Plasmid

A plasmid is a DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA. They are double stranded and, in many cases, circular. Plasmids usually occur naturally in bacteria, but are sometimes found in eukaryotic organisms (e.g., the 2-micrometre-ring in Saccharomyces cerevisiae).

Plasmid size varies from 1 to over 1,000 kilobase pairs (kbp).The number of identical plasmids within a single cell can range anywhere from one to even thousands under some circumstances. Plasmids can be considered to be part of the mobilome, since they are often associated with conjugation, a mechanism of horizontal gene transfer.
The term plasmid was first introduced by the American molecular biologist Joshua Lederberg in 1952.
Plasmids are considered transferable genetic elements, or "replicons", capable of autonomous replication within a suitable host. Plasmids can be found in all three major domains, Archea, Bacteria and Eukarya. Similar to viruses, plasmids are not considered a form of "life" as it is currently defined. Unlike viruses, plasmids are "naked" DNA and do not encode genes necessary to encase the genetic material for transfer to a new host, though some classes of plasmids encode the sex pilus necessary for their own transfer. Plasmid host-to-host transfer requires direct, mechanical transfer by conjugation or changes in host gene expression allowing the intentional uptake of the genetic element by transformation. Microbial transformation with plasmid DNA is neither parasitic nor symbiotic in nature, since each implies the presence of an independent species living in a commensal or detrimental state with the host organism. Rather, plasmids provide a mechanism for horizontal gene transfer within a population of microbes and typically provide a selective advantage under a given environmental state. Plasmids may carry genes that provide resistance to naturally occurring antibiotics in a competitive environmental niche, or alternatively the proteins produced may act as toxins under similar circumstances. Plasmids also can provide bacteria with an ability to fix elemental nitrogen or to degrade recalcitrant organic compounds which provide an advantage under conditions of nutrient deprivation.

Vectors:

There are two types of plasmid integration into a host bacteria: Non-integrating plasmids replicate as with the top instance; whereas episomes, the lower example, integrate into the host chromosome.

Plasmids used in genetic engineering are called vectors. Plasmids serve as important tools in genetics and biotechnology labs, where they are commonly used to multiply (make many copies of) or express particular genes. Many plasmids are commercially available for such uses. The gene to be replicated is inserted into copies of a plasmid containing genes that make cells resistant to particular antibiotics and a multiple cloning site (MCS, or polylinker), which is a short region containing several commonly used restriction sites allowing the easy insertion of DNA fragments at this location. Next, the plasmids are inserted into bacteria by a process called transformation. Then, the bacteria are exposed to the particular antibiotics. Only bacteria which take up copies of the plasmid survive, since the plasmid makes them resistant. In particular, the protecting genes are expressed (used to make a protein) and the expressed protein breaks down the antibiotics. In this way the antibiotics act as a filter to select only the modified bacteria. Now these bacteria can be grown in large amounts, harvested and lysed (often using the alkaline lysis method) to isolate the plasmid of interest.
Another major use of plasmids is to make large amounts of proteins. In this case, researchers grow bacteria containing a plasmid harboring the gene of interest. Just as the bacteria produces proteins to confer its antibiotic resistance, it can also be induced to produce large amounts of proteins from the inserted gene. This is a cheap and easy way of mass-producing a gene or the protein it then codes for, for example, insulin or even antibiotics.

However, a plasmid can only contain inserts of about 1–10 kbp. To clone longer lengths of DNA, lambda phage with lysogeny genes deleted, cosmids, bacterial artificial chromosomes or yeast artificial chromosomes could be used.

 Applications:

 Disease Models:

Plasmids were historically used to genetically engineer the embryonic stem cells of rats in order to create rat genetic disease models. The limited efficiency of plasmid based techniques precluded their use in the creation of more accurate human cell models. Fortunately, developments in Adeno-associated virus recombination techniques, and Zinc finger nucleases, have enabled the creation of a new generation of isogenic human disease models.

 Gene therapy:

The success of some strategies of gene therapy depends on the efficient insertion of therapeutic genes at the appropriate chromosomal target sites within the human genome, without causing cell injury, oncogenic mutations (cancer) or an immune response. Plasmid vectors are one of many approaches that could be used for this purpose. Zinc finger nucleases (ZFNs) offer a way to cause a site-specific double strand break to the DNA genome and cause homologous recombination. This makes targeted gene correction a possibility in human cells. Plasmids encoding ZFN could be used to deliver a therapeutic gene to a pre-selected chromosomal site with a frequency higher than that of random integration. Although the practicality of this approach to gene therapy has yet to be proven, some aspects of it could be less problematic than the alternative viral-based delivery of therapeutic genes.
Types:

one way of grouping plasmids is by their ability to transfer to other bacteria. Conjugative plasmids contain so-called tra-genes, which perform the complex process of conjugation, the transfer of plasmids to another bacterium (Fig. 4). Non-conjugative plasmids are incapable of initiating conjugation, hence they can only be transferred with the assistance of conjugative plasmids, by 'accident'. An intermediate class of plasmids are mobilizable, and carry only a subset of the genes required for transfer. They can 'parasitize' a conjugative plasmid, transferring at high frequency only in its presence. Plasmids are now being used to manipulate DNA and may possibly be a tool for curing many diseases.

It is possible for plasmids of different types to coexist in a single cell. Several different plasmids have been found in E. coli. But related plasmids are often incompatible, in the sense that only one of them survives in the cell line, due to the regulation of vital plasmid functions. Therefore, plasmids can be assigned into compatibility groups.
Another way to classify plasmids is by function. There are five main classes:

• Fertility-F-plasmids, which contain tra-genes. They are capable of conjugation (transfer of genetic material between bacteria which are touching).
• Resistance-(R)plasmids, which contain genes that can build a resistance against antibiotics or poisons and help bacteria produce pili. Historically known as R-factors, before the nature of plasmids was understood.
• Col-plasmids, which contain genes that code for (determine the production of) bacteriocins, proteins that can kill other bacteria.
• Degradative plasmids, which enable the digestion of unusual substances, e.g., toluene or salicylic acid.
• Virulence plasmids, which turn the bacterium into a pathogen (one that causes disease).

Plasmids can belong to more than one of these functional groups.
Plasmids that exist only as one or a few copies in each bacterium are, upon cell division, in danger of being lost in one of the segregating bacteria. Such single-copy plasmids have systems which attempt to actively distribute a copy to both daughter cells.
Some plasmids or microbial hosts include an addiction system or "postsegregational killing system (PSK)", such as the hok/sok (host killing/suppressor of killing) system of plasmid R1 in Escherichia coli.^[8] This variant produces both a long-lived poison and a short-lived antidote. Several types of plasmid addiction systems (toxin/ antitoxin, metabolism-based, ORT systems) were described in the literature^[9] and used in biotechnical (fermentation) or biomedical (vaccine therapy) applications. Daughter cells that retain a copy of the plasmid survive, while a daughter cell that fails to inherit the plasmid dies or suffers a reduced growth-rate because of the lingering poison from the parent cell. Finally, the overall productivity could be enhanced.

Yeast Plasmids:

Other types of plasmids, often related to yeast cloning vectors include:

• Yeast integrative plasmid (YIp), yeast vectors that rely on integration into the host chromosome for survival and replication, and are usually used when studying the functionality of a solo gene or when the gene is toxic. Also connected with the gene URA3, that codes an enzyme related to the biosynthesis of pyrimidine nucleotides (T, C);
• Yeast Replicative Plasmid (YRp), which transport a sequence of chromosomal DNA that includes an origin of replication. These plasmids are less stable, as they can "get lost" during the budding.

Plasmid DNA extraction:

As alluded to above, plasmids are often used to purify a specific sequence, since they can easily be purified away from the rest of the genome. For their use as vectors, and for molecular cloning, plasmids often need to be isolated.
There are several methods to isolate plasmid DNA from bacteria, the archetypes of which are the miniprep and the maxiprep/bulkprep. The former can be used to quickly find out whether the plasmid is correct in any of several bacterial clones. The yield is a small amount of impure plasmid DNA, which is sufficient for analysis by restriction digest and for some cloning techniques.
In the latter, much larger volumes of bacterial suspension are grown from which a maxi-prep can be performed. Essentially this is a scaled-up miniprep followed by additional purification. This results in relatively large amounts (several micrograms) of very pure plasmid DNA.
In recent times many commercial kits have been created to perform plasmid extraction at various scales, purity and levels of automation. Commercial services can prepare plasmid DNA at quoted prices below $300/mg in milligram quantities and $15/mg in gram quantities (early 2007^[update]).

Conformations:

Plasmid DNA may appear in one of five conformations, which (for a given size) run at different speeds in a gel during electrophoresis. The conformations are listed below in order of electrophoretic mobility (speed for a given applied voltage) from slowest to fastest:

• "Nicked Open-Circular" DNA has one strand cut.
• "Relaxed Circular" DNA is fully intact with both strands uncut, but has been enzymatically "relaxed" (supercoils removed). You can model this by letting a twisted extension cord unwind and relax and then plugging it into itself.
• "Linear" DNA has free ends, either because both strands have been cut, or because the DNA was linear in vivo. You can model this with an electrical extension cord that is not plugged into itself.
• "Supercoiled" (or "Covalently Closed-Circular") DNA is fully intact with both strands uncut, and with a twist built in, resulting in a compact form. You can model this by twisting an extension cord and then plugging it into itself.
• "Supercoiled Denatured" DNA is like supercoiled DNA, but has unpaired regions that make it slightly less compact; this can result from excessive alkalinity during plasmid preparation.

The rate of migration for small linear fragments is directly proportional to the voltage applied at low voltages. At higher voltages, larger fragments migrate at continually increasing yet different rates. Therefore the resolution of a gel decreases with increased voltage.
At a specified, low voltage, the migration rate of small linear DNA fragments is a function of their length. Large linear fragments (over 20kb or so) migrate at a certain fixed rate regardless of length. This is because the molecules 'resperate', with the bulk of the molecule following the leading end through the gel matrix. Restriction digests are frequently used to analyse purified plasmids. These enzymes specifically break the DNA at certain short sequences. The resulting linear fragments form 'bands' after gel electrophoresis. It is possible to purify certain fragments by cutting the bands out of the gel and dissolving the gel to release the DNA fragments.
Because of its tight conformation, supercoiled DNA migrates faster through a gel than linear or open-circular DNA.

Simulation of plasmids:

The use of plasmids as a technique in molecular biology is supported by bioinformatics software. These programmes record the DNA sequence of plasmid vectors, help to predict cut sites of restriction enzymes, and to plan manipulations. Examples of software packages that handle plasmid maps are Geneious, Lasergene, GeneConstructionKit, ApE, and Vector NTI.

Ti plasmid:

The structure of the Ti plasmid

Ti plasmid is a circular plasmid that often, but not always, is a part of the genetic equipment that Agrobacterium tumefaciens and Agrobacterium rhizogenes use to transduce its genetic material to plants. The Ti plasmid is lost when Agrobacterium is grown above 28°C. Such cured bacteria do not induce crown galls, i.e. they become avirulent. pTi and pRi share little sequence homology but are functionally rather similar. The Ti plasmids are classified into different types based on the type of opine produced by their genes. The different opines specified by pTi are octopine, nopaline, succinamopine and leucinopine.
The plasmid has 196 genes that code for 195 proteins. There is no one structural RNA. The plasmid is 206,479 nucleotides long, the GC content is 56% and 81% of the material is coding genes. There are no pseudogenes.

The modification of this plasmid is very important in the creation of transgenic plants, but only in dicotyledon plants.

Virulence Region:

Genes in the virulence region are grouped into the operons virABCDEFG, which code for the enzymes responsible for mediating transduction of T-DNA to plant cells.
 virA codes for a receptor which reacts to the presence of phenolic compounds such as acetosyringone,, syringealdehyde or acetovanillone which leak out of damaged plant tissues.

• virB encodes proteins which produce a pore/pilus-like structure.
• virC binds the overdrive sequence.
• virD1 and virD2 produce endonucleases which target the direct repeat borders of the T-DNA segment, beginning with the right border
• virG activates vir-gene expression after binding to a consensus sequence, once it has been phosphorylated by virA.

R1 plasmid:

The R1 Plasmid is a plasmid that was first isolated from Salmonella paratyphi bacteria in 1963.
The R1 plasmid imparts multi-drug antibiotic resistance to its host bacteria.
It’s known as a “low copy” plasmid, meaning that it exists in relatively few copies in any given bacteria. Because of its low copy nature, R1 must rely on “type II” segregation system to ensure that at least one copy is contained in each daughter cell after mitosis.
Some genes on the R1 plasmid are:

• ParM is a prokaryotic actin homologue which provides the force to drive copies of the R1 plasmid to opposite ends of rod shaped bacteria before mitosis.
• The Hok/sok system a postsegregational killing system of the plasmid.
• CopA-like RNA, an antisense RNA involved in replication control of the plasmid.

Plasmid preparation:

Plasmid miniprep. 0.8% agarose gel ethidium bromide-stained.

A plasmid preparation is a method used to extract and purify plasmid DNA. Many methods have been developed to purify plasmid DNA from bacteria. These methods invariably involve three steps:

• Growth of the bacterial culture
• Harvesting and lysis of the bacteria
• Purification of plasmid DNA

Growth of the bacterial culture:

Plasmids are almost always purified from liquid bacteria cultures, usually E. coli, which have been transformed and isolated. Virtually all plasmid vectors in common use encode one or more antibiotic resistance genes as a selectable marker (Ex :kanamycin,Ampicillin), which allows bacteria that have been successfully transformed to multiply uninhibited.

Harvesting and lysis of the bacteria:

When bacteria are lysed under alkaline conditions both DNA and proteins are precipitated. Some scientists reduce the concentration of NaOH used to 0.1M in order to reduce the occurrence of ssDNA. After the addition of acetate-containing neutralization buffer the large and less supercoiled chromosomal DNA and proteins precipitate, but the small bacterial DNA plasmids can renature and stay in solution.

Preparations by size:

Kits are available from varying manufacturers to purify plasmid DNA, which are named by size of bacterial culture and corresponding plasmid yield. In increasing order, these are the miniprep, midiprep, maxiprep, megaprep, and gigaprep. The plasmid DNA yield will vary depending on the plasmid copy number, type and size, the bacterial strain, the growth conditions, and the kit.

Minipreparation:

Minipreparation of plasmid DNA is a rapid, small-scale isolation of plasmid DNA from bacteria. It is based on the alkaline lysis method invented by the researchers Birnboim and Doly in 1979. The extracted plasmid DNA resulting from performing a miniprep is itself often called a "miniprep". Minipreps are used in the process of molecular cloning to analyze bacterial clones. A typical plasmid DNA yield of a miniprep is 20 to 30 µg depending on the cell strain.

Miniprep Protocols:

http://www.protocol-online.org/prot/Molecular_Biology/Plasmid/Miniprep/

Midipreparation;

The starting E. coli culture volume is 15-25 ml of LB broth and the expected DNA yield is 100-350 µg.

Maxipreparation:

The starting E. coli culture volume is 100-200 ml of LB broth and the expected DNA yield is 500-850 µg.

Megapreparation:

The starting E. coli culture volume is 500 ml – 2.5 L of LB broth and the expected DNA yield is 1.5-2.5 mg.

Gigapreparation:

The starting E. coli culture volume is 2.5-5 L of LB broth and the expected DNA yield is 7.5–10 mg.

Purification of plasmid DNA:

Addition of phenol/chloroform can dissolve and denature proteins, like DNase. This is especially important if the plasmids are to be used for enzyme digestion. Otherwise, smearing may occur in enzyme restricted form of plasmid DNA.

DNA VACCINES

DNA vaccination

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. 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 (T_C or CTL) responses, helper T-cell (T_H) 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. 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 T_H 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 converts it into pathogenic 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 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  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 of DNA vaccines

Table 1. Advantages And Disadvantages Of Nucleic Acid-Based Immunization
Advantages	Disadvantages
Subunit vaccination with no risk for infection Antigen presentation by both MHC class I and class II molecules Able to polarise T-cell help toward type 1 or type 2 Immune response focused only on antigen of interest Ease of development and production Stability of vaccine for storage and shipping Cost-effectiveness Obviates need for peptide synthesis, expression and purification of recombinant proteins and the use of toxic adjuvants Long-term persistence of immunogen In alive expression ensures protein more closely resembles normal eukaryotic structure, with accompanying post-translational modifications	Limited to protein immunogens (not useful for non-protein based antigens such as bacterial polysaccharides) Risk of affecting genes controlling cell growth Possibility of inducing antibody production against DNA Possibility of tolerance to the antigen (protein) produced Potential for atypical processing of bacterial and parasite proteins

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 then 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 pathay. 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.

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.

DNA immunization is able to raise a range of T_H 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 life-long 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.

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, 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.^[30][39] 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.

Interferons

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.^{[25][26][48][49][50]} 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 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.^]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.
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.