Association of COVID-19 mortality with serum selenium, zinc and copper: Six observational studies across Europe.

Introduction: Certain trace elements are essential for life and affect immune system function, and their intake varies by region and population. Alterations in serum Se, Zn and Cu have been associated with COVID-19 mortality risk. We tested the hypothesis that a disease-specific decline occurs and correlates with mortality risk in different countries in Europe. Methods: Serum samples from 551 COVID-19 patients (including 87 non-survivors) who had participated in observational studies in Europe (Belgium, France, Germany, Ireland, Italy, and Poland) were analyzed for trace elements by total reflection X-ray fluorescence. A subset (n=2069) of the European EPIC study served as reference. Analyses were performed blinded to clinical data in one analytical laboratory. Results: Median levels of Se and Zn were lower than in EPIC, except for Zn in Italy. Non-survivors consistently had lower Se and Zn concentrations than survivors and displayed an elevated Cu/Zn ratio. Restricted cubic spline regression models revealed an inverse nonlinear association between Se or Zn and death, and a positive association between Cu/Zn ratio and death. With respect to patient age and sex, Se showed the highest predictive value for death (AUC=0.816), compared with Zn (0.782) or Cu (0.769). Discussion: The data support the potential relevance of a decrease in serum Se and Zn for survival in COVID-19 across Europe. The observational study design cannot account for residual confounding and reverse causation, but supports the need for intervention trials in COVID-19 patients with severe Se and Zn deficiency to test the potential benefit of correcting their deficits for survival and convalescence.

Loss of GM-CSF-dependent instruction of alveolar macrophages in COVID-19 provides a rationale for inhaled GM-CSF treatment.

GM-CSF promotes myelopoiesis and inflammation, and GM-CSF blockade is being evaluated as a treatment for COVID-19-associated hyperinflammation. Alveolar GM-CSF is, however, required for monocytes to differentiate into alveolar macrophages (AMs) that control alveolar homeostasis. By mapping cross-species AM development to clinical lung samples, we discovered that COVID-19 is marked by defective GM-CSF-dependent AM instruction and accumulation of pro-inflammatory macrophages. In a multi-center, open-label RCT in 81 non-ventilated COVID-19 patients with respiratory failure, we found that inhalation of rhu-GM-CSF did not improve mean oxygenation parameters compared with standard treatment. However, more patients on GM-CSF had a clinical response, and GM-CSF inhalation induced higher numbers of virus-specific CD8 effector lymphocytes and class-switched B cells, without exacerbating systemic hyperinflammation. This translational proof-of-concept study provides a rationale for further testing of inhaled GM-CSF as a non-invasive treatment to improve alveolar gas exchange and simultaneously boost antiviral immunity in COVID-19. This study is registered at ClinicalTrials.gov (NCT04326920) and EudraCT (2020-001254-22).

Diagnostic performance of the SARS-CoV-2 S1RBD IgG ELISA (ImmunoDiagnostics) for the quantitative detection of SARS-CoV-2 antibodies on dried blood spots.

Dried Blood Spots (DBS) are broadly used in SARS-CoV-2 surveillance studies, reporting either the presence or absence of SARS-CoV-2 antibodies. However, quantitative follow-up has become increasingly important to monitor humoral vaccine responses. Therefore, we aimed to evaluate the performance of DBS for the detection of anti-spike SARS-CoV-2 antibody concentrations using a commercially available assay, reporting in a standardised unitage (International Units/mL; IU/mL). To assess the sensitivity and specificity of the ImmunoDiagnostics ELISA on serum and DBS for SARS-CoV-2 antibody detection, we analysed 72 paired DBS and serum samples. The SARS-CoV-2 S1 IgG ELISA kit (EUROIMMUN) on serum was used as the reference method. We performed a statistical assessment to optimise the cut-off value for DBS and serum and assessed the correlation between DBS and serum antibody concentrations. We found that anti-spike SARS-CoV-2 antibody concentrations detected in DBS are highly correlated to those detected in paired serum (Pearson correlation 0.98; p-value < 0.0001), allowing to assess serum antibody concentration using DBS. The optimal cut-off for antibody detection on DBS was found to be 26 IU/mL, with 98.1% sensitivity and 100% specificity. For serum, the optimal cut-off was 14 IU/mL, with 100% sensitivity and 100% specificity. Therefore, we conclude that the ImmunoDiagnostics ELISA kit has optimal performance in the detection of SARS-CoV-2 antibodies on both DBS and serum. This makes DBS ideal for large-scale follow-up of humoral SARS-CoV-2 immune responses, as it is an easy but valuable sampling method for quantification of SARS-CoV-2 antibodies, compared to serum.

A dual-antigen self-amplifying RNA SARS-CoV-2 vaccine induces potent humoral and cellular immune responses and protects against SARS-CoV-2 variants through T cell-mediated immunity.

Self-amplifying RNA vaccines may induce equivalent or more potent immune responses at lower doses compared to non-replicating mRNA vaccines via amplified antigen expression. In this paper, we demonstrate that 1 µg of an LNP-formulated dual-antigen self-amplifying RNA vaccine (ZIP1642), encoding both the S-RBD and N antigen, elicits considerably higher neutralizing antibody titers against Wuhan-like Beta B.1.351 and Delta B.1.617.2 SARS-CoV-2 variants compared to those of convalescent patients. In addition, ZIP1642 vaccination in mice expanded both S- and N-specific CD3+CD4+ and CD3+CD8+ T cells and caused a Th1 shifted cytokine response. We demonstrate that the induction of such dual antigen-targeted cell-mediated immune response may provide better protection against variants displaying highly mutated Spike proteins, as infectious viral loads of both Wuhan-like and Beta variants were decreased after challenge of ZIP1642 vaccinated hamsters. Supported by these results, we encourage redirecting focus toward the induction of multiple antigen-targeted cell-mediated immunity in addition to neutralizing antibody responses to bypass waning antibody responses and attenuate infectious breakthrough and disease severity of future SARS-CoV-2 variants.

Humoral and Cellular Responses to COVID-19 Vaccination Indicate the Need for Post-Vaccination Testing in Frail Population.

Despite the high efficacy of the BNT162b2 vaccine in the general population, data on its immunogenicity among frail elderly individuals are limited. Recently, levels of anti-SARS-CoV-2 spike IgG antibodies and serum neutralization titers were confirmed as good immune markers of protection against the virus, with evidence showing a reverse correlation between these two parameters and susceptibility to infection. Here we analyzed sera from 138 nursing home residents (median age of 88.9 years) and 312 nursing home staff (median age of 50.7 years) to determine the humoral response to two doses of the BNT162b2 vaccine, and found markedly decreased serum anti-spike antibody levels and neutralization titers in the nursing home resident (NHR) group, with over 11% non-responders compared to only 1.3% among the controls. Moreover, three months post-vaccination, a significant decrease in antibody titers was observed in COVID-19-naive nursing home residents. Subsequent flow cytometry and interferon gamma secretion analyses indicated that antibody non-responders among NHRs also failed to mount cellular responses. The presented data emphasize that additional measures are needed in the population of frail elderly individuals. Given the high proportion of non-responders among NHRs, continued monitoring should be considered in this group.

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 (PaO2:FiO2) 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.

Organ-specific genome diversity of replication-competent SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is not always confined to the respiratory system, as it impacts people on a broad clinical spectrum from asymptomatic to severe systemic manifestations resulting in death. Further, accumulation of intra-host single nucleotide variants during prolonged SARS-CoV-2 infection may lead to emergence of variants of concern (VOCs). Still, information on virus infectivity and intra-host evolution across organs is sparse. We report a detailed virological analysis of thirteen postmortem coronavirus disease 2019 (COVID-19) cases that provides proof of viremia and presence of replication-competent SARS-CoV-2 in extrapulmonary organs of immunocompromised patients, including heart, kidney, liver, and spleen (NCT04366882). In parallel, we identify organ-specific SARS-CoV-2 genome diversity and mutations of concern N501Y, T1027I, and Y453F, while the patient had died long before reported emergence of VOCs. These mutations appear in multiple organs and replicate in Vero E6 cells, highlighting their infectivity. Finally, we show two stages of fatal disease evolution based on disease duration and viral loads in lungs and plasma. Our results provide insights about the pathogenesis and intra-host evolution of SARS-CoV-2 and show that COVID-19 treatment and hygiene measures need to be tailored to specific needs of immunocompromised patients, even when respiratory symptoms cease.

Course and Survival of COVID-19 Patients with Comorbidities in Relation to the Trace Element Status at Hospital Admission.

Selenium (Se) and zinc (Zn) are essential trace elements needed for appropriate immune system responses, cell signalling and anti-viral defence. A cross-sectional observational study was conducted at two hospitals in Ghent, Belgium, to investigate whether Se and/or Zn deficiency upon hospital admission correlates to disease severity and mortality risk in COVID-19 patients with or without co-morbidities. Trace element concentrations along with additional biomarkers were determined in serum or plasma and associated to disease severity and outcome. An insufficient Se and/or Zn status upon hospital admission was associated with a higher mortality rate and a more severe disease course in the entire study group, especially in the senior population. In comparison to healthy European adults, the patients displayed strongly depressed total Se (mean ± SD: 59.2 ± 20.6 vs. 84.4 ± 23.4 µg L-1) and SELENOP (mean ± SD: 2.2 ± 1.9 vs. 4.3 ± 1.0 mg L-1) concentrations at hospital admission. Particularly strong associations were observed for death risk of cancer, diabetes and chronic cardiac disease patients with low Se status, and of diabetes and obese patients with Zn deficiency. A composite biomarker based on serum or plasma Se, SELENOP and Zn at hospital admission proved to be a reliable tool to predict severe COVID-19 course and death, or mild disease course. We conclude that trace element assessment at hospital admission may contribute to a better stratification of patients with COVID-19 and other similar infectious diseases, support clinical care, therapeutic interventions and adjuvant supplementation needs, and may prove of particular relevance for patients with relevant comorbidities.

Clinical characteristics and outcomes of COVID-19 in people living with HIV in Belgium: A multicenter, retrospective cohort.

The aim of this study was to describe the clinical characteristics and outcomes of coronavirus disease 2019 (COVID-19) among people living with HIV (PLWH) in Belgium. We performed a retrospective multicenter cohort analysis of PLWH with either laboratory-confirmed, radiologically diagnosed, or clinically suspected COVID-19 between February 15, 2020 and May 31, 2020. The primary endpoint was outcome of COVID-19. Secondary endpoints included rate of hospitalization and length of hospital stay and rate of Intensive Care Unit (ICU) admission and mechanical ventilation. One hundred and one patients were included in this study. Patients were categorized as having either laboratory-confirmed (n = 65), radiologically-diagnosed (n = 3), or clinically suspected COVID-19 (n = 33). The median age was 51.3 years (interquartile range [IQR] 41.3-57.3) and 44% were female. Ninety-four percent of patients were virologically suppressed and 67% had a CD4+ cell count more than or equal to 500 cells/µl. Overall, 46% of patients required hospitalization and the median length of hospital stay was 6 days (IQR 3-15). Age more than or equal to 50 years, Black Sub-Saharan African patients, and being on an integrase strand transfer inhibitor-based regimen were associated with being hospitalized. ICU admission and mechanical ventilation was required for 15% and 10% of all patients respectively. Overall, 9% of patients died while 78 (77%) patients made a full recovery. HIV patients with COVID-19 experienced a high degree of hospitalization despite having elevated CD4+ cell counts and a high rate of virologic suppression. Matched case-control studies are warranted to measure the impact that HIV may have on patients with COVID-19.

On the whereabouts of SARS-CoV-2 in the human body: A systematic review.

Since SARS-CoV-2 appeared in the human population, the scientific community has scrambled to gather as much information as possible to find good strategies for the containment and treatment of this pandemic virus. Here, we performed a systematic review of the current (pre)published SARS-CoV-2 literature with a focus on the evidence concerning SARS-CoV-2 distribution in human tissues and viral shedding in body fluids. In addition, this evidence is aligned with published ACE2 entry-receptor (single cell) expression data across the human body to construct a viral distribution and ACE2 receptor body map. We highlight the broad organotropism of SARS-CoV-2, as many studies identified viral components (RNA, proteins) in multiple organs, including the pharynx, trachea, lungs, blood, heart, vessels, intestines, brain, male genitals and kidneys. This also implicates the presence of viral components in various body fluids such as mucus, saliva, urine, cerebrospinal fluid, semen and breast milk. The main SARS-CoV-2 entry receptor, ACE2, is expressed at different levels in multiple tissues throughout the human body, but its expression levels do not always correspond with SARS-CoV-2 detection, indicating that there is a complex interplay between virus and host. Together, these data shed new light on the current view of SARS-CoV-2 pathogenesis and lay the foundation for better diagnosis and treatment of COVID-19 patients.