Healthcare workers without a prior history of SARS-CoV-2 infection at Tokyo-Shinagawa Hospital were enrolled. All participants were confirmed to be seronegative (< 0.1) for nucleocapsid antibodies by Elecsys Anti-SARS-CoV-2 (Roche) at enrollment. All volunteers provided written informed consent prior to enrollment. The participants received two doses of the Pfizer/BioNTech BNT162b2 vaccine. Secondary vaccination was conducted on day 21 after the primary vaccination. Blood samples were collected at five time points for longitudinal analyses: day 0 (the day of first vaccination), day 1, day 21 (the day of second vaccination), day 22, and day 47–51. The following information was collected for each participant: sex, age, body mass index (BMI), pre-existing medical conditions (hypertension, diabetes mellitus, and dyslipidemia), and history of PCR-confirmed SARS-CoV-2 infection. All studies were approved by the Institutional Review Board of the National Institute of National Institute of Infectious Diseases and Tokyo Shinagawa Hospital (Permit numbers: 1292 and 20-A-33). This study was performed in accordance with the principles of the Declaration of Helsinki.
Sample processing and cell isolation.
Blood samples were collected in Vacutainer CPT tubes (BD Biosciences), and peripheral blood mononuclear cells (PBMCs) and plasma samples were isolated via centrifugation at 1800 × g for 20 min. PBMCs and plasma samples were separated by centrifugation at 300 × g for 15 min. Plasma samples were isolated via additional centrifugation at 800 × g for 15 min and stored at -80 °C until further analysis. For ELISA and pseudotyped virus-neutralizing assays, plasma samples were used after heat-inactivation at 56 °C for 30 min. For cytokine/chemokine quantification, plasma samples were used immediately after thawing at room temperature. PBMCs were centrifuged at 300 × g for 10 min and washed twice with PBS (FUJIFILM Wako Chemicals). PBMCs were used immediately after isolation for analysis of MDSCs (e-MDSCs, M-MDSCs, and PMN-MDSCs), or cryopreserved at -135 °C using CELLBANKER 1 plus (ZENOGEN PHARMA) for additional analysis.
Adverse events questionnaire
All participants completed two questionnaires on vaccine-related adverse events that occurred during days 1–7 (primary vaccination-related adverse events) and days 22–29 (secondary vaccination-related adverse events). Questionnaires asked about the presence of three local symptoms (pain, redness, and swelling) and eight systemic symptoms (fever, fatigue, headache, chills, vomiting, diarrhea, muscle pain, and joint pain), and the symptom severities were evaluated on five grades (grade 0–4, see Supplemental Information S1). The criteria used for the clinical trial of the BNT162b2 mRNA vaccine were applied1. Local 3 symptom severity scores and systemic 8 symptom severity scores were summed for the total local symptom severity score and total systemic symptom severity score, respectively.
The human codon-optimized nucleotide sequence encoding the spike protein of the SARS-CoV-2 isolate (GenBank: MN995567) was commercially synthesized (Eurofinsgenomics). RBD (amino acids: 331-529) with the signal peptide (amino acids: 1-20; MIHSVFLLMFLLTPTESYVD) with a C-terminal histidine tag was cloned into the mammalian expression vector pCAGGS. Recombinant proteins were produced using Expi293F cells according to the manufacturer’s instructions (Thermo Fisher Scientific). Expi293F cells were maintained in Expi293 expression medium (Thermo Fisher Scientific). The supernatant from transfected cells was harvested on day 5 post-transfection, and recombinant proteins were purified using Ni-NTA agarose (QIAGEN).
A recombinant reference monoclonal antibody (CR3022) was prepared as previously described35,36. Briefly, the VH/VL genes of CR3022 were cloned into expression vectors with human IgG1 heavy chain and kappa light chain. Pairs of heavy and light chain vectors were transfected into Expi293F cells according to the manufacturer’s instructions. Thereafter, CR3022 was purified from the culture supernatant using a protein G column (Thermo Fisher Scientific) and dialyzed with PBS.
F96 Maxisorp Nunc-Immuno plates (Thermo Fisher Scientific) were coated with 2 μg/mL of RBD protein overnight at 4 °C. After washing with PBS, the plates were blocked with 1% bovine serum albumin (BSA) in PBS for 1.5 h at room temperature. Heat-inactivated plasma and monoclonal antibodies were serially diluted in PBS containing 1% BSA and 0.1% Tween-20 (eight 4-fold serial dilutions starting at 1:20 dilution for plasma, and eight 4-fold serial dilutions starting at 1 μg/mL for monoclonal antibody), then incubated overnight at 4 °C. The following day, plates were washed with PBS containing 0.05% Tween-20. HRP-conjugated goat anti-human IgG (Southern Biotech) was diluted in Can Get Signal Immunoreaction Enhancer Solution 2 (TOYOBO) and incubated for 1.5 h at room temperature. After washing the plates with PBS containing 0.05% Tween-20 and PBS, HRP-activity was visualized by the addition of OPD substrate (Sigma), and OD490 was measured using an Epoch2 microplate reader (Biotek) and iMark microplate reader (Bio-Rad). IgG titers were quantified using CR3022 as a reference antibody in each plate.
RBD IgG titers of COVID-19 convalescent patients were partially referred from data used in Figure 1C of a previous publication2. The criteria for data selection were (1) time points 23–64 days after symptom onset, and (2) severe symptom severity defined by the WHO.
Pseudotyped virus neutralization assay
VSV pseudotyped virus bearing SARS-CoV-2 spike protein was generated as described previously37. Briefly, the cDNA of the SARS-CoV-2 spike protein was synthesized (Integrated DNA Technologies Inc.) and cloned into the pCAGGS expression vector. The plasmid (pCAG-SARS-CoV-2) containing a 19 aa truncation at the C-terminus of the spike protein was constructed. The pCAG-SARS-CoV-2 expression vector was transfected into 293T cells on collagen-coated tissue culture plates. After 24 h of incubation, the cells were infected with G-complemented VSVΔG/Luc38 at a multiplicity of infection of 0.5, and thereafter, the uninfected viruses were washed. After 24 h of incubation, the culture supernatants with VSV pseudotyped virus were collected, centrifuged to remove cell debris, and then stored at -80 °C until use for the virus neutralization assay.
For the pseudotyped virus neutralization assay, SARS-CoV-2 pseudotyped virus was incubated with an equal volume of serially diluted, heat-inactivated plasma (five 5-fold serial dilutions starting at 1:10 dilution) for 1 h at 37 °C. The mixture was inoculated into VeroE6/TMPRSS2 cells (JCRB1819, JCRB Cell Bank) seeded in 96-well solid white flat-bottom plates (Corning), and then incubated for 24 h at 37 °C in a chamber supplied with 5% CO2. VeroE6/TMPRSS2 cells were maintained in DMEM (Fujifilm Wako Pure Chemical) containing 10% heat-inactivated fetal bovine serum (FBS, Biowest), 1 mg/mL geneticin (Thermo Fisher Scientific), and 100 U/mL penicillin/streptomycin (Thermo Fisher Scientific). Luciferase activity in cultured cells was measured using the Bright-Glo Luciferase Assay System (Promega) with a GroMax Navigator Microplate Luminometer (Promega). Half-maximal inhibitory concentration (IC50) was calculated using Prism 9 (GraphPad) and presented as neutralization titers.
NT titers of COVID-19 convalescent patients were partially referred from a previous publication (Moriyama et. al. Immunity 2021, in Figure 1D). The criteria of data selection were the same as described in ELISA section.
For analysis of MDSCs, after blocking non-specific antibody binding using Human TruStain FcX (1:200, BioLegend) for 5 min at room temperature, cells were stained for 2 h at 4 °C with the following antibodies: CD45-APC-Alexa Fluor 700 (HIB30, 1:300; BD Biosciences), CD3-BV605 (SK7, 1:300, BD Biosciences), CD19-BV605 (HIB19, 1:300, BioLegend), CD56-BV605 (5.1H11, 1:300 BioLegend), HLA-DR-PE-Cy7 (G46-6, 1:300, BD Biosciences), CD11b-FITC (ICRF44, 1:300 BioLegend), CD33-PE (P67.6, 1:300 BioLegend), CD14-BV421 (M5E2, 1:300, BD Biosciences), and CD15-APC (W6D3, 1:300 BioLegend). To analyze the 15 immune cell types of interest, cryopreserved PBMCs were thawed at 37 °C and washed twice with RPMI 1640 (FUJIFILM Wako Pure Chemical Corporation) containing 10% heat-inactivated fetal bovine serum (Nichirei Biosciences), 2 mM glutamine (FUJIFILM Wako Pure Chemical Corporation), 100 U/mL penicillin (FUJIFILM Wako Pure Chemical Corporation), and 100 μg/mL streptomycin (FUJIFILM Wako Pure Chemical Corporation) before use. After blocking of non-specific antibody binding as described above, cells were stained for 2 hours at 4 °C with the following antibodies: CD45-BV570 (HI30, 1:150), CD3-BUV661 (HIT3a, 1:300, BD Biosciences), CD4-PE-Cy5.5 (RPA-T4, 1:100, BD Biosciences), CD8-BB660 (RPA-T8, 1:300, BD Biosciences), CD8-BV786 (RPA-T8, 1:300, BD Biosciences), CD19-BUV563 (SJ25C1, 1:300, BD Biosciences), CD56-BB515 (B159, 1:300, BD Biosciences), HLA-DR-APC-H7 (G46-6, 1:300, BD Biosciences), CD14-BV421 (M5E2, 1:300, BD Biosciences), CD16-BUV395 (3G8, 1:300, BD Biosciences), CD11c-APC-R700 (BU15, 1:300, BD Biosciences), CD88-PE-Cy7 (S5/1, 1:300 BioLegend), CD1c-BUV737 (F10/21A3, 1:300, BD Biosciences), CD123-BUV496 (6H6, 1:300, BD Biosciences), CD141-BV605 (1A4, 1:300, BD Biosciences), CD5-BV480 (UCHT2, 1:300, BD Biosciences), CD163-PE-CF594 (GHI/61, 1:300, BD Biosciences), CD163-BV786 (GHI/61, 1:300, BD Biosciences), Siglec-6-BV650 (767329, 1:300, BD Biosciences), Axl-BUV615 (108724, 1:300, BD Biosciences). After staining, the cells were washed twice and resuspended in PBS containing 0.5% BSA, 5 mM EDTA (Thermo Fisher Scientific), and 0.25 μg/mL 7-AAD (Sigma) to detect dead cells. MDSCs were analyzed using a FACS Canto II (BD Biosciences) or FACS Aria III cytometer (BD Biosciences) and FACS Diva v.9.0 software (BD Biosciences), and 15 immune cells other than MDSCs were analyzed using a FACS Symphony S6 cytometer (BD Biosciences) and FACS Diva v.9.0 software (BD Biosciences).
Flow cytometry data analysis
FCS files were analyzed using FlowJo software (v.10.8.0, BD Biosciences). Gating strategies for identifying immune cells are shown in Extended Data Fig. 2. The frequency of each immune cell population was calculated as a proportion of CD45+ 7-AAD- cells (live CD45+ cells).
Cytokine / chemokine quantification
Plasma cytokines/chemokines were measured using a cytometric bead array kit (BD Biosciences) according to the manufacturer’s instructions. Plasma samples were diluted 4-fold for analysis. Data were acquired using a FACS Canto II cytometer (BD Biosciences) and analyzed using FCAP Array Software Version 3.0 (BD Biosciences). For plasma cytokine levels below the detection limit, the value was set to 40 pg/mL.
Primary and secondary immune cell post/pre ratios were calculated as follows: primary, (cell frequency at day 1) / (cell frequency at day 0); secondary, (cell frequency at day 22) / (cell frequency at day 21). The cytokine/chemokine post/pre ratio was calculated as follows: (concentration at day 21) / (concentration at day 22). For generating Z-score for validating adverse event-related cellular parameters, each immune cell post/pre ratio was log-2 transformed, normalized to have a mean of 0 and a variance of 1. Composite scores were then calculated by directly summing these normalized values. All other parameters were analyzed without additional data transformation. Statistical analyses and visual representation were performed using Prism 9. (GraphPad).
RBD IgG, pseudotyped virus NT titer, local or systemic symptom severity scores, cytokine/chemokine post/pre ratios, age, and BMI were compared using the Kruskal-Wallis test followed by Dunn’s post hoc test (Fig. 1b–d, 3b–e, 3 g –j) or Mann-Whitney test (Fig. 5b–c, 5e–g, Extended Data Fig. 1a–b).
Longitudinal analysis of local and systemic symptom severity scores (Fig. 1e) and plasma cytokine/chemokine concentrations (Extended Data Fig. 4) were performed using the Wilcoxon test. Fisher’s exact test was performed to examine the statistical difference in the incidence of symptoms between primary and secondary vaccinations (Fig. 1f–g).
High-dimensional analysis was performed using the scikit-learn python library. Frequency data of 18 immune cell types at 4 time points (day 0, 1, 21, and 22) were standardized by removing the mean and scaling to unit variance, and dimensionality reduction to a two-dimensional space was performed using Uniform Manifold Approximation and Projection (UMAP) (Fig. 1j, Extended Data Fig. 4a–c).
Spearman correlations between primary and secondary symptom severity scores (Fig. 1h–i), cell post/pre ratios, antibody responses, symptom severity scores, and plasma cytokine/chemokine post/pre ratios (Fig. 2, Fig. 3a, Fig. 3f, Fig. 4, Extended Data Fig. 5) were identified. Correlations were considered significant at p < 0.05.
To validate the correlations between cellular parameters and adverse events or between cellular parameters and antibody responses, cellular parameters were stratified as follows: In cases that the cell post/pre ratios inversely correlated with adverse events or antibody responses, the cell post/pre ratios below the median of the cohort were identified as "positive”, and vice versa for "negative”. In Fig. 5 and Extended Data Fig. 7, participants whose cellular parameters were all "positive” (all parameters were below the median of the cohort) were stratified as the matched group, and vice versa.
In all statistical analyses, significance was considered as follows: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.