Dna Vaccine

Author : Dr. Saurav K Sarkar Discipline : B. Tech (Biotech)

Topic : DNA Vaccines Module : BT 501 - VI

Designation : Lecturer Semester : V

Objective : The student should understand : 1. What is DNA Vaccine 2. Limitations of current vaccines 3. Advantages of DNA vaccines 4. Disadvantages of DNA vaccines Background Edward Jenner carried out the first vaccination in 1796 by injecting a young boy with cowpox. This conferred protection against a subsequent infection with the deadly smallpox virus. Through concerted worldwide vaccination campaigns, smallpox has now been eliminated. Most of the vaccines used today are based on similar principles to Jenner's original vaccine – they are live but attenuated (disabled) bacteria or viruses, which cause the body to mount a protective immune response against the target pathogen. Examples include the measles, mumps, rubella and tuberculosis vaccines. Other current vaccines are ‘killed vaccines’ – the pathogen itself is killed so it is no longer infectious but it can still stimulate the immune system. Unfortunately, vaccines against all common diseases cannot be made using the above methods and other approaches are needed. One successful strategy is the use of subunit vaccines, where the gene for one specific protein on the pathogen is expressed, and the protein used as the vaccine. The current hepatitis B and influenza vaccines are protein subunits. In 1990 J.A. Wolff and coworkers demonstrated direct transfer of plasmid DNA in saline solution into mouse muscle in vivo and subsequent expression of the gene. Mice injected with plasmids encoding the nucleoprotein of influenza A virus developed both Abs and Class I MHC restricted cytotoxic T cells (CTLs). On challenge with virulent influenza A strain, 100% of DNA injected mice survived, whereas 100% of control mice died by day 9. Within a span of a few years, several DNA vaccines protective against a wide range of viruses, bacteria, and parasites, as well as various tumors were raised for preclinical studies. A DNA delivering system, originally used for transfecting plant cells, termed the gene gun, which essentially consists of a helium gas pressure-driven device capable of delivering tiny gold particles coated with plasmid DNA through the skin into the underlying muscle of mice is adopted for administering these vaccines. In the DNA vaccines, the gene for a pathogen protein is introduced into human cells and then expressed to produce the protein inside the body. Conventional vaccines have prevented many millions of cases of killer diseases such as small-pox and polio.

But some pathogens, such as malaria, have proven to be a considerable challenge to vaccine developers. It is in such cases that DNA vaccines may prove useful. Indeed, a promising DNA vaccine candidate has been developed for malaria. DNA vaccines are also currently being developed for over 15 other human illnesses including AIDS, herpes, tuberculosis and rotavirus, a common cause of childhood diarrhoea. DNA vaccination differs from traditional vaccines in that just the DNA coding for a specific component of a disease-causing organism is injected into the body. The DNA can be administered either in a saline solution injected through a hypodermic needle or on DNA-coated gold beads propelled into the body using gene guns. The actual production of the immunizing protein takes place in the vaccinated host. This eliminates any risk of infection associated with some live and attenuated virus vaccines. Limitations of the current mode of vaccine production: • Not all infectious agents can be grown in culture, and so no vaccines have been developed for many diseases • Production of animal and human viruses requires expensive animal cell culture • Both the yield and rate of production of animal and human viruses in culture are often quite low, thereby making vaccine production costly • Extensive safety precautions are necessary to ensure that laboratory and production personnel are not exposed to a pathogenic agent • Batches of vaccine may not be killed or may be insufficiently attenuated during the production process, thereby introducing virulent organisms into the vaccine and inadvertently spreading the disease • Attenuated strains may revert, a possibility that requires continual testing to ensure that the reacquisition of virulence has not occurred • Not all diseases (e.g., AIDS) are preventable through the use of traditional vaccines • Most current vaccines have a limited shelf life and often require refrigeration to maintain potency which causes storage problems Essential Features of Nucleic Acid Vaccines : This is also known as naked DNA vaccine. DNA encoding the Ag cannot replicate in the human or animal cells. The plasmids are grown in E. coli, and their origin of replication is not suitable for mammalian cells. A promoter element suitable for high-level gene expression on mammalian cells may be included in the gene construct. The construct also has an appropriate mRNA transcript termination-polyadenylation sequence. After intramuscular injection by the gene gun, the plasmid enters the cytoplasm and then the nucleus of the 1

Author : Dr. Saurav K Sarkar Topic : DNA Vaccines Designation : Lecturer Discipline : B. Tech (Biotech) Module : BT 501 - VI Semester : V myocytes, but is not integrated into the genome. The myocytes and the dendritic cells do not divide with high rate, neither they carry genetic homology with the plasmid, so homologous recombination is rare. It was proposed that the DNA plasmids leak out of the myocytes over a period and picked up by dendritic cells for presentation with MHC Class I molecules to cytotoxic T cells. Thus, the DNA coated gold particles deposited in the epidermis and muscle cells soon find their way into Langerhans' cells, resident dendritic cells in the skin, which would then migrate to local lymph nodes. Potential advantages of nucleic acid vaccines : They can induce both humoral and CTL responses. Very small amounts of DNA, sometimes nanograms, can induce excellent CTL response. Theoretically, the persistence of Ag synthesis is likely to lessen the number of booster doses, though in preclinical studies several DNA doses were required. DNA vaccines are better alternatives to synthetic peptides or pure molecular vaccines that are liable to incorrect folding and/or glycosylation, generating additional problems. Once an appropriate DNA vaccine is engineered, it remains stable and batch variation is minimal, facilitating quality control procedures. Mass production would make DNA vaccines relatively cheap. Survival of DNA-immunized mice. Injected mice were immunizes with DNA that contained the influenza A virus nucleoprotein gene under the control of the Kous sarcoma virus promoter on an E. coli plasmid. The control mice wert injected with plasmid DNA only. The x axis represents the number of days after the animals were challenged with the live influenza virus

Disadvantages of DNA vaccines : The felicitous findings with DNA vaccines for influenza Ag in mice was not always repeatable with other kinds of Ag or other animal species. Some early attempts with genes for HIV envelope protein were quantitatively inferior. Long term chronic persistent expression of Ag by dendritic cells and myocytes may eventually lead to a state of autoimmunity. There is also the possibility that a constant leak of small quantities of Ag over a long period could lead to immunologic tolerance. The safety of introducing foreign DNA molecules at high copy number, from the view point of carcinogenicity-insertional mutagenesis, is still unknown. There is much excitement, at present, about the promise of DNA vaccines. Scientists are expected to pay more attention to augment DNA uptake, optimize expression of inserted DNA in vivo, or modulate resultant responses. Insertion of IL-12 and HIV Ag gene in a single plasmid increased remarkably the Ag specific CTL response, at the same time shifting immunity towards TH1 in a mouse model. Some limited clinical experimentations in humans have recently been allowed. Advantages of genetic immunization over conventional vaccines • Cultivation of dangerous infectious agents is not required. • Since genetic immunization does not utilize any viral or bacterial strains, there is no chance that an attenuated strain will revert to virulence. • Since no organisms are used, attenuated organisms that may cause disease in voung or immunocompromised animals will not be a problem. • Approach is independent of whether the microorganism is difficult to grow or attenuate. • Production is inexpensive because protein does not need to be produced or purified. • Storage is inexpensive because of the stability of DNA. • One plasmid could encode several Ags/vaccines, or several plasmids could be mixed together and administered at the same time.

2