IAI Accepts, published online ahead of print on 10 November 2014 Infect. Immun. doi:10.1128/IAI.02550-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.
1
Toxin-mediated paracellular transport of antitoxin antibodies facilitates protection against
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Clostridium difficile infection
3 4
Z. Zhanga, X. Chenb, L. Hernandeza, P. Liparia, A. Flatterya, S.-C. Chena, S. Kramera, J. D.
5
Polishooka, F. Racinea, H. Capea, C. P. Kellyb, A. G. Theriena#
6 7
Merck Research Laboratories, Merck & Co, Inc, Kenilworth, NJa; Division of Gastroenterology,
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Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MAb
9 10
Running head: Paracellular transport of antibodies in the gut
11 12
#
Address Correspondence to Alex G. Therien, [email protected]
13
The exotoxins TcdA and TcdB are the major virulence factors of Clostridium difficile.
14
Circulating neutralizing antitoxin antibodies are protective in C. difficile infection (CDI) as
15
demonstrated, in part, by the protective effects of actoxumab and bezlotoxumab, which
16
bind to and neutralize TcdA and TcdB, respectively. The question of how systemic IgG
17
antibodies neutralize toxins in the gut lumen remains unresolved, although it has been
18
suggested that the Fc-receptor FcRn may be involved in active antibody transport across
19
the gut epithelium. In this study, we demonstrate that genetic ablation of FcRn and excess
20
irrelevant human IgG has no impact on actoxumab/bezlotoxumab-mediated protection in
21
murine and hamster models of CDI, suggesting that Fc-dependent transport of antibodies
22
across the gut wall is not required for efficacy. Tissue distribution studies in hamsters
23
suggest, rather, that the transport of antibodies depends on toxin-induced damage to the
24
gut lining. In an in vitro two-dimensional culture system that mimics the architecture of the
25
intestinal mucosal epithelium, toxins on the apical side of epithelial cell monolayers are
26
neutralized by basolateral antibodies, and antibody transport across the cell layer is
27
dramatically increased upon addition of toxin to the apical side. Similar data were obtained
28
with F(ab’)2 fragments, which lack an Fc domain, consistent with FcRn-independent
29
paracellular, rather than transcellular, transport of antibodies. Kinetic studies show that
30
initial damage caused by apical toxin is required for efficient neutralization by basolateral
31
antibodies. These data may represent a general mechanism of humoral response-mediated
32
protection against enteric pathogens.
33 34
35
Introduction
36
The enteric pathogen Clostridium difficile is a gram-positive, anaerobic, spore-forming
37
bacterium. C. difficile infections (CDI) cause diarrhea, pseudomembranous colitis and in some
38
severe cases, colonic rupture and death (1). In recent years, CDI-associated morbidity and
39
mortality has increased significantly and the disease poses a significant healthcare threat in the
40
United States and globally (2).
41
The major virulence factors of C. difficile are the Rho-inactivating toxins A and B (TcdA and
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TcdB) that consist of large single-chain proteins with similar multidomain structures and
43
functions (3-5). It is thought that both toxins bind to target mammalian cells (typically gut
44
epithelial cells) at least in part through their C-terminal receptor-binding domains (combined
45
repetitive oligopeptide, or CROP domains) and become internalized via receptor-mediated
46
endocytosis (6, 7). Following internalization, acidification of the endosome leads to a
47
conformational change within the toxins that results in translocation of the glucosyltransferase
48
domain (GTD) across the endosomal membrane and auto-cleavage via the cysteine protease
49
domain (8-10). This releases the GTD into the cytoplasm where it inactivates rho-type GTPase
50
by covalent glucosylation, resulting in disruption of the cytoskeleton, changes in cellular
51
morphology and eventually, cell death (11, 12). The resulting disruption of the gut epithelial
52
barrier leads to the symptoms of the disease, which are exacerbated by toxin-mediated
53
recruitment of a pro-inflammatory host immune response (3-5, 13).
54
Although standard of care antibiotic therapy with metronidazole, vancomycin or fidaxomicin is
55
often effective in resolving primary cases of CDI, ~25% of patients develop one or more
56
recurrent episode of CDI even after an initial cure (1, 14). Multiple lines of evidence suggest that
57
adaptive humoral immune responses against the C. difficile toxins are protective in both primary
58
and recurrent CDI. Kyne et al. first showed that circulating levels of anti-TcdA IgG were
59
positively correlated with a lower rate of primary CDI in colonized patients (15) and with a
60
lower rate of recurrence among patients who had suffered a primary episode of CDI (16). A
61
more recent study has shown that anti-TcdB IgG levels also correlate with protection against
62
CDI recurrence (17). While correlative in nature, these studies provided the impetus to test the
63
hypothesis that antitoxin antibodies might be protective in CDI, and this has now been
64
demonstrated in multiple animal models (18-22). More significantly for human disease, passive
65
immunotherapy with the antitoxin neutralizing antibody combination consisting of actoxumab
66
and bezlotoxumab (specific for TcdA and TcdB, respectively) has been shown to reduce CDI
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recurrence in human patients (23). The combination of actoxumab and bezlotoxumab, both fully-
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human IgG1 antibodies, is currently in phase III clinical development for the prevention of
69
recurrent CDI.
70
Despite the evidence that circulating antitoxin antibodies are protective in CDI, the question of
71
how systemic IgG antibodies bind to and neutralize toxins presumably located largely in the gut
72
lumen remains unanswered. Previous studies have shown that the neonatal immunoglobulin
73
receptor FcRn mediates specific transport of IgG antibodies across the gut wall (24, 25) and
74
plays a role in antibody-mediated protection against infections of the gastrointestinal tract in
75
mice (26, 27). In this study, we use both in vitro and in vivo model systems to explore the
76
mechanism through which neutralizing antitoxin IgG antibodies cross the gut epithelium and
77
neutralize C. difficile toxins located in the lumen of the gut, thereby protecting the host against
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CDI. We show that trans-epithelial neutralization depends on non-specific paracellular transport
79
of the antibodies rather than specific transcellular Fc-receptor-mediated transport mechanisms,
80
and that toxin-induced damage paradoxically facilitates the transport of (and toxin neutralization
81
by) antitoxin antibodies.
82 83
Materials and Methods
84
Toxins and cells
85
Purified TcdA and TcdB from strain VPI 10463 were purchased from Native Antigen
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(Oxfordshire, UK). T84 cells were cultured in DMEM:F12 medium (ATCC) supplemented with
87
5% fetal bovine serum (FBS) and Penicillin-Streptomycin (P/S). Caco-2 cells were cultured in
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MEM (ATCC) supplemented with 10%FBS, nonessential amino acids, sodium-bicarbonate and
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P/S. MDCK cells were cultured in DMEM (Life Tech.) supplemented with 10%FBS plus P/S.
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Mouse primary CDI model
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The mouse antibiotic-associated CDI model has previously been described (28). Briefly, 8 week
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old, female, C57BL6 wildtype or FcRn-/- mice (purchased from Jackson Laboratory) were orally
93
administrated with an antibiotic mixture of kanamycin (40 mg/kg/d), gentamicin (3.5 mg/kg/d),
94
colistin (4.2mg/kg/d), metronidazole (21.5 mg/kg/d), and vancomycin (4.5 mg/kg/d) for 3 days
95
followed, 2 days later, by a single dose of clindamycin (10 mg/kg) intraperitoneally 24 hour prior
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to challenge with 1x105 cfu C. difficile (strain VPI 10463) by gavage. Actoxumab and/or
97
bezlotoxumab were administered intraperitoneally to mice each at a dose of 250 µg, given 1 day
98
prior to challenge with C. difficile. Statistical analysis was carried out using the Logrank test
99
with Bonferroni correction on GraphPad Prism 6 software (GraphPad Sotware, San Diego, CA).
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Mouse recurrent CDI model
101
Mice were treated with oral vancomycin (50 mg/kg, administered by gavage) once daily for 5
102
days following C. difficile challenge. Actoxumab and bezlotoxumab in combination were
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administered intraperitoneally each at a dose of 250 µg, given 1 day prior to challenge with C.
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difficile and again 1 day after discontinuing vancomycin therapy. Statistical significance was
105
determined using the Logrank test with Bonferroni correction on GraphPad Prism 6 software
106
(GraphPad Sotware, San Diego, CA).
107
Hamster CDI model
108
C. difficile B1 spores (generously provided by D. Gerding, Hines VA, Hines, IL) were prepared
109
from confluent cultures grown anaerobically on agar plates for 12 days. Cells were washed from
110
the surface of the agar in an aerobic environment using sterile ice cold diH2O, then left on ice for
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3 hrs. Spores were placed in microfuge tubes and were separated from vegetative cells by
112
centrifugation at 13,000 rpm for 3 min at 4°C, then further purified by serial washes in ice cold
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diH2O and repeat centrifugation. Spores were resuspended in diH2O, quantified by anaerobically
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incubating aliquots on Clostridium difficile Selective Agar (CDSA) supplemented with 0.12ml
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10% sodium taurocholate in sterile water, and aliquots stored at -80°C. Male Golden Syrian
116
hamsters approximately 100g (Charles River Laboratories) were preconditioned for C. difficile
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susceptibility by oral administration of clindamycin at 30 mg/kg five days prior to infectious
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spore challenge. On Day 0, hamsters were infected with a saline suspension containing ~50
119
spores of toxigenic C. difficile strain B1. Hamsters were divided into 3 treatment groups of 6
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animals each, plus one group of infected controls (n=2). Treatment groups included
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actoxumab/bezlotoxumab at 50 mg/kg each subcutaneously (SC) once-daily (qd) for 4 days
122
beginning 5 h after infectious challenge; non-specific human IgG (huIgG; Equitech-Bio., Inc.)
123
administered at 2 g/kg SC qd for 4 days beginning 4 h after infectious challenge; or a
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combination of the two above treatment regimens (actoxumab/bezlotoxumab + huIgG).
125
Hamsters were monitored at least twice-daily for morbidity, mortality and signs of disease
126
including diarrhea (“wet tail”), body weight loss, lethargy, hunched posture or distended
127
abdomen. Animals were euthanized if judged to be in a moribund state or if weight loss
128
exceeded 20%. Statistical significance was determined using the Logrank test with Bonferroni
129
correction on GraphPad Prism 6 software (GraphPad Sotware, San Diego, CA).
130
Tissue distribution of actoxumab/bezlotoxumab in hamsters
131
Male Golden Syrian hamsters were preconditioned with clindamycin on Day -5 relative to
132
infectious spore challenge, as described above. On Day 0, hamsters were divided into 2 groups,
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one left untreated and the second infected with ~50 spores of C. difficile strain B1. At 5 hours
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after spore challenge infected and uninfected hamsters received a single SC dose of
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actoxumab/bezlotoxumab at 50 mg/kg each. Hamsters were euthanized at timepoints after
136
administration of antibodies and gastrointestinal tract (GI) from the stomach to the rectum was
137
collected. The GI tract was sectioned into duodenum, jejunum, ileum, cecum and ascending
138
colon. Cecum contents were collected undiluted, and contents of other GI sections were
139
obtained from the lumen by flushing each section with 1ml sterile phosphate buffered saline
140
(PBS). GI tissue was then thoroughly rinsed with sterile PBS, weighed and homogenized in
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sterile PBS containing 10% glycerol (5 mL for cecum and 1.5 mL for other GI sections).
142
Concentration of human IgG in each tissue or lumenal contents was determined using a human
143
IgG quantitation ELISA (Bethyl Laboratories). Statistical significance was determined by first
144
converting the data to log of antibody concentration, carrying out unpaired, two-tailed t-tests
145
comparing healthy versus CDI hamsters, and applying the Bonferroni correction for multiple
146
comparisons.
147
Immunohistochemistry
148
Hamsters were preconditioned with clindamycin and infected (or not) with C. difficile strain B1
149
spores as described above and dosed with actoxumab/bezlotoxumab (50 mg/kg of each antibody)
150
subcutaneously, once 5h after spore challenge and a second time 24h later. Cecum tissues were
151
collected 24h after the second dose of actoxumab/bezlotoxumab or vehicle from healthy,
152
untreated hamsters (unchallenged and dosed with vehicle), healthy, treated hamsters
153
(unchallenged with C. difficile spores and dosed with actoxumab/bezlotoxumab) or diseased,
154
treated hamsters (challenged with C. difficile spores and dosed with actoxumab/bezlotoxumab),
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and fixed with 10% neutral buffered formalin for 22h and subsequently processed for formalin
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fixed paraffin embedded sections. Localization of injected actoxumab/bezlotoxumab was carried
157
out using a goat anti-human IgG antibody from Jackson ImmunoResearch (West Grove, PA)
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followed by sequential incubation with a goat HRP polymer and diaminobenzidine in a
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SuperPictureTM Polymer Detection Kit (Invitrogen, Grand Island, NY). Tissue sections were
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counterstained with Hematoxylin (Biocare Medical, Concord, CA) for structural analysis.
161
Generation of (Fab’)2 fragments of actoxumab and bezlotoxumab
162
F(ab’)2 fragments of actoxumab and bezlotoxumab were generated using the Pierce F(ab’)2
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Preparation Kit (Thermo Scientific, Waltham, MA, USA) according to the manufacturer's
164
instructions. Fc fragments were removed from F(ab’)2 by gel filtration chromatography using a
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Superdex-S200 column (GE Healthcare Life Sciences, Piscataway, NJ, USA) pre-equilibrated
166
with PBS. Five ml fractions were collected and the peaks corresponding to the F(ab’)2 fragment
167
were pooled. F(ab’)2 purity was ascertained under reducing and non-reducing conditions by
168
SDS-PAGE, using 4–12% Novex Tris-Glycine gels (Life Technologies, Grand Island, NY,
169
USA), followed by Coomassie brilliant blue staining. No contamination of samples by uncleaved
170
IgG was detectable (Fig. S4A and B)
171
Two-dimensional cell culture and transepithelial electrical resistance (TER) measurements
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To establish the two-dimension culture system, cells were seeded on the well insert (BD Falcon
173
HTS 24-multiwell insert, 351181; Bedford, MA, USA) at 0.5 - 1.0x105 cells per well, with 250
174
l medium in the apical chamber and 800 l medium in the basolateral chamber. Cells were
175
cultured for ≥14 days at 37oC (in 5% CO2) to ensure full differentiation and confluency, as
176
assessed by transepithelial electrical resistance (TER) measurements plateauing at ≥600 Ω.cm2
177
(Caco-2) or ≥1000 Ω.cm2 (T84), measured using the Epithelial Volt-Ohm Meter Millicell ERS-2
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(EMD Millipore, Billerica, MA, USA). For fast-growing MDCK cells, maximal TER (plateauing
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at ≥600Ωcm2) was achieved 1-2 days after seeding. For assessments of toxin neutralization,
180
actoxumab or bezlotoxumab (or F(ab’)2 fragments thereof) were added to a final concentration of
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50 g/ml to the apical chamber or 100 g/ml to the basolateral chamber, either 18h before or
182
immediately before addition of toxin to the apical chamber (see figure legends). TER
183
measurements were obtained at different time points either immediately before (t = 0h) or at
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various time points after addition of various concentrations of TcdA or TcdB to either the apical
185
or basolateral chambers. TER measurements were normalized to values obtained in the absence
186
of toxin at each time point to account for minor time-dependent variability. Ten l samples were
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taken from the apical chamber either immediately before addition of the toxin (t = 0h) or at
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various time points after addition of toxin (see figure legends) for quantitation of antibody
189
concentration. Fifty l samples were taken from the basolateral chamber 48h after addition of
190
toxin for quantitation of toxin concentration (see below). Statistical significance was determined
191
by matched two-way ANOVA with Tukey’s multiple comparison test using GraphPad Prism 6
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software (GraphPad Sotware, San Diego, CA).
193
ELISAs
194
ELISAs were carried out according to standard methodology. Briefly, high-protein-binding
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ELISA plates (Fisher Scientific, Waltham, MA, USA) were coated with capture antibody
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(chicken anti-human F(ab’)2 secondary antibody, from Fisher SA1-72043) at 10 g/ml and
197
blocked using blocking buffer (Fisher Scientific). Samples isolated from apical chamber of two-
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dimensional culture system (see above) were diluted in blocking buffer and added to wells, with
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purified actoxumab, bezlotoxumab or F(ab’)2 fragments thereof used as standards. Wells were
200
washed and HRP-linked secondary antibody (300 ng/ml; Fisher Scientific) in blocking buffer
201
was added for detection of bound antibodies. Following extensive washing, HRP substrate
202
solution (Fisher Scientific) was added and luminescent signal was read on a SpectraMax M4
203
instrument. Quantification of toxins in the basolateral chamber was carried out by ELISA using a
204
kit from tgcBIOMICS (Bingen, Germany) and following the manufacturer’s instructions.
205
Statistical significance was determined by matched two-way ANOVA with Tukey’s multiple
206
comparison test using GraphPad Prism 6 software (GraphPad Sotware, San Diego, CA).
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Ethics Statement
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All procedures in animals were performed in accordance with the highest standards for the
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humane handling, care and treatment of research animals, and adhered to the National Research
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Council’s “Guide for Care and Use of Laboratory Animals, 8th Edition”. Mouse studies were
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approved by the Beth Israel Deaconess Medical Center (BIDMC) Institutional Animal Care and
212
Use Committee (protocol number 101-2013). Hamsters studies were approved by the Merck &
213
Co., Inc. (in Kenilworth, NJ) Institutional Animal Care and Use Committee (protocol number
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0280-12).
215 216
Results
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Role of Fc-mediated transport in protection against C. difficile infection.
218
The neonatal Fc receptor FcRn has previously been implicated in transport of IgG from the
219
systemic to the lumenal side of the gut wall (24, 25, 29) and has been shown to play a role in
220
IgG-mediated protection against some forms of bacterial infections (26, 27). To assess whether
221
these observations extend to C. difficile infections, we evaluated the human IgG1 antibody
222
combination actoxumab/bezlotoxumab in a murine CDI model comparing FcRn knockout mice
223
with WT littermates. Since efficacy of actoxumab/bezlotoxumab had not previously been
224
demonstrated in mice, we first administered actoxumab, bezlotoxumab, or a combination of both
225
antibodies intraperitoneally to WT mice 24h before challenge with the toxigenic C. difficile
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strain VPI 10463. The antibody combination provided excellent protection against mortality in
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this model, while the individual antibodies alone were only partially protective (Fig. 1A).
228
Furthermore, in both primary (Fig 1B) and recurrent (Fig 1C) CDI paradigms (see “Materials
229
and Methods”), a single administration of actoxumab/bezlotoxumab increased survival to the
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same extent in WT mice compared to FcRn-null mice, demonstrating that the FcRn receptor does
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not play a significant role in antibody-mediated protection in this murine model. These data,
232
however, do not rule out the possibility that other Fc-mediated transport mechanisms may be
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involved in the efficacy of actoxumab/bezlotoxumab. To address this, we used the gold standard
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Syrian hamster model of CDI, in which actoxumab/bezlotoxumab has previously been
235
demonstrated to be protective (18). Since targeted genetic deletion is not possible in hamsters,
236
and to cover any and all possible Fc-mediated activities of actoxumab/bezlotoxumab, we co-
237
dosed the antibody combination in a therapeutic paradigm (see “Materials and Methods”) with or
238
without a 40-fold excess of irrelevant human IgG (which contains ~63% IgG1; data not shown),
239
reasoning that such antibodies should compete with actoxumab/bezlotoxumab for binding to Fc
240
receptors. As shown in Fig. 1D, co-dosing with excess human IgG had no significant impact on
241
actoxumab/bezlotoxumab-mediated protection, suggesting that such protection is independent of
242
Fc-mediated transport mechanisms.
243
Tissue distribution of actoxumab/bezlotoxumab in the hamster CDI model
244
To gain insight into whether and to what extent systemic antibodies reach the site of infection in
245
the lumen of the gut, we characterized the intestinal tissue distribution of systemically
246
administered actoxumab and bezlotoxumab in hamsters. Hamsters challenged with or without a
247
toxigenic strain of C. difficile (strain B1; see (18)) were injected with a single dose of
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actoxumab/bezlotoxumab at 50 mg/kg. Samples of intestinal tissues were collected from both
249
infected and uninfected animals at various time points after challenge and levels of human IgG
250
were measured by ELISA in lumenal contents and in the washed whole gut tissues. While no
251
significant differences were observed in actoxumab/bezlotoxumab levels within the intestinal
252
tissues between infected and uninfected hamsters (Fig. 2A), levels of actoxumab/bezlotoxumab
253
in the GI lumen were significantly higher in infected hamsters, in particular in the cecum, where
254
antibody levels were nearly undetectable in healthy animals (Figs. 2B and S1). Increased
255
antibody transport across the gut wall in diseased hamsters was confirmed at the cellular level
256
using immunohistochemistry (Figs. 2C, S1C). In healthy hamsters, antibodies are primarily
257
located in the subepithelial space (including the lamina propria) of the mucosa, with no staining
258
in the epithelial layer, which acts as a barrier preventing leakage of antibodies into the gut lumen.
259
Conversely, in hamsters infected with C. difficile, the actoxumab/bezlotoxumab signal is present
260
throughout the mucosa, including parts of the epithelial layer, which exhibits significant
261
damage/sloughing, allowing antibodies to enter the intestinal lumen.. Together, these data show
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that transport of systemic antibodies to the gut lumen is significantly facilitated by damage to the
263
gut epithelium mediated by C. difficile toxins.
264
Transepithelial toxin neutralization by antibodies in a two-dimensional culture system
265
To better understand the protective role of circulating antitoxin antibodies against toxins in the
266
gut lumen, we used a two-dimensional cell culture system wherein apical and basolateral
267
compartments are separated by a single monolayer of differentiated epithelial cells (30-33). The
268
system mimics the polarized nature of the intact intestinal mucosal epithelium which separates
269
the gut lumen (apical side) from the subepithelial/systemic space (basolateral side). The integrity
270
of the epithelial layer is monitored by measuring the transepithelial electrical resistance (TER); a
271
drop in TER indicates that the integrity of the epithelial monolayer has been compromised (31).
272
For studies aimed at understanding how neutralizing antibodies present on the basolateral/
273
systemic side can neutralize toxin on the apical/lumenal side, we first demonstrated that TcdA
274
and TcdB added to the apical chamber cause significant time- and concentration-dependent
275
decreases in TER in the colonic epithelial cell line, Caco-2 (Fig. 3A and B). To confirm that the
276
epithelial monolayer was fully differentiated and polarized, we replicated the previously-
277
published observation (33) that colonic epithelial cells are more sensitive to TcdB applied to the
278
basolateral side compared to the apical side, whereas sensitivity to TcdA is comparable on both
279
sides (Fig. S2). The effects of the neutralizing antibodies actoxumab and bezlotoxumab on toxin-
280
induced damage are shown in Fig. 3C,D, E and F for Caco-2 cells (and in Fig. S3 for T84 cells).
281
When applied to the same side (apical) as the toxins, the antibodies show a strong neutralizing
282
effect on the toxins as shown by rightwards shifts in the concentration response curves of the
283
toxins (shown 24h after addition of toxin; Figs. 3E and F). Significant, though smaller, shifts are
284
also observed when antibodies are added to the basolateral/systemic side of the epithelium,
285
approximating the protection afforded by systemically-circulating neutralizing antibodies in the
286
context of CDI. To assess the potential role of Fc-mediated transport in transepithelial
287
neutralization, we generated F(ab’)2 fragments of actoxumab and bezlotoxumab, which lack an
288
Fc region. The F(ab’)2 fragments, which were first shown to be devoid of contamination by
289
uncleaved antibody and fully neutralizing in a toxin induced cell death assay (34) (Fig. S4),
290
neutralized the effects of toxins at least as efficiently as intact antibodies (Figs. 3E and F),
291
demonstrating that the Fc regions of IgG molecules on the basolateral/systemic side of the
292
epithelial layer are not necessary for transepithelial toxin neutralization. Supporting this notion,
293
addition of excess irrelevant human IgG to the basolateral chamber had no impact on the
294
transepithelial neutralization of toxin (Fig. S5).
295
Mechanism of antibody transport across the epithelial monolayer
296
Neutralization of apical toxin by basolateral antibodies strongly suggests that antibodies are
297
transported to the apical side. To confirm this, we measured the extent to which antibody applied
298
to the basolateral chamber translocates to the apical chamber in the presence and absence of
299
toxin on the apical side. Similar to the TER assays described above, 100 g/ml antibody was
300
added to the basolateral side of a confluent monolayer of Caco-2 cells 18h prior to addition of
301
buffer or toxin at different concentrations, and antibody concentration in the apical chamber was
302
measured by ELISA at different time points. As shown in figure 4A and B (and in Fig. S6A and
303
B for T84 cells), transport of actoxumab and bezlotoxumab into the apical chamber is minimal
304
18h after addition of antibodies to the basolateral chamber (at t = 0h with respect to the time of
305
toxin addition). Transport of antibodies into the apical chamber increases with time in the
306
presence of toxin and this effect is dependent on the concentration of toxin added to the apical
307
chamber. Indeed, while the concentration of antibodies on the apical side is 40% and >20% in the presence of 64 ng/ml TcdA or 256 ng/ml TcdB,
310
respectively. The dependence of antibody transport on toxin is presumed to result from toxin-
311
dependent disruptions in the epithelial barrier function leading to increased non-specific
312
paracellular leakage of antibodies to the apical side, analogous to the high levels of antibodies
313
observed in C. difficile infected hamsters versus healthy hamsters (Fig. 2). Importantly, transport
314
of F(ab’)2 fragments of actoxumab and bezlotoxumab was at least as high as intact antibody in
315
the presence and absence of toxin (Fig. 4C and D), confirming that Fc-dependent transport
316
mechanisms such as FcRn are not involved in antibody transport in this system. Indeed,
317
concentrations of F(ab’)2 were consistently higher in the apical chamber, particularly at high
318
toxin concentration. This may be due to (i) the smaller size of F(ab’)2 fragments allowing them
319
to leak through paracellular gaps more efficiently and/or (ii) a slightly higher molar
320
concentration of F(ab’)2 added to the basolateral chamber compared to intact antibodies (since
321
100 g/ml each of intact antibodies and F(ab’)2 fragments were used in these experiments, and
322
the molecular mass of F(ab’)2 is ~1.5-fold lower than intact antibody) .
323
Antibody-induced recovery of epithelial monolayer following toxin-induced damage
324
In order to better understand how toxin neutralization might protect the gut epithelium in the
325
context of the gut wall (where damage is repaired much faster than in isolated epithelial cells
326
owing to the presence of other cells and growth factors in the intact gut wall (35)), we assessed
327
transepithelial neutralization and transport in the two-dimensional culture assay using MDCK
328
cells, which proliferate at a much faster rate in vitro than gut epithelial cells. Similar to Caco-2
329
and T84 cells (Figs. 3 and S3), we observed a robust TcdA-dependent decrease in TER and
330
significant neutralization of apical TcdA by basolateral actoxumab (Fig. 5A and B). Interestingly,
331
the protection afforded by actoxumab was biphasic, with a significant yet incomplete effect at 6h
332
and complete neutralization (except for the highest concentration of TcdA) at 24h. We confirmed
333
that toxin is indeed fully neutralized at 24h by transferring the contents of the apical chambers at
334
this timepoint (TcdA concentrations up to 64 ng/ml) to intact MDCK and Caco-2 monolayers,
335
and showing limited effects on TER after 24h (data not shown). These data are consistent with
336
the notion that toxin on the apical side must first cause damage to the epithelial layer (shown by
337
a drop in TER at 6h) in order for the antibody on the basolateral side to fully neutralize apical
338
toxin.
339
In parallel with these TER measurements, we assessed transport of antibody to the apical side
340
24h after addition of various concentration of toxin (Figs. 5C and S6C). Surprisingly, the apical
341
concentration of actoxumab was
1
Toxin-mediated paracellular transport of antitoxin antibodies facilitates protection against
2
Clostridium difficile infection
3 4
Z. Zhanga, X. Chenb, L. Hernandeza, P. Liparia, A. Flatterya, S.-C. Chena, S. Kramera, J. D.
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Polishooka, F. Racinea, H. Capea, C. P. Kellyb, A. G. Theriena#
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Merck Research Laboratories, Merck & Co, Inc, Kenilworth, NJa; Division of Gastroenterology,
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Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MAb
9 10
Running head: Paracellular transport of antibodies in the gut
11 12
#
Address Correspondence to Alex G. Therien, [email protected]
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The exotoxins TcdA and TcdB are the major virulence factors of Clostridium difficile.
14
Circulating neutralizing antitoxin antibodies are protective in C. difficile infection (CDI) as
15
demonstrated, in part, by the protective effects of actoxumab and bezlotoxumab, which
16
bind to and neutralize TcdA and TcdB, respectively. The question of how systemic IgG
17
antibodies neutralize toxins in the gut lumen remains unresolved, although it has been
18
suggested that the Fc-receptor FcRn may be involved in active antibody transport across
19
the gut epithelium. In this study, we demonstrate that genetic ablation of FcRn and excess
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irrelevant human IgG has no impact on actoxumab/bezlotoxumab-mediated protection in
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murine and hamster models of CDI, suggesting that Fc-dependent transport of antibodies
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across the gut wall is not required for efficacy. Tissue distribution studies in hamsters
23
suggest, rather, that the transport of antibodies depends on toxin-induced damage to the
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gut lining. In an in vitro two-dimensional culture system that mimics the architecture of the
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intestinal mucosal epithelium, toxins on the apical side of epithelial cell monolayers are
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neutralized by basolateral antibodies, and antibody transport across the cell layer is
27
dramatically increased upon addition of toxin to the apical side. Similar data were obtained
28
with F(ab’)2 fragments, which lack an Fc domain, consistent with FcRn-independent
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paracellular, rather than transcellular, transport of antibodies. Kinetic studies show that
30
initial damage caused by apical toxin is required for efficient neutralization by basolateral
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antibodies. These data may represent a general mechanism of humoral response-mediated
32
protection against enteric pathogens.
33 34
35
Introduction
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The enteric pathogen Clostridium difficile is a gram-positive, anaerobic, spore-forming
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bacterium. C. difficile infections (CDI) cause diarrhea, pseudomembranous colitis and in some
38
severe cases, colonic rupture and death (1). In recent years, CDI-associated morbidity and
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mortality has increased significantly and the disease poses a significant healthcare threat in the
40
United States and globally (2).
41
The major virulence factors of C. difficile are the Rho-inactivating toxins A and B (TcdA and
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TcdB) that consist of large single-chain proteins with similar multidomain structures and
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functions (3-5). It is thought that both toxins bind to target mammalian cells (typically gut
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epithelial cells) at least in part through their C-terminal receptor-binding domains (combined
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repetitive oligopeptide, or CROP domains) and become internalized via receptor-mediated
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endocytosis (6, 7). Following internalization, acidification of the endosome leads to a
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conformational change within the toxins that results in translocation of the glucosyltransferase
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domain (GTD) across the endosomal membrane and auto-cleavage via the cysteine protease
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domain (8-10). This releases the GTD into the cytoplasm where it inactivates rho-type GTPase
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by covalent glucosylation, resulting in disruption of the cytoskeleton, changes in cellular
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morphology and eventually, cell death (11, 12). The resulting disruption of the gut epithelial
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barrier leads to the symptoms of the disease, which are exacerbated by toxin-mediated
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recruitment of a pro-inflammatory host immune response (3-5, 13).
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Although standard of care antibiotic therapy with metronidazole, vancomycin or fidaxomicin is
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often effective in resolving primary cases of CDI, ~25% of patients develop one or more
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recurrent episode of CDI even after an initial cure (1, 14). Multiple lines of evidence suggest that
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adaptive humoral immune responses against the C. difficile toxins are protective in both primary
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and recurrent CDI. Kyne et al. first showed that circulating levels of anti-TcdA IgG were
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positively correlated with a lower rate of primary CDI in colonized patients (15) and with a
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lower rate of recurrence among patients who had suffered a primary episode of CDI (16). A
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more recent study has shown that anti-TcdB IgG levels also correlate with protection against
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CDI recurrence (17). While correlative in nature, these studies provided the impetus to test the
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hypothesis that antitoxin antibodies might be protective in CDI, and this has now been
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demonstrated in multiple animal models (18-22). More significantly for human disease, passive
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immunotherapy with the antitoxin neutralizing antibody combination consisting of actoxumab
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and bezlotoxumab (specific for TcdA and TcdB, respectively) has been shown to reduce CDI
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recurrence in human patients (23). The combination of actoxumab and bezlotoxumab, both fully-
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human IgG1 antibodies, is currently in phase III clinical development for the prevention of
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recurrent CDI.
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Despite the evidence that circulating antitoxin antibodies are protective in CDI, the question of
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how systemic IgG antibodies bind to and neutralize toxins presumably located largely in the gut
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lumen remains unanswered. Previous studies have shown that the neonatal immunoglobulin
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receptor FcRn mediates specific transport of IgG antibodies across the gut wall (24, 25) and
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plays a role in antibody-mediated protection against infections of the gastrointestinal tract in
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mice (26, 27). In this study, we use both in vitro and in vivo model systems to explore the
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mechanism through which neutralizing antitoxin IgG antibodies cross the gut epithelium and
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neutralize C. difficile toxins located in the lumen of the gut, thereby protecting the host against
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CDI. We show that trans-epithelial neutralization depends on non-specific paracellular transport
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of the antibodies rather than specific transcellular Fc-receptor-mediated transport mechanisms,
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and that toxin-induced damage paradoxically facilitates the transport of (and toxin neutralization
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by) antitoxin antibodies.
82 83
Materials and Methods
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Toxins and cells
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Purified TcdA and TcdB from strain VPI 10463 were purchased from Native Antigen
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(Oxfordshire, UK). T84 cells were cultured in DMEM:F12 medium (ATCC) supplemented with
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5% fetal bovine serum (FBS) and Penicillin-Streptomycin (P/S). Caco-2 cells were cultured in
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MEM (ATCC) supplemented with 10%FBS, nonessential amino acids, sodium-bicarbonate and
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P/S. MDCK cells were cultured in DMEM (Life Tech.) supplemented with 10%FBS plus P/S.
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Mouse primary CDI model
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The mouse antibiotic-associated CDI model has previously been described (28). Briefly, 8 week
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old, female, C57BL6 wildtype or FcRn-/- mice (purchased from Jackson Laboratory) were orally
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administrated with an antibiotic mixture of kanamycin (40 mg/kg/d), gentamicin (3.5 mg/kg/d),
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colistin (4.2mg/kg/d), metronidazole (21.5 mg/kg/d), and vancomycin (4.5 mg/kg/d) for 3 days
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followed, 2 days later, by a single dose of clindamycin (10 mg/kg) intraperitoneally 24 hour prior
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to challenge with 1x105 cfu C. difficile (strain VPI 10463) by gavage. Actoxumab and/or
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bezlotoxumab were administered intraperitoneally to mice each at a dose of 250 µg, given 1 day
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prior to challenge with C. difficile. Statistical analysis was carried out using the Logrank test
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with Bonferroni correction on GraphPad Prism 6 software (GraphPad Sotware, San Diego, CA).
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Mouse recurrent CDI model
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Mice were treated with oral vancomycin (50 mg/kg, administered by gavage) once daily for 5
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days following C. difficile challenge. Actoxumab and bezlotoxumab in combination were
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administered intraperitoneally each at a dose of 250 µg, given 1 day prior to challenge with C.
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difficile and again 1 day after discontinuing vancomycin therapy. Statistical significance was
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determined using the Logrank test with Bonferroni correction on GraphPad Prism 6 software
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(GraphPad Sotware, San Diego, CA).
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Hamster CDI model
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C. difficile B1 spores (generously provided by D. Gerding, Hines VA, Hines, IL) were prepared
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from confluent cultures grown anaerobically on agar plates for 12 days. Cells were washed from
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the surface of the agar in an aerobic environment using sterile ice cold diH2O, then left on ice for
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3 hrs. Spores were placed in microfuge tubes and were separated from vegetative cells by
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centrifugation at 13,000 rpm for 3 min at 4°C, then further purified by serial washes in ice cold
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diH2O and repeat centrifugation. Spores were resuspended in diH2O, quantified by anaerobically
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incubating aliquots on Clostridium difficile Selective Agar (CDSA) supplemented with 0.12ml
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10% sodium taurocholate in sterile water, and aliquots stored at -80°C. Male Golden Syrian
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hamsters approximately 100g (Charles River Laboratories) were preconditioned for C. difficile
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susceptibility by oral administration of clindamycin at 30 mg/kg five days prior to infectious
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spore challenge. On Day 0, hamsters were infected with a saline suspension containing ~50
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spores of toxigenic C. difficile strain B1. Hamsters were divided into 3 treatment groups of 6
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animals each, plus one group of infected controls (n=2). Treatment groups included
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actoxumab/bezlotoxumab at 50 mg/kg each subcutaneously (SC) once-daily (qd) for 4 days
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beginning 5 h after infectious challenge; non-specific human IgG (huIgG; Equitech-Bio., Inc.)
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administered at 2 g/kg SC qd for 4 days beginning 4 h after infectious challenge; or a
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combination of the two above treatment regimens (actoxumab/bezlotoxumab + huIgG).
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Hamsters were monitored at least twice-daily for morbidity, mortality and signs of disease
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including diarrhea (“wet tail”), body weight loss, lethargy, hunched posture or distended
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abdomen. Animals were euthanized if judged to be in a moribund state or if weight loss
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exceeded 20%. Statistical significance was determined using the Logrank test with Bonferroni
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correction on GraphPad Prism 6 software (GraphPad Sotware, San Diego, CA).
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Tissue distribution of actoxumab/bezlotoxumab in hamsters
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Male Golden Syrian hamsters were preconditioned with clindamycin on Day -5 relative to
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infectious spore challenge, as described above. On Day 0, hamsters were divided into 2 groups,
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one left untreated and the second infected with ~50 spores of C. difficile strain B1. At 5 hours
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after spore challenge infected and uninfected hamsters received a single SC dose of
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actoxumab/bezlotoxumab at 50 mg/kg each. Hamsters were euthanized at timepoints after
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administration of antibodies and gastrointestinal tract (GI) from the stomach to the rectum was
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collected. The GI tract was sectioned into duodenum, jejunum, ileum, cecum and ascending
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colon. Cecum contents were collected undiluted, and contents of other GI sections were
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obtained from the lumen by flushing each section with 1ml sterile phosphate buffered saline
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(PBS). GI tissue was then thoroughly rinsed with sterile PBS, weighed and homogenized in
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sterile PBS containing 10% glycerol (5 mL for cecum and 1.5 mL for other GI sections).
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Concentration of human IgG in each tissue or lumenal contents was determined using a human
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IgG quantitation ELISA (Bethyl Laboratories). Statistical significance was determined by first
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converting the data to log of antibody concentration, carrying out unpaired, two-tailed t-tests
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comparing healthy versus CDI hamsters, and applying the Bonferroni correction for multiple
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comparisons.
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Immunohistochemistry
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Hamsters were preconditioned with clindamycin and infected (or not) with C. difficile strain B1
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spores as described above and dosed with actoxumab/bezlotoxumab (50 mg/kg of each antibody)
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subcutaneously, once 5h after spore challenge and a second time 24h later. Cecum tissues were
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collected 24h after the second dose of actoxumab/bezlotoxumab or vehicle from healthy,
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untreated hamsters (unchallenged and dosed with vehicle), healthy, treated hamsters
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(unchallenged with C. difficile spores and dosed with actoxumab/bezlotoxumab) or diseased,
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treated hamsters (challenged with C. difficile spores and dosed with actoxumab/bezlotoxumab),
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and fixed with 10% neutral buffered formalin for 22h and subsequently processed for formalin
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fixed paraffin embedded sections. Localization of injected actoxumab/bezlotoxumab was carried
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out using a goat anti-human IgG antibody from Jackson ImmunoResearch (West Grove, PA)
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followed by sequential incubation with a goat HRP polymer and diaminobenzidine in a
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SuperPictureTM Polymer Detection Kit (Invitrogen, Grand Island, NY). Tissue sections were
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counterstained with Hematoxylin (Biocare Medical, Concord, CA) for structural analysis.
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Generation of (Fab’)2 fragments of actoxumab and bezlotoxumab
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F(ab’)2 fragments of actoxumab and bezlotoxumab were generated using the Pierce F(ab’)2
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Preparation Kit (Thermo Scientific, Waltham, MA, USA) according to the manufacturer's
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instructions. Fc fragments were removed from F(ab’)2 by gel filtration chromatography using a
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Superdex-S200 column (GE Healthcare Life Sciences, Piscataway, NJ, USA) pre-equilibrated
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with PBS. Five ml fractions were collected and the peaks corresponding to the F(ab’)2 fragment
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were pooled. F(ab’)2 purity was ascertained under reducing and non-reducing conditions by
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SDS-PAGE, using 4–12% Novex Tris-Glycine gels (Life Technologies, Grand Island, NY,
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USA), followed by Coomassie brilliant blue staining. No contamination of samples by uncleaved
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IgG was detectable (Fig. S4A and B)
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Two-dimensional cell culture and transepithelial electrical resistance (TER) measurements
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To establish the two-dimension culture system, cells were seeded on the well insert (BD Falcon
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HTS 24-multiwell insert, 351181; Bedford, MA, USA) at 0.5 - 1.0x105 cells per well, with 250
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l medium in the apical chamber and 800 l medium in the basolateral chamber. Cells were
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cultured for ≥14 days at 37oC (in 5% CO2) to ensure full differentiation and confluency, as
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assessed by transepithelial electrical resistance (TER) measurements plateauing at ≥600 Ω.cm2
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(Caco-2) or ≥1000 Ω.cm2 (T84), measured using the Epithelial Volt-Ohm Meter Millicell ERS-2
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(EMD Millipore, Billerica, MA, USA). For fast-growing MDCK cells, maximal TER (plateauing
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at ≥600Ωcm2) was achieved 1-2 days after seeding. For assessments of toxin neutralization,
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actoxumab or bezlotoxumab (or F(ab’)2 fragments thereof) were added to a final concentration of
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50 g/ml to the apical chamber or 100 g/ml to the basolateral chamber, either 18h before or
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immediately before addition of toxin to the apical chamber (see figure legends). TER
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measurements were obtained at different time points either immediately before (t = 0h) or at
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various time points after addition of various concentrations of TcdA or TcdB to either the apical
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or basolateral chambers. TER measurements were normalized to values obtained in the absence
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of toxin at each time point to account for minor time-dependent variability. Ten l samples were
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taken from the apical chamber either immediately before addition of the toxin (t = 0h) or at
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various time points after addition of toxin (see figure legends) for quantitation of antibody
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concentration. Fifty l samples were taken from the basolateral chamber 48h after addition of
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toxin for quantitation of toxin concentration (see below). Statistical significance was determined
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by matched two-way ANOVA with Tukey’s multiple comparison test using GraphPad Prism 6
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software (GraphPad Sotware, San Diego, CA).
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ELISAs
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ELISAs were carried out according to standard methodology. Briefly, high-protein-binding
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ELISA plates (Fisher Scientific, Waltham, MA, USA) were coated with capture antibody
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(chicken anti-human F(ab’)2 secondary antibody, from Fisher SA1-72043) at 10 g/ml and
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blocked using blocking buffer (Fisher Scientific). Samples isolated from apical chamber of two-
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dimensional culture system (see above) were diluted in blocking buffer and added to wells, with
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purified actoxumab, bezlotoxumab or F(ab’)2 fragments thereof used as standards. Wells were
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washed and HRP-linked secondary antibody (300 ng/ml; Fisher Scientific) in blocking buffer
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was added for detection of bound antibodies. Following extensive washing, HRP substrate
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solution (Fisher Scientific) was added and luminescent signal was read on a SpectraMax M4
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instrument. Quantification of toxins in the basolateral chamber was carried out by ELISA using a
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kit from tgcBIOMICS (Bingen, Germany) and following the manufacturer’s instructions.
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Statistical significance was determined by matched two-way ANOVA with Tukey’s multiple
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comparison test using GraphPad Prism 6 software (GraphPad Sotware, San Diego, CA).
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Ethics Statement
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All procedures in animals were performed in accordance with the highest standards for the
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humane handling, care and treatment of research animals, and adhered to the National Research
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Council’s “Guide for Care and Use of Laboratory Animals, 8th Edition”. Mouse studies were
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approved by the Beth Israel Deaconess Medical Center (BIDMC) Institutional Animal Care and
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Use Committee (protocol number 101-2013). Hamsters studies were approved by the Merck &
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Co., Inc. (in Kenilworth, NJ) Institutional Animal Care and Use Committee (protocol number
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0280-12).
215 216
Results
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Role of Fc-mediated transport in protection against C. difficile infection.
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The neonatal Fc receptor FcRn has previously been implicated in transport of IgG from the
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systemic to the lumenal side of the gut wall (24, 25, 29) and has been shown to play a role in
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IgG-mediated protection against some forms of bacterial infections (26, 27). To assess whether
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these observations extend to C. difficile infections, we evaluated the human IgG1 antibody
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combination actoxumab/bezlotoxumab in a murine CDI model comparing FcRn knockout mice
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with WT littermates. Since efficacy of actoxumab/bezlotoxumab had not previously been
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demonstrated in mice, we first administered actoxumab, bezlotoxumab, or a combination of both
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antibodies intraperitoneally to WT mice 24h before challenge with the toxigenic C. difficile
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strain VPI 10463. The antibody combination provided excellent protection against mortality in
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this model, while the individual antibodies alone were only partially protective (Fig. 1A).
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Furthermore, in both primary (Fig 1B) and recurrent (Fig 1C) CDI paradigms (see “Materials
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and Methods”), a single administration of actoxumab/bezlotoxumab increased survival to the
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same extent in WT mice compared to FcRn-null mice, demonstrating that the FcRn receptor does
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not play a significant role in antibody-mediated protection in this murine model. These data,
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however, do not rule out the possibility that other Fc-mediated transport mechanisms may be
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involved in the efficacy of actoxumab/bezlotoxumab. To address this, we used the gold standard
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Syrian hamster model of CDI, in which actoxumab/bezlotoxumab has previously been
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demonstrated to be protective (18). Since targeted genetic deletion is not possible in hamsters,
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and to cover any and all possible Fc-mediated activities of actoxumab/bezlotoxumab, we co-
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dosed the antibody combination in a therapeutic paradigm (see “Materials and Methods”) with or
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without a 40-fold excess of irrelevant human IgG (which contains ~63% IgG1; data not shown),
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reasoning that such antibodies should compete with actoxumab/bezlotoxumab for binding to Fc
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receptors. As shown in Fig. 1D, co-dosing with excess human IgG had no significant impact on
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actoxumab/bezlotoxumab-mediated protection, suggesting that such protection is independent of
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Fc-mediated transport mechanisms.
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Tissue distribution of actoxumab/bezlotoxumab in the hamster CDI model
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To gain insight into whether and to what extent systemic antibodies reach the site of infection in
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the lumen of the gut, we characterized the intestinal tissue distribution of systemically
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administered actoxumab and bezlotoxumab in hamsters. Hamsters challenged with or without a
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toxigenic strain of C. difficile (strain B1; see (18)) were injected with a single dose of
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actoxumab/bezlotoxumab at 50 mg/kg. Samples of intestinal tissues were collected from both
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infected and uninfected animals at various time points after challenge and levels of human IgG
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were measured by ELISA in lumenal contents and in the washed whole gut tissues. While no
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significant differences were observed in actoxumab/bezlotoxumab levels within the intestinal
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tissues between infected and uninfected hamsters (Fig. 2A), levels of actoxumab/bezlotoxumab
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in the GI lumen were significantly higher in infected hamsters, in particular in the cecum, where
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antibody levels were nearly undetectable in healthy animals (Figs. 2B and S1). Increased
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antibody transport across the gut wall in diseased hamsters was confirmed at the cellular level
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using immunohistochemistry (Figs. 2C, S1C). In healthy hamsters, antibodies are primarily
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located in the subepithelial space (including the lamina propria) of the mucosa, with no staining
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in the epithelial layer, which acts as a barrier preventing leakage of antibodies into the gut lumen.
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Conversely, in hamsters infected with C. difficile, the actoxumab/bezlotoxumab signal is present
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throughout the mucosa, including parts of the epithelial layer, which exhibits significant
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damage/sloughing, allowing antibodies to enter the intestinal lumen.. Together, these data show
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that transport of systemic antibodies to the gut lumen is significantly facilitated by damage to the
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gut epithelium mediated by C. difficile toxins.
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Transepithelial toxin neutralization by antibodies in a two-dimensional culture system
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To better understand the protective role of circulating antitoxin antibodies against toxins in the
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gut lumen, we used a two-dimensional cell culture system wherein apical and basolateral
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compartments are separated by a single monolayer of differentiated epithelial cells (30-33). The
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system mimics the polarized nature of the intact intestinal mucosal epithelium which separates
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the gut lumen (apical side) from the subepithelial/systemic space (basolateral side). The integrity
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of the epithelial layer is monitored by measuring the transepithelial electrical resistance (TER); a
271
drop in TER indicates that the integrity of the epithelial monolayer has been compromised (31).
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For studies aimed at understanding how neutralizing antibodies present on the basolateral/
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systemic side can neutralize toxin on the apical/lumenal side, we first demonstrated that TcdA
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and TcdB added to the apical chamber cause significant time- and concentration-dependent
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decreases in TER in the colonic epithelial cell line, Caco-2 (Fig. 3A and B). To confirm that the
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epithelial monolayer was fully differentiated and polarized, we replicated the previously-
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published observation (33) that colonic epithelial cells are more sensitive to TcdB applied to the
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basolateral side compared to the apical side, whereas sensitivity to TcdA is comparable on both
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sides (Fig. S2). The effects of the neutralizing antibodies actoxumab and bezlotoxumab on toxin-
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induced damage are shown in Fig. 3C,D, E and F for Caco-2 cells (and in Fig. S3 for T84 cells).
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When applied to the same side (apical) as the toxins, the antibodies show a strong neutralizing
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effect on the toxins as shown by rightwards shifts in the concentration response curves of the
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toxins (shown 24h after addition of toxin; Figs. 3E and F). Significant, though smaller, shifts are
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also observed when antibodies are added to the basolateral/systemic side of the epithelium,
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approximating the protection afforded by systemically-circulating neutralizing antibodies in the
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context of CDI. To assess the potential role of Fc-mediated transport in transepithelial
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neutralization, we generated F(ab’)2 fragments of actoxumab and bezlotoxumab, which lack an
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Fc region. The F(ab’)2 fragments, which were first shown to be devoid of contamination by
289
uncleaved antibody and fully neutralizing in a toxin induced cell death assay (34) (Fig. S4),
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neutralized the effects of toxins at least as efficiently as intact antibodies (Figs. 3E and F),
291
demonstrating that the Fc regions of IgG molecules on the basolateral/systemic side of the
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epithelial layer are not necessary for transepithelial toxin neutralization. Supporting this notion,
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addition of excess irrelevant human IgG to the basolateral chamber had no impact on the
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transepithelial neutralization of toxin (Fig. S5).
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Mechanism of antibody transport across the epithelial monolayer
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Neutralization of apical toxin by basolateral antibodies strongly suggests that antibodies are
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transported to the apical side. To confirm this, we measured the extent to which antibody applied
298
to the basolateral chamber translocates to the apical chamber in the presence and absence of
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toxin on the apical side. Similar to the TER assays described above, 100 g/ml antibody was
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added to the basolateral side of a confluent monolayer of Caco-2 cells 18h prior to addition of
301
buffer or toxin at different concentrations, and antibody concentration in the apical chamber was
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measured by ELISA at different time points. As shown in figure 4A and B (and in Fig. S6A and
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B for T84 cells), transport of actoxumab and bezlotoxumab into the apical chamber is minimal
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18h after addition of antibodies to the basolateral chamber (at t = 0h with respect to the time of
305
toxin addition). Transport of antibodies into the apical chamber increases with time in the
306
presence of toxin and this effect is dependent on the concentration of toxin added to the apical
307
chamber. Indeed, while the concentration of antibodies on the apical side is 40% and >20% in the presence of 64 ng/ml TcdA or 256 ng/ml TcdB,
310
respectively. The dependence of antibody transport on toxin is presumed to result from toxin-
311
dependent disruptions in the epithelial barrier function leading to increased non-specific
312
paracellular leakage of antibodies to the apical side, analogous to the high levels of antibodies
313
observed in C. difficile infected hamsters versus healthy hamsters (Fig. 2). Importantly, transport
314
of F(ab’)2 fragments of actoxumab and bezlotoxumab was at least as high as intact antibody in
315
the presence and absence of toxin (Fig. 4C and D), confirming that Fc-dependent transport
316
mechanisms such as FcRn are not involved in antibody transport in this system. Indeed,
317
concentrations of F(ab’)2 were consistently higher in the apical chamber, particularly at high
318
toxin concentration. This may be due to (i) the smaller size of F(ab’)2 fragments allowing them
319
to leak through paracellular gaps more efficiently and/or (ii) a slightly higher molar
320
concentration of F(ab’)2 added to the basolateral chamber compared to intact antibodies (since
321
100 g/ml each of intact antibodies and F(ab’)2 fragments were used in these experiments, and
322
the molecular mass of F(ab’)2 is ~1.5-fold lower than intact antibody) .
323
Antibody-induced recovery of epithelial monolayer following toxin-induced damage
324
In order to better understand how toxin neutralization might protect the gut epithelium in the
325
context of the gut wall (where damage is repaired much faster than in isolated epithelial cells
326
owing to the presence of other cells and growth factors in the intact gut wall (35)), we assessed
327
transepithelial neutralization and transport in the two-dimensional culture assay using MDCK
328
cells, which proliferate at a much faster rate in vitro than gut epithelial cells. Similar to Caco-2
329
and T84 cells (Figs. 3 and S3), we observed a robust TcdA-dependent decrease in TER and
330
significant neutralization of apical TcdA by basolateral actoxumab (Fig. 5A and B). Interestingly,
331
the protection afforded by actoxumab was biphasic, with a significant yet incomplete effect at 6h
332
and complete neutralization (except for the highest concentration of TcdA) at 24h. We confirmed
333
that toxin is indeed fully neutralized at 24h by transferring the contents of the apical chambers at
334
this timepoint (TcdA concentrations up to 64 ng/ml) to intact MDCK and Caco-2 monolayers,
335
and showing limited effects on TER after 24h (data not shown). These data are consistent with
336
the notion that toxin on the apical side must first cause damage to the epithelial layer (shown by
337
a drop in TER at 6h) in order for the antibody on the basolateral side to fully neutralize apical
338
toxin.
339
In parallel with these TER measurements, we assessed transport of antibody to the apical side
340
24h after addition of various concentration of toxin (Figs. 5C and S6C). Surprisingly, the apical
341
concentration of actoxumab was
