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Suggested Citation:"2 Assessing Our Understanding of the Threats." Institute of Medicine. 2002. Biological Threats and Terrorism: Assessing the Science and Response Capabilities: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10290.
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2
Assessing Our Understanding of the Threats

OVERVIEW

The focus of this session of the workshop was an assessment of known threats. Anthrax is a proven risk and of immediate concern. Smallpox is equally urgent because of its capability for person-to-person transmission and the large number of completely susceptible individuals in the United States and around the world. Presenters discussed details of the bioweapons potential and treatments available for each of these threats along with those of three other “high-priority” potential bioterrorist agents: plague, tularemia, and botulinum toxin. However, these are not the only credible bioterrorist agents out there. For example, the former Soviet Union is known to have weaponized at least thirty biological agents, including several vaccine- or drug-resistant strains.

There are many imaginable bioterrorist scenarios, but if the goal is to induce mass casualties, an aerosol attack is probably most likely. Aerosols exhibit wide-area coverage, and their small particle size allows them to deposit very deeply in the lung tissue which is where many agents, including anthrax, induce maximal damage. A large amount of agent disseminated under good meteorological conditions over a substantially sized city could have considerable downwind reach, resulting in large numbers of casualties.

Food-borne bioterrorism, which could encompass a variety of agents, must also be considered an equally likely threat. Agents that cause foodborne illness are easy to obtain from the environment and often have very low-dose requirements. Foodborne pathogens may in fact be the easiest bioterrorism agent to disseminate. In addition to public health risks, there are several important agri-

Suggested Citation:"2 Assessing Our Understanding of the Threats." Institute of Medicine. 2002. Biological Threats and Terrorism: Assessing the Science and Response Capabilities: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10290.
×

cultural risks which were mentioned briefly but not discussed in detail during this workshop.

Anthrax

B. anthracis is a very stable organism because of its ability to sporulate. Most naturally occurring anthrax cases are cutaneous and are transmitted from agricultural exposure. The incidence of infection is unknown; most cases occur in underdeveloped countries. Throughout the world, since the late 1930s, attenuated strains have been used as live veterinary spore vaccines and have proven to be highly effective in controlling disease in domesticated animals. Since the 1950s, one of these strains has been used as a live attenuated strain in humans in countries of the former Soviet Union. The molecular pathogenesis of anthrax, including the exact target of its lethal factor, is largely unknown. However, enough is known that we can begin to predict where second-generation vaccines and various antitoxin modalities might work.

Currently, there are three types of preventative or therapeutic countermeasures against anthrax: vaccination, antibiotics, and various adjunctive anti-toxin treatments. In terms of developing new therapeutics, initial immediate efforts should be to evaluate already licensed antibiotics. Longer-term efforts should include identifying protective antigens that are effective against modified strains; developing vaccines that act more quickly and would be more useful in a post-exposure scenario; exploring the combined use of vaccines and antibiotics; and exploring new antitoxin treatments. Critical to all of these efforts is the need for a large-scale central animal testing facility.

Smallpox

Smallpox has several features that make it an attractive bioterrorist agent: it is highly stable; it is infectious by aerosol; it is highly contagious; most clinicians lack experience recognizing the disease; and, because vaccination against smallpox ceased after eradication, most of the world’s population is highly susceptible to infection.

Even though a smallpox vaccine exists, there are several unresolved bioterrorism-related issues regarding smallpox vaccination:

It is not clear which health care providers should be immunized preceding any potential outbreak versus immediately following an outbreak.

The current supply of vaccinia immune globulin is insufficient for treating all of the expected adverse effects associated with vaccinia immunization, should all 300 million doses of cell-cultured vaccinia that are currently being produced be administered.

Suggested Citation:"2 Assessing Our Understanding of the Threats." Institute of Medicine. 2002. Biological Threats and Terrorism: Assessing the Science and Response Capabilities: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10290.
×

The cell-cultured vaccine that is currently being produced is considered only a stop gap measure since it cannot be used in immunocompromised individuals or children.

It is unclear whether the available vaccine would protect against aerosol exposure of the type and magnitude that would be expected in a bioterrorist event. Although monkey/monkeypox studies have shown that yes, the vaccine does provide adequate protection, it is unclear how applicable these studies are to smallpox in humans.

Currently, most research and development efforts for smallpox therapeutics are focused on antiviral drugs. Thus far, the leading candidate is cidofovir, which has been approved under an IND for treating disseminated vaccinia but has not been approved for treating smallpox. Despite its promise, however, cidofovir is unsuitable for mass casualty use. More effort needs to be directed toward other therapeutics, such as immunomodulators.

Plague

Plague—a deadly and highly contagious disease—was weaponized in the former Soviet Union for aerosol delivery and engineered for antimicrobial resistance and possibly enhanced virulence. In the WHO modeling scenario that was developed in 1970, a 50-kilogram release over a city of five million would cause about 150,000 cases, or 36,000 deaths, in the first wave. A secondary spread would cause a further 500,000 cases, or 100,000 deaths. Plague requires intensive medical and nursing support and isolation for at least the first fortyeight hours, followed by two to three weeks of slow convalescence. The hospitalization and isolation that would be required for this many people in a single city is nearly unimaginable.

Currently in the U.S., there is no available plague vaccine. The live vaccines that are sometimes used in other countries have unacceptable adverse effects. There are, however, a number of laboratories trying to develop a new generation vaccine, as well as new delivery methods. Several different types of antibiotics that can be used to treat plague are included in the national pharmaceutical stockpile. Antibiotic treatment must be instituted early during the course of infection, otherwise death occurs in three to six days.

Tularemia

Tularemia was weaponized as an aerosol both in the U.S. and the former Soviet Union where it was also engineered for vaccine-resistance. In the WHO modeling scenario of 1970, 50 kg over a city of 5 million would incapacitate 250,000 people and cause 19,000 deaths. Tularemia is highly infectious but not contagious. Treatment is similar to that for plague but more extensive, as is the post-prophylaxis to prevent relapses of disease. The tularemia vaccine is a live

Suggested Citation:"2 Assessing Our Understanding of the Threats." Institute of Medicine. 2002. Biological Threats and Terrorism: Assessing the Science and Response Capabilities: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10290.
×

attenuated vaccine that was previously available as an investigational drug through DoD and is now being investigated by the Joint Vaccine Acquisition Program. However, it does not offer full protection against inhalational transmission, and it takes about fourteen days for protection to develop. The vaccine has been recommended for use in people who work routinely with the organism in the laboratory, but it is unknown whether it would be useful in first responders at high risk for exposure.

Botulinum Toxin

Botulinum toxin has several features that make it an attractive bioweapon, including its extreme potency and lethality; the ease of its production, transport and misuse; and its profound impact on its victims as well as the health care infrastructure. Like tularemia, it has a very diverse mode of transmission: it can spread through foods, beverages or as an aerosol. Botulinum toxin, of which there are seven serotypes, kills by paralytic ability and is one of the most poisonous substances known.

Although an investigational vaccine exists, immunization is really not a viable option for bioweapons defense: the vaccine is still only investigational even after ten years; its components are aging and losing potency; it only protects against toxins A, B, C, D, and E, not serotypes F and G; it is very painful to receive; it requires a booster at one year; and the use of it would deprive the recipient for life of access to medicinal botulinum toxin.

The army has developed an equine antitoxin that provides coverage against all seven toxin serotypes, but the supply is limited and the drug carries the risk of serious allergic reaction. However, equine antitoxin is inexpensive to produce and could be made in large quantities if a specialized facility were available. A human-derived botulinum antitoxin has been developed as an orphan drug but is very difficult to produce in large quantities and is of limited use because it protects against only five serotypes.

DoD is developing a recombinant vaccine which is not expected to become a licensed product, however, for at least another ten years. Researchers are also developing recombinant human antibodies as an alternative therapeutic option. Antibodies have several distinct advantages as bioweapons defense agents: they induce immediate immunity; they can be produced in unlimited quantities; and they are highly potent. In fact, an unlimited supply of human recombinant antitoxin is probably the best defensive measure against botulinum toxin.

Suggested Citation:"2 Assessing Our Understanding of the Threats." Institute of Medicine. 2002. Biological Threats and Terrorism: Assessing the Science and Response Capabilities: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10290.
×

ANTHRAX

Colonel Arthur M. Friedlander*

Senior Military Research Scientist, United States Army

Medical Research Institute of Infectious Diseases

“It has now prevailed and been recognized in this neighborhood about forty years, and notwithstanding all that has been done to prevent it, by ventilation, the use of respirators and other means, it still continues, as severe and frequent as it ever was, overclouding the life of the sorter.” (Bell, 1880)

Anthrax has a long history: apocryphal accounts describe it as the fifth and sixth plagues in Exodus, when dust was cast into Pharaoh’s eyes; it was the first disease for which a microbial etiology was determined by Robert Koch; and the anthrax vaccine was one of the first live vaccines, developed by Pasteur and one of the first examples of attenuation of a fully virulent organism for use as a vaccine. Physicians in the latter part of the 19th century, particularly in England, were well aware of the clinical and pathological aspects of anthrax. It is now incumbent upon a new generation of physicians to become intimately familiar with this disease.

There are several characteristics of B. anthracis that make it a potentially very lethal bioweapon, most importantly its stability and infectivity as an aerosol and its large footprint after aerosol release. An aerosol release of anthrax could potentially affect millions of individuals.

The organism’s stability stems from its ability to sporulate. Dormant spores are estimated to have survived in some archaeological sites for hundreds of years. The spores occur in soils worldwide and infect grazing herbivores. After they enter their mammalian host, they germinate into actively replicating vegetative cells. When the mammalian host dies and its carcass is exposed to the air, the bacteria sporulate. It is unknown whether spores go through a germination-replication-sporulation cycle in the soil, or whether amplification of bacterial numbers occurs only within the host.

Under natural circumstances, humans become infected only via contact with infected animals or contaminated animal products. Anthrax is primarily a developing world disease associated with agricultural exposure causing cutaneous or gastrointestinal infection. However it emerged as “woolsorter’s disease” in the industrial world in the latter half of the 19th century. With the rise of the industrial revolution, the large quantities of contaminated animal products being processed in enclosed rooms generated aerosols of anthrax spores which caused the first known cases of inhalational anthrax. Today, inhalational anthrax is extraordinarily rare.

*  

This statement reflects the professional view of the author and should not be construed as an official position of the U.S. Army Medical Research Institute of Infectious Diseases.

Suggested Citation:"2 Assessing Our Understanding of the Threats." Institute of Medicine. 2002. Biological Threats and Terrorism: Assessing the Science and Response Capabilities: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10290.
×

The incidence of all forms of anthrax is unknown because reporting is unreliable. Large anthrax outbreaks tend to occur during breakdowns of the public health structure, for example during war. Ten thousand human cases occurred in Zimbabwe during the 1970’s and early 1980’s; only eight of these cases were reported to be inhalational anthrax although no autopsies were performed.

B. anthracis is a large gram-positive, sporeforming, non-hemolytic, non-motile bacillus. Its known virulence factors include a polyglutamic acid capsule, which is antiphagocytic and without which the organism is attenuated; and the well-known lethal and edema toxins. Like most bacterial pathogens, the virulence determinants are encoded on plasmids. The two toxins are encoded on one plasmid, and the genes responsible for synthesis of the capsule are on a smaller plasmid. It is possible, under specific laboratory conditions, to eliminate the smaller plasmid and produce an unencapsulated, attenuated strain of bacteria that still produces both toxins. Since the late 1930’s and early 1940’s, the Sterne strain and others similar to it have been used throughout the world as live veterinary spore vaccines that have proven to be highly effective in controlling disease in domesticated animals. Since the 1950’s, a similar strain has been used as a live attenuated vaccine for humans in the former Soviet Union. It is also possible to delete the toxin plasmid, resulting in an avirulent organism that produces only capsule.

Inhalational anthrax is characterized by lymphadenitis of the tracheobronchial and mediastinal lymph nodes and mediastinitis. On chest x-ray, the lungs are usually clear while there is usually mediastinal widening and often pleural effusions. The incubation period is usually a week or less. The initial symptoms are mild and non-specific and include fatigue, which can be very profound, headache, fever, chills, and sweats. There may or may not be a cough, usually non-productive. Because the disease is centered in the mediastinum, patients sometimes experience a sense of precordial discomfort. Abdominal pain has been prominent in some cases. As the infection progresses, symptoms include the abrupt onset of dyspnea, tachycardia, increased chest pain, and occasionally stridor. Pneumonia may occur but is usually absent. There is a rapid progression to cyanosis, shock, and death.

An early diagnosis is enormously important. The definitive criterion for establishing a diagnosis is isolation of the bacteria from blood, pleural or cerebral spinal fluid, or other tissue. Since there is no other gram-positive bacillus that causes sepsis in healthy individuals, the isolation of a bacillus from the blood should alert every clinical microbiology department to the diagnosis of anthrax. A chest x-ray or CT scan should also show the characteristically enlarged mediastinum. An outbreak would be characterized by large numbers of previously healthy people with these symptoms appearing in emergency rooms and physician offices.

Our knowledge of the gross pathogenesis of this disease is good but, as with many infectious diseases, we know very little about its molecular pathogenesis. The infectious spore enters the body either through a break in the skin, the GI

Suggested Citation:"2 Assessing Our Understanding of the Threats." Institute of Medicine. 2002. Biological Threats and Terrorism: Assessing the Science and Response Capabilities: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10290.
×

tract, or by the respiratory route. In the skin and GI tract, the spore germinates locally. After entry through the respiratory tract, the spore is transported from the lung via macrophages to the regional lymph nodes. It spreads from node to node producing hemorrhagic necrosis and then extending into the mediastinum. From the lymph it spreads into the systemic circulation. About fifty percent of individuals with inhalational anthrax have evidence of meningitis. Impairment of respiratory function due to interference with lymphatic and vascular outflow associated with mediastinitis and pleural effusions is likely the primary mechanism of death.

The anthrax toxin, which causes edema and cell necrosis, probably contributes to death as well. Although the anthrax toxin is similar to many other bacterial and plant toxins, it is unusual in the sense that the functional domains reside on separate proteins. The individual proteins by themselves are inactive and have no biological function. Only when protective antigen combines with lethal factor does it constitute lethal toxin, and only when it binds with edema factor does it constitute edema toxin.

From cell culture studies, it appears that the anthrax toxins function as outlined in Figure 2-1. Protective antigen binds to a cellular receptor where a cell surface protease cleaves it, releasing a small 20 kD fragment and exposing a cryptic site on the molecule; it then forms a heptamer and subsequently binds to either edema or lethal factor. This whole complex is then internalized by receptormediated endocytosis into an acidic vesicle. Under conditions of low pH, the complex inserts into the membrane and, like other toxins, the enzymatic toxin components are delivered to the cytosol. Edema factor raises cyclic AMP levels to pharmacological levels, which is clearly responsible for some of its biological effects. The exact target of lethal factor, a zinc protease, remains, to date, unknown.

As follows from this model there are several potential targets for antitoxin therapeutics (see Collier’s discussion of the most promising). The recent identification of a motor protein that controls sensitivity, or resistance to lethal toxin, will hopefully lead to additional antitoxin strategies. The current human vaccine is thought to act predominantly by inducing antibodies that block the binding of protective antigen to the cell surface receptor and block the binding of edema and lethal factor to the cell-bound protective antigen although the antibodies may also act on the organism itself.

It is unclear whether anthrax pathogenesis involves a cytokine cascade. If so, the recent licensure of activated protein C for use in sepsis could have important ramifications for anti-anthrax therapy. The possible role of cytokines in anthrax warrants further evaluation. The multiple studies that have tested adjunctive treatment for sepsis should be used to guide this effort.

There are many unresolved issues with regards to prophylaxis and treatment. For example, which antibiotics should be used? Do we need adjunctive treatments? What if the B. anthracis strain is antibiotic- or vaccine-resistant?

Suggested Citation:"2 Assessing Our Understanding of the Threats." Institute of Medicine. 2002. Biological Threats and Terrorism: Assessing the Science and Response Capabilities: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10290.
×

About ten years ago, Russian investigators reportedly produced both multidrugresistant and vaccine-resistant B. anthracis strains.

In terms of therapy, there are several points to be emphasized. First, it should be remembered that antibiotics affect only the bacillus, not the spore. Thus it is possible that sufficient numbers of ungerminated spores may persist in an exposed individual after completion of a course of antibiotics, only to cause disease upon subsequent germination when antibiotics are no longer present. The conditions which govern the germination of spores in vivo remain obscure. Secondly, the notion that inhalational anthrax is invariably fatal once symptoms occur is likely untrue as evidenced by the survival of some of the current cases. Indeed, there is experimental evidence supporting the efficacy of late-stage intensive treatment in non-human primates showing signs of bacteremia or even mediastinitis. Lastly, there are many other antibiotics that show activity in vitro that may extend the therapeutic options for prophylaxis and treatment. These need to be evaluated in animal models before consideration for human use.

The Department of Health and Human Services, with input from the Department of Defense, is currently focusing on three therapeutic issues: testing licensed antibiotics that could be used to treat anthrax; developing human antibodies against the current vaccine, which has been administered to about 500,000 individuals; and assessing combined vaccine and antibiotic use. Other issues that need to be addressed include: identifying near-term, mid-term, and long-term research goals; identifying new protective antigens that are effective against modified strains; producing vaccines that work more quickly, particularly from the perspective of a post-exposure scenario; and, critical to all of these efforts, developing a large-scale central animal testing facility as evaluation of new treatments in humans will likely be extremely difficult.

FIGURE 2-1 Anthrax Toxin Function with Protective Antigens

Suggested Citation:"2 Assessing Our Understanding of the Threats." Institute of Medicine. 2002. Biological Threats and Terrorism: Assessing the Science and Response Capabilities: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10290.
×

MEDICAL COUNTERMEASURES AGAINST THE RE-EMERGENCE OF SMALLPOX VIRUS

Peter B. Jahrling,* Ph.D.

Senior Research Scientist, United States Army Medical

Research Institute of Infectious Diseases

The recent bioterrorist attacks involving anthrax have increased awareness that biological agents are truly weapons of mass destruction. Unlike anthrax, the smallpox virus is a contagious disease with fairly high rates of human to human transmission. As such, the use of smallpox as a bioterrorist agent is considered to pose an even greater threat than anthrax.Following publication of the Institute of Medicine report Assessment of the Future Scientific Needs for Live Variola Virus in 1999, collaborative research involving the Department of Defense (DoD) and the Department of Health and Human Services (DHHS) was initiated to address the recommendations suggested by the Report. The subject of today’s presentation focuses on the recent research and our development of an animal model for Variola (smallpox) virus infection.

The desirability of animal model development is driven by the proposed Food and Drug Administration Animal Efficacy Rule, which was written to facilitate approval of countermeasures for infectious diseases, such as smallpox, which do not occur naturally in human populations. The Rule requires that pathophysiology of the animal model disease be faithful to the human disease course, and that the efficacy study endpoint must be based on reduced morbidity or mortality. Insight into the “toxemia” of human smallpox might be achieved by application of modern tools of virology and immunology to the model infection. Conventional wisdom holds that variola does not produce smallpox-like disease in any species other than humans; however, cynomolgus monkeys infected parenterally with variola strain were reported to develop non-specific, febrile disease. In studies conducted by the DoD in collaboration with the Centers for Disease Control, we tested four variola strains for virulence in monkeys exposed to high infectious doses delivered by aerosol, intravenous, or a combination of routes. Eventually, we identified a virus strain that produced a lethal disease process resembling rapidly progressive, human smallpox.

Initially, two variola strains (Yamada and Lee) were used to expose monkeys to aerosolized doses of 108 plaque-forming units (PFU). Results were disappointing, since the disease courses were mild and nondescript. Subsequent studies used different variola strains (Harper & India 7124), higher doses (109 PFU), and included intravenous inoculation, which may be critical. Summary data are shown in Table 2-1.

*  

This statement reflects the professional view of the author and should not be construed as an official position of the U.S. Army Medical Research Institute of Infectious Diseases.

Suggested Citation:"2 Assessing Our Understanding of the Threats." Institute of Medicine. 2002. Biological Threats and Terrorism: Assessing the Science and Response Capabilities: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10290.
×

TABLE 2-1 Results of primate exposures to smallpox virus

MK #

Sex

kg

inoc

strain

route

intended dose

Day death

C099

M

7.2

5/31/2001

VAR Harper

Aerosol + IV

10^9

 

C625

M

6.5

5/31/2001

VAR Harper

Aerosol + IV

10^9

4

C681

M

8.5

5/31/2001

VAR Harper

Aerosol + IV

10^9

4

C881

M

5.5

5/31/2001

VAR Harper

Aerosol + IV

10^9

6

 

 

 

 

 

 

 

 

C171

M

5.5

6/1/2001

VAR 7124

Aerosol + IV

10^9

3

C651

M

6.9

6/1/2001

VAR 7124

Aerosol + IV

10^9

4

C115

M

7

6/1/2001

VAR 7124

Aerosol + IV

10^9

3

C713

M

4.9

6/1/2001

VAR 7124

Aerosol + IV

10^9

13

 

 

 

 

 

 

 

 

C373

M

4.6

7/6/2001

VAR 7124

IV

10^9

4

C088

F

4.1

7/6/2001

VAR 7124

IV

10^9

3

C437

M

3.9

7/6/2001

VAR 7124

IV

10^9

6

C956

M

3.9

7/6/2001

VAR 7124

IV

10^9

4

 

 

 

 

 

 

 

 

C083

M

8.4

8/24/2001

VAR 7124

IV

10^9

 

C003

M

6.2

8/24/2001

VAR 7124

IV

10^9

10

 

 

 

 

 

 

 

 

57-394

F

3.2

8/24/2001

VAR 7124

IV

10^8

 

C271

M

7.2

8/24/2001

VAR 7124

IV

10^8

 

C282

F

6

8/24/2001

VAR 7124

IV

10^8

 

 

 

 

 

 

 

 

 

C835

M

4.3

8/24/2001

VAR 7124

IV

10^7

 

57-245

F

3.5

8/24/2001

VAR 7124

IV

10^7

 

C677

M

6

8/24/2001

VAR 7124

IV

10^7

 

 

 

 

 

 

 

 

 

C382

F

5

8/24/2001

VAR 7124

IV

10^6

 

C409

M

4.9

8/24/2001

VAR 7124

IV

10^6

 

48-48

F

3.3

8/24/2001

VAR 7124

IV

10^6

 

Suggested Citation:"2 Assessing Our Understanding of the Threats." Institute of Medicine. 2002. Biological Threats and Terrorism: Assessing the Science and Response Capabilities: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10290.
×

Three of four monkeys exposed to the Harper strain by a combination of aerosol and intravenous routes died rapidly, three to six days after exposure. Likewise, all four monkeys exposed to the India strain died, although one developed a more protracted disease course and died on day 13. Subsequent inoculation of four monkeys with 109 PFU India 7124 via the intravenous route alone yielded uniform rapid lethality. In subsequent attempts to obtain a more slowly evolving disease course, lower doses produced systemic infections and more protracted disease courses, but no deaths.

Hematologic evaluation of lethally infected primates revealed profound leukocytosis, with WBC > 50,000/mm3 (20% monocytes) in acutely ill animals. Platelet counts dropped to an average of 125,000, consistent with coagulation factor perturbations and fibrin deposition associated with evolving disseminated intravascular coagulation (DIC). Serum chemistry evaluations revealed profound increases in serum creatinine and blood urea nitrogen consistent with kidney lesions as well as elevations in AST and ALT consistent with hepatic damage. Infectious virus at concentrations > 108 PFU/g were retrieved from all visceral tissues obtained from acutely moribund or terminal monkeys at necropsy. This is consistent with the demonstration of viral antigens by immunohistochemistry in association with pathological lesions in these same tissues. Infectious viral burdens in monkey # C-713 (which died on day 13) were lower. Infectious virus was also retrieved from throat swabs of iv-inoculated animals within 48 hours of exposure, before the evolution of skin lesions or fevers. It is probably that these animals are contagious at this early stage of infection; isolation of virus from throat swabs of human smallpox-infected patients was never systematically attempted. Detection of viral genomes in the blood of inoculated monkeys as early as one day after inoculation was achieved using TaqMan PCR. This assay, which requires less than one hour to run, promises to detect infection during the asymptomatic prodrome, when countermeasures such as antiviral drugs are predicted to be most effective.

Additional insight into the pathogenesis of variola in lethally infected primates is being obtained by evaluation of high-density cDNA microarray data, which measures and classifies gene expression in peripheral blood cells obtained sequentially. RNA was prepared from isolated peripheral blood leukocytes, labeled as fluorescent cDNA for microarray analysis, and hybridized to arrays which include >10,000 uniquely named genes. Using a two- color comparative hybridization format, expression patterns were analyzed according to biological themes. Gene expression analysis identified dramatic response patterns that correlated with lethality and gave insight into pathogenesis. Several relevant biological themes included interference with interferon, IL-18, and TNF-alpha, inhibition of interleukin-1 beta and apoptosis. Activation of coagulation cascade factors, and down regulation of immunoglobulin response and cell mediated immunity-related genes were also related to lethality. Microarray evaluations will be extended to include tissue expression patterns, comparisons with other

Suggested Citation:"2 Assessing Our Understanding of the Threats." Institute of Medicine. 2002. Biological Threats and Terrorism: Assessing the Science and Response Capabilities: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10290.
×

virus infections (especially monkeypox) in primates, and in vitro systems with primary target cells. Potential benefits of host genome-wide expression profiling include early detection of infected individuals, recognition of variant agents and prognostic markers, identification of virulence and disease, novel therapeutic and prophylactic strategies, and determination of early signatures of a protective immune response to vaccination.

Further refinements of this primate model are necessary before it can be exploited to test antiviral drugs or other countermeasures in accordance with the FDA Animal Efficacy Rule. Ideally, a combination of viral strain, dose, and route can be identified which produces a less accelerated disease course, one that more closely reflects the temporal course of human smallpox. The observation that certain strains of variola can produce fulminent disease in monkeys is a breakthrough in the quest for effective countermeasures for smallpox. This outcome is the exact opposite of what was predicted, since until now it was assumed that the host range of variola was restricted to humans.

Evaluation of the model disease course has yielded considerable insight into the nature of the “toxemia” associated with human smallpox. In addition, clinically relevant samples of blood and tissues have been obtained and tested, to validate modern diagnostic strategies such as TaqMan PCR. Using this technique, we have demonstrated the feasibility of obtaining a definitive diagnosis of exposure to smallpox virus during the prodrome. This capability should improve the likelihood of successful intervention using antiviral drugs. The production of these clinical samples is a significant byproduct of animal model development. The samples constitute a national resource for validation of diagnostic strategies based on detection of smallpox viral genomes and antigens now, and in the future.

Further refinement of the primate model for smallpox will include evaluation of the sub lethal, yet severe, infections associated with lower doses of inoculum virus. Objective, quantifiable correlates of disease severity may eventually be substituted for lethality in efficacy evaluations of candidate antiviral drugs and other countermeasures. As model refinement continues, concurrent advances in the identification of useful antiviral drugs are anticipated. These advancements, combined with further enhancements in diagnostic strategies, are reasonable milestones projected for this collaborative DoD/DHHS research program. Effective mitigation of an adversary’s most potent biological weapon (smallpox) must be a national priority.

Suggested Citation:"2 Assessing Our Understanding of the Threats." Institute of Medicine. 2002. Biological Threats and Terrorism: Assessing the Science and Response Capabilities: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10290.
×

TULAREMIA AND PLAGUE: ASSESSING OUR UNDERSTANDING OF THE THREAT

David T. Dennis,* M.D., M.P.H.

Division of Vector-Borne Infectious Diseases

Centers for Disease Control and Prevention

Yersinia pestis and Francisella tularensis are category A critical biological agents that pose a risk to national security because they could be easily disseminated (both agents) or transmitted person-to-person (Y. pestis), could cause a high mortality, and require special action for public health preparedness (CDC, 2000). Both agents have been weaponized as aerosols, the expected mode of delivery in a bioterrorist attack (Inglesby et al., 2000; Dennis et al., 2001). Plague holds special concern because of its potential to cause panic, its contagiousness in the pulmonary form, its fulminating clinical course and high fatality, and the possibility that it could be engineered for plasmid-mediated resistance to multiple antimicrobial agents (Galimand et al., 1997). Sepsis with either agent can result in catastrophic physiological consequences of compliment and cytokine cascade (systemic inflammatory response syndrome [SIRS]). Severity of illness is expected to require intensive medical care, including respiratory and other organ support that might readily overwhelm hospital response capacity (Inglesby et al., 2001). Pneumonic plague’s contagiousness would require isolation and possible quarantine, which would complicate medical and public health management. A WHO model of the release of 50 kg of Y. pestis over a city of 5 million predicts 500,000 cases with 100,000 deaths when both primary and secondary transmission are considered; a similar model for release of F. tularensis predicts 250,000 persons incapacitated and 19,000 deaths (WHO, 1997).

BOX 2-1 Bioterrorism Aphorisms

Do not assume anything

Expect the unexpected

What we know is not much

Bioterrorism is, among other things, unnatural

We do not know what we do not know

We are not ready

*  

The information provided in this paper reflects the professional view of the author and not an official position of the U.S. Department of Health and Human Services or the Centers for Disease Control and Prevention.

Suggested Citation:"2 Assessing Our Understanding of the Threats." Institute of Medicine. 2002. Biological Threats and Terrorism: Assessing the Science and Response Capabilities: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10290.
×

Standard, classical microbiological diagnostic tests would be of limited value in a major bioterrorism event, since they are time consuming and labor intensive; unfortunately, newer, rapid testing methods for these agents, such as antigen detection and DNA amplification, are neither standardized nor widely available.

Recommendations for antimicrobial treatment of plague or tularemia patients in a bioterrorist attack have been developed for both the mass- and contained-casualty situations (Inglesby et al., 2000; Dennis et al., 2001). The principal recommended antimicrobials are available through the National Pharmaceutical Stockpile. Two of these, gentamicin and ciprofloxacin, are not FDA-approved for treating plague or tularemia. Post-exposure antimicrobial prophylaxis is recommended to protect certain populations in the event of bioterrorist use of either plague or tularemia agents, but it would be difficult to identify populations at risk and administer drugs to them in a timely fashion. In the case of plague, isolation of cases and their close contacts, and quarantine of exposed populations could be difficult to enforce and would likely create fear and chaos (Inglesby et al., 2001).

Historical vaccines for plague and tularemia, based on killed and liveattenuated preparations respectively, are currently unavailable in the United States, and a newer generation of vaccines and immunotherapeutics is greatly needed to address pre- and post-exposure prevention of disease. Recombinant protein subunit vaccines (against F1 and V antigens singly, in combination, and as fusion products) have been developed for plague (Titball and Williamson, 2001). One subunit combination product has recently undergone Phase I clinical testing in the U.K. A microencapsulated formulation shows promise for respiratory tract delivery (Eyles et al., 1998). Further, oral administration of a Salmonella typhimurium mutant expressing the F1 antigen of Y. pestis protects mice against subcutaneous inoculation of a virulent plague strain (Titball et al., 1997). Intraperitoneal administration of monoclonal antibodies to the F1 antigen protects mice against both parenteral and aerosol challenge with human pathogenic Y. pestis strains (Anderson et al., 1998). Similar vaccines and recombinants have not yet been developed for use against tularemia, but recent progress in sequencing of the F. tularensis genome may lead to identification of candidate immunoprotective proteins (Karlsson et al., 2000).

Recent advances in Y. pestis and F. tularensis strain typing, using multiplelocus, variable-number tandem repeat analyses are expected to provide rapid tracking of outbreak strains as well as providing a foundation for deciphering global genetic relationships of these organisms that could be useful in the event of a BT attack (Johansson et al., 2000; Klevytska et al., 2001; Farlow et al., 2001).

Personal experience gained by participating in CDC responses to bioterrorist use of Bacillus anthracis reinforces the need to think freely about potential misuse of Y. pestis or F. tularensis (Box 2-1), to heed lessons learned (Box 2-2), and to ensure that preparedness and response needs are met before a critical event occurs (Box 2-3).

Suggested Citation:"2 Assessing Our Understanding of the Threats." Institute of Medicine. 2002. Biological Threats and Terrorism: Assessing the Science and Response Capabilities: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10290.
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BOX 2-2 Lessons Learned

Primary response is local

Federal support must be immediately available

Better agency integration needed (DHHS, Justice, Defense, State and Local)

Surge capacity vital

We don’t know what we don’t know

BOX 2-3 Critical Public Health Preparedness and Response Needs

Pre-approved, integrated, organizational plans, policies, protocols

Systems for surveillance, case id, contact tracing (plague), rapid epidemic/environment assessment

Surge capacity for outbreak and consequence management, including epidemiology/survey sampling, lab, Rx, Px, logistics

New/improved diagnostics and molecular id

New/improved vaccines and therapeutics

BOTULINUM TOXIN AS A BIOWEAPON

Stephen S. Arnon, M.D.

Founder and Chief

Infant Botulism Treatment and Prevention Program

California Department of Health Services

Botulinum Toxin and Human Botulism

Botulinum toxin is the most poisonous substance known. One gram, evenly aerosolized and inhaled, could kill over one million people; one hundred grams, evenly distributed in a food or beverage and ingested, could also kill over one million people (Arnon et al., 2001). Botulinum toxin is considered a plausible prime bioweapon threat because of its extreme potency and lethality, its ease of transport and misuse, and its profound impact on victims and the health care infrastructure. Botulinum toxin is the only non-replicating member of the six Centers for Disease Control and Prevention (CDC)-designated Class A (highest threat) agents. Other aspects of the toxin may also make it attractive as a bioweapon (Table 2-2).

Botulinum toxin is a simple di-chain protein whose “heavy chain” (100 kD) contains the binding and internalization domains and whose “light chain” (50 kD) contains the catalytic (Zn++-proteinase) domain. The toxin binds at peripheral nerve cholinergic synapses, the most important of which clinically is the

Suggested Citation:"2 Assessing Our Understanding of the Threats." Institute of Medicine. 2002. Biological Threats and Terrorism: Assessing the Science and Response Capabilities: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10290.
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TABLE 2-2 Features making botulinum toxin attractive as a bioweapon

Attribute

Consequence

Extreme potency and lethality

Can cause clusters of 10–500 fatali ties in any city at any time

Ease of production, transport and

Convenience and accessibility

misuse

 

Profound impact on victims and health care infrastructure

Can target key small groups (e.g. Congress, Supreme Court); can overwhelm urban hospital ICU ca pacity

Aerosols degrade quickly

Enemy’s infrastructure or arms can be captured intact; no decontamina tion needed

Not person-to-person transmissible

Use does not place user at risk

Versatility in use (foods, beverages, aerosols)

Can terrorize almost at will

Rapid turnover of publicly served foods and beverages

Evidence may be discarded before illness presents; easy to escape de tection and capture

neuromuscular junction. After uptake into the neuronal cytoplasm, the toxin cleaves one or more of the “SNARE-complex” proteins, thereby preventing the release of the acetylcholine-containing vesicles that normally cause muscle contraction. The net result of the toxin’s action is a flaccid muscle paralysis. Botulinum toxin is produced by the spore-forming anaerobic bacterium Clostridium botulinum, whose natural home worldwide is the soil and dust, from which it can be isolated with undue difficulty.

Botulinum toxin exists in seven different serotypes arbitrarily assigned the letters A-G; antibody that neutralizes one toxin type does not neutralize any other serotypes (e.g., anti-A antitoxin does not neutralize toxins B-G, etc.). The toxin is stable in foods and unchlorinated beverages for extended periods of time, but is easily inactivated by heating (e.g., 85oC x 5 minutes). Consequently, foodborne botulism (whether natural or bioterrorist) can result only from eating foods that are not heated, or not heated thoroughly. Waterborne (or beverageborne) botulism has never been reported but is scientifically possible. Botulinum toxin is colorless, odorless, and as far as is known, tasteless.

Botulinum toxin is the only Class A agent that is also a licensed medicine, a fact that complicates the design of defenses against possible bioweapon use of the toxin. In the United States type A and type B botulinum toxins are licensed for the treatment of blepharospasm, strabismus and cervical dystonia. However, both toxins are extensively used “off-label” to treat a range of more widely prevalent disorders that include spasticity (from stroke, head trauma, cerebral

Suggested Citation:"2 Assessing Our Understanding of the Threats." Institute of Medicine. 2002. Biological Threats and Terrorism: Assessing the Science and Response Capabilities: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10290.
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FIGURE 2-2 Infant Botulism Patient

palsy, multiple sclerosis, etc.), headache (both migraine and tension), low back pain, benign prostatic hypertrophy, and even facial wrinkles and other cosmetic concerns. Many other therapeutic uses of the toxin are under investigation.

Human botulism has several forms: foodborne, waterborne (potentially), inhalational, infant and wound. Only the first three varieties represent bioterrorist possibilities. Recognition of a botulism outbreak depends on the astute clinician who promptly notifies public health authorities. Clinically, botulism always begins in the muscles of the head, eyes, face and throat, most probably because of their relatively greater blood flow and density of innervation per unit muscle mass. The illness then progresses as a symmetric, descending flaccid paralysis. Severe cases are resource-intensive because they require antitoxin treatment, mechanical ventilation, gastrointestinal tube or intravenous feeding, and 24-hour intensive nursing care (Figure 2-2). Death results either from obstruction of the upper airway by unswallowable secretions and flaccid pharyngeal muscles or from complications of mechanical ventilation and intensive care.

Production and Delivery of Botulinum Toxin as a Bioweapon

Botulinum toxin is readily available as a bioweapon because of the relative ease with which its source, C. botulinum, may be isolated from nature or other-

Suggested Citation:"2 Assessing Our Understanding of the Threats." Institute of Medicine. 2002. Biological Threats and Terrorism: Assessing the Science and Response Capabilities: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10290.
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wise obtained. A minimal amount of laboratory equipment and microbiological expertise is needed to cultivate C. botulinum and concentrate its toxin to weaponutilizable material for oral use. With access to an autoclave, 500–1000 human oral lethal doses could be produced for a few hundred dollars. Making effective toxin aerosols would require greater scientific sophistication and resources.

The natural occurrence of foodborne botulism brought the existence of botulinum toxin to medical attention over 200 years ago; this route of toxin dispersal remains available to terrorists today. No instances of waterborne botulism have been reported, and standard potable water treatments rapidly inactivate the toxin. One instance of accidental inhalational botulism occurred approximately 30 years ago among veterinary technicians performing animal autopsies, thereby confirming the feasibility of the aerosol delivery route for humans (Arnon et al., 2001).

Present Methods of Controlling the Botulinum Toxin Threat

The botulinum toxin threat may be analyzed by means of a three-part schema consisting of sources (the “reservoir”), terrorists (the “vectors”) and potential victims (the “population”). The sources of C. botulinum are many and uncontrollable. They include rogue nations, unemployed former bioweapons scientists, open-access microbiological culture collections in various countries, the black market, and most fundamentally, the ubiquitous presence of C. botulinum in soils worldwide. The terrorists are by definition uncontrollable but may be thwarted in whole or in part by good a priori intelligence. Hence, the potential victims represent the only point of intervention in defending against bioweapon use of botulinum toxin. The interventions currently available to protect potential victims consist of 1) surveillance and early detection of toxin release, 2) immunization with botulinum toxoid, and 3) provision of antitoxin and supportive care.

The United States has a well-established botulism surveillance and detection system directed by CDC that was recently enhanced by daily reporting of all suspected botulism cases by the states. Availability of biosensors in key locations (e.g., airports) to detect aerosol release of botulinum toxin would enhance present surveillance capabilities.

Immunization of either civilian or military populations with botulinum toxoid is not a practical defense against weaponized botulinum toxin for several reasons. The existing pentavalent botulinum toxoid contains only A-E toxoids (i.e., it lacks F and G), and it remains an Investigational New Drug that requires informed consent before administration. The pentavalent toxoid is painful and highly reactogenic, and the full series of immunizations takes a year to complete (0-2-12-52 weeks). Finally, administration of toxoid deprives the recipient of access to any of the therapeutic benefits of medicinal botulinum toxin because

Suggested Citation:"2 Assessing Our Understanding of the Threats." Institute of Medicine. 2002. Biological Threats and Terrorism: Assessing the Science and Response Capabilities: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10290.
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the toxoid induces formation of toxin-neutralizing antibodies and the immunologic memory cells that produce them.

Antitoxin and supportive hospital care constitute the mainstays of the treatment of patients with botulism. Licensed and Investigational New Drug (IND) equine antitoxins exist in limited supply, as does an Investigational (IND) human-derived antitoxin developed for the treatment of infant botulism (Arnon, 1993). The licensed equine antitoxin is only a bivalent (anti-AB toxins) product, while the Investigational (IND) equine antitoxin is a heptavalent (anti-ABCDEFG toxins) product. The human-derived antitoxin (Botulism Immune Globulin Intravenous; BIG-IV) is a pentavalent product (anti-ABCDE toxins) whose license application, recently filed with the U.S. Food and Drug Administration, requested labeling only for its anti-A and B toxins activity because virtually all U.S. infant botulism cases are caused by these two toxin types.

Equine botulinum antitoxin is relatively inexpensive to make and can be produced in relatively large amounts. Its production requires a horse farm and staff, full-time veterinarians and a plasma-fractionation facility. At present a substantial reserve of frozen equine heptavalent plasma exists under CDC management, and efforts are underway to fractionate it into an available vialed product. However, equine antitoxin has a short (ca. 1-week) half-life, and its use carries the risk of serious allergic reactions to equine proteins.

Human-derived botulinum antitoxin (BIG-IV) is made from volunteers initially immunized with pentavalent botulinum toxoid for occupational safety reasons and then boosted with toxoid to obtain hyperimmune source plasma. BIG-IV has a long (ca. 1 month) half-life and a negligible allergic risk. The pool of potential plasma donors is small and insufficient to meet national needs. Treatment with human botulism antitoxin is effective as well as safe. In a randomized, placebo-controlled, double-blinded clinical trial, use of BIG-IV to treat infant botulism patients shortened their mean hospital stay by over 50%, from 5.7 weeks to 2.6 weeks, and reduced their mean hospital charges from approximately $130,000 to approximately $60,000 per case (California Department of Health Services, presently unpublished data).

Novel Methods for Containing the Botulinum Toxin Threat

The development of recombinant botulinum vaccines by the Department of Defense (DoD) for toxin types A, B, C, E and F began several years ago by expressing fragments of toxin in yeast; the vaccine against type A toxin is highly immunogenic and protective in mice (Byrne and Smith, 2000). DoD currently projects that a licensed recombinant vaccine product may be available in about 10 years. Its use would require special consideration because recipients would be deprived thereafter of the therapeutic benefits of medicinal botulinum toxin, one of which, post-head-trauma spasticity, is a likely consequence of combat

Suggested Citation:"2 Assessing Our Understanding of the Threats." Institute of Medicine. 2002. Biological Threats and Terrorism: Assessing the Science and Response Capabilities: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10290.
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situations. In addition, an adversary who knew the toxin types contained in the vaccine might choose to weaponize toxin types D or G in disregard of the current view that technical difficulties preclude doing so.

Recombinant human antitoxin antibody represents another way to control the bioweapon threat posed by botulinum toxin. A highly potent preparation of recombinant human (biotech) antibodies that neutralizes botulinum type A was recently reported (Marks, 2001), thus establishing “proof-of-concept” for these products. The phage-display technology that underlies the development of these recombinant human antitoxin antibodies also enables neutralizing human antibodies to the remaining six (B-G) botulinum toxin types to be rapidly created. The phage-display technology is versatile as well as fast, and it could be used to make human anti-anthrax antitoxin as well as the human Vaccinia Immune Globulin (VIG) needed to support the widespread administration of some or all of the 300 million doses of smallpox vaccine that the United States government recently decided to purchase.

Recombinant human antitoxin has several important advantages over vaccines, toxoids and existing equine antitoxins: 1) recombinant human antitoxin (RHA) provides immediate immunity when given, and there is no delay in waiting for the recipient’s immune system to make its own antibody in response to the vaccine or toxoid administration; 2) RHA can be made in the substantial quantity that is needed for the United States’ and its allies’ stockpiles; 3) RHA is very potent and has a long (ca. one-month) half-life; 4) RHA is safe and non-allergenic and so can be given multiple times; 5) RHA is practical for widespread use in civilian and military populations; 6) the licensure pathway for RHA is understood, because FDA has already licensed 10 monoclonal antibody products; and perhaps most importantly, 7) RHA can be used either prophylactically or therapeutically. The ability to provide RHA prophylactically to selected populations potentially at high risk (e.g., an overseas military force, Congress, etc.) and thereby provide with a single injection an immediate and long-lasting (6–12 months) immunity to all 7 botulinum toxin serotypes might effectively remove weaponized botulinum toxin from an adversary’s arsenal, simply because the opponent would know in advance that the toxin bioweapon would not work.

Second-Generation Botulinum Toxin Bioweapons

Modern molecular biology techniques enable the gene for the enzymatic “light chain” of botulinum toxin to be fused with the gene of a targeting molecule that will take the toxic combination to various non-neuronal cell types. Secretory and other cell types that contain the substrates for botulinum toxin “light chain” may be found in the pancreas, liver, thyroid, adrenal and heart (Rosetto et al., 1996). Thus, botulinum toxin “light chain,” if redirected to the pancreas, could block the secretion of insulin and thereby cause epidemic diabetes. Such

Suggested Citation:"2 Assessing Our Understanding of the Threats." Institute of Medicine. 2002. Biological Threats and Terrorism: Assessing the Science and Response Capabilities: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10290.
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recombinant, “second-generation” toxic molecules have been made, usually with the goal of finding an improved anti-cancer or equivalent therapeutic agent.

Also, the genes for the both the light chain and heavy chain of botulinum toxin have been engineered for high efficiency expression in Escherischia coli for the stated purpose of enhancing the production of medicinal botulinum toxin, which presently commands a market of several hundred million dollars per year. However, virtually all humans carry E. coli as part of their normal intestinal microflora. Strains of E. coli that commonly cause diarrhea are often spread through contaminated foods and may be further disseminated by the fecal-oral route. An enteropathogenic strain of E. coli engineered to express the genes for both chains of botulinum toxin would have the potential to eliminate much of the human race, a potential that underscores the need for prompt development of effective countermeasures to botulinum toxin.

Priorities for Preparedness

The major preparedness needs for the United States can be arranged into public health, clinical medicine and research categories (Table 2-3). The three most important immediate needs are 1) fractionating and vialing the 7,000 liters of frozen heptavalent equine plasma for the National Pharmaceutical Stockpile, 2) ensuring an adequate surge capacity for ventilators and mobile intensive care units, and 3) rapidly developing a heptavalent human recombinant antitoxin to provide an unlimited supply of the key defensive commodity.

TABLE 2-3 Priorities for preparedness

Public Health

Fractionate, vial and IND the 7000l of frozen Army-CDC equine heptavalent plasma

Develop more surge capacity at all levels (federal, state, local); both laboratory and epi demiology

Develop rapid in vitro toxin detection methods

Produce human recombinant antitoxin for stockpiling as well as current use

Clinical Medicine

Improve communications with public health colleagues for early detection

Ensure adequate surge capacity for ventilators and mobile intensive care units and their staffing

Produce human recombinant antitoxin for stockpiling as well as current use

Research

Begin development of human recombinant antibodies against toxin serotypes B-F

Begin scale-up production of existing human recombinant anti-A antibodies to establish pathways and capabilities

Obtain intelligence on toxin-derived bioweapons research in other countries to enable defensive recombinant human antitoxin development

Suggested Citation:"2 Assessing Our Understanding of the Threats." Institute of Medicine. 2002. Biological Threats and Terrorism: Assessing the Science and Response Capabilities: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10290.
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Summary and Conclusion

Existing in vitro technologies could produce the stockpiles...needed both to deter terrorist attacks and to avoid the rationing of antitoxin that would be required in a large outbreak of botulism. A single small injection on oligoclonal human antibodies could, in theory, provide protection against toxins A-G for many months. “Until such a product becomes available, the possibilities for reducing the population’s vulnerability to the intentional misuse of botulinum toxin remain limited.” (Working Group on Civilian Biodefense, 2001)

RESEARCH CONSIDERATIONS FOR BETTER UNDERSTANDING OF BIOLOGICAL THREATS

Kenneth Alibek, M.D., Ph.D., Sc.D.

Distinguished Professor of Medical Microbiology and Executive Director, Center for Biodefense

George Mason University

President

Advanced Biosystems, Inc., a subsidiary of Hadron, Inc.

Because they are our best protection against infectious disease, it is necessary that we continue to develop, approve, and introduce new vaccines against many naturally occurring infectious diseases and against some biological weapons threat agents. Yet the U.S. tends to focus its discussion of bioweapons vaccines and therapeutics on only a handful of potential agents, even though the former Soviet Union is known to have developed at least thirty biological agents for use as bioweapons. Alarmingly, it takes only two to three years to develop a biological weapon but, in the best-case scenario, eight to ten years to develop a new vaccine.

It has been suggested that live vaccines are too reactogenic for general use. But, for the purpose of boosting our biodefense arsenal, perhaps this issue requires reevaluation. In Russia, for example, all major vaccines—including anthrax, plague, and tularemia—are live vaccines. The United States had a good live plague vaccine and has a very strong live tularemia vaccine. The latter may not be approved for human use, but its protective efficiency is very high. Bioengineered vaccines are another possibility.

The use of alternate methods of vaccine administration must also be addressed. Biodefense vaccine administration techniques should not only be safe but must also provide for the vaccination of large numbers of people in a very short amount of time. Currently the U.S. focuses on injection vaccines, but there have been many studies on aerosol, inhalational, and oral vaccines. An aerosol plague vaccine, for example, can be used to immunize more than 1,200 people per hour, while a single operator can administer injection vaccines to only 20 to 30 people per hour. Inhalable plague, anthrax, and tularemia vaccines have all

Suggested Citation:"2 Assessing Our Understanding of the Threats." Institute of Medicine. 2002. Biological Threats and Terrorism: Assessing the Science and Response Capabilities: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10290.
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been extensively studied in Russia and have not shown any significant side effects. This type of vaccine could work both systemically and locally, for example to induce mucosal immunity in the respiratory tract.

However, though vaccines have proven extremely effective against infectious diseases in general, they are of limited utility in the defense against infections caused by biological weapons. Vaccination is a successful defense only when the target population is well-defined and can be identified well in advance of an attack; when the biological threat agents in the enemy’s biological weapons arsenal are known; when vaccines for those agents have already been developed; and when the biological agents used are not genetically altered strains capable of circumventing a vaccine. Most military and nearly all terrorist scenarios will not meet all of these criteria. Therefore, vaccination of the general population against biological weapon agents is neither feasible nor advisable. In the context of biological weapons, the best use of vaccination is for troop protection, where both the target population and potential threat are more defined.

Other, non-vaccine biodefense products need to be more seriously considered. In particular, over the past twenty years there has been extensive research on immunomodulators and their role in protecting against viral and bacterial pathogens. Although several such products have been developed that could potentially resolve many of our biodefense issues, none of them have been introduced yet into the field of biological weapons defense. In a biological attack, the target population would likely be large and poorly defined, the scale or even the fact of the attack may not be immediately apparent, and the biological agent used in the attack may not be immediately identified. For either military or terrorist use of biological weapons, creation of an aerosol cloud—usually accomplished by explosion or spraying—is by far the most effective mode for deploying biological weapons. This method can be used against large target areas and with practically any biological threat agent. Therefore, effective medical defense against biological weapons must incorporate protection against aerosol deployment. The nature of a biological weapons attack also dictates that the most successful medical defenses will be prophylactic, rapid-acting, long-lasting, effective against a broad spectrum of threat agents, and relatively easy to deliver to a large population.

Finally, we need to re-examine our knowledge about the pathogenesis of bacterial and viral infections. For example, we still refer to the three major virulence factors in any discussion of anthrax. However, many other virulence factors may exist. My laboratory has conducted hundreds of experiments on anthrax lethal toxin, and thus far we have found no evidence whatsoever that it is capable of inducing normal healthy donor immune cells to produce any cytokines involved in the development of septic shock. Human endothelial cells are, however, relatively susceptible to the effect and the action of lethal toxin. It is becoming apparent that current theories on the role of anthrax toxin must be reexamined and revised. Our experiments have indicated that other overlooked

Suggested Citation:"2 Assessing Our Understanding of the Threats." Institute of Medicine. 2002. Biological Threats and Terrorism: Assessing the Science and Response Capabilities: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10290.
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anthrax virulence factors exist, including cell wall components and (possibly) hemolysins. Our work has indicated that the only factor of the anthrax bacterium capable of consistently inducing the mediators of septic shock seen in late-stage anthrax infection is a component of the cell wall skeleton, not lethal toxin, and that there exist many other overlooked exogenous and endogenous mediators which contribute in the development of anthrax sepsis and septic shock. Although these are preliminary data with which we are not able to make any final conclusions, they do highlight the need to re-examine the pathogenesis of anthrax. They may also explain why people die when antibiotics are administered in the late stages of infection: these cell wall skeleton components are very powerful inducers of septic shock mediators, and their concentration remains very high in the bloodstream after bacterial death. The preliminary work we have conducted to this point has led us to the belief that the most successful strategy for anthrax treatment would be dependant on the stage of infection (post-exposure, lymphatic, systemic, or late-stage).

It is our recommendation that a task force be established to more carefully analyze the events that occur during anthrax infection. It is very important that we re-evaluate our knowledge of pathogenesis and identify what we have missed in the field of protection and treatment of infectious diseases caused by biological weapons.

AEROSOL TECHNOLOGY AND BIOLOGICAL WEAPONS

C.J. Peters, M.D.

Professor, Departments of Microbiology and Immunology and Pathology

University of Texas Medical Branch at Galveston

Richard Spertzel, VMD, Ph.D.

Former head of the biological weapons inspection team for the UN Special Commission on Iraq

William Patrick III

President

Biothreats Assessment Co.

The threat of international terrorism to this country and others has never been as serious as it is today. The U.S. abandoned its program for offensive biological warfare in 1968, but the successful effort to weaponized infectious organisms and toxins should have educated the U.S. government to their dangers. The revelations that the Soviet state had a more extensive and similarly successful undertaking (Alibek, 1999) only increases the likelihood that the development of weapons of mass destruction lies within the reach of others. The

Suggested Citation:"2 Assessing Our Understanding of the Threats." Institute of Medicine. 2002. Biological Threats and Terrorism: Assessing the Science and Response Capabilities: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10290.
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extensive programs used by these two states are often cited as hurdles too high for other countries to surmount, but it must be remembered that they worked with multiple agents and manufactured literally tons of product in a time before many of today’s advances in biology and fermentation technology. The Gulf war brought the potential of other states to produce biological weapons home, but the weapons were not used in spite of the argument that they are intensely destabilizing (Haselkorn, 1999). The spate of anthrax laced letters in 2001 has led to a re-awakening of concern.

Numerous commissions have reviewed the threat of bioterrorism (Countering the Changing …, 2000; Second Annual Report, 2000) in recent years and uniformly concluded that the US was vulnerable and that the likelihood of such an event was high. Nations suspected of having offensive biological warfare programs have been named by the Office of Technology Assessment (1993) and these same states are often also identified as terrorist sponsors. In light of these agreed-upon threats, why has there been so little concern about this possibility in many quarters and why has so much surprise been expressed over the outcome of a handful of letters containing anthrax spores dispatched through the mail? One important component is the lack of familiarity with the concepts which were the pillar of the old US biowarfare program and also the Soviet program, particularly as refers to the danger of aerosols. Another reason may be that no one expected that the manufacturer of such a deadly powder as was placed in Congressional letters in autumn of 2001 would employ an envelope as a delivery route rather than using a more stealthy and lethal dissemination system. This complacency was reinforced by the large number of “anthrax” powder hoaxes that have occurred over the last few years, a social phenomenon worthy of study in itself.

This short essay attempts to outline why we should be concerned about use of biological weapons in terrorism, why some scenarios are more dangerous than others, and some general observations concerning what we could do to combat bioterrorism.

Nature of the Threat

When one considers the ways in which BT might be carried out, the first rule should be that we do not know who the possible terrorist will be, his or her motivation, or the wherewithal that may be available for the attack. Thus, attacks to incapacitate selected persons to gain attention, or to cause serious illness for revenge might have a very different approach than attacks designed to cause mass casualties. An effort by a disgruntled clinical laboratory worker could have a very different scope than one by a well-funded non-state organization or a state-sponsored group. Parenthetically, the failure of the Japanese Aum Shinriko cult to succeed with biological terrorism should not provide much comfort concerning the need for state sponsorship given the manifest ineptitude of the perpetrators (Smithson, 2001).

Suggested Citation:"2 Assessing Our Understanding of the Threats." Institute of Medicine. 2002. Biological Threats and Terrorism: Assessing the Science and Response Capabilities: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10290.
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The second issue is the dissemination of factual information concerning the real dangers of such an attack. Some would argue that the less said the better. Unfortunately for this approach, US society does not respond without facts and public opinion supporting programs. This means that the actual dangers must be explained to the public and responsible political leaders without inflammatory rhetoric or divulging detailed methods for the assaults, and we must take the chance that plain-speaking might motivate some to undertake the very actions we are trying to prevent. Furthermore, discussion of the facts may help the public, media, and health authorities respond in a calmer and more rational fashion than was observed with the aerosol anthrax attacks of 2001.

If we consider the methods by which microbes might be delivered to a target population there are multiple routes. Direct inoculation, infection of natural vectors or reservoirs and loosing them on the target population, or infection of a few persons and counting on their spreading the infection even further are some possibilities. If we focus on terrorist strategies that can inflict mass casualties none of these possibilities is highly feasible today with the exception of the use of smallpox, a virus that is well-known to spread from human to human after a long and successful career in that evolutionary niche. If we conclude that other organisms must be delivered directly to the target host, we should also consider water, food, and aerosols as potential vehicles of infection. Contaminated water from wells and storage containers has been associated with outbreaks of disease, but the odds are against this approach for causing mass casualties because of the dilution factor, chlorination, and the usual treatment of water before consumption in this country. Food-borne pathogens have caused many outbreaks in the US and are a major cause of morbidity and mortality. Even though our distribution system is highly centralized, food items are usually not consumed synchronously except at special events. Improved surveillance of food-borne disease and newer methods of molecular typing of offending organisms should provide a counterweight to the wide dissemination of contaminated food. If a few cases are recognized and traced to a food source, warnings and recalls may well serve to protect us.

The over-all societal impact of any one of these dissemination methods could be considerable, regardless of the actual health damage. We have case studies already, including non-lethal Salmonella infection of a few hundred citizens (Torok et al., 1999), Sarin gas attacks with only 20 deaths (Smithson, 2001), food tampering, and most recently anthrax delivered by letter or even anthrax hoaxes. Aerosols, however, are an important route of attack because of their ability to cause really large numbers of casualties. The deficiencies of aerosols such as dependence on metrological conditions, the unsuitability of most organisms for air-borne spread, and the technical demands may be counterbalanced in the hands of skillful perpetrators by the advantages of stand-off attack, silent spread of incapacitating or lethal disease, and wide-area coverage.

Suggested Citation:"2 Assessing Our Understanding of the Threats." Institute of Medicine. 2002. Biological Threats and Terrorism: Assessing the Science and Response Capabilities: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10290.
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Aerosol Infections

Aerosol infection has long been recognized as a route of microbial transmission. Measles, influenza, smallpox, and tuberculosis are all known to be transmissible between patients by aerosols and additionally in the laboratory tularemia, rickettsiae, viral hemorrhagic fevers, and many other agents are threats to the microbiologist (DHHS, 1999). Decreases in human tuberculosis and virtual elimination of diseases such as measles and smallpox from common medical experience as well as development of enhanced methods of protecting laboratory workers (ironically using technology developed during the US biowarfare program) have resulted in a loss of appreciation for this route of infection, but the US and Soviet BW programs were largely based on the properties of selected agents for causing large scale infection of human populations under the proper metrological conditions and with carefully developed methods of dissemination. Moreover, the terrorist could attack enclosed environments such as stadiums or large buildings in order to eliminate the meteorological factors that degrade a small particle aerosol.

Biological agents have not seen widespread use in warfare so it is not surprising that there is skepticism as to their efficacy. It is not appreciated that the US program in offensive biological warfare (terminated on November 28, 1969) rigorously tested each step in the link between a microorganism selected by several criteria and the delivery of a credible biological attack (US Congress, OTA, 1993a, b; Rosebury, 1947; Hersh, 1968; McDermott, 1987; Cole, 1997; Sidwell et al., 1997; Patrick, 2001). Tularemia would be an excellent example because extensive information is available in the published literature, congressional hearings, and popular press. From the initial isolation of the organism by Francis and coworkers, it was notorious for causing infections in the laboratory, a frequent hallmark of aerosol infectivity. The aerosol properties were intensively studied and methods were found to enhance its stability in storage and in aerosols. Animals and later humans were challenged with graded doses of the bacterium delivered in different particle sizes to establish the quantitative properties of these aerosols. Open air dissemination was mimicked using a surrogate organism, Serratia marscens, and this confirmed that an organism with the aerosol stability and infectivity of Francisella tularensis could cause mass casualties over large geographic areas provided attention was given to metrological conditions. The areas affected could reach thousands of square kilometers. The resulting environmental transmission from the large number of different nonhuman mammalian and arthropod species that would be infected in a tularemia attack cannot be evaluated. Thus, there is little doubt that large numbers of human casualties could be caused by efficiently weaponized organisms readily available from nature.

A relatively small number of agents are suitable for causing literally thousands or hundreds of thousands of casualties, and this may provide a basis for

Suggested Citation:"2 Assessing Our Understanding of the Threats." Institute of Medicine. 2002. Biological Threats and Terrorism: Assessing the Science and Response Capabilities: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10290.
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prioritization of medical and other measures to deny the intent of terrorists. The agents in Table 2-4 and Table 2-5 contain a substantial proportion of the organisms capable of achieving mass casualties. Biological toxins, even the highly potent botulinum toxin, and chemical agents are absent because of their relatively low potential to cause casualties whether evaluated on the basis of purified agent or the basis of the likely highest concentration practically achievable. It is impossible to list every possible agent and there are always discussions among experts as to whether some should be added or omitted. However, there is general agreement that those in Table 2-4 comprise the most deadly and the most likely to seriously destabilize governmental function and civil society. All these agents were weaponized by the USSR or the U.S. (pre-1969) or both. They grow to excellent titer for more efficient manufacture and they are highly stable and infectious in aerosols when properly prepared. Agents in Table 2-5 are of lesser threat, particularly those at the bottom of the list. Some, such as typhus or glanders may well belong in Table 2-4. Toxins are inherently of lesser efficiency because they cannot match the killing or incapacitating power of the highly infectious organisms; the toxins have to produce their effects as delivered, but the infectious agents grow and produce toxins or other effects in the recipient’s body.

According to a WHO scenario several infectious agents would be expected to produce 35,000 to >100,000 casualties if 50 kg were delivered in a line source and carried down-wind over a populated area. In the case of some of the more stable agents, down-wind reach would exceed 20 km. The Office of Technology Assessment (1993) has published similar figures. It must be borne in mind that the US and Soviet programs prepared literally metric tons, not kilograms, of agent and that appropriate devices for delivering line sources or multiple overlapping point sources were available (Alibek, 2001; Sidell et al., 1997). Impact on the infected members of the population would depend on the agent used and the nature of the response (for example, alacrity of recognition of initial patients, public health and medical infrastructure, vaccine and antibiotic stockpiles).

The additional impact that might be possible through modification of naturally occurring organisms by methods well within the reach of simple biotechnology including induction of antimicrobial resistance, enhancement of viru-

TABLE 2-4 Some diseases and their causative agents considered to be aerosol bio logical warfare threats capable of causing mass casualties (CDC Category A Biologi cal Agents/Diseases)

Variola major (Smallpox)

Bacillus anthracis (Anthrax)

Yersinia pestis (Plague)

Francisella tularensis (Tularemia)

Viral hemorrhagic fevers (Filoviruses, Arenaviruses, and Rift Valley virus)

Suggested Citation:"2 Assessing Our Understanding of the Threats." Institute of Medicine. 2002. Biological Threats and Terrorism: Assessing the Science and Response Capabilities: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10290.
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lence by addition of toxin genes, or selection of more stable or virulent organisms is formidable. Issues surrounding the more extensive engineering of threat agents are beyond the scope of this discussion, however, it is important to note that the potential exists, but that there would probably be a need for human testing of any resulting candidate.

Properties of Small Particle Aerosols

The basic properties of aerosols must be understood to appreciate the way in which such weapons could be used. The optimum aerosol particle size is thought to be 1–5 microns. This size provides two critical properties: the particles do not settle out over a several hour time period but rather are truly airborne and are carried on wind currents or through heating, ventilation, and air conditioning systems (HVAC) and they are of an optimum size to reach the terminal respiratory bronchiole or alveolus of humans and deposit in those critical areas to set up infection. Generation of small particle aerosols requires energy as evidenced by classical examples such as laryngeal tuberculosis, the wide dissemination of rubella by a disco singer (Marks et al., 1981), or the persistent cough of the index case of a nosocomial Lassa fever outbreak (Carey et al., 1972). Such particle sizes can be achieved intentionally by generating a liquid aerosol with a spray device or by using an appropriately manufactured powder. The dissemination system for a liquid must have the correct relationship between viscosity and solids content of the liquid, air pressure, orifice diameter, and other variables to attain the critical particle size, as well as have the proper stabilizers in the solution to assure that infectivity is not lost. The dry powders are difficult to manufacture, but they are extremely dangerous because they can be prepared so as to aerosolize with minimal energy input and can be manufactured in very fine particle sizes. If the skills to prepare these particles are avail-

TABLE 2-5 Some agents often mentioned as potential aerosol biological warfare or biological terrorist threat

Tick-borne flaviviruses

Typhus and other Rickettsiae

Glanders

Alphaviral encephalitidies

Brucellosis

Q fever

Melioidosis

Nipah virus

Staph Enterotoxin B

Ricin

Tricothecene mycotoxins

Suggested Citation:"2 Assessing Our Understanding of the Threats." Institute of Medicine. 2002. Biological Threats and Terrorism: Assessing the Science and Response Capabilities: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10290.
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able, it may also be possible to formulate encapsulated weapons of greater biological stability.

It is obvious that the preparation and testing of such weapons requires microbiological skills to attain the high concentrations of organisms needed. Equally as important is the expertise for dissemination of liquid or powder aerosols in a stable form in the correct particle size. Such capabilities may be available from many people in different walks of life. Nonetheless, assembling the needed skill sets implies an organization with some resources, particularly as one moves into the powders and into greater quantities of agent. Practice or trials would be important to assure success, although, as the saying goes, “the proof of the pudding …”. Persons with previous experience in offensive biowarfare programs could be extremely valuable resources to such an endeavor.

When these aerosol clouds are generated, there is a period of instability and larger particles or agglomerates fall out near the dissemination device with considerable surface contamination possible. Once the small particle aerosols are formed, they will move with wind currents and traverse the landscape, being gradually diluted by mixing and decay. Because of their dependence on wind currents, aerosols may be diverted from their planned target; warming of the earth’s surface after sunrise will result in their being carried to higher levels of the atmosphere unless inversion conditions are present. Loss of infectivity by biological decay will also occur, depending on stabilizers in the suspension medium, ultraviolet intensity, humidity, and temperature. They will enter buildings through HVAC systems, but the urban landscape profile may result in extensive disturbances of air flow. The ultraviolet light sensitivity of most of the organisms, combined with the meteorological needs, will favor the use of an evening, night time, or early morning, attack if done outdoors. Terrorist attacks may also be directed toward buildings or enclosed stadiums, making the air conditioning systems the obvious route of the delivery of the aerosol.

The extreme “fluffiness” or ease of aerosolization of the most dangerous powders is difficult to imagine. The material in the Hart Senate office building seems to offer an excellent example: all present in the room when the offending letter was simply opened had spores in their noses when tested 4 hours later; anthrax spores traveled to adjacent rooms through the HVAC; extensive surface contamination was present. Subsequent examination of the powder showed it to have a very high concentration of anthrax spores (reportedly 1012/g), finely dispersed particles, and it was readily aerosolized with the slightest disturbance. Undoubtedly, without antibiotic prophylaxis most of those in the room would have suffered inhalation anthrax. Had this material been introduced clandestinely into the air conditioning intake, there would have been no warning until the first cases were recognized, perhaps too late for therapy. The magnitude of the exposure can be seen from studies done by the Canadian Defense Forces (Kournikakis et al., 2001) in a simulation using only 1/10th the number of spores and 1/20th the estimated quantity of a readily aerosolized powder containing

Suggested Citation:"2 Assessing Our Understanding of the Threats." Institute of Medicine. 2002. Biological Threats and Terrorism: Assessing the Science and Response Capabilities: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10290.
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Bacillus globigius as a surrogate for Bacillus anthracis. In less than one minute, spores spread throughout the room in similar concentrations as observed at the site of the envelope opening. Testing of the filter from the respirator worn by the subject opening the letter yielded 80,000 infectious units, equivalent to an estimated 15-320 human LD50 for anthrax spores.

These principles governing airborne particles in the 1–5 micron range are essential in our response to the possibility of an aerosol attack. First of all, buildings with their HVAC may provide little protection against small-particle aerosols if currents of air bring a widely disseminated agent into the zone of their air intake. Indeed, the HVAC systems may provide a particular vulnerability to bioterrorism. Secondly, environmental sampling of surfaces or clothing in areas exposed to such aerosols or nasal swabs of potentially exposed persons will not predict if a person was infected with any certainty; finding agent in these situations demonstrates that an attack has taken place. Thirdly, surgical masks and similar defenses are not effective against such small particles and only give a false sense of protection; highly efficient masks that can protect against small particles (e.g., N100 masks designed for medical staff working with tuberculosis patients) and which are properly fitted on trained personnel are needed (Lowe et al., 1999). Obviously, protective gear must be worn during the risk period, but these small-particle aerosols are odorless and invisible to the eye so the general utility of personal protective gear is limited.

Secondary Aerosols

The issue of secondary aerosols is an important one. Infectious agents on solid surfaces such as soil, counter tops, machinery are thought not to be subject to aerosolization unless considerable energy is applied. Studies of Bacillus spores have shown aerosols intentionally deposited on the ground are very difficult to re-suspend with ordinary traffic or even intentional beating of the surface (Patrick, 2001). Soil subjected to high air flow yields few particles in the dangerous 1–5 micron range (Chinn et al., 1990). These field evaluations show that even when the contamination of a surface reaches 107/meter2 the concentration above the surface, even with considerable disturbance, will be extremely low. Thus, in a field biowarfare situation there is little danger from secondary aerosolization.

There is little experience with transfer of infectious powders from one solid surface to another or with the deposit of larger clumps of highly aerosolizable particles on hard solid surfaces. Surrogate infectious agents, fluorescent tracers or radioactive particles predict that highly concentrated biological agents (titers >109/g and perhaps as much as 1012/g) will extensively contaminate surfaces they impact and, if viable, pose a contact risk. Large quantities of organisms from dangerous powders can result in short-term presence of organisms in the external nares of exposed persons and the fall-out of larger particles can lead to environmental contamination near the site of dissemination. The possibility of secondary

Suggested Citation:"2 Assessing Our Understanding of the Threats." Institute of Medicine. 2002. Biological Threats and Terrorism: Assessing the Science and Response Capabilities: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10290.
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aerosols in this situation is thought to be small, but application of high energy sources or the presence of physical clumps of particles could be problematic.

The risk of aerosol infection to an exposed human would depend on the amount of material aerosolized and the infectious dose for humans. The actual amount of tularemia or Q fever required to infect 50% of exposed humans is known and is on the order of 1–10 organisms. For other organisms such as anthrax it is necessary to extrapolate from cynomolgus monkeys or other experimental animals. In this case the lethal dose for 50% of animals is 8,000 spores by aerosol. The LD50 is determined by exposing animals to graded doses of the infectious agent and calculating the linear relationship between the logarithm of the dose increment and the increase in response of the target animals (Finney, 1964). This is usually done between 20–80% lethality and the LD50 calculated. In fact the linearity can probably be extrapolated further to furnish at least an approximation of the risk from lower doses; in the case of anthrax, published values for the slope (Glassman et al., 1965; Chinn et al., 1990) suggest that inhaling a dozen spores could be risky in a small percentage of the population.

These concepts are important to the practical management of situations in which a suspicious powder is involved. The physical properties of a readily aerosolized powder will be recognized by an experienced observer or by laboratory analysis. An ordinary dried culture of, for example, anthrax will not pose a great hazard beyond the readily recognized and treated cutaneous anthrax. Decontamination of a building needs to address the dangerous states of the contaminating organism. Safety is the goal, not “sterility”. In the case of anthrax spores, significant quantities of aerosolizable particles is the criterion. Sterility is less important than being certain that any residual infectivity is earth bound.

Relative Importance of Different Agents

Consideration of the different bioterrorism agents and some of their properties is the first step to prioritize defenses against them. Each has different properties as we see them today and thus each presents different threats and different opportunities for control. This discussion has been cast in terms of the worst case scenarios (effective broad-scale aerosol dissemination) but we must recognize that, although protection against this situation is important, the most likely eventuality is a less extensive or less successful attack. Fortunately, attention to the worst case is a step toward the more general solution, although the lesser eventuality should also be in the mind of planners.

It is also important to note that biodefense efforts meld with the general struggle against infectious diseases. For example, strengthening the public health system will provide benefits regardless of whether a bioterrorist attack occurs. Money spent on communications within the public health system is long overdue. Planning will help in disaster response, regardless of the nature of the event. Perhaps much of the money spent on increasing smallpox vaccine stocks

Suggested Citation:"2 Assessing Our Understanding of the Threats." Institute of Medicine. 2002. Biological Threats and Terrorism: Assessing the Science and Response Capabilities: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10290.
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will eventually be “sunk costs” but we should not regard research and vaccine development on other agents as anything other than an benefit for human-kind.

Smallpox provides a threat whose consequences are simply unacceptable, regardless of the probability of its use. Therefore we must develop clinician awareness, diagnostic systems, and stockpiles of existing vaccine that give us a validated countermeasure to deploy in case of attack. Whether additional antiviral drug and vaccine development is justified is a matter of prioritization against other threats.

Anthrax also is a special case. It is widely distributed in nature and thus readily available to terrorists in virulent form. The spores are extraordinarily stable on storage and in aerosols obviating many of the terrorist’s research needs to develop an effective weapon. Inhalation anthrax is a fearsome disease if not treated early with effective antibiotics, and production of antibiotic-resistant anthrax is readily achieved.

Plague and tularemia are both severe diseases but they require another level of sophistication in weaponization. Their cultivation in virulent form and their dissemination in stable aerosols is more difficult than for anthrax. The viral hemorrhagic fevers are essentially without therapy, have severe psychological impact, and carry a high mortality. Their production is still more difficult, but the technology is readily accessible to an experienced microbiologist (Peters, 2000).

When considering the impact of limited or massive dissemination of the agents in tables 2-4 and 2-5, one must factor in the disruption of the health care system, the role of antibiotic resistance, the fear-factor in the population and medical staff, as well as the state of defensive preparations. One element that is often neglected is the influence of a communicable disease on travel and commerce. Any of these diseases could lead to severe disruptions in the free travel of U.S. citizens and others within the US and in international air transport systems. If the agent is also an agricultural pathogen, then internal movement of animals would be frozen and exports would be stopped, resulting in even more severe economic consequences.

Strategies to Confront the Problem

Any attempt to deal with BT should consider the entire spectrum of responses, including state and local organization supported by a comprehensive federal plan. The public health system will be the back-bone, but there will have to be participation of the entire society. Recognition by the clinician, laboratory diagnosis, and mobilization of countermeasures will all play a part. As noted above, the strategy should be tailored to each agent or group of agents.

It must be emphasized that environmental detection and patient diagnosis of the specific agent employed are keystones of an improved response to the threat. Detection suffers from the need to be active at the time and site of an attack, so economics will probably limit its future usefulness to selected high risk venues.

Suggested Citation:"2 Assessing Our Understanding of the Threats." Institute of Medicine. 2002. Biological Threats and Terrorism: Assessing the Science and Response Capabilities: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10290.
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Diagnostics are, in principle, more focused and also require the suspicions of informed clinicians; widely deployed they are a very significant expense. An additional demand on detection and diagnostics is the recognition of subversion of our defenses by inducing resistance to anti-infectives or other protective modalities.

Vaccine approaches are suitable for selected at-risk groups, particularly for specific high-priority BT agents. However, specific vaccines are not general remedies for the threat to the civilian population. The expense and difficulty of administration and the inevitable side effects will limit their widespread use. They remain important elements of our response in selected populations and specific circumstances.

Anti-infective drugs could be a very effective response if problems of drugdevelopment, drug-resistance, safety and efficacy testing, stockpiling, and distribution can be solved. Other supportive measures directed to bacterial toxins or the over-exuberant inflammatory responses induced by some viruses could be useful, as well. Further definition of the Toll-like receptor family could open the way to broadly protective remedies that could be used in the event of BT attacks.

Some agents pose sufficient problems to demand immediate and thorough attention. Smallpox, because of its track record of interhuman transmissibility and high case fatality, is clearly a first-echelon target. Anthrax, because of its ease of weaponization, deserves attention to the development of more effective therapy beyond antibiotics. Antitoxic strategies at the level of the toxin molecules as well as their down-stream effects should be developed in a very short time frame. Furthermore, the terrorist use of antibiotic-resistant strains should be anticipated.

Plague and tularemia might seem to be resolved in principle because of the existence of effective antibiotics, but their relatively short incubation periods place high demands on availability of effective antimicrobials and the facility with which antibiotic resistance can be induced has important implications for defensive strategies. This is complicated because the log-normal distribution of incubation periods is “front-loaded” (Sartwell, 1950) and because late treatment can fail even though the bacteria are eradicated.

The viral hemorrhagic fever agents would induce widespread fear and even panic among the both general population and health-care providers. The arenavirus drug ribavirin should be stockpiled in modest amounts in the mean while, but more general strategies against the arenaviruses and other viral threat pathogens should be pursued.

Of course intelligence information and any dissuasion afforded by international agreements would be most welcome. We clearly cannot depend on these modalities to protect us completely. Many of the agents are widely available and so measures designed to limit their access are illusory in their effectiveness; anthrax is a case in point. However, limiting access to certain agents such as Ebola, Marburg, and smallpox viruses should be pursued. The equipment needed to produce limited amounts of biological agents is readily available and

Suggested Citation:"2 Assessing Our Understanding of the Threats." Institute of Medicine. 2002. Biological Threats and Terrorism: Assessing the Science and Response Capabilities: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10290.
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we cannot control or monitor access, but perhaps we can develop measures to track high output equipment and the movement of particularly sensitive expertise and genetic material.

A strong research program and the industrial base to develop promising research leads into practical human countermeasures will be the best defense. One of the impediments, in addition to the perennial need for funding, is the lack of suitable containment laboratories. Furthermore, the diminution of expertise and suitable laboratories to study infectious aerosols is alarming. Another variable in play is the concern for limiting dissemination of research results; we have to be very careful not to suffocate our defensive effort with excessive secrecy unless the controls can be shown to add to our safety.

REDUCING THE RISK: FOODBORNE PATHOGEN AND TOXIN DIAGNOSTICS

Susan E. Maslanka,* Jeremy Sobel,* and Bala Swaminathan*

Foodborne and Diarrheal Diseases Branch Division of Bacterial and Mycotic Diseases

National Center for Infectious Diseases

Centers for Disease Control and Prevention

Estimates of Foodborne Illness in the United States

The spectrum of illnesses caused by consumption of contaminated foods may range from self-limiting mild gastroenteritis to life-threatening neurologic, hepatic and renal syndromes (Mead et al., 1999). recently estimated the number of illnesses, hospitalizations and deaths in the United States using data from various national surveillance systems. Their estimates indicate that contaminated foods cause approximately 76 million illnesses, 325,000 hospitalizations and 5,000 deaths in the United States each year. The economic burden is estimated to be 9 to 32 billion U.S. dollars. More than 200 known diseases are transmitted through foods; the agents of foodborne illnesses include viruses, bacteria and their toxins, fungi and their toxins, parasites, poisonous plant components, marine biotoxins, heavy metals and possibly, prions. However in 82% of foodborne illnesses the identity of the pathogen is unknown. Of 1,500 deaths each year due to known pathogens, 75% are caused by Salmonella, Listeria monocytogenes and Toxoplasma.

*  

The information provided in this paper reflects the professional view of the authors and should not be construed as an official position of the U.S. Department of Health and Human Services or the Centers for Disease Control and Prevention.

Suggested Citation:"2 Assessing Our Understanding of the Threats." Institute of Medicine. 2002. Biological Threats and Terrorism: Assessing the Science and Response Capabilities: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10290.
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Changes in the Foodborne Disease Outbreak Scenario

The epidemiology of foodborne diseases has undergone profound changes in the last 2 decades. Some factors influencing this change are the global distribution of food supplies to meet increasing consumer demands for greater diversity of foods, centralization of food production, processing and distribution to improve efficiencies and reduce costs, demographic changes occurring in industrialized nations that have resulted in increases in the proportion of the population with heightened susceptibility to severe foodborne infections, changes in food-related behavior of consumers and dramatic increases in world travel (Kaferstein et al., 1997; Swerdlow and Altekruse, 1998). One negative effect of high-degree consolidation of food production, processing and distribution is that food safety-related failures may affect large numbers of people over large geographic areas and may have disastrous public health consequences. Because of the explosive increases in international travel, new and emerging pathogens from one corner of the world are able to arrive at a location thousands of miles away in a matter of hours. The transcontinental flights themselves offer ample opportunities for transmission of foodborne disease during travel (Tauxe et al., 1987). In addition, the manufacturers and/or the distributors of the contaminated food are likely to encounter dire financial and public relations consequences following the implication of their products as a source of widespread illness.

These changes in food diversity and consumer demands have changed the way outbreaks are investigated. In the past, the majority of foodborne outbreaks occurred locally and could be readily detected by epidemiologic surveillance methods. An outbreak could be detected by an acute increase in foodborne illness and local food handling mistakes could be identified and controlled following epidemiology investigations. The “New Scenario” foodborne outbreak may involve a complex multistate investigation that may also be separated by time of onset of illness. While epidemiology investigations still provide needed information; laboratory data, particularly subtyping data, is now critical to implicate a food source and to link cases which may be geographically unrelated. A new level of quality (validation and standardization) of laboratory methods is required because of the potential adverse effects on a manufacturer of an implicated product. A once local problem, managed locally, now requires extensive resources to investigate and control.

Large Foodborne Disease Outbreaks

Examples illustrating large-scale (several thousands of cases) foodborne outbreaks are listed in Table 2-6.

The 1985 outbreak of Salmonella ser. Typhimurium infections was most likely caused by improper switching of the stainless-steel pipes in the milk processing facility, which resulted in raw milk coming in contact with pasteurized

Suggested Citation:"2 Assessing Our Understanding of the Threats." Institute of Medicine. 2002. Biological Threats and Terrorism: Assessing the Science and Response Capabilities: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10290.
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TABLE 2-6 Foodborne outbreaks

Year

Location

Etiologic agent

Food vehicle

Number of persons affected

1985

Midwestern U.S.A.

Salmonella serotype Typhimurium

2% pasteurized milk produced by a large dairy

250,000

1994

Nationwide, U.S.A.

Salmonella ser. Enteritidis

Ice cream

224,000

1997

Sakai city, Japan

E. coli O157:H7

School lunch, radish sprouts

10,000

milk (Ryan et al., 1987). Interestingly, the outbreak was first recognized as a potentially large one when clinical laboratories in the region ran out of laboratory supplies for culturing Salmonella from ill persons. The ice cream-associated outbreak of Salmonella enteritidis infections in 1994 was caused by improper cleaning and sanitation of the ice cream premix tanker that was used previously to transport raw liquid eggs (Hennessy et al., 1996). The Japanese outbreak of E. coli O157:H7 infections was most likely caused by contamination of seeds used for sprouting or contamination of water used in the sprouting process (Michino et al., 1999).

Foodborne Pathogen/Toxins as Agents for Bioterrorism

Intentional contamination of our food and water supply is a real threat. Before this year, the only acts of bioterrorism in the U.S. involved foodborne agents. In 1984, members of a religious commune in Oregon attempted to influence the outcome of a local election by intentionally contaminating salad bars in several restaurants with Salmonella ser. Typhimurium. The outbreak affected at least 750 persons and S. Typhimurium was cultured from stool specimens of 388 persons (Török et al., 1997). In 1996, 12 of 45 laboratory workers at a large medical center in Texas became infected with Shigella dysenteriae type 2; the outbreak was associated with eating pastries or doughnuts that had been placed in the staff break room on a specific day. Epidemiologic and laboratory investigations strongly suggested intentional contamination of pastries by someone who had access to the bacterial stock cultures in the medical center’s laboratory and who was familiar with the methods of culturing the bacteria (Kolavic et al., 1997).

Unlike some potential threat agents (i.e., smallpox) for which the sources are limited, many foodborne agents such as Salmonella, E. coli O157, and even botulinum toxin are relatively easy to obtain or produce. Many of the agents are stable under a variety of conditions and so could easily be added to food and

Suggested Citation:"2 Assessing Our Understanding of the Threats." Institute of Medicine. 2002. Biological Threats and Terrorism: Assessing the Science and Response Capabilities: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10290.
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water supplies before consumption. Although there was no reason to suspect foul play in any of the three foodborne outbreaks listed above, each could have easily been caused by intentional contamination by one or more persons involved in some way in food processing, preparation or transport.

Some foodborne disease agents require only a small inoculum to cause disease. Shigellosis can be caused by as few as a few hundred organisms; the infective dose of E. coli O157:H7 is thought to be even less (Hornick, 1998). Botulinum toxin is one of the most potent toxins known; it has been estimated that 1 gram of botulinum toxin is enough to kill 1.5 million people. Introduction of botulinum toxin into a food source would severely strain the resources of the health care system (e.g. antitoxin, hospital support, mechanical ventilators, etc) to adequately respond.

Although perhaps less deadly, other pathogens intentionally introduced into food and/or water supplies could also negatively affect the ability of a community to respond to the disease. Widespread disease could easily overburden the health-care system (hospitals, doctors, medical supplies), the public health system (epidemiologists, diagnostic testing laboratories), and emergency response teams (police, paramedics, decontamination crews). In addition, lack of consumer confidence in the quality of the food and water supply would be an additional burden on community governments. Capacity for early detection of intentional contamination of the nation’s food and water supply is vital to minimize the impact on community health.

Challenges to Rapid Response

There are a number of challenges to providing a rapid response to intentional or unintentional widespread foodborne outbreaks (Mead et al., 1999).

Specimen collection. Although a mundane and easily overlooked aspect of response, a standard protocol for specimen collection is needed. Different foodborne agents (bacterial, viral, parasitic, etc) have different requirements for preservation to ensure efficient recovery for laboratory detection (Kaferstein et al., 1997).

Cost-reduction initiatives in healthcare. There is a move toward non-culture diagnostic and anti-microbial susceptibility tests to reduce healthcare costs. In some cases, tests for certain agents are not performed unless specifically requested by the physician (Swerdlow and Altekruse, 1998).

Need to differentiate between sporadic and outbreak cases. Foodborne illness occurs daily in the United States. Subtyping methods are needed which can rapidly and accurately separate sporadic cases from outbreak cases (Tauxe et al., 1987).

Lack of monetary incentives for commercial companies. The development, validation, and standardization requirements needed to produce a test kit that can be used for clinical specimens are time consuming and expensive. The

Suggested Citation:"2 Assessing Our Understanding of the Threats." Institute of Medicine. 2002. Biological Threats and Terrorism: Assessing the Science and Response Capabilities: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10290.
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lack of potential for profit prevents commercial companies from developing the required subtyping methods (Ryan et al., 1987).

Demand for real-time data. Currently there is no standardized computer system that will allow real-time data exchange between laboratories. The lack of real-time data exchange increases the time for establishing interventions to disease.

PulseNet as a Model

The National Molecular Subtyping Network for Foodborne Disease Surveillance (PulseNet) is CDC’s network of public health and food regulatory agency laboratories. As a model, this network fulfills some of the needs for rapid response to outbreaks. Each state health department has the capacity to perform DNA “fingerprinting” of foodborne pathogens using CDC’s standardized pulsed-field gel electrophoresis protocols. DNA patterns are analyzed using a standard software package using parameters set by CDC. Testing laboratories are able to communicate electronically via the Internet. CDC maintains a national database of DNA “fingerprint” patterns which is updated as new patterns are confirmed. This database has allowed state health departments to have early recognition of case clusters and helps to identify or confirm potential sources of disease. PulseNet is a rapid, effective means of communication between public health laboratories.

Integrated Approach to Foodborne Diagnostics

An integrated approach is needed to respond to intentional and nonintentional outbreaks of foodborne disease. (1) Sample collection, (2) improved diagnostics (including pathogen identification without isolation, rapid characterization and subtyping without isolation, and preservation of samples for subsequent pathogen recovery if needed), and (3) implementation of a real-time communication network must be seamlessly interconnected in order to effectively apply intervention measures during widespread outbreaks.

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In the wake of September 11th and recent anthrax events, our nation’s bioterrorism response capability has become an imminent priority for policymakers, researchers, public health officials, academia, and the private sector. In a three-day workshop, convened by the Institute of Medicine’s Forum on Emerging Infections, experts from each of these communities came together to identify, clarify, and prioritize the next steps that need to be taken in order to prepare and strengthen bioterrorism response capabilities. From the discussions, it became clear that of utmost urgency is the need to cast the issue of a response in an appropriate framework in order to attract the attention of Congress and the public in order to garner sufficient and sustainable support for such initiatives. No matter how the issue is cast, numerous workshop participants agreed that there are many gaps in the public health infrastructure and countermeasure capabilities that must be prioritized and addressed in order to assure a rapid and effective response to another bioterrorist attack.

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