Monkey Models of Tuberculosis: Lessons Learned · immunopathogenesis of M. tuberculosis infection and reactiva-tion. However, there are differences between the two species. For example,

Monkey Models of Tuberculosis: Lessons Learned

Juliet C. Peña,b* Wen-Zhe Hoa,b

Animal Biosafety Level III Laboratory at the Center for Animal Experiment, State Key Laboratory of Virology, Wuhan University, Wuhan, Chinaa; Department of Pathologyand Laboratory Medicine, Temple University School of Medicine, Philadelphia, Pennsylvania, USAb

The use of animal models has been invaluable for studying the pathogenesis of Mycobacterium tuberculosis infection, as well asfor testing the efficacy of vaccines and drug regimens for tuberculosis. Among the applied animal models, nonhuman primates,particularly macaques, share the greatest anatomical and physiological similarities with humans. As such, macaque models havebeen used for investigating tuberculosis pathogenesis and preclinical testing of drugs and vaccines. This review focuses on pub-lished major studies which illustrate how the rhesus and cynomolgus macaques have enriched and may continue to advance thefield of global tuberculosis research.

About one-third of the population worldwide is infected withMycobacterium tuberculosis, while nearly 9 million cases of

active tuberculosis (TB) are reported annually (1). Hence, theneed for improved preventative and treatment strategies againstTB is ever-increasing. In an attempt to globally control TB, re-searchers have employed multiple animal models (mice, guineapigs, rabbits, cows, nonhuman primates, and others) for testingnovel experimental vaccines and therapies for TB (2, 3). The use ofnonhuman primates (NHP), especially cynomolgus macaques(CM; Macaca fascicularis, also called long-tailed macaques, a spe-cies of Old World monkeys native to Southeast Asia) and rhesusmacaques (RM; Macaca mulatta, a species of Old World monkeysnative to Asia; most experimental models are from India orChina), has led to significant advances in TB research due to theirinherent commonalities with humans, as illustrated in previousreviews in this subject area (4–6). By using the macaque model ofTB, we can gain even greater insights into ways to prevent M.tuberculosis infection and disease progression.

JUSTIFICATION FOR MACAQUES IN TB RESEARCH

Macaques exhibit remarkable similarities to humans in virtuallyevery aspect of their anatomy and physiology (7–9). As such, ma-caques respond similarly to many human immunological, patho-logical, and drug agents, providing a tremendous advantage overother animal models (6, 10). The literature shows that macaquesand humans share extensive clinical manifestations of TB, includ-ing pulmonary and extrapulmonary signs and symptoms (6, 10).Clinicians and researchers can monitor the disease course in ma-caques by measuring nearly identical parameters tested in hu-mans, ranging from skin and blood tests to radiographic imagingand body fluid samples (Table 1). In addition, multidrug chemo-therapy for TB provides effective treatment in both humans andmacaques (11, 12). Furthermore, as in humans, Mycobacteriumbovis bacillus Calmette-Guérin (BCG) vaccination exhibits vari-able efficacy in macaques of even the same species (13–15). Table1 highlights the similarities and differences in M. tuberculosis in-fection between humans and the rhesus macaques and cynomol-gus macaques.

HISTORICAL OUTLOOK ON MACAQUE MODELS

The use of NHP models to study M. tuberculosis infection tracesback to published literature from the 1960s. This “Golden Age” ofTB research using NHP, performed through the 1970s, generated

valuable data on the evolving BCG vaccine (16), as well as onereport on the TB drug efficacies of ethambutol and isoniazid (12).The majority of the studies focused on the BCG-induced immunereactions and vaccine efficacy (17–23). All pertinent studies pub-lished during this era used Indian RM models, which underwentintrabronchial (i.b.), intratracheal (i.t.), or aerosol infection withM. tuberculosis. The estimated mycobacterial retention rate inmammalian lungs after aerosol exposure was derived from prioranimal models, including macaques exposed to anthrax spores, aswell as guinea pigs and mice infected with M. tuberculosis (24).Figure 1 recaps the major published articles of NHP models withexperimental M. tuberculosis infection during this time period (12,17–23).

Not until about 20 years after the Golden Age was anotherstudy using experimentally infected macaques with M. tuberculo-sis published (25). This large research gap may be attributedto several factors, including the high maintenance costs and nec-essary space/equipment for biocontainment to properly conductsuch experiments (6). Additionally, the limited animal availabil-ity, handling difficulties, and adverse public opinions discouragedTB research with NHP. However, with greater research fundingand the emergence of compatible reagents for macaques, furtherinvestigations into M. tuberculosis infection using NHP modelsregained momentum (10). Especially amid the expansion of theNational Primate Research Centers (NPRCs), TB investigators re-visited the macaque model of experimental M. tuberculosis inoc-ulation with renewed enthusiasm. A series of subsequent investi-gations were dedicated to macaque research to assess novel TBvaccines and drugs, as well as to gain an understanding of thepathogenesis of M. tuberculosis infection and reactivation. Figures

Accepted manuscript posted online 29 December 2014

Citation Peña JC, Ho W-Z. 2015. Monkey models of tuberculosis: lessons learned.Infect Immun 83:852–862. doi:10.1128/IAI.02850-14.

Editor: A. T. Maurelli

Address correspondence to Wen-Zhe Ho, [email protected].

* Present address: Juliet C. Peña, Office of Disease Prevention and HealthPromotion, U.S. Department of Health and Human Services, Rockville, Maryland,USA.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

doi:10.1128/IAI.02850-14

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2 and 3 abridge the RM (13, 26–43) and CM (10, 15, 25, 42–59)models of experimental M. tuberculosis infection after the GoldenAge, from 2001 to 2014.

The majority of NHP models of M. tuberculosis infection haveinvolved either CM or Indian RM, except for at least five reportedresearch projects which used Chinese RM in Wuhan, China (34,35), Solna, Sweden (28, 29), and Rijswijk, the Netherlands (30). Sofar within the scientific literature illustrating macaque models ofM. tuberculosis infection, the routes of inoculation have includedi.b. instillation as well as i.t., aerosol, and intranasal (i.n.) infec-tion. Although zoonotic TB outbreaks have revealed horizontaltransmission of M. tuberculosis, an experimental macaque modelof natural M. tuberculosis infection has not been reported. Tables 2to 4 summarize by route the dose and M. tuberculosis strain ofinfection in each study design, along with the major findings of thelisted papers.

RHESUS VERSUS CYNOMOLGUS MACAQUE MODELS OF TB

Both RM and CM models have served to evaluate the efficacy ofTB vaccines/drugs, as well as improve our understanding of theimmunopathogenesis of M. tuberculosis infection and reactiva-tion. However, there are differences between the two species. Forexample, one investigation revealed that BCG provides greater

protective efficacy in CM than in RM against M. tuberculosis in-fection (42). Later investigations exploring stereological tech-niques for measuring the bacterial burden showed that RM aremore susceptible to M. tuberculosis infection than are CM (60). Asa result, RM are more often used for the study of active TB,whereas CM provide better models of latent or chronic TB (10).Depending on the route and dose of infection, as well as the strainfor the inoculum, either CM or RM can develop acute, chronic, orlatent TB. Therefore, several research endeavors have employedboth species for developing and determining the efficacy of TBscreening immunoassays (61–64) (Table 2).

RHESUS MACAQUES

RM have been used extensively to study TB (Table 3). The earlyinvestigations during the Golden Age (17–21) specifically showedthat M. tuberculosis infection in RM progresses rapidly, within 8 to9 weeks after aerosol inhalation of the H37Rv strain administeredat low doses of up to 62 CFU. In 2004, the Tulane NPRC estab-lished a model of asymptomatic, or latent, M. tuberculosis infec-tion of RM (26). The investigators there revealed that RM are agood model for not only active TB but also asymptomatic TBwhen the investigators used lower doses (30 CFU) of the H37Rvstrain. In addition to RM of Indian origin, Chinese RM can also be

TABLE 1 Comparison of TB in humans versus rhesus and cynomolgus macaquesa

Parameter Humans Rhesus macaques Cynomolgus macaques

Clinical manifestationsRate of disease progressionb Acute �� latent (10% vs 90%) Acute �� latent (90% vs 10%) Acute � latent (60% vs 40%)Presence of active/chronic infection symptom

Cough � � �Bloody sputum � � �Increased body temp � � �Wt loss � � �

Presence of latent infection symptomsNo clinical signs � � �Activated by coinfection (HIV or SIV) � � �

Clinical testsSkin tests

PPD � � �Old tuberculin test � � �

Blood testsELISA, ELISpot � � �Quantiferon-TB Gold � � �Primagram � � �CBC, ESR, CRP, LT � � �

Imaging (chest X-Ray, MRI, PET/CT) � � �Fluid sampling (BAL, gastric aspirate) � � �

PathologyCaseous granulomas � � �Calcification �/� �/� �/�Fibrous capsule �/� �/� �/�Pulmonary cavities � � �Disseminated lesions �/� �/� �/�

a The majority of macaques, particularly the rhesus species, develop acute or active TB after artificial infection, whereas 90% of infected humans have latent TB. Chronic infection isdefined as persistent signs of active disease, radiographic involvement, or culture positivity. Although the PPD and old tuberculin skin tests are used in both humans and macaques,these diagnostic exams are less reliable in macaques than in humans (69). Also, macaques exhibit a more random than apical distribution of pulmonary cavities. Abbreviations:BAL, bronchoalveolar lavage; CBC, complete blood count; CRP, C-reactive protein; LT, lymphocyte transformation; rBCG, recombinant bacillus Calmette-Guérin, �, positive forthe indicated finding or functional modality; �, absent finding or unused modality; �/�, a variable finding.b Acute disease lasts weeks to months, and latent disease lasts months to years. Percentages (in parentheses) indicate the global percentage of the species infected with M.tuberculosis.

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used as a viable model of acute TB (34). Chinese RM are highlysusceptible to M. tuberculosis infection and develop active TB re-gardless of the dose of strain H37Rv used (34). In an attempt toimprove the methods to monitor the progression of TB disease,Helke et al. (27) from the Oregon NPRC showed that use of high-resolution radiographic and fine immunologic studies helped de-fine the disease status in RM as in humans. Namely, computedtomography (CT) evaluation and M. tuberculosis-specific T cellfrequencies measured by enzyme-linked immunosorbent spot(ELISPOT) assays correlated well with the bacterial burden andseverity of disease (27).

CYNOMOLGUS MACAQUES

In addition to RM, CM have aided researchers similarly in thestudy of TB (Table 4), particularly after the so-called Golden Age(16). The first published CM investigation of controlled M. tuber-culosis infection stemmed from Walsh et al.’s (25) work in thePhilippines (Fig. 3), in collaboration with the University of Cali-fornia Los Angeles School of Medicine. In this project, the Philip-pine CM were found to make an excellent model of not only acuteTB but also chronic TB. This research pioneered the intratrachealinfection of Philippine CM in a TB model. Within 7 years,Capuano et al. (10) from The University of Pittsburgh developeda CM model representing the full spectrum of human M. tubercu-losis infection by infecting the macaques after intrabronchial in-oculation with a low dose (25 CFU) of the Erdman strain. Inter-estingly, this low dose of inoculum precipitated various reactionsin the CM, ranging from latent TB to active-chronic and evenrapidly progressive TB. The CM’s pulmonary and even extrapul-monary manifestations of disease within the different stages of TBinfection closely resembled the pathological and clinical findingsin human TB, as confirmed by laboratory assessments, includingpurified protein derivative (PPD) tests and erythrocyte sedimen-tation rate (ESR) profiling (10) (Table 1). A substantial number ofthe proceeding publications on CM models of M. tuberculosis in-fection were similarly published by researchers at The Universityof Pittsburgh (47–50, 52, 56, 57). Therefore, CM models have nowbeen used for evaluating TB drugs and vaccines (42, 46, 52).

SPECIFIC MACAQUE MODELS OF TBMacaque models for TB vaccine evaluation. Using the RMmodel, researchers from the Swedish Institute of Infectious Dis-ease Control strived to augment, broaden, and prolong immuneprotection against M. tuberculosis. They showed that a recombi-nant BCG (AFRO-1) could induce strong antigen-specific T cellresponses when combined with the TB vaccine vector rAD35 (28,29). Two other investigations using RM models of M. tuberculosisinfection also focused on evaluating TB vaccines (30, 31). TheBiomedical Primate Research Center in Rijswijk, the Netherlands,aimed to develop a model to test TB vaccines before progressing tohuman clinical trials (30). By using an RM model employing in-tratracheal inoculation with the Erdman strain of M. tuberculosis,the study revealed that prior immunization with the MVA.85-boosted BCG and an attenuated, phoP-deficient TB vaccine pro-vided effective protection against M. tuberculosis infection. Anovel aerosol challenge model effected by Sharpe et al. (31) helpedto assess the endpoints for testing the BCG/MVA.85 vaccine. ThisNHP model used a three-jet collision nebulizer in addition to amodified Henderson apparatus (a device for studying the infec-tivity and virulence of microorganisms in small air droplets; the 3components include a continuous aerosol-generating unit (colli-sion spray), exposure unit, and sampling unit), as described byBarclay et al. (18), in a head-out plethysmography chamber. Theresults of these studies indicated that the gamma interferon(IFN-�) indices—from ELISPOT assays and enzyme-linked im-munosorbent assays (ELISAs)— do not relate to protectionagainst TB; only the magnetic resonance imaging (MRI) readoutsoffered a reliable correlate, using ex vivo lung samples removed atnecropsy (18, 31). Another way to objectively measure the efficacyof TB vaccines and drugs was later developed by Luciw et al. (37)at the California NPRC. The research findings from these studiesdetermined that stereological analysis (i.e., three-dimensional in-terpretation of planar sections of materials or tissues) from MRIcould provide quantitative data, showing a significant correlationbetween bacterial load and lung granulomas.

Researchers did not use CM for testing TB vaccines until much

FIG 1 The “Golden Age” of TB research using rhesus macaques. The timeline illustrates major studies of experimental M. tuberculosis infection in Indian rhesusmacaques during the so-called Golden Age from the 1960s to 1970s (12, 17–23). Events are organized chronologically by year of publication. Thus, the 10-yearstudy by Good (17) actually began before the first reported investigation in 1966. Locations of the experiments (red) and the corresponding author (blue) areindicated. Abbreviations: ic, intracutaneous; im, intramuscular; iv, intravenous; sc, subcutaneous; M.tb, M. tuberculosis; NBL, Naval Biological Laboratories; UC,University of California.

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later than for the early RM models of TB. Between 2005 and 2012,three reported studies using CM models evaluated the efficacy ofTB vaccines that had been tested previously with smaller animalmodels, including guinea pigs and mice (44, 45). The first projectevaluated the efficacy of the 72f recombinant BCG vaccine andHASP65 plus interleukin-12 (IL-12)/HVJ vaccine (44), which an-other team of investigators also tested later in Osaka, Japan (45).Both research investigations showed that the recombinant BCGvaccines were more effective than BCG alone. In subsequent years,the multistage vaccine H56 was found to boost the effects of BCGto protect CM against active TB and the reactivation of latent TB(52). Ultimately, the macaque models have exhibited the potentialto help evaluate preventative methods and interventions beforereaching human clinical trials, particularly for TB vaccines.

Macaque models for TB drug evaluation. Finally, another re-cently reported investigation of experimental M. tuberculosis in-fection in a CM model was also published from the University ofPittsburgh School of Medicine (56, 57). The purpose of this re-search endeavor was to determine potential alternative markersfor evaluating the efficacy of TB drugs. Specifically, the studiescompared the overall metabolic and radiographic changes, as well

as the alterations within individual granulomas, in CM infectedwith M. tuberculosis. Of note, the results revealed that TB granu-lomas evolve and resolve independently within a single host andthat individual lesions respond variably to different drugs (56).Furthermore, the clinical findings concluded that the overall pos-itron emission tomography (PET) and CT signals could be used asprognostic markers to predict successful TB drug therapies. How-ever, the overall metabolic and radiographic changes reported bythis study were nonspecific indicators of metabolic activity, mea-sured from [18F]FDG-radiolabeled glucose in PET/CT imagingdata (56).

Macaque models for the study of TB pathogenesis. By gaininga better understanding of the pathogenesis of M. tuberculosis in-fection and reactivation of latent TB, researchers can further de-velop and improve treatment strategies. As such, investigators atthe Tulane NPRC profiled the TB granuloma transcriptome in anRM model to identify key immune signaling pathways that areactivated during M. tuberculosis infection (32). Previously, scien-tists from the Chicago Center for Biomedical Research character-ized gene networks in RM after only BCG vaccination/infection(65). Mechanistic studies of M. tuberculosis delineated more spe-

FIG 2 Twenty-first century TB research using rhesus macaques. The timeline shows important studies of experimental M. tuberculosis infection in rhesusmacaques during the 21st century, from 2001 to 2014 (13, 26–34, 36–43). Events are organized chronologically by year of publication. Locations of theexperiments (red) and the corresponding author (blue) are specified accordingly. Boxed events refer to comparative TB studies using both cynomolgus andrhesus macaques. All studies, except for those reported in references 28 to 30 and 34, used Indian rhesus macaques. Abbreviations: BPRC, Biomedical PrimateResearch Center; rBCG, recombinant BCG.

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cific factors employed by M. tuberculosis to successfully infect andpersist in mammalian lungs (33). The identification of a potentialtherapeutic target sparked from the discovery of the M. tubercu-losis stress response factor SigH as an important player in thegrowth/replication of M. tuberculosis (38, 39).

Lin et al. (47) characterized the clinical manifestations of TBdisease within the three stages: latent, active-chronic, and rapidlyprogressive TB. The results of these studies showed that at nec-ropsy, CM with active TB had more CD4� and CD8� T cells in the

lungs and more gamma interferon from peripheral blood mono-nuclear cells, bronchoalveolar lavage fluid, and mediastinal lymphnodes than CM with latent TB (47). Investigators from the samegroup also showed that tumor necrosis factor neutralization re-sulted in disseminated disease in both acute and latent TB withnormal granulomatous structures (48). Another CM study exam-ined the role of CD4� regulatory T cells in active TB; these cellsoccurred in response to increased inflammation rather than actingas a causative factor in the progression to active disease (49).

FIG 3 TB research using cynomolgus macaques. The timeline includes significant studies on cynomolgus macaques experimentally infected with M. tuberculosisfrom 1996 to 2014 (10, 15, 25, 42–59). Events are organized chronologically by year of publication. Locations of experiments (red) and the corresponding author(blue) are listed. Boxed events refer to comparative TB studies using both cynomolgus and rhesus macaques, as shown in Fig. 2. Abbreviations: BPRC, BiomedicalPrimate Research Center; IFN-�, gamma interferon; TNF-�, tumor necrosis factor alpha; Treg, regulatory T cells; U Pitt, University of Pittsburgh.

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In light of the discovery that TB granulomas change uniquelywithin a host (56), the latest published investigation by Lin et al.(57) aimed to define how these structures vary between CM withlatent and active TB. Interestingly, the sterilization of the granu-lomas occurred in both stages of TB, regardless of the differentialkilling of bacteria within a single host (57). Moreover, TB vaccine-related studies have elucidated possible genetic mechanisms ofhost resistance to M. tuberculosis after immunization (58), as wellas immunosuppressant mechanisms of M. tuberculosis virulence(59). While adding insight into the molecular and pathologicalpathways of TB progression, these findings may also help to eval-uate the disease status as well as spur the development of noveltherapeutic targets.

Macaque models of TB/HIV coinfection. Macaques also serveas an excellent model of TB/HIV coinfection, which is of impor-tance to understand TB latency and reactivation (36). Upon inoc-ulation with high-dose BCG (36, 66, 67) or low-dose Erdmanstrain (25 CFU, i.b.) (50), latently infected RM and CM, respec-tively, had reactivated TB when coinfected with the simian immuno-deficiency virus (SIV). Pathogenic SIV-BCG interactions facilitatedthe development of TB-like disease (67), while antiretroviralagents restored M. tuberculosis-specific T cell immune responses(36). The reactivation of latent TB in CM infected with SIV wasassociated with early T cell depletion and not the virus load (50).Another particularly innovative RM model of TB was establishedat the Southwest NPRC (40). The purpose of that study was toinstitute an NHP model for pediatric TB/HIV coinfection. New-

born macaques were infected with M. tuberculosis Erdman strainintrabronchially or via the aerosol route, using an ultrasonic neb-ulizer specifically adapted for the newborn macaque nose. Inves-tigators confirmed M. tuberculosis infection by various methods,including chest X-rays, ELISPOT, bronchoalveolar and gastric la-vages, and necropsy. Because people with HIV/AIDS carry a highrisk of M. tuberculosis infection and disease severity, it is clinicallysignificant to have a model that mimics coinfection.

KEY LESSONS AND FUTURE DIRECTIONS

Collectively, the literature on macaque models of TB has sharedfour important lessons and opportunities for improvement. First,we recognize that the biosafety requirements, cost of equipmentand maintenance, and animal availability have impeded scientistsfrom using NHP models in TB research. To address these issues,researchers may experiment with smaller genera and species of NHPsthat still recapitulate human TB, such as the common marmoset(Callithrix jacchus) model (68). Second, we found that differentanimal species, individually and as a whole, respond differentlywhen exposed to M. tuberculosis in terms of immunopathogenesisof the disease (43, 64) (Table 2). Therefore, investigators mustconsider the purpose of the study for the appropriate selection ofNHP species. Third, the strain of M. tuberculosis used for inocu-lation can significantly impact the disease outcome. Instead ofemploying merely the laboratory-adopted Erdman and H37Rvstrains, study designs should include clinical isolates of M. tuber-culosis. Although this approach may introduce more variability

TABLE 2 TB studies using both rhesus and cynomolgus macaquesa

Exptl design

Major findings ReferenceNo. of animalsM. tuberculosis strainb (inoculation route),dose(s) (CFU)

6 RM, 6 CM Erdman (i.t.), 3,000 BCG provides greater protective efficacy against TB in CM than in RM 42c

8 RM, 15 CM Erdman (i.t.), 1,000–3,000 In vitro IFN-� assays provide reproducible, reliable results whilecausing less stress than the PPD skin test

61

5 RM, 4 CM Erdman (i.t.), 100 Synthetic peptides may be used in lieu of full-length ESAT-6 protein inTB diagnostic antibody detection assays

62d

26 RM Erdman (i.t.), 30–1,000 Highest rate of TB detection achieved when skin test is combined withPrimaTB STAT-PAK immunoassay

63e

4 RM H37Rv (i.t.), 2103 RM Beijing (i.t.), 1,00016 CM Erdman (i.t.), 100–1,000

4 RM H37Rv (i.b.), 1,000 The multiplex microbead immunoassay profiles M. tuberculosisantibodies at multiple stages of infection/disease

646 CM Erdman (i.b.), 25

9 RM, 21 CM Erdman K01 (aerosol), 30–500 MRI and stereology provides most accurate, quantifiablemeasurements of TB disease burden; RM exhibit highersusceptibility to M. tuberculosis than CM

60

6 RM Erdman (i.b.), 500 M. tuberculosis antibody profiles depend on the NHP species andinfecting M. tuberculosis strain but do not significantly change withTB disease progression

434 RM H37Rv (i.b.), 1,00014 CM Erdman (i.b.), 25a All studies used Indian RM models.b The Erdman strain is a virulent set of M. tuberculosis isolates existing in two forms, including the laboratory ATCC 35801 isolate and the clinically isolated K01; this strain is mostcommonly used to study acute tuberculosis. Here, Erdman strain ATCC 35801 was used unless K01 is indicated. H37RV is an attenuated laboratory strain of M. tuberculosistypically used to study latent infection.c A TB vaccine-related study (42).d The study reported in reference 62 also used African green monkeys.e In the study reported in reference 63, additional RM, CM, and African green monkey groups were inoculated with the mycobacteria M. kansaii and M. avium.

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into the NHP studies of TB while making studies performed atdifferent sites more difficult to compare, the findings would likelyfurther mimic human TB disease and add valuable knowledge tothe field. Lastly, most of the macaque studies so far have shownonly acute TB, which is much less prevalent than latent TB inhumans. Hence, it is necessary to establish more clinically relevantNHP models that resemble human passive airway transmission.By creating an NHP model of natural M. tuberculosis infection

under experimental conditions, researchers would be able to testnew and existing TB vaccines/drugs for protection against M. tu-berculosis infection.

CONCLUDING REMARKS

Evidently, the macaque models have served as a valuable tool inTB research over the past several decades. As demonstrated in theliterature, the CM and RM TB models have revealed clinically

TABLE 3 TB studies using rhesus macaquesa

Exptl design

Major findings Reference(s)No. ofRM

M. tuberculosis strainb (inoculation route),dose(s) (CFU)

8 Erdman (i.b.), 10–150 RM are a good model for latent TB, with use of low doses of H37Rv 2612 H37Rv (i.b.), 30–6,000,000

4 H37Rv (i.b.), 1,000 High-resolution radiographic and fine immunologic studies provide definition ofTB disease progression

27

18 Erdman (i.t.), 500 Recombinant BCG (AFRO-1) induces strong antigen-specific T cell responseswith TB vaccine vector (rAD35)

28, 29c

24 Erdman (i.t.), 1,000 MVA.85 boosting of BCG and an attenuated, phoP-deficient TB vaccine showprotective efficacy against TB

30c

16 Erdman K01 (aerosol), 40–65 RM may be used as models of M. tuberculosis aerosol challenge; IFN-� (ELISpot,ELISA) does not correlate with protection against TB; only MRI offers areliable correlate

31

NP NP Early TB lesions have a highly proinflammatory environment, expressing IFN-�,TNF-�, JAK, STAT, and C-C/C-X-C chemokines; in contrast, late TB lesionshave a silenced inflammatory response

32

12 326 CDC1551 Himar 1 mutants (i.n.),100,000

Virulence mechanisms of M. tuberculosis include transport of lipid virulencefactors, biosynthesis of cell wall arabinan and peptidoglycan, DNA repair,sterol metabolism, and lung cell entry

33

9 H37Rv (i.b.), 50–3,000 Chinese RM are highly susceptible to M. tuberculosis infection and develop activeTB regardless of the dose of strain H37Rv or Erdman used

34, 3524 Erdman (i.b.), 25–500

16 CDC1551 (i.n.), 50 RM are an excellent model of TB/HIV coinfection and can be used to study TBlatency and reactivation

36

6 Erdman K01 (i.b.), 500 Stereological analysis quantitative data show a strong correlation betweenbacterial load and lung granulomas

37

13 CDC1551 (i.n.), 5,000 The M. tuberculosis stress response factor sigH is required for M. tuberculosisgrowth and replication in mammalian lungs

38

3 Erdman (i.b./i.n.), 5–50 Newborn macaques infected with aerosolized M. tuberculosis develop human-likeimmunologic responses and are a good model for pediatric TB/HIV

40

32 Erdman K01 (i.b.), 275 RM aerosol vaccination with AERAS-402 elicits transient cellular immuneresponses in blood and robust, sustained immune responses in BAL fluid butdoes not protect against high-dose M. tuberculosis infection

13c

17 CDC1551 (aerosol), 100 Clinical profiles vary considerably among RM infected with M. tuberculosis butcan help identify predictive biomarkers for TB susceptibility along with geneexpression profiles

41

a All studies, except for those reported in references 28 to 30 and 33, used Indian rhesus macaques. Abbreviations: i.d., intradermal; i.n., intranasal; NP, not provided.b The Erdman strain is most commonly used to study acute TB. It is a virulent subset of M. tuberculosis and exists in two forms, the laboratory isolate ATCC 35801 and the clinicalisolate, K01; the Erdman ATCC 35801 strain was used in most studies, except those for which the K01 strain is indicated. H37RV is an attenuated laboratory strain of M. tuberculosistypically used to study latent TB infection. CDC1551 is a clinical isolate of M. tuberculosis and exhibits a similar degree of virulence as the Erdman strain.c TB vaccine-related study.

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TABLE 4 TB studies using cynomolgus macaquesa

Exptl design

Major findings Reference(s)No. ofCM

M. tuberculosis strain (inoculation),dose(s) (CFU)

28 Erdman (i.t.), 10–100,000 Philippine CM provide an excellent model of chronic TB 25

17 Erdman (i.b.), 25 Low-dose infection of CM represents the full spectrum of human M. tuberculosis infection andprovides a model to study latent as well as active-chronic and rapidly progressive TB

10

16 Erdman (i.t.), 500 CM vaccination with the 72f rBCG vaccine provides better protective efficacy than with BCG 44b

44 Erdman (i.t.), 500 CM vaccination with the HSP65 plus IL-12/HVJ vaccine provides better protective efficacythan BCG

44, 45b

15 Erdman (i.t.), dose not reported CM vaccination with Mtb72F/AS02A provides greater protective efficacy than BCG alone 46b

24 Erdman (i.b.), 1,000 CM vaccination with mc26020 or mc26030 provides less protection than with BCG 15b

25 Erdman (i.b.), 25 At necropsy, CM with active TB have more lung T cells and more IFN-� from PBMC, BALfluid, and mediastinal lymph nodes than CM with latent TB

47

24 Erdman (i.b.), 25 Neutralization of TNF results in disseminated disease in acute and latent TB infection withnormal granuloma structure in a CM model

48

41 Erdman (i.b.), 25 Increased regulatory T cells in active TB occur in response to increased inflammation, not as acausal factor of disease progression

49

15 Erdman (i.b.), 25 Reactivation of latent TB with SIV is associated with early T cell depletion and not virus load 50

7 Erdman (i.b.), 25 M. tuberculosis-specific multifunctional T cells are better correlates of antigen load and diseasestatus than of protection

51c

5 Erdman (i.b.), 200

33 Erdman (i.t.), 25–500 The multistage vaccine H56 boosts effects of BCG to protect CM against active TB andreactivation of latent TB

52

14 Erdman (i.b.), 25–200 The CM model of M. tuberculosis infection mimics human TB, particularly in granuloma typeand structure

53

8 Erdman (i.t.), 250 M. tuberculosis may modulate protective immune responses via the use of indoleamine 2,3-dioxygenase (an immunosuppressant) found in nonlymphocytic regions of TB granulomas

14b

9 Erdman (i.b.), 25 Experimental and epidemiologic estimates of the M. tuberculosis mutation rate are comparable 54

27 Erdman (i.b.), 500 Early expansion/differentiation of V�2V�2 T effector cells during M. tuberculosis infectionincreases resistance to TB

55

26 Erdman (i.b.), 25–400 TB granulomas evolve and resolve independently within a single host; individual lesionsrespond differently to different drugs; overall PET and CT signals can predict successful TBdrug treatment

56

12 Erdman (i.b.), 1,000 Compared to nonvaccinated CM, BCG-vaccinated CM exhibit higher expression levels ofTNF-�, IL-10, IL-1b, TLR4, IL-17, IL-6, IL-12, and iNOS in lungs

58b

39 Erdman (i.b.), 25 Sterilization of TB granulomas occurs in both active and latent TB amid the differential killingof M. tuberculosis within a single host

572 SNP strains (i.b.), 34

8 Erdman (i.b.), 240–500 CM vaccination with BCG transiently increases levels of macrophages and lymphocytes inblood, with later recruitment in the lungs; however, M. tuberculosis continues to replicate inlungs

59b

a Abbreviations: SNP strains, strains with a single-nucleotide polymorphism mutation; rBCG, recombinant BCG; BAL, bronchoalveolar lavage; PBMC, peripheral bloodmononuclear cells; iNOS, inducible nitric oxygen synthase.b TB vaccine-related study.c Animals were coinfected with M. tuberculosis and SIV.

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similar manifestations of TB disease or latency through multiplediagnostic and prognostic parameters. Even the variabilities inimmune responses to M. tuberculosis infection imitate the diversehuman host reactions to the pathogen (41). Although a number ofstudies on TB vaccine evaluation have been conducted using ex-perimentally infected macaques, the majority of these studiesmerely showed the reduction of TB disease progression by BCG-based vaccines. Therefore, an urgent need still exists in order toestablish macaque models that can demonstrate protectionagainst M. tuberculosis infection besides TB disease progression.Fortunately, some investigators and funding institutions have re-vamped an interest in further developing NHP models for TBresearch. Undoubtedly, given the reemerging TB epidemic, manypeople worldwide should benefit from more advanced treatmentand prevention strategies against M. tuberculosis infection and dis-ease progression. Hence, the use of NHP models should be con-sidered a highly effective means of reaching these common goals.In essence, from a global health standpoint, there is truth in thewords of the literary science author Chris Roberson: “Everythingis improved by the judicious application of primates.”

ACKNOWLEDGMENTS

J.C.P. and W.H. were the sole contributors to the literature analysis andwritten work. Graphic illustrations were developed by J.C.P. and W.H.and enhanced by Patrick Lane at ScEYEnce Studios.

We received no additional support in the preparation of the manu-script.

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Juliet C. Peña, M.D., M.P.H., dedicated 1 year(2013 to 2014) as a postdoctoral fellow in Wen-Zhe Ho’s laboratory at the Temple UniversitySchool of Medicine in Philadelphia, PA, whereshe focused her studies on the immunopatho-genesis of tuberculosis and HIV. Her interest intuberculosis research stemmed from her medi-cal and public health background, as well as hercollaborations with Wen-Zhe Ho’s laboratoryusing monkey models of tuberculosis. Sheearned her M.D. (2012) and M.P.H. (2014)from The University of Arizona College of Medicine and the Mel and EnidZuckerman College of Public Health. Currently, Dr. Peña is completing ahealth policy fellowship at the Office of Disease Prevention and Health Pro-motion within the U.S. Department of Health and Human Services.

Wen-Zhe Ho has 30 years of research experi-ence in understanding the interactions betweenhost innate immunity and the pathogens HIV,hepatitis C virus, and M. tuberculosis. He re-ceived his M.D. and clinical training as a pedia-trician in infectious diseases at Wuhan Univer-sity, China in the 1980s. He was a postdoctoralfellow in infectious diseases at the Children’sHospital of Philadelphia and the Wistar Insti-tute of the University of Pennsylvania, where hebecame a full professor (2005). He also receivedhis M.P.H. at the University of Pennsylvania (2006), through which hegained a greater interest in public health issues of M. tuberculosis and HIVcoinfection. In 2009, Dr. Ho moved to Temple University, where he nowserves as a tenured professor in the Departments of Pathology and Cell Bi-ology. Additionally, as Director/Professor at the Center for Animal Experi-ment/ABSL-III Laboratory of Wuhan University (2010 to present), he con-tinues his research on M. tuberculosis infection using monkey models.

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