Engineering a soluble high-affinity receptor domain
that neutralizes staphylococcal enterotoxin C
in rabbit models of disease
D.M.Mattis1, A.R.Spaulding2,4, O.N.Chuang-Smith2,
E.J.Sundberg3,5, P.M.Schlievert2,4and D.M.Kranz1,6
1Department of Biochemistry, University of Illinois, Urbana, IL 61801,
USA,2Department of Microbiology, University of Minnesota Medical
School, Minneapolis, MN 55455, USA,3Boston Biomedical Research
Institute, Watertown, MA 02472, USA,4Present address: Department
of Microbiology, University of Iowa, Iowa City, IA 52242, USA and
5Present address: Institute of Human Virology, Department of Medicine,
University of Maryland School of Medicine, Baltimore, MD 21201, USA
6To whom correspondence should be addressed:
Received August 31, 2012; revised August 31, 2012;
accepted October 17, 2012
Edited by James Marks
Superantigens (SAgs) are a class of immunostimulatory
exotoxins that activate large numbers of T cells, leading
to overproduction of cytokines and subsequent inflamma-
tory reactions and systemic toxicity. Staphylococcal en-
terotoxin C (SEC), a SAg secreted by Staphylococcus
aureus, has been implicated in various illnesses including
non-menstrual toxic shock syndrome (TSS) and necrotiz-
ing pneumonia. SEC has been shown to cause TSS illness
in rabbits and the toxin contributes to lethality associated
with methicillin-resistant S.aureus (MRSA) in a rabbit
model of pneumonia. With the goal of reducing morbidity
and mortality associated with SEC, a high-affinity variant
of the extracellular variable domain of the T-cell receptor
beta-chain for SEC (∼14 kDa) was generated by directed
evolution using yeast display. This protein was character-
ized biochemically and shown to cross-react with the
homologous (65% identical) SAg staphylococcal entero-
toxin B (SEB). The soluble, high-affinity T-cell receptor
protein neutralized SEC and SEB in vitro and also signifi-
cantly reduced the bacterial burden of an SEC-positive
strain of MRSA (USA400 MW2) in an infective endocar-
ditis model. The neutralizing agent also prevented lethal-
ity due to MW2 in a necrotizing pneumonia rabbit
model. These studies characterize a soluble high-affinity
neutralizing agent against SEC, which is cross-reactive
with SEB, and that has potential to be used intravenously
with antibiotics to manage staphylococcal diseases that
involve these SAgs.
Keywords: directed evolution/staphylococcal enterotoxin B
(SEB)/staphylococcal enterotoxin C (SEC)/yeast display
The Gram-positive bacterium Staphylococcus aureus is
responsible for a wide spectrum of diseases, with infections
that can target skin and mucous membranes, heart, lungs,
bones and blood (Lowy, 1998;Mertz et al., 2007).Among the
major factors associated with severity of S.aureus illnesses is
a class of secreted exotoxins called superantigens (SAgs).
These exotoxins are a group of relatively small (19–30 kDa)
proteins variably produced by S.aureus strains (Fraser and
Proft, 2008). The term ‘superantigen’ was given to these
toxins because their major systemic toxicity depended on the
abilities of the toxins to stimulate uncharacteristically large
proportions of T cells, causing massive production of pro-
inflammatory molecules, including tumor necrosis factor-a
(TNF-a), interleukin (IL)-1, IL-2 and interferon-g (IFN-g)
(Marrack and Kappler, 1990;Li et al., 1999).
The mechanism of superantigenicity is now understood at
both biochemical and structural levels. The activity requires
SAg binding to the Vb region of the heterodimeric T-cell re-
ceptor (Vb) and simultaneously to class II products of the
major histocompatibility complex (MHC II). While conven-
tional peptide/MHC complexes stimulate relatively small
numbers of T cells in the body (usually ,0.01%), SAgs may
stimulate up to 30% of T cells (Marrack and Kappler, 1990;
Papageorgiou and Acharya, 1997; Li et al., 1999). The major
systemic effects of SAgs that result from these interactions
are fever, hypotension and skin rash production (Fraser and
The potency in hyperactivation of the immune system
makes staphylococcal SAgs capable of incapacitation and le-
thality, and appears responsible for their involvement in mul-
tiple illnesses. While perhaps best known as the direct cause of
toxic shock syndrome (TSS), in recent years SAgs have been
increasingly implicated in several other illnesses, including
airway diseases, necrotizing pneumonia and purpura fulminans
(Luppi et al., 1998; Bachert et al., 2007; Strandberg et al.,
2010).Staphylococcus aureus is also a leading cause of infect-
ive endocarditis (Sacar et al., 2010),an illness that is character-
ized by colonization of damaged heart valves by S.aureus
strains, leading to the production of cauliflower-appearing
vegetations composed of bacterial colonies and host cells. We
have shown previously that the SAg TSS toxin-1 (TSST-1),
under regulation of the SrrAB two-component regulatory
system, may be one of the virulence factors contributing to de-
velopment of infective endocarditis (Pragman et al., 2004). It
was hypothesized that the infective endocarditis hallmark of
vegetations, consisting of lesions with biofilm-appearing col-
onies of S.aureus, may be associated with SAg-mediated,
immune system dysregulation. Supporting a possible role of
SAgs, a recent analysis of methicillin-susceptible S.aureus
(MSSA) endocarditis showed that the TSST-1 gene was
among only a few genes that were significantly associated with
isolates derived from endocarditis patients, in distinct contrast
to isolates from soft tissue infections (Nienaber et al., 2011).
In the study, over 90% of the MSSA strains that caused endo-
carditis in patients from North America, Europe and Australia
expressed the TSST-1 gene.
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Protein Engineering, Design & Selection vol. 26 no. 2 pp. 133–142, 2013
Published online November 15, 2012 doi:10.1093/protein/gzs094
by guest on December 31, 2015
Methicillin-resistant S.aureus (MRSA)-producing SAgs
pose added problems in management of staphylococcal ill-
nesses, due to their increased difficulty in treatment asso-
ciated with antibiotic resistance and SAg toxicity (Resch
et al., 2009). Our recent studies with a rabbit model showed
that SAgs play an important role in the lethality associated
with pulmonary infections caused by community-associated
highly-studied CA-MRSA USA400 strain MW2 that secretes
SEC (Fey et al., 2003).
In the present study, based on success with the high-
affinity-engineered SEB antagonist Vb G5-8 (Buonpane
et al., 2007), we sought to develop a high-affinity Vb against
SEC that could be used in vivo as a potential therapeutic.
Guided by the structure of SEC in complex with several var-
iants of the Vb8, and a first-generation engineering effort
(Kieke et al., 2001; Cho et al., 2005), we took a rational
design approach, combined with directed evolution using
yeast display (Boder and Wittrup, 2000) to improve the affin-
ity of the Vb8 for SEC. Unexpectedly, based on previous
studies with SEB-reactive Vb regions (Wang et al., 2010),
our highest affinity Vb (KD¼ 2 nM for SEC) called L3 was
also able to cross-react with high affinity for the SAg SEB
(KD¼ 4 nM for SEB), which shares 65% sequence identity
with SEC (Bohach et al., 1990;McCormick et al., 2001). The
L3 Vb neutralized both SEC and SEB in an in vitro T-cell
assay. We also tested the in vivo efficacy of L3 in four differ-
ent rabbit models of SEC toxicity. L3 prevented the lethality
associated with direct exposure to purified recombinant SEC,
and it was effective in two different rabbit models with the
SEC-positive MRSA strain MW2 (USA400), including in-
fective endocarditis and necrotizing pneumonia models.
al., 2010), includingthe
Materials and methods
Yeast display libraries and cloning
The gene encoding the mouse Vb8 mutant called L2CM
(Kieke et al., 2001) was cloned into yeast display vector
pCT302, which contains an N-terminal haemagluttinin (HA)
tag and a C-terminal Myc (c-myc) tag (Fig. 1A) (Boder and
Wittrup, 2000). Libraries of mutants in L2CM were con-
structed in the CDR2 (residues 52–56) and HV4 (residues
70–74) regions by site-directed mutagenesis using overlap-
ping degenerate primers. The mutant called L3 was cloned
by introducing the mutations of clone HV7 into the mutant
CD6 using QuikChange as described by the manufacturer
(Stratagene, La Jolla, CA, USA).
Selection and analysis of Vb mutants by flow cytometry
Expression of Aga2p fusions were induced by growth of
yeast cells in medium containing galactose at 208C for
32–48 h. After induction, yeast-displayed Vb proteins were
selected by fluorescence-activated cell sorting (FACS) or ana-
lyzed by flow cytometry. Yeast cell libraries were selected by
using equilibrium or off-rate-based methodologies. For equi-
librium sorting, the yeast library was incubated with various
concentrations of biotin-SEC3 (Toxin Technology, Sarasota,
FL, USA) in phosphate-buffered saline (PBS) for 1 h on ice.
Cells were washed with 0.5 ml PBS–0.05% bovine serum
albumin (PBS–BSA) and then incubated with streptavidin–
phycoerythrin (SA–PE) (BD Biosciences) at a 1: 1000
dilution. Libraries were selected using FACS analysis
(MoFlo; Cytomation). Off-rate selection was performed by
incubation of yeast libraries with biotin-SEC3 under equilib-
rium conditions, followed by washing, incubation with non-
biotinylated SEC3 in 10-fold molar excess at 258C for
various times, followed by SA–PE staining.
Yeast cells isolated by FACS were either expanded by cul-
turing for subsequent rounds of selection, or plated on select-
ive medium to isolate yeast clones. Individual clones were
analyzed for equilibrium and off-rate binding to biotin-SAg.
Equilibrium binding was performed as described above for
sorting, except that either biotin-SEC3 or biotin-SEB was
used. Off-rate analysis was performed as described above for
sorting, except that the second incubation with unlabeled
SEC3 was performed at 378C rather than at 258C. Samples at
various time points were analyzed and percent-bound
biotin-SEC (mean fluorescence units (MFUs) of samples at
specific time point/MFUs at time point zero ? 100) was
plotted against time. C-myc and HA tag expression were
used to estimate surface expression of clones. Cells were ana-
lyzed on a Coulter Epics XL flow cytometer or a BD Accuri
C6 flow cytometer.
Cloning, expression and purification of soluble Vb proteins
Vb mutant L3 was subcloned as an NheI–BglII fragment
into the pET28 expression vector (Novagen) for expression
in bacteria. The Vb proteins L3, L2CM, G5-8 and mTCR15
were expressed in BL21 (DE3) Escherichia coli, refolded in
vitro from inclusion bodies, and purified with Ni agarose
resin (Qiagen), followed by gel filtration high-performance
liquid chromatography (BioCad Sprint) using a size exclu-
sion Superdex 200 column (Pharmacia) in PBS (pH 7.4) as
described previously (Buonpane et al., 2007). Protein pre-
parations were examined by sodium dodecyl sulfate poly-
acrylamide gel electrophoresis (SDS-PAGE) and mass
spectrometry. For mass spectrometry analysis, purified
protein was equilibrated with ammonium acetate buffer and
then analyzed at the UIUC Mass Spectrometry Laboratory
by Electrospray Ionization on a Quattro II mass spectrometer
using Masslynx software.
Binding of soluble Vb to SAg
Binding of soluble Vb to biotinylated-SAg was examined by
enzyme-linked immunosorbent assay (ELISA) and surface
plasmon resonance (SPR). For the ELISAs, Vb protein was
coated on ELISA wells (5 mg/ml), followed by addition of
various concentrations of biotin-SEC3 or biotin-SEB, fol-
lowed by streptavidin-conjugated horseradish peroxidase (BD
Biosciences), and finally substrate to yield a colorimetric
read-out at an absorbance of 450 nM wavelength. The affin-
ities and kinetic parameters of Vb : SEC3 and Vb : SEB
interactions were determined by SPR analysis using a
BIAcore 3000 instrument as described previously (Kieke
et al., 2001; Buonpane et al., 2007).
T-cell hybridoma line m6-16 cells (5 ? 105cells/ml) that
expresses a TCR with the mouse Vb8.2 region (Holler and
Kranz, 2003) were stimulated with 35 nM SAg (SEC3 or
SEB) in the presence of MHC class II-positive B cell line
LG-2 cells (105cells/ml). Soluble Vb proteins (L3, L2CM,
G5-8 or negative control mTCR15) were added at various
D.M.Mattis et al.
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concentrations and incubated at 378C for 20–24 h. Cells
were centrifuged, and supernatants were collected. IL-2
levels in supernatants were measured by ELISA.
CA-MRSA USA400 strain MW2 was originally obtained in
the Upper Midwest from a young patient who succumbed to
necrotizing pneumonia. The strain of low passage is main-
tained in the lyophilized state in the Schlievert laboratory.
Genome-sequencing studies of MW2 indicated that the strain
has the potential to produce the SAgs SEC, SEA and the
SE-like SAgs H, K, L, Q and X (Derzelle et al., 2009). Our
studies of MW2 and other S.aureus strains reported findings
show that SEC is expressed at orders of magnitude higher
levels than the other genes in certain strains and thus is likely
to be more involved in the diseases caused by SAgs. For
example, in the MW2 strain, SAg levels in culture
supernatants were determined as follows: SEC, 77 000 ng/ml;
SEA, 0.15 ng/ml; SE-like H, 0.075 ng/ml; SE-like K,
0.075 ng/ml; SE-like L, 0.075 ng/ml; SE-like Q, 30 ng/ml and
SE-like X, 0.1 ng/ml. These SAg levels were determined by
quantitative Western immunoblots, which utilizes purified
toxins tested comparably as standards. This method of toxin
quantification has been reported previously (Schlievert et al.,
All animal experiments were performed according to proto-
cols approved by the Institutional Animal Care and Use
Committee (IACUC) of the University of Minnesota.
Endotoxin enhancement model
New Zealand White (NZW) rabbits (?2 kg each) were
injected intravenously (i.v.) with 5 mg/kg of the SAg SEC3
Fig. 1. Vb8 mutants against SEC3 isolated by yeast display. (A) Vb8.2 gene (L2CM) was fused to Aga2 for expression on the surface of yeast cells. The Vb
gene was flanked by HA and C-myc epitopes. (B) Crystal structure of the Vb8 mutant L2CM (dark gray) in complex with SEC3 (dark blue). Mutations A52V
and S54N in the CDR2 region were selected previously by random PCR mutagenesis for improved affinity of L2CM to SEC (Kieke et al., 2001) and are
shown in orange. Residues mutated to generate L3 are in CDR1 (residues 28–30, magenta), in HV4 (residues 70–74, green) and individual residues Gly24
(marine blue) and Gly42 (red). Figure based on PDB file 2AQ3. (C) Model of the L2CM structure with L3 substituted mutations in the HV4 region, showing
aromatic base stacking between L3: W72 and overlay of SEB/SEC3 Y90 that may contribute to improved affinity of L3 for both SEB and SEC. Model based
on PDB files 2AQ3 and 3R8B. (D) Model of CDR1 loop of G5-8 in complex with SEB/SEC3 overlay, showing the likely interaction between L3: Y28 and
SEC/SEB: N60, contributing to improved affinity by the CDR1 mutations (Bonsor et al., 2011). Model based on PDB files 2AQ3 and 3R8B. (E) Sequences of
Vb8 mutants isolated by yeast display. Unique sequences of clones selected from the HV and CD libraries are listed. The two highest affinity mutants
identified from yeast display library selection were HV7 and CD6. HV7 contains mutations in the HV4 region (green), and CD6 contains mutations in the
CDR1 region (magenta). Combining the mutations in HV7 and CD6 generated the highest affinity mutant, L3. mTCR15 refers to a single-site mutant that had
improved display on yeast compared with the wild-type Vb8, but retains the wild-type residues that contact SEC3. G5-9 refers to a high-affinity Vb previously
developed in our laboratory against SEB (Buonpane et al., 2007).
High-affinity SEC antagonist
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in PBS either alone or in combination with 100 mg/kg
soluble Vb L3 in PBS, followed 4 h later with i.v. injections
of 0.15 mg/kg/ml endotoxin (Salmonella typhimurium). SAgs
have been shown to amplify the effects of endotoxin through
synergistic release of TNF-a (Schlievert, 1982; Dinges and
Schlievert, 2001). Temperatures were monitored for 4 h after
injection. The rabbits were monitored for up to 48 h for signs
of fever, diarrhea and death. P value determined by Fisher’s
exact test (n ¼ 9 per group).
Miniosmotic pump model
In this model, Dutch-belted rabbits receive continuously
released SEC3 from subcutaneously implanted miniosmotic
pumps (Alza, Palo Alto, CA, USA) (Parsonnet et al., 1987).
Young adult rabbits (?1–2 kg) were anesthetized with keta-
mine and xylazine, and incisions were made on the left
flanks. A subcutaneous pocket was made in each rabbit large
enough to accommodate the miniosmotic pump. The minios-
motic pumps are loaded with 200 mg SAg and implanted in
the pocket. The rabbits were sutured, allowed to wake,
returned to their cages and monitored for temperature on Day
2, and TSS symptoms and death over 8 days. In this model,
the maximum fever occurs on Day 2. Soluble 100 mg Vb or
the control PBS was administered i.v. once daily for 7 days.
P value determined by two-tailed unpaired t-test (n ¼ 3 per
group). The error bars represent standard error of mean.
MW2 necrotizing pneumonia
NZW rabbits (?2 kg each) were anesthetized with ketamine
and xylazine and incisions were made through the neck fur
and then in the trachea. The animal was placed on its right
side to insert a catheter through the trachea into the right
bronchi. Approximately 1.8 ? 109bacteria were injected, the
trachea sealed and the incision sutured. The animal was
allowed to wake and was then returned to its cage and moni-
tored. This model of necrotizing pneumonia has been
described previously (Strandberg et al., 2010). The experi-
ment used SEC-secreting USA400 CA-MRSA strain (MW2).
Rabbits were administered an i.v. bolus of either the control
PBS (pH 7.4) or 500 mg Vb L3 once daily. Rabbit tempera-
tures and survival were monitored daily over 4 days. P value
determined by log-rank (Mantel–Cox) test (n ¼ 4 per group).
NZW rabbits were anesthetized and underwent surgery to
insert catheters through the left carotid arteries to the aortic
valves, where the catheters remained for 2 h (Schlievert
et al., 1998; Pragman et al., 2004). The experiments con-
sisted of two groups of rabbits (n ¼ 4 per group). Either the
control PBS (pH 7.4) or 100 mg of Vb L3 was injected i.v.
through the marginal ear vein of the NZW rabbits (?2 kg
each), followed by the SEC-secreting microbe CA-MRSA
USA400 strain MW2 in the range of 9 ? 107–1 ? 108bac-
teria. The rabbits were administered either control PBS or
100 mg of Vb L3 twice a day for up to 4 days. The rabbits
were examined daily for survival and upon premature death,
or on Day 4 for vegetations.
Yeast display and engineering of high-affinity anti-SEC
To engineer soluble, high-affinity SEC antagonists, we used
a mouse Vb8 gene cloned as an Aga-2 fusion in the yeast
display vector pCT302 (Fig. 1A) (Boder and Wittrup, 2000).
Previously in our laboratory, random mutagenesis was used
to affinity-mature the wild-type Vb8 against SEC, resulting
in the mutant called L2CM (Kieke et al., 2001). However,
our more recent studies have shown that site-directed muta-
genesis approaches could yield significantly higher affinities
(e.g. in the 50–200 pM range) for Vb mutants against the
SAg SEB, providing more potent in vitro and in vivo neutral-
izing agents (Buonpane et al., 2007). To accomplish this
with the SEC system, L2CM was used as the starting tem-
plate for a directed evolution strategy with specific site-
directed libraries of mutants.
Based on alanine scanning mutagenesis (Churchill et al.,
2000) and crystal structures (Cho et al., 2005), Vb residues
at the interface of the Vb : SEC complex were mutated to
generate two libraries, one in hypervariable region 4 (here-
after termed HV library) and one in complementarity deter-
mining region 2 (hereafter referred to as CD library)
(Fig. 1B). Detailed description of generating, sorting and
identifying clones from each of the two libraries are provided
in Supplementary Results. The two yeast display libraries
yielded various clones with improved binding to SEC3, com-
pared with L2CM (Fig. 2A and B). The clones derived from
the HV library also showed improved off-rates, as evidenced
by a greater fraction of bound biotin-SEC3 after incubation
with excess unlabeled SEC3 for 10 min (Fig. 2C). A single
CD clone, CD6, also showed a slight improvement in
Among the HV mutants, there were four unique sequences
(Fig. 1E), all containing the same mutations at two of the
five positions in the HV4 region: S71T and H72W, suggest-
ing that these mutations were important for improved affinity.
There also appeared to be preferences for leucine at position
70 (3/4), asparagine at position 73 (2/4) and glutamic acid at
position 74 (2/4) (Fig. 1E and Supplementary Fig. S2A).
Based on modeling of the crystal structure L2CM in
complex with SEC3, we believe that mutation Trp72 in L3
could interact with Tyr90 in SEC3 (Fig. 1C).
The sequences of eight CD clones showed that, unexpect-
edly, there were no mutations in the CDR2 region but there
were mutations in or near the CDR1 region, at positions 24
and 28–30. One clone (CD6) contained an additional muta-
tion at position 42. The origin of these mutations is almost
certainly due to the original pCT302 vector which contained
the high-affinity Vb mutant called G5-9, developed against
SEB, and was used to clone the polymerase chain reaction
(PCR) products of the HV4 and CDR2 libraries (Fig. 1E)
(Buonpane et al., 2007). Thus, during the homologous re-
combination step involving yeast transformation of the librar-
ies, there was likely a low frequency of recombination
between undigested pCT302/G5-9 and the PCR-amplified
libraries of L2CM. The prevalence of the G5-9/L2CM chimera
in the SEC-selected library suggests that other CDR2 mutations
in the library were not preferred above the A52V and S54N
mutations of L2CM, and that the CDR1 of G5-9 provided some
improvement above L2CM in SEC binding (as it did with
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by guest on December 31, 2015
SEB). In fact, the recent crystal structure of a related SEB-
binding mutant called G5-8 shows that the Tyr28 mutation of
CDR1 contacts residue Asn60 that is conserved between SEB
and SEC (Fig. 1D) (Bonsor et al., 2011).
As clones HV7 and CD6 had improved binding, and we
have shown previously that such mutations can yield syner-
gistic binding when combined (Kieke et al., 2001; Moza
et al., 2006), the HV7 and CD6 mutations were introduced
into a single mutant called L3 (Fig. 1E). Yeast display titra-
tions with SEC showed that mutant L3 had an affinity that
was improved slightly above that of both HV7 and CD6
(Fig. 2D), and thus L3 was used for further studies with
soluble Vb protein and neutralization of SEC.
Analysis of L3 Vb regions for SEB binding
Since L3 shared the same CDR1 mutations as the SEB affin-
ity matured mutant G5-9, and the SAgs SEB and SEC3 share
the common residue Tyr90 that we believe interacts with L3
Trp72, we decided to test yeast display binding of L3 to
SEB. We found that yeast displayed L3 bound to 10 nM
biotin-SEB (Fig. 3). In contrast, the Vb engineered for high-
affinity binding to SEB, called G5-8, did not bind SEC3, al-
though as expected it bound to SEB (Buonpane et al., 2007).
The mutations in L3 originated from one of three sources:
CDR2 mutations in the template L2CM, HV4 mutations
from mutant HV7 or CDR1 mutations from mutant CD6
(Fig. 1E). To determine which mutations in L3 were respon-
sible for the cross-reactivity with SEB, yeast displayed Vb
regions CD6, HV7 and L2CM were also analyzed with
Fig. 2. Yeast displayed clones analyzed for SEC3 binding by flow cytometry. (A) Flow cytometry histograms of L2CM, HV7 and CD6 after staining with
0.1 nM biotin-SEC3, followed by SA–PE. (B) MFU of various Vb8 mutants stained with 0.1 nM biotin-SEC3 followed by SA–PE. (C) %SEC3 remaining
bound to various Vb8 mutants following incubation with 0.5 nM biotinylated-SEC3, 10 min after addition of 10-fold molar excess of unlabeled SEC3.
(D) L2CM, the two highest-affinity clones selected from each library (CD6 and HV7), and the combined mutant L3 were titrated with 0.01 nM to 100 nM
SEC3, followed by SA–PE. The normalized MFUs were obtained by flow cytometry and plotted against SEC3 concentration to generate the binding curves.
Fig. 3. Yeast display high-affinity Vb mutants stained with 10 nM SEC3 and
SEB. Vb mutants L3, G5-8, CD6, HV7 and L2CM were stained with 10 nM
biotin-SEC3 (left column) or 10 nM biotin-SEB (right column), followed by
SA–PE. Mutants L3 and HV7 bound both SAgs. CD6 and L2CM bound only
10 nM SEC3. G5-8 bound only 10 nM SEB. Gray peak indicates background
fluorescence. Black line indicates fluorescence of Vb mutant.
High-affinity SEC antagonist
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10 nM biotin-SEB and 10 nM biotin-SEC3 (Fig. 3). Mutants
L3, CD6, HV7 and L2CM that were selected for improved
SEC3 binding, all bound 10 nM SEC3 (Fig. 3, left-hand
column), as was originally observed in the process of yeast
display selections. Binding to 10 nM SEB was observed for
L3, G5-8 and HV7 (Fig. 3, right-hand column). Neither
L2CM nor CD6 showed detectable binding to 10 nM SEB.
The detectable binding of SEB by HV7 indicates that the
HV4 mutations, including the tryptophan at position 72,
were important in binding to SEB. As indicated, a possible
interaction might occur between Trp72 of the L3 HV4 region
and Tyr90 of SEC, and this residue (Tyr90) is also present in
SEB (Fig. 1C).
Expression, purification and SAg binding analyses
of soluble Vb proteins
To produce soluble protein for neutralization studies, the Vb
proteins, including L3, were expressed and refolded from in-
clusion bodies in E.coli. After refolding and nickel resin af-
finity purification, the L3 protein eluted as a monomer on gel
filtration chromatography and exhibited the expected sizes by
SDS-PAGE and mass spectrometry (calculated size based on
amino acid sequence, 14 566 Da; size observed on mass
spectrometry, 14 563 Da) (data not shown). The protein yield
averaged 2 mg/l of inclusion bodies.
L3, L2CM and G5-8 proteins were examined using SPR to
measure the affinity and kinetics of their interactions with
SEB and SEC3 (Fig. 4 and Table I). The affinity constants
(KD values) for SEC (Fig. 4A) were 10 nM (L2CM) and
2 nM (L3), while G5-8 showed no detectable binding, con-
sistent with the flow cytometry results. Compared with
L2CM, L3 also exhibited an ?7-fold improvement in
off-rate, suggesting that HV4 and CDR1 mutations increase
the stability of the complex with SEC3. The half-life (t1/2) of
the complex at 258C was thus predicted to be ?20 min.
Consistent with flow cytometry results, the affinity of L3
for SEB (KD¼ 4 nM) was shown to be considerably higher,
by about 100-fold, than the affinity of L2CM for SEB (KD¼
490 nM) (Fig. 4B). Thus, it is possible to generate a cross-
reactive, high-affinity Vb, by manipulating different regions
of the interface, focusing in principle on residues that are
shared between SEC and SEB.
In vitro neutralization of SAg-mediated activation of T cells
To analyze the SEC and SEB neutralization ability of mutant
L3, we monitored the release of IL-2 by a T-cell line that
expresses the Vb8 region. In this assay IL-2 is secreted when
the Vb8-positive T cell line is incubated with SAg (35 nM)
and a human class II MHC-positive B cell line called LG-2.
The soluble high-affinity Vb proteins L3, G5-8, L2CM and
mTCR15 (a wild-type Vb8 proteins that has a single muta-
tion allowing expression in soluble form; Kieke et al., 2001)
were added at various concentrations, and after ?24 h the
supernatants were assayed for IL-2 levels using a capture
ELISA. The wild-type Vb (mTCR15), which has micromolar
affinity for both SEC3 and SEB, was ineffective at neutraliz-
ing either SAg (Fig. 5). In contrast, L2CM was able to com-
pletely neutralize SEC3 activity with an IC50of 400 nM, and
L3 was able to completely neutralize SEC3 activity with an
IC50 of 40 nM (Fig. 5A). Thus, L3 exhibited ?10-fold
improved inhibitory properties, consistent with its 7-fold
enhanced lifetime of dissociation compared with L2CM. The
Vb G5-8 that was developed against SEB was unable to neu-
tralize SEC (Fig. 5A), but was effective at neutralizing SEB
activity (Fig. 5B). In contrast, L3 was able to neutralize both
SEB activity (Fig. 5B) and SEC activity, as anticipated from
the results of binding studies.
Fig. 4. Binding (SPR) of soluble Vb8 proteins. (A) SPR traces of serial 2-fold dilutions (800 nM stocks) of affinity-matured Vb8 mutants L2CM, L3 and
G5-8 injected at a flow rate of 25 ml/min over immobilized (A) SEC3 or (B) SEB, as previously described (Buonpane et al., 2007). RU, response units.
Binding affinity (KD) are shown, NB, no binding.
Table I. SPR results of high-affinity Vbs
SAg SEC3SAg SEB
L2CM 3.96 ? 105
L32.68 ? 105
3.94 ? 102310
5.75 ? 10242
6.7 ? 105
5.75 ? 105
2.57 ? 10234
1.4 ? 1024
aND, not determined.
bNB, no binding.
D.M.Mattis et al.
by guest on December 31, 2015
Neutralization of SEC by the soluble Vb L3 in an
endotoxin enhancement rabbit model
To determine whether Vb L3 could neutralize SEC in vivo,
we used SEC in an endotoxin enhancement rabbit model,
which lowers the lethal doses of both SEC and endotoxin by
orders of magnitude (Schlievert, 1982; Dinges and Schlievert,
2001). NZW rabbits were injected with 5 mg/kg SEC3 fol-
lowed by either PBS or the soluble Vb L3 at 100 mg/kg. After
4 h, each rabbit was injected i.v. with 0.15 mg/kg of endotoxin
lipopolysaccharide (LPS), which is 100 times the half-
maximal lethal dose (LD50) in rabbits pretreated with 5 mg/kg
SEC3 (the LD50of LPS alone is 500 mg/kg). After 48 h, none
of the nine rabbits that received PBS survived, while eight of
the nine rabbits that received the soluble Vb L3 survived
(Fig. 6A). The ability of the L3 protein to prevent death in this
model was highly significant (P, 0.001).
Neutralization of SEC by the soluble Vb L3 in a
miniosmotic pump rabbit model
In a bacterial infection, SAg is thought to be produced con-
tinuously by the bacteria. To simulate this, a miniosmotic
pump releases ?200 mg of SEC3 over an 8-day period. In
this model, either PBS or 100 mg of Vb L3 was administered
i.v. daily (n ¼ 3 per group). The second-day body tempera-
tures of the rabbits that received the control PBS averaged
39.68C, which was significantly greater than the rabbits that
received the Vb L3, which were 38.78C (Fig. 6B). Although
these values differ only by a degree, the difference is signifi-
cant, with a P value , 0.05. Two of the three rabbits that
received the Vb L3 survived, while none of the three rabbits
that received the control PBS survived (Fig. 6C).
Neutralization of SEC secreted by MW2 in a
necrotizing pneumonia rabbit model
To examine the ability of Vb L3 to neutralize SEC secreted
from bacteria, we used a necrotizing pneumonia rabbit
model. This model is appropriate because SEC is implicated
as playing a role in the development of necrotizing pneumo-
nia (Strandberg et al., 2010). NZW rabbits were administered
2 ? 109bacteria of the SEC-positive strain CA-MRSA
USA400 MW2, intrabronchially. Although other SAg genes
are present in this strain, it is important to note that SEC is
secreted at more than 2500-fold higher concentrations than
other SAgs in MW2 (see Materials and methods), suggesting
that among the SAgs one might expect SEC to have the most
significant hyperinflammatory effect. Treatment consisted of
i.v. injection of 500 mg Vb L3 or PBS daily. Temperatures
were measured daily, as was survival. Three of the four
rabbits that were given MW2 followed by the control PBS
died over the 4 days (Fig. 7B). The control rabbits had an in-
crease in body temperature that peaked on the second day
(Fig. 7A). All four of the rabbits that received the Vb L3 sur-
vived, and their temperatures remained normal (Fig. 7A and
B). Thus, Vb L3 was able to neutralize the SEC secreted by
MW2 and survival of the treated rabbits was significant with
Neutralization of SEC secreted by MW2 in an infective
endocarditis rabbit model
There has been evidence that TSST-1 is involved in infective
endocarditis in a rabbit model (Pragman et al., 2004), and its
prevalence in S.aureus isolates from human endocarditis sug-
gests that it is involved in the human disease (Nienaber
et al., 2011). Having already shown that L3 could neutralize
the toxin alone, and also prevent lethality caused by the
MW2 strain in the pneumonia model, we explored the ability
Fig. 5. In vitro inhibition of SEC3- and SEB-mediated T cell activity by
soluble Vb proteins. T cell cytokine (IL-2) release assay showing inhibitory
activity of soluble, high-affinity Vb mutants L3 (filled circles), L2CM
(filled squares), G5-8 (filled triangle, dotted line) and the wild-type Vb
mTCR15 (open circles) against (A) SEC3 and (B) SEB. Mutants were added
at the indicated concentrations to wells containing the Vb8-positive T cell
line m6-16, SAg and the human class II-positive B cell line LG-2. The error
bars represent SEM (n ¼ 2 per group).
Fig. 6. In vivo models testing Vb L3 neutralization of SEC3. (A) LPS
enhancement model. Rabbits were injected i.v. with either the SAg SEC
alone or in combination with the Vb L3, followed 4 h later with LPS. Zero
of nine rabbits survived when injected with SEC alone. L3 is able to
neutralize the toxin yielding survival of 8/9 rabbits. P value determined by
Fisher’s exact test (n ¼ 9 per group). (B) and (C) Miniosmotic pump model.
Rabbits were surgically implanted with a pump that releases either the SAg
SEC alone or in combination with the Vb L3. Second day body
temperatures were significantly increased and 0/3 rabbits survived when
injected with SEC alone versus survival of 2/3 rabbits and reduced body
temperatures when treated with Vb L3. (B) P value determined by
two-tailed unpaired t test (n ¼ 3 per group). The error bars represent SEM.
High-affinity SEC antagonist
by guest on December 31, 2015
of L3 to neutralize SEC in an infective endocarditis rabbit
model, with the MW2 strain (Schlievert et al., 1998). In this
model, catheters are inserted into anesthetized rabbits to the
aortic valves where the catheter damages the valves for 2 h
prior to removal. Infective endocarditis was modeled by
injecting ?108SEC-secreting bacteria USA400 strain MW2
into the marginal ear vein, allowing for formation of vegeta-
tions at the valve sites for up to 4 days, similar to that
observed in infective endocarditis. Rabbits were administered
either the control PBS (pH 7.4) or 100 mg of Vb L3 twice
daily and examined for survival four times daily and for
vegetations upon death or on Day 4 upon euthanasia.
Two of the four rabbits infected only with MW2, but
without L3 treatment, died before the fourth day (one within
24 h and the second at 48 h). The rabbit that died at 24 h
developed a single vegetation of 17 mg with 1.3 ? 107
colony-forming units (CFUs)/vegetations (Fig. 7C and
Supplementary Fig. S3C). The relatively rapid death of this
rabbit likely accounts for the smaller vegetation size com-
pared with the other rabbits. The other rabbits infected only
with MW2, but without L3 treatment, developed multiple
large vegetations (Fig. 7C and Supplementary Fig. S3A–B
and S3D) with an average weight of 84 mg and 6.2 ? 107
CFU/vegetations (Fig. 7C and D).
One of the four rabbits, which was administered Vb L3,
died at 72 h, and the other three were euthanized at Day 4
(as required by IACUC protocol). In contrast to the control
(PBS) rabbits, all four of the Vb L3-treated rabbits had
either noor onlysmall
Fig. S3E–H). In addition, the average vegetation weight of
organisms derived from the Vb L3-treated rabbits was
9.2 mg and 340 CFU/vegetations (Fig. 7C and D). Although
significant (P values of 0.038 and 0.013, respectively, for
weight and CFU/vegetations) these data were skewed some-
what because the one Vb-treated rabbit that died at 72 h had
4.7 ? 106CFU when the aortic valve surface was scraped
and analyzed (Fig. 7D), but this was still below that of any
of the control rabbits.
We show here that a small protein domain (Vb) of the extra-
cellular receptor for SEC could be engineered for high affin-
ity against SEC, and that the engineered protein (L3)
cross-reacted, unexpectedly but fortuitously, with the related
toxin SEB. Consistent with these binding properties, L3 was
able to neutralize both SEC and SEB in T cell activation
assays in vitro. L3 was also able to not only protect rabbits
from the lethality associated with exposure to the toxin SEC
itself, but it also protected rabbits from the lethality caused
by a strain of MRSA (MW2) that secretes SEC. Successful
treatment of rabbits infected in the lung with the MW2
strain, in the necrotizing pneumonia model, is likely due to
the specific anti-inflammatory properties of the neutralizing
agent. Treatment of MW2 in the infective endocarditis
model also yielded beneficial results, with a reduction in
vegetation size, although the mechanism of action of the
neutralizing agent in this model is less clear and remains to
be determined. Nevertheless, the results suggest an important
role for the SAg in this disease, and they provide further evi-
dence that these small, high-affinity protein domains repre-
sent a therapeutic strategy that could show efficacy, even in
cases where antibiotics have limited or delayed impact.
SAg genes have been identified in the majority of MRSA
strains from various clinical diseases (e.g. Campbell et al.,
2008; Lalani et al., 2008; Xiong et al., 2009; DeVries et al.,
2011; Robert et al., 2011; Sharma-Kuinkel et al., 2011) and
they have recently been found to be carried by VRSA strains
(Kos et al., 2012). In addition to their emergence in
antibiotic-resistant strains, SAgs secreted by S.aureus and
group A streptococci have been shown to contribute to mul-
tipleillnesses including TSS,
purpura fulminans and extreme pyrexia syndrome (Luppi
et al., 1998; Bachert et al., 2007; Strandberg et al., 2010).
Fig. 7. In vivo models testing Vb L3 neutralization of MW2 SEC secreting
bacteria. (A) and (B) Necrotizing pneumonia model. Rabbits were
intrabronchially administered 2 ? 109bacteria of the CA-MRSA USA400
strain MW2, which secretes SEC. Treatment consisted of either an i.v. of
500 mg Vb L3 or PBS daily. (A) Temperatures in rabbits administered Vb
remained low while rabbits that received MW2 only had an increase in
temperature. The error bars represent SEM. (B) One of four rabbits survived
when injected with MW2 alone. The Vb L3 is able to neutralize the toxin
yielding survival of 4/4 rabbits. P value of (B) (P, 0.05) determined by
log-rank (Mantel–Cox) test (n ¼ 4 per group). (C) and (D) Endocarditis
model. Vegetations were isolated and weighed either at death or on Day
4. PBS-treated rabbits (n ¼ 4) developed multiple large vegetations with a
mean weight of 84 mg (C) and 6.18 ? 107CFU/vegetations (D). The Vb
L3-treated rabbits (n ¼ 4) had either no or small vegetations with a mean
weight of 9 mg (C) and 340 CFU/vegetations (D). Both the average weight
of the vegetation and the CFU/vegetations were statistically significant with
P values ,0.05. P values determined by two-tailed unpaired t-test (n ¼ 4
per group). The error bars represent SEM.
D.M.Mattis et al.
by guest on December 31, 2015
Clinical and epidemiological features also implicate SAgs in
diseases such as atopic dermatitis and Kawasaki syndrome
(Leung et al., 2002; Schlievert et al., 2008; Macias et al.,
2011). A recent study showed that the SAg TSST-1 gene was
among the few genes significantly associated with isolates
derived from endocarditis patients (Nienaber et al., 2011).
Despite advances in acute care and antibiotics, mortality
rates due to staphylococcal infections remain high. As
neither the treatment of symptoms nor antibiotics are capable
of removing SAgs already secreted by the bacteria and
present within tissues of the host, the relatively small
(?14 kDa) agents described here may be able to penetrate
affected tissues and reduce the hyperinflammatory effects of
Our previous studies with a high-affinity neutralizing
agent against SEB showed that the Vb G5-8 could be used
to treat pulmonary disease (Strandberg et al., 2010), thereby
implicating the SAg in lung disease. It was reasonable to
assume that some of the pulmonary effects of SAgs were due
to the massive inflammation that the toxins induce, and thus
treatment with an agent that acts early in the cascade mech-
anism could impact the disease. In the infective endocarditis
model, the direct role of SAgs may be less clear, but the ob-
servation that the Vb neutralizing agent showed significant
effects implies that the process of endocarditis caused by
S.aureus involves the functional effects, or dysregulation, of
T cells at the site of the damaged valves.
Based on our previous engineering of the high-affinity Vb
G5-8 against SEB (Buonpane et al., 2007), we used G5-8 in
several rabbit models of SEB-mediated diseases, including
TSS(Buonpaneet al.,2007), necrotizing
(Strandberg et al., 2010) and atopic dermatitis (John et al.,
2009) exacerbated by SEB. However, the Vb regions previ-
ously engineered against SEB did not cross-react with high
affinity against the structurally related SAg SEC, despite the
two SAgs sharing 65% homology (Bohach et al., 1990;
McCormick et al., 2001; Wang et al., 2010). As SEC is im-
portant in disease, and often expressed by the USA400
strains of MRSA, here we elected to further engineer a high-
affinity Vb against SEC, called L2CM (Kieke et al., 2001),
to improve its affinity and its neutralizing potential. L2CM
was originally engineered by random mutagenesis, and with
improved strategies for site-directed mutagenesis we focused
on libraries within the Vb loops nearest to the SEC binding
interface, HV4 and CDR2. A strong preference for several
HV4 residues was observed in the improved Vb mutants, in-
cluding a tryptophan at position 72. It is reasonable to
suggest that part of the improved affinity for SEC and its
higher affinity for SEB is related to Trp72 proximity to the
SAg residue Tyr90, which is found in both SEB and SEC3
(Fig. 1C), perhaps through additional buried hydrophobicity
While Trp72 and the HV4 mutations may account for
some of the additional binding energy for both SEC and
SEB, it is possible that both the CDR1 and CDR2 mutations
in L3 may also act synergistically. For example, the addition-
al residue in CDR1 has been shown to position the Tyr28 to
contact Asn60 in the G5-8/SEB structure, and Asn60 is
shared between SEB and SEC (Fig. 1D). Furthermore, our
recent studies have suggested that the Arg53 mutation of the
SEB-reactive G5-8 prevents high-affinity binding to SEC
because the unique tyrosine of SEC at position 26 occupies
the pocket that Arg53 is inserted into in SEB (Wang et al.,
2010; Bonsor et al., 2011). In contrast, our previous extensive
structural and binding analyses of Val52 and Asn54 in
L2CM have shown that these mutations act through multiple
mechanisms, pre-configuring the Vb surface for binding and
also increasing the contacts with SEC (Cho et al., 2005).
Furthermore, the retention of the wild-type Gly53 in L3
likely allows the Val52 and Asn54 residues to engage SEC,
yet still interact effectively with SEB. It is possible that these
CDR1 and CDR2 interactions were not adequate to raise the
affinity above a threshold binding needed for flow cytometry
with 10 nM SEB, but that combined with the HV4 mutations
they yielded the final nanomolar affinity for SEB. Regardless
of the exact structural details, which will require crystalliza-
tion of the complexes, the results with L3 show that it is pos-
sible to engineer a protein domain that can bind with high
affinity to two different ligands by manipulating different
regions of the interface.
In summary, we show that it is possible to use an engi-
neered, high-affinity neutralizing agent against SEC to
further demonstrate the importance of SEC in rabbit models
of disease. Accordingly, the neutralizing protein L3 reduced
the lethality and severity in two disease animal models, nec-
rotizing pneumonia and infective endocarditis caused by
SEC-positive CA-MRSA USA400 strain MW2. Quantitative
analysis of bacterial burden, both by weighing scraped tissue
and colony counts revealed a significantly lower total weight
of vegetations as well as fewer CFUs/vegetations in the
L3-treated rabbits compared with the control (PBS)-treated
rabbits in the infective endocarditis model (Fig. 7C and D).
The inhibition of vegetation development further supports
the notion that SAgs play an important role in the etiology of
infective endocarditis. Although there are no doubt other
factors that are important in these disease etiologies, our evi-
dence suggests that S.aureus SAgs may provide one target
for treatment. The high-affinity and in vitro effectiveness of
the Vb L3 for both SEC and SEB indicates a potential use
as a therapeutic against bacterial strains that may secrete
either or both SAgs. While antibiotics constitute the major
approach to treatment of infective endocarditis (Sacar et al.,
2010) and necrotizing pneumonia (Shilo and Quach, 2011),
the mortality rates in these staphylococcal diseases, especial-
ly MRSA, remain high (Fowler et al., 2005) and thus it is
important to identify novel strategies to intervene.
Supplementary data are available at PEDS online.
We thank the staff of University of Illinois Biotechnology Center for assist-
ance in flow sorting and DNA sequencing.
This work was supported by National Institutes of Health
(F30-HL096352-01 to D.M.M.) and a grant from the
National Institutes of Health-supported Great Lakes Regional
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