The main type of vaccinations are delivered by injection. It is important to formulate and deliver vaccines appropriately to get better drug efficacy and reduce infections in animals and humans. Modern technologies have given researchers a new opportunity to develop safer and more effective vaccines. Edward Jenner,in 1976, was first to inoculate a young boy with cowpox when he acquired small pox (Rogan and Babiuk, 2005). Currently, the majority of vaccines are either live attenuated or killed. DNA vaccines represent an alternative to conventional vaccine delivery. This review highlights pre clinical and clinical efficacy of DNA vaccines, the advantages of such vaccines, and some of second generation vaccines.
DNA vaccination are rings of DNA containing a promoter, terminator and a gene encoding an antigen. They are a new approach for generating all types of immunity i.e. "antibodies, cytolytic T lymphocytes (CTL) and T-helper cells". DNA vaccines control gene expression in the host and induce a systemic immune response to the proteins encoded by the plasmid. It can be used for, immunotherapy, cancer, autoimmune and allergic diseases. (Liu, 2003).
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DNA Vaccines and Immunisation
The initial studies conducted were that of direct transfection in vivo with DNA that was used to express foreign proteins and induce immune response. Preclinical studies have indicated that the antigenicity of viral proteins produced by DNA vaccination are similar to that of the proteins when produced naturally during infection. DNA vaccines could be given as alternatives to live virus vaccines, as immunisation of direct transfection in vivo with plasmid DNA is efficient to form CD8 cytotoxic T lymphocytes (CTL). CTL recognises peptides of the major histocompatibility Complex (MHC). DNA vaccines produce protein antigens that gain access to pathways of antigen presentation via Class 1 MHC molecules. (Donnelly and Ulmer, 1997).
DNA vaccines have been used to raise immune responses against antigens of non viral pathogens. Protective immune responses also have been found in mice immunised against pathogens e.g L. major using plasmid DNA. Results indicated that the protection in DNA vaccines in the pathogens L. major, P. yoelii, and M. tuberculosis, are likely to be cell-mediated. Both cell-mediated and humoral immune responses are caused by using plasmid DNA vaccination as well as production of alloimmune responses that are capable of destroying tumour cells (Donnelly and Ulmer, 1997).
Attenuated bacterial strain allow the administration of recombinant vaccines via the mucosal surfaces. Intracellular bacteria e.g. Salmonella, Shigella and Listeria, have recently been used as carriers for DNA vaccines in-vitro and in-vivo. Antigen-presenting cells (APC) ed DNA vaccines with the help of carrier bacteria e.g. macrophages and dendritic cells (DC's) (Glück and Metcalf, 2002).
Delivery of DNA
Presently, plasmids are delivered by injection either intradermally, intramuscularly, or delivered by a gene gun. In a study by Fynan et al (1993) the effect of inoculation on DNA vaccination was evaluated in "murine and avian influenza virus models". In both models, the vaccine consisted of purified plasmid DNA that had been designed to express an influenza virus hemagglutinin glycoprotein. Two inoculations were tested in mice and chickens and it was shown that the parenteral routes intramuscular and intravenous achieved good protection. However the most efficient is the gene gun to deliver DNA-coated gold beads to the epidermis (Fynan et al, 1993). Plasmid is directed by the gene gun to the cell where it can function, rather than needle injection that directs it extra cellularly where the majority of the plasmids are degraded before expression of antigen. However, for humans and animals except mice, gene gun is not very effective as only a limited amount of DNA can be coated onto gold beads hence many administrations are required with in order to achieve sufficient immune responses (Baca-Estrada et al, 2000). In addition, other delivery includes a naked DNA which has also been applied to mouse skin and is taken up in hair follicles to stimulate an immune response (Ellis, 2001).
The trapping of DNA vaccines in the lung with macro aggregates, to induce mucosal immunity is under investigation in vivo. Genetic adjuvants can be used as a method of delivery where the genes are co-delivered in order to modulate immune responses to DNA vaccines. Cytokines, chemokines and co-stimulatory molecules have known to modulate DNA vaccines. Two methods used to modulate the immune response induced by DNA vaccines are: "(i) supplementing DNA vaccines with plasmids encoding cytokines and (ii) targeting the Ag encoded by DNA vaccine through genetically fusing the Ag to molecules binding cell surface receptors". Mostly plasmids encoding the Ag are mixed with plasmids encoding the genetic adjuvant which a large number of combinations can be tested without needing large numbers of new constructs (Scheerlinck, 2001).
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One of the first DNA vaccines for veterinary use is "West Nile virus vaccine, for horses and infectious haematopoietic necrosis vaccine for farm reared Atlantic salmon". Marker vaccines have been formed by manipulating DNA which allows the differentiation between infected and vaccinated animals, named DIVA vaccines. DNA in the vaccine can be manipulated to delete a specific gene so the animal does not produce antibodies to code the protein which is present in the pathogen (Chalmers, 2006).
Route and Dosage
A variety of diluents can be used in DNA vaccies including "distilled water, saline, and sucrose". A typical dose is "10 to 100 lg of plasmid DNA" required to show responses when administered. With gene gun "0.1-1 lg of plasmid DNA" is required to induce antibody or CTL response. When human volunteers were given a DNA vaccine encoding a malarial antigen, doses of plasmid DNA in the "500- to 2500-lg" range gave enhanced CTL responses. A single vaccination with DNA can induce an antibody and CTL response in several model systems where as with gene gun multiple administrations are required in humans. (Gurunathan, 2000).
DNA vaccines are safer than killed vaccines since they do not have adjuvants and have no adverse reactions. They have a broad range of immune response e.g. cell-mediated and humoral immunity. DNA vaccines can be delivered to various parts of the body to induce either mucosal or systemic. It is also found that immunity induced by DNA delivery is long lived ensuring that herd immunity does exist (Baca-Estrada, 2000).
Another advantage is that in the host as a natural form an encoded protein is expressed and cause longer expression of the antigen (Nandedkar, 2009). Furthermore, it has been reported that DNA vaccines induce "major histocompatibility complex (MHC) class I restricted CD8` T-cell responses" and mimic live attenuated vaccine effects. DNA vaccines are cost effective and stored easily, eliminating the need for refrigeration to maintain stability of a vaccine during distribution (Liu, 2003).
Table 1- Comparative analysis of various vaccine formulations (Liu, 2003).
The major problem in DNA delivery is to get the DNA into cell and to get it into the correct compartment of the cell before it is degraded. To overcome that, potency of DNA vaccines is increased. DNA is adsorbed to the surface of small particles or encapsulated within the particles. Attaching DNA to small particle protects DNA from degradation before reaching cells and antigen presentation including specialised epithelial cells e.g. M cells and DC's. In addition, polyanionic DNA forms electrostatic bonds with polycationic polymer particles that facilitates cellular uptake of DNA. The polycationic polymer, polyethyleneimine (PEI), is shown to condense DNA and is an effective transfection reagent. Delivery of DNA polymer complexes are inserted in cells where gene expression is likely to elicit an immune response, especially in DC's (Talsma et al, 2006).
Second generation vaccines
A variety of approaches are under investigation to increase the efficacy of DNA vaccines. Some devices target the DNA to specific sites or increase the transfection of cells. There are a few clinical trials of DNA vaccines for a number of diseases e.g. "HIV, malaria, influenza, hepatitis B and cancer". In most of the trials DNA vaccines are injected intramuscularly and the hepatitis B DNA vaccine has been tested by using gene gun. In clinical trials, vaccines immunised with DNA encoding a hepatitis B antigen with a gene gun "seroconverted". A mucosal jet injector device has been previously utilised in a clinical trial of an HIV DNA vaccine. HIV-infected patients responded to HIV DNA vaccines with antibody or CTL responses against antigens to which they had not previously responded. (Liu, 2003).
Aluminium salts used in DNA vaccines have shown to have high potency by maximising the expression of the encoded Ag metal (Donnelly et al, 2005). In DNA vaccines, proteins removed from cells as virus like particles, e.g. influenza nucleoprotein, have been found to be strong immunogens in animals. Ubiquitination or fusion to lysosomal associated membrane protein methods are used to express protein which is targeted to specific intracellular compartments, However using the gene gun, both of these methods increase immune responses to proteins (Donnelly et al, 2005). Attenuated bacteria are being used to deliver the plasmid into antigen presenting cells. In addition to bacteria, polymers and lipid based delivery systems are also being developed. By incorporating the plasmids into these lipid based delivery systems, they can be delivered mucosally or transdermally. Lipid based DNA vaccines by transdermal delivery in mice can induce cytokine and antibody response (Baca-Estrada, 2000). In study done by Talsma et al (2006), a combined targeting ligand DNA delivery system was developed consisting of recombinant reovirus type 3 Ïƒ1 attachment protein. In vitro results showed that PEI conjugated to nuclear localisation sequences (NLS Ïƒ1-) performed substantially better as a transfection agent than either PEI alone or PEI conjugated to Ïƒ1 (Talsma et al, 2006).
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In conclusion, DNA vaccines have moved from laboratory into clinical trials. It represents a new method for diseases needing cellular immunity and allows researchers to adopt new, more effective delivery system. Currently, DNA vaccines have been trailed in several human studies however, the immune responses in humans is lower than mice. This indicates a key aspect needing improvement for future use. On the other hand, as DNA vaccines have broad application, this delivery method offers great promise for protection against severe diseases especially the second generation DNA vaccines.