plants cells and five hundred micrograms per milliliter
(500 .quadrature.g/nml) carbenicillin to kill any remaining A. tumefaciens.
The leaf discs were then transferred to fresh MS selection medium
at two week intervals. As shoots formed at the edge of the leaf
discs and grew large enough for manual manipulation, they were excised
(usually at three to six weeks after cocultivation with transformed
A. tumefaciens) and transferred to a root-inducing medium, e.g.
MS rooting medium..sup.6 As roots appeared the plantlets were either
allowed to continue to grow under sterile tissue culture conditions
or transferred to soil and allowed to grow in a controlled environment
chamber.
D. Selection of Genetically-Engineered Plants Which Express HBsAg
Approximately three months (nine months for tomato fruit assays)
after the initial cocultivation of the putative BBsAg expressing
tomato plants (HB-plants) with A. tumefaciens, they were tested
for the presence of HBsAg.
1. Biochemical and Immunochemical Assays
Root, stem, leaf and fruit samples of the plants were excised.
Each tissue was homogenized in a buffered solution, e.g. one hundred
millimolar sodium phosphate (100 mM), pH 7.4 containing one millimolar
ethylenediamine tetraacetate (1.0 mM EDTA) and five-tenths millimolar
phenylmethylsulfonyl fluoride (0.5 mM PMSF) as a proteinase inhibitor.
The homogenate was centrifuged at five thousand times gravity (5000.times.G)
for ten minutes. A small aliquot of each supernatant was then reserved
for protein determination by the Lowry method. The remaining supernatant
was used for the determination of the level of HBsAg expression
using two standard assays: (a) a HBsAg radioimmunoassay, the reagents
for which were purchased from Abbott Laboratories and (b) immunoblotting
using a previously described method of Peng and Lam.sup.61 with
a monoclonal 10 antibody against anti-HBsAg purchased from Zymed
Laboratories. Depending upon the level of HBsAg expression in each
tissue, the supernatant may have been partially purified using a
previously described affinity chromatographic method of Pershing
et al.sup.62 using monoclonal antibody against HBsAg bound to commercially
available Affi-Gel 10 gel from Bio-Rad Laboratories, Richmond, Calif.
The purified supernatant was then concentrated by lyophilization
or ultrafiltration prior to radioimmunoassay and immunoblotting.
2. Detection of the HBsAg Gene Construct
The stable integration of the HBsAg construct (expression vector)
for plant cell transfection was tested by hybridization assays of
genomic DNA digested with Eco R1, and with a combined mixture of
Bam H1 and Sst 1 in each plant tissue for both control and HBsAg-transfected
plants with a HBsAg coding sequence probe using standard southern
blots.sup.60. In addition, seeds were collected from self-fertlized
plants, and progeny were analyzed by standard Southern analysis.
E. Regeneration of HBsAg Transgenic Tomato Plants
Once the transgenic plant has been perfected, the transgenic plant
is regenerated by growing multiples of the transgenic plant to produce
the oral vaccine. Of course, the most common method of plant propagation
is by seed. Regeneration by seed propagation, however, has the deficiency
that there is a lack of uniformity in the crop. Seeds are produced
by plants according to the genetic variances governed by Mendelian
rules. Basically, each seed is genetically different and each will
grow with its own specific traits. Therefore, it is preferred that
the transgenic plant be produced by homozygous selection such that
the regenerated plant has the identical traits and characteristics
of the parent transgenic plant, e.g. a reproduction of the vaccine.
F. Administration of HBsAg Vaccine to Humans Through Consumption
of Tomato Juice Produced from HBsAg Transgenic Tomatoes
Once the vaccine is produced through the mass regeneration of the
transgenic plant, the crop is harvested and utilized directly as
food or processed into a consumable food. Although the food may
be processed as a solid or liquid, in some cases it is preferred
that it be in liquid form for ease of consumption. The transgenic
tomatoes could be homogenized to produce tomato juice which could
be bottled for drinking. HBsAg vaccine administration is accomplished
by a human drinking the tomato juice or consuming the fruit in a
quantity and time scale (once or multiple doses over a period of
time) to confer immunity to hepatitis B virus infection.
EXAMPLE II
A.1 Construction of Hepatitis B Surface Antigen Expression Vector
pHB101
Referring to the plasmid construct illustrated in FIG. 3, the DNA
sequence encoding for HBsAg contained within restriction endonuclease
sites Pst I-Hind IH on plasmid pMT-SA (provided by Li-he Guo, Chinese
Academy of Sciences) was excised and subsequently ligated into the
unique Bam HI-Sac I site of the excised beta-Glucuronidase (GUS)
gene on plasmid pBI121 to construct the binary plasmid pB 101.
Plasmid pBI121, obtained from Clonetech Laboratories, Inc., Palo
Alto, Calif., has cleavage sites for the restriction endonucleases
Bam HI and Sac I located between the CaMV 35S promoter and the GUS
structural gene initiation sequence and between the GUS gene termination
sequence and the NOS polyadenylation signals, respectively. Plasmid
pBI121 was selected since the GUS structural gene can be excised
from the plasmid using Bam HI and Sac T. another structural gene
encoding an antigenic protein can be inserted, and the new gene
will be functionally active in plant gene expression. Plasmid pBI121
also contains a NPT II gene encoding neomycin phosphotransferase
R and conferring kanamycin resistance. The NPT II gene is flanked
by promoter and polyadenylation sequences from a Nopaline synthase
(NOS) gene.
The HBsAg DNA coding sequence.sup.64,65 (the S gene) was excised
from plasmid pMT-SA (constructed at Chinese Academy of Sciences)
as a Pst I-Hind mH fragment and isolated by electrophoresis in a
one percent (1%) agarose gel. The Pst-Hind mH fragment was visualized
in the agarose gel by staining with ethidium bromide, illuminated
with ultraviolet light (UV) and purified with a Prep-aGene kit (BioRad
Laboratories, Richmond, Calif.). The HBsAg coding region on the
Pst I-Hind III fragment was then ligated into the Pst I-Hind mH
digested plasmid pBluescript KS (Stratagene, La Jolla, Calif.) to
form the plasmid pKS-FBS. The HBsAg gene in plasmid pKS-HBS was
then opened 116 base pairs (bp) 3' to the termination codon with
BstB I and the resulting ends were blunted by filling with Klenow
enzyme and dCTP/dGTP. The entire coding region (820 bp) was then
excised with Bam HI, which is site derived from the plasmid vector
pBluescript. This results in the addition of Bam HI and Sma I sites
5' to the original HBsAg coding sequence from plasmid pMT-SA.
Plasmid pBI121.sup.66, obtained from Clonetech, Laboratories, Inc.,
Palo Alto, Calif., was digested with Sac I and the ends blunted
with mung bean nuclease. The GUS coding region was then released
from pBI121 by treatment with Bam HI and the 11 kilobase pair (kbp)
GUS-less pBI121 plasmid vector isolated. Subsequently, the HBsAg
coding fragment excised from pKS-HB was ligated into the GUS-less
plasmid pBI121 to yield plasmid pHB101 (FIG. 3). Transcription of
the HBsAg gene in this construct is driven by the cauliflower mosaic
virus 35S (CaMV 35S) promoter derived from pBI121, and the polyadenylation
signal is provided by the nopaline synthase terminator.
The plasmid vector pHB101 then contains 1) a neomycin phosphotransferase
II (NPTII) gene which provides the selectable marker for kanamycin
resistance; 2) a HBsAg gene regulated by a cauliflower mosaic virus
(CaMV 35S) promoter sequence; and 3) right and left T-DNA border
sequences which effectively cause the DNA sequences for the NOS
and HBsAg genes to be transferred to plant cells and integrated
into the plant genome. The diagrammatic structure of pHB101 is shown
in FIG. 5.
A.2 Construction of Hepatitis B Surface Antigen Expression Vector
pHB102
Plasmid pHB102, an improved expression vector, was constructed
from plasmid pHB101 by removal of the CaMV 35S promoter and insertion
of a modified 35S promoter linked to a translational enhancer element.
The CaMV 35S promoter in the plasmid pRTL2-GUS.sup.67 contains a
duplication of the upstream regulatory sequences between nucleotides
-340 and -90 relative to the transcription initiation site. Fused
to the 3' end of the promoter is the tobacco etch virus 5' nontranslated
leader sequence (TL), which acts as a translational enhancer in
tobacco cells.
As seen in FIG. 4, the promoter (with dual enhancer) was obtained
from plasmid pRTL2GUS. pRTL2GUS was digested with Nco I and the
ends were blunted with mung bean nuclease. The CaMV 35S with duplicated
enhancer linked to tobacco etch virus (IEV) 5' nontranslated leader
sequence (the promoter-leader fragment) was then released by digestion
with Hind Im, and purified by agarose gel electrophoresis. Plasmid
pHB101 was digested with Hind Im and Sma I to release the CaMV 35S
promoter fragment and the promoterless plasmid vector was purified
by agarose gel electrophoresis. This yielded a blunt end just 5'
to the HbsAg coding sequence for fusion with the blunted Nco I site
at the 3' end of the purified promoter-leader fragment from pRTL2GUS.
Then the promoter-leader fragment from pRTL2GUS was ligated into
the Hind III-Sma I site on promoter-less plasmid pHB101 to yield
plasmid pHB102.
The HBsAg coding region of plasmid pHB102 lies upstream of the
nopaline synthase (NOS) terminator. The plasmid contains the left
and right borders of the T-DNA that is integrated into the plant
genomic DNA via Agrobacterium tumefaciens mediated transformation,
as well as the neomycin phosphotransferase (NPT II) gene which allows
selection with kanamycin. Expression of the HbsAg gene is driven
by the CaMV 35S with dual transcriptional enhancer linked to the
TEV 5' nontranslated leader. The TEV leader acts as a translational
enhancer to increase the amount of protein made using a given amount
of template MRNA..sup.67
B. Transfer of Binary Vectors, pHB101 and pHB102, to A. tumefaciens
Plasmid pHB101, containing the HbsAg gene and the CaMV 35S promoter,
and plasmid pHB102, containing the HBsAg gene and CaMV 35S promoter
with dual transcription enhancer linked to the TEV 5' nontranslated
leader were then separately transferred to Agrobacterium tumefaciens.
Plasmid pHB101 or pHB102, each containing the HBsAg gene, was transferred
to the A. tumefaciens strain LBA4404 obtained from Clonetech Laboratories,
Inc. as in Example I.
A. tumefaciens was cultured in 50 milliliters (50 ml) of YEP (yeast
extract-peptone broth).sup.58 containing two-tenths milligrnms per
milliliter (0.2 mg/mi) streptomycin until the optical density (O.D.)
at 600 nanometers (nm) of the culture reaches about five tenths
(0.5). The cells were then centrifuged at 2000 times gravity (2000.times.G)
to obtain a bacterial cell pellet. The Agrobacterium pellet was
resuspended in ten milliliters of ice cold one hundred fifty millimolar
sodium chloride (150 mM NaCl.sub.2). The cells were then centrifuged
again at 2000.times.G and the resulting Agrobacterium pellet was
resuspended in one milliliter (1 ml) of ice cold twenty millimolar
calcium chloride (20 mM CaCl.sub.2). Five-tenths microgram (0.5
.quadrature.g) of plasmid pHB101 or plasmid pHB102 was added to
two tenths milliliters (0.2 ml) of the calcium chloride suspension
of A. tumefaciens cells in a one and five tenths milliliter (1.5
ml) microcentrifuge tube and incubated on ice for sixty minutes.
The plasmid pHB110 or pHB102 DNA and A. tumefaciens cells mixture
was frozen in liquid nitrogen for one minute, thawed in a twenty-eight
degree Celsius (28.quadrature.C) water bath, and then mixed with
five volumes or 1 milliliter (1 ml) of YEP (yeast extract-peptone
broth). The plasmid pHB101 or pHB102 and A. tumefaciens mixture
was then incubated at twenty-eight degrees Celsius (28.quadrature.C)
for four hours with gentle shaking. The mixture was plated on YEP
(yeast extract-peptone broth) agar medium containing fifty micrograms
per milliliter (50 .quadrature.g/ml) kanamycin. Optimum drug concentration
may differ depending upon the Agrobacterium strain in other experiments.
The plates were incubated for three days at twenty-eight degrees
Celsius (28.quadrature.C) before selection of resultant colonies
which contained the transformed Agrobacterium harboring the pHB101
or the pHB102 plasmids. These colonies were then transferred to
five milliliters (5 ml) of YEP (yeast extract-peptone broth) containing
fifty micrograms per milliliter (50 .quadrature.g/ml) of kanamycin
for three days at twenty-eight degrees Celsius (28.quadrature.C).
The presence of pHB101 or pHB102 DNA in the transformed Agrobacterium
culture was verified by restriction mapping of the plasmid DNA purified
by alkaline lysis of the bacterial cells..sup.59
C. Plant Transformation by A. tumefaciens containing the HBsAg
Gene as Part of the Ti Vector System
Tobacco plants were transformed by the leaf disc method utilizing
Agrobacterium tumefaciens containing either plasmid pHB101 or pHB102
and then the kanamycin resistant transformed tobacco plants were
regenerated.
Leaf disc transformation was performed in accordance with the procedure
of Horsch et al.sup.6. Tobacco seeds (Nicotiana tabacum L. cv Samsun)
were surface sterilized with twenty per cent (20%) household bleach
(diluted one to five from the bottle) for ten minutes and then washed
five times with sterile water. The seeds were sown on sterile MSO.sup.6
medium in GA-7 boxes (Magenta Corporation, Chicago Ill.). The seedlings
were grown under moderate light for four to six weeks, and leaf
tissue was excised with a sterile scalpel and cut into five-tenths
square centimeter (0.5 cm.sup.2) pieces.
The A. tumefaciens containing pHB101 or pHB102 which had been grown
in YEP (east extract-peptone broth) medium were diluted one to ten
with MSO.sup.6 for tobacco leaf pieces. Leaf pieces were inoculated
by immersion in the diluted transformed A. tumefaciens culture and
cocultured on regeneration medium MS 104.sup.6 for two days at twenty-seven
degrees Celsius (27.quadrature.C). Leaf pieces were then washed
with sterile water to remove the free A. tumefaciens cells and placed
on fresh MS selection medium which contained two hundred micrograms
per milliliter (200 .quadrature.g/ml) kanamycin to select for transformed
plant cells and two hundred micrograms per milliliter (200 .quadrature.g/ml)
cefotaxime to inhibit bacterial growth. Leaf pieces were subcultured
every two weeks on fresh MS selection medium until shoots appeared
at the cut edges. As shoots formed at the edge of the leaf pieces
and grew large enough for manual manipulation, they were excised
(usually at three to six weeks after cocultivation with transformed
A. tumefaciens) and transferred to a root-inducing medium, e.g.
MS rooting medium containing one hundred micrograms per milliliter
of kanamycin (100 .quadrature.g/ml). As roots appeared, the plantlets
were either allowed to continue to grow under sterile tissue culture
conditions or transferred to soil and allowed to grow in a controlled
environment chamber.
D. Analysis of RNA from Transformed Tobacco
The regenerated kanamycin-resistant pHB101 and pHB102 transformed
tobacco plants were analyzed by hybridizing RNA samples with a.sup.32
P labelled probe encompassing the HBsAg gene coding region.
Total RNA from the leaves of the p HB101 transformed tobacco plants
was isolated as described Approximately four tenths of a gram (0.4
g) of young growing leaf tissue from a transformed plant was frozen
in liquid nitrogen and ground to a powder with a cold mortar and
pestle. The powder was resuspended in five milliliters (5 ml) of
RNA extraction buffer composed of two hundred millimolar (0.2M)
Tris-HCI, pH 8.6; two hundred millimolar sodium chloride (0.2M NaCI);
twenty millimolar ethylenediaminetetraacetic acid (20 mM EDTA) and
two percent sodium dodecyl sulfate (2% SDS) and immediately extracted
with five milliliters (5 ml) of phenol saturated with ten millimolar
(10 mM) Tris-HCI, pH 8.0 per one millimole ethylenediaminetetraacetic
acid (1 mM EDTA), and five milliliters (5 ml) of chloroform. After
centrifugation at three thousand times gravity (3,000.times.G) to
separate the phases, the upper aqueous layer was removed and made
to three tenths molar (0.3M) potassium acetate, pH 5.2. The nucleic
acids in the extract were precipitated with two and a half (2.5)
volumes of ethanol, pelleted at eight thousand times gravity (8,000.times.G),
dried under reduced pressure, resuspended in one milliliter (1 ml)
of water, and reprecipitated with the addition of one milliliter
(1 ml) of six molar (6M) ammonium acetate and five milliliters (5
ml) of ethanol. The final pellet was dried and resuspended in two
tenths of a milliliter (0.2 ml) of water, and the concentration
of RNA estimated by measuring the absorbance of the samples at 260
nanometers (nm), assuming that a solution of one milligram per milliliter
(1 mg/ml) RNA has an absorbance of twenty-five (25) units.
Five micrograms of each RNA sample was denatured by incubation for
fifteen minutes at sixty-five degrees Celsius (65.quadrature.C)
in twenty millimolar (20 mM) MOPS (3-N-morpholino) propanesulfonic
acid, pH 7.0; ten millimolar (10 mM) sodium acetate; one millimolar
ethylenediaminetetraacetic acid (1 mM EDTA); six and one half percent
(6.5% w/v) formaldehyde; fifty percent (50% v/v) formamide, and
then fractionated by electrophoresis in one percent (1%) agarose
gels. The nucleic acids were transferred to a nylon membrane by
capillary blotting.sup.59 for sixteen hours in twenty-five millimolar
(25 mM) sodium phosphate, pH 6.5. Then the nucleic acids were crosslinked
to the membrane by irradiation with ultraviolet (UV) light and the
membrane pretreated with hybridization buffer twenty-five
hundredths molar (0.25M) sodium phosphate, pH 7.0; one millimolar
ethylene diamine tetraacetic acid (1 mM EDTA); seven percent (7%)
sodium dodecyl sulfate (SDS)! for one hour at sixty-eight degrees
Celsius (68.quadrature.C). The membrane was probed with 10.sup.6
counts per minute per milliliter (cpm/ml) .sup.32 P-labelled random-primed
DNA using a 700 base pair (bp) Bam HI-Acc I fragment from plasmid
pKS-HBS which includes most of the coding region for HBsAg. Blots
were hybridized at sixty-eight degrees Celsius (68.quadrature.C)
in hybridization buffer and washed twice for five hundred and fifteen
minutes with forty millimolar (40 mM) sodium phosphate, pH 7.0 per
one millimolar ethylene diaminetetraacetic acid (1 mM EDTA) per
five percent sodium dodecyl sulfate (5% SDS) at sixty-eight degrees
Celsius (68.quadrature.C) and exposed to X-OMAT AR film for twenty
hours.
The results of the RNA hybridization probe with selected transformants
harboring the plasmid pHB101construct and with a wild-type control
(wt) can be seen in FIG. 6A. The signals were highly variable between
transformants, as expected due to the effects of position of insertion
into the genomic DNA and differing copy number. The transcripts
were about 1.2 kb in length by comparison with the RNA standards,
which was consistent with the expected size. The wild-type control
leaf RNA showed no detectable signal at this stringency of hybridization.
Substantial steady-state levels of MRNA which specifically hybridized
with the HBsAg probe was present in the leaves of selected transformants
which indicated that mRNA stability was not a problem for the expression
of HBsAg in tobacco leaves.
E. Analysis of Protein from Transformed Tobacco Plants
Protein was extracted from transformed tobacco leaf tissues by
homogenization with a Ten-Broek ground glass homogenizer (clearance
0.15 mm) in five volumes of buffer containing twenty millimolar
(20 mM) sodium phosphate, pH 7.0, one hundred fifty millimolar (150
mM) sodium chloride, twenty millimolar (20 mM) sodium ascorbate,
one-tenth percent (0.1%) Triton X-100, and five tenths millimolar
(0.5 mM) PMSF, at four degrees Celsius (4.quadrature.C). The homogenate
was centrifuged at one thousand times gravity (1000.times.G) for
five minutes and the supernatant centrifuged at twenty-seven thousand
times gravity (27,000.times.G) for fifteen minutes. The 27,000.times.G
supernatant was then centrifuged at one hundred thousand times gravity
(100,000.times.G) for one hour and the pellet resuspended in extraction
buffer. The protein in the different fractions was measured by the
Coomassie dye-binding assay (Bio-Rad). HBsAg protein was assayed
by the AUSZYME Monoclonal kit (Abbott Laboratories, Abbott Park,
Ill.) using the positive control, HBsAg derived from human serum,
as the standard. The positive control was diluted to give HBsAg
protein levels of nine hundredths to one and eight tenths nanograms
(0.09-1.8 ng) per assay. After color development, the absorbance
at four hundred ninety-two nanometers (492 nm) was read and a linear
relationship was found. As seen in FIG. 6B, the weld-type control
plant contained no detectable HBsAg protein (Column 1); fairly low
levels of HBsAg protein were observed, ranging from three to ten
nanograms per milligram (3-10 ng/mg) soluble protein for the pHB101
construct (Columns 2 through 6); and from twenty-five to sixty-five
nanograms per milligram (25-65 ng/mg) for the pHB102 construct (Columns
7 through 9). The reaction was specific because the wild-type tobacco
showed no detectable HBsAg protein. HBsAg from human serum and recombinant
HBsAg (rHBsAg) from plasmid-transformed yeast occur as approximately
twenty nanometer (20 nm) spherical particles consisting of protein
embedded in a phospholipid bilayer. Ninety-five percent of the rHBsAg
in the 27,000.times.G supernatants of transgenic tobacco leaf extracts
pelleted at 2000,000.times.G for thirty minutes. This suggested
a particle form. Thus, evidence was sought to ascertain if rHBsAg
in tobacco existed as particles.
F. Immunoaffinity Purification of HBsAg from Transformed Tobacco
Plants
Transformed tobacco leaf extracts were tested for the presence
of material which reacts specifically with monoclonal antibody to
serum-derived HBsAg. Further tests were conducted to determine if
the recombinant HBsAg material in the transformed tobacco leaves
was present as particles and the size range of the particles.
Monoclonal antibody against HBsAg, clone ZMHB1, was obtained from
Zymed Laboratories (South San Francisco, Calif.). The immunogen
source for this antibody is human serum. The monoclonal antibody
was bound to Affi-Gel HZ hydrazide gel (Bio-Rad Laboratories, Richmond,
Calif.) according to the instruction supplied in the kit. The 100,000.times.G
resuspended soluble material was made to five tenths molar (0.5M)
sodium chloride and mixed with the immobilized antibody-gel by end-over-end
mixing for sixteen hours at four degrees Celsius (4.quadrature.C).
The gel was washed with ten volumes of PBS.5 ten millimolar
(10 mM) sodium phosphate, pH 7.0, five tenths molar (0.5M) sodium
chloride! and ten volumes of PBS.15 fifteen hundredths molar
(0.15M) sodium chloride! and bound BBsAg eluted with two tenths
molar (0.2M) glycine, pH 2.5. The eluate was immediately neutral
with Tris-base, and particles pelleted at one hundred and nine thousand
times gravity (109,000.times.G) for one and a half hours at five
degrees Celsius (5.quadrature.C). The pelleted material was negatively
stained with phosphotungstic acid and visualized with transmission
electron microscopy using a Phillips CMIO microscope. The presence
of rHBsAg particles were revealed by negative staining and electron
microscopy, FIG. 7. rHBsAg particles ranged in diameter between
ten and forty nanometers (10-40nm). Most particles were between
sixteen and twenty-eight nanometers (16-28 nm). These are very similar
to the particles observed in human serum,.sup.69 although no rods
were observed. The rHBsAg particles from yeast occur in a range
of sizes with a mean of seventeen nanometers (17 nm)..sup.2 Thus
rHBsAg produced in transgenic tobacco leaves has a similar physical
form to the human HBsAg.
G. Sucrose and Cesium Chloride Gradient Analysis of HBsAg from
Transgenic Tobacco
Further evidence of the particle behavior of rHBsAg was obtained
from sedimentation and buoyant density studies of the transgenic
tobacco leaf extracts.
Extras of the transgenic tobacco leaf tissue were made as described
in the protein analysis section and five tenths milliliter (0.5
ml) of the 27,000.times.G supernatants were layered on linear eleven
milliliter (11 ml) five to thirty percent (5-30%) sucrose gradients
made in ten millimolar (10 mM) sodium phosphate, pH 7.0, fifteen
hundredths molar (0.15M) sodium chloride or discontinuous twelve
milliliters (12 ml) one and one tenth to one and four tenth grams
per milliliter (1.1-1.4 g/ml) cesium chloride gradients made in
ten millimolar (10 mM) sodium phosphate, pH 7.0 three milliliters
(3 ml) each of one and one tenth, one and two tenths, one and three
tenths, and one and four tenths grams per milliliter (1.1, 1.2,
1.3 and 1.4 g/ml) cesium chloride!. Positive control HBsAg from
the AUSZYME kit was also layered on separate gradients. The sucrose
gradients were centrifuged in a Beckman SW41Ti rotor at thirty-three
thousand revolutions per minute (33,000 rpm) for five hours at five
degrees Celsius (5.quadrature.C), and fractionated into one milliliter
(1 ml) fractions while monitoring the absorbance at two hundred
and eighty nanometers (280 nm). The cesium chloride gradients were
centrifuged in a Beckman SW40Ti rotor at thirty thousand revolutions
per minute (30,000 rpm) for twenty five hours at five degrees Celsius
(5.quadrature.C), and fractionated into five tenths milliliter (0.5
ml) fractions. HBsAg in the gradient was assayed using the AUSZYME
kit as described above.
FIG. 8 shows a sucrose gradient profile of rHBsAg activity from the
transgenic tobacco leaves harboring the plasmid construct pHB102.
The transgenic tobacco rHBsAg sedimented with a peak near the 60S
ribosomal subunit, and the serum-derived HBsAg material sedimented
in a somewhat sharper peak just slightly slower. This data is consistent
with the finding that human HBsAg sediments at 55S..sup.70 The observation
that the plant rHBsAg material sedimented slightly faster and with
a broader peak than the human HBsAg is consistent with the larger
mean size of the rHBsAg plant particles and the wider range of particle
sizes.
The buoyant density of the rHBsAg particles from transgenic tobacco
plants in cesium chloride, FIG. 9, was found to be approximately
one and sixteen hundredths grams per milliliter (1.16 g/ml), while
the human HBsAg particles showed a density of about one and two
tenths grams per milliliter (1.20 g/ml). Thus, the rHBsAg from the
transgenic tobacco plants exhibits sedimentation and density properties
that are very similar to the subviral HBsAg particles obtained from
human serum. Most importantly, HBsAg in the particle form is much
more immunogenic than that found in the peptide form alone..sup.2
H. Reproduction of HBsAg Transgenic Tobacco Plants
Reproduction of transgenic plants was accomplished as stated in
Example I.
EXAMPLE III
A. Transformation of Tomato with HBsAg Gene
Tomato, Lycopersicom esculentum var. VFN8, was transformed as in
Example H. B and C by the leaf disc method using Agrobacterium tumefaciens
strain LBA4404 as a vector, McComck et al., 1986..sup.23 A. tumefaciens
cells harboring plasmid pHB102, constructed as in Example II. A.2,
which carries the HBsAg coding region fused to the tobacco etch
virus untranslated leader, Carrington & Freed, 1990,.sup.73
and the cauliflower mosaic virus 35S promoter, were used to infect
cotyledon explants from seven day old seedlings. The explants were
not preconditioned on feeder plates, but infected directly upon
cutting, and co-cultivated in the absence of selection for two days.
Explants were then transferred to medium B, McCormick et al., 1986,.sup.23
containing five-tenths milligrams per milliliter (0.5 mg/ml) carbenicillin
and one-tenth milligram per milliliter (0.1 mg/ml) kanamycin for
selection of transformed callus. Shoots were rooted in MS medium
containing one-tenth milligram per milliliter (0.1 mg/ml) kanamycin
but lacking hormones, and transplanted to soil and grown in a greenhouse.
Several independent kanamycin-resistant callus lines were obtained
after Agrobacterium-mediated transformation of the tomato variety
VFN8. One of these lines regenerated shoots with high frequency
and was rooted and grown in soil in the greenhouse. The tissues
from these plants were used for the protein and RNA analyses.
B. Quantitation of HBsAg in Leaves and Fruits
Plants tissues were extracted by grinding in a mortar and pestle
with solid carbon dioxide (CO.sub.2), and suspended in three volumes
of buffer containing twenty millimolar (20 mM) sodium phosphate,
one hundred fifty millimolar sodium chloride (150 mM NaCl), five
tenths millimolar phenylmethylsulfonyl fluoride (0.5 mM PMSF), one
tenth percent (0.1%) Triton X-100, pH 7.0. After centrifuging the
homogenate at ten thousands times gravity (10,000.times.g) for five
minutes at four degrees Celsius (4.quadrature.C), aliquots of the
supernatant were assayed for total soluble protein by the method
of Bradford.sup.74 and for HBsAg with the Auszyme II kit (Abbott
Laboratories) as described in Example II. E.
HBsAg Levels in Transformed Tomato Tissues
In order to test for accumulation of HBsAg protein in transgenic
plants, extracts of leaf and fruit were made, which were used for
HBsAg-specific ELISA. A standard curve was obtained using authentic
HBsAg which was derived from the serum of infected individuals.
Table 1 shows the levels of accumulation of HBsAg in leaves and
ripe fruit of transgenic plants. Young leaf and red fruit from greenhouse-grown
transgenic tomato plants were extracted and assayed for total soluble
protein and HBsAg as described above. Similar tissues from untransformed
control tomato plants showed very low background for HBsAg.
The level found in tomato leaves is similar to the highest level
found in leaves of transgenic tobacco by Mason et a., 1992.sup.72,
and represents 0.007% of the total soluble protein. The amount of
HBsAg in ripe fruit was somewhat lower, 0.0043%, or 87 ng/g fresh
weight. Similar extracts of untransformed tomato leaves showed negligible
amounts of anti-HBsAg reactive material, at least 50-fold lower
than the transformed plants.
The level of expression in the tomato fruit, although somewhat
lower on a total protein basis, represents a substantial proportion
of the whole plant accumulation of HBsAg because the fruit are much
more dense than the leaves. A small tomato weighing one hundred
grams would contain approximately nine micrograms (9 .quadrature.g)
of HBsAg.
Organ |
HBsAg Levels in Transgenic Tomato Leaf Fruit ng/mg |
total soluble protein (%) ng/g fresh weight |
Leaf |
70 |
(0.007%) |
Fruit |
(red) 43 |
(0.0043%) |
Table 1
C. RNA Extraction and Northern Blotting
RNA was extracted as described in Example II. D., except that the
tissues were ground with solid carbon dioxide (CO.sub.2) instead
of liquid nitrogen (N.sub.2). RNA was fractionated and blotted to
nylon membranes (Boehringer-Mannheim), fixed by irradiation on a
ultraviolet transilluminator for three minutes, and air dried. Total
RNA on the blot was visualized by staining with twenty-five hundredths
percent (0.25%) methylene blue per twenty-five hundredths molar
sodium acetate (0.25M NaOAc), pH 4.5 for five minutes and destaining
with water. The blot was then prehybridized in twenty-five hundredths
molar (0.25M) sodium phosphate, pH 7.0, ten millimolar ethylenediaminetetraacetic
acid (10 mM EDTA), seven percent sodium dodecyl sulfide (7% SDS)
for one hour at sixty-eight degrees Celsius (68.quadrature.C) and
probed with digoxygenin-labeled random-primed DNA made using the
HBsAg coding region as template according to the manufacturer's
instructions (Genius 2 Kit, Boehringer-Mannheim). After washing
the blot twice with forty millimolar (40 mM) sodium phosphate, pH
7.0, five percent sodium dodecyl sulfate (5% SDS) at sixty-eight
degrees Celsius (68.quadrature.C) and twice with forty millimolar
(40 mM) sodium phosphate, pH 7.0, one percent sodium dodecyl sulfate
(1% SDS) at sixty-eight degrees Celsius (68.quadrature.C), the hybridized
RNA was detected by probing with anti-digoxygenin-alkaline phosphatase
conjugate and developing color for sixteen hours according to the
manufacturer's instructions (Genius 2 Kit, Boehringer-Mannheim).
The activity of the HBsAg gene in transgenic plants was assessed
by RNA blotting. Total RNA isolated from transformed tomato leaves
and green fruit and from untransformed leaves was fractionated in
a denaturing agarose gel, transferred to a nylon membrane, and hybridized
with random-primed digoxygenin-labeled probe made using the HBsAg
coding sequence as template. FIG. 10A shows that RNA from transformed
tomato leaf and fruit hybridized with the HBsAg probe, while RNA
from untransformed leaf showed no detectable signal. The level of
HBsAg mRNA in leaves was approximately three to five times greater
than in fruit, on a total RNA basis. FIG. 10B shows a similar RNA
blot stained with methylene blue to reveal the total RNA pattern,
and indicates that the samples were loaded with equivalent amounts
of total RNA. Thus, the HBsAg transgene is transcribed faithfully
in transgenic tomato leaf and fruit, and accumulates to substantial
levels. The yield of RNA from ripe fruit was poor, and was not analyzed
by RNA blotting.
D. Tissue Blotting for HBsAg Detection
Leaves of transformed or untransformed tomato plants were excised
and pressed on fine-grain sandpaper before blotting abaxial side
down on nitrocellulose. Tomato fruits were sectioned with a razor
blade and pressed onto nitrocellulose for 30 sec. The blot was blocked
with 5% nonfat dry milk in 10 mM sodium phosphate, pH 7.2, 140 mM
NaCl, 0.05% Tween-20, 0.05% NaN3 (PBST)for 2 hr at 37.quadrature.C.
The blot was probed with mouse monoclonal anti-HBsAg (Zymed Laboratories)
at 1:1000 dilution in 2% nonfat dry milk in PBST for 2 hr at 23.quadrature.C.,
before washing and detection with goat anti-mouse IgG-alkaline phosphatase
conjugate (BioRad) and development with NBT and BCIP according to
manufacturer's instructions (Genius 2 Kit, Boehringer-Mannheim).
EXAMPLE IV
A. Construction of Transmissible Gastroenteritis Virus Plasmid
Expression Vector
The Transmissible Gastroenteritis Virus (TEGV) coding sequence
TGEV S-protein as described in Sanchez et al., 1992.sup.75 was obtained
from Dr. Lisa Welter (Ambico-West Los Angeles, Calif.) as a PCR
product cloned into plasmid pGEM-T (Promega Corp., Madison, Wis.).
The 5' end was truncated six base pairs (6 bp) upstream of the translation
initiation site by digestion with HincII. The 1.2 kilobase (kb)
HincII/XhoI fragment was isolated and ligated into plasmid pBluescript
KS (Stratagene, La Jolla, Calif.) which was previously digested
with SmaI and XhoI. The resulting plasmid, pTG5', was then digested
with BamHI and XhoI and the 1.2 kilobase (kb) fragment isolated.
The 3.3 kilobase (kb) Xhol/SstI fragment, representing the 3' end
of the S-protein protein coding region, was isolated and ligated
together with the 1.2 kilobase (kb) BamHI/Xhol fragment from plasmid
pTG5', representing the 5' end of the S-protein coding region, into
plasmid pBluescript KS that had been digested with BamHI and SstI.
The resulting plasmid, pKS-TG, was then digested with BamHI and
SstI to give the entire 4.5 kilobase (kb) S-protein coding sequence,
which was then ligated into the potato tuber expression vector plasmid
pPS20.sup.76 that was digested with BamHI and SstI and isolated
from the GUS coding region. Plasmid pPS20 is a derivative of pBI101.sup.77,
and contains a kanamycin resistance cassette for selection of transformed
plants. The resulting plasmid, pPS-TG, contains the S-protein coding
region downstream of the patatin promoter, which drives tuber-specific
expression in potato plants, and followed by the nopaline synthase
polyadenylation signal.
B. Potato Transformation
Agrobacterium tumefaciens LBA4404 was transformed with plasmid
pPS-TG by the freeze-thaw method of An.sup.78, and the plasmid structure
verified by restriction digestion. The Agrobacterium stain harboring
plasmid pPS-TG was used for transformation of the potato variety
"Atlantic." The potato transformation protocol was as
described in Wenzler.sup.79 and shoots were regenerated on media
containing fifty milligrams per liter (50 mg/L) kanamycin. Microtubers
were induced on nodal stem segments as described by Wenzler..sup.79
C. Analysis of S-protein Expression in Microtubers
Total RNA was extracted from microtubers using the method of Mason
and Mullet.sup.80 , except that the microtubers were homogenized
in three volumes of buffer in microcentrifuge tubes with pellet
pestles, rather than grinding with liquid nitrogen (N.sub.2). The
RNA samples were assayed for S-protein mRNA by RNA dot blotting.sup.81
and hybridization with a digoxygenin-labeled probe made by random-primed
DNA synthesis (Genius 2 Kit, Boehringer-Mannheim, Indianapolis,
Ind.). The 2.2 kilobase (kb) XhoI/XbaI fragment from the coding
region of the TGEV S-protein gene was the template for probe synthesis.
Hybridization and detection were done as per kit instructions (Genius
2 Kit, Boehringer-Mannheim, Indianapolis, Ind.), except that the
hybridization buffer contained twenty-five hundredths molar (0.25M)
sodium phosphate, pH 7.0, five percent (5%) sodium lauryl sulfate,
and ten millimolar ethylenediaminetetraacetic acid (10 mM EDTA).
The results were only qualitative, but indicate that there was a
range of different levels of expression of S-protein MRNA among
the independent transformants, as is expected for a random insertion
of the foreign gene into the host plant genome.
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The foregoing description of the invention has been directed to
a particular preferred embodiments in accordance with the requirements
of the patent and statutes and for purposes of explanation and illustration.
It will become apparent to those skilled in the art that modifications
and changes may be made without departing from the scope and the
spirit of the invention.
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