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.

_________________________

1 The Committee did not consider animal models used for safety evaluation of products developed under the 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.



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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. REFERENCES Alves DA, Glynn AR, Steele KE, Lackemeyer MG, Garza NL, Buck JG, Mech C, Reed DS. 2010. Aerosol exposure to the Angola strain of Marburg virus causes lethal viral hemorrhagic fever in cynomolgus macaques. Vet Pathol 47(5):831-851. Anderson J, Tucker K. 2006. Development and validation of animal models. In: Swearengen JR, ed. Biodefense: Research Methodology and Animal Models. Boca Raton: CRC Press. p 41-60. Baize S, Leroy EM, Georges AJ, Georges-Courbot MC, Capron M, Bedjabaga I, Lansoud-Soukate J, Mavoungou E. 2002. Inflammatory responses in Ebola virus-infected patients. Clin Exp Immunol 128(1):163-168. Baize S, Leroy EM., Georges-Courbot MC, Capron M, Lansoud-Soukate J, Debré P, Fisher-Hoch SP, McCormick JB, Georges AJ. 1999. Defective humoral responses and extensive intravascular apoptosis are associated with fatal outcome in Ebola virus-infected patients. Nat Med 5(4):423-426. Barrette RW, Metwally SA, Rowland JM, Xu L, Zaki SR, Nichol ST, Rollin PE, Towner JS, Shieh WJ, Batten B, Sealy TK, Carrillo C, Moran KE, Bracht AJ, Mayr GA, Sirios-Cruz M, Catbagan DP, Lautner EA, Ksiazek TG, White WR, McIntosh MT. 2009. Discovery of swine as a host for the Reston ebolavirus. Science 325(5937):204-206. Baskerville A, Bowen ET, Platt GS, McArdell LB, Simpson DI. 1978. The pathology of experimental Ebola virus infection in monkeys. J Pathol 125(3):131-138. Baskerville A, Fisher-Hoch SP, Neild GH, Dowsett AB. 1985. Ultrastructural pathology of experimental Ebola haemorrhagic fever virus infection. J Pathol 147(3):199-209. Bausch DG, Nichol ST, Muyembe-Tamfum JJ, Borchert M, Rollin PE, Sleurs H, Campbell P, Tshioko FK, Roth C, Colebunders R, Pirard P, Mardel S, Olinda LA, Zeller H, Tshomba A, Kulidri A, Libande ML, Mulangu S, Formenty P, Grein T, Leirs H, Braack L, Ksiazek T, Zaki S, Bowen MD. Smit SB, Leman PA, Burt FJ, Kemp A, Swanepoel R; International Scientific and Technical Committee for Marburg Hemorrhagic Fever Control in the Democratic Republic of the Congo. 2006. Marburg hemorrhagic fever associated with multiple genetic lineages of virus. N Engl J Med 355(9):909-919. Beall FA, Dalldorf FG. 1966. The pathogenesis of the lethal effect of anthrax toxin in the rat. J Infect Dis 116(3):377- 389.

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