Phenotypes of disease severity in a cohort of hospitalized COVID-19 patients: results from the IMPACC study

BackgroundBetter understanding of the association between characteristics of patients hospitalized with coronavirus disease 2019 (COVID-19) and outcome is needed to further improve upon patient management. MethodsImmunophenotyping Assessment in a COVID-19 Cohort (IMPACC) is a prospective, observational study of 1,164 patients from 20 hospitals across the United States. Disease severity was assessed using a 7-point ordinal scale based on degree of respiratory illness. Patients were prospectively surveyed for 1 year after discharge for post-acute sequalae of COVID-19 (PASC) through quarterly surveys. Demographics, comorbidities, radiographic findings, clinical laboratory values, SARS-CoV-2 PCR and serology were captured over a 28-day period. Multivariable logistic regression was performed. FindingsThe median age was 59 years (interquartile range [IQR] 20); 711 (61%) were men; overall mortality was 14%, and 228 (20%) required invasive mechanical ventilation. Unsupervised clustering of ordinal score over time revealed distinct disease course trajectories. Risk factors associated with prolonged hospitalization or death by day 28 included age [≥] 65 years (odds ratio [OR], 2.01; 95% CI 1.28-3.17), Hispanic ethnicity (OR, 1.71; 95% CI 1.13-2.57), elevated baseline creatinine (OR 2.80; 95% CI 1.63-4.80) or troponin (OR 1.89; 95% 1.03-3.47), baseline lymphopenia (OR 2.19; 95% CI 1.61-2.97), presence of infiltrate by chest imaging (OR 3.16; 95% CI 1.96-5.10), and high SARS-CoV2 viral load (OR 1.53; 95% CI 1.17-2.00). Fatal cases had the lowest ratio of SARS-CoV-2 antibody to viral load levels compared to other trajectories over time (p=0.001). 589 survivors (51%) completed at least one survey at follow-up with 305 (52%) having at least one symptom consistent with PASC, most commonly dyspnea (56% among symptomatic patients). Female sex was the only associated risk factor for PASC. InterpretationIntegration of PCR cycle threshold, and antibody values with demographics, comorbidities, and laboratory/radiographic findings identified risk factors for 28-day outcome severity, though only female sex was associated with PASC. Longitudinal clinical phenotyping offers important insights, and provides a framework for immunophenotyping for acute and long COVID-19. FundingNIH RESEARCH IN CONTEXTO_ST_ABSEvidence before this studyC_ST_ABSWe did a systematic search of the PubMed database from January 1st, 2020 until April 24th, 2022 using the search terms: "hospitalized" AND "SARS-CoV-2" OR "COVID-19" AND "Pro-spective" AND "Antibody" OR "PCR" OR "long term follow up" and applying the following filters: "Multicenter Study" AND "Observational Study". No language restrictions were applied. While clinical, laboratory, and radiographic features associated with severe COVID-19 in hospitalized adults have been described, description of the kinetics of SARS-CoV-2 specific assays available to clinicians (e.g. PCR and binding antibody) and their integration with other variables is scarce for both short and long term follow up. The current literature is comprised of several studies with small sample size, cross-sectional design with laboratory data typically only recorded at a single point in time (e.g., on admission), limited clinical characteristics, variable duration of follow up, single-center setting, retrospective analyses, kinetics of either PCR or antibody testing but not both, and outcomes such as death or, mechanical ventilation that do not allow delineation of variations in clinical course. Added value of this studyIn our large longitudinal multicenter cohort, the description of outcome severity, was not limited to survival versus death, but encompassed a clinical trajectory approach leveraging longitudinal data based on time in hospital, disease severity by ordinal scale based on degree of respiratory illness, and presence or absence of limitations at discharge. Fatal COVID-19 cases had the lowest ratio of antibody to viral load levels over time as compared to non-fatal cases. Integration of PCR cycle threshold and antibody values with demographics, baseline comorbidities, and laboratory/radiographic findings identified additional risk factors for outcome severity over the first 28 days. However, female sex was the only variable associated with persistence of symptoms over time. Persistence of symptoms was not associated with clinical trajectory over the first 28 days, nor with antibody/viral loads from the acute phase. Implications of all the available evidenceThe described calculated ratio (binding IgG/PCR Ct value) is unique compared to other studies, reflecting host pathogen interactions and representing an accessible approach for patient risk stratification. Integration of SARS-CoV-2 viral load and binding antibody kinetics with other laboratory as well as clinical characteristics in hospitalized COVID-19 patients can identify patients likely to have the most severe short-term outcomes, but is not predictive of symptom persistence at one year post-discharge.

Shift of lung macrophage composition is associated with COVID-19 disease severity and recovery

Though it has been 2 years since the start of the Coronavirus Disease 19 (COVID-19) pandemic, COVID-19 continues to be a worldwide health crisis. Despite the development of preventive vaccines, very little progress has been made to identify curative therapies to treat COVID-19 and other inflammatory diseases which remain a major unmet need in medicine. Our study sought to identify drivers of disease severity and death to develop tailored immunotherapy strategies to halt disease progression. Here we assembled the Mount Sinai COVID-19 Biobank which was comprised of ~600 hospitalized patients followed longitudinally during the peak of the pandemic. Moderate disease and survival were associated with a stronger antigen (Ag) presentation and effector T cell signature, while severe disease and death were associated with an altered Ag presentation signature, increased numbers of circulating inflammatory, immature myeloid cells, and extrafollicular activated B cells associated with autoantibody formation. Strikingly, we found that in severe COVID-19 patients, lung tissue resident alveolar macrophages (AM) were not only severely depleted, but also had an altered Ag presentation signature, and were replaced by inflammatory monocytes and monocyte-derived macrophages (MoM{Phi}). Notably, the size of the AM pool correlated with recovery or death, while AM loss and functionality were restored in patients that recovered. These data therefore suggest that local and systemic myeloid cell dysregulation is a driver of COVID-19 severity and that modulation of AM numbers and functionality in the lung may be a viable therapeutic strategy for the treatment of critical lung inflammatory illnesses.

Proteomic Characterization of Acute Kidney Injury in Patients Hospitalized with SARS-CoV2 Infection

Acute kidney injury (AKI) is a known complication of COVID-19 and is associated with an increased risk of in-hospital mortality. Unbiased proteomics using biological specimens can lead to improved risk stratification and discover pathophysiological mechanisms. Using measurements of [~]4000 plasma proteins in two cohorts of patients hospitalized with COVID-19, we discovered and validated markers of COVID-associated AKI (stage 2 or 3) and long-term kidney dysfunction. In the discovery cohort (N= 437), we identified 413 higher plasma abundances of protein targets and 40 lower plasma abundances of protein targets associated with COVID-AKI (adjusted p <0.05). Of these, 62 proteins were validated in an external cohort (p <0.05, N =261). We demonstrate that COVID-AKI is associated with increased markers of tubular injury (NGAL) and myocardial injury. Using estimated glomerular filtration (eGFR) measurements taken after discharge, we also find that 25 of the 62 AKI-associated proteins are significantly associated with decreased post-discharge eGFR (adjusted p <0.05). Proteins most strongly associated with decreased post-discharge eGFR included desmocollin-2, trefoil factor 3, transmembrane emp24 domain-containing protein 10, and cystatin-C indicating tubular dysfunction and injury. Using clinical and proteomic data, our results suggest that while both acute and long-term COVID-associated kidney dysfunction are associated with markers of tubular dysfunction, AKI is driven by a largely multifactorial process involving hemodynamic instability and myocardial damage.

Circulating proteins to predict adverse COVID-19 outcomes

Predicting COVID-19 severity is difficult, and the biological pathways involved are not fully understood. To approach this problem, we measured 4,701 circulating human protein abundances in two independent cohorts totaling 986 individuals. We then trained prediction models including protein abundances and clinical risk factors to predict adverse COVID-19 outcomes in 417 subjects and tested these models in a separate cohort of 569 individuals. For severe COVID-19, a baseline model including age and sex provided an area under the receiver operator curve (AUC) of 65% in the test cohort. Selecting 92 proteins from the 4,701 unique protein abundances improved the AUC to 88% in the training cohort, which remained relatively stable in the testing cohort at 86%, suggesting good generalizability. Proteins selected from different adverse COVID-19 outcomes were enriched for cytokine and cytokine receptors, but more than half of the enriched pathways were not immune-related. Taken together, these findings suggest that circulating proteins measured at early stages of disease progression are reasonably accurate predictors of adverse COVID-19 outcomes. Further research is needed to understand how to incorporate protein measurement into clinical care.

Acute COVID-19 gene-expression profiles show multiple etiologies of long-term sequelae

Summary ParagraphTwo years into the SARS-CoV-2 pandemic, the post-acute sequelae of infection are compounding the global health crisis. Often debilitating, these sequelae are clinically heterogeneous and of unknown molecular etiology. Here, a transcriptome-wide investigation of this new condition was performed in a large cohort of acutely infected patients followed clinically into the post-acute period. Gene expression signatures of post-acute sequelae were already present in whole blood during the acute phase of infection, with both innate and adaptive immune cells involved. Plasma cells stood out as driving at least two distinct clusters of sequelae, one largely dependent on circulating antibodies against the SARS-CoV-2 spike protein and the other antibody-independent. Altogether, multiple etiologies of post-acute sequelae were found concomitant with SARS-CoV-2 infection, directly linking the emergence of these sequelae with the host response to the virus.

Divergent early antibody responses define COVID-19 disease trajectories

A damaging inflammatory response is strongly implicated in the pathogenesis of severe COVID-19 but mechanisms contributing to this response are unclear. In two prospective cohorts, early non-neutralizing, afucosylated, anti-SARS-CoV-2 IgG predicted progression from mild, to more severe COVID-19. In contrast to the antibody structures that predicted disease progression, antibodies that were elicited by mRNA SARS-CoV-2 vaccines were low in Fc afucosylation and enriched in sialylation, both modifications that reduce the inflammatory potential of IgG. To study the biology afucosylated IgG immune complexes, we developed an in vivo model which revealed that human IgG-Fc{gamma}R interactions can regulate inflammation in the lung. Afucosylated IgG immune complexes induced inflammatory cytokine production and robust infiltration of the lung by immune cells. By contrast, vaccine elicited IgG did not promote an inflammatory lung response. Here, we show that IgG-Fc{gamma}R interactions can regulate inflammation in the lung and define distinct lung activities associated with the IgG that predict severe COVID-19 and protection against SARS-CoV-2. One Sentence SummaryDivergent early antibody responses predict COVID-19 disease trajectory and mRNA vaccine response and are functionally distinct in vivo.

Fatal breakthrough infection after anti-BCMA CAR-T therapy highlights suboptimal immune response to SARS-CoV-2 vaccination in myeloma patients

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines are highly effective in healthy individuals. Patients with multiple myeloma (MM) are immunocompromised due to defects in humoral and cellular immunity as well as immunosuppressive therapies. The efficacy after two doses of SARS-CoV-2 mRNA vaccination in MM patients is currently unknown. Here, we report the case of a MM patient who developed a fatal SARS-CoV-2 infection after full vaccination while in remission after B cell maturation antigen (BCMA)-targeted chimeric antigen receptor (CAR)-T treatment. We show that the patient failed to generate antibodies or SARS-CoV-2-specific B and T cell responses, highlighting the continued risk of severe coronavirus disease 2019 (COVID-19) in vaccine non-responders. In the largest cohort of vaccinated MM patients to date, we demonstrate that 15.9% lack SARS-CoV-2 spike antibody response more than 10 days after the second mRNA vaccine dose. The patients actively receiving MM treatment, especially on regimens containing anti-CD38 and anti-BCMA, have lower antibody responses compared to healthy controls. Thus, it is of critical importance to monitor this patient population for serological responses. Non-responders may benefit from ongoing public health measures and from urgent study of prophylactic treatments to prevent SARS-CoV-2 infection.

Application of a 27-protein candidate cardiovascular surrogate endpoint to track risk ascendancy and resolution in COVID-19.

BackgroundThere is an urgent need for tools allowing the early prognosis and subsequent monitoring of individuals with heterogeneous COVID-19 disease trajectories. Pre-existing cardiovascular (CV) disease is a leading risk factor for COVID-19 susceptibility and poor outcomes, and cardiac involvement is prevalent in COVID-19 patients both during the acute phase as well as in convalescence. The utility of traditional CV risk biomarkers in mild COVID-19 disease or across disease course is poorly understood. We sought to determine if a previously validated 27-protein predictor of CV outcomes served a purpose in COVID-19. MethodsThe 27-protein test of residual CV (RCV) risk was applied without modification to n=860 plasma samples from hospitalized and non-hospitalized SARS-CoV-2 infected individuals at disease presentation from three independent cohorts to predict COVID-19 severity and mortality. The same test was applied to an additional n=991 longitudinal samples to assess sensitivity to change in CV risk throughout the course of infection into convalescence. ResultsIn each independent cohort, RCV predictions were significantly related to maximal subsequent COVID-19 severity and to mortality. At the baseline blood draw, the mean protein-predicted likelihood of an event in subjects who died during the study period ranged from 88-99% while it ranged from 8-36% in subjects who were not admitted to hospital. Additionally, the test outperformed existing risk predictors based on commonly used laboratory chemistry values or presence of comorbidities. Application of the RCV test to sequential samples showed dramatic increases in risk during the first few days of infection followed by risk reduction in the survivors; a period of catastrophically high cardiovascular risk (above 50%) typically lasted 8-12 days and had not resolved to normal levels in most people within that timescale. ConclusionsThe finding that a 27-protein candidate CV surrogate endpoint developed in multi-morbid patients prior to the pandemic is both prognostic and acutely sensitive to the adverse effects of COVID-19 suggests that this disease activates the same biologic risk-related mechanisms. The test may be useful for monitoring recovery and drug response.

Cytotoxic lymphocytes are dysregulated in multisystem inflammatory syndrome in children

Multisystem inflammatory syndrome in children (MIS-C) presents with fever, inflammation and multiple organ involvement in individuals under 21 years following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. To identify genes, pathways and cell types driving MIS-C, we sequenced the blood transcriptomes of MIS-C cases, pediatric cases of coronavirus disease 2019, and healthy controls. We define a MIS-C transcriptional signature partially shared with the transcriptional response to SARS-CoV-2 infection and with the signature of Kawasaki disease, a clinically similar condition. By projecting the MIS-C signature onto a co-expression network, we identified disease gene modules and found genes downregulated in MIS-C clustered in a module enriched for the transcriptional signatures of exhausted CD8+ T-cells and CD56dimCD57+ NK cells. Bayesian network analyses revealed nine key regulators of this module, including TBX21, a central coordinator of exhausted CD8+ T-cell differentiation. Together, these findings suggest dysregulated cytotoxic lymphocyte response to SARS-Cov-2 infection in MIS-C.

Mapping Systemic Inflammation and Antibody Responses in Multisystem Inflammatory Syndrome in Children (MIS-C)

Initially, the global outbreak of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spared children from severe disease. However, after the initial wave of infections, clusters of a novel hyperinflammatory disease have been reported in regions with ongoing SARS-CoV-2 epidemics. While the characteristic clinical features are becoming clear, the pathophysiology remains unknown. Herein, we report on the immune profiles of eight Multisystem Inflammatory Syndrome in Children (MIS-C) cases. We document that all MIS-C patients had evidence of prior SARS-CoV-2 exposure, mounting an antibody response with normal isotype-switching and neutralization capability. We further profiled the secreted immune response by high-dimensional cytokine assays, which identified elevated signatures of inflammation (IL-18 and IL-6), lymphocytic and myeloid chemotaxis and activation (CCL3, CCL4, and CDCP1) and mucosal immune dysregulation (IL-17A, CCL20, CCL28). Mass cytometry immunophenotyping of peripheral blood revealed reductions of mDC1 and non-classical monocytes, as well as both NK- and T-lymphocytes, suggesting extravasation to affected tissues. Markers of activated myeloid function were also evident, including upregulation of ICAM1 and Fc{gamma}R1 in neutrophil and non-classical monocytes, well-documented markers in autoinflammation and autoimmunity that indicate enhanced antigen presentation and Fc-mediated responses. Finally, to assess the role for autoimmunity secondary to infection, we profiled the auto-antigen reactivity of MIS-C plasma, which revealed both known disease-associated autoantibodies (anti-La) and novel candidates that recognize endothelial, gastrointestinal and immune-cell antigens. All patients were treated with anti-IL6R antibody or IVIG, which led to rapid disease resolution tracking with normalization of inflammatory markers. One Sentence SummaryThis study maps the cellular and serological immune dysfunction underlying a novel pediatric inflammatory syndrome associated with SARS-CoV-2.

A Streamlined CyTOF Workflow To Facilitate Standardized Multi-Site Immune Profiling of COVID-19 Patients

Mass cytometry (CyTOF) represents one of the most powerful tools in immune phenotyping, allowing high throughput quantification of over 40 single parameters at single-cell resolution. However, wide deployment of CyTOF-based immune phenotyping studies are limited by complex experimental workflows and the need for specialized CyTOF equipment and technical expertise. Furthermore, differences in cell isolation and enrichment protocols, antibody reagent preparation, sample staining and data acquisition protocols can all introduce technical variation that can potentially confound integrative analyses of large data-sets of samples processed across multiple labs. Here, we describe the validation of a streamlined whole blood CyTOF workflow that addresses many of these challenges and facilitates wider adoption of CyTOF immune monitoring across sites with limited technical expertise or sample-processing resources or equipment. Our workflow utilizes commercially available reagents including the Fluidigm MaxPar Direct Immune Profiling Assay, a dry tube 30-marker immunophenotyping panel, and SmartTube Proteomic Stabilizer, which allows for simple and reliable fixation and cryopreservation of whole blood samples. We validate a workflow that allows for streamlined staining of whole blood samples with minimal processing requirements or expertise at the site of sample collection, followed by shipment to a central CyTOF core facility for batched downstream processing and data acquisition. We further demonstrate the application of this workflow to characterize immune responses in a cohort of hospitalized COVID-19 patients, highlighting key disease-associated changes in immune cell frequency and phenotype.

An inflammatory cytokine signature helps predict COVID-19 severity and death

The COVID-19 pandemic caused by infection with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has led to more than 100,000 deaths in the United States. Several studies have revealed that the hyper-inflammatory response induced by SARS-CoV-2 is a major cause of disease severity and death in infected patients. However, predictive biomarkers of pathogenic inflammation to help guide targetable immune pathways are critically lacking. We implemented a rapid multiplex cytokine assay to measure serum IL-6, IL-8, TNF-, and IL-1{beta} in hospitalized COVID-19 patients upon admission to the Mount Sinai Health System in New York. Patients (n = 1484) were followed up to 41 days (median 8 days) and clinical information, laboratory test results and patient outcomes were collected. In 244 patients, cytokine measurements were repeated over time, and effect of drugs could be assessed. Kaplan-Meier methods were used to compare survival by cytokine strata, followed by Cox regression models to evaluate the independent predictive value of baseline cytokines. We found that high serum IL-6, IL-8, and TNF- levels at the time of hospitalization were strong and independent predictors of patient survival. Importantly, when adjusting for disease severity score, common laboratory inflammation markers, hypoxia and other vitals, demographics, and a range of comorbidities, IL-6 and TNF- serum levels remained independent and significant predictors of disease severity and death. We propose that serum IL-6 and TNF- levels should be considered in the management and treatment of COVID-19 patients to stratify prospective clinical trials, guide resource allocation and inform therapeutic options. We also propose that patients with high IL-6 and TNF- levels should be assessed for combinatorial blockade of pathogenic inflammation in this disease.