Delayed Antibody and T-Cell Response to BNT162b2 Vaccination in the Elderly, Germany

We detected delayed and reduced antibody and T-cell responses after BNT162b2 vaccination in 71 elderly persons (median age 81 years) compared with 123 healthcare workers (median age 34 years) in Germany. These data emphasize that nonpharmaceutical interventions for coronavirus disease remain crucial and that additional immunizations for the elderly might become necessary.

CoV-2, weekly self-collected oropharyngeal swab specimens from participants were analyzed after enrollment. Blood sampling took place immediately before the first (week 0) and second (week 3) dose of vaccination with BNT162b2, as well as at week 7-8 (4 weeks after the second vaccination). Antibody and T cell data obtained from the 123 healthcare workers have been used as a control cohort in another study (D. Hillus et al., unpub. data, https://www.medrxiv.org/content/10.1101/2021.05.19.21257334v2).

Antibody Assessment
For detection of SARS-CoV-2-specific antibodies to the spike and nucleocapsid proteins, we used the SeraSpot Anti-SARS-CoV-2 IgG microarray-based multiparameter immunoassay according to manufacturer's instructions (Seramun Diagnostica GmbH, https://www.seramun.com). In brief, this assay is based on the use of 4 recombinant SARS-CoV-2 proteins (complete spike, S1 domain, receptor-binding domain [RBD], and nucleocapsid) as capture antigens. These and test-specific controls are printed in an array arrangement on the bottom of each well. Bound antibodies from the patient serum samples are detected by horseradish peroxidase-labeled antibodies against human IgG. Color intensity at the site of formed immune complexes (pale blue to dark blue) correlates with antibody concentration. The SpotSight plate scanner was used for measurements. Results are calculated and normalized as signal-to-cutoff (S/CO) ratios by dividing the observed signal strength of a specific location by that of an internal cutoff control. Samples with an S/CO ratio >1.0 are defined as positive by the manufacturer.

Surrogate SARS-CoV-2 Neutralization Test
To detect neutralizing activity in serum samples 3 weeks after the first vaccination and 4 weeks after the second vaccination, we used the commercially available ELISA-based SARS-CoV-2 surrogate neutralization test cPass (medac GmbH, https://international.medac.de) according to the manufacturer's instructions. Serum samples and positive and negative controls were diluted 1:10 in sample dilution buffer and preincubated 1:1 with RBD-horseradish peroxidase for 30 min at 37°C. Each reaction mixture was then added to the hACE2 precoated plate and incubated at 37°C for 15 min. After a washing step, 3′3,5,5-tetramethylbenzidine solution was added to each well and the plate was incubated at room temperature for 15 min.
Following a stop solution step, the optical density (OD) at 450 nm was detected. Data were interpreted by the calculation of the relative inhibition using the following equation: inhibition Page 3 of 7 [%] = (1-OD value of sample/OD value of negative control) × 100. Samples were considered negative at an inhibition of <30% and considered positive otherwise.

IgG Avidity Assay
Maturation of IgG avidity was detected in younger (n = 30) and elderly (n = 16) participants at week 3 and week 7 by using an anti-SARS-CoV-2 S1 IgG ELISA Kit index of more than 60% were considered to be high avidity.

Interferon-γ Release of SARS-CoV-2-Specific T Cells
We applied a commercially available IGRA for assessment of interferon-γ (IFN-γ) release of SARS-CoV-2-specific T cells (Euroimmun). In parallel, 0.5 mL freshly collected Lithium-heparin blood was stimulated with a SARS-CoV-2 peptide pool from the spike S1 domain, 0.5 mL of blood was stimulated with mitogen as a positive control, and 0.5 ml of blood in a blank was used as a negative control. After 24 hours of incubation at 37°C, IFN-γ concentration in the plasma fraction of all 3 stimulation tubes was measured by ELISA. IFN-γ response in the blank served as a measure of patient-individual background IFN-γ activity and was subtracted from the IFN-γ response in the stimulation tubes. For analyses of IGRA outcome, we defined an arbitrary cutoff by using average IFN-γ activity (33.42 mIU/mL) determined in the 15 SARS-CoV-2 IgG-negative unvaccinated control multiplied by 5 as the threshold (167.1 mIU/mL) for borderline IGRA-reactive and multiplied by 10 for the threshold (334.2 mIU/mL) for IGRA-positive.

Statistical Analysis
Values are given as medians with interquartile range unless stated otherwise. GraphPad PRISM statistics version 27.0 (IBM Deutschland, https://www.ibm.com/de-de) was used for statistical analysis. Group differences were assessed in a univariate analysis by using Fisher exact test or nonparametric Mann Whitney U test. P values of <0.05 were considered statistically significant. All 95% CI for proportions were calculated by using the Wilson procedure with a correction for continuity (1).

Appendix Table.
Proportion of positive outcome and outcome values in different test systems in study of delayed antibody and T-cell response to BNT162b2 vaccination in the elderly, Germany  *p value was calculated by Fisher exact test. ACE2, angiotensin-converting enzyme 2; IGRA, interferon-γ release assay; IU, international units; NP, nucleocapsid protein; RAI, relative avidity index; RBD, receptor-binding domain; S1, spike subdomain 1; S/CO, signal-to-cutoff ratio. †p value was calculated by the nonparametric Mann Whitney U test.