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Genetic Recombination
215
understanding of the underlying mechanisms. Analgesics, especially opioids, are relatively
effective, but their use is restricted and limited by patient tolerance. Gene transfer is a novel
means of expressing identified transgenes, including those for a-aminobutyric acid, glutamic
acid decarboxylase,
-opioid receptor and neurotrophic factors. The gene delivered into the
nervous system by retrograde axonal transport, followed by its repeated intramuscular transfer
using liposomes containing the haemagglutinating virus of Japan (HVJ) has given positive
results of therapy.
7. Gene transfer in cellular immunotherapy: Adoptive cellular therapy is a potentially
powerful method of eradicating established tumours. As established in animal models and
subsequent clinical studies, the T cells have been found to be particularly potent effector cells.
It is apparent that the stimulation of certain subpopulations of T cells that are reactive to tumour
antigens can lead to more therapeutic T cells. The use of genetransfer techniques has facilitated
development of more effective and specific methods to generate these tumour-specific T cells.
Another area of tremendous interest is in the adoptive transfer of DCs manipulated to present
tumour antigen to resting naive T cells. Gene transfer techniques offer more optimal ways to
generate therapeutic dendritic cells (DCs). Adoptive immunotherapy may ultimately have its
greatest use in patients undergoing cellular rescue after ablative chemotherapy. The infusion
of immunocompetent T cells, genetically modified stem cells or the programmed DCs may
offer a possibility to direct a patient’s immune response to eliminate residual microscopic
disease.
8. Genetic disease treatment: It was well before the advent of recombinant DNA technology
that the interest in gene therapy was shown in the middle of 1960. At that
time, the first
speculation about the possible treatment of genetic disorders by introducing functional genes
via virus-mediatedgene transfer was made. In 1990, the first gene therapy clinical trial for the
treatment of patients with melanoma was conducted. The results of this study indicated that
retrovirus-mediated gene transfer in patients was safe.
Gene Transfer Technology in Other Areas
In grapevine, current lack of high-throughput genetic techniques (e.g. induced mutant collections)
and the difficulties associated with genetic mapping ( allele diversity, chimerism, and generation
time) highlight the critical role of transgenic technology for characterizing gene function. The
presence of original features in grapevine, including perennial status, vegetative architecture,
inflorescence/tendril, flower organization (corolla) and fleshy fruit of considerable acidity with
various flavonoids compounds, makes functional genomics an essential approach to link a gene to
a trait.
The gene transfer techniques are important in the production of enzyme reference materials,
which could be of great therapeutic and biochemical significance. The gene transfer techniques
are also used in the management of marine resources. Tetrodotoxin obtained from frog and
pseudopterosin extracted from Caribbean coral are used as CVS stimulant and anti-inflammatory
agent, respectively. The technique of gene transfer can systematize management of important
marine sources.
QUESTIONS
1. Define genetic recombination and describe various methods of genetic recombination.
2. Describe the applications of genetic engineering for production as pharmaceuticals.
3. Differentiate amongst transformation, conjugation and transduction.
4. Define protoplast fusion. Write a note on its applications.
5. Write short notes on screening of soil, historical development of antibiotics and Humatrope.
6. What is gene cloning? Give its applications.
7. Explain the different methods of genetic recombination.
8. Define the term ‘cloning’ and give its applications.
9. What is genetic recombination? Name three types of genetic recombination.
10. Write in brief about protoplast fusion.
11. What are gene transduction and transformation?
INTRODUCTION
Medicine evolves the science and art of healing humans. It consists of a variety of health care
practices aimed at maintaining wellness by the prevention and treatment of illness. Before
introduction of medicine, arts of healing were based on ritual practices that were developed out of
religious and cultural traditions.
Besides medical practitioners, many highly trained health professionals are involved in
the delivery of modern health care. These include nurses, emergency medical technicians and
paramedics, radiographers, pharmacists, dieticians, physiotherapists, respiratory therapists, speech
therapists, occupational therapists and bioengineers. All these segments of health care professionals
contribute to sound public health.
Public Health
WHO defines public health as the art and science of preventing disease, prolonging life and
promoting health and efficiency through organized community efforts. The spectrum of activities
in public health covers the following:
1. Education of the individuals in the principles of hygiene
2. Organization of medical and nursing services for the early diagnosis and preventive treatment
of diseases
3. Sanitational issues for effective control of communicable infections. It also involves total
development of social machinery that ensures a standard of living adequate for the maintenance
of health to every individual in the community.
The major objectives of the public health are identified as follows:
1. Promotion of health and efficiency
2. Prevention of disease and prolongation of life
Medicine and Edible Vaccines
14
CHAPTER
Introduction
Edible Vaccines
Questions
Textbook of Pharmaceutical Biotechnology
218
3. Elevation of the standard of living adequate for health maintenance
4. Provision of the right to health and longevity
Social medicine: It is defined as the science and art of preventing disease, prolonging life and
promoting health and efficiency of population by intercepting social factors that have direct or
indirect relationship with the disease process.
Community medicine: It is a branch of medical practice that is concerned with promoting,
maintaining and when necessary, restoring the health of human communities, rather than with
clinical use of individual patients. It takes into account the following:
1. Defined consumer population or community
2. Comprehensive and integrated service
3. Defined delivery system of health care
4. An epidemiological understanding of community health problem and
5. Management-oriented approach for the solution of these problems.
Basic Sciences of Medicine
The subject of medicine has gained its strength from various disciplines of biosciences. They
include the following:
1. Anatomy: It is the study of the physical structure of organisms.
2. Biochemistry: It is an interdisciplinary science, involving study of the chemistry taking place
in living organisms, especially the structure and functions of their chemical components.
3. Biomechanics: The study of the structure and function of biological systems by means of the
methods of mechanics.
4. Biostatistics: Knowledge of statistics as applied to biological studies is essential in the
planning, evaluation and interpretation of medical research. It is also essential to understand
epidemiology and evidence-based medicines.
5. Biophysics: It is an interdisciplinary science that uses the methods of physics and physical
chemistry to study biological systems.
6. Cytology: It is the microscopic study of individual cells.
7. Embryology: It refers to the study of early development of organisms.
8. Epidemiology: It covers the study of the demographics of disease processes and includes, but
is not limited to, the study of epidemics.
9. Genetics: It is the study of genes and their role in biological inheritance.
10. Histology: It is the branch of science dealing with the study of structures of biological tissues
by light microscopy, electron microscopy and immunohistochemistry.
11. Immunology: It is the study of immune system, which includes the innate and adaptive
immune system in humans.
12. Microbiology: The study of microorganisms, including protozoa, bacteria, fungi and virus.
13. Molecular biology: It is the study of molecular underpinnings of the process of replication,
transcription and translation of the genetic material.
Medicine and Edible Vaccines
219
14. Neuroscience: It includes those disciplines of science that are related to study of the nervous
system. A main focus of neuroscience is on biology and physiology of the human brain and
spinal cord.
15. Pathology: It is the science dealing with study of disease, the causes, course, progression and
resolution thereof.
16. Pharmacology: It is that branch of bioscience that deals with the study of drugs and their action
on living organism.
17. Photobiology: It is the study of interactions between nonionizing radiation and living organisms.
18. Physiology: It is the study of normal functioning of the body and the underlying regulatory
mechanisms.
19. Radiobiology: It encompasses study of the interactions between ionizing radiation and living
organisms.
20. Toxicology: It is the study of hazardous effects of drugs and poison on human beings.
EDIBLE VACCINES
Vaccines play an important role in the health care system of any country. Many diseases are
eradicated, and the spread of many ailments are effectively controlled by the use of live or attenuated
causative agents as vaccines. However, high cost of production, maintaining a chain for vaccine
distribution, cultivation of pathogens, poor reliability of the attenuation process, dangers due to
contamination attached to the production and use of many free or attenuated vaccines are the main
hurdles in the production of vaccines.
Plants may serve as useful production units for vaccines, since large amounts of antigens
could be produced at a relatively low cost using agricultural technique, instead of expensive
and sophisticated cell culture-based expression systems. Antigens expressed in a plant can be
administered orally as edible parts of the plant or by parenteral routes (IM or IV) after isolation and
purification from the plant tissue. The edible part of the plant to be used as vaccine is usually fed
in raw condition in order to prevent possible denaturation that takes place during cooking. It also
enables to avoid cumbersome purification protocols.
For production of edible vaccines, it is always desirable to select a plant whose products are
consumed raw to avoid degradation during cooking (Table 14.1).
Table 14.1 Some antigens produced by edible plants
Antigen Plant
CT-B Potato
Foot and mouth virus
Arabidopsis
Herpes virus B surface antigen Tobacco
Human cytomegalovirus glycoprotein B Tobacco
Rabies glycoprotein Tomato
Abbreviation: CT-B, cholera toxin B subunit.
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The production of edible vaccines was first reported in 1990 in tobacco (at 0.02% of total leaf
protein level). The production of vaccines in potatoes was reported in the bacterium, Agrobacterium
tumefaciens, commonly used to deliver the DNA for bacterial or viral antigens. A plasmid carrying
the antigen gene and an antibiotic resistance gene are incorporated into the bacterial cells. The cut
piece of potato leaves are exposed to an antibiotic that can kill the cells lacking the new genes. The
surviving cells can multiply and form a callus. This callus is allowed to sprout shoots and roots that
are grown in soil to generate plants. In about three weeks time, the plants bear potatoes with antigen
vaccines. The examples of antigen vaccines in plants are given in Table 14.2.
The antigens in transgenic plants are delivered through bioencapsulation. The tough outer
wall of plant cells protects them from gastric secretions and finally breaks up in the intestine.
The antigens are released, taken up by microfold cells (M cell) 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. This generates serum immunoglobulin
G ( IgG), immunoglobulin E ( IgE) responses, local immunoglobulin A ( IgA) response and memory
cells, which would promptly neutralize the attack by the real infectious agents (Fig. 14.1).
In future, the global immunization program could be possible with edible vaccines. The
distinctive advantages with the production of edible vaccines are as follows:
1. Low cost of production
2. Separation and purification is easy
3. Pathogenic contamination can be avoided, safe for use
4. Aseptic conditions are not required for oral immunization
5. No constricted criteria for its storage; it can be stored near the site of use
Important Steps in the Production of Edible Vaccines
For production of edible vaccines or antibodies, it is desirable to select a plant whose products
are consumed raw to avoid degradation during cooking. Thus, plants like tomato, banana and
cucumber are generally the plants of choice. While the expression of a gene that is stably integrated
Table 14.2 Production of antigen vaccines from transgenic plants
Protein Plant
Rabies virus glycoproteins Tomato
CT-B protein Potato, tobacco
HBsAg Tobacco
Norwalk virus capsid protein Tobacco
Protein of foot-and-mouth disease virus Tobacco
Mouse glutamate decarboxylase
Arabidopsis
Glycoproteins of swine transmissible Potato
Gastroenteritis coronavirus protein
Arabidopsis
Abbreviations: CT-B, cholera toxin B subunit; HBsAg, hepatitis B surface antigen.
Medicine and Edible Vaccines
221
into the genome allows maintenance of the material in the form of seeds, some virus-based vectors
can also be used to express the gene transiently to develop the products in a short period.
1. First generation: Foreign DNA can be introduced into plant’s genome 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 circulartumour-inducing (Ti) plasmid, which enables it to infect
plant cells, integrate into their genome and produce a hollow tumour (crown gall tumour), where
it can live. This ability can be exploited to insert foreign DNA into plant genome. Prior to this, the
plasmid needs to be disarmed by deleting the genes for auxin and cytokinin synthesis, so that it
does not produce tumour.
2. Second generation: 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 are obtained by crossing two
plant lines harbouring different antigens. Adjuvant may also be coexpressed along with the antigen
in the same plant.
Approaches to mucosal vaccine formulation include the following:
1. Gene fusion technology, creating nontoxic derivatives of mucosal adjuvants
2. Genetically inactivating antigens by deleting an essential gene
Immunogenicity
Antigen
DNA for epitope
Virus with chimeric coat protein
Plant infection
Virus infected plant
Isolation of chimeric virus
Extraction of antigen Feeding of edible vaccines
— Transformation
—Regenerated
Gene
Plasmid transformaton vector
Plant of choice
Transgenic plant
Figure 14.1 Antigens produced in transgenic plants.
Textbook of Pharmaceutical Biotechnology
222
3. Coexpression of antigen and a cytokine that modulates and controls mucosal immune response
4. Genetic material itself that allows DNA/ RNA uptake and its endogenous expression in the
host cell.
Chimeric virus: Certain viruses can be redesigned to express fragments of antigenic proteins
on their surface, such as cowpea mosaic virus (CPMV), alfalfa mosaic virus, tobacco mosaic
virus (TMV), cauliflower mosaic virus (CaMV), potato virus X and tomato bushy stunt virus.
Technologies involved are overcoat and epicoat technology.
It may even be possible to present
a cocktail of specific HIV epitopes on the surface of the plant virus. CPMV is genetically and
thermally stable and can survive in highly acidic condition (pH 1) for 1 h. Wide range of epitopes
has been expressed in CPMV. This includes HIV-1 gp41 and gp120, human rhinovirus, canine
parvovirus, foot-and-mouth disease virus, fungal epitopes, mammalian epitopes from hormones or
from colon cancer cells and protozoan epitopes from Plasmodium falciparum.
Challenges in the Production of Edible Vaccines
Three successful human clinical trials have shown that adequate doses of antigen can be achieved
with plant-based vaccines. To determine the right dosage, one needs to consider the age and weight
of the person, 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 of eat or eat all and throw it up later. Too low a
dose would fail to induce antibodies and too high a dose would instead cause tolerance. Giving 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. Attempts at boosting the
amount of antigens often lead to stunted growth of plants and reduced tuber/fruit formation, as too
much mRNA from the transgene causes gene silencing in plant genome.
Some of the techniques to overcome these limitations are as follows:
1. Optimization of coding sequence of bacterial/viral genes for expression as plant nuclear genes
2. Expression in plastids
3. Plant viruses expressing foreign genes
4. Coat-protein fusions
5. Viral-assisted expression in transgenic plants
Nonscientific Challenge
At present, small companies are undertaking most of the research on edible vaccines with markets of
developing countries in view. A few international aid organizations and some national governments
are rendering support, but the effort remains largely underfunded.
Regulatory Issues
It is still unclear whether the edible vaccines would be regulated under food, drugs or agricultural
products. The licensing of vaccine component antigen itself—genetically engineered fruit or transgenic
seeds—is not yet clear.
Medicine and Edible Vaccines
223
Clinical Trials
Antigen expression in plants has been successfully shown in the past, like heat-labile enterotoxin
subunit B (LT-B) of enterotoxigenic Escherichia coli (ETEC) in tobacco and potato, rabies virus
G protein in tomato, HBsAg in tobacco
and potato, Norwalk virus in tobacco and potato and CT-B
of Vibrio cholerae in potato. Ethical considerations usually preclude clinical trials from directly
assessing protection, except in a few cases.
Some Edible Vaccines Produced by Plants
1. Vaccines of potato: The transgenic potatoes were created
and grown by Charles Arntzen, Hugh S. Mason and their
colleagues at the Boyce Thompson Institute for Plant
Research, an affiliate of Cornell University. The volunteers
ate bite-sized pieces of raw potatoes, which had been
genetically engineered to produce part of the toxin secreted
by the E. coli. An edible vaccine could stimulate an immune
response in humans.
2. Vaccines of tobacco: In 1990, the first report of the production
of edible vaccine in tobacco—at 0.02% of total leaf protein
level—was published in the form of a patent application
under the International Patent Cooperation Treaty. Since
acute watery diarrhoea is caused by enterotoxigenic E. coli
and V
. cholerae that colonize the small intestine and produce
one or more enterotoxin, an attempt was made towards the
production of edible vaccine by expressing LT-B in tobacco.
3. Vaccines of banana: For edible vaccines or subunit
vaccinations, bananas can be conveniently used. The
advantage of bananas as desired vectors is that they can be
eaten raw as compared to potatoes or rice that needs to be
cooked and banana can also be consumed in a pure form.
4. Vaccines of tomato: Tomatoes serve as ideal candidates for
HIV antigen, unlike many other transgenic plants that carry
the protein. Tomatoes are edible and immune to any thermal
process, which helps retain its healing capabilities. The
introduction of the artificial protein gene into the tomato
germs was accomplished with the help of a needle. The germs
were then cultivated on a special nutrient medium. The plants
that grew roots were planted into the soil and grown in the
hothouse until they reached maturity and produced fruit.
The plants were then tested for the protein that could be
Textbook of Pharmaceutical Biotechnology
224
found in the leaves and the tomato plant itself.
.
5. Vaccines of maize: More than 2 billion people are infected
every year with hepatitis B, and about 350 million of these
are at high risk of serious illness and death from liver damage
and liver cancer. The Egyptian scientists have genetically
engineered maize plants to produce a protein used to make
hepatitis B virus vaccine.
6. Vaccines of rice: When the predominant T cell epitope
peptides are derived from Japanese cedar, pollen allergens
are expressed in rice seeds and are delivered to the mucosal
immune system. The development of an allergic immune
response of the allergen-specific T
h
2 cell is suppressed. It
is also observed that not only specific IgE production and
release of histamine from mast cells are suppressed, but also
inflammatory symptoms of pollinosis, such as sneezing,
are also found to be suppressed. These results indicate the
feasibility of using an oral immunotherapy agent derived
from transgenic plants that accumulate T-cell epitope
peptides of allergens for allergy treatment.
Advantages and Limitations of Edible Vaccines
Edible vaccines have many advantages over traditional injected vaccines. Edible vaccines are
cost effective and can be administered by different routes. They lower the risk of contamination
and eliminate the cost of transportation. The transgenic plants can be cost efficient in storage,
preparation, production and transportation. These transgenic plants do not require cold chain
storages. The various advantages of edible vaccines are enlisted as follows:
1. Production of vaccines with low costs
2. Easy for mass production system by breeding compared to an animal system
3. Eliminate cost of transportation
4. Edible plants are very effective as delivery vehicles for inducing oral immunization
5. Excellent feasibility of oral administration in comparison to injection
6. Easy for separation and purification of vaccines from plant materials
7. Convenience and safety in storing and transporting vaccines
8. Adjuvant for immune response is not necessary
9. Effective prevention of pathogenic contamination from animal cells
10. Effective retention of activity of vaccines by controlling the temperature in plant cultivation
11. Storage near the site of use
12. Heat stable, eliminating the need for refrigeration
13. Antigen protection through bioencapsulation is possible
14. Reduced need for medical personnel and sterile injection condition
15. Subunit vaccine (not attenuated pathogens) means improved safety