Study cohort

In total, 38 individuals receiving SARS-CoV-2 vaccinations were recruited at the Freiburg University Medical Center, Germany. Of those, blood was collected from 31 individuals vaccinated three times with the mRNA vaccines bnt162b/Comirnaty or mRNA-1273/Spikevax and 5 individuals receiving a 4th vaccination. All vaccinees did not have a history of SARS-CoV-2 infection prior to inclusion confirmed by seronegativity for anti-SARS-CoV-2 nucleocapside IgG (anti-SARS-CoV-2 N IgG). Moreover, blood was collected from 13 individuals with SARS-CoV-2 breakthrough infections after a 3rd mRNA vaccination. Breakthrough infections were confirmed by positive PCR-testing from oropharyngeal swab. All 13 individuals with breakthrough infections included in this study had mild symptoms without respiratory insufficiency (according to WHO guidelines26). Characteristics of the participants are summarized in Supplementary Table1, including the results of the HLA-genotyping performed by next-generation sequencing.

Written informed consent was obtained from all study participants. The study was conducted in accordance to federal guidelines, local ethics committee regulations (Albert-Ludwigs-Universitt, Freiburg, Germany; vote: 322/20, 21-1135 and 315/20) and the Declaration of Helsinki (1975).

PBMCs were isolated from venous blood samples collected in EDTA blood collection tubes by density centrifugation with lymphocyte separation medium (Pancoll separation medium, PAN Biotech GmbH). PBMCs were stored at 80C until further processing. The cells were thawed in prewarmed RPMI cell culture medium supplemented with 10% fetal calf serum, 1% penicillin/streptomycin, 1.5% 1M HEPES (all purchased from Thermo Scientific) and 50U/mL Benzonase (Sigma).

Sequence homology was analyzed in Geneiousversion11.0.5 (https://www.geneious.com/) using Clustal Omega version1.2.2 alignment with default settings27. Reference genome of human ancestral SARS-CoV-2 (MN908947.3) was obtained from NCBI database. Genome sequences of SARS-CoV-2 variants of concern (VOCs) B.1, B.1.1.7, B.1.351, P.1, B.1.617.2, B.1.1.529 BA.1 and B.1.1.529 BA.2 were identified via CoVariants (https://covariants.org/). Spike epitopes in ancestral strain and all VOCs were aligned according to their homology on an amino acid level.

Peptides were manufactured with an unmodified N-terminus and an amidated C-terminus with standard Fmoc chemistry (Genaxxon Bioscience). All peptides showed a purity of >70%. To generate tetramers, SARS-CoV-2 spike peptides (A*01/S865: LTDEMIAQY, A*02/S269: YLQPRTFLL) were loaded on biotinylated HLA class I (HLA-I) easYmer (immunAware) according to manufacturers instructions. Subsequently, peptide-loaded-HLA class I monomers were tetramerized with phycoerythrin (PE)-conjugated streptavidin according to the manufacturers instructions.

1.5 106 PBMCs were stimulated with the spike protein-derived peptides A*01/S865 or A*02/S269 and anti-CD28 monoclonal antibody (0.5g/mL) for 14 days in RPMI cell culture medium supplemented with rIL-2 (20 IU/ml, StemCell Technologies). At day 4, 7 and 11, 50% of the culture medium was exchanged with freshly prepared medium containing 20 IU/mL rIL-2. After 14 days, PBMCs were stimulated with peptides again, and stained for CD107a for 1h at 37C to analyze degranulation. Subsequently, brefeldin A (GolgiPlug, 0.5l/mL) and monensin (GolgiStop, 0.5l/mL) (all BD Biosciences) were added and incubation continued for four more hours, followed by surface and intracellular staining with anti-IFNy, anti-TNF and anti-IL-2-specific antibodies. For calculation of the expansion capacity and to assess the cytotoxic capacity of the expanded cells, peptide-loaded HLA class I tetramer staining was performed together with intracellular staining of Granzyme B, Granzyme K, Perforin and Granulysin.

CD8+ T cells targeting spike epitopes were enriched as described previously28. In brief, 5 106 to 20 106 PBMCs were stained with PE-coupled peptide-loaded HLA class I tetramers for 30min at room temperature followed by incubation with magnetic anti-PE microbeads. Subsequent positive selection of magnetically labelled cells was achieved by using MACS technology (Miltenyi Biotec) according to the manufacturers protocol. The enriched spike-specific CD8+ T cells were analyzed using multicolor flow cytometry. Cell frequencies were calculated as previously described28. Of note, only samples with 5 non-nave spike-specific CD8+ T cells were included in subsequent analyses. Accordingly, the detection limit of spike-specific CD8+ T cells in this study was 0.25 1 106, depending on the initial cell input. This cut-off number has been applied and validated in different studies on antigen-specific T cells and has shown to generate reproducible results3,11,29,30.

Antibodies used for multiparametric flow cytometry are listed in Supplementary Table2. To facilitate staining of intranuclear and cytoplasmic targets, FoxP3/Transcription Factor Staining Buffer Set (Thermo Fisher) and Fixation/Permeabilization Solution Kit (BD Biosciences) were used, respectively. Finally, cells were fixed in 2% paraformaldehyde (Sigma) and samples were analyzed on FACSCanto II or LSRFortessa with FACSDiva software version 10.6.2 (BD), or CytoFLEX (Beckman Coulter) with CytExpert Software version 2.3.0.84. Further analyses of the data were performed using FlowJo version 10.6.2 (Treestar). Phenotypical analyses were based on 5 106 to 20 106 PBMCs that were used as an input number for the magnetic bead-based enrichment of spike-specific CD8+ T cells.

For dimensionality reduction, flow cytometry data were analyzed with R version 4.1.1 and the Bioconductor CATALYST package (release 3.13)31. Initially, viable and tetramer-positive CD8+ T cells (or subsets of those) were identified using FlowJo 10 in two separate multiparametric flow cytometry panels (activation panel: HLA-DR, BCL-2, PD-1, CD137, Ki67, TCF-1, EOMES, T-BET, TOX, CD38, CD45RA, CCR7; differentiation panel: CD45RA, CCR7, CD27, CD28, CD127, CD11a, CD57, CXCR3, CD95, CD57, CD39, KLRG1, PD-1). To facilitate visualization of the dimensionality reduction by t-SNE and diffusion map analysis, cell counts were sampled down to at least 20 cells per sample, and marker expression intensities were transformed by arcsinh-transformation with a cofactor of 150.

Determination of SARS-CoV-2-specific antibodies was performed by using the Euroimmun assay Anti-SARS-CoV-2-QuantiVac-ELISA (IgG) for detecting anti-SARS-CoV-2 spike IgG (anti-SARS-CoV-2 S IgG; <35.2 BAU/mL: negative, 35.2 BAU/mL: positive) and the Mikrogen assay recomWell SARS-CoV-2 (IgG) for detecting anti-SARS-CoV-2 N IgG (detection limit, 24a.u.ml1) according to the manufacturers instructions. Data were collected with the SparkControl Magellan software version2.2.

Samples of vaccinated individuals and those with breakthrough infections were tested in a plaque reduction neutralization assay as previously described3. In brief, VeroE6 cells were seeded in 12-well plates at a density of 4 105 cells per well. Serum samples were diluted at ratios of 1:16, 1:32, 1:64, 1:128, 1:256, 1:512 and 1:1024 in a total volume of 50l PBS. For each sample, a serum-free negative control was included. Diluted sera and negative controls were subsequently mixed with 90 plaque-forming units (PFU) of authentic SARS-CoV-2 (either B.1, B.1.617.2 (delta) and B.1.1.529 BA.1 (omicron)) in 50l PBS (1,600 PFU/mL) resulting in final sera dilution ratios of 1:32, 1:64, 1:128, 1:256, 1:512, 1:1024 and 1:2048. After incubation at room temperature for 1h, 400l PBS was added to each sample and the mixture was subsequently used to infect VeroE6 cells 24h after seeding. After 1.5h of incubation at room temperature, inoculum was removed and the cells were overlaid with 0.6% Oxoid-agar in DMEM, 20mM HEPES (pH 7.4), 0.1% NaHCO3, 1% BSA and 0.01% DEAE-Dextran. Cells were fixed 72h after infection using 4% formaldehyde for 30min and stained with 1% crystal violet upon removal of the agar overlay. PFU were counted manually. Plaques counted for serum-treated wells were compared to the average number of plaques in the untreated negative controls, which were set to 100%. Calculation of PRNT50 values was performed using a linear regression model in GraphPad Prism 9 (GraphPad Prism Software).

GraphPad Prism software version 9.3.1 was used for statistical analysis. Statistical significance was assessed by Kruskal-Wallis test, one-way ANOVA with mixed-effects model, two-way ANOVA with full model and main model. Statistical analysis was performed for A*01/S865 (n=7) and A*02/S269 (n=8) longitudinally analyzed CD8+ T cell responses in Figs.1a, b, 3c, 4a, b and Supplementary Figs.2a, 5ac, 7ce for n=28 subjects longitudinally followed in Fig.2a, for A*01/S865 (n=2) and A*02/S269 (n=3) T cell responses longitudinally followed in Fig.2c, for n=26 subjects in Fig.2b, for n=6 prepandemic samples Supplementary Fig.1c, for n=2 subjects in Supplementary Fig.3c, for n=7 at 3 months after 2nd vaccination, n=11 at 9 months after 2nd vaccination and n=11 at 3 months after 3rd vaccination in Fig.3a and Supplementary Fig.4b, for n=4 at 3 months after 2nd vaccination, n=8 at 9 months after 2nd vaccination and n=10 at 3 months after 3rd vaccination in Supplementary Fig.4b, for A*01/S865 (n=7) and A*02/S269 (n=6) longitudinally analyzed CD8+ T cell responses in Fig.3d, for n=8 at 3 months after 2nd vaccination, n=12 at 9 months after 2nd vaccination and n=11 at 3 months after 3rd vaccination in Fig.3b, for n=4 in Supplementary Fig.6a, for A*01/S865 (n=2) and A*02/S269 (n=2) longitudinally analyzed CD8+ T cell responses in Supplementary Fig.6b, for n=10 at 3 months after 2nd vaccination, n=12 at 9 months after 2nd vaccination and n=11 at 3 months after 3rd vaccination in Fig.4c, for n=10 at 3 months after 2nd vaccination, n=11 at 9 months after 2nd vaccination and n=11 at 3 months after 3rd vaccination in Fig.4d, for n=6 at 3 months after 2nd vaccination, n=12 at 9 months after 2nd vaccination and n=10 at 3 months after 3rd vaccination in Fig.4e, for n=6 at 3 months after 2nd vaccination, n=12 at 9 months after 2nd vaccination and n=11 at 3 months after 3rd vaccination in Fig.4f, for Omicron infection n=12, Delta infection n=2 and 4th vaccination n=5 longitudinally analyzed T-cell responses in Fig.5a, for Omicron infection n=11, Delta infection n=2 and 4th vaccination n=4 analyzed T cell responses in Fig.5b and in peak response in Supplementary Fig.8a, for Omicron infection n=12, Delta infection n=2 and 4th vaccination n=3 longitudinally analyzed T cell responses in Fig.6c, for Omicron infection n=11, Delta infection n=1 and 4th vaccination n=3 in Fig.6d, for Omicron infection n=6, Delta infection n=2 and 4th vaccination n=4 analyzed T cell responses after 1 month in Supplementary Fig.8a and Supplementary Fig.9b, for Omicron infection n=6, Delta infection n=2 and 4th vaccination n=4 analyzed T cell responses in Supplementary Fig.9a.

Further information on research design is available in theNature Research Reporting Summary linked to this article.

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COVID-19 mRNA booster vaccine induces transient CD8+ T effector cell responses while conserving the memory pool for subsequent reactivation -...