Animal models of invasive aspergillosis for drug discovery

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REVIEWS Drug Discovery Today � Volume 19, Number 9 � September 2014

Animal models of invasiveaspergillosis for drug discovery

Caroline Paulussen, Gaelle A.V. Boulet, Paul Cos, Peter Delputte andLouis J.R.M. Maes

Laboratory of Microbiology, Parasitology and Hygiene (LMPH), Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp,

Universiteitsplein 1, B-2610 Antwerp, Belgium

Although Aspergillus infections pose a growing threat to immunocompromised individuals, the limited

range of existing drugs does not allow efficient management of invasive aspergillosis. Moreover, drug

resistance is becoming increasingly common. Given that drug discovery relies on high-quality animal

studies, careful design of in vivo models for invasive aspergillosis could facilitate the identification of

novel antifungals. In this review, we discuss key aspects of animal models for invasive aspergillosis,

covering laboratory animal species, immune modulation, inoculation routes, Aspergillus strains,

treatment strategies and efficacy assessment, to enable the reader to tailor specific protocols for different

types of preclinical antifungal evaluation study.

IntroductionAspergillus species are ubiquitous in the environment, particularly

in air, water and soil. Humans inhale hundreds of conidia every

day, usually without adverse effects, although several species, such

as Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus and

Aspergillus niger, can act as human pathogens [1]. Virulence factors,

such as thermotolerance, production of large numbers of small

conidia, adhesins, hydrophobicity and resistance to oxidative

stress, contribute to the pathogenic potential of A. fumigatus

[2,3]. Moreover, A. fumigatus produces gliotoxin, which affects

circulating neutrophils, inhibits phagocytosis and enhances dis-

semination [4]. The biosynthesis of the pigment melanin is an-

other virulence factor because it inactivates the C3 component of

the complement system. As such, mutants without conidial pig-

mentation show reduced virulence [5].

The clinical consequences of Aspergillus infection are deter-

mined not only by the virulence of the strain, but also by the

immune status of the host. Alveolar macrophages and neutro-

phils form the primary cellular host defense against conidia. In

established infections, neutrophils mediate the destruction

and clearance of hyphae, but T helper, natural killer and

dendritic cells also contribute to antifungal defense [3,6,7]. In

Corresponding author: Maes, Louis J.R.M. ([email protected])

1380 www.drugdiscoverytoday.com 1359-6446/06/$ - see front matt

immunocompetent individuals, A. fumigatus can cause allergic

conditions, such as asthma and sinusitis, which rarely require

antifungal therapy. When the mucosal or immunological

defenses of the lung are severely compromised, the inhaled

conidia can germinate and result in lung colonization [1]. Ap-

proximately 2% of patients with asthma and 1–15% of patients

with cystic fibrosis develop allergic bronchopulmonary aspergil-

losis, a hypersensitive response to fungal components [1]. Chron-

ic pulmonary aspergillosis (CPA) is characterized by gradual

destruction of lung tissue, potentially combined with the forma-

tion of lung cavities that might contain an aspergilloma [8].

Other noninvasive types of aspergillosis include ocular infections

and otomycosis [9]. Invasive aspergillosis (IA) causes the highest

morbidity and mortality (50–90%) [10]. Four types of IA have

been described, according to the clinical features, underlying

disease and host response: (i) invasive pulmonary aspergillosis

(IPA), the most common form of IA in immunocompromised

patients; (ii) tracheobronchitis and obstructive bronchial disease

with inflammation of the bronchial mucosa; (iii) acute invasive

rhinosinusitis; and (iv) disseminated disease that might involve

the brain and other organs [2]. Individuals receiving immuno-

suppression, such as transplant or chemotherapy patients, are the

most susceptible [11]. Also prolonged corticosteroid therapy,

HIV, chronic granulomatous disease (CGD) and hematological

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Drug Discovery Today � Volume 19, Number 9 � September 2014 REVIEWS

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malignancies, such as leukemia, place individuals at risk for IA

[12,13].

The outcome of IA largely depends on early diagnosis and

adequate antifungal therapy. However, the nonspecific symptoms,

which might not be present in all patients, complicate diagnosis.

Radiographic, computed tomographic (CT) or magnetic resonance

imaging (MRI) images of the chest showing a halo, air crescent

signs or cavities can indicate Aspergillus infection. For IA, microbi-

ological analysis of a bronchoalveolar lavage (BAL) is performed.

Aspergillus species can seldom be recovered from blood, urine or

cerebrospinal fluid [14]. Various serological tests are commercially

available to detect Aspergillus antigens, such as galactomannan

(GM), a polysaccharide cell wall component that is released into

the circulation during fungal growth, or (1,3)-b-D-glucan (BG), an

integral cell wall component. These noninvasive diagnostic tools

seem promising, but sensitivity and specificity issues still need to

be resolved [14]. PCR is a sensitive technique to detect and quan-

tify Aspergillus in blood and BAL, but no commercial or standard-

ized tests are yet available [15].

In the prevention and treatment of aspergillosis, three main

classes of antifungal are available: polyenes, azoles and echino-

candins. For years, the polyene amphotericin B (AmB) has been

considered first choice, mainly because of its potency and broad

antifungal spectrum. However, AmB is associated with adverse

effects and renal toxicity, which led to lipid-based reformulations

[16]. Among the azoles, voriconazole is currently recommended as

primary therapy for IA, whereas posaconazole, itraconazole and

the echinocandin caspofungin are mainly used as secondary ther-

apy. The azoles and echinocandins show less toxicity compared

with AmB, but their use is restricted because of pharmacokinetic

limitations and drug interactions [17]. Overall, the limited range

of existing drugs does not allow adequate management of the

different clinical manifestations of Aspergillus infection in the

diverse and complex patient populations. In addition, resistance

to azoles is becoming increasingly common [18]. Hence, the

discovery of novel antifungal agents remains a priority. To develop

efficient treatment strategies for aspergillosis, more basic and

applied mycological and clinical research has to be pursued. Ani-

mal models have a key role in the discovery of virulence factors,

unraveling of the pathogenesis and exploration of host factors, but

equally important is their role in preclinical drug research and

development (R&D) of antifungal therapy for IA, which is the

focus of this review [19].

Antifungal drug discoveryIn basic drug discovery research, medicinal chemists design novel

compounds or analogs of existing ones based on new or known

fungal targets. Although in silico screening can contribute to many

facets of drug discovery, in vitro and in vivo models remain pivotal

for the evaluation of antifungal potential [20]. In vitro analyses give

insight into drug activity at a enzymatic and cellular level, and into

pharmacokinetic properties, such as metabolic stability and mem-

brane passage. Subsequently, different fungal species and mam-

malian cells are exposed to assess potency and selectivity. The

Clinical Laboratory Standards Institute (CLSI) and the European

Committee on Antimicrobial Susceptibility Testing (EUCAST) de-

veloped standard susceptibility tests for antifungals [21,22]. In

addition, physicochemical profiling involving solubility testing

and mechanistic studies need to be performed. Active compounds

with a high selectivity and adequate in vitro potency finally move

up to the in vivo phase of preclinical evaluation. Efficacy, pharma-

cokinetic behavior and toxicity of compounds in animal models

will determine whether the drug is a suitable candidate for further

study. Therapeutic efficacy is preferably tested first in a small

laboratory animal model, because of ethical considerations and

to limit the amount of compound required. Adequate assessment

favors a course of disease with clear endpoints. The pharmacologi-

cal profiling includes the evaluation of the optimal route of

administration, tissue distribution, clearance and drug interac-

tions. Many compounds that have been identified as promising

candidates in vitro unfortunately turn out to fail once tested in vivo,

because complex processes, such as absorption, distribution, met-

abolic stability and toxicity, remain difficult to address in vitro [23].

Animal models of invasive aspergillosis for drugdiscoveryTo evaluate candidate antifungals, animal models of IA that mimic

human disease as closely as possible are required. Each model is

characterized by the animal species (strain, weight and sex) and

design, such as immunosuppressive regimen, inoculation route,

clinical manifestation and therapeutic (prophylactic versus cura-

tive) regimen. Ideally, the model should reflect most aspects of the

human pathology, such as the extent of infection, organs in-

volved, and physiological and histopathological events. Given

that each model has its own limitations, many variants have

been developed depending on the specific research question

[24]. Figure 1 gives an overview of the various parameters that

should be considered when developing an IA animal model for

antifungal evaluation. Figure 2 proposes potential approaches to

develop mouse models of disseminated and pulmonary IA for the

primary evaluation of drug efficacy.

Choice of laboratory animal speciesDifferent species, such as mice, rabbits, guinea pigs, chickens and

ducks, have been used to study IA [25–28]. For many experiments,

the mouse is the animal of choice because of its anatomical,

physiological and genetic similarity to the human system and

its attractive logistical features [29]. Mice are relatively inexpen-

sive, easy to house and handle, and the genetic background of

most strains is known. Transgenic mice or specific inbred strains

allow researchers to study closely the pathogenesis, virulence and

particular immunologic features of IA. For example, p47phox�/�

mice are used to study fungal infections related to CGD because of

their high susceptibility to infection and the pathogenesis is

similar to that seen in humans [30]. Frequently used for patho-

genesis studies is the inbred mouse strain DBA/2, which has a

deficiency in the C5 component of the complement system and is

highly susceptible to IA regardless of the portal of entry of the

conidia [31]. During drug development, outbred strains are more

commonly used because they are genetically randomized and,

thus, reflect the genetic variation found in humans. However,

considerations such as the calculation of the sample size must be

taken into account, because increased phenotypic deviation can

lead to low-powered experiments [32]. Inbred strains show genetic

homogeneity and have the advantages of being more stable, better

defined and more uniform [33]. However, results obtained for one

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InoculationIntranasal

IntratrachealIntravenousIntracerebral

AnimalRodents

NonrodentsInvertebrates

AspergillusStrain

VirulenceReporter

Aspergillosis

Clinical manifestationPulmonary IA

Disseminated IA

AdministrationRoute

Vehicle

TreatmentRegimen

combination

Fungal burdenCFU/g tissuechitin assay

qPCRGM and/or BG detection

Immune statusCompetent

Suppression

Outcome

Animal Survival

Histopathology

imaging

An

imal

mo

del

s IA

Eva

luat

ion

dru

g c

and

idat

esInoculation

IntranasalIntratrachealIntravenousIntracerebral

AnimalRodents

NonrodentsInvertebrates

AspergillusStrain

VirulenceReporter

Aspergillosis

Clinical manifestationPulmonary IA

Disseminated IA

AdministrationRoute

Vehicle

TreatmentRegimen

combination

Fungal burdenCFU/g tissuechitin assay

qPCRGM and/or BG detection

Immune statusCompetent

Suppression

Outcome

Animal Survival

Histopathology

imagin g

An

imal

mo

del

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Eva

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Drug Discovery Today

FIGURE 1

Overview of the pivotal parameters that should be considered in the development of an animal model for invasive aspergillosis: laboratory animal species and

immune status, inoculation routes, Aspergillus strain, route of administration, treatment strategy and efficacy assessment. Abbreviations: BG, (1,3)-b-D-glucan; CFU,

colony-forming unit; GM, galactomannan; IA, invasive aspergillosis; qPCR, quantitative PCR.

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inbred strain cannot be generalized to other strains [34]. The use of

small numbers of several strains of inbreds can be considered as an

alternative for outbreds [33], but the low cost of the latter makes

them the most attractive option for initial evaluation of new

compounds. In a later stage, inbred strains can be used for in-

depth study of the activity, pharmacokinetics or other properties

of the new antifungal.

Studies that require large blood volumes or serial sampling

demand the use of larger animals. Although BAL investigation

in mice is often fatal, larger animals allow BAL sampling over time.

Rabbits have been used in experimental models of Aspergillus eye

infection and models of systemic and pulmonary IA [27,35]. They

allow CT imaging to monitor disease progression [36], but their

cost, specific requirements for husbandry and limited availability

of inbred strains are important disadvantages [37]. For the evalua-

tion of voriconazole in IA, guinea pigs are most often used to

achieve long systemic exposure comparable to that in humans,

because this drug is rapidly cleared in other rodents [38]. Rats are

often used to study pulmonary IA [28,39].

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For the study of fungal infections, several invertebrate models

have been established [40]. Whole-organism screening based on

Caenorhabditis elegans, Drosophila melanogaster or Danio rerio is

gaining interest to study metabolic stability, toxicology and

distribution [41]. Obviously, the major limitation of the inverte-

brate models is the overly simplistic physiology compared with

humans.

ImmunosuppressionVarious combinations of immunosuppressive agents, schedules

and doses have been proposed to induce immunosuppression in IA

animal models to: (i) establish a good and reproducible infection;

(ii) mimic the immune status of patients with IA; or (iii) study

Aspergillus pathogenesis in relation to the host immune status.

Important risk factors for developing IA are treatment with corti-

costeroids, chemotherapy and neutropenia [42]. Cyclophospha-

mide, a DNA alkylating agent that interferes with cellular

replication, gives rise to prolonged neutropenia and, therefore,

is the most commonly used immunosuppressant [1,26].


High dose

~virulence

High dose

~virulence

InoculationAnimal Aspergillus Clinical manifestationImmune status

Animal survivalfungal count (CFU/g)

Mouse CompetentIntra-

venous Disseminated IA

Mouse

Incompetente.g. cyclophosphamide200 mg/kg 3 days pre-

infection for acutedisease course

Intra-tracheal

Invasive aspergillosis

Pulmonary IA

Acutecourse

ofdisease

CompoundPhysicochemistry

determinesadministration

Efficacy

AdministrationRoute

Vehicle

Start treatment@ day infection

High dose daily

Drug Discovery Today

FIGURE 2

Approaches to the development of mouse models of disseminated and pulmonary invasive aspergillosis (IA) for the initial evaluation of in vivo drug efficacy.Disseminated disease can be obtained in immunocompetent mice, whereas pulmonary IA generally requires immunosuppression (a cyclophosphamide dosing

scheme is suggested, but the mouse strain and Aspergillus strain used can further determine the optimal immunosuppressive conditions). Overall, a high

inoculation dose is needed to achieve reproducible infection. The physicochemical characteristics of the compound dictate route and vehicle of administration.

For the initial evaluation of the compound, it is important to start ‘high-dose’ treatment on the day of infection or even before to allow maximal drug activity.Animal survival and fungal burden in target organs are straightforward outcome parameters that accurately reflect drug efficacy in disseminated or pulmonary IA.

Abbreviation: CFU, colony-forming units.

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Neutropenia can also be induced by administration of neutrophil-

depleting monoclonal antibodies, which could be valuable in stud-

ies on IA pathogenesis, and should be monitored by counting

neutrophils in blood smears [43]. Immunosuppression with corti-

costeroids in IA models is based on their ability to affect alveolar

macrophage function, the first barrier to pulmonary infection. In

addition, steroids suppress the production of several cyto- and

chemokines, which compromise the secondary pulmonary defenses

against IA [44,45]. Corticosteroids can be combined with cyclophos-

phamide to obtain an overall immunosuppressed state [46,47].

Although immunosuppression might support the development

of IA models, it also introduces experimental complexity because it

can significantly influence fungal virulence, pathogenesis or out-

come of antifungal therapy [42,43,48]. In addition, bacterial infec-

tions pose an important risk to immunosuppressed animals and

should be prevented by prophylactic administration of antibiotics.

These disadvantages should be taken into careful consideration

before implementing immunosuppressive strategies. When an IA

systemic aspergillosis model was developed through intravenous

infection, immunosuppression with cyclophosphamide or corti-

sone-acetate led to more variation in clinical outcome and increased

lethality. By contrast, immunosuppression is known to be indis-

pensable to obtain a reproducible pulmonary infection [49].

InoculationDifferent inoculation methods have been described, including

intravenous, intratracheal, intranasal and intracerebral routes.

Intranasal inoculation by instillation or inhalation mimics the

natural route of infection and leads to the development of pulmo-

nary IA. In intranasal instillation, a fungal suspension is directly

instilled down the nares of an anesthetized mouse. Afterwards, the

animal can be placed in a (semi-)vertical position to allow the

conidia to move in the lungs [50]. Only skilled lab technicians can

perform this technique and the exact number of conidia that

ultimately reach the lung tissue cannot be determined without

sacrificing the animal. Given that the major disadvantage of

intranasal inoculation is the variable infection rate, large groups

of animals should be included in each experiment [2]. The inha-

lation model is the best approximation of the physiologic acquisi-

tion of infection [51]. Animals are placed in an inhalation chamber

(e.g. Hinners chamber or Madison chamber) in which the conidial

suspension is nebulized [52]. This approach establishes a more

reproducible and homogenous infection in the entire lung com-

pared with intranasal instillation [53]. Intratracheal inoculation

can also be used to establish a pulmonary infection and starts with

a minor incision to expose the trachea for injection of the conidial

suspension [50]. This method circumvents lodging of conidia in

the nares, but is time consuming and requires specialized equip-

ment and personnel [34]. Oropharyngeal aspiration also delivers

fungal suspension directly to the lungs without the need for a

surgical procedure [54].

Intravenous inoculation allows the establishment of a dissemi-

nated infection [38,55] and is easier to standardize because the

fungal inoculum is directly and entirely injected in the blood



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stream, resulting in good correlation between infection dose and

mortality rate [56]. Intravenous infection leads to an overwhelm-

ing systemic infection without the need for additional immuno-

suppression. In our experience, intravenous injection of 1 � 107

colony-forming units (CFU) per mouse leads to an acute, repro-

ducible infection, while a lower inoculum size gives rise to more

variation (C. Paulussen et al., unpublished). Intravenous inocula-

tion of immunocompetent mice is a valuable system for the initial

evaluation of new antifungal compounds [57]. To establish pul-

monary infection upon intravenous inoculation, the animal must

be made immune incompetent. Given that disseminated aspergil-

losis also often affects the central nervous system, an IA model was

developed in immunosuppressed mice using intracerebral inocu-

lation in the posterior midline of the cranium, resulting in high

brain burdens and infection of several other tissues [58].

Overall, pulmonary models of IA are closest to human disease,

because the lung is the primary portal of entry and invasion.

However, systemic models of IA are more reproducible, relatively

easy to establish and, therefore, suitable for primary in vivo evalu-

ation of new antifungal compounds.

Outcome parametersA straightforward outcome parameter of disease is overall mortali-

ty, but ethical committees generally do not allow death as an

acceptable endpoint. Ideally, animals are euthanized in moribund

state. To obtain objective criteria for euthanasia, animals should be

weighed and monitored daily for overall appearance and behavior

according to a functional observational battery (FOB) [59]. Weight

loss is a fairly accurate indicator of disease progress, but even

without disease, up to 15% weight loss can be expected upon

immunosuppression.

Measurement of the fungal tissue burden is the primary myco-

logical endpoint. Tissue burden studies give insight into the

antifungal activity of a new compound and allow a significant

reduction of the number of animals compared with survival

studies; however, the choice of fungal quantification method is

pivotal. Determination of the CFU count per weight unit of target

organ is a simple but semiquantitative technique. Liver, lungs,

spleen, kidney and brain are homogenized, serially diluted and

spread on an agar plate. Given the filamentous nature of Aspergil-

lus, a large fungal mass of tangled hyphae cannot be distinguished

from single-cell conidial forms when cultivated on agar [2,51].

Disruption of the homogenized organ suspension is necessary, but

this can kill conidia and lead to underestimation of CFU counts

[48]. An alternative to determine fungal burden is based on the

quantification of chitin, a fungal cell wall component [60]. How-

ever, this assay is technically demanding and does not indicate the

viability of the organisms [37]. A sensitive alternative approach to

measure fungal burden is (real-time) quantitative PCR (qPCR),

which quantifies every cell of the fungal mass [46]. Several reports

indicate that qPCR performs better than CFU counting to monitor

infections with mycelial organisms such as A. fumigatus in untreat-

ed animals. However, both techniques allow the assessment of

drug efficacy at a single time point. Singh et al. claim that CFU

enumeration is more appropriate than qPCR to confirm cure,

because it can indicate the presence of a limited amount of residual

organisms, whereas qPCR can detect more subtle changes in fungal

burdens after treatment [61]. The detection of GM or BG in blood


or BAL can also be used to assess the fungal burden. GM detection

data correlate with those obtained with qPCR, but the tests are

expensive and insufficiently validated. In addition, qPCR and GM

detection do not discriminate between viable and dead fungi

[46,62].

Histopathological examination of the infected tissue gives in-

sight into the extent of infection, inflammation and tissue dam-

age. Lung tissue is harvested, fixed, stained with Gomori’s

methenamine silver stain to evaluate fungal invasion, and coun-

terstained with hematoxylin and eosin to assess the influx of

inflammatory cells and lung injury [34].

Bioluminescent A. fumigatus strains offer the possibility to

monitor disease progression in individual animals without the

need to sacrifice the animals and could be used to evaluate the

efficacy of antifungal drugs. However, this complex and expensive

approach is still under investigation [63].

Overall, the current best practice is to evaluate survival (i.e.

moribund state) combined with reduction of fungal burdens in

target tissues as outcome parameters. New techniques, such as GM

or BG detection and qPCR, provide accurate and reliable quantifi-

cation tools that are gaining more interest.

Route of administration and treatment regimenTo assess the antifungal potential of new compounds, different

therapeutic regimens and routes of administration can be applied

with the objective to achieve maximal drug levels in the target

tissues and/or organs. Intranasal and inhalation treatments are

mainly used in pulmonary aspergillosis, whereas oral, intravenous

and intraperitoneal treatments are applied in the other models.

Depending on the route of administration, specific formulations

should be explored. Oral gavage is generally the preferred option

because administration with food or water can result in variable

exposure, particularly if the test compound has a bad taste. A

disadvantage of oral treatment might be the limited systemic

availability because of rapid first-pass elimination. A catheter

needs to be placed if daily intravenous injections are considered;

in some instances, intraperitoneal treatment might be a good

alternative.

Given that drug clearance occurs at different rates by different

mechanisms in different animals, evaluation of new antifungals

demands careful consideration of the animal species. For initial

assessment of in vivo antifungal activity, therapy is commonly

initiated on the day of infection or even the day before, because IA

is characterized by a hyperacute course of infection/disease. Using

this strategy, compounds with only moderate to weak activity can

also be picked up; therefore, more drugs can be considered for

further evaluation. Thorough assessment of therapeutic efficacy,

including dose-titration studies and the evaluation of treatment

schedules, requires animal models that closely mimic the clinical

situation. Using these models, toxicity, pharmacodynamics and -

kinetics can be studied. Overall, the properties of the candidate

antifungal will largely determine the ideal treatment regimen and

starting point of the therapy [35,37].

Concluding remarksAlthough animal studies form an essential part of the drug discov-

ery and development process, there is no universal method or

best practice to obtain the ideal animal model for aspergillosis.


Nevertheless, identification of new antifungals for IA relies on

performing high-quality in vivo experiments. Taking the properties

of the candidate compound and the desired clinical application

into account, variables relating to animal and fungal species,

immunomodulatory approach, inoculation method, outcome

parameters and therapeutic strategy should all be carefully inte-

grated to establish the most appropriate animal protocol for each

specific application.

N

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Animal models of invasive aspergillosis for drug discovery