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Clin. Vaccine Immunol. doi:10.1128/CVI.00117-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Characterization of New Formalin Botulinum Neurotoxin Toxoids

Laboratory of Respiratory and Special Pathogens, Division of Bacterial, Parasitic and Allergenic Products, Office of Vaccines Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892

^* To whom correspondence should be addressed. Email: james.keller{at}fda.hhs.gov.

	Abstract

Antigenicity of several formalin-detoxified botulinum neurotoxin preparations was measured by inhibition and sandwich 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 ELISA analysis indicated that the in-house toxoids were two to three-fold 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 IgG titers were detected in animals that had received any of the in-house toxoids, with greater than 99% being IgG₁ and the remainder IgG₂. These immunized mice remained asymptomatic after being challenged with 50 – 1,000,000 LD₅₀ 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 did survive neurotoxin challenges with 2 LD₅₀ units, but died when challenged with 6 LD₅₀ 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 ELISA analysis 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.