Study Design. Coronavirus disease-19 (COVID-19) Vaccination in Adolescents and Children (COVAC; NCT04800133) aimed at evaluating the humoral and cellular immunogenicity in children8. This study was approved by the University of Hong Kong (HKU)/ Hospital Authority Hong Kong West Cluster Institutional Review Board (UW21-157).
Participants. This study included the healthy adolescents aged 11–17 years and adults aged 18 years or older who received three doses of BNT162b2 intramuscularly. Potential participants with stably healthy conditions, known history of COVID-19, history of severe allergy, significant neuropsychiatric conditions, immunocompromised states were included. Transfusion of blood products within 60 days, haemophilia, pregnancy or breastfeeding were excluded from this study.
Procedures. Potential participants were recruited via school, media, or referral in Hong Kong. Study physicians contacted and obtained informed consent from participants aged 18 years or above, or for underage participants, from their parents and legally acceptable representatives.
S-RBD, surrogate virus neutralization assay (sVNT) and plaque reduction neutralization test (PRNT)
Peripheral clotted blood was drawn, and the serum was stored at -80℃ after separation. The SARS-CoV-2 S receptor-binding domain (R-SBD) IgG enzyme-linked immunosorbent assay (ELISA) and PRNT were carried out as previously described and validated8. sVNT was conducted according to the manufacturer’s instructions (GenScript Inc, Piscataway, USA) and as described in our previous publication. All sera were heat-inactivated at 56℃ for 30 mins before testing27,49. Details for the detections of S-RBD IgG, sVNT and PRNT were performed by the same methods showed in our previous study8. Briefly, S-RBD IgG ELISA plates were coated overnight with 100 ng/well of purified recombinant S-RBD in PBS buffer, followed by the incubation with 100 µL Chonblock Blocking/Sample Dilution (CBSD) ELISA buffer (Chondrex Inc, Redmond, USA) at room temperature (RT) for 2 hrs. Then added the 1:100 diluted serum in CBSD ELISA buffer to the wells and incubated at 37℃ for another 2 hrs. After washing with 0.1% Tween 20 PBS (PBST), the plates were incubated with 1: 5000 diluted horseradish peroxidase (HRP)-conjugated goat anti-human IgG (Thermo Fisher Scientific) at 37℃ for 1 h and washed with PBST for 5 times. Finally, 100 µL of HRP substrate (Ncm TMB one, New Cell & Molecular Biotech. Ltd. China) was added for 15 mins before stopped this reaction by 50 µL of 2 M H2SO4. The optical density (OD) was analyzed in a Sunrise absorbance microplate reader (Tecan, Männedorf, Switzerland) at 450 nm wavelength. Each OD reading was calculated by subtracting the background OD in PBS-coated control wells with the serum of participants. Values at or above an OD450 of 0.5 were considered positive, otherwise were imputed as 0.25.
For sVNT detection, 10 µL of serum were diluted at 1:10 and incubated with an equal volume HRP conjugated to the WT SARS-CoV-2 S-RBD (6ng) at 37℃ for 30 mins, followed by the addition of 100 µL of each sample to each well of microtitre plates coated with angiotensin-converting enzyme-2 (ACE-2) receptor at 37℃ for 15 mins. After washing and drying, 100 µL of 3,3’,5,5’-tetramethylbenzidine (TMB) was added and incubated at RT far away from light for 15 mins. Finally, the reaction was terminated, and the absorbance was read at 450 nm in a microplate reader. After confirmation that the positive and negative controls provided the recommended OD450 values, the % inhibition of each serum was calculated as (1 - sample OD value/negative control OD value) ×100%. Inhibition (%) of at least 30%, the limit of quantification (LOQ), was regarded as positive, while values below 30% were imputed as 15%.
The PRNT assay was performed in duplicate under a facility with biosafety level 3 as described before8. In brief, serum was diluted from 1:10 to 1:320, and then incubated with BetaCoV/Hong Kong/VM20001061/2020 (WT strain), hCoV-19/Hong Kong/ VM21044713_WHP5047-S5/2021 (Omicron BA.1), hCoV-19/Hong Kong/VM22000135_HKUVOC0588P2/2022 (Omicron BA.2), or SARS-CoV-2/human/USA/COR-22-063113/2022 (Omicron BA.5) at 30 plaque-forming units in a culture plate (Techno Plastic Products AG, Trasadingen. Switzerland) at 37℃ for 1h. Then the virus-serum mixtures were added onto Vero E6 TMPRESS2 cell monolayers and further incubated at 37℃ for 1h. The plates were overlaid with 1% agarose in cell culture medium and incubated for 3 days. After fixing and staining, antibody titres were defined as the reciprocal of the highest serum dilution that resulted in > = 90% (PRNT90, a more stringent cut-off) or > 50% (PRNT50) reduction in the number of plaques. Values below the lowest dilution tested of 10 were imputed as 5.
S IgG, avidity and FcγRIIIa-binding
Detections of S IgG, avidity and FcγRIIIa-binding were carried out as previously described8. Briefly, proteins were diluted in PBS for specific antibody detection. Firstly, Plates (Nunc MaxiSorp., Thermofisher Scientific) were coated with 250 ng/mL WT (AcroBiosystems) or Omicron BA.1 (AcroBiosystems) or Omicron BA.2 (AcroBiosystems) SARS-CoV-2 S protein for IgG and IgG avidity detections, or 500 ng/mL WT (Sinobiological) or Omicron BA.1 (AcroBiosystems) S for FcγRIIIa-binding detections, or 300 ng/mL ORF8 (Masashi Mori, Ishiwaka University, Japan) at 37℃ for 2 hrs25,50. The detection of ORF8 specific IgG was used to exclude infected individuals.
For IgG detection, plates were blocked with 1% FBS in PBS for 1 h before incubated with heat-inactivated (HI) serum, which was 1:100 diluted in 0.05% Tween-20/0.1% FBS in PBS at RT for 2 hrs. For antibody avidity, plates were washed three times with 8M Urea before incubated with IgG-HRP (1:5000, G8-185, BD) for 2 hrs. HRP was revealed by stabilized hydrogen peroxide and tetramethylbenzidine (R&D systems) for 20 mins, then stopped with 2N H2SO4 and analyzed with an absorbance microplate reader at 450 nm wavelength (Tecan Life Sciences). For FcγRIIIa-binding measurement, plates were coated with 500 ng/mL S protein and incubated with 1:50 diluted HI serum at 37℃ for 1 h before incubated with 100 ng/mL biotinylated FcγRIIIa-V158 at 37℃ for 1 h, followed by the detection of S specific FcγRIIIa-V158-binding antibodies by using streptavidin-HRP (1:10000, Pierce).
T cell responses
Peripheral blood mononuclear cells (PBMCs) were isolated from the whole blood of participants by density gradient separation and stored in liquid nitrogen before use. Firstly, PBMCs were thawed in RPMI medium supplemented with 10% human AB serum, then rested in a 37℃ incubator for 2 hrs. The cells were stimulated with 1 µg/mL overlapping peptide pools representing the WT SARS-CoV-S proteins (Miltenyi Biotec, Bergisch Gladbach, Germany), or B.1.1.529/BA.1 S mutation pool (Miltenyi Biotec, Bergisch Gladbach, Germany) and WT reference pool (Miltenyi Biotec, Bergisch Gladbach, Germany), supplemented with 1 µg/mL anti-CD28 and anti-CD49d costimulatory antibodies (Clones CD28.2 and 9F10, respectively, Biolegend, San Diego, USA) at 37℃ for 16 hrs. An equal volume of sterile double-distilled water (ddH2O) was used as a negative control. This mixture was stimulated for 2 hrs, followed by the addition of Brefeldin A (BFA,10 µg/mL; Sigma, Kawasaki, Japan) 51. Secondly, the cells were washed and immunostained with a fixable viability dye (eBioscience, Santa Clara, USA, 1:60), and antibodies against-CD3 (HIT3a, 1:60), CD4 (OKT4, 1:60), CD8 (HIT8a, 1:60), followed by fixed, permeabilized and stained with antibodies against IFN-γ (B27, 1:15) and IL-2 (MQ1-17H12, 1:15). All of these antibodies were purchased from Biolegend. Finally, data acquisition was carried out using flow cytometry (LSR II, BD Biosciences, Franklin Lakes, USA) and analyzed by FlowJo v10 software (BD, Ashland, USA). Antigen-specific IFN-γ+ and IL-2+ T cell results were finalized after subtracting the background (ddH2O) data and presented as the percentage of CD4+ or CD8+ T cells44. T cell responses against a single peptide pool were considered positive when the frequency of cytokine-expressing cells was higher than 0.005% and the stimulation index was higher than 2, while negative values were imputed as 0.0025%.
Outcomes. Humoral immunogenicity (S IgG and S-RBD IgG levels, sVNT %inhibition, 90% and 50% PRNT titres, S IgG avidity and FcγRIIIa-binding) and cellular immunogenicity markers (S-, Omicron S mutation- and Omicron WT reference- specific IFN-γ+ and IL-2+ CD4+ and CD8+ T cell responses) assessed after the third dose of BNT162b2.
Statistical analyses.
Sample size
As the study was conducted during the Omicron BA.2 wave in Hong Kong, participants who were infected were excluded and some participants defaulted vaccination or follow-up clinic. All evaluable samples were tested by S-RBD IgG and sVNT, and sample sizes for more demanding assays, e.g. PRNT and T cell testing, were reduced based on laboratory capacity.
Analysis sets
The primary analysis of humoral and cellular immunogenicity outcomes was performed in the healthy adolescents and adults in the evaluable analysis population who received intramuscular injection of BNT162b2 vaccine on a per-protocol basis as described before8. All of these evaluable population remained uninfected during study visits based on self-reporting, ORF8 IgG negativity and negative baseline S-RBD IgG, had no major protocol deviations. Each immunogenicity outcome was calculated by GM, GM ratios (GMRs) were reported with a two-sided 95% CI, corresponding to a one-sided 97.5% CI, to test the non-inferiority hypothesis at the margin of 0.60. Non-inferiority analyses were further confirmed in the expanded analysis population. When both non-inferiority and inferiority were not met, the results were inconclusive. Participants with valid results at consecutive timepoints were compared by GM fold rise (GMFR). Immunogenicity outcomes data below the cut-off were imputed with half the cut-off value. Immunogenicity outcomes were analysed by an unpaired or paired t test after natural logarithmic transformation. The proportion of participants with a positive results was reported in percent with 95% CI derived from Clopper-Pearson method. The comparisons of proportions between groups were performed with the Fisher exact test.
Vaccine efficacy estimates
Neutralizing antibody titres were used to estimate the vaccine efficacies as a secondary objective as described before8,19. Briefly, the mean neutralizing levels (fold of convalescent) were derived by dividing the GMTs of PRNT50 for WT, Omicron BA.2 or BA.5 SARS-CoV-2 in healthy evaluable adolescents with that of 102 convalescent sera collected on days 28–59 post-onset of illness in patients aged ≥ 18 years. Point estimates of VE were extrapolated by the best fit of the logistic model generated from the online plot digitizer tool (https://automeris.io/WebPlotDigitizer/, version 4.5).