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Neutralization of Botulinum Neurotoxin Type E by a Humanized Antibody

View Article: PubMed Central - PubMed

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.

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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).
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toxins-08-00257-f003: 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).

Mentions: Neutralization activity of IgG hu8ELC18 was also assessed in vivo paralysis model with a range of concentrations of antibody (from 1.0 µg to 0.32 ng per dose) and pure BoNT/E3 toxin. In this assay, a sub-lethal dose of toxin was subcutaneously injected into the left inguinocrural region of mice to induce local flaccid paralysis. The results from in vivo neutralization in the mouse flaccid paralysis model against pure BoNT/E3 (1.0 LD50 per dose) recorded at 24 h post injection are represented in Figure 3. Complete neutralization of 1 LD50 of pure BoNT/E toxin was achieved with 8 ng of hu8ELC18, and partial neutralization with two further five-fold dilutions (1.6 and 0.32 ng). Animals injected with 1.0 µg of antibody in the absence of toxin did not show any sign of abnormality (data not shown). Mice injected with toxin alone, in the absence of antibody, developed visible abdominal ptosis at the site of injection, as a result of flaccid paralysis.


Neutralization of Botulinum Neurotoxin Type E by a Humanized Antibody
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).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5037483&req=5

toxins-08-00257-f003: 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).
Mentions: Neutralization activity of IgG hu8ELC18 was also assessed in vivo paralysis model with a range of concentrations of antibody (from 1.0 µg to 0.32 ng per dose) and pure BoNT/E3 toxin. In this assay, a sub-lethal dose of toxin was subcutaneously injected into the left inguinocrural region of mice to induce local flaccid paralysis. The results from in vivo neutralization in the mouse flaccid paralysis model against pure BoNT/E3 (1.0 LD50 per dose) recorded at 24 h post injection are represented in Figure 3. Complete neutralization of 1 LD50 of pure BoNT/E toxin was achieved with 8 ng of hu8ELC18, and partial neutralization with two further five-fold dilutions (1.6 and 0.32 ng). Animals injected with 1.0 µg of antibody in the absence of toxin did not show any sign of abnormality (data not shown). Mice injected with toxin alone, in the absence of antibody, developed visible abdominal ptosis at the site of injection, as a result of flaccid paralysis.

View Article: PubMed Central - PubMed

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.

No MeSH data available.


Related in: MedlinePlus