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.