Study design

For this study, 67 individuals were recruited by the occupational healthcare department of the University Hospital Bonn in Germany. The initial contact was either established by telephone or during the regular examination at the occupational healthcare center. A consent form was signed on-site before the collection of samples. 29 individuals were recruited to study the effect of the XBB.1.5 booster vaccine, of those 9 refused the vaccination but were further monitored to control for possible changes in SARS-CoV-2 immunity due to the infections with circulating variants. 17 Omicron-infected individuals without other exposures to SARS-CoV-2 antigens were recruited as a control group positive for responses against the mutated epitopes of the Omicron surface proteins. 7 of these individuals were selected for the T-cell assays and 10 for the neutralization assays based on the magnitude of previously measured anti-SARS-CoV-2 responses4,11,13. 21 individuals who received 2-3 doses of wild-type vaccine without other exposures to SARS-CoV-2 antigens were recruited as a control group negative for responses against the mutated epitopes of the Omicron surface proteins. Of these, 11 were selected for the T-cell assays and 10 for the neutralization assays based on the magnitude of previously measured anti-SARS-CoV-2 responses4,11,13. Individuals included in control groups are healthcare workers and were monitored regularly for SARS-CoV-2 infections by RT-PCR (reverse transcription polymerase chain reaction) and antigen tests during the period between the pandemic outbreak and sample collection. Age or sex was not among the selection criteria. SARS-CoV-2 infections were confirmed by RT-PCR or antigen test. Antibodies against the nucleocapsid protein were measured as an additional control for undetected SARS-CoV-2 infections. Detailed information on the vaccination, infection, and sampling time points, as well as demographic information, is provided in Supplemental Table 1. Vaccinations of all individuals included in this study were performed at the occupational healthcare department of the University Hospital Bonn. The study was approved by the Ethics Committee of the Medical Faculty of the University of Bonn (ethics approval number 125/21). All participants provided written informed consent. No compensation was provided for the participants.

Sample collection and storage

Three EDTA (ethylenediaminetetraacetic acid) blood collection tubes (Sarstedt, 02.1066.001) of peripheral blood (total volume of 10–25 ml) were collected from each study participant by venipuncture. Blood samples were centrifuged for 10 min at 600 × g, after which plasma was harvested and stored until analysis at −80 °C. PBMC (peripheral blood mononuclear cells) were isolated from the leftover fraction by density gradient centrifugation using SepMate™ (Stemcell, 85450) tubes with density gradient medium (Pancoll, PAN-Biotech, P04-60500) following the manufacturer’s directions. First, the blood was diluted 1:1 with PBS (phosphate-buffered saline) containing 2% FCS, then carefully layered on top of the density gradient medium, and centrifuged at 1200 × g for 10 min. The top layer containing the PBMCs was decanted and washed twice with 30 ml PBS containing 2% FCS (fetal calf serum). Each aliquot containing 10 million isolated PBMC was resuspended in 1 ml FCS containing 10% DMSO (dimethyl sulfoxide) and frozen at -80°C overnight. For long-term storage, frozen PBMC samples were transferred to liquid nitrogen.

Assessment of XBB.1.5-neutralizing antibodies in plasma

The plasma neutralization capacity against the XBB.1.5 variant was determined by a plaque reduction neutralization assay. First, plasma was heat-inactivated for 30 min at 56 °C and serially two-fold diluted in OptiPRO (Gibco, 12309-019) serum-free cell culture medium. A total of 12 dilutions starting with 2-fold were measured for each sample. No further technical replicates were performed. Plasma dilutions were then combined 1:1 with 80 plaque-forming units of Omicron SARS-CoV-2 (XBB.1.5 in OptiPRO serum-free cell culture medium, incubated for 1 h at 37 °C, and added to Vero E6 cells (ATCC, CRL-1586). 24 h before the infection, the cells were seeded in 24-well plates at a density of 1.25 × 105 cells/well in D10 media (DMEM supplemented with 10% heat-inactivated fetal calf serum, penicillin [100 U/ml], and streptomycin [100 g/ml]). The media was aspirated before the addition of the plasma/virus mix. Following 1 h incubation at 37 °C, the inoculum was aspirated, and cells were overlaid with a 1:1 mixture of 1.5% (w/v) carboxymethylcellulose in 2x MEM supplemented with 4% FCS. After incubation at 37°C for three days, the overlay was removed, and the cells were fixed using a 6% formaldehyde solution. Subsequently, cells were stained with a 1% solution of crystal violet in ethanol to reveal the formation of plaques. The plaque count was plotted against the plasma dilutions, and the half-maximal inhibitory concentration (IC50) was determined using GraphPad Prism software version 9.4.1. (681).

Measurement of neutralizing antibodies specific for the mutated epitopes of the XBB.1.5 surface proteins

To measure the proportion of neutralizing antibodies that recognize mutated regions of the XBB.1.5 surface proteins, we developed a competitive plaque reduction neutralization assay. Initially, plasma was heat-inactivated for 30 min at 56 °C and diluted in OptiPRO (Gibco, 12309-019) serum-free cell culture medium. The plasma dilutions were calculated based on the previous measurement of plasma neutralization capacity against the XBB.1.5 variant to achieve the 80% neutralization effect. Accordingly, prepared dilutions were then incubated with 11 serial 2-fold dilutions of wild-type SARS-CoV-2 surface proteins, spike (Acro Biosystems, SPN-C52H7), membrane (RayBiotech, YP_009724393) and envelope (Acro Biosystems, ENN-C5128) in OptiPRO serum-free media starting with 10ug/ml each and incubated overnight at 4 °C. Sample dilutions, standard dilutions (pooled plasma from donors seropositive for SARS-CoV-2 spike), and negative controls (media without plasma) were combined 1:1 with 80 plaque-forming units of XBB.1.5 variant in OptiPRO serum-free cell culture medium, incubated for 1 h at 37 °C, and added to Vero E6 cells (ATCC, CRL-1586) in a final volume of 200 µl. 24 h before the infection, the cells were seeded in 24-well plates at a density of 1.25 × 105 cells/well in D10 media (DMEM supplemented with 10% heat-inactivated fetal calf serum, penicillin [100 U/ml], and streptomycin [100 g/ml]). The media was aspirated before the addition of the plasma/virus mix. Following 1 h incubation at 37 °C, the inoculum was aspirated, and cells were overlaid with a 1:1 mixture of 1.5% (w/v) carboxymethylcellulose in 2x MEM supplemented with 4% FCS. After incubation at 37 °C for 3 days, the overlay was removed, and the cells were fixed using a 6% formaldehyde solution. Subsequently, cells were stained with a 1% solution of crystal violet in ethanol to reveal the formation of plaques. The plaque count was plotted against the concentration of the surface proteins, and a sigmoidal curve was interpolated using GraphPad Prism software version 9.4.1. (681). The top plateau (representing the signal from XBB.1.5-not-wild-type-neutralizing antibodies) and bottom plateau (representing the signal from total XBB.1.5-neutralizing antibodies) of each curve were interpolated from a standard curve and divided to obtain the proportion of XBB.1.5-not-wild-type-neutralizing antibodies relative to the total XBB.1.5-neutralizing antibodies. This fraction was then multiplied with the corresponding measurement of plasma neutralization capacity against the XBB.1.5 to obtain the level of XBB.1.5-not-wild-type-neutralizing antibodies in plasma.

Stimulation of T cells with overlapping peptide pools

Cryopreserved PBMC samples were thawed at 37 °C and transferred to warm R10 media (RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum, 2 mM l-glutamine, penicillin [100 U/ml], and streptomycin [100 g/ml]). Cells were then centrifuged for 10 min at 300 × g, and the supernatant was decanted. This washing step was repeated two times, after which cells were rested overnight at 37°C. The next morning, PBMC were counted, seeded in 96-well U bottom plates at a density of 1 million/well and stimulated with a pool of overlapping peptides covering the entire sequence of either wild-type SARS-CoV-2 spike protein (PepMix™ SARS-CoV-2 (Spike Glycoprotein), JPT, PM-WCPV-S-1) or the XBB.1.5 spike protein (PepMix™ SARS-CoV-2 (Spike XBB.1.5), JPT, PM-SARS2-SMUT15-1). The final concentration was 1 µg/ml per peptide for both peptide pools in 250 µl R10 media. An equally treated negative control without the peptides was included for each sample. A positive control where cells were stimulated with PMA (20 ng/ml) (Sigma-Aldrich, P1585-1MG) and ionomycin (1 μg/ml) (Sigma-Aldrich, I3909-1ML) was included for each experiment. Stimulation was performed at 37 °C for 24 h.

Detection of SARS-CoV-2-spike-specific T cells by flow cytometry

Following stimulation, cells were washed with PBS and stained for viability in 100 µl of 1% solution of ZombieAqua dye (Biolegend, 423102) in PBS for 15 min at 4 °C. Subsequently, samples were washed with FACS buffer (PBS supplemented with 2% FCS, 0.05% NaN3, and 2 mM EDTA), and stained for surface markers in 100 µl FACS buffer containing the following antibodies; anti-CD3-APC-Cy7 (clone UCHT1, Biolegend, 300426, diluted 1:40), anti-CD4-BV786 (clone SK3, BD Bioscience, 344642, diluted 1:40), anti-CD8-AF700 (clone SK1, Biolegend, 344724, diluted 1:40), anti-CD69-FITC (clone FN50, Biolegend, 310904, diluted 1:80), anti-4-1BB-APC (clone 4B4-1, Biolegend, 309810, diluted 1:80), and anti-OX40-PE-Cy7 (clone ACT35, Biolegend, 350012, diluted 1:80). All antibodies were checked for performance and titrated before use. After staining for 15 min at 4 °C, cells were washed with FACS buffer and acquired on a BD FACS Celesta with BD FACSDiva™ Software Version 8.0 (BD Bioscience). The frequencies of antigen-specific T cells were calculated as negative-control-subtracted data. Possible longitudinal fluctuations in laser intensity were monitored before every experiment using fluorescent beads (Rainbow beads, Biolegend, 422905). PMT voltages were adjusted accordingly to ensure constant signal intensity over time. The data were analyzed using the FlowJo Software version 10.0.7 (TreeStar). No technical replicates were performed due to the scarcity of the samples.

Statistical analysis

Statistical analysis and graphing were performed using GraphPad Prism software version 9.4.1. (681) or RStudio 2021.09.0 Build 351 software14. Differences between the two time points were assessed by the Wilcoxon matched-pairs signed rank test and differences between the groups were assessed using the Mann–Whitney test with Holm’s correction for multiple testing. All tests were performed two-sided. Statistical significance is indicated by the following annotations: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.