DNA Vaccines and the Gene Gun Introduction Whether you are watching "Star Trek" or are slowly becoming engrossed in your new science fiction novel, you inevitably will stumble upon a fantastic vision of futuristic medicine placed before you by the author or screenwriter. In many of these imaginings of future medicine, the patient is treated with a futuristic medical device that delivers a simple, needle-free shot into the neck or arm, bringing medication with little unpleasantness to the patient, and producing miraculous results. You understand the incredible value of being vaccinated, and are aware of the effectiveness of the current and past vaccines for typhus, tetanus, measles, smallpox, and hepatitis A and B, yet you wished that this device had been around when you had to go in for your latest group of vaccine shots that were required of you before going to college, or maybe when you punctured your hand with that rusty nail and had to get a tetanus shot. You certainly would be a lot less reluctant to get that yearly flu shot. Well, this futuristic scenario is not as far off as you might think! Vaccines In order to vaccinate people and therefore protect them from a particular disease, you have to let their immune system come in contact with the disease-causing organism so that their immune system will recognize the pathogen at a much later date and build defenses against it. In fact, scientists describe the cells and proteins of the immune system in terms of possessing "immunologic memory," for after the immune system is done waging war against an invading pathogen, it leaves behind memory cells that stand at the ready in case this pathogen makes another offensive on the body at a later date. The most effective vaccines activate both humoral and cellular immunity to the pathogen. The humoral arm of the immune system protects against an invading pathogen by producing antibodies, which are proteins that bind to the protein parts of the pathogen, thereby neutralizing the pathogen's ability to enter and infect cells and also flagging them so macrophages can engulf and destroy them. The cellular arm of the immune system protects by directing cytotoxic (cell-killing) T cells to kill pathogen-infected cells in the body, for these infected cells are often turned into pathogen-producing factories that would rapidly overwhelm the host's body, if unchecked. The earliest vaccines were derived from organisms that were closely related to the pathogen being vaccinated against, but actually infected another mammalian species. Because the two pathogens shared many similarities, the immune system would become "wary" and protect against both organisms (for example, the cowpox virus was used to protect against smallpox). Later vaccines were made from organisms that had been made innocuous by treating them so they were "dead" or "inactivated," and thus no threat to the person being vaccinated, or from a pathogen that had been either bred or genetically manipulated so as to be very weak and easily eradicated by the immune system (attenuated). Examples of these types of vaccines are the early Salk vaccine for polio, which uses the inactivated polio virus, and the later Sabin vaccine for polio, which uses an attenuated virus. More modern vaccine approaches include subunit vaccines, which just use purified proteins from the outside of the pathogen, or DNA vaccines. All of the above approaches are expensive, technically difficult, and extremely labor-intensive to produce, as well as being hard to transport and store, which introduces problems in developing countries. In addition, these methods have been tried for other illnesses, such as malaria, AIDS, herpes, and hepatitis C, but have either been ineffective or have proven too dangerous to implement. DNA Vaccines Traditional vaccines are based upon the premise of injection of the pathogen or some of its proteins into the body, where the immune system can "learn" how to recognize it and prepare itself for future exposure. Yet due to the failure and difficulties associated with these traditional vaccine approaches, researchers are beginning to look for other more novel methods of vaccination. Some postulated that it might be possible to simply introduce DNA that encoded for some of the proteins of a disease-causing organism, and that a person so inoculated with the pathogen's genes would somehow express those genes to make the proteins encoded by the DNA vaccine. This, in turn, would reveal parts of the pathogen to the immune system and confer future protection against it. Understandably, this idea was thought almost ludicrous by the mainstream vaccine researchers; even if the genes from the vaccine could make it inside a cell's nucleus and be transcribed and translated, the chances that the levels of protein that could be expressed could protect against a healthy dose of the live pathogen seemed slim indeed. Despite the lack of faith from their peers, these unconventional scientists explored DNA vaccination, and to their amazement (as well as the rest of the scientific community), simply injecting naked DNA encoding a pathogen's proteins seemed to work beautifully. More substantial animal trials ensued, and the mounting evidence for the success of DNA vaccines could no longer be ignored. DNA vaccines could indeed protect an animal against future infection with a live pathogen. DNA Vaccine Mechanism At right is a diagram illustrating the mechanism behind these successful DNA vaccines. First, the DNA vaccine is introduced into the person to be immunized. This is done by injection or delivery by a "gene gun" (see below), with muscle typically the targeted tissue. The DNA vaccine is a plasmid that contains one or more genes of the pathogen that is being immunized against behind a strong eukaryotic promoter. Transcription and translation occur from the vaccine plasmids that find their way into the nucleus of the muscle cells, to make the pathogen-derived protein. Some of this protein makes it outside of the cell, where it is either bound by antibody molecules on B cells or phagocytosed by macrophages. Either way, the protein gets digested inside these cells into small peptides and placed in the binding groove of a cell surface protein called the class II major histocompatibility complex (MHC II), much like a hot dog fits into a bun. T cell receptors (TCRs) on the surface of helper T cells can recognize these peptides as being foreign to the body, and therefore from an invading pathogen. Once the peptides are bound and recognized as foreign by the TCR, the helper T cell releases a variety of interleukin (IL) proteins to stimulate both arms of the immune system (humoral and cellular) to kick into gear. This IL release has a number of effects. The first is to autostimulate the helper T cell that detected the foreign protein, so that it can proliferate to fight off the disease. Another secreted IL protein ramps up humoral immunity by causing the B cells whose membrane-bound antibodies were able to bind the foreign protein to differentiate (multiply and change) into antibody-secreting plasma cells for the production of massive quantities of serum antibodies reactive to this pathogen's protein. The last effect of the IL release is to promote the cellular arm of the immune system to a state of war-readiness. The warriors of the cellular arm are the cytotoxic T lymphocytes (CTLs). They are activated by the IL proteins released by the helper T lymphocytes, and their job is to search for and destroy any cells that have foreign matter inside them, such as proteins from a replicating virus. You might ask: How do the CTLs know if a cell has an invader inside it? The answer lies in the fact that all nucleated cells of the body take a small sampling of all of the proteins being synthesized in the cytosol and break them down into small peptides. Similar to the B cells and macrophages before, these peptides are placed in the binding groove of another major histocompatibility complex, this time called class I MHC. CTLs that have been activated by ILs, and that recognize the "displayed" peptide as being of foreign origin, will target and destroy the cell with cytotoxins. These cells of the immune system that have become activated by introduction of foreign matter from an infection or vaccine, whether B cell, helper T cell, or CTL, also create "memory cells" when they proliferate. It is these memory cells that protect you later in life from infection by the same pathogen you were once exposed to, and are the reason you only get chicken pox once and can never get the flu twice from the same strain of influenza virus. It is this protection that vaccines seek to offer, by tricking the immune system into thinking it has been infected, with the introduction of innocuous proteins derived from a harmful pathogen, so that they bring forth the cellular and molecular arsenal against the foreign matter and leave behind these memory cell sentries to deal with future invasion. Human Vaccine Trials There are many current ongoing trials of genetic vaccines in humans, most of which are in the early stages (Stage I), which simply measure how well the treatments are tolerated by people, and do not measure the efficacy of the vaccine. The DNA vaccines include those for hepatitis B, herpes, HIV, malaria, adenocarcinoma (cancer of breast and colon), B cell lymphoma (cancer of the blood and lymph cells that provide humoral, or antibody-mediated, immunity), and T cell lymphoma (cancer of blood and lymph cells that provide cellular, or cell-mediated, immunity), all of which have been relatively well tolerated in the Stage I trials. Enter the Gene Gun What about the futuristic methods for delivering vaccines without needles promised earlier? Well, early DNA vaccines were simple needle and syringe injections of DNA, with the most successful trials involving injection of DNA into the muscle of the animals. However, researchers have recently come up with a more modern way to deliver genetic vaccines: the gene gun. The gene gun works by quickly shooting into the skin of the vaccinated person (or animal) DNA that has been coated onto microcarriers. Typically, the DNA is coated onto ultrasmall gold beads and propelled out the "barrel" of the gene gun by a shock wave from high-pressure gas hitting a cartridge full of the DNA-coated gold microcarriers. Because of their small size, these DNA-covered beads penetrate the skin with no discomfort, and deliver the DNA vaccine to the underlying tissues, where transgenic expression of the protein is encoded, and priming of the immune system by the vaccine begins. DNA Vaccine Web Site Links DNA Vaccine Web - Compendium of DNA vaccine Web resources. Scientific American - Great article on genetic vaccines. The Institute for Human Gene Therapy - General info, seminars, basic science, clinical trials, education... All the Virology on the WWW - Vast virology resource. Introduction to Gene Therapy Home Page - Intro to gene therapy lectures. Gene Gun Test Run - Testing the gene gun at UCSD. Proceedings of the National Academy of Sciences - Abstract: Gene gun-mediated skin transfection with interleukin 12 gene results in regression of established primary and metastatic murine tumors. (full article PDF file) Copyright © 1999, Harcourt College Publishers, A Harcourt Higher Learning Company. All rights reserved. Use of this site indicates acceptance of the Terms and Conditions of Use. For problems or suggestions concerning this service, please contact Chemistry Webmaster.