Abstract Human cytotoxic lymphocytes kill infected cells through release of lytic proteins contained in cytotoxic granules. The cytotoxic granules of human NK and CD8 T cells contain perforin, granzyme and granulysin (GNLY), and GNLY has direct antimicrobial activity against Mycobacterium tuberculosis (Mtb). Murine studies suggest that CD8 T cells have only a modest role in immunity to tuberculosis (TB). We hypothesize that mouse CD8 T cells are ineffective at containing Mycobacterium tuberculosis (Mtb) as they don’t express GNLY. To test this hypothesis, GNLY-transgenic mice that express human granulysin protein under the control of human regulatory elements were infected with Mtb. GNLY-Tg and non-tg control mice control Mtb similarly. There was no difference in the ability of GNY-Tg and non-tg CD8 T cells to transfer protection to T cell deficient mice. Anti-CD3 stimulation led to expression of granulysin in human CD8 T cells, but not in murine CD8 T cells. However, after Mtb infection, GNLY was expressed in NK cells but not CD8 T cells. We conclude that GNLY-expressing NK cells do not alter resistance to Mtb. Importantly, GNLY-Tg mice are inadequate model to study CD8 function during TB. We are developing a new strategy to express granulysin in CD8 T cells. We expressed GNLY (isoform 2) in primary murine CD8 T cells by retroviral gene transfer and detected GNLY expression in 60% of CD8 T cells. Future directions We are assessing the cytolytic activity of GNLY-expressing CD8 T cells and their ability to kill intracellular pathogens in vitro and in vivo. We expect to develop a new GNLY-Tg model that can be used to study the function of CD8 T cells and granulysin in many different diseases. This work is supported by NIH AI159374. This work is supported by NIH grant R21 AI159374.

There is growing evidence that genetic diversity in Mycobacterium tuberculosis, the causative agent of tuberculosis, contributes to the outcomes of infection and public health interventions, such as vaccination. Epidemiological studies suggest that among the phylogeographic lineages of M. tuberculosis, strains belonging to a sublineage of Lineage 2 (mL2) are associated with concerning clinical features, including hypervirulence, treatment failure, and vaccine escape. The global expansion and increasing prevalence of this sublineage has been attributed to the selective advantage conferred by these characteristics, yet confounding host and environmental factors make it difficult to identify the bacterial determinants driving these associations in human studies. Here, we developed a molecular barcoding strategy to facilitate high-throughput, experimental phenotyping of M. tuberculosis clinical isolates. This approach allowed us to characterize growth dynamics for a panel of genetically diverse M. tuberculosis strains during infection and after vaccination in the mouse model. We found that mL2 strains exhibit distinct growth dynamics in vivo and are resistant to the immune protection conferred by Bacillus Calmette-Guerin (BCG) vaccination. The latter finding corroborates epidemiological observations and demonstrates that mycobacterial features contribute to vaccine efficacy. To investigate the genetic and biological basis of mL2 strains' distinctive phenotypes, we performed variant analysis, transcriptional studies, and genome-wide transposon sequencing. We identified functional genetic changes across multiple stress and host response pathways in a representative mL2 strain that are associated with variants in regulatory genes. These adaptive changes may underlie the distinct clinical characteristics and epidemiological success of this lineage. IMPORTANCE Tuberculosis, caused by the bacterium Mycobacterium tuberculosis, is a remarkably heterogeneous disease, a feature that complicates clinical care and public health interventions. The contributions of pathogen genetic diversity to this heterogeneity are uncertain, in part due to the challenges of experimentally manipulating M. tuberculosis, a slow-growing, biosafety level 3 organism. To overcome these challenges, we applied a molecular barcoding strategy to a panel of M. tuberculosis clinical isolates. This novel application of barcoding permitted the high-throughput characterization of M. tuberculosis strain growth dynamics and vaccine resistance in the mouse model of infection. Integrating these results with genomic analyses, we uncover bacterial pathways that contribute to infection outcomes, suggesting targets for improved therapeutics and vaccines.

Human CD8 T cells express the antimicrobial peptide granulysin in their cytotoxic granules, andin vitroanalysis suggest that it restricts growth ofMycobacterium tuberculosisand other intracellular pathogens. The murine model of tuberculosis cannot assess granulysin’s rolein vivo, as rodents lack the granulysin gene.

CD8 T cells provide limited protection against Mycobacterium tuberculosis (Mtb) infection in the mouse model. As Mtb causes chronic infection in mice and humans, we hypothesize that Mtb impairs T cell responses as an immune evasion strategy. TB10.4 is an immunodominant antigen in people, nonhuman primates, and mice, which is encoded by the esxH gene. In C57BL/6 mice, 30-50% of pulmonary CD8 T cells recognize the TB10.44-11 epitope. However, TB10.4-specific CD8 T cells fail to recognize Mtb-infected macrophages. We speculate that Mtb elicits immunodominant CD8 T cell responses to antigens that are inefficiently presented by infected cells, thereby focusing CD8 T cells on nonprotective antigens. Here, we leverage naturally occurring polymorphisms in esxH, which frequently occur in lineage 1 strains, to test this "decoy hypothesis". Using the clinical isolate 667, which contains an EsxHA10T polymorphism, we observe a drastic change in the hierarchy of CD8 T cells. Using isogenic Erd.EsxHA10T and Erd.EsxHWT strains, we prove that this polymorphism alters the hierarchy of immunodominant CD8 T cell responses. Our data are best explained by immunodomination, a mechanism by which competition for APC leads to dominant responses suppressing subdominant responses. These results were surprising as the variant epitope can bind to H2-Kb and is recognized by TB10.4-specific CD8 T cells. The dramatic change in TB10.4-specific CD8 responses resulted from increased proteolytic degradation of A10T variant, which destroyed the TB10.44-11epitope. Importantly, this polymorphism affected T cell priming and recognition of infected cells. These data support a model in which nonprotective CD8 T cells become immunodominant and suppress subdominant responses. Thus, polymorphisms between clinical Mtb strains, and BCG or H37Rv sequence-based vaccines could lead to a mismatch between T cells that are primed by vaccines and the epitopes presented by infected cells. Reprograming host immune responses should be considered in the future design of vaccines.

Immune responses following Mycobacterium tuberculosis (Mtb) infection or vaccination are frequently assessed by measuring T-cell recognition of crude Mtb antigens, recombinant proteins, or peptide epitopes. We previously showed that not all Mtb-specific T cells recognize Mtb-infected macrophages. Thus, an important question is what proportion of T cells elicited by Mtb infection recognize Mtb-infected macrophages. We address this question by developing a modified elispot assay using viable Mtb-infected macrophages, a low multiplicity of infection and purified T cells. In C57BL/6 mice, CD4 and CD8 T cells were classically MHC restricted. Comparable frequencies of T cells that recognize Mtb-infected macrophages were determined using interferon-γ elispot and intracellular cytokine staining, and lung CD4 T cells more sensitively recognized Mtb-infected macrophages than lung CD8 T cells. Compared to the relatively high frequencies of T cells specific for antigens such as ESAT-6 and TB10.4, low frequencies of total pulmonary T cells elicited by aerosolized Mtb infection recognize Mtb-infected macrophages. Finally, we demonstrate that BCG vaccination elicits T cells that recognize Mtb-infected macrophages. We propose that the frequency of T cells that recognize infected macrophages could correlate with protective immunity and may be an alternative approach to measuring T-cell responses to Mtb antigens.

Containment of Mycobacterium tuberculosis (Mtb) infection requires T cell recognition of infected macrophages. Mtb has evolved to tolerate, evade, and subvert host immunity. Despite a vigorous and sustained CD8(+) T cell response during Mtb infection, CD8(+) T cells make limited contribution to protection. Here, we ask whether the ability of Mtb-specific T cells to restrict Mtb growth is related to their capacity to recognize Mtb-infected macrophages. We derived CD8(+) T cell lines that recognized the Mtb immunodominant epitope TB10.4(4-11) and compared them to CD4(+) T cell lines that recognized Ag85b(240-254) or ESAT6(3-17). While the CD4(+) T cells recognized Mtb-infected macrophages and inhibited Mtb growth in vitro, the TB10.4-specific CD8(+) T cells neither recognized Mtb-infected macrophages nor restricted Mtb growth. TB10.4-specific CD8(+) T cells recognized macrophages infected with Listeria monocytogenes expressing TB10.4. However, over-expression of TB10.4 in Mtb did not confer recognition by TB10.4-specific CD8(+) T cells. CD8(+) T cells recognized macrophages pulsed with irradiated Mtb, indicating that macrophages can efficiently cross-present the TB10.4 protein and raising the possibility that viable bacilli might suppress cross-presentation. Importantly, polyclonal CD8(+) T cells specific for Mtb antigens other than TB10.4 recognized Mtb-infected macrophages in a MHC-restricted manner. As TB10.4 elicits a dominant CD8(+) T cell response that poorly recognizes Mtb-infected macrophages, we propose that TB10.4 acts as a decoy antigen. Moreover, it appears that this response overshadows subdominant CD8(+) T cell response that can recognize Mtb-infected macrophages. The ability of Mtb to subvert the CD8(+) T cell response may explain why CD8(+) T cells make a disproportionately small contribution to host defense compared to CD4(+) T cells. The selection of Mtb antigens for vaccines has focused on antigens that generate immunodominant responses. We propose that establishing whether vaccine-elicited, Mtb-specific T cells recognize Mtb-infected macrophages could be a useful criterion for preclinical vaccine development.

Mycobacterium tuberculosis (Mtb), the cause of tuberculosis (TB), is an intracellular bacterium that evades antibacterial immunity. As control of Mtb requires T cell immunity, most vaccine strategies focus on eliciting protective T cells. How Mtb benefits from promoting T cell activation or evading T cell recognition is not certain. Here, we took advantage of a naturally occurring Mtb polymorphism to address this question in mouse model. EsxH elicits an immunodominant CD8 T cell response. At the peak of immunity against Mtb in C57BL/6 mice, 30–50% of CD8 T cells are specific for EsxH4–11. Clinical Isolate 667 (CI667) is an Mtb strain containing a polymorphism in EsxH that has a single amino acid change of WT EsxH4–11 epitope from IMYNYPAM into IMYNYPTM (A10T). We hypothesized that this naturally occurring polymorphism would alter the hierarchy of CD8 T cell responses elicited by Mtb. Following Erdman (WT) Mtb infection of C57BL/6 mice, CD8 T cells respond to three distinct epitopes, including the reported immunodominant EsxH4–11 epitope. In contrast, there was no response to any of EsxH epitopes after CI667 infection. Instead, the subdominant response to the 32C antigen became dominant. In BALB/c mice, CI667 stimulated a reduced but detectable CD8 response to EsxH20–28; the CD4 response to EsxH74–88 was similar to WT Mtb infection. These results show that naturally occurring polymorphisms can have drastic effects on T cell immunity. We hypothesize that EsxH acts as a decoy antigen, and distracts the CD8 response from focusing on antigens that are presented by infected macrophages and protective. We are in the process of testing this hypothesis by developing isogenic Mtb strains that express EsxHErdman or EsxHCI667.

The goal of most vaccines is the induction of long-lived memory T and B cells capable of protecting the host from infection by cytotoxic mechanisms, cytokines and high-affinity antibodies. However, efforts to develop vaccines against major human pathogens such as HIV and HCV have not been successful, thereby highlighting the need for novel approaches to circumvent immunoregulatory mechanisms that limit the induction of protective immunity. Here, we show that mouse natural killer (NK) cells inhibit generation of long-lived virus-specific memory T- and B cells as well as virus-specific antibody production after acute infection. Mechanistically, NK cells suppressed CD4 T cells and follicular helper T cells (T-FH) in a perforin-dependent manner during the first few days of infection, resulting in a weaker germinal centre (GC) response and diminished immune memory. We anticipate that innovative strategies to relieve NK cell-mediated suppression of immunity should facilitate development of efficacious new vaccines targeting difficult-to-prevent infections.

Epstein-Barr virus (EBV), an oncogenic gammaherpesvirus, causes acute infectious mononucleosis (AIM) and is linked to the development of several human malignancies. There is an urgent need for a vaccine that is safe, prevents infection and/or limits disease. Unique among human herpesviruses, glycoprotein (gp)350/220, which initiates EBV attachment to susceptible host cells, is the major ligand on the EBV envelope and is highly conserved. Interaction between gp350/220 and complement receptor type 2 (CR2)/CD21 and/or (CR1)/CD35 on B-cells is required for infection. Potent antibody responses to gp350/220 occur in animal models and humans. Thus, gp350/220 provides an attractive candidate for prophylactic subunit vaccine development. However, in a recent Phase II clinical trial immunization with soluble recombinant gp350 reduced the incidence of AIM, but did not prevent infection. Despite various attempts to produce an EBV vaccine, no vaccine is licensed. Herein we describe a sub-unit vaccine against EBV based on a novel Newcastle disease virus (NDV)-virus-like particle (VLP) platform consisting of EBVgp350/220 ectodomain fused to NDV-fusion (F) protein. The chimeric protein EBVgp350/220-F is incorporated into the membrane of a VLP composed of the NDV matrix and nucleoprotein. The particles resemble native EBV in diameter and shape and bind CD21 and CD35. Immunization of BALB/c mice with EBVgp350/220-F VLPs elicited strong, long-lasting neutralizing antibody responses when assessed in vitro. This chimeric VLP is predicted to provide a superior safety profile as it is efficiently produced in Chinese hamster ovary (CHO) cells using a platform devoid of human nucleic acid and EBV-transforming genes.

Abstract Pathogen-specific neutralizing antibodies (nAbs) are a key component of protective immunity after infection or vaccination. However, broadly-specific nAbs that can prevent infections with highly mutable viruses like HIV are rarely seen in infected patients and are poorly elicited by current vaccines. Therefore, new vaccine strategies are needed to enhance the induction of nAbs, potentially by augmenting the germinal center (GC) reactions that facilitate somatic hypermutation, affinity maturation and development of high-affinity nAbs. Here, we demonstrate that natural killer (NK) cells impair humoral immunity by contributing to a weak GC response and subdued generation of virus-specific nAbs after acute lymphocytic choriomeningitis virus (LCMV) infection of mice. The magnitude and duration of the LCMV-induced GC response was substantially reduced by NK cells, which constricted the number of both follicular helper T cells (Tfh) and GC B cells present in lymphoid tissues. NK cell-mediated suppression of GC responses was also observed in different stains of mice and after infection with a variety of dissimilar pathogens, suggesting that NK cell suppression of humoral immunity is a universal feature of infection. Notably, NK cell inhibition of the GC response during LCMV infection resulted in a markedly delayed and relatively weak nAb response. Thus, targeting of immunosuppressive NK cells could represent a revolutionary method to enhance the efficacy of future vaccine regimens.