Seroprevalence of anti-SARS-CoV-2 antibodies in COVID-19 patients and healthy volunteers up to 6 months post disease onset

Abstract

SARS-CoV-2 has emerged as a human pathogen, causing clinical signs, from fever to pneumonia-COVID-19-but may remain mild or asymptomatic. To understand the continuing spread of the virus, to detect those who are and were infected, and to follow the immune response longitudinally, reliable and robust assays for SARS-CoV-2 detection and immunological monitoring are needed. We quantified IgM, IgG, and IgA antibodies recognizing the SARS-CoV-2 receptor-binding domain (RBD) or the Spike (S) protein over a period of 6 months following COVID-19 onset. We report the detailed setup to monitor the humoral immune response from over 300 COVID-19 hospital patients and healthcare workers, 2500 University staff, and 198 post-COVID-19 volunteers. Anti-SARS-CoV-2 antibody responses follow a classic pattern with a rapid increase within the first three weeks after symptoms. Although titres reduce subsequently, the ability to detect anti-SARS-CoV-2 IgG antibodies remained robust with confirmed neutralization activity for up to 6 months in a large proportion of previously virus-positive screened subjects. Our work provides detailed information for the assays used, facilitating further and longitudinal analysis of protective immunity to SARS-CoV-2. Importantly, it highlights a continued level of circulating neutralising antibodies in most people with confirmed SARS-CoV-2.

Keywords: COVID-19; SARS-CoV-2; Seroprevalence; neutralizing antibodies.

Figure 1

SARS‐CoV‐2 ELISA setup. SARS‐CoV‐2 IgG antibody detection in serum samples from SARS‐CoV‐2 PCR‐positive subjects or pre‐COVID‐19 controls using Immulon 4HBX 96‐well plates coated with RBD protein for ELISA. Absorbance (optical density, OD) was evaluated at 450 nm. (A) Three serum samples, pre‐COVID‐19 control, medium, and high titer were treated by the indicated methods to inactivate virus particles. (B) Isolated RBD monomer, RBD dimer, pooled RBD and Spike protein were used for coating at the same conditions, and four COVID‐19 serum samples (colored) were tested versus four pre‐COVID‐19 (open symbols). (C and D) 96‐well plate was coated with (C) RBD or (D) Spike protein at indicated concentrations and three COVID‐19 (colored) and pre‐COVID‐19 (black) sera were tested. (E) Secondary antibody dilution titration anti‐IgG, at indicated dilution on 96‐well plate coated with 2 μg/ml RBD protein for three sera. (F) Quality control (QC) and sample serum serial dilution. QC sample is a mix of the serum samples tested from four healthcare workers (S1–S4). Dashed line indicates blank values. Data show individual sample values.

Figure 2

SARS‐CoV‐2 ELISA testing. SARS‐CoV‐2 IgG antibody detection in serum samples from SARS‐CoV‐2 PCR‐positive subjects (colored) or pre‐COVID‐19 controls (open) using Immulon 4HBX 96‐well plates coated with RBD (circles) or Spike (squared) proteins for ELISA. Absorbance was evaluated at 450 nm. (A) Serum at 1/50 dilution from 19 SARS‐CoV‐2 PCR‐positive healthcare workers were assessed for anti‐SARS‐CoV‐2 RBD and Spike IgG and compared with 100 pre‐COVID‐19 sera. Bars indicate mean ± SD. (B and C) ROC analysis, plotting sensitivity against the specificity of (B) RBD or (C) Spike samples as shown in (A). D) Serum at 1/50 dilution from pre‐COVID‐19 cohorts, healthy (100 donors, open symbols), food allergies (61 donors, dark grey symbols), and bee/wasp allergies (20 donors, light grey symbols), were tested for RBD and Spike protein reactivity. (E) Example of cross‐reactivity on RBD or Spike protein from pre‐COVID‐19 serum as used in (D). Statistical analysis was performed using Kruskal–Wallis test with Graphpad Prism software. ^* p < 0.05, ^*** p < 0.001. Dashed lines indicate Black, blank values; Red, RBD cutoff; Green, Spike cutoff. Data show individual sample values.

Figure 3

Seroconversion in hospitalized patients. SARS‐CoV‐2 IgG antibody detection in serum samples from SARS‐CoV‐2 PCR‐positive hospital patients (colored) or pre‐COVID‐19 controls (open) using Immulon 4HBX 96‐well plates coated with RBD (circles) or Spike (squared) proteins for ELISA. Absorbance was evaluated at 450 nm. (A) Overview of all over 300 SARS‐CoV‐2 PCR‐positive tested subjects from Hospital Santa Maria and accumulated 181 pre‐COVID‐19 controls. (B) Selected samples presented in (A) post‐day 14 of the initial reported COVID‐19 symptoms (n = 73 COVID‐19 donors, n = 100 pre‐COVID‐19 healthy controls). (C) Selected samples presented in (A) pre‐day 7 of the initial reported COVID‐19 symptoms (n = 78 COVID‐19 donors, n = 100 pre‐COVID‐19 healthy controls). (D) Longitudinal follow‐up of patients sampled at the indicated week of COVID‐19 symptom onset and re‐sampling 7 days later (n = 76 COVID‐19 donors). Blue indicates continued high signal, Green those that seroconverted at the second sampling in week 2, Orange those that seroconverted at the second sampling past week 2, Red those in which no seroconversion was detected in first and second sampling. (E) Selected samples presented in (A) without reported COVID‐19 symptoms (n = 40 COVID‐19 donors, n = 100 pre‐COVID‐19 healthy controls). (F) Longitudinal follow‐up of asymptomatic patients sampled in the first week of COVID‐19 symptom onset and re‐sampling 7 days later, colors as used as in (D) (n = 10 donors). Dashed lines indicate Black, blank values; Red, RBD cutoff; Green, Spike cutoff. Data show individual sample values, bars indicate mean ± SD.

Figure 4

Seroconversion in subgroups and time. SARS‐CoV‐2 IgG antibody detection in serum samples from SARS‐CoV‐2 PCR‐positive hospital patients or pre‐COVID‐19 controls using Immulon 4HBX 96‐well plates coated with RBD (circles) or Spike (squared) proteins for ELISA. Absorbance was evaluated at 450 nm. (A and B) IgG OD signals were plotted of (A) female (n = 42) and male (n = 28) or (B) by age at the time of blood sampling, of those subjects 14‐days post first COVID‐19 symptoms. Red line marks the mean. (C and D) IgG OD signals of all subjects were plotted by (C) severity of symptoms or (D) over time since the day of first symptoms. (E and F) Hospital patients receiving (E) immunomodulatory medication (orange, n = 31) or (F) antiviral medication (purple, n = 12) who tested SARS‐CoV‐2 PCR‐positive were assessed for IgG antibodies and compared with pre‐COVID‐19 controls (open symbols, n = 100). Statistical analysis was performed using Kruskal–Wallis test using Graphpad Prism software. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001. Data show individual sample values, bars indicate mean ± SD.

Figure 5

Largescale seroconversion testing of Lisbon University staff. SARS‐CoV‐2 IgG antibody detection in 1/50 diluted serum samples from University of Lisbon staff using Immulon 4HBX 96‐well plates for ELISA. Absorbance was evaluated at 450 nm. (A) Schedule of screening plates, coated with 2 μg/ml RBD, accommodating 90 samples/plate, and includes two blanks, two quality control at high concentration (QC_hi), and two at low concentration (QC_lo). B) Overview of tested staff from Lisbon University (n = 2571 donors). Red symbols indicate negative scores, purple symbols indicate ODs above the cutoff. (C) Schedule of re‐screening plates, coated with 2 μg/ml RBD (left) or Spike (right), accommodating 21 samples/plate, and includes two dilutions per sample (1/50 and 1/150), two blanks, two QC_hi, and two QC_lo per protein used. (D) Screening results from the re‐screen (n = 68 donors), showing samples tested on Spike protein. Green depicts those samples above the cut off for RBD and Spike at both 1/50 and 1/150 dilution (n = 38); Red indicates those samples below the cut off on the second screen for either RBD or Spike (n = 30). (E–G) Showing RBD and Spike protein signals for the re‐assessed samples and an additional 10 negative samples, (E) all samples, (F) samples assessed negative (open to red symbols) and an additional 10 that were originally negative (red to red symbols), (G) samples assessed as positive (open to green symbols). (H) Quality control signals for 12 sequential plates, showing QC_hi (circles) and QC_lo (squares). Dotted lines indicate average signal ± SD for QC_hi (red, 200ng/ml) and QC_lo (blue (10 ng/ml)) of human anti‐SARS‐CoV‐2. Data show individual sample values.

Figure 6

Longitudinal SARS‐CoV‐2 antibody titers. SARS‐CoV‐2 antibodies were assessed in 1/50 diluted serum samples from donors in Portugal using Immulon 4HBX 96‐well plates for ELISA. Absorbance was evaluated at 450 nm. (A–C) Anti‐SARS‐CoV‐2 RBD antibody titers plotted over time for (A) IgM, (B) IgG, and (C) IgA (n = 356 total donors). Red line marks the geometric mean. (D–I) Anti‐SARS‐CoV‐2 antibody titers for males and females during (D, F, and H) early, days 7–40 (females n = 32, males n = 29) or (E, G, and H) late response, days 40–150 (females n = 60, males n = 114) for (D and E) IgM, (F and G) IgG or (H and I) IgA. Red line marks the mean. (J–L) Anti‐SARS‐CoV‐2 RBD antibody titers plotted by severity of COVID‐19 symptoms experienced for (J) IgM, (K) IgG, and (L) IgA. Red line indicates the mean (Asymptomatic n = 13, Mild n = 28, moderate n = 54, severe n = 8). (M and N) SARS‐CoV‐2 neutralizing activity was determined in sera (n = 84 total donors) and plotted against (M) time since SARS‐CoV‐2 PCR+ or N) IgG titer. Red lines indicate geographic mean. Statistical analysis was performed using Mann–Whitney U‐test (d–i) or Kruskal–Wallis test using Graphpad Prism software. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001. Data show individual sample values.