Leif E. Sander

Two elementary parameters for quantifying viral infection and shedding are viral load and whether samples yield a replicating virus isolate in cell culture. We examined 25,381 cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Germany, including 6110 from test centers attended by presymptomatic, asymptomatic, and mildly symptomatic (PAMS) subjects, 9519 who were hospitalized, and 1533 B.1.1.7 lineage infections. The viral load of the youngest subjects was lower than that of the older subjects by 0.5 (or fewer) log10 units, and they displayed an estimated ~78% of the peak cell culture replication probability; in part this was due to smaller swab sizes and unlikely to be clinically relevant. Viral loads above 109 copies per swab were found in 8% of subjects, one-third of whom were PAMS, with a mean age of 37.6 years. We estimate 4.3 days from onset of shedding to peak viral load (108.1 RNA copies per swab) and peak cell culture isolation probability (0.75). B.1.1.7 subjects had mean log10 viral load 1.05 higher than that of non-B.1.1.7 subjects, and the estimated cell culture replication probability of B.1.1.7 subjects was higher by a factor of 2.6.

Live vaccines have long been known to trigger far more vigorous immune responses than their killed counterparts. This has been attributed to the ability of live microorganisms to replicate and express specialized virulence factors that facilitate invasion and infection of their hosts. However, protective immunization can often be achieved with a single injection of live, but not dead, attenuated microorganisms stripped of their virulence factors. Pathogen-associated molecular patterns (PAMPs), which are detected by the immune system, are present in both live and killed vaccines, indicating that certain poorly characterized aspects of live microorganisms, not incorporated in dead vaccines, are particularly effective at inducing protective immunity. Here we show that the mammalian innate immune system can directly sense microbial viability through detection of a special class of viability-associated PAMPs (vita-PAMPs). We identify prokaryotic messenger RNA as a vita-PAMP present only in viable bacteria, the recognition of which elicits a unique innate response and a robust adaptive antibody response. Notably, the innate response evoked by viability and prokaryotic mRNA was thus far considered to be reserved for pathogenic bacteria, but we show that even non-pathogenic bacteria in sterile tissues can trigger similar responses, provided that they are alive. Thus, the immune system actively gauges the infectious risk by searching PAMPs for signatures of microbial life and thus infectivity. Detection of vita-PAMPs triggers a state of alert not warranted for dead bacteria. Vaccine formulations that incorporate vita-PAMPs could thus combine the superior protection of live vaccines with the safety of dead vaccines.

The Omicron variant of SARS-CoV-2 is causing a rapid increase in infections across the globe. This new variant of concern carries an unusually high number of mutations in key epitopes of neutralizing antibodies on the viral spike glycoprotein, suggesting potential immune evasion. Here we assessed serum neutralizing capacity in longitudinal cohorts of vaccinated and convalescent individuals, as well as monoclonal antibody activity against Omicron using pseudovirus neutralization assays. We report a near-complete lack of neutralizing activity against Omicron in polyclonal sera from individuals vaccinated with two doses of the BNT162b2 COVID-19 vaccine and from convalescent individuals, as well as resistance to different monoclonal antibodies in clinical use. However, mRNA booster immunizations in vaccinated and convalescent individuals resulted in a significant increase of serum neutralizing activity against Omicron. This study demonstrates that booster immunizations can critically improve the humoral immune response against the Omicron variant.

Lymphocytes circulate through lymph nodes (LN) in search for antigen in what is believed to be a continuous process. Here, we show that lymphocyte migration through lymph nodes and lymph occurred in a non-continuous, circadian manner. Lymphocyte homing to lymph nodes peaked at night onset, with cells leaving the tissue during the day. This resulted in strong oscillations in lymphocyte cellularity in lymph nodes and efferent lymphatic fluid. Using lineage-specific genetic ablation of circadian clock function, we demonstrated this to be dependent on rhythmic expression of promigratory factors on lymphocytes. Dendritic cell numbers peaked in phase with lymphocytes, with diurnal oscillations being present in disease severity after immunization to induce experimental autoimmune encephalomyelitis (EAE). These rhythms were abolished by genetic disruption of T cell clocks, demonstrating a circadian regulation of lymphocyte migration through lymph nodes with time-of-day of immunization being critical for adaptive immune responses weeks later.

Acute-phase proteins (APPs) are an evolutionarily conserved family of proteins produced mainly in the liver in response to infection and inflammation. Despite vast pro- and antiinflammatory properties ascribed to individual APPs, their collective function during infections remains poorly defined. Using a mouse model of polymicrobial sepsis, we show that abrogation of APP production by hepatocyte-specific gp130 deletion, the signaling receptor shared by IL-6 family cytokines, strongly increased mortality despite normal bacterial clearance. Hepatic gp130 signaling through STAT3 was required to control systemic inflammation. Notably, hepatic gp130–STAT3 activation was also essential for mobilization and tissue accumulation of myeloid-derived suppressor cells (MDSCs), a cell population mainly known for antiinflammatory properties in cancer. MDSCs were critical to regulate innate inflammation, and their adoptive transfer efficiently protected gp130-deficient mice from sepsis-associated mortality. The hepatic APPs serum amyloid A and Cxcl1/KC cooperatively promoted MDSC mobilization, accumulation, and survival, and reversed dysregulated inflammation and restored survival of gp130-deficient mice. Thus, gp130-dependent communication between the liver and MDSCs through APPs controls inflammatory responses during infection.

Children have reduced severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection rates and a substantially lower risk for developing severe coronavirus disease 2019 compared with adults. However, the molecular mechanisms underlying protection in younger age groups remain unknown. Here we characterize the single-cell transcriptional landscape in the upper airways of SARS-CoV-2-negative (n = 18) and age-matched SARS-CoV-2-positive (n = 24) children and corresponding samples from adults (n = 44), covering an age range of 4 weeks to 77 years. Children displayed higher basal expression of relevant pattern recognition receptors such as MDA5 (IFIH1) and RIG-I (DDX58) in upper airway epithelial cells, macrophages and dendritic cells, resulting in stronger innate antiviral responses upon SARS-CoV-2 infection than in adults. We further detected distinct immune cell subpopulations including KLRC1 (NKG2A)+ cytotoxic T cells and a CD8+ T cell population with a memory phenotype occurring predominantly in children. Our study provides evidence that the airway immune cells of children are primed for virus sensing, resulting in a stronger early innate antiviral response to SARS-CoV-2 infection than in adults.

Macrophages tightly scale their core metabolism after being activated, but the precise regulation of the mitochondrial electron-transport chain (ETC) and its functional implications are currently unknown. Here we found that recognition of live bacteria by macrophages transiently decreased assembly of the ETC complex I (CI) and CI-containing super-complexes and switched the relative contributions of CI and CII to mitochondrial respiration. This was mediated by phagosomal NADPH oxidase and the reactive oxygen species (ROS)-dependent tyrosine kinase Fgr. It required Toll-like receptor signaling and the NLRP3 inflammasome, which were both connected to bacterial viability-specific immune responses. Inhibition of CII during infection with Escherichia coli normalized serum concentrations of interleukin 1β (IL-1β) and IL-10 to those in mice treated with dead bacteria and impaired control of bacteria. We have thus identified ETC adaptations as an early immunological-metabolic checkpoint that adjusts innate immune responses to bacterial infection.

The functional relevance of preexisting cross-immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a subject of intense debate. Here, we show that human endemic coronavirus (HCoV)–reactive and SARS-CoV-2–cross-reactive CD4+ T cells are ubiquitous but decrease with age. We identified a universal immunodominant coronavirus-specific spike peptide (S816-830) and demonstrate that preexisting spike- and S816-830–reactive T cells were recruited into immune responses to SARS-CoV-2 infection and their frequency correlated with anti–SARS-CoV-2-S1-IgG antibodies. Spike–cross-reactive T cells were also activated after primary BNT162b2 COVID-19 messenger RNA vaccination and displayed kinetics similar to those of secondary immune responses. Our results highlight the functional contribution of preexisting spike–cross-reactive T cells in SARS-CoV-2 infection and vaccination. Cross-reactive immunity may account for the unexpectedly rapid induction of immunity after primary SARS-CoV-2 immunization and the high rate of asymptomatic or mild COVID-19 disease courses.

Macrophages can be niches for bacterial pathogens or antibacterial effector cells depending on the pathogen and signals from the immune system. Here we show that type I and II IFNs are master regulators of gene expression during Legionella pneumophila infection, and activators of an alveolar macrophage-intrinsic immune response that restricts bacterial growth during pneumonia. Quantitative mass spectrometry revealed that both IFNs substantially modify Legionella-containing vacuoles, and comparative analyses reveal distinct subsets of transcriptionally and spatially IFN-regulated proteins. Immune-responsive gene (IRG)1 is induced by IFNs in mitochondria that closely associate with Legionella-containing vacuoles, and mediates production of itaconic acid. This metabolite is bactericidal against intravacuolar L. pneumophila as well as extracellular multidrug-resistant Gram-positive and -negative bacteria. Our study explores the overall role IFNs play in inducing substantial remodeling of bacterial vacuoles and in stimulating production of IRG1-derived itaconic acid which targets intravacuolar pathogens. IRG1 or its product itaconic acid might be therapeutically targetable to fight intracellular and drug-resistant bacteria.

ABSTRACT Mycobacterium bovis Bacille Calmette-Guérin (BCG) is the only licensed vaccine against tuberculosis (TB), yet its moderate efficacy against pulmonary TB calls for improved vaccination strategies. Mucosal BCG vaccination generates superior protection against TB in animal models; however, the mechanisms of protection remain elusive. Tissue-resident memory T (T RM ) cells have been implicated in protective immune responses against viral infections, but the role of T RM cells following mycobacterial infection is unknown. Using a mouse model of TB, we compared protection and lung cellular infiltrates of parenteral and mucosal BCG vaccination. Adoptive transfer and gene expression analyses of lung airway cells were performed to determine the protective capacities and phenotypes of different memory T cell subsets. In comparison to subcutaneous vaccination, intratracheal and intranasal BCG vaccination generated T effector memory and T RM cells in the lung, as defined by surface marker phenotype. Adoptive mucosal transfer of these airway-resident memory T cells into naive mice mediated protection against TB. Whereas airway-resident memory CD4 + T cells displayed a mixture of effector and regulatory phenotype, airway-resident memory CD8 + T cells displayed prototypical T RM features. Our data demonstrate a key role for mucosal vaccination-induced airway-resident T cells in the host defense against pulmonary TB. These results have direct implications for the design of refined vaccination strategies. IMPORTANCE BCG remains the only licensed vaccine against TB. Parenterally administered BCG has variable efficacy against pulmonary TB, and thus, improved prevention strategies and a more refined understanding of correlates of vaccine protection are required. Induction of memory T cells has been shown to be essential for protective TB vaccines. Mimicking the natural infection route by mucosal vaccination has been known to generate superior protection against TB in animal models; however, the mechanisms of protection have remained elusive. Here we performed an in-depth analysis to dissect the immunological mechanisms associated with superior mucosal protection in the mouse model of TB. We found that mucosal, and not subcutaneous, BCG vaccination generates lung-resident memory T cell populations that confer protection against pulmonary TB. We establish a comprehensive phenotypic characterization of these populations, providing a framework for future vaccine development.

Histamine is known as a regulator of gastrointestinal functions, such as gastric acid production, intestinal motility, and mucosal ion secretion. Most of this knowledge has been obtained from animal studies. In contrast, in humans, expression and distribution of histamine receptors (HR) within the human gastrointestinal tract are unclear.We analysed HR expression in human gastrointestinal tissue specimens by quantitative reverse transcription-polymerase chain reaction and immunostaining.We found that H1R, H2R, and H4R mRNA were expressed throughout the gastrointestinal tract, while H3R mRNA was absent. No significant differences in the distribution of HR were found between different anatomical sites (duodenum, ileum, colon, sigma, and rectum). Immunostaining of neurones and nerve fibres revealed that H3R was absent in the human enteric nervous system; however, H1R and H2R were found on ganglion cells of the myenteric plexus. Epithelial cells also expressed H1R, H2R and, to some extent, H4R. Intestinal fibroblasts exclusively expressed H1R while the muscular layers of human intestine stained positive for both H1R and H2R. Immune cells expressed mRNA and protein for H1R, H2R, and low levels of H4R. Analysis of endoscopic biopsies from patients with food allergy and irritable bowel syndrome revealed significantly elevated H1R and H2R mRNA levels compared with controls.We have demonstrated that H1R, H2R and, to some extent, H4R, are expressed in the human gastrointestinal tract, while H3R is absent, and we found that HR expression was altered in patients with gastrointestinal diseases.

Live attenuated vaccines are generally highly efficacious and often superior to inactivated vaccines, yet the underlying mechanisms of this remain largely unclear. Here we identify recognition of microbial viability as a potent stimulus for follicular helper T cell (TFH cell) differentiation and vaccine responses. Antigen-presenting cells (APCs) distinguished viable bacteria from dead bacteria through Toll-like receptor 8 (TLR8)-dependent detection of bacterial RNA. In contrast to dead bacteria and other TLR ligands, live bacteria, bacterial RNA and synthetic TLR8 agonists induced a specific cytokine profile in human and porcine APCs, thereby promoting TFH cell differentiation. In domestic pigs, immunization with a live bacterial vaccine induced robust TFH cell and antibody responses, but immunization with its heat-killed counterpart did not. Finally, a hypermorphic TLR8 polymorphism was associated with protective immunity elicited by vaccination with bacillus Calmette-Guérin (BCG) in a human cohort. We have thus identified TLR8 as an important driver of TFH cell differentiation and a promising target for TFH cell–skewing vaccine adjuvants. Sander and colleagues show that antigen-presenting cells detect bacterial RNA from live bacteria via TLR8 and promote TFH cell differentiation and vaccine responses through the induction of a specific cytokine profile.

Abstract Objective to assess reactogenicity and immunogenicity of heterologous prime-boost immunisations of ChAdOx1-nCoV19 (Vaxzevria, ChAdOx) followed by BNT162b2 (Comirnaty, BNT) compared to homologous BNT/BNT immunisation. Design prospective, observational cohort study. Setting unicenter study in a cohort of health care workers at a tertiary care center in Berlin, Germany. Participants 340 health care workers immunised between 27 December 2020 and 21 May 2021 at Charité - Universitätsmedizin Berlin, Germany Main outcome measures the main outcomes were reactogenicity assessed on days one, three, five and seven post prime and boost vaccination, and immunogenicity measured by serum SARS-CoV-2 full spike-, spike S1-, and spike RBD-IgG, virus neutralisation capacity, anti-S1-IgG avidity, and T cell reactivity measured by Interferon gamma release assay at 3-4 weeks post prime and boost immunisation. Results Heterologous ChAdOx/BNT booster vaccination was overall well-tolerated and reactogenicity was largely comparable to homologous BNT/BNT vaccination. Systemic reactions were most frequent after prime immunisation with ChAdOx (86%, 95CI: 79-91), and less frequent after homologous BNT/BNT (65%, 95CI: 56-72), or heterologous ChAdOx/BNT booster vaccination (48%, 95CI: 36-59). Serum antibody responses and T cell reactivity were strongly increased after both homologous and heterologous boost, and immunogenicity was overall robust, and comparable between both regimens in this cohort, with slightly increased S1-IgG avidity and T cell responses following heterologous booster immunisation. Conclusions Evidence of rare thrombotic events associated with ChAdOx has led to recommendation of a heterologous booster with mRNA vaccines for certain age groups in several European countries, despite a lack of robust safety and immunogenicity data for this vaccine regimen. This interim analysis provides evidence that the currently recommended heterologous ChAdOx/BNT immunisation regimen with 10-12 week vaccine intervals is well tolerated and slightly more immunogenic compared to homologous BNT/BNT vaccination with three week vaccine intervals. Heterologous prime-boost immunisation for COVID-19 may be generally applicable to optimise logistics and improve immunogenicity and to mitigate potential intermittent supply shortages for individual vaccines.

Global healthcare systems are challenged by the COVID-19 pandemic. There is a need to optimize allocation of treatment and resources in intensive care, as clinically established risk assessments such as SOFA and APACHE II scores show only limited performance for predicting the survival of severely ill COVID-19 patients. Additional tools are also needed to monitor treatment, including experimental therapies in clinical trials. Comprehensively capturing human physiology, we speculated that proteomics in combination with new data-driven analysis strategies could produce a new generation of prognostic discriminators. We studied two independent cohorts of patients with severe COVID-19 who required intensive care and invasive mechanical ventilation. SOFA score, Charlson comorbidity index, and APACHE II score showed limited performance in predicting the COVID-19 outcome. Instead, the quantification of 321 plasma protein groups at 349 timepoints in 50 critically ill patients receiving invasive mechanical ventilation revealed 14 proteins that showed trajectories different between survivors and non-survivors. A predictor trained on proteomic measurements obtained at the first time point at maximum treatment level (i.e. WHO grade 7), which was weeks before the outcome, achieved accurate classification of survivors (AUROC 0.81). We tested the established predictor on an independent validation cohort (AUROC 1.0). The majority of proteins with high relevance in the prediction model belong to the coagulation system and complement cascade. Our study demonstrates that plasma proteomics can give rise to prognostic predictors substantially outperforming current prognostic markers in intensive care.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilises the angiotensin-converting enzyme 2 (ACE2) transmembrane peptidase as cellular entry receptor. However, whether SARS-CoV-2 in the alveolar compartment is strictly ACE2-dependent and to what extent virus-induced tissue damage and/or direct immune activation determines early pathogenesis is still elusive.Spectral microscopy, single-cell/-nucleus RNA sequencing or ACE2 "gain-of-function" experiments were applied to infected human lung explants and adult stem cell derived human lung organoids to correlate ACE2 and related host factors with SARS-CoV-2 tropism, propagation, virulence and immune activation compared to SARS-CoV, influenza and Middle East respiratory syndrome coronavirus (MERS-CoV). Coronavirus disease 2019 (COVID-19) autopsy material was used to validate ex vivo results.We provide evidence that alveolar ACE2 expression must be considered scarce, thereby limiting SARS-CoV-2 propagation and virus-induced tissue damage in the human alveolus. Instead, ex vivo infected human lungs and COVID-19 autopsy samples showed that alveolar macrophages were frequently positive for SARS-CoV-2. Single-cell/-nucleus transcriptomics further revealed nonproductive virus uptake and a related inflammatory and anti-viral activation, especially in "inflammatory alveolar macrophages", comparable to those induced by SARS-CoV and MERS-CoV, but different from NL63 or influenza virus infection.Collectively, our findings indicate that severe lung injury in COVID-19 probably results from a macrophage-triggered immune activation rather than direct viral damage of the alveolar compartment.

Abstract Prolonged lung pathology has been associated with COVID-19, yet the cellular and molecular mechanisms behind this chronic inflammatory disease are poorly understood. In this study, we combine advanced imaging and spatial transcriptomics to shed light on the local immune response in severe COVID-19. We show that activated adventitial niches are crucial microenvironments contributing to the orchestration of prolonged lung immunopathology. Up-regulation of the chemokines CCL21 and CCL18 associates to endothelial-to-mesenchymal transition and tissue fibrosis within these niches. CCL21 over-expression additionally links to the local accumulation of T cells expressing the cognate receptor CCR7. These T cells are imprinted with an exhausted phenotype and form lymphoid aggregates that can organize in ectopic lymphoid structures. Our work proposes immune-stromal interaction mechanisms promoting a self-sustained and non-resolving local immune response that extends beyond active viral infection and perpetuates tissue remodeling.

Recently, nitric oxide (NO) has been shown to be vital in inflammatory processes. Nitric oxide synthase (NOS) exists in three different isoforms, two constitutively produced with physiological roles, and an inducible form, iNOS, which is involved in inflammation. This study examined the localisation of iNOS in biopsies from patients with periodontitis using immunohistochemistry, and compared these with healthy tissue biopsies. Biopsies were obtained from 16 periodontitis patients undergoing periodontal surgery and from clinically healthy tissues of 5 patients having crown lengthening procedures. The periodontitis diseased tissue demonstrated a greater level of iNOS expression than the healthy tissue. The source of iNOS in the periodontal tissues was determined by our monoclonal antibody to be the macrophage, with the endothelial cells also contributing. A role for NO in the inflammatory response of periodontal tissues is suggested, but the precise role requires further elucidation.

Mediator release from mast cells (MC) is a crucial step in allergic and non-allergic inflammatory disorders. However, the final events in response to activation leading to membrane fusion and thereby facilitating degranulation have hitherto not been analyzed in human MC. Soluble N-ethyl-maleimide-sensitive factor attachment protein receptors (SNARE) represent a highly conserved family of proteins that have been shown to mediate intracellular membrane fusion events. Here, we show that mature MC isolated from human intestinal tissue express soluble N-ethylmaleide sensitive factor attachment protein (SNAP)-23, Syntaxin (STX)-1B, STX-2, STX-3, STX-4, and STX-6 but not SNAP-25. Furthermore, we found that primary human MC express substantial amounts of vesicle associated membrane protein (VAMP)-3, VAMP-7 and VAMP-8 and, in contrast to previous reports about rodent MC, only low levels of VAMP-2. Furthermore, VAMP-7 and VAMP-8 were found to translocate to the plasma membrane and interact with SNAP-23 and STX-4 upon activation. Inhibition of SNAP-23, STX-4, VAMP-7 or VAMP-8, but not VAMP-2 or VAMP-3, resulted in a markedly reduced high-affinity IgE receptor-mediated histamine release. In summary, our data show that mature human MC express a specific pattern of SNARE and that VAMP-7 and VAMP-8, but not VAMP-2, are required for rapid degranulation.

Live vaccines historically afford superior protection, yet the cellular and molecular mechanisms mediating protective immunity remain unclear. Here we found that vaccination of mice with live, but not dead, Gram-negative bacteria heightened follicular T helper cell (Tfh) differentiation, germinal center formation, and protective antibody production through the signaling adaptor TRIF. Complementing the dead vaccine with an innate signature of bacterial viability, bacterial RNA, recapitulated these responses. The interferon (IFN) and inflammasome pathways downstream of TRIF orchestrated Tfh responses extrinsically to B cells and classical dendritic cells. Instead, CX3CR1+CCR2- monocytes instructed Tfh differentiation through interleukin-1β (IL-1β), a tightly regulated cytokine secreted upon TRIF-dependent IFN licensing of the inflammasome. Hierarchical production of IFN-β and IL-1β dictated Tfh differentiation and elicited the augmented humoral responses characteristic of live vaccines. These findings identify bacterial RNA, an innate signature of microbial viability, as a trigger for Tfh differentiation and suggest new approaches toward vaccine formulations for coordinating augmented Tfh and B cell responses.

Abstract The Omicron variant of SARS-CoV-2 is causing a rapid increase in infections across the globe. This new variant of concern carries an unusually high number of mutations in key epitopes of neutralizing antibodies on the viral spike glycoprotein, suggesting potential immune evasion. Here we assessed serum neutralizing capacity in longitudinal cohorts of vaccinated and convalescent individuals, as well as monoclonal antibody activity against Omicron using pseudovirus neutralization assays. We report a near-complete lack of neutralizing activity against Omicron in polyclonal sera from individuals vaccinated with two doses of the BNT162b2 COVID-19 vaccine and from convalescent individuals, as well as resistance to different monoclonal antibodies in clinical use. However, mRNA booster immunizations in vaccinated and convalescent individuals resulted in a significant increase of serum neutralizing activity against Omicron. The presented study demonstrates that booster immunizations may be critical to substantially improve the humoral immune response against the Omicron variant.Authors Henning Gruell, Kanika Vanshylla, Florian Kurth, Leif E. Sander, and Florian Klein contributed equally to this work.

The systemic immune response to viral infection is shaped by master transcription factors, such as NF-κB, STAT1, or PU.1. Although long noncoding RNAs (lncRNAs) have been suggested as important regulators of transcription factor activity, their contributions to the systemic immunopathologies observed during SARS-CoV-2 infection have remained unknown. Here, we employed a targeted single-cell RNA sequencing approach to reveal lncRNAs differentially expressed in blood leukocytes during severe COVID-19. Our results uncover the lncRNA PIRAT (PU.1-induced regulator of alarmin transcription) as a major PU.1 feedback-regulator in monocytes, governing the production of the alarmins S100A8/A9, key drivers of COVID-19 pathogenesis. Knockout and transgene expression, combined with chromatin-occupancy profiling, characterized PIRAT as a nuclear decoy RNA, keeping PU.1 from binding to alarmin promoters and promoting its binding to pseudogenes in naïve monocytes. NF-κB–dependent PIRAT down-regulation during COVID-19 consequently releases a transcriptional brake, fueling alarmin production. Alarmin expression is additionally enhanced by the up-regulation of the lncRNA LUCAT1, which promotes NF-κB–dependent gene expression at the expense of targets of the JAK-STAT pathway. Our results suggest a major role of nuclear noncoding RNA networks in systemic antiviral responses to SARS-CoV-2 in humans.

Global healthcare systems continue to be challenged by the COVID-19 pandemic, and there is a need for clinical assays that can help optimise resource allocation, support treatment decisions, and accelerate the development and evaluation of new therapies.We developed a multiplexed proteomics assay for determining disease severity and prognosis in COVID-19. The assay quantifies up to 50 peptides, derived from 30 known and newly introduced COVID-19-related protein markers, in a single measurement using routine-lab compatible analytical flow rate liquid chromatography and multiple reaction monitoring (LC-MRM). We conducted two observational studies in patients with COVID-19 hospitalised at Charité - Universitätsmedizin Berlin, Germany before (from March 1 to 26, 2020, n=30) and after (from April 4 to November 19, 2020, n=164) dexamethasone became standard of care. The study is registered in the German and the WHO International Clinical Trials Registry (DRKS00021688).The assay produces reproducible (median inter-batch CV of 10.9%) absolute quantification of 47 peptides with high sensitivity (median LLOQ of 143 ng/ml) and accuracy (median 96.8%). In both studies, the assay reproducibly captured hallmarks of COVID-19 infection and severity, as it distinguished healthy individuals, mild, moderate, and severe COVID-19. In the post-dexamethasone cohort, the assay predicted survival with an accuracy of 0.83 (108/130), and death with an accuracy of 0.76 (26/34) in the median 2.5 weeks before the outcome, thereby outperforming compound clinical risk assessments such as SOFA, APACHE II, and ABCS scores.Disease severity and clinical outcomes of patients with COVID-19 can be stratified and predicted by the routine-applicable panel assay that combines known and novel COVID-19 biomarkers. The prognostic value of this assay should be prospectively assessed in larger patient cohorts for future support of clinical decisions, including evaluation of sample flow in routine setting. The possibility to objectively classify COVID-19 severity can be helpful for monitoring of novel therapies, especially in early clinical trials.This research was funded in part by the European Research Council (ERC) under grant agreement ERC-SyG-2020 951475 (to M.R) and by the Wellcome Trust (IA 200829/Z/16/Z to M.R.). The work was further supported by the Ministry of Education and Research (BMBF) as part of the National Research Node 'Mass Spectrometry in Systems Medicine (MSCoresys)', under grant agreements 031L0220 and 161L0221. J.H. was supported by a Swiss National Science Foundation (SNSF) Postdoc Mobility fellowship (project number 191052). This study was further supported by the BMBF grant NaFoUniMedCOVID-19 - NUM-NAPKON, FKZ: 01KX2021. The study was co-funded by the UK's innovation agency, Innovate UK, under project numbers 75594 and 56328.

Antiviral CD8+ T cell immunity depends on the integration of various contextual cues, but how antigen-presenting cells (APCs) consolidate these signals for decoding by T cells remains unclear. Here, we describe gradual interferon-α/interferon-β (IFNα/β)-induced transcriptional adaptations that endow APCs with the capacity to rapidly activate the transcriptional regulators p65, IRF1 and FOS after CD4+ T cell-mediated CD40 stimulation. While these responses operate through broadly used signaling components, they induce a unique set of co-stimulatory molecules and soluble mediators that cannot be elicited by IFNα/β or CD40 alone. These responses are critical for the acquisition of antiviral CD8+ T cell effector function, and their activity in APCs from individuals infected with severe acute respiratory syndrome coronavirus 2 correlates with milder disease. These observations uncover a sequential integration process whereby APCs rely on CD4+ T cells to select the innate circuits that guide antiviral CD8+ T cell responses.

The cyclic GMP-AMP synthase (cGAS)-STING pathway is central for innate immune sensing of various bacterial, viral and protozoal infections. Recent studies identified the common HAQ and R232H alleles of TMEM173/STING, but the functional consequences of these variants for primary infections are unknown. Here we demonstrate that cGAS- and STING-deficient murine macrophages as well as human cells of individuals carrying HAQ TMEM173/STING were severely impaired in producing type I IFNs and pro-inflammatory cytokines in response to Legionella pneumophila, bacterial DNA or cyclic dinucleotides (CDNs). In contrast, R232H attenuated cytokine production only following stimulation with bacterial CDN, but not in response to L. pneumophila or DNA. In a mouse model of Legionnaires' disease, cGAS- and STING-deficient animals exhibited higher bacterial loads as compared to wild-type mice. Moreover, the haplotype frequency of HAQ TMEM173/STING, but not of R232H TMEM173/STING, was increased in two independent cohorts of human Legionnaires' disease patients as compared to healthy controls. Our study reveals that the cGAS-STING cascade contributes to antibacterial defense against L. pneumophila in mice and men, and provides important insight into how the common HAQ TMEM173/STING variant affects antimicrobial immune responses and susceptibility to infection. Trial registration ClinicalTrials.gov DRKS00005274, German Clinical Trials Register

The SARS-CoV-2 pandemic prompted a global vaccination effort and the development of numerous COVID-19 vaccines at an unprecedented scale and pace. As a result, current COVID-19 vaccination regimens comprise diverse vaccine modalities, immunogen combinations, and dosing intervals. Here, we compare vaccine-specific antibody and memory B cell responses following two-dose mRNA, single-dose Ad26.COV.2S, and two-dose ChAdOx1, or combination ChAdOx1/mRNA vaccination. Plasma-neutralizing activity, as well as the magnitude, clonal composition, and antibody maturation of the RBD-specific memory B cell compartments, showed substantial differences between the vaccination regimens. While individual monoclonal antibodies derived from memory B cells exhibited similar binding affinities and neutralizing potency against Wuhan-Hu-1 SARS-CoV-2, there were significant differences in epitope specificity and neutralizing breadth against viral variants of concern. Although the ChAdOx1 vaccine was inferior to mRNA and Ad26.COV.2S in several respects, biochemical and structural analyses revealed enrichment in a subgroup of memory B cell neutralizing antibodies with distinct RBD-binding properties resulting in remarkable potency and breadth.

Objective The development of sufficient COVID-19 vaccines has been a big breakthrough in fighting the global SARS-CoV-2 pandemic. However, vaccination effectiveness can be reduced in patients with autoimmune rheumatic diseases (AIRD). The aim of this study was to identify factors that lead to a diminished humoral vaccination response in patients with AIRD. Methods Vaccination response was measured with a surrogate virus neutralisation test and by testing for antibodies directed against the receptor-binding-domain (RBD) of SARS-CoV-2 in 308 fully vaccinated patients with AIRD. In addition, 296 immunocompetent participants were investigated as a control group. Statistical adjusted analysis included covariates with a possible influence on antibody response. Results Patients with AIRD showed lower antibody responses compared with immunocompetent individuals (median neutralising capacity 90.8% vs 96.5%, p<0.001; median anti-RBD-IgG 5.6 S/CO vs 6.7 S/CO, p<0.001). Lower antibody response was significantly influenced by type of immunosuppressive therapy, but not by rheumatic diagnosis, with patients under rituximab therapy developing the lowest antibody levels. Patients receiving mycophenolate, methotrexate or janus kinase inhibitors also showed reduced vaccination responses. Additional negative influencing factors were vaccination with AZD1222, old age and shorter intervals between the first two vaccinations. Conclusion Certain immunosuppressive therapies are associated with lower antibody responses after vaccination. Additional factors such as vaccine type, age and vaccination interval should be taken into account. We recommend antibody testing in at-risk patients with AIRD and emphasise the importance of booster vaccinations in these patients.

Upon microbial infection, cells of the innate immune system undergo profound metabolic reprogramming in order to eradicate pathogens, promote inflammation, and eventually restore tissue homeostasis. Mitochondria are at the core of these adaptations, given their dual role as metabolic hubs and innate immune signaling platforms. The mitochondrial electron transport chain (ETC) is very well characterized at the genetic, molecular, structural, and biochemical level. In contrast, the role for mitochondrial ETC and metabolites beyond fulfilling cellular ATP synthesis in innate immune cell biology was not understood until recently. Here we discuss the latest advances in our understanding of immune functions of mitochondria and particularly the mitochondrial respiratory chain.

Despite two years of intense global research activity, host genetic factors that predispose to a poorer prognosis of COVID-19 infection remain poorly understood. Here, we prioritise eight robust (e.g., ELF5) or suggestive but unreported (e.g., RAB2A) candidate protein mediators of COVID-19 outcomes by integrating results from the COVID-19 Host Genetics Initiative with population-based plasma proteomics using statistical colocalisation. The transcription factor ELF5 (ELF5) shows robust and directionally consistent associations across different outcome definitions, including a >4-fold higher risk (odds ratio: 4.88; 95%-CI: 2.47-9.63; p-value < 5.0 × 10-6) for severe COVID-19 per 1 s.d. higher genetically predicted plasma ELF5. We show that ELF5 is specifically expressed in epithelial cells of the respiratory system, such as secretory and alveolar type 2 cells, using single-cell RNA sequencing and immunohistochemistry. These cells are also likely targets of SARS-CoV-2 by colocalisation with key host factors, including ACE2 and TMPRSS2. In summary, large-scale human genetic studies together with gene expression at single-cell resolution highlight ELF5 as a risk gene for severe COVID-19, supporting a role of epithelial cells of the respiratory system in the adverse host response to SARS-CoV-2.

The level of neutralising capacity against Omicron BA.1 and BA.2 after third COVID-19 vaccination in patients on paused or continuous methotrexate (MTX) therapy is unclear.In this observational cohort study, neutralising serum activity against SARS-CoV-2 wild-type (Wu01) and variant of concern Omicron BA.1 and BA.2 were assessed by pseudovirus neutralisation assay before, 4 and 12 weeks after mRNA booster immunisation in 50 rheumatic patients on MTX, 26 of whom paused the medication. 44 non-immunosuppressed persons (NIP) served as control group.While the neutralising serum activity against SARS-CoV-2 Wu01 and Omicron variants increased 67-73 fold in the NIP after booster vaccination, the serum activity in patients receiving MTX increased only 20-23 fold. Patients who continued MTX treatment during vaccination had significantly lower neutralisation against all variants at weeks 4 and 12 compared with patients who paused MTX and the control group, except for BA.2 at week 12. Patients who paused MTX reached comparably high neutralising capacities as NIP, except for Wu01 at week 12. The duration of the MTX pause after-not before-was associated with a significantly higher neutralisation capacity against all three variants, with an optimal duration at 10 days after vaccination.Patients pausing MTX after COVID-19 booster showed a similar vaccine response to NIP. Patients who continued MTX demonstrated an impaired response indicating a potentially beneficial second booster vaccination. Our data also suggest that a 1 week MTX break is sufficient if the last administration of MTX occurs 1-3 days before vaccination.

Rationale: Pulmonary sarcoidosis is generally presumed to be a T-helper cell type 1- and macrophage-driven disease. However, mouse models have recently revealed that chronically inflamed lung tissue can also comprise T follicular helper (Tfh)-like cells and represents a site of active T-cell/B-cell cooperation. Objectives: To assess the role of pulmonary Tfh- and germinal center-like lymphocytes in sarcoidosis. Methods: BAL fluid, lung tissue, and peripheral blood samples from patients with sarcoidosis were analyzed by flow cytometry, immunohistology, RNA sequencing, and in vitro T-cell/B-cell cooperation assays for phenotypic and functional characterization of germinal center-like reactions in inflamed tissue. Measurements and Main Results: We identified a novel population of Tfh-like cells characterized by high expression of the B helper molecules CD40L and IL-21 in BAL of patients with sarcoidosis. Transcriptome analysis further confirmed a phenotype that was both Tfh-like and tissue resident. BAL T cells provided potent help for B cells to differentiate into antibody-producing cells. In lung tissue, we observed large peribronchial infiltrates with T and B cells in close contact, and many IgA+ plasmablasts. Most clusters were nonectopic; that is, they did not contain follicular dendritic cells. Patients with sarcoidosis also showed elevated levels of PD-1high CXCR5- CD40Lhigh ICOShigh Tfh-like cells, but not classical CXCR5+ Tfh cells, in the blood. Conclusions: Active T-cell/B-cell cooperation and local production of potentially pathogenic antibodies in the inflamed lung represents a novel pathomechanism in sarcoidosis and should be considered from both diagnostic and therapeutic perspectives.

Report28 September 2022Open Access Transparent process The human host response to monkeypox infection: a proteomic case series study Ziyue Wang Ziyue Wang orcid.org/0000-0002-4121-4799 Department of Biochemistry, Charité – Universitätsmedizin Berlin, Berlin, Germany Contribution: Formal analysis, Visualization, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Pinkus Tober-Lau Pinkus Tober-Lau orcid.org/0000-0002-9148-3663 Department of Infectious Diseases and Respiratory Medicine, Charité – Universitätsmedizin Berlin, Berlin, Germany Contribution: Data curation, Writing - original draft, Writing - review & editing Search for more papers by this author Vadim Farztdinov Vadim Farztdinov orcid.org/0000-0003-4441-2552 Core Facility High Throughput Mass Spectrometry, Charité – Universitätsmedizin Berlin, Berlin, Germany Contribution: Formal analysis, Visualization, Writing - original draft, Writing - review & editing Search for more papers by this author Oliver Lemke Oliver Lemke Department of Biochemistry, Charité – Universitätsmedizin Berlin, Berlin, Germany Contribution: Formal analysis, Visualization, Writing - original draft, Writing - review & editing Search for more papers by this author Torsten Schwecke Torsten Schwecke orcid.org/0000-0002-9439-3508 Department of Biochemistry, Charité – Universitätsmedizin Berlin, Berlin, Germany Contribution: Methodology, Writing - original draft Search for more papers by this author Sarah Steinbrecher Sarah Steinbrecher orcid.org/0000-0001-8760-3899 Department of Infectious Diseases and Respiratory Medicine, Charité – Universitätsmedizin Berlin, Berlin, Germany Contribution: Data curation Search for more papers by this author Julia Muenzner Julia Muenzner orcid.org/0000-0002-5402-5890 Department of Biochemistry, Charité – Universitätsmedizin Berlin, Berlin, Germany Contribution: Formal analysis, Writing - original draft Search for more papers by this author Helene Kriedemann Helene Kriedemann orcid.org/0000-0003-2209-6368 Department of Infectious Diseases and Respiratory Medicine, Charité – Universitätsmedizin Berlin, Berlin, Germany Contribution: Data curation Search for more papers by this author Leif Erik Sander Leif Erik Sander orcid.org/0000-0002-0476-9947 Department of Infectious Diseases and Respiratory Medicine, Charité – Universitätsmedizin Berlin, Berlin, Germany Berlin Institute of Health, Berlin, Germany Contribution: Project administration Search for more papers by this author Johannes Hartl Johannes Hartl orcid.org/0000-0001-8470-5355 Department of Biochemistry, Charité – Universitätsmedizin Berlin, Berlin, Germany Contribution: Data curation, Formal analysis, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Michael Mülleder Michael Mülleder orcid.org/0000-0001-9792-3861 Core Facility High Throughput Mass Spectrometry, Charité – Universitätsmedizin Berlin, Berlin, Germany Contribution: Conceptualization, Supervision, Funding acquisition, Methodology, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Markus Ralser Corresponding Author Markus Ralser [email protected] orcid.org/0000-0001-9535-7413 Department of Biochemistry, Charité – Universitätsmedizin Berlin, Berlin, Germany Berlin Institute of Health, Berlin, Germany The Wellcome Centre for Human Genetics, Nuffield Department of Medicine, University of Oxford, Oxford, UK Contribution: Conceptualization, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Florian Kurth Florian Kurth orcid.org/0000-0002-3807-473X Department of Infectious Diseases and Respiratory Medicine, Charité – Universitätsmedizin Berlin, Berlin, Germany Contribution: Conceptualization, Data curation, Supervision, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Ziyue Wang Ziyue Wang orcid.org/0000-0002-4121-4799 Department of Biochemistry, Charité – Universitätsmedizin Berlin, Berlin, Germany Contribution: Formal analysis, Visualization, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Pinkus Tober-Lau Pinkus Tober-Lau orcid.org/0000-0002-9148-3663 Department of Infectious Diseases and Respiratory Medicine, Charité – Universitätsmedizin Berlin, Berlin, Germany Contribution: Data curation, Writing - original draft, Writing - review & editing Search for more papers by this author Vadim Farztdinov Vadim Farztdinov orcid.org/0000-0003-4441-2552 Core Facility High Throughput Mass Spectrometry, Charité – Universitätsmedizin Berlin, Berlin, Germany Contribution: Formal analysis, Visualization, Writing - original draft, Writing - review & editing Search for more papers by this author Oliver Lemke Oliver Lemke Department of Biochemistry, Charité – Universitätsmedizin Berlin, Berlin, Germany Contribution: Formal analysis, Visualization, Writing - original draft, Writing - review & editing Search for more papers by this author Torsten Schwecke Torsten Schwecke orcid.org/0000-0002-9439-3508 Department of Biochemistry, Charité – Universitätsmedizin Berlin, Berlin, Germany Contribution: Methodology, Writing - original draft Search for more papers by this author Sarah Steinbrecher Sarah Steinbrecher orcid.org/0000-0001-8760-3899 Department of Infectious Diseases and Respiratory Medicine, Charité – Universitätsmedizin Berlin, Berlin, Germany Contribution: Data curation Search for more papers by this author Julia Muenzner Julia Muenzner orcid.org/0000-0002-5402-5890 Department of Biochemistry, Charité – Universitätsmedizin Berlin, Berlin, Germany Contribution: Formal analysis, Writing - original draft Search for more papers by this author Helene Kriedemann Helene Kriedemann orcid.org/0000-0003-2209-6368 Department of Infectious Diseases and Respiratory Medicine, Charité – Universitätsmedizin Berlin, Berlin, Germany Contribution: Data curation Search for more papers by this author Leif Erik Sander Leif Erik Sander orcid.org/0000-0002-0476-9947 Department of Infectious Diseases and Respiratory Medicine, Charité – Universitätsmedizin Berlin, Berlin, Germany Berlin Institute of Health, Berlin, Germany Contribution: Project administration Search for more papers by this author Johannes Hartl Johannes Hartl orcid.org/0000-0001-8470-5355 Department of Biochemistry, Charité – Universitätsmedizin Berlin, Berlin, Germany Contribution: Data curation, Formal analysis, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Michael Mülleder Michael Mülleder orcid.org/0000-0001-9792-3861 Core Facility High Throughput Mass Spectrometry, Charité – Universitätsmedizin Berlin, Berlin, Germany Contribution: Conceptualization, Supervision, Funding acquisition, Methodology, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Markus Ralser Corresponding Author Markus Ralser [email protected] orcid.org/0000-0001-9535-7413 Department of Biochemistry, Charité – Universitätsmedizin Berlin, Berlin, Germany Berlin Institute of Health, Berlin, Germany The Wellcome Centre for Human Genetics, Nuffield Department of Medicine, University of Oxford, Oxford, UK Contribution: Conceptualization, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Florian Kurth Florian Kurth orcid.org/0000-0002-3807-473X Department of Infectious Diseases and Respiratory Medicine, Charité – Universitätsmedizin Berlin, Berlin, Germany Contribution: Conceptualization, Data curation, Supervision, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Author Information Ziyue Wang1,†, Pinkus Tober-Lau2,†, Vadim Farztdinov3,†, Oliver Lemke1, Torsten Schwecke1, Sarah Steinbrecher2, Julia Muenzner1, Helene Kriedemann2, Leif Erik Sander2,4, Johannes Hartl1, Michael Mülleder3,†, Markus Ralser *,1,4,5,† and Florian Kurth2,† 1Department of Biochemistry, Charité – Universitätsmedizin Berlin, Berlin, Germany 2Department of Infectious Diseases and Respiratory Medicine, Charité – Universitätsmedizin Berlin, Berlin, Germany 3Core Facility High Throughput Mass Spectrometry, Charité – Universitätsmedizin Berlin, Berlin, Germany 4Berlin Institute of Health, Berlin, Germany 5The Wellcome Centre for Human Genetics, Nuffield Department of Medicine, University of Oxford, Oxford, UK † These authors contributed equally to this work *Corresponding author. Tel: +49 30 45052814; E-mail: [email protected] EMBO Mol Med (2022)14:e16643https://doi.org/10.15252/emmm.202216643 PDFDownload PDF of article text and main figures.PDF PLUSDownload PDF of article text, main figures, expanded view figures and appendix. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The rapid rise of monkeypox (MPX) cases outside previously endemic areas prompts for a better understanding of the disease. We studied the plasma proteome of a group of MPX patients with a similar infection history and clinical manifestation typical for the current outbreak. We report that MPX in this case series is associated with a strong plasma proteomic response among nutritional and acute phase response proteins. Moreover, we report a correlation between plasma proteins and disease severity. Contrasting the MPX host response with that of COVID-19, we find a range of similarities, but also important differences. For instance, CFHR1 is induced in COVID-19, but suppressed in MPX, reflecting the different roles of the complement system in the two infectious diseases. Of note, the spatial overlap in response proteins suggested that a COVID-19 biomarker panel assay could be repurposed for MPX. Applying a targeted protein panel assay provided encouraging results and distinguished MPX cases from healthy controls. Hence, our results provide a first proteomic characterization of the MPX human host response and encourage further research on protein-panel assays in emerging infectious diseases. Synopsis Plasma proteome analysis of patients with monkeypox (MPX) reveals key features of the host response to MPX infection. A COVID-19-derived biomarker panel was repurposed for this different viral disease. Distinct changes, especially in proteins associated with the acute phase and nutritional response, distinguish MPX cases from healthy controls. Several proteins, including SERPINA3, SAA1, LRG1, and apolipoproteins, correlate well with disease severity measured by the number of skin lesions. Due to the overlap in host response proteins, the possibility of repurposing biomarker panels across different viral infections was explored to improve pandemic preparedness. The paper explained Problem Until the recent outbreak, monkeypox was mainly confined to endemic areas in West and Central Africa, gaining little research interest. Aiming to breach the knowledge gap, we applied state-of-the-art plasma proteomics to a group of six patients with similar disease history and severity. Results Applying a recent proteomic method, ZenoSWATH-MS, on plasma samples obtained from a small but characteristic case series, we report distinct changes in proteins involved in the acute phase and nutritional response. Several proteins correlated with the number of skin lesions, indicating a potential use as disease severity markers. Comparing the proteomes to those of matched patients with COVID-19, we found numerous similarities. Moreover, we explored the usefulness of applying a proteomic COVID-19 biomarker panel assay to monkeypox cases and obtained a classification of the different disease groups. Impact This study is the first characterization of the human host response to monkeypox infection, offering insights into the pathophysiology. Moreover, we speculate that there is a thus far untapped potential for accelerating the response to disease outbreaks through the repurposing of biomarker assays. Introduction The outbreak of monkeypox (MPX) with currently more than 40,000 confirmed infections worldwide, is exceptional in scale and spread (Kraemer et al, 2022), and has been declared a global emergency by the WHO (World Health Organisation, 2022a). MPX is caused by the zoonotic monkeypox virus (MPXV), a member of the genus Orthopoxvirus (World Health Organisation, 2022b). The first human MPX case was reported in 1970 in the Democratic Republic of the Congo (DRC), which is still the region with the highest level of endemicity in Africa (Bunge et al, 2022). Several outbreaks have been reported from African countries during the past decades, but research on MPX has largely been neglected. The clinical presentation often includes typical skin lesions, fever, and swollen lymph nodes. MPX is usually self-limiting, but severe cases can occur and a case fatality rate of 1–10% has been reported from Africa, with generally higher case fatality associated with infections from the Central African viral clade compared to the West African virus clade (Bunge et al, 2022). The molecular epidemiology of the current MPX outbreak suggests that the current strain is closely related to that of a 2018–2019 outbreak in the United Kingdom and may have been circulating in the human population for some time, possibly with adaptation to the human host (Isidro et al, 2022; World Health Organisation, 2022c). In the current outbreak, there is a clear predominance of infections among men who have sex with men (MSM), and several large public events have been associated with the rapid emergence of cases in different parts of the world. Currently, transmission via close skin and mucosal contact, possibly including sexual transmission, seems likely (Dye & Kraemer, 2022; European Centre for Disease Prevention and Control, 2022; Pfäfflin et al, 2022; Thornhill et al, 2022). Even though the current outbreak is still in its early stages, a self-limiting course cannot be assumed; rather, it is a longer-term public-health problem that will hopefully bring diagnostic and therapeutic benefits to endemic African countries. The COVID-19 pandemic has reminded us of the need to create infrastructure and methodologies to respond rapidly to emerging pathogens. Mass spectrometry-based proteomics is one of the emerging technologies in this regard, which due to the technical and analytical advances during the last years is increasingly moving into clinical applications (Liotta et al, 2001; Messner et al, 2020; Struwe et al, 2020; He et al, 2022). In the early phase of the COVID-19 pandemic, proteomic analyses provided rapid insights into the nature of the human response to SARS-CoV-2 and captured hallmarks of its immune evasion strategies and pathophysiology, including its impact on the complement system, coagulation cascade, and inflammatory and nutritional response machinery (D'Alessandro et al, 2020; Messner et al, 2020; Shen et al, 2020; Demichev et al, 2021; Overmyer et al, 2021; Nuñez et al, 2022). Furthermore, proteomic signatures turned out to classify disease severity in COVID-19 and allow for outcome prediction weeks in advance (Völlmy et al, 2021; Demichev et al, 2022; Nuñez et al, 2022). Recently, we were able to show the strength of mass spectrometry-based proteomics for rapid translation to medical care by generating a routine-applicable proteomic biomarker panel which predicted COVID-19 severity and outcome in a multicohort study (Wang et al, 2022a). While such proteomic assays are currently primarily used to monitor clinical trials, they are increasingly being considered for their potential to optimize treatment and resource allocation, as well as to aid navigation of difficult triaging situations in the event of a pandemic. Here, we describe the proteomic changes in a case series, a small but characteristic group of patients hospitalized due to MPXV infection that share a similar disease and infection history. We detect significant and consistent proteomic changes caused by MPXV infection, enabling us to characterize the MPX host response at the proteomic level despite the moderate cohort size of a case series, in a timely manner. We report several protein markers that correlate with disease severity in the tested cases, that classify the disease proteome, and that contrast the human host response of MPXV to that of SARS-CoV-2 infection. Because we detected a partial overlap between the MPX and COVID-19 host response proteome, we also used a targeted proteomic panel developed for COVID-19 (Wang et al, 2022a) to explore the possibility of repurposing existing biomarker panels for classifying newly emerging infections. Although our results are derived from a small number of cases, they nonetheless suggest that repurposing of multiplex panel assays might be a viable strategy to improve pandemic preparedness. Our case series study provides a biochemical characterization of the MPX host response and reveals correlation of host proteins with MPX disease severity, and expands knowledge on protein panel testing for emerging infections. Results MPX patient case series and clinical presentation A group of five patients were hospitalized at Charité University Hospital between 26th and 31st May 2022 for treatment of MPX, detected by PCR from cutaneous blisters. Interestingly, all patients had attended the same social event 10–14 days before developing symptoms, three of whom considered it most likely to have been infected on that occasion. We then included a 6th patient with an unrelated infection history who was hospitalized in mid-June 2022, but that otherwise had a related disease history. All six patients were of European descent, and all self-identified as men having sex with men (MSM) having practiced receptive anal sexual intercourse within 14 days prior to hospitalization. The group of patients was therefore notably homogeneous regarding history and time course of infection, triggering our interest in a case series study. Overall, MPX patients exhibited mild to moderate symptoms, and no severe systemic affections such as encephalitis, myocarditis, or kidney failure were observed. Prodromes included fever, myalgia, and fatigue, and had already subsided in all patients by the time of admission to the hospital. The number of MPX skin lesions ranged from 5 to 36 and there were no clinical or laboratory signs of organ dysfunction. In all patients, the chief complaint and cause of hospitalization was severe anal or perianal pain requiring systemic analgesics in addition to topical treatment. Samples for proteome measurements were taken at a median of 8 days after symptom onset. Comorbidities included HIV (n = 2, both well controlled on antiretroviral therapy), other STIs (n = 1), and hepatitis C (n = 1). Patients were discharged with alleviated symptoms after 3–6 days. A summary of clinical characteristics is given in Table 1. Table 1. Patient characteristics. MPX cases (n = 6) Male, n (%) 6 100% Age, years 31 IQR: 27–41; range: 26–49 BMI, kg/m2 22.0 IQR: 19.6–23.4; range: 17.6–25.1 Comorbidities, n (%) 3 50% HIV, n (%) 2 33% Hepatitis C, n (%) 1 17% Other STIsa, n (%) 1 17% ∆ symptom onset to sample, days 8 IQR: 5–14; range: 5–17 ∆ PCR to sample, days 3.5 IQR 1.5–5; range: 0–5 Fever, n (%) 6 100% Number of lesions 9 IQR: 5–20; range: 5–36 Duration of hospital stay, days 3.5 IQR: 3–5; range: 3–6 C-reactive protein at admission, mg/l 20.0 IQR: 10.4–57.9; range: 8.7–120.8 Leukocytes at admission, per nl 9.7 IQR: 8.3–11.7; range: 8.1–12.9 Lymphocytes at admission, per nl 3.1 IQR: 1.6–3.7; range: 1.4–3.8 Lactate dehydrogenase, U/l 214 IQR: 203–273; range: 181–381 BMI, body mass index; HIV, human immunodeficiency virus infection; STI, sexually transmitted infection. Data are presented as median and IQR; range, unless otherwise specified. a Other STIs: co-infection with Neisseria gonorrhoeae, Ureaplasma, and Mycoplasma hominis. The partial sequence of the genome of the MPXV isolate obtained from one of the patients was determined and is available on GenBank (ON813251.2). To gain maximum information from the case series cohort, we assembled two control cohorts. The first consisted of 15 age- and sex-matched healthy volunteers (Table EV2). Ten patients with SARS-CoV-2 infection, hospitalized due to moderate COVID-19 (grade 3 on the 8-point WHO ordinal scale, i.e., without the need for supplemental oxygen therapy), constituted the second control group. Their proteomes were measured within the same batch on our MS platforms, but had also been analyzed by us as part of a previous study (Demichev et al, 2021). A plasma proteomic signature of MPXV infection Because of the moderate size of the case series study, we focused on obtaining maximally precise proteomic measurements and contrasted against both control groups. For obtaining proteomic measurements, we prepared tryptic digests from the MPX cases, matched healthy controls, and patients with moderate COVID-19, and included a broad panel of stable-isotope-labeled internal standards (PQ500, Biognosys). The tryptic digests obtained were then recorded using an online coupling of microflow chromatography and Zeno SWATH DIA, a latest generation of DIA proteomic technology (preprint: Wang et al, 2022b). Indeed, to our knowledge, the present study represents the first biomedical application of Zeno SWATH MS. After data were recorded as a single batch, raw data were processed with DIA-NN (Demichev et al, 2020), and data were post-processed to detect differentially concentrated proteins as well as the enrichment of pathway terms using pathway definitions from REACTOME (Croft et al, 2014). A workflow diagram of the procedures is provided (Fig 1A). Considering the relatively mild severity of clinical symptoms and skin manifestation, the data revealed a substantial proteomic response to MPXV infection within the abundant “functional fraction” of the plasma proteome. This proteome fraction constitutes more than 99% of the plasma proteomic mass and is composed of around 300 proteins, most of which directly function in the plasma (Anderson & Anderson, 2002). As 200–300 of them are consistently quantified using high-throughput proteomics in neat plasma (Messner et al, 2020), and because this fraction contains more than 50 typical protein biomarkers (Demichev et al, 2021) that capture host physiological parameters (Vernardis et al, 2022), this functional fraction of the plasma proteome is of special interest for the development of clinical assays (Wang et al, 2022a). After pre-processing, 226 of the highly abundant proteins were found consistently quantified in the neat plasma sample. We detected low within-group coefficients of variation, below 25% for MPX and control, and about 34% for COVID-19 cases, indicating a high quantitative precision of the measurements, but also the presence of a biological signal (Fig EV1C). Indeed, we found 56 of the major plasma proteins to be differentially abundant in MPX patients compared to healthy controls. Twenty-four of these were lower concentrated in MPX, and 32 detected at a higher concentration (Fig 1B). The nature of the affected proteins indicated the molecular processes affected by MPX, as revealed by an enrichment analysis. For example, we see “immune system” and “regulation of complement cascade” mostly enriched among upregulated pathways. Among downregulated pathways, “plasma lipoprotein assembly” and “metabolism of fat-soluble vitamins” are enriched (Fig 1C). Figure 1. The human host response to monkeypox virus infection determined at the level of the plasma proteome Schematic overview of the workflow using discovery proteomics (Zeno SWATH MS (Wang et al, 2022b)) in parallel to a targeted proteomic assay that quantifies COVID-19 severity biomarkers (Wang et al, 2022a) to characterize the plasma proteome in an MPX case series, and compare the proteomes to those of healthy volunteers and COVID-19 patients. Volcano plot of contrast MPX vs healthy controls; α <= 0.015 and ¦logFC¦ >= 1.35 were used for selection of regulated proteins. Gene set analysis (GSEA) of REACTOME (Croft et al, 2014) terms enrichment for contrast MPX vs control. Y-axis shows –log10 of adjusted P-value (fdr) for Normalized Enrichment Score (x-axis) for each term. Terms with fdr <= 0.3 are labeled. Boxplots illustrating key proteins that differ between patients with MPX and controls (P-values and fdr for corresponding contrast MPX vs Control are provided in brackets): TTR (P-value = 9E-10, fdr = 2E-7), LBP (P-value = 2E-4, fdr = 2E-3), APOC1 (P-value = 5E-8, fdr = 3E-6), and C9 (P-value = 2E-8, fdr = 2E-6). Here, as usual, the central bar marks the median (second quartile), the bottom edge of the box marks the first quartile, the top edge of the box marks the third quartile, and the bottom and top whiskers mark the minimum and maximum values that are not outliers. The specific values of the protein expressions are also shown. Provided P-values are obtained from moderated statistics implemented in limma, dfrs were calculated according to Benjamini-Hochberg. Correlation between MPX severity (NSkin lesions) and protein expression (y-axis). One MPX patient had an unclear additional skin condition (not a pure case of MPX) and therefore was excluded from the regression analysis that compares the number of skin lesions with the proteome; however, the proteome of this patient was largely in agreement with those of the other MPX cases (Fig EV3). As a measure of MPX severity, the log2(1 + NLesions / 15) was used. Here NLesions is the number of lesions. R2 shows squared correlation coefficient. MPX patients are colored orange, control patients green. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Samples quality control (QC) QC chart of standardized median expression of proteins the study sample. QC chart of standardized interquartile range (IQR) of protein expressions in the study samples. Box plot of within group coefficient of variation of protein intensities. Download figure Download PowerPoint At the level of individual proteins, the greatest differences between cases and controls were found in proteins associated with the acute phase response. These included significantly lower levels of the negative acute phase proteins TTR, ALB, and RBP4, as well as higher levels of acute phase proteins CRP, SAA1, SERPINA3, LBP, CP, and LRG1. Of note, various proteins involved in hepatic lipid metabolism and nutrient transport (APOA1, APOA2, APOC1, APOC2, APOC3) were lower in MPX patients than in controls, a known but not fully understood phenomenon also observed in other infections (Hardardóttir et al, 1995) (Fig 1D). Compared to controls, MPX patients exhibited a significantly higher level of complement component 9, the main element of the channel part of the membrane attack complex. Also, TTR in combination with the differentially expressed apolipoproteins is noteworthy, as it is a marker for malnutrition (Dellière et al, 2018), and we recently found it as a rapid responder in a caloric-restriction experiment conducted with healthy volunteers (Vernardis et al, 2022). We first speculated that acute MPX could result in a reduced caloric intake in affected patients. However, this picture was not confirmed by the clinical records of our patients, indicating that TTR is also part of the host response. We did not observe a significant influence of the concomitant conditions such as HIV or hepatitis C on the plasma proteomes. Results of the plasma proteomic response in patients with and without concomitant HIV infection are shown in Fig EV2. Both patients with HIV had immunologically well-controlled infections with suppressed viral load. Nevertheless, these patients can exhibit signs of ongoing immune activation, but if this response to HIV infection was present, it was masked by the acute response of the plasma proteome to the acute MPXV infection. Click here to expand this figure. Figure EV2. Exploratory analysis of monkeypox samples clustering according to comorbidities Hierarchical clustering using top 20% of most variable proteins. It is seen that HIV samples do not cluster together. PCA score plot using full proteome. It is seen that HIV is not a driving factor of samples variance. Its largest contribution is into the second principal component. Download figure Download PowerPoint Next, we tested whether there is a relationship between the proteomic response and the number of skin lesions observed in our patients, determined as a proxy of disease severity. Several peptides showed a statistically robust correlation with the number of lesions, including the upregulated acute phase proteins SERPINA3, SAA1, and LRG1, as well as the downregulated apolipoproteins APOA1, APOA2, and APOC3 (Fig 1E). In particular, LRG1, an upstream modifier of TGF-beta signaling, is being increasingly recognized as an important contributor to disease pathogenesis and hence as a potential therapeutic target in a range of inflammatory conditions (Camilli et al, 2022). Despite the moderate size of the case series, our data

Immune detection of microbial viability is increasingly recognized as a potent driver of innate and adaptive immune responses. Here we describe recent mechanistic insights into the process of how the immune system discriminates between viable and non-viable microbial matter. Accumulating evidence suggests a key role for microbial RNA as a widely conserved viability associated PAMP (vita-PAMP) and a molecular signal of increased infectious threat. Toll-like receptor 8 (TLR8) has recently emerged as a critical sensor for viable bacteria, ssRNA viruses, and archaea in human antigen presenting cells (APC). We discuss the role of microbial RNA, and other potential vita-PAMPs in antimicrobial immunity and vaccine responses.

ABSTRACT The clinical course of COVID-19 is highly variable, however, underlying host factors and determinants of severe disease are still unknown. Based on single-cell transcriptomes of nasopharyngeal and bronchial samples from clinically well-characterized patients presenting with moderate and critical severities, we reveal the different types and states of airway epithelial cells that are vulnerable for SARS-CoV-2 infection. In COVID-19 patients, we observed a two- to threefold increase of cells expressing the SARS-CoV-2 entry receptor ACE2 within the airway epithelial cell compartment. ACE2 is upregulated in epithelial cells through Interferon signals by immune cells suggesting that the viral defense system may increase the number of potentially susceptible cells in the respiratory epithelium. Infected epithelial cells recruit and activate immune cells by chemokine signaling. Recruited T lymphocytes and inflammatory macrophages were hyperactivated and showed a strong interaction with epithelial cells. In critical patients, increased expression of CCL2, CCL3, CCL5, CXCL9, CXCL10, IL8, IL1B and TNF in macrophages was identified as a likely cause of a hyperinflammatory lung pathology. Moreover, we observed exacerbated epithelial cell death, likely leading to lung injury and respiratory failure in fatal cases. Our study provides novel insights into the pathophysiology of COVID-19 and suggests an immunomodulatory therapy along the CCL2, CCL3/CCR1 axis as promising option to prevent and treat critical course of COVID-19.

The human malaria parasite Plasmodium falciparum relies on lipids to survive; this makes its lipid metabolism an attractive drug target. The lipid phosphatidylserine (PS) is usually confined to the inner leaflet of the red blood cell membrane (RBC) bilayer; however, some studies suggest that infection with the intracellular parasite results in the presence of this lipid in the RBC membrane outer leaflet, where it could act as a recognition signal to phagocytes. Here, we used fluorescent lipid analogues and probes to investigate the enzymatic reactions responsible for maintaining asymmetry between membrane leaflets, and found that in parasitised RBCs the maintenance of membrane asymmetry was partly disrupted, and PS was increased in the outer leaflet. We examined the underlying causes for the differences between uninfected and infected RBCs using fluorescent dyes and probes, and found that calcium levels increased in the infected RBC cytoplasm, whereas membrane cholesterol was depleted from the erythrocyte plasma membrane. We explored the resulting effect of PS exposure on enhanced phagocytosis by monocytes, and show that infected RBCs must expend energy to limit phagocyte recognition, and provide experimental evidence that PS exposure contributes to phagocytic recognition of P. falciparum-infected RBCs. Together, these findings underscore the pivotal role for PS exposure on the surface of Plasmodium falciparum-infected erythrocytes for in vivo interactions with the host immune system, and provide a rationale for targeted antimalarial drug design.

Abstract The Omicron variant of SARS-CoV-2 is causing a rapid increase in infections in various countries. This new variant of concern carries an unusually high number of mutations in key epitopes of neutralizing antibodies on the spike glycoprotein, suggesting potential immune evasion. Here we assessed serum neutralizing capacity in longitudinal cohorts of vaccinated and convalescent individuals, as well as monoclonal antibody activity against Omicron using pseudovirus neutralization assays. We report a near-complete lack of neutralizing activity against Omicron in polyclonal sera after two doses of the BNT162b2 vaccine, in convalescent individuals, as well as resistance to different monoclonal antibodies in clinical use. However, mRNA booster immunizations in vaccinated and convalescent individuals resulted in a significant increase of serum neutralizing activity against Omicron. Our study demonstrates that booster immunizations will be critical to substantially improve the humoral immune response against the Omicron variant.

ABSTRACT Streptococcus pneumoniae is a frequent colonizer of the upper respiratory tract and a leading cause of bacterial pneumonia. The innate immune system senses pneumococcal cell wall components, toxin, and nucleic acids, which leads to production of inflammatory mediators to initiate and control antibacterial defense. Here, we show that the cGAS (cyclic GMP-AMP [cGAMP] synthase)-STING pathway mediates detection of pneumococcal DNA in mouse macrophages to primarily stimulate type I interferon (IFN) responses. Cells of human individuals carrying HAQ TMEM173 , which encodes a common hypomorphic variant of STING, were largely or partly defective in inducing type I IFNs and proinflammatory cytokines upon infection. Subsequent analyses, however, revealed that STING was dispensable for restricting S. pneumoniae during acute pneumonia in mice. Moreover, explorative analyses did not find differences in the allele frequency of HAQ TMEM173 in nonvaccinated pneumococcal pneumonia patients and healthy controls or an association of HAQ TMEM173 carriage with disease severity. Together, our results indicate that the cGAS/STING pathway senses S. pneumoniae but plays no major role in antipneumococcal immunity in mice and humans.

Abstract Children are consistently reported to have reduced SARS-CoV-2 infection rates and a substantially lower risk for developing severe COVID-19. However, the molecular mechanisms underlying protection against COVID-19 in younger age groups remain widely unknown. Here, we systematically characterized the single-cell transcriptional landscape in the upper airways in SARS-CoV-2 negative and age-matched SARS-CoV-2 positive children (n=42) and corresponding samples from adults (n=44), covering an age range of four weeks to 77 years. Children displayed higher basal expression of the relevant pattern recognition receptor (PRR) pathways in upper airway epithelial cells, macrophages, and dendritic cells, resulting in stronger innate antiviral responses upon SARS-CoV-2 infection compared to adults. We further detected distinct immune cell subpopulations with an overall dominance of neutrophils and a population of cytotoxic T cells occurring predominantly in children. Our study provides evidence that the airway epithelial and mucosal immune cells of children are pre-activated and primed for virus sensing, resulting in a stronger early innate antiviral responses to SARS-CoV-2 infection compared to adults.

Disease-modifying therapies (DMTs) are widely used in neuroimmunological diseases such as multiple sclerosis (MS) and neuromyelitis optica spectrum disorder (NMOSD). Although these treatments are known to predispose patients to infections and affect their responses to vaccination, little is known about the impact of DMTs on the myeloid cell compartment. In this study, we use mass cytometry to examine DMT-associated changes in the innate immune system in untreated and treated patients with MS (n = 39) or NMOSD (n = 23). We also investigated the association between changes in myeloid cell phenotypes and longitudinal responsiveness to homologous primary, secondary, and tertiary SARS-CoV-2 mRNA vaccinations. Multiple DMT-associated myeloid cell clusters, in particular CD64+HLADRlow granulocytes, showed significant correlations with B and T cell responses induced by vaccination. Our findings suggest the potential role of myeloid cells in cellular and humoral responses following vaccination in DMT-treated patients with neuroimmunological diseases.

Abstract Macrophages specialize in phagocytosis, a cellular process that eliminates extracellular matter, including microbes, through internalization and degradation. Despite the critical role of phagocytosis during bacterial infection, the fate of phagocytosed microbial cargo and its impact on host cell is poorly understood. Here, we reveal that ingested bacteria constitute an alternative nutrient source that skews immunometabolic host responses. Tracing stable isotope-labelled bacteria, we found that phagolysosomal degradation of bacteria provides carbon atoms and amino acids that are recycled into various metabolic pathways, including glutathione and itaconate biosynthesis, and satisfy macrophage bioenergetic needs. Metabolic recycling of microbially-derived nutrients is regulated by the nutrient sensing mTORC1 and intricately tied to microbial viability. Dead bacteria, as opposed to live ones, sustain the cellular adenosine monophosphate (AMP) pool and subsequently activate AMP protein kinase (AMPK) to inhibit mTORC1. Consequently, killed bacteria strongly fuel metabolic recycling, but elicit decreased reactive oxygen species (ROS) production and a reduced IL-1β secretion compared to viable bacteria. These results reveal a novel insight into the fate of engulfed microbes and highlights a microbial viability-associated metabolite that triggers host metabolic and immune responses. Our findings hold promise for shaping immunometabolic intervention in various immune-related pathologies.

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Federal Ministry of Education and Research. Background/Introduction To increase vaccine safety, adverse events of COVID-19 vaccines are continuously monitored. This also includes rare complications of the cardiovascular system. However, it has so far been scarcely investigated whether chronic and subtle, clinically inapparent myocardial and/or vascular injury occurs in a COVID-19 vaccinated population on a broader basis. Purpose The aim of this study was to analyse chronic and subclinical adverse effects of COVID-19 vaccines on the cardiovascular system. Vaccine-associated myocardial involvement was examined by measuring high-sensitive troponin T (hsTnT) while mid-regional pro-adrenomedullin (MR-proADM) levels were evaluated to assess endothelial dysfunction. Methods This was a prospective observational study with a vulnerable population of healthcare workers (HCWs) and elderly (&amp;gt; 70 years) patients who were vaccinated with either the ChAdOx1 nCov-19 adenoviral vector vaccine (AZ) and the BNT162b2 mRNA vaccine (BNT), or with BNT/BNT between the 12th of January and 30th of November 2021 (Figure 1). HsTnT and MR-proADM was measured in blood samples at three visits (V1: 1st baseline, immediately before vaccination; V2: 3-4 weeks after 1st vaccination; V3: 3-4 weeks after 2nd vaccination). HsTnT levels were further compared with a healthy reference cohort. Results 120 participants were included (V1=119, V2=120, V3=120). 50 (41.7%) received AZ/BNT. 70 (58.3%) were vaccinated with BNT/BNT of whom 20 (16.7%) were elderly and 50 (41.7%) were HCWs. Median hsTnT levels were at 4ng/L, 5ng/L and 4ng/L (V1-V3) for AZ/BNT and at 6ng/L, 6ng/L and 6ng/L (V1-V3) for BNT/BNT, respectively (Figure 2). Levels of MR-proADM were 0.43nmol/L, 0.46nmol/L and 0.44nmol/L (V1-V3) in the AZ/BNT cohort and 0.49nmol/L, 0.43nmol/L and 0.49nmol/L in the BNT/BNT group. Change of hsTnT and MR-proADM values between visits did not show significant increases. Two cases had a transient (7ng/L, 19ng/L, 5ng/L; male, 29 years) or permanent (15ng/L, 21ng/L, 25ng/L; male, 37 years) increase in hsTnT levels, exceeding the upper reference limit (≥ 14ng/L). Compared to the reference population (n=300), median hsTnT was significantly higher at all visits for both vaccination groups (p&amp;lt;0.05), without significant differences between the AZ/BNT and BNT/BNT cohorts. Conclusion(s) With two individual exceptions, no overall chronic myocardial injury or endothelial involvement was observed up to 4 weeks after the 2nd SARS-CoV-2 vaccination when comparing hsTnT and MR-proADM after both vaccinations to baseline values.Figure 1Vaccination scheme.Figure 2.hsTnT values at V1-V3.

Disease progression of subjects with coronavirus disease 2019 (COVID-19) varies dramatically. Understanding the various types of immune response to SARS-CoV-2 is critical for better clinical management of coronavirus outbreaks and to potentially improve future therapies. Disease dynamics can be characterized by deciphering the adaptive immune response.In this cross-sectional study we analyzed 117 peripheral blood immune repertoires from healthy controls and subjects with mild to severe COVID-19 disease to elucidate the interplay between B and T cells. We used an immune repertoire Primer Extension Target Enrichment method (immunoPETE) to sequence simultaneously human leukocyte antigen (HLA) restricted T cell receptor beta chain (TRB) and unrestricted T cell receptor delta chain (TRD) and immunoglobulin heavy chain (IgH) immune receptor repertoires. The distribution was analyzed of TRB, TRD and IgH clones between healthy and COVID-19 infected subjects. Using McFadden's Adjusted R2 variables were examined for a predictive model. The aim of this study is to analyze the influence of the adaptive immune repertoire on the severity of the disease (value on the World Health Organization Clinical Progression Scale) in COVID-19.Combining clinical metadata with clonotypes of three immune receptor heavy chains (TRB, TRD, and IgH), we found significant associations between COVID-19 disease severity groups and immune receptor sequences of B and T cell compartments. Logistic regression showed an increase in shared IgH clonal types and decrease of TRD in subjects with severe COVID-19. The probability of finding shared clones of TRD clonal types was highest in healthy subjects (controls). Some specific TRB clones seems to be present in severe COVID-19 (Figure S7b). The most informative models (McFadden´s Adjusted R2=0.141) linked disease severity with immune repertoire measures across all three cell types, as well as receptor-specific cell counts, highlighting the importance of multiple lymphocyte classes in disease progression.Adaptive immune receptor peripheral blood repertoire measures are associated with COVID-19 disease severity.The study was funded with grants from the Berlin Institute of Health (BIH).

The rapid emergence of multidrug-resistant pathogens, especially Gram-negative bacteria and mycobacteria, represents one of the major medical challenges of the 21st century. The gradual loss of effective classical antibiotics for many bacterial pathogens, combined with an increasing population density and mobility, urgently calls for the development of novel treatments. Here, we discuss the potential of adjuvant immunotherapies to selectively stimulate protective immune responses as a treatment option for bacterial infections. In order to elicit appropriate immune responses and to avoid unwanted inflammatory tissue damage, it is essential to identify ligands and receptor pathways that specifically control protective responses at the site of infection. We summarize existing data and discuss suitable candidate targets for future immunotherapies of infectious diseases.

A vaccine adjuvant elicits broad protection against influenza in animals

Type 2 diabetes mellitus (T2DM) and chronic obstructive pulmonary disease (COPD) often co-exist, yielding increased risk of cardiovascular (CV) complications including heart failure (HF). We assessed risk of cardiorenal outcomes, mortality and safety in patients with versus without COPD in the EMPA-REG OUTCOME trial.Patients (n = 7,020) with T2DM and CV disease (CVD) were treated with empagliflozin (10 mg or 25 mg) or placebo. Cox regression was used to assess COPD subgroup (placebo only) associations with, and treatment effects of empagliflozin versus placebo on first hospitalization for HF (HHF), CV death, all-cause mortality, incident/worsening nephropathy, and all-cause hospitalization.At baseline, patients with COPD (n = 707) had more HF and used insulin more frequently than those without COPD. During follow-up in the placebo group, those with baseline COPD had increased risk of HHF (HR 2.15 [95% CI 1.32, 3.49]), HHF/CV death (1.60 [1.10, 2.33]), incident/worsening nephropathy (1.68 [1.26, 2.24]), all-cause hospitalization (1.44 [1.19, 1.74]) and all-cause death (1.60 [1.09, 2.35]) compared to those without COPD. Empagliflozin consistently reduced all clinical outcomes, irrespective of COPD status (interaction p-values 0.14 to 0.96), with a confirmed safety profile.In patients with T2DM and CVD, COPD increased the risk of mortality and cardiorenal outcomes, including HF. Empagliflozin consistently reduced these outcomes versus placebo regardless of COPD, suggesting that empagliflozin's benefits in patients with T2DM and CVD are not mitigated by the presence of COPD.

Abstract Glycoprotein 90K, encoded by the interferon-stimulated gene LGALS3BP , displays broad antiviral activity. It reduces HIV-1 infectivity by interfering with Env maturation and virion incorporation, and increases survival of Influenza A virus-infected mice via antiviral innate immune signaling. Its antiviral potential in SARS-CoV-2 infection remains largely unknown. Here, we analyzed the expression of 90K/ LGALS3BP in 44 hospitalized COVID-19 patients at multiple levels. We quantified 90K protein concentrations in serum and PBMCs as well as LGALS3BP mRNA levels. Complementary, we analyzed two single cell RNA-sequencing datasets for expression of LGALS3BP in respiratory specimens and PBMCs from COVID-19 patients. Finally, we analyzed the potential of 90K to interfere with SARS-CoV-2 infection of HEK293T/ACE2, Calu-3 and Caco-2 cells using authentic virus. 90K protein serum concentrations were significantly elevated in COVID-19 patients compared to uninfected sex- and age-matched controls. Furthermore, PBMC-associated concentrations of 90K protein were overall reduced by SARS-CoV-2 infection in vivo, suggesting enhanced secretion into the extracellular space. Mining of published PBMC scRNA-seq datasets uncovered monocyte-specific induction of LGALS3BP mRNA expression in COVID-19 patients. In functional assays, neither 90K overexpression in susceptible cell lines nor exogenous addition of purified 90K consistently inhibited SARS-CoV-2 infection. Our data suggests that 90K/ LGALS3BP contributes to the global type I IFN response during SARS-CoV-2 infection in vivo without displaying detectable antiviral properties in vitro.

Commercially available ELISA-based antibody tests are used to approximate vaccination success against SARS-CoV-2 in at-risk patients, but it is unclear whether they correlate with neutralization of the Omicron variant. 269 serum samples of a cohort of 44 non-immunosuppressed participants and 65 MTX-treated rheumatic patients taken before and after COVID-19 booster vaccinations were measured using COVID-19 antibody testing systems with wild-type and Omicron BA.1 antigens developed by three different manufacturers (surrogate virus neutralization test cPass, and binding antibody tests QuantiVac and SeraSpot), as well as with a pseudovirus neutralization test (pVNT). The pVNT was considered the gold standard for determining the presence and level of anti-SARS-CoV-2 antibodies. All three wild-type ELISAs showed excellent test performance compared with wild-type neutralization in pVNT. However, out of 56 samples without Omicron BA.1 neutralization in pVNT, 71.4% showed positive results in at least one and 28.6% in all three wild-type ELISAs at the manufacturer-defined cut-offs. Omicron ELISAs showed either decreased specificity (57.1% and 55.4% for binding ELISAs) or sensitivity (51.2% in cPass) compared to Omicron neutralization in pVNT. The proportion of any false positive results among all samples decreased from 26.5% before to 3.2% after booster vaccination, however binding antibody test specificities remained below 70%. We found a poorer test performance of new Omicron antibody test systems compared to wild-type tests in detecting neutralizing antibodies against the corresponding SARS-CoV-2 variants. Decisions for booster vaccination or passive immunization of at-risk patients should not be based solely on antibody test results.

Secondary immune events likely aggravate Covid-19 pathology. In inflammation and homeostasis macrophages are indispensable regulators of pulmonary tissue integrity. We investigated pulmonary macrophage states in two independent cohorts of patients suffering from Covid-19&nbsp;ARDS&nbsp;employing functional single cell transcriptomics and multiplexed histopathological methods. CD163+ macrophage numbers were increased in lung tissues of deceased Covid-19 patients. Transcriptomes of broncheoalveolar lavage macrophages dispersed in between 3 poles in integrated gene spaces. Next to a monocytic pole, Macrophages were either mainly of conventional alveolar macrophage differentiation with elevated lipid/cholesterol processing, or exhibited a third gene profile, of likely recent hematopoietic origin. Strikingly, these CD163+/LGMN+ cells highly expressed gene signatures of macrophage polarizations found in pulmonary fibrotic disease such as Idiopathic Pulmonary Fibrosis (IPF). Furthermore, in primary human in-vitro cultured monocytes, SARS-CoV-2 co-incubation directly&nbsp;induced the expression of these fibrosis-associated&nbsp;programs, which were distinct from inflammatory or antiviral responses. These findings were&nbsp;present&nbsp;in proteomic as well as single cell transcriptomic analyses. Moreover, we present radiological, ultra-structural, and lung mechanical evidence of Covid-19-triggered pulmonary fibrosis. In conclusion, macrophages in pulmonary tissue of&nbsp;Covid-19 ARDS patients expressed fibrosis-associated gene modules which were inducible by direct in-vitro stimulation of monocytes with SARS-CoV-2.

While evidence for pre-existing SARS-CoV-2-cross-reactive CD4 + T cells in unexposed individuals is increasing, their functional significance remains unclear. Here, we comprehensively determined SARS-CoV-2-cross-reactivity and human coronavirus-reactivity in unexposed individuals. SARS-CoV-2-cross-reactive CD4 + T cells were ubiquitous, but their presence decreased with age. Within the spike glycoprotein fusion domain, we identified a universal immunodominant coronavirus-specific peptide epitope ( iCope ). Pre-existing spike- and iCope -reactive memory T cells were efficiently recruited into mild SARS-CoV-2 infections and their abundance correlated with higher IgG titers. Importantly, the cells were also reactivated after primary BNT162b2 COVID-19 mRNA vaccination in which their kinetics resembled that of secondary immune responses. Our results highlight the functional importance of pre-existing spike-cross-reactive T cells in SARS-CoV-2 infection and vaccination. Abundant spike-specific cross-immunity may be responsible for the unexpectedly high efficacy of current vaccines even with single doses and the high rate of asymptomatic/mild infection courses.

Abstract Recent advances have highlighted the outstanding role of the innate immune system for instructing adaptive immunity. Translating this knowledge into successful immunotherapies like vaccines, however, has proven to be a difficult task. This essay is based on the hypothesis that immune responses are tightly scaled to the infectious threat posed by a given microbial stimulus. A meticulous immunological risk‐assessment process is therefore instrumental for eliciting well‐balanced responses and maintaining immune homeostasis. The immune system makes fine distinctions, for example, between live and dead bacteria, or pathogenic and non‐pathogenic microorganisms. Here, I discuss recent evidence for some of the mechanisms underlying these distinctions and speculate on strategies for therapeutically targeting the immunological risk‐assessment machinery.

The threonine phosphatase eyes absent (EYA) has been identified as a novel regulator of innate immune responses to cytosolic nucleic acids and undigested DNA from apoptotic cells. EYA regulates responses of yet unidentified DNA sensors and enhances interferon-beta and CXCL10 transcription.

Severe COVID-19 is marked by activated, highly cytotoxic

Cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl - channel and ABC transporter; its mutations cause the clinical picture of cystic fibrosis (CF). Of late, CFTR has emerged as an important regulator of platelet function, as CFTR dysfunction causes agonist-induced platelet hyperactivation. These findings are reminiscent of platelets from SARS-CoV-2 infected patients since thromboembolic complications represent hallmarks of severe COVID-19 that may critically contribute to morbidity and mortality. CFTR modulators have recently been introduced as a treatment for patients with various CFTR mutations, but have also been reported by us and others to enhance channel function of wild type CFTR. We therefore postulated that CFTR modulators may exert anti-coagulant effects on platelets of healthy donors (HD) and COVID-19 patients.We recruited 36 COVID-19 patients with moderate, and 34 COVID-19 patients with severe disease course (all w/o anti-platelet drugs), and 38 HDs. In line with our hypothesis, we observed significant reductions in platelet agonists adenosine diphosphate (ADP)- or thrombin receptor activating protein-6 (TRAP6)-induced CD62p/CD63 expression, Ca 2+ -mobilization, aggregation, and adhesion of platelets from HDs by pre-treatment with ivacaftor. In blood from COVID-19 patients, platelet activation correlates with disease severity, as demonstrated by a 5-fold and 8-fold increase in the proportion of CD62p + platelets from patients with moderate and severe disease, respectively, relative to HDs. Similarly, the proportion of CD63 + platelets in patients with severe COVID-19 was 2-fold higher than in HDs. Retrospective analysis of clinical data from a total of 4,050 CF patients with COVID-19 receiving single or combination therapy of ivacaftor, lumacaftor, tezacaftor, or elexacaftor in comparison to an untreated cohort revealed that CF therapy reduced the relative risk to suffer thromboembolism-associated cardiovascular events such as heart attack or deep vein thrombosis by 50.0% or 61.1%, respectively, suggesting an anti-thrombotic effect of CFTR modulators in CF COVID-19 patients. In line with this observation, ex vivo pre-treatment of platelets from acute COVID-19 patients with ivacaftor reduced Ca 2+ mobilization, adhesion, and aggregation of platelets .Our results demonstrate an anticoagulant effect of CFTR potentiators on platelets from HDs and severe COVID-19 patients and thus, suggest CFTR potentiators as a promising strategy to reduce the risk of thrombotic events in the clinical management of COVID-19 and similar pro-thrombotic disease states. F. Behrens received funding from the Berlin Institute of Health (BIH). L. Michalick reports grants from the BIH and the German Centre for Cardiovascular Research (DZHK). A. Haghikia is participant in the BIH-Charité Advanced Clinician Scientist Pilotprogram funded by the Charité - Universitätsmedizin Berlin and the BIH and reports a research grant within the BIH &amp; MDC Focus Area Translational Vascular Biomedicine. R. Preissner reports partial funding of this work by the German Research Foundation (KFO339, TRR295). M. Witzenrath reports grants from the German Research Foundation (SFB-TR84 C06 and C09, SFB 1449 B02), and from the German Ministry of Education and Research (BMBF) in the framework of CAPSyS (01ZX1604B, 01ZX1304B), SYMPATH (01ZX1906A), PROVID (01KI20160A), Phage4Cure (16GW0141), MAPVAP (16GW0247) and NUM-NAPKON. W. M. Kuebler reports grants from the German Research Foundation (SFB-TR84 A2 and C9, SFB 1449 B1, SFB 1470 A4, KU1218/9-1, KU1218/11-1, and KU1218/12-1), the BMBF in the framework of SYMPATH (01ZX1906A) and PROVID (01KI20160A), and the DZHK. S. Simmons reports grants from the DZHK and the German Foundation for Heart Research (F-09-19). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The COVID-19 pandemic has spurred large-scale, inter-institutional research efforts. To enable these efforts, researchers must agree on dataset definitions that not only cover all elements relevant to the respective medical specialty but that are also syntactically and semantically interoperable. Following such an effort, the German Corona Consensus (GECCO) dataset has been developed previously as a harmonized, interoperable collection of the most relevant data elements for COVID-19-related patient research. As GECCO has been developed as a compact core dataset across all medical fields, the focused research within particular medical domains demands the definition of extension modules that include those data elements that are most relevant to the research performed in these individual medical specialties.To (i) specify a workflow for the development of interoperable dataset definitions that involves a close collaboration between medical experts and information scientists and to (ii) apply the workflow to develop dataset definitions that include data elements most relevant to COVID-19-related patient research regarding immunization, pediatrics, and cardiology.We developed a workflow to create dataset definitions that are (i) content-wise as relevant as possible to a specific field of study and (ii) universally usable across computer systems, institutions, and countries, i.e., interoperable. We then gathered medical experts from three specialties (infectious diseases with a focus on immunization, pediatrics, and cardiology) to the select data elements most relevant to COVID-19-related patient research in the respective specialty. We mapped the data elements to international standardized vocabularies and created data exchange specifications using HL7 FHIR. All steps were performed in close interdisciplinary collaboration between medical domain experts and medical information specialists. The profiles and vocabulary mappings were syntactically and semantically validated in a two-stage process.We created GECCO extension modules for the immunization, pediatrics, and cardiology domains with respect to the pandemic requests. The data elements included in each of these modules were selected according to the here developed consensus-based workflow by medical experts from the respective specialty to ensure that the contents are aligned with the respective research needs. We defined dataset specifications for a total number of 48 (immunization), 150 (pediatrics), and 52 (cardiology) data elements that complement the GECCO core dataset. We created and published implementation guides and example implementations as well as dataset annotations for each extension module.These here presented GECCO extension modules, which contain data elements most relevant to COVID-19-related patient research in infectious diseases with a focus on immunization, pediatrics and cardiology, were defined in an interdisciplinary, iterative, consensus-based workflow that may serve as a blueprint for the development of further dataset definitions. The GECCO extension modules provide a standardized and harmonized definition of specialty-related datasets that can help to enable inter-institutional and cross-country COVID-19 research in these specialties.

Abstract Background The severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) pandemic causes a high burden of acute and long-term morbidity and mortality worldwide despite global efforts in containment, prophylaxis, and therapy. With unprecedented speed, the global scientific community has generated pivotal insights into the pathogen and the host response evoked by the infection. However, deeper characterization of the pathophysiology and pathology remains a high priority to reduce morbidity and mortality of coronavirus disease 2019 (COVID-19). Methods NAPKON-HAP is a multi‐centered prospective observational study with a long‐term follow‐up phase of up to 36 months post-SARS-CoV-2 infection. It constitutes a central platform for harmonized data and biospecimen for interdisciplinary characterization of acute SARS-CoV-2 infection and long-term outcomes of diverging disease severities of hospitalized patients. Results Primary outcome measures include clinical scores and quality of life assessment captured during hospitalization and at outpatient follow-up visits to assess acute and chronic morbidity. Secondary measures include results of biomolecular and immunological investigations and assessment of organ-specific involvement during and post-COVID-19 infection. NAPKON-HAP constitutes a national platform to provide accessibility and usability of the comprehensive data and biospecimen collection to global research. Conclusion NAPKON-HAP establishes a platform with standardized high-resolution data and biospecimen collection of hospitalized COVID-19 patients of different disease severities in Germany. With this study, we will add significant scientific insights and provide high-quality data to aid researchers to investigate COVID-19 pathophysiology, pathology, and chronic morbidity.

Abstract The clinical course of COVID-19 is variable and often unpredictable. To test the hypothesis that disease progression and inflammatory responses associate with alterations in the microbiome and metabolome, we analyzed metagenome, metabolome, cytokine, and transcriptome profiles of repeated samples from hospitalized COVID-19 patients and uninfected controls, and leveraged clinical information and post-hoc confounder analysis. Severe COVID-19 was associated with a depletion of beneficial intestinal microbes, whereas oropharyngeal microbiota disturbance was mainly linked to antibiotic use. COVID-19 severity was also associated with enhanced plasma concentrations of kynurenine and reduced levels of several other tryptophan metabolites, lysophosphatidylcholines, and secondary bile acids. Moreover, reduced concentrations of various tryptophan metabolites were associated with depletion of Faecalibacterium , and tryptophan decrease and kynurenine increase were linked to enhanced production of inflammatory cytokines. Collectively, our study identifies correlated microbiome and metabolome alterations as a potential contributor to inflammatory dysregulation in severe COVID-19.

Abstract Background Erythema multiforme (EM) is an acute, immune-mediated mucocutaneous disease, most often preceded by herpes simplex virus (HSV) infection or reactivation. Mycoplasma pneumoniae (Mp) is considered the second major trigger of EM and is often associated with an atypical and more severe presentation of disease, characterized by prominent mucosal involvement. However, contrary to HSV-associated Erythema multiforme (HAEM), immunological mechanisms of Mp-associated EM remain unclear. Case presentation We present the case of a 50-year-old male patient presenting with community-acquired pneumonia (CAP) and erythema multiforme majus (EMM). Acute Mp infection was diagnosed by seroconversion, with no evidence of HSV infection as a cause of EMM. We performed immune phenotyping of blister fluid (BF) and peripheral blood (PB) T cells and detected a clonally expanded TCRVβ2 + T cell population that was double positive for CD4 and CD8, and expressed the cytotoxic markers granulysin and perforin. This CD4 + CD8 + population comprised up to 50.7% of BF T cells and 24.9% of PB T cells. Two years prior to the onset of disease, the frequency of PB CD4 + CD8 + T cells had been within normal range and it gradually returned to baseline levels with the resolution of symptoms, suggesting an involvement of this population in EMM disease pathophysiology. Conclusions This report is the first to provide a phenotypic description of lesional T cells in Mp-associated EMM. Characterizing the local immune response might help to address pathophysiological questions and warrants further systematic research.

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ABSTRACT Objective To determine the immediate need for a fourth COVID-19 vaccination based on the neutralizing capacity in patients on methotrexate (MTX) therapy after mRNA booster immunization. Methods In this observational cohort study, neutralizing serum activity against SARS-CoV-2 wildtype (Wu01) and variant of concern (VOC) Omicron BA.1 and BA.2 were assessed by pseudovirus neutralization assay before, 4 and 12 weeks after mRNA booster immunization in 50 rheumatic patients on MTX, 26 of whom paused the medication. 44 non-immunosuppressed persons (NIP) served as control group. Results While the neutralizing serum activity against SARS-CoV-2 Wu01 and Omicron variants increased 67-to 73-fold in the NIP after booster vaccination, the serum activity in patients receiving MTX increased only 20-to 23-fold. As a result, significantly lower neutralizing capacities were measured in patients on MTX compared to the NIP at week 4. Patients who continued MTX treatment during vaccination had significantly lower neutralizing serum titres against all three virus strains at week 4 and 12 compared to patients who paused MTX and the control group, except for BA.2 at week 12. Patients who paused MTX reached comparably high neutralization titres as the NIP, except for Wu01 at week 12. Neutralization of omicron variants was significantly lower in comparison to wildtype in both groups. Conclusion Patients pausing MTX showed a similar vaccine response to NIP. Patients who continued MTX demonstrated an impaired booster response indicating a potential benefit of a second booster vaccination.

Background: In face of the global spread of Corona Virus Disease (COVID)-19, best practice for mechanical ventilation in COVID-19 associated Acute Respiratory Distress Syndrome (ARDS) is intensely debated. Specifically, the rationale for high positive end-expiratory pressure (PEEP) and prone positioning in early COVID-19 ARDS has been questioned. Methods: The first 15 consecutive patients with COVID-19 associated respiratory failure transferred to a single ICU were assessed over the first 15 days of mechanical ventilation. Best PEEP was defined by maximal oxygenation, and was determined by decremental PEEP trials with monitoring of oxygenation, dead space fraction, airway pressures and trans-pulmonary pressures. In nine patients the impact of prone positioning on oxygenation was investigated. As well, the effects of invasive mechanical ventilation with PEEP and prone positioning on pulmonary opacities were determined. Findings: Patients responded to initiation of invasive high PEEP ventilation with markedly improved oxygenation, which was accompanied by reduced pulmonary opacities within 6h of mechanical ventilation. Decremental PEEP trials confirmed the need for high PEEP (17·9 ± 3·9 mbar) for optimal oxygenation in early COVID-19 ARDS, while driving pressures remained low. Prone positioning substantially increased oxygenation in COVID-19 ARDS.Interpretation: In early COVID-19 ARDS, substantial PEEP values were required for optimizing oxygenation. In our patient cohort, pulmonary opacities resolved during invasive mechanical ventilation with high PEEP suggesting recruitment of lung volume. Prone positioning improved oxygenation in early COVID-19 ARDS.Trial Registration: Currently registered in the German clinical trials register and the WHO International Clinical Trials Registry Platform, ID: DRKS00021688Funding Statement: German Research Foundation (DFG), German Federal Ministry of Education and Research (BMBF). This study was supported by the Clinical Study Center of Berlin Institute of Health, Charité – Universitätsmedizin BerlinDeclaration of Interests: All authors declare no conflict of interest. Ethics Approval Statement: The study was approved by the ethics committee of the Charité - Universitätsmedizin Berlin (EA2/066/20) and was performed according to the Declaration of Helsinki and Good Clinical Practice principles (ICH 1996). Written informed consent was obtained from all patients, or their legal representatives.

Summary Severe COVID-19 is linked to both dysfunctional immune response and unrestrained immunopathogenesis, and it remains unclear if T cells also contribute to disease pathology. Here, we combined single-cell transcriptomics and proteomics with mechanistic studies to assess pathogenic T cell functions and inducing signals. We identified highly activated, CD16 + T cells with increased cytotoxic functions in severe COVID-19. CD16 expression enabled immune complex-mediated, T cell receptor-independent degranulation and cytotoxicity not found in other diseases. CD16 + T cells from COVID-19 patients promoted microvascular endothelial cell injury and release of neutrophil and monocyte chemoattractants. CD16 + T cell clones persisted beyond acute disease maintaining their cytotoxic phenotype. Age-dependent generation of C3a in severe COVID-19 induced activated CD16 + cytotoxic T cells. The proportion of activated CD16 + T cells and plasma levels of complement proteins upstream of C3a correlated with clinical outcome of COVID-19, supporting a pathological role of exacerbated cytotoxicity and complement activation in COVID-19.

Das metabolische Syndrom und damit oft verbunden die Entwicklung einer Fettleber stellen ein zunehmendes klinisches Problem dar. Pathogenetisch spielen bei der Progression der Fettleber zur NASH (nicht-alkoholische Steatohepatitis) und schliesslich zur Leberfibrose entzündliche Prozesse eine entscheidende Rolle. Untersucht wurde hier die signalwegspezifische (gp130/STAT/ras) Rolle des pro-inflammatorischen Zytokins IL–6 in einem neuen Maus-Model der NASH Entwicklung. Hierzu wurden hepatozyten-spezifische konditionale knockout-Mäuse mit einer defizienten gp130 (Δgp130), STAT (ΔSTAT) oder ras (Δras) Aktivierung mit einer Cholin-defizienten Diät und Ethionin Trinkwasserzusatz (CDE) behandelt. Ergebnisse: Bereits nach zwei Wochen zeigten Δgp130 und ΔSTAT Mäusen eine beginnende und im Verlauf sich weiter verstärkende grobtropfige Leberverfettung. Konkordant zeigten diese Tiere einen erhöhten Lebertriglyzeridgehalt sowie erhöhte Transaminasen gegenüber WT und Δras Tieren. Als weitere metabolische Komponente und Ausdruck einer gestörten Glukosetoleranz wiesen Δgp130 und ΔSTAT Mäuse erhöhte Nüchternblutglukosewerte auf. Die Analyse der Entzündungsreaktion des Modells zeigte in diesen beiden Gruppen eine signifikant erhöhte Infiltration mit CD4 und CD11b positiven Zellen. Westernblotanalysen domonstrierten eine Aufhebung der STAT3 Aktivierung in Δgp130 und ΔSTAT Mäusen, sowie andererseits eine Verstärkung der STAT3-Induktion in Tieren mit defizienter ras-Aktivierung. Mittels real-time PCR konnten weiterhin erhöhte TNFa Spiegel und erniedrigte Adiponectinwerte in Δgp130 Tieren gemessen werden. Zur Analyse einer evtl. Fibroseentwicklung im hier vorgestellten NASH-Model wurden nach 12-wöchiger Behandlung Sirius-Rot Färbungen durchgeführt. Diese zeigten eine deutliche Einlagerung von Kollagen-Fasern selektiv in Δgp130 und ΔSTAT Tieren, bei nahezu fehlender Reaktion in der WT und Δras Gruppe. Schlussfolgerung: Wir konnten in unserem Mausmodel einer NASH-Entwicklung zeigen, dass gp130– vermittelt über STAT3–Schutz vor gestörter Glukosetoleranz und Progression der Leberverfettung bietet. Dabei kommt es offensichtlich durch fehlende hepatozytäre Schutzmechanismen im Verlauf zur Entwicklung von Entzündungsprozessen, welche in einer Leberfibrosierung resultieren.

IL-6 is known to play a crucial role in the pathogenesis of chronic intestinal inflammation by modulating T cell functions. Here we investigated the role of gp130, the common signal transducer for all IL-6 cytokines, in a murine model of acute T-cell independent colitis in order to better characterize the impact of gp130 on innate immune cells and the early stages of inflammation. Experimental colitis was induced by dextran sulfate sodium treatment of mice with inducible systemic deletion of gp130 (MxCre/gp130), macrophage/neutrophil specific gp130-deficiency (LysCre/gp130) or bone marrow chimeric mice. Systemic deletion of gp130 (MxCre/gp130) protected mice from severe colitis and wasting and attenuated the mucosal inflammatory infiltrate as well as local cytokine, chemokine and adhesion molecule expression. Experiments in newly generated Macrophage/neutrophil-specific gp130-deleted animals (LysCre/gp130) and gp130 bone marrow chimeric mice, revealed a dual mechanism of proinflammatory effects mediated by gp130. Leukocyte recruitment was impaired in gp130-deleted animals and gp130-deleted recipients of wt bone marrow, demonstrating a central role of gp130-dependent signals in non-myeloid cells for directing leukocytes to sites of inflammation, which was further confirmed in a model of sterile peritonitis. In contrast, macrophage/neutrophil specific gp130 deficiency delayed and attenuated the disease but only marginally affected the inflammatory infiltrate, indicating a defective activation of mucosal leukocytes. We provide evidence that IL-6 cytokines acting via gp130 are required in the acute stages of intestinal inflammation by modulating the dynamics of innate immune cell recruitment and activation.

After injury, the liver has the ability to restore its mass and function through a process called “liver regeneration“. In resting cells, NF-κB is confined inactive in the cytoplasm, bound to its inhibitor IκB. After stimulation, IκB is phosphorylated by the IKK complex (composed of two catalytic subunits IKK1 and IKK2 and a regulatory subunit NEMO) leading IκB to a proteasomal degradation and thus liberating NF-κB to translocate into the nucleus. As NF-κB is involved in early events of hepatocyte proliferation, we therefore studied the impact of hepatocyte-specific IKK2 deletion on liver regeneration. IKK2 constitutive knock-out (ko) mice die in-utero. Hence we generated C57/BL6 hepatocyte-specific IKK2 deletion (IKK2Δhepa) mouse using the Cre/LoxP system. 70% partial hepatectomy (PH) was performed on IKK2f/f mice (wt) and IKK2Δhepa mice (knock-out), and liver regeneration was studied. All mice survived after PH. However, we observed impaired NF-κB activation in hepatocytes of IKK2Δhepa mice. IL–6 and TNF-α are two main cytokines driving the priming of hepatocytes after PH. Whereas IL–6 showed similar protein levels in wt and ko mice, TNF-α protein expression showed a earlier and stronger up-regulation 1h after PH in ko mice, while wt animals reached similar result at only 6h after PH. TNF induces matrix remodelling via MMP–9 and we observed earlier MMP–9 protease activity in ko mice. These data suggest earlier priming in the ko animals. Serum Amyloid A, marker of the Acute Phase Response in mice exhibited a strong increase in mRNA expression as early as 6h in ko animals compared to wt. Neutrophil chemoattractant chemokine CXCL–1 mRNA expression was analysed by real time PCR and the kinetic revealed an overexpression of CXCL–1 in ko mice with a peak of expression at 6h. This result directed us to analyse the involvement of the innate immune response after PH. We examined blood and liver cells 12h after PH via flow cytometry, and ko mice exhibited higher number of polymorphonuclear (PMN) cells in both blood and liver compared to wt animals. As a result of all these early events, ko animals presented earlier hepatocytes proliferation compared to wt mice, confirmed by BrdU analysis and cyclins expression. Our data revealed that deleting IKK2 in hepatocyte caused a faster priming phase, an increased acute phase response and a stronger PMN cells recruitment in the liver leading to an earlier hepatocyte proliferation in IKK2Δhepa mice during liver regeneration.

Abstract Glycoprotein 90K, encoded by the interferon-stimulated gene LGALS3BP , displays broad antiviral activity. It reduces HIV-1 infectivity by interfering with Env maturation and virion incorporation, and increases survival of Influenza A virus-infected mice via antiviral innate immune signaling. Here, we analyzed the expression of 90K/ LGALS3BP in 44 hospitalized COVID-19 patients. 90K protein serum levels were significantly elevated in COVID-19 patients compared to uninfected sex- and age-matched controls. Furthermore, PBMC-associated concentrations of 90K protein were overall reduced by SARS-CoV-2 infection in vivo, suggesting enhanced secretion into the extracellular space. Mining of published PBMC scRNA-seq datasets uncovered monocyte-specific induction of LGALS3BP mRNA expression in COVID-19 patients. In functional assays, neither 90K overexpression in susceptible cell lines nor exogenous addition of purified 90K consistently inhibited SARS-CoV-2 infection. Our data suggests that 90K/ LGALS3BP contributes to the global type I IFN response during SARS-CoV-2 infection in vivo without displaying detectable antiviral properties.

Abstract Monkeypox (MPX) is caused by the homonymous orthopoxvirus (MPXV) known since the 1970s to occur at low frequency in West and Central Africa. Recently, the disease has been spreading quickly in Europe and the US. The rapid rise of MPX cases outside previously endemic areas and the different clinical presentation prompt for a better understanding of the disease, including the development of clinical tests for rapid diagnosis and monitoring. Here, using Zeno SWATH MS - a latest-generation proteomic technology - we studied the plasma proteome of a group of MPX patients with a similar infection history and clinical severity typical for the current outbreak. Moreover, we compared their proteomes to those of healthy volunteers and COVID-19 patients. We report that MPX is associated with a strong and characteristic plasma proteomic response and describe MPXV infection biomarkers among nutritional and acute phase response proteins. Moreover, we report a correlation between plasma protein markers and disease severity, approximated by the degree of skin manifestation. Contrasting the MPX host response with that of COVID-19, we find a range of similarities, but also important differences. For instance, Complement factor H-related protein 1 (CFHR1) is induced in COVID-19, but suppressed in MPX, reflecting the different role of the complement system in the two infectious diseases. However, the partial overlap between MPX and COVID-19 host response proteins allowed us to explore the repurposing of a clinically applicable COVID-19 biomarker panel assay, resulting in the successful classification of MPX patients. Hence, our results provide a first proteomic characterization of the MPX human host response based on a case series. The results obtained highlight that proteomics is a promising technology for the timely identification of disease biomarkers in studies with moderate cohorts, and we reveal a thus far untapped potential for accelerating the response to disease outbreaks through the repurposing of multiplex biomarker assays.

Abstract Background Nirmatrelvir/ritonavir is an effective therapy against SARS-CoV-2. Patients with end-stage renal disease (ESRD) are at high risk for severe COVID-19 and show impaired vaccine responses underlining the importance of antiviral therapy. However, use of nirmatrelvir/ritonavir is not recommended in these patients due to lack of clinical and pharmacokinetic data. Objective To investigate pharmacokinetics and hepatic tolerance of nirmatrelvir/ritonavir in patients with ESRD and haemodialysis (HD). Patients and methods Four patients diagnosed with SARS-CoV-2 infection received nirmatrelvir/ritonavir 150/100mg twice daily as recommended for renal impairment; HD ran in two- to three-day intervals. Plasma and serum samples were drawn before and after each HD during the 5-day treatment and for ensuing 3-5 days. Results Median peak levels of nirmatrelvir obtained two hours after medication pre-HD in three patients were 7745ng/mL on day 3 and 6653ng/mL on day 5; median post-HD levels (C 6h ) declined to 5765ng/mL (74%) and 5521ng/mL (83%), on days 3 and 5 of treatment, respectively. Three days after end of treatment, median levels were 365ng/mL pre-HD and 30ng/mL post-HD. Measurements of the fourth patient, six hours after drug intake pre-HD showed nirmatrelvir-levels of 3704ng/mL on treatment day 3 which fell to 2308ng/mL post-HD, at one hour before intake of the next dose (C min ). Conclusion Use of nirmatrelvir/ritonavir in patients with ESRD results in high nirmatrelvir blood concentrations, which are still within the range known from patients without renal failure. No accumulation of nirmatrelvir took place and levels declined to zero within few days after end of treatment.

Abstract The clinical course of the 2019 coronavirus disease (COVID-19) is variable and to a substantial degree still unpredictable, especially in persons who have neither been vaccinated nor recovered from previous infection. We hypothesized that disease progression and inflammatory responses were associated with alterations in the microbiome and metabolome. To test this, we integrated metagenome, metabolome, cytokine, and transcriptome profiles of longitudinally collected samples from hospitalized COVID-19 patients at the beginning of the pandemic (before vaccines or variants of concern) and non-infected controls, and leveraged detailed clinical information and post-hoc confounder analysis to identify robust within- and cross-omics associations. Severe COVID-19 was directly associated with a depletion of potentially beneficial intestinal microbes mainly belonging to Clostridiales, whereas oropharyngeal microbiota disturbance appeared to be mainly driven by antibiotic use. COVID-19 severity was also associated with enhanced plasma concentrations of kynurenine, and reduced levels of various other tryptophan metabolites, lysophosphatidylcholines, and secondary bile acids. Decreased abundance of Clostridiales potentially mediated the observed reduction in 5-hydroxytryptophan levels. Moreover, altered plasma levels of various tryptophan metabolites and lower abundances of Clostridiales explained significant increases in the production of IL-6, IFNγ and/or TNFα. Collectively, our study identifies correlated microbiome and metabolome alterations as a potential contributor to inflammatory dysregulation in severe COVID-19. Graphical Abstract

<b>Single-cell RNA-Seq of airway samples of COVID-19 patients and healthy controls</b><br>This dataset comprises single-cell RNA-Seq data of nasopharyngeal, protected specimen brush, and bronchial lavage samples of 19 COVID-19 patients (eight moderate and eleven critical according to the WHO classification) and five healthy controls, for a total of 36 samples. <br>An in-depth description is presented in the manuscript "Cross-talk between the airway epithelium and activated immune cells defines severity in COVID-19" (https://www.medrxiv.org/content/10.1101/2020.04.29.20084327v1). <br>The data is uploaded as two .rds files of Seurat objects that can be imported into R. The _main file contains all samples from the nasopharynx, while the _loc file contains data from nasopharyngeal, protected specimen brush, and bronchial lavage samples of two patients. <br>A quantification of viral RNA reads (as CPM, in total over cells and background) is provided as .xlsx file. Please note that these values may differ from viral load estimates obtained from diagnostic procedures and may be less accurate.<br>

This dataset comprises single-cell RNA-Seq data of nasopharyngeal samples of 32 COVID-19 patients and 16 healthy controls, for a total of 48 samples. 25 COVID-19 patients and 10 controls were diagnosed with hypertension and treated with either ACE inhibitors (ACEi) or angiotensin receptor blockers(ARB) (SARS-CoV-2-positive: 10 ACEi and 15 ARB; SARS-CoV-2-negative: 6 ACEi and 4 ARB).<br>An in-depth description is presented in the manuscript "Delayed viral clearance and exacerbated airway hyperinflammation in hypertensive COVID-19 patients" (https://www.medrxiv.org/content/10.1101/2020.09.22.20199471v1).

<b>Single-cell RNA-Seq of airway samples of COVID-19 patients and healthy controls</b><br>This dataset comprises single-cell RNA-Seq data of nasopharyngeal, protected specimen brush, and bronchial lavage samples of 19 COVID-19 patients (eight moderate and eleven critical according to the WHO classification) and five healthy controls, for a total of 36 samples. <br>An in-depth description is presented in the manuscript "Cross-talk between the airway epithelium and activated immune cells defines severity in COVID-19" (https://www.medrxiv.org/content/10.1101/2020.04.29.20084327v1). <br>The data is uploaded as two .rds files of Seurat objects that can be imported into R. The _main file contains all samples from the nasopharynx, while the _loc file contains data from nasopharyngeal, protected specimen brush, and bronchial lavage samples of two patients. <br>A quantification of viral RNA reads (as CPM, in total over cells and background) is provided as .xlsx file. Please note that these values may differ from viral load estimates obtained from diagnostic procedures and may be less accurate.Raw count values (cellranger output) are provided in the file count_matrices_NBT.tar.<br>

RATIONALE: IL-3, tL-5 and GM-CSF exert overlapping biological functions in basophils and eosinophils, because their receptors have common signaling beta-chain.However, no comprehensive study has been performed to simultaneously compare the molar effects of IL-3, IL-5 and GM-CSF between different functions of these cells in the same experiment.METHODS: Survival enhancement, CDllb expression and CD69 expression were analyzed in both basophils and eosinophils.The enhancement of histamine release was studied in basophils.We also investigated the protein and mRNA expression levels of IL-3Ralpha, IL-5Ralpha and GM-CSFRalpha using flow cytometry and real-time PCR.RESULTS: The rank order of potency in basophils was IL-3 >> IL-5=GM-CSF for degranulation, survival and CDI lb expression.The ED50 of IL-3 was 0.5-1 pM, 0.03 pM and 7 pM, respectively, for these three functions.In eosinophils, the rank order was IL-5=GM-CSF > IL-3 for survival and CDI lb expression.The ED50 of IL-5 was 0.6 pM and 100pM for survival and CDI lb expression, respectively.However, in both cell types, CD69 expression was most potently induced by IL-3.Compared with eosinophils, basophils expressed a much higher level of IL-3Ralpha, similar or slightly lower level of IL-5Ralpha, and an apparently lower level of GM-CSFRaIpha.CONCLUSIONS: IL-3 is the most potent stimulator for basophils, whereas IL-5 and GM-CSF are most potent for eosinophils.In general, the rank order of potency of these cytokines corresponded exactly to their receptor expression levels.However, CD69 expression differed completely from this paradigm.

<pre>This dataset comprises single-cell RNA-Seq data of nasopharyngeal samples of of adults and children with COVID-19 (n=24 and n=21, respectively) and SARS-CoV-2-negative controls in each age group (n=23 and 18, respectively).<br>An in-depth description is presented in the manuscript "Pre-activated anti-viral innate immunity in the upper airways controls early SARS-CoV-2 infection in children" (https://www.medrxiv.org/content/10.1101/2021.06.24.21259087v1).</pre>

<pre>This dataset comprises single-cell RNA-Seq data of nasopharyngeal samples of COVID-19 patients and 16 healthy controls, for a total of 48 samples. 25 COVID-19 patients and 10 controls were diagnosed with hypertension and treated with either ACE inhibitors (ACEi) or angiotensin receptor blockers(ARB) (SARS-CoV-2-positive: 10 ACEi and 15 ARB; SARS-CoV-2-negative: 6 ACEi and 4 ARB).<br>An in-depth description is presented in the manuscript "Delayed viral clearance and exacerbated airway hyperinflammation in hypertensive COVID-19 patients" (https://www.medrxiv.org/content/10.1101/2020.09.22.20199471v1).</pre>