Species differences in plasma protein binding of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease inhibitor nirmatrelvir.

Plasma protein binding (PPB) studies on the SARS-CoV-2 main protease inhibitor nirmatrelvir revealed considerable species differences primarily in dog and rabbit, which prompted further investigations into the biochemical basis for these differences.The unbound fraction (fu) of nirmatrelvir in dog and rabbit plasma was concentration (2-200 µM)-dependent (dog fu,p 0.024-0.69, rabbit fu,p 0.010-0.82). Concentration (0.1-100 µM)-dependent binding in serum albumin (SA) (fu,SA 0.040-0.82) and alpha-1-acid glycoprotein (AAG) (fu,AAG 0.050-0.64) was observed in dogs. Nirmatrelvir showed minimal binding to rabbit SA (1-100 µM: fu,SA 0.70-0.79), while binding to rabbit AAG was concentration-dependent (0.1-100 µM: fu,AAG 0.024-0.66). In contrast, nirmatrelvir (2 µM) revealed minimal binding (fu,AAG 0.79-0.88) to AAG from rat and monkeys. Nirmatrelvir showed minimal-to-moderate binding to SA (1-100 µM; fu,SA 0.70-1.0) and AAG (0.1-100 µM; fu,AAG 0.48-0.58) from humans across tested concentrations.Nirmatrelvir molecular docking studies using published crystal structures and homology models of human and preclinical species SA and AAG were used to rationalise the species differences to plasma proteins. This suggested that species differences in PPB are primarily driven by molecular differences in albumin and AAG resulting in differences in binding affinity.

Brief Research Report: Serum clara cell 16 kDa protein levels are increased in patients hospitalized for severe SARS-CoV-2 or sepsis infection.

Background: Clara cell 16 kDa protein (CC16) is a secretory protein primarily expressed in epithelial cells in the lungs. Previous studies show that CC16 exerts anti-inflammatory and immune-modulatory properties in both acute and chronic pulmonary diseases. However, despite the evidence of CC16's high biomarker potential, evaluation of its role in infectious diseases is yet very limited. Methods: Serum CC16 concentrations were measured by ELISA and assessed in two different types of severe infections. Using a case-control study design, patients treated for either severe SARS-CoV-2 or severe non-pulmonary sepsis infection were compared to age- and sex-matched healthy human subjects. Results: Serum CC16 was significantly increased in both types of infection (SARS-CoV-2: 96.22 ± 129.01 ng/ml vs. healthy controls: 14.05 ± 7.48 ng/ml, p = 0.022; sepsis: 35.37 ± 28.10 ng/ml vs. healthy controls: 15.25 ± 7.51 ng/ml, p = 0.032) but there were no distinct differences between infections with and without pulmonary focus (p = 0.089). Furthermore, CC16 serum levels were positively correlated to disease duration and inversely to the platelet count in severe SARS-CoV-2 infection. Conclusions: Increased CC16 serum levels in both SARS-CoV-2 and sepsis reinforce the high potential as a biomarker for epithelial cell damage and bronchoalveolar-blood barrier leakage in pulmonary as well as non-pulmonary infectious diseases.

Evaluation of In Vitro Distribution and Plasma Protein Binding of Selected Antiviral Drugs (Favipiravir, Molnupiravir and Imatinib) against SARS-CoV-2.

There are a number of uncertainties regarding plasma protein binding and blood distribution of the active drugs favipiravir (FAVI), molnupiravir (MOLNU) and imatinib (IMA), which were recently proposed as therapeutics for the treatment of COVID-19 disease. Therefore, proton dissociation processes, solubility, lipophilicity, and serum protein binding of these three substances were investigated in detail. The drugs display various degrees of lipophilicity at gastric (pH 2.0) and blood pH (pH 7.4). The determined pKa values explain well the changes in lipophilic character of the respective compounds. The serum protein binding was studied by membrane ultrafiltration, frontal analysis capillary electrophoresis, steady-state fluorometry, and fluorescence anisotropy techniques. The studies revealed that the ester bond in MOLNU is hydrolyzed by protein constituents of blood serum. Molnupiravir and its hydrolyzed form do not bind considerably to blood proteins. Likewise, FAVI does not bind to human serum albumin (HSA) and α1-acid glycoprotein (AGP) and shows relatively weak binding to the protein fraction of whole blood serum. Imatinib binds to AGP with high affinity (logK' = 5.8-6.0), while its binding to HSA is much weaker (logK' ≤ 4.0). The computed constants were used to model the distribution of IMA in blood plasma under physiological and 'acute-phase' conditions as well.

Exosome Proteomics Reveals the Deregulation of Coagulation, Complement and Lipid Metabolism Proteins in Gestational Diabetes Mellitus.

Exosomes are small extracellular vesicles with a variable protein cargo in consonance with cell origin and pathophysiological conditions. Gestational diabetes mellitus (GDM) is characterized by different levels of chronic low-grade inflammation and vascular dysfunction; however, there are few data characterizing the serum exosomal protein cargo of GDM patients and associated signaling pathways. Eighteen pregnant women were enrolled in the study: 8 controls (CG) and 10 patients with GDM. Blood samples were collected from patients, for exosomes' concentration. Protein abundance alterations were demonstrated by relative mass spectrometric analysis and their association with clinical parameters in GDM patients was performed using Pearson's correlation analysis. The proteomics analysis revealed 78 significantly altered proteins when comparing GDM to CG, related to complement and coagulation cascades, platelet activation, prothrombotic factors and cholesterol metabolism. Down-regulation of Complement C3 (C3), Complement C5 (C5), C4-B (C4B), C4b-binding protein beta chain (C4BPB) and C4b-binding protein alpha chain (C4BPA), and up-regulation of C7, C9 and F12 were found in GDM. Our data indicated significant correlations between factors involved in the pathogenesis of GDM and clinical parameters that may improve the understanding of GDM pathophysiology. Data are available via ProteomeXchange with identifier PXD035673.

Prognostic value of heparin-binding protein for mortality in severe COVID-19 pneumonia.

Aim: The study investigated heparin-binding protein (HBP) levels in patients with severe COVID-19 pneumonia and their relation to prognosis. Methods: A total of 134 patients with serious COVID-19 pneumonia were prospectively analyzed. HBP levels were statistically compared between both the patient and healthy control groups and within the patient group itself. Results: HBP was defined to be significantly higher in the patient group compared with the control group. There was a statistically significant distinction between the patients who survived and those who died with regard to HBP levels. When the cutoff value of HBP was >13.47, sensitivity (89.8%), specificity (74.1%) had area under the curve values of 0.806 (p < 0.001). Conclusion: HBP level may be used for prognosis prediction of patients with COVID-19.

Association of extracellular vesicle inflammatory proteins and mortality.

Even before the COVID-19 pandemic declines in life expectancy in the United States were attributed to increased mortality rates in midlife adults across racial and ethnic groups, indicating a need for markers to identify individuals at risk for early mortality. Extracellular vesicles (EVs) are small, lipid-bound vesicles capable of shuttling functional proteins, nucleic acids, and lipids. Given their role as intercellular communicators and potential biomarkers of disease, we explored whether circulating EVs may be markers of mortality in a prospective, racially, and socioeconomically diverse middle-aged cohort. We isolated plasma EVs from 76 individuals (mean age = 59.6 years) who died within a 5 year period and 76 surviving individuals matched by age, race, and poverty status. There were no significant differences in EV concentration, size, or EV-associated mitochondrial DNA levels associated with mortality. We found that several EV-associated inflammatory proteins including CCL23, CSF-1, CXCL9, GDNF, MCP-1, STAMBP, and 4E-BP1 were significantly associated with mortality. IL-10RB and CDCP1 were more likely to be present in plasma EVs from deceased individuals than in their alive counterparts. We also report differences in EV-associated inflammatory proteins with poverty status, race, and sex. Our results suggest that plasma EV-associated inflammatory proteins are promising potential clinical biomarkers of mortality.

Novel aspects of sepsis pathophysiology: NETs, plasma glycoproteins, endotheliopathy and COVID-19.

In 2016, sepsis was newly defined as life-threatening organ dysfunction caused by a dysregulated host response to infection. Sepsis remains one of the crucial medical problems to be solved worldwide. Although the world health organization has made sepsis a global health priority, there remain no specific and effective therapy for sepsis so far. Indeed, over the previous decades almost all attempts to develop novel drugs have failed. This may be partly ascribable to the multifactorial complexity of the septic cascade and the resultant difficulties of identifying drug targets. In addition, there might still be missing links among dysregulated host responses in vital organs. In this review article, recent advances in understanding of the complex pathophysiology of sepsis are summarized, with a focus on neutrophil extracellular traps (NETs), the significant role of NETs in thrombosis/embolism, and the functional roles of plasma proteins, histidine-rich glycoprotein (HRG) and inter-alpha-inhibitor proteins (IAIPs). The specific plasma proteins that are markedly decreased in the acute phase of sepsis may play important roles in the regulation of blood cells, vascular endothelial cells and coagulation. The accumulating evidence may provide us with insights into a novel aspect of the pathophysiology of sepsis and septic ARDS, including that in COVID-19.

Proteome-wide Mendelian randomization identifies causal links between blood proteins and severe COVID-19.

In November 2021, the COVID-19 pandemic death toll surpassed five million individuals. We applied Mendelian randomization including >3,000 blood proteins as exposures to identify potential biomarkers that may indicate risk for hospitalization or need for respiratory support or death due to COVID-19, respectively. After multiple testing correction, using genetic instruments and under the assumptions of Mendelian Randomization, our results were consistent with higher blood levels of five proteins GCNT4, CD207, RAB14, C1GALT1C1, and ABO being causally associated with an increased risk of hospitalization or respiratory support/death due to COVID-19 (ORs = 1.12-1.35). Higher levels of FAAH2 were solely associated with an increased risk of hospitalization (OR = 1.19). On the contrary, higher levels of SELL, SELE, and PECAM-1 decrease risk of hospitalization or need for respiratory support/death (ORs = 0.80-0.91). Higher levels of LCTL, SFTPD, KEL, and ATP2A3 were solely associated with a decreased risk of hospitalization (ORs = 0.86-0.93), whilst higher levels of ICAM-1 were solely associated with a decreased risk of respiratory support/death of COVID-19 (OR = 0.84). Our findings implicate blood group markers and binding proteins in both hospitalization and need for respiratory support/death. They, additionally, suggest that higher levels of endocannabinoid enzymes may increase the risk of hospitalization. Our research replicates findings of blood markers previously associated with COVID-19 and prioritises additional blood markers for risk prediction of severe forms of COVID-19. Furthermore, we pinpoint druggable targets potentially implicated in disease pathology.

A synthetic protein as efficient multitarget regulator against complement over-activation.

The complement system constitutes the innate defense against pathogens. Its dysregulation leads to diseases and is a critical determinant in many viral infections, e.g., COVID-19. Factor H (FH) is the main regulator of the alternative pathway of complement activation and could be a therapy to restore homeostasis. However, recombinant FH is not available. Engineered FH versions may be alternative therapeutics. Here, we designed a synthetic protein, MFHR13, as a multitarget complement regulator. It combines the dimerization and C5-regulatory domains of human FH-related protein 1 (FHR1) with the C3-regulatory and cell surface recognition domains of human FH, including SCR 13. In summary, the fusion protein MFHR13 comprises SCRs FHR11-2:FH1-4:FH13:FH19-20. It protects sheep erythrocytes from complement attack exhibiting 26 and 4-fold the regulatory activity of eculizumab and human FH, respectively. Furthermore, we demonstrate that MFHR13 and FHR1 bind to all proteins forming the membrane attack complex, which contributes to the mechanistic understanding of FHR1. We consider MFHR13 a promising candidate as therapeutic for complement-associated diseases.

Identification of Robust Protein Associations With COVID-19 Disease Based on Five Clinical Studies.

Multiple studies have investigated the role of blood circulating proteins in COVID-19 disease using the Olink affinity proteomics platform. However, study inclusion criteria and sample collection conditions varied between studies, leading to sometimes incongruent associations. To identify the most robust protein markers of the disease and the underlying pathways that are relevant under all conditions, it is essential to identify proteins that replicate most widely. Here we combined the Olink proteomics profiles of two newly recruited COVID-19 studies (N=68 and N=98) with those of three previously published COVID-19 studies (N=383, N=83, N=57). For these studies, three Olink panels (Inflammation and Cardiovascular II & III) with 253 unique proteins were compared. Case/control analysis revealed thirteen proteins (CCL16, CCL7, CXCL10, CCL8, LGALS9, CXCL11, IL1RN, CCL2, CD274, IL6, IL18, MERTK, IFNγ, and IL18R1) that were differentially expressed in COVID-19 patients in all five studies. Except CCL16, which was higher in controls, all proteins were overexpressed in COVID-19 patients. Pathway analysis revealed concordant trends across all studies with pathways related to cytokine-cytokine interaction, IL18 signaling, fluid shear stress and rheumatoid arthritis. Our results reaffirm previous findings related to a COVID-19 cytokine storm syndrome. Cross-study robustness of COVID-19 specific protein expression profiles support the utility of affinity proteomics as a tool and for the identification of potential therapeutic targets.

Heparin-Functionalized Adsorbents Eliminate Central Effectors of Immunothrombosis, including Platelet Factor 4, High-Mobility Group Box 1 Protein and Histones.

Inflammation and thrombosis are closely intertwined in numerous disorders, including ischemic events and sepsis, as well as coronavirus disease 2019 (COVID-19). Thrombotic complications are markers of disease severity in both sepsis and COVID-19 and are associated with multiorgan failure and increased mortality. Immunothrombosis is driven by the complement/tissue factor/neutrophil axis, as well as by activated platelets, which can trigger the release of neutrophil extracellular traps (NETs) and release further effectors of immunothrombosis, including platelet factor 4 (PF4/CXCL4) and high-mobility box 1 protein (HMGB1). Many of the central effectors of deregulated immunothrombosis, including activated platelets and platelet-derived extracellular vesicles (pEVs) expressing PF4, soluble PF4, HMGB1, histones, as well as histone-decorated NETs, are positively charged and thus bind to heparin. Here, we provide evidence that adsorbents functionalized with endpoint-attached heparin efficiently deplete activated platelets, pEVs, PF4, HMGB1 and histones/nucleosomes. We propose that this elimination of central effectors of immunothrombosis, rather than direct binding of pathogens, could be of clinical relevance for mitigating thrombotic complications in sepsis or COVID-19 using heparin-functionalized adsorbents.

Multiple early factors anticipate post-acute COVID-19 sequelae.

Post-acute sequelae of COVID-19 (PASC) represent an emerging global crisis. However, quantifiable risk factors for PASC and their biological associations are poorly resolved. We executed a deep multi-omic, longitudinal investigation of 309 COVID-19 patients from initial diagnosis to convalescence (2-3 months later), integrated with clinical data and patient-reported symptoms. We resolved four PASC-anticipating risk factors at the time of initial COVID-19 diagnosis: type 2 diabetes, SARS-CoV-2 RNAemia, Epstein-Barr virus viremia, and specific auto-antibodies. In patients with gastrointestinal PASC, SARS-CoV-2-specific and CMV-specific CD8+ T cells exhibited unique dynamics during recovery from COVID-19. Analysis of symptom-associated immunological signatures revealed coordinated immunity polarization into four endotypes, exhibiting divergent acute severity and PASC. We find that immunological associations between PASC factors diminish over time, leading to distinct convalescent immune states. Detectability of most PASC factors at COVID-19 diagnosis emphasizes the importance of early disease measurements for understanding emergent chronic conditions and suggests PASC treatment strategies.

Endothelial dysfunction contributes to severe COVID-19 in combination with dysregulated lymphocyte responses and cytokine networks.

The systemic processes involved in the manifestation of life-threatening COVID-19 and in disease recovery are still incompletely understood, despite investigations focusing on the dysregulation of immune responses after SARS-CoV-2 infection. To define hallmarks of severe COVID-19 in acute disease (n = 58) and in disease recovery in convalescent patients (n = 28) from Hannover Medical School, we used flow cytometry and proteomics data with unsupervised clustering analyses. In our observational study, we combined analyses of immune cells and cytokine/chemokine networks with endothelial activation and injury. ICU patients displayed an altered immune signature with prolonged lymphopenia but the expansion of granulocytes and plasmablasts along with activated and terminally differentiated T and NK cells and high levels of SARS-CoV-2-specific antibodies. The core signature of seven plasma proteins revealed a highly inflammatory microenvironment in addition to endothelial injury in severe COVID-19. Changes within this signature were associated with either disease progression or recovery. In summary, our data suggest that besides a strong inflammatory response, severe COVID-19 is driven by endothelial activation and barrier disruption, whereby recovery depends on the regeneration of the endothelial integrity.

Next generation plasma proteome profiling of COVID-19 patients with mild to moderate symptoms.

BACKGROUND: COVID-19 has caused millions of deaths globally, yet the cellular mechanisms underlying the various effects of the disease remain poorly understood. Recently, a new analytical platform for comprehensive analysis of plasma protein profiles using proximity extension assays combined with next generation sequencing has been developed, which allows for multiple proteins to be analyzed simultaneously without sacrifice on accuracy or sensitivity. METHODS: We analyzed the plasma protein profiles of COVID-19 patients (n = 50) with mild and moderate symptoms by comparing the protein levels in newly diagnosed patients with the protein levels in the same individuals after 14 days. FINDINGS: The study has identified more than 200 proteins that are significantly elevated during infection and many of these are related to cytokine response and other immune-related functions. In addition, several other proteins are shown to be elevated, including SCARB2, a host cell receptor protein involved in virus entry. A comparison with the plasma protein response in patients with severe symptoms shows a highly similar pattern, but with some interesting differences. INTERPRETATION: The study presented here demonstrates the usefulness of "next generation plasma protein profiling" to identify molecular signatures of importance for disease progression and to allow monitoring of disease during recovery from the infection. The results will facilitate further studies to understand the molecular mechanism of the immune-related response of the SARS-CoV-2 virus. FUNDING: This work was financially supported by Knut and Alice Wallenberg Foundation.

Mapping the proteo-genomic convergence of human diseases.

Characterization of the genetic regulation of proteins is essential for understanding disease etiology and developing therapies. We identified 10,674 genetic associations for 3892 plasma proteins to create a cis-anchored gene-protein-disease map of 1859 connections that highlights strong cross-disease biological convergence. This proteo-genomic map provides a framework to connect etiologically related diseases, to provide biological context for new or emerging disorders, and to integrate different biological domains to establish mechanisms for known gene-disease links. Our results identify proteo-genomic connections within and between diseases and establish the value of cis-protein variants for annotation of likely causal disease genes at loci identified in genome-wide association studies, thereby addressing a major barrier to experimental validation and clinical translation of genetic discoveries.

Metabolic Perturbation Associated With COVID-19 Disease Severity and SARS-CoV-2 Replication.

Viruses hijack host metabolic pathways for their replicative advantage. In this study, using patient-derived multiomics data and in vitro infection assays, we aimed to understand the role of key metabolic pathways that can regulate severe acute respiratory syndrome coronavirus-2 reproduction and their association with disease severity. We used multiomics platforms (targeted and untargeted proteomics and untargeted metabolomics) on patient samples and cell-line models along with immune phenotyping of metabolite transporters in patient blood cells to understand viral-induced metabolic modulations. We also modulated key metabolic pathways that were identified using multiomics data to regulate the viral reproduction in vitro. Coronavirus disease 2019 disease severity was characterized by increased plasma glucose and mannose levels. Immune phenotyping identified altered expression patterns of carbohydrate transporter, glucose transporter 1, in CD8+ T cells, intermediate and nonclassical monocytes, and amino acid transporter, xCT, in classical, intermediate, and nonclassical monocytes. In in vitro lung epithelial cell (Calu-3) infection model, we found that glycolysis and glutaminolysis are essential for virus replication, and blocking these metabolic pathways caused significant reduction in virus production. Taken together, we therefore hypothesized that severe acute respiratory syndrome coronavirus-2 utilizes and rewires pathways governing central carbon metabolism leading to the efflux of toxic metabolites and associated with disease severity. Thus, the host metabolic perturbation could be an attractive strategy to limit the viral replication and disease severity.

Aging whole blood transcriptome reveals candidate genes for SARS-CoV-2-related vascular and immune alterations.

The risk of severe COVID-19 increases with age as older patients are at highest risk. Thus, there is an urgent need to identify how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) interacts with blood components during aging. We investigated the whole blood transcriptome from the Genotype-Tissue Expression (GTEx) database to explore differentially expressed genes (DEGs) translated into proteins interacting with viral proteins during aging. From 22 DEGs in aged blood, FASLG, CTSW, CTSE, VCAM1, and BAG3 were associated with immune response, inflammation, cell component and adhesion, and platelet activation/aggregation. Males and females older than 50 years old overexpress FASLG, possibly inducing a hyperinflammatory cascade. The expression of cathepsins (CTSW and CTSE) and the anti-apoptotic co-chaperone molecule BAG3 also increased throughout aging in both genders. By exploring single-cell RNA-sequencing data from peripheral blood of SARS-CoV-2-infected patients, we found FASLG and CTSW expressed in natural killer cells and CD8 + T lymphocytes, whereas BAG3 was expressed mainly in CD4 + T cells, naive T cells, and CD14 + monocytes. In addition, T cell exhaustion was associated with increased expression of CCL4L2 and DUSP4 over blood aging. LAG3, PDCD1, TIGIT, VCAM1, HLA-DRA, and TOX also increased in individuals aged 60-69 years old; conversely, the RGS2 gene decreased with aging. We further identified a distinct gene expression profile associated with type I interferon signaling following blood aging. These results revealed changes in blood molecules potentially related to SARS-CoV-2 infection throughout aging, emphasizing them as therapeutic candidates for aggressive clinical manifestation of COVID-19. KEY MESSAGES: • Prediction of host-viral interactions in the whole blood transcriptome during aging. • Expression levels of FASLG, CTSW, CTSE, VCAM1, and BAG3 increase in aged blood. • Blood interactome reveals targets involved with immune response, inflammation, and blood clots. • SARS-CoV-2-infected patients with high viral load showed FASLG overexpression. • Gene expression profile associated with T cell exhaustion and type I interferon signaling were affected with blood aging.

PABPC4 Broadly Inhibits Coronavirus Replication by Degrading Nucleocapsid Protein through Selective Autophagy.

Emerging coronaviruses (CoVs) can cause severe diseases in humans and animals, and, as of yet, none of the currently available broad-spectrum drugs or vaccines can effectively control these diseases. Host antiviral proteins play an important role in inhibiting viral proliferation. One of the isoforms of cytoplasmic poly(A)-binding protein (PABP), PABPC4, is an RNA-processing protein, which plays an important role in promoting gene expression by enhancing translation and mRNA stability. However, its function in viruses remains poorly understood. Here, we report that the host protein, PABPC4, could be regulated by transcription factor SP1 and broadly inhibits the replication of CoVs, covering four genera (Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus) of the Coronaviridae family by targeting the nucleocapsid (N) protein through the autophagosomes for degradation. PABPC4 recruited the E3 ubiquitin ligase MARCH8/MARCHF8 to the N protein for ubiquitination. Ubiquitinated N protein was recognized by the cargo receptor NDP52/CALCOCO2, which delivered it to the autolysosomes for degradation, resulting in impaired viral proliferation. In addition to regulating gene expression, these data demonstrate a novel antiviral function of PABPC4, which broadly suppresses CoVs by degrading the N protein via the selective autophagy pathway. This study will shed light on the development of broad anticoronaviral therapies. IMPORTANCE Emerging coronaviruses (CoVs) can cause severe diseases in humans and animals, but none of the currently available drugs or vaccines can effectively control these diseases. During viral infection, the host will activate the interferon (IFN) signaling pathways and host restriction factors in maintaining the innate antiviral responses and suppressing viral replication. This study demonstrated that the host protein, PABPC4, interacts with the nucleocapsid (N) proteins from eight CoVs covering four genera (Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus) of the Coronaviridae family. PABPC4 could be regulated by SP1 and broadly inhibits the replication of CoVs by targeting the nucleocapsid (N) protein through the autophagosomes for degradation. This study significantly increases our understanding of the novel host restriction factor PABPC4 against CoV replication and will help develop novel antiviral strategies.

Identification of immune correlates of fatal outcomes in critically ill COVID-19 patients.

Prior studies have demonstrated that immunologic dysfunction underpins severe illness in COVID-19 patients, but have lacked an in-depth analysis of the immunologic drivers of death in the most critically ill patients. We performed immunophenotyping of viral antigen-specific and unconventional T cell responses, neutralizing antibodies, and serum proteins in critically ill patients with SARS-CoV-2 infection, using influenza infection, SARS-CoV-2-convalescent health care workers, and healthy adults as controls. We identify mucosal-associated invariant T (MAIT) cell activation as an independent and significant predictor of death in COVID-19 (HR = 5.92, 95% CI = 2.49-14.1). MAIT cell activation correlates with several other mortality-associated immunologic measures including broad activation of CD8+ T cells and non-Vδ2 γδT cells, and elevated levels of cytokines and chemokines, including GM-CSF, CXCL10, CCL2, and IL-6. MAIT cell activation is also a predictor of disease severity in influenza (ECMO/death HR = 4.43, 95% CI = 1.08-18.2). Single-cell RNA-sequencing reveals a shift from focused IFNα-driven signals in COVID-19 ICU patients who survive to broad pro-inflammatory responses in fatal COVID-19 -a feature not observed in severe influenza. We conclude that fatal COVID-19 infection is driven by uncoordinated inflammatory responses that drive a hierarchy of T cell activation, elements of which can serve as prognostic indicators and potential targets for immune intervention.

Comprehensive Immunologic Evaluation of Bronchoalveolar Lavage Samples from Human Patients with Moderate and Severe Seasonal Influenza and Severe COVID-19.

Infection with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) or seasonal influenza may lead to respiratory failure requiring intubation and mechanical ventilation. The pathophysiology of this respiratory failure is attributed to local immune dysregulation, but how the immune response to viral infection in the lower airways of the human lung differs between individuals with respiratory failure and those without is not well understood. We used quantitative multiparameter flow cytometry and multiplex cytokine assays to evaluate matched blood and bronchoalveolar lavage (BAL) samples from control human subjects, subjects with symptomatic seasonal influenza who did not have respiratory failure, and subjects with severe seasonal influenza or SARS-CoV-2 infection with respiratory failure. We find that severe cases are associated with an influx of nonclassical monocytes, activated T cells, and plasmablast B cells into the lower airways. Cytokine concentrations were not elevated in the lower airways of moderate influenza patients compared with controls; however, 28 of 35 measured cytokines were significantly elevated in severe influenza, severe SARS-CoV-2 infection, or both. We noted the largest elevations in IL-6, IP-10, MCP-1, and IL-8. IL-1 family cytokines and RANTES were higher in severe influenza infection than severe SARS-CoV-2 infection. Interestingly, only the concentration of IP-10-correlated between blood and BAL during severe infection. Our results demonstrate inflammatory immune dysregulation in the lower airways during severe viral pneumonia that is distinct from lower airway responses seen in human patients with symptomatic, but not severe, illness and suggest that measurement of blood IP-10 concentration may predict this unique dysregulation.