Antibody Response against SARS-CoV-2 and Seasonal Coronaviruses in Nonhospitalized COVID-19 Patients

Study participants and sample processing.Blood was drawn from potential blood donors for reconvalescent plasma therapy after written consent at the Clinical Transfusion Medicine, Tübingen, Germany, between 4 April and 12 May 2020, under the guidelines of the local ethics committees, 222/2020BO. The study cohort consisted of 49 patients older than 18 years, who either provided a PCR-confirmed diagnosis of SARS-CoV-2 (n = 46) or were symptomatic and close contacts of positively diagnosed COVID-19 patients (partners tested positive) (n = 3). All patients were nonhospitalized with asymptomatic to mild courses of disease, and they were fully convalescent, showing no symptoms on the day of blood donation. Basic demographic information was collected including age and sex, as well as self-perceived symptoms (cough, fever, limb pain and headache, diarrhea, and loss of taste). In addition, blood from four healthy donors and one hospitalized patient was collected (see Table S1 in the supplemental material). Serum samples were stored at −80°C.

Cell culture.Caco-2 (human colorectal adenocarcinoma) cells were cultured at 37°C with 5% CO₂ in Dulbecco modified Eagle medium (DMEM) containing 10% fetal calf serum (FCS), with 2 mM l-glutamine, 100 μg/ml penicillin-streptomycin, and 1% nonessential amino acids (NEAA).

Viruses.A throat swab sample collected in March 2020 at the diagnostic department of the Institute for Medical Virology and Epidemiology of Viral Diseases, University Hospital Tübingen, from a SARS-CoV-2-positive patient was used to isolate the virus (200325_Tü1). Fifty microliters of patient material was diluted in medium, sterile filtrated, and used directly to inoculate 200,000 Caco-2 cells in a 6-well plate. At 48 hpi (hours postinfection) the supernatant was collected, centrifuged, and stored at −80°C. Supernatant as well as cell lysates from infected cells was tested by Western blotting using a SARS-CoV-2 anti-nucleocapsid protein (NP) specific antibody (GeneTex). The identity of the virus was confirmed by qRT-PCR via S and E gene amplification (RealStar SARS-CoV-2 RT-PCR; Altona, Germany) with a high viral load (cycle threshold [C_T] values <17). An aliquot of the isolate was used to amplify the virus in a medium flask of Caco-2 cells (2 × 10⁶ cells) in 13 ml DMEM plus supplements and 5% FCS. At 48 hpi, the supernatant was centrifuged and stored in aliquots at −80°C.

The recombinant SARS-CoV-2 expressing mNeonGreen (icSARS-CoV-2-mNG) (26) was obtained from the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA) at the UTMB (University of Texas Medical Branch). To generate icSARS-CoV-2-mNG stocks, Caco-2 cells were infected as described above, and the supernatant was harvested 48 hpi, centrifuged, and stored at −80°C.

Next-generation sequencing (NGS) was used to fully characterize isolates 200325_Tü1 and icSARS-CoV-2-mNG. Briefly, RNA was isolated from viral stocks by using the Qiagen DSP virus pathogen minikit on a QIAsymphony instrument (Qiagen, Hilden, Germany). The sequencing was performed at the NGS Competence Center Tübingen, UKT, Germany, and the data analysis was carried out at the Institute of Medical Genetics and Applied Genomics, University of Tübingen. The clinical isolate 200325_Tü1 was identified as belonging to the B.1.126 SARS-CoV-2 lineage while icSARS-CoV-2-mNG belongs to the A lineage, as expected.

For multiplicity of infection (MOI) determination, a titration using serial dilutions of both virus stocks (200325_Tü1 and mNG) was conducted. The number of infectious virus particles per milliliter was calculated as (MOI × cell number)/(infection volume), where MOI = −ln(1 − infection rate). To reach an infection rate of ∼20%, an MOI of 0.3 was used for SARS-CoV-2-200325_Tü1 and 1.1 for SARS-CoV-2-mNG.

Enzyme-linked immunosorbent assays (ELISAs).(i) Euroimmun. The Euroimmun SARS-CoV-2 ELISA (IgG) (Euroimmun, Lübeck, Germany) with the recombinant S1 target antigen of SARS-CoV-2 was performed according to manufacturer’s instructions in serum. Patient samples are diluted 1:101 in sample buffer. The included controls and calibrator in the test kit were used with each run. Results are given as ratios (optical density [OD] of control or clinical sample/OD of calibrator). According to the manufacturer, ratios were classified as negative (<0.8), borderline (≥0.8 to <1.1), and positive (≥1.1).

(ii) Mediagnost. IgG antibody detection directed to the S1-RBD SARS-CoV-2 in human sera using the Mediagnost test system was performed according to the manufacturer’s instructions. Briefly, these tests are two-step enzyme-linked immunosorbent assays. The solid phase consists of a 96-well microtiter plate (Greiner, Bio-One, Frickenhausen, Germany) that is coated with the recombinant SARS-CoV-2 spike protein S1. The antibodies from patients that are directed against SARS-CoV-2 S1 protein bind to the solid-phase-coated S1 protein. Next, a horseradish peroxidase (HRP)-conjugated goat anti-human IgG binds to the human IgG antibodies. The following step involves the substrate for the HRP being added, by which the substrate is converted from colorless into blue, and after addition of a stop solution, the color changes to yellow. The extinction of the yellow solution can be measured at a wavelength of 450 nm with reference at 620 nm. Increasing extinctions represent increasing amounts of antibodies to SARS-CoV-2 S1 protein. Samples showing extinctions that were three times higher than the negative control can be interpreted as being positive for anti-SARS-CoV-2 S1. IgA and IgM antibody detection directed to the SARS-CoV-2 S1 protein was made in analogy to the above-described IgG detection system except that the HRP-labeled detection antibody was directed against human IgA or human IgM antibodies. According to the manufacturer, negative, borderline, and positive ratios were classified as follows: <0.42, ≥0.42 to 0.7, and ≥0.7 for IgG, <0.33, ≥0.33 to 0.7, and ≥0.7 for IgA, and <0.87, ≥0.87 to 1.47, and ≥1.47 for IgM, respectively.

Elecsys anti-SARS-CoV-2 (Roche).For qualitative detection of anti-SARS-CoV-2 (IgG + IgM) antibodies, the electrochemiluminescence immunoassay (ECLIA) was performed using the fully automated Cobas E 6000/601 immunoassay analyzer (Roche Diagnostics, Mannheim, Germany). This assay targets recombinant SARS-CoV-2 nucleocapsid (NC) protein. Two calibrators are used (Cal1 nonreactive, cutoff index [COI] of 0.101; and Cal2 reactive, COI of 1.2) in the double antigen-sandwich-based assay (SARS-CoV-2 recNC biotin label, and SARS-CoV-2 recNC ruthenium complex label). Twenty microliters each of sera and reference solutions were used, and immune complexes were fixed to streptavidin-coated microparticles. Readout is given in relative light units in the form of cutoff index (COI, signal/cutoff). The Elecsys reagents derived from lot 49500101. For negative control (<150% Cal1), we used pooled sera from 100 mothers at birth of the 2012 Tuebingen congenital cytomegalovirus (CMV) study. Furthermore, we used a negative-control serum from a direct COVID-19 contact person, repeatedly negatively tested for SARS-CoV-2 RNA and nucleocapsid-specific antibodies without any symptoms during and after a 1-week close exposure. For positive control, we used a dilution series of a serum from a reconvalescent student infected symptomatically (fever, cough, loss of smell) who tested positive for viral RNA and NC-specific antibodies. The COIs ranged from 100 to 1. If the numeric COI result was ≥1.0, the serum was diagnosed as reactive, and COIs of <1.0 were attributed as nonreactive. COI values of the positive controls were stable over at least 2 months.

Multiplexed detection of anticoronavirus antibodies.Whole-viral-protein lysates from 229E, OC43, and NL63 (ZeptoMetrix Corp.) and from SARS-CoV-2 were used for DigiWest as described previously (16). Viral protein lysates were used for denaturing gel electrophoresis and Western blotting using the NuPAGE system. Blot membranes were washed with PBST (0.1% Tween 20, phosphate-buffered saline [PBS]), and membrane-bound proteins were biotinylated by adding 50 μM N-hydroxysuccinimide (NHS)–PEG12–biotin (Thermo Fisher Scientific) in PBST for 1 h. After washing in PBST, membranes were dried overnight. Subsequently, the Western blot lanes were cut into 96 strips of 0.5-mm width and were transferred to a 96-well plate (Greiner Bio-One). For protein elution, 10 μl of elution buffer was added to each well (8 M urea, 1% Triton X-100 in 100 mM Tris-HCl, pH 9.5). The protein eluates were diluted with 90 μl dilution buffer (5% bovine serum albumin [BSA] in PBST, 0.02% sodium azide). Neutravidin-coated MagPlex beads (Luminex) of a distinct color identity (ID) were added to the protein eluates, and binding was allowed overnight; 500 μM PEG12-biotin in PBST was added to block remaining neutravidin binding sites. The bead-containing fractions were pooled, and thereby the original Western blot lanes were reconstituted. Beads were washed in PBST and resuspended in storage buffer (1% BSA, 0.05% azide, PBS). The generated bead set represents the proteomes of the four coronaviruses (SARS-CoV-2, OC43, 229E, NL63), and reactivity against all proteins can be tested in one assay.

For serum incubation, 5 μl of the bead mix was equilibrated in 50 μl serum assay buffer (blocking reagent for ELISA [Roche] supplemented with 0.2% milk powder, 0.05% Tween 20, and 0.02% sodium azide, 25% Low Cross buffer [Candor Bioscience], 25% IgM-reducing agent buffer [immunochemistry]). Serum assay buffer was discarded, and 30 μl of diluted patient serum (1:200 in serum assay buffer) was added and incubated for 2 h at room temperature (RT) on a shaker. After washing in PBST, 30 μl of phycoerythrin-labeled anti-human IgG secondary antibody (diluted 1:200 in serum assay buffer; Dianova) was added and incubated for 45 min at 23°C. The beads were washed twice with PBST, and readout was performed on a Luminex FlexMAP 3D.

The DigiWest analysis tool was used to assess serum reactivity against the viral proteins (16). Virus protein-specific peaks were identified, and average fluorescence intensity (AFI) values were calculated by integration of peak areas.

Neutralization assay.For neutralization experiments, 1 × 10⁴ Caco-2 cells/well were seeded in 96-well plates the day before infection in medium containing 5% FCS. Cells were coincubated with SARS-CoV-2 clinical isolate 200325_Tü1 at an MOI of 0.3 and patient sera in serial 2-fold dilutions from 1:20 up to 1:2,560. At 48 hpi cells were fixed with 80% acetone for 5 min, washed with PBS, and blocked for 30 min at room temperature (RT) with 10% normal goat serum (NGS). Cells were incubated for 1 h at RT with 100 μl of serum from a hospitalized convalescent donor in a 1:1,000 dilution and washed 3 times with PBS. One hundred microliters of goat anti-human Alexa 594 (1:2,000) in PBS was used as secondary antibody for 1 h at RT. Cells were washed 3 times with PBS and counterstained with 1:20,000 DAPI solution (2 mg/ml) for 10 min at RT. For quantification of infection rates, images were taken with the Cytation3 (BioTek) and DAPI-positive and Alexa 594-positive cells were automatically counted by the Gen5 software (BioTek).

Alternatively, Caco-2 cells were coincubated with the SARS-CoV-2 strain icSARS-CoV-2-mNG at an MOI of 1.1 and patient sera in serial 2-fold dilutions from 1:40 up to 1:5,120. At 48 hpi cells were fixed with 2% PFA and stained with Hoechst 33342 (1-μg/ml final concentration) for 10 min at 37°C. The staining solution was removed and exchanged for PBS. For quantification of infection rates, images were taken with the Cytation3 (BioTek) and Hoechst-positive and mNG-positive cells were automatically counted by the Gen5 software (BioTek). Virus-neutralizing titers (VNT₅₀s) were calculated as the half-maximal inhibitory dose (ID₅₀) using 4-parameter nonlinear regression (GraphPad Prism).

Software and statistical analysis.GraphPad Prism 8.0 was used for statistical and correlation analyses and to generate graphs. Figures were generated with CorelDrawX7. Other software used included Gen5 v.3.04.