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International Journal of Research in Biomedicine and Biotechnology
Universal Research Publications. All rights reserved

Review Article
DNA VACCINE: A MODERN BIOTECHNOLOGICAL APPROACH
TOWARDS HUMAN WELFARE AND CLINICAL TRIALS.
Uttam Kumar1*, Sumit Kumar2, Shiju Varghese2, Rohit Chamoli3, Priyanka Barthwal4
2
Doon College of Agriculture Science and Technology Dehradun, Uttarakhand, India
3
Chinmaya Degree College, Haridwar, Uttarakhand, India
4
Govind Ballabh Pant College of Engineering, Garhwal, Uttarakhand, India
Corresponding author: Uttam Kumar, 1*Forest Research Institute, Dehradun, Uttarakhand. India. Cellular contact:
+919458105299, Email: ut.uttam@gmail.com
Received 07 March 2013; accepted 14 March 2013
Abstract
Vaccination is need of todays world for prevention of diseases. Vaccines reduce the mortality rates in the world from
infectious diseases such as measles, polio and diphtheria. The concept of vaccination is very old. Conventional vaccines
(First generation vaccines) are composed of Live or Attenuated microorganisms. But they may have some problems, so
further research is going on for development of a vaccine which cost-effective and having specific immune responses.
DNA vaccines are third generation vaccines and protect an organism from diseases by injecting it with genetically
engineered DNA. DNA vaccines are made up of bacterial plasmid. DNA vaccine is able to produce both humoral and cell-
mediated immunity. A single DNA vaccine is able to produce immunity for two or more diseases. In this review we are
discussing about the preparation, insertion, mechanism, advantages and disadvantages of DNA vaccines. DNA vaccines
have a bright future ahead.
2013 Universal Research Publications. All rights reserved
KEYWORDS: DNA Vaccine, Gene gun method, Mechanism of Action, Future.
INTRODUCTION immunization and has been worked on the vaccines for
WHO estimated that 80% of illness in the world is due to Anthrax and Rabies. Pasteur created an attenuated form of
various diseases which cause more than 20 million deaths virus and used for immunization [5, 6]. Vaccines are
per year [1]. Vaccines play a major role in prevention of composed of Antigens that artificially induces the bodys
diseases. Vaccination is a cost-effective measure for immune system to produce antibodies, so that body become
disease prevention. Vaccines reduce the mortality rates in resistant against particular disease.
the world from infectious diseases such as measles, polio Conventional vaccines (First generation vaccines) are
and diphtheria. composed of Live or Attenuated microorganisms. They
The concept of vaccination has been around for centuries. requires whole microorganism for vaccine preparation.
Edward Jenner and Louis Pasteur were made first attempt Although vaccines are very useful for disease prevention,
for human diseases. A powder was derived from crusts of but the conventional vaccines also have some problems
small pox lesions and used first time in 15 th century. This such as the attenuated forms of a pathogen can revert to a
powder was inserted into body with the help of a pin or harmful form and may still be able to cause disease. So, to
poking device [2]. This process was called as variolation. reduce the risk rates second generation vaccines were
These practices were not meant to save human lives but developed. These are subunit vaccines, composed of
used for preserve the beauty of a young woman. After that defined protein antigens or recombinant protein
vaccination was originated, when Edward Jenner created components [7].
the first successful vaccine against small pox in 1796. He DNA vaccines are called as third generation vaccines.
injected the infectious material from a woman with cowpox Recombinant DNA technology plays an important role in
into the arm of young boy; the boy became resistant from preparation of these vaccines. DNA vaccines are made up
life-threatening viruses [3]. Smallpox was the first disease of small, circular piece of bacterial DNA that has been
which has been prevented by scientists by intentionally genetically engineered for the production of two or more
inoculating individuals at risk with infecting agent [4]. antigens from a single pathogen. When vaccine DNA is
In 1885, Louis Pasteur became involved in the practices of inserted into the host cells of body, the inner-machinery

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17
of cells converts that DNA into the pathogenic proteins.
These proteins are recognized as foreign, then the immune
system triggers a range of immune responses [8, 9].
MOLECULAR TOOLS FOR DNA VACCINES
DNA vaccines are prepared on the basis of Recombinant
DNA technology. RDT is used for preparation of gene
sequences which are not found in biological organisms.
These sequences are prepared by transferring genetic
materials (DNA sequences) one organism to another
organism. In this technology basically following tools are
using:-
Plasmid (Vectors):- Plasmid is a DNA molecule, which
has capacity of replication independently of the
chromosomal DNA [22]. Plasmids are double stranded in
nature and usually are circular. Plasmids occur naturally in
bacteria, but sometimes are present in eukaryotic
organisms. Plasmid is an important tool for preparation of
DNA vaccines. Plasmids are generally using for
multiplication of gene of interest by inserting the desired
gene into the plasmid.
Nucleases: - Nucleases degrade DNA molecules by
breaking the phosphodiester bonds that binds nucleotides to
each-other in a DNA strand. These are of two types-
Endonucleases and Exonucleases. Exonuclease removes
one nucleotide at a time from the end of a DNA molecule.
While Endonucleases are able to break internal
phosphodiester bonds within a DNA molecule.
Ligases: - DNA ligase enzyme uses for repairing of single-
stranded breaks in double-stranded DNA and also joins
together two individual fragments of double-stranded
DNA.
DNA modifying enzymes: - Several enzymes modify
DNA molecules by addition or removal of specific
chemical groups. Some are as following:-
a. Alkaline Phosphatase- It removes the phosphate
group present at 5 terminus of a DNA molecule.
b. Polynucleotide kinase- It has the opposite effect to
alkaline phosphatase.
c. Terminal deoxinucleotidyl transferase- It adds one
or more deoxirybonucleotides onto the 3 terminus of a
DNA molecule.
Topoisomerases: - The final tool for recombinant DNA
technology is topoisomerase. These enzymes are able to
change the conformation of covalently bind-circular DNA
(plasmid) by introducing or removing supercoiling.
Fig1. STRUCTURE OF DNA VACCINE [23].
International Journal of Research in Biomedicine and Biotechnology 2013; 3(1): 17-20
18
PREPARATION OF DNA VACCINES
DNA vaccines are made up of bacterial plasmid [Fig 1].
Plasmid which is used in DNA-based vaccines normally
has two units: Antigen expression unit and The Production
unit. Antigen expression unit is made up of promoter
sequences, followed by antigen-coding and polyadenylation
sequences. The Production unit is composed of bacterial
sequences which are useful for amplification and selection
of plasmid [10]. Once the vaccine plasmid is constructed,
then it is transferred into bacteria. Then this DNA acts as
the vaccines [11].
INSERTION OF DNA VACCINE INTO ANIMALS
Vaccinated DNA can be transferred into animal tissues by
several methods. Two most useful methods are injection of
DNA in saline, by using a hypodermic needle and Gene
gun method. DNA in saline is normally injected
intramuscularly (i.m.) in skeletal muscle, or intradermally
(i.d.) to the extracellular spaces. This procedure can be
done by Electroporation [12], by temporarily damaging
muscle fibers with myotoxins like bupivacaine; or by using
saline or sucrose solutions [8]. Immune response evoked by
DNA vaccine can be affected by several factors, such as
needle type [13], needle alignment, speed of injection,
volume of injection, muscle type, age, sex and
physiological conditions of animal being injected [8].
Gene gun method is the most common method of plasmid
DNA insertion. In this method plasmid DNA is absorbed
on to Gold or tungsten micro particles into the target cells
[8,14].This process is also calledas Micro projectile [Fig-2].
Various amounts of DNA can be transferred according to
the method of insertion. By saline injection 10g-1mg and
by gene gun method 100 to 1000 times less then
intramuscular saline injection can be injected [15].
Fig.2. INSERTION OF DNA VACCINE INTO ANIMALS
[23].
MECHANISM OF ACTION OF DNA VACCINES
When a plasmid DNA is injected into skin or muscle, then
protein is produced endogenously and intracellular as small
antigenic peptides by the proteases of the host. These
peptides then transferred to the lumen of the endoplasmic
reticulum (E.R.) by membrane associated transporters. In
the E.R., these peptides bind with class-I MHC molecules.
Then these peptides are presented on the cell surface of
class-I MHC molecules and stimulate CD8+ cytotoxic T-
cells (CTL) and they evoke cell mediated immunity. CTL
inhibit viruses through two processes- cytolysis of infected
cells and non-cytolysis like cytokinin production [16].
The antigenic proteins can also be presented by class-II
MHC molecules. In this process antigen presenting cells
stimulates the CD4+ helper T-cells. These CD4+ cells can
recognize the peptides of exogenous proteins that were
endocytosed by APCs, then these peptides degraded into
fragments and loaded onto class-II MHC molecules. By the
CD4+ cells, B-cells are stimulated and enhance the
antibody production [10].
Fig.3. MECHANISM OF ACTION OF DNA VACCINE
[23].
ADVANTAGES OF DNA VACCINES
DNA vectors play an important role in clinical applications,
where large-scale production is not easily managed by
conventional vaccines and other vaccines [17].
Viral mediated gene transfer by genetically modified
lentiviruses, adenoviruses and retroviruses is very useful
because it has high transfection efficiency and stability
[18].
DNA vaccines have no risks like conventional vaccines.
Immune response can be evoked by both class-I and class-
II molecules. This is a unique feature of DNA vaccines
[19].
It is possible to make a single DNA vaccine which can
encodes for several antigens or proteins.
DNA vaccines are safe than live attenuated which can
cause infection in vivo. It is studied that after multiple
immunization anti-DNA antibodies are not produced [20].
Oligonucleotides can also be transferred by DNA vaccines.
These oligonucleotides can alter gene splicing or gene
expression such as siRNA [21].
LIMITATIONS OF DNA VACCINES
Major limitation of DNA vaccines is that these are useful
only for protein antigens. DNA vaccines cannot be used for
non-protein based antigens.
A major risk with DNA vaccine is that they can affect the
genes which control the growth of cells.
It may be possible to inducing antibody production against
DNA.
Tolerance can be generated to the antigens produced by
DNA vaccines.
FUTURE OF DNA VACCINES AND CONCLUSIONS
The field of DNA vaccines is big and it will continue to
advance and new approaches to enhance the
immunogenicity of DNA vaccines. DNA vaccines could
help in improving applications for gene therapy and gene
vaccination. Improved gene expression and better genetic
engineering of plasmid DNA may increase antibody
response to the gene products.
The DNA vaccination is a new branch of medical sciences.
Studies and research are going on but some DNA vaccines
International Journal of Research in Biomedicine and Biotechnology 2013; 3(1): 17-20
19
have moved to clinical trials. In this review we have
discussed that how DNA vaccines are advanced then
conventional and second generation vaccines. Antigen
expression of DNA vaccines can be improved by inclusion
of Adjuvant in the formulation, or as immune modulators to
improve the immunogenicity. If immunotherapy is
combined with chemotherapy and radiotherapy, then it
could yield systemic or additive therapeutic results.
In future, DNA vaccines may prepare by noval approaches
in the form of microspheres, nanobeads or micro-
nanoprojection. So vaccination will become painless,
effective and safe needle-free routes such as the intranasal
or the oral route. These nanovaccines are in experimental
stage at present but may have a great future ahead.
DNA vaccines are able to produce more than one antigen,
so we can make one medicine for many diseases. DNA
vaccines against lethal diseases such as AIDS, Cancer,
Rabies, Malaria and Diabetes can be available. Now-a-days
DNA vaccines are in their early phase but one day is going
to be the vaccines of next generation.
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