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.
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

'ABSOLUTELY AMAZING':
Yerkes' Harriet Robinson notes that
research in the field has grown at an
extraordinarily rapid rate.


'EARLY STAGES': Apollon's Richard
Ciccarelli notes that researchers need to
determine how DNA vaccines work in
humans.

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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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.
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5,500 members
Executive director: M. Michele
Hogan
President: Charles A. Janeway,
Jr.
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.
American Society for
Microbiology
1325 Massachusetts Ave., N.W.
Washington, D.C. 20005
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Fax: (202) 942-9367
http://www.asmusa.org
43,000 members
President: Stanley Falkow
"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).
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.
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

COMMERCIAL POTENTIAL:
Vical's Alain Schreiber predicts that
DNA vaccine products could
generate "hundreds of millions of
dollars in annual sales."


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.

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.