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Biological Threats and Terrorism: Assessing the Science and Response Capabilities: Workshop Summary (2002)

Chapter: 4 The Research Agenda: Implications for Therapeutic Countermeasures to Biological Threats

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Suggested Citation:"4 The Research Agenda: Implications for Therapeutic Countermeasures to Biological 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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4
The Research Agenda: Implications for Therapeutic Countermeasures to Biological Threats

OVERVIEW

As with vaccines, not only are therapeutics an integral component of our biodefense arsenal, but making it publicly known that we are producing a constant stream of new, innovative antimicrobials would serve as a very strategic form of deterrence. Several issues related to antibiotic, antiviral, antitoxin, and antibody research and development were identified and discussed during this session of the workshop. In light of the plethora of bioterrorist agents that could be used against us, of utmost importance is deciding whether we should focus our efforts on the development of broad-spectrum or agent-specific antimicrobials. For example, one possible antiviral strategy is the development of family-specific antivirals. Increasing evidence suggests that common antiviral targets exist.

Our antibiotic arsenal is limited to only a handful of old antibiotics. Unfortunately, the general confidence in existing antibiotics and the complacency that was associated with infectious diseases in the 1960’s resulted in a lag in producing new classes of antibiotics. There are about twenty-five antibiotics currently in the early phases of clinical development. However, none of these are new classes of antibiotics, and none are broad-spectrum. In fact, there has been only one new class of antibiotic developed in the past two decades, and resistance to it emerged before it came to market. This is alarming given the increasing accessibility of the tools and knowledge needed to develop antibiotic-resistant strains of bioterrorist agents.

There is concern that the situation will become ever worse with the recent FDA changes in clinical trial design requirements. It is expected that the increased cost associated with larger clinical trials will discourage companies from

Suggested Citation:"4 The Research Agenda: Implications for Therapeutic Countermeasures to Biological 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.
×

pursuing new antibiotic development, especially when there are other therapeutic interests vying for the same resources. Although the FDA attempts to balance the demands of a public health emergency with their needs as a regulatory agency and offers several accelerated routes to licensure, including the proposed animal efficacy rule, there is still a sense that these regulatory processes need to be streamlined even more in order to accelerate drug discovery and development efforts and provide more incentive for the pharmaceutical industry.

Our antiviral amamentarium is even more limited than our antibiotic arsenal. Cidofovir, for example, can only be administered intravenously and is highly nephrotoxic, making it unsuitable for mass casualty use. The clinical utility of ribavirin as an antiviral drug strategy for bioterrorist agents remains unclear.

Antibiotics and antivirals are not the only potential therapeutic defense against bioterrorist agents. Basic research on the anthrax toxin system has led to some exciting prospects for antitoxin targeting. The most promising are the dominant negative inhibitors (DNIs), mutant forms of the protective antigen that block translocation of the virulence factors across the plasma membrane. Currently, DNIs are a very late stage product. If they can be proven efficacious in infected animal models, they could be produced and deployed very rapidly. There are several other approaches in much earlier stages of development.

The use of recombinant monoclonal antibodies is another option which has been implicated for use against several biothreat agents, including anthrax, smallpox, and botulinum neurotoxins. For example, recent research has shown that a small mixture of recombinant monoclonal antibodies provides complete protection in mice against botulinum neurotoxin type A. Antibodies have several advantages as a bioweapons defense tool: they have been shown in multiple studies to be safe; ten have already been approved by the FDA and seventy more are in clinical trials, so their route to licensure is known; the technology and knowledge needed for production are readily available; their overall course through the discovery and approval process is much quicker than those of other types of therapeutics; and the technology platform used to produce and manufacture antibodies could be applicable to multiple agents.

Finally, scaling up research and development of all of these various potential therapeutics will require an evaluation of the availability of and need for additional laboratory capacity. In particular, there are a very limited number of BSL-3 and 4 labs where nonhuman primate studies can be conducted. Hope was expressed that in the future the FDA will accept rodent data in lieu of nonhuman primate data, if it can be demonstrated that the efficacy is the same in rodents as in nonhuman primates. This would allow for more testing in a greater number of facilities, although it would still require at least BSL-3 capability. Aerosol testing requires BSL-4 capabilities, as well as trained, vaccinated personnel.

Suggested Citation:"4 The Research Agenda: Implications for Therapeutic Countermeasures to Biological 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.
×

COUNTERMEASURES TO BIOLOGICAL THREATS: THE CHALLENGES OF DRUG DEVELOPMENT

Gail H. Cassell,* Ph.D.

Vice President, Infectious Diseases

Drug Discovery Research and Clinical Investigation Eli Lilly and Company

The Problem

The diversity of existing biological weapons and the ever increasing possibilities preclude simple therapeutic countermeasures to bioterrorism. Furthermore, response possibilities are rather limited even for known threats. Although there are 13 viruses on the current list of potential biothreats, there is only one indicated antiviral—cidofovir—and it both requires intravenous administration and is highly nephrotoxic. More broadly, there are no truly broad spectrum antivirals, and only a limited number of antivirals for routine pathogens like influenza, herpes, hepatitis-B, and HIV.

The situation is somewhat better but still worrisome with respect to antibiotics. There has been only one new class of antibiotics developed in the past two decades, and resistance emerged to this class before it entered the commercial market. This is a clear challenge to developing an armamentarium against biological pathogens.

At first glance, the situation with respect to antibiotics currently in clinical development looks encouraging. About 25 antibiotics are in the first 3 stages of development, with several billion dollars devoted to their development. However, there are no new classes being pursued, nor are new broad spectrum antibiotics. Furthermore, most are quinolones, and 50 percent or more of the strains of E. coli in Beijng are resistant to quinolones as are many foodborne pathogens. In addition, quinolones are contraindicated for children, and neither quinolones nor tetracycline are acceptable for pregnant women.

The Challenges

So it would be mistaken to be sanguine about current antibiotic therapies to counter bioterrorism. Nor can one be optimistic about near-term prospects. Eli Lilly recently conducted a competitive analysis revealing that most of the large pharmaceutical companies, with the possible exception of Pfizer, are reducing their investment in antibiotic development. There are probably several reasons.

A decade ago, we looked at new technologies like high throughput screening, combinatorial chemistry, and microarray assays, and anticipated a golden

*  

The information provided in this paper reflects the professional view of the author and not an official position of Eli Lilly and Company.

Suggested Citation:"4 The Research Agenda: Implications for Therapeutic Countermeasures to Biological 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.
×

age of antibiotics. But today we have no new classes of antibiotics as a result of those efforts.

It has become painfully apparent that discovering new antibiotics is not as easy as once believed. We have, for example, a plethora of targets. Numerous targets have been found with documented in vivo expression of antigens. But they are not necessarily what are called “drugable targets.” A target can be validated and essential for bacterial viability, but if there is not a chemical entity that will penetrate the bacterial cell wall and inhibit growth, you don’t have a real target. In addition, the chemical entity must be safe and not highly toxic. There is a 90 percent failure rate from the discovery of a target to the launch of a new antibiotic. This lack of success has likely damped further spending in this area.

Moreover, there has been increased investment in antivirals. And, with the sequencing of the human genome, competition for resources within pharmaceutical companies has turned to other therapeutic areas where there are tremendous opportunities and great unmet medical needs with bigger market opportunities. In fact, infectious diseases, specifically those requiring antibiotic therapy, do not fare too well in financial analyses.

Conclusion

In short, our antibiotic armamentarium is limited, there is growing concern about an increasing number of potential new weapons, and there has been a marked increase in resistance to existing antibiotics.

It seems clear that no public health response to bioterrorism is likely to prove effective without addressing the overall problem of antimicrobial resistance and the challenges of drug discovery and development.

Finally, the best deterrent against the use of a biological weapon of mass destruction may be a constant stream of new, innovative antibiotics and antivirals. Knowledge of such commitment and successful developments would surely dissuade the efforts of our enemies in such an arena.

THE FDA AND THE END OF ANTIBIOTICS

David M. Shlaes, M.D., Ph.D.

Vice President for Infectious Diseases

Wyeth-Ayerst Research

Robert C. Moellering Jr., M.D.

Physician-in-Chief, Beth Israel Deaconess Medical Center

Herrman Blumgart Professor of Medicine

Harvard Medical School

Antibacterial research has, for almost two decades now, been the “Cinderella” area in the pharmaceutical industry. The market for these products, used to

Suggested Citation:"4 The Research Agenda: Implications for Therapeutic Countermeasures to Biological 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 4-1

Cure Rate

Delta

90%

10%

80–89%

15%

< 80%

20%

treat acute, not chronic disease, is modest. There are many products available including many generics. For the most part, the market is growing only slowly and, except for the problem of resistance, is largely satisfied.

Recently, when presenting proposals for Phase III trial designs for antibacterial compounds to the FDA and European regulatory bodies, a number of companies, both small and large, were told that the designs for equivalence studies had to target a 10% delta for the lower limit of the confidence interval. This requirement, seemingly innocent and technical, threw the pharmaceutical industry into a panic and probably contributed to the recent withdrawals by Lilly and Bristol-Myers-Squibb from the antibacterial discovery business. Why?

Most clinical trials leading to approval of antibacterial drugs are based on equivalence studies. The delta statistically defines the boundary for equivalence. In the past, based on the 1992 points to consider document from the FDA (FDA, 1992), a step function for the delta has been used (Table 4-1).

These boundaries have meant the study results must show there is a 95% (97.5 % one sided) probability that true cure rate for the new drug is not more than 10 to 20 % lower than the cure rate for the approved drug. In most reasonable sized studies the new drug has had to be as good as or better than the old one to be successful.

The European regulatory authorities and the FDA are now suggesting that a 10% delta be used routinely in drug development (FDA, 2001b), and the FDA has now “disclaimed” the old step function on their web site (FDA, 2001a). It is not completely clear upon what data this suggestion is based, other than purely statistical considerations. In fact, a quick calculation will show that two independent trials successful at a 15% delta would result in approving a drug inferior at the 10% delta only 2% of the time. The concern that the FDA has expressed is over something called “biocreep.” In this concept, a slightly inferior experimental drug becomes the comparator for the next generation of compounds and so on until the experimental drugs of the future asymptotically approach the efficacy of placebo. However, one must wonder whether, for serious infections, this is any more than a theoretical concern, especially when most recent approvals (Synercid, Zyvox) have been based on comparison with standard therapy using older agents. One of the regulatory agency’s best weapons against biocreep is their control over the choice of comparators in clinical trials.

Suggested Citation:"4 The Research Agenda: Implications for Therapeutic Countermeasures to Biological 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 deltas used in the step-function of the 1992 points to consider document were chosen, not based on scientific reasoning, but based on the size of trial that would be required given the cure rate (Pharmaceutical Research and Manufacturers of America [PhRMA], personal communication). The trial size required is very sensitive to efficacy rate, evaluability, b error (power) and the delta. For typical trials with an injectable antibiotic, the patient numbers under various assumptions are shown in Table 4-2. The increased numbers have implications for the ability to run a trial in a reasonable length of time, time of availability of the new drug to patients and physicians, time to market and overall costs of development. For example, Bristol-Myers-Squibb was told that they would be required to run a trial in acute bacterial meningitis at a 10% delta requiring enrolling over 700 patients (Roger Echols, BMS, personal communication). That would be the largest meningitis trial ever done, require about five years to accomplish this enrollment and would require enlisting over 90% of the patients from outside the United States. They declined based on the impracticality of the design and the fact that in the later years of the study, it was not clear that their comparator would still be considered the standard of care in the medical community. Similar concerns exist regarding our ability to run trials at a 10% delta for infections caused by resistant bacteria like vancomycin-resistant enterococci.

TABLE 4-2 Number of patients for each indication with a one-sided 97.5% CI (assumes 75% evaluability)

Indication

Cure Rate

90% Power 10% delta

90% Power 15% delta

A

85%

 

 

Number of Studies: 2

 

1532

688

B

80%

 

 

Number of Studies: 2

 

2248

1000

C

70%

 

 

Number of Studies: 2

 

2948

1316

D

65%

 

 

Number of Studies: 1

 

1598

710

Related to indication C

 

 

 

TOTAL

 

8326

3714

 

 

80% Power 10% delta

80% Power 15% delta

TOTAL

 

6226

2770

Suggested Citation:"4 The Research Agenda: Implications for Therapeutic Countermeasures to Biological 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 4-2 also shows the increase in numbers required if a 10% delta is required for all indications. Costs of a Phase III trial are directly related to the number of patients enrolled. Therefore, in the scenario above, the costs increase more than 100% going from a 15% to a 10% delta design and much more if one was starting at the old step function plan. This can be ameliorated to some extent by decreasing the b power to 80% from 90%. However, doing that results in a 32% risk of falsely concluding that the experimental compound is inferior to the comparator—a risk not acceptable for most companies.

One might argue that large pharmaceutical companies can easily absorb these costs and, that if they want to sell a product, they should do so. However, just as in government agencies like NIH, proposed research in the pharmaceutical industry is subject to prioritization. In the case of the industry, business considerations play a large role in the process. Therefore, in most companies, programs with modest potential markets and large costs are automatically deprioritized unless there is some other, overriding strategic issue to be considered. Thus, one unintended result of promulgating these guidelines will be a decrease in the number of companies carrying out antibacterial research as was seen in the late 1980s and is occurring again now.

PhRMA has suggested a number of alternate approaches to the FDA and the industry is more than willing to work with FDA, IDSA and other interested parties to address their concerns regarding clinical trial design in antibacterial development. However, the attempt by regulatory authorities to implement an across-the-board requirement for 10% delta trial designs has already wreaked irreparable damage to our ability to provide a reliable pipeline of new antibiotics for serious infections. We hope that the advisory committee of the FDA will understand these concerns and act appropriately. We would also ask that the European regulatory authorities reconsider their stance for the same reasons.

THE ROLE OF ANTIVIRALS IN RESPONDING TO BIOLOGICAL THREATS

C.J. Peters, M.D.

Professor, Departments of Microbiology and Immunology and Pathology

University of Texas Medical Branch at Galveston

Many available viruses could be used to cause harm to others under many different scenarios. It is important to try to focus on some specific priorities to attempt to limit the problem to a tractable scope, yield maximum benefit in the short term and develop more comprehensive goals that we can hope to attain in the longer term. It is important to consider vaccines and drugs together as part of an overall antiviral strategy.

Suggested Citation:"4 The Research Agenda: Implications for Therapeutic Countermeasures to Biological 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 Threat

To ameliorate the adverse consequences of a bioterrorist (BT) attack, the scenario is everything and, equally, the scenario for every possible attack is unknowable. However, aerosol attacks have the greatest potential to cause mass casualties and also lend themselves to stealthy application (see chapter on aerosols). Only a limited number of viruses are known that grow to high titer and are stable and infectious in aerosols and thus lend themselves to this form of attack. Tables one and two list a number of human-pathogenic viruses that have often been considered as aerosol threats (Alibek, 2001; Ferguson, 1999; Alibek, 1998). The viral hemorrhagic fevers (VHF) (Table 4-3) are among the most dangerous (Peters, 2000). The VHF agents are lipid-enveloped RNA viruses with a genome size of around 1–2 million Daltons belonging to four different virus families (Peters and Zaki, 1999). They are zoonotic viruses and all are aerosol-infectious, either shown through formal studies in the laboratory and/or by the observation of frequent “unexplained” laboratory infections. As might be expected from their taxonomic diversity, they differ in individual strategies for maintenance in nature and in their pathogenesis of human disease. Several of these viruses were developed by the Soviets for use as strategic weapons for mass destruction, including Machupo, Lassa, Rift Valley fever, and Marburg viruses. At this time, technical difficulties may limit the prospects for weaponization of Crimean Congo HF and the hantaviruses, but like most problems, these are subject to solution.

TABLE 4-3 Viral hemorrhagic fevers commonly mentioned in association with biological warfare or biological terrorism

PRIMARY HEMORRHAGIC FEVERS (HF)

ARENAVIRIDAE

 

 

Lassa Fever

 

 

South American HF (Argentine, Bolivian, etc)

BUNYAVIRIDAE

 

 

Phlebovirus

Rift Valley fever

 

Nairovirus

Crimean Congo HF

 

Hantavirus

HF with renal syndrome

 

 

Hantavirus pulmonary syndrome

FILOVIRUS

 

 

 

Marburg HF

 

 

Ebola HF

 

FLAVIVIRUS

 

 

Yellow fever

 

 

Tick-borne HF (Kyasanur forest disease, Omsk, etc)

Suggested Citation:"4 The Research Agenda: Implications for Therapeutic Countermeasures to Biological 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 4-4 Other viruses suggested to have potential in biological warfare or biological terrorism

 

Smallpox

 

Monkeypox

 

Nipah

 

“Viral encephalitides”

 

-Venezuelan equine encephalitis

 

-Other alphaviruses

 

-Tick-borne encephalitis virus

 

“Eradicated”: polio and measles

 

Influenza A: 1918 strain, Hong Kong H5N1, others

The other candidate aerosol infectious viruses (Table 4-4) also are largely zoonotic with the exception of the very important BT agent, smallpox. The zoonotic agents can spread to close family contacts and to health care staff, but continuing chains of transmission are not a threat. Smallpox is quite different because its natural history is one of continuous inter-human transmission. The lack of a reservoir outside human-kind, the moderately higher transmissibility, and the existence of a highly effective vaccine that can be efficiently delivered combined to allow the eradication of the virus as a cause of natural disease. Monkeypox is another poxvirus which shares high aerosol stability and infectivity with smallpox but which has a much lower interhuman transmissibility and case fatality (Jezek and Fenner, 1988).

Nipah virus is representative of a newly proposed genus of a very well-established family, Paramyxoviridae. Before the other human pathogen in this genus, Hendra virus, emerged in Australia in 1995 (Murray et al., 1998) the existence of the genus was unsuspected, but now its members are seen to be widely distributed among flying foxes (Macrochiroptera) and at least these two members have the potential to cross over into domestic animal species and also infect humans (Chua et al., 2000). Their future behavior is unpredictable, but analysis of the state of emerging infections in the world and the recent recognition of two serious episodes in the recent past suggest they are highly dangerous (Peters, 2001). The spread of Nipah virus among swine in Malaysia was progressive and could have resulted in an enormous economic, human, and political disruption if it had extended into Thailand and China. Stopping the march of the virus entailed destruction of more than one million pigs after it caused 265 human cases with a 40% case fatality and significant residual morbidity among survivors (Parashar, et al., 2000). The aerosol properties of this virus are unknown, but it appears to spread among pigs by small-particle aerosols or droplets. The great majority of human cases had close contact with living swine, but the occurrence of a small number of cases in persons living near pig farms but without actual contact with pigs also raises the question of aerosol infection of

Suggested Citation:"4 The Research Agenda: Implications for Therapeutic Countermeasures to Biological 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.
×

humans. Where will the next unexpected virus come from and what taxon will it belong to?

Several alphaviruses causing viral encephalitis have been regarded as potential biological agents. The best established is VEE which was weaponized by the US and the Soviets. VEE also has the potential to establish itself as an endemic mosquito-borne disease in North America based on past epidemiological evidence (Weaver, 1997).

Other less well-established threats will undoubtedly become more important in the future. These could even include common viruses such as polio and measles should they be eradicated (previous Forum). The problem is exemplified by smallpox virus. When vaccine was widely used and substantial immunity was maintained in the population, the threat of smallpox epidemics arising from isolated cases was less than it is today. Polio epidemics would be highly disruptive, but measles would probably be the worst threat. For example, measles epidemics during the U.S. Civil War were among the greatest impediments to expanding armies because of their heavy impact in both morbidity and mortality among recruits and staging centers for the two armies (Steiner, 1968).

Influenza virus is infectious by aerosols and is capable of propagating efficiently among humans by aerosols even though other routes are also important (Kilbourne, 1975). In general, the impact of aerosol spread on control is huge. In the US we can deal effectively with fecal-oral, fomite, and large droplet transmission through our general level of sanitation or by using simple mask, eye protection, and hand washing measures. However, aerosols must be controlled with efficient filters on breathing air and the filters must be well-fitting and in use during the time of risk; any society would have difficulty dealing with an aerosol spread epidemic. The ability to produce recombinant influenza strains from natural strains or using synthesized genes is an accomplished feat (Neumann et al., 1999). The prediction of which viruses will be highly transmissible and lethal will come in the near future. Whether such viruses would be produced and could actually spread among human populations is another matter.

The Solutions

Biological warfare (BW) and terrorism present different challenges. BW scenarios could involve the use of biological weapons on the battlefield and would target a selected group which could therefore be immunized using their training schedules, different operational missions, and on-going military service to select and prioritize.

A strategic biological warfare effort could also be directed against civilian populations, as was envisaged by the Soviets, and this scenario could possibly be countered through vaccine protection. However, a civilian population facing an ill-defined bioterrorist threat would be much harder to protect by immunization because of the problems of vaccine coverage and the inevitable adverse

Suggested Citation:"4 The Research Agenda: Implications for Therapeutic Countermeasures to Biological 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.
×

events associated with all vaccines. The example of smallpox vaccine would be illustrative. Use of this vaccine in the defined and limited group of military persons in basic training was accomplished in the 1970’s with little morbidity among the vaccine recipients and little risk to non-military. If this program had been expanded to the entire civilian population with the consequent adverse effects (Koplan and Hicks, 1974), there would have been a huge backlash. The “swine flu “ vaccination program in 1976 provides an excellent example of the likely outcome (Neustadt and Fineberg, 1978). Widespread vaccination against an ill-defined threat would be associated with the adverse effects that will ensue from any vaccine and would bring the effort would be brought to a stop with serious negative medical and political consequences. Even a perfect vaccine would be tarred by the unfortunate events that occur by chance in a large, healthy population receiving no treatment at all.

Thus, vaccines against viruses could be very useful but would likely only be used on a large scale in civilian populations in the face of a clear and present danger. Nevertheless, they are an important part of an over-all antiviral strategy because they provide protection for particular groups, including those studying the virus in the laboratory, antiviral drug developers, and those working with the virus in regions where it occurs naturally. Furthermore, availability of attenuated strains can be essential to expanding research activities, including antiviral drug development, to laboratories with lower levels of containment.

Antiviral drugs could provide protection, subject to all the same problems of stockpiling and delivery as antibacterial agents now considered for use against such threats such as anthrax, plague, and tularemia. It is now clear that, with an adequate molecular and structural biological base, drug development capability, and financing, effective antivirals can be discovered and indeed even designed. Antivirals inhibiting enzymes active in nucleic acid synthesis or protein cleavage have been highly effective although with some price in toxicity. Other targets exist which might be less closely allied to host cell constituents and which might provide a greater therapeutic index, just as drugs such as penicillin inhibit gram positive bacterial cell wall synthesis, which has no counterpart target in mammalian cells. Monoclonal antibody strategies are also possible, but many of the protective targets are the neutralizing antibody epitopes, which are commonly virus specific. This can be overcome with multi-virus cocktails, but this in turn demands development and production efforts of multiple antibodies. Fortunately, the neutralizing epitopes are highly conserved and usually linked to virulence on the individual viruses of interest. The success of any antiviral strategy—drugs (including antibodies), vaccines, or combinations—will depend critically on the context in which it is used. Stockpiles of remedies will be essential, as well as expectant use of some vaccines. Equally important will be plans to deliver emergency vaccines or drugs in a timely fashion where they are needed. And most importantly, it will be incumbent on the physician to recognize the diseases and

Suggested Citation:"4 The Research Agenda: Implications for Therapeutic Countermeasures to Biological 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.
×

request definitive laboratory evaluation of the provisional diagnosis. Finally, the definitive laboratory diagnosis must be quickly available.

Both antiviral vaccines and drugs suffer from major development problems. They would require an expensive developmental effort that has never been able to attract industrial support based on disease activity in endemic areas, even when the U.S. Department of Defense has expressed an interest and provided an additional market. A large number of viruses are involved (Table 4-3, Table 4-4), further multiplying the problem. Finally, the utility of these medical countermeasures is severely limited if they cannot be tested in adequate numbers of humans for efficacy and safety. The safety testing would be extremely important in prophylactic use or if large numbers of people are to receive the substance.

The only feasible solution is to provide public support for the discovery and testing of BT related antiviral vaccines and drugs. As a practical matter, the approach would have to be directed toward finding solutions which will apply to the most important individual viruses or to broad groups of agents, perhaps at the generic or family level. We should look very carefully, without being shackled by our past attitudes, at new technologies for translating the advances of biotechnology into human products; the way we test, develop, and approve drugs and vaccines for different uses; and the actual increment in safety derived from additional requirements on manufacturers.

A Case Study

A vaccine against Argentine hemorrhagic fever (Junin virus) provides insight into some of the obstacles. This vaccine was desired by DoD because of suspicions, later confirmed, that the Soviet Union was developing Junin and the related Machupo virus as biological weapons. Immunity against Junin virus was shown to protect against Machupo infection in experimental animals, thus providing the expectation that a Junin vaccine could protect against both arenaviruses.

A laboratory attenuated strain of Junin virus had been shown to be safe and immunogenic in at-risk laboratory workers in Argentina. This strain was too reactogenic and insufficiently characterized for general use, but it provided a very important bench-mark for development of a further-attenuated vaccine for humans. The availability of limited testing of prototype vaccines in humans before undertaking definitive work can be an extremely important step in facilitating vaccine development with the potential to save years or decades in development time and costs.

Vaccine development was pursued as a joint US-Argentine project with participation of Argentine colleagues at each step and with efforts to brief Argentine public health officials at regular intervals over the decade-long period. This was extremely important when the time for field-testing of the vaccine arrived because charges of “scientific imperialism” could have blocked the actual evaluation of the vaccine against the disease even though it only occurred naturally in

Suggested Citation:"4 The Research Agenda: Implications for Therapeutic Countermeasures to Biological 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.
×

Argentina. After the vaccine met FDA standards and had undergone preliminary evaluation for safety and immunogenicity in US volunteers it was shown to be protective in double-blind, placebo-controlled trials in the endemic area for Argentine hemorrhagic fever (Maiztegui et al., 1998; Barrera-Oro and McKee, 1991). Subsequent use in Argentina brought the number of inoculated humans above 200,000, further establishing the safety in large numbers of recipients.

Among the lessons is that useful drugs and vaccines can be developed in the US outside the private sector if there is a substantial long-term financial and institutional investment, if they can be tested overseas, and if there is forethought and participation of the target test nation’s scientific and political establishment. Other issues such as change in political and economic circumstances in the participating country and temporary or permanent shifts in disease incidence during vaccine development unpredictable and difficult to control hurdles to success.

Existing Measures for Specific Agents

The number of potentially deployable antiviral measures for recognized viral threats is limited (Table 4-3). All the viruses in the arenavirus family are inhibited in cell culture by the antiviral drug ribavirin and there are substantial data to support the use of intravenous ribavirin in the treatment of Lassa fever (McCormick et al., 1986). Preclinical and anecdotal human data for the use of the drug in other arenavirus infections strongly suggests it is efficacious (Peters, 2002). Emergence of resistance under therapy has not been seen (Kenyon et al., 1986). The drug has undergone extensive preclinical evaluation and is licensed in the U.S. for aerosol and oral use in respiratory syncytial virus pneumonia and hepatitis C respectively. Its use in pregnant women and children is restricted because of preclinical data suggesting teratogenicity and growth retardation. Because of the lack of a market and the expense of New Drug Applications, additional studies or licensure of intravenous ribavirin for use in arenavirus infections have not been pursued. The drug has also been used in Crimean Congo HF (family Bunyaviridae, genus Nairovirus) in South Africa and a modest clinical experience supports the positive preclinical data for its efficacy.

Rift Valley fever (family Bunyaviridae, genus Phlebovirus) presents an important problem (Peters, 2000). In addition to its potential use as a BW agent, it is also a threat for importation. It is a mosquito-borne disease that usually depends on the presence of sheep or cattle to support its transmission, but humans may well be able serve as substitute amplifiers in the proper circumstances. Aerosol-mediated human infection is the basis for the BW potential of the virus and also the infections commonly occurring among laboratory workers. Thus, both human and veterinary vaccines would be desirable for Rift Valley fever control if the virus were introduced into the U.S. by bioterrorists or natural spread. Its propensity for spread outside its natural range in sub-Saharan Africa

Suggested Citation:"4 The Research Agenda: Implications for Therapeutic Countermeasures to Biological 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.
×

has been demonstrated by introductions into Egypt, Saudi Arabia, and Yemen in past years. The veterinary involvement brings the additional dimension of economic disruption of the livestock industry; export of meat products would be interrupted for months or years and the movement of animals within the US would be greatly restricted. There is no satisfactory veterinary vaccine, but two human vaccines have been developed. The inactivated, Salk-type vaccine has a long lag before induction of immunity and is available in limited supply, not positive attributes to deal with a rapidly moving disease such as Rift Valley fever has proven to be (Pittman et al., 1999). In addition, the infrastructure for producing additional inactivated vaccine does not exist in the U.S. any longer. The other human Rift Valley fever vaccine is a live-attenuated product that has been tested in 60 persons with a good safety profile and the elicitation of antibodies expected to be protective, based on preclinical studies and the experience with the inactivated vaccine in laboratory workers (Pittman PR; Morrill J, Peters CJ, unpublished observations). There are no on-going efforts in further development and testing of this product.

Yellow fever (family Flaviviridae) presents a hazard either through direct aerosol delivery, spread by artificially infected vectors, or by importation into cities where human-mosquito cycles could support further transmission. The US presumably has a low receptivity to mosquito amplification of the virus, but many tropical metropolises are thought to be highly vulnerable. The vaccine is one of the best known and best studied viral vaccines and has been in use for more than 60 years; but in recent months has been associated, for the first time with fatal adverse reactions in several older vaccinees (Martin et al., 2001). Furthermore, the vaccine is not manufactured in the U.S. and world stocks are not sufficient in some areas to meet today’s needs, let alone an unforeseen surge (Nathan, et al, 2001).

Tick-borne encephalitis is another flavivirus disease that has been considered as a potential BW threat. Inactivated vaccines against the virus are widely available in Europe and are thought to be safe and protective against the virus as delivered by tick bite. None are available in the U.S., even in investigational status, nor are they protective against other tick-borne flaviviruses that cause VHF.

Smallpox is one of the most serious viral threats (see chapter on BW threats or on smallpox). Although there is no information as to who possesses the agent beyond the two authorized laboratories, it has to be noted that the virus could be disseminated directly by aerosol, based on its stability and infectiousness by that route; and it can also spread from person-to-person. The extent of this interhuman spread is controversial but modeling using selected sets of parameters (O’Toole, et al, 1999; Meltzer, et al, 2001) suggests that the outcome of even a limited dissemination could be disastrous. The amount of vaccine to contain an outbreak of smallpox in our mobile world could be substantial, given the need to ring-vaccinate contacts, medical staff, and many others. This is best met by production of large quantities of vaccine above current stocks. The safety and effi-

Suggested Citation:"4 The Research Agenda: Implications for Therapeutic Countermeasures to Biological 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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cacy of the inexpensive calf-produced vaccine was excellent and hopefully the desire to manufacture the newer vaccine stocks in cell culture will not impede the availability of the older, proven modality of control.

In the case of smallpox, the vaccine has some of the attributes of an antiviral drug because vaccination within 4 days or perhaps even longer after exposure prevents smallpox. Alternate approaches are discussed in the smallpox chapter, but it is worth pointing out that no further-attenuated protective vaccine can be evaluated for efficacy in either normal or immune deficient hosts, no vaccine can be proven to be safer without inoculating literally millions of normal persons, no vaccine can be shown to be safer in immunosuppressed hosts without performing unacceptable experiments, and no smallpox drug can be shown to be effective in humans. Surrogate markers are just surrogates and the mainstay of protection of the world’s population must remain the proven vaccine. An effort to provide a perfect solution to protection and treatment of all possible exposed persons may well siphon off resources needed for protection from other threat agents.

The classical smallpox vaccine faces additional hurdles for its effective use today because of the much larger number of immunosuppressed persons. Accidental vaccination as well as transmission from vaccinees pose hazards, and the management of an immunosuppressed patient exposed to smallpox would be a challenge. An effective drug against vaccinia virus would be life-saving for immunosuppressed persons. Drug efficacy could be shown in normal humans receiving smallpox vaccine and its relative efficacy in immunocompetent and immunodeficient animals studied in the laboratory.

Research Directions

It would be desirable to have two things in our portfolio for all the viruses known to pose BW threats. First, we should understand how to make vaccine candidates that would be protective in realistic animal models against all the threat agents and that would utilize a technology feasible for human vaccine production. Second, we should have broadly reactive antiviral drugs or other therapeutic approaches that would be effective in families or at least genera of threat agents. The priorities for advanced development would depend on the perceived level of threat and would be leavened by the natural occurrence of the target diseases, which would determine both the availability of a test bed and the additional benefit to society from developing the viral countermeasure.

In the field of vaccines, we need a revolutionary approach to vaccine development that attempts to move research on human immunogens forward to another quantum level. The history of dengue vaccine research is an example of where we do not want to be: time line stretching over decades; under-funded and marginally focused efforts; candidate vaccines are classical live-attenuated and poorly characterized in modern terms; and some promising results but no end in sight. Approaches to live vaccines could be greatly simplified with a

Suggested Citation:"4 The Research Agenda: Implications for Therapeutic Countermeasures to Biological 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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flexible, standardized, safe vector; research dedicated to resolving this problem in humans could be critical to our ability to respond to the threat of viral terrorism. Alphavirus replicons provide one of several hopeful possibilities (Polo et al., 2000; Smerdon and Liljestrom, 1999). An alternative would be a protein sub-unit based vaccine, and the best immunogenicity is generally obtained with a self-assembling particle such as with hepatitis B surface antigen or core particles. Purification of proteins could be improved through variations of approaches such as the histidine-tagged proteins employed for laboratory immunogens. The immunology of most of the viral agents listed is not well-understood and should have more work, particularly in primates. Among the viruses listed, Ebola stands out as the single agent that requires much more indepth understanding of its immunology. To date no simple approach has protected primates against death after exposure to the Zaire subtype of Ebola virus (Sullivan et al., 2000).

In the field of antivirals, there is a good deal of basic research needed on the viruses themselves, although structural and molecular biology are providing increasingly well-defined opportunities among the better-studied taxons. Both alphavirus and flavivirus structure is understood at a high level of resolution (Pletnev, et al.; Perera, et al., 2001) and the construction of the particles provides opportunities to intervene in surface assembly or capsid-nucleocapsid interactions that would be completely independent of viral polymerases. Inhibition of receptor binding through structural knowledge is another approach. The fusion peptides of filoviruses and arenaviruses are now better understood (Gallaher, 1996; Gallaher et al., 2001; Weissenhorn et al., 1998), and it may be possible to prevent infection with inhibitors designed using the same principles as for an apparently successful HIV drug (LaBranche et al., 2001).

Among the VHF there is often extensive endothelial cell involvement and modulation of their metabolism; taking advantage of insights gained through extensive work on septic shock and the “sepsis syndrome” may be useful. One promising approach has been the design of inhibitors of NF kappa B intranuclear binding (Yang et al., 1999) to prevent the over exuberant inflammatory reaction thought to be a major pathogenetic mechanism of Lassa fever and other arenavirus diseases (Mahanty et al., 2001; Marta, et al., 1999).

Conclusions

The approach to civilian biodefense against viruses consists of three elements. In the short-term we should obtain adequate stocks of smallpox vaccine, as well as the ancillary materials to deploy it. We also need to develop both policies for immunosuppressed persons and treatment strategies if they become infected with vaccinia. In the medium vista we should bring to fruition the projects that are already well along the way but are not actually ready for deployment (see Table 4-5): modest stockpiles of intravenous ribavirin, assuring avail-

Suggested Citation:"4 The Research Agenda: Implications for Therapeutic Countermeasures to Biological 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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ability of yellow fever vaccine, and further testing of a live-attenuated Rift Valley fever vaccine. These goals are both good bioterrorism policy and good public health preparedness. Other medium term goals can be addressed depending on priorities and resources. Long-term goals should be focused on developing better ways to translate the findings from molecular virology into drugs and vaccines for human use; to understand the immunology, structure, and molecular biology of viruses that are threats; and to apply basic science to development of broad spectrum solutions.

TABLE 4-5 Existing potentially deployable antiviral measures

VIRUS

MODALITY

COMMENTS

NEEDED IF TO BE USED

Arenaviruses

Junin vaccine, live-attenuated

Not licensed in U.S. but preclinical studies met FDA requirements for IND. Extensive experience on safety and efficacy in Argentina. DoD has master seed virus and has manufactured in the past. Current stocks limited.

Re-manufacture from existing master seed. Additional testing of new lots, licensure.

 

Ribavirin, intravenous

Aerosol and oral forms licensed in U.S. DoD holds IND. Overseas studies support efficacy in Lassa fever. Preclinical and anecdotal human data support use in other arenavirus HF. Intravenous form not readily available.

Assessment of amount of powdered drug actually available. Manufacture of additional stocks of intravenous solution. Licensure of iv formulation.

Rift Valley fever

Vaccine, inactivated

DoD holds IND. Effective in preventing laboratory infections and safe in a few thousand humans. Requires 2–3 weeks for protection as well as annual boosters. Limited availability.

No facility to manufacture new lots; containment and vaccinated staff required to work with live virus.

Suggested Citation:"4 The Research Agenda: Implications for Therapeutic Countermeasures to Biological 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.
×

 

Vaccine, live-attenuated

Under IND. Administered to >60 humans with safety and immune response suggesting efficacy. Not under active development. Limited availability. DoD has master seed.

Needs additional phase II testing. Manufacture from existing master seed. Will need decision as to whether to license based on preclinical data, human immune response (compared to experience with inactivated vaccine in lab workers) or field trial.

Crimean Congo HF

Ribavirin, intravenous

Drug not licensed in U.S. for iv use. Preclinical and anecdotal human data support its use but no definitive data. Employed routinely in South Africa. DoD has limited stocks and holds IND.

Needs more clinical data, presumably a trial. As for arenaviruses.

Yellow fever

Vaccine, live-attenuated

Licensed vaccine. Currently not manufactured in U.S. Stocks, surge capacity are in doubt.

Manufacture, stockpiles.

Tick-borne encephalitis

Vaccine, inactivated

Not licensed or produced in U.S. but widely available in Europe

Development of manufacturing capability.

Smallpox

Vaccine, live attenuated

U.S. and world stocks substantial but in face of multifocal epidemic would be inadequate. Manufacture of additional vaccine underway

Stockpile underway.

Monkeypox

Vaccine, live attenuated

Same vaccine as smallpox. Threat mainly direct delivery of virus; limited interhuman transmission.

Smallpox vaccine stockpile underway.

Suggested Citation:"4 The Research Agenda: Implications for Therapeutic Countermeasures to Biological 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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NEW RESEARCH IN ANTITOXINS

R. John Collier, Ph.D.

Maude and Lillian Presley Professor of Microbiology and Molecular Genetics, Harvard Medical School

Inhalational anthrax is a deadly disease. Based on recent events, the case fatality rate with supportive care and appropriate antibiotics is approximately fifty percent. However, in the case of massive attack and according to previous knowledge, the case fatality rate could easily approach one hundred percent. Although an anthrax vaccine exists, there are currently no plans to implement mass immunization. At least for the foreseeable future, the civilian population is completely susceptible to infection.

Currently, antibiotic therapy is our only therapeutic countermeasure. Because death from anthrax is largely, if not solely, due to the action of the anthrax toxin, antitoxins may prove to be a valuable ancillary treatment. An added advantage of antitoxins is that they not only inhibit toxin action and the progression of symptoms but, because toxins are aimed at immune system cells, antitoxins also boost the immune system by protecting those cells.

There are several viable antitoxin options based on what we have learned in these last few years about the structure and action of anthrax toxin. Three antixoxin approaches in particular appear very promising: dominant negative inhibitors (DNI), which are mutant forms of the protective antigen that block translocation; polyvalent inhibitors (PVI), which are chemically synthesized inhibitors that block toxin assembly; and soluble forms of the toxin receptor, ATR, which block toxin attachment to cells. There is also a fourth approach that is based on the fact that, because lethal factor (LF) is a zinc protease, some of the number of metallic protease inhibitors that are already in use in other drugs may be effective inhibitors of LF as well. At least one major pharmaceutical company is currently screening their large library of metallic protease inhibitors for activity in this regard.

Anthrax toxin consists of three large proteins: edema factor (EF), protective antigen (PA), and lethal factor (LF). None of these proteins alone is toxic, but a combination of EF and PA induces an edematous response, and a combination of PA and LF causes lethality. LF and EF are enzymes that act on target molecules in the cytosol of mammalian cells. As is commonly known, proteins generally do not penetrate membranes. But B. anthracis has devised a way to do this. In particular, PA serves as the vehicle for delivering EF and LF into the cytosol.

The model diagrammed in Friedlander, Chapter 2 p. 50, Figure 2-1 provides a basis for understanding each of the three inhibitors that have been developed. First, the growing bacteria release EF, LF and PA as monomeric proteins into its environment. Then, the proteins diffuse to the cell surface and undergo a rather intricate assembly process which results in the formation of toxic complexes.

Suggested Citation:"4 The Research Agenda: Implications for Therapeutic Countermeasures to Biological 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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These complexes pass into the cell and then into the endosomal compartment, where LF and EF are released into the cytosol.

The toxic complex that enters the cell is formed by the assembly of an activated PA heptamer which begins when monomeric PA binds to the toxin receptor, ATR. When the bound PA encounters cell-bound feron, a protease, a sub-domain of the PA molecule is released as fragment PA-20, leaving only the PA-63 fragment bound to the receptor. There are about 10,000 receptors per cell, which means that there are about 10,000 PA molecules being activated on the cell surface, where they diffuse, collide, recognize each other, and spontaneously form a heptameric ring known as the pre-pore, or pre-insertion, form. Unlike the native PA molecule, heptameric PA is capable of binding EF and LF with very high affinity.

The crystallographic structures of the native PA, heptameric PA, EF, and LF are all known. Native PA has a four-domain structure. Part of domain 1 is cut off by furin, and the remainder of domain 1 presents a site to which EF and LF bind; domain 2 is the pore-forming domain; domain 3 is involved in the ligamerization; and domain 4 is involved in receptor binding.

Dominant Negative Inhibitors (DNIs)

DNIs are mutant forms of PA that block EF and LF from entering the cytosol through the membrane. Normally, there are loops on the subunits of the poreforming domain 2 that undergo massive confirmational changes in acidic conditions and move in such a way that they form an aqueous pore across the membrane. The lumen of the pore may be the passageway for EF and LF, although this has yet to be proven. It is clear, however, that the translocation of EF and LF bound to the heptameric PA occurs in concert with pore formation. There are sites in PA, in the loops in contact with the lumen of the pre-pore, that when mutated result in an inactive form of PA. It is not completely understood how these mutated residues actually block translocation, but clearly they are potent inhibitors of toxin action. In cell culture, a one-to-one ratio of mutant to wild-type PA almost completely inhibits toxin action. This has also been demonstrated in rat models. Even with a wild-type PA/LF mixture that is ten times lethal dose, if as little as a quarter as much of the dominant negative form of PA is coinjected into the rat, the animal survives indefinitely with no symptoms whatsoever.

Of all of the approaches described here, DNIs are likely to prove the most effective. It is remarkable that by introducing a small change in only one amino acid out of some 700, it is possible to convert a toxin subunit into a very effective inhibitor of toxin action. The dead and inactive complexes that are formed by DNI action are probably channeled off to the lysosomes where they are destroyed. So this mode of action creates a sink, or destruction pathway, for the normal toxin subunits that the bacteria produces. Because many toxins are oli-

Suggested Citation:"4 The Research Agenda: Implications for Therapeutic Countermeasures to Biological 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.
×

gomeric and involve pore formation, this approach could also be generalized and applied to other toxins as well.

Currently, DNIs are a very late stage product. If they can be proven efficacious in infected animal models, they could be produced and deployed very rapidly. The product should be safe since PA is nontoxic and acts at very low levels relative to the toxin. Also, these mutations do not appear to affect the immunogenicity of PA. With regard to post-exposure prophylaxis, because it would not be possible to identify individuals had inhaled a lethal dose of spores, it would not be unreasonable to inject all exposed individuals with DNIs. For individuals who are in danger of experiencing a delayed form of anthrax, the antitoxin in conjunction with appropriate adjuvants would hopefully induce an active immunity to PA and thereby stop infection and the progression of disease.

Polyvalent Inhibitors (PVIs)

PVIs block toxin action by prohibiting EF and LF from even binding to heptameric PA in the first place. PVIs are polyacrylamide polymers that act at multiple sites on the heptamer. This is a very early stage product, although proof of principle experiments have validated the approach. PVIs have been shown to inhibit toxicity in cell culture as well as rescue rats that have been injected with lethal doses of toxin. This approach could be generalized and used in the development of inhibitors for other oligomeric virulence factors. However, much more work needs to be done before PVIs could ever seriously be considered for therapy.

Soluble Forms of the Toxin Receptor, ATR

This type of inhibitor is still on the drawing board. It is a logical ramification of the discovery of the identity of ATR, a type 1 membrane protein with an extracellular domain that binds directly to PA. Not much is known about ATR’s function except that it is closely related to the TEM8 protein that is upregulated on colorectal cancer endothelial cells. A soluble version of one of the domains of ATR, generated by eliminating the transmembrane part of the protein and its cytoplasmic tail, has been shown to protect cells in culture by competing with the normal receptor for PA. The potency of the soluble form of ATR has yet to be tested in vivo. But once positive results are achieved, development could proceed rapidly.

Suggested Citation:"4 The Research Agenda: Implications for Therapeutic Countermeasures to Biological 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 HUMAN ANTIBODY: IMMEDIATE IMMUNITY FROM BOTULINUM NEUROTOXIN AND OTHER CLASS A BIOTHREAT AGENTS.

James D. Marks, M.D., Ph.D.

Departments of Anesthesia and Pharmaceutical Chemistry

University of California, San Francisco

Botulinum Neurotoxins as Biothreat Agents

The spore forming bacteria Clostridium botulinum secrete botulinum neurotoxin (BoNT), the most poisonous substance known (Gill, 1982). The protein toxin consists of a heavy and light chain, which contain three functional domains (Simpson, 1980; Montecucco and Schiavo, 1995; Lacy et al., 1998). The C-terminal portion of the heavy chain (HC) comprises the binding domain, which binds to cellular receptors on presynaptic neurons, resulting in toxin endocytosis (Dolly et al., 1984, Montecucco, 1986). The N-terminal portion of the heavy chain (HN) comprises the translocation domain, which allows the toxin to escape the endosome. The light chain is a zinc endopeptidase that cleaves different members of the SNARE complex, depending on serotype, resulting in blockade of neuromuscular transmission (Schiavo et al., 1992; Lacy and Stevens, 1999).

There are 7 BoNT serotypes (A-G) (9), four of which (A, B, E, and F) cause the human disease botulism (Arnon et al., 2001). Botulism is characterized by prolonged paralysis, which if not immediately fatal requires prolonged hospitalization in an Intensive Care Unit (ICU) and mechanical ventilation. The potent paralytic ability of the toxin has resulted in its use in low doses as a medicine to treat a range of overactive muscle conditions including cervical dystonias, cerebral palsy, post-traumatic brain injury, and post-stroke spasticity (Mahant et al., 2000). BoNTs are also classified by the Centers for Disease Control (CDC) as one of the 6 highest-risk threat agents for bioterrorism (the Class A agents), due to their extreme potency and lethality, ease of production and transport, and need for prolonged intensive care (Arnon et al., 2001). It is likely that any one of the seven BoNT serotypes can be used as a biothreat agent. Both Iraq and the former Soviet Union produced BoNT for use as weapons (United Nations Security Council, 1995; Bozheyeva et al., 1999) and at least 3 additional countries (Iran, North Korea, and Syria) have developed or are believed to be developing BoNT as instruments of mass destruction. Iraq produced 19,000 L of concentrated BoNT of which 10,000 L were weaponized in missile warheads or bombs (United Nations Security Council, 1995; Zilinskas, 1997). The 19,000 L are not fully accounted for and represent an amount of toxin capable of killing the world’s population three times over. The Japanese cult Aum Shinrikyo at-

Suggested Citation:"4 The Research Agenda: Implications for Therapeutic Countermeasures to Biological 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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tempted to use BoNT for bioterrorism by dispersing toxin aerosols at multiple sites in Tokyo (Arnon et al., 2001).

Exposure of even a small number of civilians would paralyze the health care delivery system of any metropolitan center. Treatment of botulism requires prolonged ICU hospitalization and mechanical ventilation for up to six weeks. With the downsizing and closing of hospitals, most ICUs run at 80–100% occupancy. In San Francisco, for example, there are approximately 210 ICU beds, with an average occupancy rate of greater than 90%. As few as thirty cases of botulism would fill all empty ICU beds and occupy them for up to 6 weeks. This would eliminate availability of ICU beds for post-operative patients requiring ICU care, such as organ transplantation, neurosurgery, cardiac surgery, and traumatic injuries. Patients requiring such operations would represent ‘collateral damage’, with necessary surgery postponed, or transferred to outlying hospitals. Major civilian exposure to BoNT would have catastrophic effects. It has been estimated that aerosol exposure of 100,000 individuals to toxin, as could occur with an aerosol release over a metropolitan area, would result in 50,000 cases with 30,000 fatalities (St. John et al., 2001). Such exposure would result in 4.2 million hospital days and an estimated cost of $8.6 billion. In this study, the most important factors reducing mortality and cost were early availability of antitoxin and mechanical ventilation (St. John et al., 2001). Such treatment could reduce deaths by 25,000 and costs by $8.0 billion.

Prevention and Treatment of Botulism

No specific small molecule drugs exist for prevention or treatment of botulism, but an investigational pentavalent vaccine is available from the CDC (Siegel, 1988) and a recombinant vaccine is under development (Byrne and Smith, 2000). Regardless, mass civilian or military vaccination is unlikely due to the rarity of disease or exposure and the fact that vaccination would deny subsequent medicinal use of BoNT. Post-exposure vaccination is useless, due to the rapid onset of disease.

Antibodies for Prevention and Treatment of Botulism

Neutralizing antibody (Ab) can be used for both prevention and treatment of botulism. Historically, such Ab has been made by hyperimmunzing either horses (equine antitoxin) or human volunteers (human botulinum immune globulin). After immunization, the serum is collected and antibody prepared. The resulting antibody is polyclonal, consisting of hundreds to thousands of different antibodies that bind to many different parts of the toxin. Equine antitoxin has been shown to protect against the development of botulism in multiple animal models when administered prior to or after exposure to toxin (Franz et al., 1993). Antibody therapy is also the standard of care for botulism in humans, with equine anti-toxin and hyperimmune human globulin used to treat adult (Black and

Suggested Citation:"4 The Research Agenda: Implications for Therapeutic Countermeasures to Biological 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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Gunn, 1980; Hibbs et al., 1996) and infant botulism (Arnon, 1993) respectively. The best evidence for the value of antibody in treating botulism comes from a prospective randomized comparison of human botulinum immune globulin to non-immune globulin (Arnon, 1993). In this study, infants treated with human botulinum immune globulin had their ICU stays reduced by 2 weeks and their hospital stay reduced by 3 weeks compared to treatment with non-immune globulin. Treatment with immune globulin was beneficial even when administered 1 week after hospitalization.

Polyclonal antibodies typically neutralize toxin with high potency. Their manufacture, however, requires immunization and plasmapheresis, making large-scale production not feasible. As a result only a minimal number of doses of equine antitoxin and human immune globulin exist. These supplies would be inadequate to treat a major neurotoxin exposure. In addition, polyclonal antibodies suffer from batch to batch variability and potential infectious risk associated with an animal or human product. Horse antitoxin is also immungenic when administered to humans and can cause serum sickness and anaphylactic shock.

Monoclonal Antibody Technologies

Since 1975 it has been possible to make antibodies derived from a single antibody producing B-lymphocyte, so called monoclonal antibodies (mAbs) (Kohler and Milstein, 1975). Mabs consist of a single Ab and are made by fusing a B-cell to an immortal cell line, which can be expanded without limit. Mabs can be manufactured in unlimited supply, do not require a source of immune donors, are consistent batch to batch, and have no infectious risk. Initial mAbs were derived from mice or other rodents and were immunogenic when administered to humans, limiting their clinical development. Unfortunately, hybridoma technology has not proven generally adaptable to making human antibodies by fusing B-cells from immunized humans to an immortal cell line. With the advent of modern molecular biology techniques, however, it has become possible to make monoclonal antibodies that are far less immunogenic. Chimeric antibodies are made by grafting human antibody constant domains onto the murine variable domains, yielding antibodies which are approximately 75% human in sequence (Morrison et al., 1984). Humanized antibodies are made by grafting only the antigen binding antibody loops from the variable domains onto human variable domain frameworks, yielding an antibody which is 90% human in sequence (Jones et al., 1986). Such antibodies are far less immunogenic in humans than murine mAbs. The human constant domains also impart a long serum half life of up to one month. As a result, a single dose of antibody can provide 3 to 6 months of protection against pathogens.

Recently, it has proven possible to make antibodies that are entirely human in sequence either by immunizing mice transgenic for the human immunoglobulin locus and making hybridomas (Mendez et al., 1997) or by using phage dis-

Suggested Citation:"4 The Research Agenda: Implications for Therapeutic Countermeasures to Biological 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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play (Marks and Marks, 1996 ). For phage display, repertoires of antibody genes are cloned into a bacterial phage vector where the antibodies are displayed on the surface of the bacteriophage fused to one of the phage coat proteins (Marks et al., 1991). While it is not technically possible to display full length IgG antibodies on phage, it is possible to display smaller single chain Fv (scFv) or Fab antibody fragments. Such antibody fragments contain the antigen recognition of the IgG (the variable domains), but lack the constant regions.

To perform phage display, repertoires of antibody heavy and light chain variable domain genes are assembled and cloned into a phage vector to create libraries of scFv or Fabs displayed on the phage surface. The source of the variable region genes can be any species, including immunized humans. Once phage libraries are constructed, binding phage antibodies can be isolated from non-binding phage by a variety of types of affinity chromatography. Binding phage antibodies can be detected by ELISA, and the antibodies characterized with respect to affinity, epitope recognized, sequence, and biologic activity. Given the high transformation efficiency of bacteria, it is possible to make libraries of millions to billions of different antibodies, allowing immortalization of the entire immune response to an antigen. As a result, hundreds to thousands of antibodies are generated, allowing isolation of high affinity antibodies to rare epitopes. In contrast, generation of hybridomas captures only a fraction of the immune response, due to the inefficiency of the fusion process. Other advantages of antibody phage display include the ability to make antibodies from immunized humans and to engineer antibody affinities to values not achievable by hybridoma technology (Schier et al., 1996).

Neutralization of Botulinum Neurotoxin by Monoclonal and Oligoclonal Antibody

Recombinant mAb could provide an unlimited supply of antitoxin free of infectious disease risk and not requiring a source of human donors for plasmaphoresis. Such mAb must be of high potency in order to provide an adequate number of doses at reasonable cost. In some instances, the potency of polyclonal antibody can be recapitulated in a single mAb (Lang et al., 1993). In the case of BoNT, potent neutralizing mAb have yet to be produced: single mAb neutralizing at most 10 to 100 times the 50% lethal dose (LD50) of toxin in mice (Pless et al, 2001; Hallis et al., 1993).

To generate mAb capable of neutralizing BoNT serotype A (BoNT/A), we generated scFv phage antibody libraries from immunized humans, mice, and mice transgenic for the human immunoglobulin locus (Amersdorfer et al., 1997; Amersdorfer et al., in press). After screening more than 100 unique mAb from these libraries, three groups of scFv were identified which bound non-overlapping epitopes on the BoNT/A binding domain (HC) and which neutralized toxin in vitro (prolonged the time to neuroparalysis in a murine hemidia-

Suggested Citation:"4 The Research Agenda: Implications for Therapeutic Countermeasures to Biological 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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phragm model) (Amersdorfer et al., 1997; Amersdorfer et al., in press). In vitro toxin neutralization increased significantly when two scFv binding non-overlapping epitopes were combined. The small size of the scFv, however, precluded study of in vivo toxin neutralization, due to the rapid clearance of the 25 kDa scFv from serum (Colcher et al., 1990).

To evaluate in vivo BoNT neutralization, IgG were constructed from the variable domain genes of three BoNT/A scFv that neutralized toxin in vitro. No single IgG significantly neutralized toxin in vivo, but combining mAb led to potent toxin neutralization. The most potent mAb pair protected mice challenged with 1500 LD50s of toxin, while combining all three mAb protected mice challenged with 20,000 LD50s of toxin (per 50 mg of antibody administered) (Nowakowski et al., 2002). The potency of the three antibody combination (oligoclonal Ab) was formally titered using the standard mouse neutralization bioassay and was determined to be 45 International Units/mg of Ab. One International Unit (IU) neutralizes 10,000 LD50s of BoNT/A toxin, yielding a potency of 450,000 LD50s /mg of Ab. This is 90 times more potent than the hyperimmune human globulin used to treat infant botulism (Arnon, 1993) and approaches the potency of hyperimmune mono-serotype horse type A anti-toxin (Sheridan et al., 2001).

The increase in potency appears to result primarily from a large increase in the affinity of the oligoclonal Ab for toxin compared to the individual mAb (Nowakowski et al., 2002), and also to greater blockade of the toxin surface which interacts with cellular receptors (Mullaney et al., 2001). Such mechanisms may be generally applicable to many antigens in solution, suggesting that oligoclonal Ab may offer a general route to more potent antigen neutralization than mAb. The precise contribution of these two mechanisms to the increase in potency is unknown. It is also unknown as to whether engineering the affinity of one of the mAbs to a value approaching that of the oligoclonal Ab would yield a similar increase in potency as combining mAbs.

Conclusions, Obstacles, and Future Research Needs

Recombinant oligoclonal Ab offers a safe and unlimited supply of drug for prevention and treatment of BoNT/A intoxication. With an elimination half-life of up to 4 weeks, Ab would provide months of protection against toxin. Since the current oligoclonal Ab consists of either chimeric or human IgG, production could be immediately scaled to produce a stockpile of safe anti-toxin. Alternatively, we have already replaced the chimeric S25 IgG with a fully human IgG and increased potency of the oligoclonal Ab more than 2 fold. Work is ongoing to replace chimeric C25 with a fully human homologue. Chimeric, humanized, and human mAb represent an increasingly important class of therapeutic agents whose means of production are known. The high potency of the oligoclonal Ab makes it possible to manufacture millions of doses of antitoxin from a single manufacturing facility which could be stockpiled for future use. Ten mAb have

Suggested Citation:"4 The Research Agenda: Implications for Therapeutic Countermeasures to Biological 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 approved by the FDA for human therapy and more then 70 other Mab therapeutics are in clinical trials (Reichert, 2001). As a result, the process of scaling production and manufacturing, as well as the necessary toxicology and clinical safety testing requirements are known. This should result in a rapid development timeline, especially compared to vaccines or small molecule drugs. The major challenges and obstacles to development are FDA regulatory issues related to combining mAbs and a predicted worldwide shortage of IgG manufacturing capacity (Reichert, 2001).

Oligoclonal Ab would be applicable to the other BoNT toxin serotypes and these antibodies should be generated as rapidly as possible. Oligoclonal antibody may also be able to potently neutralize other class A agents as well. Anthrax toxicity is toxin mediated, and polyclonal Ab has been shown to be protective for this agent (Little et al., 1997; Beedham et al., 2001). Vaccinia immunoglobulin can be used to prevent or treat smallpox or complications arising from vaccination of immunocompromised hosts (Feery, 1976). Ab may also be useful for plague and the hemorrhagic fevers (Hill et al., 1997; Wilson et al., 2000). Given the threats posed by these agents, rapid generation and evaluation of oligoclonal Ab for their neutralization is warranted.

MEETING THE REGULATORY AND PRODUCT DEVELOPMENT CHALLENGES TO ADDRESS TERRORISM

Andrea Meyerhoff,* M.D.

Director, Anti-terrorism Programs

Office of the Commissioner, Food and Drug Administration

FDA’s mandate in anti-terrorism warrants a balance between its requirements as a regulatory agency and the demands of a public health emergency. We attempt to achieve this balance by facilitating the availability of safe, effective drugs, vaccines, and medical devices in a manner that is consistent with our legal responsibilities as a regulatory agency.

Organization of FDA Anti-Terrorism Programs

The FDA is divided into five centers which are organized based on the type of products that are regulated. Three of these centers regulate products that deal with medical care: CDER (Center for Drugs); CBER (Center for Biologics), which regulates vaccines; and CDRH (Center for Devices and Radiation Health), which regulates a range of medical devices from diagnostic assays to mechanical ventilators.

*  

This statement reflects the professional view of the author and should not be construed as an official position of the Food and Drug Administration.

Suggested Citation:"4 The Research Agenda: Implications for Therapeutic Countermeasures to Biological 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 Director of Anti-terrorism Programs is housed in the Office of the Commissioner, which is not housed in any particular center but rather coordinates across these centers. There is a designated anti-terrorism point of contact (POC) within each center and with whom the director liaisons on antiterrorism issues.

For many products under development, there is already a relationship established with the appropriate regulating center. New products that are seeking regulatory guidance and old products that may have an anti-terrorism application are often routed through the Anti-terrorism Programs first and then passed on to the appropriate POC. Anti-terrorism Programs works with the POC to coordinate all efforts that are relevant to each particular stage of product development.

Existing Regulatory Mechanisms for Enhanced Product Availability

There are several existing regulatory mechanisms that can be invoked to address issues of anti-terrorism product availability. They apply to a number of different phases of product development, from the early pre-IND (i.e., before the drug is introduced into human trials) to the review of the NDA (i.e., new drug application for marketing approval):

Pre-IND meeting is an attempt to begin early dialogue between the sponsor and the review division and provide regulatory guidance in preparing IND (investigational new drug) applications. IND applications include a set of data that are shown to the agency before the product is used for the first time in humans. The entire body of data are reviewed by all of the various disciplines that are brought to bear at that stage, and missing pieces of data are identified. Pre-IND meetings are regarded as resources for developers. There is no set period in the pre-IND phase when this meeting must occur. Some sponsors approach the FDA quite early; others meet immediately before they submit the IND just to make sure that everything is okay.

IND regulations refer to the set of regulations that determine how a product will be used when it is initially introduced into a human population. IND regulations may also be viewed as a mechanism for making an investigational product available. IND regulations have three basic components: an informed consent form; review of the protocols for planned use by an institutional review board; and a plan for the collection of safety and efficacy data from the human population in which the product is going to be used.

“Streamlined” IND is not an official regulatory term, but it serves the purpose of addressing the requirements of the IND regulations while simultaneously making a product available to a large population in an emergency setting. Streamlined INDs are in place for both biologic and drug products, and the template can be used for other products as well if the need should arise.

The animal efficacy rule was proposed and published in the Federal Register in October 1999. It is intended to apply when a disease cannot be studied in

Suggested Citation:"4 The Research Agenda: Implications for Therapeutic Countermeasures to Biological 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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humans, that is when the disease is either very rare or it would be unethical to introduce the disease into a human population. Clearly, diseases due to biological agents of intentional use would fit into this category. This rule provides the framework for which efficacy data could be derived from an animal model of disease and is intended to address efficacy only. The safety of the drug still needs to be studied in the human population. The rule is based on the use of a scientifically valid animal model and generally requires the use of two species. In cases where a well-established species is already recognized as a scientifically valid model for disease, it would be decided on a case-by-case basis whether efficacy data is needed from a second species as well. Currently, this rule is still only proposed and has not been used (the approval of ciprofloxacin for anthrax invoked accelerated approval, not the animal rule); finalization is anticipated within the next few months.

Accelerated approval (sometimes referred to as subpart H regulation) refers to a set of regulations that permit the use of a surrogate marker for the purposes of demonstrating efficacy of a product if the product is considered reasonably likely to provide an improvement in mortality or serious morbidity. Still, postmarketing data would need to be collected to verify the surrogate. This is the regulatory approach that was taken for ciprofloxacin for anthrax which was initially approved for human use in the mid-1980s, so there was already a fairly well-developed set of human pharmacokinetic data and a very large safety database. The surrogates in this case were human serum levels of ciprofloxacin which have been shown to be associated with improved survival in monkeys that have been exposed to aerosolized B. anthracis spores. Serum level in humans have been shown to reach or exceed weight-adjusted levels in monkeys. The labeled regimen for post-exposure inhalational anthrax is a sixty-day dosing period. Safety databases of patients who received the drug for more than sixty days, patients who received the drug for sixty days, patients who received it for less fewer than sixty days, and patients who received other antibiotics all show similar adverse event rates. GI events are the most common, with a slightly incidence of higher abdominal pain and rash in the sixty-day group. However, patients who received the drug for sixty days showed no previously unidentified adverse events associated with the shorter, more usual seven to fourteen day dosing periods. There is also a substantial pediatric safety database which supports the approval of ciprofloxacin for post-exposure inhalational anthrax indication in pediatric patients.

Priority review is a request that is made by the applicant at the time of NDA filing. It is generally used for products that are considered to have special public health significance and results in a review process that is shortened to six months rather than the usual ten or twelve. In addition to accelerated approval, ciprofloxacin for anthrax also received priority review.

Suggested Citation:"4 The Research Agenda: Implications for Therapeutic Countermeasures to Biological 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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Recent FDA Anti-Terrorism Initiatives: Drug Development

Recent FDA antiterrorism-specific initiatives, most of which involve anthrax, include:

In early November, 2001, the FDA published a Federal Register notice recognizing that doxycycline and penicillin are also approved for anthrax. The Federal Register notice was published because product labels do not contain specific dosing information for post-exposure inhalational anthrax, even though scientific data support this labeling. The Federal Register notice states this, provides the dosing recommendations, and invites applications from manufacturers of these drugs to request labeling supplements. This was done as a way to expand the options of products available to manage what was clearly a growing population of people who had been exposed to aerosolized spores of B. anthracis. Because of potential side effects, drug intolerance, other medications, and any of a number of other reasons why people cannot take a particular class of drugs, having more available options expands our ability to manage large populations of exposed individuals.

There are a number of ongoing efforts among several government agencies to provide regulatory guidance for the development of animal models to be used in the evaluation of drugs specifically for diseases related to bioterrorism. There has been much ongoing collaboration with DOD laboratories and the NIH to establish guidelines and goals for studying these products in animal models.

The FDA has been considering other products besides antimicrobials that could be made available for the treatment of clinically apparent inhalational anthrax.

REGULATION AND PRODUCTION OF RECOMBINANT HUMAN ANTIBODIES

Kathryn E. Stein,* Ph.D.

Director, Division of Monoclonal Antibodies

Center for Biologics Evaluation and Research Food and Drug Administration

The FDA is fully prepared to deal with the issue of monclonal antibody cocktails and, in fact, has had relevant policies in place since 1994. At that time, it was anticipated that manufacturers might develop antibody cocktails directed at either different epitopes on a particular antigen or different antigens on a particular organism. From a safety and efficacy perspective, these policies consider

*  

This statement reflects the professional view of the author and should not be construed as an official position of the Food and Drug Administration.

Suggested Citation:"4 The Research Agenda: Implications for Therapeutic Countermeasures to Biological 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.
×

cocktails as single products. However, there must be a rationale for the use of each component in the cocktail as well as a means for determining the dose of each component. These data could come from preclinical animal models, for example, or in vitro neutralization or other tests.

The real question is, what kinds of antibodies should be included in the cocktails? For example, because the murine Fc region is the most immunogenic part of a monoclonal, both chimeric and humanized antibodies, with human Fc regions, have been engineered and shown to exhibit much less immunogenicity in humans than whole murine antibodies. With regards to how antibodies are produced, there is some concern that phage display may create combinations of heavy and light chain genes that would raise unusual issues regarding immunogenicity.

One could envision a mixed antibacterial and antiviral cocktail comprised of antibodies to a diverse assortment of potential bioterrorist agents. However, more research is needed to identify protective factors and determine which virulence factors the antibodies should target. (See Marks for further discussion on antibody options.)

There are many antibodies currently being researched in academe that could be developed into cocktails. There needs to be greater partnerships among academe, government, and industry in order to bring the intellectual property to the antibody engineers so that these products can be developed.

There are ways to lyophilize monoclonal antibodies such that the cocktails could be stable at room temperature and on the shelf of every emergency room, although formulation needs to be further studied. The FDA is willing to consider any proposed products.

Limitations on monoclonal cocktails pertain mostly to production. The worldwide capacity for mammalian cell culture has reached its maximum. In order to increase production to build a stockpile for prophylaxis or treatment in the event of a large-scale bioterrorist attack, we must either build more manufacturing facilities or buy or renovate already existing facilities. Such large-scale production will likely require government assistance and funding.

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Suggested Citation:"4 The Research Agenda: Implications for Therapeutic Countermeasures to Biological 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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Suggested Citation:"4 The Research Agenda: Implications for Therapeutic Countermeasures to Biological 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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Suggested Citation:"4 The Research Agenda: Implications for Therapeutic Countermeasures to Biological 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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Suggested Citation:"4 The Research Agenda: Implications for Therapeutic Countermeasures to Biological 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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Suggested Citation:"4 The Research Agenda: Implications for Therapeutic Countermeasures to Biological 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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Suggested Citation:"4 The Research Agenda: Implications for Therapeutic Countermeasures to Biological 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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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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