Mycobacterium tuberculosis (Mtb) remains one of the successful human bacterial pathogen, yet litt... more Mycobacterium tuberculosis (Mtb) remains one of the successful human bacterial pathogen, yet little is known about the factors that influence infection outcome. The Cynomolgus macaque model of tuberculosis (TB) recapitulates many of the manifestations of human TB and provides an excellent platform to study early disease progesssion, which is not tractable in the humans. Macaques (n=38) were infected with low dose of Mtb (Erdman strain, ~25CFU) and whole blood samples were obtained for transcriptional analysis at serial time-points (pre infection to 6 months post infection) when the animals were declared to have clinically active TB or latent infection. Linear Mixed Modeling Analysis (LMMA) revealed increased activity of transcript clusters associated with interferon response, inflammation, hematopoiesis, early immune regulation, innate, and acquired immune responses between 20-56 days post Mtb infection in all animals. Despite considerable overlap in the transcriptional signatures o...
The only licensed vaccine (BCG) for tuberculosis (TB) has variable efficacy and does not protect ... more The only licensed vaccine (BCG) for tuberculosis (TB) has variable efficacy and does not protect against pulmonary TB. New vaccines are in development and in human clinical trials. However, vaccine trials are long and expensive without useful surrogate markers of protection. A problem thus emerges: what does a protective response to TB look like? This project’s goal is to identify markers of vaccine efficacy that can further be developed as new clinical endpoints. We posit that exploiting differences in vaccine outcome (protected vs. unprotected) will identify novel markers that correlate with outcome, and that serial PET/CT will predict outcome early in infection, as well as identify sites of protection. Cynomolgus macaques vaccinated with BCG and a multistage vaccine (H56 in CAF01) have more sterile thoracic lymph nodes (LN) 6 months post-Mtb challenge, compared to unvaccinated controls, indicating that LN play an important role in H56-conferred protection. BCG/H56 prevents dissem...
Lung granulomas are the pathologic hallmark of Tuberculosis (TB). T cells are a major cellular co... more Lung granulomas are the pathologic hallmark of Tuberculosis (TB). T cells are a major cellular component of tuberculosis lung granulomas and are known to play an important role in containment and progression of Mtb infection. We used cynomolgus macaques, a non-human primate model that recapitulates human TB, with clinically active disease and latent infection, to understand the functional characteristics and dynamics of T cells in individual granulomas and to correlate T cell cytokine response of granulomas to its bacterial burden and its systemic response. Our results suggest that each granuloma within an individual host is independent with respect to total cell numbers, proportion of T cells, pattern of cytokine response, and bacterial burden. The spectrum of these components overlaps greatly amongst various clinical status of these animals, indicating that a diversity of granulomas exist in individual hosts. Multi-parametric flow cytometry analysis of functional T cells in granul...
Co-infection with Mycobacterium tuberculosis (Mtb) and human immunodeficiency virus (HIV) is a wo... more Co-infection with Mycobacterium tuberculosis (Mtb) and human immunodeficiency virus (HIV) is a worldwide public health concern, leading to worse clinical outcomes caused by both pathogens. We used a non-human primate model of simian immunodeficiency virus (SIV)-Mtb co-infection, in which latent Mtb infection was established prior to SIVmac251 infection. The evolutionary dynamics of SIV env was evaluated from samples in plasma, lymph nodes, and lungs (including granulomas) of SIV-Mtb co-infected and SIV only control animals. While the diversity of the challenge virus was low and overall viral diversity remained relatively low over 6–9 weeks, changes in viral diversity and divergence were observed, including evidence for tissue compartmentalization. Overall, viral diversity was highest in SIV-Mtb animals that did not develop clinical Mtb reactivation compared to animals with Mtb reactivation. Among lung granulomas, viral diversity was positively correlated with the frequency of CD4+ T...
There is pressing urgency to understand the pathogenesis of the severe acute respiratory syndrome... more There is pressing urgency to understand the pathogenesis of the severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2) which causes the disease COVID-19. SARS-CoV2 spike (S)-protein binds ACE2, and in concert with host proteases, principally TMPRSS2, promotes cellular entry. The cell subsets targeted by SARS-CoV-2 in host tissues, and the factors that regulate ACE2 expression, remain unknown. Here, we leverage human, non-human primate, and mouse single-cell RNA-sequencing (scRNA-seq) datasets across health and disease to uncover putative targets of SARS-CoV-2 amongst tissue-resident cell subsets. We identify ACE2 and TMPRSS2 co-expressing cells within lung type II pneumocytes, ileal absorptive enterocytes, and nasal goblet secretory cells. Strikingly, we discover that ACE2 is a human interferonstimulated gene (ISG) in vitro using airway epithelial cells, and extend our findings to in vivo viral infections. Our data suggest that SARS-CoV-2 could exploit species-specific interferon-driven upregulation of ACE2, a tissue-protective mediator during lung injury, to enhance infection.
Human immunodeficiency virus infection is the most common risk factor for severe forms of tubercu... more Human immunodeficiency virus infection is the most common risk factor for severe forms of tuberculosis (TB), regardless of CD4 T cell count. Using a well-characterized cynomolgus macaque model of human TB, we compared radiographic, immunologic and microbiologic characteristics of early (subclinical) reactivation of latent M. tuberculosis (Mtb) infection among animals subsequently infected with simian immunodeficiency virus (SIV) or who underwent anti-CD4 depletion by a depletion antibody. CD4 depleted animals had significantly fewer CD4 T cells within granulomas compared to Mtb/SIV co-infected and Mtb-only control animals. After 2 months of treatment, subclinical reactivation occurred at similar rates among CD4 depleted (5 of 7 animals) and SIV infected animals (4 of 8 animals). However, SIV-induced reactivation was associated with more dissemination of lung granulomas that were permissive to Mtb growth resulting in greater bacterial burden within granulomas compared to CD4 depleted...
For many pathogens, including most targets of effective vaccines, infection elicits an immune res... more For many pathogens, including most targets of effective vaccines, infection elicits an immune response that confers significant protection against reinfection. There has been significant debate as to whether natural Mycobacterium tuberculosis (Mtb) infection confers protection against reinfection. Here we experimentally assessed the protection conferred by concurrent Mtb infection in macaques, a robust experimental model of human tuberculosis (TB), using a combination of serial imaging and Mtb challenge strains differentiated by DNA identifiers. Strikingly, ongoing Mtb infection provided complete protection against establishment of secondary infection in over half of the macaques and allowed near sterilizing bacterial control for those in which a secondary infection was established. By contrast, boosted BCG vaccination reduced granuloma inflammation but had no impact on early granuloma bacterial burden. These findings are evidence of highly effective concomitant mycobacterial immuni...
Whole blood transcriptional profiling offers great diagnostic and prognostic potential. Although ... more Whole blood transcriptional profiling offers great diagnostic and prognostic potential. Although studies identified signatures for pulmonary tuberculosis (TB) and transcripts that predict the risk for developing active TB in humans, the early transcriptional changes immediately following Mycobacterium tuberculosis infection have not been evaluated. We evaluated the gene expression changes in the cynomolgus macaque model of TB, which recapitulates all clinical aspects of human M. tuberculosis infection, using a human microarray and analytics platform. We performed genome-wide blood transcriptional analysis on 38 macaques at 11 postinfection time points during the first 6 mo of M. tuberculosis infection. Of 6371 differentially expressed transcripts between preinfection and postinfection, the greatest change in transcriptional activity occurred 20–56 d postinfection, during which fluctuation of innate and adaptive immune response–related transcripts was observed. Modest transcriptional...
Mycobacterium tuberculosis infection presents across a spectrum in humans, from latent infection ... more 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 granu...
Although recent studies in mice have shown that components of B cell and humoral immunity can mod... more Although recent studies in mice have shown that components of B cell and humoral immunity can modulate the immune responses against Mycobacterium tuberculosis , the roles of these components in human and nonhuman primate infections are unknown. The cynomolgus macaque ( Macaca fascicularis ) model of M. tuberculosis infection closely mirrors the infection outcomes and pathology in human tuberculosis (TB). The present study used rituximab, an anti-CD20 antibody, to deplete B cells in M. tuberculosis- infected macaques to examine the contribution of B cells and humoral immunity to the control of TB in nonhuman primates during the acute phase of infection. While there was no difference in the overall pathology, disease profession, and clinical outcome between the rituximab-treated and untreated macaques in acute infection, analyzing individual granulomas revealed that B cell depletion resulted in altered local T cell and cytokine responses, increased bacterial burden, and lower levels o...
Non-human primates, primarily macaques, have been used to study tuberculosis for decades. However... more Non-human primates, primarily macaques, have been used to study tuberculosis for decades. However, in the last 15 years, this model has been refined substantially to allow careful investigations of the immune response and host-pathogen interactions in Mycobacterium tuberculosis infection. Low-dose challenge with fully virulent strains in cynomolgus macaques result in the full clinical spectrum seen in humans, including latent and active infection. Reagents from humans are usually cross-reactive with macaques, further facilitating the use of this model system to study tuberculosis. Finally, macaques develop the spectrum of granuloma types seen in humans, providing a unique opportunity to investigate bacterial and host factors at the local (lung and lymph node) level. Here, we review the past decade of immunology and pathology studies in macaque models of tuberculosis.
Tuberculosis remains a significant global health burden. Little is understood regarding the criti... more Tuberculosis remains a significant global health burden. Little is understood regarding the critical pathogen-driven factors and host immune responses that result in latent infection and reactivation. The Wayne model is an in vitro model of latent infection in which Mycobacterium tuberculosis undergoes a hypoxia induced nonreplicating persistent state of metabolism. Using this model, important findings in genomic and proteomic factors involved in newly defined metabolic pathways and drug susceptibility have been identified. The mouse remains the most popular in vivo model latent infection. The Cornell model, reflecting chronic infection altered by antibiotic treatment, and the low to moderate dose chronic infection model have been used. Studies using this model have revealed important insights regarding TNF, IFN, reactive nitrogen intermediates, IL-10, CD4 and CD8 function during latent infection. However, the immune responses in the murine model most likely reflect chronic infection rather than true latency. The non-human primate model is the only animal model in which a true latent state occurs. However, its limited availability, high cost and support required are impractical for frequent use. Disparate data from multiple studies can be used to predict complex biologic interactions through mathematical simulations (in silico model). Mathematical modeling can be use to foresee important insights into host–pathogen interactions that can then be confirmed by in vivo experiments. Since no single perfect model of latent infection exists, the use of multiple models has and will continue to provide significant contributions in our understanding of latency and reactivation of tuberculosis.
Mycobacterium tuberculosis (Mtb) remains one of the successful human bacterial pathogen, yet litt... more Mycobacterium tuberculosis (Mtb) remains one of the successful human bacterial pathogen, yet little is known about the factors that influence infection outcome. The Cynomolgus macaque model of tuberculosis (TB) recapitulates many of the manifestations of human TB and provides an excellent platform to study early disease progesssion, which is not tractable in the humans. Macaques (n=38) were infected with low dose of Mtb (Erdman strain, ~25CFU) and whole blood samples were obtained for transcriptional analysis at serial time-points (pre infection to 6 months post infection) when the animals were declared to have clinically active TB or latent infection. Linear Mixed Modeling Analysis (LMMA) revealed increased activity of transcript clusters associated with interferon response, inflammation, hematopoiesis, early immune regulation, innate, and acquired immune responses between 20-56 days post Mtb infection in all animals. Despite considerable overlap in the transcriptional signatures o...
The only licensed vaccine (BCG) for tuberculosis (TB) has variable efficacy and does not protect ... more The only licensed vaccine (BCG) for tuberculosis (TB) has variable efficacy and does not protect against pulmonary TB. New vaccines are in development and in human clinical trials. However, vaccine trials are long and expensive without useful surrogate markers of protection. A problem thus emerges: what does a protective response to TB look like? This project’s goal is to identify markers of vaccine efficacy that can further be developed as new clinical endpoints. We posit that exploiting differences in vaccine outcome (protected vs. unprotected) will identify novel markers that correlate with outcome, and that serial PET/CT will predict outcome early in infection, as well as identify sites of protection. Cynomolgus macaques vaccinated with BCG and a multistage vaccine (H56 in CAF01) have more sterile thoracic lymph nodes (LN) 6 months post-Mtb challenge, compared to unvaccinated controls, indicating that LN play an important role in H56-conferred protection. BCG/H56 prevents dissem...
Lung granulomas are the pathologic hallmark of Tuberculosis (TB). T cells are a major cellular co... more Lung granulomas are the pathologic hallmark of Tuberculosis (TB). T cells are a major cellular component of tuberculosis lung granulomas and are known to play an important role in containment and progression of Mtb infection. We used cynomolgus macaques, a non-human primate model that recapitulates human TB, with clinically active disease and latent infection, to understand the functional characteristics and dynamics of T cells in individual granulomas and to correlate T cell cytokine response of granulomas to its bacterial burden and its systemic response. Our results suggest that each granuloma within an individual host is independent with respect to total cell numbers, proportion of T cells, pattern of cytokine response, and bacterial burden. The spectrum of these components overlaps greatly amongst various clinical status of these animals, indicating that a diversity of granulomas exist in individual hosts. Multi-parametric flow cytometry analysis of functional T cells in granul...
Co-infection with Mycobacterium tuberculosis (Mtb) and human immunodeficiency virus (HIV) is a wo... more Co-infection with Mycobacterium tuberculosis (Mtb) and human immunodeficiency virus (HIV) is a worldwide public health concern, leading to worse clinical outcomes caused by both pathogens. We used a non-human primate model of simian immunodeficiency virus (SIV)-Mtb co-infection, in which latent Mtb infection was established prior to SIVmac251 infection. The evolutionary dynamics of SIV env was evaluated from samples in plasma, lymph nodes, and lungs (including granulomas) of SIV-Mtb co-infected and SIV only control animals. While the diversity of the challenge virus was low and overall viral diversity remained relatively low over 6–9 weeks, changes in viral diversity and divergence were observed, including evidence for tissue compartmentalization. Overall, viral diversity was highest in SIV-Mtb animals that did not develop clinical Mtb reactivation compared to animals with Mtb reactivation. Among lung granulomas, viral diversity was positively correlated with the frequency of CD4+ T...
There is pressing urgency to understand the pathogenesis of the severe acute respiratory syndrome... more There is pressing urgency to understand the pathogenesis of the severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2) which causes the disease COVID-19. SARS-CoV2 spike (S)-protein binds ACE2, and in concert with host proteases, principally TMPRSS2, promotes cellular entry. The cell subsets targeted by SARS-CoV-2 in host tissues, and the factors that regulate ACE2 expression, remain unknown. Here, we leverage human, non-human primate, and mouse single-cell RNA-sequencing (scRNA-seq) datasets across health and disease to uncover putative targets of SARS-CoV-2 amongst tissue-resident cell subsets. We identify ACE2 and TMPRSS2 co-expressing cells within lung type II pneumocytes, ileal absorptive enterocytes, and nasal goblet secretory cells. Strikingly, we discover that ACE2 is a human interferonstimulated gene (ISG) in vitro using airway epithelial cells, and extend our findings to in vivo viral infections. Our data suggest that SARS-CoV-2 could exploit species-specific interferon-driven upregulation of ACE2, a tissue-protective mediator during lung injury, to enhance infection.
Human immunodeficiency virus infection is the most common risk factor for severe forms of tubercu... more Human immunodeficiency virus infection is the most common risk factor for severe forms of tuberculosis (TB), regardless of CD4 T cell count. Using a well-characterized cynomolgus macaque model of human TB, we compared radiographic, immunologic and microbiologic characteristics of early (subclinical) reactivation of latent M. tuberculosis (Mtb) infection among animals subsequently infected with simian immunodeficiency virus (SIV) or who underwent anti-CD4 depletion by a depletion antibody. CD4 depleted animals had significantly fewer CD4 T cells within granulomas compared to Mtb/SIV co-infected and Mtb-only control animals. After 2 months of treatment, subclinical reactivation occurred at similar rates among CD4 depleted (5 of 7 animals) and SIV infected animals (4 of 8 animals). However, SIV-induced reactivation was associated with more dissemination of lung granulomas that were permissive to Mtb growth resulting in greater bacterial burden within granulomas compared to CD4 depleted...
For many pathogens, including most targets of effective vaccines, infection elicits an immune res... more For many pathogens, including most targets of effective vaccines, infection elicits an immune response that confers significant protection against reinfection. There has been significant debate as to whether natural Mycobacterium tuberculosis (Mtb) infection confers protection against reinfection. Here we experimentally assessed the protection conferred by concurrent Mtb infection in macaques, a robust experimental model of human tuberculosis (TB), using a combination of serial imaging and Mtb challenge strains differentiated by DNA identifiers. Strikingly, ongoing Mtb infection provided complete protection against establishment of secondary infection in over half of the macaques and allowed near sterilizing bacterial control for those in which a secondary infection was established. By contrast, boosted BCG vaccination reduced granuloma inflammation but had no impact on early granuloma bacterial burden. These findings are evidence of highly effective concomitant mycobacterial immuni...
Whole blood transcriptional profiling offers great diagnostic and prognostic potential. Although ... more Whole blood transcriptional profiling offers great diagnostic and prognostic potential. Although studies identified signatures for pulmonary tuberculosis (TB) and transcripts that predict the risk for developing active TB in humans, the early transcriptional changes immediately following Mycobacterium tuberculosis infection have not been evaluated. We evaluated the gene expression changes in the cynomolgus macaque model of TB, which recapitulates all clinical aspects of human M. tuberculosis infection, using a human microarray and analytics platform. We performed genome-wide blood transcriptional analysis on 38 macaques at 11 postinfection time points during the first 6 mo of M. tuberculosis infection. Of 6371 differentially expressed transcripts between preinfection and postinfection, the greatest change in transcriptional activity occurred 20–56 d postinfection, during which fluctuation of innate and adaptive immune response–related transcripts was observed. Modest transcriptional...
Mycobacterium tuberculosis infection presents across a spectrum in humans, from latent infection ... more 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 granu...
Although recent studies in mice have shown that components of B cell and humoral immunity can mod... more Although recent studies in mice have shown that components of B cell and humoral immunity can modulate the immune responses against Mycobacterium tuberculosis , the roles of these components in human and nonhuman primate infections are unknown. The cynomolgus macaque ( Macaca fascicularis ) model of M. tuberculosis infection closely mirrors the infection outcomes and pathology in human tuberculosis (TB). The present study used rituximab, an anti-CD20 antibody, to deplete B cells in M. tuberculosis- infected macaques to examine the contribution of B cells and humoral immunity to the control of TB in nonhuman primates during the acute phase of infection. While there was no difference in the overall pathology, disease profession, and clinical outcome between the rituximab-treated and untreated macaques in acute infection, analyzing individual granulomas revealed that B cell depletion resulted in altered local T cell and cytokine responses, increased bacterial burden, and lower levels o...
Non-human primates, primarily macaques, have been used to study tuberculosis for decades. However... more Non-human primates, primarily macaques, have been used to study tuberculosis for decades. However, in the last 15 years, this model has been refined substantially to allow careful investigations of the immune response and host-pathogen interactions in Mycobacterium tuberculosis infection. Low-dose challenge with fully virulent strains in cynomolgus macaques result in the full clinical spectrum seen in humans, including latent and active infection. Reagents from humans are usually cross-reactive with macaques, further facilitating the use of this model system to study tuberculosis. Finally, macaques develop the spectrum of granuloma types seen in humans, providing a unique opportunity to investigate bacterial and host factors at the local (lung and lymph node) level. Here, we review the past decade of immunology and pathology studies in macaque models of tuberculosis.
Tuberculosis remains a significant global health burden. Little is understood regarding the criti... more Tuberculosis remains a significant global health burden. Little is understood regarding the critical pathogen-driven factors and host immune responses that result in latent infection and reactivation. The Wayne model is an in vitro model of latent infection in which Mycobacterium tuberculosis undergoes a hypoxia induced nonreplicating persistent state of metabolism. Using this model, important findings in genomic and proteomic factors involved in newly defined metabolic pathways and drug susceptibility have been identified. The mouse remains the most popular in vivo model latent infection. The Cornell model, reflecting chronic infection altered by antibiotic treatment, and the low to moderate dose chronic infection model have been used. Studies using this model have revealed important insights regarding TNF, IFN, reactive nitrogen intermediates, IL-10, CD4 and CD8 function during latent infection. However, the immune responses in the murine model most likely reflect chronic infection rather than true latency. The non-human primate model is the only animal model in which a true latent state occurs. However, its limited availability, high cost and support required are impractical for frequent use. Disparate data from multiple studies can be used to predict complex biologic interactions through mathematical simulations (in silico model). Mathematical modeling can be use to foresee important insights into host–pathogen interactions that can then be confirmed by in vivo experiments. Since no single perfect model of latent infection exists, the use of multiple models has and will continue to provide significant contributions in our understanding of latency and reactivation of tuberculosis.
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