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.