Potent Neutralization of Botulinum Neurotoxin/B by Synergistic Action of Antibodies Recognizing Protein and Ganglioside Receptor Binding Domain
et al. (2012) Potent Neutralization of Botulinum Neurotoxin/B by Synergistic Action of Antibodies
Recognizing Protein and Ganglioside Receptor Binding Domain. PLoS ONE 7(8): e43845. doi:10.1371/journal.pone.0043845
Potent Neutralization of Botulinum Neurotoxin/B by Synergistic Action of Antibodies Recognizing Protein and Ganglioside Receptor Binding Domain
Changchun Chen 0
Shuhui Wang 0
Huajing Wang 0
Xiaoyan Mao 0
Tiancheng Zhang 0
Guanghui Ji 0
Xin Shi 0
Tian Xia 0
Weijia Lu 0
Dapeng Zhang 0
Jianxin Dai 0
Yajun Guo 0
Michel R. Popoff, Institute Pasteur, France
0 1 School of Pharmacy, The Center for Antibody Medicine of Ministry of Education, Shanghai Jiao Tong University , Shanghai , People's Republic of China, 2 International Joint Cancer Institute, The Second Military Medical University , Shanghai , People's Republic of China, 3 National Engineering Research Center for Antibody Medicine, State Key Laboratory of Antibody Medicine and Targeting Therapy and Shanghai Key Laboratory of Cell Engineering , Shanghai , People's Republic of China, 4 Lanzhou Institute of Biological Products , Lanzhou, Gansu , People's Republic of China, 5 PLA General Hospital Cancer Center , Beijing , People's Republic of China
Background: Botulinum neurotoxins (BoNTs), the causative agents for life-threatening human disease botulism, have been recognized as biological warfare agents. Monoclonal antibody (mAb) therapeutics hold considerable promise as BoNT therapeutics, but the potencies of mAbs against BoNTs are usually less than that of polyclonal antibodies (or oligoclonal antibodies). The confirmation of key epitopes with development of effective mAb is urgently needed. Methods and Findings: We selected 3 neutralizing mAbs which recognize different non-overlapping epitopes of BoNT/B from a panel of neutralizing antibodies against BoNT/B. By comparing the neutralizing effects among different combination groups, we found that 8E10, response to ganglioside receptor binding site, could synergy with 5G10 and 2F4, recognizing non-overlapping epitopes within Syt II binding sites. However, the combination of 5G10 with 2F4 blocking protein receptor binding sites did not achieve synergistical effects. Moreover, we found that the binding epitope of 8E10 was conserved among BoNT A, B, E, and F, which might cross-protect the challenge of different serotypes of BoNTs in vivo. Conclusions: The combination of two mAbs recognizing different receptors' binding domain in BoNTs has a synergistic effect. 8E10 is a potential universal partner for the synergistical combination with other mAb against protein receptor binding domain in BoNTs of other serotypes.
Funding: This work was supported by grants from National Nature Science Foundation for China, Ministry of Science & Technology of China (973 and 863
Program Projects), Shanghai Commission of Science & Technology (Key Laboratory and projects) as well as special program projects for infectious diseases and
new drug development from Ministry of Science & Technology of China (2010ZX09401-407). Dr. Jianxin Dai is recipient of Pujiang Scholar Award from Shanghai
Commission of Education. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
. These authors contributed equally to this work.
Botulinum neurotoxins (BoNTs) comprise a group of highly
lethal toxins consisting of 7 serotypes (BoNT/A-G) produced by
the anerobic bacteria, Clostridium botulinum [1,2]. Four of the
BoNT serotypes (A, B, E, and F) cause human botulism, a
neuroparalytic disease which results from ingestion of pre-formed
toxin present in contaminated food and from toxin produced in
vivo from infected wounds . Owing to their extreme potency and
lethality, BoNTs are included in the list of category A select agents
and toxins . Each BoNT isoform is synthesized as a single
polypeptide chain with a molecular mass of ,150 kDa. The
inactive precursor protein is cleaved either by clostridial or tissue
proteases into a 50-kDa light chain (LC) and a 100 kDa heavy
chain (HC) linked by an essential interchain disulfide bridge and
by the belt, a loop from the HC that wraps around the LC .
The LCs act as zinc metallopeptidases, which solely hydrolyze one
of three SNARE proteins depending on the serotype: BoNT A and
E cleave synaptosomal-associated protein of 25 kDa (snap-25) and
BoNT/B and F cleave the vesicle associated membrane protein
(VAMP) [6,7,8,9]. resulting in a blockade of neurotransmission
and flaccid paralysis . The heavy chain is divided into two
functionally distinct regions: a C terminal binding domain (Hc)
and a N terminal translocation domain (HN) . The binding
domain initially interacts with low affinity to a group of
gangliosides on the presynaptic plasma membrane , after
which it binds to a protein acceptor. Interestingly, the BoNTs
serotypes that exhibit highest sequence similarity share the same
protein receptor, i.e., BoNT types A, E, and F bind SV2
[13,14,15], whereas BoNT types B bind SytI and II . The
existence of two classes of binding sites distinguished by different
affinities and the discovery of protease-sensitive binding to neurons
resulted in a double-receptor concept. In a first step complex
polysialogangliosides accumulate BoNTs on the plasma
membrane surface; and in a second step, protein receptors mediate
Monoclonal antibodies (mAbs) have been intensely explored as
inhibitors of the recognition step between BoNTs and their cellular
receptors.  However, the two-receptor model makes it difficult
to development of a mAb-based antitoxin for botulism. Single
mAb recognizing only one epitope can hardly block the binding
between BoNT and cell completely. It might be the reason why
single mAb can only neutralize at most 10 to 100 times the 50%
lethal dose (LD50) of toxin in mice . Combination of two or
three mAbs recognizing nonoverlapping epitopes can neutralize
BoNT A very potently . Although the binding areas of
antibodies were mapped, however, the epitopes which these
antibodies bind to are not finely defined. However, not all
randomly paired mAbs have the potent synergistical neutralizing
function. It is more difficult to select mAb pairs with desirable
synergistical function from a panel of neutralizing mAbs whose
epitopes are not clear. Because both the protein receptor and
ganglioside receptor are essential for the entrance of BoNTs into
neurons , we predicted that two monoclonal antibodies, which
recognized protein and ganglioside receptor binding domain
respectively, can neutralize synergistically against BoNTs.
In this study, we selected three neutralizing mAbs, which
recognize different non-overlapping epitopes of BoNT/B, and
compare the neutralizing effects among different antibody
combinations. We found that 8E10, response to ganglioside
receptor binding site, could collaborate with 5G10 and 2F4,
recognizing non-overlapping epitopes within Syt II binding sites.
However, the combination between two mAbs, 5G10 and 2F4,
blocking protein receptor binding sites, did not achieve
synergistical effects. We also found that the binding epitope of 8E10 was
conserved among BoNT A, B, E and F. It could cross-protect the
challenge of different serotypes of BoNTs in vivo. The results
provide a potential universal partner for the synergistical
combination with other mAb against protein receptor binding
domain in BoNTs of other serotypes.
1. A panel of neutralizing antibodies specific for BoNT/BHc
To generate mAbs capable of neutralizing BoNT/B, gene
encoding protective antigen Hc fragment of botulism neurotoxin
serotype B (BoNT/B-Hc) was optimized and synthesized. The
reconstructed Hc proteins were expressed in E.coli BL21 (DE3) in
soluble form and purified for preparation of a toxoid immunogen.
Sixteen hybridomas were obtained and screened for the
production of anti-BoNT/B Hc mAbs, using ELISA with purified
BoNT/B Hc (Fig. 1A). Cell culture medium corresponding to the
positive wells was subjected to western blot assay for testing the
binding specificity of these mAbs (Fig. 1B). The results showed that
all of the positive hybridomas were able to bind to native or
denatured BoNT/B Hc specifically. The supernatants of these
clones were then tested for their ability to protect mice against 4
LD50 of BoNT/B for 4 days. Among them, 7 mAbs (8D1, 8E10,
1F4, 2F4, 5G10, 2H12 and 2B2) could neutralize BoNT/B in vivo
2. Blocking activities of the neutralizing mAbs
The first step in BoNT attachment to nerve membranes starts
with C-terminus of Hc binding to gangliosides of neuron
membranes. Ganglioside binding could either bring the toxin
and the protein receptor into close proximity or cause a
conformational change at the protein receptor binding site to
enhance interactions . To identify binding domain which was
blocked by the seven neutralizing mAbs, we used ELISA-based
analysis of GT1b or SytII binding with BoNT/B Hc pre-incubated
with or without mAbs. The results indicated that mAb 8E10, 1F4
and 2B2 blocked the interaction between BoNT/B Hc and GT1b
(Fig. 2A), whereas 8D1, 2F4, 5G10 and 2H12 interrupted the
binding between BoNT/B Hc and Syt II (Fig. 2B).
To further investigate if the epitopes recognized by the
antibodies were overlapped, we tested mAbs using competitive
binding ELISAs. The optimal dilution of each HRP-conjugated
mAb was determined by testing serial two-fold dilution in BoNT B
Hc-coated ELISA plates and selecting this as the working dilution
to generate an optical density of around 1.5. The selected working
dilution was 1/8000 for mAbs 2B2 and 2F4, 1/16000 for mAbs
8D1, 1F4, 8E10, 5G10 and 1/4000 for 1F4, 2H12. The results of
competitive ELISAs were reported in Table 1.
HRP-conjugated 8E10 inhibited the binding of 1F4 or 2B2 to
the antigen and vice versa, showing that 8E10, 1F4 and 2B2
recognized overlapping epitopes. HRP-conjugated 5G10 inhibited
the binding of 2H12 to the antigen and vice versa, indicating that
5G10 and 2H12 recognized overlapping epitopes. The same
competition was also observed between 2F4 and 8D1. MAbs were
thus categorized into 3 groups. MAbs binding to the same epitopes
or overlapping epitopes were put into the same group (Table 2).
Group 1 consisted of mAbs 8E10, 1F4 and 2B2, group 2 included
mAbs 5G10 and 2H12 and group 3 included mAbs 2F4 and 8D1.
Because the antigen binding affinity of the mAbs 8E10, 5G10 and
2F4 was higher than that of other 4 neutralizing mAbs (Table 1),
they were selected for fine epitope mapping based on
3. Epitope mapping of mAb 8E10, 5G10 and 2F4
After three successive rounds of panning on 8E10, 5G10 and
2F4 respectively, we randomly selected and purified 100 positive
phages. DNA sequence analysis of the phage-encoded peptides
revealed 46 amino-acid consensus sequences, which was aligned
with the sequence of BoNT/B displayed in Table 3. All phages
selected by 8E10 have a common motif of SXWY. Alignment of
this sequence on the sequence of BoNT/B provides a best fit with
1259SKWY1262. 5G10 and 2F4 bind to SDXFY and KXSP,
which correspond exactly to residues 1201SDEFY1205 and
1114KDSP1117 of BoNT/B respectively. The peptides containing
1259SKWY1262, 1201SDEFY1205 and 1114KDSP1117 motif in
the BoNT/B were synthesized and denoted as P1, P2 and P3
respectively. The ELISA assay results showed that mAb 8E10,
5G10 and 2F4 bound to KLH-conjugated P1, P2 and P3 peptides
(P1, P2, P3-KLH) specifically since these mAbs did not cross-react
with the other peptides recognized by the other two mAbs and
control peptide-KLH (CP-KLH)(Fig. 3A). The capability of the
synthesized peptides to block the binding of mAb 8E10, 5G10 and
2F4 to BoNT/B Hc was also determined. The results revealed that
peptide P1, P2, P3 effectively inhibited the binding of mAb 8E10,
5G10, 2F4 to BoNT/B Hc respectively. In contrast, control
peptide had not inhibitory effect on their interaction (Fig. 3B).
These results further demonstrated that the motifs listed above
were the epitopes of 8E10, 5G10 and 2F4 respectively.
BoNT/B Hc has been crystalized in complex with trisaccharide
sialyllactose (a mimic of GT1b) (PDB 1F31) and with SytII peptide
(PDB 2NM1). The crystal structure reveals two distinguished
domains which bind to GT1b and SytII respectively. Therefore, it
is possible to compare the binding site of protein and ganglioside
receptor to BoNT/B Hc with the localization of the mAb 8E10,
5G10 and 2F4 epitope identified in this study. The illustration of
Results were expressed as percent binding of HRP-conjugated mAbs. The amount of binding obtained in the absence of unlabeled antibody was set at 100% for each
HRP conjugated mAb. First column indicates the group assignments of mAbs based on competition binding assay. mAbs binding to overlapping epitopes are grouped
together, while non-competing mAbs are grouped individually.
structure of BoNT/B Hc is showed in Fig. 4. The epitopes of
mAbs were colored and overlaid on the three-dimensional
structure of BoNT/B. The epitope of 8E10 (colored in red) is
just located inside the GT1b-binding domain (colored in yellow)
(Fig. 4 A). The residues 1114KDSP1117 recognized by 2F4
(Fig. 4B, blue) cover part of the Syt II binding pocket formed by
residues K1114, S1116, P1117, V1118, Y1183, E1191, K1192,
F1194 and F1204 (Fig. 4B, green). The 5G10 epitope comprises 5
amino acids 1201SDEFY1205 (Fig. 4B, cyan), which overlaps
another side of Syt II binding site formed by residues P1117,
W1178, Y1181, F1194, A1196, P1197 and F1204 (Fig. 4B, green).
However, the epitopes of 5G10 and 2F4 are not overlapping with
each other. These results provide the structure basis of neutralizing
functions of these mAbs. The mAb 8E10 neutralizes BoNT/B
through blocking the interaction between BoNT/B Hc and its
ganglioside receptor on neurons. 5G10 and 2F4 inhibit the
binding of BoNT/B Hc with protein receptor SytII.
4. Binding inhibition assay in vitro
In order to determine the toxin neutralization by IgG in vitro, we
used SytII transfected and ganglioside treated PC12 cells, a
neuroendocrine cell line of which wild type is lack of functional
toxin receptors , to serve as a target cell model. Flow
cytometry results showed that FITC-labeled recombinant BoNT/
B Hc could bind to SytII transfected and ganglioside treated
PC12. 8E10 could completely block the interaction between
BoNT/B Hc and ganglioside treated PC12 (Fig. 5A). 5G10 and
2F4 could inhibit BoNT/B Hc to bind to the SytII transfected
PC12 effectively (Fig. 5B). However, as to the SytII transfected and
ganglioside treated PC12, none of the mAbs above could
completely block its binding to BoNT/B Hc. In contrast with
the single mAb, the combination of 8E10 with 5G10 or 2F4 could
completely inhibit the binding of BoNT/B Hc to the double
treated PC12, while the combination of 5G10 with 2F4 could not
block the binding of PC12 to BoNT/B Hc (Fig. 5C). The results of
confocal microscope showed that the combination of 5G10 and
2F4 could only decrease the fluorescence signal on the membrane
of double treated PC12 similar to the effects of single antibody
treatment. In contrast, the mixture of 8E10 and 5G10 or 2F4
abrogated the fluorescence signal from the cell membrane
completely (Figure S1). These results demonstrated that mAbs,
which recognized ganglioside or SytII binding domain
respectively, had the potent to neutralize BoNT/B synergistically.
5. Protection of mice from BoNT/B toxicity using neutralizing mAbs
In vivo toxin neutralization was studied using a standard mouse
protection assay. We incubated 100 mg mAbs with BoNT/B for
1 hour prior to intraperitoneal injection into BALB/c mice and
the groups of mice with 6 animals for each group were used to test
dose level. Mice were observed for morbidity and mortality over
30 days. Complete protection was observed with doses up to 20
LD50s for 8E10, and 40 LD50s for 5G10 and 2F4. Partial
protection, as indicated by increased survival compared with
antibody-free control mice, was afforded with higher doses
(Table 4). At 80 LD50s, mice receiving 5G10 and 2F4 survived
72 hours, compared to 6 hours for the control mice. As
combination of mAbs have demonstrated synergy in BoNT/A
neutralization , the different pairs of mAb were studied at
increasing doses of toxin to explore potent combination. We mixed
50 mg each of the mAbs (total of 100 mg) with different LD50s
BoNT/B and tested the combination by intravenous injection. At
640 LD50s, only 1 of 6 mice receiving the pair of 5G10+2F4
survived whereas none of mice receiving two of the pairs of mAbs
(8E10+5G10 or 8E10+2F4) died. 3 of 6 mice that received the pair
of 8E10+5G10, and 2 of 6 mice that received the pair of
8E10+2F4 survived for 96 hours in the group challenged with
1,280 LD50s. In contrast, the control mice died in less than
Phage-displayed peptide sequence
Absolute and relative frequency
--1259SKWY1262--Phage-displayed consensus amino acids are shown in boldface.
Figure 4. Molecular model overlay of neutralizing epitopes within the BoNT/B Hc binding domain. The model was established using the
software Discovery Studio 2.0 (Accelrys, San Diego, CA) based on the crystal structure of BoNT/B Hc (PDB 1F31) from the Protein Data Bank. BoNT/B
Hc is shown in a surface representation. (A) The residues reported as GT1b-binding site are colored yellow (Nat. Struct. Biol. 1998), and the residues
recognized by 8E10 are colored red. (B) The SytII-binding site residues are colored green, and the residues of 5G10 and 2F4 are indicated in cyan and
3 hours. The survival rate of the group received 8E10+5G10+2F4
is the same as that of the group received the pair of 8E10+5G10.
No mice survived in the group challenged with 2,560 LD50s (Data
6. Neutralizing potency of mAb 8E10 against different serotypes of BoNTs
Given the binding epitope of 8E10 is located at the conservative
motif of BoNT A, B, E, F (Fig. 6A), we wondered whether it could
bind and neutralize across these serotypes. To evaluate this, we
first expressed the Hc domain of BoNT A, E and F, and
determined the binding kinetics of the 8E10 antibody to the
BoNT/A, B, E, and F Hc by surface plasmon resonance (SPR)
and showed the results in Table 5 and Figure S2. The results show
that the binding affinity between 8E10 and BoNT/A or F is a little
bit lower than the binding affinity between 8E10 and BoNT/E,
whereas the affinity between 8E10 and BoNT/B is the highest.
The fact that all four serotypes of BoNTs bind 8E10 with
nanomolar efficiency demonstrates that all serotypes tested are of
high affinity. The results of ELISA (Fig. 6B) and western blotting
(Fig. 6C) showed that 8E10 could bind with native and denatured
Hc of BoNT A, B, E, F, indicating that 8E10 recognized the
conserved linear epitope among these BoNTs. In addition, we
compared the potency of BoNT/A, B, E and F neutralization in
vivo by mAbs 8E10. All of 6 mice challenged with 20 LD50s of
BoNT/B were protected by 100 mg of 8E10. Same dosage of 8E10
protected 5/6 of mice challenged with 20 LD50s of BoNT/A or F,
respectively and 4 of 6 mice also survived under the challenge of
20 LD50s of BoNT/E (Fig. 6D). In contrast, none of the mice
survived longer than 24 hours after challenged with BoNTs
without the injections of mAbs.
For the last two decades, the relationship between the structure
and function of BoNT has been studied intensively. With the
complex structure of BoNT/B and its receptors, protein receptor
Syt-II  and its ganglioside receptor GT1b , we can
combine these data to provide the structure basis of the double
receptor interaction proposed by Montecucco et al. .
Biofunctional assays also provided the evidence that both protein
receptor SytII and ganglioside co-receptor were necessary for the
infective process of BoNT/B . This led us to the hypothesis
that a combination comprised of a pair of neutralizing antibodies
that bound to different receptors binding domains of the toxin
would be more effective neutralizers than either member of the
pair alone. We generated three mAbs, 8E10, 5G10 and 2F4 which
could neutralize the challenge of 20 LD50 of BoNT/B in vivo.
Finely epitope mapping revealed that 8E10 recognized ganglioside
binding domain in BoNT/B, 5G10 and 2F4 bound with 2
nonoverlapping epitopes surround the SytII binding domain
respectively. In addition, we found that the combination between 8E10
(recognizing ganglioside binding domain) and 5G10 or 2F4
(recognizing protein receptor binding domain) could result in a
more than 3060 folds increase in potency compared with that of
any the single mAb. However, for the combination of 5G10 and
2F4, which both blocked protein receptor binding sites by
nonoverlapping epitopes, no synergistic effect was observed. This
indicates that double blocking the ganglioside and protein receptor
binding domain simultaneously with two mAbs is helpful for the
synergistic effects of the two antibodies combination against
The combinations of antibodies, which recognize
nonoverlapping epitopes, synergistically cooperate in neutralization potency
have been reported previously . In that study, random
combinations between 2 of the 3 neutralizing mAbs significantly
prolonged the time to neuroparalysis compared with single mAbs.
However, in our study, 2 mAbs, which bind to the same function
domain of BoNT/B, had much less synergistic effects than those
whose epitopes located in different function domains of BoNT/B.
In addition, a BoNT/B-specific triplex antibody combination
exhibited cooperative neutralizing effects to the toxin in vivo that
were no better than those of the pairs of antibodies (Table 4).
These findings are different from the conclusion of the previous
report. Fine epitope mapping showed that the two mAbs (3D12
and S25) which bind the BoNT/A HCC (C terminal of Hc) overlap
the putative sialoganglioside binding site and cover a large portion
of HCC. The other mAb C25 bound a conformational epitope that
consisted of the sequence from the N- and C-terminal subdomains
of BoNT/A Hc, which overlapped with a putative inositol
phosphate binding site that may be important for attachment to
the lipid membrane. This function domain is essential for the
binding to anionic lipid in the environment of lipid raft, which is
important for the translocation processing of the toxin. [25,26].
Interestingly, the neutralizing potency of the pair of mAbs
(C25+3D12), which bind to translocation domain and
sialoganglioside binding domain respectively, is 10 times higher than that
of S25+3D12, whose epitopes are located at the same function
domain. This finding provides evidence that the synergetic effects
of mAbs, which recognize different function domains of BoNT/A,
are better than the effects of those recognizing the same function
domain. Although we didnt observe the cooperative effects
between the mAbs binding to SytII binding domain of BoNT/B,
we did prove that the combination of two mAbs recognizing
different receptors binding domain in BoNT/B has a synergistic
effect. Accordingly, the data indicate that the combination
between 2 mAbs recognizing different function domains in BoNTs
could be the general principle for the potent synergistic effect.
Neutralizing mAbs binding multiple serotypes of botulinum
neurotoxin are rare have been reported previously . The
crossreactive mAbs bound to a relatively conserved epitope at the tip of
the BoNT HN. This is a functionally important epitope for
intoxication, as mAb binding leads to potent BoNT neutralization.
In this study, a neutralizing mAb 8E10 binding the conservative
domain on Hc was reported. The structure of Hc from BoNT/B
(PDB 2NM1) , BoNT/E (PDB 3FFZ)  and BoNT/F (PDB
3FUQ)  supports the view that the Hc fold is highly conserved.
GT1b binds on a cleft formed by W1266 and Y1267 on one face
Survival time (hours)
and E1203, H1253, and F1252 on the other for BoNT/A .
The structure of Hc from BoNT/B in complex with the
trisaccharide sialyllactose, a mimic of GT1b (PDB 1F31), displays
a similar binding cavity with corresponding residues W1261,
Y1262, and H1240 . In addition, the crystal structure of Hc
from BoNT/F displays a ganglioside-binding pocket with
corresponding residues W1250, Y1251, and H1241 . Amino acid
sequence alignment shows that S1264, W1266 and Y1267,
conserved among all BoNT serotypes , constitute key residues
of a lactose-binding motif (H. . .SXWY. . .G) that contribute to the
crevice binding with GT1b. The implication is that the
GT1bbinding pocket for all these BoNTs is similar. We found in this
study that the motif 1259SKWY1262 is the recognizing epitope of
8E10, which could also cross react with the Hc of BoNT A/B/E/F
(Fig. 6). This conserved epitope may partially explain how 8E10
could cross-protect the challenge of different serotype of BoNTs in
vivo. Although its synergistic effect with the mAbs blocking protein
receptor binding domain in BoNT A, E or F has not been
detected, we can predict that 8E10 could act synergistically with
the mAbs recognizing protein receptor binding domain against
BoNT A, E or F. This study provides a potential universal partner
for the synergistical combination with other mAb against protein
receptor binding domain in BoNTs of other serotypes.
Materials and Methods
1. Ethics Statements
Mice were purchased from Animal Center of Chinese Academy
of Sciences and maintained under pathogen-free conditions. All
animal experimental procedures were carried out in strict
accordance with the guidelines of the Animal Experiment
Committee of the International Joint Cancer Institute, and were
approved by the Animal Experiment Committee of the
International Joint Cancer Institute.
2. Holotoxin, Antigens, protein and peptides
BoNT/A, BoNT/B, BoNT/E and BoNT/F were provided by
Lanzhou Institute of Biological Products (Lanzhou, China). DNA
encoding BoNT/A Hc (residues 868 through 1296), BoNT/B Hc
(residues 853 through 1291), BoNT/E Hc (817 through 1255),
BoNT/F Hc (847 through 1280), and Syt II-LD fragment (residue
37 through 86) were synthesized and subcloned into a pET
expression vector, expressed in E. coli BL21(DE3). The
recombinant histidine tagged BoNT/A, B, E, F Hc and Syt II-LD
fragment were isolated and purified by nickel affinity gel column
chromatography and their molecular weight and purity were
verified by gel electrophoresis [31,32].
Peptides used in this study, P1 (YFCISKWYLKEV),
P2 (PISDSDEFYNTI), P3 (IKLKKDSPVGEI), P4
(THPHLPRALMRS), were synthesized by Yeli Bio-Scientific
Inc. (Shanghai, China).
3. Animals and cell lines
6- to 8-week-old female BALB/c mice were purchased from
Animal Center of Chinese Academy of Sciences and maintained
under pathogen-free conditions. PC12 (rat adrenal
pheochromocytoma cells) was purchased from Cell Bank of Chinese Academy
of Science (Shanghai, China). To generate cells that express syt II
(syt II), full-length mouse syt II (Genechem. Shanghai, China) was
subcloned into pCDNA3.1 (CLONTECH, Mountain View, CA)
and transfected into PC12 cells via electroporation. Transfected
cells were selected with 1 mg/ml G418, and several independent
monoclonal cell lines were established. For experiment in which
cells were preloaded with gangliosides (Merk Chemicals,
Darmstadt, Germany), cells were grown to 80% confluence followed by
incubation in serum-free media plus 250 mg/ml gangliosides. 24 h
later, the serum-free/ganglioside media was replaced with
complete media, and the cells were incubated with FITC labeled
4. mAb preparation
6-week-old female BALB/c mice were subcutaneously
immunized twice at 3-week intervals with 10 `g of BoNT/B Hc
emulsified in Freunds complete or incomplete adjuvant
(SigmaAldrich, Shanghai, China). Three days after a final immunization
with BoNT/B Hc antigen alone, spleen cells from the mice and
mouse myeloma NS1 cells (Cell Bank, Chinese Academy of
Science, Shanghai, China) were fused and maintained according
to the standard procedure . The hybridomas producing
antiBoNT/B antibodies were screened by an indirect enzyme-linked
immunosorbent assay (ELISA), using the purified recombinant
BoNT/B Hc as a coated antigen. For ascites production, 56106
hybridoma cells were injected intraperitoneally into BALB/c mice,
which had been primed with 0.5 ml pristane (Sigma-Aldrich,
Shanghai, China). Ascites fluid was collected, and the mAbs
purified, using HiTrap Protein G coupled to Sepharose 4B
(Amersham Bioscience, Uppsala, Sweden). mAb isotypes were
determined by the Mouse Typer Isotyping Panel Kit (Bio-Rad,
Hercules, CA). The binding specificities of the mAbs were
determined by western blotting. The binding strength of
neutralizing mAbs to the BoNT/B Hc was analyzed by surface
plasmon resonance technology using a Biacore T100 instrument
(Amersham Bioscience; Uppsala, Sweden) .
5. Binding specificity and cross-reactivity assay by western blot and ELISA
0.1 mg of purified BoNT/B Hc were mixed with 26 SDS
sample buffer (125 mM Tris pH 6.8, 4% SDS, 10% glycerol,
0.006% bromophenol blue, 1.8% -mercaptoethanol), boiled and
loaded onto 10% polyacrylamide gels. After electrophoresis, the
samples were transferred on nitrocellulose sheets, probed with all
of these 16 positive binding mAbs respectively (1:1000), and
stained with a goat anti-mouse IgG coupled to horseradish
peroxidase (HRP) (BD Bioscience, San Jose, CA) and developed
with ECL Plus.
To determine the cross-reactivity of 8E10, Easy Wash 96-well
plates (Corning, Corning, NY) were coated at 4uC overnight with
100 ml/well purified BoNT A/B/E/F Hc (at 5 mg/ml) respectively
(BSA was set as negative control). After blocked with blocking
buffer (5% non fat dry milk in PBS) for 2 hours at room
temperature, 1 ng of 8E10 in 100 ul PBS was added and
incubated for 2 hour at 37uC. The wells were washed and
incubated at 37uC for 1 h with HRP-conjugated goat anti-mouse
IgG at a 1:3000 dilution. After extensive washing, 3, 3, 5,
5tetramethylbenzidine (TMB) was added (50 ul/well) and
incubated for 10 min at room temperature in the dark and the reaction
was stopped by the addition of 2 M H2SO4 (50 ul/well).
Absorbance of the samples in the plates was read in an automated
ELISA microplate reader at 450 nm. For western blot, purified
BoNT A/B/E/F Hc were subjected to SDS PAGE, transfer to
nitrocellulose membrane and probed with 8E10 as described
6. Kinetic analysis
BIAcore measurements were performed with the Biacore T200
instrument (GE Healthcare) at 25uC in running buffer (10 mM
Tris pH 8.0, 100 mM NaCl, 0.005% surfactant P20, 50 mM
NiCl). CM5 chips were coated with BoNT/B Hc. 7 neutralizing
mAbs were passed over the chip surface at concentrations ranging
from 4.4 nM to 22.2 nM for 240 seconds at a flow rate of 30 ul/
min and dissociation was recorded during 60 minutes. The chip
was regenerated with 20 ml of 35 mM EDTA at 50 ml/min.
Binding kinetics were evaluated using the BiaEvaluation software
package (GE Healthcare) using a Langmuir model 1:1.
To determine the binding abilities of mAb 8E10 and BoNT/A/
B/E or F Hc, Protein A was cross-linked to the dextran surface of
a CM5 sensor chip. Protein A was immobilized using amine
coupling with 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide
hydrochloride and N-hydrosuccinimide to a density of 1000
2000 response units (RU). mAb 8E10 was captured to
approximately 100 RU. The analyte BoNT/A/B/E or F Hc was passed
over the chip surface at concentrations ranging from 4.4 nM to
22.2 nM as indicated for 240 seconds at a flow rate of 30 ml/min
and dissociation was recorded during 60 minutes. Binding kinetics
were evaluated as described above.
7. Competition ELISAs
To identify the binding domain which was blocked by the
neutralizing mAbs, BoNT/B Hc was conjugated with horseradish
peroxidase (HRP) using HRP Plus Activated Conjugation Kit
(Thermo, Rockford, IL) according to the instruction of the kit.
Easy Wash 96-well plates (Corning, Corning, NY) were coated at
4uC overnight with 100 ml/well recombinant syt II-LD (at 5 mg/
ml) or GT1b (at 10 mg/ml) in PBS, washed with PBS/0.05%
Tween-20 (Sigma-Aldrich, Shanghai, China) and then blocked for
1 hour at 37uC with PBS/0.05% Tween-20/5% bovine calf
serum/3% goat serum (Sigma-Aldrich, Shanghai, China).
HRPconjugated BoNT/B Hc was pre-incubated with neutralizing
mAbs at molar ratio of 1:100 for 1 hour at 37uC before added into
coated plate. 3, 3, 5, 5-tetramethylbenzidine (TMB) was used as a
substrate (Sigma-Aldrich, Shanghai, China) to detect bound
To determine the binding epitope of the mAbs were
overlapping or not, competition was tested by the competition-ELISA
 with some modifications. Mixtures of constant quantities of
the HRP-mAbs (8D1, 8E10, 1F4, 5G10, 2H12, 2B2 and 2F4)
conjugate in the optimal working dilutions with dilutions of the
competing non-labelled mAb were incubated on BoNT/B Hc
coated plates for 1 hour at 37uC. Follow a final wash cycle, 50 ul
of substrate and stopping solution were added and absorbance
values were determined as previously described. A control without
competing antibody (PBS) and a control containing the same mAb
(self-competition) were included in the test. Results were expressed
as percent binding of HRP-conjugated mAb. The amount of
binding obtained in the absence of unlabeled antibody was set at
100% for each HRP-conjugated mAb.
8. Epitope mapping by biopanning
Anti-BoNT/B antibodies (8E10, 5G10 and 2F4) were separately
immobilized on 96-well plate. Phages (1.561011 pfus) from Ph.D.
-12 Phage Display Peptide Library (New England Biolabs, Beijing,
China) were pre-absorbed by immobilized mouse IgG. The
phages, diluted in Tris-buffered saline, were incubated at 4uC for
1 h with immobilized mAbs. The wells were washed for five times
with TBST (0.1% Tween 20/Tris-buffered saline). Then, the
phages that bound with mAbs were amplified by direct infection
with E. coli ER2738. The amplified phages were purified by
precipitation with 20% PEG8000, 2.5 M NaCl and used in the
next cycle. Three rounds of selection were routinely performed.
The indirect binding assay to test the reactivity of anti-BoNT/B
Hc mAbs with synthetic peptides was performed in 96-well plates
as described previously  with minor modifications. The plates
were coated with KLH-peptide 1, 2, 3 respectively. Following 2
washings and blockade of free protein-binding sites, mAbs were
added to each well respectively. Mabs binding to peptide were
detected by sequential addition of an appropriate dilution of
HRPconjugated goat anti-mouse IgG. To confirm the specific of the
epitopes identified, anti-BoNT/B mAbs were incubated with
synthetic peptides (P1, P2, P3) respectively, and then the mixture
was respectively added to plates precoated with BoNT/B Hc.
After 2 h incubation, the wells were washed and detected with an
appropriate dilution of HRP-conjugated secondary antibody.
After the addition of TMB and stop solution, absorbance was
read at 450 nm by a microplate reader.
9. Flow cytometry assay
The inhibition of mAbs for cell binding activity of BoNT/B was
examined by performing flow cytometry . BoNT/B Hc was
labeled with the FITC labeling kit according to the instruction
(Pierce Biotechnology, Rockford, IL).The single or pairs of mAbs
were mixed with FITC-BoNT/B Hc at 37uC for 1 h.
Gangliosidetreated or Syt II transfected (Syt II+) or double positive PC12 cells
were labeled by overnight incubation with the mixture at 4uC and
washed with cold PBS, and examined by flow cytometry (BD
Biosciences, San Jose, CA). The PC12 cells alone and
FITClabeled Hc fragment of BoNT/B were used as systemic control
and negative control, respectively.
10. Confocal microscopy
FITC labeled BoNT/B Hc (1 mg) was incubated with control
medium, monoclonal antibody (100 mg) or pairs of mAbs (50 mg of
each Ab) for one hour at room temperature. Sub-confluent
Ganglioside-treated SytII+-PC12 cells, plated on glass cover slips,
were incubated with the BoNT/B Hc-mAbs mixtures at 4uC for
one hour and then for 2 hours at 37uC. Cells were then washed
with PBS for 15 minutes, fixed with 4% paraformaldehyde for
15 mins, and washed again before mounting. Confocal laser
scanning was performed on a Olympus Fluoview 500 system using
the 406objective, and Fluoview software was used for image
11. Neutralization of BoNT/B activity in vivo
For the initial screening of the positive hybridoma that
produced the neutralizing antibody, 0.5 ml of culture supernatant
from hybridomas grown in a 24-well tissue culture flask for 35
days, was pre-incubated with 4 LD50 of BoNT/B for 1 h at room
temperature, and the reaction mixtures intraperitoneally (i.p.)
injected to each of six mice. More than four survivals in four days
were considered as positive. In order to define the activity of the
positive neutralizing mAbs and synergistic effect of pairs of mAbs,
100 mg of the positive neutralizing mAbs were added to the
indicated number of mouse LD50 of BoNT/B neurotoxin in a
total volume of 0.5 ml of gelatin phosphate buffer and incubated
at RT for 60 min. For pairs of mAbs, 50 mg of each mAb was
added. The mixture was then injected i.p. into female BALB/c
mice (1622 g). Mice were studied in groups of six and were
observed at least daily. The morbidity and mortality of mice were
determined over 30 days. For the cross-protection assays, 100 mg
of 8E10 was pre-incubated with 20 LD50s of BoNT/A, B, E, F for
1 hour respectively. The mixtures were injected into mice, and the
final death tally was determined 4 days after injection.
Figure S1 The effect of BoNT/B-specific antibodies on binding
of BoNT/B Hc with ganglioside-treated Syt II+PC12 cells.
Ganglioside-treated Syt II+PC12 cells were cultured with
FITClabeled BoNT/B Hc (green), with or without mAbs as indicated
and visualized by confocal microscopy.
Figure S2 Binding affinity of mAb 8E10 and BoNT A/B/E/F
Hc. The 8E10 was immobilized on the surface of a Protein A
cross-linked CM5 chip. Purified BoNT/A/B/E/F Hc at
concentrations of 22.2 nM, 14.8 nM, 9.9 nM, 6.6 nM, 4.4 nM were
directly injected over the target and the reference surface. The
signal obtained from the reference surface was subtracted to avoid
the binding of non-specificity.
Conceived and designed the experiments: JXD YJG CCC HJW SHW.
Performed the experiments: CCC SHW HJW XYM GHJ. Analyzed the
data: JXD CCC HJW SHW YJG TCZ. Contributed reagents/materials/
analysis tools: CCC SHW XYM TCZ GHJ XS TX WJL DPZ. Wrote the
paper: JXD YJG CCC.
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