Seven-month kinetics of SARS-CoV-2 antibodies and role of pre-existing antibodies to human coronaviruses

Unraveling the long-term kinetics of antibodies to SARS-CoV-2 and the individual characteristics influencing it, including the impact of pre-existing antibodies to human coronaviruses causing common cold (HCoVs), is essential to understand protective immunity to COVID-19 and devise effective surveillance strategies. IgM, IgA and IgG levels against six SARS-CoV-2 antigens and the nucleocapsid antigen of the four HCoV (229E, NL63, OC43 and HKU1) were quantified by Luminex, and antibody neutralization capacity was assessed by flow cytometry, in a cohort of health care workers followed up to 7 months (N = 578). Seroprevalence increases over time from 13.5% (month 0) and 15.6% (month 1) to 16.4% (month 6). Levels of antibodies, including those with neutralizing capacity, are stable over time, except IgG to nucleocapsid antigen and IgM levels that wane. After the peak response, anti-spike antibody levels increase from ~150 days post-symptom onset in all individuals (73% for IgG), in the absence of any evidence of re-exposure. IgG and IgA to HCoV are significantly higher in asymptomatic than symptomatic seropositive individuals. Thus, pre-existing cross-reactive HCoVs antibodies could have a protective effect against SARS-CoV-2 infection and COVID-19 disease.

specific IgGs in the "Sustainers/Increasers'' group. Participants were grouped based on their antibody levels at M6 compared to the previous visit, individuals were labelled for each isotype-antigen pair as "Decayers" when the ratio of antibody levels between both visits was <1 and as "Sustainers/Increasers" when the ratio was ≥ 1. Here, we only represent participants who classify as "Sustainers/Increasers'' for IgG against each of the studied antigens (n=34). The numbers of sustainers/increasers seropositive for IgG against the indicated antigen are between parentheses. Supplementary Figure 3. Comparisons of serological and clinical characteristics between sustainers/increasers and decayers. a) Differences in antibody levels at seroconversion between sustainers/increasers and decayers (median fluorescence intensity, MFI) of IgA, IgG and IgM against each antigen (Nucleocapsid (N), and its Cterminal domain, the Receptor Binding Domain (RBD), full S protein and its subregions S1 and S2)) (n = 110). b) Differences in antibody increase index (represented in log scale) between seropositive participants who reported symptoms and those who did not in month 6 (M6) after recovering from a previous SARS-CoV-2 infection (n=53) c) Differences in antibody increase index (represented in log scale) between seropositive symptomatic participants who reported less than 10 days of symptom duration and those who reported more than 10 days (n=53). The center line of boxes depicts the median; the lower and upper hinges correspond to the first and third quartiles; the distance between the first and third quartiles corresponds to the interquartile range (IQR); whiskers extend from the hinge to the highest or lowest value within 1.5 × IQR of the respective hinge. Wilcoxon rank test was used to assess statistically significant differences in antibody levels between groups. . Spearman test was used to calculate the p-value and R correlation coefficient. Shaded areas represent 95% confidence intervals.

Quantification of antibodies to SARS-CoV-2 by Luminex
The levels of IgG, IgM and IgA were assessed in single replicates by high-throughput multiplex quantitative suspension array technology (qSAT). The assay was performed in 6 plates of 384 wells with samples from the same individual in the same plate (samples from visit M0 to M6).The SARS-CoV-2 antigens included were the spike full protein (S) (aa 1-1213 expressed in Expi293 and His tag-purified), the S1 (aa 1-681, expressed in Expi293 and His tag-purified) and S2 (purchased from SinoBiologicals), the receptor binding domain (RBD) (fused with C-terminal 6xHis and StrepTag purification sequences and purified from supernatant of lentiviral-transduced CHO-S cells cultured under a fed-batch system), the nucleocapsid full protein (N) and the specific N C-terminal region, and the four HCoV N full length proteins (expressed in E. coli and His tag-purified). Assay performance was previously established as 100% specificity and 95.78% sensitivity for seropositivity 14 days after symptoms onset [1].

Coupling of proteins to microspheres
MagPlex® polystyrene 6.5 μm COOH-microspheres (Luminex Corp, Austin, TX, USA) were washed, sonicated and activated with Sulfo-NHS (N-hydroxysulfosuccinimide) and EDC (1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) (Thermo Fisher Scientific Inc.,Waltham USA). Next, microspheres were washed and resuspended in 50 mM MES pH 5.0 (MilliporeSigma, St. Louis, USA). The recombinant proteins were then incubated with the microspheres at the optimal concentrations (from 10 to 50 μg/mL) and left at 4°C on a shaker overnight. Coupled microspheres were resuspended in PBS with 1% BSA to covalently block the free carboxylic group (-COOH) absorbing most of the non-specific binding to secondary or tertiary antibodies during assay steps [2] and heterophilic antibody binding seen in previous systems [3].
Microspheres recovery were quanti ed on a Guava® easyCyte™ Flow Cytometer (Luminex Corporation, Austin, USA). Equal amounts of each antigen-coupled microspheres were multiplexed and stored at 2000 microspheres/μL at 4°C, protected from light.

qSAT assay
Antigen-coupled microspheres were added to a 384-well μClear ® flat bottom plate (Greiner Bio-One, Frickenhausen, Germany) in multiplex (2000 microspheres per analyte per well) in a volume of 90 μL of Luminex Buffer (1% BSA, 0.05% Tween 20, 0.05% sodium azide in PBS) using 384 channels Integra Viaflo semi-automatic device (96/384, 384 channel pipette). Two hyperimmune pools (one for IgG and IgA, and another one for IgM) were used as positive controls in each plate assay for QA/QC purposes and were prepared at 2-fold, 8 serial dilutions from 1:12.5. Pre-pandemic samples were used as negative controls to estimate the cut off of seropositivity.

Neutralizing antibodies
The stable cell line 300.19-ACE2 was obtained by transfecting 300.19 cells with a plasmid encoding human ACE2 cDNA (SinoBiological) with an Amaxa cell line Nucleofector kit V, followed by hygromycin selection and subsequent subcloning. RBD-mFc fusion protein, containing RBD fused to the Fc region of murine IgG1 was obtained by cloning RBD amplified from the pcDNA3-SARS-CoV-2-S-RBD-Fc (Addgene) into the PFUSE-mIGg1-Fc1 (InvivoGen). HEK-293T cells were transiently transfected with the RBD-mFc plasmid using polyethylenimine as previously described [4]. The supernatant containing the RBD-mFc protein was collected 7 days after transfection, and concentrated 4-fold using an Amicon Ultra-15 Centrifugal Filter Unit with an Ultracel-30 membrane (Millipore). A total of 1.2 x10 3 300.19-ACE2 cells per well in a 96-well plate were incubated for 30 min at 4°C with 4 mg/mL of RBD-mFc fusion protein previously exposed to diluted plasma (1:50) for 30 min at 4°C. Samples were stained with anti-mouse IgG-PE 1:200 (Jackson ImmunoResearch), washed, and analyzed by Flow cytometry using standard procedures. Samples were acquired with a FACSCanto II (BD Biosciences) and analyzed with FlowJo Xv10.0.7 (Tree Star, Inc) software [4]. Pseudovirus-based neutralization assay using HIV-based pseudovirus and ACE2 expressing 293T cells is described in Pradenas et al. [5]. This assay has been validated by direct comparison of IC50 neutralization values obtained using pseudoviruses infecting ACE2 expressing 293 cells and replicative viruses infecting Vero cells in Trinité et al. [6].