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.
Many believe 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. 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. 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 vaccine recipients (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. 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 pathogen. The vaccine DNA is injected into the cells of the body, where the "inner machinery" of the host cells "reads" the DNA and uses it to synthesize the pathogen's proteins. Because these proteins are recognised as foreign, when they are processed by the host cells and displayed on their surface, the immune system is alerted, which then triggers a range of immune responses. These DNA vaccines developed from “failed” gene therapy experiments. 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.
Current use 
Thus far, few experimental trials have evoked a response 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  and a veterinary DNA vaccine to protect horses from West Nile virus has been approved. In August 2007, a preliminary study in DNA vaccination against multiple sclerosis was reported as being effective.
Advantages and disadvantages 
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. 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 the gene sequence of the immunogen to reflect the codons more commonly used in the target species may improve its expression.
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. 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 than CTE-alone vector.  Additional 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. An example of DNA vaccine plasmid is pVAC, it uses SV40 promoter.
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.
Delivery methods 
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. Injection in saline is normally conducted intramuscularly (IM) in skeletal muscle, or intradermally (ID), with DNA being delivered to the extracellular spaces. This can be assisted by electroporation; by temporarily damaging muscle 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, needle alignment, speed of injection, volume of injection, muscle type, and age, sex and physiological condition of the animal being injected.
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.
Alternative delivery methods have included aerosol instillation of naked DNA on mucosal surfaces, such as the nasal and lung mucosa, and topical administration of pDNA to the eye and vaginal mucosa. Mucosal surface delivery has also been achieved using cationic liposome-DNA preparations, biodegradable microspheres, attenuated Shigella or Listeria vectors 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 μg-1 mg, whereas gene gun deliveries require 100 to 1000 times less DNA than intramuscular saline injection to raise an effective immune response. Generally, 0.2 μg – 20 μg are required, although quantities as low as 16 ng have been reported. These quantities vary from species to species, with mice, for example, requiring approximately 10 times less DNA than primates. Saline injections require more DNA because the DNA is delivered to the extracellular spaces of the target tissue (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 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.
|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|
|Method of Delivery||Advantage||Disadvantage|
|Intramuscular or Intradermal injection||
Immune response raised by DNA vaccines 
Helper T-Cell responses 
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. 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. Generally, saline needle injections (either IM or ID) tend to induce TH1 responses, while gene gun delivery raises TH2 responses. 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.
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. However, Mor et al.. (1995) 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. 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). pDNA injected by needle is endocytosed into the dendritic cell, which is then stimulated to differentiate for TH1 cytokine production, 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 models and somewhat successful in shifting the response for an already established disease.
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, as well as subdominant CTL epitopes, 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). 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, 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.
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, or GM-CSF for DNA vaccines against the murine malaria model P. yoelii. 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.
Humoral (antibody) response 
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 lifelong maintenance of protective response to influenza haemagglutinin has been demonstrated in mice after gene gun delivery. Antibody-secreting cells migrate to the bone marrow and spleen for long-term antibody production, and are generally localised there after one year.
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.
|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, 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). However, subsequent research revealed that other cells (such as keratinocytes, fibroblasts and epithelial Langerhans cells) could also internalize DNA. 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, or through specific receptors. 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. 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 
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 (APC) Iwasaki et al., 1997). After gene gun inoculation to the skin, transfected Langerhans cells migrate to the draining lymph node to present antigen. After IM and ID injections, dendritic cells have also been found to present antigen in the draining lymph node and transfected macrophages have been found in the peripheral blood.
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 in the context of MHC class 1. This can prime cytotoxic T-cell responses and seems to be important for a full primary immune response.
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.
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.
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. 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. IFN-γ is also critically important in controlling malaria infections, 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. 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. Plasmid encoding 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. This can be further enhanced by first priming with a pPyCSP and pGM-CSF mixture, and later boosting with a recombinant poxvirus expressing PyCSP. 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.
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). 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.
Immunostimulatory CpG motifs 
Plasmid DNA itself appears to have an adjuvant effect on the immune system. Bacterially derived DNA has been found to trigger innate immune defence mechanisms, the activation of dendritic cells, and the production of TH1 cytokines. This is due to recognition of certain CpG dinucleotide sequences which are immunostimulatory. 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. The optimal immunostimulatory sequence has been found to be an unmethylated CpG dinucleotide flanked by two 5’ purines and two 3’ pyrimidines. 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. 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.
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. 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. This can be further enhanced by priming with a mixture of plasmids encoding PyCSP and murine GM-CSF, before boosting with recombinant vaccinia virus. An effective prime-boost strategy for the simian malarial model P. knowlesi has also been demonstrated. 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. This has been demonstrated by coating biodegradable cationic microparticles (such as poly(lactide-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 300 nm in diameter appear to be most efficient for uptake by antigen presenting cells.
Alphavirus vectors 
Recombinant alphavirus-based vectors have also been used to improve DNA vaccination efficiency. 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 
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- PowderMed pdf report
- DyNAVacS, an Integrative Tool for Optimised DNA Vaccine Design from the Institute of Genomics and Integrative Biology.
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