Validating a Proteomic Signature of Severe COVID-19.

COVID-19 is a heterogenous disease. Biomarker-based approaches may identify patients at risk for severe disease, who may be more likely to benefit from specific therapies. Our objective was to identify and validate a plasma protein signature for severe COVID-19. DESIGN: Prospective observational cohort study. SETTING: Two hospitals in the United States. PATIENTS: One hundred sixty-seven hospitalized adults with COVID-19. INTERVENTION: None. MEASUREMENTS AND MAIN RESULTS: We measured 713 plasma proteins in 167 hospitalized patients with COVID-19 using a high-throughput platform. We classified patients as nonsevere versus severe COVID-19, defined as the need for high-flow nasal cannula, mechanical ventilation, extracorporeal membrane oxygenation, or death, at study entry and in 7-day intervals thereafter. We compared proteins measured at baseline between these two groups by logistic regression adjusting for age, sex, symptom duration, and comorbidities. We used lead proteins from dysregulated pathways as inputs for elastic net logistic regression to identify a parsimonious signature of severe disease and validated this signature in an external COVID-19 dataset. We tested whether the association between corticosteroid use and mortality varied by protein signature. One hundred ninety-four proteins were associated with severe COVID-19 at the time of hospital admission. Pathway analysis identified multiple pathways associated with inflammatory response and tissue repair programs. Elastic net logistic regression yielded a 14-protein signature that discriminated 90-day mortality in an external cohort with an area under the receiver-operator characteristic curve of 0.92 (95% CI, 0.88-0.95). Classifying patients based on the predicted risk from the signature identified a heterogeneous response to treatment with corticosteroids (p = 0.006). CONCLUSIONS: Inpatients with COVID-19 express heterogeneous patterns of plasma proteins. We propose a 14-protein signature of disease severity that may have value in developing precision medicine approaches for COVID-19 pneumonia.

Association of vancomycin plus piperacillin-tazobactam with early changes in creatinine versus cystatin C in critically ill adults: a prospective cohort study.

PURPOSE: Although dozens of studies have associated vancomycin + piperacillin-tazobactam with increased acute kidney injury (AKI) risk, it is unclear whether the association represents true injury or a pseudotoxicity characterized by isolated effects on creatinine secretion. We tested this hypothesis by contrasting changes in creatinine concentration after antibiotic initiation with changes in cystatin C concentration, a kidney biomarker unaffected by tubular secretion. METHODS: We included patients enrolled in the Molecular Epidemiology of SepsiS in the ICU (MESSI) prospective cohort who were treated for ≥ 48 h with vancomycin + piperacillin-tazobactam or vancomycin + cefepime. Kidney function biomarkers [creatinine, cystatin C, and blood urea nitrogen (BUN)] were measured before antibiotic treatment and at day two after initiation. Creatinine-defined AKI and dialysis were examined through day-14, and mortality through day-30. Inverse probability of treatment weighting was used to adjust for confounding. Multiple imputation was used to impute missing baseline covariates. RESULTS: The study included 739 patients (vancomycin + piperacillin-tazobactam n = 297, vancomycin + cefepime n = 442), of whom 192 had cystatin C measurements. Vancomycin + piperacillin-tazobactam was associated with a higher percentage increase of creatinine at day-two 8.04% (95% CI 1.21, 15.34) and higher incidence of creatinine-defined AKI: rate ratio (RR) 1.34 (95% CI 1.01, 1.78). In contrast, vancomycin + piperacillin-tazobactam was not associated with change in alternative biomarkers: cystatin C: - 5.63% (95% CI - 18.19, 8.86); BUN: - 4.51% (95% CI - 12.83, 4.59); or clinical outcomes: dialysis: RR 0.63 (95% CI 0.31, 1.29); mortality: RR 1.05 (95%CI 0.79, 1.41). CONCLUSIONS: Vancomycin + piperacillin-tazobactam was associated with creatinine-defined AKI, but not changes in alternative kidney biomarkers, dialysis, or mortality, supporting the hypothesis that vancomycin + piperacillin-tazobactam effects on creatinine represent pseudotoxicity.

Signaling Through FcγRIIA and the C5a-C5aR Pathway Mediate Platelet Hyperactivation in COVID-19.

Patients with COVID-19 present with a wide variety of clinical manifestations. Thromboembolic events constitute a significant cause of morbidity and mortality in patients infected with SARS-CoV-2. Severe COVID-19 has been associated with hyperinflammation and pre-existing cardiovascular disease. Platelets are important mediators and sensors of inflammation and are directly affected by cardiovascular stressors. In this report, we found that platelets from severely ill, hospitalized COVID-19 patients exhibited higher basal levels of activation measured by P-selectin surface expression and had poor functional reserve upon in vitro stimulation. To investigate this question in more detail, we developed an assay to assess the capacity of plasma from COVID-19 patients to activate platelets from healthy donors. Platelet activation was a common feature of plasma from COVID-19 patients and correlated with key measures of clinical outcome including kidney and liver injury, and APACHEIII scores. Further, we identified ferritin as a pivotal clinical marker associated with platelet hyperactivation. The COVID-19 plasma-mediated effect on control platelets was highest for patients that subsequently developed inpatient thrombotic events. Proteomic analysis of plasma from COVID-19 patients identified key mediators of inflammation and cardiovascular disease that positively correlated with in vitro platelet activation. Mechanistically, blocking the signaling of the FcγRIIa-Syk and C5a-C5aR pathways on platelets, using antibody-mediated neutralization, IgG depletion or the Syk inhibitor fostamatinib, reversed this hyperactivity driven by COVID-19 plasma and prevented platelet aggregation in endothelial microfluidic chamber conditions. These data identified these potentially actionable pathways as central for platelet activation and/or vascular complications and clinical outcomes in COVID-19 patients. In conclusion, we reveal a key role of platelet-mediated immunothrombosis in COVID-19 and identify distinct, clinically relevant, targetable signaling pathways that mediate this effect.

Elevated Plasma Levels of Matrix Metalloproteinase-3 and Tissue-Inhibitor of Matrix Metalloproteinases-1 Associate With Organ Dysfunction and Mortality in Sepsis.

BACKGROUND: Matrix Metalloproteinases (MMP) respond to tissue damage during sepsis. Higher plasma concentrations of MMPs and the tissue-inhibitor of matrix metalloproteinases (TIMP) have been reported in sepsis compared with healthy controls. The objective of this study was to examine if plasma levels of MMP-3, MMP-9, and TIMP-1 associate with mortality and organ dysfunction during sepsis. METHODS: We conducted a prospective cohort study of critically ill patients with sepsis adjudicated per Sepsis-3 criteria at a tertiary academic medical center. We measured plasma concentrations of MMP-3, MMP-9, and TIMP-1 on intensive care unit admission. We phenotyped the subjects for shock, acute respiratory distress syndrome (ARDS), acute kidney injury (AKI), and mortality at 30 days. We used logistic regression to test the associations between the MMPs and TIMP-1 with shock, ARDS, AKI, and mortality. RESULTS: Higher plasma TIMP-1 levels were associated with shock (odds ratio [OR] 1.51 per log increase [95% CI 1.25, 1.83]), ARDS (OR 1.24 [95% CI 1.05, 1.46]), AKI (OR 1.18 [95% CI 1.01, 1.38]), and mortality (OR 1.20 [95% CI 1.05, 1.46]. Higher plasma MMP-3 concentrations were associated with shock (OR 1.40 [95% CI 1.12, 1.75]) and mortality (OR 1.24 [95% CI 1.03, 1.48]) whereas MMP-9 levels were not associated with outcomes. Higher plasma TIMP-1 to MMP-3 ratios were associated with shock (OR 1.41 [95% CI 1.15, 1.72], P = 0.02). CONCLUSION: Elevated plasma concentrations of TIMP-1 associate with organ dysfunction and mortality in sepsis. Higher plasma levels of MMP-3 associate with shock and mortality. Plasma MMP and TIMP-1 may warrant further investigation as emerging sepsis theragnostic biomarkers.

Erythrocytes identify complement activation in patients with COVID-19.

COVID-19, the disease caused by the SARS-CoV-2 virus, can progress to multisystem organ failure and viral sepsis characterized by respiratory failure, arrhythmias, thromboembolic complications, and shock with high mortality. Autopsy and preclinical evidence implicate aberrant complement activation in endothelial injury and organ failure. Erythrocytes express complement receptors and are capable of binding immune complexes; therefore, we investigated complement activation in patients with COVID-19 using erythrocytes as a tool to diagnose complement activation. We discovered enhanced C3b and C4d deposition on erythrocytes in COVID-19 sepsis patients and non-COVID sepsis patients compared with healthy controls, supporting the role of complement in sepsis-associated organ injury. Our data suggest that erythrocytes may contribute to a precision medicine approach to sepsis and have diagnostic value in monitoring complement dysregulation in COVID-19-sepsis and non-COVID sepsis and identifying patients who may benefit from complement targeted therapies.

Signaling through FcγRIIA and the C5a-C5aR pathway mediates platelet hyperactivation in COVID-19.

Patients with COVID-19 present with a wide variety of clinical manifestations. Thromboembolic events constitute a significant cause of morbidity and mortality in patients infected with SARS-CoV-2. Severe COVID-19 has been associated with hyperinflammation and pre-existing cardiovascular disease. Platelets are important mediators and sensors of inflammation and are directly affected by cardiovascular stressors. In this report, we found that platelets from severely ill, hospitalized COVID-19 patients exhibit higher basal levels of activation measured by P-selectin surface expression, and have a poor functional reserve upon in vitro stimulation. Correlating clinical features to the ability of plasma from COVID-19 patients to stimulate control platelets identified ferritin as a pivotal clinical marker associated with platelet hyperactivation. The COVID-19 plasma-mediated effect on control platelets was highest for patients that subsequently developed inpatient thrombotic events. Proteomic analysis of plasma from COVID-19 patients identified key mediators of inflammation and cardiovascular disease that positively correlated with in vitro platelet activation. Mechanistically, blocking the signaling of the FcγRIIa-Syk and C5a-C5aR pathways on platelets, using antibody-mediated neutralization, IgG depletion or the Syk inhibitor fostamatinib, reversed this hyperactivity driven by COVID-19 plasma and prevented platelet aggregation in endothelial microfluidic chamber conditions, thus identifying these potentially actionable pathways as central for platelet activation and/or vascular complications in COVID-19 patients. In conclusion, we reveal a key role of platelet-mediated immunothrombosis in COVID-19 and identify distinct, clinically relevant, targetable signaling pathways that mediate this effect. These studies have implications for the role of platelet hyperactivation in complications associated with SARS-CoV-2 infection. ONE-SENTENCE SUMMARY: The FcγRIIA and C5a-C5aR pathways mediate platelet hyperactivation in COVID-19.

CD8+ T cells contribute to survival in patients with COVID-19 and hematologic cancer.

Patients with cancer have high mortality from coronavirus disease 2019 (COVID-19), and the immune parameters that dictate clinical outcomes remain unknown. In a cohort of 100 patients with cancer who were hospitalized for COVID-19, patients with hematologic cancer had higher mortality relative to patients with solid cancer. In two additional cohorts, flow cytometric and serologic analyses demonstrated that patients with solid cancer and patients without cancer had a similar immune phenotype during acute COVID-19, whereas patients with hematologic cancer had impairment of B cells and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific antibody responses. Despite the impaired humoral immunity and high mortality in patients with hematologic cancer who also have COVID-19, those with a greater number of CD8 T cells had improved survival, including those treated with anti-CD20 therapy. Furthermore, 77% of patients with hematologic cancer had detectable SARS-CoV-2-specific T cell responses. Thus, CD8 T cells might influence recovery from COVID-19 when humoral immunity is deficient. These observations suggest that CD8 T cell responses to vaccination might provide protection in patients with hematologic cancer even in the setting of limited humoral responses.

CD8 T cells compensate for impaired humoral immunity in COVID-19 patients with hematologic cancer.

Cancer patients have increased morbidity and mortality from Coronavirus Disease 2019 (COVID-19), but the underlying immune mechanisms are unknown. In a cohort of 100 cancer patients hospitalized for COVID-19 at the University of Pennsylvania Health System, we found that patients with hematologic cancers had a significantly higher mortality relative to patients with solid cancers after accounting for confounders including ECOG performance status and active cancer status. We performed flow cytometric and serologic analyses of 106 cancer patients and 113 non-cancer controls from two additional cohorts at Penn and Memorial Sloan Kettering Cancer Center. Patients with solid cancers exhibited an immune phenotype similar to non-cancer patients during acute COVID-19 whereas patients with hematologic cancers had significant impairment of B cells and SARS-CoV-2-specific antibody responses. High dimensional analysis of flow cytometric data revealed 5 distinct immune phenotypes. An immune phenotype characterized by CD8 T cell depletion was associated with a high viral load and the highest mortality of 71%, among all cancer patients. In contrast, despite impaired B cell responses, patients with hematologic cancers and preserved CD8 T cells had a lower viral load and mortality. These data highlight the importance of CD8 T cells in acute COVID-19, particularly in the setting of impaired humoral immunity. Further, depletion of B cells with anti-CD20 therapy resulted in almost complete abrogation of SARS-CoV-2-specific IgG and IgM antibodies, but was not associated with increased mortality compared to other hematologic cancers, when adequate CD8 T cells were present. Finally, higher CD8 T cell counts were associated with improved overall survival in patients with hematologic cancers. Thus, CD8 T cells likely compensate for deficient humoral immunity and influence clinical recovery of COVID-19. These observations have important implications for cancer and COVID-19-directed treatments, immunosuppressive therapies, and for understanding the role of B and T cells in acute COVID-19.

Comprehensive mapping of immune perturbations associated with severe COVID-19.

Although critical illness has been associated with SARS-CoV-2-induced hyperinflammation, the immune correlates of severe COVID-19 remain unclear. Here, we comprehensively analyzed peripheral blood immune perturbations in 42 SARS-CoV-2 infected and recovered individuals. We identified extensive induction and activation of multiple immune lineages, including T cell activation, oligoclonal plasmablast expansion, and Fc and trafficking receptor modulation on innate lymphocytes and granulocytes, that distinguished severe COVID-19 cases from healthy donors or SARS-CoV-2-recovered or moderate severity patients. We found the neutrophil to lymphocyte ratio to be a prognostic biomarker of disease severity and organ failure. Our findings demonstrate broad innate and adaptive leukocyte perturbations that distinguish dysregulated host responses in severe SARS-CoV-2 infection and warrant therapeutic investigation.

Deep immune profiling of COVID-19 patients reveals distinct immunotypes with therapeutic implications.

Coronavirus disease 2019 (COVID-19) is currently a global pandemic, but human immune responses to the virus remain poorly understood. We used high-dimensional cytometry to analyze 125 COVID-19 patients and compare them with recovered and healthy individuals. Integrated analysis of ~200 immune and ~50 clinical features revealed activation of T cell and B cell subsets in a proportion of patients. A subgroup of patients had T cell activation characteristic of acute viral infection and plasmablast responses reaching >30% of circulating B cells. However, another subgroup had lymphocyte activation comparable with that in uninfected individuals. Stable versus dynamic immunological signatures were identified and linked to trajectories of disease severity change. Our analyses identified three immunotypes associated with poor clinical trajectories versus improving health. These immunotypes may have implications for the design of therapeutics and vaccines for COVID-19.

Deep immune profiling of COVID-19 patients reveals patient heterogeneity and distinct immunotypes with implications for therapeutic interventions.

COVID-19 has become a global pandemic. Immune dysregulation has been implicated, but immune responses remain poorly understood. We analyzed 71 COVID-19 patients compared to recovered and healthy subjects using high dimensional cytometry. Integrated analysis of ~200 immune and >30 clinical features revealed activation of T cell and B cell subsets, but only in some patients. A subgroup of patients had T cell activation characteristic of acute viral infection and plasmablast responses could reach >30% of circulating B cells. However, another subgroup had lymphocyte activation comparable to uninfected subjects. Stable versus dynamic immunological signatures were identified and linked to trajectories of disease severity change. These analyses identified three "immunotypes" associated with poor clinical trajectories versus improving health. These immunotypes may have implications for therapeutics and vaccines.

Immunologic perturbations in severe COVID-19/SARS-CoV-2 infection.

Although critical illness has been associated with SARS-CoV-2-induced hyperinflammation, the immune correlates of severe COVID-19 remain unclear. Here, we comprehensively analyzed peripheral blood immune perturbations in 42 SARS-CoV-2 infected and recovered individuals. We identified broad changes in neutrophils, NK cells, and monocytes during severe COVID-19, suggesting excessive mobilization of innate lineages. We found marked activation within T and B cells, highly oligoclonal B cell populations, profound plasmablast expansion, and SARS-CoV-2-specific antibodies in many, but not all, severe COVID-19 cases. Despite this heterogeneity, we found selective clustering of severe COVID-19 cases through unbiased analysis of the aggregated immunological phenotypes. Our findings demonstrate broad immune perturbations spanning both innate and adaptive leukocytes that distinguish dysregulated host responses in severe SARS-CoV-2 infection and warrant therapeutic investigation. One Sentence Summary: Broad immune perturbations in severe COVID-19.

Erythrocytes Reveal Complement Activation in Patients with COVID-19.

COVID-19, the disease caused by the SARS-CoV-2 virus, can progress to multi-organ failure characterized by respiratory insufficiency, arrhythmias, thromboembolic complications and shock. The mortality of patients hospitalized with COVID-19 is unacceptably high and new strategies are urgently needed to rapidly identify and treat patients at risk for organ failure. Clinical epidemiologic studies demonstrate that vulnerability to organ failure is greatest after viral clearance from the upper airway, which suggests that dysregulation of the host immune response is a critical mediator of clinical deterioration and death. Autopsy and pre-clinical evidence implicate aberrant complement activation in endothelial injury and organ failure. A potential therapeutic strategy warranting investigation is to inhibit complement, with case reports of successful treatment of COVID-19 with inhibitors of complement. However, this approach requires careful balance between the host protective and potential injurious effects of complement activation, and biomarkers to identify the optimal timing and candidates for therapy are lacking. Here we report the presence of complement activation products on circulating erythrocytes from hospitalized COVID-19 patients using flow cytometry. These findings suggest that novel erythrocyte-based diagnostics provide a method to identify patients with dysregulated complement activation.