Download Transgenic Plants Created for Oral Immunization Against Diarrheal

REVIEW ARTICLE
Transgenic Plants Created for Oral Immunization
Against Diarrhea1 Diseases
Liz Richter, Hugh S. Mason, and Charles] 4vntzen
.
1
We have created transgenic plants that express subunit antigens of infectious bacteria and viruses.These proteins have been isolated and characterized to demonstrate
that they retain immunogenic properties. When raw
potatoes containing the reconibinant inimunogens were
fed to mice, the animals were orally ininiunized. Research
completed to date has demonstrated the feasibhty of using
a genetically-engineered food as an inexpensive oral
vaccine production and delivery system for diarrheal
disease. The system could be a convenient means to
deliver vaccines to travelers. It also has great potential as
an appropriate technology for producing vaccines “in
country” in the developing world. This review focuses
on work from our group on recombinant subunit vaccines in potato tubers against enterotoxigenic Escherickia
coli and Nonvalk virus.
labile (LT) and/or heat stabile (ST) enterotoxin, are the
most common causes of travelers’ diarrhea world wide.’
Cholera and ETEC are the primary agents of bacterial
diarrhea in the developing world. Based upon previous
oral immunization and cross-protection studies using
candidate vaccines,*immunity to one of these diseases will
cause at least partial protection against the other since the
toxins are immunologically related.-’The most important
cause of severe viral gastroenteritis in human and animal
is rotavirus; it infects almost every child in the early years4
and causes the highest amount of diarrheal mortality
among ~ h i l d r e nRotavirus-caused
.~
diarrhea rarely occurs
more than once. It is, therefore, a good candidate disease
to be controlled through vaccination. Studies aimed at
developing a rotavirus vaccine include,oral administration
of recombinant virus core and capsid proteins produced
in cultured insect cells.This complex appears to be protective against rotavirus infection in rabbits.6
Recent advances in molecular biology have allowed
characterization of Norwalk virus (NV) as an agent of
acute epidemic gastroenteritis in humans.’ United States
epidemiology studies have demonstrated that Nonvalklike virus infection correlated to 67% of gastroenteritis
outbreaks in nursing homes, 60% in summer camps, and
28% on cruise ships.xCurrently a commercially-available
vaccine for NV does not exist. However, a candidate subunit vaccine, based upon recombinant protein expression
from cultured insect cells, is being investigated’ and
human clinical trials have been initiated (personal communication, M. K. Estes).
Background
In the developing world, diarrheal disease is a leading cause of death, especially among children. Travelers
to these areas are frequently exposed to the bacteria and
viruses that cause diarrheal disease, resulting in symptoms
ranging from discomfort to severe debilitation.Vaccines
to prevent diarrheal disease would be of value to travelers from developed countries, who lack protective immunity, and especially important to health care providers in
the developing world, who could use them to decrease
infant and adult mortality.
Endemic bacterial pathogens, including enterotoxigenic Esckrvickia coli (ETEC), which produce a heat
Mucosal Immunology
Mucosal antibodies are a primary defense against
many infectious diseases, including enteric diseases.The
virulence of diseases may be mitigated or prevented by
secretory IgA antibodies in the gut, respiratory tract, or
other mucosal membranes. Mucosal immune responses
are most easily induced by oral immunization. Optimally,
an oral vaccine should induce both mucosal and serum
antibodies. Immunogenicity of oral antigens must be
maintained despite the low pH and hostile environment
of the gut towards the protein.The M cells in the Peyer’s
Liz Richter, PbD, Hugh S. Mason, PhD, a n d Charles J.
Amtzen, PbD: Boyce Thompson Institute for Plant Research
at Cornell University, Ithaca, New York, U.S.A.
This research has been supported by the Thrasher Research
Fund, NIH Grant No. 1-R01A136519-01 and Texas Advanced
Technology Program Grant No. 999902-084.
Reprint requests: Charles J. Amtzen, P k D r Boyce Thompson
Institute for Plant Research Incorporated, Tower Road,
Ithaca, NY 14853-1801
J Travel M e d 1996; 3:52-56.
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R i c h t e r et al, Edible Vaccines f r o m T r a n s g e n i c P l a n t s
patches of the intestinal epithelia can phagocytize particles from the intestinal lumen and transfer them to
underlying lymphocytes.Therefore,bindmg to the M cells
may enhance imniunogenicity of an antigen. Proteins that
are complexed in particulate structures may be more resistant to proteases and seem to be more orally immunogenic than single peptides.There is much current research
focused on oral adjuvants that can be added to an antigen to increase the immune response against fed antigens. In general, an oral inoculation requires up to 1000
times the amount of antigen to promote an immune
response than does parenteral inoculation. '","
Technology Advancements and Cost-Related
limitations in Modern Vaccines
53
control of infectious diseases have been made. In particular, new techniques have generated a deeper understanding of human immune responses to infectious
agents, and identification of the immunogens of important bacteria and viruses. Cloned genes for many of
these iimnunogens are now available.
Currently, only one recombinant vaccine has been
approved by the Food and Drug Administration for
commercial production; Merck, Sharpe and Dohme
markets this vaccine against hepatitis B, and it is based
upon the recombinant hepatitis B surface antigen (rHBsAg).This protein is produced and purified from yeast.
The availability of this vaccine, at present, is largely limited to developed countries because of its cost.
Transgenic Plants for Vaccine Production
With the advent of modern molecular biology and
biotechnology, enormous advances in our understanding of the molecular events underlying the onset and
A.
Flc
LT-B from enterotoxlgenlc E. coll
NVCP from Nonvalk VINS
I
Plant
transformation
vector
for foreign gene
expression
Agrobacteriummediated
genetic transformation
and regenerationof
transgenic plants
D.
I
s@J1
E.
m
G.
1
Extraction
and analysis
of foreign
In a recent issue of Science,'* our group reported
that mice could be orally immunized by feeding them
I
/
J
Stable integration
f aene into nuclear
chromosome
plant tissue
A. Optimal factors for choosing a candidate
antigen
Purified protein is sufficient to promote imniunity
to the disease.
Purifird protein is orally iniinunogenic.
The gene for the protein is cloned.
The protein maintains its antigenic epitopes and
assembles into its native form when expressed in a
heterologous system.
B. Factors to consider in selecting a gene for
plant expression systems
Modification of the gene with plant specific regulatory elements.
Analysis and reconstruction of antigen-coding
sequence for optimal plant expression.
C. Create a plant transformation vector
D. Transform plant tissue
E. Generate plants for analysis
F. Analyze recombinant protein for antigenic
epitopes and proper assembly within plant tissue
G. Optimize production of recombinant protein
in plant tissue by redesign of gene or transformation vector
H. Test oral immunogenicity of the vaccine-containing transgenic food
Test for induction of an immune response to the
recombinant protein.
Challenge immunized animals (if possible) with the
disease to deterniine if the candidate vaccine can
induce a protective immune response.
IMMUNOGENICIN STUDIES
Figure 1 Production of candidate vaccines in plants is shown schematically along with factors t o consider for various steps.
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raw transgenic potatoes containing a recombinant antigen. The animals demonstrated an immune response
specific for the recombinant protein, the E. coli heat-labile
enterotoxin B subunit (LT-B). Essentially, the potato
plant was genetically engineered to produce LT-B in the
tuber tissue, which could simply be eaten to promote an
immune response. Although raw potatoes are not anticipated to be the ultimate vaccine delivery system, they
were chosen for these studies because they had proved
suitable as food for mice in initial studies.
Among the various methods that cause plants to produce a desired protein, plants that are permanently transformed and that can be propagated for expansion to
large-scale production and evaluation of a potential vaccine are required.This method is illustrated in Figure 1.
For a review, including transient expression of candidate
vaccines in plants, see Mason et al.I3Plants are also being
investigated for the manufacture of other products,
including recombinant monoclonal a n t i b o d i e ~ , ' ~and
.'~
biodegradable plastic, polyhydroxyalkanoates (PHB). I h
Enterotoxigenic E. coli and Vibrio cholerae Vaccine
Research and vaccine development against enterotoxigenic E. coli (ETEC) and Vibrio cholerae are aimed at
preventing the severe diarrhea caused by the heat-labile
enterotoxin, LT, and cholera toxin, CT.These toxins are
structurally, functionally, and immunologically related
(see reference 3 for a review).The toxins are composed
of one A and five B subunits.The toxic activity is due to
the A1 fragment of the A subunit, whereas the pentameric B subunit allows binding to a ganglioside G,,
membrane receptor on cells in the small intestine. One
strategy for a vaccine against these diseases would induce
antibodies that block binding of the B pentamer to the
intestinal epithelial cells.
LT-B was one of the first candidate antigens we chose
to evaluate for production of a vaccine in plants, because
it fit several of the criteria listed in Figure 1 for optimal
candidates.Early studies showed the efEicacy of the B subunit of cholera toxin as an oral immunogen to induce
mucosal immunity to cholera toxin'."; both LT-A and
LT-B have been cloned, and when expressed in recombinant E. coli, the recombinant LT-B (rLT-B) subunit
assembled into a pentamer."
In initial studies, we used tobacco plants to test the
capacity of transgenic plants to express LT-B." As tobacco
has been a model system for much work in plants, the
LT-B gene expression could easily be compared to other
protein expression studies.We found that the tobacco rLTB was the same size as the bacterially expressed rLT-B
and oligomerized to the molecular niass that corresponds to a pentamer. In order to test the antigenicity
of the tobacco rLT-B, it was partially purified to remove
J o u r n a l o f Travel M e d i c i n e , V o l u m e 3 , N u m b e r 1
toxic alkaloids. A modification at the carboxyl terminus
of the rLT-B increased the accumulation of the recombinant protein and did not affect its oligomerization.
When mice were orally inoculated by intubation with
the tobacco-derived rLT-B, both serum and mucosal
antibodies were induced.
Potato plants were also transformed to produce the
same modified rLT-B.This recombinant protein was not
purified from the plant material, but tubers were fed
directly to mice for their inoculation.After a regimen of
four such inoculations, the mice developed both serum
IgG and mucosal IgA antibodies to LT-B."These results
validate the concept of using edible plants for vaccine production and delivery.
Optimization of the system is our current research
effort. The titers of antibodies in mice eating the transformed potatoes were not as high as when the mice were
intubated with bacterially produced rLT-B. The concentration of LT-B in the tubers needs to be increased
so that the mice can be given a smaller dose of raw potato
to ingest. In our published studies, mice were given 20
pg of rLT-B in a 5 g tuber per dose;whereas, human feeding trials have used 1 nig or more B subunit per dose.',"
There are many factors that can influence accumulation of a recombinant protein within a plant tissue. Some
of these involve targeting the protein to cellular compartments. In these studies, modifications to the protein
must be analyzed for their effect on that protein's ability to function as an oral vaccine. Current work on LTB is focused on increasing the concentration of the
rLT-B in tubers and inimunologic testing with other
species of animals.We anticipate human clinical trials to
evaluate the feasibility of using transgenic plants for vaccine delivery.
Norwalk Virus Vaccine
We have also chosen the Nonvalk virus capsid protein (NVCP) as a candidate antigen for a plant-produced vaccine.The genome of NV has been cloned,' and
the NVCP has been expressed in an insect cell culture
system where the recombinant protein, rNV, assembles
into an empty capsid or virus-like particle (VLP) coniposed of 90 dimers of capsid protein molecules.2'1These
VLPs can be seen with electron microscopy and appear
very similar to native Nonvalk virus." Studies using the
insect cell rNV for oral immunization of mice have
been successful in inducing serum and mucosal antibodies
against NVCP with as little as 50 p g purified rNV per
inoculation."
Our group has used the gene for NVCP to transform tobacco and potato plants. Physical characteristics,
including particle formation as determined by electron
microscopy, of purified material fi-om transformed tobacco
Richter e t al, E d i b l e Vaccines from Transgenic Plants
55
plants are comparable to the rNV from cultured insect
cells. It is encouraging that the rNV produced in plant
tissue also assembles into aVL€?Thisstructure is expected
to be immunogenic as it should present similar antigenic
sites as the native whole virus and may be more resistant to proteases. The rNV has also been produced in
potato plants, and feeding studies with mice receiving 4050 pg per dose induced serum IgG specific for NVC€?*’
Because mice are not infected by NV,human feeding trials of transgenic tubers will be necessary for ultimate
determination of protective immunity. Currently, studies on the optimization of expression level and subcellular localization for potato tuber rNV are underway in
our laboratories.
Conclusion
With the development of an inexpensive production system, recombinant vaccines may be more readily
available to developing countries.The results summarized
here suggest low cost “edible vaccines” from plants are
feasiblewe have initiated studies with candidate vaccines
for both bacterial and viral pathogens.
The development of oral vaccines and an understanding of mucosal immunology are areas of intense
re~earch.’~
Oral vaccines are desired for ease of administration and to eliminate the need for injection equipment.
In the March 1995 issue of this journal, a study of a potential oral vaccine for travelers’ diarrhea was rep0rted.A purified, bacterially produced recombinant B subunit from
cholera was mixed with phosphate buffered saline and
sodium bicarbonate in water and ingested by subjects.This
resulted in protective eficacy in the prevention of travelers’ diarrhea seven days after the second inoculation.”
As information about more dseases and potential vaccines
becomes available,potential recombinant subunit vaccines
will certainly be proposed for many diseases.
Figure 3 Simplicity of local banana distribution in Nigeria illustrates the usefulness of vaccine production in bananas.
The primary advantage of plant-produced edible vaccines is their potential for vastly decreasing the expense
of recombinant subunit vaccines. One potential target of
major world-wide importance would be an inexpensive
vaccine for hepatitis B (Figure 2). The knowledge that
parenteral inoculation provides protective immunity in
humans has offered encouragement that recombinant
HBsAg is a candidate antigen for plant vaccine production.
Research of plant-based production and delivery of
vaccines is at an early stage. Several factors have yet to
be studied, such as stability of antigens upon storage of
the edible plant tissue. If transformed plants are grown
in the area or country where they will be used, shipping
costs can be minimized and refrigeration may not be necessary. It may be possible to combine several “vaccines”
in one plant or produce vaccines composed of several proteins that form an antigenic complex, such as the recombinant rotavirus capsid and core proteins.2hThe ability
to manipulate plants as protein manufacturing systems
24325
Figure 2 Edible vaccine for hepatitis B in banana is a goal
for controlling the disease in developing countries.
56
will become easier as plant biotechnology expands, thus
predicting success in multimeric vaccines.
With the work summarized here, we have shown the
feasibility of a food plant used to produce and deliver a
vaccine. In an effort to define an optimal food delivery
system,our group recently reported a method for genetic
transformation of banana plants.” Bananas or plantains
are important nutritional staples for many tropical countries, and they satisfy the criterion that an “edible vaccine”cou1d be produced“in country” The ability to grow,
harvest, and distribute bananas is already in place for many
of the most impoverished areas of the world (Figure 3 ) .
Also, uncooked banana fruit is readily eaten by human
infants who have the most to gain by large scale inexpensive immunizations. Current research with banana
plants involves determining how to cause accumulation
of recombinant proteins in the banana fruit.While these
efforts are underway, other plants (including potatoes) d
continue to be used for essential preliminary studies on
feasibility and efficacy of plant produced oral vaccines.
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