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We present an integrated analysis of the clinical measurements, immune cells, and plasma multi-omics of 139 COVID-19 patients representing all levels of disease severity, from serial blood draws collected during the first week of infection following diagnosis. We identify a major shift between mild and moderate disease, at which point elevated inflammatory signaling is accompanied by the loss of specific classes of metabolites and metabolic processes. Within this stressed plasma environment at moderate disease, multiple unusual immune cell phenotypes emerge and amplify with increasing disease severity. We condensed over 120,000 immune features into a single axis to capture how different immune cell classes coordinate in response to SARS-CoV-2. This immune-response axis independently aligns with the major plasma composition changes, with clinical metrics of blood clotting, and with the sharp transition between mild and moderate disease. This study suggests that moderate disease may provide the most effective setting for therapeutic intervention.

Host immune responses play central roles in controlling SARS-CoV2 infection, yet remain incompletely characterized and understood. Here, we present a comprehensive immune response map spanning 454 proteins and 847 metabolites in plasma integrated with single-cell multi-omic assays of PBMCs in which whole transcriptome, 192 surface proteins, and T and B cell receptor sequence were co-analyzed within the context of clinical measures from 50 COVID19 patient samples. Our study reveals novel cellular subpopulations, such as proliferative exhausted CD8 + and CD4 + T cells, and cytotoxic CD4 + T cells, that may be features of severe COVID-19 infection. We condensed over 1 million immune features into a single immune response axis that independently aligns with many clinical features and is also strongly associated with disease severity. Our study represents an important resource towards understanding the heterogeneous immune responses of COVID-19 patients and may provide key information for informing therapeutic development.

Patients infected with influenza are at high risk of secondary bacterial infection, which is a major proximate cause of morbidity and mortality. We have shown that in mice, prior infection with influenza results in increased inflammation and mortality upon Staphylococcus aureus infection, recapitulating the human disease. Lipidomic profiling of the lungs of superinfected mice revealed an increase in CYP450 metabolites during lethal superinfection. These lipids are endogenous ligands for the nuclear receptor PPARα, and we demonstrate that Ppara-/- mice are less susceptible to superinfection than wild-type mice. PPARα is an inhibitor of NFκB activation, and transcriptional profiling of cells isolated by bronchoalveolar lavage confirmed that influenza infection inhibits NFκB, thereby dampening proinflammatory and prosurvival signals. Furthermore, network analysis indicated an increase in necrotic cell death in the lungs of superinfected mice compared to mice infected with S. aureus alone. Consistent with this, we observed reduced NFκB-mediated inflammation and cell survival signaling in cells isolated from the lungs of superinfected mice. The kinase RIPK3 is required to induce necrotic cell death and is strongly induced in cells isolated from the lungs of superinfected mice compared to mice infected with S. aureus alone. Genetic and pharmacological perturbations demonstrated that PPARα mediates RIPK3-dependent necroptosis and that this pathway plays a central role in mortality following superinfection. Thus, we have identified a molecular circuit in which infection with influenza induces CYP450 metabolites that activate PPARα, leading to increased necrotic cell death in the lung which correlates with the excess mortality observed in superinfection.

Toll-like receptors (TLRs) on macrophages sense microbial components and trigger the production of numerous cytokines and chemokines that mediate the inflammatory response to infection. Although many of the components required for the activation of the TLR pathway have been identified, the mechanisms that appropriately regulate the magnitude and duration of the response and ultimately restore homeostasis are less well understood. Furthermore, a growing body of work indicates that TLR signaling reciprocally interacts with other fundamental cellular processes, including lipid metabolism but only a few specific molecular links between immune signaling and the macrophage lipidome have been studied in detail. Oxysterol-binding protein (Osbp) is the founding member of a family of lipid-binding proteins with diverse functions in lipid sensing, lipid transport, and cell signaling but its role in TLR responses is not well defined. Here, we demonstrate that altering the state of Osbp with its natural ligand, 25-hydroxycholesterol (25HC), or pharmacologically, sustains and thereby amplifies Tlr4-induced cytokine production in vitro and in vivo. CRISPR-induced knockdown of Osbp abrogates the ability of these ligands to sustain TLR responses. Lipidomic analysis suggested that the effect of Osbp on TLR signaling may be mediated by alterations in triglyceride production and treating cells with a Dgat1 inhibitor, which blocks triglyceride production and completely abrogates the effect of Osbp on TLR signaling. Thus, Osbp is a sterol sensor that transduces perturbations of the lipidome to modulate the resolution of macrophage inflammatory responses. [ABSTRACT FROM AUTHOR]

To investigate how host and pathogen diversity govern immunity against Mycobacterium tuberculosis (Mtb), we performed a large-scale screen of vaccine-mediated protection against aerosol Mtb infection using three inbred mouse strains [C57BL/6 (B6), C3HeB/FeJ (C3H), Balb/c x 129/SvJ (C129F1)] and three Mtb strains (H37Rv, CDC1551, SA161) representing two lineages and distinct virulence properties. We compared three protective modalities, all of which involve inoculation with live mycobacteria: Bacillus Calmette-Gue'rin (BCG), the only approved TB vaccine, delivered either subcutaneously or intravenously, and concomitant Mtb infection (CoMtb), a model of pre-existing immunity in which a low-level Mtb infection is established in the cervical lymph node following intradermal inoculation. We examined lung bacterial burdens at early (Day 28) and late (Day 98) time points after aerosol Mtb challenge and histopathology at Day 98. We observed substantial heterogeneity in the reduction of bacterial load afforded by these modalities at Day 28 across the combinations and noted a strong positive correlation between bacterial burden in unvaccinated mice and the degree of protection afforded by vaccination. Although we observed variation in the degree of reduction in bacterial burdens across the nine mouse/bacterium strain combinations, virtually all protective modalities performed similarly for a given strain-strain combination. We also noted dramatic variation in histopathology changes driven by both host and bacterial genetic backgrounds. Vaccination improved pathology scores for all infections except CDC1551. However, the most dramatic impact of vaccination on lesion development occurred for the C3H-SA161 combination, where vaccination entirely abrogated the development of the large necrotic lesions that arise in unvaccinated mice. In conclusion, we find that substantial TB heterogeneity can be recapitulated by introducing variability in both host and bacterial genetics, resulting in changes in vaccine-mediated protection as measured both by bacterial burden as well as histopathology. These differences can be harnessed in future studies to identify immune correlates of vaccine efficacy. [ABSTRACT FROM AUTHOR]

Rationale: Contacts of patients with tuberculosis (TB) constitute an important target population for preventive measures because they are at high risk of infection with Mycobacterium tuberculosis and progression to disease.Objectives: We investigated biosignatures with predictive ability for incident TB.Methods: In a case-control study nested within the Grand Challenges 6-74 longitudinal HIV-negative African cohort of exposed household contacts, we employed RNA sequencing, PCR, and the pair ratio algorithm in a training/test set approach. Overall, 79 progressors who developed TB between 3 and 24 months after diagnosis of index case and 328 matched nonprogressors who remained healthy during 24 months of follow-up were investigated.Measurements and Main Results: A four-transcript signature derived from samples in a South African and Gambian training set predicted progression up to two years before onset of disease in blinded test set samples from South Africa, the Gambia, and Ethiopia with little population-associated variability, and it was also validated in an external cohort of South African adolescents with latent M. tuberculosis infection. By contrast, published diagnostic or prognostic TB signatures were predicted in samples from some but not all three countries, indicating site-specific variability. Post hoc meta-analysis identified a single gene pair, C1QC/TRAV27 (complement C1q C-chain / T-cell receptor-α variable gene 27) that would consistently predict TB progression in household contacts from multiple African sites but not in infected adolescents without known recent exposure events.Conclusions: Collectively, we developed a simple whole blood-based PCR test to predict TB in recently exposed household contacts from diverse African populations. This test has potential for implementation in national TB contact investigation programs.

Our understanding of mechanisms underlying progression from Mycobacterium tuberculosis infection to pulmonary tuberculosis disease in humans remains limited. To define such mechanisms, we followed M. tuberculosis-infected adolescents longitudinally. Blood samples from forty-four adolescents who ultimately developed tuberculosis disease (“progressors”) were compared with those from 106 matched controls, who remained healthy during two years of follow up. We performed longitudinal whole blood transcriptomic analyses by RNA sequencing and plasma proteome analyses using multiplexed slow off-rate modified DNA aptamers. Tuberculosis progression was associated with sequential modulation of immunological processes. Type I/II interferon signalling and complement cascade were elevated 18 months before tuberculosis disease diagnosis, while changes in myeloid inflammation, lymphoid, monocyte and neutrophil gene modules occurred more proximally to tuberculosis disease. Analysis of gene expression in purified T cells also revealed early suppression of Th17 responses in progressors, relative to M. tuberculosis-infected controls. This was confirmed in an independent adult cohort who received BCG re-vaccination; transcript expression of interferon response genes in blood prior to BCG administration was associated with suppression of IL-17 expression by BCG-specific CD4 T cells 3 weeks post-vaccination. Our findings provide a timeline to the different immunological stages of disease progression which comprise sequential inflammatory dynamics and immune alterations that precede disease manifestations and diagnosis of tuberculosis disease. These findings have important implications for developing diagnostics, vaccination and host-directed therapies for tuberculosis.Trial registrationClincialtrials.gov, NCT01119521.

Bioactive lipid mediators play a crucial role in the induction and resolution of inflammation. To elucidate their involvement during influenza infection, liquid chromatography/mass spectrometry lipidomic profiling of 141 lipid species was performed on a mouse influenza model using two viruses of significantly different pathogenicity. Infection by the low-pathogenicity strain X31/H3N2 induced a proinflammatory response followed by a distinct anti-inflammatory response; infection by the high-pathogenicity strain PR8/H1N1 resulted in overlapping pro- and anti-inflammatory states. Integration of the large-scale lipid measurements with targeted gene expression data demonstrated that 5-lipoxygenase metabolites correlated with the pathogenic phase of the infection, whereas 12/15-lipoxygenase metabolites were associated with the resolution phase. Hydroxylated linoleic acid, specifically the ratio of 13- to 9-hydroxyoctadecadienoic acid, was identified as a potential biomarker for immune status during an active infection. Importantly, some of the findings from the animal model were recapitulated in studies of human nasopharyngeal lavages obtained during the 2009-2011 influenza seasons.

Although host genetics influences susceptibility to tuberculosis (TB), few genes determining disease outcome have been identified. We hypothesized that macrophages from individuals with different clinical manifestations of Mycobacterium tuberculosis (Mtb) infection would have distinct gene expression profiles and that polymorphisms in these genes may also be associated with susceptibility to TB. We measured gene expression levels of >38,500 genes from ex vivo Mtb-stimulated macrophages in 12 subjects with 3 clinical phenotypes: latent, pulmonary, and meningeal TB (n = 4 per group). After identifying differentially expressed genes, we confirmed these results in 34 additional subjects by real-time PCR. We also used a case-control study design to examine whether polymorphisms in differentially regulated genes were associated with susceptibility to these different clinical forms of TB. We compared gene expression profiles in Mtb-stimulated and unstimulated macrophages and identified 1,608 and 199 genes that were differentially expressed by >2- and >5-fold, respectively. In an independent sample set of 34 individuals and a subset of highly regulated genes, 90% of the microarray results were confirmed by RT-PCR, including expression levels of CCL1, which distinguished the 3 clinical groups. Furthermore, 6 single nucleotide polymorphisms (SNPs) in CCL1 were found to be associated with TB in a case-control genetic association study with 273 TB cases and 188 controls. To our knowledge, this is the first identification of CCL1 as a gene involved in host susceptibility to TB and the first study to combine microarray and DNA polymorphism studies to identify genes associated with TB susceptibility. These results suggest that genome-wide studies can provide an unbiased method to identify critical macrophage response genes that are associated with different clinical outcomes and that variation in innate immune response genes regulate susceptibility to TB.

Despite widespread use of the bacille Calmette-Guérin (BCG) vaccine, tuberculosis (TB) remains a leading cause of global mortality from a single infectious agent (Mycobacterium tuberculosis or Mtb). Here, over two independent Mtb challenge studies, we demonstrate that subcutaneous vaccination of rhesus macaques (RMs) with rhesus cytomegalovirus vectors encoding Mtb antigen inserts (hereafter referred to as RhCMV/TB)--which elicit and maintain highly effector-differentiated, circulating and tissue-resident Mtb-specific CD4[sup.+] and CD8[sup.+] memory T cell responses--can reduce the overall (pulmonary and extrapulmonary) extent of Mtb infection and disease by 68%, as compared to that in unvaccinated controls, after intrabronchial challenge with the Erdman strain of Mtb at [similar]1 year after the first vaccination. Fourteen of 34 RhCMV/TB-vaccinated RMs (41%) across both studies showed no TB disease by computed tomography scans or at necropsy after challenge (as compared to 0 of 17 unvaccinated controls), and ten of these RMs were Mtb-culture-negative for all tissues, an exceptional long-term vaccine effect in the RM challenge model with the Erdman strain of Mtb. These results suggest that complete vaccine-mediated immune control of highly pathogenic Mtb is possible if immune effector responses can intercept Mtb infection at its earliest stages., Author(s): Scott G Hansen [1]; Daniel E Zak [2]; Guangwu Xu [1]; Julia C Ford [1]; Emily E Marshall [1]; Daniel Malouli [1]; Roxanne M Gilbride [1]; Colette M Hughes [...]

Recombinant adenoviral vectors (rAds) are lead vaccine candidates for protection against a variety of pathogens, including Ebola, HIV, tuberculosis, and malaria, due to their ability to potently induce T cell immunity in humans. However, the ability to induce protective cellular immunity varies among rAds. Here, we assessed the mechanisms that control the potency of CD8 T cell responses in murine models following vaccination with human-, chimpanzee-, and simian-derived rAds encoding SIV-Gag antigen (Ag). After rAd vaccination, we quantified Ag expression and performed expression profiling of innate immune response genes in the draining lymph node. Human-derived rAd5 and chimpanzee- derived chAd3 were the most potent rAds and induced high and persistent Ag expression with low innate gene activation, while less potent rAds induced less Ag expression and robustly induced innate immunity genes that were primarily associated with IFN signaling. Abrogation of type I IFN or stimulator of IFN genes (STING) signaling increased Ag expression and accelerated CD8 T cell response kinetics but did not alter memory responses or protection. These findings reveal that the magnitude of rAd-induced memory CD8 T cell immune responses correlates with Ag expression but is independent of IFN and STING and provide criteria for optimizing protective CD8 T cell immunity with rAd vaccines., Introduction CD8 T cells play a critical role in mediating protection to a variety of intracellular pathogens, including Ebola, HIV, tuberculosis, and malaria. Replication-defective recombinant adenoviral vectors (rAds) are promising [...]