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Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library

View Article: PubMed Central - PubMed

ABSTRACT

Tetanus neurotoxin (TeNT) produced by Clostridiumtetani is one of the most poisonous protein substances. Neutralizing antibodies against TeNT can effectively prevent and cure toxicosis. Using purified Hc fragments of TeNT (TeNT-Hc) as an antigen, three specific neutralizing antibody clones recognizing different epitopes were selected from a human immune scFv antibody phage display library. The three antibodies (2-7G, 2-2D, and S-4-7H) can effectively inhibit the binding between TeNT-Hc and differentiated PC-12 cells in vitro. Moreover, 2-7G inhibited TeNT-Hc binding to the receptor via carbohydrate-binding sites of the W pocket while 2-2D and S-4-7H inhibited binding of the R pocket. Although no single mAb completely protected mice from the toxin, they could both prolong survival when challenged with 20 LD50s (50% of the lethal dose) of TeNT. When used together, the mAbs completely neutralized 1000 LD50s/mg Ab, indicating their high neutralizing potency in vivo. Antibodies recognizing different carbohydrate-binding pockets could have higher synergistic toxin neutralization activities than those that recognize the same pockets. These results could lead to further production of neutralizing antibody drugs against TeNT and indicate that using TeNT-Hc as an antigen for screening human antibodies for TeNT intoxication therapy from human immune antibody library was convenient and effective.

No MeSH data available.


Inhibition of TeNT-Hc binding to gangliosides by 2-7G, 2-2D, and S-4-7H antibodies (Ab). (A) The ability of Ab-mediated inhibition of TeNT-Hc binding to GM1a; (B) The capacity of Ab-mediated inhibition of TeNT-Hc binding to GD3; (C) The ability of Ab-mediated inhibition of the TeNT-Hc binding to GT1b. The dotted lines represented the half-maximal ELISA signal of TeNT-Hc binding to ganglioside.
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toxins-08-00266-f005: Inhibition of TeNT-Hc binding to gangliosides by 2-7G, 2-2D, and S-4-7H antibodies (Ab). (A) The ability of Ab-mediated inhibition of TeNT-Hc binding to GM1a; (B) The capacity of Ab-mediated inhibition of TeNT-Hc binding to GD3; (C) The ability of Ab-mediated inhibition of the TeNT-Hc binding to GT1b. The dotted lines represented the half-maximal ELISA signal of TeNT-Hc binding to ganglioside.

Mentions: For determining the Ab-mediated inhibition of the TeNT-Hc binding to the receptor-dependent carbohydrate-binding site, three different assays were performed. The ability of Ab-mediated inhibition of the TeNT-Hc binding to GT1b, GM1a, and GD3 were analyzed individually (Figure 5). It was found that, for GM1a, in the presence of 2-7G, 2-2D, and S-4-7H, the percentage binding of the TeNT-Hc was reduced to 7.3%, 98.3%, and 97.7%, respectively, when compared to the binding of TeNT-Hc in PBS (absent of Abs), which was regarded as 100% binding (Figure 5A). Thus, the binding inhibitory activity of 2-7G, 2-2D, and S-4-7H was 92.7%, 1.7%, and 2.3%, respectively. It was also shown that there was a 2-7G inhibition of TeNT-Hc binding to the receptor via the carbohydrate-binding sites of the W pocket in which the toxin binding domain of the 2-7G epitopes belong. For GD3, the binding inhibitory activity of 2-7G, 2-2D, and S-4-7H was 2.5%, 85.6%, and 88.1%, respectively. This suggested that the 2-2D and S-4-7H inhibited the binding of TeNT-Hc to the receptor via the carbohydrate-binding sites of the R pocket (Figure 5B). For GT1b, the binding inhibitory activity of 2-7G, 2-2D, S-4-7H, and a various combination of the three Abs ranged from 78.6% to 99.6%. The inhibitory activities for the combination of the Abs were higher for the Ab alone, and the greatest inhibitory activity was demonstrated in the group for the combination of the three Abs combined (Figure 5C). In order to facilitate the analysis of a concentration dependent inhibition of TeNT-Hc binding to gangliosides by antibodies, half-maximal binding of TeNT-Hc to gangliosides was calculated. The half-maximal binding of TeNT-Hc to gangliosides GM1a, GD3 and GT1b were 24.2 nM, 24.6 nM and 26.0 nM respectively (Figure 5A–C).


Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library
Inhibition of TeNT-Hc binding to gangliosides by 2-7G, 2-2D, and S-4-7H antibodies (Ab). (A) The ability of Ab-mediated inhibition of TeNT-Hc binding to GM1a; (B) The capacity of Ab-mediated inhibition of TeNT-Hc binding to GD3; (C) The ability of Ab-mediated inhibition of the TeNT-Hc binding to GT1b. The dotted lines represented the half-maximal ELISA signal of TeNT-Hc binding to ganglioside.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5037492&req=5

toxins-08-00266-f005: Inhibition of TeNT-Hc binding to gangliosides by 2-7G, 2-2D, and S-4-7H antibodies (Ab). (A) The ability of Ab-mediated inhibition of TeNT-Hc binding to GM1a; (B) The capacity of Ab-mediated inhibition of TeNT-Hc binding to GD3; (C) The ability of Ab-mediated inhibition of the TeNT-Hc binding to GT1b. The dotted lines represented the half-maximal ELISA signal of TeNT-Hc binding to ganglioside.
Mentions: For determining the Ab-mediated inhibition of the TeNT-Hc binding to the receptor-dependent carbohydrate-binding site, three different assays were performed. The ability of Ab-mediated inhibition of the TeNT-Hc binding to GT1b, GM1a, and GD3 were analyzed individually (Figure 5). It was found that, for GM1a, in the presence of 2-7G, 2-2D, and S-4-7H, the percentage binding of the TeNT-Hc was reduced to 7.3%, 98.3%, and 97.7%, respectively, when compared to the binding of TeNT-Hc in PBS (absent of Abs), which was regarded as 100% binding (Figure 5A). Thus, the binding inhibitory activity of 2-7G, 2-2D, and S-4-7H was 92.7%, 1.7%, and 2.3%, respectively. It was also shown that there was a 2-7G inhibition of TeNT-Hc binding to the receptor via the carbohydrate-binding sites of the W pocket in which the toxin binding domain of the 2-7G epitopes belong. For GD3, the binding inhibitory activity of 2-7G, 2-2D, and S-4-7H was 2.5%, 85.6%, and 88.1%, respectively. This suggested that the 2-2D and S-4-7H inhibited the binding of TeNT-Hc to the receptor via the carbohydrate-binding sites of the R pocket (Figure 5B). For GT1b, the binding inhibitory activity of 2-7G, 2-2D, S-4-7H, and a various combination of the three Abs ranged from 78.6% to 99.6%. The inhibitory activities for the combination of the Abs were higher for the Ab alone, and the greatest inhibitory activity was demonstrated in the group for the combination of the three Abs combined (Figure 5C). In order to facilitate the analysis of a concentration dependent inhibition of TeNT-Hc binding to gangliosides by antibodies, half-maximal binding of TeNT-Hc to gangliosides was calculated. The half-maximal binding of TeNT-Hc to gangliosides GM1a, GD3 and GT1b were 24.2 nM, 24.6 nM and 26.0 nM respectively (Figure 5A–C).

View Article: PubMed Central - PubMed

ABSTRACT

Tetanus neurotoxin (TeNT) produced by Clostridiumtetani is one of the most poisonous protein substances. Neutralizing antibodies against TeNT can effectively prevent and cure toxicosis. Using purified Hc fragments of TeNT (TeNT-Hc) as an antigen, three specific neutralizing antibody clones recognizing different epitopes were selected from a human immune scFv antibody phage display library. The three antibodies (2-7G, 2-2D, and S-4-7H) can effectively inhibit the binding between TeNT-Hc and differentiated PC-12 cells in vitro. Moreover, 2-7G inhibited TeNT-Hc binding to the receptor via carbohydrate-binding sites of the W pocket while 2-2D and S-4-7H inhibited binding of the R pocket. Although no single mAb completely protected mice from the toxin, they could both prolong survival when challenged with 20 LD50s (50% of the lethal dose) of TeNT. When used together, the mAbs completely neutralized 1000 LD50s/mg Ab, indicating their high neutralizing potency in vivo. Antibodies recognizing different carbohydrate-binding pockets could have higher synergistic toxin neutralization activities than those that recognize the same pockets. These results could lead to further production of neutralizing antibody drugs against TeNT and indicate that using TeNT-Hc as an antigen for screening human antibodies for TeNT intoxication therapy from human immune antibody library was convenient and effective.

No MeSH data available.