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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.
n 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.
n 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.
n 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 Th 2 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 Th 1 cytokines produced by helper T-cells. The cytokines once bind the killer T-cells get activated and become mature cytotoxic Tcells.
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.
n n 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: n n 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 Hbs. Ag and IL-2 on the same plasmid induced marked increase of Ab responses and T-cell proliferation compared to a plasmid encoding Hbs. Ag 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 Th 2 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 Th 1 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