Defibrotide mitigates endothelial cell injury induced by plasmas from patients with COVID-19 and related vasculopathies.

BACKGROUND AND OBJECTIVES: COVID-19 progression is characterized by systemic small vessel arterial and venous thrombosis. Microvascular endothelial cell (MVEC) activation and injury, platelet activation, and histopathologic features characteristic of acute COVID-19 also describe certain thrombotic microangiopathies, including atypical hemolytic-uremic syndrome (aHUS), thrombotic thrombocytopenic purpura (TTP), and hematopoietic stem cell transplant (HSCT)-associated veno-occlusive disease (VOD). We explored the effect of clinically relevant doses of defibrotide, approved for HSCT-associated VOD, on MVEC activation/injury. METHODS: Human dermal MVEC were exposed to plasmas from patients with acute TMAs or acute COVID-19 in the presence and absence of defibrotide (5µg/ml) and caspase 8, a marker of EC activation and apoptosis, was assessed. RNAseq was used to explore potential mechanisms of defibrotide activity. RESULTS: Defibrotide suppressed TMA plasma-induced caspase 8 activation in MVEC (mean 60.2 % inhibition for COVID-19; p = 0.0008). RNAseq identified six major cellular pathways associated with defibrotide's alteration of COVID-19-associated MVEC changes: TNF-α signaling; IL-17 signaling; extracellular matrix (ECM)-EC receptor and platelet receptor interactions; ECM formation; endothelin activity; and fibrosis. Communications across these pathways were revealed by STRING analyses. Forty transcripts showing the greatest changes induced by defibrotide in COVID-19 plasma/MVEC cultures included: claudin 14 and F11R (JAM), important in maintaining EC tight junctions; SOCS3 and TNFRSF18, involved in suppression of inflammation; RAMP3 and transgelin, which promote angiogenesis; and RGS5, which regulates caspase activation and apoptosis. CONCLUSION: Our data, in the context of a recent clinical trial in severe COVID-19, suggest benefits to further exploration of defibrotide and these pathways in COVID-19 and related endotheliopathies.

Machine Learning Highlights Downtrending of COVID-19 Patients with a Distinct Laboratory Profile

Background New York City (NYC) experienced an initial surge and gradual decline in the number of SARS-CoV-2-confirmed cases in 2020. A change in the pattern of laboratory test results in COVID-19 patients over this time has not been reported or correlated with patient outcome. Methods We performed a retrospective study of routine laboratory and SARS-CoV-2 RT-PCR test results from 5,785 patients evaluated in a NYC hospital emergency department from March to June employing machine learning analysis. Results A COVID-19 high-risk laboratory test result profile (COVID19-HRP), consisting of 21 routine blood tests, was identified to characterize the SARS-CoV-2 patients. Approximately half of the SARS-CoV-2 positive patients had the distinct COVID19-HRP that separated them from SARS-CoV-2 negative patients. SARS-CoV-2 patients with the COVID19-HRP had higher SARS-CoV-2 viral loads, determined by cycle threshold values from the RT-PCR, and poorer clinical outcome compared to other positive patients without the COVID12-HRP. Furthermore, the percentage of SARS-CoV-2 patients with the COVID19-HRP has significantly decreased from March/April to May/June. Notably, viral load in the SARS-CoV-2 patients declined, and their laboratory profile became less distinguishable from SARS-CoV-2 negative patients in the later phase. Conclusions Our longitudinal analysis illustrates the temporal change of laboratory test result profile in SARS-CoV-2 patients and the COVID-19 evolvement in a US epicenter. This analysis could become an important tool in COVID-19 population disease severity tracking and prediction. In addition, this analysis may play an important role in prioritizing high-risk patients, assisting in patient triaging and optimizing the usage of resources.

Premortem Skin Biopsy Assessing Microthrombi, Interferon Type I Antiviral and Regulatory Proteins, and Complement Deposition Correlates with Coronavirus Disease 2019 Clinical Stage.

Apart from autopsy, tissue correlates of coronavirus disease 2019 (COVID-19) clinical stage are lacking. In the current study, cutaneous punch biopsy specimens of 15 individuals with severe/critical COVID-19 and six with mild/moderate COVID-19 were examined. Evidence for arterial and venous microthrombi, deposition of C5b-9 and MASP2 (representative of alternative and lectin complement pathways, respectively), and differential expression of interferon type I-driven antiviral protein MxA (myxovirus resistance A) versus SIN3A, a promoter of interferon type I-based proinflammatory signaling, were assessed. Control subjects included nine patients with sepsis-related acute respiratory distress syndrome (ARDS) and/or acute kidney injury (AKI) pre-COVID-19. Microthrombi were detected in 13 (87%) of 15 patients with severe/critical COVID-19 versus zero of six patients with mild/moderate COVID-19 (P < 0.001) and none of the nine patients with pre-COVID-19 ARDS/AKI (P < 0.001). Cells lining the microvasculature staining for spike protein of severe acute respiratory syndrome coronavirus 2, the etiologic agent of COVID-19, also expressed tissue factor. C5b-9 deposition occurred in 13 (87%) of 15 patients with severe/critical COVID-19 versus zero of six patients with mild/moderate COVID-19 (P < 0.001) and none of the nine patients with pre-COVID-19 ARDS/AKI (P < 0.001). MASP2 deposition was also restricted to severe/critical COVID-19 cases. MxA expression occurred in all six mild/moderate versus two (15%) of 13 severe/critical cases (P < 0.001) of COVID-19. In contrast, SIN3A was restricted to severe/critical COVID-19 cases co-localizing with severe acute respiratory syndrome coronavirus 2 spike protein. SIN3A was also elevated in plasma of patients with severe/critical COVID-19 versus control subjects (P ≤ 0.02). In conclusion, the study identified premortem tissue correlates of COVID-19 clinical stage using skin. If validated in a longitudinal cohort, this approach could identify individuals at risk for disease progression and enable targeted interventions.

Measurements of SARS-CoV-2 antibody dissociation rate constant by chaotrope-free biolayer interferometry in serum of COVID-19 convalescent patients.

Kinetics measurements of antigen-antibody binding interactions are critical to understanding the functional efficiency of SARS-CoV-2 antibodies. Previously reported chaotrope-based avidity assays that rely on artificial disruption of binding do not reflect the natural binding kinetics. This study developed a chaotrope- and label-free biolayer interferometry (BLI) assay for the real-time monitoring of receptor binding domain (RBD) binding kinetics with SARS-CoV-2 spike protein in convalescent COVID-19 patients. An improved conjugation biosensor probe coated with streptavidin-polysaccharide (SA-PS) led to a six-fold increase of signal intensities and two-fold reduction of non-specific binding (NSB) compared to streptavidin only probe. Furthermore, by utilizing a separate reference probe and biotin-human serum albumin (B-HSA) blocking process to subtracted NSB signal in serum, this BLI biosensor can measure a wide range of the dissociation rate constant (koff), which can be measured without knowledge of the specific antibody concentrations. The clinical utility of this improved BLI kinetics assay was demonstrated by analyzing the koff values in sera of 24 pediatric (≤18 years old) and 63 adult (>18 years old) COVID-19 convalescent patients. Lower koff values for SARS-CoV-2 serum antibodies binding to RBD were measured in samples from children. This rapid, easy to operate and chaotrope-free BLI assay is suitable for clinical use and can be readily adapted to characterize SARS-CoV-2 antibodies developed by COVID-19 patients and vaccines.

Rapid, robust, and sustainable antibody responses to mRNA COVID-19 vaccine in convalescent COVID-19 individuals.

Longitudinal studies are needed to evaluate the SARS-CoV-2 mRNA vaccine antibody response under real-world conditions. This longitudinal study investigated the quantity and quality of SARS-CoV-2 antibody response in 846 specimens from 350 patients, comparing BNT162b2-vaccinated individuals (19 previously diagnosed with COVID-19, termed RecoVax; and 49 never diagnosed, termed NaiveVax) with 122 hospitalized unvaccinated (HospNoVax) and 160 outpatient unvaccinated (OutPtNoVax) COVID-19 patients. NaiveVax experienced delay in generating SARS-CoV-2 total antibodies (TAb) and surrogate neutralizing antibodies (SNAb) after the first vaccine dose (D1) but rapid increase in antibody levels after the second dose (D2). However, these never reached RecoVax's robust levels. In fact, NaiveVax TAb and SNAb levels decreased 4 weeks after D2. For the most part, RecoVax TAb persisted, after reaching maximal levels 2 weeks after D2, but SNAb decreased significantly about 6 months after D1. Although NaiveVax avidity lagged behind that of RecoVax for most of the follow-up periods, NaiveVax did reach similar avidity by about 6 months after D1. These data suggest that 1 vaccine dose elicits maximal antibody response in RecoVax and may be sufficient. Also, despite decreasing levels in TAb and SNAb over time, long-term avidity may be a measure worth evaluating and possibly correlating to vaccine efficacy.

Cytokine signatures of end organ injury in COVID-19.

Increasing evidence has shown that Coronavirus disease 19 (COVID-19) severity is driven by a dysregulated immunologic response. We aimed to assess the differences in inflammatory cytokines in COVID-19 patients compared to contemporaneously hospitalized controls and then analyze the relationship between these cytokines and the development of Acute Respiratory Distress Syndrome (ARDS), Acute Kidney Injury (AKI) and mortality. In this cohort study of hospitalized patients, done between March third, 2020 and April first, 2020 at a quaternary referral center in New York City we included adult hospitalized patients with COVID-19 and negative controls. Serum specimens were obtained on the first, second, and third hospital day and cytokines were measured by Luminex. Autopsies of nine cohort patients were examined. We identified 90 COVID-19 patients and 51 controls. Analysis of 48 inflammatory cytokines revealed upregulation of macrophage induced chemokines, T-cell related interleukines and stromal cell producing cytokines in COVID-19 patients compared to the controls. Moreover, distinctive cytokine signatures predicted the development of ARDS, AKI and mortality in COVID-19 patients. Specifically, macrophage-associated cytokines predicted ARDS, T cell immunity related cytokines predicted AKI and mortality was associated with cytokines of activated immune pathways, of which IL-13 was universally correlated with ARDS, AKI and mortality. Histopathological examination of the autopsies showed diffuse alveolar damage with significant mononuclear inflammatory cell infiltration. Additionally, the kidneys demonstrated glomerular sclerosis, tubulointerstitial lymphocyte infiltration and cortical and medullary atrophy. These patterns of cytokine expression offer insight into the pathogenesis of COVID-19 disease, its severity, and subsequent lung and kidney injury suggesting more targeted treatment strategies.

TOP-Plus Is a Versatile Biosensor Platform for Monitoring SARS-CoV-2 Antibody Durability.

BACKGROUND: Low initial severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody titers dropping to undetectable levels within months after infection have raised concerns about long-term immunity. Both the antibody levels and the avidity of the antibody-antigen interaction should be examined to understand the quality of the antibody response. METHODS: A testing-on-a-probe "plus" panel (TOP-Plus) was developed to include a newly developed avidity assay built into the previously described SARS-CoV-2 TOP assays that measured total antibody (TAb), surrogate neutralizing antibody (SNAb), IgM, and IgG on a versatile biosensor platform. TAb and SNAb levels were compared with avidity in previously infected individuals at 1.3 and 6.2 months after infection in paired samples from 80 patients with coronavirus disease 2019 (COVID-19). Sera from individuals vaccinated for SARS-CoV-2 were also evaluated for antibody avidity. RESULTS: The newly designed avidity assay in this TOP panel correlated well with a reference Bio-Layer Interferometry avidity assay (r = 0.88). The imprecision of the TOP avidity assay was <10%. Although TAb and neutralization activity (by SNAb) decreased between 1.3 and 6.2 months after infection, the antibody avidity increased significantly (P < 0.0001). Antibody avidity in 10 SARS-CoV-2 vaccinated individuals (median: 28 days after vaccination) was comparable to the measured antibody avidity in infected individuals (median: 26 days after infection). CONCLUSIONS: This highly precise and versatile TOP-Plus panel with the ability to measure SARS-CoV-2 TAb, SNAb, IgG, and IgM antibody levels and avidity of individual sera on one sensor can become a valuable asset in monitoring not only patients infected with SARS-CoV-2 but also the status of individuals' COVID-19 vaccination response.

Postconvalescent SARS-CoV-2 IgG and Neutralizing Antibodies are Elevated in Individuals with Poor Metabolic Health.

PURPOSE: Comorbidities making up metabolic syndrome (MetS), such as obesity, type 2 diabetes, and chronic cardiovascular disease can lead to increased risk of coronavirus disease-2019 (COVID-19) with a higher morbidity and mortality. SARS-CoV-2 antibodies are higher in severely or critically ill COVID-19 patients, but studies have not focused on levels in convalescent patients with MetS, which this study aimed to assess. METHODS: This retrospective study focused on adult convalescent outpatients with SARS-CoV-2 positive serology during the COVID-19 pandemic at NewYork Presbyterian/Weill Cornell. Data collected for descriptive and correlative analysis included SARS-COV-2 immunoglobin G (IgG) levels and history of MetS comorbidities from April 17, 2020 to May 20, 2020. Additional data, including SARS-CoV-2 IgG levels, body mass index (BMI), hemoglobin A1c (HbA1c) and lipid levels were collected and analyzed for a second cohort from May 21, 2020 to June 21, 2020. SARS-CoV-2 neutralizing antibodies were measured in a subset of the study cohort. RESULTS: SARS-CoV-2 IgG levels were significantly higher in convalescent individuals with MetS comorbidities. When adjusted for age, sex, race, and time duration from symptom onset to testing, increased SARS-CoV-2 IgG levels remained significantly associated with obesity (P < 0.0001). SARS-CoV-2 IgG levels were significantly higher in patients with HbA1c ≥6.5% compared to those with HbA1c <5.7% (P = 0.0197) and remained significant on multivariable analysis (P = 0.0104). A positive correlation was noted between BMI and antibody levels [95% confidence interval: 0.37 (0.20-0.52) P < 0.0001]. Neutralizing antibody titers were higher in COVID-19 individuals with BMI ≥ 30 (P = 0.0055). CONCLUSION: Postconvalescent SARS-CoV-2 IgG and neutralizing antibodies are elevated in obese patients, and a positive correlation exists between BMI and antibody levels.

Association of Age With SARS-CoV-2 Antibody Response.

Importance: Accumulating evidence suggests that children infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are more likely to manifest mild symptoms and are at a lower risk of developing severe respiratory disease compared with adults. It remains unknown how the immune response in children differs from that of adolescents and adults. Objective: To investigate the association of age with the quantity and quality of SARS-CoV-2 antibody responses. Design, Setting, and Participants: This cross-sectional study used 31 426 SARS-CoV-2 antibody test results from pediatric and adult patients. Data were collected from a New York City hospital from April 9 to August 31, 2020. The semiquantitative immunoglobin (Ig) G levels were compared between 85 pediatric and 3648 adult patients. Further analysis of SARS-CoV-2 antibody profiles was performed on sera from 126 patients aged 1 to 24 years. Main Outcomes and Measures: SARS-CoV-2 antibody positivity rates and IgG levels were evaluated in patients from a wide range of age groups (1-102 years). SARS-CoV-2 IgG level, total antibody (TAb) level, surrogate neutralizing antibody (SNAb) activity, and antibody binding avidity were compared between children (aged 1-10 years), adolescents (aged 11-18 years), and young adults (aged 19-24 years). Results: Among 31 426 antibody test results (19 797 [63.0%] female patients), with 1194 pediatric patients (mean [SD] age, 11.0 [5.3] years) and 30 232 adult patients (mean [SD] age, 49.2 [17.1] years), the seroprevalence in the pediatric (197 [16.5%; 95% CI, 14.4%-18.7%]) and adult (5630 [18.6%; 95% CI, 18.2%-19.1%]) patient populations was similar. The SARS-CoV-2 IgG level showed a negative correlation with age in the pediatric population (r = -0.45, P < .001) and a moderate but positive correlation with age in adults (r = 0.24, P < .001). Patients aged 19 to 30 years exhibited the lowest IgG levels (eg, aged 25-30 years vs 1-10 years: 99 [44-180] relative fluorescence units [RFU] vs 443 [188-851] RFU). In the subset cohort aged 1 to 24 years, IgG, TAb, SNAb and avidity were negatively correlated with age (eg, IgG: r = -0.51; P < .001). Children exhibited higher median (IQR) IgG levels, TAb levels, and SNAb activity compared with adolescents (eg, IgG levels: 473 [233-656] RFU vs 191 [82-349] RFU; P < .001) and young adults (eg, IgG levels: 473 [233-656] RFU vs 85 [38-150] RFU; P < .001). Adolescents also exhibited higher median (IQR) TAb levels, IgG levels, and SNAb activity than young adults (eg, TAb levels: 961 [290-2074] RFU vs 370 [125-697]; P = .006). In addition, children had higher antibody binding avidity compared with young adults, but the difference was not significant. Conclusions and Relevance: The results of this study suggest that SARS-CoV-2 viral specific antibody response profiles are distinct in different age groups. Age-targeted strategies for disease screening and management as well as vaccine development may be warranted.

Testing-on-a-probe biosensors reveal association of early SARS-CoV-2 total antibodies and surrogate neutralizing antibodies with mortality in COVID-19 patients.

The association of mortality with the early humoral response to SARS-CoV-2 infection within the first few days after onset of symptoms (DAOS) has not been thoroughly investigated partly due to a lack of sufficiently sensitive antibody testing methods. Here we report two sensitive and automated testing-on-a-probe (TOP) biosensor assays for SARS-CoV-2 viral specific total antibodies (TAb) and surrogate neutralizing antibodies (SNAb), which are suitable for clinical use. The TOP assays employ an RBD-coated quartz probe using a Cy5-Streptavidin-polysacharide conjugate to improve sensitivity and minimize interference. Disposable cartridges containing pre-dispensed reagents require no liquid manipulation or fluidics during testing. The TOP-TAb assay exhibited higher sensitivity in the 0-7 DAOS window than a widely used FDA-EUA assay. The rapid and automated TOP-SNAb correlated well with two well-established SARS-CoV-2 virus neutralization tests. The clinical utility of the TOP assays was demonstrated by evaluating early antibody responses in 120 SARS-CoV-2 RT-PCR positive adult hospitalized patients. Higher TAb and SNAb positivity rates and more robust antibody responses at patient's initial hospital presentation were seen in inpatients who survived COVID-19 than those who died in the hospital. Survival analysis using the Cox Proportional Hazards Model showed that patients who had negative TAb and/or SNAb at initial hospital presentation were at a higher risk of in-hospital mortality. Furthermore, TAb and SNAb levels at presentation were inversely associated with SARS-CoV-2 viral load based on concurrent RT-PCR testing. Overall, the sensitive and automated TAb and SNAb assays allow the detection of early SARS-CoV-2 antibodies which associate with mortality.

Routine Laboratory Blood Tests Predict SARS-CoV-2 Infection Using Machine Learning.

BACKGROUND: Accurate diagnostic strategies to identify SARS-CoV-2 positive individuals rapidly for management of patient care and protection of health care personnel are urgently needed. The predominant diagnostic test is viral RNA detection by RT-PCR from nasopharyngeal swabs specimens, however the results are not promptly obtainable in all patient care locations. Routine laboratory testing, in contrast, is readily available with a turn-around time (TAT) usually within 1-2 hours. METHOD: We developed a machine learning model incorporating patient demographic features (age, sex, race) with 27 routine laboratory tests to predict an individual's SARS-CoV-2 infection status. Laboratory testing results obtained within 2 days before the release of SARS-CoV-2 RT-PCR result were used to train a gradient boosting decision tree (GBDT) model from 3,356 SARS-CoV-2 RT-PCR tested patients (1,402 positive and 1,954 negative) evaluated at a metropolitan hospital. RESULTS: The model achieved an area under the receiver operating characteristic curve (AUC) of 0.854 (95% CI: 0.829-0.878). Application of this model to an independent patient dataset from a separate hospital resulted in a comparable AUC (0.838), validating the generalization of its use. Moreover, our model predicted initial SARS-CoV-2 RT-PCR positivity in 66% individuals whose RT-PCR result changed from negative to positive within 2 days. CONCLUSION: This model employing routine laboratory test results offers opportunities for early and rapid identification of high-risk SARS-CoV-2 infected patients before their RT-PCR results are available. It may play an important role in assisting the identification of SARS-CoV-2 infected patients in areas where RT-PCR testing is not accessible due to financial or supply constraints.

SARS-CoV-2 antibody characterization in emergency department, hospitalized and convalescent patients by two semi-quantitative immunoassays.

BACKGROUND: In the ongoing COVID-19 pandemic, there is an urgent need for comprehensive performance evaluation and clinical utility assessment of serological assays to understand the immune response to SARS-CoV-2. METHODS: IgM/IgG and total antibodies against SARS-CoV-2 were measured by a cyclic enhanced fluorescence assay (CEFA) and a microsphere immunoassay (MIA), respectively. Independent performance evaluation included imprecision, reproducibility, specificity and cross-reactivity (CEFA n = 320, MIA n = 364). Clinical utility was evaluated by both methods in 87 patients at initial emergency department visit, 28 during subsequent hospitalizations (106 serial samples), and 145 convalescent patients. Totally 916 patients and 994 samples were evaluated. RESULTS: Agreement of CEFA and MIA was 90.4%-94.5% (Kappa: 0.81-0.89) in 302 samples. CEFA and MIA detected SARS-CoV-2 antibodies in 26.2% and 26.3%, respectively, of ED patients. Detection rates increased over time reaching 100% after 21 days post-symptom onset. Longitudinal antibody kinetic changes by CEFA and MIA measurements correlated well and exhibited three types of seroconversion. Convalescent sera showed a wide range of antibody levels. CONCLUSION: Rigorously validated CEFA and MIA assays are reliable for detecting antibodies to SARS-CoV-2 and show promising clinical utility when evaluating immune response in hospitalized and convalescent patients, but are not useful for early screening at patient's initial ED visit.