Persistence of viral RNA in lymph nodes in ART-suppressed SIV/SHIV-infected Rhesus Macaques.

The establishment of a long-lived viral reservoir is the key obstacle for achieving an HIV-1 cure. However, the anatomic, virologic, and immunologic features of the viral reservoir in tissues during antiretroviral therapy (ART) remain poorly understood. Here we present a comprehensive necroscopic analysis of the SIV/SHIV viral reservoir in multiple lymphoid and non-lymphoid tissues from SIV/SHIV-infected rhesus macaques suppressed with ART for one year. Viral DNA is observed broadly in multiple tissues and is comparable in animals that had initiated ART at week 1 or week 52 of infection. In contrast, viral RNA is restricted primarily to lymph nodes. Ongoing viral RNA transcription is not the result of unsuppressed viral replication, as single-genome amplification and subsequent phylogenetic analysis do not show evidence of viral evolution. Gag-specific CD8+ T cell responses are predominantly observed in secondary lymphoid organs in animals chronically infected prior to ART and these responses are dominated by CD69+ populations. Overall, we observe that the viral reservoir in rhesus macaques is widely distributed across multiple tissue sites and that lymphoid tissues act as a site of persistent viral RNA transcription under conditions of long-term ART suppression.

IL-10 Impairs Local Immune Response in Lung Granulomas and Lymph Nodes during Early Mycobacterium tuberculosis Infection.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, continues to be a major global health problem. Lung granulomas are organized structures of host immune cells that function to contain the bacteria. Cytokine expression is a critical component of the protective immune response, but inappropriate cytokine expression can exacerbate TB. Although the importance of proinflammatory cytokines in controlling M. tuberculosis infection has been established, the effects of anti-inflammatory cytokines, such as IL-10, in TB are less well understood. To investigate the role of IL-10, we used an Ab to neutralize IL-10 in cynomolgus macaques during M. tuberculosis infection. Anti-IL-10-treated nonhuman primates had similar overall disease outcomes compared with untreated control nonhuman primates, but there were immunological changes in granulomas and lymph nodes from anti-IL-10-treated animals. There was less thoracic inflammation and increased cytokine production in lung granulomas and lymph nodes from IL-10-neutralized animals at 3-4 wk postinfection compared with control animals. At 8 wk postinfection, lung granulomas from IL-10-neutralized animals had reduced cytokine production but increased fibrosis relative to control animals. Although these immunological changes did not affect the overall disease burden during the first 8 wk of infection, we paired computational modeling to explore late infection dynamics. Our findings support that early changes occurring in the absence of IL-10 may lead to better bacterial control later during infection. These unique datasets provide insight into the contribution of IL-10 to the immunological balance necessary for granulomas to control bacterial burden and disease pathology in M. tuberculosis infection.

Publisher Correction: Antibody and TLR7 agonist delay viral rebound in SHIV-infected monkeys.

In Fig. 4b of this Article, the x-axis labels 'PGT121' and 'GS-9620' were inadvertently swapped in both graphs. In Fig. 5a, b, 'TLR7' should have been 'GS-9620'. These figures have been corrected online.

Lymph nodes are sites of prolonged bacterial persistence during Mycobacterium tuberculosis infection in macaques.

Tuberculosis is commonly considered a chronic lung disease, however, extrapulmonary infection can occur in any organ. Even though lymph nodes (LN) are among the most common sites of extrapulmonary Mycobacterium tuberculosis (Mtb) infection, and thoracic LNs are frequently infected in humans, bacterial dynamics and the effect of Mtb infection in LN structure and function is relatively unstudied. We surveyed thoracic LNs from Mtb-infected cynomolgus and rhesus macaques analyzing PET CT scans, bacterial burden, LN structure and immune function. FDG avidity correlated with the presence of live bacteria in LNs at necropsy. Lymph nodes have different trajectories (increasing, maintaining, decreasing in PET activity over time) even within the same animal. Rhesus macaques are more susceptible to Mtb infection than cynomolgus macaques and this is in part due to more extensive LN pathology. Here, we show that Mtb grows to the same level in cynomolgus and rhesus macaque LNs, however, cynomolgus macaques control Mtb at later time points post-infection while rhesus macaques do not. Notably, compared to lung granulomas, LNs are generally poor at killing Mtb, even with drug treatment. Granulomas that form in LNs lack B cell-rich tertiary lymphoid structures, disrupt LN structure by pushing out T cells and B cells, introduce large numbers of macrophages that can serve as niches for Mtb, and destroy normal vasculature. Our data support that LNs are not only sites of antigen presentation and immune activation during infection, but also serve as important sites for persistence of significant numbers of Mtb bacilli.

Antibody and TLR7 agonist delay viral rebound in SHIV-infected monkeys.

The latent viral reservoir is the critical barrier for the development of a cure for HIV-1 infection. Previous studies have shown direct antiviral activity of potent HIV-1 Env-specific broadly neutralizing antibodies (bNAbs) administered when antiretroviral therapy (ART) was discontinued, but it remains unclear whether bNAbs can target the viral reservoir during ART. Here we show that administration of the V3 glycan-dependent bNAb PGT121 together with the Toll-like receptor 7 (TLR7) agonist vesatolimod (GS-9620) during ART delayed viral rebound following discontinuation of ART in simian-human immunodeficiency virus (SHIV)-SF162P3-infected rhesus monkeys in which ART was initiated during early acute infection. Moreover, in the subset of monkeys that were treated with both PGT121 and GS-9620 and that did not show viral rebound after discontinuation of ART, adoptive transfer studies and CD8-depletion studies also did not reveal virus. These data demonstrate the potential of bNAb administration together with innate immune stimulation as a possible strategy for targeting the viral reservoir.

Concurrent infection with Mycobacterium tuberculosis confers robust protection against secondary infection in macaques.

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 immunity in the lung, which may inform TB vaccine design and development.

Profiling the airway in the macaque model of tuberculosis reveals variable microbial dysbiosis and alteration of community structure.

BACKGROUND: The specific interactions of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), and the lung microbiota in infection are entirely unexplored. Studies in cancer and other infectious diseases suggest that there are important exchanges occurring between host and microbiota that influence the immunological landscape. This can result in alterations in immune regulation and inflammation both locally and systemically. To assess whether Mtb infection modifies the lung microbiome, and identify changes in microbial abundance and diversity as a function of pulmonary inflammation, we compared infected and uninfected lung lobe washes collected serially from 26 macaques by bronchoalveolar lavage over the course of infection. RESULTS: We found that Mtb induced an initial increase in lung microbial diversity at 1 month post infection that normalized by 5 months of infection across all macaques. Several core genera showed global shifts from baseline and throughout infection. Moreover, we identified several specific taxa normally associated with the oral microbiome that increased in relative abundance in the lung following Mtb infection, including SR1, Aggregatibacter, Leptotrichia, Prevotella, and Campylobacter. On an individual macaque level, we found significant heterogeneity in both the magnitude and duration of change within the lung microbial community that was unrelated to lung inflammation and lobe involvement as seen by positron emission tomography/computed tomography (PET/CT) imaging. By comparing microbial interaction networks pre- and post-infection using the predictive algorithm SPIEC-EASI, we observe that extra connections are gained by Actinomycetales, the order containing Mtb, in spite of an overall reduction in the number of interactions of the whole community post-infection, implicating Mtb-driven ecological reorganization within the lung. CONCLUSIONS: This study is the first to probe the dynamic interplay between Mtb and host microbiota longitudinally and in the macaque lung. Our findings suggest that Mtb can alter the microbial landscape of infected lung lobes and that these interactions induce dysbiosis that can disrupt oral-airway boundaries, shift overall lung diversity, and modulate specific microbial relationships. We also provide evidence that this effect is heterogeneous across different macaques. Overall, however, the changes to the airway microbiota after Mtb infection were surprisingly modest, despite a range of Mtb-induced pulmonary inflammation in this cohort of macaques.

Rhesus Macaques Are More Susceptible to Progressive Tuberculosis than Cynomolgus Macaques: a Quantitative Comparison.

In the past 2 decades, it has become increasingly clear that nonhuman primates, specifically macaques, are useful models for human tuberculosis (TB). Several macaque species have been used for TB studies, and questions remain about the similarities and differences in TB pathogenesis among macaque species, which can complicate decisions about the best species for a specific experiment. Here we provide a quantitative assessment, using serial positron emission tomography and computed tomography (PET-CT) imaging and precise quantitative determination of bacterial burdens of low-dose Mycobacterium tuberculosis infection in cynomolgus macaques of Chinese origin, rhesus macaques of Chinese origin, and Mauritian cynomolgus macaques. This comprehensive study demonstrates that there is substantial variability in the outcome of infection within and among species. Overall, rhesus macaques have higher rates of disease progression, more lung, lymph node, and extrapulmonary involvement, and higher bacterial burdens than Chinese cynomolgus macaques. The small cohort of Mauritian cynomolgus macaques assessed here indicates that this species is more similar to rhesus macaques than to Chinese cynomolgus macaques in terms of M. tuberculosis infection outcome. These data provide insights into the differences among species, providing valuable data to the field for assessing macaque studies of TB.

Heterogeneity in tuberculosis.

Infection with Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), results in a range of clinical presentations in humans. Most infections manifest as a clinically asymptomatic, contained state that is termed latent TB infection (LTBI); a smaller subset of infected individuals present with symptomatic, active TB. Within these two seemingly binary states, there is a spectrum of host outcomes that have varying symptoms, microbiologies, immune responses and pathologies. Recently, it has become apparent that there is diversity of infection even within a single individual. A good understanding of the heterogeneity that is intrinsic to TB - at both the population level and the individual level - is crucial to inform the development of intervention strategies that account for and target the unique, complex and independent nature of the local host-pathogen interactions that occur in this infection. In this Review, we draw on model systems and human data to discuss multiple facets of TB biology and their relationship to the overall heterogeneity observed in the human disease.

Digitally Barcoding Mycobacterium tuberculosis Reveals In Vivo Infection Dynamics in the Macaque Model of Tuberculosis.

Infection with Mycobacterium tuberculosis causes a spectrum of outcomes; the majority of individuals contain but do not eliminate the infection, while a small subset present with primary active tuberculosis (TB) disease. This variability in infection outcomes is recapitulated at the granuloma level within each host, such that some sites of infection can be fully cleared while others progress. Understanding the spectrum of TB outcomes requires new tools to deconstruct the mechanisms underlying differences in granuloma fate. Here, we use novel genome-encoded barcodes to uniquely tag individual M. tuberculosis bacilli, enabling us to quantitatively track the trajectory of each infecting bacterium in a macaque model of TB. We also introduce a robust bioinformatics pipeline capable of identifying and counting barcode sequences within complex mixtures and at various read depths. By coupling this tagging strategy with serial positron emission tomography coregistered with computed tomography (PET/CT) imaging of lung pathology in macaques, we define a lesional map of M. tuberculosis infection dynamics. We find that there is no significant infection bottleneck, but there are significant constraints on productive bacterial trafficking out of primary granulomas. Our findings validate our barcoding approach and demonstrate its utility in probing lesion-specific biology and dissemination. This novel technology has the potential to greatly enhance our understanding of local dynamics in tuberculosis.IMPORTANCE Classically, M. tuberculosis infection was thought to result in either latent infection or active disease. More recently, the field has recognized that there is a spectrum of M. tuberculosis infection clinical outcomes. Within a single host, this spectrum is recapitulated at the granuloma level, where there can simultaneously be lesional sterilization and poorly contained disease. To better understand the lesional biology of TB infection, we digitally barcoded M. tuberculosis to quantitatively track the fate of each infecting bacterium. By combining this technology with serial PET-CT imaging, we can dynamically track both bacterial populations and granuloma trajectories. We demonstrate that there is little constraint on the bacterial population at the time of infection. However, the granuloma imposes a strong bottleneck on dissemination, and the subset of granulomas at risk of dissemination can be distinguished by physical features.

Very Low Doses of Mycobacterium tuberculosis Yield Diverse Host Outcomes in Common Marmosets (Callithrix jacchus).

Identifying and refining small-animal models of tuberculosis that recapitulate aspects of human Mycobacterium tuberculosis infection can contribute to advancing our understanding of critical facets of the disease. To study the effects of very low-dose infections with 2 strains of M. tuberculosis on disease progression and survival in common marmosets, animals were challenged with strains Erdman and CDC1551 at doses ranging from 1 to 12 cfu. These data revealed that the susceptibility of marmosets to M. tuberculosis infection is influenced by strain virulence and initial dose. Marmoset infection with the Erdman strain, even at very low doses, resulted in rapid disease progression associated with severe weight loss, extensive pathology, and poor survival. By contrast, challenge with the less virulent CDC1551 strain resulted in slower disease progression, delayed weight loss, and prolonged survival. One marmoset infected with CDC1551 at a very low dose (approximately 1 cfu) was able to contain and control M. tuberculosis infection in a subclinical state that persisted as long as 300 d. These findings underscore the critical importance of understanding the heterogeneity in host outcome that can arise in association with different infectious doses and strains in the marmoset model of tuberculosis.

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.

The Importance of First Impressions: Early Events in Mycobacterium tuberculosis Infection Influence Outcome.

Tuberculosis remains a major health threat in much of the world. New vaccines against Mycobacterium tuberculosis are essential for preventing infection, disease, and transmission. However, the host immune responses that need to be induced by an effective vaccine remain unclear. Increasingly, it has become clear that early events in infection are of major importance in the eventual outcome of the infection. Studying such events in humans is challenging, as they occur within the lung and thoracic lymph nodes, and any clinical signs of early infection are relatively nonspecific. Nonetheless, clinical studies and animal models of tuberculosis have provided new insights into the local events that occur in the first few weeks of tuberculosis. Development of an effective vaccine requires a clear understanding of the successful (and detrimental) early host responses against M. tuberculosis, with the goal to improve upon natural immune responses and prevent infection or disease.