Transcript Genetic Vaccines

Genetic Vaccines
Dr. Ziad Jaradat
INTRODUCTION
Despite the marked advances in public
health measures and antimicrobial
medications over the last half century,
infectious diseases remain one of the leading
causes of morbidity and mortality worldwide.
The most powerful and cost effective way to
control such infectious diseases remains the
prophylactic vaccines.
Vaccines constitute the greatest
achievement of modern medicine. They have
eradicated small pox, pushed polio to the
brink of extinction and spared countless
people from typhus, tetanus, measles
hepatitis A & b and many other dangerous
infections.
The World Health Organization estimates
that vaccination against diptheria, tetanus,
whooping cough, measles, polio and
tuberculosis prevents approximately 3 million
deaths a year making vaccination the most
effective public health measure in decreasing
morbidity and mortality in humans.
Traditional vaccines such as live, attenuated
or whole inactivated agents have been very
successful in the past. However, for many
microorganisms that still lack an effective
vaccine.
The traditional vaccines may not be
appropriate either due to safety issues in
which some attenuated pathogens revert
back to their active stage or due to a lack in
immune potency. Therefore, genetic
immunization also known as DNA vaccines
might be the alternative strategy for solving
such problems.
Types of Traditional Vaccines
Killed vaccines: Vaccination with killed
pathogen such as hepatitis A or antigens
isolated from a pathogen such as parts of
hepatitis B can not make their way into cells,
they therefore give rise to primarily humoral
responses and do not activate killer T cells.
Such responses are ineffective against many
organisms that infiltrate cells. Even when
non-living vaccines do prevent a disease, the
protection often wears off after a time,
consequently, recipients may need periodic
booster shots.
Attenuated live vaccines:
usually viruses, do inter cells and make
antigens that are displayed by the inoculated
cells. They thus spur attack by killer T
lymphocytes as well as by antibodies.
This dual activity is necessary for blocking
infection by many viruses. Due to this dual
activation of both humoral and cellular
immunity, live vaccines such as measles,
mumps, rubella and polio provide long life
immunity.
Genetic Vaccines
History:
1- Stansey and Parchkis (1955) and Ito et al
(1957) performed DNA transfer experiments
and were able to induce tumor and antibody
formation.
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2- Atanasiu (1962), Ortho, et al (1964), and
Israel et al, 1979 ; demonstrated that the
administration of polyoma viral DNA either
subcataneously or IP to a rodent induced the
production of antibodies against the virus and
also led to the production of tumor.
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3- Similar experiments by Will et al (1982),
Debensky et al, (1984) and Wolff et al, (1990)
detailed the expression of plasmids encoding
hepatitis B proteins , insulin and reporter genes.
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4- Tang et al, (1992) described the ability of
plasmids coated onto gold beads and
delivered into mice to derive the expression of
a foreign protein and stimulate an antibody
response to influenza virus. (These authors
coined the term genetic immunization).
Definition of DNA Vaccines and
Basic Concept:
Genes encoding antigen(s) specific to a
particular pathogen are cloned into a
plasmid with an appropriate promoter, and
the plasmid DNA is administered to the
vaccine recipient.
The DNA is taken up by the host cells and
the gene is expressed. The resultant
foreign protein antigens is produced in the
cell and then processed and presented
appropriately to the immune system.
How Does DNA Vaccines Work:
DNA vaccines elicit protective immunity
against an infectious agent or pathogen
primarily by activating two branches of the
immune sysem: the humoral arm, which
attacks pathogens outside of cells, and the
cellular arm which eliminates cells that are
colonized by an invader. Immunity is achieved
when such activity generates long lasting
memory cells.
Vaccines induction of immunity begins with
the entry of a DNA vaccine into a targeted
cell, such as muscle and the subsequent
production of the antigens normally found
on the pathogen of interest.
In the humoral response, B cells bind to
released copies of antigenic proteins and
then multiply.
Many of the progeny secrete antibody
molecules that during an infection would
glom (jump and confiscate) onot the
pathogen and mark it for destruction.
Other offspring become the memory cells
that will quell the pathogen if it circulates
outside cells.
Meanwhile display of antigenic protein fragments
or peptides on inoculated cells (within grooves on
MHC class I molecules) can trigger a cellular
response .
Binding to the antigenic complexes induces
cytotoxic (killer cells) to multiply and kill the bound
cells and others displaying those same peptides in
the same way. Some activated cells will also
become memory cells ready to eliminate cells
invaded by the pathogen in the future.
In actuality, several preliminary steps
must occur before such response can
occur.
To set the stage for B cell activation the
following steps occur:
Professional antigen presenting cells
(APCs) must ingest antigen molecules that
are secreted into the extracellular space,
chop them, and display the resulting
peptides on MHC class II molecules.
Helper T-cells in turn, must recognize both
the peptide complexes and “ a costimulatory molecule” found only on APCs.
The helper cells secrete signaling
molecules known as Th2 cytokines which
help to activate B cells bound to antigens.
To activate the cytotoxic T cells the following
steps occur:
APCs have to take up the vaccine plasmid,
synthesize the encoding antigens and exhibit
fragments of the antigens on MHC class I
molecules along with co-stimulatory
molecules.
The killer T cells recognizes those signals at
the same time displays receptors for Th1
cytokines produced by helper T-cells.
The cytokines once bind the killer T-cells get
activated and become mature cytotoxic T-cells.
DNA vaccines also yield memory helper T
cells that are needed to support the
defense activities of other memory cells.
Methods and Location of
Immunization
One feature of genetic immunization that has
become apparent over the past few years is
that the way a DNA vaccine is delivered may
have an effect on the type of immune
response generated.
It was reported that both the site of
inoculation and the method by which the
plasmid is delivered may independently
affect the induced immunity in a qualitative
and may be in a quantitative manner.
Successful DNA vaccination has been
demonstrated via a number of different routes
including:
- intravenous
- intramuscular
- intra epidermal
- intra spleenic
- intra hepatic
with the majority of DNA vaccines so far being
administered through skin or muscle.
Studies in rodents on the transfection
efficiency of injected DNA have
demonstrated that muscle is 100-1000 times
more permissive than other tissues for the
uptake and expression of DNA.
Tissues are also differ in the efficiency with
which they present antigens to the immune
system.
Tissues such as skin and the mucosal linings
of the respiratory tract and the gut that serve
as barriers against the entry of pathogens
have associated lymphoid tissues that provide
high levels of local immune surveillance.
These tissues also contain cells that are
specialized for MHC class II restricted
presentation of antigens to helper T-cells. So
it is apparent that:
 muscles rout of administration supports efficient
transfection.
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Intraperitonal and subcotaneous , are the
traditional routs of administration, however
they do not support efficient transfection.
Skin and muscle tissues, support less efficient
transfection but deliver DNA to tissues with
immune surveillance.
Methods of Administration
Plasmid delivery at these sites is usually
accomplished by one of two methods:
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1- needle injection of DNA suspended in saline
2- Gene gun, this method has more commonly used for
epidermal rather than intramascular administration.
Several researchers have reported that the gene
gun mediated immunization is far more efficient than
needle injection, eliciting similar levels of antibody
and cellular responses with 100-5000 fold less DNA.
It was reported that as little as 16 ng of plasmid
DNA delivered epidermally via gene gun could
induce antibody and CTL responses in mice,
wherase intradermal injection of the same plasmid
requires 10-1000 µg of DNA to elicit comparable
responses.
With regard to the immunization regimens,
there has not been any regimen that is shown
to be superior to others, it seems that each
disease and each vaccine construct differs
from the other, therefore, the best regimen of
DNA vaccine administration yet to be
determined.
Enhancement of DNA
vaccines action
The most promising method of vaccine
enhancement is the co-administration of
plasmid encoding cytokines along with a
plasmid encoding an antigen.
Cytokines are molecules secreted mainly
by bone marrow derived cells , they induce
specific response in cells expressing a
receptor for a particular cytokine.
There are several cytokines that can be
co-administration with the gene to
enhance the immune response to genetic
immunization.
Only the major ones will be discussed
IL-2 : a potent stimulator of cellular immunity
that induces proliferation and differentiation
of T cells as well as B cell and NK cell
growth.
 Watanable et al...... reported a five fold
increase in antibody response when IL-2
plasmid was co- injected with the plasmid
encoding the antigen.
Chow et al...... Demonstrated that injection of a
vector that encoded HbsAg and IL-2 on the
same plasmid induced marked increase of Ab
responses and T-cell proliferation compared to a
plasmid encoding HbsAg alone.
Taken these results and results from other
studies, it is suggested that IL-2 gene coinjection can increase both humoral and cellular
immunity .
IL-4:
induces differentiation of T-helper cells into
Th2 subtype, enhances B cell growth, and
mediates Ig class switching. It was reported
that injection of a plasmid encoding IL-4 3
days before immunization with a protein
antigen increased Ag specific antibody
levels compared to protein immunization
alone.
However, studies showed that IL-4 inhibits
Th1 mediated responses, thus put
limitation on using it as adjuvant in viral or
tumor vaccines or immunotherapy.
Granulocyte-monocyte colonystimulating factor (GM-CSF):
This cytokine increases production of
granulocytes and macrophages and induces
maturation and activation of APCs such as
dendritic cells.
Xiang and Ertl tested this theory in vivo by
co-inoculating mice with plasmids encoding
GM-CSF and rabies glycoprotein.
Co expression of GM-CSF and rabies
glycoproeins increased Ab response in a
dose dependant manner and enhanced Thelper cell responses compared to injection
with plasmid encoding rabies protein alone.
Same results were obtained with DNA
vaccines against HIV-I, influenza,
encephalomyocarditis virus and HCV.
Advantages and properties of
DNA vaccines
Plasmid vectors can be constructed and
tested rapidly.
Rapid and large-scale manufacturing
procedures are available.
DNA is more temperature stable than live
preparations.
Microgram quantities of expression vector
can induce immune response.
Unlike killed vaccines, DNA vaccines can produce
diverse and persistent immune response (both
humoral and cellular arms of the immune
response)
Protection can be achieved in large primate
models of human infections
Multiple vectors encoding several antigens can be
delivered in a single administration
Unlike the live attenuated vaccines, who posses
the risk of reversion to pathogenic state while
replicating inside the host, DNA vaccines are
safe and do not encode for genes that cause
diseases
Unlike the killed vaccines, who induce short
immunity and need frequent boosting, DNA
vaccines cause long lasting immunity with
minimum boosts
Thank you