Differential Neutralizing Activities of a Single Domain Camelid Antibody (VHH) Specific for Ricin Toxin’s Binding Subunit (RTB)
Mantis NJ (2014) Differential Neutralizing Activities of a Single Domain Camelid Antibody (VHH) Specific
for Ricin Toxin's Binding Subunit (RTB). PLoS ONE 9(6): e99788. doi:10.1371/journal.pone.0099788
Differential Neutralizing Activities of a Single Domain Camelid Antibody (VHH) Specific for Ricin Toxin's Binding Subunit (RTB)
Cristina Herrera 0
David J. Vance 0
Leslie E. Eisele 0
Charles B. Shoemaker 0
Nicholas J. Mantis 0
Ellen R. Goldman, Naval Research Laboratory, United States of America
0 1 Division of Infectious Disease, Wadsworth Center, New York State Department of Health , Albany , New York, United States of America, 2 Department of Biomedical Sciences, University at Albany School of Public Health , Albany , New York, United States of America, 3 Scientific Cores, Wadsworth Center, New York State Department of Health , Albany , New York, United States of America, 4 Department of Infectious Disease and Global Health, Tufts Cummings School of Veterinary Medicine , North Grafton, Massachusetts , United States of America
Ricin, a member of the A-B family of ribosome-inactivating proteins, is classified as a Select Toxin by the Centers for Disease Control and Prevention because of its potential use as a biothreat agent. In an effort to engineer therapeutics for ricin, we recently produced a collection of alpaca-derived, heavy-chain only antibody VH domains (VHH or ''nanobody'') specific for ricin's enzymatic (RTA) and binding (RTB) subunits. We reported that one particular RTB-specific VHH, RTB-B7, when covalently linked via a peptide spacer to different RTA-specific VHHs, resulted in heterodimers like VHH D10/B7 that were capable of passively protecting mice against a lethal dose challenge with ricin. However, RTB-B7 itself, when mixed with ricin at a 1:10 toxin:antibody ratio did not afford any protection in vivo, even though it had demonstrable toxin-neutralizing activity in vitro. To better define the specific attributes of antibodies associated with ricin neutralization in vitro and in vivo, we undertook a more thorough characterization of RTB-B7. We report that RTB-B7, even at 100-fold molar excess (toxin:antibody) was unable to alter the toxicity of ricin in a mouse model. On the other hand, in two well-established cytotoxicity assays, RTB-B7 neutralized ricin with a 50% inhibitory concentration (IC50) that was equivalent to that of 24B11, a well-characterized and potent RTB-specific murine monoclonal antibody. In fact, RTB-B7 and 24B11 were virtually identical when compared across a series of in vitro assays, including adherence to and neutralization of ricin after the toxin was prebound to cell surface receptors. RTB-B7 differed from both 24B11 and VHH D10/B7 in that it was relatively less effective at blocking ricin attachment to receptors on host cells and was not able to form high molecular weight toxin:antibody complexes in solution. Whether either of these activities is important in ricin toxin neutralizing activity in vivo remains to be determined.
Funding: CH gratefully acknowledges the support of a Carson Carr Diversity Scholarship from the University at Albany. This project was supported in part by a
grant from the National Institutes of Health (AI097688) to NJM. The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: Co-author Nicholas Mantis is a PLOS ONE Editorial Board member. This does not alter the authors adherence to PLOS ONE Editorial
policies and criteria.
There are ongoing initiatives to develop countermeasures
against ricin, a Select Toxin, as classified by the Centers for
Disease Control and Prevention (CDC), and which has been the
subject of a number of recent high profile bioterrorism incidents in
the United States [1,2]. Ricin is a glycoprotein derived from the
castor bean plant, Ricinus communis, and a member of the medically
important family of A-B toxins . Ricins enzymatic subunit
(RTA) is an RNA N-glycosidase that inactivates eukaryotic
ribosomes by catalyzing the hydrolysis of a universally conserved
residue within the so-called sarcin/ricin loop (SRL) of 28S rRNA
[4,5]. Ricins B subunit (RTB) is a galactose- and
N-acetylgalactosamine (Gal/GalNAc)-specific lectin that has two important
functions in cytotoxicity. First, RTB promotes ricin attachment
and endocytosis of ricin into all mammalian cell types, including
epithelial cells, sinusoidal endothelial cells, and macrophages [6,7].
Second, following endocytosis, RTB mediates the retrograde
transport of RTA from the plasma membrane to the trans-Golgi
network (TGN) and endoplasmic reticulum (ER), where RTA is
liberated from RTB and retro-translocated into the cell cytoplasm
Considering its essential role in toxin uptake and trafficking,
RTB is an appealing target for antibody-based therapeutics.
Structurally, RTB is composed of two globular domains (1 and 2)
each containing three homologous sub-domains (a, b and c),
although only the external sub-domains (1a and 2c) retain
functional carbohydrate recognition activity (Figure 1) [6,10].
Sub-domain 1a (residues 1759) is Gal-specific and is considered a
low affinity carbohydrate recognition domain (CRD), whereas
sub-domain 2c (residues 228262) binds both Gal and GalNAc
and is considered a high affinity CRD . RTB has four
intramolecular disulfide bonds, in addition to the single
intermolecular disulfide bond that joins it to RTA [14,15]. Finally, RTB
has two N-linked mannose side chains that have been postulated to
interact with mannose-binding protein(s) during ricin intracellular
transport and/or influence the intracellular stability of RTB [16
A number of RTB-specific murine monoclonal antibodies
(mAbs) have been described in the literature, although only a
handful have been shown to have toxin-neutralizing activity [20
26]. The two most well characterized mAbs from our laboratory
are SylH3 and 24B11. Although SylH3 and 24B11 are each able
to neutralize ricin in vitro and in vivo, they apparently do so by
different mechanisms . SylH3 virtually abolishes ricins
ability to adhere to host cell receptors, and is therefore postulated
to neutralize ricin by steric hindrance. 24B11 also affects ricin
binding to host cell surfaces, although this activity alone does not
fully account for 24B11s potent toxin-neutralizing activity.
Rather, we recently reported that 24B11 is able to associate with
ricin in solution or after the toxin has adhered cell surface
receptors. Surface-bound 24B11 is subsequently endocytosed as a
toxin:antibody complex and interferes with retrograde transport of
ricin to the TGN . We have also reported that SylH3 and
24B11 Fab fragments were as effective as the full length IgGs at
neutralizing ricin in vitro and in vivo, indicating that in the case of
SylH3 and 24B11, neither bivalency nor Fc-domains are necessary
determinants of toxin-neutralizing activity .
However, the relationships between affinity, avidity, epitope
specificity, Fc-mediated effector functions, and toxin-neutralizing
activity in vitro and in vivo remains poorly understood in the case of
ricin. Defining mechanisms of toxin-neutralizing activity by
RTBspecific antibodies has been particularly challenging because only
a limited number of conventional murine mAbs besides 24B11
and SylH3 are available [21,23,2527,29]. Moreover,
conventional mAbs like 24B11 and SylH3 are not easily reengineered or
modified to permit a systematic analysis of the factors that render
an antibody effective at neutralizing ricin. Such versatility can only
be achieved with single-domain camelid-derived antibodies,
referred to as VHHs or simply nanobodies, which are small,
generally highly stable, and easily expressed in Escherichia coli or on
the surface of filamentous bacteriophages like M13 . For
example, RTA- and RTB-specific single chain nanobodies were
affinity isolated from a phage-displayed semisynthetic llama library
and have proven useful for a number of diagnostic applications
. Camelid-derived, single domain antibodies against Shiga
toxin, botulinum neurotoxins (BoNT) and Clostridium difficile toxins
have also been described .
We recently produced and partially characterized a collection of
ricin-specific VHHs from alpacas . We identified 11 unique
RTA-specific VHHs and 9 unique RTB-specific VHHs. Among
the nine unique RTB-specific VHHs, only one, RTB-B7, had
demonstrable toxin-neutralizing activity (TNA) in a Vero cell
cytotoxicity assay, although a number of others like RTB-D12 had
apparent affinities for ricin that were equal to or less than
RTBB7s . RTB-B7 was covalently linked via a short peptide spacer
(GGGGS)3 to three different RTA-specific VHHs, including
RTAD10, resulting in three different VHH heterodimers that each
proved capable of passively protecting mice against a lethal dose
ricin challenge. We have subsequently characterized two of the
three RTA-specific VHH components of the three heterodimers in
great detail, including solving the X-ray crystal structures of the
VHH monomers in complex with RTA (MJ Rudolph, DJ Vance, J
Cheung, MC Franklin, F Burshteyn, MS Cassidy, EN Gary, C
Herrera, CB Shoemaker, and NJ Mantis, manuscript in revision).
However, very little is known about RTB-B7. It is not known if
RTB-B7, at high doses, is able to passively protect mice from ricin
toxin, or how RTB-B7 compares to RTB-specific murine mAbs in
terms of toxin-neutralizing activity. As only a few RTB-specific
toxin-neutralizing antibodies have been identified, we deemed it
important to characterize each of them as fully as possible as a
means to identify which specific attributes (e.g., affinity, epitope
specificity, inhibition of attachment) are critical for antitoxin
activity. Therefore, in this study we undertook a detailed in vitro
characterization of RTB-B7.
Materials and Methods
2.1 Chemicals, Biological Reagents and Cell Lines
Ricin toxin (Ricinus communis agglutinin II), FITC (Fluorescein
isothiocyanate)-labeled ricin, biotinylated ricin toxin, Ricinus
communis agglutinin I (RCA-I) and ricin toxin B subunit (RTB)
were purchased from Vector Laboratories (Burlingame, CA).
Ricin was dialyzed as described  against phosphate buffered
saline (PBS) at 4uC in 10,000 MW cutoff Slide-A-Lyzer dialysis
cassettes (Pierce, Rockford, IL), prior to use in cytotoxicity studies.
D-(+)- Lactose, was obtained from J.T. Baker (Center Valley, PA)
and asialofetuin (ASF) from Sigma-Aldrich (St. Louis, MO). Goat
serum was purchased from Gibco-Invitrogen (Carlsbad, CA).
Anti-E-tag Horseradish peroxidase (HRP) conjugated mAb was
purchased from Bethyl Laboratories, Inc (Montgomery, TX) and
goat-anti-mouse IgG HRP conjugated and streptavidin HRP
conjugated were purchased from Fisher Scientific (Pittsburgh, PA).
Unless noted otherwise, all other chemicals were obtained from
Sigma-Aldrich (St. Louis, MO). Cell lines and cell culture media
were obtained from the tissue culture media core facility at the
Wadsworth Center. THP-1 cells were grown in RPMI +10% Fetal
Bovine Serum (FBS) and Vero cells, a fibroblast-like kidney cell
line derived from the African green monkey, were grown in
DMEM +10% FBS. All cell lines were maintained in 37uC with
5% CO2 incubators, unless noted otherwise. Single chain camelid
antibodies (VHHs) which were E-tagged for ELISA purposes have
been previously described  (Table 1).
2.2 Mouse Strains, Animal Care and Ricin Toxin Challenge Studies
Mouse experiments were performed as described . Briefly,
groups of female BALB/c mice (5 mice per group) approximately
810 weeks of age were purchased from Taconic Labs (Hudson,
NY). Animals were housed under conventional, specific
pathogenfree conditions and were treated in compliance with the
Wadsworth Centers Institutional Animal Care and Use Committee
(IACUC) guidelines. For the challenge experiments, mice were
injected by the intraperitoneal (i.p.) route on day 0 with pre-mixed
ricin toxin (RT; 2 mg per mouse) and the corresponding VHH
(RTB-B7 at 20 mg and 100 mg per mouse; RTB-D8 and
RTBD12 at 20 mg per mouse; D10/B7 at 30 mg per mouse) in a final
volume of 0.4 mL PBS. Following challenge mice were monitored
at least two times per day for overt signs of discomfort, including
lethargy, hunching, and failure to resume normal feeding
behavior, ruffled fur and absence of grooming. In addition, every
24 h, mice were assessed for the onset of hypoglycemia, a
wellestablished surrogate marker of ricin intoxication. A drop of blood
(5 ml) was collected from the lateral tail vein of each animal and
blood glucose levels were measured using a hand-held glucometer
(Accu-Chek Advantage, Roche, Indianapolis, IN). Mice were
euthanized by carbon dioxide (CO2) asphyxiation when they
became overtly moribund and/or blood glucose levels fell below
25 mg/dL. At no point in the study were the animals administered
analgesics or anesthetics so as not to confound the effects of the
antibody treatment. Statistical analysis was carried out using
GraphPad Prism 5 (GraphPad Software, San Diego, CA). For
Figure 1. Previously identified and characterized epitopes on RTB recognized by neutralizing and non-neutralizing mAbs. X-ray
crystal structure of ricin holotoxin visualized using PyMOL and based on PDB file 2AAI . RTA (grey), RTB (black), ricins N-linked mannose side
chains (yellow sticks) and lactose moieties (white sticks) are shown in the upper panel. Confirmed and putative epitopes (*) recognized by
neutralizing (triangles) and non-neutralizing (squares) RTB-specific mAbs are color-coded on the holotoxin structure to match RTBs linear subdomain
organization in the lower panel. This figure was modified from an earlier version .
survival studies, statistical significance was determined using the
Log-Rank (Mantel-Cox) test.
2.3 Ethics Statement
Experiments described in this study that involved mice were
reviewed and approved by the Wadsworth Centers IACUC under
protocol #13-384. The Wadsworth Center complies with the
Public Health Service Policy on Humane Care and Use of
Laboratory Animals and was issued assurance number A3183-01.
Moreover, the Wadsworth Center is fully accredited by the
Association for Assessment and Accreditation of Laboratory
Animal Care (AAALAC). Obtaining this voluntary accreditation
status reflects that Wadsworth Centers Animal Care and Use
Program meets all of the standards required by law, and goes
beyond the standards as it strives to achieve excellence in animal
care and use.
2.4 ELISAs for Determining VHHs Specificity
ELISAs were performed as described . Briefly, Nunc
Immuno MicroWell 96 well plates from ThermoFisher Scientific
(Rochester, NY) were coated overnight with 0.1 mg/well of ricin
(15 nM), RTB (29 nM), RCA-I (16 nM) or 0.4 mg/well of ASF
(82 nM) all in PBS (pH 7.4). The following day the plates were
blocked with 2% PBS-goat serum (pH 7.4) for 2 h, then plates
were treated with antibodies (VHHs at 10 mg/mL, 330 nM; 24B11
at 22.75 mg/mL, 151.67 nM; TFTB-1 at 2 mg/mL, 13.33 nM) in
aGenBank accession numbers; bVero cytotoxicity assay performed as described in Materials and Methods; value shown are in nM; cPre-bound and post-bound THP-1
cytotoxicity assays performed in Materials and Methods; values shown are in nM. dAnti-RTB mAbs previously characterized in [26,29]. Abbreviations: ND, not
determined; NA, not applicable.
Figure 2. Monomeric RTB-B7 does not passively protective mice from ricin challenge. Antibodies D10/B7, RTB-B7, RTB-D8 or RTB-D12
were mixed with ricin (2 mg; equivalent to 10xLD50) and then administered to adult BALB/c mice (n = 5 per group) by i.p. injection. Mice were
monitored for seven days for (A) survival and (B) hypoglycemia.
5-or 2-fold serial dilutions or with 0.1 mg/well of bovine serum
albumin (BSA) (8 nM) for 1 h. For competition ELISAs, the
antibodies were pre-mixed with ricin at 200 mg/mL and incubated
for at least 30 min prior to adding to the plates for 1 h. Secondary
antibodies, HRP-anti-E-tag, HRP-goat-anti-mouse IgG, or
avidinHRP were incubated for 1 h and developed using SureBlue
Peroxidase Substrate, TMB (KPL, Gaithersburg, MD). The
reactions were stopped using 1 M phosphoric acid and absorbance
was read at 450 nm using the VersaMax Microplate Reader with
Softmax Pro 5.2 software (Molecular Devices, Sunnyvale, CA). All
samples were performed at least in triplicate.
2.5 Vero Cell Cytotoxicity Assays
Vero cell cytotoxicity assays were performed as described .
In brief, Vero cells were trypsinized, adjusted to ,5 6104 cells per
mL and seeded (100 ml/well) into white bottom 96-well plates
(Corning Life Sciences, Corning, NY), and allowed to adhere
overnight. Vero cells were then treated with ricin (0.01 mg/mL;
154 pM), ricin:Ab mixtures, or medium alone (negative control)
for 2 h at 37uC. Cells were washed to remove non-internalized
toxin or ricin:Ab mixtures, and 100 ml of fresh medium was added
to the wells. Fresh medium was allowed to incubate for 48 h and
cell viability was measured using CellTiter-Glo (Promega,
Madison, WI). All samples were performed in quadruplicate and
100% viability was defined as the average value obtained from
wells in which cells were treated with medium only.
2.6 THP-1 Cell Cytotoxicity Assays
THP-1 cell cytotoxicity assays were done as described .
Briefly, THP-1 cells were spun (5 min at 4006g) and adjusted to
,5 6104 cells per mL and seeded (100 ml/well) into clear
Ubottom 96-well plates (BD Bioscience, San Jose CA) and allowed
to grow overnight. The next day, THP-1 cells were spun to
remove medium and were then treated with ricin (0.01 mg/mL;
154 pM), ricin:Ab mixtures, or medium alone for 2 h at 37uC.
Cells were then subjected to centrifugation and washed to remove
non-internalized toxin or ricin:Ab mixtures. Fresh medium was
added to the wells and allowed to incubate for 48 h. Cell viability
was determined using CellTiter-Glo after the content of each plate
was transferred into white bottom 96-well plates. In order to
Figure 3. RTB-B7 neutralizes ricin in a dose-dependent manner. Ricin was mixed with 2-fold serial dilutions of indicated antibodies and then
applied to (A) Vero cells or (B) THP-1 cells for 2 h. The cells were then washed and cell viability was measured 48 h later, as described in Materials and
Methods. The data (mean 6 SD) represent a single experiment in which each sample was done in quadruplicate. The experiment was repeated at
least twice with identical results.
address post-attachment experiments, THP-1 cells were kept on
ice at 4uC, to allow for ricin attachment but prevented
internalization of the toxin prior to antibody treatment. The
cytotoxicity assay was performed as described above but cells were
kept on ice until they were transferred to 37uC for the 48 h
incubation period. All samples were performed in quadruplicate
and 100% viability was defined as the average value obtained from
wells in which cells were treated with medium only.
2.7 Ricin Binding Assays using THP-1 Cells and Flow Cytometry
Ricin binding to cell surfaces was performed as described .
In brief, THP-1 cells were collected by gentle, low speed
centrifugation (5 min at 4006g). The resulting cell pellets were
suspended to ,5 6106 cells per mL and then seeded (200 ml/well)
into clear U-bottom 96-well plates (BD Bioscience, San Jose CA).
FITC-labeled ricin (3 mg/mL) was mixed with Abs or lactose
(30 mg/mL) for 30 min on ice in the dark prior to being added to
THP-1 cells. Cells were then washed twice with PHEM buffer to
remove unbound toxin:Ab complexes and fixed with 4%
paraformaldehyde (PFA) in PHEM for 15 min. Ricin binding to
the surfaces of THP-1 cells was measured using FACS Calibur
flow cytometer (BD Bioscience, San Jose CA). A minimum of
10,000 events was analyzed per sample.
2.8 Analytical Ultracentrifugation (AUC)
Ricin and Ab samples were dialyzed overnight in PBS (pH 7.4)
at 4uC in 10,000 MW cutoff Slide-A-Lyzer dialysis cassettes
(Pierce, Rockford, IL) prior to being subject to AUC.
Sedimentation Velocity (SV) experiments were conducted in a Beckman
Optima XL-I analytical ultracentrifuge at 20uC at a rotor speed of
50,000 rpm. Double-sector charcoal-filled epon centerpieces were
filled with a sample volume of 400 ml and the reference volume of
dialysis buffer was 420 ml. Absorption measurements were made at
and TFTB-1 at 2 mg/mL) overnight. Serial dilutions of lactose (5 mg/mL) were incubated with biotinylated ricin before being applied to plates and
developed. Binding was normalized to ricin bound to antibody in the absence of lactose. The data shown represent a single experiment in which
each sample was done in triplicate and repeated at least twice. Data are expressed as the mean 6 SD.
280 nm or 230 nm, dependending on the protein concentration.
Samples were run in an An-60 Ti four-hole rotor with zero time
between scans. Care was taken to have the rotor at thermal
equilibrium for an hour before accelerating directly to the speed of
the experiment. The data were analyzed by the c(s) method found
in SEDFIT . The experimentally calculated sedimentation
coefficients were converted to s20,w values within the SEDFIT
software and graphed using Origins (OriginLab Corporation,
2.9 Statistical Analyses and Modeling Software
Statistical analysis was carried out using GraphPad Prism 5
(GraphPad Software, San Diego, CA). The open-source molecular
visualization software PyMOL (DeLano Scientific LLC, Palo Alto,
CA) was used for modeling of ricin.
3.1 Passive Protection Studies with VHH RTB-B7
We previously reported that three different heterodimeric
VHHs, each containing RTB-B7, were capable of passively
protecting mice against ricin toxin . However, in that same
study we reported that monomeric RTB-B7 (10 mg per mouse;
10:1 VHH:toxin molar ratio) afforded no benefit to mice in the
face of a 10xLD50 ricin challenge. To investigate whether the
failure of RTB-B7 to passively protect mice was simply an issue of
insufficient antibody, we repeated the passive protection studies
using 2-fold (20 mg) and 10-fold (100 mg) higher amounts of
RTBB7. For comparison purposes, two other RTB-specific VHHs were
tested in parallel: RTB-D8 and RTB-D12. We chose RTB-D12
because its apparent affinity (EC50) for ricin toxin, as determined
by ELISA, is virtually identical to that of RTB-B7s (0.8 nM versus
0.6 nM, respectively). RTB-D8 was chosen because it binds to
ricin with a relatively low affinity (EC50 3.6 nM) and was therefore
not expected to provide an in vivo toxin-neutralizing activity. As a
positive control for these studies, groups of mice were treated with
the VHH heterodimer D10/B7. Following ricin challenge, animals
were monitored for a period of 7 days for the onset of
hypoglycemia, a well-established surrogate marker of ricin
intoxication, as well as mortality.
As expected, mice that received the heterodimer D10/B7
(30 mg; 20:1 VHH heterodimer:toxin molar ratio) survived ricin
challenge and experienced only minor declines in blood glucose
levels (Fig. 2). Conversely, neither RTB-D8 nor RTB-D12 (each
at 20 mg; 20:1 VHH:toxin molar ratio) afforded any protection
against ricin toxin, as evidenced by the fact that the VHH-treated
mice experienced a rapid decline in blood glucose levels and died
within 24 h (Fig. 2). Surprisingly, even relatively high amounts of
RTB-B7 (20 mg and 100 mg, equivalent to 20:1 and
100:1 VHH:toxin molar ratio, respectively) had no impact on
survival (0/5 mice survived in both groups) following ricin
challenge (Fig. 2). Overall, these data demonstrate that
RTBaFITC-ricin (46 nM) was incubated with lactose or indicated antibodies for 30 min before being applied to THP-1 cells and then subjected to flow cytometry, as
described in Materials and Methods. The percent (%) inhibition of ricin binding was calculated by dividing the experimental geometric mean fluorescence intensity (MFI)
by the control (FITC-ricin only) geometric MFI and then multiplying by 100.
B7, as a monomer, does not elicit protection in vivo, but as a
heterodimer (D10/B7) is capable of passively protecting mice
against 10xLD50 of ricin.
3.2 RTB-B7 Neutralizes Ricin in vitro as Effectively as 24B11
The inability of RTB-B7 (even at 100 fold molar excess over
ricin) to passively protect mice against a 10xLD50 ricin challenge
was unexpected, considering that we previously reported that
RTB-B7 was highly effective at neutralizing ricin in a Vero cell
cytotoxicity assay . We therefore revisited the Vero cell
cytotoxicity assay and compared RTB-B7 side-by-side with 24B11,
as well as the non-neutralizing VHHs, RTB-D8 and RTB-D12. As
reported previously, we found that RTB-B7 neutralized ricin in a
dose-dependent manner. Moreover, RTB-B7s estimated IC50
(,1.5 nM) was virtually identical to 24B11s IC50 when the two
antibodies were compared in the same assay (Fig. 3A). In contrast,
neither RTB-D8 nor RTB-D12 (which has the same apparent
affinity for ricin as RTB-B7) had any detectable toxin-neutralizing
activity (Fig. 3A).
We next examined the capacity of RTB-B7 to neutralize ricin
in vitro using THP-1 cells, a human
monocyte/macrophagederived cell line that is potentially more representative of ricins
primary target cell in vivo [42,43]. In the THP-1 assay, RTB-B7
also neutralized ricin with a dose-dependent profile that was nearly
identical to 24B11s (Fig. 3B), thereby demonstrating that
RTBB7 is among the most potent in vitro toxin-neutralizing antibodies
described to date. In the THP-1 assay, RTB-D8 and RTB-D12
had some detectable toxin-neutralizing activity at very high
concentrations, suggesting these two VHHs should be classified
as partially neutralizing (not non-neutralizing) antibodies (Fig. 3B).
3.3 Characteristics of RTB-B7 Recognition of Ricin and RTB
The capacity of RTB-B7 to neutralize ricin in vitro but not in vivo
led us to investigate in more detail the specific in vitro properties of
RTB-B7, particularly with respect to specificity of toxin binding
and recognition. In an effort to define the epitope recognized by
RTB-B7, we subjected RTB-B7 (as well as RTB-D8 and
RTBD12) to the following previously established assays: RCA-I ELISA,
pepscan analysis , panning with a 12-mer phage-displayed
peptide library [28,44], Western blot analysis, and competitive
ELISAs with a collection of well-characterized RTB-specific
neutralizing and non-neutralizing murine mAbs .
RCA-I is a tetrameric glycoprotein from Ricinus communis
consisting of two ricin-like heterodimers whose B subunit (RCB)
shares 84% sequence identity with RTB [29,45,46]. We found that
RTB-B7 bound ricin and RCA-I with similar EC50s,
demonstrating that RTB-B7s epitope is likely conserved between the two
closely related proteins (Fig. S1). RTB-D8 and RTB-D12 were
similar to RTB-B7 in that they bound equally well to RCA-I and
ricin. However, RTB-B7s epitope is likely discontinuous in
nature, as neither pepscan or affinity enrichment using a 12-mer
phage-displayed library identified peptides that specifically bound
RTB-B7. Moreover, Western blot analysis indicated that RTB-B7
reactivity was abolished when ricin (or RTB) was treated with
bmercaptoethanol (BME) (data not shown).
It was previously reported that neither SylH3 nor 24B11, two
RTB-specific neutralizing mAbs, were able to competitively inhibit
RTB-B7 from binding to ricin . We extended these previous
findings by performing competitive ELISAs with additional
neutralizing (JB4) and non-neutralizing (TFTB-1, B/J F9, C/M
A2, SA3, CB12, and JB11) murine mAbs. Ricin-coated ELISA
plates were incubated with saturating amounts of murine mAbs
and then probed with the RTB-B7, RTB-D8 or RTB-D12 (1 mg/
mL; 33 nM). The EC50s of RTB-B7, RTB-D8 and RTB-D12
were unaffected by any of the murine mAbs tested (data not
shown), suggesting that the three VHHs recognize distinct
epitopes on RTB.
To determine whether RTB-B7 recognizes an epitope on RTB
that is influenced by ligand engagement, we performed ricin
ELISAs in the absence or presence of saturating amounts of
lactose (10 mg/mL). The apparent affinity of RTB-B7 for ricin
was unchanged (or even slightly enhanced) in the presence of
lactose (Fig. 4A, B). This is in contrast to the non-neutralizing
murine mAb, TFTB-1, whose capacity to recognize ricin was
negatively impacted (,20%) by lactose (Fig. 4B). The effect of
ligand binding was even more pronounced when the antibodies
were tested for their ability to capture soluble, biotinylated ricin
in the presence of increasing concentrations of lactose (Fig. 4C).
Lactose concentrations greater than 0.1 mg/mL resulted in a
precipitous drop in TFTB-19s ability to capture soluble ricin,
whereas the abilities of VHHs RTB-B7, RTB-D8 and RTB-D12 to
capture ricin were not altered (Fig. 4C). These data indicate that
RTB-B7 recognizes an epitope on RTB that is not influenced by
We have recently noted that certain mAbs recognize
platebound ricin with high affinity, but bind poorly to ricin in solution
(J. OHara, A. Yermakova, E. Sully, and N. Mantis, manuscript in
preparation). Based on these and other observations, we speculate
that adsorption of ricin to polystyrene microtiter plates results in
the exposure of normally cryptic or subdominant epitopes
(Rform). To examine accessibility of RTB-B7s epitope, we
performed ELISAs in the absence or presence of soluble ricin
toxin (Figure 5). Antibodies 24B11 and RTA-G12, an
RTAspecific VHH with toxin-neutralizing activity, were examined in
parallel. We confirmed that RTB-B7, RTB-D8 and RTB-D12, as
well as 24B11 and RTA-G12, recognized plate-bound ricin with
roughly equal EC50s (Figure 5A). On the other hand, attachment
of RTB-B7, 24B11 and RTA-G12 to plate bound ricin was
completely inhibited by very low amounts of soluble native ricin
(IC50,0.1 mg/mL), whereas inhibition of RTB-D8 and RTB-D12
required very high concentrations (.50 mg/ml) of toxin
(Figure 5B). These data demonstrate that RTB-B7s epitope is
presented on the surface of native ricin, whereas the epitopes
3.4 RTB-B7 is Able to Neutralize Ricin after the Toxin has attached to Host Cell Receptors
There is evidence that RTB-specific toxin-neutralizing
antibodies can be loosely classified into one of two categories based on the
degree to which they prevent ricin from binding to cellular
receptors and whether or not they neutralize ricin after the toxin
has pre-bound to host cells [21,23,2529]. For example, SylH3 is a
potent inhibitor of ricin-receptor interactions but only marginally
effective at neutralizing ricin when pre-bound to cell surfaces,
while 24B11 only partially inhibits toxin-receptor interactions but
is highly effective at neutralizing ricin when toxin is pre-bound to
cells [27,28]. In an effort to better characterize RTB-B7, we first
examined its ability to inhibit ricin from binding to the surrogate
receptor ASF. Biotinylated ricin was mixed with RTB-B7 at a
range of concentrations and then applied to ELISA plates coated
with ASF. SylH3 and 24B11, as well as RTB-D8 and RTB-D12,
were examined in parallel. As expected, SylH3 almost completely
blocked (.90%; EC50 ,1 nM) the ability of ricin to bind to ASF,
while 24B11 only marginally (,30%) impacted toxin-ASF
interactions (Fig. 6). In contrast, neither RTB-B7 nor the partially
neutralizing VHHs, RTB-D8 and RTB-D12, affected the ability of
ricin to associate with ASF, even at the highest concentrations
To assess antibody effects on ricins interactions with cell
surfaces, FITC-ricin was incubated with approximately equimolar
amounts of RTB-B7 or other VHHs/mAbs and then applied to
THP-1 cells at 4uC. The cells were then washed and subjected to
flow cytometry to quantitate the amount of FITC-ricin bound to
the cells. Lactose (30 mg/mL; 88 mM) was used as a positive
control for these studies, as saturating amounts of the disaccharide
are known to occupy RTBs carbohydrate recognition domains
and competitively inhibit ricin binding to cell surfaces . The
non-neutralizing mAb TFTB-1 was used as a negative control
. As expected, lactose inhibited ricin from binding to THP-1
cells, whereas TFTB-1 did not (,5%) (Table 2). VHHs RTB-D8,
RTB-D12 and RTB-B7 partially (2030%) inhibited ricin from
binding to THP-1 cells, while D10/B7 and 24B11 reduced ricin
binding to THP-1 cells by .85% and .65%, respectively. Thus,
RTB-B7 does not inhibit ricin binding to cell surfaces as efficiently
We next examined the ability of RTB-B7 to recognize
receptorbound ricin and to neutralize toxin when pre-bound to cell
surfaces. ELISA plates coated with ASF were incubated with a
saturating concentration of ricin and then probed with RTB-B7
across a range of concentrations. RTB-B7 (Fig. 7A), as well as
RTB-D8, RTB-D12 and D10/B7 (data not shown), recognized
ASF-ricin in a dose-dependent manner. RTB-B7 was next tested
for the ability to neutralize ricin after the toxin was pre-bound to
cell surfaces. In order to do so, THP-1 cells were cooled on ice to
arrest toxin endocytosis and treated with ricin for 30 min before
the addition of RTB-B7, RTB-D8, RTB-D12 or 24B11, as
described in the Materials and Methods. We found that 24B11
(IC50 0.25 nM) and RTB-B7 (IC50 1.4 nM) were each able to
effectively neutralize ricin in this assay (Fig. 7B), whereas
RTBD8 and RTB-D12 were relatively ineffective, as they had
detectable toxin-neutralizing activities only at very high
concentrations (.330 nM) (Fig. 7B). These data demonstrate that
RTBB7, like 24B11, has the capacity to neutralize ricin after the toxin
has adhered to cell surface receptors.
Figure 8. Sedimentation coefficients for ricin and ricin:antibody complexes. Ricin and ricin:Ab complexes were subject to AUC, as
described in the Materials and Methods. (A) Sedimentation coefficients of ricin were determined at indicated toxin concentrations (0.10.4 mg/ml).
(B) Sedimentation coefficients of ricin:Ab complexes. Ricin was mixed with 24B11 (3.5 mM), D10/B7 (3.5 mM) and RTB-B7 (7 mM) to achieve an
equimolar ratio between ricin and ricin-binding sites on each of the three different antibodies. The mixtures were incubated at room temperature for
120180 min and then subjected to AUC (20uC). For convenience, the corrected sedimentation coefficients (s20,w) are denoted above each
sedimentation distribution in the plot. The inset in panel B is simply a magnification of the figure sedimentation profiles to enhance resolution of the
3.6 Antibodies D10/B7 and 24B11 Promote Ricin
Aggregation in Solution, Whereas RTB-B7 does not
VHH heterodimer D10/B7 consists of two ricin-specific single
chain VHH monomers, RTA-D10 and RTB-B7, joined by a
flexible peptide linker . By virtue of its ability to bind epitopes
on RTA and RTB, D10/B7 can theoretically crosslink ricin and
promote the formation of heterogeneous toxin-antibody
complexes. Although monomeric in nature, we cannot formally exclude
the possibility that RTB-B7 may homodimerize and therefore
promote ricin aggregation to some degree. Because the formation
of toxin:antibody complexes may contribute to toxin-neutralizing
activity, we subjected our antibody samples to AUC analysis.
Sedimentation coefficients for ricin, 24B11, D10/B7 and RTB-B7
were examined at a range of concentrations and were determined
to be 4.6S, 6.7S, 2.9S and 2.3S, respectively (Fig. 8 A; S2AC).
The sedimentation coefficient for ricin (s20,w = 4.6S) was identical
to what is reported in the literature .
In order to characterize the aggregation potential of the
individual antibodies, RTB-B7, D10/B7 and 24B11 were mixed
at a 1:1 molar ratio with ricin and then subjected to AUC. As
shown in Figure 8B, D10/B7 and 24B11 promoted ricin
aggregation, whereas RTB-B7 did not. Specifically, ricin:24B11
mixtures resulted in three defined sedimentation coefficient
distributions at 6.9S, 9.4S and 12.8S, which we interpreted as
corresponding to 24B11 (s20,w = 6.9S), a 1:1 ricin:24B11 complex
(s20,w = 9.4S) and 2:1 ricin:24B11 complex (s20,w = 12.8S). The
sedimentation coefficients for ricin:D10/B7 mixture revealed
distributions at 4.5S and 7.3S, followed by a long tail with a
larger complex identified at 9.7S. We interpret this distribution
profile as corresponding to free ricin (s20,w = 4.5S), a 1:1 mixture
of ricin:D10/B7 (s20, w = 7.3S), and heterogeneous ricin:D10/B7
aggregates of various sizes. Due to RTB-B7s small size, its effect
on ricin aggregation was difficult to discern. However, the peak
distribution around 4.6S had a much broader shoulder than the
ricin control sample, suggesting the presence of free ricin plus a 1:1
ricin:RTB-B7 complexes, not higher in molecular weight
The antibody response to ricin toxin is surprisingly complex and
the specific correlates of protection to this biothreat agent remain
poorly defined, especially as compared to other protein toxins. For
example, tetanus and diphtheria vaccines have been in clinical use
for decades and it is well established that the primary correlate of
protection is associated with a specific threshold of serum IgG
toxin-neutralization activity . In the case of ricin, however,
protection (in mice) does not necessarily correlate with either
preexisting toxin-neutralizing activities or total antitoxin antibody
levels [49,50]. Similarly, analysis of serum IgG responses in
humans that received a candidate ricin toxin subunit vaccine
revealed a disconnect between total antibody titers and
toxinneutralizing activities (i.e., several individuals with high
ricinspecific serum IgG titers had low or undetectable
toxin-neutralizing activities) [51,52]. The absence of definitive correlates of
immunity to ricin has hindered evaluation of candidate ricin
subunit vaccines and made the development of
immunotherapeutics challenging. Therefore, as a strategy to deconvolve the
polyclonal antibody response to ricin, we have focused on
characterizing a large number of murine mAbs and camelid
nanobodies with the goal of identifying the specific determinants
that are associated with toxin-neutralizing activity in vivo [39,53].
In this study, we undertook a comprehensive in vitro
characterization of the camelid antibody RTB-B7. RTB-B7 is of interest as
it was the only antibody with toxin-neutralizing activity among a
collection of nine recently described RTB-specific VHHs .
Moreover, heterodimers consisting of RTB-B7 linked to one of
three different RTA-specific VHHs (i.e., RTA-D10) were able to
passively protect mice against ricin challenge, even though
100fold molar excess of monomeric RTB-B7 does not alter the toxicity
of ricin in vivo (Figure 2). The main findings of the current study
are that RTB-B7 (i) has potent in vitro toxin-neutralizing activity in
two well-established cell-based cytotoxicity assays; (ii) recognizes a
conformational epitope that is conserved between RTB and RCB
(see below); (iii) binds to and neutralizes ricin in its soluble and
receptor-bound forms; but, (iv) only partially inhibits ricin
attachment to cell surface receptors. Thus, at least in vitro,
RTBB7 and 24B11 are virtually indistinguishable. Yet, 24B11 is able to
passively protect mice against ricin toxin, whereas RTB-B7 is not
. The reason why RTB-B7 is devoid of detectable
toxinneutralizing activity in vivo remains unknown, but the fact that
24B11 Fab fragments are able to passively immunize mice
demonstrates that neither antibody avidity or Fc-mediated
clearance is absolutely required for protection in vivo . Based
on these data, we postulate that epitope specificity, and not affinity
or avidity per se is the primary determinant of ricin
toxinneutralizing activity. The importance of epitope specificity in ricin
toxin neutralization in vivo is supported as well by other studies
with murine mAbs and VHH heterodimers [26,39,54].
While RTB-B7s epitope was not definitively identified in this
study, it can be tentatively localized to a limited region on RTB.
For example, RTB-B7 bound equally well to RTB and RCB,
indicating that RTB-B7s epitope is conserved between the two
proteins, which are only 84% identical at the amino acid level
. The differences between RTB and RCB are concentrated
within domain 1, particularly within subdomains 1a and 1b,
suggesting that RTB-B7 may recognize an epitope in RTBs
domain 2. Antibody competition studies with a collection of
neutralizing (24B11, SylH3 and JB4) and non-neutralizing mAbs
(TFTB-1, B/J F9, C/M A2, SA3, CB12 and JB11) is also
consistent with RTB-B7 being targeted to domain 2, particularly a
stretch of residues (200240) within subdomain 2c that is
conserved between RTB and RCB. The fact that RTB-B7 was
not particularly effective at inhibiting ricin attachment to cell
surfaces would suggest that RTB-B7s epitope is spatially distinct
from key residues of RTB involved in Gal/GalNAc recognition
(e.g., residues 248 and 234 in domain 2). Finally, in taking in
account solvent accessibility, we postulate that RTB-B7 recognizes
a conformational epitope localized within residues 200230 of
It is interesting to note that the heterodimer VHH D10/B7,
which consists of RTB-B7 covalently linked via a short peptide
spacer to RTA-D10, was highly effective (.85%) at blocking
attachment of ricin to the surface of THP-1 cells and, as AUC
analysis revealed, promoting ricin aggregation in solution. It is
tempting to speculate that one or both of these attributes are
important in neutralizing ricin in vivo. RTA-D10 has moderate
toxin-neutralizing activity in vitro, but like RTB-B7 is unable to
protect mice against ricin challenge. We recently solved the X-ray
crystal structure of RTA-D10 in complex with RTA (MJ Rudolph,
DJ Vance, J Cheung, MC Franklin, F Burshteyn, MS Cassidy, EN
Gary, C Herrera, CB Shoemaker, and NJ Mantis, manuscript
resubmitted), revealing that RTA-D10 makes contact with a face of
RTA that is almost diametrically opposed to RTB. Considering
that the spacer between RTB-B7 and RTA-D10 is (at least)
theoretically too short to enable intra-molecular RTA-RTB
interaction, it is likely that D10/B7 promotes the formation of
ricin intermolecular crosslinking, which is consistent with the
aggregation seen in the AUC experiments. It is unclear at this
point whether toxin aggregation and inhibition of toxin binding to
cell surfaces are separate phenomena or whether aggregation itself
results in the inability of ricin to attach to membrane bound Gal/
GalNAc moieties. In future studies we will systematically construct
and characterize additional anti-ricin VHH heterodimers and
compare them with their respective monomeric constitutes as a
means to dissect the functional properties of antibodies that are
important in toxin neutralization in vivo. Only then will it be
possible to rationally design effective therapeutics against ricin.
Figure S1 Reactivity of RTB-B7, RTB-D8 and RTB-D12
with RCA-I. ELISA plates were coated with ricin or RCA-I
overnight. (A) RTB-B7, (B) RTB-D8 and (C) RTB-D12 were serial
diluted and added to the wells and reactivity was determined as
mentioned in Materials and Methods. BSA wells were used to
control for background. The data shown represent a single
experiment in which each sample was done in triplicate and
repeated at least twice. Data are expressed as the mean 6 SD.
Figure S2 Sedimentation coefficients for 24B11, D10/B7
and RTB-B7. AUC was used to determine sedimentation
coefficients for antibodies (A) RTB-B7, (B) D10/B7 and (B)
24B11 at indicated concentrations, as described in the Materials
and Methods. For convenience, the corrected sedimentation
coefficients (s20,w) are denoted above each sedimentation
We would like to acknowledge Drs. Anastasiya Yermakova and Joanne
OHara for technical assistance and providing valuable feedback and
scientific insight related to this project. We thank Jacqueline M. Tremblay
(Tufts University) for providing us with purified VHHs. We thank Mr.
Renjie Song of the Wadsworth Center Immunology Core for assistance
with flow cytometry, the Wadsworth Center Biochemistry Core for
assistance with analytical ultracentrifugation, and Dr. Karen Chave of the
Wadsworth Center Protein Expression Core for murine monoclonal
Conceived and designed the experiments: CH DJV LEE NJM. Performed
the experiments: CH DJV LEE. Analyzed the data: CH DJV LEE NJM.
Contributed reagents/materials/analysis tools: CBS. Wrote the paper: CH
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