Generation of High-Titer Neutralizing Antibodies against Botulinum Toxins A, B, and E by DNA Electrotransfer

Abstract

Botulinum neurotoxins are known to be among the most toxic known substances. They produce severe paralysis by preventing the release of acetylcholine at the neuromuscular junction. Thus, new strategies for efficient production of safe and effective anti-botulinum neurotoxin antisera have been a high priority. Here we describe the use of DNA electrotransfer into the skeletal muscle to enhance antiserum titers against botulinum toxin serotypes A, B, and E in mice. We treated animals with codon-optimized plasmid DNA encoding the nontoxic but highly immunogenic C-terminal heavy chain fragment of the toxin. By employing both codon optimization and the electrotransfer procedure, the immune response and corresponding neutralizing antiserum titers were markedly increased. The cellular localization of the antigen and the immunization regimens were also shown to increase neutralizing titers to >100 IU/ml. This study demonstrates that DNA electrotransfer is an effective procedure for raising neutralizing antiserum titers to remarkably high levels.

Efficacy of a Potential Trivalent Vaccine Based on Hc Fragments of Botulinum Toxins A, B, and E Produced in a Cell-Free Expression System

“…The slightly reduced ELISA titers induced by the trivalent vaccine were not reflected by reduced protection. Equal or reduced potencies have been measured when other multivalent Hc vaccines have been compared to their monovalent counterparts, depending on the nature of vaccine and mode of administration (38,48,56). Our results suggest that the multivalent HcA, HcB, and HcE vaccine protected mice to the same extent as the monovalent preparations.…”

Vaccines against botulism

“…Plasmid-based HC expression is enhanced with codon optimization for the host vaccinated [16][17][18][19][20][21]. A gene encoding a signal peptide was added to the N terminus of HC to promote HC secretion and enhance immune response [16,[18][19][20]. Jathoul et al found the human ubiquitin C (UbC) promoter elicited high expression of HC/F and subsequently high HC/F antibody titers, which protected against a challenge by 10,000 MLD 50 Units of BoNT/F [17] Plasmid-based vaccination may require multiple vaccinations to elicit protective antibody titers [20].…”

DNA electroporation in rabbits as a method for generation of high-titer neutralizing antisera

“…As already shown in ref. 18, animals treated with two electroporation of plasmid led to a neutralizing titer of 20 IU/ml. When a third immunization boost was performed, the total antibody titer increased from 2800 to 5500 if this third treatment was a DNA plasmid electroporation boost, and to 10 900 if it was a recombinant FcBoNT/A protein boost (1 μg in alum, intraperitoneally).…”

“…Neutralizing antisera are have been codon-optimized to fit the mouse usage of the genetic code and fused to the mouse erythropoietin secretion signal as described in ref. 18. In this previous study, we have shown that immunizing mice by electroporating these DNA plasmids into the skeletal tibialis anterior skeletal muscle led to generation of high titer total antibodies in mice against botulinum toxins types A, B, and E. The best neutralizing titer against type A toxin was 20 international units/ml (IU/ml) after two electroporation treatments, knowing that 1 IU corresponds to neutralization of 5000 mouse lethal dose (MLD).…”

“…All plasmid DNA electroporation in mice were performed using the electrical conditions usually used in our group and already described in ref. 18, i.e., eight square wave electric pulses of 200 V/cm and 20 ms duration at a frequency of 5 Hz, using plate electrodes. Neutralizing titers were determined by mouse protection assay at day 75.…”