Genetic Vaccines

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Genetic Vaccines

• Dr. Ziad Jaradat

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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.

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• 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.

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• 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.

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• 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.

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• 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.

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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.

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• 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.

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• 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.

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• 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.

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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).

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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.

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• 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.

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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.

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• 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.

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• 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.

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• 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.

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• In actuality, several preliminary steps must
  occur before such response can occur.
• To set the stage for B cell activation the
  following steps occur

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• 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.

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• Helper T-cells in turn, must recognize both the
  peptide complexes and a co-stimulatory
  molecule found only on APCs.
• The helper cells secrete signaling molecules
  known as Th2 cytokines which help to activate B
  cells bound to antigens.

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• 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.

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• 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.

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• DNA vaccines also yield memory helper T cells
  that are needed to support the defense activities
  of other memory cells.

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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.

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• 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.

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• 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.

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• 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.

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• 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.

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• 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.

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Methods of Administration

• Plasmid delivery at these sites is usually
  accomplished by one of two methods
• 1- needle injection of DNA suspended in saline
• 2- Gene gun, this method has more commonly used
  for epidermal rather than intramascular
  administration.

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• 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.

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• 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.

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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.

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• Cytokines are molecules secreted mainly by bone
  marrow derived cells , they induce specific
  response in cells expressing a receptor for a
  particular cytokine.

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• 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

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• 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.

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• 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
  co-injection can increase both humoral and
  cellular immunity .

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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.

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• 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.

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Granulocyte-monocyte colony-stimulating 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.

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• Co expression of GM-CSF and rabies glycoproeins
  increased Ab response in a dose dependant manner
  and enhanced T-helper 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.

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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.

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• 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

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• 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

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• Thank you