Infectious diseases are the biggest cause of mortality and morbidity in humans, especially in poor and developing countries, and new and emerging infectious agents are also being added to the list, with more than 29 infectious agents since 1973, there were new and 20 emerging diseases. At the same time, trying to get the vaccine against many infectious agents with conventional vaccines has been declining, and for many years there has not been a new vaccine, which indicates the limitations of the development of common vaccines, including destruction or inactive vaccines, weakened vaccines toxoids known as first-generation vaccines, as well as second-generation or recombinant vaccines, are prepared by genetic engineering. From the second generation, only hepatitis B vaccine has been widely used, and in addition, a recombinant vaccine against Lyme disease has been confirmed in humans. However, efforts to provide recombinant vaccines against many infectious agents, and in particular emerging infectious agents, have not yielded a favourable result because these vaccines mainly induce a humoural immune system and their cellular immunity is not optimal. At the same time, vaccines are very expensive and also widely used, making it difficult for poor and developing countries which have high rates of infectious diseases. After many years of research, spending on large-scale costs and evaluating the types of vaccines provided by the aforementioned vaccines has not been provided with a vaccine against infectious agents such as AIDS, malaria, hepatitis C  and many other factors until now. Therefore, new vaccines are needed with better quality and fewer side effects.
Inactive vaccines create an immune response through the induction of CD4+ T cells and haemorrhage, but this is not long-term immunity. In contrast to weakened live vaccines, both immune cells and haemorrhages are stimulated and the immune system generates more time. However, the amount of attenuation reduces the immunisation of the live vaccine, and there is a problem with producing a weak vaccine which is supposed to target several types of subtypes of the viruses or pathogens. There are too many concerns about the safety of both non-live and weaken vaccines. These restrictions have created a need for a new generation of vaccines with wider immune-building.
The study on DNA vaccines first began in the 1990s, when the plasmid DNA which was injected into the skin or muscle was reported to induce antibody responses to antigens. The simplicity and compromise of this vaccine approach have attracted much attention in conducting studies on viral and non-viral antigens. In the theory of DNA, vaccines (without the need for a proliferating pathogen) can cause widespread immune responses, such as viral vaccines.
TYPES OF VACCINES
Killed vaccine (inactivated vaccine)
Inactivated vaccine (or killed vaccine) is a vaccine consisting of virus particles, bacteria or other pathogens that have been grown in culture and then killed using a method such as heat or formaldehyde. These vaccines include hepatitis A and hepatitis B vaccine, poliovirus vaccine and ticks encephalitis.
Live attenuated (weakened) vaccine
This is the use of non-pathogenic pathogens of microbial pathogens. To produce a weakened microbial pathogen that is not capable of causing disease or a very non-severe disease, various physical and chemical methods, or mutational radiation and genetic engineering are used. This vaccine series is highly beneficial due to the viability of the microbes and the imitation of actual illness, and sometimes, a very mild illness stimulates both immune systems. Of these vaccines: rubella, mumps, measles, chickenpox, tuberculosis, cholera, typhoid  and yellow fever. However, given the liveliness of microbes in children, the elderly and those with a weak immune system can cause dangerous infections. These people can even get the disease from healthy people who have received these vaccines recently.
Second-generation vaccines or recombinant vaccines
The recombinant vaccines made by genetic engineering and they are two types. In the first type, those used to provide a live vaccine are used to modify genetic engineering methods to non-agonise an infectious agent by eliminating or causing mutations in part of one or more genes responsible for pathogens or viral microbes. The next approach is to isolate one or more gene encoding pathogenic proteins and importing into another harmless or weakened virus or bacterium.
Third-generation vaccines (gene vaccine)
These vaccines are direct injected plasmids that have the ability to express the desired gene within the body's cells. By inserting the plasmid into the body, the recombinant protein is produced in the body and placed in the immune system. DNA suspension vaccines are from bacterial plasmids carrying immunogenic protein genes. The plasmid, which was genetically engineered for gene delivery, is cloned into Escherichia coli bacteria and after purification by various means such as intramuscular injection, intraperitoneal injection, immersion, transfer through nanoparticles and mucosal to the body sign in 
The first demonstration of the usefulness of the vaccine DNA was done by injection of a human growth hormone-encoding plasmid (HGH) to the mouse. In this first test, the hGH gene was injected into the skin of the ear of the mouse to produce its protein for treatment. Several immunised mice showed remarkable levels of antibodies. Similar to the immune response observed in the viral infection or the weakened virus vaccination, intracellular production of peritoneal or peptide antigen induces a high Th1 cellular response. However, due to low protein production (coded by a DNA vaccine), the Th2 immune response is low. Considering the usefulness of DNA vaccines in small animal models, further clinical trial studies were carried out. One of several Phase I trials, which began about two decades ago, is about evaluating the DNA vaccine of the HIV-1 virus for the purpose of therapeutic and preventative use. Further studies have included DNA vaccines for other HIV-1 antigens, cancer antigens, influenza, HPV, hepatitis and malaria. However, the results of these early studies were not satisfactory. DNA vaccines are safe and well tolerated, but proved to be poorly immunised. The induced antibody titre is very low or absent, the CD + 8 T-cell response is rarely present, and the response of T-type CD + 4s is also low. However, these studies have shown that DNA vaccines can safely produce a human immune response. A DNA vaccine has been approved to protect horses against the West Nile Virus for use in veterinary medicine. Klinman et al. have been able to tolerate specific immunity to the antigen by using a DNA-based myelin-based protein-coding vaccine (in patients with multiple sclerosis. DNA vaccines have many benefits to poor immune responses in humans. The first successful vaccine project with this method is the MenB strain of MC58, which was approved by the European Medicines Agency and branded by Bexsero.
SAFE AND TOLERABLE DNA VACCINES
DNA vaccines seem to be more harmless and more stable than ordinary vaccines. Plasmids are non-viable and do not multiply, and therefore have a low risk of developing secondary disease and infection. The main concern about their potential DNA vaccines has been integrated into the host genome and generated immune responses to agent anti-DNA. Extensive surveys have found little evidence of integration, and the merger risk appears to be less than normal mutation.
ADVANTAGES AND DISADVANTAGES
Significant advantages of these vaccines include the cheapness, simplicity of production and consumption, transport and higher resistance. The other important feature of these vaccines is the ability to put several antigens in the plasmid, resulting in immunisation against all of agent. Furthermore, studies have shown that antagonisms made from vaccine plasmids in the host body for glycation and post-translational changes are the same as the main microbial protein. One of the main concerns about DNA vaccines is the risk of developing anti-DNA antibodies, which studies have shown that the use of a gun due to the more accurate and lesser need for plasmid can be effective in resolving this problem. Studies have shown that immunisation of DNA vaccines over time has been used to overcome this problem by using different adjuvants, including the use of interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) genes in the plasmid.
MECHANISM OF DNA VACCINE
DNA vaccination is an interesting method of immunisation for its ability to induce CTL strong responses, and it also can activate other immune systems. The DNA vaccine achieves this goal by mimicking natural viral infections. The expression of foreign genes transmitted to an in vivo (such as the body of the mouse) results in the production of proteins, these proteins, such as proteins produced by viral genes, are processed and delivered to the immune system. The ultimate outcome of DNA vaccination is the production of non-living, non-proliferating and non-inflammatory antigens that not only stimulate the immunity of TCD8+ and TCD4+ cells, but also stimulate immune B cells. Details of the mechanisms of each DNA components of the vaccine are still being investigated and are still is not fully understood. Somatic cells (myocyte or keratinocyte) and antigen-presenting progenitor cells are two major types of cells that are at the onset of the immune response generated by the DNA vaccine. For example, after intramuscular injection of the plasmid, DCs and myocytes are transfected. After the plasmid is inserted into the transfected cell nucleus, the plasmid genes are expressed, and the external antigen is produced as a peptide chain by the host cell. These peptides can act as dendritic cells or myositis in conjunction with Class I or II MHCs or cross-linking pathways, which can lead to immune function.
ESSENTIAL REQUIREMENTS OF A PLASMID
The plasmid used for the DNA vaccine should have a strong promoter for expression in the cells, which is usually used for viral promoters. In some cases, the use of a polyadenylation sequence (poly-A) is also effective in stabilising the transcripts. Approaches that increase the transcription and DNA translation of the vaccine may improve the immunisation of the vaccine in humans.
Other factors that are necessary, may be the origin sequence of the plasmid, which is required to begin assimilation in a bacterium, an antibiotic resistance gene used during purification, and a transcriptional stop region that is greater than the region 3 The Humuor region of Bovin's growth hormone is used.
PROMOTERS AND ENHANCERS
Sometimes several promoter or additive such as enhancer or silencer to improve the stability of mRNA and protein expression vector to come inside. Plasmids also have a strong termination signal. Some multicistronic vectors are designed to express several immunogens or to express a number of immune stimulatory proteins. For maximise protein expression, the vector should be effectively designed. For example, optimising codon usage of mRNA in eukaryotic cells is the best way to increase protein expression.
Another important factor in the production of DNA vaccine is the selection of promoter. Although the SV40 promoter has been widely used, the DNA expression rate of vaccines has increased with the use of the promoter of the immediate genesis of cytomegalovirus (CMV). This promoter is one of the strongest promoters. Studies on env gene in the HIV-1 virus have shown that Promoter power is expressed in terms of expression and thus has a significant effect on the immunogenicity of the vaccine. Some studies have shown that the promoter of the MHC class II is also effective in mice and also prevents anti-immunostimulants such as interferons (INFs), anti-tumour necrosis factors, as well as anti-infective. In addition, several studies have shown that combined promoter (CMR) with intramuscular chicken beta-actin (CAG promoter) causes multiple expressions of the protein CMV.
ENTERING SEQUENCES AND OPTIMISING CODONS
For various cellular components, DNA sequence sequencing or optimisation of DNA codons can be performed and improve TH1 or TH2 responses. Adding a ubiquitin signal to the N-terminal region of gene increased in immunity of cell. Sometimes spatial changes of amino acids in the protein enhance the immune response. Optimising codons also increase protein expression. There are now automatic algorithms to optimise codons to help vaccinate.
PLASMID TRANSFER METHODS
Various methods are used to immunise animals with DNA vaccines. The type of T cell response is influenced by the type of transmission method and the type of expression of the immunogen. Each method has its own special privilege to enable safety. However, no single method is successful in increasing the protective effect and proper regulation of the induction of immune response.
USE OF EPIGENETIC AND IRNAS IN GENETIC VACCINES
The use of post-translation changes to genetic vaccines is under consideration. For example, these inhibitory RNAs can be used to inhibit genes that reduce immune responses to the vaccine, for example, inhibition of caspase 12 in the receiving vaccine cells have prevented cell death and increased immunogenicity with T CD8 for HIV-gp120.
Researches show that epigenetic changes, such as methylation of genes or changes in histones, can affect the immunogenicity and also change the duration of vaccine expression. In a study on adenovirus vectors for expression of the gene for the growth of human fibroblast growth factor (hFGF-4) In rats, it was shown that the level of expression of this gene was significantly reduced over several days after use in rats by the CMV promoter methylation.
Needle injections are used to induce Th1 responses. In other studies, they have proven that the nucleoprotein encoding the plasmid DNA protects the animal from the cytotoxic activity. Many studies have shown that injection of DNA vaccines with needles against viral infections is very effective in animal models. Generally, for injection of DNA vaccines, topical anaesthetics (bupivacaine), a hypotonic solution, or salt, and occasionally electroporation are used. Immunisation is usually done by injection into a skeletal muscle or intradermal injection, and the DNA is transmitted to the intracellular or intercellular region.
Transfer DNA into the cell by electroporation or through temporary damage to muscle fibres by an anaesthetic (bupivacaine) or hypertonic solutions. It is difficult to obtain a stable immune response in needle-based immunisation. The immune response by needle injection is influenced by various factors including the type of needle, type of muscle, the age of the animal and injection rate.
The gun is often used to increase Th2 responses. Plasmid DNA is coated on the surface of gold and is introduced into the cells by compressed helium gas as an accelerator. The use of a gun due to its effect on the progenitor and Langerhans cells is a particular importance.
The gun's method has the advantage of having a lower amount of DNA into the cell, and it also has the benefit of golden safety. In salt injection methods, 2–20 μg of plasmid DNA is needed for each mouse, while a gene gun with 1–3 μg plasmid DNA produces an effective immune response. Most of these results are observed in mice.
For clinical trials in humans, it is important to reduce the dose of plasmid DNA, since they vary in size from one species to another. For example, primates (such as monkeys) require about 10 times more plasmid DNA than mice. The immunisation of the plasmid DNA is weak, so several immunisations with these methods and injections at different sites are required to produce a strong immune response.
Another method is breathing through the nose and transferring plasmid DNA to the nasal and lung nasal surfaces. Vanniasinkam et al. investigated the tissue release of DNA plasmids in nasal breathing using in situ hybridisation method. They determined that DNA plasmids exist in the lung, liver, spleen, lymph nodes, kidneys, embryos and mouse oesophagus alveoli. HIV plasmids have been identified 2–4 weeks after respiration. The HIV env gene mRNA has been detected in the lungs, liver and spleen, while HIV-1 proteins have been identified in the lung. Vaccination of plasmids containing HIV genes through the nose and also from the intravaginal route induces a high level of Th2 response to HIV viral antigens.
In addition, intranasal immunisation with plasmid DNA containing the haemagglutinin gene of the influenza virus has caused immune response to the influenza virus in the mouse model.
TRANSMISSION WITH BIOJECTOR AND CO2 POWER
Transferring a DNA vaccine using a non-needle biojector device induces a Th2 immune response, as well as the response of the T-cell CD8+ and INF-gamma (when recombinant adenovirus or vector vaccine virus is used as a booster). Transmission of DNA without needle using a CO2 biojector is a useful method for immunisation.
The electroporation method induces one of the strongest Th1 cell responses. This method induces an immunity of ten times or more to the response induced by other plasmid DNA vaccination techniques in animals. However, the disadvantage of this method is the use of high voltage, which delayed its use in medicine. Although some modified electroporation procedures have been reported recently, more effective methods are needed. Donate et al. recently developed a non-invasive electrode known as multi electrode array (MEA). They used MEA to vaccinate DNA for hepatitis B infection. The surface-active anti-hepatitis B plasmid was transmitted by MEA intradermal into guinea pigs. The results showed an increase in the expression of the protein after the transfer of the plasmid by MEA compared to the injection.
The topical application of the DNA vaccine on the skin is a useful method of immunisation for being simple, painless and cost effective. However, the level of immune response is relatively low. Liu et al. used the HIV-1 vaccine, as well as cytokine-expressing plasmids, locally on the skin of the mouse. Their findings showed that topical use of the DNA of the HIV-1 vaccine induces an immune response against the env virus antigen.
Okuda et al. showed the expression of the matrix gene (M) of the influenza virus using several times the DNA vaccine used on the skin of the mouse (after removing the keratinocyte layer). DNA vaccination against HSV-2 has also been reported on vaginal mucosa.
OTHER USES OF GENE VACCINES
In addition to the effectiveness of this technology in the provision of vaccine against infectious agents, many studies have been carried out for other applications and have achieved favourable results. These include the prevention and treatment of various diseases, in particular types of cancers, allergic treatments and other autoimmune diseases, treatment chronic viral infections such as (HBV, HCV, HSV, HIV, HPV), gene therapy, the production of essential immunological agents such as monoclonal antibodies, growth factors and slow transmission of drugs whose genes have been identified, immunisation and the structure and activity of proteins and peptides.
IMPROVEMENT OF THE GENE VACCINE
DNA immunisation by inducing the production of various cytokines leads to a wide range of TH reactions. An important advantage of DNA vaccination the convenient adjustment of the type of T cell response (Th1 or Th2) is the addition of several cytokine plasmids. To create a stronger DNA vaccine, the regulatory effect of immunosuppression has been evaluated by the use of IL2, GM-CSF, IL-12, INF-gamma or different expression plasmids.
When the vaccine and expression plasmids were placed inside expressive liposomes and used in mice, the response to the increased sensitivity of antigen and lymphocyte T-cytotoxic activity increased significantly. When the vaccine is used in the form of a plasmid, the expression of cytokines has increased for 1 week or more. For example, an IgG antibody analysis in a group that received a combination of vaccine and cytokine plasmids showed Indicating increased levels of IgG1/IgG2a. These results proved that IL-2 expressive plasmid strongly promotes the specific immune response of HIV-1 by activating Th-1 cells.
The combined use of cationic liposomes plus DNA vaccines greatly increases immune responses and antibodies against HIV-1 persist for a long time. The combined use of the DNA vaccine with IL-12 and GM-CSF expressing plasmids induces high levels of HIV-specific CTLs and when used in the rat (i.n.), increases the sensitivity of delayed sensitivity.
Sasaki et al. developed a DNA vaccine env virus-encoding vaccine HIV-1 and assessed the effect of saponin vaccine adjuvant QS-21. Vaccination from the nasal passageway and intramuscular injection induces significant systemic immune responses and QS-21 increases the production of antigen-specific IgG2a antibodies, the response to delayed sensitivity and cellulolytic activity of splenocytes. In the study of Toda et al., the effect of 8-bromocyclic AMP adjuvant on the HIV-1 vaccine DNA was investigated. Applying the combination DNA vaccine and 8-bromocyclic AMP from the intramuscular pathway and nasal route in mice increased the immunity of cellular and humoural proteins compared to the immunisation of the only DNA vaccine.
CpG stimulatory sequences (CpG motifs)
Non-mammalian CpG sequences are abundant in bacteria but do not exist in the vertebral genome. Mycobacterium DNA has been reported to increase the response of the adjuvant to cancer. Oligo deoxyribonucleic acid-containing CpG sequences activates host defence mechanisms including inherent and acquired immune responses. CpG sequences of bacterial stimulation contribute to increased immune responses. In the DNA of bacteria, non-methylated double nucleotides are present in the CpG that increase immune responses. These single-stranded DNA sequences are immmunostimulatory DNA sequences (ISS), and in the immune system called pathogens associated molecular patterns and are also identified with the TLR-9 by the intrinsic immune system. The varieties of these sites have been identified in the order of GACGTC-3, 5-AGCGCT-3, 5-AACGTT-3 and it has been determined that their abundance The genome of bacteria is twenty times more common than the mammalian genome. Investigating the role of the presence and absence of this structure in the gene vaccine plasmid with the methylation of these sites indicates a decrease in immunity level and in comparison to introducing more of this site in the design of the vaccine, causes a significant increase in cellular and blood immune responses, increased activity of macrophages and NK-cell, followed by the production of various cytokines such as INF-alpha, beta and gamma, a variety of ILs and IgM antibodies, which play an important role in controlling intracellular bacterial infections. Injection of bacterial DNA or a single-stranded short-staple (oligo) fragment containing the CpG site can protects the experimental animal against the lethal dose of Francisella tularensis and the Listeria monocytogenes bacteria. Any change in this position or its metabolism will result in the elimination of immunisation. In fact, these sites act like a supporter, and its use in the plasmid structure of the vaccine can increase its immunogenicity. With regard to these new cases, further research has been done to provide better carriers for vaccines. The discovery of these structures and their role in Arthur Krieg's (1994) at the University of Ottawa, Canada, has led many research to be used as an advocate for vaccines against infectious diseases and for the treatment of autoimmune diseases, allergies, cancer and asthma. Based on this finding, in 1997, a company called CpG ImmunoPharmaceuticals was founded by Gurunathan et al. in the United States and Germany to take advantage of the medical use of this discovery. To evaluate the effectiveness of this assistant, the researchers conducted a clinical trial of one of the materials produced by the company at the University of Toronto, Canada in April 1999, on 48 healthy volunteers, which will continue for 2 years. In this study, a plasmid containing a specific CpG gene was used to evaluate the immunogenicity of vaccines in comparison with non-this vaccine vaccines. The plasmid, called CpG 7909, is actually tested as an aiding with recombinant hepatitis B vaccine in terms of safety and lack of tolerance. The initial results of the clinical trials of this gene support have shown very valuable results in increasing the immune response to hepatitis B vaccine. It is hoped that this vaccine will be tested in addition to its immunogenicity against hepatitis in terms of proper stimulation of the immune system against cancer, allergy, nouns and other infectious agents, and can be used as immunotherapeutic agents. In addition to the creation of this cytoprotective agent, the CpG 8916, CpG 8954 has been developed, which alone or in combination with pathogenic antigens in primary clinical trials has shown strong anti-cancer and anti-infectious agents. The type of marker in the plasmid also plays a role in the immune response. Comparison of the presence of the gene of resistance to Ampicillin and Kanamycin kanR in the CMV-derived vaccine revealed that the presence of the ampR gene induced a stronger reaction than kanamycin, and this increase was not due to the difference in the amount of antigen production, but rather the existence of two transcripts The ISS palindromic CpG stimulator (hexamer 5 'AACGTT 3') is an agent of the ampR gene, while the Kanamycin resistance gene lacks that row. Plasma administration containing two copies of the AACGTT row in the rat produces a stronger immunity than the plasmid lacking this gene.
The presence of plasmid in the tissue and its entry into the cell has a direct relation with the amount of gene induction and ultimately the level of immune response. Intercellular environment Due to the presence of various endonuclease enzymes, no enzymes for circular plasmids are available. The presence of these enzymes makes the vaccine of the gene, which is a pure circular plasmid, is prepared after injection in less than a few minutes to form a linear plasmid and then become small DNA fragments. Adding serum to a circular plasmid in the laboratory also results in the decomposition of a gene vaccine plasmid. Therefore, to increase the efficiency of the vaccine, it is necessary to use methods that cause the plasmid to be more stable in the tissue before it enters the cell, or to increase the rate of arrival of the plasmid and also reduce the rate of degradation of the plasmid. The use of liposomes, polymers and various types of plasmid encoding methods within different materials can increase the half-life of the tissue, which we will mention in other sections. These materials also increase the amount of plasmid transfer into cells. Using specific cellular receptors can also increase the specific binding of the vaccine to the cells. As an example, asialoglycoprotein receptors (plasmid) can be used to transfer specific liver vaccines to liver cells, and it has been shown that intravenous administration of this mixture leads to the specific introduction of plasmid into liver cells.
Other methods of increasing endocytosis indicate that certain proteins of the influenza virus and Sendai virus increase the endocytosis. The use of immunosuppressive agents (adjuvants) and immune-stimulating factors can also increase the immune response in gene vaccines.
Due to the novelty of this field and its vast capabilities, extensive research is underway to optimise these vaccines: optimise vaccine plasmids for proper and adequate antigen induction, appropriate vaccine delivery methods, auxiliary agents and related materials, especially CpG, removal of unnecessary sequences and the storage of smaller plasmids, and the proper formulation of the vaccine. Studies have been conducted to use a combination of the vaccine in the first administration and then to the administration of a recombinant vaccine called Prime Boost Vaccination.
Given the importance of this technology in the provision of cheap infectious vaccines in the world, and especially in low income and developing countries, the WHO specialised committee has put this technology at the heart of its research and other affiliated centres.
In the case of hepatitis C gene vaccine, due to the high prevalence of this virus in the world and the absence of appropriate vaccine and the same dispersion of different genotypes of this virus in different regions of the Core gene isolated from a single patient after determining the row in clonal gene vaccine plasmid and is under investigation in experimental animals.
According to the clinicaltrials.gov database, recently, 54 DNA viral and non-viral vaccines are being evaluated at the clinical trial. Most of these clinical trials study are viral vaccines (25%) or vaccine cancers (21%) [Figure 1].
DISCUSSION AND CONCLUSION
Immunisation of the vaccine in the medical sciences has a prominent place. Removal (Immunisation) by DNA plasmid has had significant advances over the course of 23 years of study. Good studies have been reported using various animal models against many microbial infections. Since DNA vaccines are easily designed and manufactured, they are easier to keep and cost less, as one of the most desirable types of vaccine. However, the efficacy and safety of these vaccines have not been thoroughly investigated, which in this regard is the most worrisome carcinogenicity and anti-DNA antibodies. Despite the introduction of the first DNA vaccine, information on the vaccination method, adjuvant and the genetic structure of the vaccine is still not complete. Currently, one of the best combinations of DNA vaccination is the use of electroporation with appropriate genetic adjuvants and after using viral vector vaccines as boosters. However, more clinical trials are needed to prove the immune responses that immune to DNA vaccine in humans. DNA vaccines are currently inducing strong Th1 immune responses, but Th2 responses are poor. To overcome this problem, you can use some of these adjuvants or combine a booster vaccine. The following Table 1 summarises the advantages and disadvantages of the methods used in DNA vaccine.
Financial support and sponsorship
We gratefully acknowledge the Vice-Chancellor for Research and Technology, Kermanshah University of Medical Sciences, for financial support of this study resulting from Grant No. 96515.
Conflicts of interest
There are no conflicts of interest.
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