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.

Do Memory CD4 T Cells Keep Their Cell-Type Programming: Plasticity versus Fate Commitment?

“…Human data support the same conclusion but the antigen-specific data are limited (Locci et al 2013;Bancroft et al 2016;Da Silva Antunes et al 2017). No longitudinal data on antigen-specific resting memory CD4 T-cell phenotypes from individual human donors are available at singlecell flow-cytometric resolution, which is much needed to demonstrate memory CD4 T-cell subset stability.…”

Th1/Th17 polarization persists following whole-cell pertussis vaccination despite repeated acellular boosters

“…Of 785 peptides, 16-mers overlapping by 8 residues, the top epitopes recognized by more than 5% of donors corresponding to 132 peptides were chosen (43). Epitopes from TT were also selected as recently described (79). A total of 122 previously defined epitopes of EBV/CMV were also used as a control for antigens not contained in the vaccine (84).…”

“…This corresponds to 10 mg/ml of each peptide in 100% DMSO, and a final concentration of 2.5 μl/ml in an assay corresponds to 0.05% DMSO. This approach has been used to develop megapools specific for Timothy grass, tuberculosis, dengue virus (DENV), pertussis, and tetanus (43,79,(85)(86)(87). All the peptides used in this study are listed in Supplemental Table 2.…”

Development and Validation of a Bordetella pertussis Whole-Genome Screening Strategy

“…Candidates for Deconvolution. In the next series of experiments, we evaluated the feasibility of a screening modality utilizing an ex vivo AIM assay [10][11][12] similar to the approach used in Figure 1(b). The results of AIM assays where PBMC from a representative donor out of 6 donors were stimulated with Figure 5(a).…”

“…In previous studies [9,10], we have investigated potential immune correlates of this waning immunity, by dissecting immune responses in young adults, originally primed with either aP or wP vaccines. These studies were enabled by the definition of the T cell epitopes contained in the BP antigens contained in the aP vaccines (pertussis toxin, PtTox; two serotypes of fimbriae, Fim2/3; filamentous hemagglutinin, FHA; and pertactin, PRN), which was achieved following the systematic analysis of responses following the expansion of antigen-specific T cells in short-term in vitro culture [9], and the development of the activation-induced marker (AIM) assays, which allowed measurement of responses directly ex vivo without any further manipulation [11,12].…”