Neutralization of Botulinum Neurotoxin Type E by a Humanized Antibody

Abstract

Botulinum neurotoxins (BoNTs) cause botulism and are the deadliest naturally-occurring substances known to humans. BoNTs have been classified as one of the category A agents by the Centers for Disease Control and Prevention, indicating their potential use as bioweapons. To counter bio-threat and naturally-occurring botulism cases, well-tolerated antibodies by humans that neutralize BoNTs are relevant. In our previous work, we showed the neutralizing potential of macaque (Macaca fascicularis)-derived scFv-Fc (scFv-Fc ELC18) by in vitro endopeptidase immunoassay and ex vivo mouse phrenic nerve-hemidiaphragm assay by targeting the light chain of the botulinum neurotoxin type E (BoNT/E). In the present study, we germline-humanized scFv-Fc ELC18 into a full IgG hu8ELC18 to increase its immunotolerance by humans. We demonstrated the protection and prophylaxis capacity of hu8ELC18 against BoNT/E in a mouse model. A concentration of 2.5 ng/mouse of hu8ELC18 protected against 5 mouse lethal dose (MLD) in a mouse protection assay and complete neutralization of 1 LD50 of pure BoNT/E toxin was achieved with 8 ng of hu8ELC18 in mouse paralysis assay. Furthermore, hu8ELC18 protected mice from 5 MLD if injected up to 14 days prior to intraperitoneal BoNT/E administration. This newly-developed humanized IgG is expected to have high tolerance in humans.

Keywords: antibody; botulinum neurotoxin type E; botulism.

Figure 1

Sequence of the macaque framework regions and those coded by the most similar human germline genes. Based on the physiochemical classes of the amino acids (AA), differences in the framework regions are classified into very similar AA (green), similar AA (blue), dissimilar AA (orange), and very dissimilar AA (red).

Figure 2

ELISA assay of all humanized ELC18 variants (from hu1ELC18 to hu8ELC18) and non-humanized ELC18 (No reactivity against BSA, not shown).

Figure 3

Neutralization activity of hu8ELC18 in mouse flaccid paralysis. Pure BoNT/E3 toxin (1.0 LD50 per dose) was pre-mixed with a range of antibody concentration (from 1.0 µg per dose). Antibody-toxin mixtures were left for 30 min at room temperature before injecting subcutaneously 0.1 mL (n = 4) into the left inguinocrural region of female, MF1 strain of mice. Animals were scored at 24 h post injection. Results are expressed as mean score of 4 mice ± SEM. The positive control group of mice (n = 4) was injected with 1 LD50 of BoNT/E3 toxin alone, and the negative control group (n = 2) were given 1.0 µg of antibody in the absence of toxin (data not shown).