Characterization of the antibody response to the receptor binding domain of botulinum neurotoxin serotypes A and E

Abstract

Clostridium botulinum neurotoxins (BoNTs) are the most toxic proteins for humans. The current clostridial-derived vaccines against BoNT intoxication have limitations including production and accessibility. Conditions were established to express the soluble receptor binding domain (heavy-chain receptor [HCR]) of BoNT serotypes A and E in Escherichia coli. Sera isolated from mice and rabbits immunized with recombinant HCR/A1 (rHCR/A1) from the classical type A-Hall strain (ATCC 3502) (BoNT/A1) and rHCR/E from BoNT serotype E Beluga (BoNT/E(B)) neutralized the homologous serotype of BoNT but displayed differences in cross-recognition and cross-protection. Enzyme-linked immunosorbent assay and Western blotting showed that alpha-rHCR/A1 recognized epitopes within the C terminus of the HCR/A and HCR/E, while alpha-rHCR/E recognized epitopes within the N terminus or interface between the N and C termini of the HCR proteins. alpha-rHCR/E(B) sera possessed detectable neutralizing capacity for BoNT/A1, while alpha-rHCR/A1 did not neutralize BoNT/E. rHCR/A was an effective immunogen against BoNT/A1 and the Kyoto F infant strain (BoNT/A2), but not BoNT serotype E Alaska (BoNT/E(A)), while rHCR/E(B) neutralized BoNT/E(A), and under hyperimmunization conditions protected against BoNT/A1 and BoNT/A2. The protection elicited by rHCR/A1 to BoNT/A1 and BoNT/A2 and by rHCR/E(B) to BoNT/E(A) indicate that immunization with receptor binding domains elicit protection within sub-serotypes of BoNT. The protection elicited by hyperimmunization with rHCR/E against BoNT/A suggests the presence of common neutralizing epitopes between the serotypes E and A. These results show that a receptor binding domain subunit vaccine protects against serotype variants of BoNTs.

FIG. 1.

Purification of recombinant HCR/A1. (A) BoNT/A1 is cleaved by Clostridial proteases into a dichain toxin that are linked by a disulfide bond. The N-terminal light chain encodes a zinc protease. The C-terminal heavy chain includes a translocation domain (HCT), and a C-terminal receptor binding domain which can be subdivided into an N-terminal (HCR_N) and C-terminal domain (denoted ΔA). (B) rHCR/A1 was purified from E. coli cell paste by a three-column strategy. The clarified extract was purified sequentially using nickel affinity, gel filtration and ion exchange chromatography. rHCR/A (5 μg) was separated by SDS-PAGE under reducing conditions and visualized by staining with silver.

FIG. 2.

Immunological characterization of rabbit sera against rHCR/A1 and rHCR/E_B. (A) ELISA of rabbit antisera to rHCR/A1 (left panel) or rHCR/E_B (right panel), using 100 ng rHCR/A1, 100 ng rHCR/A2, 100 ng rHCR/ΔA, 100 ng rHCR/E_B, and 100 ng rHCR/ΔE as capture antigens. (B) Antigens (500 ng) were separated by SDS-PAGE under reducing conditions and visualized by Western blotting with anti-rHCR/A1 sera (left panel) or anti-rHCR/E_B (right panel).

FIG. 3.

Immunological characterization of mouse sera against rHCR/A1 and rHCR/E_B. (A) ELISA of mouse antisera to rHCR/A1 (left panel) or rHCR/E_B (right panel), using 100 ng rHCR/A1, 100 ng rHCR/A2, 100 ng rHCR/ΔA, 100 ng rHCR/E_B, and 100 ng rHCR/ΔE as capture antigens. (B) Antigens (500 ng) were separated by SDS-PAGE under reducing conditions and visualized by Western blotting with anti-rHCR/A1 sera (left panel) or anti-rHCR/E_B (right panel).

FIG. 4.

Protein modeling of HCR/A2 (Kyoto F) and HCR/E (Beluga). (A) Using the structures of BoNT/A (pdb:3bta), BoNT/B (pdb:1epw), and tetanus HCR (pdb:1doh) as templates, the three-dimensional structures of HCR/A2 and HCR/E_B were generated using Swiss-Model. Ribbon diagrams of HCR/A1 (blue), HCR/A2 (red), and HCR/E_B (black) are displayed in the upper panels. Molecular surface electrostatic potentials of each protein were computed using the Coulomb method and are displayed in the lower panels (blue, positive charge; red, negative charge; white, neutral). The regions of lowest structural homology between HCR/A1, HCRA2, and HCR/E are circled and labeled 1 through 4. (B) Enlarged view of region 5 highlighting the primary residues contributing to the electrostatic surface of the molecule (left panel). Sequence alignment of the peptides forming this region are displayed on the right with conserved charge residues highlighted.