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A Novel Panel of Rabbit Monoclonal Antibodies and Their Diverse Applications Including Inhibition of Clostridium perfringens Epsilon Toxin Oligomerization - PubMed

  • ️Mon Jan 01 2018

A Novel Panel of Rabbit Monoclonal Antibodies and Their Diverse Applications Including Inhibition of Clostridium perfringens Epsilon Toxin Oligomerization

Jennifer R Linden et al. Antibodies (Basel). 2018.

Abstract

The pore-forming epsilon toxin (ETX) produced by Clostridium perfringens is among the most lethal bacterial toxins known. Sensitive antibody-based reagents are needed to detect toxin, distinguish mechanisms of cell death, and prevent ETX toxicity. Using B-cell immuno-panning and cloning techniques, seven ETX-specific monoclonal antibodies were generated from immunized rabbits. ETX specificity and sensitivity were evaluated via western blot, ELISA, immunocytochemistry (ICC), and flow cytometry. ETX-neutralizing function was evaluated both in vitro and in vivo. All antibodies recognized both purified ETX and epsilon protoxin via western blot with two capable of detecting the ETX-oligomer complex. Four antibodies detected ETX via ELISA and three detected ETX bound to cells via ICC or flow cytometry. Several antibodies prevented ETX-induced cell death by either preventing ETX binding or by blocking ETX oligomerization. Antibodies that blocked ETX oligomerization inhibited ETX endocytosis and cellular vacuolation. Importantly, one of the oligomerization-blocking antibodies was able to protect against ETX-induced death post-ETX exposure in vitro and in vivo. Here we describe the production of a panel of rabbit monoclonal anti-ETX antibodies and their use in various biological assays. Antibodies possessing differential specificity to ETX in particular conformations will aid in the mechanistic studies of ETX cytotoxicity, while those with ETX-neutralizing function may be useful in preventing ETX-mediated mortality.

Keywords: Clostridium perfringens; antibodies; epsilon protoxin; epsilon toxin; neutralizing; oligomerization; pore formation.

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Conflict of interest statement

The antibodies described in the publication are the subject of granted and pending patent applications. This work was funded in part through a sponsored research agreement from Biogen Inc. and employees of this company including E.C., G.A., A.D., T.W., and W.M. helped design, collect, and analyze data from B-cell panning until antibody production. In addition, these authors reviewed the final manuscript. Evaluation of antibody performance in various bio-assays were performed by Weill Cornell Medical College employees including J.R.L., K.T., S.S., P.W., S.H. and T.V.

Figures

Figure 1
Figure 1

Overview of Rabbit immunization and production of anti-ETX monoclonal antibodies. (A) Timeline for rabbit immunization with pETX and blood collection. (B) Overview of rabbit B-cell isolation and cloning procedure for production of anti-ETX monoclonal antibodies. For detailed information, please referee to the methods section.

Figure 2
Figure 2

Detection of pETX, ETX, and ETX-oligomer via western blot analysis. (A) 5 ng of ETX and pETX were loaded onto gels. To detect ETX-pore, whole-cell lysates from rMAL-CHO cells (CHO-ETX) were used. Whole-cell lysates from untreated rMAL-CHO cells (CHO-CT) were used as a negative control. Antibodies JL001.1 and JL006 only detected purified pETX and ETX. JL1001.2 and JL008 were able to detect pETX and ETX, as well as bound ETX monomer of treated rMAL-CHO cells. JL005 was able to detect pETX and ETX as well as the ETX-oligomer. JL004 was able to pETX, ETX, ETX monomer bound to rMAL-CHO cells, and the ETX-oligomer. JL004 was used to determine a dose response (B) and time course (C) for ETX pore formation. For the dose response curve, cells were treated for 30 min at indicated ETX doses. For the time course, cells were incubated with 50 nM of ETX for indicated time points. 0 min time point is untreated control. Results for time course are shown in duplicate. (D) Densitometry measurements for ETX pore formation over time. (E) Densitometry measurements for bound ETX monomer over time. (F) Densitometry measurements for total ETX (Pore + Monomer) detected over time. Results are mean ± SD.

Figure 3
Figure 3

Detection of ETX via indirect ELISA. (A) ELISA plates were incubated with indicated amounts of ETX and then probed with 1 μg/mL of indicated anti-ETX monoclonal antibody. Displayed results are adjusted for background absorbance. (B) ELISA plates were coated with indicated amounts of pETX and probed with 1 μg/mL of the specified anti-ETX monoclonal. (C) Alternatively, ELISA plates were coated with 10 nM pETX and probed with the specified concentrations of anti-ETX antibodies. Wells probed with peroxidase-conjugated anti-rabbit antibody only (2AB Only) were used as negative controls. Results are mean ± SD.

Figure 4
Figure 4

Detection of pETX via Sandwich ELISA. (A) To test possible capture antibodies, JL001.2 with the rabbit Fc regions (JL001.2-Rb) and JL004 with the rabbit Fc region (JL004-Rb) were coated on ELISA plates and incubated with 0.6 nM and 7.5 nM pETX. JL001.2 with the mouse Fc region (JL001.2-Ms) was used as the detection antibody. (B) Sensitivity of pETX detection using indirect ELISA (JL001.2-Rb) was compared to the sensitivity of the sandwich ELISA using JL004-Rb as capture antibody and JL001.2-Ms as the detection body. The sandwich ELISA was more sensitive than the indirect ELISA method. The limit of detection (LOD) for the sandwich ELISA (red-dotted line) was 2.65 pM and 13.68 pM for the indirect ELISA (black dotted line). Results are mean ± SD.

Figure 5
Figure 5

Evaluation of ETX detection via immunocytochemistry (ICC). (A) rMAL-CHO cells expressing MAL-GFP fusion protein (green) were treated with or without 50 nM ETX for 30 min. Cells were washed, fixed, blocked, and probed with anti-ETX antibodies. Antibody binding was detected using at Cy-3 conjugated anti-rabbit antibody (red) and visualized by fluorescent microscopy. ETX was detected using JL011.2, J002, and JL008 and colocalized with rMAL-GFP. ETX was not detected with JL001.1 or JL004, JL005, and JL006 (images not shown). (B) Relative ETX fluorescence detected on 50 nM ETX-treated rMAL-CHO cells with individual antibodies. JL001.1 was used as a negative control. Results are mean ± SD. ** p < 0.01 determined by one-way ANOVA with post-hoc Tukey HSD.

Figure 6
Figure 6

Evaluation of ETX detection via flow cytometry. rMAL-CHO cells were treated with ETX, trypsinized, fixed, blocked, and then probed with anti-ETX antibodies. Bound antibody was detected using a PE conjugated anti-rabbit antibody. (A) Histograms of ETX fluorescence for different antibodies. A non-specific polyclonal IgG isotype control (PC IgG) was used a negative control for affinity-purified polyclonal (AP204). A monoclonal IgG isotype control (MC IgG) was used as a negative control for monoclonal antibodies JL001.1, JL001.2, JL002, JL004, JL005, JL006, and JL008. Representative examples of experimental triplicates. (B) Representative dot blots of control and ETX-treated rMAL-CHO cells probed with JL008. (C) Percent of cells positive for ETX when probed with indicated antibodies. Control treated cells were compared to ETX-treated cells. Results are mean ± SD. * p < 0.002 determined by t-test.

Figure 7
Figure 7

In vitro neutralizing ability of anti-ETX antibodies against ETX. (A) Media containing 100 nM of ETX was pre-incubated with the indicated anti-ETX antibodies at 30μg/mL for 30 min prior to treatment of rMAL-CHO cells for 1 h. Media containing antibodies alone were used to evaluate possible antibody toxicity to rMAL-CHO cells (CT). Cells treated without antibodies (no AB) were used as controls. Cell death was evaluated by PI exclusion. Antibodies JL001.2, JL002, JL004, and JL008 prevented cell death similar to non-ETX-treated controls. None of the antibodies exhibited cytotoxic effects on rMAL-CHO cells. * p < 0.01 compared to No AB ETX, determined by t-test. (B) Media containing 100 nM of ETX were pretreated with indicated doses of anti-ETX antibodies before treating cells for 4 h and evaluating cell death by PI exclusion. Results are mean ± SD with a smooth-fit line.

Figure 8
Figure 8

Anti-ETX antibody inhibition of ETX binding and oligomerization in rMAL-CHO cells. (A) Alexa-647 conjugated epsilon protoxin (pETX-647) was pre-incubated with indicated anti-ETX antibodies prior to treatment of rMAL-CHO cells. Medium containing pETX-647 alone was used as a positive control for pETX binding (Pos. CT). Medium without pETX or antibodies was used as a negative control (Neg CT). Only antibody JL004 inhibited pETX-647 binding to rMAL-CHO cells. (B) Alexa-594 conjugated epsilon toxin (ETX-594) was pre-incubated with indicated anti-ETX antibodies prior to treatment of rMAL-CHO cells. Media containing ETX-594 alone was used as a positive control for ETX binding (Pos. CT). Media without ETX-594 or antibodies was used as a negative control (Neg CT). In the negative control cells, note that the majority of rMAL-GFP is located at the plasma membrane, especially in areas of cell-to-cell contact (white triangles). In comparison, cells treated with ETX-594 alone (Pos. CT) or JL001.1 show evidence of rMAL-GFP internalization, visualized as punctate dots (white arrows). Internalized ETX-594 also colocalizes with rMAL-GFP. No ETX-594 binding was observed in the presence of JL004. JL001.2, JL002, and JL008 inhibited ETX internalization, but still allowed ETX-594 binding to cells and colocalization with plasma membrane-associated rMAL-GFP (white asterisks). (C) Western blot analysis for ETX oligomerization in rMAL-CHO cells via detection of the ~150 kDA ETX-oligomer complex. ETX was pretreated with indicated antibodies before treating cells. rMAL-CHO cells treated with ETX only was used as a positive control for ETX oligomerization. ETX oligomerization was detected by probing membranes with JL004, an antibody that has been determined to detect the approximately 150 kDa ETX-oligomer (Figure 1). (D) Full-length western blots of rMAL-CHO cells treated with ETX and ETX pretreated with indicated antibodies. Bands observed at ~50 kDa are believed to be the heavy chains of the anti-ETX antibodies used for pretreatment. (E) ETX-induced vacuolation and formation of Rab7 positive late endosomes were evaluated in rMAL-CHO cells. Cells were treated with ETX for indicated time points and stained for Rab7. Note at later time points, vacuole membranes stain positive for Rab7+ (white arrows). (F) JL001.1, JL001.2, JL004, and JL008 were evaluated for their ability to inhibit cellular vacuolation in ETX-treated rMAL-CHO cells via live cell imaging. Media containing ETX were pretreated with indicated antibodies prior to cell treatment.

Figure 9
Figure 9

Post-exposure antibody treatment protects against ETX-induced cytotoxicity in vitro. rMAL-CHO cells were treated with 100 nM ETX for four (A) or 24 h (B). Cells were treated with ETX without antibodies as a positive control. Cells treated without ETX or antibodies were used as a negative control. Anti-ETX antibody JL004 or JL008 were added to cells treated with ETX post treatment at indicated time points; 0, 5, 10, 15, 20, or 30 min. Cell death was determined by PI exclusion. Percent cell death (% Cell Death) was determined by dividing the number of PI+ cells in experimental conditions by the number of PI+ cells when cells were treated with ETX alone. * p < 0.01 compared to ETX-treated controls determined by ANOVA with post-hoc Tukey HSD.

Figure 10
Figure 10

Anti-ETX antibody protects mice from ETX-induced central nervous system (CNS) symptoms in vivo. Mice were treated ETX via intraperitoneal injection. To determine if anti-ETX antibody JL008 could protect against ETX-induced CNS symptoms and death, mice were either treated with (A) JL008 30 min prior to ETX treatment (PRE-AB, n = 3) or (B) one hour post-treatment via intravenous injection (POST-AB, n = 3). Mice treated with ETX and intravenously injected with saline were used as controls (ETX, n = 4). Animal were observed for up to 360 min post-ETX treatment. Antibody treatment prior to and post-ETX treatment prevented ETX-induced CNS symptoms and death compared to ETX-treated control animals. The same control ETX group was used for both comparisons. * p < 0.02 determined by Log-Rank (Mantel-Cox) test.

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