The Centers for Disease Control and Prevention in Atlanta classify anthrax as a Category A agent, meaning it is considered a high threat to national security due to its ease of transmission, the associated high rate of death or serious illness and its potential for causing "public panic and social disruption."

Certainly, the anthrax postal attacks in 2001 fit into that description, creating fear and significantly disrupting the workings of Congress, as well as several media organizations.

So far, at least, the number of bioterrorism victims cannot hold a candle to those killed by more conventional approaches, such as hijacked jetliners, explosives or even fertilizer plus fuel oil. But the anthrax postal attacks resulted in a total of 22 infections and five deaths, according to CDC figures, and although attempts in other countries have failed altogether - such as the release of anthrax spores by Japanese radicalists Aum Shinrikyo in 1993 - it seems likely such attempts will continue.

Researchers since 2001 have been making good use of their time, with advances reported in multiple areas. The genome of Bacillus anthracis has been sequenced and the 3-dimensional structure of the complex between anthrax toxin and its host-cell receptor determined. There also has been research into therapeutics and vaccine strategies. (See BioWorld Today, Oct. 2, 2003.)

While a vaccine exists - Anthrax Vaccine Adsorbed, or AVA - that is used to vaccinate high-risk individuals, there are concerns about its side effects, as well as its incomplete characterization and complex immunization schedule. Now, in the Sept. 14, 2004, print edition of the Proceedings of the National Academy of Sciences, researchers from San Diego biotechnology company Vical Inc., the Columbus campus of Ohio State University and the Naval Medical Research Center in Silver Spring, Md., report on vaccine progress. Titled "A cationic lipid-formulated plasmid DNA vaccine confers sustained antibody-mediated protection against aerosolized anthrax spores," the paper describes a novel plasmid DNA vaccine that has shown promise in preclinical testing.

Before their published work, the researchers first constructed a variety of vaccines consisting of two genes coding for parts of the anthrax toxin, namely protective antigen (PA) and lethal factor (LF), known as lethal toxin (Letx) when complexed.

"The idea was to use the strengths of DNA vaccines to make a lot of different constructs and test them in mice, and only move forward with the best ones," Gary Hermanson, senior research manager, bacterial and immunogen optimization at Vical and first author of the study, told BioWorld Today.

Those constructs then were used to immunize rabbits, and the group tested their ability to generate an antibody response to the separate anthrax toxin proteins, as well as the Letx complex. The researchers also tested different vaccine formulations. In a separate set of studies, rabbits were exposed to aerosolized anthrax to test survival rates after different types of vaccinations and evidence of sterile immunity - that is, the ability to completely block spore germination.

When antibody titers were measured several weeks after the second immunization, animals in each group generated specific antibodies to either PA or LF, depending on their vaccination. All groups (except controls) generated antibodies to Letx; titers of animals vaccinated with LF pDNA were lower than those of animals vaccinated with either PA pDNA or a combination.

All of the rabbits vaccinated with PA pDNA, either alone or in combination with LF pDNA, survived exposure to aerosolized anthrax, as did all animals vaccinated with AVA. In contrast, about half of the animals vaccinated with LF pDNA alone and all controls died.

Some groups of animals immunized with PA pDNA alone or in combination with LF pDNA showed evidence of sterile immunity, meaning no increase in antibody titer to either PA or LF was observed following exposure to anthrax.

"Really powerful vaccines will prevent the pathogen from entering and infecting at all," Hermanson explained. "Certain of our formulations did not allow the bacteria to gain any sort of foothold."

Animals vaccinated with either AVA or LF showed a significant increase in Letx antibody titer, indicating a less complete immune protection. When PA pDNA or combination vaccinated rabbits were exposed to aerosolized anthrax seven months instead of five weeks after vaccination, they also showed some change in antibody titer, suggesting that the protection afforded by the vaccine declined somewhat over time.

Bivalent Vaccine Could Double Trouble For Anthrax

In the studies reported in PNAS, combining PA and LF pDNA conferred no extra protection when compared to the PA pDNA vaccines alone, in terms of either survival or antibody titer, since the PA vaccine alone already was at ceiling for both measurements. The authors, nevertheless, believe that a combination vaccine has advantages.

"The investigational vaccines that are currently in the clinic are all recombinant PA," David Kaslow, chief scientific officer at Vical and a co-author of the study, told BioWorld Today. "Some evil person could probably figure out how to mutate PA to get around a recombinant vaccine. By including LF in the bivalent vaccine, we include an extra layer of protection."

According to Vical's scientists, the approach of using a DNA vaccine also has several advantages. First, they can be developed quickly.

"Usually, cloning the gene is the first step in vaccine production - it's basically the first and last step for us," Kaslow said. Indeed, the paper reports that the vaccine development project described progressed from initial plasmid design to investigational new drug application allowance in only 28 months. The vaccine now is being evaluated in a Phase I trial.

Kaslow added, "DNA as a molecule is inherently very stable, and so we believe it will perform very well in a stockpile situation."

That's one comforting thought, at least.

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