We designed an observational, longitudinal prospective study to evaluate the immune response elicited by the BNT162b2 vaccine against SARS-CoV-2. The efficacy end-point were the development of SARS-CoV-2-specific neutralizing antibody and T-cell response. The efficacy evaluation are conducted at short-term (after the complete vaccination schedule), medium-term (6 months after vaccination); long-term (12 months after vaccination). Here we report results of the short-term evaluation.
Heparin-treated and untreated blood samples were collected before vaccination (T0), 21 days after the first dose (T1, before second dose administration) and 21 days after the second dose (T2). Blood lymphocyte counts, SARS-CoV-2 S-specific T-cells, RBD-binding antibody levels and SARS-CoV-2 NT serum titers were determined at each time point.
The primary end-point of the study was the frequency of “full responders” (i.e. subjects developing both T cells specific for SARS-CoV2 S protein and NT antibody) after the second dose of BNT162b2 vaccine. Secondary end-points were the following: i) the frequency of “full responders” after the first dose of BNT162b2 vaccine; ii) the magnitude of T cells specific for SARS-CoV2 S protein, RBD-binding and NT antibodies after the first and the second dose of BNT162b2 vaccine; iii) the correlation between T-cell response, RBD-binding antibodies and NT antibodies; iv) the changes in total CD4+ and CD8+ T cells, B cells and NK cells after the first and second dose of BNT162b2 vaccine; 5) the frequency of T-cell and NT antibody responders against SARS-CoV-2 variant strains; 6) the persistence of T-cell response, RBD-binding antibodies and NT antibodies. This last end-point will be analyzed at a later time.
Data about the frequency of subjects developing T cells and NT antibodies in response to the BNT162b2 vaccine were limited, thus this was an exploratory study. The phase 2/3 clinical trial  showed a 95% (95% CI: 90-98%) efficacy of the vaccine in protection from SARS-CoV-2 infection. Thus, adopting a conservative approach, we hypothesized a 90% rate of “full responders” developing both T cells specific for S protein and NT antibody. With a sample size of 150 subjects, the 95% confidence interval of the expected frequency is 84-94%. This correspond to a minimum number 126 “full responders” which should have been detected.
The study (CoVax) was approved by the local Ethics Committee (Comitato Etico Area Pavia) and Institutional Review Board (P-20210000232). All the subjects signed informed written consent.
Total lymphocytes subpopulation count was determined on whole blood by flow cytometry using BD Lyric flow cytometer, (BD Biosciences, San Jose,CA, USA). After lysis of red blood cells, CD3+CD4+, and CD3+CD8+ T cells, CD16+/56+NK and CD19+B cells/μl were determined by flow cytometry, using BD Multitest™ 6-color TBNK reagent and TruCOUNT tubes (BD Biosciences). Gating strategy was set up on CD45+ and side scatter (SSC).
SARS-CoV-2 variants isolation and viral titration
SARS-CoV-2 strains, including wild type China-derived strain (D614), European strain (D614G), UK strain (501Y.V1 lineage B.1.1.7), Brazilian strain (501Y.V3 lineage P.1) and South Africa strain (501Y.V2 lineage B.1.351) were isolated from infected patients’ nasal swabs; in detail, 200 µl of each sample was inoculated and propagated into Vero E6 (VERO C1008 (Vero 76, clone E6, Vero E6; ATCC1CRL-1586TM) permissive cell line and titrated to prepare cell free virus for neutralization assay. All the strains were sequenced in order to confirm the presence of variant-defining mutations. Complete genome sequencing was performed in order to confirm the presence of variant-defining mutations and sequences were submitted to GISAID under the following reference numbers (EPI_ISL_568579; EPI_ISL_1403609-11).
Half‐area 96‐well microplates were coated for 1 h with 5µg/ml recombinant RBDs  of the B1 (China/European) strain, B.1.1.7/N501Y.V1 (UK) strain, or the B.1.351/501Y.V2 (South African) strain. After overnight (or 1 h) blocking with 5% (wt/vol) skimmed milk, the plates were washed and incubated for 1 h with human serum four fold serial dilutions (starting from 1:50), then for 45 min with horseradish peroxidase-labeled goat IgG to human IgG and, finally, for 25 min with 5mg/ml orthophenylendiamine before the addition of 4 N sulphuric acid. The optical density (OD) value of the serum incubated without RBD was subtracted from the OD value of the serum incubated with RBD. Cutoff of 0.100 net OD was calculated based on mean+2SD results of SARS-CoV-2 naive subjects at the serum dilution 1:50. Serum dilution yielding 0.100 net OD value was considered as the RBD-binding serum titer.
Peripheral blood mononuclear cells (PBMC) were isolated from heparin-treated blood by standard density gradient centrifugation. The number of IFNγ-producing spot forming cells (SFC) was determined by ELISpot as previously reported . Briefly, PBMC (2x105/100μl culture medium per well) were stimulated in duplicate for 24 h in 96-well plates (coated with anti-IFN-γ monoclonal capture antibody) with peptide pools (15mers, overlapping by 10 aminoacids, Pepscan, Lelystad The Netherlands) representative of the S and peptide pool (15 mers, overlapping 11 aminoacids; JPT Peptide Technologies GmbH, Germany) representative of Nucleocapsid (N) proteins, at the final concentration of 0.25 µg/ml. Phytoheamagglutinin (PHA; 5 µg/mL) was used as positive control, and medium alone as negative control. For the evaluation of cell-mediated response against the SARS-CoV-2 variants, supernatant of Vero E6 cells cultured in 25cm2 flasks and infected for 72 h with 100 TCID50/ml of different SARS-CoV-2 strains was UV-inactivated for 1 hour and used as antigen formulation. PBMC (4x105/100μl culture medium per well) were cultured in duplicate in the presence of inactivated SARS-CoV-2-infected (or mock-infected as negative control) cell supernatant diluted 1:10 in culture medium. Culture medium was RPMI 1640 supplemented with 2mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin, and 10% of heat inactivated fetal bovine serum (FBS, Euroclone). Spots were counted using an automated AID ELISPOT reader system (AutoImmun Diagnostika GmbH, Strasburg, Germany). The mean number of spots from duplicate cultures were adjusted to 1 x 106 PBMC. The net spots per million PBMC was calculated by subtracting the number of spots in response to negative control from the number of spots in response to the S or N antigen. Responses ≥10 net spots/million PBMC were considered positive based on background results obtained with negative control (mean SFC+2SD).
Characterization of CD4+, CD4+ follicular helper (TFH), and CD8+ T-cell proliferative response
To evaluate T-cell subsets proliferation, PBMC (600,000/200μl culture medium per well) collected from 19 vaccinated subjects, 9 subjects who experienced mild SARS-CoV-2 infection and 5 unexposed control subjects were stimulated in triplicate in 96-well round-bottom plates with peptide pools representative of the S and N proteins, at the final concentration of 0.1 µg/ml for 7 days. Peptide pool from human actin, was used as negative control antigen. Culture medium was RPMI 1640 supplemented with 2mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin, 5% of heat inactivated human serum AB, 1 mM Sodium Pyruvate, 100 μM non-essential amino acids and 50µM 2-Mercaptoethanol. After culture, cells were washed with PBS 0,5µM EDTA and stained in PBS with Live/Dead Fixable Violet Dye (Invitrogen) at 4°C. After washing, cells were stained at room temperature in PBS 5% FCS with anti-CXCR5, followed by anti-IgG2b (biotinylated) and, subsequently, with Streptavidin BV421, CD3 PerCP 5.5, CD4 APC Cy7, CD8 FITC, CD25 PECy7, CD278 (ICOS) APC antibodies. Finally, cells were washed and suspended in 1% paraformaldehyde. The frequency of CD25+ICOS+ expanded CD3+CD4+, CD3+CD4+CXCR5+ and CD3+CD8+ T-cells was determined by subtracting the frequency of PBMC incubated with actin peptides from the frequency of PBMC incubated with SARS-CoV-2 S and N peptides. Flow-cytometry analyses were performed with a FACS Canto II flow cytometer and DIVA software (BD Biosciences). A representative pseudocolor plot analysis is shown in Supplementary figure 2.
Antibody response was determined using the chemiluminescent assay Elecsys Anti-SARS-CoV-2 S (Roche Diagnostics Rotkreuz, Switzerland), which provides quantitative measures of mainly IgG (but also IgA and IgM) specific for SARS-CoV-2 RBD. Results are given as units (U)/ml and are considered positive when ≥0.8 U/ml. Neutralizing antibody serum titre was determined as previously reported . Results were considered positive if higher or equal to 1:10 serum titre.
Percentages with the 95% confidence interval (95%CI), median, range and interquartile range were reported. Levels of SARS-CoV-2-specific iCammunological parameters detected at T1 and T2 in naive subjects was performed with the Wilcoxon matched paired test, whereas levels of SARS-CoV-2-specific immunological parameters detected at T0, T1 and T2 in experienced subjects were compared with the Friedman test for repeated measures (with correction for multiple comparisons and Dunn’s post-test). Number of lymphocytes subpopulations at T0, T1 and T2 were given as mean number/µL ±SD and compared with ANOVA for repeated measures (with correction for multiple comparisons and Tuckey post-test). Two-group unpaired data were compared with the Mann-Whitney U-test. Spearman’s correlation (and 95%CI) between the different immunological assays, and between immune response and subjects age were calculated at each time point analyzed. All analyses will be performed using GraphPad 8.3.0