Subunit vaccine against the seven serotypes of botulism

Abstract

Botulinum neurotoxins (BoNTs) are the most toxic proteins for humans and are classified as category A toxins. There are seven serotypes of BoNTs defined by the lack of cross-serotype toxin neutralization. Thus, an effective vaccine must neutralize each BoNT serotype. BoNTs are organized as dichain A-B toxins, where the N-terminal domain (light chain) is a zinc metalloprotease targeting soluble NSF attachment receptor proteins that is linked to the C-terminal domain (heavy chain [HC]) by a disulfide bond. The HC comprises a translocation domain and a C-terminal receptor binding domain (HCR). HCRs of the seven serotypes of BoNTs (hepta-HCR) were engineered for expression in Escherichia coli, and each HCR was purified from E. coli lysates. Immunization of mice with the E. coli-derived hepta-serotype HCR vaccine elicited an antibody response to each of the seven BoNT HCRs and neutralized challenge by 10,000 50% lethal doses of each of the seven BoNT serotypes. A solid-phase assay showed that the anti-hepta-serotype HCR sera inhibited the binding of HCR serotypes A and B to the ganglioside GT1b, the first step in BoNT intoxication of neurons. This is the first E. coli-derived vaccine that effectively neutralizes each of the seven BoNT serotypes.

FIG. 1.

Structure-function properties of the BoNT. Ribbon (A) and line (B) diagrams of BoNT/A (Protein Data Bank entry no. 3BTA) are shown. BoNTs are organized into three domains: the N terminus, LC, encodes a zinc protease (red), and the C terminus, HC, encodes an HCT (yellow) and an HCR (blue). (C) Purification of HCR/A to HCR/G. Individual plasmids encoding HCR (serotypes A to G) were expressed as six-His-tag fusion proteins in E. coli. Proteins were purified by affinity- and size-exclusion chromatography. Five micrograms of each HCR was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gel was stained with Coomassie blue and is shown. To the left are the migrations of three molecular size markers (kDa).

FIG. 2.

Immunoreactivity of mouse anti-hepta-serotype HCR vaccine in mice. An ELISA was performed, using 100 ng each of the individual rHCRs (serotypes A to G). The plates were incubated for 1 h at 37°C with serial dilutions of sera from mice immunized with the hepta-serotype HCR vaccine obtained prior to BoNT challenge or with preimmunization sera. The plates were then incubated for 1 h at 37°C with goat anti-mouse IgG-HRP (1:12,000; Pierce), washed with PBS, and incubated with TMB as the substrate. Reactions were terminated with 0.2 M H₂SO₄, and absorbance at 450 nm (A450) was read. The serum titer represents the inverse of the serum dilution used in the analysis. Data presented are representative of two independent immunization challenge experiments.

FIG. 3.

Anti-hepta-serotype HCR blocks the binding of HCR/A and HCR/B to GT1b. Gangliosides (0.1 μg in 100 μl methanol) were added to individual wells of an ELISA and incubated overnight at RT. The plates were washed with PBS and blocked for 1 h at RT with 2% (wt/vol) BSA in binding buffer. The plates were then washed and incubated for 1 h at 37°C with the indicated HCR alone (▪), with preimmune sera (□), or with mouse anti-hepta-serotype HCR sera () in 1% (wt/vol) BSA in Tris-buffered saline. Following a washing step, the plates were incubated with an anti-3 FLAG M2 monoclonal IgG-HRP conjugate (1:12,000) in binding buffer. The plates were washed and then incubated with TMB as the substrate. Reactions were terminated with 0.2 M H₂SO₄. Absorbance at 450 nm (A450) was read on a Victor 3V plate reader.