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2
Evaluation of Current and Future
TMT-Used Animal Models
This chapter examines how well specific animal models against biothreats of interest to the
Transformational Medical Technologies (TMT) reflect various aspects of the human diseases for which
medical countermeasures are being developed. As explained in the Introduction, the TMT seeks to
identify and develop new or repurposed medical countermeasures that may have broad-spectrum
capability, that is, target a number of pathogens with similar mechanisms of disease causation and
pathogenesis. This approach is focused on two major groups, hemorrhagic fever viruses and
intracellular bacterial pathogens. The Committee on Animal Models for Assessing Countermeasures to
Bioterrorism Agents thinks that currently available animal models for these biothreats, while necessary,
are imperfect representations of every aspect of human-pathogen interaction especially with regards to
their substitution for “adequate and well-controlled efficacy studies in humans” (FDA 2002, p 37989).
Given the ethical mandate of the Animal Rule to not harm human participants in clinical trials that
“would involve administering a potentially lethal or permanently disabling toxic substance or
organism” (ibid.), these models most likely represent the best approach to develop and test
countermeasures and the current efforts have performed as well as could be expected given the
limitations listed below. These limitations are critical components to be considered when evaluating the
utility of an animal model for efficacy studies1 of the known or unknown pathogens of interest to the
TMT:
• Lack of sufficient human clinical data (that is, reliable and sophisticated human clinical markers)
and knowledge of the natural history2 of these diseases or threats of interest may hinder the
successful correlation of the animal models to the infectious diseases of interest. The more scant
the human data, the greater the uncertainty of relevance of the animal model.
The Committee did not consider animal models used for safety evaluation of products developed under the
1
Animal Rule, as “safety evaluation of products is not addressed in this rule” (FDA 2002, p 37989).
2 Natural history refers to the progression of a disease without any intervention.
15
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16 ANIMAL MODELS FOR ASSESSING COUNTERMEASURES
• Both interspecies and intraspecies variability and the constraints imposed by working in
biocontainment facilities lead to methodological differences and results that may not be
translatable or comparable across different animal models of the same disease. This is
particularly relevant to the anticipated clinical experience of human patients.3
• Experience with product development and clinical trials for some conventional diseases indicate
that animal models often are unreliable surrogates for, or predictors of, efficacy and safety.4,5
Historically, animal models have been relied upon to provide preliminary efficacy data for
therapeutics against infectious diseases in support and justification of subsequent definitive efficacy
studies in human participants to obtain regulatory approval by the Food and Drug Administration
(FDA). Because the preclinical data would be evaluated in the context of knowledge from human
studies, any deficiencies in data correlation and extrapolation from the animal models to the human
condition would presumably be compensated for by the actual data collected during the human
studies. Biothreats represent a special problem in that efficacy studies before an actual event are
unlikely to take place. In addition, the actual risk of a biothreat attack is difficult to ascertain. These
difficulties are even more pronounced in the case of the “unknown-unknowns”.6
Comparing the evaluation process for bioterrorism countermeasures following the preclinical
development stage with that for drugs for which human efficacy studies are possible puts in better
perspective the regulatory challenges with which the countermeasure development for TMT (or other
biodefense) products is beset. Under optimal circumstances, the current process from drug discovery to
FDA approval takes an average of 10 to 15 years and costs more than $1 billion (Tamimi and Ellis 2009).
According to some estimates the developmental cost of a single drug has soared from $1.1 billion in
1995 to $1.7 billion in 2002, factoring in the costs of failed prospective drugs (Crawford 2004; Mundae
and Östör 2010). Those figures apply equally to biopharmaceuticals and small molecules (DiMasi and
Grabowski 2007). To date only about 8% of drugs that successfully enter phase 1 studies eventually are
granted market approval by the FDA as compared with 14% in the 1980s. The success rate of
pharmaceuticals from the first phase 1 study in humans to market is less than 10% (DiMasi et al. 2010).
The main causes of failure in the clinical trial setting are safety problems, which account for
about 20% of the attrition rate, and lack of effectiveness, which accounts for about 40% (Kola and
Landis 2004; Peck 2007). Inability to predict these failures before human testing or early in clinical trials
dramatically escalates costs. In the infectious disease arena, data from the 10 largest pharmaceutical
corporations in the period of 1991-2000 showed a success rate of about 15%, while the average success
rate for all indications was 11% (Gilbert et al. 2003). Similarly, DiMasi and colleagues (2010) showed a
success rate for systemic infectious disease of 15.6% during 1994 and 2003. It is useful to note that from
1981 to 1992 the success rate of anti-infective drugs was 28.1% and that large biopharmaceutical
companies appeared to have a higher success rate of 30.2% for all indications (DiMasi 2001). A key
3 Lack of data sharing further compounds differences in methods or lack of reproducibility of results across
models (see chapter 5 for further discussion).
4 The limitations of animal models for other disease indications (in addition to those encountered in emerging
infectious diseases or biothreats research) have been documented in a number of meta-analyses (see Macleod
2011; Perel et al. 2007; Suntharalingam et al. 2006; van der Worp et al. 2010).
5 As discussed in Developing Animal Models for Use in Animal Rule Licensure: The NIAID Approach (Appendix C, p
111-112), developing animal models in biocontainment requires substantial financial and infrastructure
investment.
6 As defined in the introduction, the term “unknown-unknown(s)” refers to pathogen(s) that may not be known or
knowable because they currently may not exist. Due to the current or future possibility that they may exist, they
are considered potential threats (e.g., a novel, genetically engineered, or created pathogen).
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EVALUATION OF CURRENT AND FUTURE TMTI-USED ANIMAL MODELS 17
question is whether medical countermeasures against emerging infectious diseases and other biothreats
have a higher likelihood of success in a (theoretical) human trial. Several facts argue against this
possibility and support the notion that achieving a success rate close to that of noncountermeasure drug
development can only be considered a best-case scenario:
• The pathogenesis of these rare or even unknown infections is mostly unknown and cannot,
therefore, guide the development process.
• The causative pathogens could be optimized to withstand interventions (e.g., via introduced
antibiotic resistance).
• The clinical setting is probably one of mass infection (which may even be caused by more than
one infectious agent) and thus is not comparable to randomized clinical trials of hospitalized
patients.
• Most product development occurs with less than average financial support by entities not
experienced in full clinical drug development.
• The restrictions imposed by biocontainment and the strong reliance on nonhuman primates
limit the number of animal studies that could be done.
ANIMAL MODELS ARE ANALOGOUS, NOT HOMOLOGOUS SYSTEMS
On a number of occasions the Animal Rule has been misread resulting in the unrealistic expectation
that animal efficacy studies accurately and completely reflect the human condition. Indeed, the term
“model” implies that it is not intended to completely replicate the human pathophysiology but rather
to provide insight into different aspects of the host-pathogen dynamic. In fact, the Animal Rule is based
on the notion that there is enough similarity in the response of animals of different species to a
pathogen or a group of pathogens to permit a reasoned method to evaluate product efficacy among
those different species (humans being the final target). Prior knowledge of the natural history and
progression of the human infection shows that the interplay between host and pathogen may or may
not mimic what occurs in humans. Animal models are analogous and not homologous and, by their
very nature, display a number of limitations both during different stages of the development process
and in the design of the experimental protocols that are applied to these models. For the purpose of this
report, homology refers to the similarity in evolutionary origin and physiological function. Analogy
refers to the quality of resemblance or similarity in function or appearance but not to the similarity in
origin or development (Anderson and Tucker 2006).
Although animal models incorporate a variable degree of homology and analogy, the only
homologous model for a human is a human (and even among humans genetic differences affect
responses and safety for vaccines and therapeutics; He et al. 2011). Most regular drugs and vaccines are
tested for both safety and efficacy in clinical trials where the conditions or diseases of concern are
endemic in a population, providing the opportunity to use a truly homologous model. Although
efficacy data from animals have been used for decades to drive the exploration of new countermeasures
to biological agents and toxins, only in the last decade has there been a need to use research data
collected exclusively from analogous models (animals belonging to nonhominid taxa) for the same
regulatory approval process as data from humans.
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18 ANIMAL MODELS FOR ASSESSING COUNTERMEASURES
CONSIDERATIONS FOR ANIMAL MODELS FOR COUNTERMEASURE DEVELOPMENT
Two of the conditions of the Animal Rule that have to be met for the FDA to use evidence of efficacy
derived from animal studies are the following:
1. There is a reasonably well-understood pathophysiological mechanism of the pathogenicity of
the infectious agent and its prevention or reduction of symptoms by the product.
2. The effect is demonstrated in more than one animal model (animals belonging to at least two
different species) expected to react with a response predictive for humans unless the effect is
demonstrated in animals belonging to a single animal species that represent a sufficiently well-
characterized animal model for predicting the response in humans (FDA 2002).
These two conditions often provide some of the biggest hurdles in developing an animal model
for countermeasure development. For example, the first condition infers that a large amount of human
clinical and pathophysiological data is available to compare with the data derived from the animal
model. In many cases, there are sparse to no data on some of the biothreat infections because of their
rare geographic distribution and infrequent rate of occurrence. Although autopsy data may be
available, they provide little information about the natural history of disease and may be influenced
during the terminal stages of infection by a variety of epiphenomena, such as the lack of supportive
treatment or the presence of secondary systemic failure. Pathogens with tropism for animals of a single
species make the fulfillment of the second condition particularly difficult. Variola virus, the causative
agent of smallpox, is a prime example of this problem because in nature it infects only humans.
Developing working animal models for variola to replicate the natural progression of smallpox is very
difficult if not impossible. Furthermore, although in some cases the model may reflect different aspects
of the pathophysiology of smallpox, the actual progression of the illness in animals may be quite
different from that observed in humans. The rabbit model for pulmonary anthrax is an example of the
latter; the difference in progression can create significant problems for protocols related to product
development (see further discussion on page 31).
The significance of the majority of pathogens currently viewed as priorities for biodefense
research changed over the last ten years in response to the September 11, 2001, events. Despite the
changed status, funds for research of these pathogens were minimal, numbers of researchers
specializing in this field were low, and overall research progress was slow. Impeding progress even
further, a considerable number of these agents are categorized as Risk Group 3 and 4 pathogens for
biosafety and security reasons (Select Agents Regulations; 7 CFR Part 331; 9 CFR Part 121; 42 CFR Part
73), therefore requiring biosafety level 3 or 4 (BSL-3 or -4) containment facilities for any research to be
conducted in the United States (ibid.). Accordingly, animals can be experimentally infected with these
pathogens only in the appropriate animal biosafety level containment facilities (ABSL-3 or -4).
The following review of several pathogens provides a broad representation of the current status
of animal models being developed for efficacy testing and highlights specific challenges common
among other models in the context of the Animal Rule, as depicted in Table 2-1.
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EVALUATION OF CURRENT AND FUTURE TMTI-USED ANIMAL MODELS 19
TABLE 2-1 Current State of Animal Model Development for Selected Pathogens in the Context
of the Animal Rule
Bacillus
Francisella tularensis
Filoviruses Variola virus
anthracis
Research and product Rodent and Surrogate models Predominantly Large body of
discovery nonhuman primate used with other murine models data
models (Falzarano poxviruses
et al. 2011)
Proof of principle Yes in rodent Yes for surrogate Historical Large body of
models models information from data
human challenges
FDA Animal Rule Applied for Product Transition
1. Well-understood Limited Limited Strong pathology but Toxin-
pathophysiology understanding due understanding of basic mechanistic mediated
to lack of human humans (Stanford et information lacking bacteremia
data al. 2007)
2. Animals of more than Mouse, guinea pig, Specific human Mouse, rat, Rabbit,
one species hamster, tropism of smallpox nonhuman primates nonhuman
nonhuman challenging primates
primates, but
limited by #1
3. Endpoint clearly related Survival, but Survival for Survival Survival and
to human benefit limited by #1 surrogate models decreased
morbidity
4. Information for Not applicable at Yes for specific Correlates of Reasonable
effective human dosing this time antibody responses protection not well correlates
defined
FILOVIRUSES
Among viruses, TMTI focuses on those that cause viral hemorrhagic fevers (VHFs), and among those
primarily on VHF-causing filoviruses (marburg-, ebola-, and “cuevaviruses”; see Table 2-2 for virus
names and abbreviations). All filoviruses, except Reston virus (RESTV) and Lloviu virus (LLOV), are
endemic in Central Africa. RESTV is found in the Philippines and LLOV appears to be endemic in
Spain. Human filovirus disease outbreaks are rare events, limited in scope, still unpredictable, and
usually occur in rural and underdeveloped areas without sophisticated medical or epidemiological
infrastructure. Outbreak intervention often occurs weeks or months after index cases7 are reported to
local authorities, and Western-style medical treatment is often hindered not only by nonexistent
infrastructure and the lack of trained personnel but also by cultural and especially religious, spiritual
constraints. Taken together, these obstacles explain the reasons for the current paucity of available
human clinical data on diseases caused by filoviruses.
The lack of basic human pathophysiological information raises the disconcerting possibility that
current animal systems for filovirus infections could be only crude approximations of the human
First disease case in an epidemic within a population (NIH 2011).
7
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20 ANIMAL MODELS FOR ASSESSING COUNTERMEASURES
clinical condition rather than truly analogous models. Currently available animal “models” usually rely
on the identification of particular animals that, after infection, develop a disease that has some
prominent clinical or pathological markers in common with those observed in infected humans rather
than on the thorough characterization of host responses that can be compared directly with those of
sick humans. Thus, in the case of filoviruses, the dearth of information on the human patient prevents
the development of a clinically defendable animal model. Furthermore, additional collection of human
clinical data may render these animals ill-suited for the evaluation of pharmaceuticals or vaccines
under the premises of the Animal Rule.
TABLE 2-2 Transformational Medical Technologies Viral Pathogen Focus Group: Filovirusesa
New Taxonomy Outdated Taxonomy (Eighth ICTV Report)
Order Mononegavirales Order Mononegavirales
Family Filoviridae Family Filoviridae
Genus Marburgvirus Genus Marburgvirus
Species Marburg marburgvirus Species Lake Victoria marburgvirus
Virus 1: Marburg virus (MARV) Virus: Lake Victoria marburgvirus (MARV)
Virus 2: Ravn virus (RAVV)
Genus Ebolavirus Genus Ebolavirus
Species Taï Forest ebolavirus Species Côte d’Ivoire ebolavirus [sic]
Virus: Taï Forest virus (TAFV) Virus: Côte d’Ivoire ebolavirus [sic] (CIEBOV)
Species Reston ebolavirus Species Reston ebolavirus
Virus: Reston virus (RESTV) Virus: Reston ebolavirus (REBOV)
Species Sudan ebolavirus Species Sudan ebolavirus
Virus: Sudan virus (SUDV) Virus: Sudan ebolavirus (SEBOV)
Species Zaire ebolavirus Species Zaire ebolavirus
Virus: Ebola virus (EBOV) Virus: Zaire ebolavirus (ZEBOV)
Species Bundibugyo ebolavirus
Virus: Bundibugyo virus (BDBV)
Genus “Cuevavirus”
Species “Lloviu cuevavirus”
Virus: Lloviu virus (LLOV)
a Taxa
not yet approved by the International Committee on Taxonomy of Viruses (ICTV) are in quotation marks.
SOURCE: Kuhn et al. 2010.
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EVALUATION OF CURRENT AND FUTURE TMTI-USED ANIMAL MODELS 21
Filovirus Infection in Humans
The description of the clinical presentation of humans infected with filoviruses is limited. There are at
least eight filoviruses, and the diseases caused by them differ substantially in case numbers, case
distribution, and case fatality rates. Moreover, there are few reported cases of some of the viruses. For
instance, the clinical presentation of the human disease caused by Bundibugyo virus (BDBV) was
reported only once (MacNeil et al. 2010). Similarly, the paucity of information on human infection with
Taï Forest virus (TAFV) (only one case described thus far and the patient survived) makes it difficult to
extrapolate the symptoms and clinical progression of the disease as observed in a single patient to the
population at large (Formenty et al. 1999). It remains uncertain whether humans were ever infected
with RESTV or LLOV, as neither has to date been isolated from humans. However, the frequent contact
of humans with RESTV-infected swine in the Philippines and the possible frequent exposure of tourists
to LLOV-infected bats in Spanish caves suggest that, if humans do get infected by these ebolaviruses,
the infections might be without clinical consequences (Barrette et al. 2009). Clinical presentation data on
Sudan virus (SUDV) infections have yet to be statistically analyzed (Okware et al. 2002; Smith et al.
1978; WHO 1978). To date, the best-characterized filovirus diseases in human patient cohorts are those
caused by Marburg virus (MARV), BDBV, and Ebola virus (EBOV), as shown in Tables 2-3, 2-4, and 2-5
(see table references, pages 22-24). It remains to be seen whether these different viruses cause
fundamentally different disease pathogenesis.
Symptoms of filovirus disease are unspecific, are easily confused with many other diseases, and
lack a pathognomonic marker that allows for the unequivocal diagnosis of filovirus infection.
Unfortunately, autopsies of fatally infected humans have only rarely been performed, partly due to
cultural constraints and partly due to safety concerns. For instance, of the 1,912 fatal filovirus infections
documented between 1967 and 2010, only 31 have been pathologically examined: eight people infected
with MARV/ Ravn virus (RAVV) (five in 1967 and one each in 1975, 1980, and 1987; Gear et al. 1975;
Gedigk et al. 1968; Geisbert and Jaax 1998; Smith et al. 1982); 21 people infected with EBOV (three in
1976 and 18 in 1995; Murphy 1978; Zaki and Goldsmith 1999); and two people infected with SUDV in
1976 (Dietrich et al. 1978; Ellis et al. 1978). The autopsies mostly addressed gross anatomy, pathology,
and standard histology and did not expand into molecular markers. The collection of more detailed
clinical data has been attempted multiple times in the past and failed for numerous reasons, including
lack of accessibility to patients, knowledge of ongoing outbreaks, or resistance of patients to be
evaluated.
Autopsies of MARV/RAVV-infected patients revealed hemorrhagic diathesis into the skin
(maculopapular rash), mucous membranes, and soft tissues. The gallbladders appeared normal, spleens
were slightly enlarged, and lymph nodes were swollen. Focal necroses in all organs except lungs,
skeletal muscles, and bones were typical findings, but inflammatory reactions were absent with the
exception of testes and ovaries. MARV/RAVV was detected in macrophages, fibroblasts, hepatocytes,
Kupffer cells, adrenal cells, neuroendocrine cells of the adrenal medulla, and alpha and beta pancreatic
islet cells (Gear et al. 1975; Gedigk et al. 1968; Geisbert and Jaax 1998; Kuhn 2008; Smith et al. 1982). The
autopsy findings in EBOV-infected patients were similar to those described for MARV/RAVV
infections (Murphy 1978; Zaki and Goldsmith 1999), whereas findings in the two autopsied SUDV-
infected humans remain controversial because of concomitant parasitic (trematode and nematode)
infections (Dietrich et al. 1978; Ellis et al. 1978).
Relatively thorough state-of-the-art molecular analyses of filovirus-infected patients are limited
to only a few studies for EBOV- and SUDV-infected patients (Baize et al. 1999, 2002; Hutchinson and
Rollin 2007; Leroy et al. 2000, 2001, 2011; Rollin et al. 2007; Sanchez et al. 2004; Wauquier et al. 2010
Attempts to identify disease progression markers have shown that EBOV disease survivors mounted an
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22 ANIMAL MODELS FOR ASSESSING COUNTERMEASURES
TABLE 2-3 Symptoms of Marburg Virus-Infected Humans
Frequency Observed in Frequency Observed in
Clinical Symptom
Survivors (%) Fatal Cases (%)
59 57
Abdominal pain
77 72
Anorexia
55 55
Arthralgia or myalgia
0 7
Bleeding from puncture sites
23 36
Bleeding from the gums
59 71
Bleeding from any site
18 4
Chest pain
14 42
Conjuctival infection
9 5
Cough
59 56
Diarrhea
36 58
Difficulty breathing
18 34
Epistaxis
100 92
Fever
73 79
Headaches
68 76
Hematemesis
0 3
Hematoma
9 4
Hemoptysis
18 44
Hiccups
5 8
Lumbar pain
86 83
Malaise or fatigue
41 58
Melena
77 76
Nausea and vomiting
9 7
Petechiae
43 43
Sore throat, odynophagia, or dysphagia
SOURCE: Adapted from Bausch et al. 2006.
early robust antibody (IgG) response directed against the viral nucleoprotein (NP) and matrix protein
VP40, followed by clearance of viral antigen and activation of cytotoxic T cells; in fatal cases, no
antibody response was observed concomitant with massive activation of monocytes and macrophages
and subsequent massive lymphocyte apoptosis. Moreover, the presence of interleukins IL-1β and IL-6
during symptomatic infections could be used as predictor for nonfatal infections, whereas release of IL-
10, IL-1RA, and neopterin could be used as predictor for fatal infections (Leroy et al. 2000; Wauquier et
al. 2010). In SUDV patients, the interleukin profile was different; survivors had higher concentrations of
interferon α (IFN-α) and fatal cases had higher concentrations of IL-6, IL-8, IL-10, and macrophage
inflammatory protein 1β (MIP-1β; Hutchinson and Rollin 2007; Rollin et al. 2007; Sanchez et al. 2004).
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EVALUATION OF CURRENT AND FUTURE TMTI-USED ANIMAL MODELS 23
TABLE 2-4 Symptoms of Ebola Virus-Infected Humans
Frequency Observed in Frequency Observed in
Clinical Symptom
Survivors (%) Fatal Cases (%)
68 62
Abdominal pain
5 2
Abortion
47 43
Anorexia
0 7
Anuria
79 50
Arthralgia or myalgia
95 85
Asthenia
5 8
Bleeding from puncture sites
0 15
Bleeding from the gums
5 7
Bloody stools
5 10
Chest pain
47 42
Conjuctival infection
0 2
Convulsions
26 7
Cough
84 86
Diarrhea
5 0
Dysesthesia
0 2
Epistaxis
95 93
Fever
74 52
Headaches
11 5
Hearing loss
0 13
Hematemesis
0 2
Hematoma
16 7
Hematuria
11 0
Hemoptysis
5 2
Hepatomegaly
5 17
Hiccups
26 12
Lumbar pain
16 14
Maculopapular rasha
16 8
Melena
68 73
Nausea and vomiting
0 8
Petechiae
58 56
Sore throat, odynophagia, or dysphagia
5 2
Splenomegaly
0 31
Tachypnea
11 1
Tinnitus
a variable
detection may be attributed to skin color
SOURCE: Adapted from Bwaka et al. 1999.
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24 ANIMAL MODELS FOR ASSESSING COUNTERMEASURES
TABLE 2-5 Symptoms of Bundibugyo Virus-Infected Humans
Frequency Observed in Frequency Observed in
Clinical Symptom
Survivors (%) Fatal Cases (%)
88 93
Abdominal pain
83 80
Anorexia or weight loss
83 86
Arthralgia or myalgia
92 87
Diarrhea
26 57
Difficulty breathing
96 100
Fatigue
100 100
Fever
84 93
Headaches
17 40
Hiccups
35 33
Maculopapular rasha
92 87
Nausea and vomiting
43 60
Sore throat, odynophagia,or dysphagia
a variable
detection may be attributed to skin color
SOURCE: Adapted from MacNeil et al. 2010.
Experimental Filovirus Infection in Animals
The animals currently used in experimental filovirus research are mostly nonhuman primates and
rodents (see Table 2-6). The majority of published data from well-established animal models,8 including
detailed data on pathogenesis and pathology of disease from African green and rhesus monkeys and
cynomolgus macaques, stem from experiments with EBOV or MARV strains (Ebola virus references:
Alves et al. 2010; Baskerville et al. 1978, 1985; Bowen et al. 1978; Bray et al. 1998; Connolly et al. 1999;
Dadaeva et al. 2006; Geisbert 2003a,b; Jaax et al. 1996; Johnson et al. 1995; Kolesnikova et al. 1997;
Pereboeba 1993; Ryabchikova et al. 1993, 1996a, 1998, 1999a, 2004; Vogel et al. 1997; Marburg virus
references: Bechtelsheimer et al. 1970; Haas et al. 1968a,b; Korb and Slenczka 1971; Lub et al. 1995;
Murphy et al. 1971; Oehlert 1971; Robin et al. 1971; Ryabchikova et al. 1994, 1996b, 1999b; Simpson 1969;
Simpson et al. 1968; Warfield et al 2007; Zlotnik 1971; Zlotnik and Simpson 1969). Table 2-7 compares
hematological disturbances and mean time to death observed in various nonhuman primate species
following EBOV infection. With the possible exception of the hematological responses, nonhuman
primates infected with MARV or EBOV roughly reflect the human disease, without significant
contradictions between clinical signs and gross pathology.
8 “Well-established” refers to animal models that are in use in several BSL-4 facilities and are referred to
repeatedly in publications on animal use in filovirus research.
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EVALUATION OF CURRENT AND FUTURE TMTI-USED ANIMAL MODELS 25
TABLE 2-6 Animals Used for the Development of Animal Models for Filovirus Disease
Virus Animal Status of Model
Common marmoset (Callithrix jacchus)
Marburg virus Model under evaluation, supposedly lethal,
unpublished
(MARV)
African green monkey (Chlorocebus aethiops) Well-established lethal model, published
Anecdotal lethal “model,” uncharacterized,
Common squirrel monkey (Saimiri sciureus)
unpublished
Rhesus monkey (Macaca mulatta) Well-established lethal model, published
Well-established lethal model (requires virus
adaptation), published (strain 2 guinea pigs
Dunkin Hartley and strain 13 guinea pigs
are sometimes also used but their pathology
has not been described in detail)
Historical lethal model (requires virus
Syrian (golden) hamsters
adaptation), basically uncharacterized
Recently established model, lethal (requires
BALB/c and SCID BALB/c laboratory mice
virus adaptation), published
Cynomolgus macaque (Macaca fascicularis) Uncharacterized model, mentioned in
Ravn virus
(RAVV) publications
Rhesus monkey (Macaca mulatta) Established lethal model, published
Recently established model, lethal (requires
BALB/c and SCID-BALB/c laboratory mice
virus adaptation), published
Bundibugyo Cynomolgus macaque (Macaca fascicularis) Model under evaluation, lethal
virus (BDBV)
Rhesus monkey (Macaca mulatta) Model under evaluation, thus far
unsuccessful, unpublished
Taï Forest virus Cynomolgus macaque (Macaca fascicularis) Established partially lethal model, published
(TAFV)
Rhesus monkey (Macaca mulatta) Model under evaluation, no data available
Reston virus Cynomolgus macaque (Macaca fascicularis) Well-established model, infrequently lethal,
(RESTV) published
Domestic pig (Sus scrofa) Model under evaluation, no data available
Not well-established model, often nonlethal,
African green monkey (Chlorocebus aethiops)
published
Sudan virus African green monkey (Chlorocebus aethiops) Not well-established model, lethal, published
(SUDV)
Cynomolgus monkey (macaca fascicularis) Established lethal model, published
Rhesus monkey (Macaca mulatta) Not well-established model, lethal, published
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28 ANIMAL MODELS FOR ASSESSING COUNTERMEASURES
rarely detectable (Hensley et al. 2002). A different study using RESTV, rather than a clinically relevant
filovirus known to infect humans, revealed a different cytokine activation profile than that shown in
human EBOV or SUDV infections (Hutchinson et al. 2001). It is also important to note that RESTV, one
of two filoviruses that thus far are thought apathogenic in humans, is virulent in cynomolgus
macaques, but not in African green monkeys. The results in cynomolgus macaques raise the question of
whether they are indeed valuable heterotypic approximations of humans, given that they should
succumb only to EBOV but not to RESTV.
To date, five filoviruses (MARV, RAVV, BDBV, EBOV, and SUDV) are being studied for
countermeasure development. Although some of the animal models for the most commonly studied of
those viruses, MARV and EBOV, are well established and published, data from animal experiments
with the other three have not yet been satisfactorily evaluated for studies of pathogenesis or evaluation
of pharmaceuticals or vaccines. Moreover, it is apparent that rodents are not good approximations for
human disease for the following reasons: (1) the virus needs to be genetically altered (adapted by serial
passage) before it is administered to the animals so that they will succumb; (2) disseminated
intravascular coagulation (DIC), which is a prominent symptom of infected humans, does not seem to
be a hallmark symptom of their disease; and (3) the typical maculopapular rash is absent.
TULAREMIA
The development of animal models for tularemia is interesting because of the availability of both
clinical information regarding direct challenge into humans and data regarding the efficacy of the
current investigational new drug (IND) vaccine Live-Vaccine Strain (LVS) to protect human volunteers
against direct pulmonary challenges with virulent strain Schu S4 of Francisella tularensis (Hornich and
Eigelsbach 1966; McCrumb et al. 1957; Saslaw and Carlisle 1961). Consequently, endpoints (diagnostic
and clinical) are available that can be used to judge the worthiness and relevance of a tularemia animal
model and possibly refine the experiments in this line of research. On the basis of these data, any
comparable animal model would be expected to be (1) very sensitive to infections with Schu S4 (Biovar
A) serotypes of Francisella; (2) resistant to infection by high doses of the LVS; and (3) protected from
significant morbidity and mortality by prevaccination with LVS.
Nonhuman primates and mice are the most prevalent animal models for primary pulmonary
tularemia. Laboratory mice have been extremely useful for dissecting the immune response to F.
tularensis and understanding some of the pathophysiology (Coriell et al. 1947; Downs et al. 1949;
Ruchman and Foshay 1949). Indeed, the pathology of pyrogranulomae and the primary organ
involvement of lung, spleen, and liver are consistent between humans and laboratory mice. However,
unlike the human, mice are sensitive to LVS infection, and low doses of LVS do not reproducibly
protect these animals from subsequent challenge by Schu S4 (Conlan et al. 2003; Wu et al. 2005); these
facts diminish the use of this model for vaccine development. Recently a model based on Fischer 344
rats was shown to be resistant to LVS administration. Further, vaccination with LVS by any route
protects these animals against subsequent challenge with relatively high doses of Schu S4 (Wu et al.
2009).
The nonhuman primate model for pulmonary tularemia exhibited similar pathology to that of
humans in the course of primary infection, while LVS administration elicited a strong protection
against challenge with the Schu S4 strain (Lyons and Wu 2007). If these nonhuman primate models are
reproducible, then it is possible that vaccines against F. tularensis could be developed. However, little
work has been done to decipher the basic mechanism of protection and immunity in these animals and
to determine absolute or relative immune responses as correlates of protection. Because of limited
understanding of how the human cellular responses develop antibacterial defenses, it remains hard to
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EVALUATION OF CURRENT AND FUTURE TMTI-USED ANIMAL MODELS 29
develop correlates of protection in humans not only to predict clinical benefits but also to increase
confidence in the protection afforded by vaccination. If correlates of protection are known, they may
further help advance the research to determine an “effective dose” in humans based on animal
experimentation, which is a required element of the Animal Rule.
ANTHRAX9
The challenges facing the production of countermeasures may be highlighted by a discussion of the
process applied to the biothreat posed by Bacillus anthracis. B. anthracis has been studied for decades,
and the details related to the life cycle of the bacterium are well known (Hugh-Jones and Blackburn
2009); therefore, the development of new products for treatment is expected to be straightforward. The
aerosolization of B. anthracis spores is the greatest biothreat risk associated with this pathogen, as
pulmonary anthrax is the most lethal form of the disease. Once the spores are inhaled, they are
phagocytosed by alveolar macrophages and taken to local lymph nodes where they germinate and
disseminate as vegetative bacilli to surrounding tissues via the bloodstream. The timing of this
dissemination is unpredictable because it depends on the generation of virulence factors, such as the
capsule, which engulfs and protects the bacilli, and the intracellular constitution of the tripartite
anthrax toxin. The role of these factors has been well described (Makino et al. 2002; Moayeri and Leppla
2004).
Although the rabbit and many nonhuman primate species are considered the primary animal
models for therapeutic product development against B. anthracis, a lot of information has been collected
through studies in rodent models. Laboratory mice have always been an attractive model because of (1)
the plethora of available tools to dissect the host responses that develop against the aerosol challenge
with B. anthracis; (2) their small “footprint” and necessary housing area; and (3) the minimal costs
associated with their procurement, care, and use. The murine repertoire of antibodies and T-cell
reactivity in response to B. anthracis challenge is generated in a process very similar to that of humans.
Across several B. anthracis studies in laboratory mice, the primary difference with the human disease is
the dominant virulent factor, which in mice is the capsule (Chand et al. 2009). Encapsulated strains of B.
anthracis that do not express toxin remain virulent and lethal in most murine models of anthrax except
for the susceptible A/J strain. A/J mice deficient in complement protein C5 die from a toxin-mediated
death following infection with low doses of the nonencapsulated Sterne strain (Welkos and Friedlander
1988). In this animal model, where the toxin is the target, the current Anthrax Vaccine-Adsorbed (AVA)
vaccine provides robust protection, as do other antitoxin modalities, such as antiserum to recombinant
protective antigen (Pitt et al. 2001). On the basis of the limited role for B. anthracis toxins in the infection
of laboratory mice, these animals are considered a poor model for human anthrax, whose pathogenesis
depends on the virulence of toxin (Heninger et al. 2006).
The rat model is thought to be inadequate because of the high baseline resistance of these
animals to infection with B. anthracis spores. Some strains of rats (e.g., Fischer 344), however, display
high sensitivity to injected purified toxin and are therefore routinely used to screen antitoxin
candidates, such as human monoclonal antibodies (Beall and Dalldorf 1966; Sawada-Hirai et al. 2004).
Guinea pigs were used in some seminal studies to describe the trafficking of spores delivered via the
lung before dissemination (Ross 1957). Guinea pigs are used in potency assays for the licensed AVA
The National Institute for Allergy and Infectious Diseases’ efforts to fund research into the standardization of
9
biodefense-related animal models for product development under the Animal Rule deserve credit for advancing
the anthrax model in particular and for raising awareness of all models more generally (see Appendix C).
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30 ANIMAL MODELS FOR ASSESSING COUNTERMEASURES
vaccine based on the protection observed following challenge with parenterally administered spores
(FDA 1973). The understanding of the efficacy of this vaccine dates back to data derived from
vaccinated workers in wool processing plants in the 1950s (Brachman et al. 1962). Analysis of these data
coupled with the fact that guinea pigs challenged by aerosolized anthrax spores are not reliably
protected by the AVA vaccine (Fellows et al. 2001) demonstrate that this animal model is not optimal
for vaccine testing and screening. Such a priori knowledge of the expected efficacy of a vaccine in
humans is unlikely to be available for the majority of current biothreats.
Rabbits and nonhuman primates, such as rhesus monkeys, are sensitive to pulmonary anthrax
and demonstrate many of the pathological findings observed in humans (Vasconcelos et al. 2003;
Zaucha et al. 1998). Moreover, the gross lesions seen in the cynomolgus macaque pulmonary anthrax
model are similar to those seen in infected humans, including splenomegaly, lymph node enlargement,
and hemorrhages in several different organs. Mediastinitis was observed in approximately 30% of the
infected animals (Vasconcelos et. al. 2003). As both rabbit and macaque species are well-protected by
the AVA vaccine, they have been very useful in the development of prophylactic therapeutics against
anthrax (Phipps et al. 2004).
In contrast to the nonhuman primate models, the rabbit provided few, if any, clues to the
disease progression. Thus, it has been challenging to develop a reproducible rabbit model for
therapeutics to be administered during the dissemination stage of the disease for the following reasons:
(1) the rabbit shows very few to no clinical symptoms postinfection, thus the timing for postinfection
intervention is not easily discernible; and (2) due to the unpredictable timing of dissemination from the
lung into the bloodstream, each animal may need therapeutic intervention at different time points.
Because the time from infection to death typically occurs within 48-72 hours, this experiment presents
enormous logistical challenges.
Although the rabbits and nonhuman primates appear to be the best models for medical
countermeasure development under the regulatory provisions of the Animal Rule, their use poses
significant challenges with regard to housing capacity, number of animals needed for statistically
meaningful results, and cost of procurement and care. Moreover, societal sensitivities toward the use of
nonhuman primates in research pose additional impediments to the continued use of these animals in
the development and production of medical countermeasures for emerging infections and biothreats.
LESSONS LEARNED FROM DEVELOPING ANIMAL MODELS FOR
THERAPEUTIC PURPOSES AGAINST BIOTHREAT AGENTS
The search for prophylactics and therapeutics against infection of B. anthracis, as summarized in the
previous pages, has encountered a number of hurdles, some of which stem from trying to force the
animal model to fit the experimental protocol instead of selecting the most appropriate model based on
the desired experimental outcome (for an expanded discussion on this issue see Chapter 4, page 56).
Furthermore, the process of developing animal models and medical countermeasures has been
intimately linked in such a manner that the development and subsequent fitness of the model is
determined solely in the context of the countermeasure rather than in a product-neutral fashion. It is
important to realize that the value of animal models depends on the context of the scientific question to
be investigated.
A number of currently used animal models do not translate well to the human condition.
Furthermore, most models are complex and therefore costly to develop. High levels of biocontainment
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EVALUATION OF CURRENT AND FUTURE TMTI-USED ANIMAL MODELS 31
are necessary to safely perform research with these pathogens, and additional restrictions are imposed
by their classification as Select Agents.10 These facts coupled with the large numbers of animals
necessary for the research and development of countermeasures point to the need to reevaluate the
ways these models are developed and used. It would be more beneficial to develop models with a
broader application profile that can be used to develop more than one countermeasure.11 Such an
approach might address not only the conundrum of over-relying on analogous systems to predict
efficacy of products in humans, but may be of significance in an encounter with an “unknown-
unknown”.
As previously stated, one of the potential unintended consequences of the Animal Rule is the
ambitious expectation that animal efficacy studies predict the human condition. This expectation is
daunting for two reasons: (1) there is not enough primary data from humans to which animal data can
be compared, and (2) the ability of animal models to reflect the human disease is not absolute. As
discussed on page 19, the collection of more detailed clinical data for filovirus infections has been
attempted multiple times in the past and failed. At this time, there is no reason to believe that collection
of data from filovirus disease outbreaks may improve in the immediate future. Further, while
potentially more detailed human data on tularemia and anthrax exists, it is far from comprehensive.
The animal models currently available may be the only avenue to accrue some data on pathogenesis,
perhaps on correlates of protection, and, through that, on efficacy of pharmaceuticals or vaccines.
These circumstances also reflect the TMT’s other concern, namely, the deliberate attack on warfighters
with an “unknown-unknown,” that is, an agent for which human clinical data are not available at the
time of attack. Therefore, the collection of human clinical data is of utmost importance in order to verify
the usefulness and augment the strengths of available models.
The previous sections described the variable results obtained by using animals belonging to
different species, a fact also encountered in other fields of biomedical research (e.g., see Craig 2009;
Mogil 2009). In addition to factors such as host susceptibility and clinical pathology, the progression of
the disease in the different animal species may not resemble that of humans, possibly resulting in failed
translational efforts, as evidenced by the increase in attrition rates of products in the later stages of
clinical development. In a recent meta-analysis of the potential reasons that animal experiments fail to
translate into clinical trials, van der Worp and colleagues (2010) identified recurring themes across
animal studies that may prevent them from providing a “correct basis for generalizations to the human
condition” as represented in clinical trials (what the authors define as external validity: “the extent to
which the results of an animal experiment provide a correct basis for generalizations to the human
condition”, p 3; see Table 2-9).
Table 2-9 presents important and common methodological deficiencies, some of which are
further discussed in Chapter 5. An additional consideration may be the choice of animals that are
young and otherwise healthy, whereas the human patients may have co-morbidities (van der Worp et
al. 2010). Addressing some of these issues may be as simple as thoroughly studying the literature. As
elaborated further in Chapter 5, however, systematic sharing of data with the wider research
community will improve the predictive capacity of animal models. In summary, the more
approximation exists between the animals and the conditions under which they are used in efficacy
“Select Agents” and toxins are agents that the Department of Health and Human Services “considers to have
10
the potential to pose a severe threat to human health. A list of these agents are found in the Select Agents
regulation (42 CFR 73).” See http://www.selectagents.gov/FAQ_General.html#sec1q3.
11 The platform technology approach adopted by the TMT fits well with the product-neutral approach. As further
discussed in Appendix C, the National Institute of Allergy and Infectious Diseases (NIAID) is similarly focused
on product-independent and product-dependent (i.e., product-neutral) models until such time as the product is
ready for the final efficacy studies (also known as pivotal studies).
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32 ANIMAL MODELS FOR ASSESSING COUNTERMEASURES
studies and the characteristics of the human population for which the countermeasures are intended
(including clinical status), the better the chances that the countermeasure would be successful.
TABLE 2-9 Common Causes of Reduced External Validity of Animal Studies
• Assessment of the effect of a treatment in a homogeneous group of animals versus a
heterogeneous group of patients.
• The use of either male or female animals only, whereas the disease occurs in male and female
patients alike.
• The use of models for inducing a disease or injury with insufficient similarity to the human
condition.
• Delays to start of treatment that are unrealistic in the clinic; the use of doses that are toxic or not
tolerated by patients.
• Differences in outcome measures and the timing of outcome assessment between animal studies
and clinical trials.
Source: Adapted from van der Worp et al. 2010.
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