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Infection and Immunity, January 2001, p. 570-574, Vol. 69, No. 1
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.1.570-574.2001
High-Affinity, Protective Antibodies to the Binding
Domain of Botulinum Neurotoxin Type A
Dorothy D.
Pless,
Edna R.
Torres,
Emily K.
Reinke, and
Sina
Bavari*
Department of Cell Biology and Biochemistry,
U.S. Army Medical Research Institute of Infectious Diseases,
Frederick, Maryland 21702-5011
Received 7 July 2000/Returned for modification 31 August
2000/Accepted 3 October 2000
 |
ABSTRACT |
Monoclonal antibodies (MAbs) were prepared against the putative
binding domain of botulinum neurotoxin A (BoNT/A), a nontoxic 50-kDa
fragment. Initially, all fusion products were screened against the
holotoxin BoNT/A and against the binding fragment, BoNT/A
HC. Eleven neutralizing hybridomas were cloned, and their specific binding to BoNT/A HC was demonstrated by surface
plasmon resonance, with dissociation constants ranging from 0.9 to
<0.06 nM. Epitope mapping by real-time surface plasmon resonance
showed that the antibodies bound to at least two distinct
regions of the BoNT/A HC fragment. These MAbs will be
useful tools for studying BoNT/A interactions with its receptor,
and they have potential diagnostic and therapeutic applications.
| |
TEXT |
The anaerobic bacterium
Clostridium botulinum produces seven immunologically
distinct but structurally similar neurotoxins designated BoNT/A to
BoNT/G that are associated with food-borne, infant, and wound botulism
(12, 15, 16). Due to their unique properties, BoNTs have
been used to treat a variety of human muscle disorders
(13). After synthesis, highly active neurotoxin is generated by proteolytic cleavage of the clostridial neurotoxins. The
active neurotoxin contains two polypeptide chains connected via a
disulfide linkage. The location of the enzymatic subunit of the CNTs
has been mapped to the light chain (~50 kDa), which has Zn
endopeptidase activity (3, 12, 17). On the other hand, the
binding and translocation motifs are located within the heavy (H) chain
(~100 kDa).
Probably due to the unusually high toxicity of BoNTs, previous attempts
to produce large numbers of high-affinity neutralizing monoclonal
antibodies (MAbs) against these neurotoxins have been unsuccessful.
Since vaccination with the nontoxic binding fragment (the 50-kDa
carboxy-terminal fragment of the heavy chain [HC]) of
BoNT/A can induce protective immunity in mice (5), we
reasoned that it should be possible to generate neutralizing
antibodies by using this fragment. We report herein that
vaccination with BoNT/A HC elicited neutralizing MAbs. We
have characterized these antibodies in detail, demonstrated their
biochemical detection of BoNT/A and its binding fragment, determined
their ability to neutralize the neurotoxin, measured their affinity,
and mapped their epitope binding sites.
Antigens.
BoNT/A was purchased from the University
of Wisconsin Food Research Institute (Madison, Wis.), and
BoNT/A HC, BoNT/B HC, and BoNT/E HC
were made and purified to homogeneity at our institute (U.S. Army
Medical Research Institute of Infectious Diseases, Frederick,
Md.). The BoNT/A HC preparation was analyzed by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12%
polyacrylamide) under reducing conditions and was at least 95% pure.
Laboratory animals.
Pathogen-free BALB/c
(H-2d) mice, 10 to 12 weeks old, were obtained
from the Frederick Cancer Research and Development Center (Frederick,
Md.). The mice were maintained under pathogen-free conditions and fed
laboratory chow and water ad libitum.
Vaccination and hybridoma production.
The mice were vaccinated
intraperitoneally five times at 4-week intervals with 0.1 to 2 µg of
BoNT/A HC in 100 µl of phosphate-buffered saline. Splenic
mononuclear cells from the mice with the highest titers were collected
and fused with myeloma cells, and after the HAT (hypoxanthine,
aminopterin, and thymine) selection, the supernatants of the hybridoma
cells were tested by enzyme-linked immunosorbent assay (ELISA) for the
presence of antibodies to BoNT/A and BoNT/A HC. Some of the
positive hybridoma supernatants were tested for their ability to
neutralize BoNT/A, as described below. Limiting dilution was used to
clone the hybrids that produced neutralizing antibodies.
Screening of hybridoma supernatants, antibody purification, and
quantification of MAbs.
The binding of the cell-free hybridoma
supernatants to BoNT/A or BoNT/A HC were measured by ELISA.
The bound antibody was detected with horseradish peroxidase-labeled
goat anti-mouse immunoglobulin G (IgG) (Cappel/Organon Teknika Corp.,
West Chester, Pa.), and the mean of duplicate optical density (OD)
measurements of each sample at 620 nm was obtained after incubation
with 3,3',5,5'-tetramethylbenzidine peroxidase substrate (Kirkegaard & Perry Laboratories, Gaithersburg, Md.). Wells were scored as positive
if the OD was greater than twice that of the negative controls in which
antigen or serum was omitted.
MAbs were purified by using protein G conjugated to Sepharose
(Pharmacia Biotech, Uppsala, Sweden), and the IgG was quantitated as
specified by the manufacturer. The isotype and subclass of each MAb
were determined by standard direct ELISA as specified by the
manufacturer (Bio-Rad Laboratories, Inc., Melville, N.Y.).
Neutralization assays.
Serial dilutions of MAbs were incubated
with various lethal doses (5 to 20 mouse 50% lethal doses
[LD50]) of BoNT/A for 1 h at room temperature. The
toxin-antibody mixture was administered intraperitoneally at a dose of
0.2 ml per mouse. Five days after challenge, the mice were scored for
survivors. In initial neutralization assays, mice were observed for up
to 20 days.
Measurement of binding kinetics using SPR.
The affinities of
the MAbs were determined with an optical biosensor using real-time
surface plasmon resonance technology (SPR) (BIAcore 1000 with upgrade;
Pharmacia Biacore, Piscataway, N.J.). Briefly, anti-mouse Fc antibody
was coupled to the chip as specified by the manufacturer and MAb was
captured and immobilized by the chip. Kinetic analyses were carried out
at a flow rate of 25 µl/min with 5 to 200 nM BoNT/A HC in
HEPES-buffered saline containing 3 mM EDTA and 0.005% Tween (HBS).
Values for the apparent equilibrium dissociation constant
(KD) were calculated from the ratio of the
dissociation (koff) and association
(kon) rate constants obtained with the
BIAevaluation 2.1 software package supplied by the vendor. Two 30-s
pulses of 10 mM glycine (pH 1.8) were used to remove bound MAbs and
regenerate the biosensor chips with anti-IgG Fc antibody.
Epitope mapping with biosensor technology.
Epitope mapping of
the MAbs was carried out by SPR at a flow rate of 5 µl/min.
Affinity-purified antibody to mouse IgG Fc (8,045 response units
[RU]) was immobilized onto the chip, and a series of
reagents were each passed over the chip for 3 min with a 2-min wash
with HBS between injections. First, purified BoNT/A HC MAb
was captured by the Fc-specific antibody. Second, nonspecific sites
were blocked by passing a saturating concentration (0.10 mg/ml) of an
unrelated MAb over the matrix surface. Third, the antigen (200 nM
BoNT/A HC in HBS) was allowed to bind to the captured MAb.
Finally the second MAb was injected, and its binding was determined.
The biosensor chip was regenerated as described above, and the process
was repeated to test the ability of all MAbs to bind as a second MAb by
using each as the first MAb. Thus, all antibody pairs were tested in
both directions. The use of controls demonstrated that binding of
BoNT/A HC required the presence of bound first MAb and that
binding of the second MAb required the presence of bound BoNT/A
HC.
Generation of neutralizing MAbs to BoNT/A.
We hypothesized
that neutralizing antibodies against BoNT/A might best be generated if
we vaccinated animals with BoNT/A HC, the protective,
nontoxic receptor binding fragment of BoNT/A. To test this hypothesis,
spleen cells from the highly seropositive (titers, >105)
mice were fused with myeloma cells. To avoid isolation of antibodies with little or no reactivity to the whole toxin, all of the fusion product supernatants were screened by a direct ELISA on 96-well plates
containing intact BoNT/A or BoNT/A HC. This vaccination approach produced 660 hybridoma fusions that recognized BoNT/A HC, and 488 of these 660 hybridoma fusions recognized the
native toxin in an ELISA-based assay.
To increase the possibility of finding neutralizing antibodies, we
examined the protective ability of some of the fusion products
that
recognized the toxin in an in vivo mouse lethality model
before cloning
and subsequently subcloning the hybrids. The cells
corresponding to the
positive wells were transferred to six-well
plates and cultured for 7 to 10 days. The culture supernatants
were tested for their ability to
protect mice against 10 LD
50 of BoNT/A. We obtained 33 hybrids that produced neutralizing antibody,
and these fusion products
were cloned. Candidate hybridomas were
subjected to a second round of
expansion and screening and were
subcloned. Eleven hybridomas were
cloned, and antibodies were
immunopurified on protein G columns. The
names and isotypes of
all MAbs were determined and are listed in Table
1. The isotypes
of MAbs 6B2-2 and 6C2-4
were IgG1, and those of the remaining
nine MAbs were IgG2a. When MAbs
were tested against 5 to 10 LD
50 of BoNT/A, all the mice
were protected (Table
1).
Next, we analyzed the specificity of the MAbs by ELISA. After
preliminary characterization of all 11 MAbs against BoNT/A,
five of
them (4A2-2, 4A2-4, 6B2-2, 6C2-4, and 6E9-1) that represented
both
isotypes were selected for further evaluation against two
heterologous
BoNTs. All antibodies recognized BoNT/A H
C and did
not bind
to the H
C fragment of the other BoNTs, nor did they protect
against either BoNT/B or BoNT/E (Table
1). These results indicate
that
vaccination with the protective fragment of BoNT/A elicited
the
production of substantial numbers of homologous neutralizing
MAbs.
Characterization of the neutralizing MAbs.
To explore the
binding characteristics of the MAbs, we examined the interaction of
the neutralizing MAbs with intact BoNT/A and BoNT/A HC
under a variety of conditions, including Western immunoblotting and
immunoprecipitation. All of the MAbs recognized a 50-kDa protein
corresponding to BoNT/A HC after the protein was Western
immunoblotted, and the antibodies did not recognize other portion of
BoNT/A (data not shown).
We also evaluated the abilities of the MAbs to detect BoNT/A
H
C under more physiological conditions. BoNT/A
H
C was incubated
with the neutralizing MAbs, and the immune
complexes were captured
by protein A-Sepharose. After being washed,
they were eluted,
subjected to sodium dodecyl sulfate-polyacrylamide
gel electrophoresis,
and detected with rabbit anti-BoNT/A
H
C. All MAbs immunoprecipitated
BoNT/A H
C with
similar relative intensities, suggesting that their
affinities may be
similar (data not
shown).
To characterize the kinetic interactions between the neutralizing MAbs
and BoNT/A H
C, we used the SPR biosensor technology.
Each
antibody was captured by anti-mouse IgG Fc on a biosensor
chip, various
concentrations of BoNT/A H
C were passed through
the flow
cell, and the binding kinetics were recorded. The kinetic
rate
constants
kon and
koff
were determined from the ascending
rate of the BIAcore signal during
binding and the descending rate
during the wash-off interval,
respectively. Examples of the binding
kinetics are shown in Fig.
1A for MAb 6E9-4 and Fig.
1B for MAb
6B2-2. We calculated the apparent
KDs of each
MAb for BoNT/A H
C from the ratio
koff/
kon. As seen in
Table
2, all of the MAbs
had similar on
rates, and their overall values for
KD ranged
from
about 1 to 0.1 nM. The
KD for one MAb,
6B2-2, was significantly
lower (<0.06 nM) but was difficult to resolve
accurately due to
its very low rate of dissociation (Fig.
1B) compared
with the
other MAbs. Although we observed differences between the MAbs
in overall affinity, it is noteworthy that a feature common to
all of
the neutralizing MAbs is their very high affinity.
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FIG. 1.
Kinetic analyses of MAb binding to BoNT/A HC
by SPR. Biosensor chips with immobilized anti-mouse Fc were used to
capture 104 RU of MAb 6E9-4 (A) or 190 RU of MAb 6B2-2 (B). After
equilibration, a series of concentrations of the antigen BoNT/A
HC (30, 60, 100, 200, and 300 nM) was passed over the chip
for 2 min, and this was followed by a washout phase with HBS buffer.
Controls for a small amount of binding of BoNT/A HC when no
MAb was bound were run, and the results have been subtracted for each
concentration of antigen. Kinetic constants were calculated using
BiaEvaluation software version 2.1.
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|
Next, we used SPR to characterize the binding sites of the MAbs. The
BoNT/A H
C MAb was captured by anti-mouse Fc, and any
remaining anti-mouse binding sites were blocked with an unrelated
MAb.
After binding the antigen BoNT/A H
C, a second MAb was
injected
and its binding was determined. This experiment was repeated
to
examine the ability of all MAbs to bind as the second MAb by using
each as the first MAb, thus testing all pairs of antibodies in
both
directions. As seen in an example in Fig.
2, MAb 4A2-2 was
captured by anti-mouse
Fc on the sensor chip and then allowed
to bind its antigen, BoNT/A
H
C. As expected, when the same antibody
4A2-2 was injected,
no additional binding was observed, since
the BoNT/A
H
C epitope for this MAb was already occupied by
interaction
with chip-immobilized MAb 4A2-2. Other heterologous
antibodies,
i.e., 6B2-2, 6E9-1, or 6E10-8, were tested similarly, and
only
MAb 6B2-2 bound, showing that its epitope on the antigen was
distinct
from that of MAb 4A2-2. We observed no binding of MAbs 6E9-1
and
6E 10-8, which indicated that their binding epitopes were the
same or similar to that of MAb 4A2-2. All combinations of antibodies
were tested likewise, and the data obtained are summarized in
Fig.
3.
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FIG. 2.
Epitope-mapping analyses of neutralizing MAbs by SPR. A
biosensor chip with immobilized anti-mouse Fc was equilibrated with HBS
buffer, and the following solutions were passed sequentially over the
chip with a 2-min injection of HBS buffer between each reagent: MAb
4A2-2 for 3 min (a), unrelated mouse MAb for 3 min (to block all
remaining anti-mouse Fc sites) (b), 10 µg of antigen BoNT/A
HC per ml for 3 min (c), and the second MAb for 3 min,
either 4A2-2, 6B2-2, 6E9-1, or 6E10-10 as indicated (d). There is a
response, sometimes large, at each injection due to the change in the
refractive index of the medium.
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FIG. 3.
Summary of epitope-mapping studies of neutralizing
MAbs by SPR. We propose at least two distinct neutralizing epitopes
from the MAb-mapping studies. One site is defined by MAb 6B2-2. Another
site is defined by MAb 6E9-1 and is shared by all other MAbs tested
except 6B2-2 and 6C2-4. The epitope of MAb 6C2-4 overlaps that
defined by MAbs 4A2-2 and 4A2-4, which must therefore be overlapping
rather than identical to the 6E9-1 binding site.
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The epitope-mapping studies defined three groups of MAbs,
corresponding to two distinct and one overlapping protective
epitope
regions on BoNT/A H
C. One particular region of
the antigen was
defined by MAb 6B2-2, while MAbs 4A2-2, 4A2-4, 6E9-1,
6E9-3, 6E9-4,
6E10-4, 6E10-5, 6E10-8, and 6E10-10 bound to a distinct
site,
clearly defining two protective epitopes. However, the
results
with MAb 6C2-4 were more complex. When captured as first
antibody,
6C2-4 bound and presented BoNT/A H
C in a way that
it bound all
other MAbs except 4A2-2 and 4A2-4. When 6C2-4 was tested
as second
MAb, it bound well (a level typical of all other
interactions)
only to antigen presented by 6B2-2. It bound to a lower
extent
to antigens presented by 6E9-1, 6E9-3, 6E9-4, 6E10-4, 6E10-5,
6E10-8, and 6E10-10, and it did not bind significantly to antigens
presented by 4A2-2 and 4A2-4. Thus, the MAb 6C2-4 epitope was
distinct from the binding epitope of 6B2-2 as well as from that
of
6E9-1 and all other 6E9 and 6E10 MAbs, but it overlapped with
that of
MAbs 4A2-2 and 4A2-4.
Interpretation of the results.
A major obstacle in obtaining
protective antibodies against BoNTs is the extreme toxicity of these
toxins. To circumvent this, various approaches have been employed
(4, 7, 8). Here we used BoNT/A HC as the
antigen and obtained numerous neutralizing MAbs against BoNT/A.
Amersdorfer et al. (1) have also vaccinated mice with
BoNT/A HC to generate a large number of MAbs. Spleens were
obtained from the vaccinated mice, and phage libraries were constructed
from their Ig VH and VK genes. After using
phage display to create and screen millions of antibodies for binding,
they characterized 28 single-chain MAb fragments. The single-chain antibodies were found by SPR to bind to four distinct epitopes with
dissociation constants ranging from 1 to 70 nM. Some of the constructs
showed partial antagonism of toxin-induced neuroparalysis, indicating
that two of the four epitopes may be somewhat protective in vitro.
However, these small antibody fragments are rapidly cleared from the
bloodstream and could not be meaningfully tested in vivo.
The MAbs reported here recognized neither BoNT/B H
C nor
BoNT/E H
C and are specific for BoNT/A H
C and
BoNT/A holotoxin. Although
the various BoNT serotypes have significant
amino acid sequence
homology, there is little to no cross-protection
among heterologous
neurotoxins. This may be explained by the lack of
sequence conservation
within the last ~150 C-terminal residues of the
H
C region (
9-11,
18).
Previous attempts to identify linear, protective epitopes of BoNT/A
suggest that these epitopes are easily denatured and may
be
discontinuous or conformational (
1,
2,
4,
6). Results
using a display library to identify epitopes to the MAbs described
here were also consistent with their binding to discontinuous
epitopes (M. Segal and S. Bavari, unpublished results). We have
used the MAbs to define at least two (possibly three) distinct,
protective epitopes on BoNT/A H
C. MAbs 6C2-4 and 6E
family bound
to distinct epitopes, but both interfered with the
binding of
MAb 4A2-2. Thus, these three MAbs may have bound to sites
clustered
in one region of the toxin that appeared to contain two
distinct
epitopes and may be part of one large protective
epitope. MAb
6B2-2 bound to a fully independent site. For each of
the three
epitopes, at least one MAb bound with very high affinity,
ranging
from 0.2 to 0.06 nM. Further characterization of these
epitopes
and identification of their locations within the BoNT/A
H
C sequence
may help to create a more effective
peptide-based
vaccine.
A second benefit of defining the protective epitopes on BoNT/A
H
C antigen will be to improve in vitro diagnostic assays.
The
current ELISA is not sufficiently reliable to predict protective
immunity because it measures the antibody response to the whole
antigen, which can include both protective and nonprotective antibodies
(
14). The MAbs to defined, protective epitopes on the
BoNT/A
H
C antigen described in this study could enable the
development
of competitive ELISAs to determine the magnitude and
duration
of a protective immune response in mice. The goal is to
develop
an in vitro correlate of immunity in mice and ultimately an in
vitro correlate of immunity in humans. In addition, one or more
of the
high-affinity MAbs described here may be useful in monitoring
food for
contamination with BoNT/A. For example, a rapid, sensitive
assay
specific for BoNT/B was recently reported that uses a serospecific
MAb
to capture the toxin from food samples (
19).
To summarize, we obtained neutralizing MAbs by vaccination with the
nontoxic binding fragment BoNT/A H
C. These MAbs have very
high affinity and recognized at least two neutralizing epitopes.
They will be important tools in attempts to monitor immunity developed
in vaccinations, to transfer passive immunity, and to further
characterize the protective epitopes on the binding domain of
BoNT/A.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Cell Biology and Biochemistry, U.S. Army Medical Research Institute of Infectious Diseases, 1425 Porter St., Frederick, MD 21702-5011. Phone:
(301) 619-4246. Fax: (301) 619-2348. E-mail:
Sina.Bavari{at}AMEDD.Army.Mil.
Editor:
J. D. Clements
| |
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Infection and Immunity, January 2001, p. 570-574, Vol. 69, No. 1
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.1.570-574.2001
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Pless, D. D., Ruthel, G., Reinke, E. K., Ulrich, R. G., Bavari, S.
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