Six months after the infection, none of the conducted six ELISAs/chemiluminescence assays detected any anti-SARS-CoV-2 antibodies, while a SARS-CoV-2-specific Th1 cell response persisted (Physique 1BCF)
Six months after the infection, none of the conducted six ELISAs/chemiluminescence assays detected any anti-SARS-CoV-2 antibodies, while a SARS-CoV-2-specific Th1 cell response persisted (Physique 1BCF). on antibody detection might have limitations in individual patients. for 10 min and stored at 8 C thereafter at the study site. For FACS analysis, blood was collected into Na-Heparin tubes and stored at room heat and in darkness until purification of PBMCs. PBMCs were separated on a Biocoll answer (Bio&SELL GmbH, Feucht, Germany) by centrifugation at 800?at room temperature (RT) for 20 min without brakes. Intermitting phase made up of PBMCs was washed with PBS two times and slowly chilled to ?80 C in a Cryo Freezing Container (Thermo Fisher Scientific/Nalgene, MA, USA, rate: ?1 C/min) in a medium containing 10% DMSO (Sigma-Aldrich, Burlington, MA, USA) and 50% FCS (Sigma-Aldrich, Burlington, MA, USA). Subsequently, PBMC samples were cryo-conserved in liquid nitrogen until analysis. 2.2. Restimulation of SARS-CoV-2-Specific Th Cells and Cytokine Detection Cryo-conserved PBMC were thawed at RT and immediately washed with cell culture medium (supplemented with 10% human AB serum (PAN Biotech, Aidenbach, Germany), penicillin/streptomycin). Upon a resting period at 37 C for 1h, a maximum number of 5 106 PBMCs was restimulated in cell culture medium made up of 1 g/mL recombinant anti-human CD28 antibody (clone CD28.2, BioLegend, San Diego, CA, USA, RRID:AB_314303) with either 0.2% DMSO (negative control), SARS-CoV-2 Spike glycoprotein PepMix 1 (S1, Fluvastatin N-terminal protection) or 2 (S2, C-terminal protection) (both JPT, Berlin, Germany). In total, 106 PBMCs were restimulated with 1 g/mL TSST1 and 1 g/mL SEB (both Sigma-Aldrich, Burlington, MA, USA) in the presence of 1 g/mL recombinant anti-human CD28, or with anti-human CD3/CD28 beads (Gibco/Thermo Fisher Scientific, Vilniaus apskritis, Lithuania) at a ratio of one bead/PBMC as high controls. All samples were incubated for 2 h, and Brefeldin A (BioLegend, San Diego, CA, USA) was added for another 14h of incubation (total 16 h). Upon centrifugation at 300?at RT for 10 min, cells were recovered in 1 mg/mL beriglobin and stained with anti-human CD3 Pacific Blue (clone UCHT1, BioLegend, San Diego, CA, USA, RRID:AB_1595437) and anti-human CD4 Brilliant Violet 605 (clone RPA-T4, BioLegend, San Diego, CA, USA, RRID:AB_2564391). After 5 min, Zombie Aqua fixable lifeless cells stain (BioLegend, San Diego, CA, USA) was added and incubated for another 10 min. Staining was halted with PBA/E, and the cells were fixed in 2% Formaldehyde/PBS at RT for 20 min. Intracellular staining with anti-human CD154 APC (clone 24C31, BioLegend, San Diego, CA, USA, RRID:AB_314832), anti-human CD137 PE/Cy7 (clone 4B4-1, BioLegend, San Diego, CA, USA, RRID:AB_2207741), anti-human IFN APC/Cy7 (clone 4S.B3, BioLegend, San Diego, CA, USA, AB_10663412) and anti-human TNF PerCP/Cy5.5 (clone MAb11, BioLegend, San Diego, CA, USA, AB_2204081) in 0.5% Saponine (Sigma-Aldrich, Burlington, MA, USA) in PBA/E was performed at 4 C for 20 min. Upon washing, the cells were recovered in PBA/E and analyzed with a FACS-Canto-Plus circulation cytometer (BD, Ashland, OR, USA). Data were analyzed with FlowJo V10.7 (BD, Ashland, OR, USA). 2.3. Anti-SARS-CoV-2 IgG Detection For the serological assessment of anti-SARS-CoV-2 antibodies, the following assays were performed as reported in Weis et al., 2021 : EDI Novel Coronavirus SARS-CoV-2 IgG ELISA kit (Epitope Diagnostics Inc., San Diego, CA, USA) for detecting anti-nucleocapsid antibodies, SARS-CoV-2 IgG ELISA kit (Euroimmun, Lbeck, Germany) for detecting anti-S1 domain name antibodies, SARS-CoV-2 S1/S2 CTSB IgG CLIA kit (DiaSorin, Saluggia, Italy) for detecting anti-S1/S2 domain name antibodies, 2019-nCoV IgG kit (Snibe Co., Ltd., Shenzhen, China) for detecting anti-2019-nCoV recombinant antigen Fluvastatin (expressing full-length spike and nucleocapsid proteins) antibodies, SARS-CoV-2 IgG CMIA kit (Abbott, Chicago, IL, USA) for detecting anti-nucleocapsid antibodies and Elecsys Anti-SARS-CoV-2 kit (Roche, Basel, Switzerland) for detecting anti-nucleocapsid antibodies. Sensitivities and specificities were evaluated as explained by the manufacturers. 3. Case Statement and Conversation During the SARS-CoV-2 outbreak in the Thuringian village of Neustadt am Rennsteig, Germany (explained in Weis et al. ), in March and April 2020, a 64-year-old male individual with a known history of Diabetes mellitus Type 2, hypertension, chronic heart Fluvastatin failureknown risk factors for severe disease courseand prior Hepatitis C contamination tested positive for SARS-CoV-2 by PCR obtained by throat swab. In March 2020, the participant developed a sore throat, a congested nose and mild headaches (Table 1). Fever, dyspnea, cough, anosmia or ageusia were not reported. The symptoms lasted approximately three weeks. The Fluvastatin patient fully recovered with no indication for Long-COVID or any other sequelae. His concurrent medication was moxonidine, amlodipine, benazepril, metoprolol and empagliflozin. Table 1 PCR test results and reported symptoms. thead th align=”center”.