High-Throughput IgG Epitope Mapping of Tetanus Neurotoxin: Implications for Immunotherapy and Vaccine Design

Abstract

Tetanus is an acute, fatal disease caused by exotoxins released from Clostridium tetani during infections. A protective humoral immune response can be induced by vaccinations with pediatric and booster combinatorial vaccines that contain inactivated tetanus neurotoxin (TeNT) as a major antigen. Although some epitopes in TeNT have been described using various approaches, a comprehensive list of its antigenic determinants that are involved with immunity has not been elucidated. To this end, a high-resolution analysis of the linear B-cell epitopes in TeNT was performed using antibodies generated in vaccinated children. Two hundred sixty-four peptides that cover the entire coding sequence of the TeNT protein were prepared in situ on a cellulose membrane through SPOT synthesis and probed with sera from children vaccinated (ChVS) with a triple DTP-vaccine to map continuous B-cell epitopes, which were further characterized and validated using immunoassays. Forty-four IgG epitopes were identified. Four (TT-215-218) were chemically synthesized as multiple antigen peptides (MAPs) and used in peptide ELISAs to screen post-pandemic DTP vaccinations. The assay displayed a high performance with high sensitivity (99.99%) and specificity (100%). The complete map of linear IgG epitopes induced by vaccination with inactivated TeNT highlights three key epitopes involved in the efficacy of the vaccine. Antibodies against epitope TT-8/G can block enzymatic activity, and those against epitopes TT-41/G and TT-43/G can interfere with TeNT binding to neuronal cell receptors. We further show that four of the epitopes identified can be employed in peptide ELISAs to assess vaccine coverage. Overall, the data suggest a set of select epitopes to engineer new, directed vaccines.

Keywords: B-cell linear epitopes; immunological diagnostic; peptide ELISA; tetanus neurotoxin.

Figure 1

Mapping of DTP vaccine induced IgG epitopes within TeNT. A library of 264, 15-mer peptides (listed in Table S1) with a 10 residue overlap and covering the coding sequence of TeNT (P04958; 1315aa) was prepared using SPOT synthesis. (A) Image of the chemiluminescence signal from the library after an incubation with a pooled of sera from children vaccinated with DTP (n = 15) followed by labeling with a goat anti-human IgG alkaline phosphatase-labeled secondary antibody. (B) Graph of the signal intensity normalized to 100% with the positive control and 0% with the negative control. Epitopes were identified within consecutive reactive peptides (Table 1).

Figure 2

Reactivity of serum from DTP vaccinated children against TeNT-MAP4 by an in-house ELISA. (A) Individual sera (n = 92) were tested through peptide ELISAs that utilized either Peptide 215 (MAP4-VPERYEFGTKPEDFN), 216 (MAP4-EYVPTFDNVIENTTS), 217 (MAP4-EKTLNDYKFQ FDSNG), or 218 (MAP4-GTVNTQFQYEYKIYS). The dashed lines represent the calculated cut-off values of each assay. The ROC analysis (B) showed that the sensitivity of the in-house peptide ELISAs were 76% for TeNT-215, 100% for TeNT-216, 100% for TeNT-217, and 100% for TeNT-218 (100%). All MAPs displayed 100% specificity except TeNT-218 (71%). Statistically significant (* p < 0.05; *** p < 0.001; **** p < 0.0001) and ns: not significant.

Figure 3

Reactivity of sera from vaccinated children (ChVS, n = 93), distributed into three different age groups, evaluated using a commercial ELISA kit. The activity of IgG antibodies is expressed in international units per mL (UI/mL). Cut-off (<0.1 UI/mL; red dashed line) was based on manufacturer’s manual according to standard curve and four-parameter logistic (4PL) regression.

Figure 4

Three-dimensional structure of TeNT (A) with the position of the 43 IgG epitopes identified through SPOT synthesis. Molecular model overlay of neutralizing epitopes within the TeNT protein. The image was constructed using PyMol based on the crystal structure of the neurotoxin (PDB: 1xdt). The catalytic (cyan), translocation (green), and receptor-binding (yellow) domains are depicted in cyan, green and yellow, respectively, with the epitopes in magenta.

Figure 5

The Hc fragment of tetanus toxin interacting with ganglioside GT1b analog (PDB: 1fv3). Identified epitopes TT-41/G and TT-43/G (orange) show close contact with ganglioside receptor analog of GT1b (N-acetyl-alpha-neuraminic acid-(2-3)-beta-D-galactopyranose-(1-3)-2-acetamido-2-deoxy-beta-D-galactopyranose-(1-4)-[N-acetyl-alpha-neuraminic acid-(2-8)-N-acetyl-beta-neurami nic acid-(2-3)]beta-D-galactopyranose-(1-4)-beta-D-glucopyranose) interacting with Asp 1221 and His 1271 residues.

Figure 6

List of the TeNT IEDB-deposited B-cell linear epitopes reanalyzed using SPOT synthesis. (A) Image of peptide reactivity using the same pool (n = 15) of ChVS used in the previous analysis. (B) The ten-epitope peptides were ordered with 15 residues, and those with fewer residues were synthesized with the addition of two Gly or GGAGG residues (shaded in gray) in the C-terminus until the complete 15 depositions were completed.

Figure 7

Identification of the subclasses of immunoglobulins that bind to TT-41/G (YPKDGNAFN NLDRIL) and TT-43/G (HNGQIGNDPNRDGGG) peptides through ELISA.