Biological Terrorism

One warning came in black-and-white in 1993: A U.S. Congressional Office of Technology Assessment report projected that releasing 100 kilograms of aerosolized anthrax spores upwind of the U.S. capital could kill between 130,000 and 3 million people-a lethality at least matching that of a hydrogen bomb. Last year, a U.S. Justice Department exercise revealed that discharging pneumonic plague in Denver could create 3,700 or more cases, with an estimated 950 or more deaths within a week.1 Then, according to recent results from a U.S. Air Force exercise, if smallpox were released in Oklahoma City, it would take less than two months to kill 1 million people-throughout the world. While the deadly repercussions of these incidents is clear, the probability of them occurring remains debatable, even after the recent albeit less deadly, anthrax incidents in Florida, New York, New Jersey, and the nation's capital area. The amounts of material required, the skill and technology involved, the delivery methods-all are difficult objectives to realize, experts say. Such attacks require high levels of expertise and organization, plus time, money, and trial and error, says Raymond Zilinskas, senior scientist at the Center for Nonproliferative Studies, Monterey Institute of International Studies. But no one is saying it's impossible. What they are saying is that current and future research that could result in vaccines, antigens or genes resistant to toxins, or even skin creams, would mitigate casualties and alleviate panic. And others are saying that the research community could help if some switched their current investigative gears to studying bioterrorism. "Beyond a few prominent individuals, you don't hear biologists getting up and renouncing biological weapons and emphasizing the need for international treaties and controls and the need for serious concern in the political and private sectors about what we're going to do about this growing threat," says biophysicist Steven Block, a bioterrorism expert at Stanford University. In a simpatico voice, the Federation of American Scientists last year called for universities to require students majoring in molecular biology or other fields potentially useful to biological weapons development to complete at least one course on the essentials of treaties, laws, regulations and other programs designed to control biological warfare. An Array of Weaponry Anthrax, plague, and smallpox are among the most likely of the approximately 20 bioweapons to be used in a large-scale attack, say scientists at the Center for Biodefense Studies at Johns Hopkins University and elsewhere. But few vaccines exist, their availability is limited, and diagnosis is often difficult and slow. Early symptoms mimic the flu or pneumonia, and treatment and management must be early to be effective. Although the public health sector would treat casualties in such an attack, much of the work needed to improve response, diagnosis, and treatment falls to scientists, particularly biologists working in genomics, immunology, and biotechnology. Increased attention to, and financial support for, bioweapons research would result not only in better security but also would have numerous indirect benefits. These benefits, says Block, include rapid, improved diagnostics for disease, improved vaccine capacity and antibiotic supply, and better mechanisms for dealing with natural outbreaks of emerging diseases. Biologist Carol Shoshkes Reiss of New York University is a member of the Defense Advanced Research Project Agency (DARPA) scientific advisory panel on unconventional pathogen counter- measures; she reviews applications for bioweapons defense research funding. DARPA pays for studies on pathogens as well as detection technology and environmental sampling. Reiss notes that a wide range of projects are under way, including diagnostic and therapeutic studies and efforts to manipulate the immune system. Potentially revolutionary projects include one to train bees to recognize the scent of chemical or biological agents; another, using a sensor and electronic chip, to detect if rat brain tissue had been exposed to a harmful substance; and another to develop a skin cream made of novel materials to soak up pathogens from the body or the environment. Recent bioweapons-related research from Harvard Medical School included findings that identified a mouse gene, which, in certain forms, renders mice resistant to anthrax. Another Harvard research group developed a protective antigen against the toxin that makes anthrax lethal. The findings might serve as the basis for a potential new vaccine or treatment for human anthrax, according to the study's authors. "What's most important is to determine how the pathogen causes disease," Reiss says. "If in the case of bacteria, such as cholera or anthrax, where it's a toxin that causes the disease, you want to develop a specific vaccine against the toxin." Block notes that vaccine development is mostly an unknown process. "We don't have a general way of making a general vaccine that gets an arbitrary pathogen that lasts for any length of time. We have anecdotal ways of doing it, but this is not an easy problem, and the fact of the matter is that making a vaccine is still very much a black art," Block says. Developing eight new vaccines to potential bioagents is estimated to cost $3.2 billion, according to a Pentagon advisory panel, plus take years before they can be developed and used; FDA approval for a new vaccine protocol requires at least two years alone. Currently, the United States cannot produce enough of the vaccines that are available; however, efforts are under way to stockpile more anthrax and smallpox vaccine. At the Armed Forces Institute of Pathology, biologist Amy Krafft is developing a fluorescent, dye-labeled probe technology for detection purposes. She worked on the TaqMan probe, which, she says, spots unique microbial genes for pathogen identification during DNA or RNA amplification in a miniaturized Light Cycler (RAPID, Idaho Technology). The instrument identifies a bioagent in about 30 minutes, compared to a day or more with classic culture, and it can be used on environmental or human samples, she says. And, earlier this year, as part of a federal initiative, microbiologists launched an online genomics and bioinformatics resource center, funded by the National Institute of Allergy and Infectious Diseases and DARPA. The center plans to foster research on smallpox and other poxviruses, such as vaccinia and monkeypox. Overseen by investigators at the University of Alabama at Birmingham and St. Louis University in Missouri, the project will feature repositories of genetic information about poxviruses, information that will be freely available to anyone with Web access.2 The Odds of an Attack Though the possibility exists that a large-scale bioweapons attack could occur, it's not a sure bet.3 Using anthrax as an example, Zilinskas explains the numerous steps required for a successful assault. First, a terrorist group must get the virulent strain and then grow the organism in a lab. Then, the organism must be turned into spores and suspended in a solution that keeps it alive and allows it to be spread through a nozzle without clogging, techniques that require some microbiological knowledge. Then, the group must acquire the right spray mechanism and then adapt it for distribution at the proper pressure and proper particle size that is required for infection. Other equipment needed for bioweapons development, testing and manufacture includes large fermenters, centrifugal separators, cross-flow filtration apparatus, freeze dryers, aerosol inhalation changers, and microencapsulation equipment, Zilinskas says. Additionally, ecological conditions must be just right: Too much or too little wind, too much sun, rain-all could damage an attack's effectiveness, he says. Zilinskas notes that between 1990 and 1995, the Japanese cult Aum Shinrikyo spent millions developing biological and chemical weapons with the help of a few biologists. The group reportedly attempted at least nine biological weapons attacks with anthrax and botulinum toxin, none of them successful, before resorting to sarin gas, which they released in a Tokyo subway station in 1995, killing 12. One attack, which involved spraying anthrax from the ninth floor of a building, failed because the group used a nonvirulent strain, and also because the nozzle clogged, Zilinskas says. Superagent creation-which requires experienced molecular biologists-is even more difficult, but certainly possible, he adds. The Russians, for example, are known to have genetically engineered anthrax and tularemia to be antibiotic-resistant. "But it takes a substantial program to do that with [well-educated] scientists," he says. Among other complications, he notes, are the unwanted pleiotropic effects that accompany genetic engineering, which could require four or five cycles of genetic engineering before the agent can be weaponized. A large-scale bioweapons terrorist attack might also fail for reasons unrelated to science and technology. Historically, one of its main barriers is defection. "Inevitably, somebody says, 'Hey, I didn't sign up to do this,'" says Brad Roberts, a research staff member at the Institute for Defense Analyses in Alexandria, Va. "It only takes one defector to take down an organization," he says. Reportedly, when the Aum Shinrikyo finally figured out how to effectively release a biological warfare agent, the person charged with doing so disappeared, Roberts says. Details of secret bioweapons work by the former Soviet Union only became known when Ken Alibek, formerly Colonel Kanatjan Alibekov, defected.4 Alibekov, the deputy director of the state agency charged with carrying out bioweapons research, supervised as many as 32,000 people out of 60,000 involved in the bioweapons program, Block says. Some fear that scientists from this old Soviet program will lend their expertise to terrorist groups or other governments. For example, Iran once actively recruited scientists to work on its biological warfare program, offering $7,000 a month in wages compared to the $60 to $110 a month that scientists earn in Russia and the newly independent states, says Zilinskas, who is currently writing a book assessing proliferation of biological warfare in Russia. One organization, the International Science and Technology Center, promotes the nonproliferation of weapons technology of mass destruction by providing weapons scientists from the former U.S.S.R. with opportunities to redirect their talents to peaceful science. "It's absolutely essential that we jack up [their]...efforts," he says. Encouraging awareness and emphasizing increased research could alleviate a primary goal of terrorism: panic. Obtaining data through rapid detection and diagnosis techniques goes a long way toward minimizing fright and relieving pressure on the public health system. When Aum Shinrikyo released sarin gas in Tokyo, 5,510 people sought treatment from local hospitals and clinics, even though only 15 percent of them were harmed, according to reports. Similar panic has imbued the public over the recent anthrax cases, with people rushing out for antibiotic prescriptions and protective garments. A member of the Center for Strategic and International Studies who coordinated the Air Force exercise in Oklahoma City says that it taught the importance of quickly gathering medical and scientific data to guide decisions and advise the public before panic develops. Timely and accurate information on potential bioweapons, she notes, only comes from the scientific community's continued efforts. Generally, though, biologists have ignored the issue of biological weapons, says Block, who listed prominent scientists Donald Henderson, Joshua Lederberg, and Matthew Meselson as some exceptions. Block would like to see a replay of what happened after the Hiroshima bombing, when many physicists took up the challenges posed by nuclear weapons and worked at national and international levels to limit their destructive potential. Jennifer Fisher Wilson (jfwilson@snip.net) is a contributing editor for The Scientist. References 1. See also, E. Russo, "Bioterrorism preparedness," The Scientist, 15[1]:1, Jan. 8, 2001. 2. www.genome.uab.edu/pox 3. See also, R. Lewis, "Bioweapons research proliferates," The Scientist, 12[9]:1, April 27, 1998. 4.T. Hollon, "Ken Alibek: For the biodefense," The Scientist, 14[8]:18, April 17, 2000. New Targets in the Battle Against Anthrax Photo: AP/Wide World Photos Biohazard? Testing a sample from a suspicious package Currently, antibiotics are the only treatment for anthrax, but they are nonspecific and target the bacterium, not its deadly toxins. This leaves little hope for people who have contracted anthrax's most insidious form. New work published last month in Nature on the structure and function of the anthrax toxin and its membrane receptor provides timely information on potential targets. John A.T. Young, the Howard M. Temin professor of cancer research, University of Wisconsin, has captured the gene for the elusive anthrax toxin receptor.1 Within its sequence, Young found a von Willdebrand factor, typically a region where protein and protein interactions occur. Young constructed a soluble peptide of that region alone, "a decoy receptor," that interferes with toxin-binding to receptors in intact cells. Robert C. Liddington, director, cell adhesion-extracurricular matrix Biology program, Burnham Institute, solved the three-dimensional structure of the lethal factor (LF), the deadliest anthrax toxin.2 Liddington identified LF's active site, which selectively destroys a particular kinase by breaking a single bond-"a smart bomb with nanometer precision." Insight into this structure provides new strategies for interfering with LF activity. In addition, earlier this year, R. John Collier, a professor of microbiology and molecular genetics, Harvard Medical School, elaborated on two other ways to interfere with anthrax toxins.3,4 Collier and Harvard chemist George Whiteside pulled a dodecimer from a peptide library that blocks the toxin's activation. Collier also produced a mutant toxin that forms an inactive toxin-receptor complex. Both of Collier's inhibitors show activity in animal models of anthrax. For each new treatment approach, the key experiment shouldn't take long to complete, Collier says. "We can get a good inkling of how well these drugs are going to do within a week." -Laura DeFrancesco References 1. K. Bradley et al., "Identification of the cellular receptor for anthrax toxin," Nature, published online Oct. 23, 2001. (www.nature.com/nature/anthrax/young.pdf) 2. A. D. Pannifer et al., "Crystal structure of the anthrax lethal factor," Nature, published online Oct. 23, 2001. (www.nature.com/nature/anthrax/liddington.pdf) 3. M. Mourez et al., "Designing a polyvalent inhibitor of anthrax toxin," Nature Biotechnology, 19:958-61, October 2001. 4.B.R. Sellman et al., "Dominant-negative mutants of a toxin subunit: an approach to therapy of anthrax," Science, 292:695-7, 2001.

Biological Terrorism

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