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5
Alternative Approaches to Animal Testing for Biodefense
Countermeasures
This chapter examines alternative approaches to the use of animals in the development of medical
countermeasures against biothreats. It presents the concept of the Three Rs as a basis to safeguard good
science while improving laboratory animal welfare. It briefly describes a number of in vitro and in vivo
methods that support the Three Rs and humane endpoints. The Committee on Animal Models for
Assessing Countermeasures to Bioterrorism Agents concludes that absolute replacement of animal
models in countermeasure development is not possible at this time and that in vitro and in silico
methods are not advanced enough yet (in part due to absence of human data) to reliably replace
animals in biodefense research. Recognizing that the premise of the Animal Rule is the use of animals
and that the Animal Welfare Act (AWA; 7 USC § 2131-2159) requires the consideration of alternatives,
the Committee recommends to embrace and further develop alternative options to (1) take advantage
of new (clinical and epidemiological) data; (2) correlate those new findings with outcomes from
established animal models; (3) improve the welfare of animals used in countermeasure development
and testing; and (4) strive, where possible, to replace nonhuman primates as the animal of choice in
biodefense research. The Committee reiterates the fundamental need for data from human populations
(versus laboratory animal species) as the crucial driver for the development of in vitro and in silico
pathways. Further, the Committee concludes that changing the standard practice of animal
experimentation to approximate the clinical course of treatment that humans may receive could
provide a more reasonable expectation of the usefulness of certain countermeasures during
development.
61
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62 ANIMAL MODELS FOR ASSESSING COUNTERMEASURES
GENERAL PRINCIPLES OF ALTERNATIVE APPROACHES
In 1959, Russell and Burch formulated three principles to reduce the numbers of animals used in
experimentation. These are known as the Three Rs: refinement, reduction, and replacement (absolute or
relative).1 The validation process of regulatory testing has over the years incorporated many methods
that support one or more of these principles for two main reasons: (1) only regulated and standardized
animal tests that are repeatedly carried out over a long period of time (typical timeframe 12 years) and
tests that have enormous costs warrant a formal validation process to be accepted as such; and (2) in the
area of safety assessments, it is especially difficult to abandon an established test because safety
standards could be lowered. In other areas of research and development, especially in the agent
discovery phase, replacement of older with more advanced methods is more common due to constant
pressure for more predictive and less costly tests.
In the case of developing countermeasures for bioterrorism agents where absolute dependence
on animal models for efficacy testing serves to replace human clinical trials, the Three Rs provide a
good framework to reduce animal use and minimize the animals’pain and distress.2 Because adoption
of alternatives was driven by both animal welfare considerations and scientific advances in our
understanding of biological phenomena, their utilization is often in the best interest of the study.
Although alternatives do not compensate for the lack of clinical efficacy trials with human participants,
they can enable technologies from which more information is gained than that gained by animal tests
alone. In addition, they reduce the use of precious and expensive resources and reduce animal pain and
distress, often resulting in improved quality of research outcomes (NCR 2008, 2009, 2011; Wolfer et al.
2004).
Applying the Principle of Refinement
The best possible treatment of animals starts with attention to husbandry (AWA; 7 USC § 2131-2159).
Social housing and enriched environment within the cages, especially for the highly social nonhuman
primates often used in these studies, while taking into consideration the scientific needs of the study,
represent key strategies to avoid or reduce distress3 (NRC 2008). The use of analgesics and anesthetics is
mandatory, not only for the alleviation of pain and distress, but also because it represents a more
realistic approximation to the treatment of human patients.4,5
The development and use of protocol-appropriate humane endpoints, especially the early
termination of studies at time points that indicate that animals are unlikely to recover, minimizes pain
The utility and applicability of the Three Rs has been described in various documents. For additional
1
information, see the Guide for the Care and Use of Laboratory Animals, Eighth Edition and references therein (NRC
2011), and the website for the National Centre for the Replacement, Refinement and Reduction of Animals in
Research (http://www.nc3rs.org.uk/).
2 The usefulness of the Three Rs and the employment of alternatives in regulatory safety testing are not discussed
here as safety testing does not fall under the auspices of the Animal Rule.
3 The ability to provide enriched environment or social housing in biocontainment facilities may be difficult,
extremely limited, or impossible. Although it is a necessary husbandry and animal welfare provision, methods to
enrich the environment or house social animals in a biocontainment facility have not been studied.
4 It should be noted that the quality of care for human patients is not universally identical and that analgesics and
anesthetics may not be available in natural outbreak settings. Therefore, different animal research protocols may
be needed for the development of treatments under these conditions.
5 Non-administration of analgesics should be scientifically justifiable and accepted by an Institutional Animal
Care and Use Committee (IACUC; Animal Welfare Act; 7 USC § 2131-2159) and employed as rarely as possible.
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and distress without compromising the result of the study6 (Chapter 5 of Recognition and Alleviation of
Pain in Laboratory Animals (NRC 2008); Nemzek et al. 2004; NRC 2011; Olfert and Godson 2000).
Conversely, insistence on death or even moribundity as an endpoint is questionable, as signs of
irreversible decline are well established for all common laboratory animal species.7 It is important that
early termination studies include complete necropsies and histopathological examination accompanied
by appropriate agent isolation from tissues to determine if the killed animal was, in fact, unlikely to
survive. Further, early endpoints ought to be verified before embarking on larger studies to ensure that
studies are not needlessly repeated. At a minimum, if natural history or descriptive studies require an
understanding of events proximate to death or a time-to-death estimate, then these data should be used
where possible as a historical benchmark to estimate fatal outcome without needing to actually follow a
full disease course in a moribund animal.
Looking at the Numbers: Reduction
Most measures to reduce the number of animals used are often justified in terms of avoiding pain or
distress and of saving resources, especially in studies conducted in biocontainment facilities. Well-
chosen statistical methods, such as appropriate power calculations of group numbers, should always be
part of the experimental design, and knowledge of historical data about the variance of the anticipated
results can help to select the appropriate sample size. Tiered testing strategies (e.g., treating individual
animals or small groups sequentially and not in parallel) and use of qualified pilot studies allow for
studies to be terminated early if no effect is observed in the first few animals.8 As discussed in the
previous section, it may be possible to avoid the use of untreated contemporary control groups if
historical data can be employed. Similarly, sharing control groups among multiple experiments
performed at the same time may also lower the number of animals required. Careful selection of
dosages and omission of unrealistic treatment groups further reduces animal use.
The broader use of inbred murids has helped to reduce the variability of experimental results
and thus the size and number of groups required. Consequently, inbred animals are often selected for
this reason, although their use does not reflect the variability of the human population (also stated in
Chapter 2, Table 2-9; for an approach that embraces genetically diverse murine models, see page 69).
The use of early, informative endpoints derived from complementary in vitro methods (e.g., estimation
of effective or maximal tolerated doses by effective concentrations in vitro and in vitro metabolism
studies with hepatocytes or microsomes to exclude the use of species that do not reflect human
metabolism) can further reduce animal numbers and refine the experiments by minimizing pain and
distress. Other noninvasive methods, such as imaging technologies and telemetry, that allow
Biomarkers and signatures of toxicity that are often derived from nonanimal models are useful tools that make
6
animal studies more sensitive or facilitate earlier termination (with humane endpoints) without loss of
information.
7 The development of (early termination) endpoints and guidelines for when animals should be euthanized is a
highly desirable animal welfare practice, and as such discussed in laboratory animal care and use regulations.
Under the AWA, such provisions are part of the research protocol and subject to IACUC approval. The
Committee recognizes that the above can be at odds with the 3rd criterion of the Animal Rule that “the animal
study endpoint is clearly related to the desired benefit in humans, which is generally the enhancement of survival
or prevention of major morbidity” (FDA 2002, p 37989). That is why pilot studies and determination of
biomarkers or endpoints through small sample sizes are important. Further, careful and focused clinical
observation of animals can identify entrance into an irreversible state of decline followed by immediate
euthanasia.
8 Such approaches may require larger group sizes for statistical power.
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64 ANIMAL MODELS FOR ASSESSING COUNTERMEASURES
monitoring of the same animal over the entire course of an experiment, instead of sacrificing more
animals at different time-points, also reduce group-sizes while preserving statistical power.9
Focusing on Replacement
While opportunities for in vitro replacements are available to a greater extent in toxicology and safety
assessments (e.g., NICEATM and ICCVAM test method evaluations;
http://iccvam.niehs.nih.gov/methods/methods.htm), non-sentient test systems in infectious disease
research generally and in studies under the Animal Rule particularly are limited. Some opportunities
lie in ex vivo approaches where animals or human volunteers are treated with the product in
development and only tissues or blood are subsequently exposed to pathogens or used for
measurements. Tissue engineering methods, including artificial organs and organotypic cultures, can
sometimes reproduce the physiological environment to study aspects of the course of infection, but
extrapolation to the in vivo conditions and the systemic multifactorial components of host defense are
limited. For further exploration of this topic as it pertains to studies under the Animal Rule, see section
In vitro tools and replacement strategies (below).
ANIMAL EFFICACY STUDIES ARE CLINICAL TRIALS
Clinical trial designs for efficacy mandate that human subjects must be protected from undue risk when
participating in clinical research activities (45 CFR 46 [2009]; National Commission for the Protection of
Human Subjects of Biomedical and Behavioral Research 1979). This protection includes the provision of
clinical standard of care in addition to the product being evaluated (unless the standard of care is
contraindicated). However, efficacy testing of a new drug or vaccine in animals routinely involves the
administration of only the test product. Although this has been the standard practice in animal research
protocols, such reliance solely on the test article to demonstrate efficacy may produce a misleading
model of what the human counterpart may actually experience and lead to false or incomplete data on
the effectiveness of the test product. Patients with dyspnea and acid and base imbalances due to
pulmonary insufficiency, for example, would have to be provided with oxygen and intravenous
electrolytes while enrolled in a trial to test the efficacy of a novel bronchodilator. Similarly, patients
with congestive heart failure participating in an efficacy trial for a drug intended to increase the
strength of myocardial contractions, would concomitantly be given diuretics (presuming sufficient
kidney function) to minimize pulmonary congestion. In either example, although the test product could
be efficacious for neutralizing or reversing the initial insult, the patient could still succumb from
underlying or preexisting complications that were not treated by the test product (Miller and Silverman
2004).
The same clinical standard of supportive care certainly applies to persons exposed to a
bioterrorism agent, regardless of whether they are lightly exposed and asymptomatic or suffering from
organ failure. Severe dehydration and hypotension resulting from a highly infectious pathogen (e.g.,
acute diarrhea, septic shock, and hemorrhagic fever) would be treated with blood volume and blood
pressure restoratives even though the origin of disease was microbial and the test product being
evaluated was an antibiotic or antiviral drug.
The incorporation of supportive veterinary care in animal efficacy testing of countermeasures
against biothreats is recognized by regulators as both reasonable and informative. In the 2009 Draft
The use for in vivo imaging strategies in product development for biothreats remains largely unrealized. For
9
these techniques to be used in the preclinical arena, validation of imaging as a correlate of bacterial or viral
burden is necessary.
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Guidance for Industry concept paper, the FDA described important components to be included in
preclinical protocols for demonstrating efficacy under the Animal Rule. In that document, it is advised
that “studies should be designed to mimic the clinical scenario and achieve meaningful outcomes
comparable to the endpoints desired in humans. In some instances, supportive care should be
administered to the animals as part of the study design” (FDA 2009a; see Appendix B). Not providing
similar medical interventions to an animal subject when assessing preclinical efficacy may result in false
(negative) conclusions about corresponding efficacy in humans. A test product could be sufficiently
effective when combined with reasonable supportive care, but it could “fail” if evaluated alone. The
acute need for additional biodefense medical countermeasures is not served when candidate drugs and
vaccines are abandoned that may, indeed, prove efficacious when tested in a comprehensive medical
fashion.
Employing an expanded spectrum of clinical care rather than relying on a single test product for
efficacy testing has other advantages beyond not prematurely discarding promising drugs and
vaccines. By more closely mimicking the broader scope of clinical care provided to patients, one may
identify which specific ancillary care regimens, if any, contribute most to the efficacy of the test
product. This information might lead to a critical component for the final label of the approved drug. In
addition, longer survival of animal subjects due to an expanded repertoire of clinical support could
result in better predictive models. If animals die too quickly,10 the pharmacokinetics and drug
metabolism of the test product or the absence of effects of increased time on an effective immune
response may not replicate or approximate the expected timelines in patients, resulting in misleading
findings.11 Furthermore, subtle yet important differences in test products or dosages may become
evident over a longer time frame of therapy due to prolonged animal survival. Objections to including
an expanded array of clinical care for animal subjects in efficacy testing protocols usually involve one of
two themes. First, anything administered to the animal besides the test product could interfere with the
“true” efficacy properties of the product in question, possibly leading to (false) positive conclusions
when the test product is not otherwise strong enough as a therapeutic or preventative agent. A counter-
argument to that objection is that efficacy should address only the specific effects of that pathogen or
toxin; if nonspecific complications represent the actual disease, then one should focus on efficacy
testing specific to those sequelae.12
The second objection is that supportive-care components are not compatible with 21 CFR Part 58
(Good Laboratory Practice for Nonclinical Laboratory Studies; GLP) because the components may
introduce high levels of variation that cannot withstand a quality-assurance audit of that study, or they
may create many expensive complexities. If, however, quantitative thresholds are established for
anticipated clinical signs (e.g., fever) and standardized supportive interventions in advance, such an
Lethality in animals may be due to secondary causes, such as severe dehydration or hypothermia as a
10
consequence of being too sick to eat, drink, or move around rather than specific or primary effects of the disease
or the product tested.
11 Metabolism and thus pharmacokinetics can differ between humans and animals (Martignoni et al. 2006). The
difference is relevant not only for extrapolations to human kinetics but also for its impact on drug efficacy studies,
i.e., where, when, and for how long are effective tissue concentrations reached. Furthermore, drug
pharmacokinetics may change under disease conditions and may be affected by the severity of disease.
12 As discussed in chapter 3, the application for licensure of a human monoclonal antibody against inhalational
anthrax by Human Genome Sciences is on hold pending additional studies. One of the concluding remarks of the
FDA-appointed committee of experts was that “there was no study with the antibiotic as the control arm” (FDA
2009b). While standard clinical care against inhalational anthrax is primarily the administration of antibiotics, the
implication is that it should be a necessary component of the animal efficacy trial, both as a separate arm of the
trial and as a combination treatment with the test product. The same trial design should apply when the standard
of care in humans is supportive therapy alone.
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66 ANIMAL MODELS FOR ASSESSING COUNTERMEASURES
expanded study design can be carried out in keeping with GLP principles. An example of this approach
would be to combine body-temperature monitoring via a standard operating procedure in anticipation
of fever as a clinical sign and initiation of antipyretic therapy in accordance with that standard
operating procedure when the body temperature rises above a predetermined threshold as documented
in the veterinary literature.
The Committee recognizes that the above is a different approach to using animals and
generating data from animal trials. It is meant to be more comprehensive and apply considerations
from human clinical trials (or treatment in the field) to animal studies in order to make more
meaningful contributions to the interpretation of data as might be applied to humans. This strategy, i.e.,
provision of supportive care to animals subjected to severe disease, is not only more humane but may
allow fewer animal numbers to be used in accordance with the principles of the Three Rs, as these are
among the common causes of reduced validity of animal studies presented in Chapter 2, Table 2-9.
Even though these actions can be performed in multiple grades of moderation without converting the
laboratory into an intensive care unit, the practical difficulties of establishing this methodology in
biocontainment facilities indicate the need for careful deliberation and study of the basic principles of
such an approach and the creation of guidelines for the care and use of animals in research done under
biocontainment conditions. The safety requirements of working in a biocontainment environment and
the potential increased costs of implementing such types of animal trials are of considerable
importance, especially for long-term studies.
IN VITRO TOOLS AND REPLACEMENT STRATEGIES
In the United States, a discussion of the future of the field of toxicology was prompted by the National
Research Council report Toxicity Testing in the 21st Century: A Vision and A Strategy (NRC 2007). In 2008,
several U.S. agencies, including the FDA, announced a coalition to facilitate this reports’
implementation: “We propose a shift from primarily in vivo animal studies to in vitro assays, in vivo
assays with lower organisms, and computational modeling for toxicity assessments” (Collins et al. 2008,
p 906).
In Vitro and In Silico Methods
Biothreat agents are prime candidates for accelerated development and regulatory clearance of
countermeasures by using animal models as alternatives to human clinical trials. This acceleration
suggests the need to develop new and innovative strategies for collecting data and observations about
how humans respond to these pathogens. Without this information the relevance of the animal models
cannot be adequately ascertained. The same need exists for information obtained from the animal
models to help develop and interpret new in vitro and computational in silico (IV/IS) methods.
Advances in molecular characterization and in computational power have made it possible to consider
approaches that do not even require the use of living systems, or at a minimum, accelerate the capacity
of these artificial systems.
Many of the elements for a fully integrated IV/IS product development and approval strategy
exist today, especially for anti-infectives against known agents that can be cultured in vitro. Standard
techniques include high-throughput screening for drug discovery, in vitro testing of antimicrobial
efficacy and drug resistance in many bacterial and viral pathogens, testing of some aspects of toxicity
and pharmacokinetics and pharmacodynamics and computer modeling of structure-activity
relationships. While in vitro assays for preclinical toxicity testing have been used extensively for several
decades (reviewed in Judson et al. 2010), reliable assays for systemic toxicities, although improving,
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remain a challenge (Adler et al. 2011; Hartung et al. 2011). Similarly, advances in computational
capacity allow for increasingly complex modeling, but the animal models remain a critical bridge to test
and confirm biosignatures (biomarkers) and other effects identified through studies of the natural
history of the disease, relevant human clinical trials, or other animal model work. These biosignatures
and pathways so identified and tested could then be a bridge to IV/IS models. However, despite
technological advances the absence of suitable high-quality comparative data impedes the realization of
this process (see previous discussion in Chapters 2 and 4 on the importance of human data).
The use of biomarkers in drug discovery and development is a related nascent area with great
promise (pharmacogenomics; Hamburg and Collins 2010).13 Suitable biomarkers, such as gene
polymorphisms or gene expression profiles, can be determined in vivo (in animal models, natural
infections, or clinical trials), and be used to predict whether a new candidate therapeutic is likely to be
effective or, based on markers associated with adverse events, which individuals might be at increased
risk for adverse reactions. This approach of moving from in vivo identification to in vitro testing of
biomarkers has been used in the last few years for several biodefense-related agents, including
monkeypox and anthrax. Time course studies of biomarker expression (i.e., gene expression arrays)
following experimental infection of animals suggest that it may be possible (at least in some situations)
to determine how long the patient has been infected, and whether optimal treatment varies depending
on time after infection (Alkhalil et al. 2010; Das et al. 2008).
The immune system is a prime example of complex (and still incompletely understood)
interactions of multiple cell types not yet amenable to IV/IS testing or modeling. An “artificial”
functional in vitro immune system could facilitate the identification of candidate vaccines and
therapeutics for immunosuppression or immune enhancement and could also help eliminate candidate
therapeutics with undesirable immunologic properties. Although this work is still in the nascent stages,
progress has been reported (Gaucher et al. 2008; Schanen and Drake 2008). In theory, this system, which
depends on cell migration and maturation, will mimic what occurs in vivo and its output will more
reliably reflect anticipated outcomes. Once created, this engineered tissue system can be manipulated
and dynamic endpoints determined. For example, the Modular Immune In Vitro Constructs (MIMIC®)
System, a simulated human immune system, enables testing of the adaptive immune response to
vaccine antigens directly on microtiter plates and can provide multiple replicates of immune system
activation and response from a single individual to different antigens ex vivo (Higbee et al. 2009). In
recent years, a number of cell assemblages have been “engineered” in vitro to functionally mimic
corresponding organs in the body (Fernandez and Khademhosseini 2010; Ingber et al. 2006; Mammoto
and Ingber 2010). For instance, the development of micropatterned cocultures of human hepatocytes
and supportive stromal cells permitted the growth of Hepatitis C virus (HCV) in vitro for the first time
and, serving as a high-throughput platform, could allow rapid in vitro screening of candidate anti-HCV
therapeutics for both efficacy and toxicity (Ploss et al. 2010). However, for this technology to assist in
the development of countermeasures, the system must demonstrate that it accurately reflects human
infections by using pathogens for which large amounts of (preferably) human in vivo data exist to test
its reliability. Until such data are available, the system may be used to explore differences between
multiple species (including humans) to further refine animal models or point to more accurate in silico
representations of the human system.
The term “pharmacoepigenetics” is sometimes used when gene expression, rather than DNA gene sequence
13
variants, is the biomarker (Baer-Dubowska et al. 2011).
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Computational Modeling
At present, computational approaches are most useful in the basic science phase of drug and vaccine
development, specifically in identifying targets and biomarkers. These approaches are often helpful in
the selection of the appropriate animal model because they allow the pathology and response to an
agent to be defined in great detail. In this context, the computational approaches reduce animal testing
by focusing future studies on the most promising leads and potentially by identifying biomarkers to
develop humane endpoints for follow-up studies.
In their current form, IV/IS tools and strategies cannot serve as complete replacements for
animal models. For complete replacements to be possible, it will be necessary to further define the
functional and regulatory networks within the mammalian host and develop modeling approaches that
allow prediction of how those networks (and ultimately the host’s physiology) will behave when
perturbed by infection or toxin. For infectious diseases, interactions of significance comprise pathogen
and host responses, including the role of specific and nonspecific immune responses. The development
of the first protease inhibitor against HIV-1 was a dramatic advance about two decades ago, and is
probably the best known example of a new therapeutic developed by computational methods from
structural information alone, but the hope is for many more examples in the future as both structural
biology and computational expertise advance (Miller et al. 1989; for a general review on computer-
aided drug design see Talele et al. 2010).
More recently, computational modeling has played an important role in the development of
vaccines against influenza. For instance, the identification of highly conserved epitope sequences of the
influenza virus elicited broadly reactive neutralizing antibodies that are currently pursued as potential
“universal” influenza vaccines (Ekiert et al. 2010; Fleishman et al. 2011; Kang et al. 2011; Toussaint et al.
2011; Wang et al. 2010). Further, the identification of preexisting, cross-reacting epitopes against H1N1
viruses on human T-cells were used to test candidate vaccines against not only influenza viruses but
other pathogens as well (Schanen et al. 2011).
In Vivo Tools to Improve Efficacy Testing
Surrogate animal models: smallpox
Orthopoxviruses are large DNA viruses that can infect a variety of vertebrate animals. Interestingly, a
strong tropism effect is observed among members of the family Orthopoxviridae; thus, in most cases, a
given orthopoxvirus infects only one host. Smallpox, caused by the variola virus, is an extremely
virulent respiratory infection observed only in humans. Monkeypoxvirus infects a number of animal
species, one of which is nonhuman primates. One of the primary virulence strategies observed during
both of these infections is the generation of a large variety of viral immunomodulator proteins that
prevent the host from mounting a protective immune response (Smith 1999).
Today smallpox is eradicated and no new infections of humans occur anywhere in the world
(WHO 2011). Although a large amount of clinical information, including autopsy data, is available from
past epidemics, the available scientific methods of the times did not allow for evaluation of the host
response; thus comparison of mechanistic data with information obtained from current animal models
is limited. Because of the stringent tropism effects, it is very difficult to infect animals with variola
virus. One alternative method to overcome this hurdle is to create a surrogate animal model in which to
establish, through the use of a different orthopox virus, similar pathophysiology and clinical disease to
that observed in humans with smallpox. Jahrling and colleagues (2004) developed a non-human
primate model of variola through the introduction of high doses (108 plaque-forming units; PFU) of
virus intravenously and effectively bypassing the initial oropharyngeal site of virus replication. Due to
the very limited and tightly controlled nature of variola virus research permitted by the World Health
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Organization, further refinements of this model are difficult and expensive to achieve, although efforts
are still ongoing to improve that model. To that effect, Hooper and colleagues (2004) proposed that a
nonhuman primate model of monkeypox given infective doses that closely represent the typical
exposure to this virus, would be more relevant to and accurately present smallpox than the use of
nonphysiological doses and routes of infection developed with variola virus. One reason for the
potential effectiveness of this strategy is the ability of the orthopoxviruses to produce strong, cross-
reactive immune responses in animals of different species. An additional benefit of this strategy may be
the relevance to “newer” types of human orthopoxvirus diseases, such as human monkeypox, which
have emerged as serious public health burdens in places where endemic smallpox was observed in the
past (Rimoin et al. 2010). Animals of several species can serve as natural reservoirs for the monkeypox
virus (Khodakevich et al. 1986, 1987a, 1987b); in this case, using surrogate animal models susceptible to
monkeypox virus would be a more natural approach than the persistent use of variola virus on
organisms with no natural affinity for this agent. Moreover, because the tropism effect is likely to occur
with other pathogens, the monkeypox strategy may become a paradigm with future use as well
(McFadden 2005).
Systems approaches to infectious diseases
Virtually all human diseases are a manifestation of interactions among many inherited polymorphic
genes and environmental factors (Churchill et al. 2004; Cookson et al. 2009; Kotb 2010; Kotb et al. 2008;
Thompson 1995; Villar et al. 2004; Voit et al. 2008; Williams 2006). Traditional reductionist approaches
to develop disease models based on gene-by-gene comparisons or extrapolations have been universally
applicable. Broader systems approaches may be useful in this regard because they can reveal how
disease variables influence one another within a whole organism; provide a roadmap to expedite the
discovery of networks of pathways that modulate disease susceptibility and outcomes; and reveal those
networks likely to be good candidates for the development of more targeted rapid diagnostics and
effective therapeutics.
Although animals with limited genetic diversity have several advantages (see above),
translating findings from these animals to humans is not always useful. Whereas nonhuman primates
offer sufficient genetic variation for the implementation of a systems perspective (Sasaki et al. 2009;
Wolfe et al. 1998), the number of replicate studies needed to generate these data is limited by ethical
considerations, inadequate stocks, and prohibitive cost. Inbred rodents, although useful for generating
the quantity of data needed for systems evaluation, are characterized by little genetic heterogeneity. To
address these challenges, novel animal models have been developed from which discoveries, made
with a systems genetics or biological approach, are likely to translate to humans more readily. For
example, recombinant inbred mice (Advanced, or the next generation Collaborative Cross strains) are
generated and bred to maximize the number of recombinations in each of their chromosomes thereby
diversifying their genetic context and exposing a wider spectrum of disease phenotypes (Durrant et al.
2011; Kotb 2010; Williams et al. 2001).
When infected, these strains exhibit a wide spectrum of disease phenotypes because, as is the
case in humans, random assortment of many polymorphic loci can accentuate resistance or
susceptibility to a particular disease. Accordingly, findings in these genetically diverse populations can
significantly enhance the translation of experimental research findings to the clinical setting to prevent
or improve the management of complex infectious diseases. Network-based systems approaches and
pathway-to-pathway comparisons between species are now more likely to expedite the discovery of
targets and networks and the translation of research across species than gene-by-gene comparisons (for
other comparative biological approaches see discussion on compartmentalization, Chapter 4, p 56).
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PROMISES AND CHALLENGES FOR THE FUTURE
FDA Commissioner Margaret Hamburg recently wrote “We must bring 21st century approaches to 21st
century products and problems” (Hamburg 2011). This is a time of rapid and unprecedented
development of enabling biotechnologies that hold great promise for the future, but it also presents
several serious challenges. Despite the diversity of currently available approaches and promising
technologies, no approaches can at this time fully address the shortfalls of using animal models as
complete surrogates for humans. The Committee notes the following concerns:
• As stated in Chapters 2 and 4, there is a need to develop new and innovative strategies for
collecting data about how humans will respond to pathogens of concern. Without this
information, there can be little useful comparison to animal models (or qualification thereof, see
Chapter 4), the effectiveness and predictability of biomarkers is curtailed, and the animal data to
be used for the development and interpretation of meaningful IV/IS methods will not be
accurate. Further, original data (positive or negative; human and animal) may not be
systematically shared with the wider research community (as also discussed in Chapter 3, p 44).
The lack of sharing causes the fragmentation of knowledge and prevents the comparison of
inputs and outcomes,14 which may be particularly important in the event of an “unknown-
unknown” emergency. Therefore, this information should be collected systematically,
consistently, and accurately and be made available to the research community to enable
progress toward standardization of methods and qualification of models, and to address ethical
concerns regarding the potential nonproductive or duplicative use of animals or the
unnecessary duplication of studies and waste of resources.
• The provision of supportive veterinary care during animal efficacy trials for countermeasures is
a means to improve data gathering from animal models to enhance the efficiency and
productivity of this research field. In the Draft Guidance for Industry the FDA states that “studies
should be designed to mimic the clinical scenario and achieve meaningful outcomes comparable
to the endpoints desired in humans. In some instances, supportive care should be administered
to the animals as part of the study design” (FDA 2009). The Animal Rule does not require that a
test product exhibit added benefit over conventional therapy (“…the drug product is reasonably
likely to produce clinical benefit in humans.”; FDA 2002, p 37995), but if conventional therapy is
beneficial for human patients then it is a reasonable measure to include in the study design.
Furthermore, studies that include provision of the standard of care as one arm were suggested
at the public meeting to evaluate the licensure application of raxibacumab under the Animal
Rule (FDA 2009b). Since for most countermeasures in development there is no other standard of
care than supportive therapy, it is appropriate to include it when evaluating the test products.
Experience with such study designs and experimental protocols may be helpful in the event of
an efficacy trial for a countermeasure against an “unknown-unknown”. Due to the nature of
biocontainment, defining the basic principles of such an approach —including guidelines for
the care and use of animals in research done in biocontainment facilities— is recommended.
In addition to data sharing being one of the principle tenets of responsible conduct of research (see the Office of
14
Research Integrity’s Introduction to the Responsible Conduct of Research,
http://ori.hhs.gov/education/products/RCRintro/), it also is a fundamental tool of “the economy of knowledge
production” (Nat Genet 2011).
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71
ALTERNATIVE APPROACHES TO ANIMAL TESTING
• The Public Health Emergency Medical Countermeasures Enterprise (PHEMCE) Review
emphasized the primary role for regulatory science15 in biodefense research (DHHS 2010). As
shown in Figure 5-1, a window of opportunity may exist in which regulatory science can help to
overcome the limited use of advanced in vitro and in vivo technologies in the development of
medical countermeasures. It is desirable to develop criteria for choosing the most suitable
methods, and essential to do this in a way that will allow effective utilization of IV/IS
technologies while not inhibiting advances. Steps in the product development process have a
clear potential use for IV/IS as an adjunct method but the use of the whole animal will not be
replaced in the process. A research strategy to address these gaps would be useful as well as
improve areas in which in vitro assays are already showing promise. A place to begin would be
an analysis of the discovery, development, and approval process for medical
countermeasures to identify (1) where the most important scientific gaps exist in terms of
utilizing alternative methods to animal models and how to address them; (2) the specific
areas where the use of in vitro and in silico methods could be sufficient or as an adjunct to
the use of animals; and (3) the criteria for choosing and utilizing the most suitable
technologies to replace animal use in biodefense research.
• Regulations that require humane treatment of animals in research (such as the AWA as
discussed above) do not impose principled limits on the use of animals, i.e., pain and distress
caused by the research protocols are to be minimized only when and to the extent consistent
with the needs of science (Walker and King 2011). However, the needs of science in this
research field should be weighed against the potential advances in knowledge and benefits
to the warfighters as well as against the duration and severity of animal pain and distress. In
previous sections, the report outlines the need for the development of humane endpoints and
biomarkers, for the administration of supportive clinical care, and for the alleviation of pain and
distress. Medical countermeasures research and development for biodefense currently depends
on the continued use of nonhuman primates, as discussed in chapter 2, and will probably
remain so until such time that robust alternatives (either absolute or relative) to their use are
available.16 However, the report’s conclusions and recommendations could help reduce a key
tension in animal research, namely that the animals that most resemble humans are
simultaneously viewed as most necessary for research that is impermissible in humans and as
having greater moral value because they resemble humans. The recommended comprehensive
strategy of implementing the Three Rs, utilizing compartmentalization and systems biology,
and enhancing collection and analysis of human data reduces dependency on nonhuman
primates by maximizing the value of data derived from all research.17 The Committee
recommends that, where possible, the TMT should encourage efforts to replace nonhuman
primates as the animal of choice in biodefense research. In addition, unhindered access to data
(as discussed above) and publishing of all results —including negative ones— are critical steps
to ensure that this data is indeed useful, animals are used judiciously, and unnecessary
duplication of work is avoided (Bateson 2011).
“The development and use of new tools, standards, and approaches to more efficiently develop products and to
15
more effectively evaluate product safety, efficacy, and quality” (FDA 2010).
16 The authors of the recent Review of Research Using Non-Human Primates “agreed that in many cases the use of
NHPs was justifiable even in the context of current understanding of animal welfare and advances in knowledge
that might now render some work on living animals unnecessary” (Bateson 2011, p 1).
17 To cite from the Review of Research Using Non-Human Primates, “it is an ethical imperative that maximum benefit
be derived from studies employing NHPs” (Bateson 2011, p 3).
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72 ANIMAL MODELS FOR ASSESSING COUNTERMEASURES
FIGURE 5-1 Regulatory science proceeds as a function of regulatory stringency and technological advancement.
Whereas stringency is necessary to safeguard the safety and efficacy of products, it can be better achieved as
newer technologies and reliable models provide a better approximation of the human system (or a relevant
component of the human system). Greater innovation or investment in many of the suggested approaches above
may be achieved by adjusting the real or perceived stringency of the current regulatory framework. As
technologies, models or approaches are discovered that provide better fidelity with a human system (or the
relevant component of the human system), then standardization may be achieved and stringency increased based
on demonstration of the model’s reliability.
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