Jaime Tomko

Radiologic Responses in Cynomolgus Macaques for Assessing Tuberculosis Chemotherapy Regimens

ABSTRACT Trials to test new drugs currently in development against tuberculosis in humans are impractical. All animal models to prioritize new regimens are imperfect, but nonhuman primates (NHPs) infected with Mycobacterium tuberculosis develop active tuberculosis (TB) disease with a full spectrum of lesion types seen in humans. Serial 2-deoxy-2-[18F]-fluoro-d-glucose (FDG) positron emission tomography (PET) with computed tomography (CT) imaging was performed on cynomolgus macaques during infection and chemotherapy with individual agents or the four-drug combination therapy most widely used globally. The size and metabolic activity of lung granulomas varied among animals and even within a single animal during development of disease. Individual granulomas within untreated animals had highly local and independent outcomes, some progressing in size and FDG uptake, while others waned, illustrating the highly dynamic nature of active TB. At necropsy, even untreated animals were found to have a proportion of sterile lesions consistent with the dynamics of this infection. A more marked reduction in overall metabolic activity in the lungs (decreased FDG uptake) was associated with effective treatment. A reduction in the size of individual lesions correlated with a lower bacterial burden at necropsy. Isoniazid treatment was associated with a transient increase in metabolic activity in individual lesions, whereas a net reduction occurred in most lesions from rifampin-treated animals. Quadruple-drug therapy resulted in the highest decrease in FDG uptake. The findings of PET-CT imaging may provide an important early correlate of the efficacy of novel combinations of new drugs that can be directly translated to human clinical trials.

Differential Virulence and Disease Progression following Mycobacterium tuberculosis Complex Infection of the Common Marmoset (Callithrix jacchus)

ABSTRACT Existing small-animal models of tuberculosis (TB) rarely develop cavitary disease, limiting their value for assessing the biology and dynamics of this highly important feature of human disease. To develop a smaller primate model with pathology similar to that seen in humans, we experimentally infected the common marmoset (Callithrix jacchus) with diverse strains of Mycobacterium tuberculosis of various pathogenic potentials. These included recent isolates of the modern Beijing lineage, the Euro-American X lineage, and M. africanum. All three strains produced fulminant disease in this animal with a spectrum of progression rates and clinical sequelae that could be monitored in real time using 2-deoxy-2-[18F]fluoro-d-glucose (FDG) positron emission tomography (PET)/computed tomography (CT). Lesion pathology at sacrifice revealed the entire spectrum of lesions observed in human TB patients. The three strains produced different rates of progression to disease, various extents of extrapulmonary dissemination, and various degrees of cavitation. The majority of live births in this species are twins, and comparison of results from siblings with different infecting strains allowed us to establish that the infection was highly reproducible and that the differential virulence of strains was not simply host variation. Quantitative assessment of disease burden by FDG-PET/CT provided an accurate reflection of the pathology findings at necropsy. These results suggest that the marmoset offers an attractive small-animal model of human disease that recapitulates both the complex pathology and spectrum of disease observed in humans infected with various M. tuberculosis strain clades.

Early Changes by 18Fluorodeoxyglucose Positron Emission Tomography Coregistered with Computed Tomography Predict Outcome after Mycobacterium tuberculosis Infection in Cynomolgus Macaques

ABSTRACT Cynomolgus macaques infected with low-dose Mycobacterium tuberculosis develop both active tuberculosis and latent infection similar to those of humans, providing an opportunity to study the clinically silent early events in infection. 18Fluorodeoxyglucose radiotracer with positron emission tomography coregistered with computed tomography (FDG PET/CT) provides a noninvasive method to measure disease progression. We sought to determine temporal patterns of granuloma evolution that distinguished active-disease and latent outcomes. Macaques (n = 10) were infected with low-dose M. tuberculosis with FDG PET/CT performed during infection. At 24 weeks postinfection, animals were classified as having active disease (n = 3) or latent infection (n = 6), with one “percolator” monkey. Imaging characteristics (e.g., lesion number, metabolic activity, size, mineralization, and distribution of lesions) were compared among active and latent groups. As early as 3 weeks postinfection, more pulmonary granulomas were observed in animals that would later develop active disease than in those that would develop latent infection. Over time, new lesions developed in active-disease animals but not in latent animals. Granulomas and mediastinal lymph nodes from active-disease but not latent animals consistently increased in metabolic activity at early time points. The presence of fewer lesions at 3 weeks and the lack of new lesion development in animals with latent infection suggest that innate and rapid adaptive responses are critical to preventing active tuberculosis. A greater emphasis on innate responses and/or rapid recruitment of adaptive responses, especially in the airway, should be emphasized in newer vaccine strategies.

Delayed Acquisition of Neonatal Reflexes in Newborn Primates Receiving A Thimerosal-Containing Hepatitis B Vaccine: Influence of Gestational Age and Birth Weight

This study examined whether acquisition of neonatal reflexes in newborn rhesus macaques was influenced by receipt of a single neonatal dose of hepatitis B vaccine containing the preservative thimerosal (Th). Hepatitis B vaccine containing a weight-adjusted Th dose was administered to male macaques within 24 h of birth (n = 13). Unexposed animals received saline placebo (n = 4) or no injection (n = 3). Infants were tested daily for acquisition of nine survival, motor, and sensorimotor reflexes. In exposed animals there was a significant delay in the acquisition of root, snout, and suck reflexes, compared with unexposed animals. No neonatal responses were significantly delayed in unexposed animals. Gestational age (GA) and birth weight (BW) were not significantly correlated. Cox regression models were used to evaluate main effects and interactions of exposure with BW and GA as independent predictors and time-invariant covariates. Significant main effects remained for exposure on root and suck when controlling for GA and BW, such that exposed animals were relatively delayed in time-to-criterion. Interaction models indicated there were various interactions between exposure, GA, and BW and that inclusion of the relevant interaction terms significantly improved model fit. This, in turn, indicated that lower BW and/or lower GA exacerbated the adverse effects following vaccine exposure. This primate model provides a possible means of assessing adverse neurodevelopmental outcomes from neonatal Th-containing hepatitis B vaccine exposure, particularly in infants of lower GA or BW. The mechanisms underlying these effects and the requirements for Th requires further study.

PET CT Identifies Reactivation Risk in Cynomolgus Macaques with Latent M. tuberculosis

Mycobacterium tuberculosis infection presents across a spectrum in humans, from latent infection to active tuberculosis. Among those with latent tuberculosis, it is now recognized that there is also a spectrum of infection and this likely contributes to the variable risk of reactivation tuberculosis. Here, functional imaging with 18F-fluorodeoxygluose positron emission tomography and computed tomography (PET CT) of cynomolgus macaques with latent M. tuberculosis infection was used to characterize the features of reactivation after tumor necrosis factor (TNF) neutralization and determine which imaging characteristics before TNF neutralization distinguish reactivation risk. PET CT was performed on latently infected macaques (n = 26) before and during the course of TNF neutralization and a separate set of latently infected controls (n = 25). Reactivation occurred in 50% of the latently infected animals receiving TNF neutralizing antibody defined as development of at least one new granuloma in adjacent or distant locations including extrapulmonary sites. Increased lung inflammation measured by PET and the presence of extrapulmonary involvement before TNF neutralization predicted reactivation with 92% sensitivity and specificity. To define the biologic features associated with risk of reactivation, we used these PET CT parameters to identify latently infected animals at high risk for reactivation. High risk animals had higher cumulative lung bacterial burden and higher maximum lesional bacterial burdens, and more T cells producing IL-2, IL-10 and IL-17 in lung granulomas as compared to low risk macaques. In total, these data support that risk of reactivation is associated with lung inflammation and higher bacterial burden in macaques with latent Mtb infection.

In vivo imaging in an ABSL-3 regional biocontainment laboratory

The Regional Biocontainment Laboratory (RBL) at the University of Pittsburgh is a state-of-the-art ABSL-3 facility that supports research on highly pathogenic viruses and bacteria. Recent advances in radiologic imaging provide several noninvasive, in vivo imaging modalities that can be used to longitudinally monitor animals following experimental infection or vaccination. The University of Pittsburgh RBL provides digital radiography, bioluminescence imaging, and PET/CT. Operating these platforms in an ABSL-3 poses unique challenges. This review will discuss the development and refinement of these imaging platforms in high containment, emphasizing specific challenges and how they were overcome.

A new method to evaluate macaque health using exhaled breath: A case study of M. tuberculosis in a BSL-3 setting

Breath is hypothesized to contain clinically relevant information, useful for the diagnosis and monitoring of disease, as well as understanding underlying pathogenesis. Nonhuman primates, such as the cynomolgus macaque, serve as an important model for the study of human disease, including over 70 different human infections. In this feasibility study, exhaled breath was successfully collected in less than 5 min under Biosafety Level 3 conditions from five anesthetized, intubated cynomolgus and rhesus macaques, before and after lung infection with M. tuberculosis The breath was subsequently analyzed using comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry. A total of 384 macaque breath features were detected, with hydrocarbons being the most abundant. We provide putative identification for 19 breath molecules and report on overlap between the identified macaque breath compounds and those identified in previous human studies.NEW & NOTEWORTHY To the best of our knowledge, this is the first time the volatile molecule content of macaque breath has been comprehensively sampled and analyzed. We do so here in a Biosafety Level 3 setting in the context of M. tuberculosis lung infection. The breath of nonhuman primates represents a novel fluid that could provide insight into disease pathogenesis.

Characterization of T Cells Specific for CFP-10 and ESAT-6 in Mycobacterium tuberculosis-Infected Mauritian Cynomolgus Macaques

ABSTRACT Nonhuman primates can be used to study host immune responses to Mycobacterium tuberculosis. Mauritian cynomolgus macaques (MCMs) are a unique group of animals that have limited major histocompatibility complex (MHC) genetic diversity, such that MHC-identical animals can be infected with M. tuberculosis. Two MCMs homozygous for the relatively common M1 MHC haplotype were bronchoscopically infected with 41 CFU of the M. tuberculosis Erdman strain. Four other MCMs, which had at least one copy of the M1 MHC haplotype, were infected with a lower dose of 3 CFU M. tuberculosis. All animals mounted similar T-cell responses to CFP-10 and ESAT-6. Two epitopes in CFP-10 were characterized, and the MHC class II alleles restricting them were determined. A third epitope in CFP-10 was identified but exhibited promiscuous restriction. The CFP-10 and ESAT-6 antigenic regions targeted by T cells in MCMs were comparable to those seen in cases of human M. tuberculosis infection. Our data lay the foundation for generating tetrameric molecules to study epitope-specific CD4 T cells in M. tuberculosis-infected MCMs, which may guide future testing of tuberculosis vaccines in nonhuman primates.