Memory CD8+ T cell diversity and B cell responses correlate with protection against SARS-CoV-2 following mRNA vaccination

Cohort

In total, 379 staff members of CCI were recruited for the study (Supplementary Table 1). CCI is a certified COVID-free oncological hospital. All the employers have been constantly tested for SARS-CoV-2 infection by PCR following a stringent schedule. Starting from February 2020 to date, each individual has been screened: every 2–4 weeks (subjects were chosen randomly during this timeframe to ensure a daily check in each branch of the hospital); after 5 days out of office; and after close contact with a SARS-CoV-2-infected subject. In case of close contact with an infected subject, participants were isolated in quarantine for 14 days; during this timeframe, they were PCR tested each week until December 2021 or PCR screened every day for 2 weeks (from January 2022). All participants were tested for NWT and SWT IgG levels in plasma at T0.

Participants classified as VN were classified into VNLRs, VNHRs and nonresponders (NRs). NRs were characterized by SWT IgG values <50 a.u. ml−1 at 6 weeks after vaccination and the remaining naive subjects’ IgG values were used to define a threshold to discriminate between VNLRs and VNHRs, leveraging 3 months of IgG data. The distribution of 3-month IgG values for multimodality was tested (P < 2.2 × 10−16, excess mass test) and the antimode (135 a.u. ml−1) was located with the expectation of a bimodal data distribution. This antimode value was used to discriminate between VNLRs and VNHRs (<135 a.u. ml−1 and >135 a.u. ml−1 at 3 months, respectively). In addition, subjects with IgG titers >5 a.u. ml−1 at T0 were defined as seropositive.

Subjects with a documented SARS-CoV-2 infection who recovered from COVID-19 with mild symptoms (subjects with post-acute COVID-19 syndrome, long COVID or lingering symptoms were not included in the study) were classified into VRLRs and VRHRs with the same methodology applied for naive subjects: 3-month IgG values were tested for multimodality (P = 0.49, excess mass test) and split using the antimode 112 a.u. ml−1 located with the expectation of a bimodal data distribution. For the analysis shown in Figs. 27, subjects in each class were ranked according to three criteria: (1) subjects were split into three age categories (reported in order of priority for analysis): from 40 years old (y.o.) to 50 y.o., <40 y.o. and >50 y.o.; (2) in these age categories, subjects were ranked by age (from youngest to oldest); and (3) in these age categories, subjects were ranked by measured IgG values at 3 months (from highest to lowest). In addition the gender was also taken into consideration. The top ranking subjects from each class were selected for analysis. According to the SWT IgG quantification and the ranking mentioned above, we decided to focus our analysis on 123 subjects who received the BNT162b2 vaccination (Supplementary Table 2). These subjects were divided into three groups: 43 VNLRs, 42 VNHRs and 38 VR donors. Blood samples were collected before the first BNT162b2 mRNA (Comirnaty, Pfizer Biontech) vaccine dose (T0), before the second vaccine dose (week 3) and after 6 weeks, 3 and 6 months from the first immunization. Blood samples were also collected 1 month after the third vaccine dose (10 months after T0). 96% of donors received BNT162b2 mRNA vaccine as third dose, whereas the remaining 4% received mRNA-1273 Moderna vaccine. Data shown in Fig. 2 to 7 and in Extended Data Fig. 2 to 9 were obtained from donors immunised with BNT162b2 mRNA vaccine. All donors signed informed consent forms approved by the Ethical Committee of the CCI; participants did not receive compensation.

Sample processing

Blood samples were collected into heparin tubes via phlebotomy. Tubes were centrifuged at 800g for 5 min and 4 °C to separate plasma that was used for serological analysis. Whole blood was diluted 1:1 with phosphate-buffered saline (PBS) 1× (Sigma-Aldrich) and PBMC isolation was obtained by density gradient centrifugation using Lympholyte (Cederlane). Tubes were centrifuged at 805g for 30 min at room temperature and PBMCs were collected into new tubes. Cells were washed with PBS 1×, centrifuged at 515g for 10 min and 4 °C, counted with the Burker chamber and cryopreserved in 10% dimethyl sulfoxide in fetal bovine serum (FBS; BioWest).

Spike IgM and IgG quantification

Chemiluminescence immunoassay was performed with TGS COVID-19 Control Set (Technogenetics CVCLCSGM), TGS COVID-19 IgM (CVCL100M) and TGS COVID-19 IgG (CVCL100G) according to the manufacturers’ instructions to quantify IgM and IgG.

Recombinant NWT, SWT-, SD-, SO- and SB-RBD antigen expression and purification

Recombinant SARS-CoV-2 N- and S-RBDs were produced using poly(ethyleneimine)-based transient transfection of Freestyle HEK293 Cells (HEK293-F, Life Technologies) cultivated in suspension. Briefly, cell medium containing the secreted proteins of interest was collected 6 d after transfection by centrifugation at 1,000g for 15 min. The samples were loaded on to a 5-ml His-Trap excel column (Cytiva) using a peristaltic pump and then eluted with 3–250 mM imidazole gradient using an NGC fast protein liquid chromatography system (BioRad). Peak fractions containing the antigens of interest were subjected to immediate concentration with concomitant buffer exchange with fresh PBS to remove imidazole using Amicon centrifugal filters (Merck). Quality controls during protein purification were carried out using reducing and nonreducing sodium dodecylsulfate–polyacryamide gel electrophoresis analysis and differential scanning fluorimetry with a Tycho NT.6 instrument (Nanotemper). All samples were concentrated to 1 mg ml−1, flash-frozen in liquid nitrogen and kept at −80 °C until usage.

ELISA

SARS-CoV-2 N and SARS-CoV-2 S-RBD-specific-binding IgG antibodies were longitudinally tested by ELISA. Maxisorp ELISA 96-well plates (Thermo Fisher Scientific) were coated with 1 µg ml−1 of SARS-CoV-2 nucleocapsid or 2 µg ml−1 of S -RBDWT,-RBDD or -RBDO protein in 1× DPBS and incubated at 4 °C overnight. After incubation, plates were washed 3× with PBS containing 0.1% Tween-20 (PBS-T) and blocked with PBS-T supplemented with 3% bovine serum albumin (BSA; Sigma-Aldrich) for 1 h at room temperature. After washing, plasma was added and incubated for 1.5 h at room temperature. Plasma was diluted in 1% BSA in PBS-T starting from 1:25 or 1:100 dilution and serially diluting each sample by 1:4. Plates were washed 3× with PBS-T and then the secondary antibody (anti-human IgG peroxidase, BD), in 1% BSA and PBS-T, was added and the plates were incubated for 1 h at room temperature. After three washes with PBS-T, plates were developed with TMB Substrate Kit (Thermo Fisher Scientific) at room temperature. The reaction was stopped with 1 M chloridric acid and the plates were read on a Tecan Spark plate reader at 450 nm. Optical densities (ODs) were background subtracted. A positive control standard was created by pooling plasma from six VR subjects, whereas a negative control standard was created by pooling plasma from six pre-pandemic processed plasma samples. Positive and negative control standards were run on each plate. The limit of detection (LOD) was defined as 1:25 for IgG or otherwise stated. The limit of sensitivity (LOS) was established on the basis of uninfected subjects, using plasma from donors never exposed to SARS-CoV-2. For each sample, the ELISA endpoint titer was calculated using nonlinear regression interpolation curve fitting based on the positive control standard. Titers were calculated as the reciprocal serum dilution that yields a corrected OD value of 0.1. Similar results were achieved using Sino Biological SARS-CoV-2 S-RBD proteins.

HEK293TN-hACE2 cell-line generation

HEK293TN-hACE2 (human angiotensin-converting enzyme 2) cell line was generated by lentiviral transduction of HEK293TN cells. HEK293TN cells were obtained from System Bioscience. Lentiviral vectors were produced following a standard procedure based on calcium phosphate co-transfection with third-generation helper and transfer plasmids. The following helper vectors were used (gifts from D. Trono): pMD2.G/VSV-G (Addgene, catalog no. 12259), pRSV-Rev (Addgene, catalog no. 12253) and pMDLg/pRRE (Addgene, catalog no. 12251). The transfer vector pLENTI_hACE2_HygR was obtained by cloning of hACE2 from pcDNA3.1-hACE2 (a gift from F. Li, Addgene, catalog no. 145033) into pLenti-CMV-GFP-Hygro (a gift from E. Campeau and P. Kaufman, Addgene, catalog no. 17446). The hACE2 cDNA was amplified by PCR and inserted under the cytomegalovirus promoter of the pLenti-CMV-GFP-Hygro after green fluorescent protein (GFP) excision with XbaI and SalI digestion; pLENTI_hACE2_HygR is now available through Addgene (catalog no. 155296). After transduction with hACE2 lentiviral vector, cells were subjected to antibiotic selection with hygromycin at 250 μg ml−1. Expression of hACE2 cells was confirmed by flow cytometry staining using anti-hACE2 primary antibody (AF933, R&D Systems) and rabbit anti-goat IgG secondary antibody (Alexa Fluor-647). HEK293TN-hACE2 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% FBS, 1% glutamine, 1% penicillin–streptomycin and 250 μg ml−1 of hygromycin (Gibco) and expression of hACE2 was found to be stable after multiple passages.

Plasmids and molecular cloning

The pCAGGS plasmid containing the sequence encoding for carboxy-terminal, His-tagged Wuhan SARS-CoV-2 Spike RBD (catalog no. NR_52310) was obtained from BEI Resources. Variants were generated by replacing the SARS-Cov-2 Wuhan RBD-encoding sequence with synthetic sequences (Genewiz), encoding for either SARS-CoV-2 RBD delta or SARS-CoV-2 RBD omicron variants into the pCAGGS plasmid template using the 5′-XbaI and 3′-NotI restriction sites.

Production of SARS-CoV-2 pseudoparticles

To generate lentiviral particles pseudotyped with SARS-CoV-2 S, we constructed a series of expression plasmids each encoding a SARS-CoV-2 S mutant. Briefly, for each variant, the corresponding C-terminal-deleted (19 amino acids) S complementary DNA was cloned in pcDNA3.1. Then, pLenti CMV-GFP-TAV2A-LUC Hygro was generated from pLenti-CMV-GFP-Hygro (Addgene, catalog no. 17446) by addition of T2A-Luciferase through PCR cloning. To produce the pseudotyped lentiviral particles, 5 × 106 HEK293TN cells were plated in a 15-cm dish in complete DMEM medium and co-transfected on the following day with 32 µg of plasmid pLenti CMV-GFP-TAV2A-LUC Hygro, 12.5 mg of pMDLg/pRRE (Addgene, catalog no. 12251), 6.25 mg of pRSV-Rev (Addgene, catalog no. 12253) and 9 µg of S plasmid. The medium was replaced with complete Iscove’s modification of DMEM 12 h before transfection. Some 30 h after transfection, the supernatant was collected, clarified by filtration through 0.45-μm pore-size membranes and concentrated by ultracentrifugation (SW32Ti rotor). Viral pseudoparticle suspensions were aliquoted and stored at −80 °C.

Neutralization assay

For the neutralization assay with pseudotyped particles, HEK293TN-hACE2 cells were plated 10,000 per well in white 96-well plates in complete DMEM. After 24 h, cells were transduced with 0.1 multiplicity of infection of SARS-CoV-2 pseudovirus previously incubated with a serial threefold dilution of inactivated plasma to obtain a 7-point dose–response curve. Then, 5 µl of each dilution was added to 45 µl of DMEM containing the pseudovirus and incubated for 1 h at 37 °C. The serum/pseudovirus mixture, 50 µl, was then added to each well and the plates were incubated for 24 h at 37 °C. Each point was assayed in triplicate. After 24 h of incubation, cell transduction was measured by luciferase assay using Bright-Glo Luciferase System (Promega) and Infinite F200 plate reader (Tecan). Measured relative light units were normalized with respect to controls and dose–response curves were generated and neutralization dose 50 (ND50) calculated by nonlinear regression curve fitting with GraphPad Prism.

In vitro stimulation

Frozen PBMCs were thawed in complete medium (RPMI supplemented with 2.5% human serum from Aurogene, 1% l-glutamine, 1% penicillin–streptomycin, 1% nonessential amino acids, 1% sodium pyruvate and 0.1% 2-mercaptoethanol). PBMCs were centrifuged at 515g for 10 min and 4 °C, counted with the Burker chamber and resuspended in complete medium. PBMCs were cultured for either 14 h or 24 h with peptide megapools, prepared as previously described28, consisting in overlapping 15-mers by 10 amino acids covering the complete sequence of the S protein of the WT SARS-CoV-2 (GenBank, accession no. MN_908947) as well as beta, delta and omicron SARS-CoV-2 variants (1 µg ml−1). Complete medium was used as a negative control. Dynabeads human T-activator CD3/CD28 (from Gibco, 1:1 bead:cell ratio) supplemented with human (h)IL-2 (from Novartis, 40 U ml−1) was included as a positive control. To analyze the effector and cytotoxic functions, after 14 h of incubation, PBMCs were stimulated with phorbol 12-myristate 13-acetate (Sigma-Aldrich, 20 ng ml−1) and ionomycin (Sigma-Aldrich, 1 µg ml−1). After 1 h, Brefeldin A (Sigma-Aldrich, 5 µg ml−1) was added and the combined treatment lasted 4 h more. For each condition, triplicate wells containing 3 × 105 cells in 200 µl were plated in 96-well round-bottomed plates and incubated at 37 °C with 5% CO2. The same subjects have been analyzed longitudinally.

Intra-/extracellular staining and flow cytometry analysis

After 14 h of incubation, PBMCs were stained for intracellular cytokines combined with surface markers, whereas after 24 h PBMCs were stained for only surface markers. Cells were washed with 1× PBS supplemented with 0.5% BSA and 2 mM EDTA (FACS buffer) and stained with Fixable Viability Stain 450 in 1× PBS for 20 min at 4 °C to discriminate viable from nonviable cells. Then PBMCs were labeled with the following antibodies for multiparametric flow cytometry analysis: anti-CD3 (BD, SK7, 1:200), anti-CD4 (BD, RPA-T4, 1:400), anti-CD8 (BD, SK1, 1:600), anti-CD14 (BD, MφP9, 1:200), anti-CD16 (BD, 3G8, 1:600), anti-CD19 (BD, HIB19, 1:600), anti-CD25 (BD, 2A3, 1:200), anti-CD27 (BD, M-T271, 1:50), anti-CD45RA (BD, HI100, 1:50), anti-CD45RA (BD, 5H9, 1:200), anti-CD56 (BD, NCAM16.2, 1:200), anti-CD45RO (BD, UCHL-1, 1:200), anti-CD69 (BD, FN50, 1:50), anti-CD197 (BD, 150503, 1:100), anti-IFN-γ (BD, B27, 1:50), anti-IL-2 (BD, MQ1-17H12, 1:100), anti-Granzyme B (BD, GB11, 1:100) and anti-TNF-α (BD, MAb11, 1:100). Dilutions are indicative, because each antibody batch has been titrated. Surface staining was performed in FACS buffer for 30 min at 4 °C. Then, cells were washed and fixed with 1% paraformaldehyde in 1× PBS. For the intracellular cytokines staining, after fixation, cells were permeabilized with 1× Perm/Wash buffer (BD) according to the manufacturer’s instructions and stained for 1 h at room temperature in BD Perm/Wash buffer. Cells were then washed twice and resuspended in FACS buffer for data acquisition. The same subjects have been analyzed longitudinally. Flow cytometry data were acquired on a BD LSR Fortessa X-20 instrument and analyzed with FlowJo software. The LOD for antigen-specific CD4+ and CD8+ T cell responses was calculated as the geomean twofold s.d. from the negative control (unstimulated). The LOS for antigen-specific CD4+ and CD8+ T cell responses was calculated as the median twofold s.d. from the negative control (unstimulated)28. When LOD and LOS were negative or 0, we considered as active responses only those >0.01 after background subtraction. No detected responses are labeled ND.

Detection of SARS-CoV-2 antigen-specific B cells

The analysis of SARS-CoV-2 antigen-specific B cells was performed using the SARS-CoV-2 B Cell Analysis Kit (Miltenyi) according to the manufacturer’s instructions. PBMCs were labeled with two tetramers formed from a recombinant SARS-CoV-2 SWT protein (conjugated with phycoerythrin (PE) and PE-Vio), combined with the following antibodies: anti-CD19, anti-CD27, anti-IgG, anti-IgA and anti-IgM.

Analysis of VOC prevalence

Aggregated GISAID (https://doi.org/10.1002/gch2.1018) and aggregated COVID-19 case data for the percentage distribution of VOCs in Italy was downloaded from the European Centre for Disease Prevention and Control (https://www.ecdc.europa.eu/en/publications-data/data-virus-variants-covid-19-eueea; access date 15 April 2022).

Statistical analysis

Wilcoxon’s signed-rank (nonparametric, paired), Wilcoxon’s rank-sum (nonparametric, unpaired) and Spearman’s correlation tests were used for statistical analysis and the P value was determined using Prism software (Graphpad Software, Inc.), or otherwise as indicated in the text. Significance of ratio was assessed by Wilcoxon’s signed-rank t-test compared with a hypothetical median of 1. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Reporting summary

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

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