A conformational change of C fragment of tetanus neurotoxin reduces its ganglioside-binding activity but does not destroy its immunogenicity

Abstract

The C fragment of tetanus neurotoxin (TeNT-Hc) with different conformations was observed due to the four cysteine residues within it which could form different intramolecular disulfide bonds. In this study, we prepared and compared three types of monomeric TeNT-Hc with different conformational components: free sulfhydryls (50 kDa), bound sulfhydryls (44 kDa), and a mixture of the two conformational proteins (half 50 kDa and half 44 kDa). TeNT-Hc with bound sulfhydryls reduced its binding activity to ganglioside G(T1b) and neuronal PC-12 cells compared to what was seen for TeNT-Hc with free sulfhydryls. However, there was no significant difference among their immunogenicities in mice, including induction of antitetanus toxoid IgG titers, antibody types, and protective capacities against tetanus neurotoxin challenge. Our results showed that the conformational changes of TeNT-Hc resulting from disulfide bond formation reduced its ganglioside-binding activity but did not destroy its immunogenicity, and the protein still retained continuous B cell and T cell epitopes; that is, the presence of the ganglioside-binding site within TeNT-Hc may be not essential for the induction of a fully protective antitetanus response. TeNT-Hc with bound sulfhydryls may be developed into an ideal human vaccine with a lower potential for side effects.

Fig. 1.

SDS–PAGE of TeNT-Hc proteins separated under both reducing (A) and nonreducing (B) conditions. Under reducing conditions, all proteins run as a single band at approximately 50 kDa. Under nonreducing conditions, TeNT-Hc 1# runs as a single band at approximately 50 kDa, TeNT-Hc 2# runs at two bands at approximately 50 kDa and 44 kDa, and TeNT-Hc 3# runs as almost a single band at approximately 44 kDa. M, protein marker.

Fig. 2.

G_T1b binding ELISA. Wells of microtiter plates coated with G_T1b were preincubated with different conformations of TeNT-Hc. The binding activities of 20 μg/ml and 10 μg/ml TeNT-Hc 1#, 2#, and 3# to GT1b was detected by using a goat antitetanus toxoid polyclonal antibody and a rabbit polyclonal secondary antibody to goat. Each point is the average of three wells. The error bars represent ±SD. Student's t test was employed to assess the statistical significance of difference among different conformations of TeNT-Hc. A two-sided P value of <0.05 was considered statistically significant.

Fig. 3.

Binding activities of TeNT-Hc 1#, 2#, and 3# to PC-12 cells measured by FACS. TeNT-Hc 1#, 2#, and 3# proteins used in this assay were all prelabeled by Alexa Fluor 488 dye, and the unlabeled cells were used as a negative control.

Fig. 4.

Kinetics of IgG response in BALB/c mice following vaccination. BALB/c mice were vaccinated with either TeNT-Hc 1#, 2#, or 3# adsorbed to Al(OH)₃ adjuvant on weeks 0, 2, and 4 and bled from 2 weeks after each vaccination to 24 weeks. The total IgG titers were detected by ELISA. Each point represents the average of six mice. The group mean log₁₀ values of the reciprocal endpoint dilution endpoint titers are shown. The error bars represent ±SD. Student's t test was employed to assess the statistical significance of difference between two independent sets of data. Comparisons among the groups were made using the Mann-Whitney test. A two-sided P value of <0.05 was considered statistically significant.

Fig. 5.

Kinetics of IgG1 (A) and IgG2a (B) subclass concentrations and ratio of IgG1 to IgG2a (C) in serum samples collected from mice vaccinated by TeNT-Hc 1#, 2# or 3# at weeks 2, 4, 6, 10, 15, 18, and 24. The quantities of IgG1 and IgG2a were determined by ELISA using an IgG1 (mouse) EIA kit and an IgG2a (mouse) EIA kit, respectively. Each point represents the average of six mice. Student's t test was employed to assess the statistical significance of difference between two independent sets of data, and the paired t test was used to determine the group differences. Comparisons among the groups were also made using the Mann-Whitney test. A two-sided P value of <0.05 was considered statistically significant.

Fig. 6.

Efficacy of immunization with low doses of different conformations of TeNT-Hc against 50-LD₅₀ tetanus neurotoxin challenge in BALB/c mice. The normal-dose vaccines, which contained 20 μg/ml of purified TeNT-Hc 1#, 2#, or 3# protein and 1.5 mg/ml Al(OH)₃ gel adjuvant, were 120-fold diluted with normal saline and used to immunize the mice once at a volume of 0.5 ml. All the mice were challenged with 50 LD₅₀s of tetanus neurotoxin in 0.5 ml of normal saline 4 weeks after the vaccination. The animals that died within 10 days after challenge were recorded. Survival curves were compared using a log rank (Mantel-Cox) test. A two-sided P value of <0.05 was considered statistically significant.