Determination of correlates of protection against tuberculosis in nonhuman primate models

Tuberculosis (TB) is one of the greatest global health challenges society faces. BCG, the only licensed vaccine for TB, has profoundly variable efficacy and does not prevent the spread of TB. Due to the lack of an effective vaccine, there are no correlates of protection to use in vaccine development. The goal of this dissertation was to develop new tools for pre-clinical and clinical trials of TB vaccines, including new outcome measures and predictive markers of efficacy. Development of these tools will expedite down-selection of vaccinate candidates, reducing their ultimate cost and hastening the reduction and eventual elimination of this disease. BCG afforded the best levels of protection in the rhesus macaque model of TB, which closely resembled TB disease in human infants. Boosting BCG by protein antigens or adenoviral vectored antigens did not improve, and in some cases worsened, outcome. A T cell signature in the lung-draining lymph nodes (LN) at necropsy, early gamma interferon (IFN-γ) ELISPOT and early PET-CT markers correlated with improved outcome in this model. We further characterized the protection afforded by an experimental boost to BCG, H56, which has been shown to prevent reactivation TB in cynomolgus macaques. BCG/H56 prevented establishment of disease in lung- draining LN. BCG/H56 also mitigated lung inflammation, which reduced apparent risk of reactivation TB by PET-CT. Early control of disease in the lung-draining LN, as well as a T cell signature, was associated with reduced risk of reactivation TB. Both studies provided evidence that PET-CT markers correlate with outcome. We thus built a holistic outcome score based iv strictly on quantifiable outcomes: gross pathology and bacterial burden determined at necropsy, and constructed models that robustly predict this outcome score early using early PET-CT markers. Altogether, these studies highlight the importance of the lung-draining LN as a site of bacterial persistence and the ability of PET-CT to assess disease and predict vaccine efficacy. Further work will build upon these studies to determine the best site of vaccination to prevent disease, and develop a blood signature correlate for use in clinical trials

Advanced model systems and tools for basic and translational human immunology

Abstract There are fundamental differences between humans and the animals we typically use to study the immune system. We have learned much from genetically manipulated and inbred animal models, but instances in which these findings have been successfully translated to human immunity have been rare. Embracing the genetic and environmental diversity of humans can tell us about the fundamental biology of immune cell types and the elasticity of the immune system. Although people are much more immunologically diverse than conventionally housed animal models, tools and technologies are now available that permit high-throughput analysis of human samples, including both blood and tissues, which will give us deep insights into human immunity in health and disease. As we gain a more detailed picture of the human immune system, we can build more sophisticated models to better reflect this complexity, both enabling the discovery of new immunological mechanisms and facilitating translation into the clinic

Integrating Non-human Primate, Human, and Mathematical Studies to Determine the Influence of BCG Timing on H56 Vaccine Outcomes

Tuberculosis (TB) is the leading cause of death by an infectious agent, and developing an effective vaccine is an important component of the WHO's EndTB Strategy. Non-human primate (NHP) models of vaccination are crucial to TB vaccine development and have informed design of subsequent human trials. However, challenges emerge when translating results from animal models to human applications, and connecting post-vaccination immunological measurements to infection outcomes. The H56:IC31 vaccine is a candidate currently in phase I/IIa trials. H56 is a subunit vaccine that is comprised of 3 mycobacterial antigens: ESAT6, Ag85B, and Rv2660, formulated in IC31 adjuvant. H56, as a boost to Bacillus Calmette-Guérin (BCG, the TB vaccine that is currently used in most countries world-wide) demonstrates improved protection (compared to BCG alone) in mouse and NHP models of TB, and the first human study of H56 reported strong antigen-specific T cell responses to the vaccine. We integrated NHP and human data with mathematical modeling approaches to improve our understanding of NHP and human response to vaccine. We use a mathematical model to describe T-cell priming, proliferation, and differentiation in lymph nodes and blood, and calibrate the model to NHP and human blood data. Using the model, we demonstrate the impact of BCG timing on H56 vaccination response and reveal a general immunogenic response to H56 following BCG prime. Further, we use uncertainty and sensitivity analyses to isolate mechanisms driving differences in vaccination response observed between NHP and human datasets. This study highlights the power of a systems biology approach: integration of multiple modalities to better understand a complex biological system

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

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

Disease pathology, bacterial burden and bacterial killing after TNF neutralization among reactivated and non-reactivated animals.

<p>(A) Macaques with radiographically defined reactivation have greater gross pathology at necropsy compared to animals that did not reactivate. Each symbol represents an animal. Statistics: Mann-Whitney (B) Thoracic bacterial burden (the sum of <i>Mtb</i> growth from all lung granulomas, other lung pathologies and all mediastinal lymph nodes, [MLN]) is higher in reactivated animals than in non-reactivated animals after TNF neutralization. Each symbol represents an animal. Statistics: Mann-Whitney (C) The number of granulomas among animals that developed reactivation (N = 13 macaques, 114 granulomas) are shown with the proportion of sterile granulomas reported in light green compared to animals that did not reactivate (N = 12 macaques, 100 granulomas). The p-value shown compares the proportion of sterile granulomas between groups of animals (Fisher’s Exact analysis). Up to 10 granulomas (randomized) are represented per animal to reduce bias. (D) Animals with reactivation had a smaller proportion of sterile MLNs compared to animals that did not reactivate. Proportions of sterile MLN are shown in the context of the total number of MLN in each group (reactivated group: N = 13 macaques, 85 MLNs vs non-reactivated group N = 13 macaques, 87 MLNs). The p-value shown compares the proportion of sterile granulomas between groups of animals (Fisher’s Exact analysis). Up tp 7 lymph nodes (randomized) are represented per animal to limit bias. (E) Dynamic granulomas (N = 42) had greater CFU per granuloma than stable (N = 132) and new (N = 23) granulomas after TNF neutralization. Percent sterile granulomas is noted for each group. Statistics: Kruskal-Wallis with Dunn’s multiple comparisons. (F) Dynamic lesions had less bacterial killing compared to stable lesions, as assessed by CFU/CEQ ratio. Statistics: Mann Whitney. The numbers indicated above each figure represents the p-value for the given statistical method.</p

PET CT features of reactivation tuberculosis during TNF neutralization.

<p>Axial views of the lung are shown from two representative latently infected macaques with granulomas seen before (left panels, top and bottom) and during the course of TNF antibody treatment (middle and right panels, top and bottom). Pre-existing lesions (green arrows) within the same lung lobe can increase in size and FDG avidity or remain the same during the TNF neutralization. New lesions (red arrows) can arise in the lungs in an already involved lobe (top row) and/or in a new lobe or extrapulmonary sites such as the liver (red arrows, bottom row).</p

Frequencies of <i>Mycobacterium tuberculosis</i> (Mtb) specific T cells producing cytokines from individual granulomas of LTBI controls at high (N = 9) or low risk (N = 7) of reactivation.

<p>T cells from granulomas were stimulated with ESAT-6 and CFP-10 peptides. The frequency of T cells producing only IL-10 (A), only IL-17 (B) or only IL-2 (C) was greater in granulomas from high-risk compared to low risk LTBI animals. No differences were observed in IFN- γ (D) or TNF production (E). Each symbol represents a granuloma. Statistics: Mann-Whitney.</p