Persistent antigen stimulation in chronic infections has been associated with antigen-specific T cell dysfunction and upregulation of inhibitory receptors, including programmed cell death protein 1 (PD-1). Pulmonary tuberculosis (TB) disease is characterized by high levels of Mycobacterium tuberculosis (Mtb), yet the relationship between bacterial load, PD-1 expression, and Mtb-specific T cell function in human TB has not been well-defined. Using peripheral blood samples from adults with LTBI and with pulmonary TB disease, we tested the hypothesis that PD-1 expression is associated with bacterial load and functional capacity of Mtb-specific T cell responses. We found that PD-1 was expressed at significantly higher levels on Th1 cytokine-producing Mtb-specific CD4 T cells from patients with smear-positive TB, compared with smear-negative TB and LTBI, which decreased after completion of anti-TB treatment. By contrast, expression of PD-1 on Mtb-specific CD8 T cells was significantly lower than on Mtb-specific CD4 T cells and did not differ by Mtb infection and disease status. In vitro stimulation of PBMC with Mtb antigens demonstrated that PD-1 is induced on proliferating Mtb-specific CD4 T cells and that Th1 cytokine production capacity is preferentially maintained within PD-1+ proliferating CD4 T cells, compared with proliferating Mtb-specific CD4 T cells that lack PD-1 expression. Together, these data indicate that expression of PD-1 on Mtb-specific CD4 T cells is indicative of mycobacterial antigen exposure and identifies a population of effector cells with Th1 cytokine production capacity. These studies provide novel insights into the role of the PD-1 pathway in regulating CD4 and CD8 T cell responses in Mtb infection and provide rationale for future studies to evaluate PD-1 expression on antigen-specific CD4 T cells as a potential biomarker for bacterial load and treatment response in human TB.

We previously reported that a recombinant pantothenate auxotroph of Mycobacterium bovis BCG expressing human immunodeficiency virus type 1 (HIV-1) subtype C Gag (rBCGpan-Gag) efficiently primes the mouse immune system for a boost with a recombinant modified vaccinia virus Ankara (rMVA) vaccine. In this study, we further evaluated the immunogenicity of rBCGpan-Gag in a nonhuman primate model. Two groups of chacma baboons were primed or mock primed twice with either rBCGpan-Gag or a control BCG. Both groups were boosted with HIV-1 Pr55 gag virus-like particles (Gag VLPs). The magnitude and breadth of HIV-specific cellular responses were measured using a gamma interferon (IFN-␥) enzyme-linked immunosorbent spot (ELISPOT) assay, and the cytokine profiles and memory phenotypes of T cells were evaluated by polychromatic flow cytometry. Gag-specific responses were detected in all animals after the second inoculation with rBCGpan-Gag. Boosting with Gag VLPs significantly increased the magnitude and breadth of the responses in the baboons that were primed with rBCGpan-Gag. These responses targeted an average of 12 Gag peptides per animal, compared to an average of 3 peptides per animal for the mockprimed controls. Robust responses of Gag-specific polyfunctional T cells capable of simultaneously producing IFN-␥, tumor necrosis alpha (TNF-␣), and interleukin-2 (IL-2) were detected in the rBCGpan-Gag-primed animals. Gag-specific memory T cells were skewed toward a central memory phenotype in both CD4؉ and CD8 ؉ T cell populations. These data show that the rBCGpan-Gag prime and Gag VLP boost vaccine regimen is highly immunogenic, inducing a broad and polyfunctional central memory T cell response. This report further indicates the feasibility of developing a BCG-based HIV vaccine that is safe for childhood HIV immunization.

Human immunodeficiency virus infection leads to lower frequencies of CD4+ T cells specific for Mycobacterium tuberculosis in blood and lungs. However, an influx of T cells to the lungs results in similar absolute numbers of specific CD4+ T cells, compared with uninfected individuals.

41HIV-1 infection substantially increases the risk of developing tuberculosis (TB). Some 42 mechanisms, such as defects in the Th1 response to Mycobacterium tuberculosis (M.tb) in 43HIV-infected individuals have been widely reported. However, Th1-independent mechanisms 44 also contribute to protection against TB. To identify a broader spectrum of defects in TB 45 immunity during HIV infection, we examined IL-17 and IL-22 production in response to 46 mycobacterial antigens in individuals with latent TB infection (LTBI) and HIV co-infection. 47Upon stimulating with mycobacterial antigens, we observed a distinct CD4+ T helper lineage 48 producing IL-22 in the absence of IL-17 and IFN-γ. Th22 cells were present at high 49 frequencies in response to mycobacterial antigens in blood and contributed up to 50% to the 50 CD4+ T cell response to mycobacteria, comparable in magnitude to the IFN-γ Th1 response 51 (median 0.91% and 0.55%, respectively). Phenotypic characterization of Th22 cells revealed 52 that their memory differentiation was similar to M.tb-specific Th1 cells (i.e. predominantly 53 early-differentiated CD45RO+CD27+ phenotype). Moreover, CCR6 and CXCR3 expression 54 profiles of Th22 cells were similar to Th17 cells, while their CCR4 and CCR10 expression 55 patterns displayed an intermediate phenotype between Th1 and Th17 cells. Strikingly, 56 mycobacterial IL-22 responses were three-fold lower in HIV-infected individuals compared 57 to uninfected individuals, and the magnitude of responses correlated inversely with HIV viral 58 load. These data provide important insights into mycobacteria-specific T helper subsets and 59 suggest a potential role for IL-22 in protection against TB during HIV infection. Further 60 studies are needed to fully elucidate the role of IL-22 in protective TB immunity. 61 4 INTRODUCTION 62

Poxvirus vectors represent promising human immunodeficiency virus (HIV) vaccine candidates and were a component of the only successful HIV vaccine efficacy trial to date. We tested the immunogenicity of a novel recombinant capripoxvirus vector, lumpy skin disease virus (LSDV), in combination with modified vaccinia Ankara (MVA), both expressing genes from HIV-1. Here, we demonstrated that the combination regimen was immunogenic in rhesus macaques, inducing high-magnitude, broad and balanced CD4 + and CD8 + T-cell responses, and transient activation of the immune response. These studies support further development of LSDV as a vaccine vector.