Definition of Human Epitopes Recognized in Tetanus Toxoid and Development of an Assay Strategy to Detect Ex Vivo Tetanus CD4+ T Cell Responses

Abstract

Despite widespread uses of tetanus toxoid (TT) as a vaccine, model antigen and protein carrier, TT epitopes have been poorly characterized. Herein we defined the human CD4+ T cell epitope repertoire by reevaluation of previously described epitopes and evaluation of those derived from prediction of HLA Class II binding. Forty-seven epitopes were identified following in vitro TT stimulation, with 28 epitopes accounting for 90% of the total response. Despite this diverse range of epitopes, individual responses were associated with only a few immunodominant epitopes, with each donor responding on average to 3 epitopes. For the top 14 epitopes, HLA restriction could be inferred based on HLA typing of the responding donors. HLA binding predictions re-identified the vast majority of known epitopes, and identified 24 additional novel epitopes. With these epitopes, we created a TT epitope pool, which allowed us to characterize TT responses directly ex vivo using a cytokine-independent Activation Induced Marker (AIM) assay. These TT responses were highly Th1 or Th2 polarized, which was dependent upon the original priming vaccine, either the cellular DTwP or acellular DTaP formulation. This polarization remained despite the original priming having occurred decades past and a recent booster immunization with a reduced acellular vaccine formulation. While TT responses following booster vaccination were not durably increased in magnitude, they were associated with a relative expansion of CD4+ effector memory T cells.

Fig 1. Response to tetanus toxoid is broad but characterized by a high degree of immunodominance.

(A) Epitopes ranked on the basis of magnitude of response. Dotted lines indicate the top 10 (65% of total response) and top 28 (90% of total response) epitopes. (B) Breadth of response indicated by proportion of donors who respond to the specified number of epitopes. (C) Epitopes plotted as a function of the response frequency. Black line indicates the 19 epitopes that are recognized by 10% or more.

Fig 2. Bioinformatics predictions efficiently identify the preponderance of the tetanus toxin response.

(A) Total SFC detected against known (black bar) and novel (grey bar) epitopes. (B) Percentage of the total response captured by the indicated percentile score from peptide binding predictions.

Fig 3. Equivalent levels of tetanus-specific T cell responses in recently boosted individuals as compared to non-recently boosted individuals.

Sum of T cell responses for all DTaP antigens for non-boosted donors (no boost) or donors boosted with Tdap 0.5–6 months ago (boost). Each individual data point represents one donor, median ± interquartile range is shown. ns: no significant difference by two-tailed Mann-Whitney test.

Fig 4. Differential polarization of T cell responses as a function of the original vaccine type used for childhood vaccination.

IFNγ and IL-5 responses were measured by dual color ELISPOT assays. Each data point represents the ratio of IFNγ/IL-5 responses of each positive individual peptide from all the reactive donors. Median ± interquartile range for donors originally primed with DTwP or DTaP vaccine is represented. Two-tailed Mann-Whitney test.

Fig 5. AIM assay detects tetanus-specific CD4⁺ T cells.

(A) Representative flow cytometry plots of CD25⁺OX40⁺ upregulation by CD4⁺ T cells in naïve (CD45RA⁺CCR7⁺; lower panel) and antigen-experienced memory (excluding naïve cells; upper panel) cells after stimulation with TT-megapool [TT (MP)], Tetanus Toxoid [TT (ag)], Dengue virus-megapool [DV (MP)], or PHA as a positive control. (B) Median CD25⁺OX40⁺ expression by CD4⁺ memory T cells after 24 hours. Each dot represents one donor originally primed with DTwP vaccine and not recently boosted. Kruskal-Wallis multiple comparison test, *, p<0.05. (C and D) Percentage IFNγ- (C) or IL-4-producing (D) CD4⁺ memory T cells in response to TT megapool, or PMA/Ion.

Fig 6. Similar levels of TT-specific CD4⁺ memory T cells after boosting as detected by the AIM assay.

CD25⁺OX40⁺ expression by CD4⁺ memory T cells after 24 hours of culture for donors originally primed with DTwP vaccine not boosted (no boost) or 0.5–6 months post Tdap boost (boost) after TT-megapool (A) or TT (B) stimulation. Each dot represents one donor. Median ± interquartile range is shown. ns: no significant difference by two-tailed Mann-Whitney test

Fig 7. AIM Assay detects tetanus-specific CD4⁺ effector memory T cell expansion.

(A) Representative flow cytometry plots of CD4⁺CD25⁺OX40⁺ T cells gated on CD45RA and CCR7 expression for central memory (CD45RA^- CCR7⁺), effector memory (CD45RA^- CCR7^-), naïve (CD45RA⁺ CCR7⁺), and TEMRA (CD45RA⁺ CCR7^-) cells after 24 hours of TT-megapool stimulation for a donor originally primed with DTwP vaccine and not boosted (no boost) or after Tdap booster vaccination (boost). The CD4⁺ cell response to TT-megapool (B) or TT whole protein (C) shows a significant increase after boosting of the effector memory population (p = 0.009 and p = 0.017 respectively) and concomitant decrease of the central memory population (p = 0.028 and p = 0.006 respectively). Two-tailed Mann-Whitney test. Median ± interquartile range is shown. Each dot represents one donor.