METHODS
Study design . Sera were collected from healthy subjects enrolled in a longitudinal study for monitoring population immunity to SARS-CoV-2 infection and vaccination in Hong Kong. The study was approved by the institutional review board of the Hong Kong West Cluster of the Hospital Authority of Hong Kong (Reference No.: UW20-169) and the Joint Chinese University of Hong Kong-New Territories East Cluster Clinical Research Ethics Committee (Reference No.: 2020.229). Enrolled participants were followed up every 6 months for sera collection and self-reported SARS-CoV-2 infections that have been RT-PCR confirmed. Pre- and post-vaccination sera were collected from age-matched (27-73 years old) individuals who received 3-doses of BioNTech (BNT) (n=20) or CoronaVac (CV) (n = 21) in 2021-2022. The pre-vaccination sera were collected on the day of receiving the first dose of the BNT or CV vaccine, and the post-vaccination sera were collected 4-8 week after receiving the third dose of the vaccine. The convalescent sera from SARS-CoV-2 infected subjects without (n=20) or with vaccination history (n=20 each for BNT and CV) were collected from study participants after self-reported SARS-CoV-2 infections. The archived pre-pandemic sera in 2019 from healthy blood donors (n=20) were used as controls.
Enzyme-linked immunosorbent assay (ELISA). MaxiSorp 96-well plates (Thermo Fisher) were coated with 0.1 µg recombinant spike [S1, S2, or full-length S (S1+S2), as indicated] or nucleocapsid proteins of OC43, 229E, SARS-CoV-2, or MERS-CoV (Sino Biological) per well overnight at 4°C. The plates were washed with PBST (PBS containing 0.05% Tween 20) and blocked with blocking buffer (5% non-fat milk in PBST) for 2 hours. Human sera were heat treated at 56°C for 30 minutes and were serially 3-fold diluted from 1:100 to 1:2700 with blocking buffer. Diluted sera (100 µL/ well) were added in duplicate to the plate and incubated for 1 hour, followed by detection using 1:10000 diluted HRP-conjugated goat anti-human IgG secondary antibody (100 µL/ well). TMB substrate (100 µL/ well) (Thermo Scientific) was added to the plate for colorimetric signal formation for 10 minutes and stopped by adding 50 µl/well of 2M sulphuric acid. Plates were read at wavelength of 450 nm for absorbance (OD 450nm). In each ELISA plate, the mean OD 450nm from wells without human sera (n=8 per plate) was calculated as the background. The area under the curve (AUC) were calculated for each serially diluted sera after subtracting the background.
MERS-CoV spike pseudoparticle neutralisation tests (ppNT).Luciferase expressing HlV/MERS-RBD pseudoparticles (5 ng of p24) were pre-incubated with serially diluted sera at 4°C for 30 minutes and added to Vero E6 cells in triplicates. Infection was determined by quantifying the firefly luciferase activity at 2 days post-infection (Promega Corporation,) using the Microbeta luminometer (PerkinElmer). The highest serum dilution that gave ≥90% reduction of the maximal luciferase activity (eg. in the absence of antibody) was regarded as the ppNT antibody titre (16).
Statistical analysis . The difference of grouped AUC of pre- and post-vaccination against each antigen within vaccination group was analysed with the Wilcoxon test. The individual AUC difference between pre- and post-vaccination of the same individual was calculated and compared with the AUC difference between two vaccination groups with the Mann-Whitney test. Correlation between AUC ratio of samples against SARS-CoV-2 versus other Human Coronaviruses were analysed using Spearman’s rank correlation. The statistical significance of all statistical tests was set at p < 0.05.