Effect of anti-interleukin drugs in patients with COVID-19 and signs of cytokine release syndrome (COV-AID): a factorial, randomised, controlled trial.

BACKGROUND: Infections with SARS-CoV-2 continue to cause significant morbidity and mortality. Interleukin (IL)-1 and IL-6 blockade have been proposed as therapeutic strategies in COVID-19, but study outcomes have been conflicting. We sought to study whether blockade of the IL-6 or IL-1 pathway shortened the time to clinical improvement in patients with COVID-19, hypoxic respiratory failure, and signs of systemic cytokine release syndrome. METHODS: We did a prospective, multicentre, open-label, randomised, controlled trial, in hospitalised patients with COVID-19, hypoxia, and signs of a cytokine release syndrome across 16 hospitals in Belgium. Eligible patients had a proven diagnosis of COVID-19 with symptoms between 6 and 16 days, a ratio of the partial pressure of oxygen to the fraction of inspired oxygen (PaO(2):FiO(2)) of less than 350 mm Hg on room air or less than 280 mm Hg on supplemental oxygen, and signs of a cytokine release syndrome in their serum (either a single ferritin measurement of more than 2000 μg/L and immediately requiring high flow oxygen or mechanical ventilation, or a ferritin concentration of more than 1000 μg/L, which had been increasing over the previous 24 h, or lymphopenia below 800/mL with two of the following criteria: an increasing ferritin concentration of more than 700 μg/L, an increasing lactate dehydrogenase concentration of more than 300 international units per L, an increasing C-reactive protein concentration of more than 70 mg/L, or an increasing D-dimers concentration of more than 1000 ng/mL). The COV-AID trial has a 2 × 2 factorial design to evaluate IL-1 blockade versus no IL-1 blockade and IL-6 blockade versus no IL-6 blockade. Patients were randomly assigned by means of permuted block randomisation with varying block size and stratification by centre. In a first randomisation, patients were assigned to receive subcutaneous anakinra once daily (100 mg) for 28 days or until discharge, or to receive no IL-1 blockade (1:2). In a second randomisation step, patients were allocated to receive a single dose of siltuximab (11 mg/kg) intravenously, or a single dose of tocilizumab (8 mg/kg) intravenously, or to receive no IL-6 blockade (1:1:1). The primary outcome was the time to clinical improvement, defined as time from randomisation to an increase of at least two points on a 6-category ordinal scale or to discharge from hospital alive. The primary and supportive efficacy endpoints were assessed in the intention-to-treat population. Safety was assessed in the safety population. This study is registered online with ClinicalTrials.gov (NCT04330638) and EudraCT (2020-001500-41) and is complete. FINDINGS: Between April 4, and Dec 6, 2020, 342 patients were randomly assigned to IL-1 blockade (n=112) or no IL-1 blockade (n=230) and simultaneously randomly assigned to IL-6 blockade (n=227; 114 for tocilizumab and 113 for siltuximab) or no IL-6 blockade (n=115). Most patients were male (265 [77%] of 342), median age was 65 years (IQR 54-73), and median Systematic Organ Failure Assessment (SOFA) score at randomisation was 3 (2-4). All 342 patients were included in the primary intention-to-treat analysis. The estimated median time to clinical improvement was 12 days (95% CI 10-16) in the IL-1 blockade group versus 12 days (10-15) in the no IL-1 blockade group (hazard ratio [HR] 0·94 [95% CI 0·73-1·21]). For the IL-6 blockade group, the estimated median time to clinical improvement was 11 days (95% CI 10-16) versus 12 days (11-16) in the no IL-6 blockade group (HR 1·00 [0·78-1·29]). 55 patients died during the study, but no evidence for differences in mortality between treatment groups was found. The incidence of serious adverse events and serious infections was similar across study groups. INTERPRETATION: Drugs targeting IL-1 or IL-6 did not shorten the time to clinical improvement in this sample of patients with COVID-19, hypoxic respiratory failure, low SOFA score, and low baseline mortality risk. FUNDING: Belgian Health Care Knowledge Center and VIB Grand Challenges program

Plant-based production of a protective vaccine antigen against the bovine parasitic nematode Ostertagia ostertagi

Abstract The development of effective recombinant vaccines against parasitic nematodes has been challenging and so far mostly unsuccessful. This has also been the case for Ostertagia ostertagi, an economically important abomasal nematode in cattle, applying recombinant versions of the protective native activation-associated secreted proteins (ASP). To gain insight in key elements required to trigger a protective immune response, the protein structure and N-glycosylation of the native ASP and a non-protective Pichia pastoris recombinant ASP were compared. Both antigens had a highly comparable protein structure, but different N-glycan composition. After mimicking the native ASP N-glycosylation via the expression in Nicotiana benthamiana plants, immunisation of calves with these plant-produced recombinants resulted in a significant reduction of 39% in parasite egg output, comparable to the protective efficacy of the native antigen. This study provides a valuable workflow for the development of recombinant vaccines against other parasitic nematodes

Overexpression of βENaC and reduced airway surface liquid height in βENaC-Tg mice.

<p>Immunolocalization of βENaC in airways from WT and βENaC-Tg mice. <b>(A)</b> Representative images of βENaC immunostaining of lung sections from WT and βENaC-Tg mice that were exposed to air or CS for 8 weeks. n = 5 per group. Dysregulation of steady state airway surface liquid (ASL) height on airway epithelia from βENaC-Tg mice under thin film conditions. Representative confocal images <b>(B)</b> and summary of measurements of airway surface liquid height <b>(C)</b> at t = 0, 2, 4, 8 and 24h after mucosal addition of 20 μl of PBS containing Rhodamine dextran to primary tracheal epithelial cultures from βENaC-Tg mice and WT littermates. Scale bar, 7 μm. n = 4 experiments per group. *p<0.001 compared to βENaC-Tg; ^§p<0.001 for t = 0h compared to all other time points within the same genotype; ^†p<0.05 for t = 24h wild-type compared to t = 2h wild-type; ^‡p<0.005 for t = 24h wild-type compared to t = 4h and 8h wild-type.</p

Cigarette smoke-induced mucin expression is increased in βENaC-Tg mice.

<p>mRNA expression of Muc5ac <b>(A)</b> and Muc5b <b>(B)</b> in total lung tissue upon 4 weeks of air or CS exposure. mRNA expression data were normalized for 3 reference genes (Hprt1, Gapdh, Tfrc). n = 6/group. *p<0.05, **p<0.01, ***p<0.001.</p

mRNA expression and protein levels of chemokines upon air or CS exposure.

<p>mRNA expression of Cxcl1 in total lung tissue upon 4 weeks <b>(A)</b> and 8 weeks <b>(C)</b> air or CS exposure. mRNA expression of Ccl20 in total lung tissue upon 4 weeks <b>(B)</b> and 8 weeks <b>(D)</b> air or CS exposure. mRNA expression data were normalized for 3 reference genes (Hprt1, Gapdh, Tfrc). n = 6-8/group. Protein levels of Cxcl1 in BAL fluid upon 4 weeks <b>(E)</b> and 8 weeks <b>(G)</b> air or CS exposure. Protein levels of Ccl20 in BAL fluid upon 4 weeks <b>(F)</b> and 8 weeks <b>(H)</b> air or CS exposure. Protein levels were measured by ELISA. n = 8-11/group. *p<0.05, **p<0.01, ***p<0.001.</p

Cigarette smoke-induced pulmonary inflammation is increased in βENaC-Tg mice.

<p><b>(A)</b> Total inflammatory cell count in BAL upon 4 weeks of air or CS exposure. Quantification of macrophages <b>(B)</b>, neutrophils <b>(C)</b> and lymphocytes <b>(D)</b> in BAL upon 4 weeks of air or CS exposure. n = 7-8/group. <b>(E)</b> Quantification of macrophages in total lung after 4 weeks of CS exposure. n = 7-8/group. Quantification of dendritic cells <b>(F)</b>, CD4+ T-lymphocytes <b>(G)</b> and CD8+ T-lymphocytes <b>(H)</b> in BAL upon 8 weeks of air or CS exposure. n = 8-11/group. *p<0.05, **p<0.01, ***p<0.001.</p

Cigarette smoke-induced alveolar destruction is increased in βENaC-Tg mice.

<p><b>(A)</b> Mean linear intercept (Lm) upon 4 weeks of air or CS exposure. n = 7-8/group. Representative image of WT mice: air-exposed <b>(B)</b> and CS-exposed <b>(C)</b>. Destructive index (DI) upon 4 weeks of air or CS exposure <b>(D)</b>. n = 7-8/group. Representative image of βENaC-Tg mice: air-exposed <b>(E)</b> and CS-exposed <b>(F)</b>. mRNA expression of Mmp12 in total lung upon 4 weeks of air- or CS exposure <b>(G)</b>. Normalized for 3 reference genes (Hprt1, Gapdh, and Tfrc). n = 6/group. *p<0.05, **p<0.01, ***p<0.001.</p

Goblet cell metaplasia and mucus secretion upon air or CS exposure.

<p><b>(A)</b> Goblet cell count upon 4 weeks of air or CS exposure. n = 8/group. Representative images of goblet cells in airways of CS-exposed WT mice <b>(B)</b> and CS-exposed βENaC-Tg mice <b>(C)</b> upon 4 weeks of CS exposure. Arrows indicate goblet cells. <b>(D)</b> Quantification of PAS+ mucus content in lumen of airways of non-lavaged mice upon 4 weeks of air or CS exposure n = 3/group. Representative image of PAS+ mucus content in airways of CS-exposed WT mice <b>(E)</b> and CS-exposed βENaC-Tg mice <b>(F)</b>. *p<0.05, **p<0.01, ***p<0.001.</p

Effect of CS exposure on lung function in WT and βENaC-Tg mice.

<p>Lung function was determined in WT and βENaC-Tg mice after exposure to air or CS for 4 weeks. <b>(A)</b> Resistance (R) of the entire compartment (airways, tissue and chest wall). <b>(B)</b> Tissue damping (G), related to tissue resistance. <b>(C)</b> Tissue elasticity (H). <b>(D)</b> Static compliance (C_stat). <b>(E)</b> Dynamic compliance (C_dyn). <b>(F)</b> Total lung capacity (TLC). *p<0.05, **p<0.01, ***p<0.001.</p