ABSTRACT
Background. COVID-19 disease severity and outcomes have been linked to high antibody titers and a dysregulated neutrophil immune response. Here we query associations and connections between the endogenous SARS-CoV-2 antibody response and neutrophil activation in COVID-19. Methods. Baseline serum or plasma samples from 57 patients hospitalized on oxygen with COVID-19 were used to perform;1) quantitative measurements of SARS-CoV-2 specific antibodies using a luciferase-based immunoprecipitation system assay, 2) quantitative measurements of neutrophil specific biomarkers using Luminex technology, and 3) neutrophil extracellular traps (NETs) as measured by myeloperoxidase-DNA (MPO-DNA) complexes by ELISA. Absolute neutrophil count (ANC) and immature granulocyte count (IGC) were measured from complete blood counts (CBC). Antibody levels were compared by disease severity using Wilcoxon rank-sum test and correlations were generated between antibody levels and neutrophil activation markers using Spearman's correlation (SC). Results. In a cohort of hospitalized patients, severe/critical COVID-19 was associated with higher levels of nucleocapsid-IgA (p=0.011) as well as spike-IgG (p= 0.0007) compared tomoderate disease,while spike-IgA and nucleocapsid-IgG showed similar associations, trending towards significance (Figure 1A). Levels of IgG-spike and IgG-nucleocapsid both had significant correlations with the ANC (SC 0.33, p = 0.029;SC 0.38 p = 0.012). All four antibody titers showed strong correlations with IGC, lactoferrin and lipocalin-2, evidence of emergency granulopoiesis. Further, S100A9, a component calprotectin correlated with spike-IgG and nucleocapsid-IgA levels (SC 0.29, p = 0.030, SC 0.29 p = 0.029). Lastly, we found circulating NETs correlated with spike IgA levels (SC 0.38 p = 0.006), and its correlations with IgG-spike and IgA-nucleocapsid additionally approached significance with NETs levels as well (Figure 1B). Antibody Levels Correlate with Disease Severity and Neutrophil Activation Markers Figure 1: A) Levels of anti-Spike and anti-Nucleocapsid IgA and IgG levels measured in the serum of 57 unvaccinated hospitalized COVID-19 patients. Moderate illness represents ordinal scale 5 requiring low flow oxygen, while severe/critical patients represent ordinal scale 6 and 7, requiring high flow oxygen, non-invasive or mechanical ventilation, respectively. P values are compared by a Wilcoxon ranked sum test. B) Heatmap showing Spearman correlations between levels of anti-Spike and anti-Nucleocapsid IgA and IgG and markers of neutrophil activation. P values for individual correlations are represented in parentheses. MPO (myeloperoxidase), ANC (absolute neutrophil count), S100A9 (S100 calcium binding protein A9). Conclusion. Higher anti-spike and anti-nucleocapsid IgG and IgA levels associate with more severe COVID-19 illness. Further, endogenous SARS-CoV-2 specific antibody levels associate with markers of emergency granulopoiesis and neutrophil activation. Inhibiting antibody mediated neutrophil activation may improve outcomes in COVID-19.
ABSTRACT
Background. Coronavirus Disease 2019 (COVID-19) caused by the SARS-CoV-2 virus is associated with dysregulation in the innate immune response including NK cells. NK cells are integral in the innate immune response against viral infections. Canonical NK cells are classified as CD56dim CD16+ and CD56bright CD16-. An unconventional subset of CD56dim CD16 - NK cells has previously been identified in COVID-19 that is not present in other viral infections. Here we characterize phenotypic changes in the NK cells of patients with severe COVID-19 as work towards determining the functional status of this unconventional subset. Methods. Peripheral blood mononuclear cells (PBMCs) and plasma were isolated from healthy donors (n=5) and patients with severe COVID-19 on Extra Corporeal Membrane Oxygenation (ECMO) (n =15). Primary NK cells were stimulated in vitro with plasma from patients with severe COVID-19 or healthy donors. Flow cytometry was used to phenotype the NK cells. A separate cohort of PBMC samples (n =7) from patients requiring hospitalization for COVID-19 underwent Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq) analysis. Results. The CD56bright CD16 - NK subset was expanded in PBMCs from patients with severe COVID-19 as compared to healthy controls. CITE-Seq demonstrated that NK cells without surface CD16 clustered separately based on transcriptional profiling and did express FCGR3A at the translational level. Stimulation with COVID-19 plasma recapitulated the loss of CD16 from primary human NK cells and led to increased activity of Caspase 3/7. a) Representative gating of NK cell subsets by Flow Cytometry in healthy and COVID-19 patient peripheral blood mononuclear cells (PBMCs). b) Percentage of total NK cells belonging to a particular cell subset compared between healthy donor samples (n=4) and COVID-19 patient samples (n =8). Data points represent an individual patient sample. Error bars represent the standard deviation of the mean. Differences between groups was analyzed using a two tailed t-test. *: p< 0.05, ns: not significant Figure 2. NK cells shift from the CD56dim CD16+ subset to the CD56dim CD16-subset after stimulation with COVID-19 plasma in vitro a) Representative gating of NK cell subsets by Flow Cytometry analysis in healthy donor NK cells stimulated by healthy plasma and COVID-19 patient plasma. b)Relative change in percentage of total NK cells belonging to a particular cell subset compared between healthy donor plasma (n=6)and COVID-19 patient plasma (n=15) stimulation conditions. Error bars represent the standard deviation of the mean and the difference between groups was analyzed using a two-tailed T-test. * *: p< 0.01, * * *: p< 0.001, ns: not significant. Conclusion. We demonstrate and characterize a nonclassical population of CD56dim CD16 - NK cells that are present in patients with severe COVID-19 and replicate this phenotype in vitro. Reproduction of this in vivo phenotype in an in vitro system will allow for additional studies on the functional state of NK cell subsets in COVID-19. The presence of this NK cell population may reflect a dysregulated innate immune response and immunopathogenesis of COVID-19.
ABSTRACT
Background. Patients admitted with COVID19 pneumonia often receive initial empiric antibacterial therapy (IEAT) despite a known low probability of bacterial co-infection. However, evidence supporting this practice is lacking. We studied the impact of IEAT on the risk of in-hospital mortality, clinical deterioration and antibiotic-associated risks in stable inpatients with COVID-19. Methods. Adult inpatients coded for COVID-19 pneumonia stable (no mechanical ventilation or vasopressors) on admission (+1 day) without a clear indication for antibiotics, were identified at hospitals in the Premier Healthcare Database. Patients who received IEAT, defined as the receipt of >= 1 antibacterial agent on admission (+1 day), were compared to a control group, using binomial regression with overlap weight matching and downstream adjustment for baseline characteristics (age, gender, race, admission month, surge index, Elixhauser score, any AOFS organ failure POA, ICU admission on day 0 to +2, receipt of remdesivir, corticosteroids, and tocilizumab). The primary outcome was in-hospital mortality or discharge to hospice;secondary outcomes included need for mechanical ventilation on day2+, and rates of non-POA-acute kidney injury (AKI). Results. At 221 hospitals between March-December 2020, 39,517 (74%) of 53,431 stable COVID-19 admits received IEAT. Patient and encounter characteristics are shown in Table 1. The crude mortality rates were 12.2% in IEAT recipients and 10.9% in controls. In adjusted analysis of patients who survived beyond admission day, mortality was 11.57% (95% CI 11.24-11.90%) in IEAT recipients and 11.23% (95% CI 10.72-11.74) in controls, for a difference of 0.34% (95% CI -0.23-0.91%, p = 0.24). Subsequent mechanical ventilation occurred similarly between groups (5.72% vs. 5.77%, p=0.83). The adjusted rate of AKI was 2.47% (95% CI 2.31-2.64%) in IEAT recipients, and 3.04% (95% CI 2.74-3.35%) in controls, for a difference of -0.57% (95% CI -0.92-0.22%, p = 0.0014). Conclusion. In patients with COVID19 initially admitted to the ward, IEAT was not associated with a reduction in mortality or deterioration requiring mechanical ventilation, but with a clinically insignificant reduction in AKI. Empiric antibiotics can likely be safely withheld in this population.
ABSTRACT
BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease 2019 (COVID-19), is associated with respiratory-related disease and death. Assays to detect virus-specific antibodies are important to understand the prevalence of infection and the course of the immune response. METHODS: Quantitative measurements of plasma or serum antibodies to the nucleocapsid and spike proteins were analyzed using luciferase immunoprecipitation system assays in 100 cross-sectional or longitudinal samples from patients with SARS-CoV-2 infection. A subset of samples was tested both with and without heat inactivation. RESULTS: At 14 days after symptom onset, antibodies against SARS-CoV-2 nucleocapsid protein showed 100% sensitivity and 100% specificity, whereas antibodies to spike protein were detected with 91% sensitivity and 100% specificity. Neither antibody levels nor the rate of seropositivity were significantly reduced by heat inactivation of samples. Analysis of daily samples from 6 patients with COVID-19 showed anti-nucleocapsid and spike protein antibodies appearing between days 8 and 14 after initial symptoms. Immunocompromised patients generally had a delayed antibody response to SARS-CoV-2, compared with immunocompetent patients. CONCLUSIONS: Antibody to the nucleocapsid protein of SARS-CoV-2 is more sensitive than spike protein antibody for detecting early infection. Analyzing heat-inactivated samples with a luciferase immunoprecipitation system assay is a safe and sensitive method for detecting SARS-CoV-2 antibodies.