AAC Accepted Manuscript Posted Online 26 January 2015 Antimicrob. Agents Chemother. doi:10.1128/AAC.04414-14 Copyright © 2015, American Society for Microbiology. All Rights Reserved.
1
Neutralization of Staphylococcal enterotoxin B by aptamer
2
antagonist
3
Kaiyu Wanga,b, Longjie Ganb, Li Jiangb, Xianhui Zhangb, Xiangyue Yanga,b, Min Chena,b,
4
Xiaopeng Lana,b
5
a
6
Fuzhou 350025, PR China, and Department of Laboratory Medicine, Dongfang Hospital,
7
Xiamen University, Fuzhou 350025, P.R. China
8
b
9
Fuzhou 350025, P.R. China
10 11
*
Institute of Laboratory Medicine, Fuzhou General Hospital of Nanjing Military Command,
Department of Laboratory Medicine, Fuzong Clinical College, Fujian Medical University,
Email: [email protected]; Tel: +86 591 22859490
12
Abstract
13
Staphylococcal enterotoxin B (SEB) is a major virulence factor for
14
Staphylococcal toxic shock syndrome (TSS). SEB activates a large subset of the T
15
lymphocytic
16
SEB-initiated toxicity may be an effective strategy for treating TSS. Using a
17
systematic evolution of ligands by exponential enrichment (SELEX) process, we
18
identified an aptamer that can antagonize SEB with nanomolar binding affinity (Kd =
19
64 nM). The aptamer antagonist effectively inhibits SEB-mediated proliferation and
20
cytokine secretion in human peripheral blood mononuclear cells. Moreover, a
21
PEGlyated aptamer antagonist significantly reduced mortality in a “double-hit” mouse
22
model of SEB-induced TSS, established via sensitization with D-galactosamine
23
followed by SEB challenge. Therefore, our novel aptamer antagonist may offer
24
potential therapeutic efficacy against SEB-mediated TSS.
25
Keywords: superantigens; aptamer; Staphylococcal enterotoxin B; toxic shock
population,
releasing
pro-inflammatory
cytokines.
Blocking
26
1. Introduction
27
Staphylococcal enterotoxins (SEs) are prototype superantigens excreted
28
primarily by Staphylococcus aureus and Streptococcus pyogenes. SEs are potent
29
activators of the immune system that can stimulate up to 40% of the naive T cell
30
population and cause diverse human diseases, including food poisoning, autoimmune
31
diseases, and toxic shock syndrome (TSS) (1). SEs can activate both
32
monocytes/macrophages and T lymphocytes by directly binding to MHC II molecules
33
and to the variable region of the β-chain of T-cell receptors (TCR), without being
34
taken up and presented by antigen presenting cells (2).
35
These toxins lead to the proliferation of monocytes/macrophages and T
36
lymphocytes and stimulate secretion of excess pro-inflammatory cytokines, including
37
interferon gamma (IFNγ), interleukin 2 (IL-2), and tumor necrosis factor alpha
38
(TNFα). Among these SEs, Staphylococcal enterotoxin B (SEB) is considered to be an
39
important cause of Staphylococcal TSS, which is characterized by high fever,
40
erythematous rash, hypotension, multi-organ system failure, and even death (3).
41
Remarkably, SEB is highly heat resistant, easily transformed into an aerosol, and
42
readily produced on a large scale using genetic engineering techniques. Humans are
43
extremely susceptible to this superantigen, so SEB is often referred to as an
44
"incapacitating agent" by the military who may use this toxin as an anti-personnel
45
aerosol or as a food supply contaminant (4).
46
At present, SEB is thought to be an effective treatment strategy for TSS by
47
blocking early processes prior to T cell activation and initiation of the toxic cascade.
48
Studies suggest multiple approaches for interfering with SEB binding to MHC-II
49
and/or TCR. These include vaccine immunization to induce anti-SEB antibodies (5, 6),
50
passive immunoprophylaxis with natural or artificial antibodies (7, 8), and the use of
51
synthetic peptides (9) or receptor mimics (10, 11) as antagonists to prevent the
52
formation of the MHC-II/SEB/TCR complex. Research is ongoing for new
53
compounds with greater half-lives, improved SEB binding affinity, and less
54
immunogenicity.
55
Aptamers are artificial short, single-stranded oligonucleotides (ssDNA or ssRNA)
56
that can form elaborate 3-D structures and specifically bind to desired targets with
57
high affinity (12, 13). Because of their intrinsic properties, aptamers are non-toxic and
58
non-immunogenic. In addition, aptamers composed of modified nucleotides have a
59
long in vivo half-life of hours to days.
60
During the past two decades, numerous aptamers have been developed to inhibit
61
pathogenic antigen activity and many have substantial clinical potential for treating
62
various diseases (14). Several researchers have reported on the selection of specific
63
DNA aptamers against SEB (15-17). Methods using these aptamers as capture
64
molecules have also been established to detect SEB in various samples (15, 18).
65
However, as far as we know, no aptamer has been selected to inhibit the biological
66
activity of SEB.
67
Therefore, we selected DNA aptamers directed against native SEB in vitro and
68
identified aptamer-antagonists capable of neutralizing SEBs by studying their
69
therapeutic effects on SEB-mediated toxic shock (TSS) in vitro using human
70
peripheral blood mononuclear cell (PBMC) cultures as well as a lethal murine model.
71
72
2. Material and Methods
73
2.1 SELEX library and primers
74
The ssDNA library, which contained random 60mer and fixed primer regions and
75
all primers used are described previously (ESI Table S1†) (19). All oligonucleotides
76
were synthesized and purified with HPLC (Invitrogen, Guangzhou, China). For
77
animal experiments, the aptamer candidate was conjugated at the 5’ end with 40-KDa
78
polyethylene glycol (PEG). The PEGlyated aptamer was prepared by treating 40-KDa
79
PEG N-hydroxysuccinimide ester with 5'-amino and 3'-inverted dT modified aptamer
80
candidates. All PEG-aptamer conjugates were purified by anion exchange and then by
81
reverse-phase HPLC (Seebio Biotech, Shanghai, China).
82
2.2 Aptamer screening for specific SEB binding
83
ssDNA aptamers specific to SEB were screened by a SELEX process as
84
described previously with minor modifications (20). Briefly, highly purified SEB
85
(Toxin Technology, Sarasota, USA) was coupled to M-270 carboxylic acid dynabeads
86
(DYNAL, Oslo, Norway) according to the manufacturer's instructions. Prior to
87
selection, the ssDNA library dissolved in 500 μl selection buffer (50 mM Tris. HCl
88
pH7.4, 5 mM KCl, 100 mM NaCl, 1 mM MgCl2) was denatured by heating at 95 oC
89
for 5 min and immediately placed on ice for 10 min. The denatured ssDNA library
90
and a 5-fold molar excess of yeast tRNA (Invitrogen) were incubated with SEB (100
91
ng) immobilized on magnetic beads in a 500 μl reaction, and were incubated at 37 oC
92
for 60 min while shaking.
93
Nonbinding oligonucleotides were removed by washing the DNA-bead complex
94
three times with selection buffer (0.2% BSA). SEB binding species were eluted by
95
heating at 100 oC for 5 min in 200 μl of sterile ddH2O. ssDNA was amplified by PCR
96
with FITC-labeled sense primers and biotin-labeled antisense primers (20-30 cycles of
97
0.5 min at 94 oC, 0.5 min at 52 oC, 0.5 min at 72 oC, followed by 5 min at 72 oC). All
98
PCR products were purified using a BioSpin gel extraction kit (Bioer Technology,
99
Hangzhou, China). From all purified PCR products from each SELEX round, large
100
aliquots were alkaline-denatured and used to prepare the aptamer strand using M-280
101
streptavidin dynabeads (DYNAL AS, Oslo, Norway) according to the manufacturer's
102
instructions, and small aliquots were stored at -80 oC prior to use.
103
To create aptamers with high affinity and specificity, we progressively increased
104
the selective pressure by increasing the number of washes and KCl, and by decreasing
105
the ratio of ssDNA to SEB and the incubation time (ESI Table S2†). The
106
counter-selection (depletion of aptamers that bind to non-target molecules) was
107
introduced against BSA-or Staphylococcal protein A (SPA)-coupled M-270 beads in
108
the 7th, the 9th, and the 11th round, respectively. BSA was the blocking protein for SEB
109
coupling, and SPA is a major protein of Staphylococcus aureus(21). To reduce the
110
matrix binders, reaction tubes were blocked with 1% BSA-PBS, and preselection
111
steps with uncoupled streptavidin beads were performed.
112
2.3 Cloning, sequencing and bioinformatic analysis
113
After rounds of SELEX selection, the selected ssDNA pool was PCR-amplified
114
using unlabeled primers under the same conditions described for screening. PCR
115
products were purified, and then cloned into pGEM-T vector (Promega, Madison,
116
USA) according to the manufacturer’s instructions. The resulting products were
117
transformed into Escherichia coli DH5α. Individual cultured colonies were chosen
118
randomly and their inserts were sequenced by Invitrogen Company. The aptamer
119
sequences were analyzed by ClustalX software and the secondary structure was
120
predicted by free-energy minimization algorithm according to Zuker (22) using the
121
internet-tool mfold (http://mfold.bioinfo.rpi.edu/cgi-bin/dna-form1.cgi).
122
2.4 Aptamer binding assay for SEB
123
A fluorescently labeled ssDNA binding assay was performed to monitor the
124
enrichment of each SELEX round and to evaluate aptamer binding affinity. In brief,
125
fluorescently labeled ssDNA was thermally denatured in 200 μl of selection buffer
126
and then incubated in the dark with SEB immobilized on magnetic beads at 37 oC for
127
1 h while shaking. After incubation, non-bound ssDNA was collected, and SEB-bound
128
ssDNA was eluted by heating at 100 oC for 5 min in 200 μl of selection buffer. The
129
fluorescent intensity of applied, non-bound, and eluted ssDNA was measured
130
respectively using a TBS-380 Mini-Fluorometer (Turner Biosystems, Sunnyvale, CA).
131
All binding assays were repeated three times. To monitor the enrichment of each
132
SELEX round, the percent of ssDNA (200 nM) binding with SEB (20 μM) was
133
calculated.
134
To determine the binding affinity of a selected aptamer, the binding assay was
135
performed as described above but with increasing amounts of ssDNA aptamer (0 to
136
400 nM) and a constant amount of SEB (20 μM) for each assay. To calculate the
137
dissociation constants (Kd) of the aptamers, the aptamer quantity bound to targets was
138
plotted and data points were fitted to the equation Y=BmaxX/(Kd+X) via non-linear
139
regression analysis, using Graphpad-Prism-V5.01 (Graphpad, San Diego, CA).
140
2.5 Human PBMC proliferation assays
141
To study the effect of aptamers on SEB-mediated T cell proliferation, an MTT
142
cell proliferation assay was performed with human PBMCs as described previously
143
(23). Human PBMCs were isolated from heparinized venous blood by Ficoll-Hypaque
144
density gradient centrifugation from healthy human donors. After lysing erythrocytes,
145
PBMCs were cultured in RPMI 1640 medium (Gibco, Grand Island, USA)
146
supplemented with 2 mM L-glutamine, 100 U/ ml penicillin, 100 mg/ml streptomycin,
147
10 mM HEPES and 10% (v/v) fetal bovine serum (Gibco) at 37 oC. PBMCs (105
148
cells/well) were plated with SEB (200 ng/ml) alone or combined with varying
149
concentrations of aptamer candidates in triplicate in a 96-well plate.
150
PBMCs cultivated in RPMI-1640 medium alone served as negative control.
151
Plates were incubated in a 5% CO2 humidified incubator at 37 oC for 48 h. After
152
stimulation, cell proliferation was measured by adding 20 μl of MTT solution (Sigma,
153
St. Louis, USA), prepared in PBS at 5 mg/ml (pH 7.2), to each well. After 2 h of
154
incubation, 100 μl of sodium dodecyl sulfate-dimethylformamide (SDS-DMF)
155
solution was added to each well and the assay was allowed to develop overnight. The
156
plates were read at 570 nm. Data are expressed as values of the stimulation index (SI).
157
The SI value was calculated as the ratio of the absorbent value at 570 nm of pools
158
incubated with SEB compared to pools incubated with medium only.
159
2.6 Cytokine quantitation
160
To measure cytokines, a standard curve was first prepared using known
161
concentrations of recombination cytokines. After stimulation with SEB with/without
162
the addition of the candidate aptamer, human TNFα, IL-1β, IL-6, IL-2, and IFNγ in
163
culture supernatants from PBMCs were measured with ELISA according to the kit
164
instructions (R&D Systems, Minneapolis, USA). Cytokines in each sample were
165
measured from the standard curve and the detection limit of all assays was 20 pg/ml.
166
2.7 Aptamer binding assay for the MHC II molecules
167
The binding ability of aptamer A11 to the MHC II molecules was measured with
168
a competition assay as described previously (16). Human Raji cells (2 × 105 cells)
169
were incubated in 100 μl of selection buffer (1% BSA) with varying final
170
concentration of SEB (0, 25, 50, 75, or 100 μg/ml) and with or without 5-fold molar
171
of A11 for 40 min on ice. Cells were washed in selection buffer and incubated with
172
FITC-conjugated SEB (FITC-SEB, 50 μg/ml), prepared using a FluoReporter® FITC
173
Protein Labeling Kit (Amresco, Ohio, USA). After extensive washing, Raji cells were
174
resuspended and analyzed by flow cytometry. The surface fluorescence of Raji cells
175
was measured as mean fluorescence of three independent experiments.
176
2.8 Murine experiments
177
To investigate the therapeutic efficacy of aptamers in vivo on SEB-induced TSS,
178
a “double-hit” murine model was established (24). Pathogen-free female BALB/c
179
mice of 10–12 weeks-of-age were purchased from the Experimental Animal Centre of
180
Fuzhou General Hospital of Nanjing Military Command (Fuzhou, China). The mice
181
were housed under controlled conditions and fed commercial mouse chow and water.
182
Groups of mice (N=19) were sensitized by intraperitoneal injection of
183
D-galactosamine (GalN, 20 mg/animal, Sigma, St. Louis, USA), and then challenged
184
by intraperitoneal injection of SEB (20 μg/animal) at 4 h. After treatment, mice were
185
fasted but allowed ad libitum access to water.
186
For therapeutic investigations, PEGlyated aptamer (14 nmol) was injected
187
intravenously 1 h before or 2 h after SEB injection. All agents were dissolved in
188
sterile nonpyrogenic saline solution (pH=7.2) and were injected intravenously via the
189
tail vein at 10 μl/g. Mouse survival was calculated by Kaplan-Meier survival analyses.
190
The IACUC of Fuzhou General Hospital of Nanjing Military Command approved all
191
experimental procedures.
192
2.9 Statistical analysis
193
Results are presented as means ± S.D. and analyzed for significant differences by
194
the Student's t-test. Kaplan-Meier methods were used to compute survival analyses. A
195
value of P < 0.05 was considered statistically significant. All data were processed by
196
SPSS 12.0 for Windows.
197
198
3. Results
199
3.1 In vitro selection and sequence analysis of SEB binding aptamers
200
3.1.1 Relative binding rate of ssDNA and SEB
201
Eleven rounds of in vitro selection were used to isolate aptamers that could
202
favorably bind to SEB. Figure 1 depicts the binding ratio of the ssDNA pool to SEB
203
which increased gradually with increasing number of selection rounds, and then
204
remained nearly constant in the last three rounds. This suggested that SEB binding
205
aptamers with good binding affinity were enriched, and the binding ability of
206
selection pools plateaued after about ten rounds of selection.
207
3.1.2 Cloning, sequencing, and sequence analysis of oligonucleotide aptamers
208
The enriched ssDNA pool of the 11th round was cloned into E. coli DH5α using
209
the pGEM-T vector system. Forty individual clones were chosen randomly and
210
sequenced. Multiple sequence alignments revealed that the primary clone structures
211
were modestly enriched. In total, 6 nonredundant sequences were identified, and 13
212
sequences were identical to aptamer A1 and 8 variants of aptamer A1, which were
213
highly homologous with aptamer A1, with only one or two base differences. Of all
214
sequences, 5 A2, 3 A3, 2 A4, 1 A5, and 1 A6 were identified. Although each aptamer
215
had the characteristic stem-loop or hairpin, their secondary structures predicted by
216
software were significantly different. Although all aptamers can bind with SEB (ESI
217
Figure S1 †), and some had dissociation constants in the low nanomolar range, none
218
could inhibit SEB function except aptamer A2, which had a weak inhibitory effect
219
(Figure 2A).
220
3.2 Screening functional aptamers by PBMC proliferation assay
221
To identify functional aptamers that can inhibit SEB superantigen activity, 40
222
individual clones from the 10th round were sequenced further as mentioned above. In
223
total, 29 nonredundant sequences were identified, and A1-A6 was from the 11th round
224
and A7-A29 was from the 10th round. Because superantigens can polyclonally activate
225
T cells, these 29 oligonucleotides were all chemically synthesized, and were tested for
226
inhibition of SEB-mediated proliferation of human PBMCs. Data show that
227
SEB-mediated proliferation of PBMCs decreased most significantly by A11 in a
228
dose-response manner, with maximal inhibition (93%) achieved at 20 times the
229
molarity of SEB compared to controls incubated with toxin alone (P < 0.05). At the
230
same concentration, A2 achieved 34% inhibition (P < 0.05). However, the other 27
231
did not significant inhibit binding (Fig. 2). A2 and A11 both bound with high affinity
232
to SEB, with dissociation constants of 26 nM and 64 nM respectively (Fig. 3A).
233
Although no significant between-aptamer sequence similarity was found, secondary
234
structures of A11 and A2 were predicted to be similar, and both had the characteristic
235
stem-loop (Fig. 3B). Thus, the candidate aptamer A11 could potentially inhibit
236
superantigen activity of SEB in vitro.
237
3.3 Inhibition of SEB-mediated cytokine release from human PBMCs
238
To confirm the inhibitory role of A11, the effect of A11 on the production of
239
pro-inflammatory cytokines was examined with ELISA in human PBMCs incubated
240
with SEB. Fig. 4 shows that A11 dose dependently reduced cytokine production from
241
PBMCs incubated with SEB, compared to controls incubated with toxin alone
242
(P
1
Neutralization of Staphylococcal enterotoxin B by aptamer
2
antagonist
3
Kaiyu Wanga,b, Longjie Ganb, Li Jiangb, Xianhui Zhangb, Xiangyue Yanga,b, Min Chena,b,
4
Xiaopeng Lana,b
5
a
6
Fuzhou 350025, PR China, and Department of Laboratory Medicine, Dongfang Hospital,
7
Xiamen University, Fuzhou 350025, P.R. China
8
b
9
Fuzhou 350025, P.R. China
10 11
*
Institute of Laboratory Medicine, Fuzhou General Hospital of Nanjing Military Command,
Department of Laboratory Medicine, Fuzong Clinical College, Fujian Medical University,
Email: [email protected]; Tel: +86 591 22859490
12
Abstract
13
Staphylococcal enterotoxin B (SEB) is a major virulence factor for
14
Staphylococcal toxic shock syndrome (TSS). SEB activates a large subset of the T
15
lymphocytic
16
SEB-initiated toxicity may be an effective strategy for treating TSS. Using a
17
systematic evolution of ligands by exponential enrichment (SELEX) process, we
18
identified an aptamer that can antagonize SEB with nanomolar binding affinity (Kd =
19
64 nM). The aptamer antagonist effectively inhibits SEB-mediated proliferation and
20
cytokine secretion in human peripheral blood mononuclear cells. Moreover, a
21
PEGlyated aptamer antagonist significantly reduced mortality in a “double-hit” mouse
22
model of SEB-induced TSS, established via sensitization with D-galactosamine
23
followed by SEB challenge. Therefore, our novel aptamer antagonist may offer
24
potential therapeutic efficacy against SEB-mediated TSS.
25
Keywords: superantigens; aptamer; Staphylococcal enterotoxin B; toxic shock
population,
releasing
pro-inflammatory
cytokines.
Blocking
26
1. Introduction
27
Staphylococcal enterotoxins (SEs) are prototype superantigens excreted
28
primarily by Staphylococcus aureus and Streptococcus pyogenes. SEs are potent
29
activators of the immune system that can stimulate up to 40% of the naive T cell
30
population and cause diverse human diseases, including food poisoning, autoimmune
31
diseases, and toxic shock syndrome (TSS) (1). SEs can activate both
32
monocytes/macrophages and T lymphocytes by directly binding to MHC II molecules
33
and to the variable region of the β-chain of T-cell receptors (TCR), without being
34
taken up and presented by antigen presenting cells (2).
35
These toxins lead to the proliferation of monocytes/macrophages and T
36
lymphocytes and stimulate secretion of excess pro-inflammatory cytokines, including
37
interferon gamma (IFNγ), interleukin 2 (IL-2), and tumor necrosis factor alpha
38
(TNFα). Among these SEs, Staphylococcal enterotoxin B (SEB) is considered to be an
39
important cause of Staphylococcal TSS, which is characterized by high fever,
40
erythematous rash, hypotension, multi-organ system failure, and even death (3).
41
Remarkably, SEB is highly heat resistant, easily transformed into an aerosol, and
42
readily produced on a large scale using genetic engineering techniques. Humans are
43
extremely susceptible to this superantigen, so SEB is often referred to as an
44
"incapacitating agent" by the military who may use this toxin as an anti-personnel
45
aerosol or as a food supply contaminant (4).
46
At present, SEB is thought to be an effective treatment strategy for TSS by
47
blocking early processes prior to T cell activation and initiation of the toxic cascade.
48
Studies suggest multiple approaches for interfering with SEB binding to MHC-II
49
and/or TCR. These include vaccine immunization to induce anti-SEB antibodies (5, 6),
50
passive immunoprophylaxis with natural or artificial antibodies (7, 8), and the use of
51
synthetic peptides (9) or receptor mimics (10, 11) as antagonists to prevent the
52
formation of the MHC-II/SEB/TCR complex. Research is ongoing for new
53
compounds with greater half-lives, improved SEB binding affinity, and less
54
immunogenicity.
55
Aptamers are artificial short, single-stranded oligonucleotides (ssDNA or ssRNA)
56
that can form elaborate 3-D structures and specifically bind to desired targets with
57
high affinity (12, 13). Because of their intrinsic properties, aptamers are non-toxic and
58
non-immunogenic. In addition, aptamers composed of modified nucleotides have a
59
long in vivo half-life of hours to days.
60
During the past two decades, numerous aptamers have been developed to inhibit
61
pathogenic antigen activity and many have substantial clinical potential for treating
62
various diseases (14). Several researchers have reported on the selection of specific
63
DNA aptamers against SEB (15-17). Methods using these aptamers as capture
64
molecules have also been established to detect SEB in various samples (15, 18).
65
However, as far as we know, no aptamer has been selected to inhibit the biological
66
activity of SEB.
67
Therefore, we selected DNA aptamers directed against native SEB in vitro and
68
identified aptamer-antagonists capable of neutralizing SEBs by studying their
69
therapeutic effects on SEB-mediated toxic shock (TSS) in vitro using human
70
peripheral blood mononuclear cell (PBMC) cultures as well as a lethal murine model.
71
72
2. Material and Methods
73
2.1 SELEX library and primers
74
The ssDNA library, which contained random 60mer and fixed primer regions and
75
all primers used are described previously (ESI Table S1†) (19). All oligonucleotides
76
were synthesized and purified with HPLC (Invitrogen, Guangzhou, China). For
77
animal experiments, the aptamer candidate was conjugated at the 5’ end with 40-KDa
78
polyethylene glycol (PEG). The PEGlyated aptamer was prepared by treating 40-KDa
79
PEG N-hydroxysuccinimide ester with 5'-amino and 3'-inverted dT modified aptamer
80
candidates. All PEG-aptamer conjugates were purified by anion exchange and then by
81
reverse-phase HPLC (Seebio Biotech, Shanghai, China).
82
2.2 Aptamer screening for specific SEB binding
83
ssDNA aptamers specific to SEB were screened by a SELEX process as
84
described previously with minor modifications (20). Briefly, highly purified SEB
85
(Toxin Technology, Sarasota, USA) was coupled to M-270 carboxylic acid dynabeads
86
(DYNAL, Oslo, Norway) according to the manufacturer's instructions. Prior to
87
selection, the ssDNA library dissolved in 500 μl selection buffer (50 mM Tris. HCl
88
pH7.4, 5 mM KCl, 100 mM NaCl, 1 mM MgCl2) was denatured by heating at 95 oC
89
for 5 min and immediately placed on ice for 10 min. The denatured ssDNA library
90
and a 5-fold molar excess of yeast tRNA (Invitrogen) were incubated with SEB (100
91
ng) immobilized on magnetic beads in a 500 μl reaction, and were incubated at 37 oC
92
for 60 min while shaking.
93
Nonbinding oligonucleotides were removed by washing the DNA-bead complex
94
three times with selection buffer (0.2% BSA). SEB binding species were eluted by
95
heating at 100 oC for 5 min in 200 μl of sterile ddH2O. ssDNA was amplified by PCR
96
with FITC-labeled sense primers and biotin-labeled antisense primers (20-30 cycles of
97
0.5 min at 94 oC, 0.5 min at 52 oC, 0.5 min at 72 oC, followed by 5 min at 72 oC). All
98
PCR products were purified using a BioSpin gel extraction kit (Bioer Technology,
99
Hangzhou, China). From all purified PCR products from each SELEX round, large
100
aliquots were alkaline-denatured and used to prepare the aptamer strand using M-280
101
streptavidin dynabeads (DYNAL AS, Oslo, Norway) according to the manufacturer's
102
instructions, and small aliquots were stored at -80 oC prior to use.
103
To create aptamers with high affinity and specificity, we progressively increased
104
the selective pressure by increasing the number of washes and KCl, and by decreasing
105
the ratio of ssDNA to SEB and the incubation time (ESI Table S2†). The
106
counter-selection (depletion of aptamers that bind to non-target molecules) was
107
introduced against BSA-or Staphylococcal protein A (SPA)-coupled M-270 beads in
108
the 7th, the 9th, and the 11th round, respectively. BSA was the blocking protein for SEB
109
coupling, and SPA is a major protein of Staphylococcus aureus(21). To reduce the
110
matrix binders, reaction tubes were blocked with 1% BSA-PBS, and preselection
111
steps with uncoupled streptavidin beads were performed.
112
2.3 Cloning, sequencing and bioinformatic analysis
113
After rounds of SELEX selection, the selected ssDNA pool was PCR-amplified
114
using unlabeled primers under the same conditions described for screening. PCR
115
products were purified, and then cloned into pGEM-T vector (Promega, Madison,
116
USA) according to the manufacturer’s instructions. The resulting products were
117
transformed into Escherichia coli DH5α. Individual cultured colonies were chosen
118
randomly and their inserts were sequenced by Invitrogen Company. The aptamer
119
sequences were analyzed by ClustalX software and the secondary structure was
120
predicted by free-energy minimization algorithm according to Zuker (22) using the
121
internet-tool mfold (http://mfold.bioinfo.rpi.edu/cgi-bin/dna-form1.cgi).
122
2.4 Aptamer binding assay for SEB
123
A fluorescently labeled ssDNA binding assay was performed to monitor the
124
enrichment of each SELEX round and to evaluate aptamer binding affinity. In brief,
125
fluorescently labeled ssDNA was thermally denatured in 200 μl of selection buffer
126
and then incubated in the dark with SEB immobilized on magnetic beads at 37 oC for
127
1 h while shaking. After incubation, non-bound ssDNA was collected, and SEB-bound
128
ssDNA was eluted by heating at 100 oC for 5 min in 200 μl of selection buffer. The
129
fluorescent intensity of applied, non-bound, and eluted ssDNA was measured
130
respectively using a TBS-380 Mini-Fluorometer (Turner Biosystems, Sunnyvale, CA).
131
All binding assays were repeated three times. To monitor the enrichment of each
132
SELEX round, the percent of ssDNA (200 nM) binding with SEB (20 μM) was
133
calculated.
134
To determine the binding affinity of a selected aptamer, the binding assay was
135
performed as described above but with increasing amounts of ssDNA aptamer (0 to
136
400 nM) and a constant amount of SEB (20 μM) for each assay. To calculate the
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dissociation constants (Kd) of the aptamers, the aptamer quantity bound to targets was
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plotted and data points were fitted to the equation Y=BmaxX/(Kd+X) via non-linear
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regression analysis, using Graphpad-Prism-V5.01 (Graphpad, San Diego, CA).
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2.5 Human PBMC proliferation assays
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To study the effect of aptamers on SEB-mediated T cell proliferation, an MTT
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cell proliferation assay was performed with human PBMCs as described previously
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(23). Human PBMCs were isolated from heparinized venous blood by Ficoll-Hypaque
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density gradient centrifugation from healthy human donors. After lysing erythrocytes,
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PBMCs were cultured in RPMI 1640 medium (Gibco, Grand Island, USA)
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supplemented with 2 mM L-glutamine, 100 U/ ml penicillin, 100 mg/ml streptomycin,
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10 mM HEPES and 10% (v/v) fetal bovine serum (Gibco) at 37 oC. PBMCs (105
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cells/well) were plated with SEB (200 ng/ml) alone or combined with varying
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concentrations of aptamer candidates in triplicate in a 96-well plate.
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PBMCs cultivated in RPMI-1640 medium alone served as negative control.
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Plates were incubated in a 5% CO2 humidified incubator at 37 oC for 48 h. After
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stimulation, cell proliferation was measured by adding 20 μl of MTT solution (Sigma,
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St. Louis, USA), prepared in PBS at 5 mg/ml (pH 7.2), to each well. After 2 h of
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incubation, 100 μl of sodium dodecyl sulfate-dimethylformamide (SDS-DMF)
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solution was added to each well and the assay was allowed to develop overnight. The
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plates were read at 570 nm. Data are expressed as values of the stimulation index (SI).
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The SI value was calculated as the ratio of the absorbent value at 570 nm of pools
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incubated with SEB compared to pools incubated with medium only.
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2.6 Cytokine quantitation
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To measure cytokines, a standard curve was first prepared using known
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concentrations of recombination cytokines. After stimulation with SEB with/without
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the addition of the candidate aptamer, human TNFα, IL-1β, IL-6, IL-2, and IFNγ in
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culture supernatants from PBMCs were measured with ELISA according to the kit
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instructions (R&D Systems, Minneapolis, USA). Cytokines in each sample were
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measured from the standard curve and the detection limit of all assays was 20 pg/ml.
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2.7 Aptamer binding assay for the MHC II molecules
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The binding ability of aptamer A11 to the MHC II molecules was measured with
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a competition assay as described previously (16). Human Raji cells (2 × 105 cells)
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were incubated in 100 μl of selection buffer (1% BSA) with varying final
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concentration of SEB (0, 25, 50, 75, or 100 μg/ml) and with or without 5-fold molar
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of A11 for 40 min on ice. Cells were washed in selection buffer and incubated with
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FITC-conjugated SEB (FITC-SEB, 50 μg/ml), prepared using a FluoReporter® FITC
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Protein Labeling Kit (Amresco, Ohio, USA). After extensive washing, Raji cells were
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resuspended and analyzed by flow cytometry. The surface fluorescence of Raji cells
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was measured as mean fluorescence of three independent experiments.
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2.8 Murine experiments
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To investigate the therapeutic efficacy of aptamers in vivo on SEB-induced TSS,
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a “double-hit” murine model was established (24). Pathogen-free female BALB/c
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mice of 10–12 weeks-of-age were purchased from the Experimental Animal Centre of
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Fuzhou General Hospital of Nanjing Military Command (Fuzhou, China). The mice
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were housed under controlled conditions and fed commercial mouse chow and water.
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Groups of mice (N=19) were sensitized by intraperitoneal injection of
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D-galactosamine (GalN, 20 mg/animal, Sigma, St. Louis, USA), and then challenged
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by intraperitoneal injection of SEB (20 μg/animal) at 4 h. After treatment, mice were
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fasted but allowed ad libitum access to water.
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For therapeutic investigations, PEGlyated aptamer (14 nmol) was injected
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intravenously 1 h before or 2 h after SEB injection. All agents were dissolved in
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sterile nonpyrogenic saline solution (pH=7.2) and were injected intravenously via the
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tail vein at 10 μl/g. Mouse survival was calculated by Kaplan-Meier survival analyses.
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The IACUC of Fuzhou General Hospital of Nanjing Military Command approved all
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experimental procedures.
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2.9 Statistical analysis
193
Results are presented as means ± S.D. and analyzed for significant differences by
194
the Student's t-test. Kaplan-Meier methods were used to compute survival analyses. A
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value of P < 0.05 was considered statistically significant. All data were processed by
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SPSS 12.0 for Windows.
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3. Results
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3.1 In vitro selection and sequence analysis of SEB binding aptamers
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3.1.1 Relative binding rate of ssDNA and SEB
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Eleven rounds of in vitro selection were used to isolate aptamers that could
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favorably bind to SEB. Figure 1 depicts the binding ratio of the ssDNA pool to SEB
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which increased gradually with increasing number of selection rounds, and then
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remained nearly constant in the last three rounds. This suggested that SEB binding
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aptamers with good binding affinity were enriched, and the binding ability of
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selection pools plateaued after about ten rounds of selection.
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3.1.2 Cloning, sequencing, and sequence analysis of oligonucleotide aptamers
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The enriched ssDNA pool of the 11th round was cloned into E. coli DH5α using
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the pGEM-T vector system. Forty individual clones were chosen randomly and
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sequenced. Multiple sequence alignments revealed that the primary clone structures
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were modestly enriched. In total, 6 nonredundant sequences were identified, and 13
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sequences were identical to aptamer A1 and 8 variants of aptamer A1, which were
213
highly homologous with aptamer A1, with only one or two base differences. Of all
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sequences, 5 A2, 3 A3, 2 A4, 1 A5, and 1 A6 were identified. Although each aptamer
215
had the characteristic stem-loop or hairpin, their secondary structures predicted by
216
software were significantly different. Although all aptamers can bind with SEB (ESI
217
Figure S1 †), and some had dissociation constants in the low nanomolar range, none
218
could inhibit SEB function except aptamer A2, which had a weak inhibitory effect
219
(Figure 2A).
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3.2 Screening functional aptamers by PBMC proliferation assay
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To identify functional aptamers that can inhibit SEB superantigen activity, 40
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individual clones from the 10th round were sequenced further as mentioned above. In
223
total, 29 nonredundant sequences were identified, and A1-A6 was from the 11th round
224
and A7-A29 was from the 10th round. Because superantigens can polyclonally activate
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T cells, these 29 oligonucleotides were all chemically synthesized, and were tested for
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inhibition of SEB-mediated proliferation of human PBMCs. Data show that
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SEB-mediated proliferation of PBMCs decreased most significantly by A11 in a
228
dose-response manner, with maximal inhibition (93%) achieved at 20 times the
229
molarity of SEB compared to controls incubated with toxin alone (P < 0.05). At the
230
same concentration, A2 achieved 34% inhibition (P < 0.05). However, the other 27
231
did not significant inhibit binding (Fig. 2). A2 and A11 both bound with high affinity
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to SEB, with dissociation constants of 26 nM and 64 nM respectively (Fig. 3A).
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Although no significant between-aptamer sequence similarity was found, secondary
234
structures of A11 and A2 were predicted to be similar, and both had the characteristic
235
stem-loop (Fig. 3B). Thus, the candidate aptamer A11 could potentially inhibit
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superantigen activity of SEB in vitro.
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3.3 Inhibition of SEB-mediated cytokine release from human PBMCs
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To confirm the inhibitory role of A11, the effect of A11 on the production of
239
pro-inflammatory cytokines was examined with ELISA in human PBMCs incubated
240
with SEB. Fig. 4 shows that A11 dose dependently reduced cytokine production from
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PBMCs incubated with SEB, compared to controls incubated with toxin alone
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(P
