Download Definition DNA vaccination is a third generation novel technique

 Definition
DNA vaccination is a third generation novel technique which includes introducing
genetically engineered DNA to produce immune reaction so as to immunize the population
against certain disease[1]
DNA vaccines contain the nucleotides encoding an antigenic portion of the virus such as
the viral core region or envelope region. The DNA is taken up into the host cell, translated,
and the protein product expressed[3]
offers new approaches for the prevention and therapy of several diseases of
both bacterial and viral origin. DNA immunisation has also emerged in the last
decade as a strikingly novel approach to immunoprophylaxis, and has been
widely used in laboratory animals and non-human primates over the last
decade to induce antibody and cellular immune responses[4]
 History
Experiments outlining the transfer of DNA into cells of living animals were reported as early
as 1950 [2].
In 1983, Enzo Paoletti and Dennis Panicali developed a strategy to produce recombinant
DNA vaccines by using genetic engineering to transform smallpox vaccine into vaccines that
may be able to prevent other diseases. They altered the DNA of cowpox virus by inserting a
gene from other viruses (namely Herpes simplex virus, hepatitis B and influenza) [1].
In 1992, scientists Tang and Johnson reported that the delivery of human growth hormone
in a expression cassette in vivo resulted in production of detectable levels of the growth
hormone in host mice. They found that these inoculated mice developed antibodies against
the human growth hormone; they termed this immunization procedure "genetic
immunization", which describes the ability of inoculated genes to be individual immunogens
[2].
In 2016 a DNA vaccine for the Zika virus began testing at the National Institutes of Health.
The study was planned to involve up to 120 subjects between 18 and 35. Separately, Inovio
Pharmaceuticals and GeneOne Life Science began tests of a different DNA vaccine against
Zika in Miami. The NIH vaccine is injected into the upper arm under high pressure.
Manufacturing the vaccines in volume remains unsolved[1]
 Plasmid Vector Design
These are plasmids that usually consist of a strong viral promoter to drive the in vivo
transcription and translation of the gene (or complementary DNA) of interest.[11] Intron A
may sometimes be included to improve mRNA stability and hence increase protein
expression.[12] Plasmids also include a strong polyadenylation/transcriptional termination
signal, such as bovine growth hormone or rabbit beta-globulin polyadenylation
sequences.[1][2][13] Multicistronic vectors are sometimes constructed to express more
than one immunogen, or to express an immunogen and an immunostimulatory protein.[14]
Because the plasmid is the “vehicle” from which the immunogen is expressed, optimising
vector design for maximal protein expression is essential.[14] One way of enhancing protein
expression is by optimising the codon usage of pathogenic mRNAs for eukaryotic cells.
Pathogens often have different AT-contents than the target species, so altering the gene
sequence of the immunogen to reflect the codons more commonly used in the target
species may improve its expression.[15]
Another consideration is the choice of promoter. The SV40 promoter was conventionally
used until research showed that vectors driven by the Rous Sarcoma Virus (RSV) promoter
had much higher expression rates.[1] More recently, expression rates have been further
increased by the use of the cytomegalovirus (CMV) immediate early promoter. Inclusion of
the Mason-Pfizer monkey virus (MPV)-CTE with/without rev increased envelope expression.
Furthermore, the CTE+rev construct was significantly more immunogenic than CTE-alone
vector.[16] Additional modifications to improve expression rates include the insertion of
enhancer sequences, synthetic introns, adenovirus tripartite leader (TPL) sequences and
modifications to the polyadenylation and transcriptional termination sequences.[1] An
example of DNA vaccine plasmid is pVAC, which uses SV40 promoter.
Structural instability phenomena are of particular concern for plasmid manufacture, DNA
vaccination and gene therapy.[17] Accessory regions pertaining to the plasmid backbone
may engage in a wide range of structural instability phenomena. Well-known catalysts of
genetic instability include direct, inverted and tandem repeats, which are conspicuous in a
many commercially available cloning and expression vectors. Therefore, the reduction or
complete elimination of extraneous noncoding backbone sequences would pointedly
reduce the propensity for such events to take place and consequently the overall plasmid's
recombinogenic potential
Once constructed, the vaccine plasmid is transformed into bacteria, where bacterial growth
produces multiple plasmid copies. The plasmid DNA is then purified from the bacteria, by
separating the circular plasmid from the much larger bacterial DNA and other bacterial
impurities. This purifies DNA acts as the vaccine
 Mechanism of action
Antigen peptides expressed after DNA immunisation are usually presented by antigenpresenting cells (APCs) in the context of either MHC class II or class I molecules to CD4+
and CD8+ T cells, respectively. There are at least three means by which MHC class I–
restricted cytotoxic T-lymphocyte (CTL) might be elicited following administration of
plasmid DNA:
transfection of professional APCs,
i.
After uptake of the plasmid, the protein is produced endogenously and intracellularly
processed into small antigenic peptides by the host proteases. The peptides then enter
the lumen of the endoplasmic reticulum (E.R.) by membrane-associated transporters. In
the E.R., peptides bind to MHC class I molecules. These peptides are presented on the
cell surface in the context of the MHC class I. Subsequent CD8+ cytotoxic T cells (CTL)
are stimulated and they evoke cell-mediated immunity. CTLs inhibit viruses through
both cytolysis of infected cells and noncytolysis mechanisms such as cytokine
production (Encke et al, 1999).
The foreign protein can also be presented by the MHC class II pathway by APCs which
elicit helper T cells (CD4+) responses. These CD4+ cells are able to recognize the
peptides formed from exogenous proteins that were endocytosed or phagocytosed by
APC, then degraded to peptide fragments and loaded onto MHC class II molecules.
Depending on the the type of CD4+ cell that binds to the complex, B cells are stimulated
and antibody production is stimulated. This is the same manner in which traditional
vaccines work

Advantages of DNA vaccines
significant advantages over standard vaccines. They can express antigenic epitopes
which more closely resemble native viral epitopes and could therefore be more
effective. With live attenuated vaccines and killed vaccines the manufacturing process
can alter the secondary and tertiary structure of the proteins and therefore the
antigenicity of the vaccine; with naked DNA vaccines the host cell is manufacturing the
viral epitope. DNA vaccines would be safer than live virus vaccines, especially in
immunocompromised patients, such as those infected with HIV.[12]DNA vaccines may
be constructed to include genes against several different pathogens, thus decreasing the
number of vaccinations necessary to fully immunize children. Construction and
manufacture of DNA vaccines would be simple. Finally DNA vaccines may hold promise
in treating those already infected with chronic viral infections (ie, HCV, HIV or HSV)[3]
Logistic advantages of DNA vaccines include the relative ease and low cost of production
and transportation making them more suited to production in the developing world
than other systems[4]

Disadvantages
The possibility of insertional mutagenesis is a concern that needs to be more rigorously
tested. While there is no evidence that the introduced DNA integrates into the host
genome, if it were to occur, it would raise the specter of carcinogenesis; oncongenes
may be turned on or tumor suppressor genes inhibited. What if DNA circulated
throughout the body after injection and integrated into germ cells? Might subsequent
generations express the antigen from birth and develop tolerance, instead of immunity,
to the pathogen? Anti-DNA antibody formation and the possibility of autoimmune
diseases is another concern.[3]
These are important issues because unlike other forms of gene therapy, which target
very ill patients, DNA vaccines are targeted at the young and the healthy. If host cells
express antigen for a prolonged period, what effect would that have on the immune
response? Could it lead to host tolerance or an exaggerated, damaging attack on tissues
expressing antigen? What is the exact nature of the gene transfer and antigen
processing? While injections are given intramuscularly it may not be myocytes that are
actually presenting antigens to T cells. What cells are taking up the gene? Initially it was
thought that myocytes were expressing the DNA product and stimulating a cellular
response, but further work indicates that dendritic cells found throughout the body
(except in the brain) may be the antigen presenting cells.[1] Because there is a brisk
humoral response, it seems that some vaccine product is being delivered to B cells,
macrophages or other MHC class II cells[3]
 Delivery methods
 Saline injections
The two most popular approaches are injection of DNA in saline, using a
standard hypodermic needle and gene gun delivery.[24 Injection in saline is
normally conducted intramuscularly (IM) in skeletal muscle, or intradermally
(ID), delivering DNA to extracellular spaces. This can be assisted by
electroporation;[25] by temporarily damaging muscle fibres with myotoxins such
as bupivacaine; or by using hypertonic solutions of saline or sucrose.[1] Immune
responses to this method can be affected by factors including needle type,[10]
needle alignment, speed of injection, volume of injection, muscle type, and age,
sex and physiological condition of the recipient

Advantages of this method
a) No special delivery mechanism
b) Permanent or semi-permanent expression
c) pDNA spreads rapidly throughout the body

disadvantages
a) Inefficient site for uptake due to morphology of muscle tissue
b) Relatively large amounts of DNA used
c) Th1 response may not be the response required

Gene gun method
propels the DNA-coated gold particles into the epidermis[83 and 84] resulted in
a more Th2 biased antibody isotype response and efficient humoral and cellular
responses[4]

Advantages
a) DNA bombarded directly into cells
b) Small amounts DNA
 Disadvantages
a) Th2 response may not be the response required
b) Requires inert particles as carrier

Liposome mediated delivery
 Advantages
a) High levels of immune response can be generated
b) Can increase transfection of intravenously delivered pDNA
c) Intravenously delivered liposome-DNA complexes can potentially
transfect all tissues
d) Intranasally delivered liposome-DNA complexes can result in
expression in distal mucosa as well as nasal muscosa and the
generation of IgA antibodies

Disadvantages