Booster vaccination against tetanus and diphtheria: insufficient protection against diphtheria in young and elderly adults

87 healthy, elderly adult volunteers (median age 71 years, range 66–92 years; 47 females), who had received one dose of Repevax® (Sanofi Pasteur MSD) in 2005 [11], were vaccinated once more against tetanus, diphtheria and pertussis (Boostrix®, GlaxoSmithKline) 5 years later in 2010 [12]. Pre- and 28 days post-vaccination Ab concentrations of the 2010 vaccination were now compared with corresponding data from a group of 46 healthy, young adult volunteers (median age 29 years, range 24–40 years; 29 females) which was recruited in 2010 and received one booster vaccination against tetanus, diphtheria, pertussis and polio (Boostrix®-Polio, GlaxoSmithKline). Their previous booster shot was well documented and dated back more than 10 years. All vaccinations were performed in accordance with the official recommendations by the Austrian health authorities [5]. Young and old participants were recruited from the general population and all participants of the old cohort were community-dwelling. Persons with chronic viral infection (Human Immunodeficiency virus, Hepatitis B virus, Hepatitis C virus), transplant recipients and patients under immunosuppressive or chemotherapy were not included in the study. Routine laboratory parameters (liver and kidney function, blood count) were determined at the time point 2010 and all participants were shown to be in good health [12]. All participants were invited back 5 years later, in 2015, for the analysis of the level of protection against tetanus and diphtheria by measuring Abs as well as T cell function. Due to various reasons (such as unwillingness to participate, change of address, ill health, death or meeting one of the exclusion criteria), only 27 elderly and 17 young adults could be studied in 2015. Exclusion criteria were the same for both groups in 2010 and 2015, namely chronic viral infections (HIV, HBV, HCV), transplant reception, cancer as well as immunosuppressive therapy. Pregnant or lactating females were also excluded from the study. We checked whether the participants re-recruited in 2015 were representative for the original larger cohorts, and found that neither age nor sex were different. We also compared pre- and post-vaccination Ab concentrations measured in 2010 between the total cohort and the sub-cohorts available for further analysis in 2015 and did not find any statistically significant difference.

Heparinized blood was drawn from the arm vein and fractionized by density gradient centrifugation using Ficoll-Paque™ Plus (GE Healthcare) to collect plasma and PBMCs. PBMCs were used freshly and plasma was stored at −20 °C.

Microtiter plates were coated with 1 μg/ml tetanus or diphtheria toxoid (Statens Serum Institute) and blocked with 0.01 M Glycin. Plasma samples were tested in duplicates. Peroxidase-labeled rabbit anti-human IgG Ab (Chemicon/Millipore) was used as secondary Ab. IgG Abs were quantified in IU/ml using standard human anti-tetanus and anti-diphtheria sera (National Institute for Biological Standards and Control). The detection limit of the assays used was 0.01 IU/ml, and values below this concentration were set to 0.005 IU/ml for calculation of geometric mean concentrations (GMC). Ab concentrations above 0.1 IU/ml were considered as protective [3, 4].

Plasma samples were incubated for one hour with 0.1 M 2-mercaptoethanol in order to eliminate IgM Abs. After heat inactivation at 56 °C for 30 min serial dilutions ranging from 1:20 to 1:4800 were prepared using Dulbecco’s modified eagle’s medium (Sigma Aldrich) containing 2 % fetal calf serum (FCS; Sigma Aldrich) and 1 % Penicillin-Streptomycin (Sigma Aldrich). The pre-diluted samples were incubated with diphtheria toxin (8 ng/ml; Sigma Aldrich) for 90 min. This mixture was transferred into 96-well tissue culture plates seeded with 15.000 Vero cells 24 h earlier. After 48 h Vero cells were stained with crystal violet. Living cells were stained violet, indicating that the Abs were able to neutralize the diphtheria toxin. All incubations were performed at 37 °C and 5 % CO₂.

Cytokine production of antigen-specific CD4⁺ T cells was induced by stimulation of PBMCs with 10 μg/ml tetanus- or diphtheria-toxoid at 37 °C for 6 h with 10 μg/ml Brefeldin A added after the first hour of stimulation. PBMCs were washed with PBS and stained with Zombie Violet™ Fixable Viability dye (Biolegend) for 20 min at RT and washed with FACS buffer (PBS + 2 mM EDTA + 2 % FCS + NaN₃). Cells were stained with anti-CD3-BV510 (clone: UCHT1; BD Biosciences), anti-CD4-PE-Cy™7 (clone: SK3; BD Biosciences) and anti-CD45RO-PerCP-Cy™5.5 (clone: UCHL1; BD Biosciences) for 20 min at 4 °C. After washing with FACS buffer, cells were fixed and permeabilized using the BD Cytofix/Cytoperm™ kit (BD Biosciences) following manufacturer’s instructions. Cells were then stained with anti-IFN-γ-FITC (clone: B27; BD Biosciences), anti-TNF-α-PE (Mab11; BD Biosciences), anti-IL2-APC (clone: MQ1-17H12; BD Biosciences), anti-IL4-PE (clone: 8D4-8; BD Biosciences), anti-IL10-APC (clone: JES3-19 F1; Biolegend), anti-IL17-PE (clone: eBio64DEC17; eBioscience), anti-IL21-Alexa Fluor®647 (clone: 3A3-N2.1; BD Biosciences), anti-TGF-β1-PE (clone: TW4-9E7; BD Biosciences) and anti-GM-CSF- Alexa Fluor®647 (clone: BVD2-21C11; BD Biosciences) for 30 min in BD Cytoperm™ (BD Biosciences) at 4 °C. After washing with PBS, cells were measured using the FACS canto II cytometer (BD) and analyzed using FlowJo software (V 10.0.7.). CD4⁺ memory cells were gated as CD3⁺CD4⁺CD45RO⁺. The unstimulated controls were subtracted from the antigen-specific samples for each cytokine and donor.

Group wise comparisons for tetanus- and diphtheria-specific Abs as well as for antigen-specific cytokine-producing T cells were performed using the Wilcoxon test. The Wilcoxon-signed rank test was applied for paired data and the Wilcoxon rank-sum test for unpaired data.

The frequency of persons protected/unprotected against tetanus and diphtheria in the young and the elderly group was compared using the chi-square test.

Spearman rank correlations were applied to study relations between neutralizing capacity and total concentrations of diphtheria-specific Abs, Ab concentrations and the time since the last vaccination, as well as to study the relationship between Abs and cytokine production in T cells.

The level of significance for all tests was α = 0.05. Accordingly, the critical value for the chi-square test was x ² = 3.84.

SPSS version 23 (SPSS Inc., Chicago, USA) was used for all statistical analyzes.

The geometric mean concentrations (GMCs) of tetanus- and diphtheria-specific Abs at three different time points are shown in Fig. 1: Pre-vaccination Ab concentrations in 2010, post-vaccination Ab concentrations on day 28 in 2010 and 5 years later in 2015. For tetanus (Fig. 1a) Ab concentrations were generally high, well above the protective level, and were not different in the two age groups. For diphtheria (Fig. 1b), GMCs were around the protective level in both age groups before vaccination in 2010, but well in the protective range 28 days after vaccination. Five years later, expectedly Ab concentrations had decreased in both age groups, but were significantly higher in the young than in the elderly cohort. In 2015 Ab concentrations in the young group were also higher than the pre-vaccination values in 2010. In contrast, Ab concentrations in the old cohort were back to the pre-vaccination level in 2010 (Fig. 1b).

Corresponding to the high Ab concentrations against tetanus, 100 % of both age groups were protected against this antigen. A small proportion of elderly persons who had been unprotected before vaccination in 2010 were protected after the vaccination and were still protected 5 years later (Fig. 2a).

The situation was different for diphtheria (Fig. 2b). In both age groups, about half of the cohort did not have protective Ab concentrations before vaccination in 2010. Most participants from both age groups had protective Ab concentrations 4 weeks after vaccination, but a small number of persons (<10 %) from each age group remained unprotected. After 5 years, the percentage of unprotected persons had dropped in both age groups, with 54 % of the elderly group and 24 % of the young group being unprotected, which was a significant difference (Fig. 2b).

In view of the fact that Ab concentrations against diphtheria were quite low in some persons, we analyzed the functionality of diphtheria-specific Abs in young and older adults. We therefore measured the neutralizing capacity of diphtheria-specific Abs from 27 elderly and 17 young adults at three time points: immediately before and 28 days after vaccination (2010) and 5 years later (2015). The neutralizing capacity and diphtheria-specific Ab concentrations measured by ELISA were highly correlated (p <0.0001, r_s >0.821 in both age groups at all time points, Fig. 3).

Recent vaccination history was synchronized for the older cohort, as they had received tetanus and diphtheria vaccinations in the context of our studies in 2005 and 2010 [11, 12]. In contrast, the time since the last vaccination before the recruitment in 2010 varied considerably within the young group. Correlations between pre- and post-vaccination Ab concentrations in 2010 and the time since the last vaccination were therefore only analyzed in the young group (Fig. 4). For tetanus there was no correlation between Ab concentrations and the time point of the last vaccination (Fig. 4a). In contrast, for diphtheria, there was a significant correlation between Ab concentrations and the time since the last vaccination (Fig. 4b). This correlation was most pronounced for the Ab concentrations 28 days after vaccination, indicating that regular booster vaccinations against diphtheria are important not only for the maintenance of Ab levels, but also for the success of booster vaccinations.

In a previously published study on elderly adults [12] we found a weak correlation between IL-5-producing T cells measured by Elispot and diphtheria-specific Abs. In the present study, a detailed analysis of cytokine production by CD4⁺ memory cells was performed at the 2015 time point using flow cytometry. The production of 9 cytokines (IFN-γ, TNF-α, IL-2, IL-4, IL-10, IL-17, IL-21, TGF-β, GM-CSF) following in vitro stimulation of PBMCs with tetanus (Fig. 5a and c) and diphtheria (Fig. 5b and d) toxoid was analyzed, and was found to be similar in young (Fig. 5a and b) and elderly (Fig. 5c and d) adults. The production of more than one cytokine was detected in CD4⁺ memory cells of all donors. Tetanus-specific T cells of young and old donors produced 5.8 ± 1.2 (mean ± SD) and 5.4 ± 1.8 cytokines, respectively (n.s.; Wilcoxon rank-sum test). 4.2 ± 1.0 and 4.0 ± 1.4 cytokines were detected after stimulation with diphtheria toxoid in T cells of young and old donors, respectively (n.s.; Wilcoxon rank-sum test). The frequency of all antigen-specific cytokine-producing T cells was similar in both age groups for tetanus and diphtheria (n.s.; Wilcoxon rank-sum test). Correlations between Ab concentrations and cytokine production were also performed (Table 1). There was no correlation between tetanus Ab concentrations and cytokine production by tetanus-specific CD4⁺ memory T cells, whereas a weak correlation was found between diphtheria-specific Abs and diphtheria-specific IL-2-, IL-21- and GM-CSF-producing CD4⁺ memory T cells. All these correlations were performed with pooled data from both age groups (n = 44) due to the low sample sizes in each group. Results were similar when both age groups were analyzed separately, but did not reach statistical significance.