Enhanced Neutralization Potency of Botulinum Neurotoxin Antibodies Using a Red Blood Cell-Targeting Fusion Protein
et al. (2011) Enhanced Neutralization Potency of Botulinum Neurotoxin Antibodies Using a Red
Blood Cell-Targeting Fusion Protein. PLoS ONE 6(3): e17491. doi:10.1371/journal.pone.0017491
Enhanced Neutralization Potency of Botulinum Neurotoxin Antibodies Using a Red Blood Cell-Targeting Fusion Protein
Sharad P. Adekar 0
Andrew T. Segan 0
Cindy Chen 0
Rodney Bermudez 0
M. D. Elias 0
Bernard H. Selling 0
B. P. Kapadnis 0
Lance L. Simpson 0
Paul M. Simon 0
Scott K. Dessain 0
Olivier Neyrolles, Institut de Pharmacologie et de Biologie Structurale, France
0 1 Lankenau Institute for Medical Research, Wynnewood, Pennsylvania, United States of America , 2 Immunome , Inc., Wynnewood, Pennsylvania, United States of America, 3 Division of Infectious Diseases and Environmental Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America , 4 Impact Biologicals , Inc. Wallingford, Pennsylvania, United States of America, 5 Department of Microbiology, University of Pune , Pune , India , 6 Augmenta Biologicals , LLC , Wynnewood, Pennsylvania , United States of America
Botulinum neurotoxin (BoNT) potently inhibits cholinergic signaling at the neuromuscular junction. The ideal countermeasures for BoNT exposure are monoclonal antibodies or BoNT antisera, which form BoNT-containing immune complexes that are rapidly cleared from the general circulation. Clearance of opsonized toxins may involve complement receptor-mediated immunoadherence to red blood cells (RBC) in primates or to platelets in rodents. Methods of enhancing immunoadherence of BoNT-specific antibodies may increase their potency in vivo. We designed a novel fusion protein (FP) to link biotinylated molecules to glycophorin A (GPA) on the RBC surface. The FP consists of an scFv specific for murine GPA fused to streptavidin. FP:mAb:BoNT complexes bound specifically to the RBC surface in vitro. In a mouse model of BoNT neutralization, the FP increased the potency of single and double antibody combinations in BoNT neutralization. A combination of two antibodies with the FP gave complete neutralization of 5,000 LD50 BoNT in mice. Neutralization in vivo was dependent on biotinylation of both antibodies and correlated with a reduction of plasma BoNT levels. In a postexposure model of intoxication, FP:mAb complexes gave complete protection from a lethal BoNT/A1 dose when administered within 2 hours of toxin exposure. In a pre-exposure prophylaxis model, mice were fully protected for 72 hours following administration of the FP:mAb complex. These results demonstrate that RBC-targeted immunoadherence through the FP is a potent enhancer of BoNT neutralization by antibodies in vivo.
Funding: This work was supported by a grant from the National Institutes of Health, NIAID R01 AI065967 to S.K.D. and institutional support from the Lankenau
Institute for Medical Research. Augmenta, LLC, provided support for PMS and contracted Impact Biologicals, Inc. for production of the FP. PMS, as a corresponding
author, participated in study design, data collection and analysis, decision to publish, and preparation of the manuscript.
Competing Interests: The authors have read the journals policy and have the following conflicts: SPA and RB are part-time employees of Immunome, Inc., a
company that has optioned rights to the antibodies described in this report. BHS is the president of Impact Biologicals, Inc. PMS is the inventor of the FP and the
President of Augmenta, LLC. SKD is a founder and Chief Scientific Officer of Immunome, Inc. and has an equity interest in the company. SKD does not receive
research support or consulting fees from Immunome, Inc., Augmenta Biologicals, LLC, or Impact Biologicals, Inc. A provisional US Patent application has been filed
on the experiments described, "Toxin Clearance", Inventors SPA, SKD, PMS, and has been assigned to LIMR and Augmenta, LLC. These relationships do not alter
the authors adherence to all the PLoS ONE policies on sharing data and materials.
Botulinum neurotoxin is one of the most potent lethal substances
known. It is produced by organisms of the genus Clostridium and
produces peripheral neuromuscular and autonomic paralysis through
inactivation of cholinergic signaling at the neuromuscular synapse.
Intoxication with BoNT proceeds by a series of steps, in which BoNT
first enters the body, transits across an epithelium, travels through the
bloodstream, and interacts with the surface of cholinergic neurons
[1,2,3]. Once bound to the neuromuscular junction, BoNT is
internalized via binding to secretory vesicle proteins and transported
into a vesicular compartment. The catalytic domain of BoNT, the
light chain (LC), acquires proteolytic activity as it is transported across
the vesicle membrane into the neuron cytosol [4,5]. Through
cleavage of tethering proteins, the BoNT LC prevents the neuron
from releasing acetylcholine in response to neural stimulation.
Passive immune therapies for BoNT intoxication have been
shown to be effective clinically and in laboratory studies, with
either antisera or oligoclonal combinations of monoclonal
antibodies [6,7,8]. Within the bloodstream, BoNT-containing
immune complexes that contain three or more antibodies are
rapidly sequestered in the spleen and liver [3,8]. Such clearance is
sufficient to provide high level neutralization (.10,000 LD50
BoNT), even if the antibodies do not have intrinsic neutralizing
activity [9,10]. Immune complexes formed between BoNT and
only one or two antibodies stably circulate in the bloodstream and
are therefore much less potent in BoNT neutralization (L.L.S.,
data not shown).
A general feature of the handling of immune complexes in vivo is
immunoadherence, i.e., attachment to red blood cells (RBC) .
The precise mechanism for BoNT clearance by immune
complexes has not been elucidated, but it may involve multiple,
redundant systems for antigen capture by Fcc receptor-bearing
reticuloendothelial cells in the liver and spleen [8,12,13]. One
aspect of this process utilizes the complement system, in which
C3bopsonized immune complexes bind to complement receptor type 1
(CR1) on RBCs in primates or to complement factor H in rodents
[14,15]. The ability of a monoclonal antibody to utilize this pathway
can be enhanced by linking it to another antibody specific for CR1,
to create a bispecific heteropolymer [16,17].
Heteropolymer:antigen complexes bound to RBCs can be directly taken up by
macrophages and are rapidly cleared from the circulation.
Methods that enhance the immunoadherence of antibodies to
RBCs may be useful for BoNT prophylaxis and treatment.
Antibody immunoadherence may be enhanced using a novel
fusion protein (FP), created by Augmenta Biologicals
(Wynnewood, PA). The FP is a recombinant protein that links streptavidin
 to an scFv derived from a monoclonal antibody specific for
GPA, the predominant protein on the RBC surface . The FP
was developed as a delivery system to adhere biotinylated
molecules to the RBC surface, which may enhance the
immunogenicity of biotinylated vaccine antigens and the clearance of
biotinylated antibody-antigen complexes. We previously described
a panel of human monoclonal antibodies specific for BoNT
serotypes A and B (BoNT/A, BoNT/B) [20,21,22]. In this study,
we examined the ability of the FP to augment the neutralizing
capability of these antibodies in vivo.
Binding of FP:mAb:BoNT complexes to red blood cells in
The FP is a recombinant protein that joins an N-terminal scFv
specific for GPA to a C-terminal streptavidin moiety (Figure 1a).
GPA is expressed exclusively on the RBC membrane, at
approximately 106 copies per cell, where its primary role is to
provide negatively charged sialic acid residues that limit
RBCRBC aggregation . The murine scFv sequence specific for
GPA was derived from the antibody TER-119 . Streptavidin is
a tetrameric protein that binds biotin with high affinity . The
FP also contains a C-terminal polyhistidine tag to facilitate
purification following expression in E. coli. Figure 1b shows a
poly-acrylamide gel in which urea-solubilized and refolded FP
samples were analyzed. Two bands were seen in the refolded FP
sample, consistent with existence of tetrameric (164 kDa) and
monomeric (41 kDa) forms. We have observed that the
monomeric FP is unable to bind biotin (P.M.S., data not shown),
consistent with the observations of others .
As depicted in Figure 1a, the FP was designed as a molecular
bridge to link biotinylated molecules, such as antigens and
antibodies, to the RBC membrane. We analyzed binding of the
FP to the surface membrane of murine RBCs in vitro using flow
cytometry, labeling the FP with biotinylated fluorescein.
Figure 2a shows near complete labeling of the RBCs mediated
by the FP molecule. FP binding was specific for GPA, since its
binding was completely inhibited by the TER-119 IgG, but not by
an isotype control antibody (rat IgG2b). Next, we tested RBC
binding of complexes containing FP, the BoNT/A-specific MAb
6A, and BoNT/A 50 kDa C-terminal domain (HC50). The HC50
was labeled with Alexa Fluor 488, and the biotinylated 6A MAb
was detected with an anti-human IgG-APC secondary antibody. In
vitro incubation of this complex with RBCs resulted in almost
complete co-labeling of RBCs with APC and Alexa-488
(Figure 2b, panels A and B). This binding was also inhibited
by TER-119, but not by the isotype control antibody (Figure 2b,
panels C and D).
In vivo neutralizing ability of single antibodies bound to
We previously reported three human antibodies that are
specific for BoNT [20,21]. 4LCA binds to the catalytic light
chain domain of BoNT/A, and can neutralize 25 LD50 BoNT/
A in the standard mouse protection assay. The 6A and 13A
mAbs bind to overlapping epitopes on the BoNT/A heavy chain
C-terminal 50 kDa domain (HC50). The 6A MAb can
neutralize 2.5 LD50 BoNT/A in vivo, while the 13A MAb is
essentially non-neutralizing. The 30B MAb binds serotype B
BoNT (BoNT/B) with high affinity, but it is not neutralizing.
We biotinylated these mAbs and tested them in vivo alone or in
combination with excess FP. The most effective complex
contained 6 mg FP and 1.5 mg 4LCA, which was able to
completely neutralize up to 100 LD50 BoNT, 4-fold greater
than could be neutralized by 100 mg naked 4LCA MAb
(Table 1). The FP also enhanced the activity of the 6A MAb,
allowing neutralization of 25 LD50 BoNT/A, a 10-fold increase
over the 2.5 LD50 neutralized by 100 mg naked 6A. Control
Figure 1. The RBC-targeting fusion protein (FP). (a) Schematic representation of the FP. The FP is comprised of an scFv, specific for the RBC
surface protein glycophorin A (GPA), fused to streptavidin (StAv). The latter is capable of binding biotinylated mAbs specific for BoNT. (b) SDS-PAGE
of the FP performed without heating the samples prior to loading. Lane 1: FP after expression in E. coli and purification in 8M urea (monomer). Lane 2:
refolded FP following dialysis for removal of urea showing the tetramer and residual monomer.
Figure 2. Binding of FP and FP:mAb complexes to GPA on murine RBCs. (a) In-vitro RBC binding by the FP complex is specific for
glycophorin A. FP with or without biotinylated-fluorescein (BIOT-FLUO) was incubated with excess competitor TER-119 antibody (TER) or an isotype
control (IgG) and analyzed by FACS. NL, no label. (b) RBC binding of the FP:6A:BoNT complex. A) Unlabeled RBCs. B) FP, biotinylated 6A and
Alexa488labeled HC50A were added to RBCs and detected with an anti-human-APC antibody. C) Competitor TER-119 inhibited binding of the complex to
RBCs. D) An IgG isotype control antibody did not affect complex binding.
experiments with un-biotinylated 4LCA and 6A mAbs,
administered with FP, showed no enhancement in activity (data not
shown). Incorporated into an FP complex, the non-neutralizing
13A MAb protected mice up to a dose of 10 LD50. Similarly,
the non-neutralizing 30B MAb could neutralize BoNT/B in vivo
when bound to the FP, although the dose of complex was larger
(12 mg FP, 3 mg 30B) and the amount of toxin was smaller (5
LD50). These observations indicate that the FP can enhance the
neutralizing activity of BoNT mAbs. In addition, while FP
quantitatively enhanced the potency of mAbs with intrinsic
neutralizing activity, it also converted qualitatively
non-neutralizing mAbs to neutralizing ones.
FP:mAb neutralization with MAb combinations and
reduction in plasma BoNT concentration
A recurrent finding of BoNT neutralization in vivo is that
oligoclonal antibody mixtures are more potent than single
antibodies. We tested a combination of the 4LCA and 6A mAbs,
which together, in their unmodified forms, can neutralize up to
1000 LD50 . Using 0.75 mg each MAb with 6 mg FP, we
observed complete survival with up to 2000 LD50 BoNT, whereas
50 mg each of naked 4LCA and 6A was not protective (Table 2).
Increasing the quantity of FP:Ab 4-fold (24 mg FP with 3 mg each
MAb), provided complete survival with BoNT doses of up to 5000
Antibodies and FP:mAb complexes were tested for their ability to protect mice from lethality induced by botulinum neurotoxin (BoNT). mAbs were tested alone,
without modification, or biotinylated and in combination with the fusion protein (FP) by mixing with the toxin in vitro and intravenous injection one hour later. Mice
were observed for 5 days. The amounts of mAb and FP used per mouse (mg), the serotype of each BoNT (A or B), and the percent of surviving mice for each dose (LD50)
administered are shown. Blank spaces indicate dose levels that were not tested.
LD50. Mice that received this dose and 10,000 LD50 BoNT/A
survived one day following the injection, indicating partial
We next used the 2000 LD50 dose to test the importance of
linking the MAb to the RBC surface through the FP.
Unbiotinylated 4LCA, at either 1.5 mg or 100 mg, did not contribute to
neutralization by biotinylated 6A and the FP (Table 2). When both
mAbs were biotinylated, but given in combination with streptavidin,
rather than FP, no protection was seen. These results suggest that
efficacy in vivo requires the formation of a complex that anchors both
mAbs to the RBC surface by binding to one or more FP molecules.
To explore this further, we assessed the plasma BoNT levels in mice
injected with 6 mg of detoxified BoNT/A and the 4LCA and 6A
FP:mAb complexes (3 mg each antibody, 24 mg FP) (Figure 3).
Ninety minutes after injection, we tested the levels of BoNT/A in
undiluted plasma using ELISA. The presence of the biotinylated
mAbs alone reduced the BoNT levels slightly, but this effect was
significantly enhanced by the presence of the FP. Taken together,
these results demonstrate the collaborative effects of a pair of mAbs
in combination with the FP, and suggests that the neutralizing
activity requires BoNT immunoadherence to RBCs in vivo.
Post-exposure and pre-exposure neutralization of BoNT
by FP:mAb complexes
We next assessed the FP:Ab mixture in post-exposure and
preexposure models, testing 6 mg FP with 0.75 mg each of the 4LCA
and 6A antibodies. In the post-exposure model, BoNT (10 LD50)
was delivered by intraperitoneal (i.p.) injection and FP:mAb
complexes were subsequently administered at hourly intervals by
intravenous (i.v.) injection. Mice were monitored for survival for 5
days. Complete survival was provided by FP:Ab given up to
2 hours following the BoNT injection, with partial survival at 3
and 4 hours post-BoNT (87.5% and 62.5% survival, respectively;
Figure 4). We also performed a pre-exposure challenge with this
dose of FP:mAb. Mice first received an i.v. injection of the
FP:mAb and were then challenged at daily intervals with 10 LD50
BoNT/A i.p. (Figure 5). FP:mAb complexes were able to provide
complete survival for mice receiving BoNT up to 72 hours after
FP:mAb administration. Mice receiving BoNT 96 hours after the
FP:mAb were partially protected (40% survival). Thus, the
FP:mAb combination can provide protection against a lethal dose
of BoNT in both post-exposure and pre-exposure conditions.
The primary obstacles to the development of a comprehensive
immune therapeutic for BoNT exposure are the extreme potency
of the toxin and the broad diversity of BoNT serotypes.
Experiments with polyclonal antibodies and oligoclonal antibody
combinations have shown that effective clearance of BoNT from
the circulation is one of the most important contributors to
neutralization [1,3,8,22,25,26]. The development of single or
2,000 LD50 % Survival
The 6A and 4LCA mAbs were tested in un-modified and biotinylated forms, alone and in combination with the fusion protein (FP) or streptavidin (SA). The combinations
were tested by mixing and incubation in vitro, with 2,000 LD50 BoNT/A, followed by intravenous injection. The doses of each component are given in mg, and the
outcomes are reported as the percentage of mice surviving (% Survival).
double molecule therapeutics for BoNT will require enhancement
of the ability of mAbs to clear BoNT from the blood circulation.
In this study, we tested the ability of a novel FP to enhance the
neutralizing capacity of BoNT-specific mAbs at the level of the
blood circulation. The FP is a bifunctional molecule that allows
the adherence of biotin-conjugated immune complexes to the
RBC surface. The FP was able to significantly increase the
neutralization potency of BoNT-specific mAbs. When combined
with the FP, the non-neutralizing 13A and 30B mAbs were able to
fully neutralize lethal doses of BoNT/A and BoNT/B,
respectively. The FP increased the quantity of BoNT/A that could be
neutralized by the 6A mAb, the 4LCA mAb, and the 6A/4LCA
mAb combination. These levels were achieved with much lower
quantities of mAbs than we had previously required for protection
with naked mAbs (1.5 mg vs. 50-100 mg). The NIH and the FDA
have not yet established a standard for protective efficacy for
BoNT passive immune therapies, but we can compare the potency
of our FP combinations with the approved BoNT anti-toxins. The
human antiserum for use in infants, BabyBig (MassBioLogics,
Boston, MA), has a BoNT/A-specific potency of 3,000 LD50/mg
. Two of our single FP:mAb combinations (with 6A or 4LCA)
and our double MAb combination (with both 6A and 4LCA) were
equivalent or better in potency (3,333266,666 LD50/mg,
Table 3). The investigational heptavalent botulinum antitoxin
(HBAT, Cangene Corporation, Winnipeg, Manitoba, CA) has a
potency of 7,500 IU/dose (1 IU equals 10,000 murine LD50s)
. An equivalent dose of the FP:mAb combination would
amount to 281 mg, or 4 mg/kg in a 70 kg person. Thus, the
potency of our FP:mAb combination is within the range required
for a human therapeutic.
Animal testing of a BoNT countermeasure needs to address
protection of an asymptomatic individual (who has absorbed a
sub-lethal dose, but may still be absorbing toxin) or an individual
at risk for exposure (such as a first responder to a contaminated
area). We found that the FP combined with 6A and 4LCA is
sufficient to provide neutralization of 10 LD50 BoNT/A for
72 hours and partial protection at 96 hours. This result indicates
the presence of physiologically relevant concentrations of the
FP:mAb combination in vivo during this time.
We also tested the FP:mAb combination in a post-exposure
model, in which the FP:Ab was administered following a lethal
dose of toxin. An intravenous injection of the combination was
able to provide complete protection from a lethal intraperitoneal
BoNT dose at 2 hours and partial protection at 4 hours. BoNT
distribution experiments have demonstrated that the window of
opportunity of exposure of a lethal dose of BoNT is determined by
a first-order reaction that depends on the amount of BoNT
administered and the period of time that elapses before the
countermeasure is administered (L.L.S., manuscript in
preparation). Thus, anti-toxins that are sufficient to neutralize an entire
circulating dose of BoNT should give the same window of
opportunity for post-exposure salvage, and our post-exposure
results are comparable to results obtained by others [10,28,29].
The proposed mechanism through which the FP augments
antibody neutralization activity involves binding of FP:mAb
complexes to the RBC surface. Flow cytometry experiments in
vitro demonstrated that the FP:mAb complexes are able to bind to
erythrocytes and that the complexes serve as a molecular bridge to
adhere BoNT to the RBC. Experiments with un-biotinylated
antibodies and the FP did not show any enhancement of
neutralization. Streptavidin alone did not improve neutralizing
activity of biotinylated BoNT antibodies, in comparison to the
RBC-targeted FP. Lastly, the levels of BoNT circulating in the
plasma of mice that had received the FP in combination with the
4LCA and 6A were lowered. These results together support the
model that the enhancement of neutralization in vivo required
biotin-dependent interaction of the cloned mAbs with the FP and
adherence of the FP-containing complexes to the surface of the
RBCs (Figure 1a).
The observation that the intrinsic neutralization capacity of the
naked mAbs correlated directly with the potency level achieved
when bound to the FP supports the idea that the FP:mAb:BoNT
complexes remain in circulation for a significant period of time
before they are definitively removed. In this model, relatively rapid
adherence of FP:mAb:BoNT to the RBC membrane would be
followed by a slower phase, in which either complex-bound toxin
is removed from the RBC surface or the BoNT-bound RBCs are
removed from the circulation.
This is distinct from clearance of C3b-opsonized immune
complexes, which are definitively taken up by the liver and spleen
in less than 15 minutes [8,13]. While circulating and adherent to
RBCs, the FP:mAb complexes would be in competition with the
neuromuscular junction for BoNT. Intoxication may result from
dissociation of BoNT from the antibody complex, or of the
FP:mAb:BoNT complex from the surface of the RBC. The high
potency of the FP:6A/4LCA complex may partly result from
stabilization of BoNT on the RBC surface through cooperative
mAb avidity effects, as maximal neutralization with the 4LCA and
6A antibodies was only observed when both were biotinylated.
Accelerated RBC destruction is not likely to be a factor in BoNT
clearance, as mice treated with FP do not exhibit a reduced
hematocrit (data not shown).
Our study has shown the value of immunoadherence as an
effective mechanism for improving the neutralizing ability of
BoNT mAbs. The potency of the FP:mAb complexes and their
utility in the pre-exposure setting demonstrated that
immunoadherent immune complexes could be used to protect those at risk of
BoNT exposure, in addition to those already exposed. In practice,
the FP could be held in a biodefense stockpile as a non-specific
immune adjuvant, to be combined with biotinylated MAb specific
for the toxin(s) to which people have been exposed. Alternatively,
FP sequences could be used to create hybrid MAb molecules that
combine, in a single polypeptide, RBC immunoadherent and
antitoxin activities in a single construct. An important advantage of the
FP is that it can be ligated quickly and irreversibly to any molecule
that has been biotinylated. This allows the creation of
immunoadherent complexes without having to synthesize novel fused
polypeptides or add synthetic linkers. Such experimental flexibility
will facilitate the study of factors that affect the potency,
distribution and metabolism of different FP-containing complexes
in vivo in order to optimize their function as an accessory
Materials and Methods
Fusion protein construction and purification
DNA for the fusion protein scFv (provided by Dr. James
Atkinson, Washington Univ., St. Louis, MO) was fused in frame
with coding sequences for core streptavidin (provided by Dr.
Stephan Dubel, Technical Univ. of Braunschweig, Germany,
based on Pahler, et al. ), followed by a polyhistidine sequence
and inserted into pET 21a(+). BL21(DE3)pLysS cells (Invitrogen,
Carlsbad, CA) were transformed with the resultant plasmid and
expression of the recombinant protein was induced with IPTG. FP
was purified from bacterial lysate using an SP-sepharose and a
His-Select Nickel Affinity gel (Sigma-Aldrich) and eluted with
TUB/100 buffer (60 mM Tris-HCl, 8 M urea, 100 mM
imidazole, pH 8.0). FP was dialyzed in a series of buffers containing
50 mM Tris-HCl and 0.4 M arginine, slowly decreasing the urea
concentration. The final preparation is in a buffer of PBS-Arg,
pH 7.4 (5 mM NaH2PO4, 70 mM NaCl, 0.4 M arginine).
Biotinylation and fluorescent labeling of mAbs
6A, 4LCA, and 13A antibodies were biotinylated using a
FluoReporter Mini-Biotin-XX Protein Labeling Kit (Molecular
Probes, Eugene, OR). HC50A, produced as in , was
conjugated to Alexa488 using a DyLight 488 Antibody Labeling
Kit and inactivated BoNT/A holotoxin (mBoNT/A-488), was
labeled with a DyLight 488 Microscale Antibody Labeling Kit
(Thermo Fisher Scientific, Rockford, IL).
in vitro analysis of FP complexes binding to RBCs via flow
Heparinized RBCs collected from female Swiss Webster mice
(Taconic Farms, Hudson, NY) were diluted 1:2 in PBS:heparin
(100 U/ml) and aliquoted at 106 per well and washed with 200 ml
PBSA (PBS/1% BSA). RBCs were incubated with or without
10fold excess (4.4 mg) rat anti-mouse TER-119 or rat IgG2b isotype
control (eBiosciences, San Diego, CA) and incubated at room
temperature (RT) for 30 min. Cells were spun down at 2000 rpm
in an Allegra 6R centrifuge with a GH3.8 rotor
(BeckmanCoulter, Brea, CA) 5 min and incubated with 400 ng FP in
100 ml PBSA for 45 min. The mAb 6A, biotinylated or
unbiotinylated, was incubated with A1-Alexa488 for 1 hr at RT, and
then added to RBCs and incubated for 30 min. Cells were washed
twice in PBSA and F(ab)2 donkey anti-human IgG APC (Jackson
ImmunoResearch, West Grove, PA) added at 1:10,000 and
incubated at RT for 30 min. Cells were washed twice in PBSA and
resuspended in a final volume of 1 ml PBSA and analyzed on a
BD FACSCantoII (Becton Dickson, Franklin Lakes, NJ) using
FlowJo 8.8.6. software (Tree Star, Ashland, OR). Competition
experiments were performed as above by incubating 400 ng FP
with biotin-fluorescein (Thermo Fisher Scientific) in 10-fold molar
excess for 50 min, adding to RBCs pre-incubated with TER-119
as described previously for 30 min, washing twice and analyzing
Plasmid construction and bacterial expression of
detoxified botulinum toxin type A (BoNT/A) protein
A codon-optimized BoNT/A igene was constructed (GenScript
USA, Piscataway, NJ). The gene contained 4 point mutations,
R363A and Y365F, which abolish the catalytic activity of BoNT/
A , and W1266L and Y1267S, which prevent binding of
BoNT/A at the neuromuscular junction . The gene also
encoded an N-terminal polyhistidine tag followed by an
enterokinase site. The gene was inserted into the pTRCHisA
vector, yielding the expression plasmid pTRC-detoxBoNT/A, and
expressed in E. coli BL21-codon plus(DE3)-RIL (Agilent
Technologies, Santa Clara, CA). Cells were grown in Terrific Broth
(1.2% peptone, 2.4% yeast extract, 0.94% K2HPO4 and 0.22%
KH2PO4) (Difco; Sparks, MD) at 37uC to ,0.8 OD600, at which
time IPTG was added, the culture was cooled to 20uC in an ice
bath, and the cells were incubated, shaking, for 12 hours.
Purification of protein
Bacterial cells from 1 liter of culture were suspended in 200 ml
of bacterial protein extract reagent, B-PER (Pierce; Rockford, IL)
at 4uC. Lysozyme (Sigma; St. Louis, MO) at a final concentration
of 0.1 mg/ml, DNASE (Sigma) at a final concentration of
0.01 mg/ml, and protease inhibitor cocktail tablet (Roche;
Manheim, Germany) were added to the cell suspension and
incubated on a rotating shaker for 2 hr. Four hundred ml of
50 mM sodium phosphate containing 300 mM NaCl, pH 8.0, was
added to the lysed cell suspension and allowed to stand for 30 min.
The suspension was centrifuged at 27,000 x g for 40 min to
The clear supernatant was loaded onto a 5 ml column of
NiNTA superflow (Qiagen) which was equilibrated with 50 mM
sodium phosphate containing 300 mM NaCl, pH 8.0. The
column was washed with 50 volumes of washing buffer (50 mM
sodium phosphate containing 300 mM NaCl, and 20 mM
imidazole, pH 8.0). Bound protein was eluted from the column
with a gradient of increasing imidazole (100 ml of 50 mM sodium
phosphate containing 300 mM NaCl and 20 mM imidazole, and
100 ml of 50 mM sodium phosphate containing 300 mM NaCl
and 250 mM imidazole, pH 8.0). The active fractions (at
,80 mM imidazole) were pooled and dialyzed against 50 mM
sodium phosphate, pH 6.8. The dialysate was centrifuged at
27,000 x g for 30 min to remove precipitate.
The clear supernatant was loaded onto a 4 ml cation exchange
column of CM Sepharose fast flow (Amersham Bioscience;
Piscataway, NJ) equilibrated with 50 mM sodium phosphate,
pH 6.8. The column was washed with 50 volumes of 50 mM
sodium phosphate, pH 6.8. Bound protein was eluted from the
column with a stepwise increasing of NaCl concentration (10, 20,
40, 60, 100, 150, 200, 300 and 500 mM of NaCl with 50 mM
sodium phosphate pH 6.8). The active fractions (at ,200 mM
NaCl) that correspond to about 150 kDa protein on a 10% SDS
polyacrylamide gel electrophoresis were pooled and dialyzed
against PBS. The purity of detoxified BoNT/A was confirmed on
the SDS-PAGE and found to be more than 98% homogeneous.
The identity of the BoNT/A was confirmed by Western blot
analysis using rabbit polyclonal antibodies raised separately
against the catalytic domain (LC) and the heavy chain domain
(HC50) of pure BoNT/A toxin.
Digestion with enterokinase and generation of dichain
C. botulinum produces BoNT/A as a di-chain active protein
molecule (nicked). Recombinant detoxified BoNT/A purified
from E. coli was treated with a protease enterokinase (EK) to
make it a di-chain nicked molecule. One milligram of purified
detoxified BoNT/A was incubated with five units of enterokinase
in a 1.5 ml of EK-Max buffer as described in EK-MaxTM kit
(Invitrogen) for O/N at 25uC. EK was then removed by
EKaway TM resign (Invitrogen). The digested protein sample was
then diluted with 5 bed volumes of 50 mM sodium phosphate,
300 mM NaCl, and 10 mM imidazole (pH 8.0). The solution
was centrifuged at 27,000 x g for 40 min to remove any
The clear supernatant was passed through a 2 ml column of
NiNTA superflow (Qiagen) equilibrated with 50 mM sodium
phosphate, 300 mM NaCl, pH 8.0. The N-terminal polyhistidine
tag that was cleaved of from the detoxified BoNT/A protein
molecule was trapped by Ni-NTA resin and the remaining
digested protein molecule passed through the column. The
passthrough sample was dialyzed against PBS and concentrated using
30K ultracentricon (Millipore). The di-chain nicked detoxified
BoNT/A was compared with the native BoNT/A by reducing
SDS-PAGE and found to have two bands (a 50 kDA light chain
and a 100 kDa heavy chain).
Blood plasma BoNT ELISA determination
For measurement of clearance of BoNT from the circulation
by FP, the detoxified BoNT/A was used. 6 mg of detoxified
BoNT/A was incubated with 3 mg biotinylated 6A, 3 mg
biotinylated 4LCA, and 24 mg FP for 1 hr at RT prior to tail
vein injection in 25 g female Swiss Webster mice. Control mice
were given toxin only or no injection. Mice were anesthetized
under isofluorane 90 mins after toxin administration and whole
blood was collected by cardiac puncture with a heparinized
syringe. Blood was separated into microfuge tubes and spun for 5
minutes at 3,000 RPM in a microcentrifuge for 5 minutes. The
plasma was aliquoted and stored at 220uC until use. To assay
BoNT/A in plasma, black 96-well flat bottom (Nunc Maxisorp)
plates were coated at 4uC overnight with 100 ml/well of 3B3, a
human mAb that binds BoNT/A, at 2 mg/ml in PBS. Plates were
washed with PBS/0.05% Tween-20 (Sigma-Aldrich, St. Louis)
and then blocked for 1 h at 37uC with PBS/0.05% Tween-20/
5% non fat dry milk. Undiluted samples were added at 100 ml/
well, incubated for 2 hours at 37uC, washed, and followed by
addition of rabbit anti-HC50A serum at 1:100 dilution. After one
hour, goat anti-rabbit-HRP was used at 100 ml/well (1:10,000
dilution) and incubated for an additional hour. OPD was
used as the colorimetric substrate; optical density at 490 nm
BoNT/A1 and BoNT/B were obtained from Metabiologics
(Madison, WI). LD50 eqivalents were 2.5 pg/LD50 for BoNT/A1
and 5 pg/LD50 for BoNT/B.
All animal work was conducted according to all relevant
guidelines in a protocol approved by the Institutional Animal Care
and Use Committee of the Lankenau Institute for Medical
Research, covered by protocol number A08-2692, Approval Date:
August 26, 2008, last amendment approval date, July 15, 2009,
Animal Welfare Assurance number A3550-01.
We are grateful to the laboratories of Lance Simpson at Thomas Jefferson
University and of George Prendergast and Scott Dessain and to the
Lankenau Institute for Medical Research.
Conceived and designed the experiments: SPA ATS MDE BHS LLS PMS
SKD. Performed the experiments: SPA ATS CC RB MDE BHS PMS.
Analyzed the data: SPA ATS MDE BPK LLS PMS SKD. Contributed
reagents/materials/analysis tools: SPA MDE BHS LLS PMS. Wrote the
paper: SPA ATS MDE PMS SKD. Edited the manuscript: BHS BPK
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