DNA Vaccines

ABSTRACT
Vaccination is historically one of the most successful strategies for the prevention of infectious diseases. For safety reasons, modern vaccinology tends toward the usage of inactivated or attenuated microorganisms and uses predominantly subunit vaccines.

The field of DNA vaccines continues to advance and several new strategies to augment the immunogenicity of DNA vaccines are under evaluation. The majority of these studies are in the early preclinical stage, but some DNA vaccines have moved into clinical trials. In this review, we describe some of the more recent efforts aimed at increasing the immunogenicity of DNA vaccines, including the use of genetic adjuvants and plasmid-based expression of viral replicons. In addition, we discuss the possibility of using DNA vaccines to address emerging infectious agents where they may provide an advantage over other vaccine strategies and we review some areas where DNA vaccines have been used to target self-antigens. In this overview, current knowledge of the host immune response to DNA vaccines is summarized in the introduction. The subsequent sections discuss techniques for enhancing DNA vaccine efficacy, such as DNA delivery to specific tissues, delivery of DNA to the cell cytoplasm or nucleus, and enhancement of the immune response using molecular adjuvants. Finally, the prospects of DNA vaccination and ongoing clinical trials with various DNA vaccines are discussed.

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INTRODUCTION

Vaccination protects a recipient from pathogenic agents by establishing an immunological resistance to infection. An injected or oral vaccine induces the host to generate Abs against the disease-causing organism; therefore, during future exposures, the infectious agent is inactivated (neutralized, or killed), its proliferation is prevented, and the disease state is not established. Communicable diseases such as tuberculosis, smallpox, cholera, typhoid, bubonic plague, and poliomyelitis have in the past been a scourge for humankind. Active immunization prevented several million cases of measles and many deaths. Diseases like diphtheria, polio, or pertussis have been controlled near to 100%, in developed counties like the United States through mass immunization programmes. Today, over 2 billion humans suffer from diseases that, theoretically, could be curtailed by vaccination. Modern vaccines typically consist of either a killed (inactivated) or a live, avirulent (attenuated) form of an infectious agent. Traditionally, the infectious agent is grown in culture, purified, and either inactivated or attenuated without, of course, losing the ability to evoke an immune response that is effective against the virulent form of the infectious organism. Limitations of the current mode of vaccine production: Not all infectious agents can be grown in culture, and so no vaccines have been developed for many diseases. Production of animal and human viruses requires expensive animal cell culture. Both the yield and rate of production of animal and human viruses in culture are often quite low, thereby making vaccine production costly. Extensive safety precautions are necessary to ensure that laboratory and production

personnel are not exposed to a pathogenic agent.

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Batches of vaccine may not be killed or may be insufficiently attenuated during the

production process, thereby introducing virulent organisms into the vaccine and inadvertently spreading the disease. Attenuated strains may revert, a possibility that requires continual testing to ensure that the reacquisition of virulence has not occurred. Not all diseases (e.g., AIDS) are preventable through the use of traditional vaccines. Most current vaccines have a limited shelf life and often require refrigeration to maintain

potency which causes storage problems. PASSIVE IMMUNIZATION: Passive immunization involves providing exogenous Abs from another individual of the same or a different species in an attempt to prevent or to attenuate an anticipated infection. Protection is immediate. But the transferred Abs are used up by combination with the Ag or catabolized in the normal way and so the protection is short lived. Pooled human serum immunoglobulins consist of a wide variety of Abs against different microbial agents. Previously, there was some problem in using -globulin preparations because the spontaneous formation of small aggregates that could cause severe anaphylactic reactions. So, -globulin was always injected intramuscularly. The increasing availability of intravenous preparations that can be safely administered in high doses has broadened the use of this treatment. This is regularly provided to congenital immunoglobulin deficient individuals and patients on steroid treatment. This treatment has also been extended experimentally to persons at a high risk of sepsis, such as persons with Human Ig is preferable to horse anti-toxin which sometimes causes serious serum sickness. Severe burns or in intensive care. Routinely it has been included in the immunization programme for travellers. For passive immunization, human monoclonal Abs to selected organisms will be much in demand as and when they will be produced in mass scale. Sometimes the passive immunization may interfere with immune responses to some Ags, as with the measles vaccine.

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NATURAL PASSIVE IMMUNIZATION: Maternal Abs acquired transplacentally during fetal life and by intestinal absorption of colostral Igs is considered as natural passive immunization that provides protection to the newborn during several months of initial life. The intestine of the baby does not usually absorb secretory IgA in mothers milk; rather it provides protection of the mucosal surface against bacteria and viruses.

ACTIVE IMMUNIZATION : VACCINATION

Active immunization is to generate effective immunity towards a specific infecting agent through Abs and a primed population of immunocompetent cells that can rapidly expand on renewed contact with the specific Ag. It has long lasting immunologic memory; sometimes life-long. Vaccination is done by introducing minimal amount of Ag as vaccine that should not be injurious or pathogenic to the host. Active immunization provides immune protection to the individual vaccine, and in consequence reduces the circulation of the infecting agent in the population and thereby protects unvaccinated persons as well. This phenomenon is known as herd immunity, which is significant in many Ration programmes.

Live, Attenuated Bacterial and Viral Vaccines : Live but attenuated viruses or bacteria become avirulent but retain the ability of transient growth within inoculated hosts. Thus, Attenuated live vaccines mimic the original microbes without causing full fledged disease. The living microbes often present the Ag to the immunocompetent cells in a much better fashion than the dead ones; for example, budding viruses on infected cells are better for CTL stimulation. Furthermore, attenuated live organisms can initiate immune responses at the site of natural infection. Many such viral vaccines have an efficiency of greater than 90%, and protection frequently lasts for many years. Among the attenuated bacterial vaccines in use today are an attenuated strain of Mycobacterium bovis bacillus Calmette-Gurin (BCG), avirulent mutants of Salmonella typhi, inactivated Vibrio cholerae, and inactivated Bordetella pertusis. These attenuated bacterial vaccines are effective for relatively short periods. It is usually advantageous to develop a live vaccine, because they are generally much more effective than killed or subunit vaccines. The major requirement for a live vaccine is that no virulent forms be present in the inoculation material. With this objective in mind, a live cholera vaccine has been developed. 1
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The Sabin polio vaccine consists of three attenuated strains of poliovirus, and is administered orally to children in sugar solution. The attenuated viruses colonize the intestine and induce protective immunity to the virulent forms of all three strains of poliovirus. After colonization, Sabin vaccine induces production of secretory IgA, which provides defense against naturally infecting poliovirus. It also induces IgM and IgG. Most other attenuated viral vaccines require a single immunizing dose; the schedule of the Sabin polio vaccine requires at least two booster doses in addition to the primary one. This is because the three strains of attenuated polio vaccine interfere with each other's replication in the intestine. Usually growth of one strain predominates with the first immunization and induces host immunity to the strain. The immunity developed towards this first strain will limit its growth at the time of subsequent immunizations when one of the two remaining strains will predominate in growth. Thus, third time immunization brings in immunity to all three strains finally.

DNA VACCINE:

HISTORY 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 producng 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 response.

The use of genetic material to deliver genes for therapeutic purposes has been practiced for many years. Experiments outlining the transfer of DNA into cells of living animals were reported as early as 1950. Later experiments using purified genetic material only further confirmed that 1
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the direct DNA gene injection in the absence of viral vectors results in the expression of the inoculated genes in the host. There have been additional experiments that extend these findings to recombinant DNA molecules, further illustrating the idea that purified nucleic acids could be directly delivered into a host and proteins would be produced. In 1992, scientists Tang and Johnson reported that the delivery of human growth hormone in a expression cassette in vivo resulted in production of detectable levels of the growth hormone in host mice. They also found that these inoculated mice developed antibodies against the human growth hormone; they termed this immunization procedure "genetic immunization", which describes individual imunogen.the ability of inoculated genes to be individual.

Many vaccines began as serendipitou discoveries. Louis Pasteur discovered attenuated vaccines when old cholera cultures lost their virulence. When chickens were inoculated with aged cultures, they unexpectedly developed immunity to cholera.

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Almost 20 years ago, Malone and Felgner at Vical Incorporated, and Wolff and colleagues at the University of Wisconsin, demonstrated that mRNA and closed loops of double-stranded DNA (plasmids) injected into muscle tissue could be taken up by cells at the administration site (transfection) resulting in the production (expression) of proteins not normally made by the host cell. It was soon realized that this approach could be utilized for both gene therapy as well as vaccine applications, and thus the field of DNA vaccines was born.. It was observed that mouse skeletal muscle can take up naked DNA and express proteins encoded by the DNA. Naked DNA was actually used as a controlin their experiments whose objective was to identify lipids that enhance DNA delivery into skeletal muscle. When DNA encoding an influenza virus protein was injected into the skeletal muscle of mice, synthesis of the virus protein in the mouse muscle triggered an immune response resulting in protection of the mice from a subsequent influenza infection.These start to secrete cytokines, migrate to lymphatic tissues and initiate immune response. Since, the dendritic cells have a finite life span, the muscle cells being poor target for CTLs, might serve as a reservoir of antigen, providing a constant reminder to the immune system.These results,published in Science in the year 1993, marked the beginning of DNA vaccines also known as nucleic acid vaccines or genetic vaccines. Since 1993, the principle of DNA vaccination has been demonstrated for a variety of bacterial, viral and parasitic diseases. Development in this area has greatly advanced over the years and human clinical trials of DNA vaccines have now been conducted against various infectious pathogens including the malaria parasite, dengue viruses, cytomegalovirus (CMV), Ebola virus, seasonal influenza viruses, avian or pandemic influenza viruses, West Nile virus (WMV), SARS coronavirus, hepatitis B virus, and HIV. The first published reports from India indicate of modest success in the development of DNA vaccines against rabies and Japanese Encepahlitis Virus in experimental animals. Interestingly, the efficacy of DNA vaccine (G protein) against rabies is correlated to levels of neutralizing antibodies, whereas in the case of JEV (envelope protein), cell-mediated immunity appears to be the major mechanism of protection.

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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 vivotranscription and translation of the gene (or complementary DNA) of interest.Intron A may sometimes be included to improve mRNA stability and hence increase protein expression Plasmids also include a strong polyadenylation/transcriptional termination signal, such as bovine growth hormone or rabbit beta-globulin polyadenylation sequences.Multicistronic vectors are sometimes constructed to express more than one immunogen, or to express an immunogen and an immunostimulatory protein. Because the plasmid is the vehicle from which the immunogen is expressed, optimising vector design for maximal protein expression is essential. One way of enhancing protein expression is by optimising the codon usage of pathogenic mRNAs for eukaryotic cells. Pathogens often have different AT contents than the species being immunized, so altering thegene sequence of the immunogen to reflect the codons more commonly used in the target species may improve its expression..

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-terminal ubiquitin signals. 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.

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DNA VACCINE DELIVERY METHODS

There are numerous ways to introduce DNA vaccine to the animal tissue, of which two important methods are:

Injection of DNA in saline, using a standard hypodermic needle :

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 temporarily damaging muscle fibres 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, alignment, speed of injection, volume of injection, muscle type, and age, sex and physiological condition of the animal being injected.

Gene Gun delivery:

Gene gun delivery, the other commonly used method of delivery, ballistically accelerates plasmid DNA (pDNA) that has been adsorbed onto gold or tungsten microparticles into the 1
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target cells, using compressed helium as an accelerant. Alternative delivery methods have included aerosol instillation mucosa. Mucosal surface of naked delivery DNA has on mucosal surfaces, also been such as using the nasal and lung mucosa, and topical administration of pDNA to the eye and vaginal achieved cationic liposomeDNApreparations,biodegradablemicrospheres attenuated Shigella or Listeria ve ctors for oral administration to the intestinal mucosa, and recombinant adenovirus vectors. 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 g-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 g 20 g 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 (normally muscle), where it has to overcome physical barriers (such as the basal lamina and large amounts of connective tissue, to mention a few) before it is taken up by the cells, while gene gun deliveries bombard DNA directly into the cells, resulting in less wastage. 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 pathogens genes induce a protective response. This has been tested with Mycoplasma pulmonis, a murine lung pathogen with a relatively small genome, and it was found that even partial expression libraries can induce protection from subsequent challenge.

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Advantages and disadvantages of commonly used DNA vaccine delivery methods Method Delivery of

Advantage

Disadvantage Inefficient site for uptake due Relatively large amounts of

No special delivery mechanism Permanent or semi-permanent

to morphology of muscle tissue

Intramuscular Intradermal injection

or

expression

DNA used spreads rapidly

pDNA

throughout the body DNA bombarded directly into

Th1 response may not be the Th2 response may not be the

response required

Gene Gun

cells

response required

Small amounts DNA

Requires

inert

particles

as

carrier

Significant shearing of DNA 10-fold lower expression, and

No particles required DNA can be delivered to cells

after high-pressure expulsion

Jet injection

lower immune response

mm to cm below skin surface

Requires large amounts of

DNA (up to 300 g)

High

levels

of

immune

response can be generated

Can increase transfection of

Toxicity Ineffectiveness in serum Risk of disease or immune

Liposomemediated delivery

intravenously delivered pDNA

Intravenously

delivered complexes can

liposome-DNA

reactions

potentially transfect all tissues 1

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How DNA Vaccine Works?

DNA vaccine produces immune response in the body mainly by two methods. They are Endogenous pathway or exogenous pathway.

ENDOGENOUS-PATHWAY:
DNA vaccine is delivered into the muscle cell by any of the delivery methods. This plasmid DNA are absorbed into the nucleus of the cell and pathogenic genes are transcribed to form mRNA then these mRNA are translocated into the cytoplasm of the cell to produce proteins with the help of ribosome along with many translational proteins and enzymes. These produced antigenic peptides are then expressed in the MHC-1 receptor molecules. T-Helper cells identify these antigenic peptides presented by antigen presenting cells like MHC-1 and then elicit immunological response in the body. T-Helper cells multiply themselves and also produce Memory T-cells.

EXOGENOUS PATHWAY:
After producing viral peptides in the cell these viral peptides come outside of the cell and are phagocytosed by antigen presenting cell these activated T-Helper cells and then they produce different types of cytokines against the pathogen. T-Helper cells in turn activate the B-cells, activated plasma B-cell produces the antibody against the pathogen and also produces the memory B-cells. When actually pathogen attacks the body memory T-cells and the antibodies produced from the memory B-cell help the body to fight against these pathogens.

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DNA VACCINES: ADVANTAGES, DISADVANTAGES AND SAFETY CONCERNS ADVANTAGES

The report lists a number of other advantages DNA vaccines have over classic vaccine methods: DNA vaccination provides long-lived immune responses, unlike many component vaccines that require multiple innoculations to maintain immunity. Vaccines for multiple diseases can all be given in a single inoculation. Currently, in the United States, the full course of childhood immunizations requires 18 visits to the doctor or clinic. All DNA vaccines can be produced using similar techniques. The ability to use generic production methods greatly simplifies the vaccine development and production process. They are extremely stable. Unlike many conventional vaccines that must be held at a constant temperature, DNA vaccines can be stored under a vast array of conditions either dried or in a solution. This eliminates the need for the "cold chain" -the series of refrigerators required to maintain a vaccine during distribution. This will greatly improve the ability to deliver vaccines to remote areas in developing countries. Candidate vaccines can be recovered from diseased tissue. Microbial DNA can be isolated from the tissue of an infected animal, purified, amplified and screened for vaccine candidates.

DISADVANTAGES:
Although DNA can be used to raise immune responses against pathogenic proteins, certain microbes have outer capsids that are made up of polysaccharides. This limits the extent of the usage of DNA vaccines because they cannot substitute for polysaccharide-based subunit vaccines. Thus far, the limitations of DNA vaccines mostly involve a lack of research, which will likely be remedied in the future when they become a more important area of interest. At present, there is a limitation in regards to microbial activity. While DNA is successful for providing an immune response when the target involves disease-causing proteins, there are some microbes that have an outer shell made of polysaccharides. Unfortunately, DNA vaccines are unsuccessful

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and instead, subunit vaccines that have a polysaccharide foundation are required. Other possible disadvantages could be, that DNA vaccines are: 1. Limited to protein immunogens (not useful for non-protein based antigens such as bacterial polysaccharides). 2. Risk of affecting genes controlling cell growth. 3. Possibility of inducing antibody production against DNA. 4. Possibility of tolerance to the antigen (protein) produced. 5. Potential for atypical processing of bacterial and parasite proteins. 6. Have potential for integration of DNA into host chromosome. 7. Have a risk of affecting the expression of genes controlling cell growth. 8. Cost per test for may exceed traditional testing methods such as microscopy. 9. Most rapid tests have limited shelf lives that place increased demands on procurement and distribution systems. 10. They are mainly qualitative, producing only "yes/no" answers that may yield less information than the existing laboratory-based quantitative tests. 11. They require subjective interpretation, which may result in reader variation in results. 12. In many cases, rapid tests are less sensitive or less accurate compared to existing reference-level laboratory tests. 13. Are not amenable for high throughput testing. 14. Requires extensive and robust quality control and quality assurance mechanisms.Given below is a tabular form of the advantages and disadvantages of using DNA vaccines:

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Table 1. Advantages And Disadvantages Of Nucleic Acid-Based Immunization Advantages Disadvantages Subunit vaccination with no risk for infection.

Antigen presentation by both MHC class I and class II molecules. Able to polarise T-cell help toward type 1 or type 2. Immune response focused only on antigen of interest

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

Ease of development and production Stability of vaccine for storage and shipping Cost-effectiveness Obviates need for peptide synthesis,

production against DNA Possibility of tolerance to the antigen (protein) produced

expression and purification of recombinant proteins and the use of toxic adjuvants.

Long-term persistence of immunogen. In vivo expression ensures protein more closely structure, resembles with normal eukaryotic postaccompanying

Potential for atypical processing of bacterial and parasite proteins.

translational modifications.

SAFETY CONCERNS
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Safety concerns of DNA vaccines relate to genetic, immunologic, toxic, and environmental effects. In this review we provide an overview of findings related to the safety of DNA vaccines, obtained so far. We conclude that the potential risks of DNA vaccines are minimal. However, their safety issues may differ case-by-case, and they should be treated accordingly.

1. GENETIC EFFECTS
In all administration methods and at all doses applied, DNA vaccines were well-tolerated and nontoxic and following effects were observed. BIODISTRIBUTION AND PERSISTENCE. The bio-distribution and persistence of a plasmid is dependent on the route of administration and delivery method. Shortly after intramuscular injection, plasmid DNA was detected in many organs remote from the site of injection in mice. In rat, plasmid DNA was detected in the lymph nodes. Several weeks after injection, plasmid DNA could only be detected at the site of injection where it persisted for the time of the study. Occasionally, plasmid was detected in gonads, but it dissipated rapidly. The level of plasmid DNA at the injection site was below 100 copies g DNA after initial injection with 100200 g DNA.( Kim et al) have shown that 30 minutes after injection 33% of the initial concentration was present. Ninety minutes after injection less than 1% remained. The amount of plasmid DNA in the organs remote from the injection site was 23 orders of magnitude lower than at the injection site. After intravenous administration in mice and rat, plasmid DNA was initially distributed at a relatively low amount to all tissues examined except the gonads and brain, in which no plasmid DNA was detected. However, plasmid DNA was rapidly cleared. Less than 1% of the initial concentration was detected in blood at 30 minutes post-administration in mice, and no plasmid was detected 60 minutes post-administration. Bureau et al studied radiolabeled plasmid DNA to study its biodistribution after intramuscular injection; between 5 minutes and 3 hours after injection more than 90% of the plasmid had been cleared. A small part of the injected DNA seemed to be relatively protected from DNase I and persisted, probably because it is involved in the transfection process. INTEGRATION INTO THE HOST GENOME.

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Integration of plasmid DNA into the recipients genome appears the major point of attention of DNA vaccination. Integration may occur randomly or by homologous recombination. Integration could lead to activation of oncogenes, inactivation of tumor suppressor genes, or, when integrated into the chromosomal DNA of germ line cells, to vertical transmission. Suicide vectors have been developed that induce apoptotic cell death of transfected cells. Such suicide vectors may be important to alleviate the concerns of potential integration and cell transformation. The results of a study by Wang et al demonstrates that integration events at a very low frequency cannot be neglected. When new technologies improve the efficiency of DNA delivery an increase in integration events can be expected. Therefore, for each new plasmid DNA to be used as a clinical vaccine integration should be considered.

2.IMMUNE EFFECTS:
INDUCTION OF AUTO-IMMUNITY. There is concern that DNA vaccines might induce auto-immunity. The immunostimulatory activity of unmethylated CpG motifs in the plasmid backbone can lead to the formation of antiDNA antibodies, which might accelerate the development of auto-immune diseases.The introduction of other immunomodulatory molecules such as cytokines, may also result in the induction of auto-immune responses to these molecules.Also the attachment of peptides, for example a nuclear localization signal, to the DNA vectors might induce auto-immunity. Another mechanism by which auto-antibodies might arise is through destruction of injected muscle cells as a result of DNA vaccination. However, in this respect, it is unlikely that DNA vaccines would pose any greater risk than conventional vaccines. INDUCTION OF IMMUNOLOGICAL TOLERANCE. Evidently, vaccination is usually applied to induce immunity. However there appears to be a fine line between the induction of immunity and the induction of tolerance. Most vaccines intended for human use are administered to infants and children. Because of the immaturity of their immune system, vaccinated newborns may develop tolerance rather than immunity safety testing of each new DNA vaccine that will be used in children or newborns is extremely important.

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MODULATION OF IMMUNE RESPONSE. When plasmids encode cytokines, they might affect the hosts immune capacity and therewith lead to long-term safety risks. Co-administration of immunostimulatory molecules, either as protein or gene, can result in their release into the circulation, potentially causing undesirable, systemic, effects. TOXICITY AND IMMUNOTOXICITY. An unanticipated safety problem has been reported recently after DNA vaccination against tuberculosis in mice. In an immunotherapeutic vaccination study with mice that were infected with Mycobacterium tuberculosis, vaccination with a DNA vaccine encoding the 65-kDa heat shock protein of Mycobacterium leprae caused pulmonary necrosis.54 Similar severe reactions were seen in mice given a DNA vaccine encoding the Ag85 antigen of M. tuberculosis.

3.ENVIRONMENTAL EFFECTS
Environmental risks of DNA vaccines received little attention so far in literature, probably because the risks are considered very low. Possible environmental risks of DNA vaccines that can be anticipated are: (1) environmental spread of recombinant plasmid vectors by shedding or by consumption of vaccinated animals (2) recombination with viruses, bacteria, or parasites inside the vaccinated host, and consequently the generation and spread of genetically modified organisms (GMOs) (3) recombination with viruses, bacteria, or parasites outside the vaccinated host after shedding. (4) integration in the genome of germ line cells. Injected plasmids might spread accidentally. e.g., by the transfer of plasmids to other species via excreted body fluids, or when the immunized animal or human dies. Plasmid DNA can also be spread by consumption of vaccinated animals. The fate of ingested DNA was examined in several studies and it was found that food-ingested DNA could be traced to several organs. Safety data accumulated so far indicate that DNA vaccines have a good safety profile in preclinical and clinical phase I studies. DNA vaccines may be intrinsically more safe than 1
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conventional vaccines, because they are non- life non-replicating, and non-spreading. They also appear well-tolerated, i.e., nontoxic. In conclusion, DNA vaccines promise to become a flexible and easy way to design and produce vaccines against important public health threats, especially when their immunogenicity will be enhanced. DNA vaccines are considered to be safe and this is supported by data from literature. Additional safety issues may originate from their encoded sequences or added substances, and these should be assessed on a case-by-case base

DNA VACCINES: APPLICATIONS

The current treatment options for autoimmune diseases typically only alleviate the symptoms; nothing is on the market that can actually stop the cause of the disease. Though experiments in mice and small mammals have had very encouraging results, the effectiveness of DNA vaccines has been weaker than anticipated in trials with primates and humans. In human trials small doses have been used mainly as a safety precaution 1. DNA vaccination and Tuberculosis: two novel tuberculosis (TB) vaccines: a DNA vaccine combination expressing mycobacterial heat shock protein 65 (Hsp65) and interleukin-12 (IL-12) by using the hemagglutinating virus of Japan (HVJ)-liposome (HSP65 + IL-12/HVJ) and a recombinant BCG harboring the 72f fusion gene (72f rBCG) have been developed. These vaccines provide remarkable protective efficacy in mouse and guinea pig models, as compared to the current available BCG vaccine. A cynomolgus monkey model, which is currently the best animal model of human tuberculosis, to evaluate the HSP65 + IL-12/HVJ and 72f rBCG vaccines was studied. Vaccination with HSP65 + IL-12/HVJ as well as 72f rBCG vaccines provided better protective efficacy as assessed by the Erythrocyte Sedimentation Rate, chest X-ray findings and immune responses than BCG. Most importantly, HSP65 + IL-12/HVJ resulted in an increased survival for over a year. This is the first report of successful DNA vaccination and recombinant BCG vaccination against M. tuberculosis in the monkey model.

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A DNA vaccine against tuberculosis based on the 65 kDa heat-shock protein differentially activates human macrophages and dendritic cells has been developed: A number of reports have demonstrated that rodents immunized with DNA vaccines can produce antibodies and cellular immune responses presenting a long-lasting protective immunity. the prophylactic and therapeutic effects of a DNA vaccine encoding the Mycobacterium leprae 65 kDa heat shock protein (DNA-HSP65) in a murine model of tuberculosis have been studied. As DNA vaccines are often less effective in humans, the stimulation of human immune responses by DNA-HSP65 has also been studied (Luis H F et al.).

2. DNA vaccination and malaria: The DNA vaccine designed to protect against malaria has been designed to induce protective CD8+ T cell responses against Plasmodium falciparum infected hepatocytes. This study involved the rationale behind the development of vaccines that induce protective CD8+ T cells, the strategy for the development of a DNA vaccine designed to protect against falciparum malaria, and the experimental data in rodent models and nonhuman primates which has provided the foundation for trials of DNA vaccines against P. falciparum malaria in humans. Physicians
conducting the first human trial of a DNA vaccine against malaria reported that the vaccine was welltolerated and safe, and that preliminary analysis of specimens from trial participants suggested a good cellular immune response with features they believe may be important in preventing the disease. The Phase I clinical trial was designed to test the safety of an experimental naked DNA vaccine against the parasite that causes malaria and to evaluate immune responses in approximately 20 human volunteers.

3. DNA Vaccine against Multiple Sclerosis: Phase 2 trial of a DNA vaccine encoding myelin basic protein for multiple sclerosis have been carried out.

4.New Cancer Vaccine Starves Tumours of Blood: 1

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ScienceDaily (May 25, 2010) A DNA-vaccine that restricts the supply of blood to tumours has been developed by scientists at the Swedish medical university Karolinska Institutet. The vaccine slows the growth of breast cancer tumours in mice.If a cancer tumour is to become larger than a few millimetres it must be able to stimulate the formation of new blood vessels, in order to secure the supply of oxygen and nutrients. Drugs that prevent the growth of blood vessels are thus a potential treatment alternative for tumours.A protein known as 'Delta-like ligand 4' (DLL4) has recently been identified as an important component in regulating the formation of new blood vessels. When a new blood vessel starts to grow from an existing vessel, DLL4 is expressed in the tip cells at the end of the new vessel sprout, and prevents neighbouring cells from forming new vessels. If the expression of DLL4 is blocked in a tumour, there is a large increase in the formation of new, but non-functional, blood vessels, and this leads to the tumour growing more slowly. A research group at Karolinska Institutet has developed a DNA-vaccine against DLL4 and the blood vessel tip cells. They have shown that vaccination against DLL4 causes an immunological antibody response to DLL4, and this hinders the growth of breast cancer in mice. Tumours from vaccinated mice had a tightly packed network of non-functional blood vessels, and poor blood supply. The vaccination did not cause any undesired effects and did not affect the animal's capacity for wound healing. 5. DNA vaccination and HIV: The earliest Phase I clinical trial for a DNA vaccine has been of an HIV-1 candidate tested in individuals infected by HIV-1, followed by studies in volunteers who were not infected by HIV1. Other prophylactic and therapeutic DNA vaccine trials followed, including trials that tested DNA vaccines against cancer influenza, malaria, hepatitis B, and other HIV-1 candidates. These trials demonstrated that the DNA vaccine platform is well tolerated and safe, as no adverse events were reported and all studies went to completion.To further increase immunogenicity of DNA vaccine, the use of molecular adjuvants such as cytokines and immunomodulatory molecules has been extensively employed in clinical trials. 6. DNA vaccination and Liver and prostrate cancer: 1
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Clinical trials conducted over the last few years have led promising results, particularly when DNA vaccines were used in combination with other form of vaccines, as demonstrated in prostate and liver cancer clinical trials. Delivery of gene-based vaccines by physical methods, that is, electroporation and gene gun, has demonstrated to amplify the immune responses induced by therapeutic vaccines against cancer. 7. DNA vaccination and Lymphoma: DNA vaccination is an attractive and effective approach for active therapeutic vaccination against B-cell malignancies given the ease of production compared to Id protein vaccines. Patient-specific DNA vaccines for therapy of B cell lymphomas and multiple myelomas based on scFv encoding a chimeric immunoglobulin molecule consisting of VH and VL genes derived from each patientss tumor have been shown to be effective in animal models. 8. DNA vaccination and Prostate Carcinoma Assessment of the feasibility and safety of vaccination with increasing doses of xenogenic DNA coding for the Rhesus Prostate Specific Antigen (rhPSA), a protein that is 89% homologous to human PSA, administered in patients with relapsed prostate cancer has been done. Results of a phase I/II trial, conducted with DNA vaccine encoding human prostatic acid phosphatase (PAP) coadministered intradermally with GM-CSF, in prostate cancer patients (stage D0) are associated with an increased PSA doubling time (PSADT), 6.5 months pretreatment versus 9.3 months in the 1 year posttreatment. A longer PSADT is associated with an extremely low risk of death from prostate cancer. Besides, 14% of patients developed PAPspecific IFN gamma-secreting CD8+T-cells immediately after the treatment course, and 41% of patients developed PAP-specific CD4+ and/or CD8+T-cell proliferation, confirming the preclinical studies. 9. DNA vaccination and Melanoma DNA vaccine platform is a promising therapeutic approach also for the treatment of malignant melanoma, as demonstrated by already completed and ongoing clinical trials. In stage IV melanoma patients, a phase I/II pilot study of intranodal delivery of Synchrotope MA2M plasmid DNA vaccine has been found to induce both humoral and CTL responses 1
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against cells expressing tumor two melanoma-associated antigen. Synchrotope MA2M plasmid is a bivalent DNA vaccine encoding epitopes for both Melan-A (MART-1) and tyrosinase with potential antineoplastic activity. The same approach was used in a improved trial conducted with the Synchrovax SEM plasmid DNA vaccine containing a plasmid pSEM that encodes 4 peptide epitope sequences, Melan-A (2635), Melan-A (3196), tyrosinase (19), and tyrosinase (369377), resulting in antigenspecific immunity even though not induce regression of established disease. 10. DNA vaccination and Cervical Cancer Current vaccination strategies are based on the induction of neutralizing antibodies against the major and minor capsid proteins, L1 and L2, of human papillomavirus, and Gardasil is only effective against a subset of HPV genotypes. Further therapeutic interventions for early-stage and late-stage cervical cancers or HPV-related disease are ineffective so far.

SOME OTHER APPLICATIONS:11. DNA vaccination used in Liver Cancer 12. DNA vaccination used in Breast Cancer 13. DNA vaccines and their applications in veterinary practice 14. DNA vaccines used in Influenza 15. There are certain Topical application of DNA vaccines 16. DNA vaccines are used in aquaculture.

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DNA VACCINES: FUTURE PROSPECTS

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 (Raz, E., 1998).

Plasmid with multiple genes provide immunity against many diseases in one booster DNA vaccines against infectious diseases such as AIDS, Rabies, Malaria can be available

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References

Protection against anthrax and plague (NMRC/Porton Down/NTC), John Palmer et al., Abstract Biodefense Nov 07 31 07 (2) (3). pDNAVACCUltra rabies DNA vaccine targeting.pdf, Kaur et al. GPI anchored anthrax vaccine.pdf, Midha and Bhatnagar Anthrax protective antigen administered by DNA vaccination to distinct subcellular locations potentiates humoral and cellular immune.pdf, Midha and Bhatnagar NTC eRNA DNA Vaccines. Coexpressed RIG-I agonist enhances antigen-specific immune response to Influenza DNA Vaccine,James Williams Ph.D., Jeremy Luke, B.A., and Clague Hodgson Ph.D. pDNAVACCUltra Vector family: high throughput intracellular targeting, 2006. James Williams, Jeremy Luke, Lance Johnson and Clague Hodgson Doherty, P.C., Knowles, B.B. & Wettstein, P.J. Immunological surveillance of tumours in the context of major histocompatability restriction of T-cell function. Adv. Cancer Res. 42, 165 (1984). | PubMed | ISI | ChemPort | Yewdell, J.W. & Bennink, J.R. Cell biology of antigen processing and presentation to MHC class I molecule restricted T-lymphocytes. Adv. Immunol. 52, 142 (1992). | PubMed | ISI | ChemPort | Tang, D.C., DeVit, M. & Johnston, S.A. Genetic immunization is a simple method for eliciting an immune response. Nature 356, 152154

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