We have produced three
antitoxins consisting of the variable domains of camelid heavy chain-only
antibodies (VH H) by expressing the genes in the chloroplast of green algae. These
antitoxins accumulate as soluble
proteins capable of binding and neutralizing
botulinum neurotoxin. Furthermore, they accumulate at up to 5% total soluble
protein, sufficient expression to easily produce these
antitoxins at scale from algae. The genes for the three different
antitoxins were transformed into Chlamydomonas reinhardtii chloroplasts and their products purified from algae lysates and assayed for in vitro
biological activity using toxin protection assays. The produced antibody domains bind to
botulinum neurotoxin serotype A (
BoNT/A) with similar affinities as camelid
antibodies produced in Escherichia coli, and they are similarly able to protect primary rat neurons from intoxication by
BoNT/A. Furthermore, the camelid
antibodies were produced in algae without the use of solubilization tags commonly employed in E. coli. These camelid antibody domains are potent
antigen-
binding proteins and the heterodimer fusion
protein containing two VH H domains was capable of neutralizing
BoNT/A at near equimolar concentrations with the toxin. Intact antibody domains were detected in the gastrointestinal (GI) tract of mice treated orally with
antitoxin-producing microalgae. These findings support the use of orally delivered
antitoxins produced in green algae as a novel treatment for
botulism.