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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.

No MeSH data available.


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).
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toxins-08-00257-f001: 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).

Mentions: To compare ELC18 to human germline genes, we performed analysis using the IMGT/V-QUEST tool. For VH, the identified V, D, and J gene segments were IGHV4-28*01, IGHD2-21*01, and IGHJ3*01, respectively. For VL, the V and J gene segments were IGKV1-9*01 and IGKJ2*03, respectively. The GI for VH and VL were calculated using IMGT/DomainGapAlign and provided an indication of the identity between framework regions of ELC18 and those encoded by the most similar human germline genes, as a percentage. The GI-value for VH was 85.7% and for VL 89.9%. Furthermore, we located and evaluated the differences in sequence between ELC18 framework regions and those coded by the ELC18 most similar human germline genes. In total, 22 of the 180 residues of the eight framework regions differed between ELC18 and those of the selected human germline gene segments (Figure 1 and Table 1).


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

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

toxins-08-00257-f001: 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).
Mentions: To compare ELC18 to human germline genes, we performed analysis using the IMGT/V-QUEST tool. For VH, the identified V, D, and J gene segments were IGHV4-28*01, IGHD2-21*01, and IGHJ3*01, respectively. For VL, the V and J gene segments were IGKV1-9*01 and IGKJ2*03, respectively. The GI for VH and VL were calculated using IMGT/DomainGapAlign and provided an indication of the identity between framework regions of ELC18 and those encoded by the most similar human germline genes, as a percentage. The GI-value for VH was 85.7% and for VL 89.9%. Furthermore, we located and evaluated the differences in sequence between ELC18 framework regions and those coded by the ELC18 most similar human germline genes. In total, 22 of the 180 residues of the eight framework regions differed between ELC18 and those of the selected human germline gene segments (Figure 1 and Table 1).

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.