Enhancing toxin-based vaccines against botulism

Abstract

Botulinum neurotoxins (BoNT) are the most toxic proteins for humans. BoNTs are single chain proteins with an N-terminal light chain (LC) and a C-terminal heavy chain (HC). HC comprises a translocation domain (HC_N) and a receptor binding domain (HC_C). Currently, there are no approved vaccines against botulism. This study tests a recombinant, full-length BoNT/A1 versus LCHC_N/A1 and HC_C/A1 as vaccine candidates against botulism. Recombinant, full-length BoNT/A1 was detoxified by engineering 3-amino acid mutations (E224A/R363A/Y366F) (M-BoNT/A1) into the LC to eliminate catalytic activity, which reduced toxicity in a mouse model of botulism by >10⁶-fold relative to native BoNT/A1. As a second step to improve vaccine safety, an additional mutation (W1266A) was engineered in the ganglioside binding pocket, resulting in reduced receptor binding, to produce M-BoNT/A1^W. M-BoNT/A1^W vaccination protected against challenge by 10⁶ LD₅₀ Units of native BoNT/A1, while M-BoNT/A1 or M-BoNT/A1^W vaccination equally protected against challenge by native BoNT/A2, a BoNT subtype. Mice vaccinated with M-BoNT/A1^W surviving BoNT challenge had dominant antibody responses to the LCHC_N domain, but varied antibody responses to HC_C. Sera from mice vaccinated with M-BoNT/A1^W also neutralized BoNT/A1 action on cultured neuronal cells. The cell- and mouse-based assays measured different BoNT-neutralizing antibodies, where M-BoNT/A1^W elicited a strong neutralizing response in both assays. Overall, M-BoNT/A1^W, with defects in multiple toxin functions, elicits a potent immune response to BoNT/A challenge as a vaccine strategy against botulism and other toxin-mediated diseases.

Keywords: Botulinum neurotoxin; Botulinum neurotoxin A1; Botulinum neurotoxin A2; Botulism; ELISA; Vaccine.

Figure 1. Schematic of the recombinant proteins used as vaccines and/or antigens to assess the host immune response to vaccination

(Upper panel) BoNT-derivatives used in this study are shown. His₆ and Strep epitopes were used for protein purification, while 3X-FLAG and two sequential hemagglutinin, 2HA, epitopes were included for cellular studies. Domain junctions were defined, using the crystal structure of BoNT/A1 (PDB:3BTA). Single amino acid designations indicate amino acid substitutions used to reduce catalysis (LC) or receptor binding (HC_C). Note, single chain BoNT and LCHC_N were used for vaccination. (Lower panel) Four μg of the indicated proteins were subjected to SDS-PAGE and Coomassie blue staining. Lanes: 1, M-BoNT/A1; 2, M-BoNT/A1 trypsin nicked and reduced; 3, M-LCHC_N/A1; 4. M-LCHC_N/A1 trypsin nicked and reduced; 5, LC/A1^RY; 6, HC_C/A1^W; and 7, TeNT^RY. Migration of molecular weight marker proteins (kDa) are shown in left lane. Note, in lane 2 nicked HC runs at ~ 80 kDa, which other experiments showed was due to cleavage of the belt region of HC by trypsin.