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Caspase Activation as a Versatile Assay Platform for Detection of Cytotoxic 1

Bacterial Toxins 2

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Angela M. Payne*, Julie Zorman, Melanie Horton, Sheri Dubey, Jan terMeulen, 4

Kalpit A Vora* 5

Vaccines Basic Research, Merck Research Laboratories 6

West Point, PA 19486 7

Running Title: Caspase induction for detection of bacterial toxins 8

9

*Corresponding Authors 10

Address: Merck and Co., Inc. 11

770 Sumneytown Pike 12

P.O. Box 4 13

West Point, PA 19486 14

Phone: 215-652-8892 15

FAX: 215-652-2142 16

e-mail: angela_payne2@merck.com 17

e-mail: kalpit.vora@merck.com 18

19

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Copyright © 2013, American Society for Microbiology. All Rights Reserved.J. Clin. Microbiol. doi:10.1128/JCM.01161-13 JCM Accepts, published online ahead of print on 3 July 2013

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Abstract 21

Pathogenic bacteria produce several virulence factors that help them establish infection in permissive 22

hosts. Bacterial toxins are a major class of virulence factors and hence are attractive therapeutic targets 23

for vaccine development. Here we describe the development of a rapid sensitive and high-throughput 24

assay that could be used as a versatile platform to measure the activity of bacterial toxins. We have 25

exploited the ability of toxins to cause cell death via apoptosis of sensitive cultured cell lines as a read 26

out to measure toxin activity. Caspases are induced early in the apoptotic pathway and hence we used 27

their induction to measure the activity of Clostridium difficile toxins A (TcdA), B (TcdB), binary (CDTa-28

CDTb), Corynebacterium diphtheria (DT) and Pseudomonas aeruginosa exotoxin A (PEA). The caspase 29

induction in cell lines, upon exposure to toxins, was optimized for toxin concentration and intoxication 30

time, and the specificity of caspase activity was established using genetically mutated toxin and use of a 31

pan-caspase inhibitor. In addition, we demonstrate the utility of the assay to measure toxin potency as 32

well as neutralizing antibody (NAb) activity against C. difficile toxins. Furthermore, the caspase assay 33

showed excellent correlation with the F-actin polymerization assay to measure TcdA and TcdB 34

neutralization titers upon vaccination of hamsters. These results demonstrate that the detection of 35

caspase induction due to toxin exposure using a chemiluminescence readout would be able to support 36

potency and clinical immunogenicity testing for bacterial toxin vaccine candidates in development. 37

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Introduction 39

Microorganisms cause pathogenesis by means of a wide variety of molecules called virulence factors. A 40

large number of divergent microbial pathogens synthesize toxins recognized as primary virulence factors 41

which affect the metabolism and cause damage to eukaryotic cells, many times with lethal effects to the 42

host (1, 2). Major symptoms associated with diseases such as diphtheria, whooping cough, cholera, 43

anthrax and dysentery, are all related to activities of toxins produced by bacteria. In recognizing the 44

central role of toxins in these and other diseases, bacterial toxins have become attractive targets for the 45

development of vaccines (1, 3). Bacterial toxins affect susceptible host cells by a variety of modes of 46

action: damage of cell membranes, inhibition of protein synthesis, activation of immune response 47

leading to cellular damage, resulting in direct cell lysis, and facilitating bacterial spread through tissues 48

(4). Organisms such as C. difficile, C. diphtheriae and P. aeruginosa, secrete toxins involved in different 49

ways in the pathogenesis of disease. C. difficile toxins, for example, cause cellular toxicity through 50

glucosylation of Rho G-protein, and ADP-ribosylation of actin, while C. diphtheriae and P. aeruginosa 51

toxins catalyze the transfer of ADP-ribose to elongation factor 2 to block host cell protein synthesis, 52

leading to target cell death(5-8). The clostridial toxin TcdB of C. difficile inactivates the small GTPases 53

Rho, Rac and Cdc42, which has been shown to trigger cell death via apoptosis (2, 9-11). 54

Apoptosis is a fundamental feature of all animal cells, and is essential for normal development and 55

tissue homeostasis, whereas unregulated apoptosis can create an imbalance in the normal cell 56

proliferation processes (4, 7). Apoptosis is characterized by the presence of distinct morphological and 57

biochemical features (12). Morphologically, it can be characterized by DNA fragmentation, membrane 58

blebbing, cell rounding, cytoskeletal collapse and the formation of membrane-bound apoptotic vesicles 59

that are rapidly eliminated by phagocytosis (13). Biochemical features of apoptotic cell death include 60

the activation of a family of intracellular cysteine endopeptidases known as caspases, that specifically 61

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cleave target proteins at a cysteine amino acid that follows an aspartic acid residue (14, 15). Caspases 62

are synthesized as inactive pro-enzymes, which are converted into active heterodimers by proteolytic 63

cleavage, and are responsible for the deliberate disassembly of the cells into apoptotic bodies(16). Their 64

activation indicates progression of the pathway of cellular apoptosis. The initiator caspases 8 and 9, and 65

the executioner caspase 3, are positioned at crucial junctions in the apoptosis pathways. The activation 66

of the initiator caspases, in response to extracellular cytotoxic agents, activates the executioner caspase 67

3, resulting in a series of events leading eventually to cell lysis and disruption of the normal cell 68

processes (8, 12, 16-18). 69

Bacterial toxins can activate the apoptotic pathways and hence, caspases are molecules of particular 70

interest in assay development as potential indicators of apoptosis due to cell exposure to toxins. A 71

number of cultured cell lines undergo apoptosis when exposed to various cytotoxic signals from 72

pathogens or other sources. Caspase activation occurs early in the programed cell death pathway, and 73

thus allows for early detection from exposure to these toxins. Measurements of caspase activation due 74

to bacterial toxin exposure, or its inhibition, may be used as potency or release tests in vaccine 75

development. The ability to inhibit toxin-induced caspase activation in vitro, by animal and human 76

serum neutralizing antibodies is valuable in evaluation of vaccine efficacy. Conventionally, cell 77

susceptibility to bacterial toxins and neutralizing antibody responses in vitro rely on radioactive 78

cytotoxicity measurements for protein synthesis, or are evaluated by microscopic observation of 79

intoxicated cell monolayers. These methods may be subjective, time consuming and inherently low 80

throughput, due to its requirement for manual observation and counting of cells (5, 7, 9, 19-22). 81

Because of these limitations, we sought to develop an alternative assay which may be used as a versatile 82

platform for measuring bacterial toxin activity, and to evaluate the immunogenicity of toxin-based 83

vaccines. Here we describe that cytotoxic activity of several unrelated bacterial toxins can be easily and 84

reliably quantified by measuring in-cell caspase activation. The assay is sensitive and makes use of 85

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multi-well plates and automated reagent handling systems, allowing high throughput quantification of 86

cellular apoptosis due to toxin. 87

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MATERIALS AND METHODS 89

Bacterial Toxins: Native C. difficle toxins, VPI ribotype 087 (TcdA from VPI10463, product number 01A01 90

and TcdB from VPI10463, product number 01A02) were purchased from tgcBIOMICS GmbH (Mainz, 91

Germany) and stored at 4oC lyophilized and -70oC after reconstitution in pyrogen-free sterile water, with 92

limited (≤3 times) freeze-thawing. The TcdA and TcdB toxins were mutated as described in the 93

literature. These mutations included W101A, D287A and W519A for TcdA and W102A, D288A and 94

W520Afor TcdB (23-25). These point mutations are demonstrated to destroy the enzyme activity 95

(glucosylase) and substrate binding of the toxin. The muted toxins were expressed in Baculovirus and 96

purified from cell lysates using Ceramic Hydroxyapatite Type II (Biorad, Hercules, CA) column 97

chromatography, and stored at -70oC. Recombinant, his-tagged C. difficile binary CDTa-CDTb toxins 98

were expressed in E.coli and purified from E.coli lysates by affinity chromatography, then buffer 99

exchanged into 50mM Hepes, pH 7.5, 150mM NaCL, and stored at -70oC. Pseudomonas aeruginosa 100

Exotoxin A (PEA) and Corynebacterium diphtheria toxin (DT) were purchased from EMD Millipore 101

Corporation (Billerica, MA), re-constituted in sterile water to 1mg/ml, and stored at 4oC according to 102

vendor’s recommendation. CRM197, the mutated form of DT, produced from a single missense 103

mutation (Gly52 to Glu) within the fragment A region(26-28), was purchased from MBL International 104

Corporation (Woburn, MA), in solution of 1mg/ml, and stored at -70oC. All toxins had ≥90% purity as 105

indicated by accompanying literature of purchased toxins, and SDS analysis of internally produced 106

material (data not shown). 107

Cell lines and cell culture: Vero cells (African green monkey kidney) were obtained from the American 108

Type Culture Collection (ATCC, Manassas, VA) and cultured in Dulbecco’s minimum essential medium 109

(DMEM) supplemented with 10% heat-inactivated fetal bovine sera (FBS) per ATCC procedures. HeLa 110

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cells (human carcinoma epithelial) were obtained from ATCC and cultured in Eagle’s minimum essential 111

medium (EMEM) supplemented with 10% heat inactivated FBS per ATCC instructions. 112

Caspase inhibition reagents: Z-VAD-FMK, a cell-permeant pan caspase inhibitor that irreversibly binds to 113

the catalytic site of caspase proteases and inhibits induction of apoptosis (Promega, Madison, WI), was 114

re-constituted in DMSO at 20mM, stored at -20oC, and used in the assay at a final concentration of 115

20µM for inhibition of caspase, per vendor’s recommendation. Sera from hamsters immunized with 116

inactivated TcdA and TcdB toxins, as previously described (29) were used for C. difficile toxin 117

neutralization assays. 118

Caspase 3/7 Assay Reagent: The Caspase-Glo 3/7 Assay reagent (Promega, Madison, WI) was used for 119

caspase detection in treated cells in vitro. The reagent provides a proluminescent caspase-3/7 120

substrate, which contains the tetrapeptide sequence DEVD, in combination with luciferase and a cell 121

lysing agent. The addition of the Caspase-Glo® 3/7 reagent, directly, to the assay well results in cell lysis, 122

followed by caspase cleavage of the DEVD substrate, and generation of luminescence. The resulting 123

luminescence read out is proportional to the amount of caspase activity in the sample. 124

Caspase Assay optimization and Toxin evaluation: The caspase assay was optimized for several 125

parameters which included toxin concentrations, cell seeding density, and sera-toxin pre-incubation 126

time for C. difficile toxins TcdA, TcdB, binary CDTa-CDTb, and DT and PA. Vero cells were used to assess 127

caspase activation by TcdA, TcdB, and binary CDTa-CDTb. HeLa cells were used as target cells for DT and 128

PA. Varying cell input experiments (data not shown) lead to the selection of a seeding density of 129

2.5X104cells/well in a total volume of 100µl/well of black, glass bottom (Thermo Scientific, Rochester, 130

NY) 96-well tissue culture plates, or 50µl cell suspension at 3X104 cells/ml in 384-well plate. Cells were 131

incubated at 37oC, 5% CO2, and allowed to grow to ~90% confluence. The following day, cell 132

supernatants were replaced by toxins diluted in tissue culture medium at a different range of 133

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concentrations for each toxin (as shown in Figure 1), and incubated overnight at 37oC, 5% CO2. Caspase-134

Glo reagent was added directly to individual wells at 30μl/well in 96-well plates (15 μl/well in 384-well 135

plates), mixed gently on an orbital shaker for about 30 seconds, and further incubated at 37oC, 5% CO2 136

for 30-60 minutes prior to reading. Luminescence measurements were obtained using Perkin Elmer’s 137

2030 Multilabel Reader Victor 4X plate reader. The toxin concentrations used for the time course 138

studies were EC90 and were 20 ng/ml (TcdA), 80pg/ml (TcdB), 1µg/ml (DT) and 10µg/ml PAE. In these 139

time course experiments, the Caspase –Glo reagent was added as described above at various times after 140

intoxication, depending on toxin and cells being evaluated, ranging from 2-48 hours, to determine 141

optimum time for toxin exposure and caspase activation. To demonstrate the specificity of the toxin-142

induced caspase activation, the genetically modified toxins were tested alongside the corresponding 143

active toxins at the same concentrations, or the pan-caspase inhibitor (Z-Val-ALa-Asp- fmk [Z-VAD-fmk]) 144

was added to the active toxin wells at 20µM. 145

F-actin polymerization assay: Detection of F-actin polymerization was employed as a means to 146

correlate results to the Caspase activation assay data. The F-actin assay was previously described in 147

detail by Xie et al (29). Briefly, Vero cell suspensions were added to 384-well plates, and incubated 148

overnight at 37oC, 5% CO2. Following incubation, supernatants were replaced by toxin pre-incubated 149

with or without neutralizing serum and plates returned to the incubator for an additional 48 hours (see 150

below for toxin concentrations used in our assay comparisons). Wells were stained following a series of 151

centrifugations, washes, and incubations, as described in Xie et al (29) to enable detection and imaging 152

of F-actin polymerization in the treated cells using a scanning cytometer (Molecular Device, Sunnyvale, 153

CA). 154

Toxin neutralization assays: EC90 concentrations for TcdA and TcdB, (4ng/ml TcdA and 40pg/ml TcdB) 155

were used for demonstrating neutralization of toxins by pre-incubation of toxins with sera from animals 156

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previously hyper-immunized with toxins as described earlier (29). For both caspase and F-actin 157

polymerization assays, Vero cells were seeded at 3X104cells/ml in 50μl/well of a 384-well plate, and 158

incubated at 37oC, 5% CO2 for 24hr. The following day, supernatants were replaced by pre-incubated 159

toxins with 1:2 serially diluted immune sera, and cells were further incubated for 24hr for the caspase 160

assay, or 48hr for the F-actin assay. The cytometric F-actin neutralization assay was conducted over a 161

period of four consecutive days, and the data acquisition for the assay involves an image of the 162

monolayer acquired using the scanning cytometer. The caspase assay was conducted over a period of 163

three consecutive days, and data acquisition involves the luminescence measurements obtained from 164

the addition of caspase substrate, using a luminescence–capable plate reader. 165

The EC50 value was calculated by four parameter regression fitting of the titration curve using GraphPad 166

Prism 5 for Windows computer software, version 5.04, for both the Caspase-Glo and the cytometric 167

assays. 168

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RESULTS 172

Toxin-induced caspase activation over a range of toxin concentrations was demonstrated in Vero cells 173

for C. difficile toxins (TcdA, TcdB, and binary CDTa and CDTb), and in HeLa cells for C. diphtheria and P. 174

aeruginosa toxins as shown in Figure 1. The average ED50 of 5 replicate wells across 2 assays for TcdA 175

and TcdB were calculated to be approximately 1.26ng/mL and 12.5pg/mL respectively. The average ED50 176

for binary CDTa-CDTb was 3.0ng/ml. The average ED50 of 5 replicate wells across 2 assays for DT and PA 177

were found to be approximately, 0.0026µg/ml and 0.42µg/ml, respectively (Figure 1). 178

To further optimize the assay we determined the time course of caspase activation upon toxin exposure. 179

Appropriate cell monolayers (seeded at 2.5X104cells/well, 24 hours prior) were treated with individual 180

toxin at EC90 concentrations shown to produce high caspase activation signal (80pg/ml of TcdB, 20ng/ml 181

of TcdA, 1µg/ml of DT and 10µg/ml PA). Caspase levels were assessed at various time intervals ranging 182

from 2-48 hours post intoxication. Preliminary time courses were conducted at fewer, shorter 183

incubation times (2, 4, 5, 7h for DT and PA, and 4, 8, 16 h for TcdA and TcdB, data not shown). Results 184

showed that levels of caspase activation increased in a time-dependent manner and the kinetics varied 185

for each toxin (Figure 2). TcdA and TcdB showed significant increase at 16hr, peaking at 24hr, 186

decreasing at 28hr, and considerable loss of activity by 48hr. DT-induced caspase activation was seen as 187

early as 4hr, with peak levels at 6hr, and decreasing thereafter with a significant drop of activity by 16hr. 188

PEA-induced caspase activation peaked around 10hr, noticeably decreased by 14hr, followed by almost 189

a complete loss of activity at 24hr. 190

To test the specificity of the toxins’ ability to induce caspase, we assayed genetically inactivated toxins 191

(Figure 3). Toxin mutagenesis has been described in peer-reviewed literature as a means to decrease or 192

inactivate bacterial toxins, and thus enable their use in development of vaccines. Previous studies have 193

shown that, introduction of specific mutations in bacterial toxin genes lead to inactivation or decrease of 194

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toxicity (23, 27, 30, 31). To test the specificity of the toxins ability to induce caspase, we assayed 195

genetically altered toxins. All mutant-toxins were evaluated using the same titrations as their 196

corresponding active toxins, and none were assayed for nuclease activity. Genetically inactivated TcdA 197

and TcdB were unable to induce caspase activity at concentrations where the wild type toxins clearly 198

demonstrated caspase induction (Figure 3A, 3B). Similar data were also obtained with CRM197, a 199

mutant DT (Figure 3C). Furthermore, we were able to inhibit the caspase signal induced by the four wild 200

type toxins in the assay by the addition of a caspase inhibitor at all concentrations of toxin tested (Figure 201

3A-D). These data demonstrate the specificity of caspase induction by individual toxins. 202

We next compared the caspase assay performance with an established cytometric method (29) to 203

evaluate cellular toxicity caused by the toxins. Both assays were conducted to assess the ability of 204

serum antibodies induced by immunization with TcdA and TcdB toxoids to neutralize the pro-apoptotic 205

activity of the wild type toxins. Pre-incubation of TcdA or TcdB toxins with serial dilutions of 206

hyperimmune hamster sera (as described in Materials and Methods section) protected the cells from 207

toxin-induced cellular toxicity, as measured by inhibition of caspase activation (Figure 4A), and inhibition 208

of cell rounding via F-actin depolymerization (data not shown). The assays were conducted head to 209

head using identical conditions for both in terms of cell numbers, toxin concentrations, serum dilutions 210

and pre-incubation of toxins with immune-sera. Following incubation of sera and toxins on the cells, 211

development of the plates and data acquisition were conducted using parameters optimized for each 212

individual assay. The study evaluated toxin neutralization from sera collected from 20 individual animals 213

previously immunized with inactivated toxins. Both methods used 384 well plates, and toxin 214

concentration previously determined (29) to cause 90% of cytotoxicity in the well. Comparable 215

neutralization titers were obtained by both methods with R2 value of 0.807 for anti-TcdA titers and 216

0.904 for anti-TcdB titers (Figure 4B). 217

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DISCUSSION: 218

Bacterial toxins are attractive targets for drug and vaccine development. Hence, several 219

methods have been reported to assess their functional activity on eukaryotic cells in vitro. 220

Moreover, functional assays are increasingly used in determining the type of immune response 221

induced by toxins, quality control of toxins as vaccine antigens, and as potency release tests in 222

toxin-based vaccine development. This has led to an increased demand for high throughput cell 223

based assays with decreased cycle times. Several assays reported in the literature rely on visual 224

observations of cytopathic effects of toxins e.g. rounding of cells or loss of viability (5, 7, 9, 19-225

22). Recently, an elegant high throughput assay has been reported that can quantitate actin 226

polymerization to measure the effects of C. difficile toxins TcdA and TcdB on Vero cells(29). 227

Other alternative methods available for quantifying toxins biological (enzymatic) activity within 228

a cell are often cumbersome, low throughput, and require costly instruments for acquiring 229

measurements related to the effects of toxin on eukaryotic cells. There are several reports in the 230

literature linking C. difficile toxins to apoptosis and activation of caspases. Guerrant et al have 231

shown caspase 3 and 9 induction by western blotting in human intestinal epithelial cell line T84 232

with TcdA exposure (32). Similar data has been reported for TcdB in HeLa cells by Qa’dan et al 233

(10). Moreover, Hippensteil et al have shown that TcdA and TcdB mediated rho-inactivation 234

leads to caspase induction and apoptosis(33). Since other bacterial toxins are known to cause 235

death of the target cells via apoptosis, we postulated that measuring caspase activation could 236

not only be used to detect the cytotoxic effects of a majority of bacterial toxins, but also 237

developed into a simple luminescent assay allowing for an earlier readout and higher 238

throughput compared to standard cell-based assays which rely on quantifying morphological 239

changes. 240

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The cell lines were chosen for their sensitivity to the respective toxin and for historical reasons. 241

Vero cells have been shown to be very sensitive to C. difficile toxins and have been used in the 242

published F-actin polymerization assay (19, 29, 33, 34). Since we were comparing the caspase 243

assay to the F-actin polymerization assay, Vero cells were selected for our caspase studies with 244

TcdA and TcdB. Similarly, diphtheria toxin and Pseudomonas enterotoxins were historically 245

tested on HeLa cells in our institution, and the use of HeLa cells for studying caspase activation 246

and thus, apoptosis, have been reported in the published literature (12, 27, 35, 36). 247

Additionally, these cell lines provided the sensitivity to carry out the assay rapidly and reliably, 248

leading to their selection as preferred cell substrates. Ultimately, the choice of cell lines will 249

depend on the individual toxin source and user needs. We were able to observe caspase 250

induction by TcdA, TcdB, and CDTa-CDTb in Vero cells, and by DT and PEA in HeLa cells. 251

Interestingly, the kinetics of the caspase induction varied with the type of toxins used. DT was 252

able to demonstrate peak caspase induction upon intoxication within 6h, followed by PEA at 253

10h. TcdA and TcdB were found to have slower kinetics (peak around 20h) of caspase induction. 254

This differential kinetics could reflect on their modes of action. That is, DT and PEA act on 255

elongation factor thus, shutting down protein synthesis, producing early induction of caspases. 256

In contrast, C. difficile toxins are known to interfere with actin polymerization, which results in 257

detachment and rounding, and may require more time to induce cell death. Hence, it was 258

notably important to determine optimal time for caspase detection after exposure to individual 259

toxins. 260

We further used a pan-caspase inhibitor Z-VAD-fmk to ascertain the specificity of the signals 261

induced upon toxin exposure. The pan-caspase inhibitor was able to inhibit the caspase activity 262

at all concentrations of toxins used. Furthermore, the cells appeared healthy upon visualization 263

suggesting that the inhibitor was able to prevent cell death induced by TcdA, TcdB, PEA and DT 264

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toxins (data not shown). We also determined the mutant toxin’s ability to induce caspase. 265

Mutations that abolished the ADP-ribosylation activity of DT resulted in toxin that was incapable 266

of inducing caspase(28). Similarly, mutations that lead to loss of glucosytransferase activity of 267

TcdA and TcdB resulted in toxins that failed to induce caspase activity (24, 34, 37-39). The data 268

strongly suggests biological/enzymatic activity of the toxins is primarily responsible for caspase 269

induction which results in loss of viability and cell death. As demonstrated by our studies and 270

results, the assay allows for differentiation of specific toxins at given concentrations, and at 271

different levels of biological activity. 272

Natural infection with Clostridium difficile or immunization with toxins TcdA and TcdB results in 273

antibody responses capable of neutralizing the toxins in vitro, which have shown to be 274

protective against disease in vivo (40). Therefore, we evaluated the ability of the caspase assay 275

to quantify the neutralization titers generated in hamsters upon immunization with toxin. We 276

observed that caspase activation was inhibited by hyperimmune sera raised against TcdA and 277

TcdB, demonstrating specificity of the assay to reliably detect caspase signal due to bacterial 278

toxins and to detect neutralization of toxin by specific antibodies. Furthermore, we compared 279

the neutralizing antibody titers generated in the animal study as measured by caspase assay 280

with those measured in the established neutralizing antibody assay (actin polymerization 281

cytometric assay described earlier), and observed excellent correlation between the two assays 282

with shorter cycle time of 3 days for caspase assay. 283

In summary, we present a versatile platform of quantifying the cytotoxic activity of several 284

common bacterial toxins via measurement of caspase activation as an early indicator for cell 285

death. The signal obtained for caspase activity and therefore, apoptosis, is directly proportional 286

to toxin dose, and can be specifically blocked by anti-toxin antibodies, at a magnitude relative to 287

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antibody titer. The assay can be performed in high throughput format. It is a fast, efficient, and 288

reliable method for evaluating cell apoptosis in response to bacterial toxins, as well as a means 289

to measure neutralizing antibody titers. This assay provides a valuable tool in the study of toxin-290

based vaccine candidates, their efficacy in clinical trials, and in establishing immune correlate to 291

protective antibody levels for bacterial toxins causing disease. 292

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ACKNOWLEDGEMENTS 295

296

The authors would like to recognize the contributions of Rachel Xoconostle for the purification 297

efforts of C. difficile recombinant toxins. We are also grateful to Andy Xie, Tony Kanavage and 298

Suzanne Cole for helpful discussions and Joe Joyce and Jon Heinrichs for critical reading of the 299

manuscript. All authors are current employees of Merck and co. Inc., and may own stocks for the 300

company 301

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303 304 Figure 1 305

Dose response of in vitro toxin-induced caspase activity:(A) C. difficile TcdA on Vero cells, (B) C. difficile 306

TcdB on Vero cells, (C) C. difficile binary toxin CDTa-CDTb on Vero cells, (D) C. diphtheria toxin on HeLa 307

cells and (E) P. aeruginosa Exotoxin A on HeLa cells. Caspase induction was measured in toxin-treated 308

cell cultures using Caspase-Glo 3/7 Assay kit (Promega) and results shown as mean luminescence (RLU) 309

plus standard deviation for 5 replicate wells across 2 assays. EC50 (50% effective toxin concentration) 310

was calculated by four-parameter logistic regression of the titration curve. 311

312

Figure 2 313

Time course of in vitro toxin-induced caspase activity: (A) C. difficile TcdA at 20 ng/ml, (B) C. difficile TcdB 314

at 80 pg/ml, (C) C. diphtheria toxin at 1 μg/ml and (D) P. aeruginosa Exotoxin A at 10 μg/ml. Caspase 315

induction was measured in toxin-treated cell cultures using Caspase-Glo 3/7 Assay kit (Promega) and 316

results shown as mean luminescence (RLU) for duplicate wells. 317

318

Figure 3 319

Specificity of toxin-induced caspase activity in vitro was demonstrated by addition of 20 μM caspase 320

inhibitor Z-VAD-FMK (open circles) or genetic inactivation of toxin (open triangles) compared to active 321

toxins (closed symbols) for (A) C. difficile TcdA, (B) C. difficile TcdB , (C) C. diphtheria toxin, and (D) P. 322

aeruginosa Exotoxin A . Genetically inactivated C. difficile toxins were produced from point mutations in 323

the enzymatic domains of TcdA and TcdB. Inactive C. diphtheria toxin CRM197 was produced by a single 324

missense mutation (Gly52 to Glu) within the fragment A region. Caspase induction was measured in 325

toxin-treated cell cultures using Caspase-Glo 3/7 Assay kit (Promega) and results shown as mean 326

luminescence (RLU) plus standard deviation for 3 replicate wells. 327

328

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Figure 4 331

Neutralization of C. difficile toxin-induced caspase activity in vitro by pre-incubation of toxin with 332

hamster serum containing anti-toxin neutralizing antibodies: Titration of sera from a subset of four 333

hamsters vaccinated with inactivated TcdA (A) and TcdB (B) showed dose-dependent inhibition of 334

caspase induction by both toxins. Comparison of TcdA (C) and TcdB (D) neutralizing antibody titers in 335

sera from 20 vaccinated hamsters determined by the caspase assay (Y axis) correlates well with titers 336

determined in the F-actin cytometric assay (X-axis) for both toxins. Results are shown as antibody titers 337

determined from inverse of antibody dilution that achieved 50% inhibition of toxin activity at an EC90 338

dose, calculated using a four-parameter logistical fit of each serum titration curve. Caspase induction 339

was measured in toxin-treated cell cultures using Caspase-Glo 3/7 Assay kit (Promega) and F-actin was 340

measured by a scanning cytometer referenced in Methods. Both assays were conducted once each at 341

the same time with the same cell cultures, toxins and sera. 342

343 344 345

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348

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