The HyVac4 Subunit Vaccine Efficiently Boosts BCG-Primed Anti-Mycobacterial Protective Immunity

BACKGROUND: The current vaccine against tuberculosis (TB), BCG, has failed to control TB worldwide and the protective efficacy is moreover limited to 10-15 years. A vaccine that could efficiently boost a BCG-induced immune response and thus prolong protective immunity would therefore have a significant impact on the global TB-burden. METHODS/FINDINGS: In the present study we show that the fusion protein HyVac4 (H4), consisting of the mycobacterial antigens Ag85B and TB10.4, given in the adjuvant IC31® or DDA/MPL effectively boosted and prolonged immunity induced by BCG, leading to improved protection against infection with virulent M. tuberculosis (M.tb). Increased protection correlated with an increased percentage of TB10.4 specific IFNγ/TNFα/IL-2 or TNFα/IL-2 producing CD4 T cells at the site of infection. Moreover, this vaccine strategy did not compromise the use of ESAT-6 as an accurate correlate of disease development/vaccine efficacy. Indeed both CD4 and CD8 ESAT-6 specific T cells showed significant correlation with bacterial levels. CONCLUSIONS/SIGNIFICANCE: H4-IC31® can efficiently boost BCG-primed immunity leading to an increased protective anti-M.tb immune response dominated by IFNγ/TNFα/IL-2 or TNFα/IL2 producing CD4 T cells. H4 in the CD4 T cell inducing adjuvant IC31® is presently in clinical trials

The Mycobacterium tuberculosis Phagosome Is a HLA-I Processing Competent Organelle

Mycobacterium tuberculosis (Mtb) resides in a long-lived phagosomal compartment that resists maturation. The manner by which Mtb antigens are processed and presented on MHC Class I molecules is poorly understood. Using human dendritic cells and IFN-γ release by CD8+ T cell clones, we examined the processing and presentation pathway for two Mtb–derived antigens, each presented by a distinct HLA-I allele (HLA-Ia versus HLA-Ib). Presentation of both antigens is blocked by the retrotranslocation inhibitor exotoxin A. Inhibitor studies demonstrate that, after reaching the cytosol, both antigens require proteasomal degradation and TAP transport, but differ in the requirement for ER–golgi egress and new protein synthesis. Specifically, presentation by HLA-B8 but not HLA-E requires newly synthesized HLA-I and transport through the ER–golgi. Phenotypic analysis of the Mtb phagosome by flow organellometry revealed the presence of Class I and loading accessory molecules, including TAP and PDI. Furthermore, loaded HLA-I:peptide complexes are present within the Mtb phagosome, with a pronounced bias towards HLA-E:peptide complexes. In addition, protein analysis also reveals that HLA-E is enriched within the Mtb phagosome compared to HLA-A2. Together, these data suggest that the phagosome, through acquisition of ER–localized machinery and as a site of HLA-I loading, plays a vital role in the presentation of Mtb–derived antigens, similar to that described for presentation of latex bead-associated antigens. This is, to our knowledge, the first description of this presentation pathway for an intracellular pathogen. Moreover, these data suggest that HLA-E may play a unique role in the presentation of phagosomal antigens

Ag85-focused T-cell immune response controls Mycobacterium avium chronic infection

CD4+ T cells are essential players for the control of mycobacterial infections. Several mycobacterial antigens have been identified for eliciting a relevant CD4+ T cell mediated-immune response, and numerous studies explored this issue in the context of Mycobacterium tuberculosis infection. Antigen 85 (Ag85), a highly conserved protein across Mycobacterium species, is secreted at the early phase of M. tuberculosis infection leading to the proliferation of Ag85-specific CD4+ T cells. However, in the context of Mycobacterium avium infection, little is known about the expression of this antigen and the elicited immune response. In the current work, we investigated if a T cell receptor (TCR) repertoire mostly, but not exclusively, directed at Ag85 is sufficient to mount a protective immune response against M. avium. We show that P25 mice, whose majority of T cells express a transgenic TCR specific for Ag85, control M. avium infection at the same level as wild type (WT) mice up to 20 weeks post-infection (wpi). During M. avium infection, Ag85 antigen is easily detected in the liver of 20 wpi mice by immunohistochemistry. In spite of the propensity of P25 CD4+ T cells to produce higher amounts of interferon-gamma (IFNγ) upon ex vivo stimulation, no differences in serum IFNγ levels are detected in P25 compared to WT mice, nor enhanced immunopathology is detected in P25 mice. These results indicate that a T cell response dominated by Ag85-specific T cells is appropriate to control M. avium infection with no signs of immunopathology.This work was developed under the scope of the project NORTE-01-0145-FEDER-000013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). Fellowships from the Portuguese Foundation for Science and Technoloy (FCT) were attributed to BCR (SFRH/BD/80352/2011; QREN-POPH through the Fundo Social Europeu (FSE) and national funds from MEC] and to CN (SFRH/BPD/112001/2015; POPH through FSE and national funds from MCTES). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

The Duration of Antigen-Stimulation Significantly Alters the Diversity of Multifunctional CD4 T Cells Measured by Intracellular Cytokine Staining

The assessment of antigen-specific T cell responses by intracellular cytokine staining (ICS) has become a routine technique in studies of vaccination and immunity. Here, we highlight how the duration of in vitro antigen pre-stimulation, combined with the cytokine accumulation period, are critical parameters of these methods. The effect of varying these parameters upon the diversity and frequency of multifunctional CD4 T cell subsets has been investigated using a murine model of TB vaccination and in cattle naturally infected with Mycobacterium bovis. We demonstrate a substantial influence of the duration of the antigen pre-stimulation period on the repertoire of the antigen-specific CD4 T cell responses. Increasing pre-stimulation from 2 to 6 hours amplified the diversity of the seven potential multifunctional CD4 T cell subsets that secreted any combination of IFN-γ, IL-2 and TNF-α. However, increasing pre-stimulation from 6 to 16 hours markedly altered the multifunctional CD4 T cell repertoire to a dominant IFN-γ+ only response. This was observed in both murine and cattle models

Human and murine clonal CD8+ T cell expansions arise during tuberculosis because of TCR selection

The immune system can recognize virtually any antigen, yet T cell responses against several pathogens, including Mycobacterium tuberculosis, are restricted to a limited number of immunodominant epitopes. The host factors that affect immunodominance are incompletely understood. Whether immunodominant epitopes elicit protective CD8+ T cell responses or instead act as decoys to subvert immunity and allow pathogens to establish chronic infection is unknown. Here we show that anatomically distinct human granulomas contain clonally expanded CD8+ T cells with overlapping T cell receptor (TCR) repertoires. Similarly, the murine CD8+ T cell response against M. tuberculosis is dominated by TB10.44-11-specific T cells with extreme TCRß bias. Using a retro genic model of TB10.44-11-specific CD8+ Tcells, we show that TCR dominance can arise because of competition between clonotypes driven by differences in affinity. Finally, we demonstrate that TB10.4-specific CD8+ T cells mediate protection against tuberculosis, which requires interferon-? production and TAP1-dependent antigen presentation in vivo. Our study of how immunodominance, biased TCR repertoires, and protection are inter-related, provides a new way to measure the quality of T cell immunity, which if applied to vaccine evaluation, could enhance our understanding of how to elicit protective T cell immunity.This work was supported by the Portuguese Foundation for Science and Technology individual fellowship (CNA) www.fct.pt, a National Institutes of Health Grant R01 AI106725 (SMB) www.nih.gov, and a Center for AIDS Research Grant P30 AI 060354 (SMB) www.nih.gov. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

A Critical Role for CD8 T Cells in a Nonhuman Primate Model of Tuberculosis

The role of CD8 T cells in anti-tuberculosis immunity in humans remains unknown, and studies of CD8 T cell–mediated protection against tuberculosis in mice have yielded controversial results. Unlike mice, humans and nonhuman primates share a number of important features of the immune system that relate directly to the specificity and functions of CD8 T cells, such as the expression of group 1 CD1 proteins that are capable of presenting Mycobacterium tuberculosis lipids antigens and the cytotoxic/bactericidal protein granulysin. Employing a more relevant nonhuman primate model of human tuberculosis, we examined the contribution of BCG- or M. tuberculosis-elicited CD8 T cells to vaccine-induced immunity against tuberculosis. CD8 depletion compromised BCG vaccine-induced immune control of M. tuberculosis replication in the vaccinated rhesus macaques. Depletion of CD8 T cells in BCG-vaccinated rhesus macaques led to a significant decrease in the vaccine-induced immunity against tuberculosis. Consistently, depletion of CD8 T cells in rhesus macaques that had been previously infected with M. tuberculosis and cured by antibiotic therapy also resulted in a loss of anti-tuberculosis immunity upon M. tuberculosis re-infection. The current study demonstrates a major role for CD8 T cells in anti-tuberculosis immunity, and supports the view that CD8 T cells should be included in strategies for development of new tuberculosis vaccines and immunotherapeutics