Methods
Study design
The study population consisted of 59 healthcare workers (HCWs) who had
received their primary vaccinations and were part of our ongoing
prospective cohort as described previously.12 Whole
blood was collected at two timepoints: September/October 2022 (T1) and
December 2022 (T2). In between these timepoints the majority of HCWs
received an ancestral/Omicron BA.1 bivalent booster vaccination. Blood
samples were collected in heparin tubes via venipuncture and were
processed as described previously.13 This study
received approval from the Medical Research Ethical Committee United
(protocol number R20.030) and was performed according to the Declaration
of Helsinki as revised in 2013.
SARS-CoV-2 S1 IFN-ɣ ELISpot
Interferon-gamma (IFN-y) T cell responses after stimulation with spike
protein subunit 1 (S1) and nucleocapsid (N) peptides were assessed using
the T-spot®.COVID (Oxford Immunotec, UK) kit as described
previously.13
SARS-CoV-2 Omicron BA.5 IFN-ɣ ELIspot
An in-house-developed ELISpot was applied to detect IFN-y T cell
responses against Omicron BA.5 spike peptides. On day 1, polyvinylidene
fluoride membranes precoated with a monoclonal anti-IFN-γ antibody (mAb
1-D1K, Mabtech, Sweden) were washed thrice with phosphate-buffered
saline (PBS) and were conditioned with AIM-V (AIM-V® + AlbuMAX® (BSA);
Gibco, USA) for 30 min at room temperature. The following stimulations
were separately added, each in a volume of 50 µL per well: AIM-V medium
as negative control, anti-CD3 (1:1000, mAb CD3-2, Mabtech) as positive
control, Omicron BA.5 mutation peptides, and corresponding ancestral
strain peptides (PepTivator® SARS-CoV-2 Prot_S B.1.1.529/BA.5, Miltenyi
Biotec, Germany). These peptide pools consisted of 15-mer peptides with
11 amino acids overlap and were added to a final 0.66 µg/mL
concentration. An amount of 2.5 × 105 PBMCs in 100 µL
AIM-V was added to each well, whereafter the microtiter plate was
incubated for 16–20 h at 37°C with 5% CO2 in a
humidified atmosphere. On day 2, the PBMCs were washed off the plate
with PBS, and 100 µL alkaline phosphatase-conjugated antibody (1:200,
7-B6-1-ALP, Mabtech) was added and incubated for two hours at room
temperature. Subsequently, the microtiter plate was washed with PBS and
100 µL substrate (BCIP-NBT-plus; Mabtech) was added and incubated at
room temperature for 7–12 min, after which the reaction was stopped
with demineralized water.
ELISpot Image Processing and Spot Quantification
For the spot quantification, we used the method previously
described.14 In short, images of the ELISpot
membranes were made with the Dx1 microscope (Veho, UK) using Plugable
Digital Viewer v3.1.07 for Windows and an intensity threshold of 95 was
applied instead of the previously described threshold of 75 to enhance
spot detection sensitivity. The number of spots in the negative control
was subtracted from the number of spots in peptide-stimulated conditions
for each individual sample. Samples were excluded if the positive
control resulted in less than 20 spots.
SARS-CoV-2 Anti-RBD IgG Quantitative ELISA
The anti-RBD (ancestral strain) IgG serum concentrations were determined
using a quantitative enzyme-linked immunosorbent assay (ELISA) (Beijing
Wantai Biological Pharmacy Enterprise, China) as described
previously.13
SARS-CoV-2 ancestral and Omicron BA.5 sVNT
Surrogate virus neutralization tests (sVNT) were performed to assess the
neutralizing activity of serum anti-RBD antibodies. The neutralizing
activity against the ancestral strain RBD was determined using the kit
(Genscript Biotech, US) and protocol as previously
described.15 A second sVNT kit (ACROBiosystems, US)
was used to determine the neutralizing activity of serum antibodies
against the Omicron BA.5 RBD. The sVNT was performed according to the
manufacturer’s guidelines using a fully automatic ETI-MAX (Diasorin,
Italy) system. Serum samples, as well as the positive and negative
control, were diluted 1:9 with a dilution buffer. These dilutions were
added 1:1 to RBD-horseradish peroxidase (HRP-RBD) in a pre-coated well,
whereafter this was incubated for 1 hour at 37°C. After washing the
wells with a washing buffer, a substrate solution was added and
incubated for 20 min at 37°C. Lastly, a stop solution was added and the
absorbance was measured at 450 nm and 620 nm to reduce background noise.
The neutralizing activity was calculated as the percentage of inhibition
using the following formula: Inhibition (%) = (1 – (OD450nm –
OD620nm)) x 100.
Statistical analyses
All data obtained in this study were expressed as median with
interquartile range (IQR) and statistical analyses were performed using
GraphPad Prism v9 (GraphPad Software). The Wilcoxon signed-rank test was
applied to compare paired datasets and the Mann–Whitney U test was
applied to compare two independent data sets. The Kruskal–Wallis test
with Dunn’s multiple comparison test was performed to compare three or
more independent groups. All statistical tests were performed at a
two-tailed α-level of 0.05.
Results
SARS-CoV-2-specific T cell and antibody responses in
prior-infected and infection-naive individuals 10 months
after previous monovalent mRNA vaccinations
First, we investigated whether a prior SARS-CoV-2 infection in addition
to primary and booster vaccinations results in prolonged increased
SARS-CoV-2-specific T cell and antibody responses. Therefore, we
determined these immune responses in HCWs who tested SARS-CoV-2 PCR
positive more than three months ago (previous) or within three months
(recent) and in HCWs who never tested SARS-CoV-2 positive (naive) until
the time of blood collection. All HCWs had received mRNA or viral vector
COVID-19 primary vaccinations, and HCWs received no (n=4), one (n=40),
or two (n=7) booster mRNA vaccinations. The last vaccination was
received at median 307 (IQR 301-314.5) days before the first blood
collection in this study. Here, we observed higher spike S1-specific T
cell responses in previously infected HCWs than in infection-naive HCWs
(p = 0.0351), whereas responses were comparable between the recently
infected and infection-naive HCWs (Fig. 1A) . Nucleocapsid
protein (N)-specific T cell responses were only observed in prior
infected HCWs since immunological responses against the N protein are
not elicited by mRNA vaccines (Fig. 1B) .
For the humoral immunity component, anti-SARS-CoV-2-RBD IgG (of
ancestral virus) was detectable in all HCWs and the serum concentrations
were comparable between all groups (Fig. 1C) . Also, we
investigated the neutralizing activity of serum antibodies against
Omicron BA.5 spike-RBD (Fig. 1D) . According to the
manufacturer’s cut-off value of ≥20% inhibition, 83.3% of previously
infected, 76.9% of recently infected, and 38.5% of infection-naive
HCWs were considered positive for the presence of Omicron BA.5 nAbs. We
observed considerable intragroup variations, but no significant
differences between the groups.