Characterization of New Formalin-Detoxified Botulinum Neurotoxin Toxoids

Abstract

Antigenicities of several formalin-detoxified botulinum neurotoxin preparations were measured by inhibition and sandwich enzyme-linked immunosorbent assay (ELISA), and immunogenicity was studied in mice. The toxoids were derived primarily from the serotype A 150-kDa neurotoxin protein, while one toxoid was derived from the naturally occurring 900-kDa toxin-hemagglutinin complex. Antigenicity was severely compromised in two commercially available toxoids. A variety of new toxoids were synthesized in-house by optimizing formaldehyde reaction conditions. Three of the resulting toxoids were found to be antigenically identical to the native toxin, as measured by inhibition ELISA, in spite of showing a reduction of toxicity by more than 100,000-fold. Sandwich ELISAs indicated that the in-house toxoids were two- to threefold less antigenic than the neurotoxin compared to commercial toxoids, which were about 100-fold less antigenic. Mice were immunized twice, on day 0 and day 14. By day 28, relatively high toxin-specific immunoglobulin G (IgG) titers were detected in animals that had received any of the in-house toxoids, with greater than 99% being IgG1 and the remainder being IgG2. These immunized mice remained asymptomatic after being challenged with 50 to 1,000,000 50% lethal dose (LD(50)) units of the 900-kDa neurotoxin. In contrast, animals immunized with several different batches of commercially available toxoids did not develop measurable toxin-specific antibody titers. However, these mice survived neurotoxin challenges with 2 LD(50) units but died when challenged with 6 LD(50) units. Neutralizing titers measured from pools of sera generated with the in-house toxoid preparations ranged from 2.5 to 5 U/ml. In terms of predicting immunogenicity, inhibition ELISAs comparing each formalin toxoid to the parent toxin provided good insight for screening the new toxoids as well as for estimating their relative in vivo potencies. Inhibition ELISA data indicate that those toxoids that most closely resemble the native toxin are highly immunogenic and protective. The superior quality of these new toxoids makes them useful tools for continued use in ELISA development and for antitoxin production.