Natural Polymorphism of Mycobacterium tuberculosis and CD8 T Cell Immunity

Coevolution between Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, and the human host has been documented for thousands of years. Interestingly, while T cell immunity is crucial for host protection and survival, T cell antigens are the most conserved region of the Mtb genome. Hypothetically, Mtb adapts under immune pressure to exploit T cell responses for its benefit from inflammation and tissue destruction for ultimately transmission. EsxH, a gene encoding immunodominant TB10.4 protein, however, contains polymorphic regions corresponding to T cell epitopes. Here, I present two complementary analyses to examine how Mtb modulates TB10.4 for immune evasion. First, I use a naturally occurring esxH polymorphic clinical Mtb isolate, 667, to investigate how A10T amino acid exchange in TB10.4 affect T cell immunity. To verify and identify the cause of the immunological differences, I construct isogenic strains expressing EsxHA10T or EsxHWT. In combination with our recent finding that TB10.44-11-specific CD8 T cells do not recognize Mtb-infected macrophages, we hypothesize that TB10.4 is a decoy antigen as it distracts host immunity from inducing other potentially protective responses. I examine whether an elimination of TB10.44-11-specific CD8 T cell response leads to a better host protective immunity. The studies of in vivo infection and in vitro recognition in this dissertation aim to provide a better understanding of the counteraction between immune evasion and protective immunity

A natural polymorphism of Mycobacterium tuberculosis in the esxH gene disrupts immunodomination by the TB10.4-specific CD8 T cell response

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

Mycobacterium tuberculosis-specific CD4+ and CD8+ T cells differ in their capacity to recognize infected macrophages

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 TB10.44-11 and compared them to CD4+ T cell lines that recognized Ag85b240-254 or ESAT63-17. 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

A chimeric EBV gp350/220-based VLP replicates the virion B-cell attachment mechanism and elicits long-lasting neutralizing antibodies in mice

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

Generation of cellular immune memory and B-cell immunity is impaired by natural killer cells.

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 like HIV and HCV have not been successful, thereby highlighting the need for novel approaches to circumvent immunoregulatory mechanisms that limit 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 center (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

Probing potential mechanisms for lack of recognition by TB10Rg3 T cells.

<p>(a-c) EsxH (TB10.4) and its partner EsxG were overexpressed together in H37Rv to determine whether increasing TB10.4 abundance would lead to recognition of infected macrophages (esxGH-OE.Mtb). (a) Tetracycline treatment of esxGH-OE.Mtb in broth culture induces protein expression of EsxH (TB10.4) as measured by western blot. A different strain, fbpB-OE.Mtb, in which fbpB (e.g., Ag85b) is induction by tetracycline, does not result in greater EsxH (TB10.4) expression. Protein signal was normalized to that of GroEL2, a chaperonin protein. (b) Tetracycline treatment of esxGH-OE.Mtb in broth culture induces esxG and esxH, but not fbpB (which encodes for Ag85b) and sigA (which encodes for RNA polymerase factor sigma A), mRNA as measured by qPCR. Fold-induction was normalized to baseline (i.e., uninduced). IFNγ production by (c) P25 or (d) TB10Rg3 T cells after co-culture with macrophages infected with uninduced or induced esxGH-OE.Mtb. (e-h) MHC class I and II expression by Mtb infected-macrophages. Representative histograms (e) and fold-change (f) of MHC class I or representative histograms (g) and fold-change (h) of MHC class II expression on infected cells. (i) P25 and (j) TB10Rg3 production of IFNγ after co-culture with macrophages pulsed with titrated amounts of γ-irradiated (non-viable) H37Rv. Data is representative of 3 experiments. Statistical testing by one-way ANOVA with Dunnett posttest. *, p<0.05; **, p<0.01; and ***, p<0.005.</p

Polyclonal CD8⁺ T cells from the lungs of Mtb-infected mice recognize infected macrophages.

<p>IFNγ production by polyclonal CD4⁺ (a) or CD8⁺ (b) T cells after co-culture with either MHC-matched (H-2^b) or MHC-mismatched (H-2^k), Mtb-infected macrophages. IFNγ production by TB10.4₄₋₁₁-tetramer-depleted (c) or tetramer-enriched (d) polyclonal CD8⁺ T cells after co-culture with either MHC-matched (H-2^b) or MHC-mismatched (H-2^k), Mtb-infected macrophages. Data is representative of at least 2 experiments. Statistical testing by a two-tailed, unpaired Student’s T test. *, p<0.05; **, p<0.01; and ***, p<0.005.</p

TB10Rg3 and P25 T cells can recognize macrophages infected with <i>Listeria monocytogenes</i> expressing TB10.4 and Ag85b proteins, respectively.

<p>(a) Representative flow plots showing Nur77 induction by TB10Rg3 T cells after co-culture with macrophages infected with △ActA.TB10 (top row) or △LLO.TB10 (bottom row) Listeria. (b) Analysis of the frequency of Nur77-expressing TB10Rg3 T cells (b, d), or normalized MFI (c, e), after co-culture with △ActA.TB10 (b, c) or △LLO.TB10 (d, e) infected macrophages. (f) Representative flow plots showing Nur77 induction by P25 population after co-culture with macrophages infected with △ActA.TB10 (top row) or △LLO.TB10 (bottom row) Listeria. (g) Analysis of the frequency of Nur77-expressing P25 T cells and (h) normalized MFI of P25 T cells. Representative of at least two experiments. Statistical testing by one-way ANOVA with Dunnett posttest. *, p<0.05; **, p<0.01; and ***, p<0.005.</p

TB10Rg3 CD8⁺ T cells do not recognize lung APCs from infected mice.

<p>(a-d) T cell proliferation after coculture with lung APC from infected mice, with or without cognate peptide, or uninfected TGPM, based on eFluor450 fluorescence dilution after 72 hours. Representative flow plot (a) and quantification (b) of C7 T cell proliferation. Representative flow plot (c) and quantification (d) of TB10Rg3 T cell proliferation. (e) Bacterial burden in the lung APCs during <i>in vitro</i> culture over the course of the experiment in the absence of T cells. Representative of 4 (TB10Rg3) or 2 (C7) experiments.</p

TB10.4-specific CD8⁺ (TB10Rg3) and Ag85b-specific CD4⁺ (P25) T cells both recognize their cognate peptides.

<p>(a) Representative histogram of Nur77 expression in P25 T cells after 2 hours of co-culture with macrophages and (b) time course of Nur77 MFI in P25 T cells. (c) Representative histogram of CD69 in P25 T cells after 2 hours of co-culture with macrophages and (d) time course of CD69 MFI in P25 T cells. (e) Representative histogram of Nur77 in TB10Rg3 T cells after 2 hours of co-culture and (f) time course of Nur77 MFI in TB10Rg3 T cells. (g) Representative histogram of CD69 in TB10Rg3 T cells at 2 hours of co-culture with macrophages and (h) time course of CD69 MFI in TB10Rg3 T cells. (i) CD69 MFI and (j) IFNγ production by P25 T cells after 72 hours of co-culture. (k) CD69 MFI and (l) IFNγ production by TB10Rg3 cells after 72 hours of co-culture. MFI, mean fluorescence intensity; mφ, macrophage.</p