A "naked DNA" vaccine for malaria has promoted an immune response in healthy volunteers (Science 282:476, 1998), challenging skepticism that DNA alone injected into muscle can mobilize cytotoxic T (CD8+) lymphocytes (CTLs) to kill such intracellular parasites as Plasmodium falciparum, the cause of malaria. As well as serving as a proof of principle for "naked DNA," the results are an important step in the fight against malaria, for which there is no vaccine.

The purpose of the study—a collaborative effort between Vical (San Diego, CA), the Naval Medical Research Center (NMRC, Bethesda, MD), and Pasteur Mérieux Connaught (Swiftwater, PA)—was to determine if immunization with DNA alone was safe, well-tolerated, and immunogenic in normal humans, says lead investigator Stephen Hoffman, director of the NMRC malaria program.

"Naked DNA" is, in essence, a plasmid loop that contains the relevant coding and control regions to allow the expression of a pathogen gene inside human cells. The DNA is also "naked" in the sense that it is delivered without the aid of vehicles such as liposomes or virus vectors. Direct intramuscular injection of naked DNA evoked a dose-dependent CTL response in 11 of 17 healthy human volunteers. As required by the US Food and Drug Administration (Rockville, MD), the vaccine incorporated only one gene from the malaria pathogen, which is not sufficient to confer full immunity in disease-naïve individuals but is adequate to test whether it could induce antigen-specific CD8+ T-cell responses.

There are at least seven other malaria vaccines in development, but this is the first example of the use of naked DNA for this disease. Many vaccine experts, including David Weiner of the University of Pennsylvania (Philadelphia) and Stephen Johnston of the University of Texas Southwest Medical Center (Dallas), believe that naked DNA is the best approach to use with the malaria parasite as well as other infectious agents because it can mobilize CTLs. "With an extremely complex life cycle, and the 6,000 genes of the malaria parasite, there is no other technology as well suited to dealing with malaria," asserts Hoffman, who is part of the multicenter team that recently determined the sequence of chromosome 2 of P. falciparum (Science 282:1126, 1998).

Many consider DNA vaccines to be conceptually third-generation technology, says Shaefer Price, CEO of PowderJect Vaccines (Madison, WI). PowderJect signed a $321 million deal in March with Glaxo Wellcome (London) for gene-gun DNA vaccine development (Nat. Biotechnol. 16:316, 1998), initially for hepatitis B virus infections. The first-generation vaccines were attenuated live pathogens. They could mobilize CTLs but carried a risk of infection, says Price. The second-generation vaccines are much safer and based on killed pathogens or their purified polymeric components. However, such vaccines usually are not effective in producing cellular immunity (they produce only antibodies), and patients need multiple doses. The third-generation vaccines consist of a pathogen's isolated DNA that functions as part of the intracellular production machinery encoding particular antigens presented to the immune system via the major histocompatibility class I pathway, which produces CTLs.

"DNA vaccines are particularly well-suited to deal with viral diseases where other live vaccines have failed or cannot be constructed due to fear of infection," says Richard Ciccarelli, vice president of vaccines at Wyeth-Lederle's (Radnor, PA, part of American Home Products). Wyeth-Lederle has invested much in DNA vaccines, especially for the treatment of sexually transmitted diseases, says Ciccarelli. To that end, in April 1998, Wyeth-Lederle acquired Apollon (Malvern, PA) for an undisclosed sum, gaining three vaccines, now in phase I/II development, against HIV, herpes simplex, and human papillomavirus. The company is also investigating the use of viral and nonviral vectors to improve the vaccines' effect.

Margaret Liu, vice president of gene therapy and vaccine research at Chiron (Emeryville, CA), once called the ability to generate CTLs without using a live vector "the immunologist's grail." Until recently, she says, vaccine investigators measured only antibody response because they believed that it was impossible to mobilize CTLs in any significant quantity. Since HIV, scientists have come to understand the importance of the cellular response, and have begun moving away from an "all or nothing" approach to treating infections with vaccines, she adds.

DNA vaccines have other potential advantages, too: They can stimulate long-lived immune responses; they can address several diseases in one vaccine; they are cheap and easy to produce; and they are extremely stable and have no special cold storage requirement. Furthermore, candidate vaccines can be purified from diseased tissue. In addition, both Weiner and Johnston see value in the technology as a research tool to select and test antigens.

However, critics claim (and even some supporters admit) that naked DNA vaccines require too much genetic material, and that the vaccines could be more effective if combined with adjuvants, lipids, or polymers. Consequently, most companies and academic teams working with DNA vaccines—including Vical and Apollon—are also experimenting with a variety of vectors to boost the effect of the DNA and reduce the amount needed.

The next step for NMRC "will be to construct a five-gene vaccine with Vical based on an irradiated sporozoite model we know works, but which is impractical to produce as a vaccine," says Hoffman. The group will begin next summer to vaccinate healthy volunteers with the new vaccine, and then will challenge them with a strain of malaria that is treatable. The Vical/Navy group has worked with Epimune (San Diego, CA) to determine peptides that would make the best T-cell epitopes, says Navy staff scientist William Rogers. The third phase will be the construction of a 15-gene vaccine containing 10 antigens from the blood-stage of malaria infection, and five from the liver-stage. If effective, this could address the anticipated need to develop two distinct vaccines, one for uninfected people such as travelers and military personnel, and another for those from areas of endemic infection, Hoffman says. (The UN and World Bank announced in October Operation Roll Back Malaria, an effort to encourage research by companies, which have largely perceived this area as lacking profit.)

Chiron is working only with HIV and hepatitis C to compare refinements of DNA technology, says Liu. Her group is working on second-generation gene vaccines using alpha (RNA) viruses to improve delivery because "they don't replicate to cause disease like viruses do, but make many copies of themselves and target the follicular dendritic cells in lymph nodes." Chiron has also developed "lipitoids"—polymers, more potent than cationic lipids, that improve delivery and reduce the DNA load needed, she says.