Final Report Summary - TILIM (The T cell immune response against latent infection with Mycobacterium tuberculosis) There are approximately 9 million cases of active tuberculosis (TB) annually worldwide resulting in 1-2 million deaths per year (1). One third of the world’s population is infected with latent TB, which therefore constitutes the largest reservoir for TB. TB is a chronic infection of Mycobacterium tuberculosis (Mtb) and is crucially controlled by a T cell-mediated immune response in people and animal models. Latent infection is established via a constant and continuous balance between bacterial growth and immune control. Still, 5% of latently infected individuals TB will eventually re-activate, and it is believed that most new TB cases is indeed due to re-activation of latent TB. Interestingly, during the long interaction with the host, the immune response against Mtb antigens tends to focus on only a few dominant epitopes of the bacteria’s proteins. The idea for the project was that while a response against the dominant epitopes leads to partial protection, it is not enough to eradicate M.tb - resulting in a lifelong latent infection. Furthermore, protection provided by an immune response against dominant epitopes will wane with time, resulting in re-activation of TB and spreading of the disease. As a consequence, an immune response against only dominant epitopes within M.tb antigens is not enough to fully control the infection and stop the spread of the disease. Moreover, recent genetic analysis of Mtb found in TB patients around the world has shown that the same epitopes that are targeted by the host T cells during infection are actually highly conserved in the bacterial genome. This suggests that not only are T cells targeting dominant epitopes not fully protective, but the induction/activation of these T cells is actually beneficial to the bacteria (2). Therefore, we investigated the potential protection against tuberculosis disease via vaccination that induces T cell responses targeting ‘subdominant epitopes’ of well-defined Mtb proteins targeted during natural infection. Here ‘subdominant epitopes’ are defined as portions of the protein that are not targeted during Mtb infection alone. Our goal was to vaccinate animals already infected with Mtb to generate subdominant-specific T cell immune responses and assess the capacity of these cells to enhance the control of infection and disease. Furthermore, we wished to understand how these T cells would develop over the course of an ongoing Mtb infection and what unique capacity they may have that can enhance protection against infection/disease.Using overlapping peptides and truncated recombinant protein, we successfully identified subdominant epitopes of Mtb protein antigens ESAT-6 and TB10.4 in a mouse model system. Vaccine induction of subdominant epitope-specific T cells was influenced by MHC diversity, wherein the increased diversity of B6xBALB/c F1 mice allowed recognition of an additional subdominant epitope over the parental strains. Prophylactic (pre-infection) vaccination successfully induced subdominant epitope-specific T cells and enhanced protection against Mtb infection (equally well as dominant epitope vaccination). Post-exposure subdominant epitope vaccination in the Cornell model of latent TB infection did not enhance protection from re-activation of infection. However, this was determined to be due to a lack of significant induction of subdominant, and could not be attributed to a failure of subdominant epitope-specific T cells themselves.Prophylactic vaccine experiments allowed for tracking and characterization of the vaccine-induced subdominant-specific T cells over the course of chronic Mtb infection. Following Mtb-infection, vaccine-induced dominant epitope-specific T cells have an improved cytokine profile, displaying a memory-like polyfunctionality that is associated with protection, and display a less differentiated surface phenotype (KLRG1/PD-1). However, a significant proportion of these cells develop a terminally differentiated phenotype, typical of Mtb-driven exhaustion and ultimate loss of control of Mtb-infection. In contrast, vaccine-induced subdominant epitope-specific T cells maintained a significantly more memory-like polyfunctionality and reduced terminal differentiation during the same timecourse of infection. Importantly, this was true also true in mice that received vaccinations that induced both dominant and subdominant T cells – showing that the differential T cell development during infection was an intrinsic property of the subdominant T cell population during infection, and not a consequence of differential bacterial growth and disease development. Overall, we have found that protective subdominant epitope-specific T cells acquire a less differentiated phenotype and maintain a more memory-like cytokine profile over the course of Mtb infection. This has strong implications for considering the incorporation of subdominant epitopes in future vaccine development against TB and other chronic infections.References1. 2010. WHO global tuberculosis control report 2010. Summary. Central European journal of public health 18: 237.2. Comas, I., J. Chakravartti, P. M. Small, J. Galagan, S. Niemann, K. Kremer, J. D. Ernst, and S. Gagneux. 2010. Human T cell epitopes of Mycobacterium tuberculosis are evolutionarily hyperconserved. Nature genetics 42: 498-503.