Genetic VaccinesGenetic Vaccines

Dr. Ziad JaradatDr. Ziad Jaradat

INTRODUCTIONINTRODUCTION

Despite the marked advances in public Despite the marked advances in public health measures and antimicrobial health measures and antimicrobial medications over the last half century, medications over the last half century, infectious diseases remain one of the leading infectious diseases remain one of the leading causes of morbidity and mortality worldwide. causes of morbidity and mortality worldwide. The most powerful and cost effective way to The most powerful and cost effective way to control such infectious diseases remains the control such infectious diseases remains the prophylactic vaccines.prophylactic vaccines.

Vaccines constitute the greatest Vaccines constitute the greatest achievement of modern medicine. They have achievement of modern medicine. They have eradicated small pox, pushed polio to the eradicated small pox, pushed polio to the brink of extinction and spared countless brink of extinction and spared countless people from typhus, tetanus, measles people from typhus, tetanus, measles hepatitis A & b and many other dangerous hepatitis A & b and many other dangerous infections. infections.

The World Health Organization estimates The World Health Organization estimates that vaccination against diptheria, tetanus, that vaccination against diptheria, tetanus, whooping cough, measles, polio and whooping cough, measles, polio and tuberculosis prevents approximately 3 million tuberculosis prevents approximately 3 million deaths a year making vaccination the most deaths a year making vaccination the most effective public health measure in decreasing effective public health measure in decreasing morbidity and mortality in humans.morbidity and mortality in humans.

Traditional vaccines such as live, attenuated Traditional vaccines such as live, attenuated or whole inactivated agents have been very or whole inactivated agents have been very successful in the past. However, for many successful in the past. However, for many microorganisms that still lack an effective microorganisms that still lack an effective vaccine. vaccine.

The traditional vaccines may not be The traditional vaccines may not be appropriate either due to safety issues in appropriate either due to safety issues in which some attenuated pathogens revert which some attenuated pathogens revert back to their active stage or due to a lack in back to their active stage or due to a lack in immune potency. Therefore, genetic immune potency. Therefore, genetic immunization also known as DNA vaccines immunization also known as DNA vaccines might be the alternative strategy for solving might be the alternative strategy for solving such problems.such problems.

Types of Traditional VaccinesTypes of Traditional Vaccines

Killed vaccinesKilled vaccines:: Vaccination with killed Vaccination with killed pathogen such as hepatitis A or antigens pathogen such as hepatitis A or antigens isolated from a pathogen such as parts of isolated from a pathogen such as parts of hepatitis B can not make their way into cells, hepatitis B can not make their way into cells, they therefore give rise to primarily humoral they therefore give rise to primarily humoral responses and do not activate killer T cells.responses and do not activate killer T cells.

Such responses are ineffective against many Such responses are ineffective against many organisms that infiltrate cells. Even when organisms that infiltrate cells. Even when non-living vaccines do prevent a disease, the non-living vaccines do prevent a disease, the protection often wears off after a time, protection often wears off after a time, consequently, recipients may need periodic consequently, recipients may need periodic booster shots.booster shots.

Attenuated live vaccines:Attenuated live vaccines:

usually viruses, do inter cells and make usually viruses, do inter cells and make antigens that are displayed by the inoculated antigens that are displayed by the inoculated cells. They thus spur attack by killer T cells. They thus spur attack by killer T lymphocytes as well as by antibodies. lymphocytes as well as by antibodies.

This dual activity is necessary for blocking This dual activity is necessary for blocking infection by many viruses. Due to this dual infection by many viruses. Due to this dual activation of both humoral and cellular activation of both humoral and cellular immunity, live vaccines such as measles, immunity, live vaccines such as measles, mumps, rubella and polio provide long life mumps, rubella and polio provide long life immunity. immunity.

Genetic VaccinesGenetic Vaccines

History: History: 1- Stansey and Parchkis (1955) and Ito et al 1- Stansey and Parchkis (1955) and Ito et al

(1957) performed DNA transfer experiments (1957) performed DNA transfer experiments and were able to induce tumor and antibody and were able to induce tumor and antibody formation.formation.

2- Atanasiu (1962), Ortho, et al (1964), and 2- Atanasiu (1962), Ortho, et al (1964), and Israel et al, 1979 ; demonstrated that the Israel et al, 1979 ; demonstrated that the administration of polyoma viral DNA either administration of polyoma viral DNA either subcataneously or IP to a rodent induced the subcataneously or IP to a rodent induced the production of antibodies against the virus and production of antibodies against the virus and also led to the production of tumor. also led to the production of tumor.

3- Similar experiments by Will et al (1982), 3- Similar experiments by Will et al (1982), Debensky et al, (1984) and Wolff et al, (1990) Debensky et al, (1984) and Wolff et al, (1990) detailed the expression of plasmids encoding detailed the expression of plasmids encoding hepatitis B proteins , insulin and reporter genes.hepatitis B proteins , insulin and reporter genes.

4- Tang et al, (1992) described the ability of 4- Tang et al, (1992) described the ability of plasmids coated onto gold beads and plasmids coated onto gold beads and delivered into mice to derive the expression of delivered into mice to derive the expression of a foreign protein and stimulate an antibody a foreign protein and stimulate an antibody response to influenza virus. (These authors response to influenza virus. (These authors coined the term coined the term genetic immunizationgenetic immunization).).

Definition of DNA Vaccines and Definition of DNA Vaccines and Basic Concept:Basic Concept:

Genes encoding antigen(s) specific to a Genes encoding antigen(s) specific to a particular pathogen are cloned into a particular pathogen are cloned into a plasmid with an appropriate promoter, and plasmid with an appropriate promoter, and the plasmid DNA is administered to the the plasmid DNA is administered to the vaccine recipient.vaccine recipient.

The DNA is taken up by the host cells and The DNA is taken up by the host cells and the gene is expressed. The resultant the gene is expressed. The resultant foreign protein antigens is produced in the foreign protein antigens is produced in the cell and then processed and presented cell and then processed and presented appropriately to the immune system.appropriately to the immune system.

How Does DNA Vaccines WorkHow Does DNA Vaccines Work::

DNA vaccines elicit protective immunity DNA vaccines elicit protective immunity against an infectious agent or pathogen against an infectious agent or pathogen primarily by activating two branches of the primarily by activating two branches of the immune sysem: immune sysem: the humoral armthe humoral arm, which , which attacks pathogens outside of cells, and the attacks pathogens outside of cells, and the cellular armcellular arm which eliminates cells that are which eliminates cells that are colonized by an invader. Immunity is colonized by an invader. Immunity is achieved when such activity generates long achieved when such activity generates long lasting memory cells.lasting memory cells.

Vaccines induction of immunity begins Vaccines induction of immunity begins with the entry of a DNA vaccine into a with the entry of a DNA vaccine into a targeted cell, such as muscle and the targeted cell, such as muscle and the subsequent production of the antigens subsequent production of the antigens normally found on the pathogen of normally found on the pathogen of interest. interest.

In the humoral responseIn the humoral response, B cells bind to , B cells bind to released copies of antigenic proteins and released copies of antigenic proteins and then multiply. then multiply. Many of the progeny secrete antibody Many of the progeny secrete antibody molecules that during an infection would molecules that during an infection would glom (jump and confiscate) onot the glom (jump and confiscate) onot the pathogen and mark it for destruction. pathogen and mark it for destruction. Other offspring become the memory cells Other offspring become the memory cells that will quell the pathogen if it circulates that will quell the pathogen if it circulates outside cells. outside cells.

MeanwhileMeanwhile display of antigenic protein fragments display of antigenic protein fragments or peptides on inoculated cells (within grooves on or peptides on inoculated cells (within grooves on MHC class I molecules) can trigger a cellular MHC class I molecules) can trigger a cellular response .response .

Binding to the antigenic complexes induces Binding to the antigenic complexes induces cytotoxic (killer cells) to multiply and kill the bound cytotoxic (killer cells) to multiply and kill the bound cells and others displaying those same peptides in cells and others displaying those same peptides in the same way. Some activated cells will also the same way. Some activated cells will also become memory cells ready to eliminate cells become memory cells ready to eliminate cells invaded by the pathogen in the future.invaded by the pathogen in the future.

In actuality,In actuality, several preliminary steps several preliminary steps must occur before such response can must occur before such response can occur. occur.

To set the stage for B cell activation the To set the stage for B cell activation the following steps occur:following steps occur:

Professional antigen presenting cells Professional antigen presenting cells (APCs) must ingest antigen molecules that (APCs) must ingest antigen molecules that are secreted into the extracellular space, are secreted into the extracellular space, chop them, and display the resulting chop them, and display the resulting peptides on MHC class II molecules.peptides on MHC class II molecules.

Helper T-cells in turn, must recognize both Helper T-cells in turn, must recognize both the peptide complexes and “ a co-the peptide complexes and “ a co-stimulatory molecule” found only on APCs.stimulatory molecule” found only on APCs.

The helper cells secrete signaling The helper cells secrete signaling molecules known as Th2 cytokines which molecules known as Th2 cytokines which help to activate B cells bound to antigens.help to activate B cells bound to antigens.

To activate the cytotoxic T cells the following To activate the cytotoxic T cells the following steps occur:steps occur:

APCs have to take up the vaccine plasmid, APCs have to take up the vaccine plasmid, synthesize the encoding antigens and exhibit synthesize the encoding antigens and exhibit fragments of the antigens on MHC class I fragments of the antigens on MHC class I molecules along with co-stimulatory molecules along with co-stimulatory molecules.molecules.

The killer T cells recognizes those signals at The killer T cells recognizes those signals at the same time displays receptors for Th1 the same time displays receptors for Th1 cytokines produced by helper T-cells. cytokines produced by helper T-cells.

The cytokines once bind the killer T-cells get The cytokines once bind the killer T-cells get activated and become mature cytotoxic T-activated and become mature cytotoxic T-cellscells..

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

Methods and Location of Methods and Location of ImmunizationImmunization

One feature of genetic immunization that One feature of genetic immunization that has become apparent over the past few has become apparent over the past few years is that the way a DNA vaccine is years is that the way a DNA vaccine is delivered may have an effect on the type of delivered may have an effect on the type of immune response generated.immune response generated.

It was reported that both the site of It was reported that both the site of inoculation and the method by which the inoculation and the method by which the plasmid is delivered may independently plasmid is delivered may independently affect the induced immunity in a qualitative affect the induced immunity in a qualitative and may be in a quantitative manner.and may be in a quantitative manner.

Successful DNA vaccination has been Successful DNA vaccination has been demonstrated via a number of different routes demonstrated via a number of different routes including:including:- intravenous- intravenous- intramuscular- intramuscular- intra epidermal- intra epidermal- intra spleenic- intra spleenic- intra hepatic- intra hepatic

with the majority of DNA vaccines so far being with the majority of DNA vaccines so far being administered through skin or muscle.administered through skin or muscle.

Studies in rodents on the transfection Studies in rodents on the transfection efficiency of injected DNA have efficiency of injected DNA have demonstrated that muscle is 100-1000 times demonstrated that muscle is 100-1000 times more permissive than other tissues for the more permissive than other tissues for the uptake and expression of DNA.uptake and expression of DNA.

Tissues are also differ in the efficiency with Tissues are also differ in the efficiency with which they present antigens to the immune which they present antigens to the immune system. system.

Tissues such as skin and the mucosal linings Tissues such as skin and the mucosal linings of the respiratory tract and the gut that serve of the respiratory tract and the gut that serve as barriers against the entry of pathogens as barriers against the entry of pathogens have associated lymphoid tissues that provide have associated lymphoid tissues that provide high levels of local immune surveillance. high levels of local immune surveillance.

These tissues also contain cells that are These tissues also contain cells that are specialized for MHC class II restricted specialized for MHC class II restricted presentation of antigens to helper T-cells. So presentation of antigens to helper T-cells. So it is apparent that:it is apparent that: muscles rout of administration supports efficient muscles rout of administration supports efficient

transfection.transfection.

Intraperitonal and subcotaneous , are the Intraperitonal and subcotaneous , are the traditional routs of administration, however traditional routs of administration, however they do not support efficient transfection. they do not support efficient transfection.

Skin and muscle tissues, support less efficient Skin and muscle tissues, support less efficient transfection but deliver DNA to tissues with transfection but deliver DNA to tissues with immune surveillance. immune surveillance.

Methods of AdministrationMethods of Administration

Plasmid delivery at these sites is usually Plasmid delivery at these sites is usually accomplished by one of two methods:accomplished by one of two methods:

1- needle injection of DNA suspended in saline 1- needle injection of DNA suspended in saline 2- Gene gun, this method has more commonly used for 2- Gene gun, this method has more commonly used for

epidermal rather than intramascular administration.epidermal rather than intramascular administration.

Several researchers have reported that the gene Several researchers have reported that the gene gun mediated immunization is far more efficient than gun mediated immunization is far more efficient than needle injection, eliciting similar levels of antibody needle injection, eliciting similar levels of antibody and cellular responses with 100-5000 fold less DNA. and cellular responses with 100-5000 fold less DNA.

It was reported that as little as 16 ng of plasmid It was reported that as little as 16 ng of plasmid DNA delivered epidermally via gene gun could DNA delivered epidermally via gene gun could induce antibody and CTL responses in mice, induce antibody and CTL responses in mice, wherase intradermal injection of the same plasmid wherase intradermal injection of the same plasmid requires 10-1000 µg of DNA to elicit comparable requires 10-1000 µg of DNA to elicit comparable responses.responses.

With regard to the immunization regimens, With regard to the immunization regimens, there has not been any regimen that is shown there has not been any regimen that is shown to be superior to others, it seems that each to be superior to others, it seems that each disease and each vaccine construct differs disease and each vaccine construct differs from the other, therefore, the best regimen of from the other, therefore, the best regimen of DNA vaccine administration yet to be DNA vaccine administration yet to be determined.determined.

Enhancement of DNA Enhancement of DNA vaccines actionvaccines action

The most promising method of vaccine The most promising method of vaccine enhancement is the co-administration of enhancement is the co-administration of plasmid encoding cytokines along with a plasmid encoding cytokines along with a plasmid encoding an antigen.plasmid encoding an antigen.

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

There are several cytokines that can be There are several cytokines that can be co-administration with the gene to co-administration with the gene to enhance the immune response to genetic enhance the immune response to genetic immunization.immunization.

Only the major ones will be discussed Only the major ones will be discussed

IL-2 :IL-2 : a potent stimulator of cellular immunity a potent stimulator of cellular immunity that induces proliferation and differentiation that induces proliferation and differentiation of T cells as well as B cell and NK cell of T cells as well as B cell and NK cell growth.growth.

Watanable et al...... reported a five fold Watanable et al...... reported a five fold increase in antibody response when IL-2 increase in antibody response when IL-2 plasmid was co- injected with the plasmid plasmid was co- injected with the plasmid encoding the antigen.encoding the antigen.

Chow et al...... Demonstrated that injection of a Chow et al...... Demonstrated that injection of a vector that encoded HbsAg and IL-2 on the vector that encoded HbsAg and IL-2 on the same plasmid induced marked increase of Ab same plasmid induced marked increase of Ab responses and T-cell proliferation compared to a responses and T-cell proliferation compared to a plasmid encoding HbsAg alone.plasmid encoding HbsAg alone.

Taken these results and results from other Taken these results and results from other studies, it is suggested that IL-2 gene co-studies, it is suggested that IL-2 gene co-injection can increase both humoral and cellular injection can increase both humoral and cellular immunity .immunity .

IL-4:IL-4:

induces differentiation of T-helper cells into induces differentiation of T-helper cells into Th2 subtype, enhances B cell growth, and Th2 subtype, enhances B cell growth, and mediates Ig class switching. It was reported mediates Ig class switching. It was reported that injection of a plasmid encoding IL-4 3 that injection of a plasmid encoding IL-4 3 days before immunization with a protein days before immunization with a protein antigen increased Ag specific antibody antigen increased Ag specific antibody levels compared to protein immunization levels compared to protein immunization alone.alone.

However, studies showed that IL-4 inhibits However, studies showed that IL-4 inhibits Th1 mediated responses, thus put Th1 mediated responses, thus put limitation on using it as adjuvant in viral or limitation on using it as adjuvant in viral or tumor vaccines or immunotherapy.tumor vaccines or immunotherapy.

Granulocyte-monocyte colony-Granulocyte-monocyte colony-stimulating factor (GM-CSF):stimulating factor (GM-CSF):

This cytokine increases production of This cytokine increases production of granulocytes and macrophages and induces granulocytes and macrophages and induces maturation and activation of APCs such as maturation and activation of APCs such as dendritic cells.dendritic cells.

Xiang and Ertl tested this theory in vivo by Xiang and Ertl tested this theory in vivo by co-inoculating mice with plasmids encoding co-inoculating mice with plasmids encoding GM-CSF and rabies glycoprotein. GM-CSF and rabies glycoprotein.

Co expression of GM-CSF and rabies Co expression of GM-CSF and rabies glycoproeins increased Ab response in a glycoproeins increased Ab response in a dose dependant manner and enhanced T-dose dependant manner and enhanced T-helper cell responses compared to injection helper cell responses compared to injection with plasmid encoding rabies protein alone.with plasmid encoding rabies protein alone.

Same results were obtained with DNA Same results were obtained with DNA vaccines against HIV-I, influenza, vaccines against HIV-I, influenza, encephalomyocarditis virus and HCV.encephalomyocarditis virus and HCV.

Advantages and properties of Advantages and properties of DNA vaccinesDNA vaccines

Plasmid vectors can be constructed and Plasmid vectors can be constructed and tested rapidly.tested rapidly.

Rapid and large-scale manufacturing Rapid and large-scale manufacturing procedures are available.procedures are available.

DNA is more temperature stable than live DNA is more temperature stable than live preparations.preparations.

Microgram quantities of expression vector Microgram quantities of expression vector can induce immune response.can induce immune response.

Unlike killed vaccines, DNA vaccines can produce Unlike killed vaccines, DNA vaccines can produce diverse and persistent immune response (both diverse and persistent immune response (both humoral and cellular arms of the immune humoral and cellular arms of the immune response)response)

Protection can be achieved in large primate Protection can be achieved in large primate models of human infectionsmodels of human infections

Multiple vectors encoding several antigens can be Multiple vectors encoding several antigens can be delivered in a single administrationdelivered in a single administration

Unlike the live attenuated vaccines, who posses Unlike the live attenuated vaccines, who posses the risk of reversion to pathogenic state while the risk of reversion to pathogenic state while replicating inside the host, DNA vaccines are replicating inside the host, DNA vaccines are safe and do not encode for genes that cause safe and do not encode for genes that cause diseasesdiseases

Unlike the killed vaccines, who induce short Unlike the killed vaccines, who induce short immunity and need frequent boosting, DNA immunity and need frequent boosting, DNA vaccines cause long lasting immunity with vaccines cause long lasting immunity with minimum boostsminimum boosts

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