Considerable effort has been directed toward controlling tuberculosis, which kills almost two million people yearly. High on the research agenda is the discovery of biomarkers of active tuberculosis (TB) for diagnosis and for monitoring treatment outcome. Rational biomarker discovery requires understanding host-pathogen interactions leading to biomarker expression. Here we report a systems immunology approach integrating clinical data and bacterial metabolic and regulatory information with high-throughput detection in human serum of antibodies to the entire Mycobacterium tuberculosis proteome. Sera from worldwide TB suspects recognized approximately 10% of the bacterial proteome. This result defines the M. tuberculosis immunoproteome, which is rich in membraneassociated and extracellular proteins. Additional analyses revealed that during active tuberculosis (i) antibody responses focused on an approximately 0.5% of the proteome enriched for extracellular proteins, (ii) relative target preference varied among patients, and (iii) responses correlated with bacillary burden. These results indicate that the B cell response tracks the evolution of infection and the pathogen burden and replicative state and suggest functions associated with B cell-rich foci seen in tuberculous lung granulomas. Our integrated proteome-scale approach is applicable to other chronic infections characterized by diverse antibody target recognition.biomarkers | humoral immunity | protein microarray | granuloma

Suppression of major histocompatibility complex (MHC) class II antigen presentation is believed to be among the major mechanisms used by Mycobacterium tuberculosis to escape protective host immune responses. Through a genome-wide screen for the genetic loci of M. tuberculosis that inhibit MHC class II-restricted antigen presentation by mycobacteria-infected dendritic cells, we identified the PE_PGRS47 protein as one of the responsible factors. Targeted disruption of the PE_PGRS47 (Rv2741) gene led to attenuated growth of M. tuberculosis in vitro and in vivo, and a PE_PGRS47 mutant showed enhanced MHC class II-restricted antigen presentation during in vivo infection of mice. Analysis of the effects of deletion or over-expression of PE_PGRS47 implicated this protein in the inhibition of autophagy in infected host phagocytes. Our findings identify PE_PGRS47 as a functionally relevant, non-redundant bacterial factor in the modulation of innate and adaptive immunity by M. tuberculosis, suggesting strategies for improving antigen presentation and the generation of protective immunity during vaccination or infection.

We document the neuropathologic findings of a 73-year old man who died from acute cerebellar hemorrhage in the context of relatively mild SARS-CoV2 infection. The patient developed sudden onset of headache, nausea, and vomiting, immediately followed by loss of consciousness on the day of admission. Emergency medical services found him severely hypoxemic at home, and the patient suffered a cardiac arrest during transport to the emergency department. The emergency team achieved return of spontaneous circulation after over 17 min of resuscitation. A chest radiograph revealed hazy bilateral opacities; and real-time-PCR for SARS-CoV-2 on the nasopharyngeal swab was positive. Computed tomography of the head showed a large right cerebellar hemorrhage, with tonsillar herniation and intraventricular hemorrhage. One day after presentation, he was transitioned to comfort care and died shortly after palliative extubation. Autopsy performed 3 h after death showed cerebellar hemorrhage and acute infarcts in the dorsal pons and medulla. Remarkably, there were microglial nodules and neuronophagia bilaterally in the inferior olives and multifocally in the cerebellar dentate nuclei. This constellation of findings has not been reported thus far in the context of SARS-CoV-2 infection.

SummaryMany hematopoietic cell types express CD1d and are capable of presenting glycolipid antigens to invariant natural killer T cells (iNKT cells). However, the question of which cells are the principal presenters of glycolipid antigens in vivo remains controversial, and it has been suggested that this might vary depending on the structure of a particular glycolipid antigen. Here we have shown that a single type of cell, the CD8α+ DEC-205+ dendritic cell, was mainly responsible for capturing and presenting a variety of different glycolipid antigens, including multiple forms of α-galactosylceramide that stimulate widely divergent cytokine responses. After glycolipid presentation, these dendritic cells rapidly altered their expression of various costimulatory and coinhibitory molecules in a manner that was dependent on the structure of the antigen. These findings show flexibility in the outcome of two-way communication between CD8α+ dendritic cells and iNKT cells, providing a mechanism for biasing toward either proinflammatory or anti-inflammatory responses.

SUMMARY Infection with Mycobacterium tuberculosis causes a variety of clinical conditions ranging from life-long asymptomatic infection to overt disease with increasingly severe tissue damage and a heavy bacillary burden. Immune biomarkers should follow the evolution of infection and disease because the host immune response is at the core of protection against disease and tissue damage in M. tuberculosis infection. Moreover, levels of immune markers are often affected by the antigen load. We review how the clinical spectrum of M. tuberculosis infection correlates with the evolution of granulomatous lesions and how granuloma structural changes are reflected in the peripheral circulation. We also discuss how antigen-specific, peripheral immune responses change during infection and how these changes are associated with the physiology of the tubercle bacillus. We propose that a dynamic approach to immune biomarker research should overcome the challenges of identifying those asymptomatic and symptomatic stages of infection that require antituberculosis treatment. Implementation of such a view requires longitudinal studies and a systems immunology approach leading to multianalyte assays.

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Analysis of antigen-specific CD4+ T cells in mycobacterial infections at the transcriptome level is informative but technically challenging. While several methods exist for identifying antigen-specific T cells, including intracellular cytokine staining, cell surface cytokine-capture assays, and staining with peptide:MHC-II multimers, all of these have significant technical constraints that limit their usefulness. Measurement of activation-induced expression of CD154 has been reported to detect live, antigen-specific CD4+ T cells but this approach remains underexplored, and to our knowledge has not previously been applied in mycobacteria-infected animals. Here we show that CD154 expression identifies adoptively transferred or endogenous antigen-specific CD4+ T cells induced by Mycobacterium bovis Bacillus Calmette-Guérin (BCG) vaccination. We confirmed that antigen-specific cytokine production was positively correlated with CD154 expression by CD4+ T cells from BCG-vaccinated mice, and show that high quality microarrays can be performed from RNA isolated from CD154+ cells purified by cell sorting. Analysis of microarray data demonstrated that the transcriptome of the CD4+ CD154+ cells was distinct from that of CD154− cells, and showed major enrichment of transcripts encoding multiple cytokines and pathways of cellular activation. One notable finding was the identification of a previously unrecognized subset of mycobacteria-specific CD4+ T cells characterized by the production of interleukin-3. Our results support the use of CD154 expression as a practical and reliable method to isolate live, antigen-specific CD4+ T cells for transcriptomic analysis and potentially for a range of other studies in infected or previously immunized hosts.

Recombinant Mycobacterium bovis bacillus Calmette-Guèrin (rBCG) has been explored as a vector for vaccines against HIV because of its ability to induce long lasting humoral and cell mediated immune responses. To maximize the potential for rBCG vaccines to induce effective immunity against HIV, various strategies are being employed to improve its ability to prime CD8+ T cells, which play an important role in the control of HIV infections. In this study we adopted a previously described approach of incorporating glycolipids that activate CD1d-restricted natural killer T (NKT) cells to enhance priming of CD8+ T cells by rBCG strains expressing an SIV Gag antigen (rBCG-SIV gag). We found that the incorporation of the synthetic NKT activating glycolipid α-galactosylceramide (α-GC) into rBCG-SIV gag significantly enhanced CD8+ T cell responses against an immunodominant Gag epitope, compared to responses primed by unmodified rBCG-SIV gag. The abilities of structural analogues of α-GC to enhance CD8+ T cell responses to rBCG were compared in both wild type and partially humanized mice that express human CD1d molecules in place of mouse CD1d. These studies identified an α-GC analogue known as 7DW8-5, which has previously been used successfully as an adjuvant in non-human primates, as a promising compound for enhancing immunogenicity of antigens delivered by rBCG.vectors. Our findings support the incorporation of synthetic glycolipid activators of NKT cells as a novel approach to enhance the immunogenicity of rBCG-vectored antigens for induction of CD8+ T cell responses. The glycolipid adjuvant 7DW8-5 may be a promising candidate for advancing to non-human primate and human clinical studies for the development of HIV vaccines based on rBCG vectors.