SARS-CoV-2 Antibody Levels Associate with Neutrophil Activation

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.

Fostamatanib Therapy in Severe ARDS Requiring Extracorporeal Membrane Oxygenation (ECMO) Support

Introduction: Neutrophil extracellular traps (NETs) are extrusions of intracellular DNA and granular material released by neutrophils as part of the host immune response. While intended as a defense mechanism, excessive production of NETs may play a role in the pathogenesis of ARDS. Fostamatinib appears to limit NET formation. A phase 2 study of fostamatinib for COVID-19 associated acute lung injury found fostamatinib to be associated with improved clinical outcomes. No patients in the clinical trial were on extracorporeal membrane oxygenator (ECMO) support. In this we report our experience with two critically ill patients on veno-venous (VV) ECMO treated with fostamatinib. Case 1: A 46-year-old male with no significant PMH was admitted with COVID-19 associated ARDS (CARDS). He required intubation on hospital day (HD) 10. Due to refractory hypoxemia, he was cannulated for VV ECMO that same day. By day 19, he had improved and was decannulated from ECMO. Following decannulation, he continued to struggle. He developed a pneumothorax which was addressed with a chest tube. Despite this he had refractory hypoxemia requiring neuromuscular blockade (NMB). Broad spectrum antibiotics were initiated. No superinfection was identified. He was again cannulated for VV ECMO on HD 30. On HD 36, fostamatinib was initiated at a dose of 150 mg bid for 14 days. The patient demonstrated fairly rapid improvement by HD 39, allowing for minimization of ECMO support. He was decannulated from VV ECMO on HD 46. He currently resides at home and has no need for oxygen. Case 2: A 53- year-old male with a PMH of psoriasis on etanercept was admitted with CARDS. He was intubated HD 1, but continued to require substantial support including prone positioning and NMB. On HD 5 he was cannulated for VV ECMO. He had early improvement and was decannulated on HD 10;however, he developed Staph aureus pneumonia resulting in marked clinical decline. On HD 12 he was placed back on VV ECMO support. He was also initiated on fostamatanib 150 mg twice daily for 14 days. He demonstrated fairly rapid improvement in oxygenation but required prolonged ECMO support for CO2 clearance. He was successfully decannulated from VV ECMO on HD 45. He is currently living at home. Conclusion: Fostamatinib appears safe to administer to COVID patients on ECMO. While it is speculative to make inferences with regards to efficacy, it is noteworthy that both critically ill COVID-19 patients treated with fostamatinib survived.

Automated Digital Quantification of Pulmonary Fibrosis in Human Histopathology Specimens

RATIONALE: Pulmonary fibrosis is a progressive disease characterized by abnormal accumulation of extracellular matrix protein, mesenchymal cells, and immune cells in the alveolar interstitium. Methods quantifying fibrosis severity in lung histopathology samples are semi-quantitative, subjective, and analyze only portions of sections. We sought to determine whether automated computerized imaging analysis shown to continuously measure fibrosis in mice could be applied in human samples to quantify pulmonary fibrosis severity and immunostained cells in whole tissue sections. METHODS: Digital images of entire tissue sections from lung explants or biopsies from patients with Hermansky-Pudlak syndrome pulmonary fibrosis (HPSPF) or idiopathic pulmonary fibrosis (IPF) stained with picrosirius red and alcian blue or immunostained with anti-CD68 antibody were analyzed using a dedicated software program to quantify fibrosis, collagen, and macrophage content. Automated fibrosis quantification was compared to pulmonary function measurements or Ashcroft score. RESULTS: Automated quantification of pulmonary tissue density and fibrosis scores of HPSPF lung explants (n=3) was significantly higher than that of IPF lung explants (n=3) or biopsies (n=4). These values were also significantly higher in IPF lung explants than in IPF biopsies. Mean Ashcroft score in HPSPF or IPF explants was significantly higher than that of IPF biopsies. In contrast to automated quantification, Ashcroft scores of HPSPF and IPF explants did not differ significantly from each other. Automated tissue density and fibrosis scores correlated with lung function values and Ashcroft scores. Automated quantification of collagen content was similar in the three groups. Immune cell aggregates were identified in hematoxylin and eosin stained tissue;automated quantification of CD68 immunolabeled cells was significantly higher in HPSPF explants than in IPF biopsies. CONCLUSIONS: Severity of pulmonary fibrosis in stained human tissue can be automatically quantified in entire lung sections using a dedicated software program. This reader-independent method provides a more accurate assessment of pulmonary fibrosis than Ashcroft scores. Automated quantification of fibrosis, collagen content, and immunostained cells in fibrotic lung tissue sections can be performed simultaneously. Robust automated digital image analysis of human lung samples enhances the available tools to quantify and study pulmonary fibrosis and has potential application in preclinical studies and clinical investigations focusing on fibrotic lung disease and other diffuse pulmonary diseases, such as sarcoidosis, hypersensitivity pneumonitis, pneumoconiosis, or COVID-19.

Administration of COVID-19 convalescent plasma in a metropolitan health care system

Rationale:The outbreak of COVID-19 in March of 2020 led to the emergent search for treatment options. Passive immune therapy with COVID-19 convalescent plasma (CCP) therapy was utilized as an investigational therapy. We designed an open label study and received an investigational new drug (IND) number from the Food and Drug Administration for the study on April 11, 2020. Methods:Patients with severe COVID-19 were enrolled and identical ABO CCP was administered. CCP was collected from recovered COVID-19 patients by our hospital blood donor services in line with FDA guidelines. Patients could receive up to three doses of CCP and each dose came from a different donor to potentiate therapeutic response. Subjects were followed for 28 days after infusion of COVID-19 convalescent plasma (CCP) in hospital or with weekly phone calls if discharged home. Results:45 patients received CCP through the study period of April 24th through August 21st. The median age was 60 (range: 16-87), and two thirds were male (n=30) with Hispanic predominance (58%). Patients also received supplemental therapies such as remdesivir, tocilizumab, dexamethasone, and inhaled nitric oxide. Twenty-six recipients received 1 dose, 12 received 2 doses, and 7 received 3 doses of CCP all of which were collected internally by the hospital blood donor center. 22 recipients were O positive, 17 were A positive, and 6 were B positive. During their hospital course, 18 patients were on mechanical ventilation, 3 of which were on ECMO, 19 were on high-flow nasal cannula, and 8 were on low-flow nasal cannula as the highest level of oxygen therapy. At the 28 day follow up, 14 patients were deceased (31%), 4 (9%) were still hospitalized, and 27 (60%) were discharged home. Two patients had a suspected transfusion related reaction that resolved with supportive care including diphenhydramine and furosemide. Conclusion:We designed and implemented a pragmatic study to provide a treatment option for patients hospitalized with COVID-19. Sixty percent of our very sick study population survived to hospital discharge and there were only two transient infusion reactions. While larger studies with a control group are necessary to more clearly evaluate the benefit of CCP in COVID-19, our study lays the foundation for the rapid implementation of a Convalescent Plasma Program for possible future pandemics as a bridge to vaccine and therapeutic trials.

Recombinant human pentraxin-2 (RHPTX-2;PRM-151) for idiopathic pulmonary fibrosis: Study design of starscape, a phase iii randomized double blind placebo controlled trial

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is a chronic, fibrosing, interstitial pneumonia which ultimately leads to an irreversible loss of lung function and respiratory compromise. The anti-fibrotic agents, pirfenidone and nintedanib have been shown to slow the rate of decline in forced vital capacity (FVC) but, neither treatment halts disease progression. Pentraxin-2 plays important biologically relevant roles in wound repair and prevention of fibrosis. Pentraxin-2, inhibits monocyte differentiation into pro-fibrotic fibrocytes and pro-inflammatory macrophages. Plasma pentraxin-2 concentrations are reduced in patients with IPF and correlate with disease severity. Recombinant human pentraxin-2 (rhPTX-2;also known as PRM-151) was evaluated for its therapeutic potential within a phase II trial (NCT02550873). This trial demonstrated statistically significant and clinically meaningful outcomes of rhPTX-2 treatment in patients with IPF. Here we report the phase III study design to further evaluate these findings. METHODS: STARSCAPE (NCT04552899) is a phase III, multi-center, randomized, double-blind, placebo controlled trial. 658 patients with IPF will be randomized (1:1) to receive either intravenous rhPTX-2 or matching placebo administered on Days 1, 3, 5 and every 4 weeks thereafter through 48 weeks. The primary endpoint is absolute change from baseline to Week 52 in FVC [mL]. The key secondary endpoint is absolute change from baseline to Week 52 in 6-minute walk distance. Eligible patients are 40-85 years, with a documented diagnosis of IPF confirmed centrally by high resolution computed tomography scan (and lung biopsy if available). Patients must demonstrate FVC ≥ 45%, FEV1/FVC ratio > 0.70 and DLCO ≥ 30% and ≤ 90% during screening. Patients are permitted to take background therapy with nintedanib or pirfenidone. Initiating a global phase III trial during the COVID-19 pandemic brings unique and unprecedented challenges. A large number of countries and sites will be included in order to mitigate potential regional recruitment challenges that may arise during the pandemic. In addition, SARS-CoV-2 serology testing will be conducted to allow exploratory analyses on the impact of COVID-19 on lung function parameters in patients with IPF. CONCLUSIONS: rhPTX-2 has demonstrated preliminary evidence of clinical efficacy on top of approved standard of care. The phase III STARSCAPE trial aims to confirm the therapeutic potential of rhPTX-2 through evaluation of a broad range of efficacy, safety, quality of life, pharmacokinetic and biomarker assessments over 52 weeks. Patients that complete this 52-week trial may be eligible to enroll into an open label extension trial.