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Genetic Vaccines Sci Am Jul 99
GROWING NEW ORGANS Sci Am Apr 99
Edible Vaccines Sci Am Sep 2000

Edible Vaccines Sci Am Sep 2000

by William H. R. Langridge

........... Vaccines have accomplished near miracles in the fight against infectious disease. They have consigned smallpox to history and should soon do the same for polio. By the late 1990s an international campaign to immunize all the world's children against six devastating diseases was Still To Be reportedly reaching 80 percent of infants (up from about Accomplished 5 percent in the mid-1970s) and was reducing the annual death toll from those infections by roughly three million. Fighting Autoimmunity Yet these victories mask tragic gaps in delivery. The 20 percent of infants still missed by the six vaccines--against diphtheria, pertussis (whooping cough), polio, measles, tetanus and tuberculosis--account for about two million unnecessary deaths each year, especially in the most remote and impoverished parts of the globe. Upheavals in many developing nations now threaten to erode the advances of the recent past, and millions still die from infectious diseases for which immunizations are nonexistent, unreliable or too costly. Edible Vaccine This situation is worrisome not only for the places that How Edible lack health care but for the entire world. Regions Vaccines harboring infections that have faded from other areas are Provide like bombs ready to explode. When environmental or social Protection disasters undermine sanitation systems or displace communities--bringing people with little immunity into Moving Against contact with carriers--infections that have been long Malnutrition gone from a population can come roaring back. Further, as international travel and trade make the earth a smaller Stopping place, diseases that arise in one locale are increasingly Autoimmunity popping up continents away. Until everyone has routine access to vaccines, no one will be entirely safe.

Case Study: The Norwalk Virus In the early 1990s Charles J. Arntzen, then at Texas A&M University, conceived of a way to solve many of the problems that bar vaccines from reaching all too many children in developing nations. Soon after learning of a World Health Organization call for inexpensive, oral vaccines that needed no refrigeration, Arntzen visited Bangkok, where he saw a mother soothe a crying baby by offering a piece of banana. Plant biologists had already devised ways of introducing selected genes (the blueprints for proteins) into plants and inducing the FURTHER altered, or "transgenic," plants to manufacture the READING: encoded proteins. Perhaps, he mused, food could be genetically engineered to produce vaccines in their edible parts, which could then be eaten when inoculations were needed.

The advantages would be enormous. The plants could be grown locally, and cheaply, using the standard growing methods of a given region. Because many food plants can be regenerated readily, the crops could potentially be produced indefinitely without the growers having to purchase more seeds or plants year after year. Homegrown vaccines would also avoid the logistical and economic problems posed by having to transport traditional preparations over long distances, keeping them cold en route and at their destination. And, being edible, the vaccines would require no syringes-which, aside from costing something, can lead to infections if they become contaminated.

Efforts to make Arntzen's inspired vision a reality are still quite preliminary. Yet studies carried out in animals over the past 10 years, and small tests in people, encourage hope that edible vaccines can work. The research has also fueled speculation that certain food vaccines might help suppress autoimmunity--in which the body's defenses mistakenly attack normal, uninfected tissues. Among the autoimmune disorders that might be prevented or eased are type I diabetes (the kind that commonly arises during childhood), multiple sclerosis and rheumatoid arthritis.

By Any Other Name

Regardless of how vaccines for infectious diseases are delivered, they all have the same aim: priming the immune system to swiftly destroy specific disease-causing agents, or pathogens, before the agents can multiply enough to cause symptoms. Classically, this priming has been achieved by presenting the immune system with whole viruses or bacteria that have been killed or made too weak to proliferate much.

On detecting the presence of a foreign organism in a vaccine, the immune system behaves as if the body were under attack by a fully potent antagonist. It mobilizes its various forces to root out and destroy the apparent invader-targeting the campaign to specific antigens (proteins recognized as foreign). The acute response soon abates, but it leaves behind sentries, known as "memory" cells, that remain on alert, ready to unleash whole armies of defenders if the real pathogen ever finds its way into the body. Some vaccines provide lifelong protection; others (such as those for cholera and tetanus) must be readministered periodically.

Classic vaccines pose a small but troubling risks that the vaccine microorganisms will somehow spring back to life, causing the diseases they were meant to forestall. For that reason, vaccine makers today favor so-called subunit preparations, composed primarily of antigenic proteins divorced from a pathogen's genes. On their own, the proteins have no way of establishing an infection. Subunit vaccines, however, are expensive, in part because they are produced in cultures of bacteria or animal cells and have to be purified out; they also need to be refrigerated.

Food vaccines are like subunit preparations in that they are engineered to contain antigens but bear no genes that would enable whole pathogens to form. Ten years ago Arntzen understood that edible vaccines would therefore be as safe as subunit preparations while sidestepping their costs and demands for purification and refrigeration. But before he and others could study the effects of food vaccines in people, they had to obtain positive answers to a number of questions. Would plants engineered to carry antigen genes produce functional copies of the specified proteins? When the food plants were fed to test animals, would the antigens be degraded in the stomach before having a chance to act? (Typical subunit vaccines have to be delivered by injection precisely because of such degradation.) If the antigens did survive, would they, in fact, attract the immune system's attention? And would the response be strong enough to defend the animals against infection?

Additionally, researchers wanted to know whether edible vaccines would elicit what is known as mucosal immunity. Many pathogens enter the body through the nose, mouth or other openings. Hence, the first defenses they encounter are those in the mucous membranes that line the airways, the digestive tract and the reproductive tract; these membranes constitute the biggest pathogen-deterring surface in the body. When the mucosal immune response is effective, it generates molecules known as secretory antibodies that dash into the cavities of those passageways, neutralizing any pathogens they find. An effective reaction also activates a systemic response, in which circulating cells of the immune system help to destroy invaders at distant sites.

Injected vaccines initially bypass mucous membranes and typically do a poor job of stimulating mucosal immune responses. But edible vaccines come into contact with the lining of the digestive tract. In theory, then, they would activate both mucosal and systemic immunity. That dual effect should, in turn, help improve protection against many dangerous microorganisms, including, importantly, the kinds that cause diarrhea.

Those of us attempting to develop [Image] food vaccines place a high priority on combating diarrhea. Together the main causes--the Norwalk virus, rotavirus, Vibrio cholerae (the cause of cholera) and enterotoxigenic Escherichia coli (a toxin-producing source of "traveler's diarrhea")--account for some three million infant deaths a year, mainly in developing nations. These pathogens disrupt cells of the small intestine in ways that cause water to flow from the blood and tissues into the intestine. The resulting dehydration may be combated by delivering an intravenous or oral solution of electrolytes, but it often turns deadly when rehydration therapy is not an option. No vaccine practical for wide distribution in the developing nations is yet available to prevent these ills.

By 1995 researchers attempting to answer the many questions before them had established that plants could indeed manufacture foreign antigens in their proper conformations. For instance, Arntzen and his colleagues had introduced into tobacco plants the gene for a protein derived from the hepatitis B virus and had gotten the plants to synthesize the protein. When they injected the antigen into mice, it activated the same immune system components that are activated by the virus itself. (Hepatitis B can damage the liver and contribute to liver cancer.)

Green Lights on Many Fronts

But injection is not the aim; feeding is. In the past five years experiments conducted by Arntzen (who moved to the Boyce Thompson Institute for Plant Research at Cornell University in 1995) and his collaborators and by my group at Loma Linda University have demonstrated that tomato or potato plants can synthesize antigens from the Norwalk virus, enterotoxigenic E. coli, V. cholerae and the hepatitis B virus. Moreover, feeding antigen-laced tubers or fruits to test animals can evoke mucosal and systemic immune responses that fully or partly protect animals from subsequent exposure to the real pathogens or, in the case of V. cholerae and enterotoxigenic E. coli, to microbial toxins. Edible vaccines have also provided laboratory animals with some protection against challenge by the rabies virus, Helicobacter pylori (a bacterial cause of ulcers) and the mink enteric virus (which does not affect humans).

It is not entirely surprising that antigens delivered in plant foods survive the trip through the stomach well enough to reach and activate the immune system. The tough outer wall of plant cells apparently serves as temporary armor for the antigens, keeping them relatively safe from gastric secretions. When the wall finally begins to break up in the intestines, the cells gradually release their antigenic cargo.

Of course, the key question is whether food vaccines can be useful in people. The era of clinical trials for this technology is just beginning. Nevertheless, Arntzen and his collaborators obtained reassuring results in the first published human trial, involving about a dozen subjects. In 1997 volunteers who ate pieces of peeled, raw potatoes containing a benign segment of the E. coli toxin (the part called the B subunit) displayed both mucosal and systemic immune responses. Since then, the group has also seen immune reactivity in 19 of 20 people who ate a potato vaccine aimed at the Norwalk virus. Similarly, after Hilary Koprowski of Thomas Jefferson University fed transgenic lettuce carrying a hepatitis B antigen to three volunteers, two of the subjects displayed a good systemic response. Whether edible vaccines can actually protect against human disease remains to be determined, however.

Still to Be Accomplished

In short, the studies completed so far in animals and people have provided a proof of principle; they indicate that the strategy is feasible. Yet many issues must still be addressed. For one, the amount of vaccine made by a plant is low. Production can be increased in different ways--for instance, by linking antigen genes with regulatory elements known to help switch on the genes more readily. As researchers solve that challenge, they will also have to ensure that any given amount of a vaccine food provides a predictable dose of antigen. [Image] Additionally, workers could try to HOW EDIBLE enhance the odds that antigens will VACCINES PROVIDE activate the immune system instead of PROTECTION passing out of the body unused. General stimulators (adjuvants) and better targeting to the immune system might compensate in part for low antigen production.

One targeting strategy involves linking antigens to molecules that bind well to immune system components known as M cells in the intestinal lining. M cells take in samples of materials that have entered the small intestine (including pathogens) and pass them to other cells of the immune system, such as antigen-presenting cells. Macrophages and other antigen-presenting cells chop up their acquisitions and display the resulting protein fragments on the cell surface. If white blood cells called helper T lymphocytes recognize the fragments as foreign, they may induce B lymphocytes (B cells) to secrete neutralizing antibodies and may also help initiate a broader attack on the perceived enemy.

It turns out that an innocuous segment of the V. cholerae toxin--the B subunit--binds readily to a molecule on M cells that ushers foreign material into those cells. By fusing antigens from other pathogens to this subunit, it should be possible to improve the uptake of antigens by M cells and to enhance immune responses to the added antigens. The B subunit also tends to associate with copies of itself, forming a doughnut-shaped, five-membered ring with a hole in the middle. These features raise the prospect of producing a vaccine that brings several different antigens to M cells at once--thus potentially fulfulling an urgent need for a single vaccine that can protect against multiple diseases simultaneously.

Researchers are also grappling with the reality that plants sometimes grow poorly when they start producing large amounts of a foreign protein. One solution would be to equip plants with regulatory elements that cause antigen genes to turn on--that is, give rise to the encoded antigens--only at selected times (such as after a plant is nearly fully grown or is exposed to some outside activator molecule) or only in its edible regions. This work is progressing. [Image] Further, each type of plant poses its own challenges. Potatoes are ideal in MOVING AGAINST many ways because they can be MALNUTRITION propagated from "eyes" and can be stored for long periods without refrigeration. But potatoes usually have to be cooked to be palatable, and heating can denature proteins. Indeed, as is true of tobacco plants, potatoes were not initially intended to be used as vaccine vehicles; they were studied because they were easy to manipulate. Surprisingly, though, some kinds of potatoes are actually eaten raw in South America. Also, contrary to expectations, cooking of potatoes does not always destroy the full complement of antigen. So potatoes may have more practical merit than most of us expected.

Bananas need no cooking and are grown widely in developing nations, but banana trees take a few years to mature, and the fruit spoils fairly rapidly after ripening. Tomatoes grow more quickly and are cultivated broadly, but they too may rot readily. Inexpensive methods of preserving these foods--such as drying--might overcome the spoilage problem. Among the other foods under consideration are lettuce, carrots, peanuts, rice, wheat, corn and soybeans.

In another concern, scientists need to be sure that vaccines meant to enhance immune responses do not backfire and suppress immunity instead. Research into a phenomenon called oral tolerance has shown that ingesting certain proteins can at times cause the body to shut down its responses to those proteins. To determine safe, effective doses and feeding schedules for edible vaccines, manufacturers will need to gain a better handle on the manipulations that influence whether an orally delivered antigen will stimulate or depress immunity.

A final issue worth studying is whether food vaccines ingested by mothers can indirectly vaccinate their babies. In theory, a mother could eat a banana or two and thus trigger production of antibodies that would travel to her fetus via the placenta or to her infant via breast milk.

Nonscientific challenges accompany the technical ones. Not many pharmaceutical manufacturers are eager to support research for products targeted primarily to markets outside the lucrative West. International aid organizations and some national governments and philanthropies are striving to fill the gap, but the effort to develop edible vaccines remains underfunded.

In addition, edible vaccines fall under the increasingly unpopular rubric of "genetically modified" plants. Recently a British company (Axis Genetics) that was supporting studies of edible vaccines failed; one of its leaders lays at least part of the blame on investor worry about companies involved with genetically engineered foods. I hope, however, that these vaccines will avoid serious controversy, because they are intended to save lives and would probably be planted over much less acreage than other food plants (if they are raised outside of greenhouses at all). Also, as drugs, they would be subjected to closer scrutiny by regulatory bodies.

Fighting Autoimmunity

Consideration of one of the challenges detailed here--the risk of inducing oral tolerance--has recently led my group and others to pursue edible vaccines as tools for quashing autoimmunity. Although oral delivery of antigens derived from infectious agents often stimulates the immune system, oral delivery of "autoantigens" (proteins derived from uninfected tissue in a treated individual) can sometimes suppress immune activity--a phenomenon seen frequently in test animals. No one fully understands the reasons for this difference. [Image] Some of the evidence that ingesting STOPPING autoantigens, or "self-antigens," AUTOIMMUNITY might suppress autoimmunity comes from studies of type I diabetes, which results from autoimmune destruction of the insulin-producing cells (beta cells) of the pancreas. This destruction progresses silently for a time. Eventually, though, the loss of beta cells leads to a drastic shortage of insulin, a hormone needed to help cells take up sugar from the blood for energy. The loss results in high blood sugar levels. Insulin injections help to control diabetes, but they are by no means a cure; diabetics face an elevated risk of severe complications.

In the past 15 years, investigators have identified several beta cell proteins that can elicit autoimmunity in people predisposed to type I diabetes. The main culprits, however, are insulin and a protein called GAD (glutamic acid decarboxylase). Researchers have also made progress in detecting when diabetes is "brewing." The next step, then, is to find ways of stopping the underground process before any symptoms arise.

To that end, my colleagues and I, as well as other groups, have developed plant-based diabetes vaccines, such as potatoes containing insulin or GAD linked to the innocuous B subunit of the V. cholerae toxin (to enhance uptake of the antigens by M cells). Feeding of the vaccines to a mouse strain that becomes diabetic helped to suppress the immune attack and to prevent or delay the onset of high blood sugar.

Transgenic plants cannot yet produce the amounts of self-antigens that would be needed for a viable vaccine against human diabetes or other autoimmune diseases. But, as is true for infectious diseases, investigators are exploring a number of promising schemes to overcome that and other challenges.

Edible vaccines for combating autoimmunity and infectious diseases have a long way to go before they will be ready for large-scale testing in people. The technical obstacles, though, all seem surmountable. Nothing would be more satisfying than to protect the health of many millions of now defenseless children around the globe. ----------------------------------------------------------

Further Information:

Oral Immunization with a Recombinant Bacterial Antigen Produced in Transgenic Plants. Charles J. Arntzen in Science, Vol. 268, No. 5211, pages 714716; May 5, 1995.

Immunogenicity in Humans of a Recombinant Bacterial Antigen Delivered in a Transgenic Potato. C. O. Tacket et al. in Nature Medicine, Vol. 4, No. 5, pages 607609; May 1998.

A Plant-Based Cholera Toxin B Subunit-Insulin Fusion Protein Protects against the Development of Autoimmune Diabetes. Takeshi Arakawa, Jie Yu, D. K. Chong, John Hough, Paul C. Engen and William H. R. Langridge in Nature Biotechnology, Vol. 16, No. 10, pages 934938; October 1998.

Plant-Based Vaccines for Protection against Infectious and Autoimmune Diseases. James E. Carter and William H. R. Langridge in Critical Reviews in Plant Sciences (in press).

The Author

WILLIAM H. R. LANGRIDGE , a leader in the effort to develop edible vaccines for infectious and autoimmune diseases, is professor in the department of biochemistry and at the Center for Molecular Biology and Gene Therapy at the Loma Linda University School of Medicine. After receiving his Ph.D. in biochemistry from the University of Massachusetts at Amherst in 1973, he conducted genetic research on insect viruses and plants at the Boyce Thompson Institute for Plant Research at Cornell University. In 1987 he moved to the Plant Biotechnology Center of the University of Alberta in Edmonton, and he joined Loma Linda in 1993. ------------------------------------------------------------------------

GROWING NEW ORGANS Sci Am Apr 99

Researchers have taken the first steps toward creating semisynthetic, living organs that can be used as human replacement parts

by David J. Mooney and Antonios G. Mikos

Every day thousands of people of all ages are admitted to hospitals because of the malfunction of some vital organ. Because of a dearth of transplantable organs, many of these people will die. In perhaps the most dramatic example, the American Heart Association reports only 2,300 of the 40,000 Americans who needed a new heart in 1997 got one. Lifesaving livers and kidneys likewise are scarce, as is skin for burn victims and others with wounds that fail to heal. It can sometimes be easier to repair a damaged automobile than the vehicle's driver because the former may be rebuilt using spare parts, a luxury that human beings simply have not enjoyed.

An exciting new strategy, however, is poised to revolutionize the treatment of patients who need new vital structures: the creation of man-made tissues or organs, known as neo-organs. In one scenario, a tissue engineer injects or places a given molecule, such as a growth factor, into a wound or an organ that requires regeneration. These molecules cause the patient's own cells to migrate into the wound site, turn into the right type of cell and regenerate the tissue. In the second, and more ambitious, procedure, the patient receives cells--either his or her own or those of a donor--that have been harvested previously and incorporated into three-dimensional scaffolds of biodegradable polymers, such as those used to make dissolvable sutures. The entire structure of cells and scaffolding is transplanted into the wound site, where the cells replicate, reorganize and form new tissue. At the same time, the artificial polymers break down, leaving only a completely natural final product in the body--a neo-organ. The creation of neo-organs applies the basic knowledge gained in biology over the past few decades to the problems of tissue and organ reconstruction, just as advances in materials science make possible entirely new types of architectural design.

Science-fiction fans are often confronted with the concept of tissue engineering. Various television programs and movies have pictured individual organs or whole people (or aliens) growing from a few isolated cells in a vat of some powerful nutrient. Tissue engineering does not yet rival these fictional presentations, but a glimpse of the future has already arrived. The creation of tissue for medical use is already a fact, to a limited extent, in hospitals across the U.S. These groundbreaking applications involve fabricated skin, cartilage, bone, ligament and tendon and make musings of "off-the-shelf" whole organs seem less than far-fetched.

Indeed, evidence abounds that it is at least theoretically possible to engineer large, complex organs such as livers, kidneys, breasts, bladders and intestines, all of which include many different kinds of cells. The proof can be found in any expectant mother's womb, where a small group of undifferentiated cells finds the way to develop into a complex individual with multiple organs and tissues with vastly different properties and functions. Barring any unforeseen impediments, teasing out the details of the process by which a liver becomes a liver, or a lung a lung, will eventually allow researchers to replicate that process.

A Pinch of Protein

Cells behave in predictable ways when exposed to particular biochemical factors. In the simpler technique for growing new tissue, the engineer exposes a wound or damaged organ to factors that act as proponents of healing or regeneration. This concept is based on two key observations, in bones and in blood vessels.

In 1965 Marshall R. Urist of the University of California at Los Angeles demonstrated that new, bony tissue would form in animals that received implants of powdered bone. His observation led to the isolation of the specific proteins (the bone morphogenetic proteins, or BMPs) responsible for this activity and the determination of the DNA sequences of the relevant genes. A number of companies subsequently began to produce large quantities of recombinant human BMPs; the genes coding for BMPs were inserted into mammalian cell lines that then produced the proteins.

Various clinical trials are under way to test the ability of these bone growth promoters to regenerate bony tissue. Applications of this approach that are currently being tested include healing acute bone fractures caused by accidents and boosting the regeneration of diseased periodontal tissues. Creative BioMolecules in Hopkinton, Mass., recently completed clinical trials showing that BMP-7 does indeed help heal severe bone fractures. This trial followed 122 patients with leg fractures in which the sections failed to rejoin after nine months. Patients whose healing was encouraged by BMP-7 did as well as those who received a surgical graft of bone harvested from another part of their body.

A critical challenge in engineering neo-organs is feeding every cell. Tissues more than a few millimeters thick require blood vessels to grow into them and supply nutrients. Fortunately, investigations by Judah Folkman have shown that cells already in the body can be coaxed into producing new blood vessels. Folkman, a cancer researcher at Harvard Medical School's Children's Hospital, recognized this possibility almost three decades ago in studies aimed, ironically, at the prevention of cellular growth in the form of cancerous tumors.

Folkman perceived that developing tumors need to grow their own blood vessels to supply themselves with nutrients. In 1972 he proposed that specific molecules could be used to inhibit such vessel growth, or angiogenesis, and perhaps starve tumors. (This avenue of attack against cancer became a major news story in 1998.) Realizing that other molecules would undoubtedly abet angiogenesis, he and others have subsequently identified a number of factors in each category.

That work is now being exploited by tissue engineers. Many angiogenesis-stimulating molecules are commercially available in recombinant form, and animal studies have shown that such molecules promote the growth of new blood vessels that bypass blockages in, for example, the coronary artery. Small-scale trials are also under way to test this approach in the treatment of similar conditions in human subjects.

Scientists must surmount a few obstacles, however, before drugs that promote tissue and organ formation become commonplace. To date, only the factors responsible for bone and blood vessel growth have been characterized. To regenerate other organs, such as a liver, for example, the specific molecules for their development must be identified and produced reliably.

An additional, practical issue is how best to administer the substances that would shape organ regeneration. Researchers must answer these questions: What specific concentrations of the molecules are needed for the desired effect? How long should the cells be exposed? How long will the factors be active in the body? Certainly multiple factors will be needed for complex organs, but when exactly in the development of the organ does one factor need to replace another? Controlled drug-delivery technology such as transdermal patches developed by the pharmaceutical industry will surely aid efforts to resolve these concerns.

In particular, injectable polymers may facilitate the delivery of bioactive molecules where they are needed, with minimal surgical intervention. Michael J. Yaszemski of the Mayo Clinic, Alan W. Yasko of the M. D. Anderson Cancer Center in Houston and one of us (Mikos) are developing new injectable biodegradable polymers for orthopedic applications. The polymers are moldable, so they can fill irregularly shaped defects, and they harden in 10 to 15 minutes to provide the reconstructed skeletal region with mechanical properties similar to those of the bone they replace. These polymers subsequently degrade in a controlled fashion, over a period of weeks to months, and newly grown bone fills the site.

We have also been studying the potential of injectable, biodegradable hydrogels--gelatinlike, water-filled polymers--for treating dental defects, such as poor bonding between teeth and the underlying bone, through guided bone regeneration. The hydrogels incorporate molecules that both modulate cellular function and induce bone formation; they provide a scaffold on which new bone can grow, and they minimize the formation of scar tissue within the regenerated region.

An intriguing variation of more conventional drug delivery has been pioneered by Jeffrey F. Bonadio, Steven A. Goldstein and their co-workers at the University of Michigan. (Bonadio is now at Selective Genetics in San Diego.) Their approach combines the concepts of gene therapy and tissue engineering. Instead of administering growth factors directly, they insert genes that encode those molecules. The genes are part of a plasmid, a circular piece of DNA constructed for this purpose. The surrounding cells take up the DNA and treat it as their own. They turn into tiny factories, churning out the factors coded for by the plasmid. Because the inserted DNA is free-floating, rather than incorporated into the cells' own DNA, it eventually degrades and the product ceases to be synthesized. Plasmid inserts have successfully promoted bone regrowth in animals; the duration of their effects is still being investigated.

One of us (Mooney), along with Lonnie D. Shea and our other aforementioned Michigan colleagues, recently demonstrated with animals that three-dimensional biodegradable polymers spiked with plasmids will release that DNA over extended periods and simultaneously serve as a scaffold for new tissue formation. The DNA finds its way into adjacent cells as they migrate into the polymer scaffold. The cells then express the desired proteins. This technique makes it possible to control tissue formation more precisely; physicians might one day be able to manage the dose and time course of molecule production by the cells that take up the DNA and deliver multiple genes at various times to promote tissue formation in stages.

A Dash of Cells

Promoting tissue and organ development via growth factors is obviously a considerable step forward. But it pales in comparison to the ultimate goal of the tissue engineer: the creation from scratch of whole neo-organs. Science fiction's conception of prefabricated "spare parts" is slowly taking shape in the efforts to transplant cells directly to the body that will then develop into the proper bodily component. The best way to sprout organs and tissues is still to rely on the body's own biochemical wisdom; the appropriate cells are transferred, in a three-dimensional matrix, to the desired site, and growth unfolds within the person or organism rather than in an external, artificial environment. This approach, pioneered by Ioannis V. Yannas, Eugene Bell and Robert S. Langer of the Massachusetts Institute of Technology, Joseph P. Vacanti of Harvard Medical School and others in the 1970s and 1980s, is now actually in use in some patients, notably those with skin wounds or cartilage damage.

The usual procedure entails the multiplication of isolated cells in culture. These cells are then used to seed a matrix, typically one consisting of synthetic polymers or collagen, the protein that forms the natural support scaffolding of most tissues. In addition to merely delivering the cells, the matrix both creates and maintains a space for the formation of the tissue and guides its structural development. Once the developmental rules for a given organ or tissue are fully known, any of those entities could theoretically be grown from a small sample of starter cells. (A sufficient understanding of the developmental pathways should eventually allow the transfer of this procedure from the body to the laboratory, making true off-the-shelf organs possible. A surgeon could implant these immediately in an emergency situation--an appealing notion, because failing organs can quickly lead to death--instead of waiting weeks or months to grow a new organ in the laboratory or to use growth factors to induce the patient's own body to grow the tissues.)

In the case of skin, the future is here. The U.S. Food and Drug Administration has already approved a living skin product--and others are now in the regulatory pipeline. The need for skin is acute: every year 600,000 Americans suffer from diabetic ulcers, which are particularly difficult to heal; another 600,000 have skin removed to treat skin cancer; and between 10,000 and 15,000 undergo skin grafts to treat severe burns.

The next tissue to be widely used in humans will most likely be cartilage for orthopedic, craniofacial and urological applications. Currently available cartilage is insufficient for the half a million operations annually in the U.S. that repair damaged joints and for the additional 28,000 face and head reconstructive surgeries. Cartilage, which has low nutrient needs, does not require growth of new blood vessels--an advantage for its straightforward development as an engineered tissue.

Genzyme Tissue Repair in Cambridge, Mass., has received FDA approval to engineer tissues derived from a patient's own cells for the repair of traumatic knee-cartilage damage. Its procedure involves growing the patient's cells in the lab, harvested from the same knee under repair when possible, and then implanting those cells into the injury. Depending on the patient and the extent of the defect, full regeneration takes between 12 and 18 months. In animal studies, Charles A. Vacanti of the University of Massachusetts Medical School in Worcester, his brother, Joseph Vacanti, Langer and their colleagues have shown that new cartilage can be grown in the shapes of ears, noses and other recognizable forms.

The relative ease of growing cartilage has led Anthony J. Atala of Harvard Medical School's Children's Hospital to develop a novel approach for treating urological disorders such as incontinence. Reprogenesis in Cambridge, Mass., which supports Atala's research, is testing whether cartilage cells can be removed from patients, multiplied in the laboratory and used to add bulk to the urethra or ureters to alleviate urinary incontinence in adults and bladder reflux in children. These conditions are often caused by a lack of muscle tone that allows urine to flow forward unexpectedly or, in the childhood syndrome, to back up. Currently patients with severe incontinence or bladder reflux may undergo various procedures, including complex surgery. Adults sometimes receive collagen that provides the same bulk as the cartilage implant, but collagen eventually degrades. The new approach involves minimally invasive surgery to deliver the cells and grow the new tissue.

Walter D. Holder, Jr., and Craig R. Halberstadt of Carolinas Medical Center in Charlotte, N.C., and one of us (Mooney) have begun to apply such general tissue-engineering concepts to a major women's health issue. We are attempting to use tissue from the legs or buttocks to grow new breast tissue, to replace that removed in mastectomies or lumpectomies. We propose to take a biopsy of the patient's tissue, isolate cells from this biopsy and multiply these cells outside the body. The woman's own cells would then be returned to her in a biodegradable polymer matrix. Back in the body, cell growth and the deterioration of the matrix would lead to the formation of completely new, natural tissue. This process would create only a soft-tissue mass, not the complex system of numerous cell types that makes up a true breast. Nevertheless, it could provide an alternative to current breast prostheses or implants.

Optimism for the growth of large neo-organs of one or more cell types has been fueled by success in several animal models of human diseases. Mikos recently demonstrated that new bone tissue can be grown by transplanting cells taken from bone marrow and growing them on biodegradable polymers. Transplantation of cells to skeletal defects makes it possible for cells to produce factors locally, thus offering a new means of delivery for growth-promoting drugs.

Recipes for the Future

In any system, size imposes new demands. As previously noted, tissues of any substantial size need a blood supply. To address that requirement, engineers may need to transplant the right cell types together with drugs that spur angiogenesis. Molecules that promote blood vessel growth could be included in the polymers used as transplant scaffolds. Alternatively, we and others have proposed that it may be possible to create a blood vessel network within an engineered organ prior to transplantation by incorporating cells that will become blood vessels within the scaffold matrix. Such engineered blood vessels would then need only to connect to surrounding vessels for the engineered tissue to develop a blood supply.

In collaboration with Peter J. Polverini of Michigan, Mooney has shown that transplanted blood vessel cells will indeed form such connections and that the new vessels are a blend of both implanted and host cells. But this technique might not work when transplanting engineered tissue into a site where blood vessels have been damaged by cancer therapy or trauma. In such situations, it may be necessary to propagate the tissue first at another site in the body where blood vessels can more readily grow into the new structure. Mikos collaborates with Michael J. Miller of the M. D. Anderson Cancer Center to fabricate vascularized bone for reconstructive surgery using this approach. A jawbone, for instance, could be grown connected to a well-vascularized hipbone for an oral cancer patient who has received radiation treatments around the mouth that damaged the blood supply to the jawbone.

On another front, engineered tissues typically use biomaterials, such as collagen, that are available from nature or that can be adapted from other biomedical uses. We and others, however, are developing new biodegradable, polymeric materials specific to this task. These materials may accurately determine the size and shape of an engineered tissue, precisely control the function of cells in contact with the material and degrade at rates that optimize tissue formation.

Structural tissues, such as skin, bone and cartilage, will most likely continue to dominate the first wave of success stories, thanks to their relative simplicity. The holy grail of tissue engineering, of course, remains complete internal organs. The liver, for example, performs many chemical reactions critical to life, and more than 30,000 people die every year because of liver failure. It has been recognized since at least the time of the ancient Greek legend of Prometheus that the liver has the unique potential to regenerate partially after injury, and tissue engineers are now trying to exploit this property of liver cells.

A number of investigators, including Joseph Vacanti and Achilles A. Demetriou of Cedars-Sinai Medical Center in Los Angeles, have demonstrated that new liverlike tissues can be created in animals from transplanted liver cells. We have developed new biomaterials for growing liverlike tissues and shown that delivering drugs to transplanted liver cells can increase their growth. The new tissues grown in all these studies can replace single chemical functions of the liver in animals, but the entire function of the organ has not yet been replicated.

H. David Humes of Michigan and Atala are using kidney cells to make neo-organs that possess the filtering capability of the kidney. In addition, recent animal studies by Joseph Vacanti's group have demonstrated that intestine can be grown--within the abdominal cavity--and then spliced into existing intestinal tissue. Human versions of these neointestines could be a boon to patients suffering from short-bowel syndrome, a condition caused by birth defects or trauma. This syndrome affects overall physical development because of digestion problems and subsequent insufficient nutrient intake. The only available treatment is an intestinal transplant, although few patients actually get one, again because of the extreme shortage of donated organs. Recently Atala has also demonstrated in animals that a complete bladder can be formed with this approach and used to replace the native bladder.

Even the heart is a target for regrowth. A group of scientists headed by Michael V. Sefton at the University of Toronto recently began an ambitious project to grow new hearts for the multitude of people who die from heart failure every year. It will very likely take scientists 10 to 20 years to learn how to grow an entire heart, but tissues such as heart valves and blood vessels may be available sooner. Indeed, several companies, including Advanced Tissue Sciences in La Jolla, Calif., and Organogenesis in Canton, Mass., are attempting to develop commercial processes for growing these tissues.

Prediction, especially in medicine, is fraught with peril. A safe way to prophesy the future of tissue engineering, however, may be to weigh how surprised workers in the field would be after being told of a particular hypothetical advance. Tell us that completely functional skin constructs will be available for most medical uses within five years, and we would consider that reasonable. Inform us that fully functional, implantable livers will be here in five years, and we would be quite incredulous. But tell us that this same liver will be here in, say, 30 years, and we might nod our heads in sanguine acceptance--it sounds possible. Ten millennia ago the development of agriculture freed humanity from a reliance on whatever sustenance nature was kind enough to provide. The development of tissue engineering should provide an analogous freedom from the limitations of the human body.

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Vaccines at Work

The merits of genetic immunization become most apparent when the actions of traditional vaccines are understood. Traditional preparations consist primarily of a killed or a weakened version of a pathogen (disease-causing agent) or of some piece (subunit) of the agent. As is true of most genetic vaccines under study, standard types aim to prime the immune system to quash dangerous viruses, bacteria or parasites quickly, before the pathogens can gain a foothold in the body. They achieve this effect by tricking the immune system into behaving as if the body were already beset by a microorganism that was multiplying unabated and damaging tissues extensively.

When responding to a real infection, the immune system homes in on foreign antigen--substances (usually proteins or protein fragments) that are produced uniquely by the causative agent and not by a host. Two major arms can come into play, both of which receive critical help from white blood cells known as helper T lymphocytes. The humoral arm, led by B lymphocytes, acts on pathogens that are outside cells. These B cells secrete antibody molecules that latch onto infectious agents and thereby neutralize them or tag them for destruction by other parts of the immune system. The cellular arm, spearheaded by cytotoxic (killer) T lymphocytes, eradicates pathogens that colonize cells. Infected cells display bits of their attacker's proteins on the cell surface in a particular way. When cytotoxic T lymphocytes "see" those flags, they often destroy the cells--and the infiltrators within.

Beyond eliminating invaders, activation of the immune system against a specific pathogen leads to the creation of memory cells that can repel the same pathogens in the future. Vaccines confer protection by similarly inducing immune responses and the consequent formation of memory cells.

But standard vaccines vary in the kind and duration of security they provide. Those based on killed pathogens (such as the hepatitis A and the injected, or Salk, polio vaccines) or on antigens isolated from disease-causing agents (such as the hepatitis B subunit vaccine) cannot make their way into cells. They therefore give rise to primarily humoral responses and do not activate killer T cells. Such responses are ineffective against many microorganisms that infiltrate cells. Also, even when nonliving preparations do block disease, the protection often wears off after a time; consequently, recipients may need periodic booster shots.

Attenuated live vaccines, usually viruses, do enter cells and make antigens that are displayed by the inoculated cells. They thus spur attack by killer T lymphocytes as well as by antibodies. That dual activity is essential for blocking infection by many viruses and for ensuring immunity when investigators do not know whether a humoral immune response would be sufficient by itself. What is more, live vaccines--such as the measles, mumps, rubella, oral polio (Sabin) and smallpox types--frequently confer lifelong immunity. For those reasons, they are considered the "gold standard" of existing vaccines.

Live vaccines can be problematic in their own way, however. Even they can fail to shield against some diseases. Those that work can cause full-blown illness in people whose immune system is compromised, as in cancer patients undergoing chemotherapy, AIDS sufferers and the elderly. Such individuals may also contract disease from healthy people who have been inoculated recently. Moreover, weakened viruses can at times mutate in ways that restore virulence, as has happened in some monkeys given an attenuated simian form of HIV, the virus that causes AIDS. For some diseases, the risks of reversion to virulence are intolerable.

Whole-organism vaccines, whether live or dead, have other drawbacks as well. Being composed of complete pathogens, they retain molecules that are not involved in evoking protective immunity. They can also include contaminants that are unavoidable by-products of the manufacturing process. Such extraneous substances sometimes trigger allergic or other disruptive reactions.

The Best of All Worlds

Genetic vaccines are quite different in structure from traditional ones. The most studied consist of plasmids--small rings of double-stranded DNA originally derived from bacteria but totally unable to produce an infection. The plasmids used for immunization have been altered to carry genes specifying one or more antigenic proteins normally made by a selected pathogen; at the same time, they exclude genes that would enable the pathogen to reconstitute itself and cause disease.

The vaccines usually are delivered by injection or by a device known as a gene gun. Injection, commonly into muscle, puts genes directly into some cells and also leads to uptake by cells in the vicinity of the inserted needle. The gene gun propels plasmids into cells near the surface of the body--typically those of the skin or mucous membranes. Once inside cells, some of the recombinant plasmids make their way to the nucleus and instruct the cell to synthesize the encoded antigenic proteins. Those proteins can elicit humoral (antibody-type) immunity when they escape from cells, and they can elicit cellular (killer-cell) immunity when they are broken down and properly displayed on the cell surface (just as occurs when cells harbor an active pathogen).

Such features raise hopes that, once perfected for use in people, DNA vaccines will preserve all the positive aspects of existing vaccines while avoiding their risks. In addition to activating both arms of the immune system, they will be unable to cause infection, because they will lack the genes needed for a pathogen's replication. As a bonus, they are easy to design and to generate in large quantities using now commonplace recombinant DNA technology, and they are as stable as other vaccines (perhaps more so) when stored. They should therefore be relatively inexpensive to manufacture and to distribute widely. Further, because they can be engineered to carry genes from different strains of a pathogen, they can potentially provide immunity against several strains at once, something that should be very helpful when the microorganism is highly variable, as in the case of influenza viruses and HIV.

Some investigators are testing vaccines composed of RNA, a single-stranded relative of DNA. RNA in cells leads readily to synthesis of any encoded proteins. RNA, however, is less stable than DNA, a property that can be problematic for vaccine manufacture and distribution. These difficulties are probably surmountable. Nevertheless, because RNA vaccines have been studied much less extensively than the DNA types, we will concentrate our discussion on DNA vaccines.

Lemonade from Lemons

The idea that genes might serve as vaccines grew in part out of research begun almost half a century ago. In the 1950s and 1960s experiments unrelated to vaccine development showed that delivery of genetic material into an animal's cells could trigger some synthesis of the encoded proteins as well as of antibodies targeted against those proteins. Thereafter, workers occasionally assessed antibody manufacture as an easy way to demonstrate that a given gene was generating a protein.

In the 1970s and early 1980s the ability of inserted genes to prompt an immune response gained attention from other researchers, this time as a disappointing phenomenon. Scientists trying to develop gene therapy (the delivery of genes to correct inherited and other disorders) noted that proteins made from therapeutic genes were sometimes destroyed in animals receiving the genes. The reason: an immune reaction to unfamiliar proteins.

By the early 1990s a handful of laboratories had begun exploring whether the unwanted immune responses to the protein products of foreign genes might be put to good use--for vaccination. Many others were dubious at first, skeptical, for instance, that the immunity elicited would be strong enough to spare people from infection by a living pathogen.

Yet in 1992 a cluster of animal studies done by independent groups demonstrated resoundingly that the concept was sound. Those groups included teams led by Stephen A. Johnston of the University of Texas Southwestern Medical Center in Dallas; by Philip Felgner of Vical in San Diego and Margaret Liu, then at Merck in West Point, Pa.; by Harriet L. Robinson, then at the University of Massachusetts; and by one of us (Weiner) at the University of Pennsylvania.

Collectively, those studies and a host of others conducted over the next few years revealed that DNA vaccines delivered into cells could stimulate the immune system of rodents and primates to generate B cell, cytotoxic T cell and helper T cell responses against many different pathogens and even against certain cancers. The research showed as well that immune responses and disease protection could be elicited when different routes of administration were used. The responses, moreover, could be enhanced by a variety of methods for facilitating DNA uptake by cells.

Since the mid-1990s many more researchers have turned their attention to DNA vaccines, and the technology has advanced to the first rung of human trials, focused on safety. The earliest trial began in 1995, when plasmids containing HIV genes were delivered to patients already infected by that virus. Bigger trials initiated in 1996 made history in another way. For the first time, physicians put new genes (coding for HIV or influenza proteins) into healthy people, instead of into those afflicted by some disorder.

So far human tests are examining vaccines designed to prevent various infections (by HIV, herpes, influenza, hepatitis B and Plasmodium--the parasite responsible for malaria), to bolster the impaired immunity of patients already infected with HIV and to treat a number of cancers (among them lymphomas and malignancies of the prostate and colon). Although cancer is not an infectious disease, much evidence indicates that harnessing the body's immune defenses may help combat it.

The safety trials ask such questions as, are the plasmids toxic, and does DNA delivered as a drug incite an immune response against the body's own DNA? Encouragingly, the studies have not identified any serious side effects to date.

Such trials do not assess disease prevention or amelioration, but many are monitoring the vaccines' effects on the immune system. Preliminary findings hint that useful immune responses can be achieved. Notably, HIV vaccines have generated both humoral and cellular responses; plasmids bearing Plasmodium antigens have evoked significant cellular immune responses; and a vaccine against hepatitis B has resulted in levels of antibodies that should be high enough to prevent infection. In common with traditional vaccines, though, current genetic approaches will probably have to be combined in many cases with generalized immune stimulators (adjuvants) in order to elicit the strong immune responses required to shield recipients from future infections.

How Do the Vaccines Work?

As clinical trials continue, bench scientists are seeking deeper insight into exactly how genetic immunization stimulates immunity, especially by the often crucial cellular arm of the defensive system. A detailed understanding should offer clues to enhancing effectiveness.

In truth, for many years immunologists faced a paradox. DNA vaccines obviously activated killer T cells. Yet simply putting DNA into skin or muscle cells and prompting those cells to display fragments of the encoded antigens should not have produced that outcome. Before such display can activate cytotoxic T cells, the killers must be primed, or switched on, in part by interacting in a specific way with what are called "professional" antigen-presenting cells. In particular, the T cells must bind to the same antigenic fragments they will detect on inoculated nonimmune cells (such as muscle) and, simultaneously, to a second, co-stimulatory molecule (a "second signal") ordinarily found only on antigen-presenting cells.

At one time, biologists thought DNA vaccines had no way of getting into antigen-presenting cells and therefore that those cells had no way of synthesizing and displaying the antigens encoded by those vaccines. Recent discoveries by several groups have shown, however, that the original view was mistaken. Some of the plasmids do in fact make their way into professional antigen- presenting cells. These cells then display antigens alongside the critical co-stimulatory molecules and help to prepare the T cells for action [see illustration on next two pages]. Such findings indicate that to induce a powerful cellular immune response, DNA vaccines must be delivered in a way that will yield good uptake by antigen-presenting cells, not only by other cell types.

Separate work suggests that the plasmid DNA surrounding antigenic genes is more than a mere gene-delivery vehicle; it strengthens the immune response evoked by the antigens. This effect apparently stems from the high frequency of CG sequences in plasmids. Each strand in the DNA double helix is built from units called nucleotides that are distinguished by the bases they contain--either adenine (A), cytosine (C), guanine (G) or thymine (T). Plasmid DNA, derived from bacteria, has a greater frequency of CG sequences than does the DNA in vertebrates. Moreover, the CG units in bacterial plasmids tend to have no methyl group attached, whereas those in vertebrates generally are methylated.

Investigators have proposed that the vertebrate body interprets a high frequency of unmethylated CG pairs as a danger signal. In response, a relatively primitive part of the immune system (one not dependent on antigen recognition) attempts to destroy or wall off the foreign intruder.

Engineering for Optimal Effect

A long with analyzing the natural behavior of genetic vaccines in the body, immunologists are looking ahead, exploring ideas for increasing overall immune reactivity and for optimizing the ratio of cellular to humoral responses. One proposal for amplifying responsiveness has emerged from studying the DNA around CG sequences.Researchers have demonstrated that plasmid DNA yields the most potent immune response when CG sequences are flanked by two purines (adenine or guanine) to their "C" side and two pyrimidines (thymine or cytosine) to their "G" side. In mice, plasmids containing such "immunostimulatory sequences" induced more vigorous antibody and cytotoxic T cell activity than did an otherwise identical vaccine. Hence, increasing the number of immunostimulatory sequences in plasmids might well amplify the immunogenicity of the antigenic codes in a DNA vaccine.

A different approach is incorporating genes for signaling molecules called cytokines into antigen-carrying plasmids or into separate plasmids. Cells of the immune system release these molecules to regulate their own, and one another's, activities. As an example, a molecule named granulocyte-macrophage colony-stimulating factor stimulates the proliferation of antigen-presenting cells, among other actions. Inclusion of its gene has been shown to boost overall responses to DNA vaccines.

To ensure that genetic vaccines trigger a strong cellular response when needed, researchers are experimenting specifically with genes for cytokines that are known to promote killer-cell activity. In mice, scientists have found that helper T cells called Th1 cells secrete cytokines that favor cellular responses at the expense of humoral (antibody) ones, whereas other helper cells (Th2 cells) secrete cytokines that favor humoral activity. In humans, helper T cells seem to come in more varieties, but a preponderance of Th1-type cytokines still promotes a cellular response, and a preponderance of Th2-type cytokines stimulates a humoral response.

One such project showed that a vaccine including genes for HIV antigens and for interleukin-12 (a classic Th1 cytokine) reduced production of anti-HIV antibodies in mice and markedly enhanced the responsiveness of cytotoxic T cells to HIV antigens. This bias toward a cellular response is particularly encouraging, because recent findings by HIV researchers indicate that a potent killer T cell response to HIV is critically important for combating HIV replication.

Genes for substances known as chemokines might be incorporated as well. Chemokines are small molecules that attract both antigen-presenting cells and T cells to damaged or infected tissues. Like cytokines, these substances differ in the mix of cells on which they act and in the precise effects they exert. As their individual actions are better understood, carefully combining specific chemokine genes with selected cytokine genes could go far toward customizing both the type and the extent of immune responses elicited.

DNA vaccines could in theory even sidestep the need for classical antigen-presenting cells to prime cytotoxic T cells. If a gene for an antigen were bundled with a gene for a co-stimulatory molecule normally made by an antigen-presenting cell, then inoculated skin, muscle or other cells would themselves display both the antigen and the crucial "second signal," thereby facilitating both the priming and the activation of cytotoxic T cells.

Getting from Here to There

If first-generation genetic vaccines do well in clinical trials, they may sometimes be combined initially with more traditional vaccines to achieve even better effects. Let us say, for example, that a subunit vaccine (consisting of a protein) evoked a good antibody response against a pathogen but that a cellular response was needed as well. Meanwhile a new DNA vaccine proved able to induce a cellular response but did not excite an ideal antibody response. In a so-called prime-boost strategy, physicians might deliver the DNA vaccine and then boost the antibody response by later delivering the subunit vaccine as well. Eventually, though, as vaccine makers learn how to optimize responses to genetic immunization (such as through the techniques described above), manufacturers may be able to achieve the needed effects by constructing genetic vaccines alone.

As the exciting, futuristic possibilities of genetic immunization are being considered, those of us who are captivated by this technology also have to roll up our sleeves and grapple with a great many details. For instance, most DNA vaccines stop yielding much protein after about a month. Would finding a way to extend plasmid survival lead to stronger immunity, or would it backfire and encourage attacks against unvaccinated, healthy tissue? How long does immunity last in human beings? How much do people vary in their responses? Which doses are most effective and what kinds of delivery schedules are best? We also need to know which substances are most useful for targeting genetic material to specific cells (including to antigen-presenting cells) and for enhancing the cellular uptake of plasmids. And which genes, out of the sometimes thousands, in a given pathogen should be selected for maximal power?

Clinical trials answering these questions and assessing the effectiveness of the first generation of DNA vaccines may not be completed for five or 10 years. Whether those specific versions reach the market, though, genetic immunization technologies are likely to prove extremely valuable for research into the basic biology of the immune response and for the design of even better vaccines.

Vaccine makers today often have little idea of which components of the immune system need to be activated most strongly against a given pathogen and which antigens and other substances can achieve that stimulation. Now, however, they can readily mix and match antigenic and other genes (such as those for cytokines and chemokines) in experimental DNA vaccines and compare the success of different combinations in small animals quite quickly. In that way, they can simultaneously gain a handle on the immune responses that are needed for protection and on the antigens and other proteins that can generate them. As part of this testing, some researchers are creating "libraries" of a pathogen's genes; an individual library contains every gene in the organism, with each gene spliced into its own plasmid. They then deliver subsets of such libraries to animals, which are also exposed to the live pathogen. Next, they identify the subsets that work best, further subdivide the groups and do more testing, until the most useful mix of antigens emerges.

As the years go by, the inherent manipulability of DNA should make it a vehicle of choice for teasing apart the body's complex immune responses to different disease-causing agents. With such information in hand, vaccine makers should be able to design vaccines that will channel immune responses down selected pathways. In the past, manufacturers had no way to custom-tailor their products easily and inexpensively. In the future, such "rationally" designed genetic vaccines are likely to provide new immune therapies for cancer and powerful ways to prevent or minimize any number of devilish infections that elude human control today.