Study design and clinical characteristics of study participants
The study was conducted between November 2020 and January 2021 and is part of a larger survey. Patients were recruited in Wershofen (Germany) and Zagreb (Croatia). A total of 50 COVID-19 patients (29 men and 21 women) were included in the study. Ethical approval for the study was granted by local ethics committees. All included COVID-19 patients had been reported positive by SARS-CoV-2 RT-PCR in official COVID-19 testing centers. 3 (6%) out of the 50 COVID-19 patients had been hospitalized during the acute phase of infection. The majority, 41 (82%), had mild symptoms, whereas 6 (12%) were fully asymptomatic (Table 1). Samples from healthy blood donors, kindly provided by the Haematology Department of the University Hospital of Bonn, were used as controls. Healthy blood donors were all negative at SARS-CoV-2 RT-PCR and serology and included 13 men and 17 women (mean age= 32.5 ±12.37) (Table 1).
After sample collection, peripheral blood mononuclear cells (PBMCs) isolated from venous blood of patients and controls were stimulated with SARS-CoV-2 peptide pools in the presence or absence of antigen extracts from Onchocerca volvulus (OvAg), Brugia malayi (BmAg), or Ascaris lumbricoides (AlAg). Culture medium (Med) and helminth antigens alone were used as stimulation controls. After 24 hours of incubation, culture supernatants were collected for cytokines expression and cell pellets for FACS staining.
Helminth antigen preparation
Soluble extracts from adult worms of Onchocerca volvulus, Brugia malayi, and Ascaris lumbricoides were prepared as previously described (19). Briefly, 20 frozen adult worms were thawed and transferred to a Petri dish pre-filled with sterile PBS. Following several washes in PBS, worms were placed inside a glass mortar (VWR, Langenfeld, Germany). 3–5 ml of RPMI-medium were added, and worms were crushed until a homogenous solution was obtained. The extracts were then centrifuged for 10 minutes at 300 g and 4°C to remove insoluble material. Supernatants were transferred to a new tube. Protein concentrations were then measured using the Pierce Coomassie Plus (Bradford) Assay Kit (ThermoFisher Scientific, San Diego, CA, USA) according to the manufacturer’s instructions. Aliquots were stored at -80°C until use. The optimal concentration for cell stimulation was defined using a titration assay, and the endotoxin level was determined using the Pierce Limulus amoebocyte lysate (LAL) Chromogenic quantification kit (ThermoFisher Scientific). The endotoxin level was routinely below the detection limit of 0.1 EU/ml.
PBMC isolation
PBMCs were isolated using a Ficoll gradient as previously described (6). In brief, 15 ml of heparinized blood was diluted 1:2 in PBS (Gibco, Life Technologies, Carlsbad, USA), transferred on 15 ml of Ficoll (PAN Biotech GmbH, Aidenbach, Germany), and centrifuged for 20 min at 800g, 4°C and without brake. Cell suspensions were then washed twice, 8 min at 300g, 4°C, and re-suspended in fetal bovine serum (FBS)-supplemented RPMI 1640 medium (Gibco). The cells were finally counted using the trypan blue (ThermoFisher Scientific) exclusion method and diluted for stimulation and culture. 100 µL of cell suspension at 2x107 cells/ml were plated in each well.
Stimulation and cell culture
To test the impact of helminth antigens on the immune reactivity to SARS-CoV-2, PBMCs from COVID-19 patients (N=50) were isolated and stimulated with 1µg/ml (50µl working solution at 5µg/ml) of SARS-CoV-2-Spike-protein-peptide-pools (SPP) (Miltenyi Biotech, Bergisch Gladbach, Germany), in the presence of culture medium or 10 µg/ml (50µl working solution at 50µg/ml) of Brugia malayi, Onchocerca volvulus or Ascaris lumbricoides extracts. The total volume for each well was adjusted to 250µl with culture medium (10%FCS in RPMI). After 24 hours of culture (at 37°C, 5% CO2), 200µl supernatants were collected and frozen until use, and cells were harvested and prepared for FACS staining.
FACS staining
All reagents were obtained from ThermoFisher Scientific, and staining was performed according to the manufacturer's instructions. Briefly, cells were harvested and washed with FACS buffer (PBS/2% FCS) at 300 g for 8 min. 1x106 cells were then resuspended in 100 µl of FACS buffer and blocked with 1 µl of FC- block (ThermoFisher Scientific) for 15 min. 5µg/1x105 cells of anti-human CD3-PerCP (clone: S4.1-7D6), CD4-eF450 (clone: RPA-T4), CD8-FITC (clone: RPA-T8), CD69-APC (clone: FN50) and CD137-PE (clone: 4B4), were added and the cell suspension was incubated for 30 min at 4°C. Cells were then washed twice with FACS buffer and fixed in 300 µl PFA (4%). To correct spectral overlaps, fluorescence compensation was done using UltraComp ebeads (ThermoFisher Scientific). Data were acquired using a Cytoflex-S flow cytometer (Beckmann Coulter, Krefeld, Germany) and analyzed with FlowJo v10 software (LLC, Ashland, OR, USA).
Luminex assay
To quantify cytokine levels in culture supernatants, ProcartaPlex Human Cytokines Panels (ThermoFisher Scientific) were used according to the manufacturer's instructions. Briefly, anti-IL-6, TNFα, IFNγ, and IL-10-coated magnetic beads were incubated with 25 µl of assay buffer, kit standards, or diluted supernatant (1:2) for 1 hour. 25 µl of biotin-labeled detection antibodies mix was then added. The plates were incubated on an orbital shaker (Stuart, Staffordshire, UK) at 500 rpm for 30 min, and 50 µl of diluted Streptavidin-PE was added. Plates were then incubated for an additional 30 minutes and washed using a hand-held magnetic plate washer. Afterward, the beads were re-suspended in 120 µl reading buffer. Data were then acquired using a MAGPIX Luminex system and analyzed with ProcartaPlex Analyst software 1.0 (ThermoFisher Scientific).
Ethics statement
Each volunteer recruited for the study gave informed consent to participate. Ethics approval for the study was obtained from the ethical boards of the University Hospital Bonn (Lfd.Nr.439/20) and the Zadar General Hospital (02-3534/20-2/20).
Statistics
All graphs were generated using GraphPad Prism 8 (La Jolla, CA, USA). For cell activation data, P values were calculated using the Kruskal-Wallis test. Dunn's multiple comparison test was used to compare all settings. Two-way ANOVA and Tukey's multiple comparison tests were used to compare the modulation of T cell reactivity in healthy donors and COVID-19 patients. Asterisks indicate the level of significance: ns= non-significant; *= P≤ 0.05; **= P≤ 0.01; ***= P≤ 0.001, ****= P≤ 0.0001. Significance is accepted if P <0.05. For cytokine data, Student's t-test was used for comparisons.