By David N. Leff

One dark night, an unmarked helicopter flies over a large American city and drops a few kilograms of a fine powder. As these aerosolized anthrax spores disperse through the air, thousands or millions of people below inhale them.

In 1993, the U.S. Congressional Office of Technology Assessment estimated that between 130,000 and 3 million deaths could follow the aerosolized release of anthrax spores upwind of the Washington, D.C., area. This lethality, it noted, matched or exceeded that of a hydrogen bomb.

In the mid-1990s, Iraq acknowledged producing and weaponizing anthrax for air-drop and missile delivery. Freelance terrorists have their hand-held version.

¿Inhalation is the most dangerous mode of entry for anthrax spores,¿ observed biochemist John Collier. ¿They have to be in fine enough form to be inhaled all the way to the lungs,¿ he explained. ¿There the microparticles are taken up by macrophages, in which they germinate, become actively dividing bacteria, and then go to the lymph nodes. From these they are able to penetrate very rapidly, and fatally, into the bloodstream.

¿Inhalational anthrax,¿ Collier added, ¿produces flu-like symptoms within two or three days, rapidly progressing to prostration and death.¿ Collier, in the Department of Microbiology and Molecular Genetics at Harvard Medical School, Boston, is senior author of an article in the current issue of Science, dated April 27, 2001. Its title: ¿Dominant-negative mutants of a toxin subunit: An approach to therapy of anthrax [Bacillus anthracis].¿

¿Currently, our only way of treating the disease is with antibiotics,¿ Collier pointed out, adding, ¿Antibiotics are really ineffective if given only after symptoms have appeared. They have to be administered very early on in the infection to have an effect.

¿The symptoms of anthrax,¿ he said, ¿are due to a toxin, which is a multi-subunit protein complex. One of these subunits is called PA ¿ protective antigen ¿ and in the active complex there are seven copies of that subunit. They work together to transport the other two subunits ¿ LF (lethal factor) and EF (edema factor) ¿ which are enzymes ¿ into our cells. There these subunits catalyze reactions that are deleterious to our metabolism.¿

Seven Up, One Down Dominates

¿What we have found,¿ Collier went on, ¿is a way to mutate the PA molecule, the one that has seven units, such that it acquires dominant-negative [DN] properties. That is to say, we hypothesized that if only one molecule of that seven-subunit toxin complex is mutated, it will totally block the action of the whole complex. And having these subunits intermingle in the assembly process with the normal non-mutated subunits that are being produced by the bacteria inactivates the complexes that form. There¿s the potential then, we believe, for administering the DN subunits to an individual who has been exposed to anthrax.

¿DN in this case,¿ Collier explained, ¿means, if you have seven copies of this PA molecule, all seven of them would have to be active. If one of them is mutated, that negativity dominates. PA¿s real function is to form a pore in the plasma membrane that protects the inside of the cell from deleterious external influences. Molecules such as proteins are generally too large to penetrate that barrier. However bacteria, such as B. anthracis, being very clever and having had a long time to evolve virulent mechanisms, have found ways to produce toxic proteins that can breach this barrier.¿

Collier and his co-authors used directed mutagenesis to produce the individual aberrant proteins, particularly PA, in cloned form in Escherichia coli. They then tested these elements in vivo.

¿We used a rat intoxication model,¿ he recounted, ¿in which we injected a specific amount of normal wild-type PA plus an aliquot of the LF. This combination will kill a 250-gram rat in about 90 minutes ¿ after extremely labored breathing and initial prostration. We took that as our constant, then started adding the DN mutants to that mixture before injection. We added equal amounts of the DN mutant PA to the wild-type rats, which totally ablated their symptoms. Even when we added only one-fourth as much DN mutant as in the normal, we observed no symptoms.¿

Fortified by these results, Collier ¿envisions that one of these dominant-negative PA mutants might be developed to the point of being stockpiled in major metropolitan areas, where it could be administered together with antibiotics to individuals who are exposed. And for those who are symptomatic or presymptomatic it would act as an anti-anthrax therapeutic.

¿But, in such an anthrax outbreak,¿ he said, ¿everyone on the ground will be seeking immunization against the toxin. One could give such a vaccine to everyone, including those individuals who had inhaled a small number of spores that might germinate in a delayed fashion some weeks later, and come down with anthrax. Hopefully, by then, their immune response would have kicked in to a sufficient degree to generate a sufficient level of antibody to recognize and neutralize the DN PA antigen, and thus protect the individual against disease.¿

Collier compared his group¿s putative vaccine with the six-shot, seven-year immunizations that the U.S. military is now giving to all active-duty land, sea and air personnel. (See BioWorld Today, July 6, 2000, p. 1).

¿The vaccine that they¿re giving,¿ he observed, ¿is somewhat of a crude mixture, derived from the supernatant fraction of an attenuated anthrax bacterium. Its major components are unmutated PA plus small amounts of EF and LF. Ours would be active both as a therapeutic and as a vaccine.¿

Military To Join In Preclinical Field Trials

He and his co-authors ¿are right now collaborating with the U.S. Army Medical Research Institute of Infectious Diseases, at Ft. Dietrich, Md., to do studies of our vaccine in mouse infection models. We will first need to show efficacy in this animal, and if the DN PA proves as efficacious as we desire, then we will find some way ¿ hopefully with government support ¿ to do human safety trials. You clearly cannot do infection trials in humans,¿ Collier concluded.

Harvard University has applied to patent Collier¿s formulation. A company, PharmAthene Inc., in Cambridge, Mass., co-founded by Collier and other scientists a few weeks ago, will license the inventions.

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