2. INTRODUCTION Genetic/ DNA immunization is a novel technique used to efficiently stimulate humoral and cellular immune responses to protein antigens The direct injection of genetic material into a living host causes a small amount of its cells to produce the introduced gene products. This inappropriate gene expression within the host has important immunological consequences, resulting in the specific immune activation of the host against the gene delivered antigen . DNA vaccination is a technique for protecting an organism against disease by injecting it with genetically engineeredDNA to produce an immunological response. Nucleic acid vaccines are still experimental, and have been applied to a number of viral, bacterial and parasitic models of disease, as well as to several tumor models. DNA vaccines have a number of advantages over conventional vaccines, including the ability to induce a wider range of immune response types
3. VACCINES First generation vaccines Are whole-organism vaccines – either live and weakened, or killed forms. Live, attenuated vaccines, such as smallpox and polio vaccines, are able to induce killer T-cell (TC or CTL) responses, helper T-cell (TH) responses and antibody immunity. However, there is a small risk that attenuated forms of a pathogen can revert to a dangerous form, and may still be able to cause disease in immunocompromised people (such as those with AIDS). While killed vaccines do not have this risk, they cannot generate specific killer T cell responses, and may not work at all for some diseases. These were the earliest vaccines.
4. Second generation vaccines In order to minimize the risks of the live attenuated vaccines, so-called second generation vaccines were developed. These are subunit vaccines, consisting of defined proteinantigens (such as tetanus or diphtheriatoxoid) or recombinant protein components (such as the hepatitis B surface antigen). These, too, are able to generate TH and antibody responses, but not killer T cell responses. Third generation vaccines DNA vaccines are third generation vaccines, and are made up of a small, circular piece of bacterial DNA (called a plasmid) that has been genetically engineered to produce one or two specific proteins (antigens) from a micro-organism. The vaccine DNA is injected into the cells of the body, where the "inner machinery" of the host cells "reads" the DNA and converts it into pathogenic proteins. Because these proteins are recognized as foreign, when they are processed by the host cells and displayed on their surface, the immune system is alerted, which then triggers a range of immune responses. These DNA vaccines developed from “failed” gene therapy experiments.
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6. Genetic Immunization: Since its early applications in the 1950's, DNA-based immunization has become a novel approach to vaccine development. Direct injection of naked plasmid DNA induces strong immune responses to the antigen encoded by the gene vaccine. Once the plasmid DNA construct is injected the host cells take up the foreign DNA, expressing the viral gene and producing the corresponding viral protein inside the cell. This form of antigen presentation and processing induced both MHC and class I and class II restricted cellular and humoral immune responses
7. CONSTRUCTION Plasmid vectors for use in vaccination DNA vaccines are composed of bacterial plasmids. Expression plasmids used in DNA-based vaccination normally contain two units: The antigen expression unit composed of promoter/enhancer sequences, followed by antigen-encoding and polyadenylation sequences and the production unit composed of bacterial sequences necessary for plasmid amplification and selection . The construction of bacterial plasmids with vaccine inserts is accomplished using recombinant DNA technology. 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 purified DNA acts as the vaccine .
8. Vector design DNA vaccines elicit the best immune response when highly active expression vectors are used. These are plasmids which usually consist of a strong viral promoter to drive the in vivo transcription and translation of the gene (or complementary DNA) of interest DNA vaccines elicit the best immune response when highly active expression vectors are used. These are plasmids which usually consist of a strong viral promoter to drive the in vivo transcription and translation of the gene (or complementary DNA) of interest Because the plasmid is the “vehicle” from which the immunogen is expressed, optimizing vector design for maximal protein expression is essential. One way of enhancing protein expression is by optimizing the codon usage of pathogenic mRNAs for eukaryotic cells. Pathogens often have different AT contents than the species being immunized, so altering the gene sequence of the immunogen to reflect the codons more commonly used in the target species may improve its expression.
11. Vaccine insert design Immunogens can be targeted to various cellular compartments in order to improve antibody or cytotoxic T-cell responses. Secreted or plasma membrane-bound antigens are more effective at inducing antibody responses than cytosolic antigens, while cytotoxic T-cell responses can be improved by targeting antigens for cytoplasmic degradation and subsequent entry into the major histocompatibility complex(MHC) class I pathway.This is usually accomplished by the addition of N-terminalubiquitinsignals. The conformation of the protein can also have an effect on antibody responses, with “ordered” structures (like viral particles) being more effective than unordered structures. Strings of minigenes (or MHC class I epitopes) from different pathogens are able to raise cytotoxic T-cell responses to a number of pathogens, especially if a TH epitope is also included.
12. Delivery methods DNA vaccines have been introduced into animal tissues by a number of different methods The two most popular approaches are injection of DNA in saline, using a standard hypodermic needle, and gene gundelivery. Injection in saline is normally conducted intramuscularly (IM) in skeletal muscle, or intradermally (ID), with DNA being delivered to the extracellular spaces. This can be assisted by electroporation; by temporarily damaging muscle fibers with myotoxins such as bupivacaine; or by using hypertonic solutions of saline or sucrose. Immune responses to this method of delivery can be affected by many factors, including needle type, needle alignment, speed of injection, volume of injection, muscle type, and age, sex and physiological condition of the animal being injected.
13. Gene gun delivery, the other commonly used method of delivery, ballistically accelerates plasmid DNA (pDNA) that has been adsorbed onto gold or tungsten micro particles into the target cells, using compressed helium as an accelerant. The method of delivery determines the dose of DNA required to raise an effective immune response. Saline injections require variable amounts of DNA, from 10 μg-1 mg, whereas gene gun deliveries require 100 to 1000 times less DNA than intramuscular saline injection to raise an effective immune response.Generally, 0.2 μg – 20 μg are required, although quantities as low as 16 ng have been reported. These quantities vary from species to species, with mice, for example, requiring approximately 10 times less DNA than primates. Saline injections require more DNA because the DNA is delivered to the extracellular spaces of the target tissue
14. Another approach to DNA vaccination is expression library immunization (ELI). Using this technique, potentially all the genes from a pathogen can be delivered at one time, which may be useful for pathogens which are difficult to attenuate or culture. ELI can be used to identify which of the pathogen’s genes induce a protective response. This has been tested with Mycoplasmapulmonis, a murine lung pathogen with a relatively small genome, and it was found that even partial expression libraries can induce protection from subsequent challenge
15. MECHANISMS OF UPTAKE A plasmid vector that expresses the protein of interest (e.g. viral protein) under the control of an appropriate promoter is injected into the skin or muscle of the the host. 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.
16. 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 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
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18. ADVANTAGES Subunit vaccination with no risk for infection Antigen presentation by both MHCclass I and class IImolecules Able to polarise T-cell help toward type 1 or type 2 Immune response focused only on antigen of interest Ease of development and production Stability of vaccine for storage and shipping Cost-effectiveness Obviates need for peptide synthesis, expression and purification of recombinant proteins and the use of toxic adjuvants Long-term persistence of immunogen In vivo expression ensures protein more closely resembles normal eukaryotic structure, with accompanying post-translational modifications
19. Disadvantages Limited to protein immunogens (not useful for non-protein based antigens such as bacterial polysaccharides) Risk of affecting genes controlling cell growth Possibility of inducing antibody production against DNA Possibility of tolerance to the antigen (protein) produced Potential for atypical processing of bacterial and parasite proteins
20. FUTURE OF DNA VACCINES - It has recently been discovered that the transfection of myocytes can be amplified by pretreatment with local anesthetics or with cardiotoxin, which induce local tissue damage and initiate myoblast regeneration. Gaining a full understanding of this mechanism of DNA uptake could prove helpful in improving applications for gene therapy and gene vaccination. Both improved expression and better engineering of the DNA plasmid may enhance antibody response to the gene products and expand the applications of the gene vaccines
