DNA Vaccines Generate Excitement As Human Trials Begin

'ABSOLUTELY AMAZING': Yerkes' Harriet Robinson notes that research in the field has grown at an extraordinarily rapid rate. With more pathogens developing resistance to antibiotics, and with the increasing vulnerability of human populations to infectious diseases, vaccine research has taken on a new urgency. Among the technologies being developed for the vaccines of the 21st century is the novel idea of using naked DNA-a simple loop of DNA containing an antigen gene-to induce an immune respons

By | March 16, 1998

'ABSOLUTELY AMAZING': Yerkes' Harriet Robinson notes that research in the field has grown at an extraordinarily rapid rate.
With more pathogens developing resistance to antibiotics, and with the increasing vulnerability of human populations to infectious diseases, vaccine research has taken on a new urgency. Among the technologies being developed for the vaccines of the 21st century is the novel idea of using naked DNA-a simple loop of DNA containing an antigen gene-to induce an immune response (K.S. Brown, The Scientist, 10[7]:14, April 1, 1996).

These DNA vaccines (some researchers prefer other terms, including "genetic vaccines") offer the hope of vaccines that may be simple to develop, safe and highly effective, and easy to transport and store. Preclinical research on DNA vaccines has been highly promising, according to a 1997 report published by the American Academy of Microbiology (available on the Web at http://www.asmusa.org/acasrc/aca1.htm). And this seems to be one of those rare instances in which researchers regard the funding from both government and private sources to be more than adequate. Yet the question remains, will DNA vaccines work as well in humans as they do in mice?

If so, "they could well be the method of the development of new vaccines for the next century," maintains Harriet L. Robinson, chief of microbiology and immunology at Yerkes Primate Research Center in Atlanta.

'EARLY STAGES': Apollon's Richard Ciccarelli notes that researchers need to determine how DNA vaccines work in humans.
The field has developed rapidly, growing out of two papers that were published in 1992 and 1993 (D. Tang et al., Nature, 356:152-4, 1992; J.B. Ulmer et al., Science, 259:1745-9, 1993). "It's absolutely amazing the number of labs that are using the technology and have written papers," Robinson observes. "I now have two full file drawers of papers that have been published on DNA vaccines, and it's become a very common technique, and not necessarily for use in vaccination." She notes that it's used in studies of basic immune responses, allergies, autoimmune diseases, and cancer, and "just to produce reagents for basic research."

The enthusiasm is understandable. According to Richard B. Ciccarelli, vice president of research and development for Apollon Inc., a Malvern, Pa., company that's conducting clinical trials of several DNA vaccines, "If DNA vaccines are used in specific disease areas that cannot be approached by other types of vaccines . . . they will have a dominant role within the vaccine industry as we go forward into the next century."

Current vaccines use one of three strategies to induce an immune response that will protect against further challenges by the native pathogenic organism. Vaccines can consist of a live but weakened ("attenuated") version of the pathogen, killed pathogens, or purified or genetically engineered proteins or polysaccharides from the pathogen.

In DNA vaccines, on the other hand, a gene for a protein from the pathogen is inserted into a bacterial plasmid. These plasmids are replicated in bacterial culture, purified, and then transferred to the recipient by injection or by a gene gun that shoots tiny plasmid-coated gold beads. The recipient's protein-synthesizing machinery uses the gene to produce an antigen, to which the immune system begins mounting a defense.

DNA vaccines have many potential advantages over traditional methods. For one thing, there's no risk of infection, as there is for live attenuated vaccines. And Robinson notes that they're also superior to protein or polysaccharide vaccines because "proteins are very easily denatured when you purify them . . . whereas a DNA vaccine produces a protein right in the host you're vaccinating, so when it's produced it's right in its native form."

Robert G. Whalen, senior staff investigator at the Centre National de la Recherche Scientifique (CNRS), maintains a Web site called "The DNA Vaccine Web" at http://www.genweb.com/Dnavax/.

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    Many DNA vaccine researchers are members of the American Society for Microbiology (ASM). The American Academy of Microbiology, which published a 1997 report on DNA vaccines, is an honorific leadership group comprising 1,400 fellows within ASM.

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  • Additionally, DNA is very stable, even at temperatures close to boiling, which may have significant advantages for the use of DNA vaccines in developing countries. With traditional vaccines, great attention must be paid to the "cold chain"-the series of refrigerators between the points of production and final distribution. The cold chain may not be necessary with DNA vaccines. Despite the exciting prospect of stable vaccines, some researchers remain a bit skeptical. For example, DNA vaccine pioneer Margaret A. Liu, vice president for vaccines research at Chiron Corp. in Emeryville, Calif., cautions, "It is true that DNA is more stable than a live virus, and it is hoped that we can make a vaccine that is room-temperature stable, which will facilitate vaccines for developing countries, but I'm not sure we're there yet."

    Immunologists seem most excited about the ability of DNA vaccines to cause the immune system to produce cytotoxic T lymphocytes in addition to antibodies. Liu calls the ability to generate cytotoxic T lymphocytes without using a live vector "the immunologist's grail" (M.A. Liu, Proceedings of the National Academy of Sciences, 94:10496-8, 1997). Additionally, vaccinologists may well be able to adjust the type of immune response induced by the vaccines, designing treatments tailored for allergies or for autoimmune disease.

    Another area that's receiving a lot of attention is the technique of producing large libraries of plasmids, each containing a single gene from a pathogen, and using the entire library-or a substantial portion of it-as a kind of combination vaccine.

    Stephen Albert Johnston, a professor of medicine and biochemistry and director of the Center for Biomedical Inventions at the University of Texas Southwestern Medical Center in Dallas, has a $4.3 million grant from the Defense Advanced Research Projects Agency (DARPA) to study genetic vaccine libraries. Johnston prefers to use the term "genetic vaccine," because he believes that "DNA vaccine" can be misinterpreted to mean a vaccine intended to induce an immune response to foreign DNA, not to the foreign protein encoded by that DNA. "Probably over the next five years or so we'll have the [genome] sequences on the table of all the major pathogens in the world," Johnston points out. "So the problem is, how do you translate that sequence into useful things? And what we're trying to set up is an assembly-line system for taking all of the genes out of these genomes that have been sequenced, running them through vaccine tests in animals in a very systematic way, and basically testing all of the genes for their vaccine potential.

    "What we literally can do-and this is partly what DARPA is interested in, in terms of anti-biological warfare stuff-is that we can make a vaccine out of the whole genome at once, shoot that in as one vaccine that represents all the genes of a given pathogen, and make that animal resistant to that pathogen.

    "One of the things that was remarkable to everybody was how little DNA you need to get an immune response," continues Johnston. "We've used as little as 0.25 nanograms of DNA that we shot into a mouse and get a good immune response. We can literally shoot as many as 27,000 different plasmids into a mouse at once and get an immune response to an individual plasmid in that group."

    Researchers note that the effectiveness of DNA vaccines in singular and library forms must be proved in clinical trials on human volunteers. "The painful process of taking a hypothesis into the clinic is happening now," remarks N. Regina Rabinovich, chief of the clinical studies section of the division of microbiology and infectious diseases of the National Institute of Allergy and Infections Diseases (NIAID).

    And it's happening in at least eight institutions conducting trials on an equal number of diseases, including influenza, hepatitis B, malaria, several forms of cancer, and HIV. Apollon, for example, is sponsoring Phase I safety studies on two versions of an HIV vaccine. "We cover close to 80 percent of the HIV genome in these constructs," explains Ciccarelli. "So it's close to a live attenuated HIV vaccine in that we include most of the genes of HIV." However, he notes, because the researchers are using a DNA vector approach, "we don't have to worry about it replicating live attenuated virus." He adds that "we've mutated some of the HIV genes to make them safer. We've deleted out their activities, but we've kept them as effective antigens.

    "We need to understand how these vaccines work in humans, and that work, I would say, is in its very early stages," Ciccarelli continues. "How is the DNA vaccine related to other types of vaccines? What do we need to know about doses in humans and immunization protocols? . . . Are formulations important? Is the concept of DNA delivery to specific cell types important? Those are fairly routine things that the vaccine industry does for all their vaccines, so there's no particular hurdle that's stronger for DNA vaccines than for other types of vaccines. It's just that we have no clinical experience to date suggesting the best path to proceed forward."

    Preventive vaccines are not the only ones under study. For example, Vical Inc., the San Diego-based biopharmaceutical company that owns patents on much of the basic DNA vaccine technology, is studying a therapeutic vaccine trade-named Allovectin that's intended to help individuals fight off malignant melanoma (R. Lewis, The Scientist, 9[7]:15, April 3, 1995).

    COMMERCIAL POTENTIAL: Vical's Alain Schreiber predicts that DNA vaccine products could generate "hundreds of millions of dollars in annual sales."
    According to Alain Schreiber, president and chief executive officer of Vical, initial clinical studies on Allovectin are going well. Although the Phase I study was intended mostly to assess the vaccine's safety, not its effectiveness, "a certain percentage [of subjects] are going into objective clinical partial-and occasionally complete-remissions of good duration." Additionally, the safety profile seems favorable. "There have been no autoimmune phenomena; there has not been any clear evidence that there is a carryover, or residual DNA, or any integration" of the vaccine into the human genome, he says.

    Still, much clinical work remains to be done. Only a few DNA vaccine clinical trials have reached Phase II, and no large-scale Phase III trial has yet begun.

    FISHING FOR IMMUNITY: Oregon State's Jo-Ann Leong, shown with grad student Grant Trobridge and undergrad Kim Trev, is working on a DNA vaccine for a disease affecting salmon and rainbow trout.
    Other organisms besides humans may reap the benefits of DNA vaccines. Work is ongoing on DNA vaccines for several veterinary diseases, including infectious hematopoietic necrosis virus (IHNV), a disease of salmon and rainbow trout that kills between 2 million and 20 million fish annually.

    Workers in the laboratory of Jo-Ann C. Leong, distinguished professor and chairwoman of microbiology at Oregon State University, had previously identified an IHNV glycoprotein that produced immunity in the fish. In constructing their DNA vaccine, they included that protein's gene as well as control sequences that would instruct host cells to properly glycosylate the protein.

    Their preliminary results (E.D. Anderson et al., Molecular Marine Biology and Biotechnology, 5[2];114-22, 1996) have been dramatic. "Let's say that 80 percent of control unvaccinated fish died with a particular dose of virus," observes Leong. "With a recombinant vaccine, in some cases we would find that 20 percent of the vaccinated fish died. When you use the DNA vaccine, only 1 [percent] to 2 percent die. The difference is pretty tremendous."

    Several problems remain to be solved, though. For example, since the virus strikes fish when they're only 1 gram to 10 grams in size, individual vaccine injections clearly would not be practical. "You have to have an easy way to get the DNA in the animal," Leong explains, "so we're trying to microencapsulate the DNA in a capsule so small that it can be taken up by the gill macrophages."

    One indication of the promise of DNA vaccines is that researchers seem to have few complaints about funding, which is coming from a wide variety of sources. NIAID sponsors a good deal of the basic research through its various disease programs. Other support comes from such agencies as the World Health Organization, DARPA, and the United States Department of Agriculture.

    The salutary funding situation is a function of the importance of DNA vaccine research and the fact that the work is still largely preclinical, Rabinovich points out. "Preclinical development is relatively cheap." The expense comes when "you get into pilot production and expanded clinical trials," she says.

    Pharmaceutical firms both large and small seem to be willing to make that investment because of the promise of stupendous returns. According to Schreiber, the current vaccine industry generates $3 billion in annual sales. DNA vaccines will only add to that sum because the technology "probably will not replace vaccines that exist now, but may offer opportunities for diseases that are complex, or where current vaccination technologies are not adequate," he points out. "In that respect, we think it could have a significant commercial windfall for the companies that have pioneered the technology. . . . Most of the products have the potential of generating hundreds of millions of dollars in annual sales."

    Despite all the hoopla over the technology and its scientific and commercial potential, Schreiber cautions, "I don't think the DNA vaccine is a panacea for all infectious diseases. It will find its place, and I think it will find an important place primarily in those diseases in which you need a good component of cell-mediated immunity. . . . So from a commercial standpoint, it's now hard work, major investment, and finding the right places and the right way to use it."

    Robert Finn, a freelance science writer based in Long Beach, Calif., can be reached online at finn@nasw.org.

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