Edible Vaccines

5/8/2018 Edible Vaccines - slidepdf.com

http://slidepdf.com/reader/full/edible-vaccines-559ac18d94ca8 1/10

www.ijmm.org

93 CMYK

93April-June 2007

T h i s

P D F i s

a v a i l a

b l e f o r f r e e d o w n l

o a d f r o

m

a s i t

e h o

s t e d b y M

e d k n

o w P u b l i c a t i

o n s

( w w w

. m e d k

n o w . c o

m ) .

EDIBLE VACCINES: CURRENT STATUS AND FUTURE

P Lal, VG Ramachandran, *R Goyal, R Sharma

Abstract

Edible vaccines hold great promise as a cost-effective, easy-to-administer, easy-to-store, fail-safe and socioculturally readily

acceptable vaccine delivery system, especially for the poor developing countries. It involves introduction of selected desired

genes into plants and then inducing these altered plants to manufacture the encoded proteins. Introduced as a concept about

a decade ago, it has become a reality today. A variety of delivery systems have been developed. Initially thought to be useful

only for preventing infectious diseases, it has also found application in prevention of autoimmune diseases, birth control,

cancer therapy, etc. Edible vaccines are currently being developed for a number of human and animal diseases. There is

growing acceptance of transgenic crops in both industrial and developing countries. Resistance to genetically modiÞed

foods may affect the future of edible vaccines. They have passed the major hurdles in the path of an emerging vaccine

technology. Various technical obstacles, regulatory and non-scientiÞc challenges, though all seem surmountable, need to

be overcome. This review attempts to discuss the current status and future of this new preventive modality.

Key words: Chimeric, edible vaccines, transgenic

*Corresponding author (email: <[email protected]>)

Departments of Microbiology (PL, VGR, RG) and Pediatrics (RS),

University College of Medical Sciences, Guru Teg Bahadur Hospital, New Delhi - 110 095, India.

Received : 28-06-06

Accepted : 14-10-06

Indian Journal of Medical Microbiology, (2007) 25 (2):93-102

Vaccines have been revolutionary for the prevention of

infectious diseases. Despite worldwide immunization of

children against the six devastating diseases, 20% of infants

are still left un-immunized; responsible for approximately

two million unnecessary deaths every year, especially in the

remote and impoverished parts of the globe.1 This is because

of the constraints on vaccine production, distribution and

delivery. One hundred percent coverage is desirable, because

un-immunized populations in remote areas can spread

infections and epidemics in the immunized “safe” areas,which have comparatively low herd immunity. For some

infectious diseases, immunizations either do not exist or they

are unreliable or very expensive. Immunization through DNA

vaccines is an alternative but is an expensive approach, with

disappointing immune response.2 Hence the search is on for

cost-effective, easy-to-administer, easy-to-store, fail-safe

and socioculturally readily acceptable vaccines and their

delivery systems. As Hippocrates said, “Let thy food be thy

medicine,” scientists suggest that plants and plant viruses can

be genetically engineered to produce vaccines against diseases

such as dental caries; and life-threatening infections likediarrhea, AIDS, etc.3 This is the concept of edible vaccines.

The following discussion will address issues relating to

their commercial development, especially their usefulness in

preventing infectious diseases in developing countries.

Concept of Edible Vaccines

Creating edible vaccines involves introduction of selected

desired genes into plants and then inducing these altered

plants to manufacture the encoded proteins. This process is

known as “transformation,” and the altered plants are called

“transgenic plants.” Like conventional subunit vaccines,

edible vaccines are composed of antigenic proteins and

are devoid of pathogenic genes. Thus, they have no way

of establishing infection, assuring its safety, especially in

immunocompromised patients. Conventional subunit vaccines

are expensive and technology-intensive, need puriÞcation,

require refrigeration and produce poor mucosal response.In contrast, edible vaccines would enhance compliance,

especially in children, and because of oral administration,

would eliminate the need for trained medical personnel. Their

production is highly ef Þcient and can be easily scaled up.

For example, hepatitis-B antigen required to vaccinate whole

of China annually, could be grown on a 40-acre plot and all

babies in the world each year on just 200 acres of land!4 They

are cheaper, sidestepping demands for puriÞcation (single dose

of hepatitis-B vaccine would cost approximately 23 paise),

grown locally using standard methods and do not require

capital-intensive pharmaceutical manufacturing facilities.Mass-indeÞnite production would also decrease dependence

on foreign supply. They exhibit good genetic stability. They

are heat-stable; do not require cold-chain maintenance; can

be stored near the site of use, eliminating long-distance

transportation. Non-requirement of syringes and needles also

decreases chances of infection.5 Fear of contamination with

animal viruses - like the mad cow disease, which is a threat

in vaccines manufactured from cultured mammalian cells - is

eliminated, because plant viruses do not infect humans.

Edible vaccines activate both mucosal and systemicimmunity, as they come in contact with the digestive tract

lining. This dual effect would provide Þrst-line defense against

pathogens invading through mucosa, like Mycobacterium

Review Article



www.ijmm.org

94 CMYK

94

T h i s

P D F i s

a v a i l a


o a d f r o

m

a s i t

e h o

s t e d b y M

e d k n


o n s

( w w w

. m e d k

n o w . c o

m ) .

Indian Journal of Medical Microbiology

tuberculosis and agents causing diarrhea, pneumonia, STDs,

HIV, etc. Scientists place high priority on combating the

diarrheal agents – Norwalk virus, Rotavirus, Vibrio cholerae

and enterotoxigenic E. coli (ETEC) – responsible for about

three million infant deaths/year, mainly in developing

countries.1 Administration of edible vaccines to mothers

might be successful in immunizing the fetus-in-utero by

transplacental transfer of maternal antibodies or the infantthrough breast milk. Edible vaccines seroconvert even in the

presence of maternal antibodies, thus having a potential role in

protecting infants against diseases like group-B Streptococcus,

respiratory syncytial virus (RSV), etc., which are under

investigation.6 Edible vaccines would also be suitable against

neglected/rare diseases like dengue, hookworm, rabies, etc.

They may be integrated with other vaccine approaches, and

multiple antigens may also be delivered. Various foods under

study are banana, potato, tomato, lettuce, rice, etc. Edible

vaccines are currently being developed for a number of human

and animal diseases, including measles, cholera, foot and

mouth disease and hepatitis B, C and E.7

Mechanism of Action

The antigens in transgenic plants are delivered through

bio-encapsulation, i.e., the tough outer wall of plant cells,

which protects them from gastric secretions, and finally

break up in the intestines. The antigens are released, taken

up by M cells in the intestinal lining that overlie peyer’s

patches and gut-associated lymphoid tissue (GALT), passed

on to macrophages, other antigen-presenting cells; and local

lymphocyte populations, generating serum IgG, IgE responses,local IgA response and memory cells, which would promptly

neutralize the attack by the real infectious agent.8

Preparation of Edible Vaccines

Introduction of foreign DNA into plant’s genome can either

be done by bombarding embryonic suspension cell cultures

using gene-gun or more commonly through Agrobacterium

tumefaciens, a naturally occurring soil bacterium, which has

the ability to get into plants through some kind of wound

(scratch, etc.). It possesses a circular “Ti plasmid” (tumor

inducing), which enables it to infect plant cells, integrateinto their genome and produce a hollow tumor (crown gall

tumor), where it can live. This ability can be exploited to

insert foreign DNA into plant genome. But prior to this, the

plasmid needs to be disarmed by deleting the genes for auxin

and cytokinin synthesis, so that it does not produce tumor.

Genes for antibiotic resistance are used to select out the

transformed cells and whole plants, which contain the foreign

gene; and for expressing the desired product, which can then

be regenerated from them.9 The DNA integrates randomly

into plant genome, resulting in a different antigen expression

level for each independent line,10

so that 50-100 plants aretransformed together at a time, from which one can choose the

plant expressing the highest levels of antigen and least number

of adverse effects. Production of transgenic plants is species

dependent and takes 3-9 months. Reducing this time to 6-8

weeks is currently under investigation. Some antigens, like

viral capsid proteins, have to self-assemble into VLPs (virus-

like particles). VLPs mimic the virus without carrying DNA

or RNA and therefore are not infectious. Each single antigen

expressed in plants must be tested for its proper assembly and

can be veriÞed by animal studies, Western blot; and quantiÞed

by enzyme-linked immunosorbent assay (ELISA).11

“Second-Generation” Edible Vaccines

Successful expression of foreign genes in plant cells and/or

its edible portions has given a potential to explore further and

expand the possibility of developing plants expressing more

than one antigenic protein. Multicomponent vaccines can

be obtained by crossing two plant lines harboring different

antigens. Adjuvants may also be co-expressed along with the

antigen in the same plant. B subunit of Vibrio cholerae toxin

(VC-B) tends to associate with copies of itself, forming a

doughnut-shaped Þve-member ring with a hole in the middle.1

This feature can bring several different antigens to M cells

at one time - for example, a trivalent edible vaccine against

cholera, ETEC (Enterotoxigenic E. coli) and rotavirus could

successfully elicit signiÞcant immune response to all three.12

Global alliance for vaccines and immunization (GAVI)

accords very high priority to such combination vaccines for

developing countries.

Various Strategies

Approaches to mucosal vaccine formulation include (i)

gene fusion technology, creating non-toxic derivatives of mucosal adjuvants; (ii) genetically inactivating antigens by

deleting an essential gene; (iii) co-expression of antigen and

a cytokine, which modulates and controls mucosal immune

response; and (iv) genetic material itself, which allows DNA/

RNA uptake and its endogenous expression in the host cell.

Various mucosal delivery systems include biodegradable

micro- and nanoparticles, liposomes, live bacterial/viral

vectors and mucosal adjuvants.13 “Prime-boost” strategy

combines different routes of administration and vaccine types,

especially where multiple antigens or doses are required.14 For

example, a single parenteral dose of MV-H DNA (measlesvirus haemagglutinin) followed by multiple oral MV-H

boosters could induce greater quantities of MV-neutralizing

antibodies than with either vaccine alone.15

Chimeric Viruses

Certain viruses can be redesigned to express fragments of

antigenic proteins on their surface, such as CPMV (cowpea

mosaic virus), alfalfa mosaic virus, TMV (tobacco mosaic

virus), CaMV (cauliflower mosaic virus), potato virus X

and tomato bushy stunt virus. Technologies involved are

overcoat and epicoat technology.16

Overcoat technology permits the plant to produce the entire protein, whereas epicoat

technology involves expression of only the foreign proteins. A

plant-derived mink enteritis virus (MEV) injectable vaccine,

vol. 25, No. 2



www.ijmm.org

95 CMYK

95April-June 2007

T h i s

P D F i s

a v a i l a


o a d f r o

m

a s i t

e h o

s t e d b y M

e d k n


o n s

( w w w

. m e d k

n o w . c o

m ) .

expressed on chimeric CPMV, was shown to protect minks

against the clinical disease. Fragments of gp41 surface protein

of HIV virus put into CPMV could evoke a strong neutralizing

antibody response in mice.3 It may even be possible to present

a cocktail of speciÞc HIV epitopes on the surface of the plant

virus. CPMV is genetically and thermally stable. It can survive

acidity (pH 1) for one hour. Wide range of epitopes has been

expressed in CPMV. They include HIV-1 (gp41), (gp120);human rhinoviruses; foot and mouth disease virus; canine

parvovirus; mammalian epitopes from hormones or from colon

cancer cells; fungal epitopes and protozoan epitopes from

Plasmodium falciparum. Wide range of routes is available

– parenteral and nasal (puriÞed particles), oral (formulated leaf

extracts), whole and homogenized leaves, fruits or vegetable

tissues.16 In all these instances, plant viruses are engineered to

carry the desired genes and used to infect their natural hosts

such as the edible plants where the cloned genes are expressed

to varying degrees in different parts of the plant, including

their edible portions.

Challenges

There are many questions which need to be answered

before developing a plant-based vaccine (Table 1). Three

successful human clinical trials have shown that adequate

doses of antigen can be achieved with plant-based vaccines.17-

19 To determine the right dosage, one needs to consider the

person’s weight, age; fruit/plant’s size, ripeness and protein

content. The amount to be eaten is critical, especially in

infants, who might spit it, eat a part or eat it all and throw it up

later. Too low a dose would fail to induce antibodies, and toohigh a dose would, instead, cause tolerance. Concentrating the

vaccine into a teaspoon of baby food may be more practical

than administering it in a whole fruit. The transformed

plants can also be processed into pills, puddings, chips, etc.

Regulatory concerns would include lot-to-lot consistency,

uniformity of dosage and purity.

Foreign proteins in plants accumulate in low amounts

(0.01-2% of total protein) and are less immunogenic; therefore

the oral dose far exceeds the intranasal/parenteral dose. For

example, oral hepatitis-B dose is 10-100 times the parenteral

dose; and 100 gm potato expressing B subunit of labile toxinof ETEC (LT-B) is required in three different doses, to be

immunogenic.19 Attempts at boosting the amount of antigens

often lead to stunted growth of plants and reduced tuber/fruit

formation, as too much m-RNA from the transgene causes

gene-silencing in plant genome.20 Some of the techniques

to overcome these limitations are (i) optimization of coding

sequence of bacterial/viral genes for expression as plant

nuclear genes, (ii) expression in plastids,21 (iii) plant viruses

expressing foreign genes,22 (iv) coat-protein fusions,23 (v)

viral-assisted expression in transgenic plants24 and (vi)

promoter elements of bean yellow dwarf virus with reporter genes GUS (β-glucuronidase) and GFP (green ßuorescent

protein), substituted later with target antigen genes.20 Antigen

genes may be linked with regulatory elements which switch

Lal et al – Edible Vaccines

Table 1: Considerations in developing a plant-based

vaccine

Antigen selection

• Is the antigen safe and non-pathogenic in al l

circumstances?

• Can the antigen induce a protective immune response?

• Is the antigen suitable for expression in plants?

Ef ficacy in model systems

• Does the antigen accumulate in plants in sufficient

quantities?

• Is the plant-derived antigen immunogenic?

• Do trial animals develop protective immune responses?

• Possible plant cell interference with antigen presentation

• Possible induction of immune tolerance

Choice of plant species for vaccine delivery

• Best food plant?

• Ability to be eaten raw and unprocessed?

• Suitable for infants?

• Widely and easily grown?• Easily stored? Resistant to spoiling?

• Amenable to transformation and regeneration?

• Possible cost to plant of multiple transgenes

Delivery and dosing issues

• Requirement of mucosal adjuvants for protective

response?

• Can a large enough dose be delivered by simply eating the

plant?

• Number of doses required?

Safety issues

• Allergenic and toxic (e.g., glycans, nicotine, etc.) potentialof plant components

• Potential for interference

• Production of oral tolerance?

• Risk of atypical measles (in plants with cloned measles

virus genes)?

• Health and environmental risks of genetically modiÞed

organisms

• Prevention of misuse/overuse

Public perceptions and attitudes to genetic modification

• Will negative attitudes to genetically modiÞed organisms

inßuence vaccine acceptability?• Legal and ethical considerations regarding products from

plants with status like tobacco

Quality control and licensing

• Can antigen expression be consistent in crops?

• Who will control vaccine availability and production?

ModiÞed from “Webster DE et al. Appetising solutions: An edible

vaccine for measles. MJA 2002; 176: 434-437. ©Copyright 2002.

The Medical Journal of Australia” - reproduced with permission.5

on the genes more readily; or do so only at selected times

(after the plant is nearly fully grown); or only in its edible

regions. Exposure to some outside activator molecule mayalso be tried.25

To enhance immunogenicity, mucosal adjuvants, better



www.ijmm.org

96 CMYK

96

T h i s

P D F i s

a v a i l a


o a d f r o

m

a s i t

e h o

s t e d b y M

e d k n


o n s

( w w w

. m e d k

n o w . c o

m ) .

targeted to the immune system, may be used, like molecules

that bind to M cells in the intestine lining and pass them

to immune cells. These include CT-B (Cholera toxin - B

subunit), LT-B (ETEC), mammalian/viral immunomodulators

and plant-derived secondary metabolites.1,12 To decrease

toxicity and allergic potential, mutant forms of E. coli-labile

toxin, like LT-K63 and LT-R72 and hinge cleavage mutant LT-

G192, are used.26

Another challenge would be in dealing withdiseases caused by multiple serotypes (dengue) or by complex

parts from different life cycles of parasites (malaria) or by

rapidly mutating organisms (HIV, trypanosomes, inßuenza).

Each plant possesses its set of advantages and disadvantages

(Table 2).

Nonscientific Challenges

Presently, small technology companies are undertaking

Table 2: Advantages and disadvantages of different plants

Plant/fruit Advantages DisadvantagesTobacco Good model for evaluating recombinant proteins Produces toxic compounds*

Low-cost preserving system (numerous seeds, stored for long time)

Easy puriÞcation of antibodies stored in the seeds, at any location

Large harvests, number of times/year

Potato Dominated clinical trials Needs cooking, which can denature the

Easily manipulated/transformed antigens and decrease immunogenicity**

Easily propagated from its “eyes”

Stored for long periods without refrigeration

Banana Do not need cooking Trees take 2-3 years to mature

Proteins not destroyed even if cooked Transformed trees take about 12 months

to bear fruitInexpensive Spoils rapidly after ripening

Grown widely in developing countries Contains very little protein, so unlikely to

produce large amounts of recombinant

proteins

Tomato Grow quickly Spoils readily

Cultivated broadly

High content of vitamin A may boost immune response

Overcome the spoilage problem by freeze-drying technology

Heat-stable, antigen-containing powders***, made into capsules

Different batches blended to give uniform doses of antigen

Rice Commonly used in baby food because of low allergenic potential Grows slowlyHigh expression of proteins/ antigens Requires specialized glasshouse

conditions

Easy storage/transportation

Expressed protein is heat-stable

Lettuce Fast-growing Spoils readily

Direct consumption

Soybean Large harvests, number of times/year

and Alfalfa

Musk melon Fast growing

(cantaloupe) Easily propagated by seed

Easily transformedOthers Carrots, peanuts, wheat, corn

*Currently, therapeutic proteins in tobacco are being produced. **Some kinds of South American potatoes can be eaten raw. Although some

studies show that cooking does not destroy full complement of antigen in potatoes.3 ***Freeze-dried tomato powder containing NV capsid

and LT-B was found immunogenic. Same technology is also used for potatoes and carrots.

most research, as edible vaccines are targeted to markets of

developing nations. Large companies are more interested

in livestock market than human application. Only few

international aid organizations and some national governments

are rendering support, but the effort remains largely under-

funded. Some of the companies funding edible vaccines

research have failed to click due to lack of investors’

conÞ

dence in returns on investments in genetically-modiÞ

ed(GM) foods. There is also a lack of research and development

(R&D) personnel in the pharmaceutical companies. In

addition, the recombinant (injectable) vaccines against

diphtheria, tetanus, etc., are so cheap now, that there would be

little incentive to develop edible vaccines for them.

Regulatory Issues

It is still unclear whether the edible vaccines would be

Indian Journal of Medical Microbiology vol. 25, No. 2



www.ijmm.org

97 CMYK

97April-June 2007

T h i s

P D F i s

a v a i l a


o a d f r o

m

a s i t

e h o

s t e d b y M

e d k n


o n s

( w w w

. m e d k

n o w . c o

m ) .

regulated under food, drugs or agricultural products and

what vaccine component would be licensed - antigen itself,

genetically engineered fruit or transgenic seeds. They would

be subjected to a very close scrutiny by the regulatory bodies

in order to ensure that they never enter the food supply.

This would include greenhouse segregation of medicinal

plants from food crops to prevent out-crossing and would

necessitate separate storage and processing facilities. Althoughedible vaccines fall under “GM” plants, it is hoped that these

vaccines will avoid serious controversy, because they are

intended to save lives.

Clinical Trials

Antigen expression in plants has been successfully shown

in the past, like LT-B (ETEC) in tobacco and potato, rabies

virus-G protein in tomato,27 HBsAg in tobacco28 and potato,

norwalk virus in tobacco and potato; CT-B (Vibrio cholerae) in

potato.1 Ethical considerations usually preclude clinical trials

from directly assessing protection, except in a few cases.29 Incontrast, veterinary researchers can assess immune protection

more directly.

ETEC

Charles Arntzen at Boyce Thompson Institute, USA,

accomplished the Þrst published successful human trial in 1997.30

Eleven volunteers were fed raw transgenic potatoes expressing

LT-B. Ten (91%) of these individuals developed neutralizing

antibodies, and six (55%) developed a mucosal response.

Norwalk virus

Nineteen (95%) out of 20 people fed with transgenic potato

expressing norwalk virus antigen showed seroconversion.31

Attempts are underway to engineer bananas and powdered

tomatoes expressing norwalk virus.

Cholera

Transgenic potato with CT-B gene of Vibrio cholerae was

shown to be ef Þcacious in mice. Eating one potato a week for

a month with periodic boosters was said to provide immunity.32

Co-expression of mutant cholera toxin subunit A (mCT-A) and

LT-B in crop seed has been shown to be effective by nasal

administration and is extremely practical.33

Measles

Mice fed with tobacco expressing MV-H (measles

virus haemagglutinin from Edmonston strain) could attain

antibody titers Þve times the level considered protective for

humans, and they also demonstrated secretory IgA in their

faeces.34 Prime boost strategy by combining parenteral and

subsequent oral MV-H boosters could induce titers 20 times

the human protective levels. These titers were signiÞ

cantlygreater than with either vaccine administered alone.5,15 MV-H

edible vaccine does not cause atypical measles, which may

be occasionally seen with the current vaccine.35 Thus it may

prove better for achieving its eradication. The success in mice

has prompted similar experiments in primates. Transgenic

rice, lettuce and baby food against measles are also being

developed. When given with CT-B (adjuvant), 35-50 gm MV-

H lettuce is enough; however, an increased dose would be

required if given alone.7

Hepatitis B

For hepatitis B, parenteral VLPs could invoke speciÞc

antibodies in mice.36 First human trials of a potato-based

vaccine against hepatitis B have reported encouraging results.

The amount of HBsAg needed for one dose could be achieved

in a single potato. Levels of speciÞc antibodies signiÞcantly

exceeded the protective level of 10 mIU/mL in humans. When

cloned into CaMv (caulißower mosaic virus), plasmid HBsAg

subtype ayw showed higher expression in roots as compared to

leaf tissue of the transgenic potato. Further studies are required

to increase the production of antigen by using different

promoters, like patatin promoter. The resulting plant material

proved superior to the yeast-derived antigen in both priming

and boosting immunity in mice. Prime boost strategy in mice

with a single sub-immunogenic parenteral dose of yeast-

derived recombinant HBsAg and subsequent oral transgenic

potatoes led to the development of antibodies that immediately

peaked at >1,000 mIU/mL and were maintained at >200

mIU/mL for Þve months. This could be a useful immunization

strategy for developing countries.37 Tomatoes expressing

hepatitis B are being grown in guarded greenhouses. Enough

antigens for 4,000 vaccine doses were obtained from just 30

tomato plants.38 Transgenic lettuce is also being developed.

Rabies

Tomato plants expressing rabies antigens could induce

antibodies in mice.39 Alternatively, TMV may also be used.40

Transformed tomato plants using CaMV with the glycoprotein

(G-protein) gene of rabies virus (ERA strain) was shown to be

immunogenic in animals.41

HIV

Initial success in splicing HIV protein into CPMV has

been achieved.39 Two HIV protein genes and CaMV as

promoter were successfully injected into tomatoes witha needle, and the expressed protein was demonstrable by

polymerase chain reaction (PCR) in different parts of the

plant, including the ripe fruit, as well as in the second-

generation plant.42 Recently, spinach has been successfully

inoculated for Tat protein expression cloned into TMV. Each

gram of leaf tissue of spinach was shown to contain up to

300-500 µg of Tat antigen.43 Mice fed with this spinach

followed by DNA vaccinations resulted in higher antibody

titers than the controls, with the levels peaking at four weeks

post-vaccination.

STDs

Human papilloma virus type-11 (HPV-11) recombinant

VLPs produced in insect cells are immunogenic when given




www.ijmm.org

98 CMYK

98

T h i s

P D F i s

a v a i l a


o a d f r o

m

a s i t

e h o

s t e d b y M

e d k n


o n s

( w w w

. m e d k

n o w . c o

m ) .

orally to BALB/c mice. The response is dose-dependent,

conformationally-dependent and genotype-restricted. Thus,

VLPs may be effective oral immunogens for the prevention

of anogenital HPV disease.41

Anthrax

Tobacco leaves bombarded with pag gene (anthrax

protective antigen - PA) using a gene gun could express a protein structurally identical to the major protein present in

existing vaccine. Billions of units of anthrax antigen could

be produced. In addition, this vaccine was devoid of edema

factor and lethal factor, responsible for the toxic side effects.

The same anthrax antigen is now being put in tomato plants.44

Scientists are also trying to transform spinach by inoculating

it with TMV-expressing PA, as spinach might be a safer

vaccine.45

Others

Transgenic Arabidopsis thaliana plants are able to producea fusion protein consisting of LT-B and early secretory antigen

ESAT-6 of Mycobacterium tuberculosis, demonstrating both

the antigens by ELISA.46 Respiratory syncytial virus (RSV)

pneumonia takes a heavy toll of life of children below two

years of age, each year. RSV expressed in tomato and potato

plants has shown encouraging results in mice. Further clinical

trials are being planned. ‘Apple juice’-based RSV vaccine is

also being developed.47 Programs for development of new

vaccines against rotavirus and Streptococcus pneumoniae

have been instituted by program for appropriate technology

in health and GAVI, with work originating in developingcountries. For rotavirus, transgenic potatoes expressing VP7

could induce high titers of IgG and mucosal IgA in mice.48 For

parasites like malaria, advances in vaccine have been hindered

by the complex multistage life cycle of the parasite, its

inaccessibility to study and by its large genome. Nevertheless,

chimeric coat proteins of CPMV expressing malarial and foot-

and-mouth disease epitopes have been reported.39

Veterinary Sciences

The first patented edible vaccine to demonstrate

efficacy in animal trials was against the transmissible

gastroenteritis virus (TGEV) in pigs and was under planning

to be made commercially available.49 Vaccines against

porcine reproductive and respiratory syndrome (PRRS) and

other diseases like parvovirus are being investigated. Various

transgenic animal feeds are currently undergoing clinical trials

in pigs.50 Other candidates are diseases of pets, animals in

swine and poultry industries, draft and wild animals. Different

plant viral system patents exist (Table 3).

Passive Immunization

Just as any antigenic protein can be expressed in plants,

antibody molecules too can be expressed in plants. Protective

antibodies produced in plants are sometimes referred to as

“plantibodies.” To date, only four antibodies produced in

plants have the potential to be used as therapeutic agents, and

only IgG-IgA against Streptococcus mutans has been tested on

humans. Attempts are being made to formulate these secretory

antibodies into toothpaste to protect against tooth decay.

Human trials are also being planned. The other antibodies

have been tested on animals and also show great promise.Producing complete secretory antibodies was quite dif Þcult

Table 3: Plant viral system patents

Virus Target host Plant Mode of administration

Enterotoxigenic E. coli (ETEC) Humans Tobacco Oral

ETEC Humans Potato Oral

ETEC Humans Maize Oral

Vibrio cholerae Humans Potato Oral

Hepatitis B virus Humans Tobacco Injection (extracted protein)

Hepatitis B virus Humans Potato OralHepatitis B virus Humans Lettuce Oral

Norwalk virus Humans Tobacco O

Norwalk virus Humans Potato Oral (VL

Rabies virus Humans Tomato Intact glycoprotein

CMV Cytomegalo virus Humans Tobacco Immunologically related

protein

Rabbit hemorrhagic disease virus Rabbits Potato Injection

Foot-and-mouth disease Agricultural domestic animals Arabidopsis Injection

Foot-and-mouth disease Agricultural domestic animals Alfalfa Injection or oral

Transmissible gastroenteritis

coronavirus (TGEV) Pigs Arabidopsis InjectionTGEV Pigs Tobacco Injection

TGEV Pigs Maize Oral

Reproduced with permission41




www.ijmm.org

99 CMYK

99April-June 2007

T h i s

P D F i s

a v a i l a


o a d f r o

m

a s i t

e h o

s t e d b y M

e d k n


o n s

( w w w

. m e d k

n o w . c o

m ) .

because apart from four protein chains (two heavy, two light

chains), they contain two additional proteins, one of which

is added in vivo during secretion. This was Þnally achieved

by crossing four separate lines of transgenic tobacco plants.

This process occurring in plants can be exploited to great

advantage. Complex and ‘high molecular weight’ antigens can

be expressed in different lines of the same plant, optimally as

component parts, and cross-pollination between these linesof plants may yield a progeny producing the complex antigen

in its native conÞguration, obviating the need for any in vitro

manipulation. This would also make enormous economic

sense. Another advantage of IgA plantibodies is that major

proportion (57%) exists in the dimerized form, in contrast

to IgA produced in insect cells using Baculovirus, which

is mostly monomeric.51 Antibodies expressed in seeds are

preferred over antibodies in leaves, which risk early decay,

thus requiring immediate extraction. In contrast, antibodies

in seeds can be preserved for long at room temperature until

their extraction or consumption. This has been achieved

by linking antibody genes to a genetic “switch.” Injectable

materials are now being targeted. They are further associated

with challenges of puriÞcation.3 HBV antibodies in tobacco

plants are being investigated. A number of other plantibodies

like anti-HIV antibodies are under development. A cream

containing anti-HIV antibodies, which might help reduce

the risk of HIV transmission during sexual intercourse, has

been proposed. Monoclonal antibodies (Mabs) against STDs

like genital herpes have been expressed in soybean. When

compared with humanized anti-HSV-2 Mab expressed in

mammalian cell culture, they were found to be similar in

stability in human semen and cervical mucus over 24 hours,

with similar ef Þcacy in preventing vaginal HSV-2 infection

in mice.41

Other Potential Domains

Cancer therapy

Plants can make monoclonal antibodies for cancer

therapy in suf Þcient quantities. Soybean has been genetically

engineered to make monoclonal antibody (BR-96) as a

vehicle for targeting doxorubicin for breast, ovarian, colon

and lung tumors.3,39 Non-Hodgkin’s lymphoma is also beinginvestigated.

Birth control

TMV has been designed to express protein found in mouse

zona pellucida (ZB3 protein).3,39 When injected, resulting

antibodies were able to prevent fertilization of eggs in mice.

Chloroplast transformation

The chloroplast genome is maternally inherited; so

following chloroplast transformation, the protein would not be

present in pollen, thereby reducing the risk of transmission of

the transgene to neighboring crops or weed species by cross-

pollination.52 It may also result in accumulation of signiÞcantly

greater quantities of transgenic protein.

Role in autoimmune diseases

For unknown reasons, oral delivery of auto-antigens can

suppress immune activity by switching on suppressor cells

of the immune system, producing immunological tolerance.

For example, intake of collagen in arthritis patients resulted

in relief. This phenomenon is frequently seen in test animalsand is time- and dose-dependent. To induce tolerance,

antigen is required to be given repeatedly or continuously in

doses larger than the immunogenic dose. MisidentiÞcation

and misadministration of plants/fruits may unknowingly

induce tolerance. Hence it becomes essential to identify the

transgenic plant/fruit and to determine safe, effective doses

and feeding schedules, which would determine whether an

antigen would stimulate or depress immunity. Among the

autoimmune disorders that might be prevented or eased are

type 1 diabetes, multiple sclerosis, rheumatoid arthritis,

transplant rejection, etc.1,53 Potatoes expressing insulin and a

protein called GAD (glutamic acid decarboxylase), linked to

CT-B subunit, were able to suppress immune attack in a mouse

strain that would become diabetic and could delay the onset

of high blood sugar.54 Another diabetes-related protein is also

being investigated. Work on multiple sclerosis, rheumatoid

arthritis, lupus and transplant rejection is under progress. For

autoimmune diseases, production of increased amounts of

self-antigen in plants is still required.

Recombinant drugs/proteins

Apart from vaccines and antibodies, plants are also being

modiÞed by engineered virus inoculation to produce enzymes;

drugs such as albumin, serum protease and interferon, which

are otherwise dif Þcult or expensive to produce - for example,

glucocerebrosidase (hGC) production in tobacco plants for

treating Gaucher’s disease. This development would bring

down its cost thousand-fold.39 Transgenic tobacco plant

producing Interleukin-10 is being tested to treat Crohn’s

disease. Industrial processes for the large-scale production

of recombinant therapeutic proteins in plants have been

developed. Other novel compounds include an anti-viral

protein that inhibits the HIV virus in vitro, trichosanthin

(ribosome inactivator) and angiotensin-I (antihypertsensitive

drug).39 Transgenic plant-derived biopharmaceutical hirudin

(an antithrombin) is now being commercially produced. Food

crops are being increasingly used to produce pharmaceuticals

and drugs.

The Future of Edible Vaccines

The future of edible vaccines may be affected by resistance

to GM foods, which was reßected when Zambia refused GM

maize in food aid from the United States despite the threat of

famine.55 Transgenic contamination is unavoidable. Besides

pollen, transgenes may spread horizontally by sucking insects,

transfer to soil microbes during plant wounding/breakdown

of roots/rootlets and may pollute surface and ground water.56




www.ijmm.org

100 CMYK

100

T h i s

P D F i s

a v a i l a


o a d f r o

m

a s i t

e h o

s t e d b y M

e d k n


o n s

( w w w

. m e d k

n o w . c o

m ) .

Recently, a GM corn approved only for animal consumption

appeared in human foods. It was found growing from seeds

left behind from the previous plantings. Quarantine done to

prevent any further spread caused monetary loss, penalties and

possible criminal charges for alleged violation of the permit

to grow the gene-altered crops.55 This incident shows failure

at an elementary level. Overall, transgenic contamination

has cost the United States approximately $12 billion. Beforeendorsing such vaccines for human use, the WHO’s concerns

of quality assurance, efficacy and environmental impact

need to be addressed and GM crops in greenhouse facilities

should be rigidly controlled, and they need to be surrounded

by protective buffer crops. GM crops have not been able to

signiÞcantly increase yield or reduce herbicide/pesticide use,

as proposed. In a few cases, the instability of transgenic lines

and transgene inactivation have led to major crop failures.

Dangerous/harmful gene products or potent immunogens/

allergens may also be incorporated. Some are reported to

produce side effects - for example, cytokines induce sickness

and CNS toxicity; α-interferon causes dementia, neurotoxicity,

mood/cognitive changes, etc. Terminator crops with “suicide”

genes for male sterility, as a means of “containing” the spread

of transgenes, actually spread both male sterile suicide genes as

well as herbicide tolerance genes via pollen. Emerging multiple

herbicide-tolerant volunteers and weeds (super weeds) and

biotech-resistant pests demand the use of highly toxic herbicides

(atrazine, glufosinate ammonium, glyphosate).57

Few credible studies are there on the safety of GM foods.

An investigation on GM foods showing ‘growth factor’-

like effects in the stomach and small intestine of young ratswas not followed up to see whether CaMV-35S promoter

was responsible for this. This promoter is present in most

commercially grown GM crops today and is especially

unstable and prone to horizontal gene transfer, recombination

or mutations by random insertion. Random insertion of genes

can destabilize the genomes of its plant and animal hosts, and

the effects could ricochet through the neighboring ecosystem.

By facilitating horizontal gene transfer/recombination, genetic

engineering may contribute to emergence and re-emergence

of infectious, drug-resistant diseases, rise of autoimmune

diseases, cancers and reactivation of dormant viruses. Bacteriamay take up transgenic DNA in food in human gut. Antibiotic

resistance marker genes can spread from transgenic food to

pathogenic bacteria, making infections very dif Þcult to treat.

Minor genetic changes in pathogens can result in dramatic

changes in host spectrum and disease-causing potentials,

and inadvertently plants may become their unintentional

reservoirs. There is also the risk of creating altogether new

strains of infectious agents, like super viruses. By DNA

shuf ßing, geneticists can create in a matter of minutes in

the laboratory, millions of recombinant viruses that have

never existed in billions of years of evolution. This may be

misused for the intentional creation of bio-weapons. Genetic

engineering is inherently hazardous because it involves

creating vectors/carriers speciÞcally designed to cross wide

species barriers, like between plant and animal kingdoms, and

transferring genes by overcoming their defense mechanisms

which are physiologically operative against such genetic

assaults.58 Inadvertent birth of a Frankenstein would result in

unmitigated disaster. Naked DNA vaccines are perhaps the

riskiest, as these short pieces of DNA are readily taken up by

cells of all species and may become integrated into the cell’s

genome material. Unlike chemical pollutants, these smallDNA fragments can multiply and mutate indeÞnitely. Feeding

GM products like maize to animals may also carry risks, not

just for the animals but also for human beings consuming the

animal products. The ecological and environmental risks of

edible vaccines need to be considered. It is still a very crude

science and has a long way to go before it will be ready for

large-scale testing in people for combating infectious diseases

and for autoimmunity.

Increase in global area utilized in cultivating transgenic

crops from 1.7 to 44.2 million hectares from 1996 to 2000 and

the number of countries growing them from 6 to 13 reßectsthe growing acceptance of transgenic crops in both industrial

and developing countries.59 At least 350 genetically engineered

pharmaceutical products are currently under development in

the United States and Canada. Edible vaccines offer major

economic and technical beneÞts in the event of bioterrorism,

as their production can be easily scaled up for millions of

doses within a limited period of time (smallpox, anthrax,

plague, etc.).41

Conclusion

Edible plant-derived vaccine may lead to a future of safer and more effective immunization. They would overcome

some of the dif Þculties associated with traditional vaccines,

like production, distribution and delivery, and they can

be incorporated into the immunisation plans. They have

passed the major hurdles in the path of an emerging vaccine

technology. Before becoming a reality, the technical obstacles,

though all seem surmountable, need to be overcome. However,

with limited access to essential health care in much of the

world and with the scientiÞc community still struggling with

complex diseases like HIV, malaria, etc., a cost-effective, safe

and ef Þcacious delivery system in the form of edible vaccineswill become an essential component in our disease-prevention

arsenal.

References

1. Landridge W. Edible vaccines. Scienti fic Am 2000;283:66-71.

2. Ramsay AJ, Kent SJ, Strugnell RA, Suhrbier A, Thomson SA,

Ramshaw IA. Genetic vaccination strategies for enhanced

cellular, humoral and mucosal immunity. Immunol Rev

1999;171:27-44.

3. Moffat AS. Exploring transgenic plants as a new vaccine source.

Science 1995;268:658,660.

4. Available from: http://www.molecularfarming.com/plant-

derived-vaccines.html.




www.ijmm.org

101 CMYK

101April-June 2007

T h i s

P D F i s

a v a i l a


o a d f r o

m

a s i t

e h o

s t e d b y M

e d k n


o n s

( w w w

. m e d k

n o w . c o

m ) .

5. Webster DE, Thomas MC, Strugnell RA, Dry IB, Wesselingh

SL. Appetising solutions: An edible vaccine for measles. Med J

Aust 2002;176:434-7.

6. Available from: http://www.niaid.nih.gov/Þnal/infds/la_infds.

htm.

7. Giddings G, Allison G, Brooks D, Carter A. Transgenic

plants as factories for biopharmaceuticals. Nat Biotechnol

2000;18:1151-5.

8. de Aizpura HJ, Russell-Jones GJ. Oral vaccination. IdentiÞcation

of classes of proteins that provoke an immune response upon

oral feeding. J Exp Med 1988;167:440- 51.

9. Available from: http:/ /www.plantphys. net/art icle .

php?ch=21andid=216.

10. Available from: http://www.sabin.org/SVR/winter2001_9.htm.

11. Haq TA, Mason HS, Clement s JD, Arntze n CJ. Oral

Immunization with a Recombinant Bacterial antigen produced

in transgenic plants. Science 1995;268:714-6.

12. Yu J, Langridge WH. A plant-based multicomponent vaccine

protects mice from enteric diseases. Nat Biotechnol 2001;19:548-

52.

13. Nugent J, Po AL, Scott EM. Design and delivery of non-

parenteral vaccines. J Clin Pharm Ther 1998;23:257-85.

14. Ramshaw IA, Ramsay AJ. The prime-boost strategy:

Exciting prospects for improved vaccination. Immunol Today

2000;21:163-5.

15. Webster DE, Cooney ML, Huang Z, Drew DR, RamshawIA, Dry IB, et al. Successful boosting of a DNA measles

immunization with an oral plant-derived measles virus vaccine.

J Virol 2002;76:7910-2.

16. Available from: http://www.geocities.com/plantvaccines/

transgenicplants.htm.

17. Tacket CO, Mason HS, Losonsky G, Estes MK, Levine MM,

Arntzen CJ. Human immune responses to a novel Norwalk

virus vaccine delivered in transgenic potatoes. J Infect Dis

2000;182:302-5.

18. Kapusta J, Modelska A, Figlerowicz M, Pniewski T, Letellier M,Lisowa O, et al. A plant-derived edible vaccine against hepatitis

B virus. FASEB J 1999;13:1796-9.

19. Tacket CO, Mason HS, Losonsky G, Clements JD, Levine

MM, Arntzen CJ. Immunogenicity in humans of a recombinant

bacterial-antigen delivered in transgenic potato. Nat Med

1998;4:607-9.

20. Available from: http://www.biotech-info.net/edible_vaccines.

html.

21. Ruf S, Hermann M, Berger IJ, Carrer H, Bock R. Stable genetic

transformation of tomato plastids and expression of a foreign protein in fruit. Nat Biotechnol 2001;19:870-5.

22. Nemchinov LG, Liang TJ, Rifaat MM, Mazyad HM, Hadidi

A, Keith JM. Development of a plant-derived subunit vaccine

candidate against hepatitis C virus. Arch Virol 2000;145:2557-

73.

23. Modelska A, Dietzschold B, Sleysh N, Fu ZF, Steplewski K,

Hooper DC, et al. Immunization against rabies with plant-

derived antigen. Proc Natl Acad Sci USA 1998;95:2481-5.

24. Mor TS, Moon YS, Palmer KE, Mason HS. Geminivirus vectors

for high level expression of foreign proteins in plant cells.

Biotechnol Bioeng 2003;81:430-7.

25. Available from: http://www.sabin.org/SVR/winter2001_9.htm.

26. Pizza M, Giuliani MM, Fontana MR, Monaci E, Douce G,

Dougan G, et al. Mucosal vaccines: Non toxic derivatives of LT

and CT as mucosal adjuvants. Vaccine 2001;19:2534-41.

27. Tripurani SK, Reddy NS, Sambasiva Rao KR. Green revolution

vaccines, edible vaccines. Afr J Biotechnol 2003;2:679-83.

28. Mason HS, Lam DM, Arntzen CJ. Expression of hepatitis B

surface antigen in transgenic plants. Proc Natl Acad Sci USA

1992;89:11745-9.

29. Mason HS, Haq TA, Clements JD, Arntzen CJ. Edible vaccine

protects mice against Escherichia coli heat-labile enterotoxin

(LT): Potatoes expressing a synthetic LT-B gene. Vaccine

1998;16:1336-43.

30. Tacket CO, Mason HS, Losonsky G, Clements JD, Levine

MM, Arntzen CJ. Immunogenicity in humans of a recombinant

bacterial antigen delivered in a transgenic potato. Nat Med

1998;4:607-9.

31. Tacket CO, Mason HS, Losonsky G, Estes MK, Levine MM,

Arntzen CJ. Human immune responses to a novel Norwalk virus vaccine delivered in transgenic potatoes. J Infect Dis

2000;182:302-5.

32. Available from: http://www.isb.vt.edu/articles/apr9803.htm.

33. Yuki Y, Kiyono H. New generation of mucosal adjuvants for the

induction of protective immunity. Rev Med Virol 2003;13:293-

310.

34. Huang Z, Dry I, Webster D, Strugnell R, Wesselingh S. Plant-

derived measles virus hemagglutinin protein induces neutralizing

antibodies in mice. Vaccine 2001;19:2163-71.

35. Polack FP, Auwaerter PG, Lee SH, Nousari HC, Valsamakis A,Leiferman KM, et al. Production of atypical measles in rhesus

macaques: Evidence for disease mediated by immune complex

formation and eosinophils in the presence of fusion-inhibiting

antibody. Nat Med 1999;5:629-34.

36. Thanavala Y, Yang YF, Lyons P, Mason HS, Arntzen CJ.

Immunogenicity of transgenic plant-derived hepatitis B surface

antigen. Proc Natl Acad Sci USA 1995;92:3358-61.

37. Richter LJ, Thanavala Y, Arntzen CJ, Mason HS. Production

of hepatitis B surface antigen in transgenic plants for oral

immunization. Nat Biotechnol 2000;18:1167-71.

38. Available from: http://whyÞles.org/166plant_vaccines/3.html.

39. Prakash CS. Edible vaccines and antibody producing plants.

Biotechnol Develop Monitor 1996;27:10-3.




www.ijmm.org

102 CMYK

102

T h i s

P D F i s

a v a i l a


o a d f r o

m

a s i t

e h o

s t e d b y M

e d k n


o n s

( w w w

. m e d k

n o w . c o

m ) .

40. Available from: http://www.brown.edu/Courses/Bio_160/

Projects1999/rabies/vacc.html.

41. Available from: http://www.geocities.com/plantvaccines/

futureprospects.htm.

4 2 . A v a i l a b l e f r o m : h t t p : / / w w w . s c i e n c e d a i l y . c o m /

releases/2004/08/040816001548.htm.

43. Karasev AV, Foulke S, Wellens C, Rich A, Shon KJ, ZwierzynskiI, et al. Plant based HIV-1 vaccine candidate: Tat protein

produced in spinach. 2005;23:1875-80.

44. Available from: http://www.ucfalumni.com/Main/Articles.

asp?ArticleID=286andCategoryID=19andSectionID=3and.


releases/2003/03/030312072233.htm.

46. Rigano MM, Alvarez ML, Pinkhasov J, Jin Y, Sala F, Arntzen

CJ, et al. Production of a fusion protein consisting of the

enterotoxigenic Escherichia coli heat-labile toxin B subunit and

a tuberculosis antigen in Arabidopsis thaliana. Plant Cell Rep 2004;22:502-8.

47. Korban SS, Krasnyanski SF, Buetow DE. Foods as production

and delivery vehicles for human vaccines. J Am Coll Nutr

2002;21:212S-7S.

48. Wu YZ, Li JT, Mou ZR, Fei L, Ni B, Geng M, et al. Oral

immunization with rotavirus VP7 expressed in transgenic

potatoes induced high titers of mucosal neutralizing IgA.

Virology 2003;313:337-42.


releases/1999/09/990913145730.htm.

50. Available from: http://www.checkbiotech.org/blocks/dsp_

document.cfm?doc_id=3547.

51. Ma JK, Hiatt A, Hein M, Vine ND, Wang F, Stabila P, et al.

Generation and assembly of secretory antibodies in plants.

Science 1995;268:716-9.

52. Daniell H, Khan M, Allison L. Milestones in chloroplast genetic

engineering: An environmentally friendly era in biotechnology.Trends Plant Sci 2002;7:84-91.

53. Weiner HL. Oral tolerance for treatment of autoimmune diseases.

Ann Rev Med 1997;48:341-51.

54. Arakawa T, Yu J, Chong DK, Hough J, Engen PC, Langridge

WH. A plant based cholera toxin B subunit-insulin fusion protein

protects against the development of autoimmune diabetes. Nat

Biotechnol 1998;16:934-8.

55. Available from: http://www.newscientist.com/article.

ns?id=dn3073.

56. Available from: http://whyÞles.org/166plant_vaccines/4.html.

57. Available from: http://www.twnside.org.sg/title/ISPReportFinal-

AGM-FreeSustainableWorld2.pdf.

58. Ho MW, Steinbrecher R. Fatal ßaws in food safety assessment.

Environ Nutr Interact 1998;2:51-84.

59. Available from: http://www.gene.ch/genet/2001/Apr/msg00052.

html.

Indian Journal of Medical Microbiology

Source of Support: Nil, Conflict of Interest: None declared.

vol. 25, No. 2

Author Help: Reference checking facility

The manuscript system (www.journalonweb.com) allows the authors to check and verify the accuracy and style of references. The tool checksthe references with PubMed as per a predefined style. Authors are encouraged to use this facility before submitting articles to the journal.

• The style as well as bibliographic elements should be 100% accurate to get the references verified from the system. A single spellingerror or addition of issue number / month of publication will lead to error to verifying the reference.

• Example of a correct styleSheahan P, O’leary G, Lee G, Fitzgibbon J. Cystic cervical metastases: Incidence and diagnosis using fine needle aspiration biopsy.Otolaryngol Head Neck Surg 2002;127:294-8.

• Only the references from journals indexed in PubMed would be checked.

• Enter each reference in new line, without a serial number.

• Add up to a maximum 15 reference at time.

• If the reference is correct for its bibliographic elements and punctuations, it will be shown as CORRECT and a link to the correct

article in PubMed will be given.

• If any of the bibliographic elements are missing, incorrect or extra (such as issue number), it will be shown as INCORRECT and link topossible articles in PubMed will be given.

Documents

Edible Vaccines