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20-September-2008 09:55:51 - DNA vaccination The making of a DNA vaccine. The making of a DNA vaccine. DNA vaccination is a technique for protecting an organism against disease by injecting it with genetically engineered DNA 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 tumour models. DNA vaccines have a number of advantages over conventional vaccines, including the ability to induce a wider range of immune response types. Vaccines are among the greatest achievements of modern medicine - in industrial nations, they have eliminated naturally-occurring cases of smallpox, and nearly eliminated polio, while other diseases, such as typhus, rotavirus, hepatitis A and B and others are well controlled.1 Conventional vaccines, however, only cover a small number of diseases, and infections that lack effective vaccines kill millions of people every year, with AIDS, hepatitis C and malaria being particularly common. First generation vaccines are whole-organism vaccines - either live and weakened, or killed forms.2 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.2 In order to minimise these risks, so-called second generation vaccines were developed. These are subunit vaccines, consisting of defined protein antigens such as tetanus or diphtheria toxoid 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. 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 recognised as foreign, they are processed by the host cells and displayed on their surface, to alert the immune system, which then triggers a range of immune responses.21 These DNA vaccines developed from failed gene therapy experiments. The first demonstration of a plasmid-induced immune response was when mice inoculated with a plasmid expressing human growth hormone elicited antibodies instead of altering growth.3 Contents 1 Current use 2 Advantages and disadvantages of DNA vaccines 3 Plasmid vectors for use in vaccination 3.1 Vector design 3.2 Vaccine insert design 4 Delivery methods 5 Immune response raised by DNA vaccines 5.1 Helper T-Cell responses 5.1.1 Raising of different types of T-cell help 5.1.2 Mechanistic basis for different types of T-Cell help 5.1.3 Practical uses of polarised T-Cell help 5.2 Cytotoxic T-cell responses 5.3 Humoral antibody response 5.3.1 Kinetics of antibody response 6 Mechanistic basis for DNA raised immune responses 6.1 DNA Uptake Mechanism 6.2 Antigen presentation by bone marrow-derived cells 6.3 Role of the target site 6.4 Maintenance of immune response 6.5 Inteferons 7 Modulation of the immune response 7.1 Cytokine modulation 7.2 Immunostimulatory CpG motifs 7.3 Alternative boosts 7.4 Additional methods of enhancing DNA-Raised immune responses 7.4.1 Formulations of DNA 7.4.2 Alphavirus vectors 8 See also 9 References 10 External links Current use Thus far, few experimental trials have evoked a response sufficiently strong enough to protect against disease, and the usefulness of the technique, while tantalizing, remains to be conclusively proven in human trials. However, in June 2006 positive results were announced for a bird flu DNA vaccine 4 and a veterinary DNA vaccine to protect horses from West Nile virus has been approved.56 In August 2007, a preliminary study in DNA vaccination against multiple sclerosis was reported as being effective.7 Advantages and disadvantages of DNA vaccines Table 1. Advantages And Disadvantages Of Nucleic Acid-Based Immunization Advantages Disadvantages Subunit vaccination with no risk for infection1 Antigen presentation by both MHC class I and class II molecules1 Able to polarise T-cell help toward type 1 or type 21 Immune response focused only on antigen of interest Ease of development and production1 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 adjuvants8 Long-term persistence of immunogen2 In vivo expression ensures protein more closely resembles normal eukaryotic structure, with accompanying post-translational modifications2 Limited to protein immunogens Potential for atypical processing of bacterial and parasite proteins1 Plasmid vectors for use in vaccination 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.9 Intron A may sometimes be included to improve mRNA stability and hence increase protein expression.10 Plasmids also include a strong polyadenylation/transcriptional termination signal, such as bovine growth hormone or rabbit beta-globulin polyadenylation sequences.2111 Multicistronic vectors are sometimes constructed to express more than one immunogen, or to express an immunogen and an immunostimulatory protein.12 Because the plasmid is the vehicle from which the immunogen is expressed, optimising vector design for maximal protein expression is essential.12 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 the gene sequence of the immunogen to reflect the codons more commonly used in the target species may improve its expression.13 Another consideration is the choice of promoter. The SV40 promoter was conventionally used until research showed that vectors driven by the Rous Sarcoma Virus RSV promoter had much higher expression rates.2 More recently, expression rates have been further increased by the use of the cytomegalovirus CMV immediate early promoter. Inclusion of the Mason-Pfizer monkey virus MPV-CTE with/without rev increased envelope expression. Furthermore the CTE+rev construct was significantly more immunogenic then CTE alone vector. 14Additional modifications to improve expression rates have included the insertion of enhancer sequences, synthetic introns, adenovirus tripartite leader TPL sequences and modifications to the polyadenylation and transcriptional termination sequences.2 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.1 This is usually accomplished by the addition of N-terminal ubiquitin signals.1516 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.17 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.1 Delivery methods DNA vaccine and Gene therapy techniques are similar. DNA vaccine and Gene therapy techniques are similar. DNA vaccines have been introduced into animal tissues by a number of different methods. These delivery methods are briefly reviewed in Table 2, with the advantages and disadvantages of the most commonly used methods summarised in Table 3. The two most popular approaches are injection of DNA in saline, using a standard hypodermic needle, and gene gun delivery. A schematic outline of the construction of a DNA vaccine plasmid and its subsequent delivery by these two methods into a host is illustrated at Scientific American.18 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 electroporation19; by temporarily damaging muscle fibres with myotoxins such as bupivacaine; or by using hypertonic solutions of saline or sucrose.2 Immune responses to this method of delivery can be affected by many factors, including needle type,8 needle alignment, speed of injection, volume of injection, muscle type, and age, sex and physiological condition of the animal being injected.2 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 target cells, using compressed helium as an accelerant.212 Alternative delivery methods have included aerosol instillation of naked DNA on mucosal surfaces, such as the nasal and lung mucosa,12 and topical administration of pDNA to the eye20 and vaginal mucosa.12 Mucosal surface delivery has also been achieved using cationic liposome-DNA preparations,1 biodegradable microspheres,2112 attenuated Shigella or Listeria vectors for oral administration to the intestinal mucosa,22 and recombinant adenovirus vectors.12 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.23 Generally, 0.2 μg - 20 μg are required, although quantities as low as 16 ng have been reported.2 These quantities vary from species to species, with mice, for example, requiring approximately 10 times less DNA than primates.1 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.21 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.2 ELI can be used to identify which of the pathogen's 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.24 Table 2. Summary of Plasmid DNA delivery methods Method of Delivery Formulation of DNA Target Tissue Amount of DNA Parenteral Injection hypodermic needle Aqueous solution in saline IM skeletal; ID; IV, subcutaneous and intraperitoneal with variable success Large amounts approximately 100-200 μg Gene Gun DNA-coated gold beads ED abdominal skin; vaginal mucosa; surgically exposed muscle and other organs Small amounts as little as 16 ng Pneumatic Jet Injection Aqueous solution ED Very high as much as 300 μg Topical application Aqueous solution Ocular; intravaginal Small amounts up to 100 μg Cytofectin-mediated Liposomes cationic; microspheres; recombinant adenovirus vectors; attenuated Shigella vector; aerosolised cationic lipid formulations IM; IV to transfect tissues systemically; intraperitoneal; oral immunization to the intestinal mucosa; nasal/lung mucosal membranes variable Table 3. Advantages and disadvantages of commonly used DNA vaccine delivery methods Method of Delivery Advantage Disadvantage Intramuscular or Intradermal injection No special delivery mechanism Permanent or semi-permanent expression pDNA spreads rapidly throughout the body Inefficient site for uptake due to morphology of muscle tissue Relatively large amounts of DNA used Th1 response may not be the response required Gene Gun DNA bombarded directly into cells Small amounts DNA Th2 response may not be the response required Requires inert particles as carrier Jet injection No particles required DNA can be delivered to cells mm to cm below skin surface Significant shearing of DNA after high-pressure expulsion 10-fold lower expression, and lower immune response Requires large amounts of DNA up to 300 μg Liposome-mediated delivery High levels of immune response can be generated Can increase transfection of intravenously delivered pDNA Intravenously delivered liposome-DNA complexes can potentially transfect all tissues Intranasally delivered liposome-DNA complexes can result in expression in distal mucosa as well as nasal muscosa and the generation of IgA antibodies Toxicity Ineffectiveness in serum Risk of disease or immune reactions Immune response raised by DNA vaccines Helper T-Cell responses Antigen presentation stimulates T cells to become either cytotoxic CD8+ cells or helper CD4+ cells. Cytotoxic cells directly attack other cells carrying certain foreign or abnormal molecules on their surfaces. Helper T cells, or Th cells, coordinate immune responses by communicating with other cells. In most cases, T cells only recognize an antigen if it is carried on the surface of a cell by one of the body's own MHC, or major histocompatibility complex, molecules. Antigen presentation stimulates T cells to become either cytotoxic CD8+ cells or helper CD4+ cells. Cytotoxic cells directly attack other cells carrying certain foreign or abnormal molecules on their surfaces. Helper T cells, or Th cells, coordinate immune responses by communicating with other cells. In most cases, T cells only recognize an antigen if it is carried on the surface of a cell by one of the body's own MHC, or major histocompatibility complex, molecules. DNA immunization is able to raise a range of TH responses, including lymphoproliferation and the generation of a variety of cytokine profiles. A major advantage of DNA vaccines is the ease with which they can be manipulated to bias the type of T-cell help towards a TH1 or TH2 response.25 Each type of response has distinctive patterns of lymphokine and chemokine expression, specific types of immunoglobulins expressed, patterns of lymphocyte trafficking, and types of innate immune responses generated. Raising of different types of T-cell help The type of T-cell help raised is influenced by the method of delivery and the type of immunogen expressed, as well as the targeting of different lymphoid compartments.262 Generally, saline needle injections either IM or ID tend to induce TH1 responses, while gene gun delivery raises TH2 responses.2625 This is true for intracellular and plasma membrane-bound antigens, but not for secreted antigens, which seem to generate TH2 responses, regardless of the method of delivery.27 Generally the type of T-cell help raised is stable over time, and does not change when challenged or after subsequent immunizations which would normally have raised the opposite type of response in a naïve animal.2625 However, Mor et al. 19959 immunized and boosted mice with pDNA encoding the circumsporozoite protein of the mouse malarial parasite Plasmodium yoelii PyCSP and found that the initial TH2 response changed, after boosting, to a TH1 response. Mechanistic basis for different types of T-Cell help It is not understood how these different methods of DNA immunization, or the forms of antigen expressed, raise different profiles of T-cell help. It was thought that the relatively large amounts of DNA used in IM injection were responsible for the induction of TH1 responses. However, evidence has shown no differences in TH type due to dose.25 It has been postulated that the type of T-cell help raised is determined by the differentiated state of antigen presenting cells. Dendritic cells can differentiate to secrete IL-12 which supports TH1 cell development or IL-4 which supports TH2 responses.28 pDNA injected by needle is endocytosed into the dendritic cell, which is then stimulated to differentiate for TH1 cytokine production,29 while the gene gun bombards the DNA directly into the cell, thus bypassing TH1 stimulation. Practical uses of polarised T-Cell help This polarisation in T-cell help is useful in influencing allergic responses and autoimmune diseases. In autoimmune diseases, the goal would be to shift the self-destructive TH1 response with its associated cytotoxic T cell activity to a non-destructive TH2 response. This has been successfully applied in predisease priming for the desired type of response in preclinical models1 and somewhat successful in shifting the response for an already established disease.30 Cytotoxic T-cell responses One of the greatest advantages of DNA vaccines is that they are able to induce cytotoxic T lymphocytes CTL without the inherent risk associated with live vaccines. CTL responses can be raised against immunodominant and immunorecessive CTL epitopes,31 as well as subdominant CTL epitopes,21 in a manner which appears to mimic natural infection. This may prove to be a useful tool in assessing CTL epitopes of an antigen, and their role in providing immunity. Cytotoxic T-cells recognise small peptides 8-10 amino acids complexed to MHC class I molecules Restifo et al., 1995. These peptides are derived from endogenous cytosolic proteins which are degraded and delivered to the nascent MHC class I molecule within the endoplasmic reticulum ER.32 Targeting gene products directly to the ER by the addition of an amino-terminal insertion sequence should thus enhance CTL responses. This has been successfully demonstrated using recombinant vaccinia viruses expressing influenza proteins,32 but the principle should be applicable to DNA vaccines too. Targeting antigens for intracellular degradation and thus entry into the MHC class I pathway by the addition of ubiquitin signal sequences, or mutation of other signal sequences, has also been shown to be effective at increasing CTL responses.16 CTL responses can also be enhanced by co-inoculation with co-stimulatory molecules such as B7-1 or B7-2 for DNA vaccines against influenza nucleoprotein,3133 or GM-CSF for DNA vaccines against the murine malaria model P. yoelii.34 Co-inoculation with plasmids encoding co-stimulatory molecules IL-12 and TCA3 have also been shown to increase CTL activity against HIV-1 and influenza nucleoprotein antigens.3335 Humoral antibody response Schematic diagram of an antibody and antigens Schematic diagram of an antibody and antigens Antibody responses elicited by DNA vaccinations are influenced by a number of variables, including type of antigen encoded; location of expressed antigen i.e. intracellular vs. secreted; number, frequency and dose of immunizations; site and method of antigen delivery, to name a few. Kinetics of antibody response Humoral responses after a single DNA injection can be much longer-lived than after a single injection with a recombinant protein. Antibody responses against hepatitis B virus HBV envelope protein HBsAg have been sustained for up to 74 weeks without boost, while life-long maintenance of protective response to influenza haemagglutinin has been demonstrated in mice after gene gun delivery.36 Antibody-secreting cells migrate to the bone marrow and spleen for long-term antibody production, and are generally localised there after one year.36 Comparisons of antibody responses generated by natural viral infection, immunization with recombinant protein and immunization with pDNA are summarised in Table 4. DNA-raised antibody responses rise much more slowly than when natural infection or recombinant protein immunization occurs. It can take as long as 12 weeks to reach peak titres in mice, although boosting can increase the rate of antibody production. This slow response is probably due to the low levels of antigen expressed over several weeks, which supports both primary and secondary phases of antibody response. Table 4. Comparison of T-Dependent Antibody Responses raise by DNA Immunizations, Protein Inoculations and Viral Infections Method of Immunization DNA Vaccine Recombinant protein Natural Infection Amount of inducing antigen ng μg ? ng-μg Duration of Ag presentation several weeks 1 week several weeks Kinetics of Ab response slow rise rapid rise rapid rise Number of inoculations to obtain high avidity IgG and migration of ASC to bone marrow one two one Ab isotype murine models C'-dependent or C'-independent C'-dependent C'-independent Additionally, the titres of specific antibodies raised by DNA vaccination are lower than those obtained after vaccination with a recombinant protein. However, DNA immunization-induced antibodies show greater affinity to native epitopes than recombinant protein-induced antibodies. In other words, DNA immunization induces a qualitatively superior response. Antibody can be induced after just one vaccination with DNA, whereas recombinant protein vaccinations generally require a boost. As mentioned previously, DNA immunization can be used to bias the TH profile of the immune response, and thus the antibody isotype, which is not possible with either natural infection or recombinant protein immunization. Antibody responses generated by DNA are useful not just in vaccination but as a preparative tool, too. For example, polyclonal and monoclonal antibodies can be generated for use as reagents. Mechanistic basis for DNA raised immune responses DNA Uptake Mechanism When DNA uptake and subsequent expression was first demonstrated in vivo in muscle cells,37 it was thought that these cells were unique in this ability because of their extensive network of T-tubules. Using electron microscopy, it was proposed that DNA uptake was facilitated by caveolae or, non-clathrin coated pits.38 However, subsequent research revealed that other cells such as keratinocytes, fibroblasts and epithelial Langerhans cells could also internalise DNA.3930 This phenomenon has not been the subject of much research, so the actual mechanism of DNA uptake is not known. Two theories are currently popular - that in vivo uptake of DNA occurs non-specifically, in a method similar to phago- or pinocytosis,12 or through specific receptors.40 These might include a 30kDa surface receptor, or macrophage scavenger receptors. The 30kDa surface receptor binds very specifically to 4500-bp genomic DNA fragments which are then internalised and is found on professional APCs and T-cells. Macrophage scavenger receptors bind to a variety of macromolecules, including polyribonucleotides, and are thus also candidates for DNA uptake.4041 Receptor mediated DNA uptake could be facilitated by the presence of polyguanylate sequences. Further research into this mechanism might seem pointless, considering that gene gun delivery systems, cationic liposome packaging, and other delivery methods bypass this entry method, but understanding it might be useful in reducing costs e.g. by reducing the requirement for cytofectins, which will be important in the food animals industry. Antigen presentation by bone marrow-derived cells A dendritic cell. A dendritic cell. Studies using chimeric mice have shown that antigen is presented by bone-marrow derived cells, which include dendritic cells, macrophages and specialised B-cells called professional antigen presenting cells APC4233 Iwasaki et al., 1997. After gene gun inoculation to the skin, transfected Langerhans cells migrate to the draining lymph node to present antigen.1 After IM and ID injections, dendritic cells have also been found to present antigen in the draining lymph node39 and transfected macrophages have been found in the peripheral blood.43 Besides direct transfection of dendritic cells or macrophages, cross priming is also known to occur following IM, ID and gene gun deliveries of DNA. Cross priming occurs when a bone marrow-derived cell presents peptides from proteins synthesised in another cell. This can prime cytotoxic T-cell responses and seems to be important for a full primary immune response.144 Role of the target site IM and ID delivery of DNA initiate immune responses differently. In the skin, keratinocytes, fibroblasts and Langerhans cells take up and express antigen, and are responsible for inducing a primary antibody response. Transfected Langerhans cells migrate out of the skin within 12 hours to the draining lymph node where they prime secondary B- and T-cell responses. In skeletal muscle, on the other hand, striated muscle cells are most frequently transfected, but seem to be unimportant in mounting an immune response. Instead, IM inoculated DNA washes into the draining lymph node within minutes, where distal dendritic cells are transfected and then initiate an immune response. Transfected myocytes seem to act as a reservoir of antigen for trafficking professional APCs.441237 Maintenance of immune response DNA vaccination generates an effective immune memory via the display of antigen-antibody complexes on follicular dendritic cells FDC, which are potent B-cell stimulators. T-cells can be stimulated by similar, germinal centre dendritic cells. FDC are able to generate an immune memory because antibodies production overlaps long-term expression of antigen, allowing antigen-antibody immunocomplexes to form and be displayed by FDC.1 Inteferons Both helper and cytotoxic T-cells can control viral infections by secreting interferons. Cytotoxic T cells usually kill virally infected cells. However, they can also be stimulated to secrete antiviral cytokines such as INF-γ and TNF-α, which don't kill the cell but place severe limitations on viral infection by down-regulating the expression of viral components.45 DNA vaccinations can thus be used to curb viral infections by non-destructive IFN-mediated control. This has been demonstrated for the hepatitis B virus.46 IFN-γ is also critically important in controlling malaria infections,47 and should be taken into consideration when developing anti-malarial DNA vaccines. Modulation of the immune response Cytokine modulation For a vaccine to be effective, it must induce an appropriate immune response for a given pathogen, and the ability of DNA vaccines to polarise T-cell help towards TH1 or TH2 profiles, and generate CTL and/or antibody when required, is a great advantage in this regard. This can be accomplished by modifications to the form of antigen expressed i.e. intracellular vs. secreted, the method and route of delivery, and the dose of DNA delivered.4825264950 However, it can also be accomplished by the co-administration of plasmid DNA encoding immune regulatory molecules, i.e. cytokines, lymphokines or co-stimulatory molecules. These genetic adjuvants can be administered a number of ways: as a mixture of 2 separate plasmids, one encoding the immunogen and the other encoding the cytokine; as a single bi- or polycistronic vector, separated by spacer regions; or as a plasmid-encoded chimera, or fusion protein. In general, co-administration of pro-inflammatory agents such as various interleukins, tumor necrosis factor, and GM-CSF plus TH2 inducing cytokines increase antibody responses, whereas pro-inflammatory agents and TH1 inducing cytokines decrease humoral responses and increase cytotoxic responses which is more important in viral protection, for example. Co-stimulatory molecules like B7-1, B7-2 and CD40L are also sometimes used. This concept has been successfully applied in topical administration of pDNA encoding IL-10.20 Plasmid encoded B7-1 a ligand on APCs has successfully enhanced the immune response in anti-tumour models, and mixing plasmids encoding GM-CSF and the circumsporozoite protein of P. yoelii PyCSP has enhanced protection against subsequent challenge whereas plasmid-encoded PyCSP alone did not. It was proposed that GM-CSF may cause dendritic cells to present antigen more efficiently, and enhance IL-2 production and TH cell activation, thus driving the increased immune response.34 This can be further enhanced by first priming with a pPyCSP and pGM-CSF mixture, and later boosting with a recombinant poxvirus expressing PyCSP.51 However, co-injection of plasmids encoding GM-CSF or IFN-γ, or IL-2 and a fusion protein of P. chabaudi merozoite surface protein 1 C-terminus-hepatitis B virus surface protein PcMSP1-HBs actually abolished protection against challenge, compared to protection acquired by delivery of pPcMSP1-HBs alone.17 The advantages of using genetic adjuvants are their low cost and simplicity of administration, as well as avoidance of unstable recombinant cytokines and potentially toxic, conventional adjuvants such as alum, calcium phosphate, monophosphoryl lipid A, cholera toxin, cationic and mannan-coated liposomes, QS21, carboxymethylcellulose and ubenimix.121 However, the potential toxicity of prolonged cytokine expression has not been established, and in many commercially important animal species, cytokine genes still need to be identified and isolated. In addition, various plasmid encoded cytokines modulate the immune system differently according to the time of delivery. For example, some cytokine plasmid DNAs are best delivered after the immunogen pDNA, because pre- or co-delivery can actually decrease specific responses, and increase non-specific responses.52 Immunostimulatory CpG motifs Plasmid DNA itself appears to have an adjuvant effect on the immune system.21 Bacterially derived DNA has been found to trigger innate immune defence mechanisms, the activation of dendritic cells, and the production of TH1 cytokines.2953 This is due to recognition of certain CpG dinucleotide sequences which are immunostimulatory.5449 CpG stimulatory CpG-S sequences occur twenty times more frequently in bacterially derived DNA than in eukaryotes. This is because eukaryotes exhibit CpG suppression - i.e. CpG dinucleotide pairs occur much less frequently than expected. Additionally, CpG-S sequences are hypomethylated. This occurs frequently in bacterial DNA, while CpG motifs occurring in eukaryotes are all methylated at the cytosine nucleotide. In contrast, nucleotide sequences which inhibit the activation of an immune response termed CpG neutralising, or CpG-N are over represented in eukaryotic genomes.55 The optimal immunostimulatory sequence has been found to be an unmethylated CpG dinucleotide flanked by two 5' purines and two 3' pyrimidines.5349 Additionally, flanking regions outside this immunostimulatory hexamer must be guanine-rich to ensure binding and uptake into target cells. The innate system works synergistically with the adaptive immune system to mount a response against the DNA encoded protein. CpG-S sequences induce polyclonal B-cell activation and the upregulation of cytokine expression and secretion.56 Stimulated macrophages secrete IL-12, IL-18, TNF-α, IFN-α, IFN-β and IFN-γ, while stimulated B-cells secrete IL-6 and some IL-12.561257 Manipulation of CpG-S and CpG-N sequences in the plasmid backbone of DNA vaccines can ensure the success of the immune response to the encoded antigen, and drive the immune response toward a TH1 phenotype. This is useful if a pathogen requires a TH response for protection. CpG-S sequences have also been used as external adjuvants for both DNA and recombinant protein vaccination with variable success rates. Other organisms with hypomethylated CpG motifs have also demonstrated the stimulation of polyclonal B-cell expansion. However, the mechanism behind this may be more complicated than simple methylation - hypomethylated murine DNA has not been found to mount an immune response. Most of the evidence for the existence of immunostimulatory CpG sequences comes from murine studies. Clearly, extrapolation of this data to other species should be done with caution - different species may require different flanking sequences, as binding specificities of scavenger receptors differ between species. Additionally, species such as ruminants may be insensitive to immunostimulatory sequences due to the large gastrointestinal load they exhibit. Further research may be useful in the optimisation of DNA vaccination, especially in the food animal industry. Alternative boosts DNA-primed immune responses can be boosted by the administration of recombinant protein or recombinant poxviruses. Prime-boost strategies with recombinant protein have successfully increased both neutralising antibody titre, and antibody avidity and persistence, for weak immunogens, such as HIV-1 envelope protein.158 Recombinant virus boosts have been shown to be very efficient at boosting DNA-primed CTL responses. Priming with DNA focuses the immune response on the required immunogen, while boosting with the recombinant virus provides a larger amount of expressed antigen, leading to a large increase in specific CTL responses. Prime-boost strategies have been successful in inducing protection against malarial challenge in a number of studies. Primed mice with plasmid DNA encoding Plasmodium yoelii circumsporozoite surface protein PyCSP, then boosted with a recombinant vaccinia virus expressing the same protein had significantly higher levels of antibody, CTL activity and IFN-γ, and hence higher levels of protection, than mice immunized and boosted with plasmid DNA alone.59 This can be further enhanced by priming with a mixture of plasmids encoding PyCSP and murine GM-CSF, before boosting with recombinant vaccinia virus.51 An effective prime-boost strategy for the simian malarial model P. knowlesi has also been demonstrated.60 Rhesus monkeys were primed with a multicomponent, multistage DNA vaccine encoding two liver-stage antigens - the circumsporozoite surface protein PkCSP and sporozoite surface protein 2 PkSSP2 - and two blood stage antigens - the apical merozoite surface protein 1 PkAMA1 and merozoite surface protein 1 PkMSP1p42. They were then boosted with a recombinant canarypox virus encoding all four antigens ALVAC-4. Immunized monkeys developed antibodies against sporozoites and infected erythrocytes, and IFN-γ-secreting T-cell responses against peptides from PkCSP. Partial protection against sporozoite challenge was achieved, and mean parasitemia was significantly reduced, compared to control monkeys. These models, while not ideal for extrapolation to P. falciparum in humans, will be important in pre-clinical trials. Additional methods of enhancing DNA-Raised immune responses Formulations of DNA The efficiency of DNA immunization can be improved by stabilising DNA against degradation, and increasing the efficiency of delivery of DNA into antigen presenting cells.1 This has been demonstrated by coating biodegradable cationic microparticles such as polylactide-co-glycolide formulated with cetyltrimethylammonium bromide with DNA. Such DNA-coated microparticles can be as effective at raising CTL as recombinant vaccinia viruses, especially when mixed with alum. Particles 300nm in diameter appear to be most efficient for uptake by antigen presenting cells.1 Alphavirus vectors Recombinant alphavirus-based vectors have also been used to improve DNA vaccination efficiency.1 The gene encoding the antigen of interest is inserted into the alphavirus replicon, replacing structural genes but leaving non-structural replicase genes intact. The Sindbis virus and Semliki Forest virus have been used to build recombinant alphavirus replicons. Unlike conventional DNA vaccinations, however, alphavirus vectors kill transfected cells, and are only transiently expressed. Also, alphavirus replicase genes are expressed in addition to the vaccine insert. It is not clear how alphavirus replicons raise an immune response, but it is thought that this may be due to the high levels of protein expressed by this vector, replicon-induced cytokine responses, or replicon-induced apoptosis leading to enhanced antigen uptake by dendritic cells. See also Vector DNA HIV vaccine References ^ a b c d e f g h i j k l m n o p q r s t u v w Robinson, H.L.; Pertmer, T.M. 2000. DNA vaccines for viral infections: basic studies and applications.. Adv Virus Res 55: 1-74. Retrieved on 2007-11-21. ^ a b c d e f g h i j k l m n o p Alarcon, J.B.; Waine, G.W.; McManus, D.P. 1999. DNA vaccines: technology and application as anti-parasite and anti-microbial agents.. Adv Parasitol 42: 343-410. Retrieved on 2007-11-21. ^ Tang, D.; Devit, M.; Johnston, S.A.; Others, 1992. Genetic immunization is a simple method for eliciting an immune response. Nature 356 6365: 152-154. doi:10.1038/356152a0. ^ Barnes, Kirsty 2004-06-07. First positive results for DNA-based flu vaccine, in-PharmaTechnologist. ^ Fort Dodge Animal Health Announces Approval of West Nile Virus DNA Vaccine for Horses, PR Newswire 2005-07-18. Retrieved on 2007-11-21. ^ CDC and Fort Dodge Animal Health Achieve First Licensed DNA Vaccine, CDC 2005-07-18. Retrieved on 2007-11-21. ^ Stuve, O.; Eagar, T.N.; Frohman, E.M.; Cravens, P.D. 2007. DNA Plasmid Vaccination for Multiple Sclerosis. Archives of Neurology 64 10: 1385. doi:10.1001/archneur.64.10.1385. PMID 17923622. ^ a b Sedegah, M.; Hedstrom, R.; Hobart, P.; Hoffman, S.L. 1994. Protection against Malaria by Immunization with Plasmid DNA Encoding Circumsporozoite Protein. Proceedings of the National Academy of Sciences of the United States of America 91 21: 9866-9870. doi:10.1073/pnas.91.21.9866. Retrieved on 2007-11-21. ^ a b Mor, G. 1995. Complexity of the cytokine and antibody response elicited by immunizing mice with Plasmodium yoelii circumsporozoite protein plasmid DNA. 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Infection and Immunity 69 9: 5565. doi:10.1128/IAI.69.9.5565-5572.2001. PMID 11500430. External links PowderMed pdf report DyNAVacS, an Integrative Tool for Optimised DNA Vaccine Design from the Institute of Genomics and Integrative Biology. Smallpox DNA Vaccine Protects Nonhuman Primates against Lethal Monkeypox J Virol. 2004 May; 789: 4433-4443. Retrieved from http://en..org/wiki/DNA_vaccination Categories: DNA | Vaccination Views Article Discussion this page History Personal tools Log in / create account Navigation Main page Contents Featured content Current events Random article Search Go Search Interaction Community portal Recent changes Contact Donate to Help Toolbox What links here Related changes Upload file Special pages Printable version Permanent link Cite this page Languages Deutsch Polski This page was last modified on 10 July 2008, at 10:28
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