Undoubtedly, the modern era medicine has by and large succeeded in generating a couple of different vaccines to save lives of infinite number of people from hepatitis A and B, meningitis, measles, typhus, tetanus, rabies, rotavirus and other deadly infections. These vaccines have eradicated smallpox and the world is expecting the vaccines to eradicate polio in the near future.
Though these vaccines have done well to control a number of diseases, they have caused significant mortality and morbidity. In spite of this, vaccines still play an important role in fighting diseases like malaria, tuberculosis, hepatitis C, herpes and AIDS. The growth of vaccines as a remedy should be seen in the context of failure of standard immunisation methods to provide protective immunisation against the diseases mentioned above. The alternative strategies are, therefore, to make vaccines for the diseases that have no effective vaccines available so far.
One of the most promising and recently developed vaccination strategies is the use of genetic material, i.e., deoxyribonucleic acid (DNA) to make genetic vaccines. These DNA vaccines are an exciting outcome of recombinant DNA (rDNA) technology. DNA vaccines are the newest vaccines and are still at experimental stages, although to date they seem to be very effective and recorded good safety result. It might happen one day that these vaccines prevent AIDS, malaria and other fatal infections.
In the case of DNA vaccines, the DNA is injected into the muscle of the animal being vaccinated. It is usually done with a "gene gun" that uses compressed gas to blow the DNA into the muscle cells. Some muscle cells express the pathogen DNA to stimulate the immune system. Both humoral and cellular immunity have been induced by DNA vaccines.
Looking back into history, the use of genetic material to deliver genes for therapeutic purposes has been practiced for many years. From as early as1950, experiments outlining the transfer of DNA into cells of living animals were reported. Later, experiments using purified genetic material further confirmed that the direct DNA gene injection in the absence of viral vectors results in the expression of the inoculated genes in the host. Additional experiments were conducted for recombinant DNA molecules, illustrating the idea that purified nucleic acids could be directly delivered into a host and proteins would be produced.
In 1992, Tang and Johnson reported that the delivery of human growth hormone in an expression cassette in vivo resulted in production of detectable levels of the growth hormone in host mice. They also found that these inoculated mice developed antibodies against the human growth hormone. They termed this immunisation procedure as "genetic immunisation," which describes the ability of inoculated genes to be individual immunogens.
Constructing rDNA vaccines
The rDNA vaccines consist of bacterial plasmids. The expression plasmids used in DNA-based vaccination contain two units - the antigen expression unit and the production unit. While the antigen expression unit is composed of promoter/ enhancer sequences and antigen-encoding and polyadenylation sequences, the production unit comprises of bacterial sequences necessary for plasmid amplification and selection.
The construction of bacterial plasmids with vaccine plasmid is transformed into bacteria, where bacterial growth produces multiple plasmid copies. The plasmid DNA is then purified from the bacteria, by separating the circular plasmid from the much larger bacterial DNA and other bacterial impurities. This purified DNA acts as the vaccine.
Way of administering
The US Food and Drug Administration has given 111 routes of administration for human medicines. For rDNA vaccines, several possible routes of plasmid delivery have been found after many clinical research and trials. The skin and mucous membranes are considered as the best site for immunisation, largely due to the high concentrations of dendritic cells (DC), macrophages and lymphocytes. Intradermal injection of DNA coated gold particles with a gene gun has also been used. The plasmid DNA can be diluted in distilled water, saline or sucrose. There has also been positive demonstration of pro-injection or co-delivery with various drugs.
Modus operandi
The rDNA vaccines work on the same lines of traditional vaccines. A plasmid vector that expresses the protein of interest under the control of an appropriate promoter is injected into the skin or muscle of the host. After uptake of the plasmid, the protein is produced endogenously and intracellularly processed into small antigenic peptides by the host proteases. Then the peptides enter the lumen of the endoplasmic reticulum (ER) by membrane-associated transporters. In the ER, peptides bind to major histocompatibility complex (MHC) class I molecules. These peptides are presented on the cell surface in the context of the MHC class I. Subsequent CD8 + cytotoxic T cells (CTL) are stimulated and they evoke cell-mediated immunity. CTLs inhibit viruses through both cytolysis of infected cells and noncytolysis mechanisms such as cytokine production.
The foreign protein can also be presented by the MHC class II pathway by antigen presenting cells (APCs), which elicit helper T cells (CD4+) responses. These CD4+ cells are able to recognise the peptides formed from exogenous proteins that were phagocytosed by APC, then degraded to peptide fragments and loaded onto MHC class II molecules. Depending on the type of CD4+ cell that binds to the complex, B cells are stimulated and antibody production is stimulated.
In many ways rDNA vaccines are advantageous than the existing vaccines. Some of the qualities of rDNA vaccines are:
■ rDNA vaccines are designed to be produced in large quantities by use of recombinant DNA technology.
■ The same plasmid can be custom tailored to make a variety of specific vaccine for different pathogens. This helps in making different rDNA vaccines for different pathogens by using the same manufacturing technique.
■ rDNA vaccines do not require any refrigeration for handling and storage.
■ They are easier to administer by using air gun or gene gun. Hence it enables to provide vaccination to a large population without the need for massive quantities of needles and syringes.
■ There is no fear of infection or full-blown illness by the use of rDNA vaccines as these vaccines do not contain all the desired genes of the patients.
■ rDNA vaccines are able to induce the expression of antigens that resemble native viral epitopes more closely than standard vaccines do since live attenuated and killed vaccines are often altered in their protein structure and antigenicity.
■ rDNA vaccine provides effective immunity against variants at once, which is not possible by standard immunisation methods. This is due to the reason that the plasmid can genetically engineered to carry genes for different variants (types) of the same pathogen like in case of HIV or influenza.
One of the most important advantages of genetic vaccines is their therapeutic potential for ongoing chronic viral infections. DNA vaccination may provide an important tool for stimulating an immune response in HBV, HCV and HIV patients. The continuous expression of the viral antigen caused by gene vaccination in an environment containing many APCs may promote successful therapeutic immune response which cannot be obtained by other traditional vaccines. This is a topic on interest that has been focused in the last five years.
Limitations
Although DNA can be used to raise immune responses against pathogenic proteins, some microbes have outer capsids that are made up of polysaccharides. Due to this reason, rDNA vaccines cannot substitute for polysaccharide-based subunit vaccines and this limits the extent of the usage of rDNA vaccines.
Future of rDNA vaccines
The rDNA vaccines are highly promising but intensive research is still going on to understand certain aspects of it. They include:
■ Which doses of DNA vaccine are most effective
■ What will be the schedule of DNA vaccination
■ How long the immunity will last
■ How much will be the individual variations in their responses
But the most important factor is that one should fully know the exact gene out of thousands in a given pathogen, which should be selected to provide the maximum powerful immune response.
Recently it has been discovered that the transfection of myocytes can be amplified by pretreatment with local anesthetics or with cardiotoxin, which induce local tissue damage and initiate myoblast regeneration. If we fully understand this mechanism of DNA uptake, it could prove helpful in improving applications for gene therapy and gene vaccination. Both improved expression and better engineering of the DNA plasmid may enhance antibody response to the gene products and expand the applications of the gene vaccines.
rDNA vaccines in India
The drive to produce affordable vaccines for diseases such as hepatitis, diphtheria, tetanus and rabies has been the basis for Indian biotech companies to move further into research. Indian companies appear well positioned to leverage their cost-effective manufacturing capabilities to corner some of this market share and compete globally. Many classical biotech companies like Serum Institute of India, Bharath Serums and Vaccines, Panacea Biotech, Indian Immunologicals and Biological E have diversified into modern biotechnology.
India's leading vaccine manufacturer, Serum Institute of India, has produced Gene Vac-B (Recombinant Hepatitis - B Vaccine, I.P.), while Shanvac-B (Recombinant Hepatitis - B Vaccine, I.P.) rDNA is manufactured by Shantha Biotechnics. The Hyderabad-based Shantha Biotechnics was the first in India to manufacture a human hepatitis B vaccine using recombinant DNA technology. Panacea Biotech has invested more than Rs 25 crore to develop a global research and development centre (GRAND) for the development of advanced drug delivery research based products.
The New York-based Bristol-Myers, maker of the blood-thinning pill Plavix, has plans to set up a research facility in the southern Indian city of Bangalore that may host as many as 400 scientists. This proves that India is certainly becoming a hub for biotechnology research.
rDNA vaccine pipeline
■ Bharat Biotech: r Epidermal Growth Factor, r Insulin, vaccines for rabies, malaria and Diarrhea
■ Shantha Biotech: Shankinase, diagnostic kits for HIV, Hepatitis B, Hepatitis C, and AlphaFeto Proteins
■ Panacea Biotech: Hepatitis B vaccine and anti- anthrax vaccine
■ Zenotech Labs: r Human granulocyte macrophage colony stimulating factor
Martin Mueller of the German Cancer Research Centre in Heidelberg (2008), said that work on mice showed that tattooing was a more effective way to deliver a new generation of experimental DNA vaccines than standard injections into muscle.
- (The author is with Accure Labs Pvt. Ltd, Noida)