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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.


The inhibition of 2-7G, 2-2D, and S-4-7H IgG4 antibodies via the interaction between TeNT-Hc and NGF–treated PC-12 cells detected by a cellular immunofluorescence assay. (A)Phase contrast images of prefixed PC-12 cells; (B) Phase contrast images of NGF-treated PC-12 cells; (C) TeNT-Hc was incubated with prefixed PC-12 cells and detected by the mouse polyclonal anti-TeNT-Hc antibodies followed by FITC labeled anti-mouse IgG antibodies. The lighter fluorescence surrounding the cell membranes suggested that TeNT-Hc could bind to PC-12 cells; (D) Bovine serum albumin (BSA) was used instead of TeNT-Hc and detected by the mouse monoclonal anti-BSA antibodies, and no binding to PC-12 cells was observed; (E) The mouse polyclonal anti-TeNT-Hc antibodies were used as a positive control, which could effectively inhibit the interaction between PC-12 cells and TeNT-Hc in vitro; (F) 2-7G IgG4 antibodies were mixed with TeNT-Hc before incubating with the PC-12 cells, and no binding between TeNT-Hc and PC-12 cells was detected; (G) 2-2D IgG4 antibodies were mixed with TeNT-Hc before incubating with the PC-12 cells, and no binding between TeNT-Hc and PC-12 was observed; (H) S-4-7H IgG4 antibodies were mixed with TeNT-Hc before incubating with the PC-12 cells, and no binding between TeNT-Hc and PC-12 was found; (I) An anti–Yersinia pestis capsular F1 protein (F1) antibody was used as a negative control, which could not inhibit the interaction between TeNT-Hc and PC-12 cells; (J) An anti–Yersinia pestis capsular V protein (V) antibody was used as a negative control, which could not inhibit the interaction between the TeNT-Hc and PC-12 cells. The magnification of the cells is 200 times.
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toxins-08-00266-f004: The inhibition of 2-7G, 2-2D, and S-4-7H IgG4 antibodies via the interaction between TeNT-Hc and NGF–treated PC-12 cells detected by a cellular immunofluorescence assay. (A)Phase contrast images of prefixed PC-12 cells; (B) Phase contrast images of NGF-treated PC-12 cells; (C) TeNT-Hc was incubated with prefixed PC-12 cells and detected by the mouse polyclonal anti-TeNT-Hc antibodies followed by FITC labeled anti-mouse IgG antibodies. The lighter fluorescence surrounding the cell membranes suggested that TeNT-Hc could bind to PC-12 cells; (D) Bovine serum albumin (BSA) was used instead of TeNT-Hc and detected by the mouse monoclonal anti-BSA antibodies, and no binding to PC-12 cells was observed; (E) The mouse polyclonal anti-TeNT-Hc antibodies were used as a positive control, which could effectively inhibit the interaction between PC-12 cells and TeNT-Hc in vitro; (F) 2-7G IgG4 antibodies were mixed with TeNT-Hc before incubating with the PC-12 cells, and no binding between TeNT-Hc and PC-12 cells was detected; (G) 2-2D IgG4 antibodies were mixed with TeNT-Hc before incubating with the PC-12 cells, and no binding between TeNT-Hc and PC-12 was observed; (H) S-4-7H IgG4 antibodies were mixed with TeNT-Hc before incubating with the PC-12 cells, and no binding between TeNT-Hc and PC-12 was found; (I) An anti–Yersinia pestis capsular F1 protein (F1) antibody was used as a negative control, which could not inhibit the interaction between TeNT-Hc and PC-12 cells; (J) An anti–Yersinia pestis capsular V protein (V) antibody was used as a negative control, which could not inhibit the interaction between the TeNT-Hc and PC-12 cells. The magnification of the cells is 200 times.

Mentions: Tetanus neurotoxins can bind to various types of cells, including the NGF-treated neuronal-like PC-12 cells through the Hc domain. NGF can induce the differentiation of PC-12 cells which develop extended neuritis and an increased number of synaptic-like vesicles (Figure 4B). First, the binding ability of TeNT-Hc to PC-12 cells was determined by a cellular immunofluorescence assay, and the results showed that TeNT-Hc could bind to the membrane of PC-12 cells (Figure 4C), whereas BSA could not (Figure 4D). When antibodies were cultured with TeNT-Hc before being added to the PC12 cells, 2-7G, 2-2D, and S-4-7H could effectively inhibit TeNT-Hc binding to PC-12, similar to the mouse polyclonal anti-TeNT-Hc antibodies (Figure 4E–H). An anti–Yersinia pestis capsular F1 and V protein antibody could not inhibit TeNT-Hc binding to PC-12 (Figure 4I–J).


Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library
The inhibition of 2-7G, 2-2D, and S-4-7H IgG4 antibodies via the interaction between TeNT-Hc and NGF–treated PC-12 cells detected by a cellular immunofluorescence assay. (A)Phase contrast images of prefixed PC-12 cells; (B) Phase contrast images of NGF-treated PC-12 cells; (C) TeNT-Hc was incubated with prefixed PC-12 cells and detected by the mouse polyclonal anti-TeNT-Hc antibodies followed by FITC labeled anti-mouse IgG antibodies. The lighter fluorescence surrounding the cell membranes suggested that TeNT-Hc could bind to PC-12 cells; (D) Bovine serum albumin (BSA) was used instead of TeNT-Hc and detected by the mouse monoclonal anti-BSA antibodies, and no binding to PC-12 cells was observed; (E) The mouse polyclonal anti-TeNT-Hc antibodies were used as a positive control, which could effectively inhibit the interaction between PC-12 cells and TeNT-Hc in vitro; (F) 2-7G IgG4 antibodies were mixed with TeNT-Hc before incubating with the PC-12 cells, and no binding between TeNT-Hc and PC-12 cells was detected; (G) 2-2D IgG4 antibodies were mixed with TeNT-Hc before incubating with the PC-12 cells, and no binding between TeNT-Hc and PC-12 was observed; (H) S-4-7H IgG4 antibodies were mixed with TeNT-Hc before incubating with the PC-12 cells, and no binding between TeNT-Hc and PC-12 was found; (I) An anti–Yersinia pestis capsular F1 protein (F1) antibody was used as a negative control, which could not inhibit the interaction between TeNT-Hc and PC-12 cells; (J) An anti–Yersinia pestis capsular V protein (V) antibody was used as a negative control, which could not inhibit the interaction between the TeNT-Hc and PC-12 cells. The magnification of the cells is 200 times.
© Copyright Policy
Related In: Results  -  Collection

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

toxins-08-00266-f004: The inhibition of 2-7G, 2-2D, and S-4-7H IgG4 antibodies via the interaction between TeNT-Hc and NGF–treated PC-12 cells detected by a cellular immunofluorescence assay. (A)Phase contrast images of prefixed PC-12 cells; (B) Phase contrast images of NGF-treated PC-12 cells; (C) TeNT-Hc was incubated with prefixed PC-12 cells and detected by the mouse polyclonal anti-TeNT-Hc antibodies followed by FITC labeled anti-mouse IgG antibodies. The lighter fluorescence surrounding the cell membranes suggested that TeNT-Hc could bind to PC-12 cells; (D) Bovine serum albumin (BSA) was used instead of TeNT-Hc and detected by the mouse monoclonal anti-BSA antibodies, and no binding to PC-12 cells was observed; (E) The mouse polyclonal anti-TeNT-Hc antibodies were used as a positive control, which could effectively inhibit the interaction between PC-12 cells and TeNT-Hc in vitro; (F) 2-7G IgG4 antibodies were mixed with TeNT-Hc before incubating with the PC-12 cells, and no binding between TeNT-Hc and PC-12 cells was detected; (G) 2-2D IgG4 antibodies were mixed with TeNT-Hc before incubating with the PC-12 cells, and no binding between TeNT-Hc and PC-12 was observed; (H) S-4-7H IgG4 antibodies were mixed with TeNT-Hc before incubating with the PC-12 cells, and no binding between TeNT-Hc and PC-12 was found; (I) An anti–Yersinia pestis capsular F1 protein (F1) antibody was used as a negative control, which could not inhibit the interaction between TeNT-Hc and PC-12 cells; (J) An anti–Yersinia pestis capsular V protein (V) antibody was used as a negative control, which could not inhibit the interaction between the TeNT-Hc and PC-12 cells. The magnification of the cells is 200 times.
Mentions: Tetanus neurotoxins can bind to various types of cells, including the NGF-treated neuronal-like PC-12 cells through the Hc domain. NGF can induce the differentiation of PC-12 cells which develop extended neuritis and an increased number of synaptic-like vesicles (Figure 4B). First, the binding ability of TeNT-Hc to PC-12 cells was determined by a cellular immunofluorescence assay, and the results showed that TeNT-Hc could bind to the membrane of PC-12 cells (Figure 4C), whereas BSA could not (Figure 4D). When antibodies were cultured with TeNT-Hc before being added to the PC12 cells, 2-7G, 2-2D, and S-4-7H could effectively inhibit TeNT-Hc binding to PC-12, similar to the mouse polyclonal anti-TeNT-Hc antibodies (Figure 4E–H). An anti–Yersinia pestis capsular F1 and V protein antibody could not inhibit TeNT-Hc binding to PC-12 (Figure 4I–J).

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.