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) and a receptor binding domain (HC). Currently, there are no approved vaccines against botulism. This study tests a recombinant, full-length BoNT/A1 versus LCHC/A1 and HC/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 >10-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. M-BoNT/A1 vaccination protected against challenge by 10 LD Units of native BoNT/A1, while M-BoNT/A1 or M-BoNT/A1 vaccination equally protected against challenge by native BoNT/A2, a BoNT subtype. Mice vaccinated with M-BoNT/A1 surviving BoNT challenge had dominant antibody responses to the LCHC domain, but varied antibody responses to HC. Sera from mice vaccinated with M-BoNT/A1 also neutralized BoNT/A1 action on cultured neuronal cells. The cell- and mouse-based assays measured different BoNT-neutralizing antibodies, where M-BoNT/A1 elicited a strong neutralizing response in both assays. Overall, M-BoNT/A1, 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.

Immunogenicity of a Bivalent Non-Purified Recombinant Vaccine against Botulism in Cattle

“…Although these are effective on conferring protection, toxoids are time-consuming to prepare and are potentially hazardous during detoxification [ 11 ]. Therefore, the development of recombinant subunit vaccines has been evaluated as a mean of solving these problems [ 12 , 16 , 19 , 22 , 23 ].…”

Characterization of immune response induced against catalytic domain of botulinum neurotoxin type E

“…Researchers have studied holotoxin that can be exploited as potential vaccine candidate to prevent intoxication. It involves several strategies like introducing point mutations in holotoxin to eliminate catalytic activity 28 . Another successful attempt done by Webb et al, by using inactive holotoxin and their data strongly suggests that the ciBoNT HP may offer a robust immunological response that elicits greater protective immunity, particularly when challenges are performed with dissimilar toxin subtypes 27 .…”

“…Some laboratories are involved in the development of recombinant protein vaccines using holotoxins, HCs and LCs. There have been several reports available elaborating the protective potential of holotoxin based vaccines against BoNTs 25,27,28 . Smith and co-workers reported an inactive BoNT/A1 holotoxin protein that provide greater protective potential upon 1,000 LD 50 toxin challenge 29 .…”

Engineering of Botulinum Neurotoxins for Biomedical Applications

“…An E. coli optimized ORF consisting of an amino-terminal 6X His tag, 2 HA epitopes, BoNT/A1 with LC neutralizing mutations E 224 A, R 363 A, Y 366 F and a carboxy terminal ST, denoted M-BoNT/A1, was expressed in a prokaryotic expression platform, purified via the affinity tags ( Table 1 ) [ 59 ]. An identical construct with additional RBD domain ganglioside binding pocket mutation W 1266 A, previously shown to impair binding/internalization of a recombinant RBD into cultured rat neuronal cells [ 60 ], was also produced. Both protein antigens were assessed to determine their ability to elicit protective immunity against a substantial challenge against the parental toxin, subtype /A2 or a cocktail of BoNT /A2 /A3 /A5 and /A6 subtypes.…”