The latest development in vaccination - DNA vaccination, which is also called as 'nucleic acid immunisation' or 'genetic immunisation' - represents the third generation of vaccines.
Recombinant DNA (r DNA) vaccines are based on purified antigenic proteins produced in bacteria like E.Coli using recombinant DNA technology. DNA vaccines, on the other hand, are based on a circular double stranded DNA molecule, referred to as plasmid.
First and second generation of vaccines have proven their efficacy against diseases caused by extracellular pathogens, mainly mediated by long-lived humoral immune responses. DNA vaccines present the possibility of tackling a variety of infections caused not only by extracellular pathogens but also by intracellular pathogens.
They could therefore be useful in prevention of infections caused by intracellular pathogens like Plasmodium falciparum (malaria), Trypanosoma sp. (Chagas disease), Leishmania sp. (leishmaniasis), mycobacterium tuberculosis (tuberculosis) and HIV, which are predominantly controlled by cell mediated immunity (cyototoxic T lymphocytes). They are extremely safe, as they do not contain any pathogenic organism that may revert to virulence. DNA vaccines are suitable even for immunocompromised patients.
Tech advantage
DNA vaccines are highly stable compared to recombinant, live or attenuated vaccines. They can be stored either dry or in aqueous solution at room temperature. Hence the huge costs associated with the cold chain during storage and distribution may be offset, making them accessible in countries with tropical setting with limited health infrastructure. DNA vaccines can be produced using fermentation, purification and validation techniques and are easy to purify using simple and inexpensive procedures. This ability to use generic production and verification techniques simplifies vaccine development and production. Moreover, DNA vaccine plasmids can be constructed using simple recombinant DNA technique. Further, coinoculation of multiple plasmids encoding different antigens of the same pathogen or different pathogens provides multivalent approach which is especially important for diseases such as malaria, AIDS and tuberculosis, wherein a single antigen alone may not offer complete protection.
Overcoming hurdles
Naked DNA vaccines have the ability to induce immune responses without any special formulation. However, biodistribution studies showed that the number of plasmid DNA molecules surviving to transfect target cells after intramuscular injection was only a small fraction of the total DNA injected. The quest for higher immune response led to a proliferation of different approaches for formulating DNA vaccines to protect the DNA from degradation and improve transfection efficiency. A number of formulations have been studied, including classical adjuvants. Among the classical adjuvants, aluminum phosphate is noteworthy for its effectiveness and simplicity of preparation. Alum phosphate is thought to act by recruiting APCs to the site of the intramuscular injection, where a proportion of muscle cells would be expressing the antigen encoded by the DNA vaccine.
Other delivery strategies include transfection facilitating lipid complexes and microparticulates. Lipid complexes can include varying combinations of cationic lipids and cholesterol. Microparticulates include DNA adsorbed to or entrapped in biodegradable microparticles such as poly-lactide-co-glycolide or chitosan, or complexed with nonionic block copolymers or polycations such as polyethyleneimine. Microparticulates appear to improve delivery of DNA to APCs by facilitating trafficking to local lymphoid tissue via the afferent lymph and facilitating uptake by dendritic cells.
More recently, electroporation, which has the potential both to force DNA into cells and create damage to adjacent muscle cells, has emerged as the most potent method for delivering DNA intramuscularly. However, electroporation also has been found to result in increased levels of integration of plasmid into the genome of host cells, which is undesirable. Jet injection for direct targeting of mucosal cells in humans has been evaluated. Epidermal immunisation by gene gun tends to target epidermal langerhans cells, potentially favouring direct presentation to CD4+ and CD8+ T cells. The gene gun also serves as a useful platform to study the effects of protein trafficking within and among APCs on immune responses.
Basic challenges
'Antigen discovery' for the treatment of diseases caused by intracellular pathogens, is a challenge for the development of DNA vaccines. Pathogens have relatively large genomes and only a limited variety of antigens have been tested till now without knowing if they are the best possible antigens. Another major concern is integration of plasmid DNA into the human genome randomly, potentially leading to insertional mutagenesis. Auto-immunity or allergic reactions may also result. Further, DNA vaccines are not effective for diseases, where polysaccharides are the prominent antigens. Besides, DNA vaccines, though well established as a research tool in mouse models, have so far shown low immunogenicity when tested alone in human clinical trials.
Future of DNA vaccines
Today, tremendous research is carried out to improve immunogenicity of DNA vaccine. Immunogenicity of DNA vaccine can be improved by redesigning the plasmid. Optimisation of DNA vaccine, immunogenicity with immunomodulators (GM-CSF, IL-2) and chemical adjuvants like CMC and calcium phosphate are viable options. The expression of antigen from DNA vaccines as fusion proteins with a destabilising ubiquitin molecule (which enhances proteasome dependent degradation of the endogenously synthesized antigen) could result in strong cell mediated and humoral response to some extent. DNA vaccines must surmount major safety concerns, including a theoretical potential for integration into the host genome and insertional mutagenesis, induction of auto immunity, immunological tolerance or a prolonged allergic reaction to an encoded protein, in which synthesis is not readily terminated.
Route of administration of the vaccine also has an important role to play, because depending on the pathogenic invasion, both mucosal and systemic immune responses may need to be taken care of. For infections like HIV, which invade through the rectovaginal mucosa, both mucosal and systemic immunities are indispensable and must be elicited to prevent the spread of the pathogen.
A clearer understanding of the distribution, cellular uptake and expression of DNA vaccines to overcome the limitations of transfection in-situ is an important need of the hour.
While the potential benefits of DNA vaccines are enormous, they are still in their infancy. DNA vaccine technology is novel and its clinical utility is not fully proven. The academic community, pharmaceutical companies and the regulatory agencies have to work together to make this technology clinically viable. Let us hope that the extensive studies on animal models and various human clinical trials of DNA vaccines initiated in the 20th century, will be translated into a clinical reality in the 21st century.
(The authors are with Department of Pharmaceutical Sciences and Technology, University Institute of Chemical Technology, Mumbai)